Facultad de Farmacia - Universidad de...

Facu l t ad de Fa rmac i a

DESIGN, SYNTHESIS AND STUDY OF QUINOXALINE‐2‐

CARBOXAMIDE 1,4‐DI‐N‐OXIDE DERIVATIVES AS ANTI‐

TUBERCULOSIS AGENTS

Elsa Moreno de Viguri

Facu l t ad de Fa rmac i a

DESIGN, SYNTHESIS AND STUDY OF QUINOXALINE‐2‐

CARBOXAMIDE 1,4‐DI‐N‐OXIDE DERIVATIVES AS ANTI‐

TUBERCULOSIS AGENTS

Elsa Moreno de Viguri

F a c u l t a d d e F a rm a c i a

DESIGN, SYNTHESIS AND STUDY OF QUINOXALINE‐2‐CARBOXAMIDE 1,4‐DI‐N‐OXIDE DERIVATIVES AS ANTI‐TUBERCULOSIS AGENTS

Memoria presentada por Dª Elsa Moreno de Viguri para aspirar al grado de Doctor por la Universidad de Navarra.

El presente trabajo ha sido realizado bajo nuestra dirección en la Unidad I+D de Medicamentos (C.I.F.A.) y autorizamos su presentación ante el Tribunal que lo ha de juzgar.

Pamplona, 4 de abril de 2011

Dr. Antonio Monge Vega Dra. Silvia Pérez Silanes

Este trabajo ha sido desarrollado en la Unidad I+D de Medicamentos del

Centro de Investigación en Farmacobiología Aplicada (C.I.F.A.) de la

Universidad de Navarra y pertenece a uno de los proyectos de investigación

que dirige el Dr. Antonio Monge Vega, Doctor en Ciencias Químicas.

El trabajo presentado en esta memoria ha sido realizado gracias a la concesión

de una ayuda: “Ayuda Predoctoral para la Formación de Personal

Investigador” concedida por el Gobierno de La Rioja.

La estancia de tres meses en el “Environment and Life Sciences Department”

de la Università degli Studi del Piemonte Orientale “Amedeo Avogadro”.

(Alessandria, Italia) ha sido realizada gracias a la “Ayuda para estancias breves

en centros de investigación extranjeros” concedida por el Gobierno de La

Rioja.

La realización de este trabajo ha sido posible gracias a la financiación

procedente de un proyecto PIUNA (2009-2011) de la Universidad de Navarra.

A mis padres ,

A Javi

Intentar significa arriesgar, arriesgar significa probablemente perder,

perder significa aprender, aprender significa siempre ganar.

Anónimo

La verdadera ciencia enseña, por encima de todo, a dudar y a ser ignorante.

Miguel de Unamuno

AGRADECIMIENTOS / ACKNOWLEDGMENTS

La realización de esta tesis doctoral no hubiera sido posible sin el apoyo de todas

las personas que han estado a mi lado durante estos años, tanto en el plano profesional

como personal. A todos ellos, que han contribuido a que estos sean los años más felices,

deseo expresar mi más sincero agradecimiento.

Al Dr. Antonio Monge, la persona responsable de que decidiera adentrarme en el

mundo de la investigación. Gracias por haberme brindado la oportunidad de formar parte

de su grupo y por el espíritu investigador transmitido. Por depositar su confianza en mí y

transmitirme la pasión por la investigación. Por hacer siempre presente la parte más

humana de los proyectos, dándole sentido al trabajo del día a día.

A la Dra. Silvia Pérez Silanes, por su implicación e interés constantes a lo largo de

toda la tesis. Por su disponibilidad y su paciencia. Por su confianza en mí y por aportar

siempre el optimismo necesario para seguir adelante.

Al Dr. Ignacio Aldana, por su apoyo y su ayuda siempre que la he necesitado. Por

creer en mí y transmitirme que era capaz de conseguir todo lo que me propusiera.

To Dr. Philip Crawford for performing the electrochemical studies and for his help

in the discussion of the results.

Al Prof. Domenico Osella per avermi dato la possibilità di lavorare nel

"Dipartimento di Scienze della Vita e dell'Ambiente" dell'Università degli Studi del

Piemonte Orientale "Amedeo Avogadro" di Alessandria. Al Dt. Mauro Ravera per il suo

coinvolgimento nel lavoro. Alla Dt. Elisabetta Gabbano per il suo continuo interesse, lo

sforzo continuo, i consigli e le questioni che sono state di grande interesse e sostegno per la

realizzazione della tesi.

To Dr. James Platts for his helpful advices in the design of partition coefficient

study.

A todos los compañeros con los que he compartido el trabajo de laboratorio a lo

largo de los años.

A mis amigos y mi familia, especialmente a Javi y a mis padres, que siempre han

estado apoyándome para seguir adelante.

Abbreviations

i

ABBREVIATIONS

AFB Acid Fast Bacilli

AFR WHO African Region

AMR WHO Regions of the Americas

BCG Bacille Calmette Guerin

BFX Benzofuroxans

CC50 50% Inhibitory Concentration Cell Citotoxicity

CDC Centers for Disease Control and Prevention

CSU Colorado State University

DMID Division of Microbiology and Infectious Diseases

DMSO Dimethyl sulfoxide

DOTS Directly Observed Treatment Short course

E Potential

ELISA Enzyme Linked Immunosorbent Assay

EMB Ethambutol

EMR WHO Eastern Mediterranean Region

EUR WHO European Region

Fc Ferrocene

HBC High-Burden Country

HIV Human Immunodeficiency Virus

HPLC High Performance Liquid Chromatography

1H-RMN Proton Nuclear Magnetic Resonance

Hz Hertz

IC50 50% Inhibitory Concentration

IC90 90% Inhibitory Concentration

IFN-γ Interferon-gamma

INH Isoniazid

IR Infrared spectroscopy

IUPAC International Union of Pure and Applied Chemistry

Abbreviations

ii

J Coupling constant

logPo/w Octanol/water partition coefficient

LOO Leave-One-Out cross-validation procedure

MDG Millennium Development Goals

M.F. Molecular formula

MDR-TB Multidrug-resistant tuberculosis

MIC Minimum Inhibitory Concentration

MOPS 3-morpholinopropanesulfonic acid

M.tb Mycobacterium tuberculosis

M.P Melting Point

M.W. Molecular weight

NIAID National Institute of Allergy and Infectious Diseases

NMR Nuclear Magnetic Resonance

N,N-DMF N,N-dimethylformamide

OECD Organization for Economic Co-operation and Development

PAS p-aminosalicylic acid

PPD Purified Protein Derivative

PZA Pyrazinamide

RIF Rifampicin

RMSE Root Mean Squared Error

RNI Reactive Nitrogen Intermediates

ROI Reactive Oxygen Intermediates

RP-HPLC Reverse Phase High Performance Liquid Chromatography

R2 Regression coefficient

SAR Structure-Activity Relationship

SEAR WHO Shout-East Asia Region

SI Selectivity Index

SM Streptomycin

SPR Structure-Property Relationship

SRI Southern Research Institute

Abbreviations

iii

TAACF Tuberculosis Antimicrobial Acquisition & Coordinating Facility

TB Tuberculosis

TBAP Tetrabutylammonium perchlorate

TLC Thin Layer Chromatography

TNF-α Tumor Necrosis Factor-alpha

UV Ultraviolet

V Volt

WPR WHO Western Pacific Region

WHO World Health Organization

XDR-TB Extensively drug-resistant tuberculosis

δ Chemical sift

λ Wavelength

Content

v

CONTENT

INTRODUCTION I. TUBERCULOSIS 3

1. Definition 5 2. History of tuberculosis 5 3. Tuberculosis and the Mycobacterium tuberculosis complex 7 4. Epidemiology 8

4.1. Global burden of tuberculosis 8

4.2. Global strategies for fighting tuberculosis 10

5. Tuberculosis, neglected disease 12 6. Tuberculosis, the disease 13

6.1. Mycobacterium tuberculosis 13 General characteristics of M.tb 13

Cell envelope of M.tb 14

M.tb genome 15

M.tb metabolism 15

6.2. Transmission. Latent TB infection and active TB disease 17

6.3. Pathogenesis 17

6.4. Clinical tuberculosis 20

7. Symptoms and diagnostic 21 7.1. Symptoms 21

7.2. Diagnostic 21

8. Treatment and prevention 24 8.1. Treatment 24

Drugs 24

Treatment regimens 26

Drugs mechanisms and drug resistance mechanisms 26

8.2. Prevention 28 Chemoprophylaxis 28

Vaccine 28

9. New approaches to prevention and treatment of tuberculosis 29 9.1. Vaccines development 29

9.2. Drug discovery 29 Drug discovery methods 29

Content

vi

New TB drugs in clinical trials 31

9.3. Drug delivery systems 32

II. QUINOXALINES AND TUBERCULOSIS 35

10. Quinoxaline, a chemical structure with a broad range of biological activities 37

11. Quinoxaline 1,4-di-N-oxide derivatives and tuberculosis 40 III. ALTERNATIVE TOOLS IN MEDICINAL CHEMISTRY 47

12. Electrochemistry 49 13. logP 50

13.1. Introduction 50

13.2. logP. Concept 53

13.3. Measurements of logP 54 Shake-flask method 54

Reverse-phase high performance liquid chromatographic methods 54

HYPOTHESIS AND OBJECTIVES IV. HYPOTHESIS 59

V. OBJECTIVES 63 VI. WORK PLAN 67

14. Bibliographic review: state of the art 69 15. Design of new quinoxaline di-N-oxide derivatives 69

16. Synthesis of the designed compounds 71 17. Characterization of the synthesized compounds 73 18. In vitro biological evaluation of the synthesized derivatives 73 19. Study of the lipophilicity of quinoxaline di-N-oxide derivatives 73 20. Study of the electrochemical behavior of quinoxaline

di-N-oxide derivatives 73 21. Study of possible structure-property and structure-activity

relationships (SPR, SAR) 74

22. Search of new lead compounds and design of future work

plans 74

Content

vii

MATERIAL AND METHODS VII. CHEMICAL SYNTHESIS 77

23. General synthetic scheme 79 24. Chemistry of benzofuroxans 80

24.1. Synthesis of benzofuroxans 80 Synthesis of benzofuroxans through oxidative cyclization (comp. c, g) 80

Synthesis of benzofuroxans via azides (comp. d) 81

24.2. Position isomers of 5(6)-monosubstituted benzofuroxans 82

25. Chemistry of β-ketoamides 83 25.1. Synthesis of N-benzyl-3-oxo-3-phenylpropanamide (comp. 2) 83

25.2. Synthesis of 3-oxo-N-(2-phenylethyl)-3-phenylpropanamide

(comp. 3) 85

25.3. Synthesis of N-substituted 3-methyl-3-oxopropanamides

(comp. 4-10) 86

26. Chemistry of quinoxaline di-N-oxide derivatives 87 26.1. Synthesis of quinoxaline di-N-oxide derivatives. The Beirut

reaction 87

26.2. Position isomers of 6(7)-monosubstituted quinoxalines 88

26.3. Photochemical reactions of quinoxalines di-N-oxide 89

VIII. EXPERIMENTAL CHEMICAL SECTION 91 27. Material and reagents 93

28. General synthetic methods 95 28.1. Method A. General methods for synthesis of benzofuroxans 96

Method A1. General method for synthesis of benzofuroxans

(comp. c, g) 96

Method A2. General method for synthesis of 5-trifluoromethyl

benzofuroxans (comp. d) 96

28.2. Method B. General methods for synthesis of N-substituted-

3-oxo-3-phenylpropanamides 97 Method B1. General method for synthesis of N-benzyl-3-oxo-3-

phenylpropanamide (comp. 2) 97

Method B2. General method for synthesis of 3-oxo-N-(2-phenylethyl)-

3-phenylpropanamide (comp. 3) 98

28.3. Method C. General method for synthesis of N-substituted-3-

methyl-3-oxopropanamides (comp. 4-10) 98

Content

viii

28.4. Method D. General method for synthesis of quinoxaline-2-

carboxamide 1,4-di-N-oxide derivatives 99

29. Purification Methods: Automated Flash Column Chromatography 100 30.1. Introduction 100

30.2. Basic elements of advanced flash column chromatography 100

IX. BIOLOGICAL EVALUATION. ANTI-TUBERCULOSIS ACTIVITY 103 30. TAACF anti-tuberculosis evaluation 105

31.1. Primary screening (Dose response): Determination of a

90% inhibitory concentration (IC90) 106

31.2. Secondary screening: Determination of mammalian cell

cytotoxicity (CC50) 106

31. DMID anti-tuberculosis evaluation 106 32.1. MIC assay 106

X. ELECTROCHEMICAL STUDY 109 XI. DETERMINATION OF PARTITION COEFFICIENT 113

32. Shake Flask method 115 33.1. Material and reagents 115

33.2. Experimental 115

33.3. Evaluation of experimental data 115

33. Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method 116 34.1. Material and reagents 116

34.2. Experimental 116

34.3. Cross-validation of the RP-HPLC method 117

34. logP predictive approaches 118

RESULTS AND DISCUSSION XII. COMPOUNDS CHARACTERIZATION 123 XIII. PURIFICATION METHODS: AUTOMATED FLASH COLUMN

CHROMATOGRAPHY 179 XIV. ANTI-TUBERCULOSIS ACTIVITY 183

35. Anti-tuberculosis results 185 35.1. TAACF anti-tuberculosis results 185

Content

ix

Primary screening results 185

Secondary screening results 189

35.2. DMID anti-tuberculosis results 191 MIC assay results 191

36. Discussion of the anti-tuberculosis activity 192 XV. ELECTROCHEMICAL STUDY 195

37. Electrochemical results 197 38. Discussion 200

38.1. Discussion. Electrochemical behavior 200

38.2. Discussion. Relationship between electrochemical behavior

and anti-tuberculosis activity 202

XVI. PARTITION COEFFICIENT STUDIES 207 39. Shake flask method results 209

40. RP-HPLC method results 209 40.1. Correlation between logPo/w and logk’0 209

40.2. logPo/w of quinoxalines di-N-oxide 210

41. Calculated logPo/w 211 42. Discussion 213

42.1. Discussion. Comparative of experimental methods and

predictive approaches 213 42.2. Discussion. Relationship between logPo/w and

anti-tuberculosis activity 215

CONCLUSIONS 217

CONCLUSIONES 221

REFERENCES 227

APPENDIXES 247 1. Relationship of synthesized compounds 249 2. Stability of quinoxalines di-N-oxide in aqueous solution 257 3. Future perspectives 259

4. Papers 263

INTRODUCTIONINTRODUCTION

I. TUBERCULOSIS

Introduction: Tuberculosis

5

1. Definition

Tuberculosis (TB) is an infectious respiratory transmitted disease mainly caused by

Mycobacterium tuberculosis (M.tb). TB usually affects the lungs, but it can also affect other parts

of the body, such as the brain, the kidneys, or the spine.1,2

Before studying TB, it is essential to understand the difference between M.tb

infection and TB disease. It is estimated that one-third of the world’s population is infected

by M.tb. TB bacteria can remain in a dormant state for months or years without making the

person sick. In this case, the term used would be latent TB. However, one out of ten

people infected with M.tb develops active TB during his/her life time, which is referred to

as TB disease.1,2

It is also important to define resistant TB before studying TB in depth. Multidrug-

resistant tuberculosis (MDR-TB) is defined as TB caused by strains of M.tb that are

resistant to at least isoniazid (INH) and rifampicin (RIF), two of the most used drugs

against TB. Extensively drug-resistant tuberculosis (XDR-TB) is defined as TB caused by

strains that are resistant not only to INH and RIF but also to a fluoroquinolone and at least

one second-line injectable agent.2-4

2. History of tuberculosis5-8

TB has a long history. Typical TB-caused skeletal abnormalities have been found in

Egyptian mummies and TB was documented in Egypt, India, and China 5,000, 3,300 and

2,300 years ago, respectively.

From the time of Hippocrates, TB was known as “phthisis”, a term derived from

the Greek meaning consumption, wasting away. It was Hippocrates who, in 460 BC,

identified “phthisis” as the most widespread disease. Aristotle considered the disease to be

contagious, although most Greek authors believed it to be hereditary. Galen defined

“phthisis” as an ulceration of the lungs, chest or throat, accompanied by coughs, low fever,

and wasting away of the body because of pus. During these early times, the efforts to cure

the disease were based on trial and error. Depending upon the time and country, patients

were urged to rest or to practice exercise, to eat or to abstain from food.

The TB epidemic in Europe, known as “The White Plague”, probably started at the

beginning of the 17th century and it was in this century when precise pathological and

anatomical descriptions of the disease began to appear. Girolamo Fracastoro explained in


6

1546 the contagious nature of TB. In 1679, Franciscus Sylvius described the

characteristic lung nodules as “tubercula” and observed their evolution to cavities. Richard

Morton confirmed that tubercles were always present in the lungs of TB patients and

Gaspart Laurent Bayle definitely proved that tubercles were not products, or results, but

the cause of the disease. It was Benjamin Marten, in 1720, who assumed that TB could be

caused by “minute living creatures”, which could generate the lesion and symptoms of

“phthisis” and could be caught by a healthy person. Nevertheless, the evidence of the

infectious nature of TB was not obtained until 1868 when Villemin’s studies were

published.

Figure 1. Robert Koch. Source: www.nobelprize.org.

Finally, in 1882, Robert Koch (Figure 1) made a presentation to the Berlin

Physiological Society describing the tubercle bacillus, M.tb (still known as Koch’s bacillus),

and demonstrating that it was the cause of TB. Koch published four criteria (known as

Koch’s postulates) designed to establish a causal relationship between a causative microbe

and a disease: the organism must be found in all animals suffering from the disease but not

in healthy animals; the organism must be isolated from a sick animal and grown in pure

culture; the cultured organism should cause disease when introduce into a healthy animal;

and, the organism must be re-isolated from the experimentally infected animal. In 1890, he

also announced a compound, tuberculin, that cured the disease but the studies concluded

that it was not active. Nevertheless, tuberculin was proven to be valuable for the diagnosis

of TB.

In the 19th century, sanatoriums could be considered the first approach to anti-TB

treatment. In fact, sanatoriums provided a dual function. First, they protected the general

population by isolating the sick persons. Secondly, they offered TB patients bed-rest,

exercise, fresh-air, and good nutrition. Finally, during the 1960s, many sanatoria started to

close and TB treatment was substituted by active therapy.


7

The introduction of antibiotics, such as streptomycin (SM), isoniazid (INH), and p-

aminosalicylic acid (PAS), led to a TB chemotherapy revolution and TB mortality rates

were reduced considerably.

Nowadays, several strategies are being developed to globally combat TB, in terms

of diagnosis, prevention and treatment.

In relation with TB, the following Nobel Prizes have been awarded:9

• Robert Koch: Nobel Prize in Physiology or Medicine 1905 “for his investigations and

discoveries in relation to tuberculosis”.

• Paul Ehrlich: Nobel Prize in Physiology or Medicine 1908 “in recognition of their work

on immunity”.

• Selman Abraham Waksman: Nobel Prize in Physiology or Medicine 1952 “for his

discovery of streptomycin, the first antibiotic effective against tuberculosis”.

3. Tuberculosis and the Mycobacterium tuberculosis complex10

The Mycobacterium tuberculosis complex is considered to consist of seven species: M.

tuberculosis (M.tb), M. bovis, M. africanum, M. microtti, M. canettii, M. caprae and M. pinnipedii.

M.tb. is the most frequent cause of human TB, and this work will mainly refer to it.

Nevertheless, all the species will be briefly explained in this chapter.

M. bovis could be considered as the second cause of human TB. The main host of

M. bovis is cattle but it also affects other mammalians including man, becoming the main

cause of zoonotic TB in human. Nevertheless, after the introduction of milk pasteurization,

there was a clear impact on the deaths in children under five years of age because of M.

bovis.

M. africanum is predominantly isolated in Africa and it is considered to be one of the

main causes of pulmonary TB, in some areas of the continent. Considering biochemical

characteristics, two subgroups have been described and it has been reported that one is

more similar to M.tb and the other one to M.bovis.

M. microtti was first isolated in 1937 and it was considered to be no virulent for

humans, cattle and laboratory animals. Therefore, it was considered as a vaccine against

TB, its efficacy was assessed in clinical trials and it was used as a vaccine in Africa for more

than 15 years showing an efficacy similar to that of Bacille Calmette Guerin (BCG).


8

However, M. microtti has been identified as the causative agent of pulmonary TB in

immunocompromised and immunocompetent humans.

M. caprae has been found in Spain, Italy, Austria, Slovenia or the Czech Republic

and it is considered the main cause of TB in cattle and the predominant agent of TB in

humans, in central European regions.

Finally, M. pinnipedii has been isolated in New Zealand, Australia and South

America. In 2003, it was reported its ability to cause disease in guinea pigs and rabbits and

it has also been suggested that it can cause infection across a wide host range.

4. Epidemiology

4.1. Global burden of tuberculosis

According to the last report published by WHO,11 there were an estimated 9.4

million incident cases of TB in 2009. This data reveals that the absolute number of cases is

increasing slightly every year although a reduction in incidence rates can be observed due to

the increment in global population. In fact, the estimated global incidence fell to 137 cases

per 100,000 population in 2009, after peaking 142 cases per 100,000 population in 2004. Of

the 9.4 million incident cases, an estimated 1.1 million (12%) were HIV-positive and,

approximately, 80% of them were in the African region.11,12

Figure 2. Estimated TB incidence rates, by country, 2009. Source: Global Tuberculosis. Control

2010. WHO, 2010.


9

There were an estimated 14 million prevalent cases of TB and, approximately, 1.7

million people died of TB in 2009, being 1.3 million HIV-negative cases.11,12

Nowadays, there are 22 high-burden countries (HBC) that account for

approximately 80% of all new cases arising each year. The five countries with the largest

number of incident cases, in 2009, were India, China, South Africa, Nigeria and Indonesia.

India alone accounts for an estimated 21% of all TB cases worldwide and China for an

estimated 14%.

Considering the WHO regions, most of the estimated incidence cases occurred in

Asia (SEAR, 55%) and Africa (AFR, 30%); and smaller proportions occurred in the

Eastern Mediterranean Region (EMR, 7%), the European Region (EUR, 4%) and the

Region of the Americas (AMR, 3%).11 Globally, rates of incidences, prevalence and

mortality are all declining; although, regionally, incidence rates are declining in five of the

WHO’s six regions. The exception is the SEAR which incidence rate is stable (Figure 3).

Figure 3. Estimated incidence and case notifications rates by WHO region, 1990-2009.

Source: Global Tuberculosis. Control 2010. WHO, 2010.

With regard to resistant TB, there were an estimated 440,000 cases of MDR-TB in

2008 and 150,000 deaths from MDR-TB. In 2009, it was estimated that 3.3% of all new TB


10

cases presented MDR-TB. The 27 countries (15 in the European Region) that account for

86% of all cases have been termed the 27 high MDR-TB burden countries. The four

countries which presented the higher number of estimated MDR-TB cases, in 2008, were

China, India, the Russian Federation and South Africa. Finally, an important data to be

taken into account is that 58 countries had reported at least one cases of XDR-TB by July

2010 (Figure 4).4,11,12

Figure 4. Distribution of countries reporting at least one case of XDR-TB, by January

2010. Source: Multidrug and extensively drug-resistant TB (M/XDR-TB). 2010 Global report on

surveillance and response. WHO, 2010.

4.2. Global strategies for fighting tuberculosis

The consequences of TB on society are immense. Worldwide, one person out of

three is infected by M.tb. Every 20 seconds, someone in the world dies from TB and a

person is infected every second. Left untreated, a person with active TB will infect an

average of 10 to 15 other people every year.13

When a person suffers from TB disease, it usually means that he/she will be unable

to work and it involves that a family will be driven to poverty and children may have to

leave school to work themselves or care for the family. Although the direct costs of

diagnosis and treatment are significant for poor families, the greatest economic loss occurs

as a result of indirect costs, and the social consequences can not be forgotten. In addition,

there is good evidence that fighting TB is highly cost-effective.14


11

In 1993 the WHO declared TB a global public health emergency. TB resumption

has been attributed to several factors, such as the increase in drug resistance; the HIV

pandemic; changes in social structure; the increase of immigrant from high prevalence

nation to developed ones; and the aging of the world population.7

The Stop TB Partnership (www.stoptb.org/) was established in 2000 as a global

movement to accelerate social and political action to stop TB around the world. It provides

the platform for international organizations, countries, donors, governmental and non-

governmental organizations, patient organizations, and individuals to conduct a collective

campaign to stop TB. The Partnership’s goal is to eliminate TB as a public health problem,

and to secure a world free of TB. Moreover, the Millennium Development Goals (MDG)

have been endorsed by the Stop TB Partnership and two additional targets have been

included: by 2015, reduce prevalence and death rates by 50%, compared with their levels in

1990; and, eliminate TB as a public health problem, defined as a global incidence of active

TB of less that one case per 1 million population per year.13-15 In summary, the mission of

the Stop TB Partnership is to:

• Ensure that every TB patient has access to effective diagnosis, treatment and cure.

• Stop transmission of TB.

• Reduce the inequitable social and economic toll of TB.

• Develop and implement new preventive, diagnostic and therapeutic tools and strategies

to stop TB.16

In 2006, the WHO launched the Stop TB Strategy which consisted on:

• Pursue high-quality DOTS (Directly Observed Treatment Short course) expansion and

enhancement.

• Address TB-HIV, MDR-TB, and the needs of poor and vulnerable populations.

• Contribute to health system strengthening based on primary health care.

• Engage all care providers.

• Empower people with TB, and communities through partnership.

• Enable and promote research.13,15

At the end of 2010, an updated Global Plan to Stop TB 2011-2015 was published;

and it takes into account actual progress made since 2006, MDR-TB or changes in the

Partnership’s structure, among others. For achieving the Partnership’s goal, the Global

Plan to Stop TB 2011-2015 presents six objectives and associated targets:14

• Ensure early diagnosis of all TB cases.

• Ensure high-quality treatment of all diagnosed cases of TB.


12

• Strengthen monitoring and evaluation including impact measurement.

• Strengthen human resource development for TB control in the context of overall

health workforce development.

• Scale-up measures to ensure appropriate infection control.

• Coordinate global-level efforts of the DOTS Expansion Working Group.

The Stop TB strategy was developed as the successor to the DOTS strategy which

was developed in the 1990s as the internationally recommended approach to TB control

and it is still expanded worldwide. DOTS remains at the hearth of the Stop TB Strategy

and it has five components:

• Political commitment.

• Early case detection through quality-assured diagnosis.

• Standardized treatment with supervision, and patient support.

• Drug supply and management system.

• Monitoring and evaluation.

The DOTS component of the Global Plant to Stop TB 2011-2015 has been built

on the achievements of the last 15 years. In addition, the plan for DOTS includes

objectives and targets related to human resource development, infection control in health

care facilities, monitoring and evaluation, engagement of all care providers through public-

private approaches, and the share of funding that is provided from domestic sources.11,14

5. Tuberculosis, neglected disease

There is no standard definition of neglected disease; however, it can be said that

neglected diseases are diseases affecting principally poor people in developing countries for

which health interventions, as well as research and development, are inadequate to the

need. Neglected diseases are a group of diseases which are medically diverse but share the

feature of being diseases strongly linked to poverty.17

They are also poverty-promoting because of their impact on child health and

development, pregnancy outcome, and worker productivity. The neglected diseases occur

primarily in rural areas of the developing world. The neglected diseases represent a major

reason why poor people cannot lift themselves out of poverty and why the low-income

countries where they live cannot economically advance. Therefore, a global fight against

neglected diseases could be a highly productive application of medical science and public

health for repairing the world.18


13

Even though TB has attracted the attention of global organizations and projects

during the last decades, it still remains considered as a neglected disease, as published by

the WHO. Therefore, it must be considered that it is often not the lack of tools but the

lack of an appropriate health infrastructure and implementation capacity that blocks disease

control. Biological, social, economic and behavioural determinants, health systems and

other factors are of importance in the effective control of these diseases and these facts

should define the appropriate strategies to fight against TB, which is not only a global

health problem but also a worldwide social problem. So, there is an urgent requirement for

a coordinated approach involving multi-disciplinary networks of investigators, as well as

partnerships between industry and the public sector in both developed and developing

countries.19

6. Tuberculosis, the disease

6.1. Mycobacterium tuberculosis

General characteristics of M. tb

Human is the natural reservoir of M.tb. M.tb is classified as a very weakly gram-

positive and acid-resistant bacteria. M.tb is a facultative intracellular parasite that survives

and grows in phagosomes of mononuclear phagocytes. Mycobacterium typically appears as

straight or slightly curved rods; and the dimensions of the bacilli have been reported to be

1-10 µm in length, and 0.2-0.6 �m in width (Figure 5). The temperature and pH condition

rates, in which the bacillus is able to multiply, are relatively narrow being the optimum

conditions 37ºC and neutral pH. They are obligate aerobic, growing most successfully in

tissues with high oxygen content; and grow very slowly, in the most favourable conditions

M.tb divides every 18 hours.20

Figure 5. Mycobacterium tuberculosis: transmission electron micrograph. Source:

www.sciencephoto.com.


14

The microorganism macromolecular structure and physiological capabilities result

in high adaptation to the specific environment. Therefore, as the environment changes, the

bacillus is able to bring into play different physiological pathways in order to survive.20

Cell envelope of M.tb7,20,21

The cell envelope of the bacillus is composed of the plasma membrane, a cell wall,

and an outer capsule like layer.

• The lipid bilayer of the cytoplasmic membrane provides osmotic protection and

regulates the traffic of specific solutes between the cytoplasm and the environment

(Figure 6).

• The membrane is surrounded by a cell wall that protects the cell contents, provides

mechanical support and is responsible for the characteristic shape of the bacterium.

The wall is constituted by and inner peptidoglycan layer, which seems to be responsible

for the shape-forming property and the structural integrity of the bacterium. The

peptidoglycan contains N-gly-colylmuramic acid instead of the usual N-acetylmuramic

acid, found amongst most other bacteria. Covalently bound, by a phosphodiester link,

to the peptidoglycan is a polysaccharide, the arabinogalactan, whose outer ends are

esterified with high molecular weight fatty acids, called mycolic acids. These mycolic

acids are typically long and branched chains containing 60 to 90-caborn atoms (Figure

6).

• The outer layer of the cell envelope presents free lipids and traversing the whole

envelope, some glycolipids such as the phosphatidyl-myoinositol mannosides,

lipomannan and lipoarabiomanan are anchored to the plasma membrane and extend to

the exterior of the cell wall. The mycobacterial wall also contains interspersed proteins,

some are in the process of being exported, and some might be residents. Several of

these proteins are responsible for cell wall construction. There are also certain proteins

called porins forming hydrophilic channels that permit the passive passage of aqueous

solutes through the mycolic acid layer (Figure 6).


15

Figure 6. Schematic representation of the mycobacterial cell envelope. Source: Adapted

from www.johnes.org.

M.tb genome7,22,23

TB research made huge progress with the description of the complete genome

sequence of the laboratory reference M.tb strain H37Rv.24 M.tb H37Rv possess a sequence

of 4,411,529 base pairs and approximately 4000 genes. Most of its genes codes for enzymes

involved in lipolysis for bacterial survival inside its host, and lipogenesis, for cellular

envelope synthesis.

The sequence of the genome, and its comparison to sequences of other

microorganisms, allowed the assignation of precise functions to 40% of the predicted

proteins, the identification of 44% orthologues, genes in different species that present the

same function because they evolved from a common ancestral gene, and leaving 16% as

unique unknown proteins.

Currently there are only two M.tb (H37Rv and CDC1551) and two M.bovis

(AF2122/97 and BCG Pasteur) genome sequences annotated and published.

M.tb metabolism

Mycobacteria can utilize a wide range of carbon compounds for growth; they are

able to assimilate carbohydrates, lipids, proteins… for their own purposes (Figure 7).25,26

Lipids of the mycobacterial cell will, therefore, be derived by elongation and

transformation of the host’s fatty acids once these acids have been acquired and

transported into the cell.


16

In addition, M.tb could derive most of its own amino acids not by de novo synthesis

but by acquisition from the host. Indeed, it seems that these amino acids will be the

principal source of NH3 to synthesize all other nitrogenous compounds it may require.

However, it has been speculated that M.tb might use nitrate as a nitrogen source and/or as

terminal electron acceptor in the absence of oxygen. In fact, the bacilli have an enzyme

bound to the cell membrane that rapidly reduces nitrate and leads to the accumulation of

nitrite.

Figure 7. Metabolism of M.tb. Source: Adapted from Tuberculosis, Bloom.

Mycobacteria require carbon and nitrogen sources but also trace elements which

have a structural or a functional function in the cell. Magnesium and iron are essential for

life. A deficiency in these elements frequently reduces the virulence of bacterial pathogens.

As iron is not soluble in the presence of oxygen and at neutral pH, special iron systems are

required to incorporate this element into the cell. Exochelins are hydrophilic peptides

secreted into the environment for iron gathering and mycobactins are hydrophobic

compounds located within the cell wall to introduce the iron into the cytoplasm. When

M.tb faces to iron restriction conditions, the bacilli adjust its metabolism to maintain

essential cellular function and increase iron uptake.20,25,27,28


17

6.2. Transmission. Latent TB infection and active TB disease

TB is an airborne contagious disease. Bacteria are spread directly from person to

person via aerosols, which, upon inhalation, bring M.tb immediately to the site of entry and

growth in the lung tissue. Only people with active TB disease can transmit TB and it is

estimated that, left untreated, each person with active TB disease will infect on average

between 10 and 15 people every year.2,29,30 However, not everyone infected with TB

bacteria becomes sick. As a result, two TB-related conditions exist: latent TB infection and

active TB disease (Table 1). It is estimated that about 5 to 10% of infected persons will

develop active TB disease in their lives times. And, about half of those people will develop

active TB disease within the first two years of infection.2,31

Table 1. Latent TB infection vs. active TB disease.

Latent TB infection Active TB disease

Does not feel sick. May present symptoms.

Not infectious, can not spread TB bacteria. May spread TB bacterial.

Positive tuberculin skin test. Positive QuantiFERON®-TB Gold test. Normal chest X-ray. Negative sputum test.

Positive tuberculin skin test. Positive QuantiFERON®-TB Gold test. May present abnormalities in chest X-ray. May present positive sputum smear or culture.

Bacteria are inactive, dormant. Bacteria are active.

Chemoprophylaxis. Treatment to treat active TB disease.

6.3. Pathogenesis22,32-38

Tuberculosis is an airborne infectious disease and its progression is determined by

the response of the host immune system. Many factors and cellular agents are involved in

the host response as shown in Figure 8. The infection by M.tb and the evolution of the

cellular response is briefly explained in following paragraphs.


18

Figure 8. Immune response to M.tb. Source: Nature. 22

An infection by M.tb starts when the bacteria are inhaled and reach the lungs. These

bacteria are rapidly internalized by alveolar macrophages (Figure 9). An inflammatory

response occurs and neutrophiles and monocytes are accumulated in the primary infection

foci. This non specific response fails and bacteria are drained away and localized in the

draining lymph nodes and reach the bloodstream. Once in circulation, the bacteria can

reach different parts of the body; but, usually, these mycobacteria are destroyed and do not

lead to secondary foci.

Figure 9. Infection by M.tb. Source: Microbiology, an introduction. Tortora.37


19

Successful elimination of virulent mycobacterial requires the activation of infected

alveolar macrophages. This is accomplished by a cell-mediated reaction enabling

macrophages to kill and digest the ingested bacilli. The interaction between the

macrophage-presented antigen and specific T cells stimulates the macrophages to secrete

interleukines (IL) that would induce proliferation of T cells (CD4+, CD8+). The cell-

mediated immune response has two functions: CD4+ and CD8+ activate macrophages and

secrete interferon-γ (IFN- γ) that is the key activating agent that produces the

antimycobacterial effects. CD8+ are also involved in the lysis of infected macrophages in a

process mediated by perforin and granulysin. Tumor necrosis factor-alpha (TNF-α),

secreted by CD4+, seems to be ineffective by itself, but synergizes with IFN-γ. Another

mechanism responsible for the antimycobacterial activity of IFN-γ and TNF-α is the

induction of the production of reactive oxygen intermediates (ROI) and related reactive

nitrogen intermediates (RNI) by macrophages via the action of the inducible form of nitric

oxide synthase. Therefore, activated macrophages increase the amounts of lysosomes and

hydrolytic enzymes and, as a consequence, bacilli stop growing and multiplying. The result

of this accumulation of macrophages is the formation of granulomas, known as tubercles

(Figure 10).

Figure 10. Initial stage of tubercle formation. Source: Microbiology, an introduction. Tortora.37

A mature granuloma consists of a central area of necrotic tissue containing

extracellular bacilli that encompassed by macrophages keeping intracellular bacilli, and

surrounded by limphocytes, consisting mainly of T cells (CD4+, CD8+) (Figure 11). The

aerobic bacilli do not grow well in the granuloma because the acidic pH, in conjunction

with the lack of essential nutrients and low oxygen conditions, prevents the multiplication

of mycobacteria; however, many bacilli remain dormant and serve as a base for later

reactivation of the disease. If the disease is kept at this stage, the lesions will become

calcified.


20

Figure 11. Mature tubercle. Source: Microbiology, an introduction. Tortora.37

When the disease progresses, the caseous center enlarges, in a process termed as

liquefaction, and forms an air-filled tuberculous cavity in which the aerobic bacilli multiply

outside the macrophages. Granulomas destroy adjacent tissue and may necrotize at the

center, leading to cavity formation (Figure 12).

Figure 12. Tuberculous cavity. Source: Microbiology, an introduction. Tortora.37

Liquefaction continues until the tubercle bronchiole wall ruptures and the bacilli are

disseminated throughout the lung and then to the circulatory and lymphatic systems

(Figure 13). Thus, wherever the blood-borne bacteria localize, new tubercles arise.

Thousands of tubercles can appear throughout the body, and the disease is now

generalized and it can lead to damage of vital organs, which can cause death.

Figure 13. Liquefaction and dissemination of the bacilli throughout the body. Source:

Microbiology, an introduction. Tortora.37

6.4. Clinical tuberculosis

Pulmonary disease is the most common presentation in areas where TB continues

to be commonly transmitted. In areas where reactivation predominates, the distribution

appears to be quite different with a higher proportion of extra-pulmonary TB. A brief

summary of the most common sites of disease is considered:39,40


21

• Pulmonary TB: is the most important form because of its frequency and because it is

the most infectious form of the disease.

• Cervical lymph node TB: the nodes are usually asymmetrical in the neck and can be

painful; although they are not associated with systemic symptoms.

• Pleural TB: seem to be an early manifestation of TB. A unilateral pleural effusion is

usually observed.

• Skeletal TB: involves the joints and bones and its incidence increases with increasing

age.

• Spinal TB: also known as Pott’s disease.

• Central nervous system TB: Meningitis is the most frequent form of central nervous

system TB and it constitutes a small percentage of clinical TB but it is very important

because of its high mortality.

7. Symptoms and diagnostic

7.1. Symptoms

Physical findings in pulmonary TB are generally not particularly helpful. At the first

stage of the disease, symptoms can not be observed or they can be confused with flu.

When the disease develops, the patient starts to show the clinical signs of TB, such as loss

of appetite, fatigue, loss of weight, night sweats, a persistent cough, and, finally, coughed-

up sputum with blood.2,39-41

In case of disseminated TB, the symptoms and signs are generally nonspecific, such

us fever, weight loss, anorexia, and weakness. Other symptoms depend on the relative

severity of disease in the organs involved. Coughs and shortness of breath are common;

headache and mental status changes are less frequent and are usually associated with

meningeal involvements.40

7.2. Diagnostic

Nowadays, the tuberculin skin test, microscopy and culture are still the main

techniques in laboratory diagnosis of TB, and, specially, in low and middle-income

countries. However, new methods focused on detection of nucleic acids and detection of

mutations in the genes, related with resistance to anti-tuberculosis drugs, have been

developed in the last twenty years. A brief summary of the main methods is presented in

this chapter.


22

• X-ray: in developed countries, radiographic examination of the chest is usually one of

the first diagnostics. The observed abnormalities can appear in different parts of the

lungs and they can also present different size, shape and density. These abnormalities

can suggest pulmonary-TB but X-ray can not be used as a specific or conclusive

diagnostic method.40,41

• Tuberculin skin test: the tuberculin skin test consists on an intradermal injection of the

purified protein derivative (PPD), which is a protein extracted from M.tb.

Approximately three weeks after the infection has started, an infected patient develops

a hard red zone in 48 to 72 hours. A positive reaction is indicated by erithema and

induration of > 10 mm in size. However, it has been estimated that almost one third of

people positive to this test do not actually have TB infection; the sensitivity of the skin

test is estimated around 70% in active TB cases and it decreases to 30% in

immunocompromised people. Moreover, people who are regularly screened for TB

infection using the skin test become immnunized to PPD; nevertheless, the tuberculin

skin test is widely used with diagnostic and epidemiological purposes.7,32,41-44

Figure 14. Positive tuberculin skin test. Source: ADAM.

• Acid fast bacilli smear microscopy: acid fast bacilli (AFB) smear microcopy is rapid and

inexpensive and thus is a very useful method to identify highly contagious patients.

This method is based on the high lipid content of the cell wall of mycobateria which

makes them resistant to decolorization by acid-alcohol after the primary staining. This

method allows an early diagnosis of mycobacteria infections because most

mycobacteria grow slowly and culture results become available only after weeks of

incubation, and it is often the only available diagnostic method in developing countries.

However, the quality of the specimens and the laboratory services is an important fact

because many false positives and false negatives can occur.41,45,46

• Culture: AFB microscopy is easy and quick, but it does not confirm TB diagnosis

because many mycobacterias are AFB in the smear microscopic examination.


23

Moreover, culture is used to detect cases with low mycobacterial loads and it is also

requested in cases at risk of drug-resistant TB. Nevertheless, most TB control

programs do not support its widespread use due to laboratory complexity, biohazard

and cost.41,46

• Biochemical methods: these methods can be considered as the conventional ones and

include:

- niacin accumulation test

- growth in the presence of p-nitrobenzoic acid

- the nitrate reduction test, which is particularly useful for differentiating M.tb from

M.bovis

- catalase test

- the pyrazinamidase test, which is useful to differentiate M.tb from the other species

of the M.tuberculosis complex although some strains of M.tb may acquire resistance

to this enzyme and give false negatives

- growth in the presence of thiphe-2-carboxylic acid hydrazide.

• Enzyme Linked Immunosorbent Assay (ELISA): few serological tests, based on the

response to mycobacterial antigens, have been developed. ELISA test kits are quick and

need minimal training; however, there is still a need to improve the sensitivity of

commercial tests.42,44

• Interferon-gamma determination: assays based on IFN-γ determination seem to be one

of the most significant developments in TB diagnostic. Many IFN-γ assays are now

available: the enzyme-linked immunospot (ELISPOT, T SPOT-TB®), the original

QuantiFERON®-TB, and its enhanced version QuantiFERON®-TB Gold assay.

ELISPOT has been evaluated and the results have shown that it offers a more accurate

approach that the tuberculin skin test for the identification of latent TB infection. With

regard to the QuantiFERON®-TB, it seems to be comparable to the tuberculin skin

test in its ability to detect latent TB infection but it is less affected by BCG vaccination.

However, IFN-γ assays involve high cost, highly trained personnel and fresh blood

samples that became this method into unattainable alternatives in developing

countries.41,42,47-49

• Automated culture methods: automated liquid culture systems may detect growth in

half of the time required for mycobacteria to grow in solid media, but have a higher


24

rate of contamination and are more expensive, which limits their use in endemic

countries.41,47,50

• Nucleic-acid amplification methods: many nucleic-acid amplification test are

commercially available and they present high specificity and high sensitivity in smear-

positive sputum; but their sensitivity in extrapulmonary TB is moderate, so that culture

diagnosis should be done in parallel. In the last few years, several amplification

methods have been commercialized, such as Amplified MTD, Amplicor MTB Test or

BD Probe Tec ET. Moreover, its cost is too high for routine use in low and middle

income countries.48,50

Many diagnostic methods are available, and, nowadays, there is a continuous

development of new techniques. However, the diagnosis protocol presents a great

variability because many factors must be considered such us patient characteristics as age or

immunosupresion, the chance of latent or active TB, different forms of TB disease, and

health care system limitations in low and middle income countries. For these reasons, the

tuberculin skin test, microscopy and culture continue being almost the only alternatives for

diagnosis in many endemic regions.

8. Treatment and prevention

8.1. Treatment

Drugs

Drugs for treating TB are usually classified as first- and second-line drugs.

Nevertheless, this classification is changing because the use of some first-line drugs is

declining in recent years and some new drugs could be included in this group.2,7,51,52

• First-line drugs: are bactericidal and combine a high degree of efficacy with a relative

toxicity to the patient during treatment. The first-line drugs group includes isoniazid

(INH), rifampicin (RIF), streptomycin (SM), ethambutol (EMB), and pyrazinamide

(PZA) (Figure 15).


25

Figure 15. First-line drugs.

• Second-line drugs: are bacteriostatic, which have a lower efficacy and are usually more

toxic and expensive. This group includes p-aminosalicylic acid (PAS), the thioamides

ethionamide and prothionamide, the rifamycin family such as rifabutin and rifapentine,

cycloserine, capreomycin, and several fluoroquinolones such as moxifloxacin,

levofloxacin and gatifloxacin (Figure 16).

Figure 16. Second-line drugs.


26

Treatment regimens

There are many different anti-tuberculosis regimens; however guidelines published

by the World Heath Organization or the Centre for Disease Control and Prevention (CDC)

can be considered as references. In general terms, the current short-course treatment for

the complete elimination of active and dormant bacilli involves two phases:2,7,48,51,53

• The initial phase consists on an effective combination of INH, RIF, PZA and EMB for

two months.

• The continuation phase consists on a combination of INH and RIF for four months

with the aim of killing any remaining or dormant bacilli and preventing recurrence.

This regimen cure around 90% of TB cases when treatment is fully adhered and

drugs are quality-assured. For these purposes, it is essential that patients have monthly

evaluations to identify possible adverse effects on the anti-TB drugs and to assess

adherence. Although basic regimens are broadly applicable there are modifications that

should be considered under special circumstances such as HIV infection, drug resistance,

pregnant women and children.

In case of HIV-infected adults, the treatment is a 6 or 9 months regimen that

consists of an initial phase of INH, PZA, EMB and rifabutin for two months; and, a

continuation phase of INH and rifabutin for four or seven months. One of the main

factors that must be considered is drug interaction and for these reason rifabutin must be

used as an alternative to RIF that interacts with antiretroviral agents.39,51,54,55

Drug resistant testing can take weeks and, for this reason, the treatment should

start as soon as drug-resistant TB disease is suspected; and, once the results are known the

treatment should be adjusted. The treatment consists on the combination of second-line

drugs that usually have more toxic effects, the treatment is much longer and it may cost

100 times more than a first-line drug treatment. However, even with this treatment, six out

of ten patients with MDR-TB will die.2,51,55-57

Drugs mechanisms and drug resistance mechanisms51,58-61

The mechanism of action of the main anti-tuberculosis drugs is briefly considered

in the following paragraphs. A graphical representation is shown in Figure 17.

• Isoniazid: INH is a pro-drug that requires being activated by the bacterial catalase-

peroxidase enzyme (KatG) to generate reactive species. It seems that the activated drug

attacks multiple targets of M.tb although the main target is inhibition of the


27

biosynthesis of mycolic acids. Resistance to INH is mostly associated with mutations or

delections in KatG. Furthermore, mutations in other genes have been reported to be

associated with INH resistance, but the relationship is unclear.

• Thioamides: they seem to be activated by EthA, an enzyme that transforms thioamides,

such as ethionamide, into highly reactive species that inhibit cell wall synthesis. These

intermediates react with a nucleophile but the mycobacterial enzymes targeted by these

adducts are not clear.

• Cycloserine: inhibits cell wall synthesis.

• Rifamycins: rifamycins family, such as rifampicin, rifabutin and rifapentine, inhibits

gene transcription, by interaction with the beta subunit of the RNA polymerase

enzyme, inhibiting the elongation of the messenger RNA. Resistance to RIF is

associated with mutations in the gene that codifies the beta subunit of the RNA

polymerase enzyme, resulting in conformational changes that decrease the affinity of

this subunit for RIF.

• Ethambutol: EMB affects the biosynthesis of the cell wall and it has been suggested

that it contributes to increase the susceptibility of the mycobacteria to other drugs.

EMB inhibits the polymerization of cell wall arabinan of arabinogalactan and

lipoarabinomannan; although, the biochemical target remains unclear.

• Pyrazinamide: PZA is a pro-drug that requires conversion into pyrazinoic acid by the

mycobacterial pyrazinamidase enzyme (PZase), which is encoded by the pncA gene. In

most cases, resistance to PZA is associated with mutation in the pncA gene.

• Aminoglycosides: inhibit protein synthesis. Streptomycin inhibits the initiation of the

messenger RNA translation.

• p-aminosalicylic acid: the mechanism of action of PAS is still subject of research. It is

suggested that it could be associated with inhibition of DNA precursors’ synthesis or

with inhibition of the iron chelating siderophore biosynthesis.

• Fluoroquinolones: the main target of fluoroquinolones, such as moxifloxacin,

levofloxacin or gatifloxacin, is the DNA gyrase and topoisomerase II. Inhibition of

DNA gyrase disrupts DNA replication and repair, bacterial chromosome separation

during division, and other cell processes. Inhibition of topoisomerase II also affects

DNA replication.


28

Figure 17. Possible drug mechanism of anti-tuberculosis drugs. Source: www.niaid.nih.gov.

8.2. Prevention

Chemoprophylaxis

Chemoprophylaxis of TB is indicated on patients with latent TB and it helps

reducing TB incidence in both HIV-positive and HIV-negative patients. Prophylaxis

usually consists of INH monotherapy for six to twelve months. If resistance to INH is

suspected, other regimens include RIF, PZA or EMB. However, conventional

chemoprophylaxis with INH or RIF after exposure to XDR-TB is probably

ineffective.7,47,48,51

Vaccine

WHO recommends that children receive BCG (Bacille Calmette Guerin) in

endemic areas. BCG is the only vaccine available for prevention of TB in humans and it is

made from a live weakened strain of M.bovis. Many studies have been developed and they

seem to be not uniform in their conclusions. However, they all conclude that BCG

vaccines afford a high degree of protection of severe forms of TB, such as meningitis or

disseminated TB, especially in children with an efficacy ranging from 40-90%. In contrast,

its efficacy against pulmonary TB, which is more prevalent in adolescents and adults, is


29

much lower, from 0-80%. Moreover, BCG may interfere with the tuberculin skin test and it

only provides protection against primary infection.2,48,62,63

9. New approaches to prevention and treatment of tuberculosis

9.1. Vaccines development

Present vaccine candidates reduce the initial bacterial burden. However, there is an

urgent need of new postexposure vaccines to prevent reactivation and reinfection of

individuals with latent TB. Nowadays, one of the main challenges is to understand the

nature of immune response against TB, which is an essential requirement for an

appropriate approach for developing a vaccine.

Nowadays, many vaccine candidates are in clinical development and they can be

divided into four groups according to their mechanism of action:64,65

• Recombinant BCG vaccines: are based either on improvement of BCG vaccine

through addition of relevant genes or on attenuation of M.tb through deletion of

virulence genes.

