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ISSN (Print): 2220-2625 ISSN (Online): 2222-307X

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Pak. J. Chem Table of Contents

ISSN (Print): 2220-2625 ISSN (Online): 2222-307X

Author(s) - Title Page #

R. Naz, R. Azmat, N. Qamar, H. Jaffery and S. Nisar - Comparative Kinetic and Mechanistic Study of

Oxidation of -Cyclodextrin by Potassium Dichromate

1

M. Hassan, S. Nisar, S. A. Kazmi, M. Qadri, S. Imad and R. Naz- Kinetics and Mechanism of Reduction of Fe(III)-Acetohydroxamic Acid by Hydroxylamine Hydrochloride at Acidic pH

8

S. Rasool, Aziz-ur-Rehman, M. A. Abbasi, S. Z. Siddiqi, A. S. Gondal, H. A. Noor, S. Sheral and I.

Ahmad - Antibacterial and Enzyme Inhibition Study of Hydrazone Derivatives Bearing 1, 3, 4-Oxadiazole

14

M. A. Abbasi, S. Manzoor, Aziz-ur-Rehman, S. Z. Siddiqui, I. Ahmad, R. Malik, M. Ashraf

Qurat-ul-Ain and S. A. A. Shah - Synthetic N-Alkyl/aralkyl-4-methyl-N-(naphthalen-1-yl)

benzenesulfonamides as Potent Antibacterial Agents

23

A. M. Rawa'a, N. M. Tariq And S. U. Wisam - The Factors Effecting The Formation of Curcumin-Al (Iii)

Complexes 30

P. Byabartta - Ruthenium azo complexes: Synthesis, spectra and electrochemistry of dithiocyanato-bis-

{1-(alkyl)-2-(arylazo)imidazole}ruthenium(II) 36

M. A. Naveed, N. Riaz, M. Saleem, B. Jabeen, M. Ashraf, U. Alam and A.Jabbar - α-Glucosidase

Inhibitory Constituents from Ficus bengalensis 42

Pak. J. Chem. 5(1): 1-7, 2015 Full Paper

ISSN (Print): 2220-2625

ISSN (Online): 2222-307X DOI: 10.15228/2015.v05.i01.p01

*Corresponding Author Received 27th January 2015, Accepted 10th February 2015

Comparative Kinetic and Mechanistic Study of Oxidation of -Cyclodextrin by

Potassium Dichromate

*1R. Naz,

1R. Azmat,

2N. Qamar,

1H. Jaffery and

1S. Nisar

*1Department of Chemistry, University of Karachi, 75270, Karachi, Pakistan.

2Department of Chemistry, Jinnah University for Women, 74600 Karachi, Pakistan

E-mail: *[email protected]

ABSTRACT A comparative kinetic and mechanistic study of oxidation of β-Cyclodextrin (β-CD) by Potassium Dichromate (K2Cr2O7) in

presence of aqueous H2SO4 medium was monitored at λmax 350 nm spectrophotometrically. The oxidation reaction follows first

order kinetics with respect to [β-CD] and [Cr2O7-2] and markedly increased by increasing [H+]. The slow reaction was

accelerated with Fe (III) as a catalyst that enhances the rate significantly. The results of varying temperature were used to compute different activation parameters. The values of activation energy Ea were 37.70KJ mol-1 for catalysed and 50.183 KJ mol-1for

uncatalysed reactions. These clearly indicate that Fe (III) greatly reduced the activation barrier thereby increases the rate of

reaction. Oxidation product results due to the oxidation of -CH2OH group present on each glucose monomer of β-CD.A

mechanism consistent with kinetic and thermodynamic data is also proposed that involves two-electron reduction of Cr (VI).

Keywords: β-Cyclodextrin, K2Cr2O7, Fe (III) catalyst, Ea, two-electron reduction.

1. INTRODUCTION Carbohydrates are the fuel of life, being the main energy source for living organisms and the central pathway of

energy storage and supply for most cells. The study of the carbohydrates and their derivatives has greatly enriched

chemistry, particularly with respect to the role of molecular shape and conformation in chemical reactions1.β-

Cyclodextrin (β-CD) is a naturally occurring carbohydrate, formed by α 1→4 linkage of seven D-glucopyranose units.

Structurally, they are hollow truncated cones in which the inner cavity is lipophilic and exterior is hydrophilic due to

the presence of primary hydroxyl groups. These days, applications of cyclodextrins have no limits from Industrial

aspects because this outstanding constructional feature of cyclodextrins molecule makes them able to form inclusion complexes and becomes a great deal of interest in food, drugs, dyes and perfumery industries

2-9. Numerous β-CD

reaction studies involving selective modifications of primary and secondary alcoholic groups of cyclodextrin have

been conducted through different organic oxidants10-12

to improve their physiochemical behaviour (solubility, surface activity etc.). However the information about the reaction kinetics and mechanism is still lacking.

Only a very few reports are available with inorganic oxidant13, 14

. Potassium Dichromate K2Cr2O7, has been

used to oxidize a variety of organic compounds15-19

but still not reported for the oxidation of β-CD. Therefore, the

object of this research is to perform the oxidation kinetics of β-CD by Potassium dichromate in H2SO4 medium and to study reaction kinetics by conventional spectrophotometric method in order to establish stoichiometry and reaction

mechanism. The reaction kinetics is also studied in presence of Fe (III) as a catalyst and results are compared to that of

uncatalysed reaction. The crucial information about thermodynamics and spectral evidences are gathered to develop mechanistic approach consistent with product analysis.

2. EXPERIMENTAL The stock solutions for kinetics investigation were prepared in doubly distilled water. β-CD, Potassium dichromate, Iron (III) chloride, and sulfuric acid used were of AR quality (Merck and BDH). Required amount of all reagents were

mixed in a beaker placed on thermostat bath DFS KW-1000DB. The rate of disappearance of Cr2O72-

was followed

spectrophotometrically by monitoring the absorbance at known time intervals. Neither β-CD nor product showed any absorbance at 350 nm. Pseudo-first order rate constants (k1) of these kinetic runs were obtained by slopes of the plot of

ln (Absorbance) versus time.

3. RESULTS AND DISCUSSION

3.1 Stoichiometry and Product Analysis The stoichiometry of oxidation of β-cyclodextrin (β-CD) was determined by adding a warm solution of β-CD (3x10

-3

mol dm-3

, 30°C) to a warm solution of K2Cr2O7 (3x10-4

mol dm-3

) having H2SO4 (1.6 mol dm-3

) until the

decolorization of K2Cr2O7 was completed. Different sets of experiments revealed that 1 mole of K2Cr2O7 reacts with

7.5 mole of β-CD.The same procedure was repeated with reaction mixtures containing 1x10-5 mol dm

-3 Iron (III)

chloride. On successive experiments, it was found that 1 mole of K2Cr2O7 reacts with 5 mole of β-CD. The obtained

stoichiometric results were also confirmed by mole ratio method which produced same results.

To identify the oxidation product an experiment was setup, as reported earlier14

in which all solutions β-CD (3x10

-3 mol dm

-3), K2Cr2O7 (3x10

-4 mol dm

-3), and H2SO4 (1.6 mol dm

-3) were added to the reaction vessel. A separate

Pakistan Journal of Chemistry 2015

2

reaction vessel was also prepared containing Iron (III) chloride (1x10-5 mol dm

-3) in addition to the other solutions.

Both reactions were conducted at30°C.After the complete disappearance of color,100cm3

saturated solution of 2, 4

dinitrophenyl hydrazine in 2M HCl were added to the 50 cm3 reaction mixture taken from reaction flask. The resultant

mixture was left overnight in a refrigerator. The precipitates of 2, 4 dinitrophenyl hydrazone were filtered, washed,

and dried. The IR spectrum of 2, 4 dinitrophenyl hydrazone confirmed the presence of aldehyde; carbonyl stretching at 1645cm

-1, band of aldehyde stretching at 2926 cm

-1. It is obvious now that an aldehyde was formed after the oxidation

of β-CD, as the spot test for acid was negative.

3.2 Spectral evidences The reaction mixtures were also investigated by UV-Visible spectroscopy in the 300-600nm region. Spectral scan was

performed for two reaction mixtures containing β-cyclodextrin (3x10-3

mol dm-3

), K2Cr2O7 (3x10-4

mol dm-3

), and

H2SO4 (1.6 mol dm-3

) and another mixture contained same amounts with Iron (III) chloride (1x10-5

mol dm-3

)

respectively. Both spectra showed the two peak maxima at 350nm that corresponds Cr (VI), and 450nm to Cr (III). Similar

scans were taken after 2 hours of reaction (Fig. 1 and 2). It is evident from the spectra that as the reaction proceeds Cr

(VI) consumes at 350nm and Cr (III) grows at around 450nm. Thus, it was confirmed that under present experimental conditions, Cr (III) ion formed as product.

3.3 Kinetic measurements The kinetics of oxidation of β-cyclodextrin with potassium dichromate K2Cr2O7is carried out in aqueous acidic

medium. The reaction is also studied in presence of Iron (III) chloride as catalyst. Sulfuric acid (H2SO4) is used to

maintain the pH throughout the reaction.The reactions were monitored spectrophotometrically by change in absorbance of Cr2O7

2- at λmax 350nm. The rate constants were calculated via slope of the plot of ln (A)versus time. The

observed rate constants are obtained in form of pseudo-first order rate constants (k1) under sustained kinetic

conditions.

3.4 Effect of [Cr2O7-2]

The reactions were carried out at various concentration of [Cr2O7-2

] ranging from 1x10-4

– 5x10-4 mol/dm

3 keeping

other variables [β-CD], [H+] and temperature constant. The values of k1 (Tab.1) were independent of varying [Cr2O7

-2]

showing a good agreement with a first order dependence on [Cr2O7-2

].It was noted that k1 was significantly increased in presence of catalyst showing Fe (III) is accelerating rate of reaction.

3.5 Effect of [β-CD] The effect of [β-CD] was also investigated by varying concentrations 1x10

-3 – 5x10

-3 mol/dm

3 at constant parameters.

Tab. 1 shows the comparative values of rate constant which increases with increasing [β-CD], indicating first order

kinetics. The values of n, order of reaction for both catalysed and uncatalysed reaction were 0.796 and 0.759

respectively (Fig. 3).

3.6 Effect of [H+] The variation in [H

+] (1.0, 1.2, 1.4, 1.6 and1.8 mol/dm

3) at other constant parameters showed the dependence of k1for

both catalysed and uncatalysedupon [H+] (Tab. 1). Change in ionic strength maintained by K2SO4 had no effect. It

was observed that rate of reaction increases as [H+] increases i.e. the reaction is acid dependent. A plot of log k1against

log [H+] (Fig. 4) gave the value of 2.2 and 1.8 for catalysed and uncatalysed reactions respectively.

3.7 Effect of [Fe(III)] Effect of catalyst Fe(III) was evaluated by using different concentration at constant[Cr2O7

-2] (1x10

-4 mol/dm

3), [β-CD]

(1x10-4

mol/dm3) and [H

+] (1.6 mol/dm

3). It was found that k1 is independent of varying [Fe(III)]. The catalyst played

a significant role in the reduction of activation energy from 50.183 to 37.70KJ mol-1

(Tab.2) and hence increased the

rate of reaction.

3.8 Effect of Temperature Dependence of temperature on rate of reaction was also studied at 30, 35, 40, 45 and 50

oC by keeping [β-CD],

[Cr2O7-2

], and [H+] constant (Tab. 2).The influence of temperature was also checked in presence of [Fe (III)]. Both

reactions followed Arrhenius Plot (Fig. 5) from which activation energy Eawas calculated. The values of Eawere37.70

and 50.183 KJ mol-1

for catalysed and uncatalysed reaction respectively (Tab. 3). It is clear that the catalyst played a significant role in reducing Activation barrier. Tab. 3 also presents the values of other activation parameters that were

computed by plotting Ln(k/T) against 1/T (Fig. 6). The high negative values of ΔS# (-181.96 &-215.922 J mol

-1)

shows that the transition state is highly solvated.

Naz et al, 2015

3

Table-1: Observed pseudo-first order rate constants in the oxidation of β-Cyclodextrin by Dichromate ion, with and without

addition of Catalyst Iron (III) Chloride

[K2Cr2 O7] ×10-4

mol/dm3

[β-Cyclodextrin]

×10-3

mol/dm3

[H2SO4]

mol/dm3

[FeCl3] ×10-5

mol/dm3

k1 ×10-5

s-1

Catalyzed Uncatalyzed

1 3 1.6 1 2 0.8

2 3 1.6 1 2 0.8

3 3 1.6 1 2 0.9

4 3 1.6 1 2 0.9

5 3 1.6 1 3 0.8

3 1 1.6 1 1 0.6

3 2 1.6 1 2 2

3 3 1.6 1 2 3

3 4 1.6 1 3 2

3 5 1.6 1 4 2

3 3 1.0 1 0.8 0.5

3 3 1.2 1 1 0.7

3 3 1.4 1 1 0.6

3 3 1.6 1 2 0.7

3 3 1.8 1 3 2

3 3 1.6 1 2 -

3 3 1.6 2 2 -

3 3 1.6 3 2 -

3 3 1.6 4 3 -

3 3 1.6 5 3 -

Temperature = 303K

Table-2: Effect of temperature on the rate constant value for oxidation of β-Cyclodextrin, with and without addition of Iron (III)

Chloride

Temperature (K)

β-Cyclodextrin

Uncatalysed

k1 ×10-5

s-1

Catalysed

k1 ×10-5

s-1

303 1 3

308 2 3

313 2 5

318 3 6

323 4 7

[K2Cr2 O7]= 3x10-4 mol/dm3[β-Cyclodextrin] = 3×10-3 mol/dm3 [H2SO4] =1.6M

[FeCl3] = 1x10-5 mol/dm3

Table-3: Arrhenius and thermodynamic activation parameters for oxidation of β-Cyclodextrin, with and without addition of Iron

(III) Chloride.

Parameters Uncatalysed

reaction

Catalysed

reaction

Ea

(KJ mol-1) 50.183 37.70

ΔH#

(KJ mol-1) 47.78 35.23

ΔS#

(J mol-1) -181.96 -215.922

ΔG#

(KJ mol-1) 57 67.61

Temperature = 303K


4

Fig-1: An overlay showing progress of reaction at Temp = 303K of [β-CD] = 3x10-3 mol dm-3, [K2Cr2O7] 3x10-4 mol dm-3, and

[H2SO4]1.6 mol dm-3

.

Fig-2: An overlay showing progress of reaction at Temp. = 303K. [β-CD] = 3x10-3 mol dm-3, [K2Cr2O7] 3x10-4 mol dm-

3,[H2SO4] 1.6 mol dm-3, and [FeCl3] = 1x10-5 mol/dm3

Fig-3: Plot of log [β-CD] versus log k1

nm.

300.00 400.00 500.00 600.00

Abs.

0.200

0.150

0.100

0.050

0.000

nm.

300.00 400.00 500.00 600.00

Abs

.

0.200

0.150

0.100

0.050

0.000

Abs.

λ (nm)

Abs.

λ (nm)

Naz et al, 2015

5

Fig-4: Plot of log [H2SO4] versus log k1.

Fig-5: Plot of 1/T versus ln k

Fig-6: Plot of 1/T versus ln (k/T).

3.9 Mechanism As β-CD comprises of seven α-D-glucopyranose units joined by α(1-4) bonds, OH-1 of each unit is blocked by

glycosidic linkage14

. This suggests that the reactive site is to be C-6 and -CH2OH group is the one where oxidation

takes place by Cr(VI). In the light of all gathered experimental facts following mechanism is proposed:


6

O

O

H2C

OHO

HO

7

7 + Cr2O72-

O

O

H2C

OHO

7

7

O Cr

O

O

O CrO2O-

(I)

I + 2H+ IH2+

IH2+

K1

K2

k2

Slow

Cr(IV) +

O

O

OHC

OHO

7

7Cr(VI) +

Cr(IV) + CrVI 2Cr(V)

Cr(V) + Substrate Product + Cr(III)

Scheme 1.

+ H2O

Scheme 1 shows that first step involves the combination of Cr(VI) with β-CD to form an intermediate anionic complex (I) which becomes doubly protonated to form a new complex (IH2

+)

16. This resulting complex slowly

decomposes in presence of water. Here in rate determining step water molecule abstracts proton resulting in the

formation of product. In our proposed mechanism, redox reaction proceeds through two-electron steps and Cr (VI) is formed.Cr (VI) involves in fast steps, does not accumulate in the reaction [A] and finally Cr (III) is formed.

2H++ 15β-CD + 2Cr (VI) 15product + 2Cr (III)

The rate law consistent with kinetic observations can be expressed as:

_d[Cr(VI)] / dt = k2[IH2

+] (1)

Since

[IH2+] = K2 [I] [H

+]

2 (2)

Therefore eq. 1 can become: _d[Cr(VI)] / dt =k2K2 [I] [H

+]

2 (3)

As,

[I] = K1[Cr2O7-2

] [β-CD] (4)

Replacing [I] in eq. 3 _d[Cr(VI)] / dt =k2K1K2[Cr2O7

-2] [β-CD][H

+]

2 (5)

As,

[Cr (VI)]T=[Cr2O7-2

] + [I-] (6)

Then,

[Cr (VI)]T=[Cr2O7-2

] {1 + K1[β-CD]} (7)

Or,

[Cr2O7-2

] = [Cr (VI)] T/1 + K1[β-CD] (8) Therefore, eq. 5 becomes:

_d[Cr(VI)] / dt =k2K1K2 [β-CD][H

+]

2 [Cr (VI)]T (9)

1 + K1[β-CD] As catalyst also affects the rate of reaction, the rate law becomes:

_d[Cr(VI)] / dt =k2K1K2 [β-CD][H

+]

2 [Cr (VI)]T [Fe (III)] (10)

1 + K1[β-CD]

Naz et al, 2015

7

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http://dx.doi.org/10.1016/j.ijpharm.2012.06.055. 3. Lopez-Nicolas, J. M., Rodriguez-Bonilla, P., Garcia-Carmona, F. C., Rev.Food Sci. Nut. (2014), 54, 251-276,

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4. Siva, S., Thulasidhasan. J., Rajendrian, N., Spectrochm. Acta. Part A: Mol. Biomol. Spect. (2013), 115, 559-567, http://dx.doi.org/10.1016/j.saa.2013.06.079.

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6. Buschmann, H. J., Schollmeyer, E. J., Inclu. Phenom. Mol. Recog. Chem. (1997). 29, 167-174, http://dx.doi.org/10.1023/A:1007981816611.

7. Grachev, M. K., Russian Chem. Rev. (2013), 82, (11), 1034-1046,

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http://dx.doi.org/10.1021/ma400695p.

9. Bricout, H., Hapiot, F., Ponchel, A., Tilloys, S., Monflier, E., Sustain. (2013), 1, 924-945, http://dx.doi.org/10.3390/su1040924.

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6215(00)00129-4.

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165-171, http://dx.doi.org/10.1016/j.colsurfa.2006.08.048.

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Polyhed.(2006), 25, 1483-1490.

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http://dx.doi.org/10.1016/j.ijpharm.2012.06.055

http://dx.doi.org/10.1080/10408398.2011.582544

http://dx.doi.org/10.1016/j.saa.2013.06.079

http://dx.doi.org/10.1080/00405009808658641

http://dx.doi.org/10.1023/A:1007981816611

http://dx.doi.org/10.1070/RC2013v082n11ABEH004381

http://dx.doi.org/10.1021/ma400695p

http://dx.doi.org/10.3390/su1040924

http://dx.doi.org/10.1016/S0008-6215(00)00129-4

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http://dx.doi.org/10.1139/v92-258


ISSN (Print): 2220-2625


*Corresponding Author Received 23rd January 2015, Accepted 12th February 2015

Kinetics and Mechanism of Reduction of Fe(III)-Acetohydroxamic Acid by

Hydroxylamine Hydrochloride at Acidic pH

1M. Hassan,

*1S. Nisar,

1S. A. Kazmi,

1M. Qadri,

2S. Imad and

1R. Naz

*1Department of Chemistry, University of Karachi, Karachi, Pakistan.

2Chemical Metrology Division, National Physical and Standard Laboratory (PCSIR), Islamabad. Pakistan.

E-mail: *[email protected]

ABSTRACT The complexes of Fe(III)-AHA were prepared in acetate buffer of pH 4.5, 5.0 and 5.5. Stopped flow technique was used to study

the reduction of these complexes by hydroxylamine hydrochloride. The reaction shows a biphasic behavior which is significantly

pH dependent. Rate of both phases increases with hydrogen ion concentration. The rate was found to be first order with respect to

[Fe(III)]. The overall rate was neither first order nor second order but there was a pre-equilibrium in mechanism. Slight increase in

the values of kobs might be a consequence of increased reducing power of hydroxylamine hydrochloride with pH.

Keywords: Reduction, stopped flow kinetics, pre-equilibrium, Acetohydroxamic acid

1. INTRODUCTION Iron acquisition presents profound difficulties for aerobic microorganism due to insolubility of Fe(OH)3. The

equilibrium concentration of Fe(III) is 10-18

M at pH 71. As a response to this environmental stress, bacteria have

developed the strategy of secreting low molecular weight compounds, siderophores, which chelate and solubilize Fe(III) ion for transport into the cell

2. Their role in microbial metabolism is to acquire iron from the environment, a

task that involves three steps; solubilization of Fe(III) through chelation, transport to and across the cell membrane

and deposition at the appropriate site into the cell 3

. The mode of iron transport into the bacterial cell is an important

area of interest in siderophore physiology. Raymond and Carrano in 1979 first time proposed three mechanisms of microbial iron transport

4.