• Subunit and live vector-based vaccines that boost BCG prime: subunit vaccine

candidates are based on antigens that are recognized by T-cells from patients with

latent infection or whose tuberculosis has been cured.

• DNA vaccines: represent the most recent approach in vaccine development. The

vector encoding the gene of interest is injected directly, resulting in the expression of

the corresponding gene and inducing potent immune response.

• Killed whole bacterial vaccines as adjunct: this family of vaccines accelerates or

complements the effects of TB chemotherapy. Many attempts to develop this vaccine

have been made, but these have been especially cautious because of the potential risk of

disease enhancement.

9.2. Drug discovery

There is an urgent need of new drugs which could shorten and simplify treatment

of drug-sensitive active and latent TB; improve treatment of MDR-TB and XDR-TB; and,

facilitate treatment of HIV-coinfected patients.

Drug-discovery methods

Nowadays, many different methods are used for discovering new drugs against TB.


30

• Genetic approaches to target identification: the availability of the genome provides a

rapid approach to find new targets. Using genetic analysis, researchers have found

several gene products that, if inhibited, could decrease bacterial growth or increase host

survival after infection. Unfortunately, this approach has not lead to the identification

of new drug candidates.66,67

• High-throughput screening: against target proteins has an important role in modern

drug discovery and it is used by both industry and academy. In the case of M.tb, high-

throughput screening has contributed to the finding of two clinical drug candidates

(TMC207 and SQ109) (Figure 19).66,67

• Structural biology and virtual screening: in the last years, more than 260 X-ray crystal

structures have been described and selected as potential drug targets. The use of these

structures enormously facilitates medicinal chemistry efforts to rationally design new

antibiotics. Virtual screening approach can be use to identify compounds that present a

pharmacophore and to develop inhibitors of a protein considering its known

structure.67,68

• Re-modeling of existing scaffolds: this approach allows introducing chemical

modifications into the core of structure for improving bacterial activities, resistance

profiles, safety, or better pharmacokinetic and/or pharmacodynamic properties. This

approach has been very successful and many drugs are actually in clinical trials (Figure

18).66,69-72

Figure 18. Re-design of existing antibacterial drug families. Source: Nature.66


31

New TB drugs in clinical trials73

A summary of the main drugs, which are currently in clinical development, is

outlined according to the different families of structures they belong to.

• Rifamycins: the maximally tolerated doses of rifampicin and rifapentine have never

been defined, so this work is now a priority. Sanofiaventis, the developer of rifapentine,

is currently performing the necessary preclinical toxicity studies to support the

appropriate Phase I studies.74,75

• Fluoroquinolones: several members of this class have been used as second-line drugs

for the treatment of MDR tuberculosis. Gatifloxacin and moxifloxacin (Figure 19), the

most recently developed fluoroquinolones, have shown better in-vitro activity against

M.tb than the older fluoroquinolones. Thus, they are currently being evaluated for its

ability to substitute for either EMB (gatifloxacin; moxifloxacin) or INH (moxifloxacin)

in first-line TB treatment and to shorten therapy to four months from the current

standard of six to nine months.74-76

• Oxazolidinones: a trial of linezolid (Figure 19) in combination with other anti-

tuberculosis agents is being conducted currently in XDR-TB patients in South Korea.

Pfizer has another oxazolidinone, PNU100480 (Figure 19), in clinical development for

TB. Other companies are also evaluating members of this class for potential use in TB

treatment, but these compounds are in still earlier stages of characterization and

development.74-76

• Nitroimidazoles: two novel nitroimidazoles are currently in clinical development:

OPC67683 and PA-824 (Figure 19). Both are pro-drugs and inhibit mycobacterial

protein and lipid biosynthesis through a mechanism of action that is not yet fully

elucidated. PA-824 has been demonstrated to undergo nitroreduction, causing

formation of multiple highly reactive intermediates, including nitrous oxide, which

likely react with a variety of intracellular targets.74,75,77-79

• Diarylquinolines: TMC207 (Figure 19), member of the diarylquinoline chemical class, is

currently in clinical development for TB.74,79,80

• Ethylenediamines: SQ109 (Figure 19), an ethylene diamine derivative, is currently under

clinical development. SQ109’s specific intracellular target has not yet been elucidated,

but it appears to function as a cell wall synthesis inhibitor.74-76,79,81

• Pyrroles: LL3858 (Figure 19), a member of the pyrrolechemical class, is currently in

clinical development.74


32

Figure 19. TB drugs in clinical development.

9.3. Drug delivery systems

The lung is the primary portal of entry for mycobacteria that cause TB, and, for this

reason, there is a huge interest in formulating drugs for pulmonary delivery. An appropriate

delivery system would allow facilitating the administration, protecting the drug from

degradation, reducing adverse effects or shortening the treatment. Therefore, the study of

drug delivery systems is one of the most promising approaches to improve TB treatment.

During the last decades, there have been many attempts to develop anti-tuberculosis drugs

as aerosolized liposome suspensions and formulations as respirable insoluble microparticles

and nanoparticles. A summary of the main approaches is outlined bellow.82-84

• Liposomes are nano- to micro-sized vesicles that consist on phospholipid bilayer that

surrounds an aqueous core. Therefore, the core enables the encapsulation of water

soluble drugs while the hydrophobic region can entrap insoluble agents.

• Niosomes are thermodynamically stable liposome-like vesicles that can host hydrophilic

drugs within the core and lipophilic ones by entrapment in hydrophobic domains.


33

• Nanosuspensions are dispersions of pure drugs stabilized with surfactants. Reduction

of the average size of solid drug particles to the nano-scale is a useful methodology to

improve the solubility of drugs that present poor water and lipid solubility.

• Nanoemulsions are thermodynamically stable oil-water dispersions displaying drop

sizes between 10 and 100 nm. A main advantage of these systems is that they are

generated spontaneously and can be produced in a large scale without the need of high

homogenization processes.

II. QUINOXALINES AND TUBERCULOSIS

Introduction: Quinoxalines and Tuberculosis

37

10. Quinoxaline, a chemical structure with a broad range of biological activities

Quinoxalines, also known as 1,4-benzodiazines, are aromatic bicycles that present

two nitrogen atoms on positions 1 and 4 (Figure 20). Quinoxaline is described as bioisoster

of quinoline, naphtyl and some other heteroaromatic rings including pirazine (Figure 20).85

Figure 20. Quinoxaline structure and its bioisosters.

Quinoxaline derivatives are a class of compounds showing a great interest in

medicinal chemistry as they display a broad range of biological properties such as

anticancer,86-91 antimycobacterial,89-95 antithrombotic,96,97 analgesic,96-101 anti-inflammatory or

antioxidants.102,103 A selection of some quinoxaline structures studied by different research

groups is shown in Figures 21 and 22.

Figure 21. Structure of quinoxaline derivatives with biological activity. a) anticancer; b)

antimycobacterial; c) antithrombotic; d) analgesic.


38

Figure 22. Structure of quinoxaline derivatives with biological activity as anti-inflammatory

or antioxidants.

There have also been published many papers in which quinoxaline derivatives, as

quinoxalinones, are studied as potent agents in the central nervous system (Figure 23).104-107

Figure 23. a) Structure of a trycyclic quinoxalinone with anxiolytic properties; b) Structure

of quinoxalinone derivatives acting as AMPA receptors inhibitors.

Moreover, some quinoxaline derivatives have been studied as antiviral agents and

they resulted to be endowed with anti HIV-1 Reverse Transcriptase Inhibitors (Figure

24).108-110

Figure 24. Quinoxaline derivatives showing activity as anti HIV-1 Reverse Transcriptase

inhibitors.

The oxidation of both nitrogens of this heterocyclic system, carried out in order to

obtain quinoxaline 1,4-di-N-oxide derivatives (Figure 25), increases the number of

biological properties.111 It has been reported that quinoxaline 1,4-di-N-oxide derivatives

actually improve the biological results shown by their reduced analogues and are endowed


39

with antiviral, anticancer,112-114 anticandida,94,115-117 antibacterial,94,113,115,118-123 and

antiprotozoal activities.124,125

Figure 25. General structure of quinoxaline 1,4-di-N-oxide derivatives.

In fact, since the 1940s, quinoxalines 1,4-di-N-oxide were known as potent

antibacterial agents and subtherapeutic levels have been used as animal growth promoters

in feed additives.123,126,127

As a result of different research projects, our group has synthesized different series

of quinoxaline 1,4-di-N-oxide derivatives, with a great variety of substituents in positions 2,

3, 6 and 7 (Figure 25). With regard to position 2, where the main group is linked, our team

has principally worked with carbonitrile, ketone, ester and amide derivatives. Methyl,

phenyl, trifluoromethyl or piperazinyl groups are some of the substituents studied on

position 3. And finally, electron-withdrawing and electron-releasing groups have been

considered on positions 6 and/or 7 of the heterocyclic ring. These projects have allowed

identifying quinoxaline 1,4-di-N-oxide derivatives as serotonine receptor antagonist,128

kinase inhibitors,129 antihypertensive and antiagregant,130 antimycobacterial,131-142

antimalarial,143-148 antichagasic,124,125,149 antileishmanial,150 antitumoral,151-157 anti-inflammatory

and antioxidant agents.102,103 A selection of the most interesting derivatives studied by the

group is shown in following figures.

Figure 26. Structures of some of the quinoxalines 1,4-di-N-oxide acting as hypoxia-

selective agents.


40

Figure 27. Structures of some of the quinoxalines 1,4-di-N-oxide with antitumoral activity.

Figure 28. Structures of some of the quinoxalines 1,4-di-N-oxide with antimalarial activity.

Figure 29. Structures of some of the quinoxalines 1,4-di-N-oxide with antichagasic

activity.

11. Quinoxaline 1,4-di-N-oxide derivatives and tuberculosis

Our group started a project on tuberculosis many years ago. INH and PZA are still

some of the treatments par excellence; and a pyridine and a pyrazine ring can be detected in

INH and PZA structures (Figure 30). At this point, the group found the chance to develop

a new group of structures. Quinoxaline is a benzylpyrazine which is a structure similar to

INH and PZA considering steric and electronic factors. Another fact which was considered

at the beginning of this project was the family of quinoxaline 1,4-di-N-oxide derivatives

that are known to show interesting biological properties, as previously considered. For

these reasons, and taking into account that oxidation of both nitrogens of this heterocyclic


41

system enormously widens the biological properties, the quinoxaline 1,4-di-N-oxide system

was selected as the starting point (Figure 30).

Figure 30. Chemical structural resemblance of quinoxaline and anti-tuberculosis drugs.

The first studies considered 2-quinoxalinecarbonitrile derivatives (Figure 31) and

these results confirmed that the N-oxide function is essential for the anti-tuberculosis

activity of quinoxaline derivatives.139-142

Figure 31. General structures of quinoxaline-2-carbonitrile derivatives.

Two of the studied compounds showed promising results but the cytotoxicity data

drove to the replacement of the carbonitrile group with carboxamide, ketone and ester

moieties (Figure 32).139,140

Figure 32. Structures of the two lead compounds.


42

As a result of the observed cytotoxicity of the carbonitrile derivatives, a series of

thirty-one 1,4-di-N-oxide quinoxaline-2-carboxamide derivatives was synthesized and

screened against M.tb (Figure 33).138 Both 2-piperazinyl carbonyl and N-arylcarboxamide

derivatives were considered; and the latter showed very interesting results.

Figure 33. General structure of the quinoxaline-2-carboxamide derivatives.

Moreover, three compounds stood out and have been considered as the base for

defining the structural requirements in order to optimize the anti-tuberculosis activity

(Figure 34).138

Figure 34. Structures of the three N-arylcarboxamide derivatives leads.

A group of twenty-eight 2-acetyl and 2-benzoyl quinoxaline 1,4-di-N-oxide

derivatives was also studied as anti-tuberculosis agents showing very promising results

(Figure 35).137

Figure 35. General structure of the 2-acetyl and 2-benzoyl quinoxaline 1,4-di-N-oxide

derivatives.

This study revealed that a methyl group substituted in position 3 seems to be

essential for the anti-tuberculosis activity; and, if this moiety was replaced by a piperazyn

derivative, the activity was totally lost (Figure 36).137 Some of these compounds were used

to design future series.


43

Figure 36. Structures of the most active ketone derivatives.

Twenty-nine 2-carboxylate quinoxaline 1,4-di-N-oxide derivatives were evaluated

against M.tb and emerged as new lead candidates for the treatment of TB (Figure 37).136

Figure 37. General structure of the carboxilate quinoxaline 1,4-di-N-oxide derivatives.

The most potent carboxylate derivatives are shown in the following figure:

Figure 38. Structures of the most potent carboxylate derivatives.

Finally, a series of thirteen 3-phenyl quinoxaline 1,4-di-N-oxide derivatives were

designed and studied (Figure 39).133

Figure 39. General structure of 3-phenyl derivatives.


44

The insertion of a phenyl group on position 3 (Figure 40) led to high potency, low

cytotoxicity and good selectivity.

Figure 40. Structure of the most promising derivative.

Some of the studied compounds have been further evaluated in more advanced in

vitro screenings and two of them even in a series of in vivo assays (Figure 41).134,135

Figure 41. Structure of the most promising derivatives.

These studies have confirmed the hypothesis of studying quinoxaline 1,4-di-N-

oxide derivatives as anti-tuberculosis agents because it has been proved that these

derivatives are active not only on simple-drug resistant strains but also on poly-drug

resistant strains; are active on non-replicating persistent mycobacteria; are active on in vivo

models; and, seem to present a different mechanism of action to the used anti-tuberculosis

drugs including the promising bio-reductive agent PA-824.

Meanwhile, Carta’s group has also been studying quinoxaline 1,4-di-N-oxide

derivatives as anti-tuberculosis agents. They have studied the influence of a sulfur atom and

nitrogen on position 2 of the heterocyclic (Figure 42).94,111,115

Figure 42. General structures of the derivatives studied by Carta’s group.


45

In summary, more than 500 quinoxaline derivatives, synthesized by our group, have

been evaluated as anti-tuberculosis agents, and, it can be said that, the N-oxide function is

essential for the biological activity. Moreover, carboxamide or ketone substitution on

position 2 and methyl and phenyl groups on position 3 of the quinoxaline ring should be

considered to improve the anti-tuberculosis activity.

III. ALTENATIVE TOOLS IN MEDICINAL CHEMISTRY

Introduction: Alternative Tools in Medicinal Chemistry

49

12. Electrochemistry

Several antimicrobial and anticancer drugs are only active following bioactivation

within the target cell and quinoxaline di-N-oxide derivatives are a family of compounds

which are supposed to act as bio-reductive species.158

For this reason, many studies have been published in the last decade in relationship

with the ability of quinoxalines di-N-oxide to be involved in enzymatic one-electron

reduction of the heterocycle in order to generate active species. These studies have been

developed to study the hypoxic selective,114,151,152,159-162 trypanocidal,163,164 or antimicrobial

activities.158,165

Moreover, several studies have investigated and reported the electrochemical

behavior of quinoxalines di-N-oxide and demonstrated reasonable correlations between

electrochemical behavior, structure and drug activity.165,166 Not only heterocyclic di-N-

oxides but also antibacterial quinones and heterocyclic nitro derivatives have revealed that

compounds showing less negative reduction potential values exhibited more powerful

antibacterial activity.165,167,168


50

13. logP

13.1. Introduction

Quinoxalines di-N-oxide are receiving growing attention in the field of medicinal

chemistry. Therefore, it would be interesting to study the physicochemical properties of

this family of compounds. A potential drug has to cross several barriers, and the bio-

response of a compound can be the result of several types of interaction between the

compounds and the receptor which can be related to hydrophobic and electrostatic forces,

electron donor-acceptor capacity or hydrogen bonding, among others. For these reasons,

the study of absorption, distribution, metabolism and excretion should be considered in the

first stages of compounds development as this information could be of great help when

identifying new candidates or optimizing structures (Figure 43).169-172

Figure 43. Barriers to drug delivery and effect.

The ability of a drug to act is related to its concentration at the site of action and its

ability to interact with the biological target. The concentration of the drug at the site of

action is discussed in terms of pharmacokinetics whereas its pharmacodynamic properties

are relevant to the specific interaction with the biological target (Figure 44). These involve

adsorption, distribution, metabolism and elimination. In order to assess the importance of

each of these factors on drug action we have to take into account structural and

physicochemical properties of the drug.173

There are four main ways by which small molecules, such as drugs, cross cell

membranes: passive diffusion, diffusion through aqueous channels, carrier-mediated

transport and pinocytosis. In this sense, passive diffusion and carrier-mediated transport

are the more important in relation with pharmacokinetic mechanisms (Figure 45).


51

Figure 44. Main routes of drug administration and elimination. 174

Moreover, aqueous solubility and lipophilicity are the most important factors in

drug absorption by passive diffusion. Drugs that are too polar or hydrophilic often exhibit

poor transport properties, whereas non-polar or lipophilic drugs usually have low aqueous

solubility and therefore low bioavailability.174,175

Figure 45. Transport across cell membranes. Source: Kaplan, Physiology.


52

This can be explained because non-polar substances dissolve in non-polar solvents,

such as lipids, and, therefore, penetrate cell membrane by diffusion. Solubility in the

membrane and diffusivity are the main factors that contribute to the permeability of a drug.

The first one can be expressed as a partition coefficient for the substance between the

membrane phase and the aqueous environment, and diffusivity is a measure of the mobility

of molecules within the lipid and is expressed as a diffusion coefficient. The diffusion

coefficient varies only slightly among different drugs and, therefore, there is a close

correlation between the lipophilicity and the permeability of the membrane to different

substances.174,175 Lipid solubility is one of the most important determinants of the

pharmacokinetic characteristics of a drug and many properties, such as absorption,

penetration or elimination are related to lipophilicity. The biodistribution, protein binding

and metabolism of drugs may also be altered by their lipophilicity, and it has been accepted

that, in general, lipophilic compounds are preferred targets for metabolism and frequently,

lipophilicity correlates positively with a high protein binding. Moreover, non-specific

toxicity is expected to be related to a compound’s accumulation in cell membranes and

therefore, its lipophilicity.169-171,174,176

Lipophilicity is a molecular property which is related to the ability of a compound

to partition between a polar and a nonpolar solvent. Two inmiscible polar/non polar

solvents have become the standard model for studying compounds lipophilicity and it is

expressed as the logarithm of the partition coefficient (logP) of a solute between the two

phases. Nowadays, logP is a general notation and subscripts are used to indicate the solvent

system. More specifically, the octanol/water partition coefficient (logPo/w) is the parameter

most used for measuring lipophilicity as it has been shown that this partition system is a

good model for many biological processes.169,170,177-179 In fact, logPo/w is also used as one of

the standard properties identified by Lipinski in the “rule of 5” for druglike molecules.180,181

Many factors such as the nature of the membrane, the volume of the aqueous phase

adjacent to the membrane and available for dissolving the drug, or the dose of the drug

must be consider when defining the optimal logP value to ensure good absorption. For

instance, if dissolution is not a limitation, a logP value of about 2 appears to be optimal for

gastrointestinal absorption.


53

13.2. logP. Concept

The term partition coefficient is not recommended by IUPAC as synonym of

partition ratio.171,182 Nevertheless, although this term is not recommended, it is not

forbidden; partition coefficient is actually the most often used term in the scientific

community and, for these reasons, it is the nomenclature that will be used in this work.

The partition coefficient is defined as the ratio of concentration of a compound in

the two phases of a mixture of two immiscible solvents at equilibrium (Equation. 1).

Equation 1

where [ ] means concentration.

The choice of the partition solvents is one of the main questions and it is generally

accepted that the octanol/water partition coefficient (logPo/w) is a good model for many

biological processes because the octanol has been chosen as a simple model of a

phospolipid membrane.169,170,177-179 Therefore, the logPo/w is defined as the logarithm of the

ratio of concentration of an un-ionized compound between a mixture of octanol and water

at equilibrium (Equation 2).

Equation 2

where is the concentration of the compound, in its neutral form, in the

octanol phase and is its concentration in the aqueous phase when the

system is at equilibrium.

For the purpose of considering the un-ionized form of the compounds, pH should

be two units below or two units above the pKa, in case of acids or bases, respectively. In

this sense, logD is defined as the partition coefficient of a compound at a particular pH. 169,183-185 Thus, logD at pH 7.4 is considered to be a good indication of the lipophilicity of a

drug at the pH of blood plasma. In fact, if ionization in the aqueous phase is considered,

logP and logD are related as follows. In case of a monoprotic acid that is partially ionized in

the aqueous phase:

Equation 3


54

In the case of a monoprotic base that is partially protonated in the aqueous phase:

Equation 4

13.3. Measurements of logP

Due to the increasing demand for determining physicochemical properties at an

early stage of compounds development, several methods for measuring the partition

coefficients have been developed and studied.169,171,181,186 The most frequently used methods

will be briefly described.

Shake-flask method

The compound is partitioned between the two solvents in a test tube. Before

partitioning, the two solvents must be mutually saturated at the temperature of the

experiment. During the partitioning procedure, it is recommended to use a mechanical

shaker, and the separation of the two phases should be achieved by centrifugation. After

equilibrium, the concentration in each layer is determined by analytical methods such as

spectroscopic methods, gas chromatography or high performance liquid chromatography.

The total quantity of compound present in both phases should be calculated and compared

with the quantity originally introduced.169,183

Nevertheless, the shake-flask method is time consuming, tedious and requires large

amounts of pure compounds. Moreover, use of this method is limited in the logPo/w range

between -2 and 4, and it is impossible to use with surface-active materials.183 In addition,

low aqueous solubility of some compounds should be considered as it is an important

limitation in the shake-flask method.

With the aim of improving the use of the shake-flask method, several variations

have been developed as the use of flow injection analysis,187,188 the stir-flask method,189 the

micro-shake-flask method171 or an automated and miniaturized shake-flask method.190

Reverse-phase high performance liquid chromatographic methods

The use of reversed-phase liquid chromatography for the indirect determination of

octanol-water partition coefficients was proposed in the 1970s and many methods have

been studied.

HPLC is performed on analytical columns packed with a commercially available

solid phase containing long hydrocarbon chains chemically bound onto silica and the

mobile phase should contain at least 25% (v/v) water. Chemicals injected into the column

move along it by partitioning between the mobile phase and the stationary phase.


55

Compounds are retained in proportion to their hydrocarbon-water partition coefficient;

therefore, hydrophilic compounds elute first and hydrophobic compounds elute last. The

partition coefficient (logk’) is defined as the logarithm of the capacity factor (k’) as follows:

logk’ = log[(tR-t0)/t0] Equation 5

where tR is the retention time of the test compound and t0 is the dead-time.

In order to correlate the capacity factor (logk’) of a compound with its partition

coefficient (logPo/w), a calibration graph using at least six points has to be established. For

the purpose of selecting the reference compounds, they should be structurally related to the

test compounds, and their logPo/w values should be in the expected range for the test

compounds.

Finally, it should also be considered that measurements should be made on the

non-ionized compound form and this method can be used to estimate partition coefficients

in the logPo/w range between 0 and 6.191

Many attempts have been made to search the chromatographic conditions that

make this method similar to the octanol-water system. In this sense, several factors should

be considered to understand the limitations of this method. Reverse phase

chromatographic systems provide an incomplete model for the octanol-water partition

system because specific interactions can occur at the stationary phase but do not exist in

the octanol-water system.179,192 Moreover the pH operating range for silica-based column

materials is limited and the pH should be corrected to work with the un-ionized forms of

the compounds.

For these reasons, many studies have been performed to define the

chromatographic conditions that best suit the octanol-water system. Finally, several reports

have concluded that the Supecosil LC-ABZ column is suitable for estimating logP because

the studied properties appear to be similar to the octanol-water partition system

properties.171,176,177 Moreover, the difference between the addition of octanol to both

components of the organic phase has also been studied and it has been concluded that

octanol should be added to reproduce the octanol-water system.176

HYPOTHESIS HYPOTHESIS AND AND

OBJECTIVESOBJECTIVES

IV. HYPOTHESIS

Hypothesis and objectives: Hypothesis

61

The experimental work presented in this memory is based on the hypothesis that

quinoxalines di-N-oxide, considered to be the core of the structure, which present a

carboxamide moiety on position two and aliphatic linker between this group and an

aromatic system, can be proposed as potent anti-tuberculosis agents (Figure 46).

Figure 46. General model proposed for the development of the project.

V. OBJECTIVES

Hypothesis and objectives: Objectives

- 65 -

The main purpose of this project is the synthesis of new quinoxaline di-N-oxide

derivatives as anti-tuberculosis agents. The strategy consists of the design and synthesis of

several derivatives of each series, and their in vitro biological evaluation against M.tb. Some

compounds will be selected, based on their structural variability and biological results, for

an in depth study of the physicochemical properties and electrochemical behavior of this

family of derivatives.

A series of specific objectives are proposed in order to achieve the main purpose of

this project:

1. Bibliographic review: state of the art.

2. Design of new quinoxaline di-N-oxide derivatives.

3. Synthesis of the designed compounds.

4. Characterization of the synthesized compounds.

5. In vitro biological evaluation of the synthesized derivatives.

6. Study of the lipophilicity of quinoxaline di-N-oxide derivatives.

7. Study of the electrochemical behavior of quinoxaline di-N-oxide derivatives.

8. Study of possible structure-property and structure-activity relationships (SPR,

SAR).

9. Search of new lead compounds and design of future work plans.

VI. WORK PLAN

Hypothesis and objectives: Work plan

- 69 -

14. Bibliographic review: state of the art

An extensive review of the drugs in use against M.tb and the new approaches in

anti-tuberculosis drug research will be carried out. A summary is outlined in the

introduction of this memory.

15. Design of new quinoxaline di-N-oxide derivatives

According to the results obtained by the group, and previously considered, certain

structural modifications are proposed in an attempt to obtain new derivatives which

improve the activity of the previous compounds.

With regard to position 2, a carboxamide group was substituted in this position.

The promising results of N-arylcarboxamide derivatives, previously studied by the group,138

justified this decision. In addition, the carboxamide moiety is present in many of the most

known anti-tuberculosis drugs, such as INH or PZA among others. Moreover, this group

is directly linked to the pyridine or pyrazine rings, which are structural related to the

quinoxaline heterocyclic. As previously reported, the insertion of a phenyl on position 3,

instead of a methyl moiety, maintained the anti-tuberculosis activity of the studied

quinoxaline derivatives.133,136 Methyl and phenyl moieties were considered in this position

for studying the influence of the molecular volume of the substituent in position 3.

Moreover, we decided to introduce an aliphatic linker between the carboxamide

and the aromatic region.


- 70 -

The synthesis of 3-phenylquinoxaline derivatives was aborted because of the low

stability of these derivatives, difficulties associated with the synthesis and the lost of anti-

tuberculosis activity of these compounds. At this point, and taking into account recent

bibliography,115,193-195 we decided to study the influence of substituents on para position on

the phenyl ring. Several halogens, a trifluoromethyl, a methoxy and a methyl moiety were

introduced on this position for studying the influence of electronic parameters. Moreover,

a series considering a 3,4,5-trimethoxy phenyl was also prepared because this substituent

had been previously studied by the group196 and it presented a great interest in the field of

medicinal chemistry.115 Finally, biphenyl and benzodioxol were considered on the aromatic

region to study the relevance of steric parameters.


- 71 -

Figure 47. Design of new series of quinoxaline di-N-oxide derivatives as anti-tuberculosis

agents.

16. Synthesis of the designed compounds

Once the target compounds are designed, the synthetic routes will be defined.

Many factors have to be considered at this stage of the project. One of the main objetives

is to look for a convergent synthesis, a strategy that involves the individual preparation of

several intermediates which are then assembled in order to obtain the desired compound.

This approach will decrease the number of steps to be taken in the synthesis and increase

the yields. In addition, a convergent synthesis will allow the obtainment of the quinoxalines

di-N-oxide in the final step, thereby avoiding the inconveniences of working with the N-

oxide moiety (Figure 48).


- 72 -

Figure 48. Convergent vs. lineal synthesis.

Beirut reaction, generally used for preparing quinoxaline di-N-oxide derivatives, will

facilitate the use of this synthetic approach (Figure 49).

Figure 49. Beirut reaction.

Finally, it should be kept in mind that tuberculosis is a disease strongly linked to

poverty, and, for this reason, some other factors will be take into account when selecting

the synthetic methods:

• Few steps.

• Easy synthesis.


- 73 -

• Low-time consuming.

• Low cost.

17. Characterization of the synthesized compounds

All of the synthesized compounds will be chemically characterized by thin layer

chromatography (TLC), infrared spectroscopy (IR), proton nuclear magnetic resonance

(1H-NMR), and elemental microanalysis (CHN). Some of the quinoxaline derivatives will

be also studied by high performance liquid chromatography (HPLC).

18. In vitro biological evaluation of the synthesized derivatives

In vitro evaluation of the anti-tuberculosis activity of all the synthesized compounds

will be carried out within the anti-tuberculosis screening programs developed by the

National Institute for Allergy and Infectious Diseases (NIAID).

19. Study of the lipophilicity of quinoxaline di-N-oxide derivatives

A selection of quinoxaline derivatives will be made according to their structural

variety with the aim of studying the logP of these derivatives by experimental methods and

predictive approaches. The reasons for this study are to set up an experimental method for

measuring the logP of quinoxaline di-N-oxide derivatives and to study the agreement

between these experimental values and predicted logP. In addition, a possible relationship

between lipophilicity and biological activity will be study in an effort to define the best

physicochemical profile of quinoxaline di-N-oxide derivatives as anti-tuberculosis drugs.

The study of the partition coefficient of quinoxaline di-N-oxide derivatives by the shake-

flask method and the RP-HPLC method will be develop in the Environment and Life

Sciences Department, Università degli Studi del Piemonte Orientale “Amedeo Avogadro”

(Alessandria, Italia) under the direction of Prof. Domenico Osella and Prof. Mauro Ravera.

20. Study of the electrochemical behavior of quinoxaline di-N-oxide derivatives

A selection of quinoxaline derivatives will be made according to their structural

variety and biological activity diversity with the aim of studying the electrochemical


- 74 -

behavior of this family of heterocycles and gaining insight into the mechanism of action.

The electrochemical studies will be performed at the Department of Chemistry at

Southeast Missouri State University through a colaboration project with Dr. Philip W.

Crawford.

21. Study of possible structure-property and structure-activity relationships (SPR, SAR)

The relationship between the reduction potential of quinoxaline di-N-oxide

derivatives and their structures will be studied in order to gain insight into a possible

mechanism of action of these compounds as anti-tuberculosis agents.

The structure-activity relationship will be studied with the aim of determining the

structural requirements necessary for improving the anti-tuberculosis activity of

quinoxaline derivatives.

22. Search of new lead compounds and design of future work plans

A joint study of the biological results, the electrochemical data and the chemical

structure of the derivatives, will lead to the selection of new lead compounds. This study

and a continuous bibliographic review will be carried out in order to define a new future

work plan (Appendix 3).

MATERIAL AND MATERIAL AND METHODSMETHODS

VII. CHEMICAL SYNTHESIS

Material and Methods: Chemical Synthesis

- 79 -

23. General synthetic scheme

Figure 50. General synthetic scheme.

Reagents and conditions: Ia) acetic acid, diethyl ether, rt.; Ib) methanol, Zn/NH4Cl aq., rt.;

II) 2-hydroxypyridine, reflux, N2 atm.; III) methanol, 0 ºC, N2 atm.; IV) methanol, CaCl2,

ethanolamine, rt.


- 80 -

24. Chemistry of benzofuroxans

24.1. Synthesis of benzofuroxans

The synthesis of the benzofuroxans (BFXs) presented in this project was achieved

by two different routes depending on the substituents on position 5 and/or 6. BFXs with a

fluorine atom on position 5 or with two chlorine atoms on positions 5 and 6 were obtained

through an oxidative cyclization. And the BFX with a trifluoromethyl moiety was

synthesized via azide followed by thermal cyclization.

Synthesis of benzofuroxans through oxidative cyclization (comp. c,g)

This synthesis of BFXs consists on an oxidation of 2-substituted nitroanilines with

sodium hypochlorite and N,N-dimethylformamide (N,N-DMF) as solvent at low

temperature (Figure 51).

Figure 51. Scheme of synthesis of BFX through oxidative cyclization.

This oxidative cyclization seems to occur through a N-chloronitroaniline anion

intermediate that decompose to a singlet nitrene intermediate and leads to the instant

formation of the corresponding benzofuroxan (Figure 52). This step can be recognized by

the transient generation of a deep red-purple color which is seen during the hypochlorite

oxidation.197,198

Figure 52. Synthesis of BFX through oxidative cyclization. Proposed reaction

mechanism.


- 81 -

This method is recommended when the nitroazide pyrolysis fails and it is also

simpler and short time consuming. However, the oxidizing conditions sometimes destroy

the product, for instance, in the synthesis of 5-trifluoromethylbenzofuroxan which must be

carried out through azide pyrolysis.

Synthesis of benzofuroxans via azides (comp. d)

The pyrolysis of o-nitrophenylazides is the most common method for the synthesis

of BFXs.198-200 In this case, the BFX that presents a trifluoromethyl moiety has been

prepared according to the following squeme (Figure 53)

Figure 53. Scheme of synthesis of 5-trifluoromethylbenzofuroxan.

The first step is the synthesis of the aryl diazonium salt, that must be performed at

low temperature due to the low stability of diazonium ions in solution. The diazonium salt

is prepared by reaction of the aniline with nitrous acid, which is generated in situ from the

sodium nitrite. The steps are the generation of the nitrosonium ion (NO+) and the addition

of the ion to the amino group, followed by elimination of water (Figure 54).201

Figure 54. Synthesis of the diazonium salt. Proposed reaction mechanism.

The substitution of the diazonium salt can followed different mechanisms but the

most probable is that the reaction of the diazonium salt with sodium azide gives an adduct

that decompose to nitrogen (N2) and the corresponding phenylazide (Figure 55).201


- 82 -

Figure 55. Formation of the azide. Proposed reaction mechanism.

Finally, the cyclization seems to occur through a singlet nitrene intermediate

generated by the pyrolysis of the azide. The nitro group attacks the nitrene, which is an

electron deficient specie, and the following electronic reorganization affords the BFX

(Figure 56).199

Figure 56. Pyrolysis of the 2-nitrophenylazide and formation of the BFX. Proposed

reaction mechanism.

24.2. Position isomers of 5(6)-monosubstituted benzofuroxans

The interconversion of two isomeric benzofuroxans in solution is one of the most

important and characteristic features of the chemistry of these compounds. Benzofuroxans

tautomerism has been reported in several articles and this process has been studied by

multinuclear NMR methods that have led to the conclusion that this isomerization process

occurs through an ortho-dinitrosobenzene intermediate (Figure 57).198-200,202-204

Figure 57. Equilibrium of 5(6)-monosubstituted BFXs.

It is apparent that the equilibrium constants for the BFX isomerization are

determined by a combination of factors depending on the substituents and the way in


- 83 -

which they are able to interact with the two positions of the heterocyclic ring. By

comparison with the compounds with the substituents directly attached to the furoxan

ring, 5(6)-substituted benzofuroxan equilibrium should be subject to attenuated electronic

effects, and the steric effects should not be considered. In these compounds, when the

substituent is an electron-acceptor group (such as nitro, carboxy, trifluoromethyl or cyano

subtituents) the 6-substituted isomer is more stable. Halo, methoxy, acetoxy and methyl

substitutions lead to small preferences for the 5-substituted benzofuroxan.198,205

25. Chemistry of β-ketoamides

25.1. Synthesis of N-benzyl-3-oxo-3-phenylpropanamide (comp. 2)

The synthesis of compound 2 wa carried out by combination of phenylglyoxal,

benzylisocyanide and acetic acid, under N2 atmosphere, that gives the expected Passerini

adduct. Finally, the reduction of the adduct takes place using activated zinc in saturated

aqueous ammonium chloride and methanol. The total transformation is a combination of

the Passerini three-component reaction followed by zinc promoted removal of the acid

component. (Figure 58).

Figure 58. Scheme of synthesis of N-benzyl-3-oxo-3-phenylpropanamide.

The first step is a multicomponent reaction known as Passerini reaction. It is a

three-component reaction involving an aldehyde or ketone, a carboxylic acid and an

isocyanide and it was discovered in 1921 by Passerini.206 Its mechanism has been a subject

of many studies and reports and different mechanisms have been proposed attending to

kinetic studies and the influence of the solvents used in the reaction.207-210 Most of the

proposed mechanisms suggest an electrophilic activation of the carbonyl followed by a

nucleophilic attack of the isocyanide206,210 as proposed by Ugi who discovered that the

reaction is accelerated in aprotic solvents indicating a non-ionic mechanism (Figure


- 84 -

59).210,211 Finally, the efficiency of this reaction seems to be associated with the irreversible

acyl transfer.212-214

Figure 59. Three-component Passerini reaction. Proposed reaction mechanism.

The second step consists on the deacetoxylation of the Passerini adduct. The use of

metals under reducing conditions is considered an effective method for the removal of

hydroxyl or acetoxyl functions. Several papers establish the use of zinc in acetic acid as a

selective reductive process for enone systems215-218 and, moreover, the use of ultrasounds to

enhance the chemical reactivity of metal powder is well established and it has been the

subject of several studies.217-219 For these reasons, Neo et al. used ultrasounds to activate

zinc with the aim of improving the reductive deacyloxylation of the three-component

Passerini adduct.218 A mechanism through a complex between the zinc atom and the two

carbonyl groups of the adduct is also proposed. The reaction seems to occur by the

complexation of a zinc atom with the amide and ketone carbonyl groups. This complex

forces a flat conformation and a pair of electrons of the zinc atom is donated to the ketone

oxygen. As a consecuence, the enolate is formed and the acetate group is released.

Tautomerization of the enol form affords the β-ketoamide.218


- 85 -

Figure 60. Reduction of the Passerini adduct. Proposed reaction mechanism.

25.2. Synthesis of 3-oxo-N-(2-phenylethyl)-3-phenylpropanamide (comp. 3)

The synthesis of compound 3 was carried out by a condensation of the ethyl-3-oxo-

3-phenylpropanoate and 2-phenylethylamine using 2-hydroxypyridine as catalyst. The

reaction is performed under N2 atmosphere to achieve humidity absence (Figure 61).

Figure 61. Scheme of synthesis of 3-oxo-N-(2-phenylethyl)-3-phenylpropanamide.

The mechanism of reaction seems to occur through a cyclic intermediate (spiranic

cyclic). The first step consists on a nucleophilic attack of the 2-hydroxypyridine at the

carbon of the ester that presents a partial positive charge giving an intermediate stabilized

by hydrogen bonding. The amine attacks this intermediate generating a new intermediate

stabilized by hydrogen bonding that develops to the desired compound (Figure 62).220,221


- 86 -

Figure 62. Synthesis of 3-oxo-N-(2-phenylethyl)-3-phenylpropanamide. Proposed reaction

mechanism.

25.3. Synthesis of N-substituted 3-methyl-3-oxopropanamides (comp. 4-10)

The synthesis of compounds 4-10 was carried out by acetoacetylation of the

corresponding arylamine and diketene under N2 atmosphere and cooled in an ice bath until

0ºC (Figure 63).

Figure 63. Scheme of synthesis of N-substituted 3-methyl-3-oxopropanamides.

The mechanism is thought to occur through a nucleophilic attack of the amine at

the carbon that presents a partial positive charge followed by a rearrangement that gives the

desired compound (Figure 64).222


- 87 -

Figure 64. Acetoacetylation by diketene. Proposed reaction mechanism.

26. Chemistry of quinoxaline di-N-oxide derivatives

26.1. Synthesis of quinoxaline di-N-oxide derivatives. The Beirut reaction

Before 1965, the synthesis of quinoxalines di-N-oxide was carried out by the

cyclization of a suitable precursor or by direct oxidation of the parent quinoxaline. These

methods suffered from many inconveniences and the synthesis of these derivatives was

changed by a one step condensation between benzofuroxans and enamines to give the

corresponding quinoxaline 1,4-di-N-oxide (Figure 65).219 This reaction was discovered by

Haddadin and Issidorides in 1965 and it is known as Beirut reaction to acknowledge the

city in which it was discovered.198,201,219,223

Figure 65. Beirut reaction.

It has been reported that BFXs can also react with ketone or aldehyde in presence

of ammonia or a secondary amine and it has been demonstrated that these reactions

proceed through enamine intermediate formed in situ.224 Despite the general definition, the

Beirut reaction has been extended and involves any condensation between a benzofuroxan

and a ketocarboxylic acid derivative, specially β-ketoester and β-ketocarboxamide, in a base

catalyzed reaction with or without intermediary formation of the corresponding

enamines.225,226 With regard to the synthesis of quinoxalinecarboxamide 1,4-di-N-oxide, it

has been reported that an improved and efficient method is using ethanolamine as base in

presence of calcium salts.131,132,225,227


- 88 -

With the aim of optimizing the synthesis of the compounds presented in this work,

the synthesis has been carried out by the classical Beirut reaction using triethylamine as

base and also employing K2CO3 in acetone or ethanolamine as base with calcium salts. As

it has been reported, the presence of calcium salts allows increasing the yields by 2 or 3 fold

and reducing the reaction times and, for these reasons, the last method was the selected

one for this work. It is suggested that the catalytic effect of the calcium salts can be

explained by the formation of a calcium chelate which has been confirmed by 1H-NMR

(Figure 66).219,225

Figure 66. Reaction of BFXs with β-ketocarboxamides in the presence of calcium salts.

Proposed mechanism of action.

26.2. Position isomers of 6(7)-monosubstituted quinoxalines

The existence of position isomers is also observed in the case of unsymmetrical

substituted quinoxaline 1,4-di-N-oxide derivatives. When the Beirut condensation is carried

out using a mono-substituted BFX, a mixture of position isomers of the corresponding

quinoxaline 1,4-di-N-oxide is obtained. Nevertheless, the degree of regiospecificity of the

reaction depends on the nature of the substituent. It has been observed that only one of

the possible isomers is obtained when a methoxy moiety is substituted on the quinoxaline

ring, while a mixture of isomers is formed with halogens, methyl and trifluoromethyl

substituents. The proportion of each isomer in the mixture is related to the nature of the

substituent in the molecule and to the synthetic method used; however, in all cases, the

most abundant isomer is that which has a substituent in position 7 of the quinoxaline ring

according to previous studies.152,196,204


- 89 -

26.3. Photochemical reactions of quinoxalines di-N-oxide

Photochemical instability of these derivatives is a fact that must be considered. It

has been observed that the absorption spectrum of quinoxalines di-N-oxide neutral

solutions change quickly due to exposition to sunlight. Previous reports suggest that the

photolysis of these derivatives consists on the interconversion of quinoxaline 1,4-dioxide to

2H-quinoxaline 4-oxide through an oxaziridine intermediate (Figure 67).219,228,229

Figure 67. Photolysis of quinoxaline di-N-oxide derivatives.

Furthermore, it has been concluded that oxaziridine intermediate also reverts back

to quinoxaline 1,4-dioxide and the equilibrium is forced to 2-oxo-1,2-diH-quinoxaline 4-

oxide in acidic solution and alcoholic solutions. For these reasons, quinoxaline 1,4-dioxide

must be kept protected from light and humidity.

VIII. EXPERIMENTAL CHEMICAL SECTION

Material and Methods: Experimental Chemical Section

- 93 -

27. Material and reagents

Infrared spectroscopy (IR)

The infrared spectra were performed on a Thermo Nicolet FTIR Nexus Euro

(Madison, USA) with OMNIC 6.0 software using KBr pellets for solid samples and NaCl

pellets for liquid samples. Frequencies are expressed in cm-1. Signals intensities are reported

as: w (weak), m (medium) and s (strong).

Nuclear Magnetic Resonance (NMR)

The NMR spectra were recorded on a Bruker 400 UltrashieldTM (Bruker BioSpin

GmbH, Rheinstetten, Germany), using TMS as the internal standard and with DMSO-d6 as

the solvent. The chemical shifts (δ) are reported in ppm and the coupling constant (J)

values are given in Hertz (Hz). Signal multiplicities are represented by: s (singlet), bs (broad

singlet), d (doublet), dd (double doublet), ddd (double double doublet), t (triplet), tt (triple

triplet) and m (multiplet).

Elemental microanalyses (C,H,N)

Elemental microanalyses were obtained on an Elemental Analyzer LECO CHN-

900 (Michigan, USA) from vacuum-dried samples. The analytical results for C, H, and N

are within ±0.5 of the theoretical values, indicating a purity of >95%.

High Performance Liquid Chromatography (HPLC)

High Performance Liquid Chromatography technique was used with two different

purposes. For purity measures, HPLC experiments were developed on an Ultimate 3000

Chromatograph (DIONEX) with Chromeleon v.6.8 software. The measurements were

performed using an RP 18 column (LICHROSPHER 100 RP 18 E.C. 5 µm; 10x0.46;

TEKNOKROMA) as the stationary phase, at a flow rate of 1 mL/min and with

methanol/water (60:40) as the mobile phase. The retention times (tr) are expressed in

minutes and the reference wavelength is set at 254 nm.

For partition coefficient determination, HPLC experiments were developed on

Waters 2695 Separation Module system and a Waters 2487 Dual λ Absorbance Detector

with Empower 2 Software, Empower Pro Software. The measurements were performed

using a Supelcosil LC-ABZ (5µm; 15cm×4.6mm, SUPELCO Sigma-Aldrich) as the

stationary phase, at a flow rate of 1 mL/min and using methanol/water as the mobile

phase.


- 94 -

Thin Layer Chromatography (TLC)

Alugram® SIL G/UV254 (Layer 0.2mm) (Macherey-Nagel GmbH & Co. KG.

Postfach 101352. D-52313 Düren, Germany) was used for Thin Layer Chromatography.

TLCs were studied under UV wavelength at 254 and 365 nm. Several mobile phases were

used as eluent considering toluene/dioxane, dichloromethane/methanol, hexane/ethyl

acetate in different gradients. The preferred one for monitoring the reactions was a mixture

of toluene/dioxane/acetic acid (90:25:4).

Column Chromatography

Purification by column chromatography was developed using glass columns and

Silica gel 60 (0.040-0.063 mm) (Merck) as stationary phase. Several mobile phases were

used as eluent being toluene/dioxane (6:4) the most used.

Flash Column Chromatography was developed on an automated Flash

Chromatography System CombiFlash® Rf (TELEDYNE ISCO, Lincoln, USA) instrument

with Silica RediSep® Rf columns (Average particle size: 35 to 70 microns; Average pore

size: 60 Å). Purification methods were developed using dichloromethane and methanol to

run suitable gradient condition.

Melting Point (M.P.)

Melting points were determined with a Mettler FP82+FP80 apparatus (Greifense,

Switzerland). Melting points are expressed in degree centigrade (ºC).

Chemicals

All reagents and solvents were purchased from commercial sources. E. Merck

(Darmstadt, Germany), Panreac Química S.A. (Montcada i Reixac, Barcelona, Spain),

Sigma-Aldrich Química, S.A., (Alcobendas, Madrid), Acros Organics (Janssen

Pharmaceuticalaan 3a, 2440 Geel, Belgium) and Lancaster (Bischheim-Strasbourg, France).


- 95 -

28. General synthetic methods

In order to facilitate the reading and comprehension of the synthesized

compounds, the codes are explained. Compounds references have been assigned as

follows:

- Benzofuroxans are referenced as a letter related to the substituents on positions Ra and

Rb. (Figure 68)

Figure 68. General structure of benzofuroxans.

Letter a b c d e f g

Ra/Rb H/H Cl/H F/H CF3/H CH3/H OCH3/H Cl/Cl

- β-ketoamides are codified as numbers related to the substituents R and R3 that define

the ten series presented in this project. (Figure 69)

Figure 69. General structure of β-ketoamides.

Series 1 2 3

R3

R

Series 4 5 6 7 8 9 10

R3 CH3 CH3 CH3 CH3 CH3 CH3 CH3

R

- Quinoxalines 1,4-di-N-oxide are expressed as a combination of the two previous assignments.

Figure 70. General structure of quinoxaline 1,4-di-N-oxide derivatives.


- 96 -

As an example, the following final compound, that presents two chlorine atoms in

positions Ra and Rb, a methyl moiety substituted in position R3 and p-bromobenzyl linked

to the carboxamide group, is referenced as 6g.

Reference: 6g

Ra/Rb=Cl/Cl

R3= CH3

R= p-bromobenzyl

28.1. Method A. General methods for synthesis of benzofuroxans

Seven benzofuroxans (BFX) are employed in

order to synthesize the quinoxaline 1,4-di-N-oxide

derivatives. Two different synthetic methods are used

to prepare the benzofuroxans depending on the

substituents in positions Ra and Rb. Compounds a, b,

e and f are commercially available.

Table 2.

Method A1. General method for synthesis of benzofuroxans (comp. c, g)

The corresponding commercial 2-nitroaniline (20.0 mmol) is dissolved in 75 mL of

N,N-dimethylformamide and cooled down to 0 ºC in an ice bath. 500-700 mL (depending

on the substituents) of sodium hypochlorite solution 10% v/v are added slowly and the

reaction mixture is stirred for 15 minutes in an ice bath. The precipitate is filtered and

washed with cold water.

This method is used to synthesize compounds c and g which are used without

further purification.

Method A2. General method for synthesis of 5-trifluoromethylbenzofuroxan (comp. d)

Preparation of 2-nitro-4-trifluoromethylphenylazide

2-nitro-4-trifluoromethylaniline (17.0 mmol) is added to a mixture of concentrated

hydrochloric acid (20 mL) and water (60 mL) and stirred for 5 minutes at room

temperature. The mixture reaction is cooled in an ice bath and a mixture of sodium nitrite

Compound Ra/Rb

a H/H

b Cl/H

c F/H

d CF3/H

e CH3/H

f OCH3/H

g Cl/Cl


- 97 -

(29.0 mmol) and water (10 mL) is added dropwise. The mixture reaction is stirred for 15

minutes at 0 ºC and filtered. The filtered reaction is added to a stirred solution of sodium

azide (38.0 mmol) and sodium acetate (360.0 mmol) in water. The solution is extracted with

dichloromethane and the organic phase is dried with sodium sulphate and filtered. The

solvent is removed in vacuo in order to obtain black oil.

Preparation of 5-trifluoromethylbenzofuroxan

2-nitro-4-trifluoromethylphenylazide (10.0 mmol) is dissolved in toluene (25 mL)

and added dropwise to a refluxed toluene solution. The mixture is refluxed for 2 hours.

The solvent is removed in vacuo and brown oil is isolated.

28.2. Method B. General methods for synthesis of N-substituted-3-oxo-3-phenylpropanamides

2 N-substituted benzoylcetamides are

employed in order to synthesize the quinoxaline 1,4-

di-N-oxide derivatives. Two different synthetic

methods are used to prepare the benzoylcetamides

depending on the R substituent. Compound 1 is

commercially available.

Table 3.

Compound R

2

3

Method B1. General method for synthesis of N-benzyl-3-oxo-3-phenylpropanamide

(comp. 2)

Acetic acid (5.0 mmol) and phenylglyoxal are diluted in diethyl ether (25 mL) under

N2 atmosphere. Once dissolved, the benzylisocyanide (5.0 mmol) is added dropwise and

the reaction mixture is stirred at room temperature for 72 hours. The residue obtained is

filtered and washed with isopropanol.