Desferrioxamine B, a hydroxamate based siderophore, is currently used for removal of iron from the body in

the treatment of patients suffering from β-thalassemia or acute iron poisoning5. Synthetic monohydroxamic acids, such

as acetohydroxamic acid (AHA) (Figure-1), can serve as a model ligand for the investigation of the hydroxamate-

based siderophore-lron(III) interactions, which were thoroughly studied by Crumblis and his coworkers6.

Aceto hydroxamic acid

Scheme-1

Binding of iron is important in biological system. The clinical efficacy of the chelator is very much dependent on the

thermodynamic and kinetic factors of iron binding by this chelator7. The stability of Fe(II) siderophores is

considerably lower than Fe(III) siderophores, and they also tend to be kinetically labile with respect to Fe(II)

dissociation. Consequently, reduction of Fe(III) siderophore at the site of deposition is an attractive mechanistic

possibility for iron release8.

2. METHODOLOGY

In our studies acetohydroxarnic acid (AHA) has been used as Fe(III) chelator and hydroxylarnine hydrochloride as

reducing agent for the kinetic study. The reduced form of the complex, hydroxylamine hydrochlonde or its’ oxidized form have no absorbance in

the visible range whereas Fe(III)-hydroxamate complexes are highly colored. Addition of reductant to a solution of the

complex results in the decrease of absorbance of the solution, which enables us to measure the extent of reduction. Lowering of absorbance of complex is mainly due to two reasons, one is reduction and the other is dilution.

Lowering of absorbance due to dilution can be corrected by converting observed absorbance into corrected absorbance

as follows;

Acorr = Aobs x (Voxi + Vred) Voxi


9

Where,

Aobs = observed absorbance, Voxi = volume of oxidant used, Vred = volume of reductant added

3. EXPERIMENT All the reagents used were of A.R grade. Distilled deionized water was boiled to free from CO2. Analytical grade

reagents and distilled water were used in preparation of the solutions each time.

4. PREPARATION OF SOLUTIONS Fe(III) solution was prepared by dissolving a calculated, accurately weighed amount of Fe(NO3)3.9H2O in 0.05 M

HNO3. Standardization with Fe- Orthophenanthroline method was used to determine actual concentration. The

solution was found to be approximately 0.01 M with 6% error. This solution served as stock solution.

Acetohydroxamic acid AHA) solution was prepared as per requirement, by dissolving calculated amount of AHA in deionized distilled water.

Hydroxylamine hydrochloride solution was freshly prepared by dissolving calculated and accurately weighed

amount of hydroxylamine hydrochloride in buffer of desired pH. Different dilutions of the solution were prepared accordingly.

Acetate buffers of pH 4.5, 5.0, 5.5 having µ = 0.2 M were prepared. The required ionic strength was adjusted

by adding calculated amount of NaCl. Complex solution was prepared by mixing Fe (III) and AHA solution of known concentration. The concentration of

AHA was kept 5times over the concentration of Fe (III). Solutions were made up to mark with required buffer.

5. INSTRUMENTATION λmax of the complex at different pH was determined by monitoring absorption spectra at particular pH on Shimadzu

spectrophotometer UV-160A. The molar extinction coefficients (ε) of Fe(III)-AHA were calculated (Table 1).

For all pH measurements, Orion pH-meter model SA-720 was used. Absorbance change in visible region was monitored on Spectronic21. The output of Spectronic21 was read in to Pentium I computer interfaced through

“Labpro” compatible with “Logger Pro” program distributed by “Vernier”. Labpro interface and the Logger Pro

program together allowed us to save the records of individual kinetic runs in files with voltage output of Spectronic 21. Kinetic study was followed by stopped flow method. SFA-II is a fast kinetic accessory, which eliminates the

problem of long mixing time. Its mixing time is less than 20 milli seconds. This means it can measure half-life down

to about 0.05 seconds. The cell IS thermo stated.

6. RESULTS AND DISCUSSION It is evident that high spin d

5 Fe(III) has no spin-allowed d-d transitions, rather LMCT exists in Fe(III)-siderophore

complexes which are not readily interpreted as are ligand - field (d-d) transitions [9]. Fe(III)-siderophore complexes

possess high Kf, but with the reduction of Fe+3

to Fe+2

, decreases. The iron(II)-siderophore complexes are colorless because of the absence of LMCT. In the pursuit of kinetic studies, discoloration of Fe(III)-siderophore on reduction to

Fe(II) enables us to follow the reaction.

In the pursuit of kinetic studies, disappearance of color of Fe(III)-siderophore on reduction to Fe(II) enables us

to follow the reaction. Consequently these kinetic studies are helpful in the determination of electron transfer or biological actions of metalloprotiens

10.

6.1 Effect of pH Complex of 1:1 metal to ligand ratio is formed at very low pH. While increasing the pH causes 1:2 & 1:3 complex

formations those are much stable and difficult to reduce.

The general trend in reduction process, as reported in the literature is in the order FeL3< FeL2+< FeL2

+, As the

stability of Fe(III)-(AHA)3 complex increases with PH, it becomes difficult to reduce Fe(III)-(AHA)3. Thus the rate of

reaction decreases with increasing pH. The reducing power of NH2OH.HCl is also very much pH dependent. By

increasing pH it’s reducing power increases. At higher pH the reducing power is more dominant over the complexation, which is indicated by a slight increase in the values of rate constant.

pH 4.5, 5.0 and 5.5 were chosen for the present study. It has been observed that the λmax decreases with

increasing pH (Table-1).

Table-1: pH dependence of λmax values

pH ε

M-1

cm-1

λmax

(nm)

4.5 1774 449

5 1916 439

5.5 2034 429

Nisar et al, 2015

10

Fe(III) + CH3CONHOH CH3CONHO- [(CH3CONHO)nFe]

n = 1, 2 or 3. n depends upon pH.

Possibility of maximum complexion, i.e. 1:3, increases with increase in pH. Fe(III)-AHA is reduced to Fe(II)-AHA on

addition of reducing agent such as hydroxylamine hydrochloride. Fe(II)-AHA is a colorless species.

Basically in the reaction mixture, two species are involved, one is Fe(III)-AHA complex while other is reductant. Both of them have contrary effects on rate with pH rise.

By increasing pH the formation of 1:2 & 1:3 complexes prevails & 1:3 complex is less reducible than 1:2

which is less reducible than 1:1. It means that by increasing pH (or decreasing H+ concentration) the reducing ability

of Fe(III) complex decreases due to different stoichiometric complexes.

FeL33-

+ H+ FeL2

-+ HL

FeL2- + R

- Fe(II) + R + L

Where, L is a bidentate ligand.

kR at higher pH is lower than kR at lower pH because FeL3 is less reducible than FeL2 which is less reducible than FeL

+.

However nature of reluctant like NH2OH.HCl is such that, by increasing the pH its Eo decreases as shown below:

MEDIUM OXIDATION STATE (-1) OXIDATION STATE (0) ACID NH3OH

+ -181 N2

BASE NH2OH -3,04 N2

Due to less value of Eo

red, Eo is greater which is directly related to K value as shown previously.

By applying simplified Marcus equation

kAB = (kAA KAB ZAB2)

1/2

ZAA ZBB

KAB = (kAA × kBB × KAB × FAB)1/2

This is the cross relationship in terms of rate constants (12). Here kAA and KBB are the self exchange rate constants of specie A and B respectively and KAB is the equilibrium constant. If FAB = 1 then equation reduces to.

kAB = (kAA × kBB × KAB)1/2

This is called Marcus equation. If all terms kAA and kBB remain constants then KAB depends only on changing

KAB with changing Eo values at different pH. So, by increasing pH resulting kAB or rate of reduction increases.

The above two contrary effects with respect to complex & reductant cancel each other, therefore no significant

increase or decrease is observed by changing pH. But a small increase can be seen by increasing pH, which might be due to the increased reducing power of reductant at higher pH.

The recent work of Bengtsson and coworkers reveals that the results obtained from the reduction of Fe(III) by

hydroxylamine, truly in absence of siderophore, are consistent with a mechanism dependent on the relative iron(III) to

hydroxylamine concentration. It has also to be considered that the consistent mechanism identifies the mechanism with two pre-equilibriums in that case

[11].

Reduction of Fe(III)-AHA by hydroxylamine hydrochloride is a typically biphasic reaction i.e. a fast phase

which may be due to the reduction of Fe-(AHA)2 parallel with slow phase that may be due to the reduction of Fe-(AHA)3 It is very difficult to determine the interval at which fast phase terminates and slow phase starts.

However, the plot kobs vs [RED] would have been a straight line, if this reaction was first order in

[hydroxylamine]. The clear leveling off describes reaction is not first order in [hydroxylamine] but rather pre-

equilibrium exists [Fig:1, 2]. The plots of 1/ Kobs as a function of 1 / [Red] are given in figures 3 and 4.

kR

kR


11

Fig-1: Plots of the Observed Rate Constants for the Fast Phase of Reduction of Fe(III)-AHA complex at different pH,

T=30+ 0.5°C, [Fe(III)-AHA] = 5.5 × 10-4M, μ=0.2

Fig-2: Plots of the Observed Rate Constants for the Slow Phase of Reduction of Fe(III)-AHA complex at different pH,

T=30+ 0.5°C, [Fe(III)-AHA] = 5.5 × 10-4M, μ=0.2

Fig-3: Double Reciprocal Plot for Fast Phase of Reduction of Fe(III)-AHA complex at different pH,

T=30+ 0.5°C, [Fe(III)-AHA] = 5.5 × 10-4M, μ=0.2

0

0.02

0.04

0.06

0.08

0.1

0.12

0 0.05 0.1 0.15

kob

s

s-1

[R] M

pH 5

pH4.5

pH5.5

-0.003

0.002

0.007

0.012

0.017

0.022

0.027

0.032

0.037

0.042

0.047

0 0.05 0.1 0.15

kob

s

/s

[R] M

pH 4.5

pH5

pH5.5

y4.5 = 0.25x + 12.15 R² = 0.965

y5 .5= 0.645x + 8.370 R² = 0.994

y5 = 0.473x + 4.083 R² = 0.996

0

10

20

30

40

50

60

70

80

0 20 40 60 80 100 120

1/ko

bs

s

1/[R] M-1

pH4.5

pH5.5

pH5

Nisar et al, 2015

12

Fig-4: Double Reciprocal Plot for Slow Phase of Reduction of Fe (III)-AHA complex at different pH,

T=30+ 0.5°C, [Fe(III)-AHA] = 5.5 × 10-4M, μ=0.2

The results obtained help us to suggest the following mechanism for both the phases

FAST PHASE

H+ + Fe

+3(AHA)3 Fe

+3(AHA)2+ HAHA

Fe+3

(AHA)2 + NH3OH+ Fe

+2 + 2AHA

- + NH2OH

+ + H

+

RATE = k3 [Fe+3

(AHA)2] [NH3OH+]

By steady-state approximation,

kl [Fe+3

(AHA)3] [H+] = k2[Fe

+3(AHA)2] [HAHA] + k3 [Fe

+3(AHA)2] [NH3OH

+]

Rate = -d Fe+3

(AHA)3 = kl [Fe+3

(AHA)3] [H+]

[Fe

+3(AHA)2] (k2 [HAHA] + k3[NH3OH

+]) = k1 [Fe

+3(AHA)3] [H

+]

[Fe+3

(AHA)2] = kl [Fe+3

(AHA)3] [H+]

(k2 [HAHA] + k3[NH30H+])

Rate = k3 k1 [Fe+3

(AHA)3] [H+] . [NH3OH

+]

(k2[HAHA] + k3[NH3OH+])

[H+] = constant; k1 [H

+] = k4

kobs = k3 . k4 . [NH3OH+]

(k2[HAHA] + k3[NH3OH+])

if k2 [HAHA] » k3. [NH3OH+]

then kobs = k3 k4 [NH3OH+]

k2 [HAHA]

if k3[NH3OH+] » k2 [HAHA]

then kobs = k4 i.e. kobs = k1 [H+]

kobs = k3 k4 .[NH3OH+]

(k2 [HAHA] + k3 [NH3OH+])

y4.5 = 5.176x - 29.06 R² = 0.963

y5 = 3.257x + 27.42 R² = 0.911

y5.5 = 5.781x - 5.938 R² = 0.991

0

50

100

150

200

250

300

350

0 20 40 60 80 100

1/ko

bs

s

1/[R] M-1

pH 4.5

pH5

pH5.5

rds


13

1 = k2 [HAHA] + k3[NH3OH+]

kobs k3 k4 [NH3OH+] k3 k4 [NH30H

+]

1 = k2 [HAHA] + 1 + 1

kobs k3 k4 [NH30H+] K4

Intercept = 1/ k4 = 1 / k1[H+]

1 = k1[H+]

Intercept

The following changes are expected to occur during the oxidation of hydroxylamine hydrochlorides:

Fe+3

+ NH3OH+ slow Fe

+2 + NH2OH

+ + H

+

Fe+3

+ NH2OH+ fast Fe

+2 + NHO+ 2H

+

2NHO H2N2O2

In this way the overall stoichiometry of the reaction is; 4Fe

+3 + 2NH3OH

+ 4Fe

+2 + H2N2O2 + 6H

+

SLOW PHASE Following mechanism can be assumed for the slow phase of reduction of Fe (III)-AHA by hydroxylamine hydrochloride:

Fe(III)L3 + HR Fe (III)L2R + HL

Fe(III)L2R Fe(II) + 2L + R+

Where R = hydroxylamine hydrochloride

7. REFERENCES 1. Biedermann, G.., Schindler, L., Actachem. Scand, (1857), 11, 731-740,

http://dx.doi.org/10.3891/acta.chem.scand.11-0731.

2. Nieland, J. B., “Inorganic Biochemistry”, Eichhorn, G.., Ed, Elsevier., Amsterdam, (1973); pp 167-202.

3. Albrecht-Gary, A-M., Crumbliss, A. L., in Iron Transport and Storage in Microorganisms, Plants, Animals,

vol.35; Metals Ions in Biological systems; Sigel, A., Sigel, H., Eds.: M. Dekkar, Inc: New York, (1998), p239. 4. Raymond and Carrano, Acc. Chem. Res., (1979), vol 12, 187.

5. Raymond, K. N., Chang, T. D. Y., Pecoraro, V. L., Carrano, C. J., In the Biochemistry and Physiology of Iron,

Saltman, P., Sieker, L., Ed., Elsevier Biochemical Amsterdam, The Netherland, (1982). 6. Monzyk, B. and Alvin, L., Crumbliss, Jr. Am. Chem. Soc. (1979), 101, 21, 6203-6213,

http://dx.doi.org/10.1021/ja00515a009.

7. Anatoly Bez korovainy, “Biochemistry of Nonheme Iron”, (1980), Plenum press. New York and London.

8. Ivan Spasojevic, Sandra., K., Armstrong., Timothy, J,. Brickman, and Alvin, L., Crumbliss, lnorg. Chern. (1999), 38, 449-454.

9. Berthold, F. M. Gertrand and Kenneth, N. R. in “Iron Carriers and Iron Protiens”, T.M. Loehr. Ed., Portland,

(1988), pp3. 10. Andrew Lee Shorter, “Kinetics and Mechanisms of Reduction of Ferrioxamine B, Ferrioxamine E,

Ferrichrome A”, Ph.D. Thesis, Inorg. Chem. division, Chemistry Department, University of Merry land,

(1982). 11. Bengtsson, Goesta, Fronaeaus, Sure, Bengtsson-Kloo, Lars (Inorganic Chemistry, Chemical Center, Lund

University, Sweden). Journal of Chemical Society, Dalton Transactions, (2002), (12), 2548-2552,

http://dx.doi.org/10.1039/b201602h.

12. Jordan, R. B. “Reaction mechanism of inorganic & organometallic systems” (1991). 1st Ed.p.173.

13. Day, R. A., JR, A. L. Underwood “Quantitative analysis”. Prentice -Hall, Inc. Englewood Cliffs, New Jersey

2nd

ed. (1967).

14. Atkins, P. W. “Physical Chemistry”. 3rd

Ed. Oxford University Press, (1986), p750.

http://dx.doi.org/10.3891/acta.chem.scand.11-0731

http://dx.doi.org/10.1021/ja00515a009

http://dx.doi.org/10.1039/b201602h


ISSN (Print): 2220-2625


*Corresponding Author Received 29th October 2014, Accepted 23rd February 2015

Antibacterial and Enzyme Inhibition Study of Hydrazone Derivatives Bearing 1, 3, 4-

Oxadiazole

S. Rasool, *Aziz-ur-Rehman, M. A. Abbasi, S. Z. Siddiqi, A. S. Gondal, H. A. Noor, S. Sheral and

1I.

Ahmad

*Department of Chemistry, Government College University, Lahore-54000, Pakistan.

1Department of Pharmacy, The Islamia University of Bahawalpur, Bahawalpur-63100, Pakistan.

E-mail: [email protected], [email protected]

ABSTRACT The antibacterial and lipoxygenase enzyme inhibition activities of two series of compounds have been investigated in the presented work. The 4-methyl/hydroxy benzoic acids (1a & 1b) were used as starting materials to prepare corresponding esters

(2a & 2b), hydrazides (3a & 3b), 5-(4-methylphenyl/4-hydroxyphenyl)-1,3,4-oxadiazol-2-thiols (4a & 4b), S-substituted esters

(5a & 5b) and acetohydrazides (6a & 6b). The acetohydrazones, 8a-i & 9a-i, were synthesized by stirring 6a & 6b with

mono(di)substituted phenylcarboxaldehydes (7a-i) in methanol. The data of IR, 1H-NMR and EIMS spectral techniques well

confirmed the structural formulae of synthesized compounds. The molecules of 4-methyl series rendered the better results than

those of 4-hydroxy series.

Keywords: 1, 3, 4-Oxadiazole, carboxylic acids, antibacterial activity, lipoxygenase inhibition activity

1. INTRODUCTION Substituted 1,3,4-oxadiazole

1,2 and acetohydrazone compounds

3,4 individually are known to confront multiple

biological activities, that is, antimicrobial, anti-enzymatic, etc. The newly synthesized molecules were assessed for

antibacterial and lipoxygenase inhibition. The included bacterial strains were S. typhi, E. coli, P. aeruginosa, B. subtilis and S. aureus which are responsible for enteric fever

5, food poisoning

6, chronic infection

7, hypersensitivity

reactions8 and pathogenesis

9. The lipoxygenase enzymes (EC 1.13.11.12) are related to inflammatory drugs

10,11. The

multiple functionality bearing molecules are presented in the current article and such a type of molecules is under

study by our group12-14

looking forward to new drug candidates.

2. RESULTS AND DISCUSSION The two series of acetohydrazone derivatives were accomplished by Scheme 1 and endorsed by spectral study of IR, 1HNMR & EIMS and the biological activities, given in Tables 2 to 4.

2.1 Chemistry In the second step, carbohydrazide formation can be accompanied by stirring or refluxing if required. The formation of 1,3,4-oxadiazole was performed in basic medium, but the final product should be collected in slight acidic medium,

and low pH (about 1-4) has negative effect on the yield. In the fifth step, acetohydrazide formation must be carried out

at room temperature. The last step includes the reaction of acetohydrazones with different aldehydes stirring in methanol, which can be catalyzed by a few drops of glacial acetic acid (Scheme 1).

The molecules were structurally finalized through the collective data of spectral analysis of IR, 1HNMR and EIMS.

The compound 8a presented molecular formula of C20H21N5O2S (mol. mass = 395), obtained from proton integration in

1HNMR spectrum and mass fragments based on peaks in EIMS. The significant fragments in EIMS spectrum

appeared at m/z 395 (molecular ion peak), 233 (the 5-(4-methylphenyl)-1,3,4-oxadiazol-2-thiomethylcarbonyl cationic

peak) and 190 (the N'-(4-(dimethylamino)benzylidenehydrazinocarbonyl cationic peak). The mass fragmentation

pattern of this molecule is sketched in Figure 1. The IR supporting absorption band appeared at (cm-1

) 1663 (C=N) for imine group. The four doublets of two protons integrated with each other were assigned to two 1,4-disubstituted

phenyl rings. The relative positions of these doublets were nominated as δ 7.88 (d, J = 8.4 Hz, 2H, H-2' & H-6') and

7.22 (d, J = 8.0 Hz, 2H, H-3' & H-5') for methyl substituted ring; and δ 7.49 (d, J = 8.4 Hz, 2H, H-2''' & H-6''') and 6.64 (d, J = 8.0 Hz, 2H, H-3''' & H-5''') for dimethylamino substituted ring. The two singlets at δ 2.91 (s, 6H, (CH3)2N-

4''') and 2.37 (s, 3H, CH3-4') resonated for dimethylamino group & methyl group with relative intensity of six & three

protons. The one methine proton and two methylene protons resonated at δ 8.09 (s, 1H, H-7''') and 4.64 (s, 2H, H-2'')

as singlets. Finally compound 8a was written as N'-(4-(dimethylamino)benzylidene-2-(5-(4-methylphenyl)-1,3,4-Oxadiazol-2-ylthio)acetohydrazide.


15

N

O

N

SHCH2 OC2H5

O

N

O

N

SCH2 NH

O

NCH

X

(I)

X

OC2H5

O

X

NHNH2

O

X

N

O

N

S

X

CH2 NHNH2

O

N

O

N

S

X

H3C

OH

O

1a & 1b 2a & 2b

4a & 4b5a & 5b

1

34

6'

2'

4'

1''

2''

3a & 3b

6a & 6b

7'''

8a-i

N

O

N

SCH2 NH

O

NCH

HO

1

34

6'

2'

4'

1''

2'' 7'''

9a-i

R2'''

4'''6'''

2'''

4'''6'''R

(II)

(III)(IV)

(V)

(VI)

X = CH3 or OH Scheme-1: Synthesis of N'-substituted-2-(5-(4-methyl/4-hydroxy phenyl)-1,3,4-oxadiazol-2-ylthio)acetohydrazide (8a-i, 9a-i).

Reagents and conditions: (I) EtOH, Conc. H2SO4, Reflux, 7-8 hours (II) 80% N2H4.H2O, MeOH, Stir, 5-6 hours (III) CS2, KOH,

EtOH, Reflux, 6-7 hours (IV) EBA (ethyl bromoacetate), LiH, DMF, Stir, 4-5 hours (V) 80% N2H4.H2O, MeOH, Stir, 3-4 hours (VI) mono(di)substituted phenylcarboxaldehydes (7a-i), MeOH, Stir, 2-4 hours.

Figure-1: Mass fragmentation pattern of N'-(4-(Dimethylamino)benzylidene)-2-(5-(4-methylphenyl)-1,3,4-oxadiazol-2-

ylthio)acetohydrazide (8a)

O

NN

S CH2 C

O

NH

C11H13N3O

CHNS CO

[M]+ m/z = 395

m/z = 190

m/z = 233

m/z = 192

m/z = 162

m/z = 133

m/z = 65

N CH

CO NH N CH

NH N CH

N2

C4H7N

C9H12N3

m/z = 91

m/z = 119

C10H9N2OS

H3C

O

NN

S CH2 C

O

H3C

O

NN

SHH3C

C OH3C

m/z = 133

O

N

H3C

C NH3C

H3C

m/z = 51

m/z = 117

CHNOS

CHN2S

CO

C3H4

N(CH3)2

N(CH3)2

N(CH3)2

(H3C)2N

Aziz-ur-Rehman et al, 2015

16

2.2 Antibacterial activity (in vitro) The antibacterial activity of the compounds has been checked out in comparison of ciprofloxacin, the routine drug. The results are tabulated as %age inhibition and minimum inhibitory concentration (MIC) values (Table 2 & 3).

Table-1: Different substituted R-groups

Compound -R Compound -R

8a,9a 4-N(CH3)2 8f,9f 2-OCH3, 5-OCH3

8b,9b 4-N(C2H5)2 8g,9g 3-OCH3, 4-OCH3

8c,9c 4-OCH3 8h,9h 2-Cl, 4-Cl 8d,9d 2-OCH3, 3-OCH3 8i,9i 2-Cl, 6-Cl 8e,9e 2-OCH3, 4-OCH3 - -

The only two compounds of hydroxy series named 9e and 9f rendered relatively better inhibition potential against the

encountered five bacterial strains. Overall the methyl series proved to be comparatively better inhibitor of P

aeruginosa and S. aureus but hydroxy series for B. subtilis strain. Both of series remained more efficient against P. aeruginosa and S. aureus as compared to others. Among the weakly active compounds against S. typhi, 9g bearing

disubstitued 3,4-dimethoxybenzylidene moiety presented low MIC (µg/mL) of 10.0 ± 0.9 compared with 7.9 ± 0.1 of

reference. The molecule, 9f was proficient against E. coli with MIC of 10.4 ± 0.4 compared with 7.3 ± 0.6 of reference. Against P. aeruginosa, 8c and 8f presented low MICs of 10.9 ± 0.7 and 11.8 ± 0.4 respectively in

comparison of 7.9 ± 0.1. B. subtilis was inhibited less efficiently by both the series. Against S. aureus, 8b, 8e, 8f and

8g showed low MICs. The molecules of 4-methyl series rendered the better results than those of 4-hydroxy series.

Table-3: The MIC values for antibacterial activity

Compound

MIC (µg/mL)

Gram negative bacteria Gram positive bacteria

S. typhi E. coli P. aeruginosa B. subtilis S. aureus

8a - - - - 12.3±0.8

8b - - 19.9±1.1 - 11.1±0.1

8c 15.2±0.4 15.2±1.4 10.9±0.7 - 12.1±0.4 8d 17.4±1.5 18.3±1.3 19.6±0.6 - -

8e 19.4+0.0 16.8±1.2 12.5±0.7 - 10.2±0.4

8f 14.2±0.2 14.2±0.3 11.8±0.4 - 10.2±1.0

8g - 16.2±1.0 19.4±0.6 - 10.6±1.4

8h - - - 15.9±0.9 19.7±0.9

8i - 15.3±0.6 18.2±0.6 - 17.5±1.5

9a 17.5±0.3 - 18.9±0.9 15.1±0.5 13.9±0.9

9b - - - - -

9c - - - - 18.3±0.2

9d - - - - 18.1±0.5

Table-2: The %age inhibition for antibacterial activity

Compound

Percentage Inhibition (%)

Gram negative bacteria Gram positive bacteria

S. typhi E. coli P. aeruginosa B. subtilis S. aureus

8a 30.0±2.1 42.6±1.0 28.4±0.9 27.2±2.1 70.8±1.5 8b 41.5±2.1 47.0±0.1 50.2±0.4 22.2±1.5 80.0±0.7

8c 64.2±0.8 68.2±0.1 71.0±0.2 58.3±1.4 84.0±0.9

8d 56.3±0.6 54.5±0.8 50.6±0.6 57.7±0.7 46.8±1.0

8e 51.3±1.6 62.5±0.6 61.1±0.5 40.9±0.4 83.3±0.3

8f 75.8±0.6 67.8±1.5 72.7±1.6 61.6±1.0 79.4±0.4

8g 40.7±1.4 64.1±1.4 51.5±1.0 45.1±1.6 79.9±1.3

8h 25.6±1.6 51.7±0.1 40.8±1.5 42.6±0.5 50.5±0.8

8i 49.5±0.3 54.6±1.6 56.3±1.2 27.7±1.0 57.0±1.4

9a 53.8±0.6 42.8±0.5 51.8±0.4 61.5±0.9 65.0±0.6

9b 36.6±0.9 37.6±0.2 37.6±0.2 45.8±0.4 30.5±0.5

9c 45.1±0.1 38.6±0.8 33.8±0.6 40.5±0.4 51.8±0.5

9d 46.4±1.6 33.0±1.1 47.2±0.4 49.9±0.5 61.2±0.5 9e 63.6±0.0 54.5±1.2 69.3±1.0 66.0±0.6 67.1±0.9

9f 76.4±1.2 70.4±0.3 58.5±0.2 51.8±0.8 70.6±0.9

9g 71.7±1.1 64.4±1.2 73.3±0.9 63.0±1.2 42.45±0.6

9h 57.0±0.0 54.4±0.9 55.3±0.5 65.6±0.9 67.8±0.7

9i 49.5±1.0 58.6±0.8 47.2±0.3 58.6±0.3 42.1±0.7

Ciprofloxacin 92.1±0.5 91.4±0.8 91.0±0.1 91.2±1.1 92.2±1.0


17

9e 15.5±0.4 16.9±0.8 13.0±0.4 13.4±0.5 13.4±0.4

9f 11.5±0.9 10.4±0.4 15.4±0.2 19.1±0.3 15.4±0.9

9g 10.0±0.9 12.3±0.4 13.5±0.8 14.0±0.0 -

9h 15.4±0.1 18.2±0.9 17.1±0.9 13.9±0.0 19.1±0.2

9i - 17.1±0.9 - 17.0±0.6 -

Ciprofloxacin 7.9±0.1 7.3±0.6 7.9±0.1 8.0±0.0 8.1±0.1

NOTE: Minimum inhibitory concentration (MIC) was measured with suitable dilutions (5-30 µg/well) and results were calculated

using EZ-Fit Perrella Scientific Inc. Amherst USA software.

2.3 Enzyme inhibition activity (in vitro) The tabulated results of %age inhibition and IC50 values (Table 4) for lipoxygenase inhibition relative to Baicalein, the

reference standard, showed the compounds of both series remained almost inactive against this enzyme except a few.

Table-4: The IC50 values of lipoxygenase inhibition activity

Compound LOX

Conc. (mM) Inhibition (%) IC50 (µM)

8a 0.5 54.6±0.1 >500

8b 0.5 90.2±0.8 288.7±1.5

8c 0.5 84.6±0.5 132.8±0.7

8d 0.5 52.1±0.6 >500

8e 0.5 38.8±0.2 -

8f 0.5 15.7±0.1 -

8g 0.5 34.3±0.1 -

8h 0.5 56.2±0.1 >500

8i 0.5 74.6±0.4 290.3±1.2

9a 0.5 28.3±0.1 -

9b 0.5 53.3±0.1 >500 9c 0.5 46.2±0.1 >500

9d 0.5 47.3±0.4 >500

9e 0.5 10.1±0.1 -

9f 0.5 32.2±0.1 -

9g 0.5 28.5±0.1 -

9h 0.5 70.9±0.9 269.6±0.7

9i 0.5 NIL -

Baicalein 0.5 93.7±1.2 22.4±1.3

NOTE: LOX = Lipoxygenase enzyme. IC50 values (concentration for 50% inhibition) of compounds were recorded using EZ–Fit

Enzyme kinetics software (Perella Scientific Inc. Amherst, USA).

Their large size, which might be unfit to the active site may attribute to their inactivity. Only compounds 8b, 8c, 8i

and 9h showed weak inhibition potential. The best among these was 8c with IC50 of 132.8 ± 0.7 µM compared with

22.4 ± 1.3 µM of Baicalein.

3. CONCLUSION The presented compounds can be subdivided into two series including 4-methylphenyl and 4-hydroxyphenyl, each

incorporating nine molecules. The molecules presented varying activity from weak to moderate antibacterial activity

but too much weak against lipoxygenase enzyme. The compounds of 4-methyl series were relatively better for their potential.

4. EXPERIMENTAL

4.1 General Melting points (M.P.) were measured through Griffin-George apparatus with open capillary tube and were uncorrected. Spectral study included IR, recorded on Jasco-320-A spectrophotometer by KBr pellet method;

1HNMR,

recorded on Bruker spectrometer in CDCl3 at 400 MHz; and EIMS, recorded on JMS-HX-110 spectrometer. The

initial purity of compounds was verified through TLC, performed on Al-plates coated with silica gel G-25-UV254

using MeCOOEt and n-C6H14 solvent systems. The chemicals of synthetic grade were purchased from Merck, Alfa Aesar & Sigma-Aldrich and the solvents used were of analytical grade.

4.2 Ethyl 4-methyl/4-hydroxy benzoate (2a & 2b) synthesis 4-Methyl/4-hydroxy benzoic acids (1a & 1b; 0.049 mol) were homogenized in 35 mL EtOH in a 100 mL round

bottom (RB) flask and followed by addition of the catalyst, concentrated sulfuric acid (3.0 mL). The flask was set to

reflux for 7-8 hours and monitored with TLC. After maximum conversion, reaction mixture was budged to a separating funnel (125 mL) followed by 50 mL ice cold distilled water and aqueous Na2CO3 solution (10%) to adjust a

pH to 9-10. The title compounds were separated through solvent extraction with 25 mL CHCl3.


18

4.2.1 Ethyl 4-Methylbenzoate (2a) Yellow liquid; Yield: 84%; Mol. formula: C10H12O2; Mol. mass: 164 gmol

-1; IR (KBr, vmax/cm

-1): 3123 (Ar C-H), 1734

(Ester C=O), 1595 (Ar C=C); 1H-NMR (400 MHz, CDCl3, δ/ppm): 7.87 (d, J = 8.0 Hz, 2H, H-2' & H-6'), 7.29 (d, J =

8.0 Hz, 2H, H-3' & H-5'), 4.02 (q, J = 7.2 Hz, 2H, -OCH2CH3), 2.42 (s, 3H, CH3-4'), 1.01 (t, J = 7.2 Hz, 3H, -

OCH2CH3); EIMS (m/z): 164 [M]+, 119 [C8H7O]

+, 91 [C7H7]

+, 65 [C5H5]

+.

4.2.2 Ethyl 4-Hydroxybenzoate (2b) White amorphous solid; Yield: 87%; M.P.: 144-116

oC; Mol. formula: C9H10O3; Mol. mass: 166 gmol

-1; IR (KBr,

vmax/cm-1

): 3412 (O-H), 3107 (Ar C-H), 1738 (Ester C=O), 1596 (Ar C=C); 1H-NMR (400 MHz, CDCl3, δ/ppm): 7.85

(d, J = 8.4 Hz, 2H, H-2' & H-6'), 6.89 (d, J = 8.4 Hz, 2H, H-3' & H-5'), 4.07 (q, J = 7.6 Hz, 2H, -OCH2CH3), 1.02 (t, J

= 7.6 Hz, 3H, -OCH2CH3); EIMS (m/z): 166 [M]+, 121 [C7H5O2]

+, 93 [C6H5O]

+, 67 [C4H3O]

+.

4.3 4-Methyl/4-Hydroxy benzohydrazide (3a & 3b) synthesis The compounds 2a & 2b (0.043 mol) were taken in a 100 mL RB flask, already containing 25 mL MeOH. 80%

Hydrazine hydrate (0.043 mol) was instantly poured and stirred for 5-6 hours. After all starting material consumed, 50 mL ice cold distilled water was poured followed by gentle shaking to acquire the precipitates of 3a & 3b. These were

collected through filtration, washed and dried.

4.3.1 4-Methylbenzohydrazide (3a) Cream white amorphous solid; Yield: 88%; M.P.: 116-118

oC; Mol. formula: C8H10N2O; Mol. mass: 150 gmol

-1; IR

(KBr, vmax/cm-1

): 3337 (N-H), 3119 (Ar C-H), 1662 (Amide C=O), 1596 (Ar C=C); 1H-NMR (400 MHz, CDCl3,

δ/ppm): 9.39 (s, 1H, CONH), 8.68 (s, 2H, N-H), 7.86 (d, J = 8.4 Hz, 2H, H-2' & H-6'), 7.26 (d, J = 8.4 Hz, 2H, H-3' &

H-5'), 2.41 (s, 3H, CH3-4'); EIMS (m/z): 150 [M]+, 119 [C8H7O]

+, 91 [C7H7]

+, 65 [C5H5]

+.

4.3.2 4-Hydroxybenzohydrazide (3b) White amorphous solid; Yield: 86%; M.P.: 264-266

oC; Mol. formula: C7H8N2O2; Mol. mass: 152 gmol

-1; IR (KBr,

vmax/cm-1

): 3387 (O-H), 3321 (N-H), 3108 (Ar C-H), 1667 (Amide C=O), 1606 (Ar C=C); 1H-NMR (400 MHz,

CDCl3, δ/ppm): 9.31 (s, 1H, CONH), 8.72 (s, 2H, N-H), 7.89 (d, J = 8.0 Hz, 2H, H-2' & H-6'), 6.83 (d, J = 8.4 Hz, 2H, H-3' & H-5'); EIMS (m/z): 152 [M]

+, 121 [C7H5O2]

+, 93 [C6H5O]

+, 67 [C4H3O]

+.

4.4 5-(4-Methyl/4-Hydroxy phenyl)-1,3,4-oxadiazol-2-thiol (4a & 4b) synthesis The compounds, 3a & 3b (0.040 mol) were homogenized in 25 mL EtOH in a 100 mL RB flask and basified by solid

KOH (0.040 mol) on reflux. The system was cooled to RT and then 0.080 mol CS2 was poured into. After reflux of 6-

7 hours, 50 mL cold distilled water was poured into followed by dilute HCl (3-4 mL, pH = 6-7), and the mixture was stirred for 0.25 hours for proper precipitation. Thus obtained products were filtered off, washed, and dried.

4.4.1 5-(4-Methylphenyl)-1,3,4-oxadiazol-2-thiol (4a) White amorphous solid; Yield: 78%; M.P.: 172-174

oC; Mol. formula: C9H8N2OS; Mol. mass: 192 gmol

-1; IR (KBr,

vmax/cm-1

): 2548 (S-H), 3137 (Ar C-H), 1667 (C=N), 1609 (Ar C=C); 1H-NMR (400 MHz, CDCl3, δ/ppm): 7.89 (d, J

= 8.4 Hz, 2H, H-2' & H-6'), 7.23 (d, J = 8.4 Hz, 2H, H-3' & H-5'), 2.42 (s, 3H, CH3-4'); EIMS (m/z): 192 [M]+, 133

[C8H7NO]+, 119 [C8H7O]

+, 117 [C8H7N]

+, 91 [C7H7]

+, 65 [C5H5]

+.

4.4.2 5-(4-Hydroxyphenyl)-1,3,4-oxadiazol-2-thiol (4b) White amorphous solid; Yield: 83%; M.P.: 176-178

oC; Mol. formula: C8H6N2O2S; Mol. mass: 194 gmol

-1; IR (KBr,

vmax/cm-1

): 3398 (O-H), 3133 (Ar C-H), 1661 (C=N), 1599 (Ar C=C); 1H-NMR (400 MHz, CDCl3, δ/ppm): 7.88 (d, J

= 8.4 Hz, 2H, H-2' & H-6'), 6.63 (d, J = 8.4 Hz, 2H, H-3' & H-5'); EIMS (m/z): 194 [M]+, 135 [C7H5NO2]

+, 121

[C7H5O2]+, 119 [C7H5NO]

+, 93 [C6H5O]

+, 67 [C4H3O]

+.

4.5 Ethyl 2-(5-(4-methyl/4-hydroxy phenyl)-1,3,4-oxadiazol-2-ylthio)acetate (5a & 5b) synthesis The compounds, 4a & 4b, (0.035 mol) were dissolved in 15 mL dimethylformamide (DMF) in a 100 mL RB flask and

stirred for 0.5 hours with LiH (0.035 mol). Then ethyl 2-bromoacetate (EBA, 0.035 mol) was added and mixture was

further stirred for 4-5 hours. After single spot on TLC, the products were made precipitate after addition of excess cold

distilled water and removed from medium by filtration, washing and drying.

4.5.1 Ethyl 2-(5-(4-Methylphenyl)-1,3,4-oxadiazol-2-ylthio)acetate (5a) White amorphous solid; Yield: 79%; M.P.: 166-168


-1; IR (KBr,

vmax/cm-1

): 3159 (Ar C-H), 1751 (Ester C=O), 1666 (C=N), 1604 (Ar C=C); 1H-NMR (400 MHz, CDCl3, δ/ppm): 7.84

(d, J = 8.4 Hz, 2H, H-2' & H-6'), 7.29 (d, J = 8.0 Hz, 2H, H-3' & H-5'), 4.61 (s, 2H, H-2''), 3.93 (q, J = 7.2 Hz, 2H, -

OCH2CH3), 2.41 (s, 3H, CH3-4'), 0.99 (t, J = 7.2 Hz, 3H, -OCH2CH3); EIMS (m/z): 278 [M]+, 233 [C11H9N2O2S]

+, 192

[C9H8N2OS]+, 133 [C8H7NO]

+, 119 [C8H7O]

+, 117 [C8H7N]

+, 91 [C7H7]

+, 65 [C5H5]

+.


19

4.5.2 Ethyl 2-(5-(4-Hydroxyphenyl)-1,3,4-oxadiazol-2-ylthio)acetate (5b) White amorphous solid; Yield: 76%; M.P.: 180-182


-1; IR (KBr,

vmax/cm-1

): 3386 (O-H), 3153 (Ar C-H), 1752 (Ester C=O), 1676 (C=N), 1601 (Ar C=C); 1H-NMR (400 MHz, CDCl3,

δ/ppm): 7.86 (d, J = 8.4 Hz, 2H, H-2' & H-6'), 6.69 (d, J = 8.0 Hz, 2H, H-3' & H-5'), 4.62 (s, 2H, H-2''), 3.90 (q, J = 7.6 Hz, 2H, -OCH2CH3), 0.98 (t, J = 7.6 Hz, 3H, -OCH2CH3); EIMS (m/z): 280 [M]

+, 235 [C10H7N2O3S]

+, 194

[C8H6N2O2S]+, 135 [C7H5NO2]

+, 121 [C7H5O2]

+, 119 [C7H5NO]

+, 93 [C6H5O]

+, 67 [C4H3O]

+.

4.6 2-(5-(4-Methyl/4-Hydroxy phenyl)-1,3,4-oxadiazol-2-ylthio)acetohydrazide (6a & 6b) synthesis The compounds 5a & 5b (0.031 mol) were mixed with 80% hydrazine hydrate (0.031 mol) in a 100 mL RB flask

containing 20 mL MeOH. The reaction mixture was stirred for 3-4 hours at RT. After final TLC with single spot, the precipitates appeared after addition of excess cold distilled water, were filtered and washed by distilled water.