The solid is dissolved in methanol (32.0 mL) and added dropwise to a solution of

Zn dust (8.0 mmol) in saturated aqueous NH4Cl (8.0 mL) previously activated in a

sonication bath for 5 minutes. The mixture is stirred at room temperature for 30 minutes

and filtered in order to eliminate the Zn. Water (100 mL) is added to the mixture and the

solid obtained is filtered and washed with water. The solid is used without further

purification.


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Method B2. General method for synthesis of 3-oxo-N-(2-phenylethyl)-3-

phenylpropanamide (comp. 3)

Ethyl-3-oxo-3-phenylpropanoate (6.0 mmol), 2-phenylethylamine (15.0 mmol) and

2-hydroxypyridine (6.0 mmol) are refluxed at 130ºC under N2 atmosphere for 48 hours.

The mixture reaction is dissolved in dichloromethane and quenched with water. The

organic phase is dried with anhydrous sodium sulphate and filtered. The solvent is removed

in vacuo and precipitated with cold isopropanol in order to obtain a white solid. The solid

is used without further purification.

28.3. Method C. General method for synthesis of N-substituted-3-methyl-3-

oxopropanamides (comp. 4-10)

The corresponding aryl amines (20.0

mmol) are diluted in methanol (10 mL) under

N2 atmosphere and cooled in ice bath until 0 ºC.

Next, diketene (25.0 mmol) is added dropwise

and the reaction is stirred for 1-3 hours. The

obtained residue is precipitated with cold diethyl

ether and filtered in order to obtain red-brown

solid.

This method is used to synthesize

compounds 4-10 which are used without further

purification.

Table 4.

Compound R

4

5

6

7

8

9

10


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28.4. Method D. General method for synthesis of quinoxaline-2-carboxamide 1,4-di-N-oxide derivatives

The appropriate BFX (1.0 mmol) and

the corresponding β-ketoamide (1.2 mmol) are

dissolved in the minimum amount of methanol.

Next, calcium chloride (0.1 mmol) is added as

catalyst and ethanolamine (0.1 mmol) as base.

The mixture reaction is stirred at room

temperature for 1-48 hours (the reaction is

monitored by TLC until no more final

compound is obtained), filtered and washed

with cold diethyl ether. The solid is dissolved in

dichloromethane and quenched with water. The

organic phase is dried with anhydrous sodium

sulphate and filtered. The solvent is removed in

vacuo and precipitated with cold diethyl ether in

order to obtain a yellow solid. The solid is

purified by column chromatography, if

necessary.

Table 5.

Series Compound

1 1a-g

2 2a-g

3 3a; 3c

4 4a-g

5 5a; 5b; 5c; 5g

6 6a; 6b; 6c; 6g

7 7a; 7b; 7c; 7g

8 8a; 8b; 8c; 8g

9 9a; 9b; 9c; 9g

10 10a; 10b; 10c; 10g


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29. Purification Methods: Automated Flash Column Chromatography 29.1. Introduction

Traditional glass column chromatography is a practical method that allows

purifying crude reaction mixtures showing reasonable separation results. There are,

however, important disadvantages associated with this method that is time consuming, has

low resolution and gets not reproducible results.

In this sense, automated flash column chromatography systems offer an alternative

technique which makes possible to:

- reduce time consume

- reduce solvent waste

- increase productivity

- purify wide ranges of sample sizes

- automate the purification stage

- get reproducible results

29.2. Basic elements of advanced flash column chromatography

There are only a few factors to be considered when preparing for flash

chromatography purification, but they all need to be selected thoughtfully in order to

achieve a successful separation. Basic elements which must be considered in order to set up

a purification method are described in the following paragraphs.

• Stationary phase: Stationary phase selection is driven by the nature of the products to

be separated. Factors such as compound polarity and functionalities greatly influence

the media selection. A wide variety of functionalized silica gel has been developed and

commercialized in order to facilitate isolation of compounds with very different

physico-chemistry properties. Some of the options that can be considered are Normal-

Phase Silica, Amine, Alumina-Basic, Alumina-Neutral, Cyano, C-18 Reversed Phase

Silica, Diol, Strong Anion Exchange or Strong Cation Exchange.

Thin Layer Chromatography has been performed using Silica and Alumina as stationary

phase with the aim of selecting the suitable one for Flash Column Chromatography.

Finally, Silica column (Silica RediSep® Rf columns -Average particle size: 35 to 70

microns; Average pore size: 60 Å) has been the selected one for the purification

purpose.


- 101 -

• Mobile phase technique: Several techniques can be used when performing column

chromatography purification. Linear with isocratic hold is the selected technique with

the aim of decreasing overall purification time and increasing resolution. This technique

allows combining isocratic and linear technique so that a linear stage is used to

gradually get the desired mobile phase conditions and then followed by linear step.

Dichloromethane and methanol are the selected solvents to purify the compounds

which are presented in this project. At this point, it must be considered that it is not

possible to use 100% methanol as it could damage silica columns. In the same way, the

capacity of the pumps to get the established mobile phase conditions is limited and it is

recommended to use dichloromethane /methanol (80:20) as polar solvent.

• Detection wavelength: The appropriate wavelength must be selected in order to detect

the desired compound. In the case of quinoxalines the selected wavelength is 254 nm

as the structure presents aromatic rings which absorb at this wavelength.

• Sample loading technique: Due to the low solubility of the samples it is necessary to use

empty solid load cartridges in order to introduce the samples onto the column. Silica

gel (Silica gel 60 _0.040-0.063 mm) is added to the sample, previously dissolved in

dichloromethane, and the solvent is removed with a rotary evaporator providing the

sample coated on a silica bed. The sample on silica is then poured into an empty

cartridge, topped with a frit, and loaded onto the system.

IX. BIOLOGICAL EVALUATION. ANTI-TUBERCULOSIS ACTIVITY

Material and Methods: Biological Evaluation. Anti-tuberculosis Activity

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In vitro evaluation of the anti-tuberculosis activity has been carried out within the

Tuberculosis Antimicrobial Acquisition & Coordinating Facility (TAACF) screening

program for the discovery of novel drugs for the treatment of tuberculosis. The purpose of

the screening program was to provide a resource whereby new experimental compounds

can be tested for their ability to inhibit the growth of virulent Mycobacterium tuberculosis

(M.Tb.) (www.taacf.org).

The TAACF was established by the National Institute of Allergy and Infectious

Diseases (NIAID, USA) in 1994 and changed into alternative resources to assist

researchers in 2010. The Southern Research Institute (SRI) coordinated the overall

program under the direction of the NIAID and Colorado State University (CSU)

performed in vivo screening services for compounds that have shown promising in vitro

activity.

In 2009 a program with the Division of Microbiology and Infectious Diseases

(DMID) of the NIAID started to carry out the biological evaluation. The DMID supports

extramural research to control and prevent diseases caused by virtually all human infectious

agents except HIV by providing funding opportunities and a comprehensive set of services

for researchers.

30. TAACF anti-tuberculosis evaluation

The TAACF screening program considered the following tests:

• In vitro TB testing

- Primary Screen (Dose Response): Determination of a 90% Inhibitory

Concentration

- Secondary Screen: Determination of Mammalian Cell Cytotoxicity

• In vivo TB testing

- Determination of Maximum Tolerated Dose

- Bioavailability

- In vivo Evaluation of Anti-Mycobacterium tuberculosis Activity in Gamma

Knock-out (GKO) Model and Murine Aerosolized TB Model

Biological tests have been performed according to previously described methods

which are outlined bellow.


- 106 -

30.1. Primary screening (Dose response): Determination of a 90% inhibitory concentration (IC90)

The initial screening is conducted against Mycobacterium tuberculosis H37Rv (ATCC

27294) in BACTEC 12B medium using the Microplate Alamar Blue Assay (MABA). 230Compounds are tested in ten 2-fold dilutions, typically from 100 µg/mL to 0.19 µg/mL.

The IC90 is defined as the concentration effecting a reduction in fluorescence of 90%

relative to controls. This value is determined from the dose-response curve using a curve-

fitting program. Any IC90 value of ≤ 10 µg/mL is considered "Active" for antitubercular

activity.

30.2. Secondary screening: Determination of mammalian cell cytotoxicity (CC50)

The VERO cell cytotoxicity assay is done in parallel with the TB Dose Response

assay. After 72 hours exposure, viability is assessed using Promega’s Cell Titer Glo

Luminescent Cell Viability Assay, a homogeneous method of determining the number of

viable cells in culture based on quantitation of the ATP present.

Cytotoxicity is determined from the dose-response curve as the CC50 using a curve

fitting program. Ultimately, the CC50 is divided by the IC90 to calculate a SI (Selectivity

Index) value. SI values of ≥ 10 are considered for further testing.

31. DMID anti-tuberculosis evaluation

31.1. MIC assay

The resazurin MIC assay, developed by Collins and Franzblau, is a colorimetric

assay used to test compounds for antimycobacterial activity.230 A color change from blue to

pink is observed when growth occurs. Compounds are initially tested at a single point

concentration of 10 µg/mL against Mycobacterium tuberculosis H37Rv, obtained form

Colorado State University, Fort Collins, CO. If compounds are active at the 10 µg/mL

level, they are further tested in an MIC assay at 8 concentrations in a dose range between

10 to 0.078 µg/mL.

• Receipt and preparation of test compounds: upon receipt, test compounds are placed

in a -20ºC freezer. The day of the experiment, one vial from each compound is

reconstituted to achieve a stock concentration of 3.2 mg/mL.

• Inoculum preparation: H37Rv is grown in Middlebrook 7H9 broth medium (7H9

medium) supplemented with 0.2% (v/v) glycerol, 10% (v/v) ADC (albumin, dextrose,


- 107 -

catalase), and 0.05% (v/v) Tween 80. The bacteria are inoculated in 50 mL of 7H9

medium in 1 liter roller bottles that are placed on a roller bottle apparatus in an ambient

37ºC incubator. When the cells reach OD600 of 0.150 (equivalent to ~1.5×107

CFU/mL), they are diluted 200-fold in 7H9 medium.

• Single point concentration procedure: the procedure is the same that used for the MIC

procedure described below, but only the first 2 fold dilution is made that reduces the

stock solution to 1.6 mg/mL. An additional 1:10 dilution is made in water which

reduces the stock solution further to 0.16 mg/mL. Addition of 6.25 µL of the 1:10

dilution to the wells in a final volume of 100 µL will give rise to a concentration

equivalent to 10 µg/mL.

• MIC procedure

1. 20 µL of the 3.2 mg/mL test compound is added to a 96-well microtiter plate.

2. 2-fold dilutions are made by the addition of 20 µL of diluent.

3. Each dilution is further diluted 1:10 in sterile water (10 µL of dilution to 90 µL of

sterile water; the additional 10-fold dilution in water is required when DMSO is

used as solvent to minimize toxicity to the bacteria. For uniformity in the assay

procedure, this dilution step is used even if water or other solvents are used.)

4. 6.25 µL of each dilution is transferred to duplicate 96-well test plates.

5. 93.75 µL of the cell suspension (~104 bacteria) in 7H9 medium is added to the test

plates.

6. Positive, negative, sterility and resazurin controls are tested.

- positive controls include: rifampicin and isoniazid

- negative controls include:

cell culture with solvent and water

cell culture only

- sterility controls include:

media only

media with solvent and water

- resazurin control includes one plate containing the diluted compounds with

resazurin only. No bacterial suspension is added. This control plate is needed to

verify whether the compound reacts with resazurin that could possibly elicit

fluorescence.

7. The 96 well test plates are incubated in an ambient 37ºC incubator for 6 days.


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8. After the 6-day incubation, 5 µL of a 0.05% sterile resazurin solution is added to

each well of the 96-well plate. The plates are placed in an ambient 37ºC incubator

for 2 days.

9. After the 2-day incubation, a visual evaluation and fluorimetric read-out is

performed. The results are expressed as µg/mL (visual evaluation) and as IC50 and

IC90 (fluorimetric readout).

X. ELECTROCHEMICAL STUDY

Material and Methods: Electrochemical Study

- 111 -

Electrochemical studies have been performed at the Department of Chemistry at

Southeast Missouri State University through a colaboration project with Dr. Philip W.

Crawford.

Dimethylformamide and tetrabutylammonium perchlorate (TBAP) were obtained

commercially in the highest purity available and used without further purification.

Ferrocene was obtained from the Aldrich Chemical Company. Cyclic voltammetric

experiments were carried out on a CHI Instruments 630 voltammetric analyzer.

A three electrode cell is used for all electrochemical experiments, consisting of a Pt-

disk (1.6 mm diameter) electrode, a Pt-wire auxilliary electrode, and a Ag/AgNO3 (0.1 M in

acetonitrile) reference electrode (SRE). Deoxygenation of all solutions is accomplished by

passing a gentle, constant stream of prepurified nitrogen through the solution for 15

minutes and maintaining a blanket of the inert gas over the solution during the experiment.

To account for daily variations in the reference electrode and liquid junction potentials,

ferrocene (Fc) is added to each solution following measurements of the test compound,

and used as an internal reference redox system.231

Test solutions contain 0.5 mM of the desired compound and 0.1 M TBAP.

Scan rates range from 50 mV/s to 500 mV/s. Half-wave potentials (E1/2) are

measured as the average of the cathodic and anodic peak potentials.232

For first derivative cyclic voltammograms, Epc is determined at the point where the

derivative curve crosses the baseline.232

All potentials are reported versus the ferrocene/ferrocinium redox couple, i.e.

Epc, SRE – E1/2, Fc/Fc+ or E1/2, SRE – E1/2, Fc/Fc+

[Fe(C5H5)2] [Fe(C5H5)2]+ + e-

XI. DETERMINATION OF PARTITION COEFFICIENT

Material and Methods: Determination of Partition Coefficient

115

The study of the partition coefficient of quinoxaline di-N-oxide derivatives by the

shake-flask method and the RP-HPLC method has been carried out in Environment and

Life Sciences Department, Università degli Studi del Piemonte Orientale “Amedeo

Avogadro” (Alessandria, Italia) under the direction of Prof. Domenico Osella and Prof.

Mauro Ravera.

32. Shake-flask method

32.1. Material and reagents

The equipments used for the experiments were: ALC Centrifuge PK130, CWS,

ANNITA III Processign&Control Interface; Jasco V550 UV/VIS spectrophotometer, with

a wavelength ranging 190 to 900 nm provided with a deuterium lamp and a tungsten iodine

lamp; pHmeter ORION model 420A.

1-octanol high analytical grade (extrapure, purity>99%) was purchased from Sigma-

Aldrich and freshly bi-distilled water was used. Octanol and water were mutually saturated

at the temperature of the experiment (25±2 ºC) for 24 hours and the separation of the two

phases was achieved by centrifugation at 3000 rpm for 3 minutes.233

Test solutions were 0.3 mM of the desired compound in octanol-saturated water

(pH=7.4).

32.2. Experimental

Test solutions are prepared and an aliquot is taken and measure in the UV-Vis

spectrophotometer. The shake-flask experiments are carried out at 25±2 ºC, at least by

triplicate for each test substance. In a 50 mL test tube, 5 mL of the test solution are

overlaid by 5 mL of water-saturated octanol and test vessels are placed in a mechanical

shaker for 30 minutes (200 rotations in five minutes). The separation of the two phases is

achieved by centrifugation at 1000 rpm for 30 minutes at the test temperature. An aliquot

of the aqueous phase is taken and measured in the UV-Vis spectrophotometer.

32.3. Evaluation of experimental data

The partition coefficient is defined as the ratio between molar concentration of the

test substance in octanol and aqueous phase after partitioning and it is calculated as

follows:

Equation 6


116

or

Equation 7

Where [substance] is the concentration of the test substance and is considered as

the absorbance measured at the selected wavelength.

33. Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method

33.1. Material and reagents

The inorganic salts and the reference compounds were, at least, of analytical grade

(Sigma-Aldrich and Fluka) and used without further purification. HPLC-grade methanol

and 1-octanol (Sigma-Aldrich) and bi-distilled water were used to prepare the mobile phase.

The retention times (tR) were measured using a Waters 2695 Separation Module

system and a Waters 2487 Dual λ Absorbance Detector with Empower Pro Software on a

Supelcosil LC-ABZ 5µm; 15cm×4.6mm stationary phase.

Test solutions were 0.3 mM of the desired compound in HPLC-grade methanol

(pH=7.4).

33.2. Experimental

The RP-HPLC methods of Minick234 and Lombardo176 were considered for

estimating the octanol/water partition coefficients (logPo/w) of the compounds. The

Supelcosil LC-ABZ column (5 µm, 15 cm×4.6 mm)171,176,234,235 was selected to substitute the

organic phase of the shake-flask method because it has been reported that this column

affords a reasonable correlation model for a great variety of compounds.171,179,236,237 The

mobile phase consisted of 20 mM MOPS (3-morpholinopropanesulfonic acid) buffer (pH

7.4) and methanol in varying proportions, from 70 to 40%. A 0.25% of octanol was added

to methanol, and octanol-saturated water was used to prepare the buffer.176 The other

chromatographic conditions were: flow set at 1.0 mL/min-1; isocratic elution, UV-visible

detector set at the wavelength with maximum absorbance (260 and 210 nm). The test

solutions were 0.3 mM in the desired compound. All the experiments were performed at

25±2 ºC at least twice.

The retention times (tR) of each quinoxaline derivatives was measured at different

proportions of methanol (from 70 to 40%) and injections of pure methanol were used to


117

determine the column dead-time (t0). The capacity factors (logk’) were calculated according

to Equation 8.

logk’ = log[(tR-t0)/t0] Equation 8

Starting from these results, the extrapolation to 0% methanol for each compound

was calculated and the capacity factors (logk’0) were used to predict the corresponding

logPo/w.

The capacity factors of compounds, with known logPo/w 169,177,191 and accepted as

reference compounds by the OECD,191 were used to create a calibration curve (Equation

9):

logPo/w = a logk’0 + b Equation 9

The common regression coefficient of determination R2 was used to evaluate the

fitting ability of the model. Another measurement for defining the accuracy of the

proposed model is the RMSE (Root Mean Squared Error), which summarizes the overall

error of the model (Equation 10).

Equation 10

Where is the logPo/w value calculated with equation Equation 9, is the

reference value and is the number of reference compounds used to create the curve.

33.3. Cross-validation of the RP-HPLC method

In order to judge if the experimentally measured logk’0 can be used to predict the

logPo/w value, a calibration curve was created with the capacity factors of the reference

compounds and a linear regression equation between the logPo/w and logk’0 was determined.

The robustness of the model and its predictivity were evaluated by the Leave-One-

Out (LOO) cross-validation procedure.238,239 According to this procedure, the logPo/w value

of each compound in the reference data set is predicted by the equations derived from all

the other compounds except the predicted one. At the end of this procedure two values are

available for each reference compound, the reference shake-flask logPo/w value ( ) and the

predicted one ( ). With these data, two statistical parameters (R2LOO, RMSELOO) were

calculated to indicate the predictivity of the model. The regression coefficient (R2LOO) is

defined by Equation 11:


118

Equation 11

Where is the mean of the reference shake-flask logPo/w value and n is the number

of reference compounds. A high value of R2LOO indicates to a good predictive ability of the

model. The root mean square error in prediction (RMSELOO) is calculated with Equation

12:

Equation 12

34. logP predictive approaches

Quinoxaline derivatives were subjected to the module ALOGPS with the aim of

comparing experimental logPo/w values and predicted data. ALOGPS provides interactive

on-line predictions of logPo/w and aqueous solubility. Nine values from different

computation methods can be obtained by using the module ALOGPS (ALOGPS 2.1

includes ALOGPs, AC logP, AB/logP, miLogP, ALOGP, MLOGP, KOWWIN,

XLOGP2 and XLOGP3) (http://www.vcclab.org/).

The ALOGPS method is part of the ALOGPS 2.1 program used to predict

lipophilicity and aqueous solubility of compounds. The lipophilicity calculations within this

program are based on the associative neural network approach and an efficient partition

algorithm. This program also provides a possibility to include new data into the memory of

neural nets without retraining the neural networks themselves in the so-called LIBRARY

mode. The LIBRARY dramatically improves prediction of the ALOGPS program for the

logPo/w prediction using in-house data sets178,240,241

The LogKow (Kow-WIN) program estimates the logPo/w of organic compounds

and drugs using an atom/fragment contribution method developed at Syracuse Research

Corporation (http://www.syrres.com).

miLogP is calculated by the methodology developed by Molinspiration as a sum of

fragment-based contributions and correction factors. This method for logPo/w prediction is

based on group contributions which have been obtained by fitting calculated logPo/w with

experimental logPo/w for a training set of more than twelve thousand, mostly drug-like

molecules. Molinspiration methodology for logPo/w calculation is very robust and capable of


119

processing practically all organic and most organometallic molecules

(http://www.molinspiration.com/).

XLOGP2 gives logPo/w values by summing the contributions of component atoms

and correction factors. Altogether 90 atom types are used to classify carbon, nitrogen,

oxygen, sulfur, phosphorus and halogen atoms, and 10 correction factors are used for some

special substructures. The contributions of each atom type and correction factor are

derived by multivariate regression analysis of 1853 organic compounds with known

experimental logPo/w values242 The additive model implemented in XLOGP3 uses a total of

87 atom/group types and two correction factors as descriptors. It is calibrated on a training

set of 8199 organic compounds with reliable logPo/w data through a multivariate linear

regression analysis.243

RESULTS AND RESULTS AND DISCUSSIONDISCUSSION

XII. COMPOUNDS CHARACTERIZATION

Results and Discussion: Compounds Characterization

- 125 -

Compound description forms are showed in this section. For benzofuroxans and β-

ketoamides, used as starting materials, the following information is included: chemical

name, compound reference, molecular formula, molecular weight, appearance, yield,

synthetic method and infrared spectroscopy (IR). Proton nuclear magnetic resonance (1H-

NMR) is only included in case of β-ketoamides structures.

The description of the forty-seven final compounds presented in this memory

include the following information: chemical name, compound reference, molecular

formula, molecular weight, melting point, appearance, yield, synthetic method, purification

method, infrared spectroscopy (IR), proton nuclear magnetic resonance (1H-NMR) and

elemental analysis. The 1H-NMR of the position isomer presented in higher proportion is

only described. High performance liquid chromatography (HPLC) is included for some of

the derivatives.


- 126 -

5-fluorobenzofuroxan (Compound c) M.F.: C6H3FN2O2

M.W.: 154.10

Appearance: yellow solid

Yield: 44%

Synthetic method: General Method A1 starting from 4-fluoro-2-nitroaniline.

IR (KBr), ν : 3083 (w, νarC-H); 1410 (s, νN+O-); 1165 (s, νarC-F) cm-1

5-trifluoromethylbenzofuroxan (Compound d) M.F.: C7H3F3N2O2

M.W.: 204.11

Appearance: yellow solid Yield: 68%

Synthetic method: General Method A2 starting from 2-nitro-4-trifluoromethyl aniline.

IR (KBr), ν : 3109 (w, νarC-H); 1343 (s, νN+O-); 1180 (s, νarC-CF3) cm-1

5,6-dichlorobenzofuroxan (Compound g) M.F.: C6H2Cl2N2O2

M.W.: 205.00


Yield: 93%

Synthetic method: General Method A1 starting from 4,5-dichloro-2-nitroaniline.

IR (KBr), ν : 3085 (w, νarC-H); 1378 (s, νN+O-); 1079 (s, νarC-Cl) cm-1


- 127 -

3-oxo-N-benzyl-3-phenylpropanamide (Compound 2) M.F.: C16H15NO2

M.W.: 253.30

Appearance: white solid

Yield: 25%

Synthetic method: General Method B1.

IR (KBr), ν : 3290 (m, νN-H); 3060 (w, νarC-H); 1687 (s, νC=O, ketone); 1635 (s, νC=O, amide) cm-1 1H-NMR (DMSO-d6), δ : 8.66 (bs, 1H, NH); 7.99 (d, 2H, H2+H6-phCO, J2--3= 7.2 Hz); 7.66-7.52 (m, 3H, H3+H4+H5-phCO); 7.37-7.25 (m, 5H, H2+H3+H4+H5+H6-phCH2); 4.32 (d, 2H, CH2-NH, JCH2-NH= 5.9 Hz); 3.92 (s, 2H, CO-CH2-CO) ppm

3-oxo-N-(2-phenylethyl)-3-phenylpropanamide (Compound 3)

M.F.: C17H17NO2

M.W.: 267.32

Appearance: white solid

Yield: 62%

Synthetic method: General Method B2.

IR (KBr), ν : 3336 (m, νN-H); 3032 (w, νarC-H); 1614 (s, νC=O, ketone) cm-1 1H-NMR (DMSO-d6), δ : 9.01 (bs, 1H, NH); 7.43-7.35 (m, 2H, H2+H6-phCO); 7.30-7.25 (m, 2H, H3+H5-phCO); 7.24-7.14 (m, 5H, H4-phCO+H2+H3+H5+H6-phCH2); 7.05-7.03 (m, 1H, H4-phCH2); 3.82 (s, 2H, CO-CH2-CO); 3.30-3.21 (m, 2H, CH2-NH); 3.08-2.67 (m, 2H, CH2-ph) ppm


- 128 -

3-oxo-N-(p-trifluoromethylbenzyl)butanamide (Compound 4) M.F.: C12H12F3NO2

M.W.: 259.23

Appearance: bronze solid Yield: 84%

Synthetic method: General Method C starting from p-trifluoromethylbenzyl amine.

IR (KBr), ν : 3259 (m, νN-H); 3090 (w, νarC-H); 1716 (s, νC=O, ketone); 1644 (s, νC=O, amide); 1106 (m, νarC-CF3); 1111 (s, νarC-CF3); 1069 (m, νarC-CF3) cm-1 1H-NMR (DMSO-d6), δ : 8.64 (t, 1H, NH, JNH-CH2= 5.5 Hz); 7.70 (d, 2H, H3+H5, J3-2= 7.9 Hz); 7.50 (d, 2H, H2+H6); 4.38 (d, 2H, CH2-NH); 3.41 (s, 2H, CO-CH2-CO); 2.16 (s, 3H, CH3) ppm

N-(p-chlorobenzyl)-3-oxobutanamide (Compound 5)

M.F.: C11H12ClNO2

M.W.: 225.68

Appearance: bronze solid

Yield: 59%

Synthetic method: General Method C starting from 20.0 mmol of p-chlorobenzylamine.

IR (KBr), ν : 3253 (s, νN-H); 3085 (m, νarC-H); 1714 (s, νC=O, ketone); 1642 (s, νC=O, amide) cm-1 1H-NMR (DMSO-d6), δ : 8.55 (bs, 1H, NH); 7.39 (dd, 2H, H3+H5, J3-2= 8.5 Hz, J3-Cl= 1.9 Hz); 7.30 (d, 2H, H2+H6); 4.28 (d, 2H, CH2-NH, JCH2-NH= 5.9 Hz); 3.38 (s, 2H, CO-CH2-CO); 2.15 (s, 3H, CH3) ppm


- 129 -

N-(p-bromobenzyl)-3-oxobutanamide (Compound 6) M.F.: C11H12BrNO2

M.W.: 270.13


Yield: 57%

Synthetic method: General Method C starting from p-bromobenzylamine.

IR (KBr), ν : 3253 (s, νN-H); 3085 (m, νarC-H); 1714 (s, νC=O, ketone); 1642 (s, νC=O, amide); 1015 (m, νarC-Br) cm-1 1H-NMR (DMSO-d6), δ : 8.55 (bs, 1H, NH); 7.52 (d, 2H, H3+H5, J3-2= 8.2 Hz); 7.24 (d, 2H, H2+H6); 4.26 (d, 2H, CH2-NH, JCH2-NH= 5.5 Hz); 3.38 (s, 2H, CO-CH2-CO); 2.15 (s, 3H, CH3) ppm

N-(p-methylbenzyl)-3-oxobutanamide (Compound 7)

M.F.: C12H15NO2

M.W.: 205.26


Yield: 40%

Synthetic method: General Method C starting from p-methylbenzylamine.

IR (KBr), ν : 3254 (m, νN-H); 3088 (m, νarC-H); 1715 (m, νC=O,ketone); 1641 (s, νC=O,amide) cm-1 1H-NMR (DMSO-d6), δ : 8.47 (t, 1H, NH, JNH-CH2= 5.8 Hz); 7.20-7.10 (m, 4H, H2+H3+H5+H6); 4.24 (d, 2H, CH2-NH); 3.36 (s, 2H, CO-CH2-CO); 2.28 (s, 3H, CH3-ph); 2.15 (s, 3H, CH3) ppm


- 130 -

3-oxo-N-(3,4,5-trimethoxybenzyl)butanamide (Compound 8) M.F.: C14H19NO5

M.W.: 281.31


Yield: 25%

Synthetic method: General Method C starting from 3,4,5-trimethoxy benzylamine.

IR (KBr), ν : 3336 (w, νN-H); 3013 (w, νarC-H); 1726 (s, νC=O, ketone); 1673 (s, νC=O, amide) cm-1 1H-NMR (DMSO-d6), δ : 8.47 (t, 1H, NH, JNH-CH2= 5.6 Hz); 6.62 (s, 2H, H2+H6); 4.23 (d, 2H, CH2-NH); 3.77 (s, 6H, 2xOCH3-m); 3.63 (s, 3H, OCH3-p); 3.40 (s, 2H, CO-CH2-CO); 2.17 (s, 3H, CH3) ppm

N-(2,2-diphenylethyl)-3-oxobutanamide (Compound 9)

M.F.: C18H19NO2

M.W.: 281.36


Yield: 32%

Synthetic method: General Method C starting from 2,2-diphenyl ethylamine.

IR (KBr), ν : 3276 (s, νN-H); 3020 (w, νarC-H); 1710 (m, νC=O, ketone); 1668 (s, νC=O, amide) cm-1 1H-NMR (DMSO-d6), δ : 8.14 (t, 1H, NH, JNH-CH2= 5.6 Hz); 7.34-7.24 (m, 10H, 2C6H5); 4.19 (t, 1H, CH, JCH-CH2= 7.9 Hz); 3.73 (dd, 2H, CH2-NH); 3.19 (s, 2H, CO-CH2-CO); 1.96 (s, 3H, CH3) ppm


- 131 -

N-(benzo[d][1,3]dioxo-5-ylmethyl)-3-oxobutanamide (Compound 10) M.F.: C12H13NO4

M.W.: 235.24


Yield: 33%

Synthetic method: General Method C starting from benzo[d][1,3]dioxol-5-ylmethylamine.

IR (KBr), ν : 3290 (m, νN-H); 3064 (w, νarC-H); 1758 (m, νC=O, ketone); 1615 (s, νC=O, amide); 1241 (m, νC=O) cm-1 1H-NMR (DMSO-d6), δ : 8.50 (t, 1H, NH, JNH-CH2= 5.6 Hz); 7.02 (bs, 1H, H2); 6.91 (bs, 2H, H5+H6); 6.01 (s, 2H, O-CH2-O); 4.27 (d, 2H, CH2-NH); 3.37 (s, 2H, CO-CH2-CO); 2.15 (s, 3H, CH3-CO) ppm


- 132 -

N,3-diphenylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 1a) M.F.: C21H15N3O3

M.W.: 357.37

M.P.: 228-229º C


Yield: 17%

Synthetic method: General Method D starting from benzofuroxan.

Purification method: Column chromatography using toluene/dioxane (6:4) as mobile phase.

IR (KBr), ν : 3256 (w, νNH); 3077 (w, νarC-H); 1693 (s, νC=O); 1348 (s, νN+O-) cm-1 1H-NMR (DMSO-d6), δ : 10.80 (s, 1H, NH); 8.60-8.56 (m, 2H, H5+H8); 8.10-8.07 (m, 2H, H6+H7); 7.62-7.60 (m, 2H, H2+H6-ph-QX); 7.49-7.47 (m; 3H, H3+H4+H5-ph-QX); 7.39 (dd, 2H, H2+H6-ph-NH, J2-3= 8.5 Hz, J2-4=1.0 Hz); 7.33-7.28 (m, 2H, H3+H5-ph-NH); 7.10 (tt, 1H, H4-ph-NH, J4-3= 7.2 Hz) ppm

Elemental analysis (C21H15N3O3):

C% H% N% Calculated 70.58 4.23 11.76 Found 70.72 4.48 11.99


- 133 -

7(6)-chloro-N,3-diphenylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 1b)

M.F.: C21H14ClN3O3

M.W.: 391.82

M.P.: 219-220º C


Yield: 18%

Synthetic method: General Method D starting from 5-chlorobenzofuroxan.


IR (KBr), ν : 3256 (w, νNH); 3058 (w, νarC-H); 1686 (s, νC=O); 1330 (s, νN+O-) cm-1 1H-NMR (DMSO-d6), δ : 10.82 (s, 1H, NH); 8.57-8.56 (m; 2H, H5+H8); 8.11 (dd, 1H, H6, J6-5=9.0 Hz, J6-8=1.7 Hz); 7.61-7.59 (m, 2H, H2+H6-ph-QX); 7.49-7.48 (m, 3H, H3+H4+H5-ph-QX); 7.38 (d, 2H, H2+H6-ph-NH, J2-3= 7.9 Hz); 7.31 (t, 2H, H3+H5-ph-NH, J3-4= 7.9 Hz); 7.11 (t, 1H, H4-ph-NH) ppm

Elemental analysis (C21H14ClN3O3):



- 134 -

7(6)-fluoro-N,3-diphenylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 1c)

M.F.: C21H14FN3O3

M.W.: 375.36

M.P.: 217-218º C


Yield: 17%

Synthetic method: General Method D starting from 5-fluorobenzofuroxan.


IR (KBr), ν : 3244 (m, νNH); 3058 (w, νarC-H); 1658 (s, νC=O); 1339 (s, νN+O-) cm-1 1H-NMR (DMSO-d6), δ : 10.83 (s, 1H, NH); 8.66-8.62 (m, 1H, H5); 8.34-8.32 (m, 1H, H8); 8.02-7.98 (m, 1H, H6); 7.60.7.59 (m, 2H, H2+H6-ph-QX); 7.49-7.47 (m, 3H, H3+H4+H5-ph-QX); 7.39-7.37 (m, 2H, H2+H6-ph-NH); 7.32-7.28 (m, 2H, H3+H5-ph-NH); 7.12-7.09 (m, 1H, H4-ph-NH) ppm

Elemental analysis (C21H14FN3O3):



- 135 -

7(6)-trifluoromethyl-N,3-diphenylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 1d)

M.F.: C22H14F3N3O3

M.W.: 425.37

M.P.: 132-133º C


Yield: 18%

Synthetic method: General Method D starting from 5-trifluoromethyl benzofuroxan.

Purification method: Column chromatography using n-hexane/ethyl acetate (1:1) as mobile phase.

IR (KBr), ν : 3250 (w, νNH); 3058 (w, νarC-H); 1686 (s, νC=O); 1345 (s, νN+O-); 1140 (s, νarC-

CF3) cm-1 1H-NMR (DMSO-d6), δ : 10.82 (s, 1H, NH); 8.83 (s, 1H, H8); 8.77 (d, 1H, H5, J5-6= 9.0 Hz); 8.39 (dd, 1H, H6, J6-8= 1.6 Hz); 7.64-7.62 (m, 2H, H2+H6-ph-QX); 7.51-7.49 (m, 3H, H3+H4+H5-ph-QX); 7.39-7.37 (m, 2H, H2+H6-ph-NH); 7.33-7.29 (m, 2H, H3+H5-ph-NH); 7.13-7.10 (m, 1H, H4-ph-NH) ppm

Elemental analysis (C22H14F3N3O3):



- 136 -

7(6)-methyl-N,3-diphenylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 1e)

M.F.: C22H17N3O3

M.W.: 371.40

M.P.: 227-228º C


Yield: 9%

Synthetic method: General Method D starting from 5-methylbenzofuroxan.


IR (KBr), ν : 3256 (w, νNH); 3064 (w, νarC-H); 1686 (s, νC=O); 1335 (s, νN+O-) cm-1 1H-NMR (DMSO-d6), δ : 10.80 (s, 1H, NH); 8.47 (d, 1H, H5, J5-6= 8.8 Hz); 8.41-8.35 (m, 1H, H8); 7.91 (dd, 1H, H6, J6-8= 1.2 Hz); 7.63-7.58 (m, 2H, H2+H6-ph-QX); 7.51-7.44 (m, 3H, H3+H4+H5-ph-QX); 7.41-7.36 (m, 2H, H2+H6-ph-NH); 7.33-7.27 (m, 2H, H3+H5-ph-NH); 7.13-7.07 (m, 1H, H4-ph-NH); 2.65 (s, 3H, CH3) ppm




- 137 -

7-methoxy-N,3-diphenylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 1f)

M.F.: C22H17N3O4

M.W.: 387.40

M.P.: 259-260º C

Appearance: pale yellow solid

Yield: 77%

Synthetic method: General Method D starting from 5-methoxy benzofuroxan.

Purification method: The product was obtained pure enough and no purification was necessary.

IR (KBr), ν : 3250 (m, νNH); 3077 (m, νarC-H); 1685 (s, νC=O); 1335 (s, νN+O-); 1243 (s, νC-

O-C) cm-1 1H-NMR (DMSO-d6), δ : 10.81 (s, 1H, NH); 8.48 (d, 1H, H5, J5-6= 9.5 Hz); 7.87 (d, 1H, H8, J8-6= 2.7 Hz); 7.68 (dd, 1H, H6); 7.61-7.58 (m, 2H, H2+H6-ph-QX); 7.48-7.46 (m, 3H, H3+H4+H5-ph-QX); 7.38 (dd, 2H, H2+H6-ph-NH, J2-3= 8.5 Hz, J2-4= 1.1 Hz); 7.32-7.28 (m, 2H, H3+H5-ph-NH); 7.10 (tt, 1H, H4-ph-NH, J4-3=7.3 Hz); 4.04 (s, 3H, OCH3) ppm




- 138 -

6,7-dichloro-N,3-diphenylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 1g)

M.F.: C21H13Cl2N3O3.½H2O

M.W.: 435.26

M.P.: 225-226º C


Yield: 46%

Synthetic method: General Method D starting from 5,6-dichlorobenzofuroxan.


IR (KBr), ν : 3308 (m,νNH); 3071 (m,νarC-H); 1666 (s, νC=O); 1332 (s, νN+O-); 1313 (s, νN+O-

) cm-1 1H-NMR (DMSO-d6), δ : 10.88 (s, 1H, NH); 8.76 (s, 1H, H5); 8.74 (s, 1H, H8); 7.61-7.59 (m, 2H, H2+H6-ph-QX); 7.51-7.48 (m, 3H, H3+H4+H5-ph-QX); 7.38 (dd, 2H, H2+H6-ph-NH, J2-3= 8.5 Hz, J2-4= 1.0 Hz); 7.33-7.29 (m, 2H, H3+H5-ph-NH); 7.11 (tt, 1H, H4-ph-NH, J4-3= 7.8 Hz) ppm

Elemental analysis (C21H13Cl2N3O3.½H2O):



- 139 -

N-benzyl-3-phenylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 2a) M.F.: C22H17N3O3

M.W.: 371.40

M.P.: 213-214º C


Yield: 15%


Purification method: Column chromatography using ethyl acetate as mobile phase.

IR (KBr), ν : 3302 (m,νNH); 3085 (w, νarC-H); 1673 (s, νC=O); 1348 (s, νN+O-); 1339 (s, νN+O-

) cm-1 1H-NMR (DMSO-d6), δ : 9.12 (t, 1H, NH, JNH-CH2= 5.8 Hz); 8.58-8.53 (m, 2H, H5+H8); 8.06-8.04 (m, 2H, H6+H7); 7.57-7.56 (m, 3H, H3+H4+H5-ph-QX); 7.51-7.49 (m, 2H, H2+H6-ph-QX); 7.22-7.21 (m, 3H, H3-H5-ph-CH2); 6.90-6.87 (m, 2H, H2+H6-ph-CH2); 4.28 (d, 2H, CH2) ppm




- 140 -

N-benzyl-7(6)-chloro-3-phenylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 2b)

M.F.: C22H16ClN3O3

M.W.: 405.84

M.P.: 230-231º C


Yield: 22%



IR (KBr), ν : 3286 (m, νNH); 3094 (w, νarC-H); 1649 (s, νC=O); 1331 (m, νN+O-) cm-1 1H-NMR (DMSO-d6), δ : 9.12 (t, 1H, NH, JNH-CH2= 5.9 Hz); 8.56-8.54 (m, 1H, H5), 8.50 (bs; 1H, H8); 8.08 (dd, 1H, H6, J6-5= 9.2 Hz, J6-8= 2.2 Hz); 7.58-7.49 (m, 5H, H2+H3+H4+H5+H6-ph-QX); 7.21-7.20 (m, 3H, H3+H4+H5-ph-CH2); 6.89-6.87 (m, 2H, H2+H6-ph-CH2); 4.28 (d, 2H, CH2) ppm




- 141 -

N-benzyl-7(6)-fluoro-3-phenylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 2c)

M.F.: C22H16FN3O3.½H2O

M.W.: 398.40

M.P.: 217-218º C


Yield: 22%



IR (KBr), ν : 3312 (m, νNH); 3059 (w, νarC-H); 1673 (s, νC=O); 1335 (m, νN+O-) cm-1 1H-NMR (DMSO-d6), δ : 9.14 (t, 1H, NH, JNH-CH2= 5.7 Hz); 8.62-8.59 (m, 1H, H5); 8.33-8.30 (m, 1H, H8); 7.99-7.94 (m, 1H, H6); 7.94-7.49 (m, 5H, H2+H3+H4+H5+H6-ph-QX); 7.21-7.20 (m, 3H, H3+H4+H5-ph-CH2); 6.90-6.88 (m, 2H, H2+H6-ph-CH2); 4.28 (d, 2H, CH2) ppm

Elemental analysis (C22H16FN3O3.½H2O):



- 142 -

N-benzyl-7(6)-trifluoromethyl-3-phenylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 2d)

M.F.: C23H16F3N3O3

M.W.: 439.40

M.P.: 236-237º C


Yield: 6%



IR (KBr), ν : 3287 (m, νNH); 3093 (w, νarC-H); 1647 (s, νC=O); 1350 (s, νN+O-); 1319 (s,νN+O-

); 1171 (s, νar-CF3); 1125 (s, νar-CF3) cm-1 1H-NMR (DMSO-d6), δ : 9.15-9.10 (m, 1H, NH); 8.81-8.77 (m, 1H, H5); 8.44-8.28 (m, 2H, H8+H6); 7.59-7.51 (m, 5H, H2+H3+H4+H5+H6-ph-QX); 7.25-7.21 (m, 5H, H2+H3+H4+H5+H6-ph-CH2); 4.29 (d, 2H, CH2, JCH2-NH=5.9 Hz) ppm




- 143 -

N-benzyl-7(6)-methyl-3-phenylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 2e)

M.F.: C23H19N3O3.½H2O

M.W.: 394.44

M.P.: 227-228º C


Yield: 36%



IR (KBr), ν : 3224 (m, νNH); 3058 (w, νarC-H); 1682 (s, νC=O); 1355 (s,νN+O-); 1314 (s, νN+O-

) cm-1 1H-NMR (DMSO-d6), δ : 9.13 (t, 1H, NH, JNH-CH2= 5.9 Hz); 8.43 (d, 1H, H5, J5-6= 8.8 Hz); 8.37 (s, 1H, H8); 7.87 (dd, 1H, H6, J6-8= 1.8 Hz); 7.59-7.55 (m, 3H, H3+H4+H5-ph-QX); 7.51-7.48 (m, 2H, H2+H6-ph-QX); 7.21-7.19 (m, 3H, H3+H4+H5-ph-CH2); 6.90-6.87 (m, 2H, H2+H6-ph-CH2); 4.28 (d, 2H, CH2) ppm

Elemental analysis (C23H19N3O3.½H2O):



- 144 -

N-benzyl-6-methoxy-3-phenylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 2f)

M.F.: C23H19N3O4

M.W.: 401.43

M.P.: 207-208º C


Yield: 35%

Synthetic method: General Method D starting from 5-methoxybenzofuroxan.


IR (KBr), ν : 3262 (m, νNH); 3080 (w, νarC-H); 1689 (s, νC=O); 1352 (s, νN+O-); 1328 (s, νN+O-); 1256 (s, νC-O-C) cm-1 1H-NMR (DMSO-d6), δ : 9.14 (t, 1H, NH, JNH-CH2= 5.9 Hz); 8.44 (d, 1H, H5, J5-6= 9.5 Hz); 7.86 (d, 1H, H8, J8-6= 2.7 Hz); 7.65 (dd, 1H, H6); 7.58-7.54 (m, 3H, H3+H4+H5-ph-QX); 7.51-7.48 (m, 2H, H2+H6-ph-QX); 7.22-7.20 (m, 3H, H3+H4+H5-ph-CH2); 6.93-6.90 (m, 2H, H2+H6-ph-CH2); 4.27 (d, 2H, CH2); 4.03 (s, 3H, OCH3) ppm




- 145 -

N-benzyl-6,7-dichloro-3-phenylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 2g)

M.F.: C22H15Cl2N3O3

M.W.: 440.29

M.P.: 209-210ºC


Yield: 8%

Synthetic method: General Method D starting from 5,6-dichloro benzofuroxan.


IR (KBr), ν : 3280 (m, νNH); 3062 (w, νarC-H); 1649 (s, νC=O); 1327 (s, νN+O-) cm-1 1H-NMR (DMSO-d6), δ : 9.15 (t, 1H, NH, JNH-CH2= 5.9Hz); 8.75 (s, 1H, H5); 8.70 (s, 1H, H8); 7.50-7.48 (m, 5H, H2+H3+H4+H5+H6-ph-QX); 7.22-7.20 (m, 3H, H3+H4+H5-ph-CH2); 6.90-6.88 (m, 2H, H2+H6-ph-CH2); 4.27 (d, 2H, CH2) ppm

Elemental analysis (C22H15Cl2N3O3):



- 146 -

3-phenyl-N-(2-phenylethyl)quinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 3a)

M.F.: C23H19N3O3

M.W.: 385.43

M.P.: 217-218º C


Yield: 10%



IR (KBr), ν : 3269 (w, νNH); 3078 (w, νarC-H); 1679 (s, νC=O); 1328 (s, νN+O-) cm-1 1H-NMR (DMSO-d6), δ : 8.75 (t, 1H, NH, JNH-CH2= 5.7Hz); 8.55-8.52 (m, 2H, H5+H8); 8.05-8.03 (m, 2H, H6+H7); 7.57-7.51 (m, 5H, H2+H3+H4+H5+H6-ph-QX); 7.27 (t, 2H, H3+H5-ph-CH2, J3-2=J3-4= 7.3 Hz); 7.20 (t, 1H, H4-ph-CH2); 7.09 (d, 2H, H2+H6-ph-CH2); 3.27-3.21 (m, 2H, CH2-NH); 2.39 (t, 2H, CH2-ph, JCH2-CH2= 7.3Hz) ppm




- 147 -

7(6)-chloro-3-phenyl-N-(2-phenylethyl)quinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 3b)

M.F.: C23H18ClN3O3

M.W.: 419.87

M.P.: 169-170º C


Yield: 8%



IR (KBr), ν : 3304 (w, νNH); 3056 (wd, νarC-H); 1668 (s, νC=O); 1330 (s, νN+O-) cm-1 1H-NMR (DMSO-d6), δ : 8.75 (t, 1H, NH, JNH-CH2= 5.6 Hz); 8.54-8.52 (m, 2H, H5+H8); 8.07 (dd, H6, J6-5= 9.3 Hz, J6-8= 2.2 Hz); 7.56-7.51 (m, 5H, H2+H3+H4+H5+H6-ph-QX); 7.27 (t, 2H, H3+H5-ph-CH2, J3-2=J3-4= 7.3Hz); 7.19 (t, 1H, H4-ph-CH2); 7.09 (d, 2H, H2+H6-ph-CH2); 3.27-3.21 (m, 2H, CH2-NH); 2.39 (t, 2H, CH2-ph, JCH2-CH2= 7.4 Hz) ppm




- 148 -

3-methyl-N-(p-trifluoromethylbenzyl)quinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 4a)

M.F.: C18H14F3N3O3

M.W.: 377.33

M.P.: 200-201º C

Appearance: pale yellow solid Yield: 21%



IR (KBr), ν : 3205 (w, νN-H); 3039 (w, νarC-H); 1669 (s, νC=O); 1325 (s, νN+O-); 1166 (m, νarC-CF3); 1101 (m, νarC-CF3); 1068 (m, νarC-CF3) cm-1 1H-NMR (DMSO-d6), δ : 9.49 (t, 1H, NH, JNH-CH2= 5.9 Hz); 8.53-8.49 (m, 2H, H5+H8); 8.03-7.96 (m, 2H, H6+H7); 7.77 (d, 2H, H3’+H5’, J3’-2’= 8.2 Hz); 7.70 (d, 2H, H2’+H6’); 4.67 (d, 2H, CH2); 2.44 (s, 3H, CH3-C3) ppm

HPLC (RP-18, 1mL/min, methanol/water (60:40): tr: 5.31 min.; purity: 95.2 %.




- 149 -

7(6)-chloro-3-methyl-N-(p-trifluoromethylbenzyl)quinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 4b)

M.F.: C18H13ClF3N3O3

M.W.: 411.77

M.P.: 217-218º C


Yield: 37%



IR (KBr), ν : 3277 (w, νN-H); 3103 (w, νarC-H); 1650 (m, νC=O); 1328 (s, νN+O-); 1161 (m, νarC-CF3); 1109 (m, νarC-CF3); 1072 (m, νarC-CF3) cm-1 1H-NMR (DMSO-d6), δ : 9.49 (t, 1H, NH, JNH-CH2= 5.9 Hz); 8.50 (s, 2H, H5+H8); 8.04 (dd, 1H, H6, J6-5= 9.1 Hz, J6-8= 2.3 Hz); 7.77 (d, 2H, H3’+H5’, J3’-2’= 8.1 Hz); 7.69 (d, 2H, H2’+H6’); 4.66 (d, 2H, CH2); 2.43 (s, 3H, CH3-C3) ppm

HPLC (RP-18, 1mL/min, methanol/water (60:40): isomer 7:tr:6.81 min.; purity: 73.2 %. isomer 6: tr: 6.20 min.; purity: 24.0 %.

Elemental analysis (C18H13ClF3N3O3):



- 150 -

7(6)-fluoro-3-methyl-N-(p-trifluoromethylbenzyl)quinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 4c)

M.F.: C18H13F4N3O3

M.W.: 395.32

M.P.: 202-203º C


Yield: 51%



IR (KBr), ν : 3212 (w, νN-H); 3079 (w, νarC-H); 1671 (m, νC=O); 1327 (s, νN+O-); 1167 (m, νarC-CF3); 1101 (m, νarC-CF3); 1065 (m, νarC-CF3) cm-1 1H-NMR (DMSO-d6), δ : 9.50 (t, 1H, NH, JNH-CH2= 5.8 Hz); 8.58 (dd, 1H, H5, J5-6= 9.5 Hz, J5-F= 5.1 Hz); 8.25 (dd, 1H, H8, J8-F= 8.8 Hz, J8-6= 2.4 Hz); 7.93 (ddd, 1H, H6, J6-F= 9.4 Hz); 7.77 (d, 2H, H3’+H5’, J3’-2’= 8.1 Hz); 7.69 (d, 2H, H2’+H6’); 4.67 (d, 2H, CH2); 2.43 (s, 3H, CH3-C3) ppm

HPLC (RP-18, 1mL/min, methanol/water (60:40): isomer 7: tr: 6.81 min.; purity: 82.2 %. isomer 6: tr: 6.33 min.; purity: 15.5 %.