4.6.1 2-(5-(4-Methylphenyl)-1,3,4-oxadiazol-2-ylthio)acetohydrazide (6a) White amorphous solid; Yield: 85%; M.P.: 176-178


-1; IR (KBr,

vmax/cm-1

): 3376 (N-H), 3073 (Ar C-H), 1663 (Amide C=O), 1681 (C=N), 1602 (Ar C=C); 1H-NMR (400 MHz,

CDCl3, δ/ppm): 9.41 (s, 1H, CONH), 8.77 (s, 2H, N-H), 7.87 (d, J = 8.0 Hz, 2H, H-2' & H-6'), 7.27 (d, J = 8.0 Hz,

2H, H-3' & H-5'), 4.65 (s, 2H, H-2''), 2.41 (s, 3H, CH3-4'); EIMS (m/z): 264 [M]+, 233 [C11H9N2O2S]

+, 192

[C9H8N2OS]+, 133 [C8H7NO]

+, 119 [C8H7O]

+, 117 [C8H7N]

+, 91 [C7H7]

+, 65 [C5H5]

+.

4.6.2 2-(5-(4-Hydroxyphenyl)-1,3,4-oxadiazol-2-ylthio)acetohydrazide (6b) White amorphous solid; Yield: 81%; M.P.: 208-210


-1; IR (KBr,

vmax/cm-1

): 3412 (O-H), 3372 (N-H), 3075 (Ar C-H), 1665 (Amide C=O), 1687 (C=N), 1604 (Ar C=C); 1H-NMR (400

MHz, CDCl3, δ/ppm): 9.47 (s, 1H, CONH), 8.83 (s, 2H, N-H), 7.87 (d, J = 8.0 Hz, 2H, H-2' & H-6'), 6.67 (d, J = 8.0 Hz, 2H, H-3' & H-5'), 4.64 (s, 2H, H-2''); EIMS (m/z): 266 [M]

+, 235 [C10H7N2O3S]

+, 194 [C8H6N2O2S]

+, 135

[C7H5NO2]+, 121 [C7H5O2]

+, 119 [C7H5NO]

+, 93 [C6H5O]

+, 67 [C4H3O]

+.

4.7 N'-Substituted-2-(5-(4-methyl/4-hydroxy phenyl)-1,3,4-oxadiazol-2-ylthio)acetohydrazide (8a-i, 9a-i)

synthesis The compounds, 6a & 6b (0.0036 mol) were mixed with mono(di)substituted phenylcarboxaldehydes (7a-i; 0.0036

mol) in a 50 mL RB flask containing 12 mL MeOH. The mixture was simply stirred for 2-4 hours. After reaction

completion affirmed via TLC, excess cold distilled water was added and aged for precipitate formation. The formed precipitates were filtered off, washed with distilled water, and dried for biological activities.

4.7.1 N'-(4-(Dimethylamino)benzylidene)-2-(5-(4-methylphenyl)-1,3,4-oxadiazol-2-ylthio)aceto hydrazide (8a) Yellow amorphous solid; Yield: 81%; M.P.: 182-184


-1; IR

(KBr, vmax/cm-1

): 3046 (Ar C-H), 1663 (C=N), 1607 (Ar C=C); 1H-NMR (400 MHz, CDCl3, δ/ppm): 10.45 (s, 1H,

CONH), 8.09 (s, 1H, H-7'''), 7.88 (d, J = 8.4 Hz, 2H, H-2' & H-6'), 7.49 (d, J = 8.4 Hz, 2H, H-2''' & H-6'''), 7.22 (d, J

= 8.0 Hz, 2H, H-3' & H-5'), 6.64 (d, J = 8.0 Hz, 2H, H-3''' & H-5'''), 4.64 (s, 2H, H-2''), 2.91 (s, 6H, (CH3)2N-4'''), 2.37 (s, 3H, CH3-4'); EIMS (m/z): 395 [M]

+, 233 [C11H9N2O2S]

+, 192 [C9H8N2OS]

+, 190 [C10H12N3O]

+, 162 [C9H12N3]

+,

134 [C9H12N]+, 133 [C8H7NO]

+, 119 [C8H7O]

+, 117 [C8H7N]

+, 91 [C7H7]

+, 65 [C5H5]

+, 51 [C4H3]

+.

4.7.2 N'-(4-(Diethylamino)benzylidene)-2-(5-(4-methylphenyl)-1,3,4-oxadiazol-2-ylthio)aceto hydrazide (8b) Light yellow amorphous solid; Yield: 82%; M.P.: 192-194


-1; IR

(KBr, vmax/cm-1

): 3049 (Ar C-H), 1669 (C=N), 1604 (Ar C=C); 1H-NMR (400 MHz, CDCl3, δ/ppm): 10.53 (s, 1H,

CONH), 8.06 (s, 1H, H-7'''), 7.86 (d, J = 8.0 Hz, 2H, H-2' & H-6'), 7.45 (d, J = 8.0 Hz, 2H, H-2''' & H-6'''), 7.25 (d, J

= 8.0 Hz, 2H, H-3' & H-5'), 6.69 (d, J = 8.0 Hz, 2H, H-3''' & H-5'''), 4.61 (s, 2H, H-2''), 2.63 (q, J = 7.2 Hz, 4H,

(CH3CH2)2N-4'''), 2.39 (s, 3H, CH3-4'), 1.04 (t, J = 7.2 Hz, 6H, (CH3CH2)2N-4'''); EIMS (m/z): 423 [M]+, 233

[C11H9N2O2S]+, 218 [C12H16N3O]

+, 192 [C9H8N2OS]

+, 190 [C11H16N3]

+, 162 [C11H16N]

+, 133 [C8H7NO]

+, 119

[C8H7O]+, 117 [C8H7N]

+, 91 [C7H7]

+, 65 [C5H5]

+, 51 [C4H3]

+.

4.7.3 N'-(4-Methoxybenzylidene)-2-(5-(4-methylphenyl)-1,3,4-oxadiazol-2-ylthio)aceto hydrazide (8c) White amorphous solid; Yield: 79%; M.P.: 202-204


-1; IR (KBr,

vmax/cm-1

): 3054 (Ar C-H), 1675 (C=N), 1617 (Ar C=C); 1H-NMR (400 MHz, CDCl3, δ/ppm): 10.73 (s, 1H, CONH),

8.08 (s, 1H, H-7'''), 7.85 (d, J = 8.0 Hz, 2H, H-2' & H-6'), 7.73 (d, J = 8.4 Hz, 2H, H-2''' & H-6'''), 7.28 (d, J = 8.4 Hz,

2H, H-3' & H-5'), 6.57 (d, J = 8.4 Hz, 2H, H-3''' & H-5'''), 4.63 (s, 2H, H-2''), 3.81 (s, 3H, CH3O-4'''), 2.42 (s, 3H,

CH3-4'); EIMS (m/z): 382 [M]+, 233 [C11H9N2O2S]

+, 192 [C9H8N2OS]

+, 177 [C9H9N2O2]

+, 149 [C8H9N2O]

+, 133

[C8H7NO]+, 121 [C8H9O]

+, 119 [C8H7O]

+, 117 [C8H7N]

+, 91 [C7H7]

+, 65 [C5H5]

+, 51 [C4H3]

+.


20

4.7.4 N'-(2,3-Dimethoxybenzylidene)-2-(5-(4-methylphenyl)-1,3,4-oxadiazol-2-ylthio)aceto hydrazide (8d) White amorphous solid; Yield: 85%; M.P.: 198-200


-1; IR (KBr,

vmax/cm-1


8.19 (s, 1H, H-7'''), 7.89 (d, J = 8.4 Hz, 2H, H-2' & H-6'), 7.56 (d, J = 8.4 Hz, 1H, H-6'''), 7.44 (dd, J = 8.4, 1.2 Hz,

1H, H-4'''), 7.22 (d, J = 8.4 Hz, 2H, H-3' & H-5'), 7.14 (t, J = 8.4 Hz, 1H, H-5'''), 4.63 (s, 2H, H-2''), 3.82 (s, 3H,

CH3O-3'''), 3.80 (s, 3H, CH3O-2'''), 2.40 (s, 3H, CH3-4'); EIMS (m/z): 412 [M]+, 233 [C11H9N2O2S]

+, 207

[C10H11N2O3]+, 192 [C9H8N2OS]

+, 179 [C9H11N2O2]

+, 151 [C9H11O2]

+, 133 [C8H7NO]

+, 119 [C8H7O]

+, 117 [C8H7N]

+,

91 [C7H7]+, 65 [C5H5]

+, 51 [C4H3]

+.

4.7.5 N'-(2,4-Dimethoxybenzylidene)-2-(5-(4-methylphenyl)-1,3,4-oxadiazol-2-ylthio)aceto hydrazide (8e) White amorphous solid; Yield: 80%; M.P.: 214-216


-1; IR (KBr,

vmax/cm-1


8.16 (s, 1H, H-7'''), 7.86 (d, J = 8.4 Hz, 2H, H-2' & H-6'), 7.75 (d, J = 8.0 Hz, 1H, H-6'''), 7.29 (d, J = 8.4 Hz, 2H, H-

3' & H-5'), 6.63 (d, J = 2.4 Hz, 1H, H-3'''), 6.54 (dd, J = 8.0, 2.4 Hz, 1H, H-5'''), 4.64 (s, 2H, H-2''), 3.83 (s, 3H,


+, 207

[C10H11N2O3]+, 192 [C9H8N2OS]

+, 179 [C9H11N2O2]

+, 151 [C9H11O2]

+, 133 [C8H7NO]

+, 119 [C8H7O]

+, 117 [C8H7N]

+,

91 [C7H7]+, 65 [C5H5]

+, 51 [C4H3]

+.

4.7.6 N'-(2,5-Dimethoxybenzylidene)-2-(5-(4-methylphenyl)-1,3,4-oxadiazol-2-ylthio)aceto hydrazide (8f) White amorphous solid; Yield: 83%; M.P.: 222-224


-1; IR (KBr,

vmax/cm-1


8.18 (s, 1H, H-7'''), 7.86 (d, J = 8.0 Hz, 2H, H-2' & H-6'), 7.31 (d, J = 3.2 Hz, 1H, H-6'''), 7.28 (d, J = 8.0 Hz, 2H, H-

3' & H-5'), 7.11 (dd, J = 9.2, 3.2 Hz, 1H, H-4'''), 6.92 (d, J = 9.2 Hz, 1H, H-3'''), 4.59 (s, 2H, H-2''), 3.87 (s, 3H,


+, 207

[C10H11N2O3]+, 192 [C9H8N2OS]

+, 179 [C9H11N2O2]

+, 151 [C9H11O2]

+, 133 [C8H7NO]

+, 119 [C8H7O]

+, 117 [C8H7N]

+,

91 [C7H7]+, 65 [C5H5]

+, 51 [C4H3]

+.

4.7.7 N'-(3,4-Dimethoxybenzylidene)-2-(5-(4-methylphenyl)-1,3,4-oxadiazol-2-ylthio)aceto hydrazide (8g) White amorphous solid; Yield: 89%; M.P.: 228-230


-1; IR (KBr,

vmax/cm-1


8.17 (s, 1H, H-7'''), 7.86 (d, J = 8.0 Hz, 2H, H-2' & H-6'), 7.35 (d, J = 1.6 Hz, 1H, H-2'''), 7.29 (d, J = 8.0 Hz, 2H, H-3' & H-5'), 7.19 (dd, J = 8.4, 1.6 Hz, 1H, H-6'''), 6.92 (d, J = 8.4 Hz, 1H, H-5'''), 4.62 (s, 2H, H-2''), 3.81 (s, 3H,


+, 207

[C10H11N2O3]+, 192 [C9H8N2OS]

+, 179 [C9H11N2O2]

+, 151 [C9H11O2]

+, 133 [C8H7NO]

+, 119 [C8H7O]

+, 117 [C8H7N]

+,

91 [C7H7]+, 65 [C5H5]

+, 51 [C4H3]

+.

4.7.8 N'-(2,4-Dichlorobenzylidene)-2-(5-(4-methylphenyl)-1,3,4-oxadiazol-2-ylthio)aceto hydrazide (8h) White amorphous solid; Yield: 81%; M.P.: 218-220

oC; Mol. formula: C18H14Cl2N4O2S; Mol. mass: 420 gmol

-1; IR

(KBr, vmax/cm-1

): 3076 (Ar C-H), 1653 (C=N), 1596 (Ar C=C), 702 (C-Cl); 1H-NMR (400 MHz, CDCl3, δ/ppm):

10.61 (s, 1H, CONH), 8.41 (s, 1H, H-7'''), 7.88 (d, J = 8.4 Hz, 2H, H-2' & H-6'), 7.58 (d, J = 8.0 Hz, 1H, H-6'''), 7.43 (dd, J = 8.0, 1.2 Hz, 1H, H-5'''), 7.34 (d, J = 1.2 Hz, 1H, H-3'''), 7.29 (d, J = 8.0 Hz, 2H, H-3' & H-5'), 4.63 (s, 2H, H-

2''), 2.39 (s, 3H, CH3-4'); EIMS (m/z): 424 [M+4]+, 422 [M+2]

+, 420 [M]

+, 233 [C11H9N2O2S]

+, 215 [C8H5Cl2N2O]

+,

192 [C9H8N2OS]+, 187 [C7H5Cl2N2]

+, 159 [C7H5Cl2]

+, 133 [C8H7NO]

+, 119 [C8H7O]

+, 117 [C8H7N]

+, 91 [C7H7]

+, 65

[C5H5]+, 51 [C4H3]

+.

4.7.9 N'-(2,6-Dichlorobenzylidene)-2-(5-(4-methylphenyl)-1,3,4-oxadiazol-2-ylthio)aceto hydrazide (8i) White amorphous solid; Yield: 80%; M.P.: 206-208


-1; IR

(KBr, vmax/cm-1

): 3071 (Ar C-H), 1664 (C=N), 1601 (Ar C=C), 707 (C-Cl); 1H-NMR (400 MHz, CDCl3, δ/ppm):

10.57 (s, 1H, CONH), 8.42 (s, 1H, H-7'''), 7.86 (d, J = 8.4 Hz, 2H, H-2' & H-6'), 7.54 (d, J = 8.4 Hz, 2H, H-3''' & H-

5'''), 7.42 (t, J = 8.4 Hz, 1H, H-4'''), 7.23 (d, J = 8.4 Hz, 2H, H-3' & H-5'), 4.64 (s, 2H, H-2''), 2.38 (s, 3H, CH3-4'); EIMS (m/z): 424 [M+4]

+, 422 [M+2]

+, 420 [M]

+, 233 [C11H9N2O2S]

+, 215 [C8H5Cl2N2O]

+, 192 [C9H8N2OS]

+, 187

[C7H5Cl2N2]+, 159 [C7H5Cl2]

+, 133 [C8H7NO]

+, 119 [C8H7O]

+, 117 [C8H7N]

+, 91 [C7H7]

+, 65 [C5H5]

+, 51 [C4H3]

+.

4.7.10 N'-(4-(Dimethylamino)benzylidene)-2-(5-(4-hydroxyphenyl)-1,3,4-oxadiazol-2-ylthio) acetohydrazide

(9a) Light orange amorphous solid; Yield: 79%; M.P.: 212-214


-1; IR

(KBr, vmax/cm-1

): 3418 (O-H), 3039 (Ar C-H), 1673 (C=N), 1597 (Ar C=C); 1H-NMR (400 MHz, CDCl3, δ/ppm):


6'''), 6.73 (d, J = 8.4 Hz, 2H, H-3' & H-5'), 6.67 (d, J = 8.0 Hz, 2H, H-3''' & H-5'''), 4.61 (s, 2H, H-2''), 2.93 (s, 6H,


21

(CH3)2N-4'''); EIMS (m/z): 397 [M]+, 235 [C10H7N2O3S]

+, 194 [C8H6N2O2S]

+, 190 [C10H12N3O]

+, 162 [C9H12N3]

+, 135

[C7H5NO2]+, 134 [C9H12N]

+, 121 [C7H5O2]

+, 119 [C7H5NO]

+, 93 [C6H5O]

+, 67 [C4H3O]

+, 51 [C4H3]

+.

4.7.11 N'-(4-(Diethylamino)benzylidene)-2-(5-(4-hydroxyphenyl)-1,3,4-oxadiazol-2-ylthio) acetohydrazide (9b) Light orange amorphous solid; Yield: 84%; M.P.: 216-218


-1; IR

(KBr, vmax/cm-1



6'''), 6.73 (d, J = 8.4 Hz, 2H, H-3' & H-5'), 6.67 (d, J = 8.0 Hz, 2H, H-3''' & H-5'''), 4.59 (s, 2H, H-2''), 2.61 (q, J = 7.6

Hz, 4H, (CH3CH2)2N-4'''), 1.07 (t, J = 7.6 Hz, 6H, (CH3CH2)2N-4'''); EIMS (m/z): 425 [M]+, 235 [C10H7N2O3S]

+, 218

[C12H16N3O]+, 194 [C8H6N2O2S]

+, 190 [C11H16N3]

+, 162 [C11H16N]

+, 135 [C7H5NO2]

+, 121 [C7H5O2]

+, 119 [C7H5NO]

+,

93 [C6H5O]+, 67 [C4H3O]

+, 51 [C4H3]

+.

4.7.12 N'-(4-Methoxybenzylidene)-2-(5-(4-hydroxyphenyl)-1,3,4-oxadiazol-2-ylthio)aceto hydrazide (9c) White amorphous solid; Yield: 77%; M.P.: 242-244


-1; IR (KBr,

vmax/cm-1

): 3405 (O-H), 3047 (Ar C-H), 1672 (C=N), 1607 (Ar C=C); 1H-NMR (400 MHz, CDCl3, δ/ppm): 11.75 (s,

1H, CONH), 8.05 (s, 1H, H-7'''), 7.83 (d, J = 8.0 Hz, 2H, H-2' & H-6'), 7.79 (d, J = 8.4 Hz, 2H, H-2''' & H-6'''), 6.68

(d, J = 8.0 Hz, 2H, H-3' & H-5'), 6.52 (d, J = 8.4 Hz, 2H, H-3''' & H-5'''), 4.62 (s, 2H, H-2''), 3.83 (s, 3H, CH3O-4''');

EIMS (m/z): 384 [M]+, 235 [C10H7N2O3S]

+, 194 [C8H6N2O2S]

+, 177 [C9H9N2O2]

+, 149 [C8H9N2O]

+, 135 [C7H5NO2]

+,

121 [C8H9O]+, 121 [C7H5O2]

+, 119 [C7H5NO]

+, 93 [C6H5O]

+, 67 [C4H3O]

+, 51 [C4H3]

+.

4.7.13 N'-(2,3-Dimethoxybenzylidene)-2-(5-(4-hydroxyphenyl)-1,3,4-oxadiazol-2-ylthio)aceto hydrazide (9d) White amorphous solid; Yield: 86%; M.P.: 230-232


-1; IR (KBr,

vmax/cm-1


1H, CONH), 8.31 (s, 1H, H-7'''), 7.89 (d, J = 8.0 Hz, 2H, H-2' & H-6'), 7.54 (d, J = 8.4 Hz, 1H, H-6'''), 7.46 (dd, J =

8.0, 1.6 Hz, 1H, H-4'''), 7.13 (t, J = 8.4 Hz, 1H, H-5'''), 6.62 (d, J = 8.0 Hz, 2H, H-3' & H-5'), 4.65 (s, 2H, H-2''), 3.83 (s, 3H, CH3O-3'''), 3.79 (s, 3H, CH3O-2'''); EIMS (m/z): 414 [M]

+, 235 [C10H7N2O3S]

+, 207 [C10H11N2O3]

+, 194

[C8H6N2O2S]+, 179 [C9H11N2O2]

+, 151 [C9H11O2]

+, 135 [C7H5NO2]

+, 121 [C7H5O2]

+, 119 [C7H5NO]

+, 93 [C6H5O]

+, 67

[C4H3O]+, 51 [C4H3]

+.

4.7.14 N'-(2,4-Dimethoxybenzylidene)-2-(5-(4-hydroxyphenyl)-1,3,4-oxadiazol-2-ylthio)aceto hydrazide (9e) Cream amorphous solid; Yield: 84%; M.P.: 238-240


-1; IR (KBr,

vmax/cm-1


1H, CONH), 8.24 (s, 1H, H-7'''), 7.86 (d, J = 8.0 Hz, 2H, H-2' & H-6'), 7.71 (d, J = 8.4 Hz, 1H, H-6'''), 6.69 (d, J =

8.0 Hz, 2H, H-3' & H-5'), 6.61 (d, J = 2.0 Hz, 1H, H-3'''), 6.56 (dd, J = 8.4, 1.6 Hz, 1H, H-5'''), 4.63 (s, 2H, H-2''), 3.84 (s, 3H, CH3O-2'''), 3.82 (s, 3H, CH3O-4'''); EIMS (m/z): 414 [M]

+, 235 [C10H7N2O3S]

+, 207 [C10H11N2O3]

+, 194

[C8H6N2O2S]+, 179 [C9H11N2O2]

+, 151 [C9H11O2]

+, 135 [C7H5NO2]

+, 121 [C7H5O2]

+, 119 [C7H5NO]

+, 93 [C6H5O]

+, 67

[C4H3O]+, 51 [C4H3]

+.

4.7.15 N'-(2,5-Dimethoxybenzylidene)-2-(5-(4-hydroxyphenyl)-1,3,4-oxadiazol-2-ylthio)aceto hydrazide (9f) Yellow amorphous solid; Yield: 80%; M.P.: 246-248


-1; IR

(KBr, vmax/cm-1


11.74 (s, 1H, CONH), 8.36 (s, 1H, H-7'''), 7.89 (d, J = 8.4 Hz, 2H, H-2' & H-6'), 7.35 (d, J = 2.4 Hz, 1H, H-6'''), 7.07

(d, J = 8.4 Hz, 1H, H-3'''), 7.02 (dd, J = 8.0, 2.0 Hz, 1H, H-4'''), 6.68 (d, J = 8.0 Hz, 2H, H-3' & H-5'), 4.65 (s, 2H, H-

2''), 3.79 (s, 3H, CH3O-5'''), 3.76 (s, 3H, CH3O-2'''); EIMS (m/z): 414 [M]+, 235 [C10H7N2O3S]

+, 207 [C10H11N2O3]

+,

194 [C8H6N2O2S]+, 179 [C9H11N2O2]

+, 151 [C9H11O2]

+, 135 [C7H5NO2]

+, 121 [C7H5O2]

+, 119 [C7H5NO]

+, 93

[C6H5O]+, 67 [C4H3O]

+, 51 [C4H3]

+.

4.7.16 N'-(3,4-Dimethoxybenzylidene)-2-(5-(4-hydroxyphenyl)-1,3,4-oxadiazol-2-ylthio)aceto hydrazide (9g) White amorphous solid; Yield: 83%; M.P.: 238-240


-1; IR (KBr,

vmax/cm-1


1H, CONH), 8.14 (s, 1H, H-7’’’), 7.86 (d, J = 8.0 Hz, 2H, H-2’ & H-6’), 7.31 (d, J = 1.6 Hz, 1H, H-2’’’), 7.13 (dd, J

= 8.4, 1.6 Hz, 1H, H-6’’’), 6.96 (d, J = 8.4 Hz, 1H, H-5’’’), 6.67 (d, J = 8.0 Hz, 2H, H-3’ & H-5’), 4.62 (s, 2H, H-

2’’), 3.81 (s, 3H, CH3O-3’’’), 3.80 (s, 3H, CH3O-4’’’); EIMS (m/z): 414 [M]+, 235 [C10H7N2O3S]

+, 207 [C10H11N2O3]

+,

194 [C8H6N2O2S]+, 179 [C9H11N2O2]

+, 151 [C9H11O2]

+, 135 [C7H5NO2]

+, 121 [C7H5O2]

+, 119 [C7H5NO]

+, 93

[C6H5O]+, 67 [C4H3O]

+, 51 [C4H3]

+.

4.7.17 N'-(2,4-Dichlorobenzylidene)-2-(5-(4-hydroxyphenyl)-1,3,4-oxadiazol-2-ylthio)aceto hydrazide (9h) White amorphous solid; Yield: 83%; M.P.: 250-252


-1; IR

(KBr, vmax/cm-1

): 3409 (O-H), 3071 (Ar C-H), 1654 (C=N), 1591 (Ar C=C), 704 (C-Cl); 1H-NMR (400 MHz, CDCl3,

δ/ppm): 11.61 (s, 1H, CONH), 8.71 (s, 1H, H-7'''), 7.89 (d, J = 8.0 Hz, 2H, H-2' & H-6'), 7.53 (d, J = 8.4 Hz, 1H, H-

6'''), 7.45 (dd, J = 8.4, 1.6 Hz, 1H, H-5'''), 7.31 (d, J = 1.6 Hz, 1H, H-3'''), 6.69 (d, J = 8.0 Hz, 2H, H-3' & H-5'), 4.61


22

(s, 2H, H-2''); EIMS (m/z): 426 [M+4]+, 424 [M+2]

+, 422 [M]

+, 235 [C10H7N2O3S]

+, 215 [C8H5Cl2N2O]

+, 194

[C8H6N2O2S]+, 187 [C7H5Cl2N2]

+, 159 [C7H5Cl2]

+, 135 [C7H5NO2]

+, 121 [C7H5O2]

+, 119 [C7H5NO]

+, 93 [C6H5O]

+, 67

[C4H3O]+, 51 [C4H3]

+.

4.7.18 N'-(2,6-Dichlorobenzylidene)-2-(5-(4-hydroxyphenyl)-1,3,4-Oxadiazol-2-ylthio)aceto hydrazide (9i) White amorphous solid; Yield: 84%; M.P.: 244-246


-1; IR

(KBr, vmax/cm-1

): 3394 (O-H), 3078 (Ar C-H), 1659 (C=N), 1597 (Ar C=C), 706 (C-Cl); 1H-NMR (400 MHz, CDCl3,

δ/ppm): 11.57 (s, 1H, CONH), 8.72 (s, 1H, H-7'''), 7.86 (d, J = 8.4 Hz, 2H, H-2' & H-6'), 7.53 (d, J = 8.4 Hz, 2H, H-

3''' & H-5'''), 7.43 (t, J = 8.4 Hz, 1H, H-4'''), 6.73 (d, J = 8.0 Hz, 2H, H-3' & H-5'), 4.61 (s, 2H, H-2''); EIMS (m/z):

426 [M+4]+, 424 [M+2]

+, 422 [M]

+, 235 [C10H7N2O3S]

+, 216 [C8H5Cl2N2O]

+, 194 [C8H6N2O2S]

+, 188 [C7H5Cl2N2]

+,

160 [C7H5Cl2]+, 135 [C7H5NO2]

+, 121 [C7H5O2]

+, 119 [C7H5NO]

+, 93 [C6H5O]

+, 67 [C4H3O]

+, 51 [C4H3]

+.

4.8 Biological assays

4.8.1 Antibacterial assay The antibacterial activity results were obtained by employing the reported method with minor alterations

12,15.

4.8.2 Enzyme inhibition assay The enzyme inhibition results were obtained by employing the reported method with minor alterations

10,11.

4.8.3 Statistical analysis The presented results are mean ± SEM of calculations obtained after three experiments and statistically analyzed on

MS Excel 2010. The MIC and IC50 values are reported which are the result of values recorded after varying dilutions

of each sample, followed by calculation by EZ-Fit software (Perrella Scientific Inc, Amherst, USA).

5. ACKNOWLEDGEMENT Higher Education Commission (HEC) of Pakistan is highly acknowledged owing to their financial support.

6. REFERENCES 1. Dabholkar, V. V., Bhusari, N. V., Int. J. Environ. Pharm. Res. (2011), 4, 1,

http://dx.doi.org/10.1016/j.ejmech.2012.04.027.

2. Rashid, M., Husain, A., Mishra, R., Eur. J. Med. Chem. (2012), 54, 855.

3. Narsibhai, B. D., Mishra, D., Vyavahare, L.V., Singh, A., Arch. Appl. Sci. Res. (2012), 4, 1816. 4. Somani, R. R., Agrawal, A. G., Kalantri, P. P., Gavarkar, P. S., Clercq, E. D., Int. J. Drug Des. Discov.

(2011), 2, 353.

5. Bhattacharya, S. S., Das, U., Choudhury, B. K., Ind. J. Med. Res. (2011), 133, 431. 6. Vogt, R. L., Dippold, L., Public Health Rep. (2005), 120, 174.

7. Pressler, T., Bohmova, C., Conway, S., Dumcius, S., Hjelte, L., Høiby, N., Kollberg, H., Tümmler, B.,

Vavrova, V., J. Cyst. Fibros. (2011), 10, S75, http://dx.doi.org/10.1016/S1569-1993(11)60011-8.

8. Barbe, V., Cruveiller, S., Kunst, F., Lenoble, P., Meurice, G., Sekowska, A., Vallenet, D., Wang, T., Moszer, I., Medigue, C., Danchin, A., Microbiology (2009), 155, 17580, http://dx.doi.org/10.1099/mic.0.027839-0.

9. Harris, L. G., Foster, S. J., Richards, R. G., Eur. Cells Mater. (2002), 4, 39.

10. Abbasi, M. A., Ahmad, V. U., Zubair, M., Rashid, M. A., Farooq, U., Nawaz, S. A., Lodhi, M. A., Makhmoor, T., Choudhary, M. I., Atta-ur-Rahman, Proc. Pak. Acad. Sci. (2005), 42, 121.

11. Alitonou, G. A., Avlessi, F., Sohounhloue, D. K., Agnaniet, H., Bessiere, J. M., Menut, C., Int. J. Aromather.

(2006), 16, 37, http://dx.doi.org/10.1016/j.ijat.2006.01.001. 12. Aziz-ur-Rehman, Nafeesa, K., Abbasi, M. A., Kashfa, H., Rasool, S., Ahmad, I., Arshad, S., Pak. J. Chem.

(2013), 3(2), 56. http://dx.doi.org/10.15228/2013.v03.i02.p03.

13. Gul, S., Aziz-ur-Rehman, Abbasi, M. A., Nafeesa, K., Malik, A., Ashraf, M., Islmail, T., Ahmad, I., Asian J.

Chem. (2013), 25, 6231. 14. Khalid, H., Aziz-ur-Rehman, Abbasi, M. A., Malik, A., Rasool, S., Nafeesa, K., Ahmad, I., Afzal, S., J. Saudi

Chem. Soc. (2013), Doi:http://dx.doi.org/10.1016/j.jscs.2013.05.001.

15. Yang, C. R., Zang, Y., Jacob, M. R., Khan, S. I., Zhang, Y. J., Li, X. C., Antimicrob. Agents Ch. (2006), 50, 1710, http://dx.doi.org/10.1128/AAC.50.5.1710-1714.2006.

http://dx.doi.org/10.1016/j.ejmech.2012.04.027

http://dx.doi.org/10.1016/S1569-1993(11)60011-8

http://dx.doi.org/10.1099/mic.0.027839-0

http://dx.doi.org/10.1016/j.ijat.2006.01.001

http://dx.doi.org/10.15228/2013.v03.i02.p03

http://dx.doi.org/10.1016/j.jscs.2013.05.001

http://dx.doi.org/10.1128/AAC.50.5.1710-1714.2006


ISSN (Print): 2220-2625


*Corresponding Author Received 16th September 2015, Accepted 23rd February 2015

Synthetic N-Alkyl/aralkyl-4-methyl-N-(naphthalen-1-yl)benzenesulfonamides as Potent

Antibacterial Agents

*M. A. Abbasi, S. Manzoor, Aziz-ur-Rehman, S. Z. Siddiqui,

1I. Ahmad,

1R. Malik,

2M. Ashraf

2Qurat-ul-Ain and

3,4S. A. A. Shah

*Department of Chemistry, Government College University, Lahore, Pakistan. 1Department of Pharmacy, The Islamia University of Bahawalpur, Pakistan.

2Department of Biochemistry and Biotechnology, The Islamia University of Bahawalpur, Bahawalpur,

Pakistan. 3Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam,

Selangor Darul Ehsan, Malaysia. 4Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns), Level 9, FF3, Universiti Teknologi

MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia.

E-mail: *[email protected]; [email protected]

ABSTRACT The current research effort involved the reaction of napthalen-1-amine (1) with 4-methylbenzenesulfonyl chloride (2) under

dynamic pH control at 9-10, maintained with 10% aqueous Na2CO3 to obtain 4-methyl-N-(naphthalen-1-yl) benzenesulfonamide

(3). The parent molecule 3 was further substituted at N-atom with alkyl/aralkyl halides (4a-f) in polar aprotic solvent; N,N-

dimethylformamide, and lithium hydride which acts as a base, to achieve N-alkyl/aralkyl-4-methyl-N-(naphthalen-1-

yl)benzenesulfonamides (5a-f). All the synthesized compounds were structurally elucidated by IR, 1H-NMR and EIMS spectral

techniques. All the derivatives were further screened for antibacterial and anti-enzymatic potential against various bacterial strains

and enzymes, respectively, and were found to be potent antibacterial agents and moderate to weak enzyme inhibitors.

Keywords: 4-Methyl-N-(naphthalen-1-yl) benzenesulfonamide, Spectral analysis, Antibacterial and Anti-enzymatic Analysis.

1. INTRODUCTION Sulfonamides are biologically active amide derivative of sulfonic acid having general formula RSO2NH-. They are

mostly used as bacteriostatic to inhibit the growth of gram positive and gram negative bacteria1-4

. They play important

role in pharmaceutical industry and function as anticonvulsant, antiviral, antifungal agents and enzyme inhibitors. Aryl sulfonamides are used against tumor cell lines. Clinically sulfonamides are mostly used to cure various types of

gastrointestinal and urinary infections. They act as anticancer agents and inhibitors of carbonic anhydrase which is the

root cause for cancer5-6

. 1-Napthylamine belongs to a class of benzo-fused aromatic compound. The driving force in

the generation of sulfonamides is the affinity of nitrogen atom of amine for sulfonyl group of sulfonyl halide and hence is the most commonly employed method for their synthesis

7.

On the basis of aforesaid evidences documented in literature and in continuation of our research efforts8-11

, we

synthesized various N-alkyl/aralkyl-4-methyl-N-(naphthalen-1-yl)benzenesulfonamides (5a-f) by reacting 4-methyl-N-(naphthalen-1-yl)benzenesulfonamide (3) in DMF/LiH with different alkyl/aralkyl sulfonyl chlorides, 4a-f. It was

evident that amalgamation of different electrophiles with sulfonamide moiety can results in improved bioactivity of

compounds which were later found to be in concordance with the biological evaluation results of synthesized derivatives against different bacterial strains and enzymes. Moreover, the structure-activity relationship was also

established.

2. RESULTS AND DISCUSSION 2.1 Chemistry A series of N-alkyl/aralyl-4-methyl-N-(naphthalen-1-yl)benzenesulfonamides was synthesized by reaction of napthalene-1-amine (1) with 4-methylbenzene-1-sulfonyl chloride (2) at room temperature in aqueous alkaline media

at pH 9-10 to yield 4-methyl-N-(naphthalen-1-yl)benzenesulfonamide (3) which was further treated with alkyl/aralkyl

halides, 4a-f, to obtain N-alkyl/aralkyl-4-methyl-N-(naphthalen-1-yl)benzenesulfonamides (5a-f) as illustrated in Scheme 1 and Table 1. Compound, 5a, was obtained as dark purple amorphous powder having molecular formula,

C20H21NO2S, which was established by counting number of protons in 1H-NMR spectrum and appearance of

molecular ion peak at m/z 339 [M]+. The signals in aromatic region appeared at δ 8.20 (d, J = 8.4 Hz, 1H, H-8), 7.90-

7.84 (m, 2H, H-5 & H-6), 7.76 (d, J = 6.4 Hz, 2H, H-2' & H-6'), 7.74 (d, J = 6.4 Hz, 1H, H-4), 7.59 (t, J = 6.4 Hz, 1H, H-3), 7.50 (d, J =8.8 Hz , 1H, H-2), 7.48 (d, J = 8.4 Hz, 2H, H-3' & H-5'), 7.40-7.31 ( m, 1H, H-7) confirmed the

presence of naphthyl ring and 1,4-disubstituted phenyl ring. A singlet at δ 2.30 having an integration of three protons

was assigned to CH3-7' group positioned at 4-position in 1,4-disubstituted phenyl ring. A multiplet integrated for one proton at δ 4.50-3.33 (H-2'') and a doublet resonated at δ 0.93 (J = 6.8 Hz) having integration of six protons (CH3-1'' &

CH3-3') was in agreement with the substitution of 2-propyl group at nitrogen atom of the parent sulfonamide core.

mailto:[email protected]


24

Similarly, on the basis of spectral evidences, the structures of other derivatives were confirmed and their spectral data has been described in experimental section. Mass fragmentation pattern of compound 5f is sketched in Figure 1.

NH2

S

O

O

Cl

H3C

S

O

O

CH3NH

S

O

O

CH3NR

Stirring, RT3-4 hours

1 2

pH 9-10

R-X

5a-f R=Alkyl/aralkyl halides

4a-f

3

1'3'

5'

Stirring, RT, 3h

Aq. Na2CO3

1

45

7 9

1'3'

5'

1

45

7 9

Naphthalen-1-amine 4-Methylbenzenesulfonyl

chloride

4-Methyl-N-(naphthalen-1-yl)

benzenesulfonamide

N-Alkyl/aralkyl-4-methyl-N-(naphthalen-1-

yl)benzenesulfonamides

7'

DMF/LiH

7'

Scheme-1: Synthesis of N-alkyl/aralkyl-4-methyl-N-(naphthalen-1-yl)benzenesulfonamides (5a-f)

Fig-1: Mass fragmentation pattern of N-(4-Chlorobenzyl)-4-methyl-N-(naphthalen-1-yl)benzenesulfonamide (5f)

Abbasi et al, 2015

25

Table-1: List of various alkyl/aralkyl groups utilized in synthesis of 5a-f.

Codes -R Codes -R

5a CH

CH3

CH3

1''2''

3''

5d 1''

3''

5''

CH2

7''

5b CH CH2 CH3

H3C 1''

2'' 3'' 4''

5e 1

'' 3

''

5''

CH2

7''

Cl

5c CH2 CH2 CH2 CH2 CH3

1'' 2'' 3'' 4'' 5''

5f 1

'' 3

''

5''

CH2

7''

Cl

2.2 Anti-bacterial potential The screening of the N-alkyl/aralkyl-4-methyl-N-(naphthalen-1-yl) benzenesulfonamides (5a-f) was carried out

against various gram-positive and gram-negative bacterial strains which demonstrated excellent to good activity as

evident from their % age inhibition and MIC values mentioned in Table 2 & Table 3, respectively. Amongst all these synthesized molecules, 5d displayed excellent MIC values against both gram-positive (B. subtilis: 11.33±0.91 & S.

aureus: 9.26±0.45 μg/well) and gram-negative bacterial strains (S. typhi: 11.25±0.68, P. aureginosa: 10.61±0.58 & E.

coli: 12.48±0.39 μg/well). Its antibacterial potential was almost close to that of reference standard, Ciprofloxacin. The

better inhibitory action of this molecule might be attributed to the attachment of benzyl moiety at the at nitrogen atom of the parent sulfonamide core.

Table-2: Antibacterial activity (% age inhibition) of N-alkyl/aralkyl-4-methyl-N-(naphthalen-1-yl)benzenesulfonamides (5a-f).

Codes S. typhi (-) P. aeruginosa (-) E. coli (-) B. subtilis (+) S. aureus (+)

3 43.75±1.25 47.80±0.30 47.25±0.85 39.09±0.50 47.73±0.90

5a 68.50±2.13 62.56±0.35 71.29±0.95 66.00±0.65 74.69±1.43

5b 44.25±1.44 64.31±0.20 51.86±0.67 52.14±2.00 60.66±1.58

5c 60.88±1.22 43.51±0.20 64.86±1.73 39.14±0.90 67.40±0.53

5d 74.13±0.38 59.44±0.25 73.71±0.75 71.86±0.57 79.58±0.63

5e 64.63±0.87 87.38±0.25 63.29±0.68 63.86±1.02 69.95±2.81

5f 36.75±1.72 78.31±0.87 12.43±1.05 33.00±1.60 47.94±1.00

Ciprofloxacin 91.21±0.22 92.00±0.23 90.63±0.12 91.98±0.04 91.38±0.01

Table-3: Antibacterial activity (MIC μg/well) of N-alkyl/aralkyl-4-methyl-N-(naphthalen-1-yl)benzenesulfonamides (5a-f).

Codes S. typhi (-) P. aeruginosa (-) E. coli (-) B. subtilis (+) S. aureus(+)

3 - - - - -

5a 12.60±0.89 11.62±0.93 11.60±0.23 18.38±0.58 14.62±0.33

5b - 13.93±1.00 17.61±0.54 18.38±0.58 14.62±0.33

5c 14.64±0.60 - 14.23±0.86 - 12.38±0.82

5d 11.25±0.68 10.61±0.58 12.48±0.39 11.33±0.91 9.26±0.45

5e 12.13±1.06 14.82±0.22 14.83±0.67 14.61±0.49 12.60±0.53

5f - 12.25±0.79 - - -

Ciprofloxacin 7.83±0.78 7.98±0.89 8.01±0.12 7.22±0.67 8.10±1.54

Note: Minimum Inhibition concentration was measured with suitable dilutions (5-30 μg/well) and results were calculated using

EZ-fit Perrella Scientific Inc. Amherts USA Software.

2.3 Anti-enzymatic analysis The anti-enzymatic analysis was carried out against acetyl and butyrylcholinesterases and lipoxygenase enzymes. The IC50 values showed that the synthesized sulfonamides overall displayed moderate to weak inhibitory actions.

Compound 5c showed relatively better inhibition against AChE having value of 88.50±0.15 µM. Similarly, compound

5b and 5c against BChE displayed reasonable inhibition having IC50 values of 41.23±0.11 ad 51.35±0.12 µM,

respectively. The inhibitory action of 5b and 5c might be due to the substitution of butan-2-yl and pentyl groups, respectively, at the parent sulfonamide nucleus. The other compounds did not show significant activities as compared

to reference standards Eserine in case of cholinesterases and Baicalein in case of lipoxygenase enzyme (Table 4).