- 151 -

3-methyl-7-trifluoromethyl-N-(p-trifluoromethylbenzyl)quinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 4d)

M.F.: C19H13F6N3O3

M.W.: 445.32

M.P.: 203-204º C


Yield: 12%



IR (KBr), ν : 3212 (w, νN-H); 3064 (w, νarC-H); 1679 (s, νC=O); 1326 (s, νN+O-); 1168 (m, νarC-CF3); 1143 (m, νarC-CF3); 1111 (m, νarC-CF3); 1085 (m, νarC-CF3) cm-1 1H -NMR (DMSO-d6), δ : 9.52 (t, 1H, NH, JNH-CH2= 5.5 Hz); 8.76 (s, 1H, H8); 8.70 (d, 1H, H5, J5-6= 9.0 Hz); 8.29 (dd, 1H, H6, J6-8= 1.7 Hz); 7.78 (d, 2H, H3’+H5’, J3’-2’ = 8.1 Hz); 7.70 (d, 2H, H2’+H6’); 4.68 (d, 2H, CH2); 2.47 (s, 3H, CH3-C3) ppm





- 152 -

3,7(6)-dimethyl-N-(p-trifluoromethylbenzyl)quinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 4e)

M.F.: C19H16F3N3O3

M.W.: 391.35

M.P.: 202-203º C


Yield: 28%



IR (KBr), ν : 3199 (w, νN-H); 3032 (w, νarC-H); 1669 (s, νC=O); 1325 (s, νN+O-); 1165 (m, νarC-CF3); 1100 (m, νarC-CF3); 1167 (m, νarC-CF3) cm-1 1H-NMR (DMSO-d6), δ : 9.49 (t, 1H, NH, JNH-CH2= 5.5 Hz); 8.40 (d, 1H, H5, J5-6= 8.8 Hz); 8.31 (s, 1H, H8); 7.84 (d, 1H, H6); 7.76 (d, 2H, H3’+H5’, J3’-2’= 8.2 Hz); 7.70 (d, 2H, H2’+H6’); 4.66 (d, 2H, CH2); 2,59 (s, 3H, CH3-C7); 2.42 (s, 3H, CH3-C3) ppm





- 153 -

7-methoxy-3-methyl-N-(p-trifluoromethylbenzyl)quinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 4f)

M.F.: C19H16F3N3O4

M.W.: 407.35

M.P.: 224-225º C


Yield: 29%

Synthetic method: General Method D starting from 5-methoxybenzofuroxan.


IR (KBr), ν : 3212 (w, νN-H); 3040 (w, νarC-H); 1679 (m, νC=O); 1325 (s, νN+O-); 1169 (m, νarC-CF3); 1118 (m, νarC-CF3); 1066 (m, νarC-CF3) cm-1 1H-NMR (DMSO-d6), δ : 9.51 (t, 1H, NH, JNH-CH2= 5.6 Hz); 8.42 (d, 1H, H5, J5-6= 9.5 Hz); 7.81 (d, 1H, H8, J8-6= 2.60 Hz); 7.77 (d, 2H, H3’+H5’, J3’-2’= 8.1 Hz); 7.71 (d, 2H, H2’+H6’); 7.61 (dd, 1H, H6); 4.66 (d, 2H, CH2); 4.00 (s, 3H, OCH3); 2,41 (s, 3H, CH3-C3) ppm





- 154 -

6,7-dichloro-3-methyl-N-(p-trifluoromethylbenzyl)quinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 4g)

M.F.: C18H12Cl2F3N3O3

M.W.: 446.22

M.P.: 205-206º C


Yield: 12%



IR (KBr), ν : 3237 (w, νN-H); 3071 (w, νarC-H); 1670 (m, νC=O); 1323 (s, νN+O-); 1169 (m, νarC-CF3); 1109 (m, νarC-CF3); 1069 (m, νarC-CF3) cm-1 1H-NMR (DMSO-d6), δ : 9.51 (t, 1H, NH, JNH-CH2= 5.8 Hz); 8.69 (s, 1H, H5); 8.68 (s, 1H, H8); 7.76 (d, 2H, H3’+H5’, J3’-2’= 8.4 Hz); 7.68 (d, 2H, H2’+H6’); 4.66 (d, 2H, CH2); 2.43 (s, 3H, CH3-C3) ppm

HPLC (RP-18, 1mL/min, methanol/water (60:40): tr: 12.72 min.; purity: 95.7%.

Elemental analysis (C18H12Cl2F3N3O3):



- 155 -

N-(p-chlorobenzyl)-3-methylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 5a)

M.F.: C17H14ClN3O3

M.W.: 343.77

M.P.: 212-213º C


Yield: 47%



IR (KBr), ν : 3192 (w, νN-H); 3071 (w, νarC-H); 1675 (s, νC=O); 1327 (s, νN+O-); 1082 (m, νarC-Cl) cm-1 1H-NMR (DMSO-d6), δ : 9.40 (t, 1H, NH, JNH-CH2= 5.8 Hz); 8.53-8.49 (m, 2H, H5+H8); 8.01-7.97 (m, 2H, H6+H7); 7.49 (d, 2H, H3’+H5’, J3’-2’= 8.5 Hz); 7.45 (d, 2H, H2’+H6’); 4.56 (d, 2H, CH2); 2.42 (s, 3H, CH3-C3) ppm





- 156 -

7(6)-chloro-N-(p-chlorobenzyl)-3-methylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 5b)

M.F.: C17H13Cl2N3O3

M.W.: 378.22

M.P.: 214-215º C


Yield: 37%


Purification method: Flash column chromatography using dichloromethane and methanol to run a gradient condition from 100% dichloromethane to 0.5% methanol.

IR (KBr), ν : 3271 (w, νN-H); 3103 (w, νarC-H); 1650 (s, νC=O); 1326 (s, νN+O-); 1073 (m, νarC-Cl) cm-1 1H-NMR (DMSO-d6), δ : 9.40 (t, 1H, NH, JNH-CH2= 5.7 Hz); 8.50 (d, 1H, H5, J5-6= 9.3 Hz); 8.49 (d, 1H, H8, J8-6= 2.0 Hz); 8.04 (dd, 1H, H6); 7.48 (d, 2H, H3’+H5’, J3’-2’= 8.6 Hz); 7.45 (d, 2H, H2’+H6’); 4.55 (d, 2H, CH2); 2.41 (s, 3H, CH3-C3) ppm





- 157 -

N-(p-chlorobenzyl)-3,7(6)-dimethylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 5e)

M.F.: C18H16ClN3O3

M.W.: 357.80

M.P.: 201-202º C


Yield: 21%



IR (KBr), ν : 3250 (w, νN-H); 3064 (w, νarC-H); 1670 (s, νC=O); 1322 (s, νN+O-); 1068 (m, νarC-Cl) cm-1 1H-NMR (DMSO-d6), δ : 9.40 (t, 1H, NH, JNH-CH2= 5.5 Hz); 8.40 (d, H5, J5-6= 8.8 Hz); 8.31 (s, 1H, H8); 7.83 (dd, 1H, H6, J6-8= 1.5 Hz); 7.49 (dd, 2H, H3’+H5’, J3’-2’= 8.5 Hz, J3’-

Cl= 1.3 Hz); 7.45 (dd, 2H, H2’+H6’, J2’-Cl= 1.1 Hz); 4.55 (d, 2H, CH2); 2.59 (s, 3H, CH3-C7); 2.40 (s, 3H, CH3-C3) ppm





- 158 -

6,7-dichloro-N-(p-chlorobenzyl)-3-methylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 5g)

M.F.: C17H12Cl3N3O3

M.W.: 412.66

M.P.: 226-227º C


Yield: 11%



IR (KBr), ν : 3243 (w, νN-H); 3071 (w, νarC-H); 1671 (s, νC=O); 1321 (s, νN+O-); 1066 (m, νarC-Cl) cm-1 1H-NMR (DMSO-d6), δ : 9.43 (t, 1H, NH, JNH-CH2= 5.9 Hz); 8.69 (d, 1H, H5, J5-8= 0.5 Hz); 8.68 (d, 1H, H8); 7.48 (d, 2H, H3’+H5’, J3’-2’= 8.8 Hz); 7.45 (d, 2H, H2’+H6’); 4.55 (d, CH2); 2.41 (s, 3H, CH3-C3) ppm





- 159 -

N-(p-bromobenzyl)-3-methylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 6a)

M.F.: C17H14BrN3O3

M.W.: 388.22

M.P.: 206-207º C


Yield: 7%



IR (KBr), ν : 3271 (w, νN-H); 3090 (w, νarC-H); 1677 (s, νC=O); 1331 (s, νN+O-); 1073 (m, νarC-Br) cm-1 1H-NMR (DMSO-d6), δ : 9.40 (t, 1H, NH, JNH-CH2=5.8 Hz); 8.52-8.49 (m, 2H, H5+H8); 8.03-7.96 (m, 2H, H6+H7); 7.59 (d, 2H, H3’+H5’, J3’-2’= 8.3 Hz); 7.43 (d, 2H, H2’+H6’); 4.54 (d, 2H, CH2); 2.42(s, 3H, CH3-C3) ppm


Elemental analysis (C17H14BrN3O3):



- 160 -

N-(p-bromobenzyl)-7(6)-chloro-3-methylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 6b)

M.F.: C17H13BrClN3O3

M.W.: 422.67

M.P.: 209-210º C


Yield: 14%



IR (KBr), ν : 3237 (w, νN-H); 3064 (w, νarC-H); 1670 (s, νC=O); 1325 (s, νN+O-); 1071 (m, νarC-Br) cm-1 1H-NMR (DMSO-d6), δ : 9.40 (bs, 1H, NH); 8.50 (d, 1H, H5, J5-6=9.3 Hz); 8.49 (d, 1H, H8, J8-6=2.4 Hz); 8.04 (dd, 1H, H6); 7.59 (d, 2H, H3’+H5’, J3’-2’= 8.4 Hz); 7.42 (d, 2H, H2’+H6’); 4.53 (d, 2H, CH2, JCH2-NH=5.8 Hz); 2.41 (s, 3H, CH3-C3) ppm


Elemental analysis (C17H13BrClN3O3):



- 161 -

N-(p-bromobenzyl)-3,7(6)-dimethylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 6e)

M.F.: C18H16BrN3O3

M.W.: 402.25

M.P.: 210-211º C


Yield: 27%



IR (KBr), ν : 3205 (w, νN-H); 3058 (w, νarC-H); 1667 (s, νC=O); 1327 (s, νN+O-); 1068 (m, νarC-Br) cm-1 1H NMR (DMSO-d6), δ : 9.40 (bs, 1H, NH); 8.40 (d, 1H, H5, J5-6= 8.8 Hz); 8.31 (s, 1H, H8); 7.83 (dd, 1H, H6, J6-8= 1.3 Hz); 7.58 (d, 2H, H3’+H5’, J3’-2’= 8.5 Hz); 7.43 (d, 2H, H2’+H6’); 4.53 (d, 2H, CH2, JCH2-NH= 5.9 Hz); 2.59 (s, 3H, CH3-C7); 2.40 (s, 3H, CH3-C3) ppm


Elemental analysis (C18H16BrN3O3):



- 162 -

N-(p-bromobenzyl)-6,7-dichloro-3-methylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 6g)

M.F.: C17H12BrCl2N3O3

M.W.: 457.11

M.P.: 222-223º C


Yield: 14%



IR (KBr), ν : 3237 (w, νN-H); 3066 (w, νarC-H); 1670 (s, νC=O); 1320 (s, νN+O-); 1067 (m, νarC-Br) cm-1 1H-NMR (DMSO-d6), δ : 9.43 (t, 1H, NH, JNH-CH2= 5.9 Hz); 8.69 (s, 1H, H5); 8.68 (s, 1H, H8); 7.58 (d, 2H, H3’+H5’, J3’-2’= 8.3 Hz); 7.41 (d, 2H, H2’+H6’); 4.53 (d, 2H, CH2); 2.41 (s, 3H, CH3-C3) ppm


Elemental analysis (C17H12BrCl2N3O3):



- 163 -

3-methyl-N-(p-methylbenzyl)quinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 7a)

M.F.: C18H17N3O3

M.W.: 323.35

M.P.: 184-185º C


Yield: 61%



IR (KBr), ν : 3224 (w, νN-H); 3045 (w, νarC-H); 1671 (s, νC=O); 1336 (s, νN+O-) cm-1 1H-NMR (DMSO-d6), δ : 9.30 (t, 1H, NH, JNH-CH2= 5.8 Hz); 8.52-8.49 (m, 2H, H5+H8); 8.02-7.97 (m, 2H, H6+H7); 7.33 (d, 2H, H2’+H6’, J2’-3’= 7.8 Hz); 7.19 (d, 2H, H3’+H5’); 4.51 (d, 2H, CH2); 2.42 (s, 3H, CH3-C3); 2.30 (s, 3H, CH3-ph) ppm





- 164 -

7(6)-chloro-3-methyl-N-(p-methylbenzyl)quinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 7b)

M.F.: C18H16ClN3O3

M.W.: 357.80

M.P.: 189-190º C


Yield: 35%



IR (KBr), ν : 3259 (w, νN-H); 3077 (w, νarC-H); 1671 (s, νC=O); 1325 (s, νN+O-) cm-1 1H-NMR (DMSO-d6), δ : 9.30 (t, 1H, NH, JNH-CH2= 5.9 Hz); 8.50 (d, 1H, H5, J5-6= 9.1 Hz); 8.48 (s, 1H, H8); 8.03 (dd, 1H, H6, J6-8= 2.3 Hz); 7.32 (d, 2H, H2’+H6’, J2’-3’= 7.9 Hz); 7.19 (d, 2H, H3’+H5’); 4.51 (d, 2H, CH2); 2.40 (s, 3H, CH3-C3); 2.30 (s, 3H, CH3-ph) ppm





- 165 -

3,7(6)-dimethyl-N-(p-methylbenzyl)quinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 7e)

M.F.: C19H19N3O3

M.W.: 337.38

M.P.: 182-183º C


Yield: 10%



IR (KBr), ν : 3281 (m, νN-H); 3065 (w, νarC-H); 1650 (s, νC=O); 1325 (s, νN+O-) cm-1 1H-NMR (DMSO-d6), δ : 9.31 (bs, 1H, NH); 8.39 (d, 1H, H5, J5-6= 8.8 Hz); 8.30 (s, 1H, H8); 7.83 (dd, 1H, H6, J6-8= 1.8 Hz); 7.33 (d, 2H, H2’+H6’, J2’-3’= 8.1 Hz); 7.19 (d, 2H, H3’+H5’); 4.50 (d, 2H, CH2, JCH2-NH= 5.8 Hz); 2.60 (s, 3H, CH3-C7); 2.40 (s, 3H, CH3-C3); 2.30 (s, 3H, CH3-ph) ppm





- 166 -

6,7-dichloro-3-methyl-N-(p-methylbenzyl)quinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 7g)

M.F.: C18H15Cl2N3O3

M.W.: 392.24

M.P.: 202-203º C


Yield: 12%



IR (KBr), ν : 3270(m, νN-H); 3045 (w, νarC-H); 1649 (s, νC=O); 1359 (m, νN+O-) cm-1 1H-NMR (DMSO-d6), δ : 9.35 (t, 1H, NH, JNH-CH2= 5.3 Hz); 8.62 (s, 1H, H5); 8.45 (s, 1H, H8); 7.32 (d, 2H, H2’+H6’, J2’-3’= 7.3 Hz); 7.19 (d, 2H, H3’+H5’); 4.51 (d, 2H, CH2); 2.55(s, 3H, CH3-C3); 2.30 (s, 3H, CH3-ph) ppm





- 167 -

3-methyl-N-(3,4,5-trimethoxybenzyl)quinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 8a)

M.F.: C20H21N3O6.½H2O

M.W.: 408.41

M.P.: 198-199º C


Yield: 53%



IR (KBr), ν : 3285 (m, νN-H); 3096 (w, νarC-H); 1653 (s, νC=O); 1334 (s, νN+O-); 1130 (s, νC-

O) cm-1 1H-NMR (DMSO-d6), δ : 9.34 (t, 1H, NH, JNH-CH2= 5.6 Hz); 8.53-8.48 (m, 2H, H5+H8); 8.03-7.96 (m, 2H, H6+H7); 6.82 (s, 2H, H2’+H6’); 4.51 (d, 2H, CH2); 3.82 (s, 6H, 2OCH3-m); 3.65 (s, 3H, OCH3-p); 2.47 (s, 3H, CH3) ppm





- 168 -

7(6)-chloro-3-methyl-N-(3,4,5-trimethoxybenzyl)quinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 8b)

M.F.: C20H20ClN3O6

M.W.: 433.84

M.P.: 193-194º C


Yield: 26%




O) cm-1 1H-NMR (DMSO-d6), δ : 9.34 (t, 1H, NH, JNH-CH2= 5.9 Hz); 8.51 (d, 1H, H5, J5-6= 9.3 Hz); 8.48 (d, 1H, H8, J8-6= 2.3 Hz); 8.04 (dd, 1H, H6); 6.81 (s, 2H, H2’+H6’); 4.51 (d, 2H, CH2); 3.82 (s, 6H, 2OCH3-m); 3.65 (s, 3H, OCH3-p); 2.47 (s, 3H, CH3) ppm





- 169 -

3,7(6)-dimethyl-N-(3,4,5-trimethoxybenzyl)quinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 8e)

M.F.: C21H23N3O6.¼H2O

M.W.: 417.93

M.P.: 203-204º C


Yield: 27%




O) cm-1 1H-NMR (DMSO-d6), δ : 9.35 (t, 1H, NH, JNH-CH2= 5.5 Hz); 8.40 (d, 1H, H5, J5-6= 8.7 Hz); 8.28 (s, 1H, H8); 7.83 (dd, H6, J6-8= 1.3 Hz); 6.82 (s, 2H, H2’+H6’); 4.50 (d, 2H, CH2); 3.82 (s, 6H, 2xOCH3-m); 3.65 (s, 3H, OCH3-p); 2.60 (s, 3H, CH3-C7); 2.44 (s, 3H, CH3-C3) ppm


Elemental analysis (C21H23N3O6.¼H2O):



- 170 -

6,7-dichloro-3-methyl-N-(3,4,5-trimethoxybenzyl)quinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 8g)

M.F.: C20H19Cl2N3O6

M.W.: 468.29

M.P.: 206-207º C


Yield: 29%




O) cm-1 1H-NMR (DMSO-d6), δ : 9.39 (t, 1H, NH, JNH-CH2= 5.7 Hz); 8.68 (s, 1H, H5); 8.66 (s, 1H, H8); 6.80 (s, 2H, H2’+H6’); 4.50 (d, 2H, CH2); 3.81 (s, 6H, 2OCH3-m); 3.64 (s, 3H, OCH3-p); 2.45 (s, 3H, CH3) ppm





- 171 -

N-(2,2-diphenylethyl)-3-methylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 9a)

M.F.: C24H21N3O3

M.W.: 399.45

M.P.: 210-211º C




IR (KBr), v: 3237 (w, νN-H); 3064 (w, νarC-H); 1681 (s, νC=O); 1339 (m, νN+O-) cm-1 1H-NMR (DMSO-d6), δ : 8.90 (t, 1H, NH, JNH-CH2= 5.7 Hz); 8.47-8.42 (m, 2H, H5+H8); 7.99-7.92 (m, 2H, H6+H7); 7.41 (dd, 4H, 2H2’+2H6’, J2’-3’= 7.2 Hz, J2’-4’=1.3 Hz); 7.35-7.31 (m, 4H, 2H3’+2H5’); 7.20 (tt, 2H, 2H4’, J4’-3’= 7.3 Hz); 4.32 (t, 1H, CH, JCH-CH2= 8.0 Hz); 4.02 (dd, 2H, CH2); 1.97 (s, 3H, CH3-C3) ppm





- 172 -

7(6)-chloro-N-(2,2-diphenylethyl)-3-methylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 9b)

M.F.: C24H20ClN3O3

M.W.: 433.90

M.P.: 173-174º C




IR (KBr), ν : 3231 (w, νN-H); 3058 (w, νarC-H); 1679 (s, νC=O); 1326 (s, νN+O-) cm-1 1H-NMR (DMSO-d6), δ : 8.88 (bs, 1H, NH); 8.45 (d, 1H, H5, J5-6= 9.2 Hz); 8.41 (d, 1H, H8, J8-6= 2.1 Hz); 8.00 (dd, 1H, H6); 7.40 (dd, 4H, 2H2’+2H6’, J2’-3’= 7.8 Hz, J2’-4’=1.2 Hz); 7.35-7.30 (m, 4H, 2H3’+2H5’); 7.22 (tt, 2H, 2H4’, J4’-3’=7.3 Hz); 4.32 (t, 1H, CH, JCH-CH2= 7.9 Hz); 4.03 (dd, 2H, CH2); 1.98 (s, 3H, CH3-C3) ppm





- 173 -

N-(2,2-diphenylethyl)-3,7(6)-dimethylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 9e)

M.F.: C25H23N3O3

M.W.: 413.48

M.P.: 196-197º C




IR (KBr), ν : 3223 (w, νN-H); 3057 (w, νarC-H); 1678 (s, νC=O); 1328 (s, νN+O-) cm-1 1H-NMR (DMSO-d6), δ : 8.90 (bs, 1H, NH); 8.34 (d, 1H, H5, J5-6= 8.5 Hz); 8.23 (s, 1H, H8); 7.79 (dd, 1H, H6, J6-8=1.2 Hz); 7.40 (d, 4H, 2H2’+2H6’, J2’-3’= 7.5 Hz); 7.32 (t, 4H, 2H3’+2H5’, J3’-4’= 7.5Hz); 7.24-7.20 (m, 2H, 2H4’); 4.31 (t, 1H, CH, JCH-CH2= 7.9 Hz); 4.02 (dd, 2H, CH2, JCH2-NH= 5.8 Hz); 2.57 (s, 3H, CH3-C7); 1.95 (m, 3H, CH3-C3) ppm





- 174 -

6,7-dichloro-N-(2,2-diphenylethyl)-3-methylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 9g)

M.F.: C24H19Cl2N3O3.½H2O

M.W.: 477.34

M.P.: 142-143º C




IR (KBr), ν : 3212 (w, νN-H); 3083 (w, νarC-H); 1676 (s, νC=O); 1321 (s, νN+O-) cm-1 1H-NMR (DMSO-d6), δ : 8.91 (t, 1H, NH, JNH-CH2= 5.8 Hz); 8.63 (s, 1H, H5); 8.60 (s, 1H, H8); 7.40 (dd, 4H, 2H2’+2H6’, J2’-3’= 7.8 Hz, J2’-4’= 1.3 Hz); 7.32 (dd, 4H, 2H3’+2H5’, J3’-4’= 7.4 Hz); 7.24 (tt, 2H, 2H4’); 4.32 (t, 1H, CH, JCH-CH2= 8.1 Hz); 4.02 (dd, 2H, CH2); 1.07 (s, 3H, CH3-C3) ppm


Elemental analysis (C24H19Cl2N3O3.½H2O):



- 175 -

N-(benzo[1,3]dioxol-5-ylmethyl)-3-methylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 10a)

M.F.: C18H15N3O5.½H2O

M.W.: 362.34

M.P.: 173-174º C


Yield: 25%



IR (KBr), ν : 3261 (m, νN-H); 3077 (w, νarC-H); 1642 (s, νC=O); 1329 (m, νN+O-) cm-1 1H-NMR (DMSO-d6), δ : 9.29 (t, 1H, NH, JNH-CH2= 5.8 Hz); 8.52-8.38 (m, 2H, H5+H8); 8.02-7.95 (m, 2H, H6+H7); 7.03 (bs, 1H, H2’); 6.91 (bs, 2H, H5’+H6’); 6.01 (s, 2H, O-CH2-O); 4.47 (d, 2H, CH2); 2.42 (s, 3H, CH3-C3) ppm





- 176 -

N-(benzo[1,3]dioxol-5-ylmethyl)-7(6)-chloro-3-methylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 10b)

M.F.: C18H14ClN3O5

M.W.: 387.78

M.P.: 202-203º C


Yield: 18%



IR (KBr), ν : 3276 (w, νN-H); 3090 (w, νarC-H); 1649 (s, νC=O); 1326 (s, νN+O-) cm-1 1H-NMR (DMSO-d6), δ : 9.29 (t, 1H, NH, JNH-CH2= 5.6 Hz); 8.51-8.49 (m, 2H, H5+H8); 8.05-8.02 (m, 1H, H6); 7.02 (bs, 1H, H2’); 6.91 (bs, 2H, H5’+H6’); 6.01 (s, 2H, O-CH2-O); 4.46 (d, 2H, CH2); 2.40 (s, 3H, CH3-C3) ppm





- 177 -

N-(benzo[1,3]dioxol-5-ylmethyl)-3,7(6)-dimethylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 10e)

M.F.: C19H17N3O5

M.W.: 367.36

M.P.: 198-199º C


Yield: 21%



IR (KBr), ν : 3266 (w, νN-H); 3071 (w, νarC-H); 1649 (s, νC=O); 1325 (s, νN+O-) cm-1 1H-NMR (DMSO-d6), δ : 9.29 (t, 1H, NH, JNH-CH2= 5.8 Hz); 8.39 (d, 1H, H5, J5-6= 8.8 Hz); 8.31 (s, 1H, H8); 7.83 (d, 1H, H6); 7.04 (bs, 1H, H2’); 6.91 (bs, 2H, H5’+H6’); 6.02 (s, 2H, O-CH2-O); 4.47 (d, 2H, CH2); 2.59 (s, 3H, CH3-C7); 2.40 (s, 3H, CH3-C3) ppm





- 178 -

N-(benzo[1,3]dioxol-5-ylmethyl)-6,7-dichloro-3-methylquinoxaline-2-carboxamide 1,4-di-N-oxide (Compound 10g)

M.F.: C18H13Cl2N3O5

M.W.: 422.23

M.P.: 230-231º C


Yield: 22%



IR (KBr), ν : 3273 (w, νN-H); 3058 (w, νarC-H); 1647 (s, νC=O); 1326 (s, νN+O-) cm-1 1H-NMR (DMSO-d6), δ : 9.31 (t, 1H, NH, JNH-CH2= 5.8 Hz); 8.69 (s, 1H, H5); 8.67 (s, 1H, H8); 7.02 (bs, 1H, H2’); 6.90 (bs, 2H, H5’+H6’); 6.02 (bs, 2H, O-CH2-O); 4.46 (d, 2H, CH2); 2.50 (s, 3H, CH3-C3) ppm




XIII. PURIFICATION METHODS: AUTOMATED FLASH COLUMN CHROMATOGRAPHY

Results and Discussion: Purification Methods:Automated flash column chromatography

- 181 -

Several methods have been performed and studied in order to optimize and

automate the purification stage and get reproducible results. Among them, two methods

are considered suitable: one of them for the purification of compounds without chlorine

atoms substituted in positions six or/and seven of the quinoxaline ring and the other one

for the compounds that present chlorine atoms substituted in the quinoxaline ring.

Experimental conditions for the purification stage are:

− Stationary phase: Silica RediSep® Rf columns (Average particle size: 35 to 70 microns;

Average pore size: 60 Å)

− Mobile phase technique: linear with isocratic hold technique (Figure 71, 72 and Table

6, 7)

− Detection wavelength: 254 nm

− Flow rate: 30 mL/min

− Equilibration volume: 30 mL

− Loading type: solid

− Mobile phase conditions: dichloromethane/methanol. (Tables 6, 7)

Figure 71. Purification report of compound 6e.

Table 6 Conditions for compounds without substituted

chlorine atoms.

Time (min.)

Methanol (%)

Technique

5 0.0 isocratic

5 0.5 gradient

7 0.5 isocratic

Figure 72. Purification report of compound 6g.

Table 7. Conditions for compounds with substituted

chlorine atoms.

Time (min.)

Methanol (%)

Technique

10 0.0 isocratic

15 0.25 gradient

10 0.25 isocratic

10 0.5 gradient

Results and Discussion: Purification Methods:Automated flash column chromatography

- 182 -

Seventeen of the forty-seven final compounds presented in this project have been

purified by flash column chromatography.

Moreover, the use of automated flash column chromatography allows purifying

compounds that could not be purified by traditional glass-column chromatography, such as

compounds from series 6.

The use of automated flash column chromatography allows increasing purification

stage yields 2 or 3 folds, simplifying the process and decreasing the volume of solvents and

residues. These aspects make this method not only a cheaper alternative but also

environment friendly.

XIV. ANTI-TUBERCULOSIS ACTIVITY

Results and Discussion: Anti-tuberculosis Activity

- 185 -

35. Anti-tuberculosis results

35.1. TAACF anti-tuberculosis results

Compounds from Series 1-7 and 9-10 were submitted to the TAACF program for

being evaluated as anti-tuberculosis agents. Compounds from Series 11-14 have been

synthesized in our group as a part of this project.131 Their anti-tuberculosis results are also

included in this section with the aim of carrying out a more completed discussion and

justifying the studies that are developed in following sections.

Primary screening results

The synthesized compounds have been screened against M. tb H37Rv in the Dose

Response assay. The assay returns the IC90 and IC50 which is the concentration, expressed

as µg/mL, where a drug inhibits the TB strain by 90% or 50%. Compounds showing an

IC90≤10 µg/mL are considered active.

First screening values are shown in Table 8.

Table 8. First screening results for Series 1 and 2.

Series Comp. Ra/Rb IC50

(µg/mL) IC90

(µg/mL) Activity

1a H/H 19.40 26.99 Weakly active

1b Cl/H 5.86 6.71 Active

1c F/H 10.44 17.93 Weakly active

1d CF3/H 14.68 19.10 Weakly active

1e CH3/H 12.25 24.65 Weakly active

1f OCH3/H 23.69 26.84 Weakly active

1g Cl/Cl 5.84 6.63 Active

2a H/H 5.06 6.71 Active


2c F/H 2.75 3.86 Active

2d CF3/H >100 >100 Inactive



2g Cl/Cl 20.33 25.45 Weakly active

Rifampicin 0.015-0.125 Reference

Isoniazid 0.025-0.05


- 186 -

Table 8 cont. First screening results for Series 3-7.


(µg/mL)

IC90

(µg/mL) Activity


3b Cl/H 11.39 15.42 Weakly active



4c F/H 3.37 4.48 Active

4d CF3/H 3.00 3.38 Active

4e CH3/H >100 >100 Inactive

4f OCH3/H 74.28 >100 Weakly active





5g Cl/Cl 38.72 51.86 Weakly

active





7a H/H 6.19 6.76 Active



7g Cl/Cl >100 >100 Weakly

active




- 187 -

Table 8 cont. First screening results for Series 9-12.


(µg/mL)

IC90

(µg/mL) Activity




9g Cl/Cl 55.49 66.54 Weakly

active 10a H/H 11.10 22.75 Weakly

active 10b Cl/H 6.07 6.99 Active


10g Cl/Cl 19.39 34.92 Weakly active

11a H/H 1.52 2.61 Active 11b Cl/H 0.38 0.43 Active 11c F/H 0.93 1.52 Active 11d CF3/H <0.20 0.41 Active 11e CH3/H 2.70 4.21 Active


11g Cl/Cl <0.2 <0.20 Active 11h F/F 0.24 1.73 Active

11i CH3/CH3 30.65 >100 Inactive

12a H/H 1.56 2.85 Active 12b Cl/H NT NT Not Tested

12c F/H 0.97 1.51 Active 12d CF3/H 0.72 0.99 Active 12e CH3/H 5.60 8.90 Active


12h F/F 0.33 0.76 Active

12i CH3/CH3 51.55 >100 Inactive




- 188 -

Table 8 cont. First screening results for Series 13 and 14.


(µg/mL)

IC90

(µg/mL) Activity

13a H/H 1.78 3.05 Active 13b Cl/H <0.20 <0.20 Active 13c F/H 0.35 0.50 Active 13d CF3/H 0.44 1.15 Active 13e CH3/H 4.54 8.62 Active


13g Cl/Cl <0.19 <0.19 Active 13h F/F <0.19 0.52 Active

13i CH3/CH3 9.40 16.36 Weakly active

14a H/H 1.92 3.46 Active 14b Cl/H <0.19 <0.19 Active 14c F/H <0.19 <0.19 Active 14d CF3/H 0.46 1.43 Active 14e CH3/H 3.60 8.85 Active


14g Cl/Cl 0.22 0.45 Active 14h F/F 0.45 2.06 Active

14i CH3/CH3 >100 >100 Inactive Rifampicin 0.015-0.125

Reference Isoniazid 0.025-0.05


- 189 -

Secondary screening results

Thirty nine compounds have been screened in the VERO cell cytotoxicity assay.

The assay returns a CC50 which allows a Selectivity Index (SI) to be calculated. If the SI

value is ≥ 10, then the compound may be considered for further testing.

Secondary screening values are shown in Table 9.

Table 9. Secondary screening results.

Comp. Ra/Rb IC90

H37Rv (µg/mL)

CC50

VERO (µg/mL)

SI CC50/ IC90

1b Cl/H 6.71 8.97 1.34

1g Cl/Cl 6.63 5.54 0.84

2a H/H 6.71 >40 >5.96

2b Cl/H 3.39 >40 >11.79

2c F/H 3.86 17.86 4.62

4b Cl/H 6.13 >40 >6.52

4c F/H 4.48 >40 >8.94

4d CF3/H 3.38 >40 >11.82

4g Cl/Cl 6.58 >40 >6.08

6b Cl/H 5.33 >40 >7.50

6g Cl/Cl 6.92 >40 >5.78

7a H/H 6.76 >40 >5.92

10b Cl/H 6.99 >40 >5.72

11a H/H 2.61 >100 >38.37

11b Cl/H 0.43 >100 >230.94

11c F/H 1.52 >100 >65.70

11d CF3/H 0.41 >100 >246.30

11e CH3/H 4.21 >100 >23.77

11g Cl/Cl <0.2 >100 >500

11h F/F 1.73 18.38 10.64

Rifampicin 0.015-0.125 >100 >800

Isoniazid 0.025-0.050 >1000 >40000


- 190 -

Table 9 cont. Secondary screening results.

Comp. Ra/Rb IC90

H37Rv (µg/mL)

CC50

VERO (µg/mL)

SI CC50/ IC90

12a H/H 2.85 >30 >10.54

12c F/H 1.51 >30 >19.89

12d CF3/H 0.99 >30 >30.03

12e CH3/H 8.90 >30 >3.37

12h F/F 0.76 16.60 21.73

13a H/H 3.05 >100 >32.81

13b Cl/H <0.20 >100 >500

13c F/H 0.50 >100 >198.41

13d CF3/H 1.15 >30 >26.019

13e CH3/H 8.62 >100 >11.60

13g Cl/Cl <0.19 >30 >153.84

13h F/F 0.52 >30 >58.027

14a H/H 3.46 >30 >8.6605

14b Cl/H <0.19 >30 >153.84

14c F/H <0.19 >30 >153.84

14d CF3/H 1.43 >30 >21.023

14e CH3/H 8.85 >30 >3.389

14g Cl/Cl 0.45 >30 >66.079

14h F/F 2.06 >30 >14.577

Rifampicin 0.015-0.125 >100 >800

Isoniazid 0.025-0.050 >1000 >40000


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35.2. DMID anti-tuberculosis results

Compounds from Series 8 were submitted to the Division of Microbiology and

Infectious Diseases (DMID) of the NIAID for being evaluated as anti-tuberculosis agents.

MIC assay results

Compounds 8a, 8b, 8e and 8g have been initially tested at a single point

concentration of 10 µg/mL against M.tb H37Rv.

Table 10. Single point concentration results for Series 8.

Compound 8b was active at the 10 µg/mL level and tested to determine the MIC.

Table 11. MIC assay results for compound 8b.

Figure 73. MIC graph for compound 8b.

Series Comp. Ra/Rb Results

(µg/mL) Activity

8a H/H >10 Inactive

8b Cl/H 10 Active

8c CH3/H >10 Inactive

8d Cl/Cl >10 Inactive

Compound IC50

H37Rv (µg/mL)

IC90

H37Rv (µg/mL)

Activity

4.87 5.42 Active


- 192 -

36. Discussion of the anti-tuberculosis activity

As can be observed in Table 8 thirty nine of the eighty two evaluated compounds

passed the established cut off at the primary screening level and moved on to the

secondary screening level showing twenty four of them interesting results with SI values

higher than 10. Moreover, six of them showed SI values higher than 150; and two

compounds, 11g and 13b, presented a SI value in the same range as the RIF reference.

Some structure-activity relationships have been established:

• Looking at the secondary screening values (Table 9) of compounds 1b, 1g, 2b, 2c, 4b,

4c, 4d, 4g, 6b, 6g, 11b, 11c, 11d, 11g, 12c, 12d, 12h, 13b, 13c, 13g, 13h, 14b, 14c, 14g

and 14h, it can be said that the insertion of an electron-withdrawing moiety in positions

6 and/or 7, especially that of a chlorine atom, increases the anti-tuberculosis activity.

• The substitution of a phenyl in position 3 decreases the biological activity as it can be

observed comparing the primary screening results of Series 2, 3 (Table 8, pages 185

and 186) to Series 11, 13 (Table 8, pages 187 and 188). For this reason, the synthesis of

compounds from Series 3 was cancelled and the methyl moiety was kept in this

position.

• It can be observed that compounds with no aliphatic linker between the carboxamide

group and the phenyl group (1a, 1b, 1c, 1e, 1f) showed higher IC90 values than

compounds that present an aliphatic linker (2a, 2b, 2c, 2e, 2f). Looking at the

secondary screening values (Table 9) of compounds 1b versus 2b, 11d versus 13d, 11g

versus 13g and 12h versus 14h, it can be observed that lengthening the aliphatic chain

can also result in a greater value of cytotoxicity. Taking into account these results, it can

be said that the preferred length for the aliphatic linker between the carboxamide group

and the aromatic ring is one methylene group.

• With the aim of exploring the influence of the p-substitution on the phenyl ring,

electron-withdrawing and electron-releasing moieties were introduced in this position.

Looking at the primary screening results showed by compounds from Series 4-7 (Table

8, page 186) versus Series 11 (Table 8, page 187), it can be said that the insertion of a

substituent on para position of the phenyl group did not improve the anti-tuberculosis

activity. The insertion of a methoxy moiety on this position led to an increase of the

cytotoxicity of the compounds as can be observed by comparing the cytotoxicity values

of Series 11 versus Series 12 and Series 13 versus Series 14 (Table 9). The insertion of


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three methoxy moieties (Series 8) on the phenyl group (Table 10) dramatically

decreases the anti-tuberculosis activity.

• Byphenyl (Series 9) and benzodioxol (Series 10) were also considered as substituents

in the carboxamide group but this modification did not improve the anti-tuberculosis

activity suggesting that the most suitable aromatic ring is the unsubstitued phenyl.

XV. ELECTROCHEMICAL STUDY

Results and Discussion: Electrochemical Study

- 197 -

37. Electrochemical results

Thirty-seven of the quinoxaline derivatives presented in this project were selected for

an electrochemical study according to their structural variety and biological activity diversity.

This study was thought with the aim of establishing the reduction potential of the quinoxaline

dioxides and their capability to form radicals which could take part in the mechanism of action

of this family of compounds as anti-tuberculosis agents. The redox properties of these

compounds were studied using cyclic voltammetry and first derivative cyclic voltammetry in

DMF with TBAP as supporting electrolyte. Results are shown in Table 12 and representative

voltammograms are provided in Figure 74. All reductions were found to be diffusion

controlled, as indicated by constant current functions at varying scan rates.232,244

Figure 74. Cyclic voltammetric reduction of 12e in DMF at 100 mV/s (versus Ag/AgNO3

reference electrode) : (a) single scan, (b) multiple scans, (c) first derivative. (d) cyclic

voltammogram for the ferrocene redox couple used as a reference for peak potential

determination.


- 198 -

Table 12. Cyclic voltammetric data for the quinoxaline di-N-oxide derivatives.

Cyclic Voltammetry 1st Derivativeb Comp.

Epc,1a (V)

Ipc,1 (V)

Epc,2 (V)

E1/2,2 (V)

Ipc,2

(V) Ipa/Ipc Epc,1

(V) Epc,2

(V) Epa,2

(V) E1/2,2 (V)

4a -1.59 2.44 -1.90 (sh)c --- 2.79 0.25 -1.59 -1.87 -1.66 -1.76

4b -1.46 (sh)

3.74 -1.78 -1.66 3.88 0.55 -1.46 -1.77 -1.54 -1.66

4e -1.60 3.46 -1.90 (sh)c --- 5.40 0.35 -1.59 -1.86 -1.69 -1.77

5a -1.53 (sh)

2.84 -1.74 -1.68 5.72 0.48 -1.54 -1.74 -1.62 -1.68

5b -1.47 (sh)

3.46 -1.74 -1.64 4.42 0.69 -1.47 -1.74 -1.54 -1.64

5e -1.57 (sh)

4.92 -1.79 -1.73 6.35 0.42 -1.60 -1.79 -1.67 -1.73

5g -1.37 3.18 -1.58a --- 2.27 --- -1.37 -1.58 --- ---

6a -1.54 (sh)

1.09 -1.79 -1.74 4.08 1.03 -1.57 -1.79 -1.68 -1.74

6b -1.44 6.55 -1.68 -1.62 6.33 0.46 -1.45 -1.68 -1.56 -1.62

6e -1.58 (sh)

3.77 -1.81 -1.74 5.00 0.65 -1.58 -1.81 -1.68 -1.74

6g -1.36 2.95 -1.56a --- 2.20 --- -1.36 -1.57 --- ---

7a -1.58 (sh)

4.97 -1.80 -1.73 5.65 0.66 -1.59 -1.80 -1.66 -1.73

7b -1.49 2.09 -1.79 -1.66 3.56 0.83 -1.49 -1.79 -1.54 -1.66

7e -1.59 (sh)

4.24 -1.81 -1.73 4.92 0.54 -1.59 -1.81 -1.66 -1.73

9a -1.59 6.37 -1.85 -1.74 5.67 0.71 -1.58 -1.85 -1.61 -1.73

9b -1.49 4.74 -1.80 -1.66 4.58 0.80 -1.49 -1.80 -1.52 -1.66

9e -1.64 (sh)

3.87 -1.87 (sh)c --- 3.99 0.88 -1.64 -1.86 -1.68 -1.77

9g -1.41 (sh)

2.65 -1.53 -1.50 1.24 1.13 -1.40 -1.53 -1.46 -1.50

a Irreversible

b Epc determined at the point where the derivative curve crosses the baseline.

c(sh) Peak is broad and poorly defined; appears as shoulder (sh). E1/2 could not be determined

accurately from the cyclic voltammogram.


- 199 -

Table 12 cont. Cyclic voltammetric data for the quinoxaline di-N-oxide derivatives.

Cyclic Voltammetry 1st Derivativeb Comp.

Epc,1a (V)

Ipc,1 (V)

Epc,2 (V)

E1/2,2 (V)

Ipc,2

(V) Ipa/Ipc Epc,1

(V) Epc,2

(V) Epa,2

(V) E1/2,2 (V)

10a -1.61 4.99 -1.85 -1.76 3.58 0.83 -1.61 -1.85 -1.67 -1.76

10b -1.49 5.26 -1.76 -1.66 4.35 0.42 -1.50 -1.75 -1.55 -1.65

10e -1.62 5.92 -1.89 -1.79 5.18 0.61 -1.62 -1.89 -1.70 -1.80

10g -1.40 2.74 -1.52 -1.50 1.49 0.99 -1.40 -1.52 -1.46 -1.49

11b -1.48 3.89 -1.79 -1.66 3.91 0.96 -1.48 -1.78 -1.53 -1.65

11c -1.52 1.44 -1.64 -1.59 0.30d 4.33 -1.52 -1.64 -1.53 -1.58

11d -1.41 2.31 -1.52 -1.42 0.40d 0.96 -1.42 -1.50 -1.33 -1.42

11e -1.66 2.44 -1.91 (sh)c

--- 2.88 0.24 -1.65 -1.92 -1.68 -1.80

11g -1.21 1.91 -1.39a --- 2.35 --- -1.21 -1.39 --- ---

12a -1.60 5.07 -1.87 -1.76 5.53 0.65 -1.60 -1.87 -1.67 -1.77

12b -1.49 5.56 -1.79 -1.66 5.68 0.70 -1.49 -1.79 -1.53 -1.66

12e -1.62 4.61 -1.87 -1.77 5.90 0.48 -1.62 -1.87 -1.67 -1.77

13a -1.62 4.07 -1.85 -1.76 4.84 0.71 -1.62 -1.85 -1.67 -1.76

13b -1.52 3.96 -1.72 (sh)c --- 2.90 0.90 -1.52 -1.70 -1.56 -1.63

13c -1.55 1.30 -1.70 -1.62 0.55d 1.09 -1.55 -1.69 -1.55 -1.62

13d -1.44 2.83 -1.54 -1.45 0.44d 3.04 -1.44 -1.54 -1.35 -1.45

13e -1.65 4.69 -1.91 -1.80 3.97 0.79 -1.65 -1.91 -1.71 -1.81

13g -1.39 2.85 -1.56a --- 2.46 --- -1.39 -1.56 --- ---

13h -1.48 1.37 -1.66a --- 0.55d --- -1.48 -1.66 --- ---

a Irreversible

b Epc determined at the point where the derivative curve crosses the baseline.

c(sh) Peak is broad and poorly defined; appears as shoulder (sh). E1/2 could not be determined

accurately from the cyclic voltammogram.

d Ipc could not be determined accurately due to following voltammetric waves.


- 200 -

38. Discussion.

38.1. Discussion. Electrochemical behavior

For all compounds studied, two cyclic voltammetric waves were observed between -

0.4 and -1.9 V during reduction. The first voltammetric wave was irreversible under these

conditions, with peak potentials, Epc,1, ranging from -1.21 to -1.65 V (vs. Fc/Fc+). For

compounds 4b, 5a, 5e, 5b, 6a, 6b, 7a, 7e, 9e and 9g this wave appeared as a shoulder to the

second wave in the voltammogram. With a few exceptions (see following), the second

voltammetric wave was quasireversible under the conditions used in the study, with peak

potentials, Epc,2, ranging between -1.39 and -1.92 V, and half-wave potentials ranging between

-1.42 and -1.80 V. For compounds 4a, 4e, 9e, 11e and 13b this voltammetric wave was not

very well defined, appearing as a shoulder in the cyclic voltammogram. Values of ΔEp for this

wave (100 mV/s) were generally greater than theoretical for a reversible, one-electron

reduction, and increased with increasing scan rate. For compounds, 5g, 6g, 11g, 13g and 13h

the second voltammetric wave was irreversible under the conditions employed in this study.

Calculated ipa/ipc ratios for compounds 5b, 6a, 6e, 7a, 7b, 9a, 9e, 9b, 9g, 10a, 10e, 10g 11b,

11d, 12a, 12b, 13a, 13e, 13b and 13c were close to unity (0.61 to 1.24) at all scan rates,

indicating the formation of relatively stable reduction products for this process on the time

scale of the voltammetric scans. Current ratios that deviated significantly from unity (0.24 to

0.55) were observed for all other derivatives, indicative of greater kinetic or other

complications following the reduction.244 No reduction waves were observed at negative

potentials of the second voltammetric wave for these quinoxaline 1,4-dioxide derivatives

under the conditions used in this study, with the exception of compounds 5g, 6g, 9g, 10g,

11g, 11c, 11d, 13g, 13d, 13c and 13h. For the latter compounds, a third irreversible reduction

wave was observed at potentials ranging from -1.8 to -2.4 V.

Examination of the data indicates the influence of quinoxaline structure on reduction

potential. The 37 compounds can be divided in 9 different analogues based on structure, with

the mono chloro and methyl substituted quinoxalines existing as mixtures of 6- and 7-

substituted positional isomers. Within each analogous series, replacement of the 6-/7-

hydrogen atom with a single electron-withdrawing substituent group, e.g. chloro, fluoro, and

trifluoromethyl, resulted in a positive shift in the peak potentials for both voltammetric waves,

whereas replacement of the 6-/7-hydrogen atom with a single electron-releasing methyl group

resulted in a negative shift. As an example, comparison of the potentials for compounds 12a

and 12b shows that replacement of the hydrogen atom with the chloro group resulted in a

+109 mV potential shift for the first voltammetric wave, and a +76 mV shift in the second


- 201 -

voltammetric wave (+105 mV shift in E1/2). Replacing the hydrogen atom in compound 12a

with the methyl group in compound 12e resulted in a -20 mV potential shift for the first

voltammetric wave, and a -10 mV shift in the second voltammetric wave (-8 mV shift in E1/2).

Similarly, comparison of compound 13a with compounds 13c and 13d shows that

substituting the 7-hydrogen with fluoro or trifluoromethyl groups results in a +74mV and

+186 mV shift in the first voltammetric wave, respectively. The reduction potentials for the

quinoxaline derivatives within each analogue generally fit the modified Hammett equation,

ΔE1/2 = ρπRσx (correlation coefficients ranged from 0.89 to 1), where σx is the polar inductive

electronic substituent constant taken as the average of the sum of σm-x and σp-x (Table 13) and

ρ is the reaction constant.245 For quinoxaline derivatives substituted in the benzene ring, the

use o (σm-x + σp-x)/2 has been recommended.246 These effects are consistent with facilitation

of reduction by a positive charge at the site of reduction,245 in keeping with previous reports. 165,168,245 Addition of a second electron-withdrawing group, i.e. chloro or fluoro, enhances this

effect. For example, comparison of the 6,7-dichloro derivatives with the corresponding 6-/7-

chloro derivatives shows that addition of the second chloro group shifts the potential of the

first voltammetric wave positively by +81 to +271 mV, and that of the second voltammetric

wave by +121 to +395 mV. No apparent correlation was observed between substitution in

the amide side chain and potential (4e, 5e, 6e, 7e, 9e, 10e, 11e and 12e). In the latter case, the

substituents are presumably too far removed from the site of reduction.

Table 13. Hammett substituent constants.a

Compound R7/R6 σp-x σm-x (σm-x + σp-x)/2

13a H/H 0 0 0

13b Cl/H 0.23 0.37 0.30

13c F/H 0.06 0.34 0.20

13d CF3/H 0.55 0.41 0.48

13e CH3/H -0.17 -0.07 -0.12

13g Cl/Cl 0.46 0.74 0.60

13h F/F 0.12 0.68 0.40

aHammett substituent constant values are taken from reference. 245


- 202 -

38.2. Discussion. Relationship between electrochemical behavior and anti-tuberculosis activity

With regard to the mechanism as anti-tuberculosis agent, heterocyclic di-N-oxides

possessing the diiminium structure are expected to undergo one electron reduction to form

a radical anion (Figure 75),151,166,247-249 and in fact reduction of quinoxaline 1,4-di-N-oxide in

DMF has been shown via ESR and cyclic voltammetry to involve the nitrone function to

form the radical anion.250 The electrochemical reductions of quinoxalines 1, 4-di-N-oxide in

aprotic solvent systems have been reported previously.165,166,168,250,251 In most cases, a

reversible reduction wave attributed to reduction of the N-oxide functionality has been

reported in DMF in the vicinity of those observed in the present study. For the

compounds in the present study, electrochemical reduction in DMF is consistent with

reduction of the N-oxide functionality to form a radical anion as well. The second

reduction wave observed could be due to the formation of the dianion, or to reduction of

the product formed from a chemical step involving the radical anion.251 The latter is

supported by the irreversibility of the first reduction wave. Kaye and Stonehill pointed out

that an objection to the iminium theory is that some potentials observed are too negative

although several reports indicate that reduction potential in vivo may be better than in

vitro,167 and N-oxides are known to undergo bio-reduction.252,253

Figure 75. Proposed mechanism of action.