26

Table-4: Anti-enzymatic analysis of N-alkyl/aralkyl-4-methyl-N-(naphthalen-1-yl)-benzenesulfonamides (5a-f).

Codes

AChE BChE LOX

Inhibition (%) 0.5 mM

IC50

(µM) Inhibition (%)

0.5 mM IC50

(µM) Inhibition (%)

0.5 mM IC50 (µM)

3 96.00±0.85 221.60±0.16 95.15±0.36 41.62±0.13 88.43±0.34 43.08±0.16

5a 96.31±0.45 155.70±0.17 99.81±0.65 118.51±0.15 51.21±0.34 477.43±1.24

5b 94.23±0.73 329.70±0.36 94.00±0.49 47.23±0.11 63.55±0.71 341.36±0.58

5c 98.54±0.55 88.50±0.15 95.51±0.32 51.35±0.12 63.62±0.55 239.13±0.69

5d 93.70±0.56 219.60±0.18 92.85±0.37 64.18±0.19 50.09±0.49 498.92±1.74

5e 30.73±0.31 - 32.86±0.22 - 64.63±0.41 380.31±0.53

5f 86.77±0.35 227.30±0.19 69.54±0.74 375.23±0.28 68.37±0.23 215.67±0.90

Control Eserine 0.85±0.001 Eserine 0.04±0.001 Baicalein 22.41±1.3

3. EXPERIMENTAL 3.1 Measurements The chemicals utilized in research work were purchased from sigma Aldrich/Fluka and were used as such. Purity of all

synthesized compounds was checked by thin layer chromatography (TLC) on plates coated with silica gel G-25-

UV254 using different percentages of ethyl acetate and n–hexane. The IR spectra were taken in KBr on a Jasco-320-A spectrophotometer (wave number in cm

-1).

1H NMR spectra were recorded in DMSO-d6 on a Bruker spectrometer

operating at 400 MHz. The chemical shifts (δ) and coupling constant (J) are given in ppm (parts per million) and Hz

(Hertz), respectively. Melting points were recorded on a Griffin and George melting point apparatus by open capillary tube and were found to be uncorrected. Mass spectra (EIMS) were measured on Finnigan MAT-112 instrument along

with data system.

3.2 Synthesis of 4-methyl-N-(naphthalen-1-yl)benzenesulfonamide (3) Napthalene-1-amine (0.5 g; 0.0034 mol; 1) was suspended in 25 ml distilled water in a 250 ml round-bottomed flask.

The pH of suspension was adjusted till 9 with 10 % aqueous Na2CO3. 4-Methylbenzenesulfonyl chloride (0.6 g; 0.0034 mol; 2) was gradually added in the reaction mixture and was further stirred for 2 hours at room temperature.

After completion of reaction, which was monitored by TLC till single spot, the product was precipitated by adding

few drops of conc. HCl till pH 2. The precipitates were filtered, washed with distilled water and air-dried to obtain 4-

methyl-N-(naphthalen-1-yl)benzenesulfonamide (3) as purple colored solid.

3.3 Synthesis of N-alkyl/aralkyl-4-methyl-N-(naphthalen-1-yl)benzenesulfonamides (5a-f) 4-Methyl-N-(naphthalen-1-yl)benzenesulfonamide (0.2 g; 0.1 mmol; 3) was solubilized in 10 ml N,N-dimethylformamide along with (0.1 mmol) of lithium hydride. The reaction mixture was allowed to stir for half an

hour at room temperature after which alkyl/aralkyl halides, 4a-f, were added to reaction mixture and was additionally

stirred for 3 hours. After reaction completion, the precipitates of product were obtained by addition of cold distilled water, filtered and air-dried to afford respective N-alkyl/aralkyl-4-methyl-N-(naphthalen-1-yl)benzenesulfonamides

(5a-f).

3.4 Antibacterial assay Antibacterial activity was carried out in 96-wells microplates under aseptic conditions. The principle involved is that

microbial cell number increases as the microbial growth proceeds in a log phase of growth which finally results in increased absorbance of broth medium

12,13. Four gram-negative (Klebsiella pneumoniae, Escherichia coli,

Pseudomonas aeruginosa and Salmonella typhi) and two gram-positive bacteria (Bacillus subtilis, Staphylococcus

aureus) (clinical isolate) were incorporated in the study. The organisms were maintained on stock culture agar medium. Test samples in appropriate solvents and dilutions were pipetted out into wells (20 µg well

-1). Overnight

maintained fresh bacterial culture after suitable dilution with fresh nutrient broth was poured into wells (180 µL). The

initial absorbance of the culture was firmly maintained between 0.12-0.19 at 540 nm. The incubation was completed

for 16-24 h at 37 oC with lid on micro plate and absorbance was measured at 540 nm using micro plate reader. Before

and after incubation, the difference was noted as an index of bacterial growth. The percent inhibition was calculated

using the formula:

Where, X = Absorbance in control with bacterial culture

Y = Absorbance in test sample.

Results are mean of triplicate (n = 3, ± SEM). Ciprofloxacin was taken as standard. MIC was measured with suitable

dilutions (5-30 µg well-1

) and results were calculated using EZ-Fit5 Perrella Scientific Inc. Amherst USA software.

Abbasi et al, 2015

27

3.5 Cholinesterase assays The AChE and BChE inhibitory assays were performed by the reported method

14,15 with minor modifications. 100 µL

volume was made by 60 µL Na2HPO4 buffer (50 mM; pH 7.7), 10 µL test compound (0.5 mM well-1

) and 10 µL

(0.005 unit well-1

) enzyme. These 100 µL assay volume was mixed together. After that its reading was taken at 405

nm followed by pre-incubation for 10 min at 37 ºC. The initiation of reaction was performed by the 10 µL of 0.5 mM

well-1

substrate (acetylthiocholine iodide for AChE and butyrylthiocholine chloride for BChE) followed by 10 µL DTNB (0.5 mM well

-1). After incubating for 15 min at 37 ºC, Again absorbance was taken at 450 nm. All conducted

tests were performed in three folds with the particular controls. Eserine (0.5 mM well-1

) was employed as a positive

control. The %age inhibition was accounted as:

By using EZ–Fit Enzyme kinetics software (Perella Scientific Inc. Amherst, USA), IC50 values of compounds were

intended, it is the concentration at which there is 50 % of enzyme is inhibited.

3.6 Lipoxygenase assay Lipoxygenase activity was assayed using the reported method

16-17 with some modifications. 200 µL assay mixture was

prepared from 150 µL Na3PO4 buffer (100 mM; pH 8.0), 10 µL test compound and 15 µL enzyme followed by

mixing, pre reading (at 234 nm) and pre incubation (10 minutes at 25 °C). Reaction was started at 25 µL of substrate

concentration, followed by absorbance at 234 nm repeated after every 6 min. All readings were taken thrice using positive and negative controls. Bailcain (0.5 mM well

-1) was used as a positive control. The % age inhibition was work

out by the similar method as given for cholinesterase assays.

4. SPECTRAL ANALYSIS 4.1 4-Methyl-N-(naphthalen-1-yl)benzenesulfonamide (3) Dark purple amorphous Solid; Yield: 72 %; m.p.: 176 ˚C; Molecular Formula: C17H15NO2S; Molecular Mass: 297 gmol

-1; IR (KBr, cm

-1): vmax: 3500 (N-H stretching), 3046 (C-H stretching of aromatic ring), 2978 (-CH2 stretching),

1635 (C=C stretching of aromatic ring), 1385 (-SO2 stretching), 1152; 1H-NMR (DMSO-d6, 400 MHz, ppm): δ 8.21

(br.d, J = 7.4 Hz, 1H, H-8), 7.82 (br.d, J = 7.4 Hz, 1H, H-5), 7.76 (d, J = 8.4 Hz, 2H, H-2' & H-6'), 7.52 (m, 2H, H-6 & H-7), 7.48 (t, J = 8.0 Hz, 1H, H-3), 7.31 (d, J = 6.4 Hz, 1H, H-2), 6.66 (d, J = 8.4 Hz, 2H, H-3' & H-5' ), 2.3 (s,

3H, CH3-7'); EI-MS (m/z): 297 [M]+, 233 [C17H15N]

+, 208 [C10H10NO2S]

+, 196 [C9H10NO2S]

+, 170 [C7H8NO2S]

+, 155

[C7H7O2S]+, 142 [C10H8N]

+, 127 [C10H7]

+, 91 [C7H7]

+.

4.2 N-(Propan-2-yl)-4-Methyl-N-(naphthalen-1-yl)benzenesulfonamide (5a) Move Sticky Solid; Yield: 65 %; Molecular Formula: C20H21NO2S; Molecular Mass: 339 gmol

-1; IR (KBr, cm

-1): vmax:

3449 (N-H stretching), 3047 (C-H stretching of aromatic ring), 2979 (-CH2 stretching), 1636 (C=C stretching of

aromatic ring), 1383 (-SO2 stretching); 1H-NMR (DMSO-d6, 400 MHz, ppm): δ 8.20 (br.d, J = 7.2 Hz, 1H, H-8), 7.84

(br.d, J = 7.4 Hz, 1H, H-5), 7.78 (d, J = 8.4 Hz, 2H, H-2' & H-6'), 7.76-7.74 (m, 2H, H-6 & H-7),7.72 (br.d, J = 6.4

Hz, 1H, H-4), 7.59 (br.t, J = 6.4 Hz, 1H, H-3), 7.50 (br.d, J = 8.8 Hz, 1H, H-2), 7.48 (d, J = 8.4 Hz, 2H, H-3' & H-5'), 4.50-3.33 ( m, 1H, H-2''), 2.30 (s, 3H, CH3-7'), 0.93 (d, J = 6.8 Hz, 6H, CH3-1'' & CH3-3''); EI-MS (m/z): 341 [M+2]

+

(C20H21NO2S+2).+, 339 [M]

+ (C20H21NO2S)

.+, 275 [C20H21N]

.+, 157 [C7H7O2S+2]

+, 155 [C7H7O2S]

+, 127 [C10H7]

+, 101

[C8H5]+, 91 [C7H7]

+, 75 [C6H4]

+, 65 [C5H5]

+, 51 [C4H3]

+, 43 [C3H7]

+, 41 [C3H5]

+.

4.3 N-(Butan-2-yl)-4-Methyl-N-(naphthalen-1-yl)benzenesulfonamide (5b) Purple Sticky Solid; Yield: 52 %; Molecular Formula: C21H23NO2S; Molecular Mass: 353 gmol

-1; IR (KBr, cm

-1): vmax:

3345 (N-H stretching), 3049 (C-H stretching of aromatic ring), 2975 (-CH2 stretching), 1639 (C=C stretching of

aromatic ring), 1383 (-SO2 stretching); 1H-NMR (DMSO-d6, 400 MHz, ppm): δ 8.20 (br.d, J = 7.6 Hz, 1H, H-8), 7.80

(br.d, J = 7.6 Hz, 1H, H-5), 7.78 (d, J = 7.2 Hz, 2H, H-2' & H-6'), 7.74-7.72 (m, 2H, H-6 & H-7), 7.64 (br.d, J = 7.2 Hz, 1H, H-4), 7.38 (br.t, J = 8.0 Hz, 1H, H-3), 7.01 (d, J = 7.2 Hz, 2H, H-3' & H-5'), 6.96 (br.d, J = 7.6 Hz, 1H, H-2),

4.40-4.34 (m, 1H, H-2''), 2.3 (s, 3H, CH3-7'), 1.57-1.54 (m, 2H, CH2-3''), 1.04 (d, J = 6.8 Hz, 3H, CH3-1''), 0.84 (t, J =

7.2 Hz, 3H, CH3-4''); EI-MS (m/z): 355 [M+2]+ (C21H23NO2S+2)

.+, 355 [M]

+ (C21H23NO2S)

.+, 289 [C21H23N]

.+, 157

[C7H7O2S+2]+, 155 [C7H7O2S]

+, 127 [C10H7]

+, 101 [C8H5]

+, 91 [C7H7]

+, 75 [C6H4]

+, 65 [C5H5]

+, 51 [C4H3]

+, 57

[C4H9]+, 41 [C3H5]

+, 29 [C2H5]

+.

4.4 N-(Pentan-1-yl)-4-Methyl-N-(naphthalen-1-yl)benzenesulfonamide (5c) Purple Sticky Solid; Yield: 71 %; Molecular Formula: C22H25NO2S; Molecular Mass: 367 gmol

-1; vmax: 3248 (N-H

stretching), 3045 (C-H stretching of aromatic ring), 2979 (-CH2 stretching), 1640 (C=C stretching of aromatic ring),

1385 (-SO2 stretching); 1H-NMR ( DMSO-d6, 400 MHz, ppm ): δ 8.18 (br.d, J = 7.2 Hz, 1H, H-8), 7.84 (br.d, J = 7.2

Hz, 1H, H-5), 7.80 (d, J = 7.6 Hz, 2H, H-2' & H-6'), 7.76-7.74 (m, 2H, H-6 & H-7), 7.66 (br.d, J = 7.4 Hz, 1H, H-4),

7.35 (br.t, J = 8.0 Hz, 1H, H-3), 6.98 (d, J = 7.6 Hz, 2H, H-3' & H-5'), 6.94 (br.d, J = 7.6 Hz, 1H, H-2), 3.63 (t, J =


28

7.6 Hz, 2H, CH2-1''), 2.3 (s, 3H, CH3-7'), 1.20-1.11 (m, 6H, CH2-2'' to CH2-4''), 0.85 (t, J = 6.4 Hz, 3H, CH3-5''); EI-MS (m/z): 369 [M+2]

+ (C22H25NO2S+2)

.+, 367 [M]

+ (C22H25NO2S)

.+, 303 [C22H25N]

.+, 157 [C7H7O2S+2]

+, 155

[C7H7O2S]+, 127 [C10H7]

+, 101 [C8H5]

+, 91 [C7H7]

+, 75 [C6H4]

+, 71 [C5H11]

+, 65 [C5H5]

+, 51 [C4H3]

+, 43 [C3H7]

+, 41

[C3H5]+.

4.5 N-(Benzyl)-4-methyl-N-(naphthalen-1-yl)benzenesulfonamide (5d) Dark Purple Sticky Solid; Yield: 67 %; Molecular Formula: C24H21NO2S; Molecular Mass: 387 gmol

-1; vmax: 3354 (N-

H stretching), 3056 (C-H stretching of aromatic ring), 2967 (-CH2 stretching), 1649 (C=C stretching of aromatic ring),

1390 (-SO2 stretching); 1H-NMR (DMSO-d6, 400 MHz, ppm): δ 8.02 (br.d, J = 7.2 Hz, 1H, H-8), 7.84 (br.d, J = 7.2

Hz, 1H, H-5), 7.80 (d, J = 7.8 Hz, 2H, H-2' & H-6'), 7.58-7.54 (m, 2H, H-6 & H-7), 7.44 (br.d, J = 7.4 Hz, 1H, H-4), 7.28-7.26 (m, 2H, H-2 & H-3), 7.23 (br.t, J = 7.0 Hz, 2H, H-3'' & H-5''), 7.21 (br.t, J = 8.5 Hz, 1H, H-4''), 7.17 (br.d, J

= 8.0 Hz, 2H, H-2'' & H-6''), 6.80 (d, J = 7.8 Hz, 2H, H-3' & H-5'), 4.93 (s, 2H, CH2-7''), 2.3 (s, 3H, CH3-7'); EI-MS

(m/z): 389 [M+2]+ (C24H21NO2S)

.+, 387 [M]

+ (C24H21NO2S)

.+, 323 [C24H21N]

.+, 157 [C7H7O2S+2]

+, 155 [C7H7O2S]

+,

127 [C10H7]+, 101 [C8H5]

+, 91 [C7H7]

+, 75 [C6H4]

+, 65 [C5H5]

+, 51 [C4H3]

+.

4.6 N-(2-Chlorobenzyl)-4-methyl-N-(naphthalen-1-yl)benzenesulfonamide (5e) Dark Purple Sticky Solid; Yield: 53 %; Molecular Formula: C24H20CINO2S; Molecular Mass: 421 gmol

-1; vmax: 3356

(N-H stretching), 3040 (C-H stretching of aromatic ring), 2989 (-CH2 stretching), 1645 (C=C stretching of aromatic

ring), 1382 (-SO2 stretching); 1H-NMR (DMSO-d6, 400 MHz, ppm): δ 8.04 (br.d, J = 7.6 Hz, 1H, H-8), 7.80 (br.d, J =

7.4 Hz, 1H, H-5), 7.78 (d, J = 8.0 Hz, 2H, H-2' & H-6'), 7.62-7.60 (m, 2H, H-6 & H-7), 7.42 (br.d, J = 7.4 Hz, 1H, H-4), 7.32 (br.d, J = 7.0 Hz, 1H, H-3''), 7.28-7.24 (m, 2H, H-2 & H-3), 7.20 (br.d, J = 7.4 Hz, 1H, H-6''), 7.12-7.10 (m,

2H, H-4'' & H-5''), 6.81 (d, J = 8.0 Hz, 2H, H-3' & H-5'), 4.50 (s, 2H, CH2-7''), 2.3 (s, 3H, CH3-7'); EI-MS (m/z): 425

[M+4 (C24H20CINO2S)].+

, 423 [M+2 (C24H20CINO2S)].+, 421 [M (C24H20CINO2S)]

.+, 359 [C24H20ClN+2]

.+, 357

[C24H20ClN].+

, 157 [C7H7O2S+2]+, 155 [C7H7O2S]

+, 127 [C7H6Cl+2]

+, 125 [C7H6Cl]

+, 113 [C6H5Cl+2]

+, 111

[C6H5Cl]+, 101 [C8H5]

+, 91 [C7H7]

+, 75 [C6H4]

+, 65 [C5H5]

+, 51 [C4H3]

+.

4.7 N-(4-Chlorobenzyl)-4-methyl-N-(naphthalen-1-yl)benzenesulfonamide (5f) Dark Purple Sticky Solid; Yield: 64 %; Molecular Formula: C24H20CINO2S; Molecular Mass: 421 gmol

-1; vmax: 3305

(N-H stretching), 3049 (C-H stretching of aromatic ring), 2943 (-CH2 stretching), 1642 (C=C stretching of aromatic ring), 1388 (-SO2 stretching);

1H-NMR (DMSO-d6, 400 MHz, ppm ): δ 8.08 (br.d, J = 7.2 Hz, 1H, H-8), 7.82 (br.d, J

= 7.2 Hz, 1H, H-5), 7.76 (d, J = 7.8 Hz, 2H, H-2' & H-6'), 7.64-7.62 (m, 2H, H-6 & H-7), 7.54 (d, J = 8.2 Hz, 2H, H-

3'' & H-5''), 7.44 (br.d, J = 7.2 Hz, 1H, H-4), 7.26-7.24 (m, 2H, H-2 & H-3), 7.02 (d, J = 8.2 Hz, 2H, H-2'' & H-6''),

6.72 (d, J = 7.8 Hz, 2H, H-3' & H-5'), 4.74 (s, 2H, CH2-7''), 2.3 (s, 3H, CH3-7'); EI-MS (m/z): 425 [M+4]+

(C24H20CINO2S+4).+

, 423 [M+2]+ (C24H20CINO2S+2)

.+, 421 [M]

+ (C24H20CINO2S)

.+, 359 [C24H20ClN+2]

.+, 357

[C24H20ClN].+

, 157 [C7H7O2S+2]+, 155 [C7H7O2S]

+, 127 [C7H6Cl+2]

+, 125 [C7H6Cl]

+, 113 [C6H5Cl+2]

+, 111

[C6H5Cl]+, 101 [C8H5]

+, 91 [C7H7]

+, 75 [C6H4]

+, 65 [C5H5]

+, 51 [C4H3]

+.

5. CONCLUSION A series of N-alkyl/aralkyl-4-methyl-N-(naphthalen-1-yl)-benzenesulfonamides (5a-f), was synthesized by reaction of

different alkyl/aralkyl halides with 4-methyl-N-(naphthalen-1-yl)-benzenesulfonamide (3) in polar aprotic solvent in the presence of base; LiH under stirring at room temperature. The N-substituted sulfonamides were structurally

confirmed by modern spectral techniques. The studied molecules displayed tremendous antibacterial activity against

gram-negative and gram-positive strains and also showed moderate to weaker inhibition against cholinesterases and

lipoxygenase enzymes. Hence the synthesized molecules could be utilized as suitable therapeutic agents for the treatment of different bacterial diseases.

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http://dx.doi.org/10.1021/bi00396a031


ISSN (Print): 2220-2625


*Corresponding Author Received 21st October 2014, Accepted 2nd February 2015

The Factors Effecting the Formation of Curcumin-Al (III) Complexes

*A. M. Rawa'a, N. M. Tariq and S. U. Wisam

*Basic Sciences Section, College of Agriculture, University of Baghdad, Baghdad, Iraq.

E-mail: *[email protected].

ABSTRACT Curcumin has been recognized as a potential natural drug to treat the Alzheimer’s disease (AD) by chelating metal ions. In the

present paper, curcumin–Al (III) [Cur - Al (III)] complexes were synthesized in aqueous solution and characterized by UV-visible

spectroscopy. The formation of complexes between Al (III) and curcumin in acidic condition, by using citric acid as catalyst were

studied. The complex molar ratio [Cur - Al (III] was found to be 2:1 in buffer phosphate of pH 1.0, 1.5, 2.0, 3.0, 3.5 and 4.0. The

optimum pH for the complex [Cur - Al (III)] formation was at phosphate buffer of pH=2.5, at this pH the complex [Cur. - Al (III)] was more stable than the other pH values (throughout the absorbance calculation at 531nm). About 92% of Al (III) was chelated

by curcumin at pH 2.5 as compared with 1.0, 1.5, 2.0, 3.0, 3.5 and 4.0 pH values. Curcumin complexes (at pH 2.5) were thermally

stable at 100 oC as compared with 75, 50 and 25oC respectively. The chemical shifts of spectrum shows that the curcumin was

strongly interact with Al (III).

Keywords: Cur –Al (III) complex, acidic medium, temperature & time

1. INTRODUCTION Curcumin is a hydrophobic polyphenol derived from rhizome of the herb Curcuma longa has a wide spectrum of

biological and pharmacological activities1. Curcumin (MW = 368.4) comprising about 3–5% of turmeric. It has a

strong yellow color yet no flavor, and widely used as dietary spice, as a food coloring2-4.

Curcumin exhibits anti-

protozoal, antibacterial, anti-inflammatory, and anti-oxidant activities5-6

.Curcumin is a strong antioxidant offers a

good protection against injuries caused by free radicals as compared with E7.

Chemically, curcumin is a bis-R,_-unsaturated _-diketone [1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-

heptadiene-3,5-dione]3-8

(commonly called diferuloylmethane) (Fig.1), curcumin exhibits keto–enol tautomerism

having a predominant keto form in acidic and neutral solutions9.Curcumin at pH 3–7, acts as an extraordinarily potent

H-atom donor10

. Zebib et al. was found that, more than 90% of curcumin decomposed rapidly in buffer systems of neutral and basic pH conditions. The stability of curcumin was increased in acidic pH condition

11 contributed to the

conjugated diene structure. However, when the pH is adjusted to neutral-basic conditions, the proton was removed

from the phenolic group leading to the destruction of this structure9-12

. In the keto form of curcumin, the heptadienone linkage between the two methoxyphenol rings

contains a highly activated carbon atom, and the C–H bonds on this carbon are very weak, due to delocalization of the

unpaired electrons on the adjacent oxygen's (Fig. 1)13

.

Fig.1: Di-keto (A) and keto-enolic tautomeric (B) conformers of curcumin.The numbers are symbols of the C or H atom.

The 1,3-diketone moiety of curcumin can transform kinetically to a keto–enol tautomeric form, and the later is more

stable and can readily chelate the metal ions to form the complexes and scavenge the active free-radicals14

. Curcumin

possesses wide-ranging anti-inflammatory and anti-cancer properties. Many of these activities can be attributed to its


31

potent antioxidant capacity at neutral and acidic pH, its inhibition of cell signaling pathways at multiple levels, its

diverse effects on cellular enzymes and its effects on angiogenesis and cell adhesion13

.

Aluminum is the second most abundant metal in the earth crust next to iron. It is widely used in industrial applications as well as in domestic uses. Indirect intake of Al (III) into our bodies cannot be ignored as the

accumulation of Al (III) in brain, although it has been identified to cause diseases such as Alzheimer’s disease (AD)

15-17

. The analysis of aluminum, especially in food samples is very critical. The situation can be even worse in case of food prepared in aluminum pan with white vinegar added in. Regular consumption of food prepared in this way is

potentially dangerous as accumulation of Al in brain cells has been blamed for causing (AD)18

.The risk increases

through drinking of ground water with Al level at 0.10 to 0.20 μg mL-1 19

.

Complexation of curcumin with transition metals has attracted much interest over the past years as one of the useful requirements for the treatment of (AD)

2,8,20 and in vitro antioxidant activity

21. Furthermore, several

metallocomplexes of curcumin have been synthesized, characterized and evaluated for various biological activities14,

22, 23.

There are many investigations to demonstrate some metal elements involved in the (AD) development. These

metal elements include Pd (II)24

, Cr (III) , Mn (II) ,Fe (III) , Cu (II) ,Zn14,25-27

and Al8,16,28

etc. Among those metals, Al

(III) a component in the senile plaques is an important element impacting on the aggregation and toxicity of Aβ

peptides29

. Therefore, one of approaches for the (AD) treatment is searching for the agents that can chelate metal ions

30, preventing metal ions from the interaction with Aβ peptides as well as the redox reaction which leads to the

oxidative stress. So far, some chelating agents and antioxidants have been reported to treat AD31-32

.

Despite the detailed information from previous studies on curcumin structure and function, the interaction, components and structures of curcumin –Al (III) complexes have not been investigated clearly. Thus the aim of the

present is to study the formation of Curcumin –Aluminum (III)[Cur.-Al (III)] in acidic condition in addition to study

the factors effecting the complex formation.

2. EXPERIMENTAL

2.1 Instrumentations and Chemicals

The UV-Visible Spectrophotometer Shimadzu model U.V.-160A and SS-3 pH Meter have been used in the present

study. The Chemicals KH2PO4, K2HPO4, sodium potassium tartrate and AlCl3.6H2O from Fluka, citric acid and ethanol (96%) from BHD and local production deionized water.

2.2 Preparationof curcumin The curcumin (Cur.) was prepared by extraction 5grams of turmeric in 150 ml of (96%) ethanol and refluxed for 2hr

at boiling point .The hot mixture was filtered; the filtrate was evaporated at 50oC till complete dryness

33. The dry

matter was weighed to find out the curcumin percentage (it was 25%).

2.3 Preparation of curcumin- Al complex [Cur.-Al (III)]

The mixture of complex [Cur.-Al (III)] was prepared in different pH values. 5ml of 2×10-4

M (Cur.) solution, 5ml of

2×10-4

M citric acid solution (as catalyst) , 5 ml of phosphate buffer pH = 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0. 5ml of

1×10-4

M AlCl3.6H2O was added to the above mixture in molar ratio [2: 1: 2 ] of curcumin, Al(III) and citric acid

respectively; 5 drops of 0.1M sodium potassium tartrate were added to inhibit the formation of insoluble AlCl3.6H2O4.

The mixtures absorbance was determined at room temperature after 30min.

2.4 Preparation of [Cur.-Al (III)] complex in different temperatures (25, 50, 75 and 100)oC with time (0, 15,

30, 45 and 60) min. in optimum pH (2.5) of phosphate buffer The mixture value of [Cur.-Al(III)] complex was 5ml of 2×10

-4 M (Cur.) solution, 5ml of 2×10

-4 M citric acid solution

, 5 ml of phosphate buffer of pH= 2.5 which was taken as optimum pH according to the previous experiments . 5ml of

1×10-4

M AlCl3.6H2O was added to the above mixture in molar ratio [2:1: 2, curcumin: Al (III): citric acid] with 5

drops of 0.1M sodium potassium tartrate. The mixture was heated in water bath at 25, 50, 75 and 1000C for 0, 15, 30,

45 and 60 min. The mixtyres were subjected to spectrophotometer to record absorbance at 531nm, which is noticed as

λmax for Al(lll)-curcumin complex solution.

3. RESULTS AND DISCUSSION As recommended by previous study

11, we have used an acidic pH conditions which have maintained more than 90%

of curcumin, as compared with neutral and basic conditions. The stability of curcumin in acidic conditions may

attribute to the conjugated diene structure. In acidic and neutral conditions the proton will be removed from the phenolic group leading to destruction of curcumin structure

11. Fig (2) shows a direct proportional relationship between

pH value and the recorded absorbance.

Rawa’a et al 2015

32

Fig.2: The absorbance (at 423nm) of 2 x 10-4 M curcumin at different phosphate buffer pH values.

We found that the optimum pH for the best complexation [Cur. - Al (III)] was at phosphate buffer of pH=2.5. At this

pH, the complexation [Cur - Al (III)] was more stable than the other pH values. It's well known from present study (throughout the absorbance calculation at 531nm), that about 92% of Al (III) was chelated by curcumin at pH 2.5 as

compared with 1.0, 1.5, 2.0, 3.0, 3.5 and 4.0 pH values.

The UV-visible spectrum bands of curcumin showed that the maximum absorption (table 1) in 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 pH values were at 424, 423, 423, 423,422, 422 and 415 nm respectively. These bands assigned to

the π → π* of curcumin (figure 3) 11,14,24,25

. The UV –visible spectrum bands of curcumin as compared with the

complexes of [Cur. - Al (III)] (table 1 & Figure 4) show that the maximum absorption of complexes were shifted to higher wavelengths (table 1) at the same buffer pH respectively.

The interaction between curcumin and metal ions was implied by the change in optical absorbance of

curcumin upon mixing with a metal solution 2, 8, 14, 24, 34-36

. The addition of Al (III) to aqueous solution of curcumin

increases the absorbance spectrum to a higher wavelength with bathochromic shift of about 107, 109, 75, 108, 80, 80 and 96nm from the original band in the absence of Al(III). It is probable, that the bathochromic shift occurs as a result

of coordination by the lone absence and pair electrons on the oxygen (O) donor atoms with the aluminum ion site, thus

stabilizing the excited state relative to the ground state and leading to longer wavelength absorption maxima 37.

The observed bathochromic shifts are consistent with the lone pair electrons in the donor atoms (O in curcumin) and

participating in (Al ion) coordination which in turn stabilizing the excited state relative to the ground state.

Since the curcumin and its complexes were thermally stable up to 160o C

11,38, the effect of 25, 50, 75 and 100

o

C for 0, 15, 30, 45 and 60 min with a molar ratio of 2:1:2 (curcumin: Al: citric acid) were studied. Table (2) and Fig

(5) show that the complex [Cur. - Al] was highly stable at 100o C up to 60 min (the yellowish color disappear due to

the chelation activity of curcumin).

0.4

0.43

0.46

0.49

0.52

0.55

0.58

1 1.5 2 2.5 3 3.5 4 4.5

Ab

sorb

ance

pH of buffer solution

Table-1: Electronic absorption spectra U.V of curcumin and complex [Cur.-Al (III)]

pH of

buffer

Abs.maximum of

curcumin

Wavelength nm of

curcumin

Abs. maximum of

complex[Cur.-Al(III)]

Wavelength nm of complex[

Cur.-Al(III)]

1.0 0.415 424 0.046 531

1.5 0.408 423 0.034 532

2.0 0.399 423 0.045 498

2.5 0.337 423 0.027 531

3.0 0.375 422 0.063 502

3.5 0.367 422 0.063 502

4.0 0.343 415 0.048 511

Cur.

pH = 1.0

pH = 1.5

pH = 2.0

pH = 2.5

pH = 3.0

pH = 3.5

pH= 4.0


33

Fig-3: Electronic absorption spectra U.V of curcumin 2×10-4 M and citric acid 2×10-4 M at different values of buffer pH= 1.0,

1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 without Al(III).

Fig-4: Electronic absorption spectra U.V of complex of [curcumin 2×10-4 M and citric acid 2×10- 4M in different values of

buffer pH= 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 with Al(III) 1×10-4M]

Table-2: Electronic absorption spectra U.V of complex of curcumin 2×10-4 M and citric acid 2×10-4M in optimum pH (2.5) of

phosphate buffer with Al (III) 1×10-4M in different temperature (25, 50, 75 and 100oC) with time (0, 15, 30, 45 and 60) min. in λ =

531 nm.

time min. Abs. at 25 o C Abs. at 50

o C Abs. at 75

o C Abs. at 100

o C

0 0.025 0.016 0.016 0.015

15 0.026 0.016 0.015 0.013

30 0.027 0.015 0.012 0.010

45 0.027 0.014 0.011 0.018

60 0.031 0.013 0.008 0.014

Cur. pH = 1.0 pH = 1.5 pH = 2.0 pH = 2.5 pH = 3.0 pH = 3.5 pH= 4.0

pH =1.0 pH =1.5 pH =2.0 pH =2.5 pH =3.0 pH =3.5 pH =4.0

Absor.

Rawa’a et al 2015

34

Fig-5: Electronic absorption spectra U.V in λ = 531 nm of complex of curcumin 2×10-4 M and citric acid 2×10-4M in optimum pH (2.5) of phosphate buffer with Al(III) 1×10-4M in different temperature (25, 50, 75 and 100)oC with time (0, 15, 30, 45 and 60)

min.

4. CONCLUSION The optimum pH, for the best complex [Cur - Al (III)] formation was at phosphate buffer of pH=2.5 (92% of Al (III)

was chelated by curcumin). The Al (III) caused a bathochromic shift of the visible absorption bands in curcumin solution. The absorbance show that the complex of [Cur. – Al (III)] was very stable in higher degree of temperature

(100 oC) until 60 min. All chemical shifts of spectrum pointed that the curcumin was interact strongly with Al (III) ion

under different pH values and temperature degrees.

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ISSN (Print): 2220-2625


*Corresponding Author Received 21st July 2014, Accepted 23rd March 2015

Ruthenium azo complexes: Synthesis, spectra and electrochemistry of dithiocyanato-

bis-{1-(alkyl)-2-(arylazo)imidazole}ruthenium(II)

*P. Byabartta

Inorganic Chemistry Research Laboratory, Department of Chemistry, Jogesh Chandra Chaudhuri College,

30- Prince Anwar Shah Road, Kolkata-700033, India.

Email: *[email protected], [email protected]

ABSTRACT Silver assisted aquation of blue cis-trans-cis-RuCl2 (RaaiR´)2 (1) leads to the synthesis of solvento species, blue-violet cis-trans-

cis-[Ru(OH2)2(RaaiR´)2](ClO4)2 [RaaiR = p-R-C6H4-N=N-C3H2-NN, (2), abbreviated as N,N/-chelator, where N(imidazole)

and N(azo) represent N and N/, respectively; R = H (a), p-Me (b), p-Cl (c) that have been reacted with NCS− in warm EtOH

resulting in red-violet dithiocyanato complexes of the type, [Ru(NCS)2(RaaR)2] (3-5). The solution structure and stereoretentive

transformation in each step have been established from 1H n.m.r. results. All the complexes exhibit strong MLCT transitions in

the visible region. They are redox active and display one metal-centred oxidation and successive ligand-based reductions. Linkage

isomerisation was studied by changing the solvent and then UV-Vis spectral analysis.

Keywords: 1-alkyl-2-(arylazo)imidazole, thiocyanato, ruthenium(II), MLCT, NMR, CV, IR.

1. INTRODUCTION The nature of chemical reactions of organic substrates can vastly be affected by their coordination to metal ions. It is

now known that organonitriles are activated by metal coordination toward addition reactions leading to a variety of synthetic transformations of RCN species. Thiocyanates, as ligand, have attracted considerable attention in recent

years not only because of their versatile coordination abilities but also some of their transition metal complexes have

been found to be useful. The ruthenium chemistry of diimine ligands is an area of significant current interest, particularly with regard to the photophysical and photo-chemical properties exhibited by such complexes. Di-imine

ligands are strong π-acceptors and are recognized stabilizers of the +2 state of ruthenium (low-spin d6, S=0). As a

consequence, an interesting aspect of the ruthenium–diimine chemistry has been to study the remarkable π-interaction between the filled t2 orbitals of ruthenium(II) and the low-lying vacant π*-orbital of the diimine chromophore. The

extent of π-interaction in these complexes depends primarily on the nature of the diimine ligands, which again

depends on the nature of the groups linked to the two carbons and the two imine-nitrogens. The presence of other π-

acceptor ligands within the coordination sphere may also have significant influence on the π-interaction between the diimine ligands and ruthenium(II)

1-5. From the viewpoint of the principle of hard and soft acids and bases, both the

RuNCS and RuSCN isomers can occur in the ruthenium complexes. For the last few years, the search for a suitable

precursor to synthesize NCS complexes is a challenging domain and the compounds are found to be useful in this context

6-11. Recently, we have developed the arylazopyrimidine as well as arylazoimidazole chemistry of ruthenium(II)

and have synthesised dichloro componds and diaquo species. Syntheses of hetero-tris-chelates, [Ru(bpy)n(RaaiR/)3-

n](ClO4)2 [bpy = 2,2′-bipyridine; n = 1, n = 2) from the solvento complexes

[Ru(OH2)2(bpy)2]2+/[Ru(OH2)2(RaaiR/)2]2+ containing labile reaction centres are reported from Prof. Sinha´s laboratory

12-23. In this paper, I examine the reaction of NCS− towards [Ru(OH2)2(RaaiR)2]2+ and the reactions of the

complexes derived there-from and also studied the dinuclear adduct formation pathway. The complexes were well

charecterised by C.H.N, FT-I.R, U.V-Vis, and Cyclic Voltammetrically. Linkage isomerisation was studied by changing the solvent and then UV-Vis spectral analysis.

2. EXPERIMENTAL Published methods were used to prepare RaaiR

7-8, ctc-RuCl2 (RaaiR)2

7-8, ctc-[Ru(OH2)2(RaaiR)2](ClO4)2 . All other

chemicals and organic solvents used for preparative work were of reagent grade (SRL, India). The purification of

MeCN and preparation of [n-Bu4N][ClO4] respectively used as solvent and supporting electrolyte in electrochemical

experiments were done following the literature method. Microanalytical data (C, H, N) were collected using a Perkin Elmer 2400 CHN instrument. Solution electronic spectra were recorded on a JASCO UV-VIS-NIR V-570

spectrophotometer. I.r. spectra were obtained using a JASCO 420 spectrophotometer (using KBr disks, 4000-200 cm-

1). The 1H nmr spectra in CDCl3 were obtained on a Bruker 500 MHz FT n.m.r spectrometer using SiMe4 as internal

reference. Solution electrical conductivities were measured using a Systronics 304 conductivity meter with solute

concentration ∼10-3 M in acetonitrile. Electrochemical work was carried out using an EG & G PARC Versastat

computer controlled 250 electrochemical system. All experiments were performed under a N2 atmosphere at 298K

using a Pt-disk milli working electrode at a scan rate of 50 mVs-1. All results were referenced to a saturated calomel electrode (SCE). Reported potentials are uncorrected for the junction effect.



37

Preparation of cis, trans, cis-Dithiocyanato-bis-{1-methyl-2-(p-tolylazoimidazolee} ruthenium(II), ctc-Ru(NCS)2

(MeaaiMe)2 CAUTION ! Perchlorates of heavy metal ions with organic ligands are potentially explosive. The syntheses involve in

some cases the use of perchlorate ions. Three independent methods were employed to synthesise.

Method (a). To an EtOH blue-violet solution (15 cm3) of ctc-[Ru(OH2)2(MeaaiMe)2](ClO4)2 (0.1 g, 0.14

mmol) was added 0.019 g (0.27 mmol) of solid NH4NCS, and the mixture was stirred at 343-353 K for 12 h. The violet solution that resulted was concentrated (4 cm3) and kept in a refrigerator overnight (12 h). The precipitate was

collected by filtration, washed thoroughly with H2O and dried in vacuo over CaCl2. Analytically pure (7b) was

obtained after chromatography over an alumina (neutral) column on eluting the red-violet band with toluene-acetonitrile (4:1, v/v) and evaporating slowly in air. The yield was 0.088 g (80%).

Method (b). To a suspension of ctc-RuCl2(MeaaiMe)2 (4b) (0.1 g, 0.18 mmol) in EtOH (25 cm3) was added

an aqueous solution of AgNO3 and stirred at room temperature (300 K) for 2 h. The AgCl which precipitated was

filtered through a G-4 sintered crucible. An EtOH solution of NH4NCS (0.025 g, 0.35 mmol) was added to the filtrate, and the resulting mixture was stirred at room temperature for 8 h under a N2 atmosphere. The violet solution was

concentrated by slow evaporation and the precipitate was processed as in Method (a); yield, 0.047 g (45%).

Method (c). To a CH2Cl2-Me2CO (1:1, v/v, 30 cm3) solution of ctc-RuCl2(MeaaiMe)2 (4b) (0.1 g, 0.18 mmol) was added an H2O-Me2CO solution of NH4NCS (0.024 g, 0.35 mmol). The mixture was stirred at 343-353 K

for 30 h. The resulting violet solution was processed as in method (a) to give analytically pure dithiocyanato

complexes; yield, 0.021 g (20%). The high yield in method (a) has prompted us to follow this route for the syntheses of the other complexes (3b-3e). The yields varied in the range 65-85%.

3. RESULTS AND DISCUSSION Diaquo complexes ctc-[Ru(OH2)2(RaaiR)2](ClO4)2, prepared by Ag+-assisted aquation of ctc-RuCl2(RaaiR)2 , were reacted with NH4NCS (excess amount >3 mol) under stirring at 343-353 K in aqueous alcohol to give

Ru(NCS)2(RaaiR)2 (3-5) in good yield (65-85%). The synthetic routes are shown in Scheme 1. The dithiocyanato

were synthesized in low yield either directly on stirring in ethanol-acetone mixture for 30 h or in situ synthesis of the

aquo complex by AgNO3 followed by the reaction with NH4NCS. The composition of the complexes is supported by microanalytical results. Room temperature solid state magnetic susceptibility measurements show that the complexes

are diamagnetic (t2g6, S = 0). The violet dithicyanato complexes are soluble in common organic solvents but

insoluble in H2O. In MeCN, they show as non-electrolytic behaviour is found for type complexes as indicated by their very low ΛM values (10-20 Ω-1cm-1mol-1).