A relationship between reduction potential and anti-tuberculosis activity could be

suggested by the data. Different stages in the mechanism of action against tuberculosis for

these compounds should be considered. In this sense, in the first stage the quinoxalines di-

N-oxide could be activated through a bio-reduction process to form a reactive radical

anion, which could lead further to superoxide ion or other toxic oxy radical species, or to

direct macromolecular interactions in subsequent stages. (Figure 75) If such a mechanism


- 203 -

occurs for these compounds, bio-reductive activation would generally be expected to be

more facile for the more easily reduced derivatives.

Table 14. Cyclic voltammetry data and anti-tuberculosis activity of quinoxaline derivatives.

Cyclic voltammetry Anti-TB Activity Comp.

Epc,1a Epc,2 E1/2,2 IC90 CC50 SI

4a -1.59 -1.90 (sh) --- 16.81 NT NT

4b -1.46 (sh) -1.78 -1.66 6.13 >40 >6.52

4e -1.60 -1.90 (sh) --- >100 NT NT

5a -1.53 (sh) -1.74 -1.68 11.04 NT NT 5b -1.47 (sh) -1.74 -1.64 29.68 NT NT 5e -1.57 (sh) -1.79 -1.73 14.56 NT NT 5g -1.374 -1.58 --- 51.86 NT NT 6a -1.54 (sh) -1.79 -1.74 15.61 NT NT 6b -1.44 -1.68 -1.62 5.33 >40 >7.50

6e -1.58 (sh) -1.81 -1.74 78.22 NT NT

6g -1.36 -1.56 --- 6.92 >40 >5.78

7a -1.58 (sh) -1.80 -1.73 6.76 >40 >5.92

7b -1.49 -1.79 -1.66 32.04 NT NT 7e -1.59 (sh) -1.81 -1.73 99.91 NT NT 9a -1.59 -1.85 -1.74 15.99 NT NT 9b -1.49 -1.80 -1.66 60.43 NT NT 9e -1.64 (sh) -1.87 (sh) --- 16.79 NT NT 9g -1.41 (sh) -1.53 -1.50 66.54 NT NT 10a -1.61 -1.85 -1.76 22.75 NT NT

10b -1.49 -1.76 -1.66 6.99 >40 >5.72

10e -1.62 -1.89 -1.79 13.22 NT NT

10g -1.40 -1.52 -1.50 34.92 NT NT


- 204 -

Table 14 cont. Cyclic voltammetry data and anti-tuberculosis activity of quinoxaline

derivatives.

Cyclic voltammetry Anti-TB Activity Comp.

Epc,1a Epc,2 E1/2,2 IC90 CC50 SI

11b -1.48 -1.79 -1.66 0.43 >100 >230.94

11c -1.52 -1.64 -1.59 1.52 >100 >65.70

11d -1.41 -1.52 -1.42 0.41 >100 >246.30

11e -1.66 -1.91 (sh) --- 4.21 >100 >23.77

11g -1.21 -1.39 --- <0.20 >100 >500

12a -1.60 -1.87 -1.76 2.85 >30 >10.54

12b -1.49 -1.79 -1.66 NT NT NT

12e -1.62 -1.87 -1.77 8.90 >30 >3.37

13a -1.62 -1.85 -1.76 3.05 >100 >32.81

13b -1.52 -1.72 (sh) --- <0.20 >100 >500

13c -1.55 -1.70 -1.62 0.50 >100 >198.41

13d -1.44 -1.54 -1.45 1.15 >30 >26.019

13e -1.65 -1.91 -1.80 8.62 >100 >11.60

13g -1.39 -1.56 --- <0.19 >30 >153.84

13h -1.48 -1.66 --- 0.52 >30 >58.027

Examination of the results for the compounds which have shown biological activity

based on their selectivity index values (Series 11-13) (Table 14) shows that in general the

less negative the reduction potential is, the more active the compounds are. In fact, for

compounds showing a Epc,1 < -1.6V the IC90 increases almost exponentially (Figures 76 and

77). Not only that, but compounds showing a Epc,1 < -1.6V also present a selectivity index

(SI) much lower than compounds with a reduction potential that is less negative (Figure

78). Caution is obviously warranted in interpreting such electrochemical data, however,

since not all the compounds tested exhibited appreciable activity. Many other factors

besides bio-reduction must also be considered in determining the in vivo mechanism of

action of anti-tuberculosis compounds, such as solubility, metabolism, diffusion,

membrane permeability, stereochemistry, absorption, and active site binding among others.

Thus, absolute correlation between electrochemical behavior and anti-tuberculosis activity

is not reasonable. However, the results of this study seem to suggest the participation of

charge transfer processes in the overall mechanism of action of these quinoxalines di-N-

oxide against tuberculosis; however, the exact mechanism was not investigated further.


- 205 -

Figure 76. Plot of reduction potentials for the first voltammetric wave of compounds 1-12

vs IC90.


vs log IC90.


- 206 -


vs. SI.

XVI. PARTITION COEFFICIENT STUDIES

Results and Discussion: Partition Coefficient Studies

- 209 -

39. Shake-flask method results

Three of the twenty quinoxaline derivatives studied by the RP-HPLC method were

selected for the shake-flask method with the aim of establishing the concordance between

the two experimental methods.

Experiments were performed four times for each derivative but no results can be

presented. No reliable results were obtained probably due to: the low solubility of the

compounds, the low stability of these derivatives in solution and the emulsions generated

during the partition procedure.

It is well known that quinoxalines di-N-oxide are not stable in aqueous solution and

during the experiments the UV-spectrum of quinoxalines changed quickly (see Appendix

2). Not only that, but also emulsion were generated during the procedure and the two

phases could not be properly separated.

40. RP-HPLC method results

Twenty of the quinoxaline derivatives presented in this project were selected to study

the partition coefficient of quinoxaline di-N-oxide derivatives. The nitrone function is not well

configured in some computational methods employed to estimate the logPo/w. and, moreover,

the classical shake-flask method does not work for these derivatives. For these reasons,

some compounds were chosen, according to their structural variety, with the aim of building a

model in a wide range of logPo/w values using a RP-HPLC method. The selected compounds

were also submitted to different computational methods to study the wellness of these

modules with this family of compounds.

40.1. Correlation between logPo/w and logk’0

In this study 9 compounds (1-9) have been selected as reference compounds from

the Recommended Reference Compounds list published by the OECD.191 Chosen

compounds allow building a model in a quite fairly range of logPo/w values (i.e., from 0 to

4.5). The retention times of these reference compounds have been measured and expressed

as logk’0. The logk’ values have been extrapolated to 0% methanol in order to determine

the capacity factors represented as logk’0. To predict the logPo/w values using the logk’0

values, the least square regression was employed to generate Equation 13.

Equation 13


- 210 -

R2= 0.999; RMSE= 0.111; n: 9.

The detailed results of the LOO test are presented in Table 15.

Table 15. logPo/w, logk’0 and related RP-HPLC logPo/w values for the reference compounds.

Name logPo/wa logk’0 RP-HPLC

logPo/w LOO-predicted

RP-HPLC logPo/w

2-butanone 0.3 -0.06 0.21 0.15

Aniline 0.9 0.67 0.94 0.96

Acetanilide 1.0 0.88 1.15 1.19

Acetophenone 1.7 1.27 1.54 1.52

Benzene 2.1 1.87 2.14 2.14

Chlorobenzene 2.8 2.62 2.89 2.90

Bromobenzene 3.0 2.84 3.10 3.12

Naphthalene 3.6 3.15 3.42 3.36

Benzyl benzoate 4.0 3.74 4.01 4.01

a Reference logPo/w values taken from OECD Guidelines 191

The cross-validation statistical parameters determined with the Leave One Out

procedure were the following:

R2LOO= 0.987; RMSELOO= 0.140.

Taking into account these values, it can be said that Equation 13 can be used to

predict the logPo/w of 1,4-di-N-oxide quinoxaline derivatives using the logk’0 values.

40.2. logPo/w of quinoxalines di-N-oxide

Retention times of quinoxaline derivatives were measured and the capacity factors

(logk’) were calculated in varying proportions of methanol from 70 to 40%. The capacity

factors were extrapolated to 0% methanol and the resulting logk’0 values of quinoxaline

derivatives are presented in Table 16. logPo/w values were determined using Equation 13

and are also reported in Table 16.


- 211 -

Table 16. Capacity factors and related RP-HPLC logPo/w for quinoxaline derivatives.

Capacity factors

Comp logk’70 logk’60 logk’50 logk’40 logk’0 RP-HPLC

logPo/w

6a -0.24 0.11 0.48 0.85 2.30 2.57

6b 0.02 0.42 0.84 1.22 2.83 3.09

6e -0.16 0.21 0.60 0.94 2.43 2.69

6g 0.19 0.65 1.15 1.60 3.51 3.78

7a -0.44 -0.10 0.25 0.62 2.02 2.29

7b -0.25 0.15 0.54 0.93 2.51 2.77

7e -0.36 -0.01 0.34 0.66 2.02 2.29

7g 0.14 0.54 0.93 1.33 2.91 3.18

9a -0.33 0.14 0.62 1.06 2.92 3.18

9b -0.13 0.38 0.91 1.38 3.42 3.69

9e -0.12 0.33 0.80 1.21 3.02 3.28

9g 0.25 0.76 1.31 1.78 3.86 4.12

11a -0.64 -0.34 -0.05 0.28 1.49 1.76

11b -0.36 -0.05 0.30 0.65 1.98 2.25

11e -0.56 -0.24 0.07 0.35 1.58 1.85

11g -0.11 0.27 0.66 0.98 2.46 2.72

13a -0.58 -0.26 0.05 0.36 1.61 1.88

13b -0.37 0.00 0.36 0.69 2.11 2.38

13e -0.45 -0.13 0.21 0.47 1.73 2.00

13g -0.06 0.34 0.74 1.07 2.60 2.87

41. Calculated logPo/w

All compounds studied by the RP-HPLC method were subjected to the ALOGPS

online module to study the wellness of these modules with this family of compounds.

Seven values were obtained for each compound using different computational methods

included in this module. The predicted logPo/w data are reported in Table 17. RMSE was

calculated for each computational method in order to judge which method best suits

experimental RP-HPLC.


- 212 -

Table 17. Calculated logPo/w for quinoxaline derivatives and related RMSE.

Calculated logPo/w Comp

RP-HPLC logPo/w ALOGPs miLOGP ALOGP MLOGP LogKOW XLOGP2 XLOGP3

6a 2.57 1.14 0.33 1.86 -1.08 1.44 5.82 1.97

6b 3.09 1.82 0.99 2.52 -0.59 2.09 6.44 2.60

6e 2.69 1.29 0.76 2.34 -0.85 1.99 6.26 2.33

6g 3.78 2.74 1.59 3.19 -0.09 2.73 7.07 3.22

7a 2.29 0.56 -0.03 1.60 -1.47 1.10 5.46 1.64

7b 2.77 1.22 0.63 2.26 -0.97 1.74 6.08 2.27

7e 2.29 0.75 0.40 2.08 -1.24 1.65 5.90 2.01

7g 3.18 2.31 1.07 2.71 -0.36 2.16 6.71 2.90

9a 3.18 1.78 1.31 2.78 -0.35 2.16 6.67 3.19

9b 3.69 2.36 1.97 3.45 0.12 2.81 7.29 3.82

9e 3.28 1.98 1.74 3.27 -0.14 2.71 7.11 3.56

9g 4.12 3.19 2.57 4.11 0.60 3.45 7.92 4.45

11a 1.76 0.43 -0.48 1.11 -1.71 0.55 5.03 1.28

11b 2.25 1.03 0.18 1.77 -1.20 1.20 5.65 1.90

11e 1.85 0.50 -0.05 1.60 -1.47 1.10 5.46 1.64

11g 2.72 1.72 0.78 2.44 -0.70 1.84 6.27 2.53

13a 1.88 0.63 -0.07 1.43 -1.47 1.04 5.18 1.74

13b 2.38 1.25 0.58 2.09 -0.97 1.69 5.80 2.37

13e 2.00 0.80 0.35 1.92 -1.24 1.59 5.62 2.10

13g 2.87 2.03 1.19 2.76 -0.47 2.33 6.43 2.99

RMSE 1.28 1.96 0.43 3.52 0.89 3.47 0.36

In an attempt to improve the predictive capacity of the ALOGPS, the LIBRARY

mode was used, and experimental logPo/w data of compounds from Series 11 were used to

generate the library that was taken into consideration for recalculating the logPo/w of the rest

of quinoxaline derivatives (Table 18).


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Table 18. Calculated logPo/w for quinoxaline derivatives using ALOGPS with and without

LIBRARY mode.

Comp. RP-HPLC logPo/w

ALOGPs ALOGPs LIBRARY

6a 2.57 1.14 2.40

6b 2.69 1.29 2.54

6e 3.09 1.82 2.92

6g 3.78 2.74 3.54

7a 2.29 0.56 1.90

7b 2.29 0.75 2.06

7e 2.77 1.22 2.41

7g 3.18 2.31 3.19

9a 3.18 1.78 3.18

9b 3.28 1.98 3.33

9e 3.69 2.36 3.67

9g 4.12 3.19 4.32

13a 1.88 0.63 1.93

13b 2.00 0.80 2.08

13e 2.38 1.25 2.43

13g 2.87 2.03 3.04

RMSE 1.29 0.19

42. Discussion

42.1. Discussion. Comparative of experimental methods and predictive approaches

The lipophilicity of a drug is related to its ability to cross cell membranes by means

of passive diffusion. This property is usually expressed by the logarithm of the

octanol/water partition coefficient, logPo/w. The logPo/w reflects the relative solubility of the

drug in octanol (a model of the lipid bilayer of a cell membrane) and water (the fluid in and

out of cells). Traditionally, logPo/w values are measured using the ‘‘shake-flask’’ with the

octanol and water partition system.

The classical “shake-flask” method is not suitable for the measurement of logPo/w

values of quinoxaline derivatives. In fact, these compounds have very low solubility in

water; moreover, some of them degrade in solution and create emulsions during the

partition procedure. For these reasons, a RP-HPLC method was used for determining


- 214 -

logPo/w values of quinoxaline derivatives and different calculative approaches were used

with the aim of comparing the capacity of these predictive approaches to estimate the

logPo/w of quinoxaline derivatives.

With regard to the proposed RP-HPLC method and taking into account the cross-

validation statistical parameters determined with the Leave One Out procedure, it can be

said that Equation 13

Equation 13

can be used to predict the logPo/w of 1,4-di-N-oxide quinoxaline derivatives using the logk’0

values calculated by the proposed RP-HPLC method.

Once the RP-HPLC method has been validated, examination of the RP-HPLC

logPo/w data (Table 16) indicates the influence of the quinoxaline structure on the logPo/w

values. As expected, the logPo/w values of all the quinoxaline derivatives are positive in a

range between 1.5 and 4.5. The 20 compounds can be divided into 5 different series based

on structure. Within each analogues series, the insertion of a methyl group resulted in a soft

increase of the logPo/w value, and replacing the hydrogen atom with one or two chloro

groups supposed an increase of 0.5 or 1.0 unit, respectively. Increasing the aliphatic chain

between the amide group and the aromatic system resulted in an increase of the logPo/w

values as can be observed comparing derivatives from Series 11 vs. Series 13. The p-bromo

substituent (Series 6) increases the lipophilicity of the molecules. Finally, looking at the

values of Series 9, it can be observed that these compounds, which present a diphenyl

substituent on the amide chain, presented the highest logPo/w values of all the derivatives.

Concerning the calculative approaches, the RMSE calculated from experimental

and estimated logPo/w values ranged between 0.36 and 3.52 for XLOGP3 and MLOGP,

respectively. From these data, XLOGP3 can be considered as the best approach to

estimate logPo/w for the quinoxaline dioxide derivatives. The examination of the data reveals

that ALOGPS LIBRARY mode presents a RMSE of 0.19, making it the best approach for

predicting partition coefficient of quinoxaline di-N-oxide derivatives.

Therefore, the results suggest that the HPLC analysis is a suitable method for

determining the logPo/w for quinoxaline derivatives instead of classical methods which are

too slow and labour intensive. It can be said that the equation established and proposed

(Equation 13) can be used to predict the logPo/w of 1,4-di-N-oxide quinoxaline derivatives

using the logk’0 values determined by the RP-HPLC method. The RP-HPLC method

allowed us to avoid the experimental problems presented by the classical shake-flask


- 215 -

method when trying to measure the partition coefficients of quinoxaline di-N-oxide

derivatives. For all the reasons mentioned above, the RP-HPLC method is proposed as the

suitable one to determine the lipophilicity of the family of quinoxaline derivatives

presented in this work.

After a comparison among different methods for the calculation of logPo/w, in cases

where no experimental data is available, XLOGP3 is proposed as the best program to

calculate the logPo/w values for the QdO presented in this work (RMSE = 0.36). On the

other hand, the ALOGPS LIBRARY method, implemented with the experimental data,

predicts logPo/w values that suit the experimental ones with the lowest RMSE (0.19).

42.2. Discussion. Relationship between logPo/w and anti-tuberculosis activity

Since logPo/w is an important ADME parameter, a correlation with the anti-

tuberculosis activity of compounds was looked for. However, it was verified that there is

no a strong relationship (R2 < 0.2) between logPo/w and activity, expressed as log(1/IC50)

(Figure 79) or log(1/IC90) (Figure 80). However, it seems that a low value of logPo/w could

be related with better values of anti-tuberculosis activity.

Figure 79. Plot of logPo/w versus logIC50 against M.tb H37Rv.


- 216 -

Figure 80. Plot of logPo/w versus logIC90 against M.tb H37Rv.

2011 nature

This fact could be explained as a consequence of the structure of the M.tb envelope

structure. Porin presented in the membrane control the diffusion of small hydrophilic

molecules; and, therefore, M.tb is more permeable to hydrophilic drugs such as INH, PZA,

EMB or gatifloxacin that present a logP of -0.71, -0.71, -0.12 and -0.23, respectively

(ALOGPS, http://www.vcclab.org). In this sense, lipophilic molecules should be able to

easily cross the lipid bilayer; however, the bilayer’s uncommon thickness and the presence

of the mycolic acids seem to dicrease the permeability to lipophilic drugs. Nevetheless, it

has been observed that the more lipophilic the agents are, the more active they usually are

against M.tb, as for instance, PAS, RIF or rifapentine presenting logP values of 0.62, 3.85

and 4.83, respectively (ALOGPS, http://www.vcclab.org). This fact suggests that there

must be an specific pathway to lipophilic drugs transport.7

Recent studies have focused on the influence of physicochemical properties of

antibacterial drugs and they have concluded that it is not possible to establish a strong

relationship between these properties and the anti-tuberculosis activity. In this sense, it

seems that much work is needed in order to understand M.tb and its metabolism. At this

moment, recent reviews affirmed that antibacterial drugs constitute an special

physicochemical space complete different from the space studied by drugs in many other

therapeutic agents.66,254

CONCLUSIONSCONCLUSIONS

Conclusions

219

This memory includes work related to the design, synthesis and characterization of

forty-seven new 1,4-di-N-oxide quinoxaline-2-carboxamide derivatives as well as the

evaluation of the anti-tuberculosis activity of eighty-two 1,4-di-N-oxide quinoxaline-2-

carboxamide derivatives carried out by the programs developed by NIAID.

Electrochemical studies developed in the Chemistry Department at the Southeast Missouri

State University and studies of the partition coefficients carried out by the Environment

and Life Sciences Department at the Università degli Studi del Piemonte Orientale

“Amedeo Avogadro”, Italy, have also been included. This research work has led to the

following conclusions:

1. The synthetic routes needed for obtaining the target compounds were set up by means

of convergent synthesis, decreasing the number of synthetic phases and avoiding the

synthetic difficulties that arise when working with dioxidized quinoxalines.

2. The use of calcium salts as catalyst and ethanolamine as the base in the Beirut reaction

was the best method for the synthesis of 1,4-di-N-oxide quinoxaline-2-carboxamide

derivatives in terms of reaction time, isolation and purification simplicity, yield, cost, and

accessibility of reagents.

3. Two purification methods have been optimized by means of flash column

chromatography automated for the synthesized compounds. One method is for the

purification of the compounds that present a chloro atom substituted in positions 6 and/or

7 of the quinoxaline ring and the other method is for the derivatives that do not present

chloro in said positions.

4. The use of high performance liquid chromatography has permitted corroboration of

the quantification of the proportion of isomers of position 7(6) of the quinoxaline

derivatives that are monosubstituted in said positions.

5. The study of the ultraviolet spectra of the compounds has facilitated the work of

defining the profile of the absorption spectra of these derivatives.

6. Thirty-nine of the eighty-two derivatives evaluated against Mycobacterium tuberculosis have

shown an IC90 value less than 10 µg/mL. Twenty-four of the derivatives whose toxicity has

been tested on VERO cells have shown good in vitro activity/cytotoxicity relationships,

with SI values greater than 10. In addition, six of these compounds have presented SI

values greater than 150, and the compounds 11g and 13b had SI values greater than 500.

These results appear to confirm that the work hypothesis is valid for the objectives being

pursued.

Conclusions

220

7. With regard to the principal group, linked to position 2 of the quinoxaline ring, the

carboxamide group is decisive for the anti-tuberculosis activity, improving the activity

results in relation with the structures studied previously by the group.

With regard to the substitution in position 3 of the quinoxaline ring, the methyl group

is essential for obtaining good biological activity as well as stability in the system.

The results obtained indicate that the substitutions in positions 6 and/or 7 of the

quinoxaline ring by an electron withdrawing group promote anti-tuberculosis activity. More

specifically, the derivatives that present one or two chlorine atoms in said positions give

more potency and selectivity, and less cytotoxicity.

8. The study of the anti-tuberculosis activity has demonstrated that the existence of an

aliphatic linker between the carboxamide group and the aromatic system is essential. The

appropriate length of said spacer is a methylene due to the fact that, in some cases, an

increase in the aliphatic chain leads to an increase in the cytotoxicity of the compounds.

9. Modification of the benzene ring in the aromatic region, as well as the introduction of

substituents in para position, regardless of their electron withdrawing or electron donating

nature, implies a decrease in the anti-tuberculosis activity and an increase in the

cytotoxicity. Therefore, the unsubstituted benzene ring is an important structural

requirement for anti-tuberculosis activity.

10. The electrochemical studies have demonstrated that the introduction of electron

withdrawing substituents in the quinoxaline ring leads to reduction potentials that are less

negative and facilitates the bio-reduction of the compounds. The relationship found

between the electrochemical behavior and the anti-tuberculosis activity of the compounds

corroborates the hypothesis that this type of derivatives can be activated by bio-reduction

generating more active species.

11. A reversed-phase high performance liquid chromatography method has been set up for

the experimental determination of the octanol-water partition coefficients for the

dioxidized quinoxaline derivatives. It was not possible to establish a relationship between

said values and the anti-tuberculosis activity of the compounds.

12. Based on the promising results, compound 11g is proposed for a more in depth study

with regard to the biological activity and proposed as a lead compound for optimization of

the structure.

CONCLUSIONESCONCLUSIONES

Conclusiones

223

Esta memoria recoge el trabajo de diseño, síntesis y caracterización de cuarenta y

siete nuevos derivados de 1,4-di-N-óxido de quinoxalin-2-carboxamida, así como la

evaluación de la actividad antituberculosa de un total de ochenta y dos derivados de 1,4-di-

N-óxido de quinoxalin-2-carboxamida llevada a cabo por los programas desarrollados por

el NIAID. Se incluyen estudios de electroquímica desarrollados en el Department of

Chemistry at Southeast Missouri State University y estudios de los coeficiente de partición

llevados a cabo en el Environment and Life Sciences Department de la Università degli

Studi del Piemonte Orientale “Amedeo Avogadro”, Italia. El desarrollo de este proyecto de

investigación, ha dado lugar a las siguientes conclusiones:

1. Se han puesto a punto las rutas sintéticas necesarias para la obtención de los

compuestos objetivo mediante síntesis convergente, disminuyendo el número de etapas

sintéticas y evitando las dificultades sintéticas asociadas al trabajo con quinoxalinas

dioxidadas.

2. La utilización de sales de calcio como catalizador y etanolamina como base en la

reacción de Beirut, ha resultado el método de síntesis más adecuado para la síntesis de

derivados de 1,4-di-N-óxido de quinoxalin-2-carboxamida en términos de tiempo de

reacción, simplicidad de aislamiento y purificación, rendimiento, y coste y accesibilidad de

los reactivos.

3. Se han optimizado dos métodos de purificación mediante cromatografía en columna

flash automatizada para los compuestos sintetizados. Uno para la purificación de los

compuestos que presentan un átomo de cloro sustituido en posiciones 6 y/o 7 del anillo de

quinoxalina y otro método para los derivados que no presentan cloro en dichas posiciones.

4. El uso de la cromatografía líquida de alta resolución, ha permitido corroborar la

cuantificación de la proporción de isómeros de posición 7(6) de los derivados de

quinoxalina monosustituidos en dichas posiciones.

5. El estudio de los espectros de ultravioleta de los compuestos ha permitido definir el

perfil del espectro de absorción de estos derivados.

6. Treinta y nueve de los ochenta y dos derivados evaluados frente a Mycobacterium

tuberculosis, han mostrado un valor de IC90 inferior a 10 µg/mL. Veinticuatro de los

derivados cuya toxicidad ha sido testada en células VERO han mostrado buenas relaciones

actividad/citotoxicidad in vitro con valores de SI superiores a 10. Además, seis de estos

compuestos han presentado valores de SI superiores a 150, mostrando dos compuestos, el

Conclusiones

224

11g y el 13b, un valor de SI superior a 500. Estos resultados parecen confirmar que la

hipótesis de trabajo planteada está bien enfocada hacia los objetivos que se persiguen.

7. Con respecto al grupo principal, unido a la posición 2 del anillo de quinoxalina, el

grupo carboxamida se establece como determinante para la actividad antituberculosa

mejorando los resultados de actividad en relación a las estructuras estudiadas previamente

por el grupo.

En cuanto a la sustitución en posición 3 del anillo de quinoxalina, el grupo metilo es

esencial para lograr buena actividad biológica así como estabilidad en el sistema.

Los resultados obtenidos indican que la sustitución en posiciones 6 y/o 7 del anillo de

quinoxalina por un grupo electroatrayente potencian la actividad antituberculosa. En

concreto, son los derivados que presentan uno o dos átomos de cloro en dichas posiciones

los que aportan mayor potencia y selectividad con menor citotoxicidad.

8. El estudio de la actividad antituberculosa ha demostrado que es esencial la existencia de

un espaciador alifático entre el grupo carboxamida y el sistema aromático. Se establece que

la longitud apropiada de dicho espaciador es de un metileno debido a que un incremento

en la cadena alifática conduce, en algunos casos, a un incremento en la citotoxicidad de los

compuestos.

9. La modificación del anillo de benceno en la región aromática, así como la introducción

de sustituyentes en posición para, con independencia de su naturaleza electroatrayente o

electrodonante, implican un descenso en la actividad antituberculosa y un incremento en la

citotoxicidad. Por lo tanto, el anillo de benceno sin sustituir se establece como un

requerimiento estructural importante para la actividad antituberculosa.

10. Los estudios de electroquímica han demostrado que la introducción de sustituyentes

electroatrayentes en el anillo de quinoxalina conducen a potenciales de reducción menos

negativos y facilitan la bio-reducción de los compuestos. La relación encontrada entre el

comportamiento electroquímico y la actividad antituberculosa de los compuestos corrobora

la hipótesis de que este tipo de derivados puedan ser activados por bio-reducción

generando especies más activas.

11. Se ha puesto a punto un método de cromatografía líquida de alta resolución en fase

reversa para la determinación experimental de los coeficientes de partición octanol-agua

para los derivados de quinoxalina dioxidados. No se ha podido establecer una relación

entre dichos valores y la actividad antituberculosa de los compuestos.

Conclusiones

225

12. En base a los prometedores resultados, se propone el compuesto 11g para su

profundización en estudios de actividad biológica y como compuesto lider para la

optimización de la estructura.

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APPENDIXAPPENDIXESES

Appendixes: 1. Relationship of synthesized compounds

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1. Relationship of synthesized compounds

1.1. Benzofuroxans

1.2. β-ketoamides

Comp. Name Structure

c 5-fluorobenzofuroxan

d 5-trifluoromethylbenzofuroxan

g 5,6-dichlorobenzofuroxan


2 N-benzyl-3-oxo-3-phenylpropanamide

3 3-oxo-N-(2-phenylethyl)-3-phenyl propanamide

4 3-oxo-N-(p-trifluoromethylbenzyl) butanamide

5 N-(p-chlorobenzyl)-3-oxobutanamide

6 N-(p-bromobenzyl)-3-oxobutanamide

7 N-(p-methylbenzyl)-3-oxobutanamide


- 250 -

1.3. Quinoxalines di-N-oxide


8 3-oxo-N-(3,4,5-trimethoxybenzyl) butanamide

9 N-(2,2-diphenylethyl)-3-oxobutanamide

10 N-(benzo[d][1,3]dioxo-5-ylmethyl)-3-oxobutanamide


1a N,3-diphenylquinoxaline-2-carboxamide 1,4-di-N-oxide

1b 7(6)-chloro-N,3-diphenylquinoxaline-2-carboxamide 1,4-di-N-oxide

1c 7(6)-fluoro-N,3-diphenylquinoxaline-2-carboxamide 1,4-di-N-oxide

1d 7(6)-trifluoromethyl-N,3-diphenyl

quinoxaline-2-carboxamide 1,4-di-N-oxide


- 251 -


1e 7(6)-methyl-N,3-diphenylquinoxaline-2-carboxamide 1,4-di-N-oxide

1f 7-methoxy-N,3-diphenylquinoxaline-2-carboxamide 1,4-di-N-oxide

1g 6,7-dichloro-N,3-diphenylquinoxaline-2-carboxamide 1,4-di-N-oxide

2a N-benzyl-3-phenylquinoxaline-2-carboxamide 1,4-di-N-oxide

2b N-benzyl-7(6)-chloro-3-phenyl


2c N-benzyl-7(6)-fluoro-3-phenyl


2d N-benzyl-7(6)-trifluoromethyl-3-phenyl quinoxaline-2-carboxamide 1,4-di-N-

oxide

2e N-benzyl-7(6)-methyl-3-phenyl


2f N-benzyl-6-methoxy-3-phenyl



- 252 -


2g N-benzyl-6,7-dichloro-3-phenyl


3a 3-phenyl-N-(2-phenylethyl)


3b 7(6)-chloro-3-phenyl-N-(2-phenylethyl) quinoxaline-2-carboxamide 1,4-di-N-oxide

4a 3-methyl-N-(p-trifluoromethyl

benzyl)quinoxaline-2-carboxamide 1,4-di-N-oxide

4b 7(6)-chloro-3-methyl-N-(p-trifluoro

methylbenzyl)quinoxaline-2-carboxamide 1,4-di-N-oxide

4c 7(6)-fluoro-3-methyl-N-(p-

trifluoromethylbenzyl)quinoxaline-2-carboxamide 1,4-di-N-oxide

4d 3-methyl-7-trifluoromethyl-N-(p-


4e 3,7(6)-dimethyl-N-(p-trifluoro methylbenzyl)quinoxaline-2-carboxamide 1,4-di-N-oxide

4f 7-methoxy-3-methyl-N-(p-trifluoro

methylbenzyl)quinoxaline-2-carboxamide 1,4-di-N-oxide


- 253 -


4g 6,7-dichloro-3-methyl-N-(p-


5a N-(p-chlorobenzyl)-3-methyl


5b 7(6)-chloro-N-(p-chlorobenzyl)-3-methylquinoxaline-2-carboxamide

1,4-di-N-oxide

5e N-(p-chlorobenzyl)-3,7(6)-

dimethylquinoxaline-2-carboxamide 1,4-di-N-oxide

5g 6,7-dichloro-N-(p-chlorobenzyl)-3-methylquinoxaline-2-carboxamide

1,4-di-N-oxide

6a N-(p-bromobenzyl)-3-methyl


6b N-(p-bromobenzyl)-7(6)-chloro-3-methylquinoxaline-2-carboxamide

1,4-di-N-oxide

6e N-(p-bromobenzyl)-3,7(6)-dimethyl quinoxaline-2-carboxamide 1,4-di-

N-oxide

6g N-(p-bromobenzyl)-6,7-dichloro-3-methylquinoxaline-2-carboxamide

1,4-di-N-oxide


- 254 -


7a 3-methyl-N-(p-methylbenzyl)


7b 7(6)-chloro-3-methyl-N-(p-methyl benzyl)quinoxaline-2-carboxamide

1,4-di-N-oxide

7e 3,7(6)-dimethyl-N-(p-methyl


7g 6,7-dichloro-3-methyl-N-(p-methyl benzyl)quinoxaline-2-carboxamide

1,4-di-N-oxide

8a 3-methyl-N-(3,4,5-trimethoxy


8b 7(6)-chloro-3-methyl-N-(3,4,5-

trimethoxy benzyl)quinoxaline-2-carboxamide 1,4-di-N-oxide

8e 3,7(6)-dimethyl-N-(3,4,5-trimethoxy benzyl)quinoxaline-2-carboxamide

1,4-di-N-oxide

8g 6,7-dichloro-3-methyl-N-(3,4,5-

trimethoxy benzyl)quinoxaline-2-carboxamide 1,4-di-N-oxide

9a N-(2,2-diphenylethyl)-3-methyl



- 255 -


9b 7(6)-chloro-N-(2,2-diphenylethyl)-3-methylquinoxaline-2-carboxamide

1,4-di-N-oxide

9e N-(2,2-diphenylethyl)-3,7(6)-

dimethylquinoxaline-2-carboxamide 1,4-di-N-oxide

9g 6,7-dichloro-N-(2,2-diphenylethyl)-3-methylquinoxaline-2-carboxamide

1,4-di-N-oxide

10a N-(benzo[1,3]dioxol-5-ylmethyl)-3-methylquinoxaline-2-carboxamide

1,4-di-N-oxide

10b N-(benzo[1,3]dioxol-5-ylmethyl)-

7(6)-chloro-3-methylquinoxaline-2-carboxamide 1,4-di-N-oxide

10e N-(benzo[1,3]dioxol-5-ylmethyl)-

3,7(6)-dimethylquinoxaline-2-carboxamide 1,4-di-N-oxide

10g N-(benzo[1,3]dioxol-5-ylmethyl)-

6,7-dichloro-3-methylquinoxaline-2-carboxamide 1,4-di-N-oxide

Appendixes: 2. Stability of quinoxalines di-N-oxide in aqueous solution

- 257 -

2. Stability of quinoxalines di-N-oxide in aqueous solution

The selected compounds (7a, 11a and 13a) were studied following the experimental

procedure considered in the experimental section. As expected, UV spectrums of

quinoxaline di-N-oxide derivatives changed during the experiments and, for this reason, the

UV observed data can not be considered to determine the logPo/w values. Moreover, the

UV spectrums changed quickly if the pH of the aqueous solution was modified as

recommended in literature.

With the aim of studying the degradation of quinoxaline di-N-oxide derivatives,

compound 7a was selected and its HPLC chromatograms were studied. As can be

observed in Figure A.1, quinoxalines present two different wavelengths with maximum

absorbance. It can be said that the absolute maximum is within the range of 250- 260 nm;

and two maximums can be often observed in this area (220-230 and 250-260 nm). Another

characteristic maximum appears close to 360 nm. This chromatogram was developed on an

Ultimate 3000 Chromatograph (DIONEX) with Chromeleon v.6.8 software. The

measurements were performed using an RP 18 column (LICHROSPHER 100 RP 18 E.C.

5 µm; 10x0.46; TEKNOKROMA) as the stationary phase, at a flow rate of 1 mL/min and

with methanol/water (60:40) as the mobile phase. Compound 7a was dissolved in

methanol.

Figure A.1. UV spectrum of compound 7a.

Once the UV spectrum of quinoxaline di-N-oxide derivatives was studied, UV

spectrums of compound 7a were studied, with a pH ranging from 1.0 to 11.0. As can be

observed in Figure A.2, fresh aqueous solution of compound 7a presents the characteristic

spectrum previously explained.

Appendixes: 2. Stability of quinoxalines di-N-oxide in aqueous solution

- 258 -

UV spectrums were studied on Spectrophotometer BIO RAD SMARSPEC TM PLUS

with wavelength from 400 to 200 nm.

Figure A.2. UV spectrum of fresh aqueous solution of compound 7a.

If the UV spectrum is studied 48 hours later, it can be seen that the quinoxaline has

been degraded as the maximums that appeared at nearly 220 and 260 nm have disappeared.

Moreover, the more extreme the pH is, the more the UV spectrum changes (Figure A.3).

Figure A.3. UV spectrum of compound 7a (pH ranging from 1 to 11).

In conclusion, no reliable results can be obtained with the shake-flask method.

Therefore, this method is not suitable for quinoxaline di-N-oxide derivatives. Nevertheless,

this fact does not affect the experimental RP-HPLC method because it is performed at

pH=7.4 and quinoxaline di-N-oxide derivatives are more stable in neutral solutions than at

extreme pH. Moreover, the period of time in which the compounds are in contact with the

mobile phase is not long enough to degrade quinoxalines as observed when studying the

corresponding chromatograms.

Appendixes: 3. Future perspectives

- 259 -

3. Future perspectives

The development of this project and a continuous bibliographical review has led to

the proposal of a new work plan which takes many aspects into consideration:

• Biological activity. Taking into account the interesting biological data shown by

compounds from Series 11 and 13, some of these derivatives, in special 11g and 13b,

should be considered for further in vitro evaluation. This screening should include the

determination of the efficacy in TB-infected macrophage model,1 MIC against single-

drug resistant and poli-drug strains and the evaluation of the activity against non-

replicating persistent M.tb. The Low Oxygen Recovery Assay could be used for this

purpose and would reveal the ability of these compounds to combat latent TB.2 This

evaluation should be developed with the aim of confirming the antimicrobial activity of

these compounds and their activity against drug resistant strains of M.tb. In case that

these compounds were promising, they should be considered for in vivo evaluation.

• Study of the mechanism of action. Quinoxalines di-N-oxide derivatives are assumed to

act by a bio-reduction activation as many others agents such as tirapazamine, PA-824,

metronidazole or OPC67683. For this reason and with the aim of determining the

mechanism of action of these compounds, first stages could consider the possibility of

activation by bio-reductive processes. For instance, one available assay is the study of

potential cross resistant with PA-824 which could show if quinoxaline di-N-oxide

derivatives and PA-824 share the bio-activation mechanism.

• New series for synthesis. Considering series 11 as the lead series, several modifications

are proposed (Figure A.4).

- Change 1. Substitution on position 3 with a trifluoromethyl moiety which is defined

as isostere of the methyl group in volume aspects but not in electronic terms. For

this reason, the insertion of this moiety could be of great interest as it could

facilitate the bio-reduction of the quinoxaline system.

- Change 2. Insertion of electron-withdrawing substituents on positions 5, 6, 7 and 8

of the quinoxaline ring with the aim of establishing their influence not only on the

anti-tuberculosis activity but also on the bio-reduction facility.

- Change 3. Substitution of the phenyl group with several heterocyclic systems that

have shown interesting properties as anti-tuberculosis agents.3-10 Once the most

active heterocyclic is defined, the corresponding �,�-unsaturated carbonile and


- 260 -

cyclopropile derivatives should be prepared as they have become of interest in this

field.11

Figure A.4. Scheme of the design of the future series.


- 261 -

References

1. Skinner, P. S.; Furney, S. K.; Jacobs, M. R.; Klopman, G.; Ellner, J. J.; Orme, I. M. “A bone marrow-derived murine macrophage model for evaluating efficacy of antimycobacterial drugs under relevant physiological conditions.” Antimicrobial Agents and Chemotherapy; 1994, 38, 2557-2563.

2. Cho, S. H.; Warit, S.; Wan, B. J.; Hwang, C. H.; Pauli, G. F.; Franzblau, S. G. “Low-oxygen-recovery assay for high-throughput screening of compounds against nonreplicating Mycobacterium tuberculosis.” Antimicrobial Agents and Chemotherapy; 2007, 51, 1380–1385.

3. Huang, Q.; Mao, J.; Wan, B.; Wang, Y.; Brun, R.; Franzblau, S. G.; Kozikowski, A. P. “Searching for new cures for tuberculosis: design, synthesis and biological evaluation of 2-methylbenzothiazoles.” Journal of Medicinal Chemistry; 2009, 52, 6757-6767.

4. Mao, J.; Yuan, H.; Wang, Y.; Wan, B.; Pieroni, M.; Huang, Q.; van Breemen, R. B.; Kozikowski, A. P.; Franzblau, S. G. “From serendipity to rational antituberculosis drugs discovery of mefloquine-isoxazole carboxylic acid esters.” Journal of Medicinal Chemistry; 2009, 52, 6966-6978.

5. Lilienkampf, A.; Pieroni, M.; Wan, B.; Wang, Y.; Franzblau, S. G.; Kozikowski, A. P. “Rational design of 5-phenyl-3-isoxazolecarboxylic acid ethyl esters as growth inhibitors of Mycobacterium tuberculosis. A potent and selective series for further drug development.” Journal of Medicinal Chemistry; 2010, 53, 678-688.

6. Carvalho, L. P. S.; Lin, G.; Jiang, X.; Nathan, C. “Nitazoxanide kills replicating and nonreplicating Mycobacterium tuberculosis and evades resistance.”Journal of Medicinal Chemistry; 2009, 52, 5789-5792.

7. Saleh, M.; Abbott, S.; Perrot, V.; Lauzon, C.; Penney, C.; Zacharie, B. “Synthesis and antimicrobial actiivty of 2-fluorophenyl-4,6-disubstituted [1,3,5]triazines.”Bioorganic & Medicinal Chemistry; 2010, 20, 945-949.

8. Sunduru, N.; Gupta, L.; Chaturvedi, V.; Dwivedi, R.; Sinha, S.; Chauhan, P. M. S. “Discovery of new 1,3,5-triazine scaffolds with potent activity against Mycobacterium tuberculosis H37Rv.” European Journal of Medicinal Chemistry; 2010, 45, 3335-3345.

9. Zampieri, D.; Mamolo, M. G.; Laurini, E.; Fermeglia, M.; Posocco, P: Pricl, S.; Banfi, E; Scialino, G.; Vio, L. “Antimycobacteial activity of new 3,5-disubstituted 1,3,4-oxadiazol-2(3H)-one derivatives. Molecular modeling investigations.” Bioorganic & Medicinal Chemistry; 2009, 17, 4693-4707.

10. Guo, S.; Song, Y.; Huang, Q.; Yuan, H.; Wan, B.; Wang, Y.; He, R.; Beconi, M. G.; Franzblau, S. G.; Kozikowski, A. P. “Identification, synthesis, and pharmacological evaluation of tetrahydroindazole based ligands as novel antituberculosis agents.”Journal of Medicinal Chemistry; 2010, 53, 649.

11. Ajay, A.; Singh, V.; Singh, S.; Pandey, S.; Gunjan, S.; Dubey, D.; Sinha, S. K.; Singh, B. N.; Chatuvedi, V.; Tripathi, R.; Ramchandran, R.; Tripathi, R. P. “Synthesis and bio-evaluation of alkylamionaryl cyclopropyl methanones as antitubercular and antimalarial agents.”Bioorganic & Medicinal Chemistry; 2010, 18, 8289-8301.

lable at ScienceDirect

European Journal of Medicinal Chemistry 45 (2010) 4418e4426

Contents lists avai

European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

Synthesis and antimycobacterial activity of new quinoxaline-2-carboxamide1,4-di-N-oxide derivatives

Elsa Moreno, Saioa Ancizu, Silvia Pérez-Silanes*, Enrique Torres, Ignacio Aldana, Antonio MongeUnidad de Investigación y Desarrollo de Medicamentos, Centro de Investigación en Farmacobiología Aplicada (CIFA), University of Navarra, C/ Irunlarrea s/n, 31008 Pamplona, Spain

a r t i c l e i n f o

Article history:Received 15 March 2010Received in revised form25 June 2010Accepted 26 June 2010Available online 3 July 2010

Keywords:QuinoxalineN-oxidesAnti-tuberculosis agentsMycobacterium tuberculosis

* Corresponding author. Tel.: þ34 948 425653; faxE-mail address: [email protected] (S. Pérez-Silanes).

0223-5234/$ e see front matter � 2010 Elsevier Masdoi:10.1016/j.ejmech.2010.06.036

a b s t r a c t

As a continuation of our research and with the aim of obtaining new anti-tuberculosis agents which canimprove the current chemotherapeutic anti-tuberculosis treatments, forty-three new quinoxaline-2-carboxamide 1,4-di-N-oxide derivatives were synthesized and evaluated for in vitro anti-tuberculosisactivity against Mycobacterium tuberculosis strain H37Rv. Active compounds were also screened to assesstoxicity to a VERO cell line. Results indicate that compounds with a methyl moiety substituted in position3 and unsubstituted benzyl substituted on the carboxamide group provide an efficient approach forfurther development of anti-tuberculosis agents.

� 2010 Elsevier Masson SAS. All rights reserved.

1. Introduction

Tuberculosis (TB) is an infectious bacterial disease caused byMycobacterium Tuberculosis (M.Tbc). The report published by WHOin 2009 established that there were an estimated 9.27 millionincident cases of TB in 2007. This means an increase from the 9.24million cases in 2006, the 8.3 million cases in 2000 and the 6.6million cases in 1990. Although the total number of incident casesof TB is increasing, it must be said that the number of cases percapita is slowly decreasing [1]. Nevertheless, the continuingemergence of multidrug-resistant strains of M. tuberculosis (MDR-TB) will inevitably make it more difficult in the future to control TB.

The global epidemiology of drug-resistant TB, particularlyextremely drug-resistant TB (XDR-TB), is unknown and the truemagnitude of the problem is probably quite underestimated. MDR-TB, which is defined as TB caused by organisms that are resistant toisoniazid and rifampicin, continues to threaten the progress beingmade in controlling the disease. The emergence of XDR-TB, definedas a less common form of MDR-TB that is resistant not only toisoniazid and rifampicin but also to any one of the fluoroquinolonesand to at least one of the second-line drugs (amikacin, capreomycinor kanamycin), has heightened this threat [2]. The recent influx ofimmigrants from countries endemic for disease and co-infectionwith human immunodeficiency virus (HIV) turns TB into a serious

: þ34 948 425652.

son SAS. All rights reserved.

problem in developed countries [3,4]. The development of HIV co-infection with MDR-TB and XDR-TB highlights the urgent need fornew drugs to extend the range of effective TB treatment options.

Quinoxaline derivatives are a class of compounds that show veryinteresting biological properties and the interest in thesecompounds is growing within the field of medicinal chemistry.Quinoxaline-1,4-di-N-oxide derivatives even improve the biolog-ical results shown by their reduced analogues and are endowedwith antiviral, anticancer, antibacterial and antiprotozoal activities[5e9]. There are many publications regarding 1,4-di-N-oxidederivatives, and more specifically alkyl and arylcarboxamidederivatives, in which their antibacterial and antimicrobial activities[10e14] have been reported or their capability to act as antitumoralagents [15,16] has been clearly demonstrated, thereby reflecting thegrowing interest in these structures over the past forty years.

As a result of the anti-tuberculosis research project, our grouphas published several papers reporting a wide range of quinoxa-line-1,4-di-N-oxide derivatives (Fig. 1) including a great variety ofsubstituents in positions 2, 3, 6 and 7. With regard to position 2,carbonitrile derivatives appeared to be quite toxic [17e21]. More-over, ketone, carboxylate and carboxamide quinoxaline-1,4-diox-ydes derivatives were actually patented in the 70s for theirantibacterial activity [12e15].

These studies have facilitated a wide structure-activity rela-tionship (SAR) analysis which lead us to design a group of thirty-six3-methylquinoxaline-2-carboxamide 1,4-di-N-oxide derivativesthat were prepared and tested against M.Tbc and to justify thedesign of the compounds presented in this paper [22].

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http://dx.doi.org/10.1016/j.ejmech.2010.06.036



N+

N+R7

R6O-

R3

R2O-

Fig. 1. General structure and numeration of quinoxaline-1,4-di-N-oxide.

E. Moreno et al. / European Journal of Medicinal Chemistry 45 (2010) 4418e4426 4419

Continuing with the anti-tuberculosis project and in an attemptto establish the structural requirements necessary for the devel-opment of anti-tuberculosis drugs, nine series of quinoxaline-2-carboxamide 1,4-di-N-oxide derivatives were proposed (Fig. 2).Several structural modifications were designed and can besummarized as follows: a) variation in the length of the aliphaticlinker between the carboxamide group and the aromatic ring; b)modification of the substituent in position 3 by a phenyl (Series1e3) and a methyl moiety (Series 4e9); c) substitution of a varietyof aromatic rings (Series 4e9).

2. Chemistry

Forty-three new 1,4-di-N-oxide-quinoxaline-2-carboxylic acidaryl amide derivatives were prepared according to the syntheticprocess illustrated in Scheme 1:

The synthesis of the new 1,4-di-N-oxide-quinoxaline derivatives(Series 1e9) was carried out by a variation of the Beirut reaction[23], where the appropriate benzofuroxanes (BFX) react with thecorresponding b-ketoamide in the presence of calcium chloride andethanolamine as catalysts [22,24].

The starting compounds, BFX, were obtained by previouslydescribed methods [18,25]. Compound 1 was commercially avail-able whereas the rest of the b-ketoamides were synthesized asfollows: compound 2 was synthesized by Passerini reactionbetween the appropriate glyoxal and isocyanide [26]; compound 3was synthesized by condensation of the corresponding ester andthe appropriate aryl amine [27]. Compounds 4e9 were obtainedthrough the acetoacetylation of corresponding aryl amines bydiketene [22,28].

Quinoxaline derivatives were unsubstituted or substituted inpositions 6 and 7 by chloro, fluoro or trifluoromethyl moiety aselectron-withdrawing groups and by methyl or methoxy moiety aselectron-releasing groups. When quinoxalines were prepared frommonosubstituted-BFX, the formation of isomeric quinoxalines 1,4-di-N-oxide was observed. In most cases, the 7-substituted isomerprevailed over 6-substituted isomer, and when the methoxysubstituted quinoxalines were prepared, only the 7-isomer wasobtained, as previously described [29,30].

3. Pharmacology

In vitro evaluation of the anti-tuberculosis activity was carriedout within the Tuberculosis Antimicrobial Acquisition &

N+

N+R7

R6O-

R3

O-

NH

O

linkeraromaticring

Fig. 2. Design of the new series of quinoxaline-2-carboxamide 1,4-di-N-oxide.

Coordinating Facility (TAACF) screening program for the discoveryof novel drugs for the treatment of tuberculosis [31]. The SouthernResearch Institute coordinates the overall program under thedirection of the U.S. National Institute of Allergy and InfectiousDisease (NIAID).

The purpose of the screening program is to provide a resourcewhereby new experimental compounds can be tested for theirability to inhibit the growth of virulent M. tuberculosis (M.Tbc.).Biological tests have been performed according to previouslydescribed methods [32].

4. Results and discussion

Structure and biological values of new synthesized quinoxaline-1,4-di-N-oxide derivatives are reported in Table 1. Compounds areassayed against M.Tbc. H37Rv in order to determine the IC90.Compounds showing values of �10 mg/mL are considered as activeand move on to the secondary screening. Cytotoxicity is assayed inVERO cells and the CC50 is determined from the doseeresponsecurve. Next, the IC90 and CC50 values are formed into a ratio termedSelectivity Index (SI). Compounds showing a SI� 10 are consideredactive for anti-tubercular activity.

As can be observed in Table 1, thirteen of the forty-three eval-uated compounds passed the cut off established by the TAACF at theprimary screening level and moved on to the secondary screeninglevel. Compounds 2b and 4dwere identified as themost interestingwith a SI higher than 10.

Some structureeactivity relationships were established. Look-ing at the values of compounds 1b, 1g, 2b, 4b, 4c, 4d, 4g, 6b, 6g, 7band 9b, it can be said that the insertion of a halogen moiety,increases the anti-tubercular activity. Taking into account the bio-logical values reported in Table 1, it can be concluded that theinsertion of a electron-withdrawing moiety, especially that ofchlorine atom, is an essential requirement for the anti-tubercularactivity, as previously established by our group [21,22].

With the aim of corroborating previous preliminary structur-eeactivity relationship observed by our group and identifying themost suitable length for the aliphatic chain between the carbox-amide group and the aromatic ring, three series of compounds(Series 1, 2 and 3) were prepared. Comparing the biological valuesshown by these compounds, it can be said that the preferred lengthfor the aliphatic chain is one methylene group. This data corrobo-rates the hypothesis established in a previous report on analoguestructures published by our group [22].

In previous investigations carried out by our group, three seriesof 1,4-di-N-oxide-quinoxaline-2-carboxylic acid aryl amide deriv-atives were synthesized, containing a methyl moiety in position 3[21,22]. To further explore the SAR of these types of compounds,a phenyl group was substituted in position 3 of the quinoxaline ring(Series 1, 2 and 3) and reported in this paper. This modification ledto a reduction of the anti-tubercular activity as can be observed bycomparing the biological values of compounds from Series 1, 2 and3 with their analogues containing a methyl group in position 3,described in previous reports [21,22].

Taking into account the biological values of the structures whichpresent a phenyl group substituted in position 3 and thecompoundswith amethyl group in this position, [22] we decided tokeep the methyl moiety in position 3 and modify the substitutionon the aromatic ring. Different substituents were introduced onpara position of the phenyl ring considering chloro, bromo or tri-fluoromethyl moiety as electron-withdrawing groups and methylas electron-releasing group (Series 4, 5, 6, 7). In this fragment of thestructure other substituents as byphenyl or a benzodioxol havebeen considered (Series 8, 9). Taking into account the biologicalvalues showed by these derivatives, it can be said that the insertion

NON+O-

R6

R7

BFX

O

NH

O O

NH

O

O

NH

O O

NH

O

R

iv iv

iviv

2

34-9

N+

N+R7

R6O-

NH

OO-

N+

N+R7

R6O-

NH

OO-

N+

N+R7

R6O-

NH

OO-

N+

N+R7

R6O-

NH

OO-

R

Series 3

Series 1 Series 2

Series 4-9

OO

+

H2N R

iii

OH

O

NC

+iaib

NH2

O

O

+ii

1

Scheme 1. General scheme of synthesis. Reagents and conditions: ia) acetic acid, diethyl ether, rt.; ib) methanol, Zn/NH4Cl aq.; ii) 2-hydroxypyridine, reflux; iii) methanol, 0 �C, N2

atmosphere; iv) methanol, CaCl2, ethanolamine.

E. Moreno et al. / European Journal of Medicinal Chemistry 45 (2010) 4418e44264420

of a substituent on para position of the phenyl group did notimprove the anti-tubercular activity suggesting that the mostsuitable aromatic ring is the unsubstituted phenyl.

5. Conclusions

Forty-three new 1,4-di-N-oxide-quinoxaline-2-carboxylic acidaryl amide derivatives were synthesized using a variation of theBeirut reaction. All of the compoundswere evaluated againstM.Tbc.H37Rv stain; thirteenwere active in the primary screening, showingan IC90�10 mg/mL, and were then moved on to the secondaryscreening level. Two of the compounds were active at this level,showing a SI� 10.

Taking into account the biological values obtained, it can be saidthat the lead general structure for developing new anti-tubercularagents should consider the 1,4-di-N-oxide-quinoxaline ring witha carboxamide functionalized on position 2 and a methyl moiety onposition 3. The most suitable substituent on positions 6 or/and 7should be an electron-withdrawing group and a methyl moiety onposition 3. With regard to the linker and the aromatic ring attachedto it, one methylene group and an unsubstituted phenyl ring areconsidered to be the most appropriate substituents.

6. Experimental protocols

6.1. Chemistry

6.1.1. General remarksAll of the synthesized compounds were chemically character-

ized by thin layer chromatography (TLC), infrared (IR), proton

nuclear magnetic resonance (1H NMR) and elemental microanal-yses (CHN).

Alugram SIL G/UV254 (Layer: 0.2 mm) (Macherey-Nagel GmbH& Co. KG., Düren, Germany) was used for TLC, and Silica gel 60(0.040e0.063 mm,Merck) was used for Conventional Flash ColumnChromatography. Flash Column Chromatographywas developed ona CombiFlash� Rf (TELEDYNE ISCO, Lincoln, USA) instrument withSilica RediSep� Rf columns. The 1H NMR spectra were recorded ona Bruker 400 Ultrashield instrument (400 MHz), using TMS asinternal standard and with DMSO-d6 as solvent; the chemical shiftsare reported in ppm (d) and coupling constant (J) values are given inHertz (Hz). Signal multiplicities are represented by: s (singlet), bs(broad singlet), d (doublet), dd (double doublet), ddd (doubledouble doublet), t (triplet), tt (triple triplet) and m (multiplet). TheIR spectra were recorded on a Nicolet Nexus FTIR (Thermo, Madi-son, USA) in KBr pellets. Elemental microanalyses were obtained ona CHN-900 Elemental Analyzer (Leco, Tres Cantos, Spain) fromvacuum-dried samples. The analytical results for C, H and N, werewithin �0.4 of the theoretical values. Chemicals were purchasedfrom Panreac Química S.A. (Barcelona, Spain), SigmaeAldrich Quí-mica, S.A. (Alcobendas, Spain), Acros Organics (Janssen Pharma-ceuticalaan, Geel, Belgium) and Lancaster (Bischheim-Strasbourg,France).

6.1.2. General procedure of the synthesis of 3-oxo-N-benzyl-3-phenylpropanamide (2)

Acetic acid (5.0 mmol) and phenylglyoxal were diluted indiethyl ether (25 mL) under N2 atmosphere. Once dissolved, thebenzylisocyanide (5.0 mmol) was added dropwise and the reactionmixture was stirred at room temperature for 72 h. The residue

Table 1Anti-tubercular activity of compounds (Series 1e9).

N+

N+R7

R6O-

R3

O- O

NHR

R7/R6

H/H a Cl/H b F/H c CF3/H d CH3/H e OCH3/H f Cl/Cl g

Anti-tubercular activity Cytotoxicity

Comp. R3 R IC90a H37Rv CC50

b VERO SIc CC50/MIC

1a C6H5 C6H5 26.99 N.T.d N.T.1b 6.71 8.97 1.341c 17.93 N.T. N.T.1d 19.10 N.T. N.T.1e 24.65 N.T. N.T.1f 26.84 N.T. N.T.1g 6.63 5.541 0.84

2a C6H5 CH2eC6H5 6.71 >40 >5.962b 3.39 >40 >11.792c 3.86 17.86 4.622d >100 N.T. N.T.2e 13.91 N.T. N.T.2f 14.58 N.T. N.T.2g 25.45 N.T. N.T.

3a C6H5 CH2eCH2eC6H5 18.61 N.T. N.T.3b 15.42 N.T. N.T.

4a CH3 CH2eC6H5e4-CF3 16.81 N.T. N.T.4b 6.13 >40 >6.524c 4.48 >40 >8.944d 3.38 >40 >11.824e >100 N.T. N.T.4f >100 N.T. N.T.4g 6.58 >40 >6.08

5a CH3 CH2eC6H5e4-Cl 11.04 N.T. N.T.5b 29.68 N.T. N.T.5e 14.56 N.T. N.T.5g 51.86 N.T. N.T.

6a CH3 CH2eC6H5e4-Br 15.61 N.T. N.T.6b 5.33 >40 >7.506e 78.22 N.T. N.T.6g 6.92 >40 >5.78

7a CH3 CH2eC6H5e4-CH3 6.76 >40 >5.927b 32.04 N.T. N.T.7e 99.91 N.T. N.T.7g >100 N.T. N.T.

8a CH3 CH2eCHe(C6H5)2 15.99 N.T. N.T.8b 60.43 N.T. N.T.8e 16.79 N.T. N.T.8g 66.54 N.T. N.T.

9a CH3 CH2-benzo[d][1,3]dioxol 22.75 N.T. N.T.9b 6.99 >40 >5.729e 13.22 N.T. N.T.9g 34.92 N.T. N.T.

RIFe 0.015e0.125 >100 >800

a IC90 against M.tb H37Rv.b Cytotoxicity in VERO cells.c Selectivity index.d Not tested.e Rifampin.


obtained was filtered and washed with isopropanol. The solid wasdissolved in methanol (32.0 mL) and added dropwise to a solutionof Zn dust (8.0 mmol) in saturated aqueous NH4Cl (8.0 mL) previ-ously activated in a sonication bath for 5 min. The mixture was

stirred at room temperature for 30 min and filtered in order toeliminate the Zn. Water (100 mL) was added to the mixture and thesolid obtained was filtered and washed with water. The solid wasused without further purification [26].


6.1.2.1. 3-oxo-N-benzyl-3-phenylpropanamide (2). Yield: 25%. IR(KBr): 3290 (m, nNeH); 3060 (w, narCeH); 1687 (s, nC]O ketone); 1635(s, nC]O amide). 1H NMR (400 MHz, DMSO-d6) d ppm: 8.66 (bs, 1H,NH); 7.99 (d, 2H, H2þH6-phCO, J2e30 ¼7.2 Hz); 7.66e7.52 (m, 3H,H3eH5-phCO); 7.37e7.25 (m, 5H, H2eH6-phCH2); 4.32 (d, 2H,CH2eNH, JCH2eNH¼ 5.9 Hz); 3.92 (s, 2H, COeCH2eCO).

6.1.3. General procedure of the synthesis of 3-oxo-N-(2-phenylethyl)-3-phenyl propanamide (3)

Ethyl benzoylacetate (6.0 mmol), 2-phenethylamine(15.0 mmol) and 2-hydroxypyridine (6.0 mmol) were refluxed at130 �C under N2 atmosphere for 48 h. The mixture reaction wasdissolved in dichloromethane and quenched with water. Theorganic phase was dried with anhydrous sodium sulphate andfiltered. The solvent was removed in vacuo and precipitated withcold isopropanol in order to obtain awhite solid. The solid was usedwithout further purification. [27]

6.1.3.1. 3-oxo-N-(2-phenylethyl)-3-phenylpropanamide (3). Yield:62%. IR (KBr): 3336 (s, nNeH); 3032 (w, narCeH); 1614 (s, nC]O). 1H NMR(400MHz, DMSO-d6) d ppm: 9.01 (bs, 1H, NH); 7.43e7.35 (m, 2H,H2þH6-phCO); 7.30e7.25 (m, 2H, H3þH5-phCO); 7.24e7.14 (m, 5H, H4-

phCOþH2þ3þ5þ6-phCH2); 7.05e7.03 (m, 1H, H4-phCH2); 3.82 (s, 2H,COeCH2eCO);3.30e3.21(m,2H,CH2eNH);3.08e2.67(m,2H,CH2-ph).

6.1.4. General procedureof the synthesis of 3-oxobutanamidederivatives(4e9)

The corresponding aryl amines (20.0 mmol) were diluted inmethanol (10 mL) under N2 atmosphere and cooled in an ice bathuntil 0 �C. Next, diketene (25.0 mmol) was added dropwise and thereactionwas stirred for 1e3 h. The residue obtained was precipitatedwith cold diethyl ether and filtered in order to obtain a redebrownsolid. The compound was used without further purification [28].

6.1.4.1. 3-oxo-N-(p-(trifluoromethyl)benzyl)butanamide (4). Yield:84%. IR (KBr): 3259 (m, nNeH); 3090 (w, narCeH); 1716 (s, nC]O

ketone); 1644 (s, nC]O amide); 1106 (m, narCeCF3); 1111 (s, narCeCF3);1069 (m, narCeCF3). 1H NMR (400 MHz, DMSO-d6) d ppm: 8.64 (t, 1H,NH, JNHeCH2¼ 5.5 Hz); 7.70 (d, 2H, H30 þH50, J30e20 ¼ 7.9 Hz); 7.50 (d,2H, H20 þH60); 4.38 (d, 2H, CH2eNH); 3.41 (s, 2H, COeCH2eCO);2.16 (s, 3H, CH3eCO).

6.1.4.2. 3-oxo-N-(p-chlorobenzyl)butanamide (5). Yield: 59%. IR(KBr): 3253 (s, nNeH); 3085 (m, narCeH); 1714 (s, nC]O ketone); 1642(s, nC]O amide). 1H NMR (400 MHz, DMSO-d6) d ppm: 8.55 (bs, 1H,NH); 7.39 (dd, 2H, H30 þH50, J30e20 ¼ 8.5 Hz, J30eCl¼ 1.9 Hz); 7.30 (d,2H, H20 þH60); 4.28 (d, 2H, CH2eNH, JCH2eNH¼ 5.9 Hz); 3.38 (s, 2H,COeCH2eCO); 2.15 (s, 3H, CH3eCO).

6.1.4.3. 3-oxo-N-(p-bromobenzyl)butanamide (6). Yield: 57%. IR(KBr): 3253 (s, nNeH); 3085 (m, narCeH); 1714 (s, nC]O ketone); 1642(s, nC]O amide); 1015 (m, narCeBr). 1H NMR (400 MHz, DMSO-d6)d ppm: 8.55 (bs, 1H, NH); 7.52 (d, 2H, H30 þH50, J30e20 ¼ 8.2 Hz); 7.24(d, 2H, H20 þH60); 4.26 (d, 2H, CH2eNH, JCH2eNH¼ 5.5 Hz); 3.38 (s,2H, COeCH2eCO); 2.15 (s, 3H, CH3eCO).

6.1.4.4. 3-oxo-N-(p-methylbenzyl)butanamide (7). Yield: 40%. IR(KBr): 3254 (m, nNeH); 3088 (m, narCeH); 1715 (m, nC]O ketone); 1641(s, nC]O amide). 1H NMR (400 MHz, DMSO-d6) d ppm: 8.47 (t, 1H, NH,JNHeCH2¼ 5.8 Hz); 7.20e7.10 (m, 4H, H20 þH30 þH50 þH60); 4.24 (d,2H, CH2eNH); 3.36 (s, 2H, COeCH2eCO); 2.28 (s, 3H, CH3-ph); 2.15(s, 3H, CH3-CO).

6.1.4.5. N-(2,2-diphenylethyl)-3-oxobutanamide (8). Yield: 32%. IR(KBr): 3276 (s, nNeH); 3020 (w, narCeH); 1710 (m, nC]O ketone); 1668

(s, nC]O amide). 1H NMR (400 MHz, DMSO-d6) d ppm: 8.14 (t, 1H, NH,JNHeCH2¼ 5.6 Hz); 7.34e7.24 (m, 10H, 2ph); 4.19 (t, 1H, CH,JCHeCH2¼7.9 Hz); 3.73 (dd, 2H, CH2eNH); 3.19 (s, 2H,COeCH2eCO); 1.96 (s, 3H, CH3eCO).

6.1.4.6. N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-oxobutanamide(9). Yield: 33%. IR (KBr): 3290 (m, nNeH); 3064 (w, narCeH); 1758 (m,nC]O ketone); 1615 (s, nC]O amide); 1241 (m, nC]O). 1H NMR(400 MHz, DMSO-d6) d ppm: 8.50 (t, 1H, NH, JNHeCH2¼ 5.6 Hz); 7.02(bs,1H,H20); 6.91 (bs, 2H,H50 þH60); 6.01 (s, 2H, OeCH2eO); 4.27 (d,2H, CH2eNH); 3.37 (s, 2H, COeCH2eCO); 2.15 (s, 3H, CH3eCO).

6.1.5. General procedure of the synthesis of 1,4-di-N-oxide-quinoxaline-2-carboxylic acid aryl amide derivatives (Series 1e9)

The appropriate BFX (1.0 mmol) and the corresponding b-ketoamide (1.2 mmol) were dissolved in a minimum amount ofmethanol. Next, calcium chloride (0.1 mmol) and ethanolamine (5drops) were added as catalysts [22,23]. The mixture reaction wasstirred at room temperature from 1 to 48 h, depending on the BFXsubstituents used; it was then filtered andwashedwith cold diethylether. The solid was dissolved in dichloromethane and quenchedwith water. The organic phase was dried with anhydrous sodiumsulphate and filtered. The solvent was removed in vacuo andprecipitated with cold diethyl ether in order to obtain a yellowsolid. The solid was purified by column chromatography, ifnecessary.

6.1.5.1. 3-phenylquinoxaline-2-carboxylic acid phenylamide 1,4-di-N-oxide (1a). Yield: 17%. IR (KBr): 3256 (w, nNH); 3077 (w, narCeH);1693 (s, nC]O); 1348 (s, nNþOe). 1H NMR (400 MHz, DMSO-d6)d ppm: 10.80 (s, 1H, NH); 8.60e8.56 (m, 2H,H5þH8); 8.10e8.07 (m,2H,H6þH7); 7.62e7.60 (m, 2H, H2þH6eph-QX); 7.49e7.47 (m; 3H,H3eH5eph-QX); 7.39 (dd, 2H, H2þH6eph-NH, J2e3¼ 8.5 Hz,J2e4¼1.0 Hz); 7.33e7.28 (m, 2H, H3þH5eph-NH); 7.10 (tt, 1H,H4eph-NH, J4e3¼7.2 Hz). Anal. Calcd. for C21H15N3O3: C, 70.58%; H,4.23%; N, 11.76%. Found: C, 70.72%; H, 4.48%; N, 11.99%.

6.1.5.2. 7-chloro-3-phenylquinoxaline-2-carboxylic acid phenyl-amide 1,4-di-N-oxide (1b). Yield: 18%. IR (KBr): 3256 (w, nNH); 3058(w, narCeH); 1686 (s, nC]O); 1330 (s, nNþOe). 1H NMR (400 MHz,DMSO-d6) d ppm: 10.82 (s, 1H, NH); 8.57e8.56 (m; 2H, H5þH8);8.11 (dd, 1H, H6, J6e5¼ 9.0 Hz, J6e8¼ 1.7 Hz); 7.61e7.59 (m, 2H,H2þH6eph-QX); 7.49e7.48 (m, 3H, H3eH5eph-QX); 7.38 (d, 2H,H2þH6eph-NH, J2e3¼7.9 Hz); 7.31 (t, 2H, H3þH5eph-NH,J3e4¼7.9 Hz); 7.11 (t, 1H, H4eph-NH). Anal. Calcd. for C21H14ClN3O3:C, 64.38%; H, 3.60%; N,10.72%. Found: C, 64.30%; H, 3.96%; N,10.48%.

6.1.5.3. 7-fluoro-3-phenylquinoxaline-2-carboxylic acid phenylamide1,4-di-N-oxide (1c). Yield: 17%. IR (KBr): 3244 (m, nNH); 3058 (w,narCeH); 1658 (s, nC]O); 1339 (s, nNþOe). 1H NMR (400 MHz, DMSO-d6) d ppm: 10.83 (s, 1H, NH); 8.66e8.62 (m, 1H, H5); 8.34e8.32 (m,1H, H8); 8.02e7.98 (m, 1H, H6); 7.60e7.59 (m, 2H, H2þH6eph-QX);7.49e7.47 (m, 3H, H3-H5eph-QX); 7.39e7.37 (m, 2H,H2þH6eph-NH); 7.32e7.28 (m, 2H, H3þH5eph-NH); 7.12e7.09 (m,1H,H4eph-NH). Anal. Calcd. for C21H14FN3O3: C, 67.20%; H, 3.76%; N,11.20%. Found: C, 66.85%; H, 3.95%; N, 11.00%.

6.1.5.4. 7-trifluoromethyl-3-phenylquinoxaline-2-carboxylic acidphenylamide 1,4-di-N-oxide (1d). Yield: 18%. IR (KBr): 3250 (w,nNH); 3058 (w, narCeH); 1686 (s, nC]O); 1345 (s, nNþOe); 1140 (s,narCeCF3). 1H NMR (400 MHz, DMSO-d6) d ppm: 10.82 (s, 1H, NH);8.83 (s, 1H, H8); 8.77 (d, 1H, H5, J5e6¼ 9.0 Hz); 8.39 (dd, 1H, H6,J6e8¼ 1.6 Hz); 7.64e7.62 (m, 2H,H2þH6eph-QX); 7.51e7.49 (m, 3H,H3eH5eph-QX); 7.39e7.37 (m, 2H, H2þH6eph-NH); 7.33e7.29 (m,2H, H3þH5eph-NH); 7.13e7.10 (m, 1H, H4eph-NH). Anal. Calcd. for


C22H14F3N3O3: C, 62.12%; H, 3.32%; N, 9.88%. Found: C, 61.83%; H,3.37%; N, 9.58%.

6.1.5.5. 7-methyl-3-phenylquinoxaline-2-carboxylic acid phenyl-amide 1,4-di-N-oxide (1e). Yield: 9%. IR (KBr): 3256 (w, nNH); 3064(w, narCeH); 1686 (s, nC]O); 1335 (s, nNþOe). 1H NMR (400 MHz,DMSO-d6) d ppm: 10.80 (s, 1H, NH); 8.47 (d, 1H, H5, J5e6¼ 8.8 Hz);8.41e8.35 (m,1H,H8); 7.91 (dd,1H,H6, J6e8¼ 1.2 Hz); 7.63e7.58 (m,2H, H2þH6eph-QX); 7.51e7.44 (m, 3H, H3eH5eph-QX); 7.41e7.36(m, 2H, H2þH6eph-NH); 7.33e7.27 (m, 2H, H3þH5eph-NH);7.13e7.07 (m, 1H, H4-ph-NH); 2.65 (s, 3H, CH3). Anal. Calcd. forC22H17N3O3: C, 71.15%; H, 4.61%; N, 11.31%. Found: C, 71.05%; H,4.80%; N, 11.01%.

6.1.5.6. 7-methoxy-3-phenylquinoxaline-2-carboxylic acid phenyl-amide 1,4-di-N-oxide (1f). Yield: 77%. IR (KBr): 3250 (m, nNH); 3077(m, narCeH); 1685 (s, nC]O); 1335 (s, nNþOe); 1243 (s, nCeOeC). 1HNMR (400 MHz, DMSO-d6) d ppm: 10.81 (s, 1H, NH); 8.48 (d,1H,H5,J5e6¼ 9.5 Hz); 7.87 (d, 1H, H8, J8e6¼ 2.7 Hz); 7.68 (dd, 1H, H6);7.61e7.58 (m, 2H, H2þH6-ph-QX); 7.48e7.46 (m, 3H, H3eH5-ph-QX); 7.38 (dd, 2H, H2þH6-ph-NH, J2e3¼ 8.5 Hz, J2e4¼1.1 Hz);7.32e7.28 (m, 2H, H3þH5-ph-NH); 7.10 (tt, 1H, H4-ph-NH,J4e3¼7.3 Hz); 4.04 (s, 3H, OCH3). Anal. Calcd. for C22H17N3O4: C,68.21%; H, 4.42%; N, 10.85%. Found: C, 67.89%; H, 4.42%; N, 10.89%.

6.1.5.7. 6,7-dichloro-3-phenylquinoxaline-2-carboxylic acid phenyl-amide 1,4-di-N-oxide (1g). Yield: 46%. IR (KBr): 3308 (m,nNH);3071 (m,narCeH); 1666 (s, nC]O); 1332 (s, nNþOe); 1313 (s, nNþOe).1H NMR (400 MHz, DMSO-d6) d ppm: 10.88 (s, 1H, NH); 8.76 (s,1H, H5); 8.74 (s, 1H, H8); 7.61e7.59 (m, 2H, H2þH6-ph-QX);7.51e7.48 (m, 3H, H3eH5-ph-QX); 7.38 (dd, 2H, H2þH6-ph-NH,J2e3¼ 8.5 Hz, J2e4¼1.0 Hz); 7.33e7.29 (m, 2H, H3þH5-ph-NH);7.11 (tt, 1H, H4-ph-NH, J4e3¼7.8 Hz). Anal. Calcd. forC21H13Cl2N3O3.1/2H2O: C, 57.89%; H, 3.22%; N, 9.65%. Found: C,57.56%; H, 2.91%; N, 9.54%.

6.1.5.8. 3-phenylquinoxaline-2-carboxylic acid benzylamide 1,4-di-N-oxide (2a). Yield: 15%. IR (KBr): 3302 (m,nNH); 3085 (w, narCeH);1673 (s, nC]O); 1348 (s, nNþOe); 1339 (s, nNþOe). 1H NMR (400 MHz,DMSO-d6) d ppm: 9.12 (t, 1H, NH, JNHeCH2¼ 5.8 Hz); 8.58e8.53 (m,2H, H5þH8); 8.06e8.04 (m, 2H, H6þH7); 7.57e7.56 (m, 3H,H3eH5-ph-QX); 7.51e7.49 (m, 2H, H2þH6-ph-QX); 7.22e7.21 (m,3H, H3eH5-pheCH2); 6.90e6.87 (m, 2H, H2þH6-pheCH2); 4.28 (d,2H, CH2). Anal. Calcd. for C22H17N3O3: C, 71.15%; H, 4.61%; N, 11.31%.Found: C, 71.02%; H, 4.50%; N, 10.94%.

6.1.5.9. 7-chloro-3-phenylquinoxaline-2-carboxylic acid benzylamide1,4-di-N-oxide (2b). Yield: 22%. IR (KBr): 3286 (m, nNH); 3094 (w,narCeH); 1649 (s, nC]O); 1331 (m, nNþOe). 1H NMR (400 MHz, DMSO-d6) d ppm: 9.12 (t, 1H, NH, JNHeCH2¼ 5.9 Hz); 8.56e8.54 (m,1H, H5),8.50 (bs; 1H, H8); 8.08 (dd, 1H, H6, J6e5¼ 9.2 Hz, J6e8¼ 2.2 Hz);7.58e7.49 (m, 5H, H2eH6-ph-QX); 7.21e7.20 (m, 3H, H3eH5-pheCH2); 6.89e6.87 (m, 2H, H2þH6-pheCH2); 4.28 (d, 2H, CH2).Anal. Calcd. for C22H16ClN3O3: C, 65.10%; H, 3.97%; N,10.35%. Found:C, 65.41%; H, 3.97%; N, 10.16%.

6.1.5.10. 7-fluoro-3-phenylquinoxaline-2-carboxylic acid benzyla-mide 1,4-di-N-oxide (2c). Yield: 22%. IR (KBr): 3312 (m, nNH); 3059(w, narCeH); 1673 (s, nC]O); 1335 (m, nNþOe). 1H NMR (400 MHz,DMSO-d6) d ppm: 9.14 (t, 1H, NH, JNHeCH2¼ 5.7 Hz); 8.62e8.59 (m,1H, H5); 8.33e8.30 (m, 1H, H8); 7.99e7.94 (m, 1H, H6); 7.94e7.49(m, 5H, H2eH6-ph-QX); 7.21e7.20 (m, 3H, H3eH5-pheCH2);6.90e6.88 (m, 2H, H2þH6-pheCH2); 4.28 (d, 2H, CH2). Anal. Calcd.for C22H16FN3O3.1/2H2O: C, 66.26%; H, 4.27%; N, 10.54%. Found: C,66.56%; H, 4.04%; N, 10.17%.

6.1.5.11. 7-trifluoromethyl-3-phenylquinoxaline-2-carboxylic acidbenzylamide 1,4-di-N-oxide (2d). Yield: 6%. IR (KBr): 3287 (m, nNH);3093 (w, narCeH); 1647 (s, nC]O); 1350 (s, nNþOe); 1319 (s,nNþOe);1171 (s, nareCF3); 1125 (s, nareCF3). 1H NMR (400 MHz, DMSO-d6)d ppm: 9.15e9.10 (m, 1H, NH); 8.81e8.77 (m, 1H, H5); 8.44e8.28 (m,2H, H8þH6); 7.59e7.51 (m, 5H, H2eH6-ph-QX); 7.25e7.21 (m, 5H,H2eH6-pheCH2); 4.29 (d, 2H, CH2, JCH2eNH¼ 5.9 Hz). Anal. Calcd. forC23H16F3N3O3: C, 62.87%; H, 3.67%; N, 9.56%. Found: C, 63.03%; H,3.96%; N, 9.36%.

6.1.5.12. 7-methyl-3-phenylquinoxaline-2-carboxylic acid benzyla-mide 1,4-di-N-oxide (2e). Yield: 36%. IR (KBr): 3224 (m, nNH); 3058(w, narCeH); 1682 (s, nC]O); 1355 (s,nNþOe); 1314 (s, nNþOe). 1H NMR(400 MHz, DMSO-d6) d ppm: 9.13 (t, 1H, NH, JNHeCH2¼ 5.9 Hz); 8.43(d, 1H, H5, J5e6¼ 8.8 Hz); 8.37 (s, 1H, H8); 7.87 (dd, 1H, H6,J6e8¼ 1.8 Hz); 7.59e7.55 (m, 3H, H3-H5-ph-QX); 7.51e7.48 (m, 2H,H2þH6-ph-QX); 7.21e7.19 (m, 3H, H3eH5-pheCH2); 6.90e6.87 (m,2H, H2þH6-pheCH2); 4.28 (d, 2H, CH2). Anal. Calcd. forC23H19N3O3.1/2H2O: C, 69.97%; H, 5.07%; N, 10.65%. Found: C,70.35%; H, 5.03%; N, 10.65%.

6.1.5.13. 6-methoxy-3-phenylquinoxaline-2-carboxylic acid benzyla-mide 1,4-di-N-oxide (2f). Yield: 35%. IR (KBr): 3262 (m, nNH); 3080(w, narCeH); 1689 (s, nC]O); 1352 (s, nNþOe); 1328 (s,nNþOe); 1256 (s,nCeOeC). 1H NMR (400 MHz, DMSO-d6) d ppm: 9.14 (t, 1H, NH,JNHeCH2¼ 5.9 Hz); 8.44 (d, 1H, H5, J5e6¼ 9.5 Hz); 7.86 (d, 1H, H8,J8e6¼ 2.7 Hz); 7.65 (dd, 1H, H6); 7.58e7.54 (m, 3H, H3eH5-ph-QX);7.51e7.48 (m, 2H, H2þH6-ph-QX); 7.22e7.20 (m, 3H, H3eH5-ph-CH2); 6.93e6.90 (m, 2H, H2þH6-ph-CH2); 4.27 (d, 2H, CH2); 4.03(s, 3H, OCH3). Anal. Calcd. for C23H19N3O4: C, 68.82%; H, 4.77%; N,10.47%. Found: C, 68.94%; H, 4.87%; N, 10.26%.

6.1.5.14. 6,7-dichloro-3-phenylquinoxaline-2-carboxylic acid benzy-lamide 1,4-di-N-oxide (2g). Yield: 8%. IR (KBr): 3280 (m, nNH); 3062(w, narCeH); 1649 (s, nC]O); 1327 (s, nNþOe). 1H NMR (400 MHz,DMSO-d6) d ppm: 9.15 (t, 1H, NH, JNHeCH2¼ 5.9 Hz); 8.75 (s, 1H,H5);8.70 (s, 1H,H8); 7.50e7.48 (m, 5H,H2eH6-ph-QX); 7.22e7.20 (m, 3H,H3eH5-ph-CH2); 6.90e6.88 (m, 2H, H2þH6-ph-CH2); 4.27 (d, 2H,CH2). Anal. Calcd. for C22H15Cl2N3O3: C, 60.02%; H, 3.43%; N, 9.54%.Found: C, 60.07%; H, 3.56%; N, 9.46%.

6.1.5.15. 3-phenylquinoxaline-2-carboxylic acid (2-phenylethyl)amide1,4-di-N-oxide (3a). Yield: 10%. IR (KBr): 3269 (w, nNH); 3078 (w,narCeH); 1679 (s, nC]O); 1328 (s, nNþOe). 1HNMR (400 MHz, DMSO-d6)d ppm: 8.75 (t,1H, NH, JNHeCH2¼ 5.7 Hz); 8.55e8.52 (m, 2H,H5þH8);8.05e8.03 (m, 2H,H6þH7); 7.57e7.51 (m, 5H,H2eH6-ph-QX); 7.27 (t,2H, H3þH5-ph-CH2, J3e2¼ J3e4¼7.3 Hz); 7.20 (t, 1H, H4-ph-CH2); 7.09(d, 2H, H2þH6-ph-CH2); 3.27e3.21 (m, 2H, CH2eNH); 2.39 (t, 2H,CH2eph, JCH2eCH2¼ 7.3 Hz). Anal. Calcd. for C23H19N3O3: C, 71.68%;H, 4.97%; N, 10.90%. Found: C, 71.72%; H, 5.22%; N, 10.88%.

6.1.5.16. 7-chloro-3-phenylquinoxaline-2-carboxylic acid (2-phenyl-ethyl)amide 1,4-di-N-oxide (3b). Yield: 8%. IR (KBr): 3304 (w, nNH);3056 (wd, narCeH); 1668 (s, nC]O); 1330 (s, nNþOe). 1H NMR(400 MHz, DMSO-d6) d ppm: 8.75 (t, 1H, NH, JNHeCH2¼ 5.6 Hz);8.54e8.52 (m, 2H, H5þH8); 8.07 (dd, H6, J6e5¼ 9.3 Hz,J6e8¼ 2.2 Hz); 7.56e7.51 (m, 5H, H2eH6-ph-QX); 7.27 (t, 2H,H3þH5-ph-CH2, J3e2¼ J3e4¼7.3 Hz); 7.19 (t,1H,H4-ph-CH2); 7.09 (d,2H, H2þH6-ph-CH2); 3.27e3.21 (m, 2H, CH2eNH); 2.39 (t, 2H,CH2eph, JCH2eCH2¼ 7.4 Hz). Anal. Calcd. for C23H18ClN3O3: C,65.80%; H, 4.32%; N, 10.01%. Found: C, 65.95%; H, 4.46%; N, 10.09%.

6.1.5.17. 3-methylquinoxaline-2-carboxylic acid p-trifluoromethyl-benzylamide 1,4-di-N-oxide (4a). Yield: 21%. IR (KBr): 3205 (w,nNeH); 3039 (w, narCeH); 1669 (s, nC]O); 1325 (s, nNþOe); 1166 (m,


narCeCF3); 1101 (m, narCeCF3); 1068 (m, narCeCF3). 1H NMR (400 MHz,DMSO-d6) d ppm: 9.49 (t, 1H, NH, JNHeCH2¼ 5.9 Hz); 8.53e8.49 (m,2H, H5þH8); 8.03e7.96 (m, 2H, H6þH7); 7.77 (d, 2H, H30 þH50,J30e20 ¼ 8.2 Hz); 7.70 (d, 2H, H20 þH60); 4.67 (d, 2H, CH2); 2.44 (s, 3H,CH3eC3). Anal. Calcd. for C18H14F3N3O3: C, 57.30%; H, 3.74%; N,11.14%. Found: C, 57.02%; H, 3.79%; N, 11.00%.

6.1.5.18. 7-chloro-3-methylquinoxaline-2-carboxylic acid p-tri-fluoromethylbenzylamide 1,4-di-N-oxide (4b). Yield: 37%. IR (KBr):3277 (w, nNeH); 3103 (w, narCeH); 1650 (m, nC]O); 1328 (s, nNþOe);1161 (m, narCeCF3); 1109 (m, narCeCF3); 1072 (m, narCeCF3). 1H NMR(400 MHz, DMSO-d6) d ppm: 9.49 (t, 1H, NH, JNHeCH2¼ 5.9 Hz); 8.50(s, 2H, H5þH8); 8.04 (dd, 1H, H6, J6e5¼ 9.1 Hz, J6e8¼ 2.3 Hz); 7.77(d, 2H, H30 þH50, J30e20 ¼ 8.1 Hz); 7.69 (d, 2H, H20 þH60); 4.66 (d, 2H,CH2); 2.43 (s, 3H, CH3eC3). Anal. Calcd. for C18H13ClF3N3O3: C,52.51%; H, 3.18%; N, 10.20%. Found: C, 52.26%; H, 3.19%; N, 9.95%.

6.1.5.19. 7-fluoro-3-methylquinoxaline-2-carboxylic acid p-tri-fluoromethylbenzylamide 1,4-di-N-oxide (4c). Yield: 51%. IR (KBr):3212 (w, nNeH); 3079 (w, narCeH); 1671 (m, nC]O); 1327 (s, nNþOe);1167 (m, narCeCF3); 1101 (m, narCeCF3); 1065 (m, narCeCF3). 1H NMR(400 MHz, DMSO-d6) d ppm: 9.50 (t, 1H, NH, JNHeCH2¼ 5.8 Hz); 8.58(dd, 1H, H5, J5e6¼ 9.5 Hz, J5-F¼ 5.1 Hz); 8.25 (dd, 1H, H8, J8-F¼ 8.8 Hz, J8e6¼ 2.4 Hz); 7.93 (ddd, 1H, H6, J6-F¼ 9.4 Hz); 7.77 (d,2H, H30 þH50, J30e20 ¼ 8.1 Hz); 7.69 (d, 2H, H20 þH60); 4.67 (d, 2H,CH2); 2.43 (s, 3H, CH3eC3). Anal. Calcd. for C18H13F4N3O3: C, 54.69%;H, 3.31%; N, 10.63%. Found: C, 54.64%; H, 3.30%; N, 10.36%.

6.1.5.20. 3-methyl-7-trifluoromethylquinoxaline-2-carboxylic acid p-trifluoromethylbenzylamide 1,4-di-N-oxide (4d). Yield: 12%. IR(KBr): 3212 (w, nNeH); 3064 (w, narCeH); 1679 (s, nC]O); 1326 (s,nNþOe); 1168 (m, narCeCF3); 1143 (m, narCeCF3); 1111 (m, narCeCF3);1085 (m, narCeCF3). 1H NMR (400 MHz, DMSO-d6) d ppm: 9.52 (t, 1H,NH, JNHeCH2¼ 5.5 Hz); 8.76 (s, 1H, H8); 8.70 (d, 1H, H5,J5e6¼ 9.0 Hz); 8.29 (dd, 1H, H6, J6e8¼ 1.7 Hz); 7.78 (d, 2H, H30 þH50,J30e20 ¼ 8.1 Hz); 7.70 (d, 2H, H20 þH60); 4.68 (d, 2H, CH2); 2.47 (s, 3H,CH3eC3). Anal. Calcd. for C19H13F6N3O3: C, 51.25%; H, 2.94%; N,9.44%. Found: C, 51.26%; H, 2.74%; N, 9.34%.

6.1.5.21. 3,7-dimethylquinoxaline-2-carboxylic acid p-tri-fluoromethylbenzylamide 1,4-di-N-oxide (4e). Yield: 28%. IR (KBr):3199 (w, nNeH); 3032 (w, narCeH); 1669 (s, nC]O); 1325 (s, nNþOe);1165 (m, narCeCF3); 1100 (m, narCeCF3); 1167 (m, narCeCF3). 1H NMR(400 MHz, DMSO-d6) d ppm: 9.49 (t, 1H, NH, JNHeCH2¼ 5.5 Hz); 8.40(d, 1H, H5, J5e6¼ 8.8 Hz); 8.31 (s, 1H, H8); 7.84 (d, 1H, H6); 7.76 (d,2H, H30 þH50, J30e20 ¼ 8.2 Hz); 7.70 (d, 2H, H20 þH60); 4.66 (d, 2H,CH2); 2,59 (s, 3H, CH3eC7); 2.42 (s, 3H, CH3eC3). Anal. Calcd. forC19H16F3N3O3: C, 58.31%; H, 4.12%; N, 10.74%. Found: C, 58.05%; H,4.09%; N, 10.48%.

6.1.5.22. 7-methoxy-3-methylquinoxaline-2-carboxylic acid p-tri-fluoromethylbenzylamide 1,4-di-N-oxide (4f). Yield: 29%. IR (KBr):3212 (w, nNeH); 3040 (w, narCeH); 1679 (m, nC]O); 1325 (s, nNþOe);1169 (m, narCeCF3); 1118 (m, narCeCF3); 1066 (m, narCeCF3). 1H NMR(400 MHz, DMSO-d6) d ppm: 9.51 (t, 1H, NH, JNHeCH2¼ 5.6 Hz); 8.42(d,1H,H5, J5e6¼ 9.5 Hz); 7.81 (d,1H,H8, J8e6¼ 2.60 Hz); 7.77 (d, 2H,H30 þH50, J30e20 ¼ 8.1 Hz); 7.71 (d, 2H, H20 þH60); 7.61 (dd, 1H, H6);4.66 (d, 2H, CH2); 4.00 (s, 3H, OCH3); 2,41 (s, 3H, CH3eC3). Anal.Calcd. for C19H16F3N3O4: C, 56.02%; H, 3.96%; N, 10.32%. Found: C,55.90%; H, 3.84%; N, 10.14%.

6.1.5.23. 6,7-dichloro-3-methylquinoxaline-2-carboxylic acid p-tri-fluoromethylbenzylamide 1,4-di-N-oxide (4g). Yield: 12%. IR (KBr):3237 (w, nNeH); 3071 (w, narCeH); 1670 (m, nC]O); 1323 (s, nNþOe);

1169 (m, narCeCF3); 1109 (m, narCeCF3); 1069 (m, narCeCF3). 1H NMR(400 MHz, DMSO-d6) d ppm: 9.51 (t, 1H, NH, JNHeCH2¼ 5.8 Hz); 8.69(s, 1H, H5); 8.68 (s, 1H, H8); 7.76 (d, 2H, H30 þH50, J30e20 ¼ 8.4 Hz);7.68 (d, 2H, H20 þH60); 4.66 (d, 2H, CH2); 2.43 (s, 3H, CH3eC3). Anal.Calcd. for C18H12Cl2F3N3O3: C, 48.45%; H, 2.71%; N, 9.42%. Found: C,48.75%; H, 2.82%; N, 9.44%.

6.1.5.24. 3-methylquinoxaline-2-carboxylic acid p-chlor-obenzylamide 1,4-di-N-oxide (5a). Yield: 47%. IR (KBr): 3192 (w,nNeH); 3071 (w, narCeH); 1675 (s, nC]O); 1327 (s, nNþOe); 1082 (m,narCeCl). 1H NMR (400 MHz, DMSO-d6) d ppm: 9.40 (t, 1H, NH,JNHeCH2¼ 5.8 Hz); 8.53e8.49 (m, 2H, H5þH8); 8.01e7.97 (m, 2H,H6þH7); 7.49 (d, 2H, H30 þH50, J30e20 ¼ 8.5 Hz); 7.45 (d, 2H,H20 þH60); 4.56 (d, 2H, CH2); 2.42 (s, 3H, CH3eC3). Anal. Calcd. forC17H14ClN3O3: C, 59.40%; H, 4.10%; N, 12.22%. Found: C, 59.14%; H,4.10%; N, 12.58%.

6.1.5.25. 7-chloro-3-methylquinoxaline-2-carboxylic acid p-chlor-obenzylamide 1,4-di-N-oxide (5b). Yield: 37%. IR (KBr): 3271 (w,nNeH); 3103 (w, narCeH); 1650 (s, nC]O); 1326 (s, nNþOe); 1073 (m,narCeCl). 1H NMR (400 MHz, DMSO-d6) d ppm: 9.40 (t, 1H, NH,JNHeCH2¼ 5.7 Hz); 8.50 (d, 1H, H5, J5e6¼ 9.3 Hz); 8.49 (d, 1H, H8,J8e6¼ 2.0 Hz); 8.04 (dd, 1H, H6); 7.48 (d, 2H, H30 þH50,J30e20 ¼ 8.6 Hz); 7.45 (d, 2H, H20 þH60); 4.55 (d, 2H, CH2); 2.41 (s, 3H,CH3eC3). Anal. Calcd. for C17H13Cl2N3O3: C, 53.99%; H, 3.46%; N,11.11%. Found: C, 54.03%; H, 3.53%; N, 11.05%.

6.1.5.26. 3,7-dimethylquinoxaline-2-carboxylic acid p-chlor-obenzylamide 1,4-di-N-oxide (5e). Yield: 21%. IR (KBr): 3250 (w,nNeH); 3064 (w, narCeH); 1670 (s, nC]O); 1322 (s, nNþOe); 1068 (m,narCeCl). 1H NMR (400 MHz, DMSO-d6) d ppm: 9.40 (t, 1H, NH,JNHeCH2¼ 5.5 Hz); 8.40 (d, H5, J5e6¼ 8.8 Hz); 8.31 (s, 1H, H8); 7.83(dd, 1H, H6, J6e8¼ 1.5 Hz); 7.49 (dd, 2H, H30 þH50, J30e20 ¼ 8.5 Hz,J30eCl¼ 1.3 Hz); 7.45 (dd, 2H, H20 þH60, J20eCl¼ 1.1 Hz); 4.55 (d, 2H,CH2); 2.59 (s, 3H, CH3eC7); 2.40 (s, 3H, CH3eC3). Anal. Calcd. forC18H16ClN3O3: C, 60.43%; H, 4.51%; N, 11.74%. Found: C, 60.08%; H,4.46%; N, 11.51%.

6.1.5.27. 6,7-dichloro-3-methylquinoxaline-2-carboxylic acid p-chlorobenzylamide 1,4-di-N-oxide (5g). Yield: 11%. IR (KBr): 3243(w, nNeH); 3071 (w, narCeH); 1671 (s, nC]O); 1321 (s, nNþOe); 1066(m, narCeCl). 1H NMR (400 MHz, DMSO-d6) d ppm: 9.43 (t, 1H, NH,JNHeCH2¼ 5.9 Hz); 8.69 (d, 1H, H5, J5e8¼ 0.5 Hz); 8.68 (d, 1H, H8);7.48 (d, 2H, H30 þH50, J30e20 ¼ 8.8 Hz); 7.45 (d, 2H, H20 þH60); 4.55 (d,CH2); 2.41 (s, 3H, CH3eC3). Anal. Calcd. for C17H12Cl3N3O3: C,49.48%; H, 2.93%; N, 10.18%. Found: C, 49.76%; H, 2.98%; N, 10.12%.

6.1.5.28. 3-methylquinoxaline-2-carboxylic acid p-bromobenzyla-mide 1,4-di-N-oxide (6a). Yield: 7%. IR (KBr): 3271 (w, nNeH); 3090(w, narCeH); 1677 (s, nC]O); 1331 (s, nNþOe); 1073 (m, narCeBr). 1HNMR (400 MHz, DMSO-d6) d ppm: 9.40 (t, 1H, NH,JNHeCH2¼ 5.8 Hz); 8.52e8.49 (m, 2H, H5þH8); 8.03e7.96 (m, 2H,H6þH7); 7.59 (d, 2H, H30 þH50, JH30eH20 ¼ 8.3 Hz); 7.43 (d, 2H,H20 þH60); 4.54 (d, 2H, CH2); 2.42(s, 3H, CH3eC3). Anal. Calcd. forC17H14BrN3O3: C, 52.60%; H, 3.63%; N, 10.82%. Found: C, 52.23%; H,3.55%; N, 10.43%.

6.1.5.29. 7-chloro-3-methylquinoxaline-2-carboxylic acid p-bromo-benzylamide 1,4-di-N-oxide (6b). Yield: 14%. IR (KBr): 3237 (w,nNeH); 3064 (w, narCeH); 1670 (s, nC]O); 1325 (s, nNþOe); 1071 (m,narCeBr). 1H NMR (400 MHz, DMSO-d6) d ppm: 9.40 (bs, 1H, NH);8.50 (d, 1H, H5, J5e6¼ 9.3 Hz); 8.49 (d, 1H, H8, J8e6¼ 2.4 Hz); 8.04(dd, 1H, H6); 7.59 (d, 2H, H30 þH50, J30e20 ¼ 8.4 Hz); 7.42 (d, 2H,


H20 þH60); 4.53 (d, 2H, CH2, JCH2eNH¼ 5.8 Hz); 2.41 (s, 3H, CH3eC3).Anal. Calcd. for C17H13BrClN3O3: C, 48.31%; H, 3.10%; N, 9.94%.Found: C, 48.30%; H, 3.00%; N, 9.64%.

6.1.5.30. 3,7-dimethylquinoxaline-2-carboxylic acid p-bromobenzy-lamide 1,4-di-N-oxide (6e). Yield: 27%. IR (KBr): 3205 (w, nNeH);3058 (w, narCeH); 1667 (s, nC]O); 1327 (s, nNþOe); 1068 (m, narCeBr).1H NMR (400 MHz, DMSO-d6) d ppm: 9.40 (bs, 1H, NH); 8.40 (d, 1H,H5, J5e6¼ 8.8 Hz); 8.31 (s, 1H, H8); 7.83 (dd, 1H, H6, J6e8¼ 1.3 Hz);7.58 (d, 2H, H30 þH50, J30e20 ¼ 8.5 Hz); 7.43 (d, 2H, H20 þH60); 4.53 (d,2H, CH2, JCH2eNH¼ 5.9 Hz); 2.59 (s, 3H, CH3eC7); 2.40 (s, 3H,CH3eC3). Anal. Calcd. for C18H16BrN3O3: C, 53.75%; H, 4.01%; N,10.45%. Found: C, 53.41%; H, 3.86%; N, 10.07%.

6.1.5.31. 6,7-dichloro-3-methylquinoxaline-2-carboxylic acid p-bro-mobenzylamide 1,4-di-N-oxide (6g). Yield: 14%. IR (KBr): 3237 (w,nNeH); 3066 (w, narCeH); 1670 (s, nC]O); 1320 (s, nNþOe); 1067 (m,narCeBr). 1H NMR (400 MHz, DMSO-d6) d ppm: 9.43 (t, 1H, NH,JNHeCH2¼ 5.9 Hz); 8.69 (s, 1H, H5); 8.68 (s, 1H, H8); 7.58 (d, 2H,H30 þH50, J30e20 ¼ 8.3 Hz); 7.41 (d, 2H, H20 þH60); 4.53 (d, 2H, CH2);2.41 (s, 3H, CH3eC3). Anal. Calcd. for C17H12BrCl2N3O3: C, 44.67%; H,2.65%; N, 9.19%. Found: C, 44.33%; H, 2.56%; N, 8.92%.

6.1.5.32. 3-methylquinoxaline-2-carboxylic acid p-methyl-benzylamide 1,4-di-N-oxide (7a). Yield: 61%. IR (KBr): 3224 (w,nNeH); 3045 (w, narCeH); 1671 (s, nC]O); 1336 (s, nNþOe). 1H NMR(400 MHz, DMSO-d6) d ppm: 9.30 (t, 1H, NH, JNHeCH2¼ 5.8 Hz);8.52e8.49 (m, 2H, H5þH8); 8.02e7.97 (m, 2H, H6þH7); 7.33 (d, 2H,H20 þH60, J20e30 ¼7.8 Hz); 7.19 (d, 2H,H30 þH50); 4.51 (d, 2H, CH2); 2.42(s, 3H, CH3eC3); 2.30 (s, 3H, CH3eph). Anal. Calcd. for C18H17N3O3: C,66.86%; H, 5.30%; N, 13.00%. Found: C, 66.62%; H, 5.28%; N, 12.78%.

6.1.5.33. 7-chloro-3-methylquinoxaline-2-carboxylic acid p-methyl-benzylamide 1,4-di-N-oxide (7b). Yield: 35%. IR (KBr): 3259 (w,nNeH); 3077 (w, narCeH); 1671 (s, nC]O); 1325 (s, nNþOe). 1H NMR(400 MHz, DMSO-d6) d ppm: 9.30 (t,1H, NH, JNHeCH2¼ 5.9 Hz); 8.50(d, 1H, H5, J5e6¼ 9.1 Hz); 8.48 (s, 1H, H8); 8.03 (dd, 1H, H6,J6e8¼ 2.3 Hz); 7.32 (d, 2H, H20 þH60, J20e30 ¼7.9 Hz); 7.19 (d, 2H,H30 þH50); 4.51 (d, 2H, CH2); 2.40 (s, 3H, CH3eC3); 2.30 (s, 3H,CH3eph). Anal. Calcd. for C18H16ClN3O3: C, 60.43%; H, 4.51%; N,11.74%. Found: C, 60.41%; H, 4.57%; N, 11.71%.

6.1.5.34. 3,7-dimethylquinoxaline-2-carboxylic acid p-methyl-benzylamide 1,4-di-N-oxide (7e). Yield: 10%. IR (KBr): 3281 (m,nNeH); 3065 (w, narCeH); 1650 (s, nC]O); 1325 (s, nNþOe). 1H NMR(400 MHz, DMSO-d6) d ppm: 9.31 (bs, 1H, NH); 8.39 (d, 1H, H5,J5e6¼ 8.8 Hz); 8.30 (s, 1H, H8); 7.83 (dd, 1H, H6, J6e8¼ 1.8 Hz); 7.33(d, 2H, H20 þH60, J20e30 ¼ 8.1 Hz); 7.19 (d, 2H, H30 þH50); 4.50 (d, 2H,CH2, JCH2eNH¼ 5.8 Hz); 2.60 (s, 3H, CH3eC7); 2.40 (s, 3H, CH3eC3);2.30 (s, 3H, CH3eph). Anal. Calcd. for C19H19N3O3: C, 67.64%; H,5.68%; N, 12.45%. Found: C, 67.27%; H, 5.70%; N, 12.25%.

6.1.5.35. 6,7-dichloro-3-methylquinoxaline-2-carboxylic acid p-methylbenzylamide 1,4-di-N-oxide (7g). Yield:12%. IR (KBr): 3270(m,nNeH); 3045 (w, narCeH); 1649 (s, nC]O); 1359 (m, nNþOe). 1H NMR(400 MHz, DMSO-d6) d ppm: 9.35 (t, 1H, NH, JNHeCH2¼ 5.3 Hz); 8.62(s, 1H, H5); 8.45 (s, 1H, H8); 7.32 (d, 2H, H20 þH60, J20e30 ¼7.3 Hz); 7.19(d, 2H, H30 þH50); 4.51 (d, 2H, CH2); 2.55(s, 3H, CH3eC3); 2.30 (s, 3H,CH3eph). Anal. Calcd. for C18H15Cl2N3O3: C, 55.12%; H, 3.85%; N,10.71%. Found: C, 55.37%; H, 4.17%; N, 10.45%.

6.1.5.36. 3-methylquinoxaline-2-carboxylic acid 2,2-diphenylethyla-mide 1,4-di-N-oxide (8a). Yield: 8%. IR (KBr): 3237 (w, nNeH); 3064(w, narCeH); 1681 (s, nC]O); 1339 (m, nNþOe). 1H NMR (400 MHz,

DMSO-d6) d ppm: 8.90 (t, 1H, NH, JNHeCH2¼ 5.7 Hz); 8.47e8.42 (m,2H, H5þH8); 7.99e7.92 (m, 2H, H6þH7); 7.41 (dd, 4H, 2H20þ2H60,J20e30 ¼7.2 Hz, J20e40 ¼1.3 Hz); 7.35e7.31 (m, 4H, 2H30þ2H50); 7.20(tt, 2H, 2H40, J40e30 ¼7.3 Hz); 4.32 (t, 1H, CH, JCHeCH2¼ 8.0 Hz); 4.02(dd, 2H, CH2); 1.97 (s, 3H, CH3eC3). Anal. Calcd. for C24H21N3O3: C,72.17%; H, 5.30%; N, 10.52%. Found: C, 72.02%; H, 5.34%; N, 10.29%.

6.1.5.37. 7-chloro-3-methylquinoxaline-2-carboxylic acid 2,2-diphe-nylethylamide 1,4-di-N-oxide (8b). Yield: 17%. IR (KBr): 3231 (w,nNeH); 3058 (w, narCeH); 1679 (s, nC]O); 1326 (s, nNþOe). 1H NMR(400 MHz, DMSO-d6) d ppm: 8.88 (bs, 1H, NH); 8.45 (d, 1H, H5,J5e6¼ 9.2 Hz); 8.41 (d, 1H, H8, J8e6¼ 2.1 Hz); 8.00 (dd, 1H, H6); 7.40(dd, 4H, 2H20þ2H60, J20e30 ¼7.8 Hz, J20e40 ¼1.2 Hz); 7.35e7.30 (m, 4H,2H30þ2H50); 7.22 (tt, 2H, 2H40, J40e30 ¼7.3 Hz); 4.32 (t, 1H, CH,JCHeCH2¼7.9 Hz); 4.03 (dd, 2H, CH2); 1.98 (s, 3H, CH3eC3). Anal.Calcd. for C24H20ClN3O3: C, 66.44%; H, 4.65%; N, 9.68%. Found: C,66.53%; H, 5.03%; N, 9.31%.

6.1.5.38. 3,7-dimethylquinoxaline-2-carboxylic acid 2,2-diphenyle-thylamide 1,4-di-N-oxide (8e). Yield: 11%. IR (KBr): 3223 (w, nNeH);3057 (w, narCeH); 1678 (s, nC]O); 1328 (s, nNþOe). 1H NMR (400 MHz,DMSO-d6) d ppm: 8.90 (bs, 1H, NH); 8.34 (d, 1H, H5, J5e6¼ 8.5 Hz);8.23 (s, 1H, H8); 7.79 (dd, 1H, H6, J6e8¼ 1.2 Hz); 7.40 (d, 4H,2H20þ2H60, J20e30 ¼7.5 Hz); 7.32 (t, 4H, 2H30þ2H50, J30e40 ¼7.5 Hz);7.24e7.20 (m, 2H, 2H40); 4.31 (t, 1H, CH, JCHeCH2¼ 7.9 Hz); 4.02 (dd,2H, CH2, JCH2eNH¼ 5.8 Hz); 2.57 (s, 3H, CH3eC7); 1.95 (m, 3H,CH3eC3). Anal. Calcd. for C25H23N3O3: C, 72.62%; H, 5.61%; N,10.16%.Found: C, 72.26%; H, 5.84%; N, 9.77%.

6.1.5.39. 6,7-dichloro-3-methylquinoxaline-2-carboxylic acid 2,2-diphenylethylamide 1,4-di-N-oxide (8g). Yield: 15%. IR (KBr): 3212(w, nNeH); 3083 (w, narCeH); 1676 (s, nC]O); 1321 (s, nNþOe). 1H NMR(400 MHz, DMSO-d6) d ppm: 8.91 (t, 1H, NH, JNHeCH2¼ 5.8 Hz); 8.63(s, 1H, H5); 8.60 (s, 1H, H8); 7.40 (dd, 4H, 2H20þ2H60, J20e30 ¼7.8 Hz,J20e40 ¼1.3 Hz); 7.32 (dd, 4H, 2H30þ2H50, J30e40 ¼7.4 Hz); 7.24 (tt, 2H,2H40); 4.32 (t, 1H, CH, JCHeCH2¼ 8.1 Hz); 4.02 (dd, 2H, CH2); 1.07 (s,3H, CH3eC3). Anal. Calcd. for C24H19Cl2N3O3.1/2H2O: C, 60.33%; H,4.22%; N, 8.79%. Found: C, 60.10%; H, 4.25%; N, 8.60%.

6.1.5.40. 3-methylquinoxaline-2-carboxylic acid (benzo[1,3]dioxol-5-ylmethyl)amide 1,4-di-N-oxide (9a). Yield: 25%. IR (KBr): 3261 (m,nNeH); 3077 (w, narCeH); 1642 (s, nC]O); 1329 (m, nNþOe). 1H NMR(400 MHz, DMSO-d6) d ppm: 9.29 (t, 1H, NH, JNHeCH2¼ 5.8 Hz);8.52e8.38 (m, 2H, H5þH8); 8.02e7.95 (m, 2H, H6þH7); 7.03 (bs,1H,H20); 6.91 (bs, 2H,H50 þH60); 6.01 (s, 2H, OeCH2eO); 4.47 (d, 2H,CH2); 2.42 (s, 3H, CH3eC3). Anal. Calcd. for C18H15N3O5.1/2H2O: C,59.61%; H, 4.41%; N, 11.59%. Found: C, 59.72%; H, 4.32%; N, 11.56%.

6.1.5.41. 7-chloro-3-methylquinoxaline-2-carboxylic acid (benzo[1,3]dioxol-5-yl methyl)amide 1,4-di-N-oxide (9b). Yield: 18%. IR (KBr):3276 (w, nNeH); 3090 (w, narCeH); 1649 (s, nC]O); 1326 (s, nNþOe). 1HNMR (400 MHz, DMSO-d6) d ppm: 9.29 (t, 1H, NH,JNHeCH2¼ 5.6 Hz); 8.51e8.49 (m, 2H, H5þH8); 8.05e8.02 (m, 1H,H6); 7.02 (bs, 1H, H20); 6.91 (bs, 2H, H50 þH60); 6.01 (s, 2H,OeCH2eO); 4.46 (d, 2H, CH2); 2.40 (s, 3H, CH3eC3). Anal. Calcd. forC18H14ClN3O5: C, 55.75%; H, 3.64%; N, 10.84%. Found: C, 55.50%; H,3.46%; N, 10.68%.

6.1.5.42. 3,7-dimethylquinoxaline-2-carboxylic acid (benzo[1,3]dioxol-5-ylmethyl) amide 1,4-di-N-oxide (9e). Yield: 21%. IR (KBr):3266 (w, nNeH); 3071 (w, narCeH); 1649 (s, nC]O); 1325 (s, nNþOe). 1HNMR (400 MHz, DMSO-d6) d ppm: 9.29 (t, 1H, NH,JNHeCH2¼ 5.8 Hz); 8.39 (d, 1H, H5, J5e6¼ 8.8 Hz); 8.31 (s, 1H, H8);7.83 (d, 1H, H6); 7.04 (bs, 1H, H20); 6.91 (bs, 2H, H50 þH60); 6.02 (s,


2H, OeCH2eO); 4.47 (d, 2H, CH2); 2.59 (s, 3H, CH3eC7); 2.40 (s, 3H,CH3eC3). Anal. Calcd. for C19H17N3O5: C, 62.12%; H, 4.66%; N,11.44%.Found: C, 61.74%; H, 4.80%; N, 11.68%.

6.1.5.43. 6,7-dichloro-3-methylquinoxaline-2-carboxylic acid (benzo[1,3]dioxol-5-ylmethyl) amide 1,4-di-N-oxide (9g). Yield: 22%. IR(KBr): 3273 (w, nNeH); 3058 (w, narCeH); 1647 (s, nC]O); 1326 (s,nNþOe). 1H NMR (400 MHz, DMSO-d6) d ppm: 9.31 (t, 1H, NH,JNHeCH2¼ 5.8 Hz); 8.69 (s, 1H,H5); 8.67 (s, 1H,H8); 7.02 (bs, 1H,H20);6.90 (bs, 2H, H50 þH60); 6.02 (bs, 2H, OeCH2eO); 4.46 (d, 2H, CH2);2.50 (s, 3H, CH3eC3). Anal. Calcd. for C18H13Cl2N3O5: C, 51.20%; H,3.10%; N, 9.95%. Found: C, 51.22%; H, 3.07%; N, 9.62%.

6.2. Pharmacology [31]

6.2.1. Primary screening (DoseeResponse): determination of a 90%inhibitory concentration (IC90)

The initial screening is conducted against M.Tbc. H37Rv (ATCC27294) in BACTEC 12B medium using the Microplate Alamar BlueAssay (MABA) [32]. Compounds are tested in ten 2-fold dilutions,typically from 100 mg/mL to 0.19 mg/mL. The IC90 is defined as theconcentration effecting a reduction in fluorescence of 90% relativeto controls. This value is determined from the doseeresponse curveusing a curve-fitting program. Any IC90 value of �10 mg/mL isconsidered “Active” for anti-tubercular activity.

6.2.2. Secondary screening: determination of mammalian cellcytotoxicity (CC50)

The VERO cell cytotoxicity assay is carried out in parallel withthe TB DoseeResponse assay. After 72 h exposure, viability isassessed using Promega’s Cell Titer Glo Luminescent Cell ViabilityAssay, a homogeneous method for determining the number ofviable cells in culture based on quantitation of the ATP present.Cytotoxicity is determined from the doseeresponse curve as theCC50 using a curve-fitting program. Then the CC50 is divided by theIC90 for calculating a Selectivity Index (SI) value. SI values of�10 areconsidered for further testing.

Acknowledgments

This work has been carried out with the financial support of thePIUNA project from University of Navarra. We also wish to expressour gratitude to the Tuberculosis Antimicrobial Acquisition &Coordinating Facility (TAACF) for the evaluation of the anti-tuber-culosis activity through research and development contracts. E.M.

is indebted to the La Rioja Government for a grant. S.A. is indebtedto the Navarra Government for a grant.

References

[1] Global Tuberculosis Control WHO REPORT 2009, http://www.who.int/tb/publications/global_report/2009/pdf/full_report.pdf (accessed 08.03.10).

[2] NIAID MDR/XDR TB Research Agenda June 6, 2007, http://www3.niaid.nih.gov/topics/tuberculosis/ (accessed 08.03.10).

[3] http://www.who.int/tb/challenges/mdr/en/index.html. (accessed 08.03.10).[4] http://www.who.int/tb/challenges/xdr/en/index.html (accessed 08.03.10).[5] E. Vicente, R. Villar, B. Solano, A. Burguete, S. Ancizu, S. Pérez-Silanes, I. Aldana,

A. Monge, A. An. R. Acad. Nac. Farm. 73 (2007) 927e945.[6] G. Aguirre, H. Cerecetto, R. Di Maio, M. Gonzalez, M.E.M. Alfaro, A. Jaso,

B. Zarranz, M.A. Ortega, I. Aldana, A. Monge-Vega, Bioorg. Med. Chem. Lett. 14(2004) 3835e3839.

[7] C. Urquiola, M. Vieites, G. Aguirre, A. Marin, B. Solano, G. Arrambide, P. Noblia,M.L. Lavaggi, M.H. Torre, M. Gonzalez, A. Monge, D. Gambino, H. Cerecetto,Bioorg. Med. Chem. 14 (2006) 5503e5509.

[8] A. Carta, M. Loriga, G. Paglietti, A. Mattana, P.L. Fiori, P. Mollicotti, L. Sechi,S. Zanetti, Eur. J. Med. Chem. 39 (2004) 195e203.

[9] B. Ganley, G. Chowdhury, J. Bhansali, J.S. Daniels, K.S. Gates, Bioorg. Med.Chem. 9 (2001) 2395e2401.

[10] M. Abu El-Haj, T.H. Cronin, DE2035480 (1971).[11] K.L. Leverkusen, U. Eholzer, R. Nast, F. Seng, US3660398 (1972).[12] M. Abu El-Haj, DE2316765 (1973).[13] T.H. Cronin, US3728345 (1973).[14] J.W. McFarland, FR2258856 (1975).[15] J.G. Frienlink, NL6504563 (1966).[16] K.M. Amin, M.F. Ismail, E. Noaman, D.H. Soliman, Y.A. Ammar, Bioorg. Med.

Chem. 14 (2006) 6917e6923.[17] M.A. Ortega, M.E. Montoya, A. Jaso, B. Zarranz, I. Tirapu, I. Aldana, A. Monge,

Pharmazie 56 (2001) 205e207.[18] M.A. Ortega, Y. Sainz, M.E. Montoya, A. Jaso, B. Zarranz, I. Aldana, A. Monge,

Arzneim.-Forsch. 52 (2002) 113e119.[19] A. Jaso, B. Zarranz, I. Aldana, A. Monge, Eur. J. Med. Chem. 38 (2003) 791e800.[20] A. Jaso, B. Zarranz, I. Aldana, A. Monge, J. Med. Chem. 48 (2005) 2019e2025.[21] B. Zarranz, A. Jaso, I. Aldana, A. Monge, Bioorg. Med. Chem. 11 (2003)

2149e2156.[22] S. Ancizu, E. Moreno, B. Solano, R. Villar, A. Burguete, E. Torres, S. Pérez-

Silanes, I. Aldana, A. Monge, Bioorg. Med. Chem. 18 (2010) 2713e2719.[23] Jie Jack Li, Name Reactions. A Collection of Detailed Reaction Mechanism, third

ed. Springer, Berlin, Heidelberg, 2006, pp. 43e44.[24] G. Stumm, H.J. Niclas, J. Prakt. Chem. 331 (1989) 736e744.[25] M. González, H. Cerecetto, Topics in heterocyclic chemistry. in: M.T.H. Khan

(Ed.), Bioactive Heterocycles IV, Benzofuroxan and Furoxan. Chemistry andBiology, vol. 10. Springer, Berlin, Heidelberg, 2007 pp. 265.

[26] A.G. Neo, J. Delgado, et al., Tetrahedron Lett. 46 (2005) 23e26.[27] H.T. Openshaw, N. Whittaker, J. Chem. Soc. 19 (1968) 89e91.[28] R.J. Clemens, Chem. Rev. 86 (1986) 241e318.[29] G.W.H. Cheeseman, in: R.F. Cookson (Ed.), Condensed Pyrazines, J. Wiley and

Sons, New York, 1979 p. 35.[30] B. Zarranz, A. Jaso, I. Aldana, A. Monge, Bioorg. Med. Chem. 12 (2004)

3711e3721.[31] TAACF: http://www.taacf.org/Process-text.htm#assays. (accessed 26.02.10).[32] L.A. Collins, S.G. Franzblau, Antimicrob. Antimicrob. Agents Chemother. 41

(1997) 1004e1009.

http://www.who.int/tb/publications/global_report/2009/pdf/full_report.pdf


http://www3.niaid.nih.gov/topics/tuberculosis/


http://www.who.int/tb/challenges/mdr/en/index.html

http://www.who.int/tb/challenges/xdr/en/index.html

http://www.taacf.org/Process-text.htm%23assays

Bioorganic & Medicinal Chemistry 18 (2010) 2713–2719

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier .com/locate /bmc

New 3-methylquinoxaline-2-carboxamide 1,4-di-N-oxide derivatives asanti-Mycobacterium tuberculosis agents

Saioa Ancizu, Elsa Moreno, Beatriz Solano, Raquel Villar, Asunción Burguete, Enrique Torres,Silvia Pérez-Silanes *, Ignacio Aldana, Antonio MongeUnidad de Investigación y Desarrollo de Medicamentos, Centro de Investigación en Farmacobiología Aplicada (CIFA), University of Navarra, C/Irunlarrea s/n, 31008 Pamplona, Spain

a r t i c l e i n f o a b s t r a c t

Article history:Received 23 September 2009Revised 9 February 2010Accepted 12 February 2010Available online 20 February 2010

Keywords:Anti-tuberculosis agentsQuinoxaline 1,4-di-N-oxide derivativesMycobacterium tuberculosis (M.Tb.)Rifampin

0968-0896/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.bmc.2010.02.024

* Corresponding author. Tel.: +34 948 425653; fax:E-mail address: [email protected] (S. Pérez-Silanes).

Mycobacterium tuberculosis (M.Tb) is a bacillus capable of causing a chronic and fatal condition in humansknown as tuberculosis (TB). It is estimated that there are 8 million new cases of TB per year and 3.1 mil-lion infected people die annually. Thirty-six new amide quinoxaline 1,4-di-N-oxide derivatives have beensynthesized and evaluated as potential anti-tubercular agents, obtaining biological values similar to thereference compound, Rifampin (RIF).

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Tuberculosis (TB) is a respiratory disease mainly caused by thebacillus Mycobacterium tuberculosis (M.Tb.). It is responsible for 8million new cases and 3.1 million deaths per year, mostly occur-ring in developing countries, although there are over 400,000new cases annually in industrialized countries. As infection withhuman immunodeficiency virus (HIV) becomes more prevalent,tuberculosis is becoming a serious problem in developed countriesas well,1 as can be observed in Figure 1. Of particular concern is thedevelopment of drug-resistant forms of the disease, Multidrug-Resistant Tuberculosis (MDR-TB) and Extensively Drug-ResistantTuberculosis (XDR-TB). These forms of the disease are more oftenfatal and are difficult and expensive to treat. It has been estimatedthat up to 50 million people are infected with drug-resistant formsof TB.2,3

Due to the problems related to MDR-TB and XDR-TB, it is neces-sary to develop new, potent, fast-acting anti-tuberculosis drugswith low-toxicity profiles that can be given in conjunction withdrugs used to treat HIV infections.4,5 New drugs and improveddelivery methods will be integral parts of a strategy to fully controlfuture outbreaks of TB, particularly MDR-TB, which has severelychallenged the limited number of effective treatment options.3

Quinoxaline and quinoxaline 1,4-di-N-oxide derivatives displayexcellent biological activities (antiviral, anticancer, antibacterial,

ll rights reserved.

+34 948 425652.

anti-parasitic, etc.) with application in many different therapeuticareas.6–8 As a result of the anti-tuberculosis research project, ourgroup has published several papers in which synthesis and biolog-ical activity assessments of a large number of quinoxaline andquinoxaline 1,4-di-N-oxide derivatives have been described,9–17

identifying some structural requirements for optimal activity.13–19

A few years ago, a group of thirty-one 3-methylquinoxaline-2-carboxamide 1,4-di-N-oxide derivatives were prepared and testedagainst M.Tb., obtaining some interesting results.13 Three of thesecompounds presented enough selectivity and good results in mac-rophage assay so as to merit continuation of their study. With theaim of improving the previous results, new amide quinoxaline 1,4-di-N-oxide derivatives have been synthesized and evaluated as po-tential anti-tubercular agents, obtaining excellent results as can beobserved in this report.


Thirty-six new 1,4-di-N-oxide-3-methylquinoxaline-2-carbox-ylic acid aryl amide derivatives were prepared through the syn-thetic route as follows:

First, the starting benzofuroxanes (BFX) were obtained by previ-ously described methods.20,12 Next, the initial b-acetoacetamidederivatives (1–4) were synthesized through acetoacetylation ofcorresponding aryl amines by diketene, using methanol as the sol-vent as shown in Scheme 1.21

Finally, the new 1,4-di-N-oxide-quinoxaline derivatives wereprepared using a variation of the Beirut reaction, where the

http://dx.doi.org/10.1016/j.bmc.2010.02.024


http://www.sciencedirect.com/science/journal/09680896

http://www.elsevier.com/locate/bmc

Figure 1. Estimated number of new TB cases, by country, 2007. [Source, Global Tuberculosis Control WHO REPORT 2009].

H2NW

+O

H2C

NH

O O

(1-4)

n= 1, 2W= H, OCH3W

OMeOHn n

Scheme 1. Synthetic scheme of initial b-acetoacetamide derivatives (1–4).

2714 S. Ancizu et al. / Bioorg. Med. Chem. 18 (2010) 2713–2719

different BFX react with the corresponding b-acetoacetamide in thepresence of calcium chloride and ethanolamine as catalysts(Scheme 2).22

Quinoxaline derivatives were un-substituted or substituted inpositions 6 and 7 by chloro, fluoro or trifluoromethyl moiety aselectron-withdrawing groups and by methyl or methoxy moietyas electron-releasing groups. When the new quinoxalines wereprepared from monosubstituted-BFX, mixtures of positionalisomers were obtained. Generally, it could be observed that the7-substituted isomer prevailed over 6-substituted isomer.23

Table 1 summarizes the biological values of the synthesizedcompounds. The primary screening level determines the activityof the compounds against M.Tb. in H37Rv strain. Samples showinga percentage of TB growth inhibition greater than or equal to 90%are considered active and therefore, move on to the secondaryscreening. Active compounds are tested in order to determine theactual minimum inhibitory concentration (MIC), and simulta-

(1-4)n= 1, W= HR6, R7

NO

N+O-

R7

R6

+

BFX

NH W

nOO

H

Scheme 2. Synthetic route of 1,4-di-N-oxide-3-methylquino

neously cytotoxicity (IC50) in VERO cells is evaluated. Next, theMIC and IC50 values are formed into a ratio termed Selectivity In-dex (SI). If the SI level is 10 or greater, a compound is consideredactive at the second level.

As can be observed in Table 1, compounds 10 and 27 were iden-tified as the most interesting derivatives, with great anti-tubercu-lar activity and low values of cytotoxicity, as can be observed bytheir SI values. Derivatives 9, 13, 28, 29, 36 and 38 have also shownvery good results. Some structure–activity relationships could beestablished.

Looking at the values for compounds 9–13, 20–22, 27–31 and36–40, it can be said that, in general, the introduction of an elec-tron-withdrawing substituent in the quinoxaline ring results inan increment in the anti-tubercular activity of the derivatives. Onthe contrary, the insertion of an electro-releasing moiety resultsin a reduction of this activity, as can be observed by the results ob-tained for the compounds 6–8, 15–17, 24–26 and 33–35. In short, itcan be concluded that the insertion of an electron-withdrawingmoiety in the quinoxaline ring is an essential requirement in orderto improve the anti-tubercular activity, as established in previousworks reported by the group.13

To further explore the SAR of these compounds, a methoxygroup was substituted in the para position of the benzene ring. Thisderivatization led to an increase of the cytotoxicity of the

N+

N+O-

O-

R7

R6

2, OCH3= H, Cl, F, OCH3, CH3, CF3

NH

O

WnCaCl2

MeOH

(5-40)O NH2

xaline-2-carboxylic acid aryl amide derivatives (5–40).

Table 1Biological values of compounds (5–40) at secondary screening

Compd R7 R6 n W MIC (lg/mL) H37Rva IC50 (lg/mL) VEROb SIc (IC50/MIC)

5 H H 1 H 2.606 >100 >38.3726 CH3 H 1 H 4.207 >100 >23.777 CH3 CH3 1 H >100 NT NT8 OCH3 H 1 H 12.346 NT NT9 Cl H 1 H 0.433 >100 >230.9410 Cl Cl 1 H <0.2 >100 >50011 F H 1 H 1.522 >100 >65.7012 F F 1 H 1.727 18.378 10.6413 CF3 H 1 H 0.406 >100 >246.3014 H H 1 OCH3 2.847 >30 >10.53715 CH3 H 1 OCH3 8.903 >30 >3.369616 CH3 CH3 1 OCH3 >100.00 NT NT17 OCH3 H 1 OCH3 12.007 NT NT18 Cl H 1 OCH3 NT NT NT19 Cl Cl 1 OCH3 NT NT NT20 F H 1 OCH3 1.508 >30 >19.89321 F F 1 OCH3 0.764 16.605 21.73422 CF3 H 1 OCH3 0.999 >30 >30.0323 H H 2 H 3.048 >100 >32.8124 CH3 H 2 H 8.621 >100 >11.6025 CH3 CH3 2 H 16.359 NT NT26 OCH3 H 2 H 13.657 NT NT27 Cl H 2 H <0.2 >100 >50028 Cl Cl 2 H <0.195 >30 >153.8429 F H 2 H 0.504 >100 >198.4130 F F 2 H 0.517 >30 >58.02731 CF3 H 2 H 1.153 >30 >26.01932 H H 2 OCH3 3.464 >30 >8.660533 CH3 H 2 OCH3 8.852 >30 >3.38934 CH3 CH3 2 OCH3 >100.00 NT NT35 OCH3 H 2 OCH3 16.866 NT NT36 Cl H 2 OCH3 <0.195 >30 >153.8437 Cl Cl 2 OCH3 0.454 >30 >66.07938 F H 2 OCH3 <0.195 >30 >153.8439 F F 2 OCH3 2.058 >30 >14.57740 CF3 H 2 OCH3 1.427 >30 >21.023RIF — — — — 0.015–0.125 >100 >800

NT, Non tested.In the first screening, all of the compounds were evaluated against M.Tb. in H37Rv strain. All of them were active at this level. In the secondary screening, the actual minimuminhibitory concentration (MIC) in H37Rv strain and cytotoxicity (IC50) in VERO cells were determined. Finally, the SI was calculated and the compounds containing SIlevel P 10 were considered active at the second level. All of these active compounds are highlighted in green in the table, with the most active ones being marked in darkgreen.

a Actual minimum inhibitory concentration in M.Tb. H37Rv strain.b Cytotoxicity in VERO cells.c Selectivity index.

S. Ancizu et al. / Bioorg. Med. Chem. 18 (2010) 2713–2719 2715

compounds, as can be observed by comparing the following deriv-atives 5 versus 14, 6 versus 15, 11 versus 20, 13 versus 22, 24 ver-sus 33, 23 versus 32, 27 versus 36 and 29 versus 38. With the aimof improving these results, current studies are ongoing to modifynot only the substituent on the benzene ring but also the aromaticring itself.

Finally, looking at the values of the compounds 10 versus 28and 13 versus 31, it can be observed that lengthening the aliphaticchain can also result in a greater value of cytotoxicity. Taking intoaccount these results and previous ones obtained by the group,13

the most suitable linker is established as a single methylene groupbetween the carboxamide and the benzene ring.

3. Conclusions

Thirty-six new 1,4-di-N-oxide-3-methylquinoxaline-2-carbox-ylic acid aryl amide derivatives were synthesized using a variationof the Beirut reaction. Thirty-four of these compounds were evalu-ated against M.Tb. in H37Rv strain; all of them showed a TB growth

inhibition percentage greater than or equal to 90%. These com-pounds were considered active at first level and moved on to thesecondary screening, where the actual minimum inhibitory con-centration (MIC) in H37Rv strain and cytotoxicity (IC50) in VEROcells were determined. In this case, twenty three of the twenty-six evaluated derivatives showed an SI greater than 10, being con-sidered to be active at the second level.

Taking into account the SI values shown in Table 1, it can be saidthat these values are similar to the reference compound, Rifampin(RIF). In conclusion, the potency, selectivity, and low cytotoxicity ofthese compounds make them valid leads for synthesizing newcompounds that possess better activity.

4. Experimental section

4.1. General remarks

All of the synthesized compounds were chemically character-ized by thin layer chromatography (TLC), infrared (IR), proton


nuclear magnetic resonance (1H NMR) and elemental microanaly-ses (CHN).

Alugram SIL G/UV254 (Layer: 0.2 mm) (Macherey-Nagel GmbH& Co. KG., Düren, Germany) was used for TLC, and Silica gel 60(0.040–0.063 mm, Merck) was used for Flash Column Chromatog-raphy. The 1H MNR spectra were recorded on a Bruker 400 Ultra-shield instrument (400 MHz), using TMS as internal standard andwith DMSO-d6 or CDCl3 as solvents; the chemical shifts are re-ported in ppm (d) and coupling constant (J) values are given inHertz (Hz). Signal multiplicities are represented by: s (singlet), bs(broad singlet), d (doublet), dd (double doublet), ddd (double dou-ble doblet), t (triplet), q (quadruplet) and m (multiplet). The IRspectra were recorded on a Nicolet Nexus FTIR (Thermo, Madison,USA) in KBr pellets. Elemental microanalyses were obtained on aCHN-900 Elemental Analyzer (Leco, Tres Cantos, Spain) from vac-uum-dried samples. The analytical results for C, H and N werewithin ±0.4 of the theoretical values. Chemicals were purchasedfrom Panreac Química S.A. (Barcelona, Spain), Sigma-Aldrich Quí-mica, S.A. (Alcobendas, Spain), Acros Organics (Janssen Pharmaceu-ticalaan, Geel, Belgium) and Lancaster (Bischheim-Strasbourg,France).

4.2. General procedure of synthesis

4.2.1. Synthesis of b-acetoacetamide derivatives (1–4)The corresponding aryl amines (20.0 mmol) were diluted in

methanol (20.0 mL) in N2 atmosphere and cooled in ice bath until0 �C. Next, diketene (22.0 mmol) was added dropwise and the reac-tion was stirred for 2–3 h. The obtained residue was precipitatedwith cold diethyl ether and filtered in order to obtain a red–brownsolid. The compound was used without further purification.

4.2.1.1. N-Benzyl-3-oxobutanamide (1). Yield 25%. IR (KBr): 3318(m, NH); 1617 (s, CO); 1540 (s, CO). 1H MNR (400 MHz, CDCl3) dppm: 9.52 (br s, 1H, NH); 7.33–7.21 (m, 5H, Ph); 4.56–4.35 (m,4H, CH2–NH–CO–CH2–CO); 1.89 (s, 3H, CH3).

4.2.1.2. N-(4-Methoxybenzyl)-3-oxobutanamide (2). Yield 77%.IR (KBr): 3317 (s, NH); 1619 (s, CO); 1539 (s, CO). 1H MNR(400 MHz, DMSO-d6) d ppm: 8.46 (t, 1H, JNH–CH2 = 5.0 Hz, NH);7.20 (d, 2H, JH2–H3 = 8.6 Hz, H2+H6); 6.89 (d, 2H, H3+H5); 4.22 (d,2H, CH2–NH); 3.73 (s, 2H, CO–CH2–CO); 3.35 (s, 3H, OCH3); 2.15(s, 3H, CH3).

4.2.1.3. 3-Oxo-N-phenethylbutanamide (3). Yield 46%. IR (KBr):3257 (s, NH); 1714 (s, CO); 1644 (s, CO). 1H MNR (400 MHz,DMSO-d6) d ppm: 8.13 (t, 1H, JNH–CH2 = 5.2 Hz, NH); 7.34–7.14 (m,5H, Ph); 3.33–3.30 (m, 2H, CH2–NH); 3.27 (s, 2H, CO–CH2–CO);2.72 (t, 2H, JCH2–Ph = 7.2 Hz, CH2–Ph); 2.10 (s, 3H, CH3).

4.2.1.4. N-(4-Methoxyphenethyl)-3-oxobutanamide (4). Yield56%. IR (KBr): 3263 (m, NH); 1719 (m, CO); 1641 (s, CO). 1H MNR(400 MHz, DMSO-d6) d ppm: 8.10 (t, 1H, JNH–CH2 = 5.3 Hz, NH);7.13 (d, 2H, JH2–H3 = 8.4 Hz, H2+H6); 6.85 (d, 2H, H3+H5); 3.72 (s,3H, OCH3); 3.32–3.18 (m, 4H, CH2–NH–CO–CH2–CO); 2.65 (t, 2H,JCH2–Ph = 7.3 Hz, CH2–Ph); 2.10 (s, 3H, CH3).

4.2.2. Synthesis of 1,4-di-N-oxide-3-methyl-quinoxaline-2-car-boxylic acid aryl amide derivatives (5–40)

The corresponding BFX (1.0 mmol) and the correspondingb-acetoacetamide (1.2 mmol) were dissolved in the minimumamount of methanol. Next, calcium chloride (0.1 mmol) and etha-nolamine (5 drops) were added as catalyst, as described by Stummand Niclas.22 The reaction was stirred at room temperature from 1to 5 h, filtered, and washed with cold ethanol. The solid was dis-solved in dichloromethane and quenched with water. The organic

phase was dried with sodium sulphate and filtered. The solventwas removed in vacuo and precipitated with cold diethyl ether inorder to obtain a yellow solid. The solid was purified by columnchromatography, if necessary.

4.2.2.1. 1,4-Di-N-oxide-3-methylquinoxaline-2-carboxylic acid2-benzylamide (5). Yield: 47%. IR (KBr): 3218 (m, NH); 1677 (s,CO); 1324 (s, N+O�). 1H MNR (400 MHz, DMSO-d6) d ppm: 9.36(t, 1H, JNH–CH2 = 5.8 Hz, NH); 8.52–8.49 (m, 2H, H5+H8); 8.02–7.95(m, 2H, H6+H7); 7.46–7.28 (m, 5H, Ph); 4.57 (d, 2H, CH2); 2.43 (s,3H, CH3). Anal. Calcd for C17H15N3O3: C, 66.02; H, 4.85; N, 13.59.Found: C, 66.02; H, 4.96; N, 13.69.

4.2.2.2. 3,7-Dimethyl-1,4-di-N-oxidequinoxaline-2-carboxylicacid 2-benzylamide (6). Yield: 1.3%. IR (KBr): 3222 (m, NH); 1661(s, CO); 1328 (s, N+O�). 1H MNR (400 MHz, DMSO-d6) d ppm: 9.37(t, 1H, JNH–CH2 = 5.9 Hz, NH); 8.39 (d, 1H, JH5–H6 = 8.8 Hz, H5); 8.31(d, 1H, JH8–H6 = 1.5 Hz, H8); 7.83 (dd, 1H, H6); 7.46–7.28 (m, 5H,Ph); 4.56 (d, 2H, CH2); 2.59 (s, 3H, CH3–C3); 2.41 (s, 3H, CH3–C7).Anal. Calcd for C18H17N3O3: C, 66.87; H, 5.26; N, 13.00. Found: C,66.79; H, 5.26; N, 12.91.

4.2.2.3. 1,4-Di-N-oxide-3,6,7-trimethylquinoxaline-2-carboxylicacid 2-benzylamide (7). Yield: 21%. IR (KBr): 3199 (m, NH); 1679(s, CO); 1328 (s, N+O�). 1H MNR (400 MHz, CDCl3) d ppm: 8.94 (t,1H, JNH–CH2 = 5.6 Hz, NH); 8.05 (s, 1H, H5); 7.98 (s, 1H, H8); 7.49–7.34 (m, 5H, Ph); 4.67 (d, 2H, CH2); 2.65 (s, 3H, CH3–C3); 2.51 (s,3H, CH3–C7); 2.48 (s, 3H, CH3–C6). Anal. Calcd for C19H19N3O3: C,67.66; H, 5.64; N, 12.46. Found: C, 67.89; H, 5.50; N, 12.45.

4.2.2.4. 1,4-Di-N-oxide-7-methoxy-3-methylquinoxaline-2-car-boxylic acid 2-benzylamide (8). Yield: 8%. IR (KBr): 3276 (m,NH); 1655 (s, CO); 1323 (s, N+O�). 1H MNR (400 MHz, DMSO-d6)d ppm: 9.37 (t, 1H, JNH–CH2 = 5.9 Hz, NH); 8.42 (d, 1H,JH5–H6 = 9.5 Hz, H5); 7.81 (d, 1H, JH8–H6 = 2.5 Hz, H8); 7.60 (dd, 1H,H6); 7.47–7.28 (m, 5H, Ph); 4.56 (d, 2H, CH2); 4.00 (s, 3H, OCH3);2.40 (s, 3H, CH3). Anal. Calcd for C18H17N3O4: C, 63.53; H, 5.00;N, 12.35. Found: C, 63.92; H, 5.05; N, 12.48.

4.2.2.5. 7-Chloro-1,4-di-N-oxide-3-methylquinoxaline-2-car-boxylic acid 2-benzylamide (9). Yield: 35%. IR (KBr): 3235 (m,NH); 1669 (s, CO); 1321 (s, N+O�). 1H MNR (400 MHz, DMSO-d6)d ppm: 8.55–8.52 (m, 2H, H5+H8); 8.49 (t, 1H, JNH–CH2 = 5.7 Hz,NH); 7.81 (dd, 1H, JH6–H5 = 9.2 Hz, JH6–H8 = 2.2 Hz, H6); 7.45–7.32(m, 5H, Ph); 4.74 (d, 2H, CH2); 2.79 (s, 3H, CH3). Anal. Calcd forC17H14ClN3O3: C, 59.38; H, 4.07; N: 12.23. Found: C, 59.06; H,4.19; N, 12.13.

4.2.2.6. 6,7-Dichloro-1,4-di-N-oxide-3-methylquinoxaline-2-carboxylic acid 2-benzylamide (10). Yield: 61%. IR (KBr): 3274(m, NH); 1649 (s, CO); 1328 (s, N+O�). 1H MNR (400 MHz, DMSO-d6) d ppm: 8.72 (s, 1H, H5); 8.68 (s, 1H, H8); 8.41 (t, 1H, JNH–

CH2 = 5.8 Hz, NH); 7.44–7.32 (m, 5H, Ph); 4.74 (d, 2H, CH2); 2.79(s, 3H, CH3). Anal. Calcd for C18H15Cl2N3O4: C, 54.11; H, 3.44; N,11.14. Found: C, 54.08; H, 3.38; N, 10.87.

4.2.2.7. 1,4-Di-N-oxide-7-fluoro-3-methylquinoxaline-2-car-boxylic acid 2-benzylamide (11). Yield: 45%. IR (KBr): 3300 (m,NH); 1671 (s, CO); 1323 (s, N+O�). 1H MNR (400 MHz, DMSO-d6)d ppm: 8.61 (dd, 1H, JH5–H6 = 9.5 Hz, JH5–F = 5.0 Hz, H5); 8.52 (d,1H, JNH–CH2 = 5.7 Hz, NH); 8.19 (dd, 1H, JH8–F = 8.5 Hz,JH8–H6 = 2.7 Hz, H8); 7.62 (ddd, 1H, JH6–F = 7.3 Hz, H6); 7.46–7.28(m, 5H, Ph); 4.74 (d, 2H, CH2); 2.77 (s, 3H, CH3). Anal. Calcd forC17H14FN3O3: C, 62.37; H, 4.28; N, 12.84. Found: C, 62.28; H,4.32; N, 12.96.


4.2.2.8. 6,7-Difluoro-1,4-di-N-oxide-3-methylquinoxaline-2-carboxylic acid 2-benzylamide (12). Yield: 38%. IR (KBr): 3292(m, NH); 1670 (s, CO); 1529 (s, N+O�). 1H MNR (400 MHz, DMSO-d6): 9.38 (t, 1H, JNH–CH2 = 6.0 Hz, NH); 8.59–8.48 (m, 2H, H5+H8);7.46–7.26 (m, 5H, Ph); 4.56 (d, 2H, CH2); 2.42 (s, 3H, CH3). Anal.Calcd for C17H13F2N3O3: C, 59.13; H, 3.77; N, 12.17. Found: C,59.36; H, 3.62; N, 11.90.

4.2.2.9. 1,4-Di-N-oxide-3-methyl-7-trifluoromethylquinoxa-line-2-carboxylic acid 2-benzylamide (13). Yield: 28%. IR (KBr):3294 (m, NH); 1670 (s, CO); 1323 (s, N+O�). 1H MNR (400 MHz,DMSO-d6) d ppm: 8.92 (d, 1H, JH8–H6 = 1.7 Hz, H8); 8.70 (d, 1H,JH5–H6 = 9.0 Hz, H5); 8.38 (br s, 1H, NH); 8.03 (dd, 1H, H6); 7.45–7.32 (m, 5H, Ph); 4.75 (d, 2H, CH2); 2.82 (s, 3H, CH3). Anal. Calcdfor C18H14F3N3O3: C, 57.29; H, 3.71; N, 11.14. Found: C, 57.25; H,3.62; N, 11.15.

4.2.2.10. 1,4-Di-N-oxide-3-methylquinoxaline-2-carboxylic acid2-(4-methoxybenzyl)-amide (14). Yield: 82%. IR (KBr): 3174 (m,NH); 1674 (f, CO); 1329 (f, N+O�). 1H MNR (400 MHz, DMSO-d6)d ppm: 9.26 (t, 1H, JNH–CH2 = 5.7 Hz, NH); 8.52–8.50 (m, 2H,H8+H5); 8.02–7.95 (m, 2H, H6+H7); 7.37 (d, 2H, JH20–H30 = 8.7 Hz,H20+H60); 6.95 (d, 2H, H30+H50); 4.49 (d, 2H, CH2); 3.76 (s, 3H,OCH3); 2.42 (s, 3H, CH3). Anal. Calcd for C18H17N3O4: C, 63.71; H,5.05; N, 12.38. Found: C, 63.43; H, 5.11; N, 12.03.

4.2.2.11. 3,7-Dimethyl-1,4-di-N-oxidequinoxaline-2-carboxylicacid 2-(4-methoxybenzyl)-amide (15). Yield: 71%. IR (KBr): 3206(m, NH); 1674 (f, CO); 1326 (f, N+O). 1H MNR (400 MHz, DMSO-d6)d ppm: 9.27 (t, 1H, JNH–CH2 = 5.9 Hz, NH); 8.39 (d, 1H,JH5–H6 = 8.8 Hz, H5); 8.30 (s, 1H, H8); 7.82 (dd, 1H, JH6–H8 = 1.7 Hz,H6); 7.36 (d, 2H, JH20–H30 = 8.5 Hz, H20+H60), 6.94 (d, 2H, H30+H50),4.48 (d, 2H, CH2), 3.76 (s, 3H, OCH3), 2.59 (s, 3H, CH3–C7), 2.39 (s,3H, CH3–C3). Anal. Calcd for C19H19N3O4: C, 64.58; H, 5.42; N,11.89. Found: C, 64.83; H, 5.61; N, 11.50.

4.2.2.12. 1,4-Di-N-oxide-3,6,7-trimethylquinoxaline-2-carbox-ylic acid 2-(4-methoxybenzyl)-amide (16). Yield, 64%. IR (KBr),3210 (m, NH); 1677 (f, CO); 1332 (f, N+O). 1H RMN (400 MHz,DMSO-d6) d ppm, 9.28 (t, 1H, JNH–CH2 = 5.7 Hz, NH); 8.27 (s, 1H,H5); 8.25 (s, 1H, H8); 7.37 (d, 2H, JH20–H30 = 8.5 Hz, H20+H60); 6.94(d, 2H, H30+H50); 4.48 (d, 2H, CH2); 3.76 (s, 3H, OCH3); 2.50 (s, 6H,CH3–C6+CH3–C7); 2.39 (s, 3H, CH3–C3). Anal. Calcd for C20H21N3O4,C, 65.38; H, 5.76; N, 11.44. Found: C, 65.09; H, 5.62; N, 11.63.

4.2.2.13. 1,4-Di-N-oxide-7-methoxy-3-methylquinoxaline-2-carboxylic acid 2-(4-methoxybenzyl)-amide (17). Yield, 79%. IR(KBr), 3283 (m, NH); 1652 (f, CO); 1329 (f, N+O). 1H MNR(400 MHz, DMSO-d6) d ppm, 9.28 (t, 1H, JNH–CH2 = 5.7 Hz, NH);8.40 (d, 1H, JH5–H6 = 9.5 Hz, H5); 7.79 (d, 1H, JH8–H6 = 2.6 Hz, H8);7.59 (dd, 1H, H6); 7.37 (d, 2H, JH20–H30 = 8.5 Hz, H20+H60); 6.94 (d,2H, H30+H50); 4.48 (d, 2H, CH2), 3.99 (s, 3H, OCH3–C7), 3.75 (s, 3H,OCH3–C40), 2.38 (s, 3H, CH3). Anal. Calcd for C19H19N3O5, C, 61.78;H, 5.18; N, 11.38. Found: 61.58; H, 5.21; N, 11.32.

4.2.2.14. 7-Chloro-1,4-di-N-oxide-3-methylquinoxaline-2-car-boxylic acid 2-(4-methoxybenzyl)-amide (18). Yield, 30%. IR(KBr), 3270 (m, NH); 1654 (f, CO); 1330 (f, N+O). 1H MNR(400 MHz, DMSO-d6), 9.27 (t, 1H, JNH–CH2 = 5.6 Hz, NH); 8.53–8.47(m, 2H, H5+H8); 8.03 (dd, 1H, JH6–H5 = 9.2 Hz, JH6–H8 = 2.3 Hz, H6);7.36 (d, 2H, JH20–H30 = 8.7 Hz, H20+H60); 6.94 (d, 2H, H30+H50); 4.48(d, 2H, CH2); 3.76 (s, 3H, OCH3); 2.40 (s, 3H, CH3). Anal. Calcd forC18H16ClN3O4, C, 57.84; H, 4.31; N, 11.24. Found: C, 57.92; H,4.32; N, 11.22.

4.2.2.15. 6,7-Dichloro-1,4-di-N-oxide-3-methylquinoxaline-2-carboxylic acid 2-(4-methoxybenzyl)-amide (19). Yield, 18%. IR(KBr), 3199 (m, NH); 1643 (f, CO); 1323 (f, N+O). 1H MNR(400 MHz, DMSO-d6) d ppm, 9.30 (t, 1H, JNH–CH2 = 5.8 Hz, NH);8.67 (s, 2H, H5+H8); 7.35 (d, 2H, JH20–H30 = 8.5 Hz, H20+H60); 6.94 (d,1H, H30+H50), 4.48 (d, 2H, CH2), 3.75 (s, 3H, OCH3), 2.40 (s, 3H,CH3). Anal. Calcd for C18H15Cl2N3O4, C, 57.84; H, 4.31; N, 11.24.Found: C, 57.92; H, 4.32; N, 11.22.

4.2.2.16. 1,4-Di-N-oxide-7-fluoro-3-methylquinoxaline-2-car-boxylic acid 2-(4-methoxybenzyl)-amide (20). Yield, 31%. IR(KBr), 3213 (m, NH); 1677 (f, CO); 1323 (f, N+O). 1H MNR(400 MHz, DMSO-d6) d ppm, 9.28 (t, 1H, JNH–CH2 = 5.9 Hz, NH);8.57 (dd, 1H, JH5–H6 = 9.5 Hz, JH5–F = 5.2 Hz, H5); 8.24 (dd, 1H,JH8–F = 8.8, JH8–H6 = 2.8 Hz, H8); 7.92 (ddd, 1H, JH8–F = 8.0 Hz, H6);7.36 (d, 2H, JH20–H30 = 8.7 Hz, H20+H60); 6.94 (d, 2H, H30+H50); 4.49(d, 2H, CH2); 3.76 (s, 3H, OCH3); 2.40 (s, 3H, CH3). Anal. Calcd forC18H16FN3O4, C, 60.50; H, 4.51; N, 11.76. Found: C, 60.74; H,4.51; N, 11.73.

4.2.2.17. 6,7-Difluoro-1,4-di-N-oxide-3-methylquinoxaline-2-carboxylic acid 2-(4-methoxybenzyl)-amide (21). Yield, 71%. IR(KBr), 3199 (m, NH); 1645 (f, CO); 1333 (f, N+O). 1H MNR(400 MHz, DMSO-d6) d ppm, 9.29 (t, 1H, JNH–CH2 = 5.8 Hz, NH);8.56–8.50 (m, 2H, H5+H8); 7.36 (d, 2H, JH20–H30 = 8.5 Hz, H20+H60);6.94 (d, 2H, H30+H50); 4.48 (d, 2H, CH2); 3.76 (s, 3H, OCH3); 2.41(s, 3H, CH3). Anal. Calcd for C18H15F2N3O4, C, 57.60; H, 4.03; N,11.20. Found: C, 57.33; H, 3.91; N, 11.14.

4.2.2.18. 1,4-Di-N-oxide-3-methyl-7-trifluoromethylquinoxa-line-2-carboxylic acid 2-(4-methoxybenzyl)-amide (22). Yield,78%. IR (KBr), 9.88%. IR (KBr), 3276 (m, NH); 1648 (f, CO); 1133(f, N+O). 1H MNR (400 MHz, DMSO-d6) d ppm, 9.29 (t, 1H,JNH–CH2 = 6.1 Hz, NH); 8.75 (s, 1H, H5); 8.70–8.69 (m, 1H, H8);8.32–8.26 (m, 1H, H6); 7.36 (d, 2H, JH20–H30 = 8.5 Hz, H20+H60); 6.95(d, 2H, H30+H50); 4.49 (d, 2H, CH2); 3.76 (s, 3H, OCH3); 2.44 (s, 3H,CH3). Anal. Calcd for C19H16F3N3O4, C, 56.02; H, 3.93; N, 10.32.Found: C, 56.39; H, 3.85; N, 10.44.

4.2.2.19. 1,4-Di-N-oxide-3-methylquinoxaline-2-carboxylic acid2-phenylethylamide (23). Yield, 55%. IR (KBr), 3213 (m, NH);1668 (s, CO); 1334 (s, N+O�). 1H MNR (400 MHz, DMSO-d6) dppm, 8.91 (t, 1H, JNH–CH2 = 5.6 Hz, NH); 8.50–8.45 (m, 2H, H5+H8);8.02–7.95 (m, 2H, H6+H7); 7.35–7.22 (m, 5H, Ph); 3.62–3.57 (m,2H, NH–CH2); 2.87 (t, 2H, JCH2–Ph = 7.1 Hz, CH2–Ph), 2.25 (s, 3H,CH3). Anal. Calcd for C18H17N3O3, C, 66.87; H, 5.26; N, 13.00. Found:C, 66.50; H, 5.30; N, 12.79.

4.2.2.20. 3,7-Dimethyl-1,4-di-N-oxidequinoxaline-2-carboxylicacid 2-phenylethylamide (24). Yield, 67%. IR (KBr), 3203 (m, NH);1666 (s, CO); 1326 (s, N+O�). 1H MNR (400 MHz, DMSO-d6) d ppm,8.91 (t, 1H, JNH–CH2 = 5.7 Hz, NH); 8.38 (d, 1H, JH5–H6 = 8.8 Hz, H5);8.23 (s, 1H, H8); 7.82 (d, 1H, H6); 7.35–7.21 (m, 5H, Ph); 3.62–3.57 (m, 2H, NH–CH2); 2.87 (t, 2H, JCH2–Ph = 7.1 Hz, CH2–Ph); 2.59(s, 3H, CH3–C7); 2.23 (s, 3H, CH3–C3). Anal. Calcd for C19H19N3O3,C, 67.65; H, 5.64; N, 12.46. Found: C, 67.28; H, 5.87; N, 12.16.

4.2.2.21. 1,4-Di-N-oxide-3,6,7-trimethylquinoxaline-2-carbox-ylic acid 2-phenylethylamide (25). Yield, 61%. IR (KBr), 3203 (m,NH); 1663 (s, CO); 1328 (s, N+O�). 1H RMN (400 MHz, DMSO-d6) dppm, 8.93 (t, 1H, JNH–CH2 = 5.6 Hz, NH); 8.26 (s, 1H, H5), 8.23 (s, 1H,H8); 7.36–7.22 (m, 5H, Ph); 3.61–3.56 (m, 2H, NH–CH2); 2.87 (t, 2H,JCH2–Ph = 7.1 Hz, CH2–Ph); 2.50 (s, 6H, CH3–C6+CH3–C7); 2.22 (s, 3H,CH3–C3). Anal. Calcd for C20H21N3O3, C, 68.38; H, 5.98; N, 11.97.Found: C, 68.47; H, 6.08; N, 11.75.


4.2.2.22. 1,4-Di-N-oxide-7-methoxy-3-methylquinoxaline-2-car-boxylic acid 2-phenylethylamide (26). Yield, 36%. IR (KBr), 3180(m, NH); 1665 (s, CO); 1329 (s, N+O�). 1H MNR (400 MHz, DMSO-d6) d ppm, 8.93 (t, 1H, JNH–CH2 = 5.5 Hz, NH); 8.40 (d, 1H,JH5–H6 = 9.5 Hz, H5); 7.77 (d, 1H, JH8–H6 = 2.5 Hz, H8); 7.60 (dd, 1H,H6); 7.36–7.23 (m, 5H, Ph); 3.99 (s, 3H, OCH3); 3.61–3.56 (m, 2H,NH–CH2); 2.87 (t, 2H, JCH2–Ph = 7.02 Hz, CH2–Ph); 2.22 (s, 3H,CH3). Anal. Calcd for C19H19N3O4, C, 64.59; H, 5.38; N, 11.90. Found:C, 64.46; H, 5.44; N, 11.66.

4.2.2.23. 7-Chloro-1,4-di-N-oxide-3-methylquinoxaline-2-car-boxylic acid 2-phenylethylamide (27). Yield, 44%. IR (KBr), 3224(m, NH); 1667 (s, CO); 1324 (s, N+O�). 1H MNR (400 MHz, DMSO-d6) d ppm, 8.91 (t, 1H, JNH–CH2 = 5.2 Hz, NH); 8.49 (d, 1H,JH5–H6 = 9.2 Hz, H5); 8.45 (d, 1H, JH8–H6 = 2.3 Hz, H8); 8.03 (dd, 1H,H6); 7.36–7.29 (m, 5H, Ph); 3.62–3.57 (m, 2H, NH–CH2); 2.87 (t,JCH2–Ph = 7.1 Hz, 2H, CH2–Ph); 2.23 (s, 3H, CH3). Anal. Calcd forC18H16ClN3O3, C, 60.42; H, 4.47; N, 11.75. Found: C, 60.14; H,4.50; N, 11. 58.

4.2.2.24. 6,7-Dichloro-1,4-di-N-oxide-3-methylquinoxaline-2-carboxylic acid 2-phenylethylamide (28). Yield, 38%. IR (KBr),3231 (m, NH); 1666 (s, CO); 1322 (s, N+O�). 1H MNR (400 MHz,DMSO-d6) d ppm, 88.93 (t, 1H, JNH–CH2 = 5.6 Hz, NH); 8.66 (s, 1H,H5); 8.64 (s, 1H, H8); 7.34–7.22 (m, 5H, Ph); 3.62–3.57 (m, 2H,NH–CH2); 2.87 (t, 2H, JCH2–Ph = 7.0 Hz, CH2–Ph); 2.24 (s, 3H, CH3–

C3). Anal. Calcd for C18H15Cl2N3O3, C, 55.10; H, 3.82; N, 10.71.Found: C, 54.70; H, 3.92; N, 10.44.

4.2.2.25. 1,4-Di-N-oxide-7-fluoro-3-methylquinoxaline-2-car-boxylic acid 2-phenylethylamide (29). Yield, 77%. IR (KBr), 3231(m, NH); 1669 (s, CO); 1328 (s, N+O�). 1H MNR (400 MHz, DMSO-d6) d ppm, 8.92 (t, 1H, JNH–CH2 = 5.7 Hz, NH); 8.56 (dd, 1H,JH5–H6 = 9.5 Hz, JH5–F = 5.2 Hz, H5); 8.20 (dd, 1H, JH8–F = 8.8 Hz,JH8–H6 = 2.5 Hz, H8); 7.91 (ddd, 1H, JH6–F = 8.0 Hz, H6); 7.36–7.23(m, 5H, Ph); 3.62–3.57 (m, 2H, NH–CH2); 2.87 (t, 2H,JCH2–Ph = 7.0 Hz, CH2–Ph); 2.23 (s, 3H, CH3). Anal. Calcd forC18H16FN3O3, C, 63.34; H, 4.69; N, 12.32. Found: C, 63.02; H,4.80; N, 12.32.

4.2.2.26. 6,7-Difluoro-1,4-di-N-oxide-3-methylquinoxaline-2-carboxylic acid 2-phenylethylamide (30). Yield, 39%. IR (KBr),3240 (m, NH); 1668 (s, CO); 1332 (s, N+O�). 1H MNR (400 MHz,DMSO-d6) d ppm, 8.93 (t, 1H, JNH–CH2 = 5.6 Hz, NH); 8.53–8.47 (m,2H, H6+H8); 7.36–7.20 (m, 5H, Ph); 3.61–3.56 (m, 2H, NH–CH2);2.87 (t, 2H, JCH2–Ph = 7.1 Hz, CH2–Ph); 2.24 (s, 3H, CH3). Anal. Calcdfor C18H15F2N303, C, 60.17; H, 4.18; N, 11.70. Found: C, 60.32; H,4.34; N, 11.87.

4.2.2.27. 1,4-Di-N-oxide-3-methyl-7-trifluoromethylquinoxa-line-2-carboxylic acid 2-phenylethylamide (31). Yield, 9%. IR(KBr), 3243 (m, NH); 1669 (s, CO); 1323 (s, N+O�). 1H MNR(400 MHz, DMSO-d6) d ppm, 8.93 (t, 1H, JNH–CH2 = 5.4 Hz, NH);8.74 (s, 1H, H8); 8.66 (d, 1H, JH5–H6 = 9.2 Hz, H5); 8.27 (d, 1H, H6);7.33–7.31 (m, 5H, Ph); 3.58–3.63 (m, 2H, NH–CH2); 2.88 (t,JCH2–Ph = 7.0 Hz, 2H, CH2–Ph); 2.27 (s, 3H, CH3). Anal. Calcd forC19H16F3N3O3, C, 58.31; H, 4.09; N, 10.74. Found: C, 58.10; H,4.025; N, 10.76.

4.2.2.28. 1,4-Di-N-oxide-3-methylquinoxaline-2-carboxylic acid2-(4-methoxyphenyl)-ethylamide (32). Yield, 10,90%. IR (KBr),3212 (m, NH); 1670 (s, CO); 1328 (s, N+O�). 1H MNR (400 MHz,DMSO-d6) d ppm, 8.88 (t, 1H, JNH–CH2 = 5.5 Hz, NH); 8.48–8.46 (m,2H, H5+H8); 8.00–7.98 (m, 2H, H6+H7); 7.22 (d, 2H, JH20–H30 = 8.6 Hz,H20+H60); 6.88 (d, 2H, H30+H50), 3.72 (s, 3H, OCH3); 3.57–3.52 (m, 2H,NH–CH2); 2.80 (t, 2H, JCH2–Ph = 7.1 Hz, CH2–Ph); 2.28 (s, 3H, CH3).

Anal. Calcd for C19H19N3O4, C, 64.59; H, 5.38; N, 11.90. Found: C,64.25; H, 5.24; N, 11.81.

4.2.2.29. 3,7-Dimethyl-1,4-di-N-oxidequinoxaline-2-carboxylicacid 2-(4-methoxyphenyl)-ethylamide (33). Yield, 19.11%. IR(KBr), 3209 (m, NH); 1666 (s, CO); 1329 (s, N+O�). 1H MNR(400 MHz, DMSO-d6) d ppm, 8.88 (t, 1H, JNH–CH2 = 5.5 Hz, NH);8.38 (d, 1H, JH5–H6 = 8.9 Hz, H5); 8.26 (s, 1H, H8); 7.81 (d, 1H, H6);7.22 (d, 2H, JH20–H30 = 8.2 Hz, H20+H60); 6.89 (d, 2H, H30+H50), 3.72(s, 3H, OCH3); 3.56–3.52 (m, 2H, NH–CH2); 2.80 (t, 2H,JCH2–Ph = 7.0 Hz, CH2–Ph); 2.58 (s, 3H, CH3–C7); 2.25 (s, 3H, CH3–

C3). Anal. Calcd for C20H21N3O4, C, 65.40; H, 5.72; N, 11.44. Found:C, 65.62; H, 5.66; N, 11.58.

4.2.2.30. 1,4-Di-N-oxide-3,6,7-trimethylquinoxaline-2-carbox-ylic acid 2-(4-methoxyphenyl)-ethylamide (34). Yield, 52.94%.IR (KBr), 3203 (m, NH); 1665 (s, CO); 1326 (s, N+O�). 1H MNR(400 MHz, DMSO-d6) d ppm, 8.90 (t, 1H, JNH–CH2 = 5.6 Hz, NH);8.26 (s, 1H, H5); 8.22 (s, 1H, H8); 7.22 (d, 2H, JH20–H3 = 8.5 Hz,H20+H60); 6.88 (d, 2H, H30+H50); 3.73 (s, 1H, OCH3); 3.56–3.51 (m,2H, NH–CH2); 2.79 (t, 2H, JCH2–Ph = 7.1 Hz, CH2–Ph); 2.46 (s, 6H,CH3–C7+CH3–C6); 2.25 (s, 3H, CH3–C3). Anal. Calcd for C21H23N3O4,C, 66.14; H, 6.04; N, 11.02. Found: C, 66.02; H, 6.09; N, 10.99.

4.2.2.31. 1,4-Di-N-oxide-7-methoxy-3-methylquinoxaline-2-carboxylic acid 2-(4-methoxyphenyl)-ethylamide (35). Yield,20.97%. IR (KBr), 3221 (m, NH); 1666 (s, CO); 1325 (s, N+O�). 1HMNR (400 MHz, DMSO-d6) d ppm, 8.89 (t, 1H, JNH–CH2 = 5.5 Hz,NH); 8.40 (d, 1H, JH5–H6 = 9.5 Hz, H5); 7.76 (d, 1H, JH8–H6 = 2.7 Hz,H8); 7.59 (dd, 1H, H6); 7.22 (d, 2H, JH20–H30 = 8.6 Hz, H20+H60); 6.88(d, 2H, H30+H50); 3.98 (s, 3H, OCH3–C7); 3.72 (s, 3H, OCH3–C40);3.57–3.52 (m, 2H, NH–CH2); 2.80 (t, 2H, JCH2–Ph = 7.1 Hz, CH2–Ph);2.22 (s, 3H, CH3). Anal. Calcd for C20H21N3O5, C, 62.66; H, 5.48; N,10.97. Found: C, 62.37; H, 5.66; N, 10.66.

4.2.2.32. 7-Chloro-1,4-di-N-oxide-3-methylquinoxaline-2-car-boxylic acid 2-(4-methoxyphenyl)-ethylamide (36). Yield,48.00%. IR (KBr), 3287 (m, NH); 1655 (s, CO); 1328 (s, N+O�). 1HMNR (400 MHz, DMSO-d6) d ppm, 8.87 (s, 1H, NH); 8.49 (d,1H,JH5–H6 = 9.3 Hz, H5); 8.45 (d, 1H, JH8–H6 = 2.1 Hz, H8); 8.02 (dd, 1H,H6); 7.22 (d, 2H, JH20–H30 = 8.5 Hz, H20+H60); 6.88 (d, 2H, H30+H50);3.73 (s, 3H, OCH3); 3.57–3.52 (m, 2H, NH–CH2); 2.80 (t, 2H,JCH2–Ph = 7.0 Hz, CH2–Ph); 2.26 (s, 3H, CH3). Anal. Calcd forC19H18ClN3O4, C, 58.84; H, 4.65; N, 10.84. Found: C, 58.62; H,4.72; N, 10.40.

4.2.2.33. 6,7-Dichloro-1,4-di-N-oxide-3-methylquinoxaline-2-carboxylic acid 2-(4-methoxyphenyl)-ethylamide (37). Yield,28.40%. IR (KBr), 3230 (m, NH); 1667 (s, CO); 1324 (s, N+O�). 1HMNR (400 MHz, DMSO-d6) d ppm, 8.90 (t, 1H, JNH–CH2 = 5.6 Hz,NH); 8.66 (s, 1H, H5); 8.64 (s, 1H, H8); 7.22 (d, 2H, JH20–H30 = 8.5 Hz,H20+H60); 6.88 (d, 2H, H30+H50); 3.72 (s, 1H, OCH3); 3.57–3.52 (m, 2H,NH–CH2); 2.79 (t, 2H, JCH2–Ph = 6.9 Hz, CH2–Ph); 2.26 (s, 3H, CH3).Anal. Calcd for C19H17Cl2N3O4, C, 54.03; H, 4.03; N, 9.95. Found:C, 53.66; H, 3.92; N, 9.65.

4.2.2.34. 1,4-Di-N-oxide-7-fluoro-3-methylquinoxaline-2-car-boxylic acid 2-(4-methoxyphenyl)-ethylamide (38). Yield,18.76%. IR (KBr), 3223 (m, NH); 1668 (s, CO); 1330 (s, N+O�). 1HMNR (400 MHz, DMSO-d6) d ppm, 8.88 (t, 1H, JNH–CH2 = 5.6 Hz,NH); 8.56 (dd, 1H JH5–H6 = 9.4 Hz, JH5–F = 5.2 Hz, H5); 8.20 (dd, 1H,JH8–F = 8.8 Hz, JH8–H6 = 2.8 Hz, H8); 7.94–7.81 (m, 1H, H6); 7.22 (d,2H, JH20–H30 = 8.6 Hz, H20+H60); 6.88 (d, 2H, H30+H50); 3.72 (s, 3H,OCH3); 3.57–3.52 (m, 2H, NH–CH2); 2.80 (t, 2H, JCH2–Ph = 7.1 Hz,CH2–Ph); 2.28 (s, 3H, CH3). Anal. Calcd for C19H18FN3O4, C, 61.46;H, 4.85; N, 11.32. Found: C, 61.10; H, 4.78; N, 11.54.


4.2.2.35. 6,7-Difluoro-1,4-di-N-oxide-3-methylquinoxaline-2-carboxylic acid 2-(4-methoxyphenyl)-ethylamide (39). Yield,12.75%. IR (KBr), 3242 (m, NH); 1669 (s, CO); 1334 (s, N+O�). 1HMNR (400 MHz, DMSO-d6) d ppm, 8.90 (t, 1H, JNH–CH2 = 5.5 Hz,NH); 8.48–8.46 (m, 2H, H5+H8); 7.22 (d, 2H, JH20–H30 = 8.4 Hz,H20+H60); 6.88 (d, 2H, H30+H50); 3.76 (s, 3H, OCH3); 3.57–3.52 (m,2H, NH–CH2); 2.79 (t, 2H, JCH2–CH2 = 7.0 Hz, CH2–CH2–Ph); 2.26 (s,3H, CH3). Anal. Calcd for C19H17F2N3O4, C, 58.61; H, 4.37; N,10.80. Found: C, 58.86; H, 4.435; N, 10.55.

4.2.2.36. 1,4-Di-N-oxide-3-methyl-7-trifluoromethylquinoxaline-2-carboxylic acid 2-(4-methoxyphenyl)-ethylamide (40). Yield,6.79%. IR (KBr), 3243 (m, NH); 1669 (s, CO); 1324 (s, N+O�). 1HMNR (400 MHz, DMSO-d6) d ppm, 8.90 (t, 1H, JNH–CH2 = 5.7 Hz,NH); 8.74 (s, 1H, H5); 8.66 (d, 1H, JH8–H6 = 1.8 Hz, H8); 8.27 (dd,1H, JH6–H5 = 9.1 Hz, H6); 7.22 (d, 2H, JH20–H30 = 8.6 Hz, H20+H60);6.88 (d, 2H, H30+H50); 3.71 (s, 3H, OCH3); 3.58–3.53 (m, 2H, NH–CH2), 2.80 (t, 2H, JCH2–Ph = 7.0 Hz, CH2–Ph); 2.22 (s, 3H, CH3). Anal.Calcd for C20H18F3N3O4, C, 57.01; H, 4.28; N, 9.98. Found: C,57.33; H, 4.22; N, 9.88.

4.3. General procedure of anti-tuberculosis activity

In vitro evaluation of the anti-tuberculosis activity was carriedout at the GWL Hansen’s Disease Center within the TuberculosisAntimicrobial Acquisition & Coordinating Facility (TAACF) screen-ing program for the discovery of novel drugs for the treatment oftuberculosis. Under the direction of the U.S. National Institute ofAllergy and Infectious Disease (NIAID), the Southern ResearchInstitute coordinates the overall program. The purpose of thescreening program is to provide a resource whereby new experi-mental compounds can be tested for their capability to inhibitthe growth of virulent M.Tb.24

4.3.1. Determination of growth inhibition percentage via MABAThe initial screen is conducted against M.Tb. H37Rv (ATCC

27294) in BACTEC 12B medium using the Microplate Alamar BlueAssay (MABA).25 Compounds exhibiting fluorescence were testedin the BACTEC 460-radiometric system. Compounds effecting<90% inhibition in the primary screen (MIC >6.25 lg/mL) werenot further evaluated.

4.3.2. Determination of minimum inhibitory concentration(MIC) via MABA

Compounds demonstrating at least 90% inhibition in the pri-mary screen were re-tested against M.Tb. H37Rv at lower concen-trations in order to determine the actual minimum inhibitoryconcentration (MIC) in the MABA. The MIC was defined as the low-est concentration effecting a reduction in fluorescence of 90% rela-tive to controls. Rifampicin was used as the reference compound(RIF 0.015–0.125 lg/mL).

4.3.3. Determination of cytotoxicity in VERO cellsCompounds are screened by serial dilution to assess toxicity to

a VERO cell line at concentrations less than or equal to 6.25 lg/mlor 10 times the MIC for M.Tb. H37Rv if sample solubility in culturemedium permits. After 72 h of exposure, viability is assessed onthe basis of cellular conversion of MTT into a formazan productusing the Promega CellTiter 96 Non-radioactive Cell Proliferation

Assay. RIF was used as the reference compound (RIF IC50

>100 lg/mL).

4.3.4. Determination of selectivity index (SI)The selectivity index (SI) is defined as the ratio of the measured

IC50 in VERO cells to the MIC for M.Tb. H37Rv. In general, require-ments for moving a compound into in vivo testing include,MIC 6 6.25 lg/mL and SI P 10 (occasionally lower). RIF was usedas the reference compound (RIF SI >800).

Acknowledgments

This work has been carried out with the financial support of theRIDIMEDCHAG-CYTED and PIUNA project from University of Nava-rra. We also wish to express our gratitude to the Tuberculosis Anti-microbial Acquisition & Coordinating Facility (TAACF) for theevaluation of the anti-tuberculosis activity through research anddevelopment contracts. S.A. is indebted to the Navarra Governmentfor a grant. E.M. is indebted to the La Rioja Government for a grant.

References and notes

1. Milstien, J. Immunological Basis for Immunization/Module 5, Tuberculosis. WHO/EPI/GEN/93.15 (World Health Organization) Global Programme for Vaccinesand Immunization/Expanded Programme on Immunization, Switzerland, 1993.

2. TAACF, http://www.taacf.org/about-TB-background.htm.3. NIAID, http://www3.niaid.nih.gov/topics/tuberculosis/.4. Tangallapally, R. P.; Yendapally, R.; Lee, R. E.; Hevener, K.; Jones, V. C.; Lenaerts,

A. J.; McNeil, M. R.; Wang, Y.; Franzblau, S.; Lee, R. E.. J. Med. Chem. 2004, 47,5276.

5. Jaso, A.; Zarranz, B.; Aldana, I.; Monge, A. J. Med. Chem. 2005, 48, 2019.6. Jan, B.; Erik De, C.; An, C.; Vikki, B.; Jörg-Peter, K. AIDS Res. Hum. Retroviruses

2000, 16, 517.7. Vicente, E.; Villar, R.; Solano, B.; Burguete, A.; Ancizu, S.; Pérez-Silanes, S.;

Aldana, I.; Monge, A. Anuu. Rep. Acad. Nac. Farm. 2007, 73, 927.8. Waring, M.; Ben-Hadda, T.; Kotchevar, A.; Ramdani, A.; Touzani, R.; Elkadiri, S.;

Hakkou, A.; Bouakka, M.; Ellis, T. Molecules 2002, 7, 641.9. Ortega, M. A.; Sainz, Y.; Montoya, M. E.; López de Ceráin, A.; Monge, A.

Pharmazie 1999, 54, 24.10. Sainz, Y.; Montoya, M. E.; Martínez-Crespo, F. J.; Ortega, M. A.; López de Ceráin,

A.; Monge, A. Arzneim.-Forsch. 1999, 49, 55.11. Ortega, M. A.; Montoya, M. E.; Jaso, A.; Zarranz, B.; Tirapu, I.; Aldana, I.; Monge,

A. Pharmazie 2001, 56, 205.12. Ortega, M. A.; Sainz, Y.; Montoya, M. E.; Jaso, A.; Zarranz, B.; Aldana, I.; Monge,

A. Arzneim.-Forsch. 2002, 52, 113.13. Zarranz, B.; Jaso, A.; Aldana, I.; Monge, A. Bioorg. Med. Chem. 2003, 11,

2149.14. Jaso, A.; Zarranz, B.; Aldana, I.; Monge, A. Eur. J. Med. Chem. 2003, 38, 791.15. Villar, R.; Vicente, E.; Solano, B.; Pérez-Silanes, S.; Aldana, I.; Maddry, J. A.;

Lenaerts, A. J.; Franzblau, S. G.; Cho, S. H.; Monge, A.; Goldman, R. C. J.Antimicrob. Chemother. 2008, 62, 547.

16. Vicente, E.; Villar, R.; Burguete, A.; Solano, B.; Pérez-Silanes, S.; Aldana, I.;Maddry, J. A.; Lenaerts, A. J.; Franzblau, S. G.; Cho, S. H.; Monge, A.; Goldman, R.C. Antimicrob. Agents Chemother. 2008, 52, 3321.

17. Vicente, E.; Pérez-Silanes, S.; Lima, L. M.; Ancizu, S.; Burguete, A.; Solano, B.;Villar, R.; Aldana, I.; Monge, A. Bioorg. Med. Chem. 2009, 17, 385.

18. Vicente, E.; Duchowicz, P. R.; Castro, E. A.; Monge, A. J. Mol. Graphics Modell.2009, 28, 28.

19. Vicente, E.; Villar, R.; Burguete, A.; Solano, B.; Ancizu, S.; Pérez-Silanes, S.;Aldana, I.; Monge, A. Molecules 2008, 13, 86.

20. González, M.; Cerecetto, H. In Topics in Heterocyclic Chemistry; Khan, M. T. H.,Ed.; Bioactive Heterocycles IV. Benzofuroxan and Furoxan Chemistry andBiology; Springer: Berlin, Heidelberg, 2007; Vol. 10, p 265.

21. Clemens, R. J. Chem. Rev. 1986, 86, 241.22. Stumm, G.; Niclas, H. J. J. Prakt. Chem. 1989, 331, 736.23. Condensed Pyrazines; Cheeseman, G. W. H., Cookson, R. F., Eds.; Willey J. and

sons: New York, NY, USA, 1979; Vol. 35, p 35.24. TAACF, http://www.taacf.org/Process-text.htm#assays.25. Collins, L. A.; Franzblau, S. G. Antimicrob. Agents Chemother. 1997, 41, 1004.

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Electrochimica Acta 56 (2011) 3270–3275

Contents lists available at ScienceDirect

Electrochimica Acta

journa l homepage: www.e lsev ier .com/ locate /e lec tac ta

,4-Di-N-oxide quinoxaline-2-carboxamide: Cyclic voltammetry and relationshipetween electrochemical behavior, structure and anti-tuberculosis activity

lsa Morenoa, Silvia Pérez-Silanesa, Shravani Gouravaramb, Abinav Macharamb, Saioa Ancizua,nrique Torresa, Ignacio Aldanaa, Antonio Mongea, Philip W. Crawfordb,∗

Neglected Diseases Section, Drug R&D Unit, Centro de Investigación en Farmacobiología Aplicada (CIFA), University of Navarra, C/Irunlarrea s/n, 31008 Pamplona, SpainDepartment of Chemistry, Southeast Missouri State University, Cape Girardeau, MO 63701, USA

r t i c l e i n f o

rticle history:eceived 20 October 2010eceived in revised form 3 January 2011ccepted 4 January 2011

a b s t r a c t

To gain insight into the mechanism of action, the redox properties of 37 quinoxaline-2-carboxamide1,4-di-N-oxides with varying degrees of anti-tuberculosis activity were studied in dimethylformamide(DMF) using cyclic voltammetry and first derivative cyclic voltammetry. For all compounds studied,electrochemical reduction in DMF is consistent with the reduction of the N-oxide functionality to form a

vailable online 22 January 2011

eywords:uinoxaline-2-carboxamide,4-di-N-oxides

radical anion. The influence of molecular structure on reduction potential is addressed and it can be saidthat a general relationship exists between reduction potential and reported antimicrobial activity. Forthose compounds which have demonstrated promising biological activity, the more active the compoundthe less negative the reduction potential typically is. The results suggest the possible participation of

s in tto the

nti-tuberculosisyclic voltammetryeduction potential

charge transfer processeand offer new insights in

. Introduction

Tuberculosis (TB) is an infectious bacterial disease mainlyaused by Mycobacterium tuberculosis (M.tb). TB is a respiratoryransmitted disease affecting nearly 32% of the world’s population.he last data published by WHO established that there were anstimated 9.4 million incident cases and 11.1 million prevalentases of TB in 2008, killing 1.3 million people worldwide [1].he continuing emergence of multidrug-resistant and extremelyrug-resistant strains of M.tb and the increasing incidences ofhe disease in immune-compromised patients highlights thergent need for new drugs which could shorten the treatmenturation and extend the range of effective TB treatment options2,3]. Quinoxaline derivatives show very interesting biologicalroperties and several studies have been published demonstratingheir capability to act not only as antibacterial and antimicrobialut also as antitumoral agents [4–7].

As a result of the anti-tuberculosis research project, our groupas published several papers in which the synthesis and biologi-al evaluation of a large number of quinoxaline and quinoxaline
,4-di-N-oxide derivatives have been described [8–14]. These stud-
es have facilitated a wide structure–activity relationship (SAR)nalysis which lead us to design a group of seventy-seven 3-ethylquinoxaline-2-carboxamide 1,4-di-N-oxide derivatives that

∗ Corresponding author. Tel.: +1 573 651 2166; fax: +1 573 986 6433.E-mail address: [email protected] (P.W. Crawford).

013-4686/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.electacta.2011.01.030

he mechanism of action of quinoxaline di-N-oxides against tuberculosisdesign of future antitubercular drugs.

© 2011 Elsevier Ltd. All rights reserved.

were prepared and tested against M.tb [15,16]. This project allowedus to observe that the lack of one or both of the N-oxide groups gen-erally led to the loss of the antimycobacterial activity [17,18]. Thewell-known N-oxide bio-reduction process and its biological con-sequences seem to be essential for the biological activity displayedby heterocyclic di-N-oxides although little is known about theirmode of action [19,20].

Several studies have investigated and reported the electro-chemical behavior of quinoxaline di-N-oxides and demonstratedreasonable correlations between electrochemical behavior, struc-ture and drug activity [21–23]. Not only heterocyclic di-N-oxidesbut also antibacterial quinones and heterocyclic nitro derivativeshave revealed that compounds showing less negative reductionpotential values exhibited more powerful antibacterial activity[24]. These studies were performed under the hypothesis of theiminium theory. It was proposed that iminium species, which arebelieved to be generated metabolically in vivo, could take part incharge transfer (CT) processes, resulting in the formation of super-oxide and generating toxic oxy radicals. At this point we decidedto study the electrochemical properties of quinoxaline di-N-oxidederivatives which possess the suitable structural requirement andshould be easily reduced due to their electrophilic nature and thestability of the resulting radical which is stabilized by resonance
[22,24,25].
The experimental goal of this study was to determine the reduc-tion potentials for several series of quinoxaline di-N-oxides in orderto obtain evidence of the relationship between the electrochemicalbehavior and the anti-tuberculosis activity.

dx.doi.org/10.1016/j.electacta.2011.01.030

http://www.sciencedirect.com/science/journal/00134686

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dx.doi.org/10.1016/j.electacta.2011.01.030

mica A

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E. Moreno et al. / Electrochi

. Experimental

.1. Chemical synthesis and anti-tuberculosis activity

The compounds involved in this paper and their struc-ures are presented in Table 1. The methods for the synthesisf quinoxaline-2-carboxamide 1,4-di-N-oxide derivatives wereeported elsewhere [15,16].

.2. Pharmacology

In vitro evaluation of the anti-tuberculosis activity was carriedut within the Tuberculosis Antimicrobial Acquisition & Coor-

able 1tructure, cyclic voltammetry data and anti-tubercular activity of quinoxaline derivatives

General structure Cyclic v

Code R R7/R6 Epc,1

1 Benzyl CH3/H −1.6582 Cl/H −1.4823 Cl/Cl −1.2114 F/H −1.5175 CF3/H −1.414

6 2-Phenylethyl H/H −1.6257 CH3/H −1.6498 Cl/H −1.5259 Cl/Cl −1.38710 CF3/H −1.43911 F/H −1.55112 F/F −1.485

13 p-Methoxybenzyl H/H −1.59814 CH3/H −1.61815 Cl/H −1.489

16 p-Trifluoromethylbenzyl H/H −1.59417 CH3/H −1.59918 Cl/H −1.46 (

19 p-Chlorobenzyl H/H −1.53 (20 CH3/H −1.57 (21 Cl/H −1.47 (22 Cl/Cl −1.374

23 p-Bromobenzyl H/H −1.54 (24 CH3/H −1.58 (25 Cl/H −1.44526 Cl/Cl −1.363

27 p-Methylbenzyl H/H −1.58 (28 CH3/H −1.59 (29 Cl/H −1.489

30 2,2-Diphenylethyl H/H −1.59131 CH3/H −1.64 (32 Cl/H −1.49133 Cl/Cl −1.41 (

24 Benzo[d][1,3]dioxol-5-ylmethyl H/H −1.61135 CH3/H −1.62436 Cl/H −1.48837 Cl/Cl −1.403

T, not tested.a Substrate, 0.5 mM; TBAP, 0.10 M; DMF; Pt working electrode; Ag/AgNO3 reference el

urrents reported in �A.b In the first screening all the compounds were evaluated against M.tb H37Rv strain. AERO Cell cytotoxicity assay gives the CC50 which is divided by the IC90 to calculate the Sc IC90: inhibitory concentration 90% (�g/mL) against M.tb H37Rv.d CC50: Cytotoxicity concentration 50% (�g/mL) in VERO Cells.e Selectivity index (SI = CC50/IC90).f Peak is broad and poorly defined; appears as shoulder (sh). E1/2 could not be determing Irreversible.

cta 56 (2011) 3270–3275 3271

dinating Facility (TAACF) screening program for the discoveryof novel drugs for the treatment of tuberculosis. The SouthernResearch Institute coordinates the overall program under the direc-tion of the U.S. National Institute of Allergy and Infectious Disease(NIAID) [26]. The purpose of the screening program is to providea resource whereby new experimental compounds can be testedfor their ability to inhibit the growth of virulent M.tb. Biologi-cal tests have been performed according to previously described
methods.
The initial screening is conducted against M.tb. H37Rv (ATCC27294) in BACTEC 12B medium using the Microplate Alamar BlueAssay (MABA). Compounds are tested in ten 2-fold dilutions, typ-ically from 100 �g/mL to 0.19 �g/mL. The IC90 is defined as the

.

oltammetrya Anti-tuberculosis activityb

Epc,2 E1/2,2 IC90c CC50

d SIe

−1.91 (sh)f – 4.21 >100 >23.77−1.786 −1.660 0.43 >100 >230.94−1.391g – <0.20 >100 >500−1.642 −1.586 1.52 >100 >65.70−1.516 −1.424 0.41 >100 >246.30

−1.848 −1.760 3.05 >100 >32.81−1.907 −1.802 8.62 >100 >11.60−1.72 (sh)f – <0.20 >100 >500−1.557g – <0.19 >30 >153.84−1.543 −1.449 1.153 >30 >26.02−1.696 −1.621 0.504 >100 >198.41−1.664g – 0.517 >30 >58.03

−1.866 −1.766 2.85 >30 >10.54−1.876 −1.774 8.90 >30 >3.37−1.790 −1.661 NT NT NT

−1.90 (sh)f – 16.81 N.T. N.T.−1.90 (sh)f – >100 N.T. N.T.

sh) −1.778 −1.660 6.13 >40 >6.52

sh) −1.738 −1.678 11.04 N.T. N.T.sh) −1.798 −1.735 14.56 N.T. N.T.sh) −1.737 −1.639 29.68 N.T. N.T.

−1.582g – 51.86 N.T. N.T.

sh) −1.795 −1.739 15.61 N.T. N.T.sh) −1.806 −1.744 78.22 N.T. N.T.

−1.685 −1.622 5.33 >40 >7.50−1.564g – 6.92 >40 >5.78

sh) −1.798 −1.729 6.76 >40 >5.92sh) −1.809 −1.735 99.91 N.T. N.T.

−1.787 −1.661 32.04 N.T. N.T.

−1.855 −1.741 15.99 N.T. N.T.sh) −1.87 (sh)f – 16.79 N.T. N.T.

−1.802 −1.660 60.43 N.T. N.T.sh) −1.531 −1.498 66.54 N.T. N.T.

−1.854 −1.763 22.75 N.T. N.T.−1.887 −1.794 13.22 N.T. N.T.−1.760 −1.657 6.99 >40 >5.72−1.525 −1.496 34.92 N.T. N.T.

ectrode; Pt wire counter electrode; 100 mV/s; room temperature; E vs. (Fc/Fc+)/V;

ll compounds showing IC90 ≤ 10 �g/mL moved onto the secondary screening. TheI. Any SI ≥ 10 are considered active for further testing.

ed accurately from the cyclic voltammogram.

3 imica

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272 E. Moreno et al. / Electroch

oncentration effecting a reduction in fluorescence of 90% rela-ive to controls. This value is determined from the dose–responseurve using a curve-fitting program. Any IC90 value of ≤10 �g/mLs considered “Active” for antitubercular activity.

The VERO cell cytotoxicity assay is carried out in parallel withhe TB Dose Response assay. After 72 h exposure, viability isssessed using Promega’s Cell Titer Glo Luminescent Cell Viabil-ty Assay, a homogeneous method for determining the number ofiable cells in culture based on quantitation of the ATP present.ytotoxicity is determined from the dose–response curve as theC50 using a curve fitting program. Then the CC50 is divided by the

C90 for calculating a selectivity index (SI) value. SI values of ≥10re considered for further testing.

.3. Electrochemistry

Dimethylformamide (DMF) and tetrabutylammonium perchlo-ate (TBAP) were obtained commercially in the highest purityvailable and used without further purification. Ferrocene wasbtained from the Aldrich Chemical Company. Cyclic voltammetricxperiments were carried out on a CHI Instruments 630 voltam-etric analyzer. Test solutions contained 0.5 mM of the desired

ompound and 0.10 M TBAP. Scan rates ranged from 50 mV/s to00 mV/s. Half-wave potentials (E1/2) were measured as the aver-ge of the cathodic and anodic peak potentials [27]. For firsterivative cyclic voltammograms, Epc was determined at the pointhere the derivative curve crosses the baseline [27].

A three electrode cell was used for all electrochemical exper-ments, consisting of a Pt-disk (1.6 mm diameter) electrode, at-wire auxilliary electrode, and a Ag/AgNO3 (0.1 M in acetoni-rile) reference electrode. Deoxygenation of all solutions wasccomplished by passing a gentle, constant stream of prepurifiedinitrogen through the solution for 15 min and maintaining a blan-et of the inert gas over the solution during the experiment. To
ccount for daily variations in the reference electrode and liq-id junction potentials, ferrocene (Fc) was added to each solutionollowing measurements of the test compound, and used as annternal reference redox system [28]. All potentials are reportedersus the ferrocene/ferrocinium (Fc/Fc+) redox couple, i.e. or .
ig. 1. Cyclic voltammetric reduction of 14 in DMF at 100 mV/s (E vs. (Ag/AgNO3)/V): (a)he ferrocene redox couple used as a reference for peak potential determination.

Acta 56 (2011) 3270–3275


3.1. Electrochemical behavior

The present study included several series of 37 quinoxaline-di-N-oxides. The redox properties of these substances were studiedusing cyclic voltammetry and first derivative cyclic voltammetryin DMF with TBAP as supporting electrolyte. Results are shownin Table 1 and representative voltammograms are provided inFig. 1. More comprehensive results are found in the supplementaryinformation. All reductions were found to be diffusion controlled,as indicated by constant current functions at varying scan rates[27,29].

For all compounds studied, two cyclic voltammetric waves wereobserved between −0.4 and −1.9 V during reduction. The firstvoltammetric wave was irreversible under these conditions, withpeak potentials, Epc,1, ranging from −1.21 to −1.66 V (vs. Fc/Fc+). Forcompounds 18–21, 23, 24, 27, 28, 31, and 33, this wave appearedas a shoulder to the second wave in the voltammogram. With afew exceptions (see following), the second voltammetric wave wasquasireversible under the conditions used in the study, with peakpotentials, Epc,2, ranging between −1.39 and −1.92 V, and half-wavepotentials ranging between −1.42 and −1.80 V (vs. Fc/Fc+). For com-pounds 1, 8, 16, 17, and 31 this voltammetric wave was not verywell defined, appearing as a shoulder in the cyclic voltammogram.Values of �Ep for this wave (100 mV/s) were generally greater thantheoretical for a reversible, one-electron reduction, and increasedwith increasing scan rate. For compounds 3, 9, 12, 22, and 26, thesecond voltammetric wave was irreversible under the conditionsutilized in this study. Calculated ipa/ipc ratios for compounds 2,5–8, 11, 13, 15, 21, 23, 24, 27, 29–35, and 37 were close to unity(0.61–1.24) at all scan rates, indicating the formation of relativelystable reduction products for this process on the time scale of thevoltammetric scans. Current ratios that deviated significantly fromunity (0.24–0.55) were observed for all other derivatives, indicative
of greater kinetic or other complications following the reduction[29]. No reduction waves were observed at potentials negative ofthe second voltammetric wave for these quinoxaline-1,4-dioxidederivatives under the conditions used in this study, with the excep-tion of compounds 3–5, 9–12, 22, 26, 33 and 37. For the latter
single scan, (b) multiple scans, (c) first derivative and (d) cyclic voltammogram for

E. Moreno et al. / Electrochimica Acta 56 (2011) 3270–3275 3273

Table 2Hammett substituent constants.a

Compound R7/R6 �p−x �m−x (�m−x + �p−x)/2

6 H/H 0 0 07 CH3/H −0.17 −0.07 −0.128 Cl/H 0.23 0.37 0.309 Cl/Cl 0.46 0.74 0.6010 CF3/H 0.55 0.41 0.48

cp

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(a)

(b)

(c)

-1.7

-1.65

-1.6

-1.55

-1.5

-1.45

-1.4

-1.35

0.60.40.20-0.2

Ep

c,1/V

(σm-x + σp-x)/2

-2

-1.9

-1.8

-1.7

-1.6

-1.5

0.60.40.20-0.2

Ep

c,2/V

(σm-x + σp-x)/2

-1.85

-1.8

-1.75

-1.7

-1.65

-1.6

-1.55

-1.5

-1.45

-1.4

0.60.40.20-0.2

E1

/2,2

/V

(σm-x + σp-x)/2

11 F/H 0.06 0.34 0.2012 F/F 0.12 0.68 0.40

a Hammett substituent constant values are taken from Ref. [30].

ompounds, a third irreversible reduction wave was observed atotentials ranging from −1.89 to −2.42 V.

Examination of the data indicates the influence of quinoxalinetructure on potential. The 37 compounds can be broken downnto 9 different analogues based on structure, with the monohloro and methyl substituted quinoxalines existing as mixturesf 6- and 7-substituted positional isomers. Within each analo-ous series, replacement of the 6-/7-hydrogen atom with a singlelectron-withdrawing substituent group, i.e. chloro, fluoro, andrifluoromethyl, resulted in a positive shift in the peak poten-ials for both voltammetric waves, whereas replacement of the-/7-hydrogen atom with a single electron-releasing methyl groupesulted in a negative shift. As an example, comparison of theotentials for compounds 13 and 15 shows that replacement ofhe hydrogen atom with the chloro group resulted in a +109 mVotential shift for the first voltammetric wave, and a +76 mVhift in the second voltammetric wave (+105 mV shift in E1/2).eplacing the hydrogen atom in compound 13 with the methylroup in compound 14 resulted in a −20 mV potential shift forhe first voltammetric wave, and a −10 mV shift in the secondoltammetric wave (−8 mV shift in E1/2). Similarly, comparison ofompound 6 with compounds 10 and 11 shows that substitut-ng the 7-hydrogen with trifluoromethyl or fluoro groups resultsn a +186 mV and +74 mV shift in the first voltammetric wave,espectively. The reduction potentials for the quinoxaline deriva-ives within each analogue generally fit the modified Hammettquation, �E1/2 = ��R�x (correlation coefficients ranged from 0.89o 1), where �x is the polar inductive electronic substituent con-tant taken as the average of the sum of �m−x and �p−x (Table 2)nd � is the reaction constant [30] (Fig. 2) (for quinoxaline deriva-ives substituted in the benzene ring, the use of (�m−x + �p−x)/2 haseen recommended [31]). These effects are consistent with facilita-ion of reduction by a positive charge at the site of reduction [30], ineeping with previous reports [21,22,25,30,32]. Addition of a sec-nd electron-withdrawing group, i.e. chloro or fluoro, enhances thisffect. For example, comparison of the 6-,7-dichloro derivatives (3,, 22, 26, 33 and 37) with the corresponding 6-/7-chloro deriva-ives (2, 8, 21, 25, 32 and 36) shows that addition of the secondhloro group shifts the potential of the first voltammetric waveositively by +81 to +271 mV, and that of the second voltammetricave by +121 to +395 mV. No apparent correlation was observed

etween substitution in the amide side chain and potential (cf. 1,4, 17, 20, 24, 28, 31 and 35). In the latter case, the substituents areresumably too far removed from the site of reduction.

.2. Relationship between electrochemical behavior andnti-tuberculosis activity

With regards to the mechanism, heterocyclic di-N-oxides pos-
essing the diiminium structure are expected to undergo onelectron reduction to form a radical anion [21,33–36] (Fig. 3),nd in fact reduction of quinoxaline 1,4-di-N-oxide in DMF haseen shown via ESR and cyclic voltammetry to involve the nitroneunction to form the radical anion [37]. The electrochemical reduc-
Fig. 2. Plot of (a) peak potential (E vs. Fc/Fc+) for the first voltammetric wave, (b) peakpotential (E vs. Fc/Fc+) for the second voltammetric wave, and (c) half-wave poten-tials (E vs. Fc/Fc+) for the second voltammetric wave of quinoxaline-di-N-oxides6–12 against (�m−x + �p−x)/2.

tions of quinoxaline 1,4-di-N-oxides in aprotic solvent systemshave been reported previously [21,22,25,32,37]. In most cases, areversible reduction wave attributed to reduction of the N-oxidefunctionality has been reported in DMF in the vicinity of thoseobserved in the present study [21,22,25,37]. For the compoundsin the present study, electrochemical reduction in DMF is consis-tent with reduction of the N-oxide functionality to form a radicalanion as well. The second reduction wave observed could be due tothe formation of the dianion, or to reduction of the product formedfrom a chemical step involving the radical anion [32]. The latter issupported by the irreversibility of the first reduction wave. Kayeand Stonehill pointed out that an objection to the iminium theoryis that some potentials observed are too negative although sev-eral reports indicate that reduction potential in vivo may be betterthan in vitro [24], and N-oxides are known to undergo bioreduction[19,20].

A relationship between reduction potential and antitubercu-losis activity could be suggested by the data. Different stagesin the mechanism of action against tuberculosis for these com-pounds should be considered. In this sense, in the first stage thequinoxaline di-N-oxide could be activated through a bioreduction

3274 E. Moreno et al. / Electrochimica Acta 56 (2011) 3270–3275

ig. 3.

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200

400

600

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1600

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rocess to form a reactive radical anion, which could lead fur-her to superoxide ion or other toxic oxy radical species, or toirect macromolecular interactions in subsequent stages (Fig. 3).

f such a mechanism occurs for these compounds, bioreductivectivation would generally be expected to be more facile for theore easily reduced derivatives. Examination of the results for the

ompounds which have shown biological activity based on theirelectivity index values (1–13) (Table 1) shows that in generalhe less negative the reduction potential is, the more active theompounds are. In fact, for compounds showing a Epc,1 < −1.6 V
he IC90 increases almost exponentially (Fig. 4). Not only that,ut compounds showing a Epc,1 < −1.6 V also present a selectivity
ndex (SI) much lower than compounds with a reduction poten-ial that is less negative (Fig. 5). Caution is obviously warranted innterpreting such electrochemical data, however, since not all the

(a)

(b)

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-1.2-1.3-1.4-1.5-1.6-1.7

Epc,1/V

Fig. 5. Plot of reduction potentials (E vs. Fc/Fc+) for the first voltammetric wave ofcompounds 1–13 against selectivity index (SI).

compounds tested exhibited appreciable activity. Many other fac-tors besides bioreduction must also be considered in determiningthe in vivo mechanism of action of antituberculosis compounds,such as solubility, metabolism, diffusion, membrane permeability,stereochemistry, absorption, and active site binding among others.Thus, absolute correlation between electrochemical behavior andanti-tuberculosis activity is not reasonable. However, the resultsof this study seem to suggest the participation of charge transferprocesses in the overall mechanism of action of these quinoxalinedi-N-oxides against tuberculosis; however, the exact mechanismwas not investigated further.

4. Conclusions

Thirty-seven quinoxaline di-N-oxides have been studied and allof them showed two cyclic voltammetric waves between −0.4 and−1.9 V (vs. Fc/Fc+) during reduction. The first voltammetric wavewas irreversible for all the studied compounds under the conditionsof the study. A relationship between electrochemical behavior andquinoxaline structure can be established and it can be said thatthe insertion of an electron-withdrawing group on the quinoxalinering results in a less negative reduction potential and makes the
bio-reduction more facile. Nevertheless, no relationship could befound between substitution on the amide chain and the reductionpotential. These results are expected to offer new insights into thestudy of the mechanism of action for future antitubercular drugs.

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E. Moreno et al. / Electrochi

cknowledgments

This work has been carried out with the financial support ofhe PIUNA project from University of Navarra. We also wish toxpress our gratitude to the Tuberculosis Antimicrobial Acquisition

Coordinating Facility (TAACF) for the evaluation of the anti-uberculosis activity through research and development contracts.. M. is indebted to the La Rioja Government for a grant.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.electacta.2011.01.030.

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