I.r. spectra of the complexes, Ru(NCS)2(RaaiR)2 (3-5) show a 1:1 correspondence to the spectra of the dichloro analogue, ctc-RuCl2(RaaiR)2 except the appearance of intense stretching at 1300-1335 and 1250-1280 cm-1 with

concomitant loss of ν(Ru-Cl) at 320-340 cm-1. They are assigned to ν(NCS)as and ν(NCS)s, respectively [5,6,15].

The ν(N=N) and ν(C=N) appear at 1365-1380 and 1570-1600 cm-1, respectively. The present series of Ru-NO complexes are assumed to contain linear NCS group. Other important frequencies are ν(H2O) at 3350-3400 cm-1 The

solution electronic spectra of these new complexes were recorded in dry acetonitrile. Dithiocyanato complexes (3-5)

Byabartta et al, 2015

38

exhibit multiple transitions in the uv-visible region (Table 1, Figure 1). They display intense MLCT transition in the

550-560 nm range. The transitions are blue shifted by ~ 40 nm as compared with corresponding dichloro derivatives, RuCl2(RaaiR)2 [6,8,11]. The 1H n.m.r. spectra of Ru(NCS)2(RaaiR)2 (3-5) complexes were unambiguously assigned

(Table 2) on comparing with RuCl2(RaaiR)2 [7-9].

Fig-1: Complete UV-Vis

spectra of the complex Ru(NCS)2 (p-Me-aaiBz)

2, 5b

Table-1: Microanalytical (C.H.N) a

and FT-IR spectroscopicb

data

Complexes C H N ν(N=N)ν(C=N)ν(C=C)ν(NCS)

Ru(NCS)2

(H-aaiMe)2 ,3a 42.4 3.5 24.8

(42.3) (3.4) (24.7) 1365 1570 1600 1300 2100

Ru(NCS)2

(p-Me-aaiMe)2,

3b 44.4 4.0 23.6

(44.5) (4.1) (23.7) 1367 1580 1602 1320 2110

Ru(NCS)2

(p-Cl-aaiMe)2, 3c 37.8 2.84 22.1

(37.7) (2.7) (22.0) 1370 1590 1610 1325 2120

Ru(NCS)2

(H-aaiEt)2, 4a 44.3 4.2 23.5

(44.5) (4.1) (23.7) 1375 1585 1613 1320 2105

Ru(NCS)2

(p-Me-aaiEt)2, 4b 37.8 4.84 22.1

(37.7) (2.7) (22.0) 1380 1590 1609 1310 2109

[Ru(NCS)2(p-Cl-aaiEt)2], 4c 34.1 4.3 19.9

(34.0) (2.4) (19.8) 1370 1570 1609 1300 2108

Ru(NCS)2

(H-aaiBz)2, 5ª 34.0 3.1 18.0

(34.1) (3.0) (18.1) 1365 1575 1606 1310 2122

Ru(NCS)2

(p-Me-aaiBz)2, 5b 31.1 3.81 18.1

(31.2) (1.82) (18.2) 1380 1570 1609 1320 2133

Ru(NCS)2

(p-Cl-aaiBz)2, 5c 34.2 3.2 18.1

(34.1) (3.0) (18.1) 1370 1575 1609 1310 2113

a

Calculated values are in parenthesis; On KBr disk The aryl protons (7-H—11-H) are downfield shifted by 0.1-0.7 ppm as compared to those of the parent dichloro

derivatives. They are affected by substitution; 8- and 10-H are severely perturbed due to changes in the electronic properties of the substituents in the C(9) and C(10)-position. The aryl protons resonate asymmetrically indicative of a


39

magnetically anisotropic environment even in the solution phase. The proton movement upon substitution (9-R) is

corroborated with the electromeric effect of R. Imidazole 4- and 5-H appear as doublet at the lower frequency side of the spectra (7.0-7.2 ppm for 4-H; 6.9-7.1 ppm for 5-H). The aryl-Me (R = Me) in Ru(NCS)2(MeaaiR)2 (3-5) appears

as a single signal at 2.30 ppm and is in consonance with stereoretentive nucleophilic substitution during synthesis of

dithiocyanato complexes from ctc-RuCl2(RaaiR)2 via aquo derivatives. Isomerisation of the ctc-isomer may lead to

ccc-configuration belonging to C1-symmetry and would give two equally intense Ar-Me signals, which is however not the case here. The potential values of the complexes in dry acetonitrile solution are set out in Table 1. The

dithiocyanato complexes exhibit a quasi-reversible (ΔEP ≥ 100 mV) oxidative response in the potential range 1.0-1.3

V. This is assigned to the Ru(III)/Ru(II) couple. The one-electron stoichiometry of this couple is confirmed by constant potential electrolysis vs SCE and the electron count ratio equals 0.94. The potential E1/2M is dependent on

the substituent type R. The present series of complexes show higher E1/2M values than that of precursor dichloro

derivatives by ~ 0.4 V. The better electron withdrawing property of NCS- over Cl- stabilises the dπ shell of the metal

and thus shifts the metal-centred redox process to more anodic values. The stronger π-acidic nature of RaaiR/ compared to α-diimine system leads to better stabilisation of Ru(II) in the present series of complexes. The cyclic

voltammogram of Ru(NCS)2(RaaiR/)2 exhibit some unusual behaviour on repetitive cycles. The reduction sweep

shows a new wave that has a counter oxidative wave on the second sweep. The second and consecutive cycles increase the peak height with subsequent decrease of the primary couple. The assignment is based on earlier

observations of similar Ru-bipyridine [21] and Ru-azopyridine [11] systems. The potential values of the present set of

complexes lie between bipyridine and azopyridine analogous complexes and follow the order azopyridine > azoimidazole > bipyridine. This is in line of π-acidity order of these different ligand systems. The one-electron

stoichiometry of the couples is assigned by comparison of current heights in differential pulse voltammetry

experiments. Successive reductions on the negative side of SCE were observable and one-electron nature was

confirmed by comparing the current heights of these process with that of couple II in the differential pulse voltammetry experiments and are assigned to the reduction of coordinated ligand. The azo group in RaaiR/ may

accommodate two electrons and hence two coordinated ligands should exhibit four reductive responses. However,

within the available potential window two reductions were clearly observable.

Table-2: UV-Visa

and cyclic voltammetricb

data

Cyclic Voltammetric data E/V (ΔEP

/ mV) UV-Vis spectra λ

max(nm)(10

-3

ε/dm3

mol-1

cm-1

) Compound

-E

L

EM1

0.388(80), 0.651(130) 1.188(110) 551(8.379),421(8.914)d

, 373(18. 3) (3a)

0.407(95), 0.691(120) 1.101(115) 548(6.773), 421(12.271)d

, 379(17.791) (3b)

0.344(80), 0.673(100) 1.201(107) 555(13.919), 424(13.416)d

, 384(40.721) (3c)

0.377(80), 0.632(75) 1.108(120) 550(8.796), 416(10.081)d

, 376(20.755) (4a)

0.383(85), 0.647(100) 1.011(80) 547(8.752), 423(7.149)d

, 380(23.694) (4b)

0.401(80) 0.711(120) 1.010(110) 550(3.996)d

, 408(10.616), 258(12.403) (4c)

0.351(85), 0.621(120) 1.032(120) 555(3.118)d

, 412(13.016), 260(14.469)d

(5a)

0.401(80), 0.711(120) 1.010(110) 550(3.996)d

, 408(10.616), 258(12.403) (5b)

0.351(85), 0.621(120), 1.032(120) 555(3.118)d

, 412(13.016), 260(14.469)d

(5c) a

Solvent dry MeCN; d

shoulder; b

Solvent dry MeCN, supporting electrolyte [n

Bu4N][ClO

4] (0.1M), w.e. Pt-disk, a.e. Pt-wire, r.e.

SCE, solute conc. ~10-3

M, scan rate 50 mVs-1

, EM

: metal oxidation, eqn (3), EL

: ligand reductions, ΔEP

= | Epa

- Epc

| V where Epa

= anodic peak potential and Epc

= cathodic peak potential.

3.1 Synthesis and Separation of the Linkage Isomers. [RuCl2(RaaiR)2] was prepared according to a previous report in the literature. The violet product of

[Ru(NCS)2(RaaiR)2] was synthesized by a method as mentioned above. To a DMSO solution of [RuCl2(RaaiR)2] ( 0.2

mmol) was added an aqueous solution containing 2 equiv of AgNO3 (0.068 g) and the reaction mixture stirred at 100

C for 2 h. A gray precipitate of AgCl was removed by filtration. To the resulting yellow filtrate was added an

aqueous solution of excess KSCN (0.104 g, 1 mmol) at ca. 5 C resulting in the immediate formation of the grey-

violet precipitate. The precipitate was filtered and repeatedly washed with water to completely remove all traces of DMSO. This process of washing is very important to avoid the conversion. The violet crystals thus obtained were

confirmed to be mostly [Ru(NCS)2(RaaiR)2] on the basis of UV-Vis spectral analysis. Recrystallization of the violet

product from acetone, acetone/DMSO (99/1 v/v), and DMF formed violet crystals of [Ru(SCN)2(RaaiR)2], grey-violet

Byabartta et al, 2015

40

crystals of [Ru(SCN)(NCS)(RaaiR)2], and red-violet crystals of [Ru(NCS)2(RaaiR)2], respectively. Due to the change

of hard donar centre, N to soft donar centre, S, the HOMO and LUMO gap changes, which shift the MLCT band from high energy to low energy value.

Table-3: 1

H-n.m.r. spectral data, δ (J/Hz), ppm of the complexes in CDCl3

Compd 4-Hc

5-Hc

7-Hc

11-H 8-H 10-H

(3ª)a

7.15 (7.5) 7.06 (7.5) 8.03 (8.1) 7.88 (8.91) 7.80 (8.1) 7.86 (6.1)

(3b) 6.98 (7.5) 6.86 (7.5) 8.17 (8.1) 8.09 (8.1) 8.04 (8.1) 8.01 (8.01)

(3c) 7.13 (8.1) 7.02 (8.1) 8.15 (7.8) 7.99 (7.0) 7.95 (7.8) 7.92 (8.8)

(4ª)a

7.14 (7.5) 7.00 (7.5) 8.01 (7.8) 7.95 (7.1) 7.85 (7.8) 7.89 (7.8)

(4b) 7.02 (8.1) 6.94 (8.1) 8.11 (7.5) 8.04 (6.5) 8.04 (7.5) 8.07 (6.5)

(4c) 7.13 (8.1) 7.06 (8.1) 8.14 (7.5) 8.06 (8.5) 8.06 (7.5) 8.0 (7.7)

(5ª)a

7.06 (7.8) 6.98 (7.8) 8.08 (8.1) 8.10 (8.3) 8.00 (8.1) 7.00 (8.1)

(5b) 6.97 (8.1) 6.85 (8.1) 8.21 (8.1) 7.80 (8.1) 8.10 (8.1) 8.50 (8.7)

(5c) 7.11 (7.8) 7.02 (7.8) 8.15 (8.1) 8.00 (6.1) 8.05 (8.1) 7.05 (8.01) a

δ(9-H) 7.60 ppm(m); b

δ (9-Me); c

doublet; d

triplet; e N-Bz,

AB type sextet, geminal coupling constant,4.98,4.78,; f

1-Me, singlet,

1.98; g N-Et,

AB type quartet, geminal coupling constant, 4.44,4.12.

4. CONCLUSIONS Dithiocyanato complexes of ruthenium(II)–azoimidazole, ctc-Ru(NCS)2(RaaiR)2 have been synthesised by stereoretentive reaction of diaquo complex [Ru(OH2)2(RaaiR)2]2+ with thiocyanate ion. The complexes exhibit

strong MLCT transitions. Voltammetric study shows Ru(III)/Ru(II) couple along with successive ligand-based

reductions. Linkage isomerisation was studied by changing the solvent and then UV-Vis spectral analysis.

5. ACKNOWLEDGEMENT Department of Science and Technology (DST) is thanked for financial support. ( FAST TRACK Grand No.

SERB/F/4888/2012-13 Dated 30.11.2012, Project Title: GOLD (I) & GOLD(III) ARYLAZOIMIDAZOLE (N, N

DONAR) & OXO COMPLEXES : SYNTHESIS, STRUCTURE, SPECTRAL STUDY, ELECTROCHEMISTRY AND CHEMICAL REACTIVITY ).

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Full Paper

ISSN (Print): 2220-2625

ISSN (Online): 2222-307X

*Corresponding Author Received 5th September 2014, Accepted 22nd May 2015

α-Glucosidase Inhibitory Constituents from Ficus bengalensis M. A. Naveed, N. Riaz*, M. Saleem, B. Jabeen,

1M. Ashraf,

1U. Alam and A.Jabbar

*Department of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur,

Bahawalpur 63100, Pakistan 1Department of Biochemistry & Biotechnology, Baghdad-ul-Jadeed Campus, The Islamia University of

Bahawalpur, Bahawalpur 63100, Pakistan

E-mail: *[email protected]; [email protected]

ABSTRACT Chromatographic purification of the methanolic extract of the aerial roots of Ficus bengalensis resulted the purifiaction of 3β-

acetoxyurs-9(11),12-diene (1), a new triterpene together with lupeol (2), lupeol acetate (3), conrauidienol (4), β-sitosterol (5),

alpinum isoflavone (6), methyl 4-hydroxybenzoate (7), 4-hydroxymellein (8), 4-hydroxybenzoic acid (9), p-coumaric acid (10),

oleanolic acid (11) and β-sitosterol 3-O-β-D-glucopyranoside (12). These compounds (1-12) were characterized by using 1D- (1H, 13C) and 2D-NMR (HMQC, HMBC, COSY, NOESY) spectroscopy and mass spectrometry (EI-MS, HR-EI-MS, FAB-MS, HR-

FAB-MS), and in comparison with the data reported for related compounds in literature. These isolates (1-12) showed inhibitory

activity against enzyme α-glucosidase with IC50 values ranging between 26.3-297.7 µM.

Key words: Ficus bengalensis, Methanolic extract, Secondary metabolites, α-Glucosidase inhibition.

1. INTRODUCTION The Genus Ficus belongs to the plant family Moraceae consists of 800 species and most of which are native to the old

world tropics1. Ficus bengalensis is a large evergreen tree distributed throughout subcontinent

2. In English, it is known

by the name “Banyan” because the Banias (Hindu merchants) used to assemble their business under this tree. Various species of the genus Ficus are used in local medicine as astringents, carminatives, stomachaches, vermonicides,

hypotensives, antihelmintics and anti-dysentery drugs3. The aerial roots of F. bengalensis are used for the healing of

wounds formed during urine flow, dysentery, diarrhea, conjunctivitis, scabies, diabetes, styptic, useful in syphilis, biliousness and inflammation of liver

4. The aqueous extract of its aerial roots is used to increase immunity against

various diseases in Ayurvedic system of medicine5 to heal skin cracks and its young twigs are used as tooth brushes

6.

Its stem latex is used as aphrodisiac, tonic, vulnerary, maturant, lessens inflammations and is useful in piles, nose-

diseases and gonorrhea in Unani system of medicine7. Its bark is used to decrease cholesterol levels

8. Various extracts

of bark of F. bengalensis possess anti-allergic and anti-stressive potential against asthma9. This plant contains

phenolics which are used for preventing cardiovascular diseases, neurodegenerative diseases and cancer10

. The various

fruit extracts of F. bengalensis exhibited antitumor activity11

. Previously, we reported the cholinesterase inhibitory constituents from the aerial roots of F. bengalensis

12. Now, we are reporting the purification, characterization and α-

glucosidase inhibition of the isolated compounds (1-12) from this source.

2. RESULTS AND DISCUSSION The methanolic extract of aerial roots of F. bengalensis was divided into n-hexane, ethyl acetate and n-butanol soluble

fractions. The chromatographic purification of the ethyl acetate soluble fraction resulted in the purification of twelve

compounds (1-12) which were characterized by using spectroscopic techniques such as UV, IR 1D- (1H,

13C) and 2D-

NMR (HSQC, HMBC, COSY, NOESY), and mass spectrometric techniques (EI-MS, HR-EI-MS, FAB-MS, HR-FAB-MS).

Compound (1) was purified as colorless amorphous powder. Its IR spectrum showed peaks for ester C=O

(1735 cm-1

) and C=C (1650 cm-1

). The HR-EI-MS showed molecular ion peak at m/z 466.3800 corresponding to the molecular formula C32H50O2. The

1H-NMR spectrum of 1 (Table 1) showed two olefinic doublets at δ 5.59 (1H, d, J =

5.8 Hz) and 5.45 (1H, d, J = 5.8 Hz) coupled together in COSY spectrum and an oxygenated methine at δ 4.47 (1H,

dd, J = 11.1, 5.2 Hz). In addition, six tertiary methyls were observed at δ 1.22, 1.17, 0.90, 0.89, 0.86, and 0.84 (3H each, s) and two secondary methyls at δ 0.92 (3H, d, J = 6.4 Hz) and 0.78 (3H, d, J = 6.0 Hz). It also showed the

signal for acetyl group at δ 2.05 (3H, s). The above data was consistent with ursane type triterpene with two double

bonds and an acetyl group13

. The 13

C-NMR spectrum (BB and DEPT) of 1 (Table 1) revealed the presence of 32

carbon signals for nine methyl, eight methylene, seven methine and eight quaternary carbon atoms. The downfield signals at δ 170.3 could be assigned to ester carbonyl while the signals at δ 154.1, 141.3, 122.9 and 115.5 confirming

the presence of two double bonds and an oxymethine at δ 80.5. The presence of double bonds at alternate position in

ring C was confirmed by HMBC correlations in which Me-25 & 26 (δ 1.22, 1.17) correlated with C-9 (δ 154.1) and Me-27 & 28 (δ 0.89, 0.84) with C-13 (δ 141.3), respectively and characteristic EI-MS fragments at m/z 313 and 255

due to cleavage of ring B and D indicating the presence of cisoid diene at C-9(11),12 of a pentacyclic triterpene14

. The

presence of acetyl group at C-3 was confirmed due to its downfield NMR shifts (δH 4.67; δC 80.5) and HMBC

correlation of H-3 (δ 4.67) with ester carbonyl at δ . The stereochemistry at C-3 was determined by 1H-NMR

Pak. J. Chem. 5(1): 42-49, 2015




spectrum through coupling constant in which the larger J = 11.1, 5.2 Hz values confirming it as axial and α-orientation

and the NOESY correlation between H-3 (δ 4.67) with Me-23 (δ 0.90). Based on above evidences 1 could be 3β-acetoxyurs-9(11),12-diene, which is reported synthetically

14,15 but isolated for the first time from any natural source.

Table-1: 1H- and 13C-NMR spectral data and HMBC correlations of 1 (CDCl3; 400, 100 MHz)

Position δH, (J in Hz) δC HMBC (H→C)

1 1.32, m

1.27, m 37.4 C-2, C-3, C-5, C-10, C-25

2 2.30, m

1.71, m 24.2 C-3, C-4, C-10

3 4.66, dd (11.1, 5.2) 80.5 C-1, C-4, C-23,C-1

4 - 38.5 -

5 0.86, m 51.1 C-3, C-7, C-23, C-24, C-25

6 1.60, m 18.2 C-4,C-8, C-10

7 1.44, m 31.9 C-5, C-9, C-26

8 - 40.6 -

9 - 154.1 -

10 - 37.8 -

11 5.59, d (5.8) 115.5 C-8, C-10, C-13,

12 5.45, d (5.8) 122.9 C-9, C-14, C-18

13 - 141.3 -

14 - 43.0 -

15 1.84, m

1.10, m 28.6 C-13, C-17, C-27

16 1.43, m

1.30, m 41.4 C-14, C-18, C-28

17 - 32.9 -

18 1.49, d (6.5) 57.2 C-12, C-14, C-20, C-22, C-29,

19 1.26, m 39.4 C-13, C-17, C-21, C-29, C-30

20 0.89, m 38.9 C-18, C-22, C-29

21 1.34, m

1.18, m 30.8 C-17, C-19, C-30

22 1.26, m

1.44, m 41.3 C-18, C-20, C-28

23 0.90, s 27.6 C-3, C-4, C-5, C-24

24 0.86, s 16.1 C-3, C-4, C-5, C-24

25 1.22, s 18.6 C-1, C-5, C-9, C-10

26 1.17, s 22.9 C-7, C-8, C-9, C-14

27 0.89, s 17.7 C-8, C-13, C-14, C-15

28 0.84, s 28.6 C-16, C-17, C-18, C-22

29 0.78, d (6.0) 17.3 C-18, C-19, C-20

30 0.92, d (6.4) 19.3 C-19, C-20, C-21

CH3CO 2.05, s 170.3, 21.2 C=O

Compound 2 was isolated as white amorphous solid and its molecular formula C30H50O was established on the basis of

HR-EI-MS by a peak at m/z 426.3851. Its 1H-NMR spectrum displayed seven methyl singlets at δ 1.70, 1.03, 0.97,

0.94, 0.83, 0.81 and 0.76 (3H each, s) and two broad singlets (1H each) at δ 4.69 and 4.57 is an indication of lupeol

type triterpene16

. It also showed an oxymethine at δ 3.19 (1H, dd, J = 11.6, 4.8 Hz, H-3). The larger coupling constant

indicates the axial and α-orientation of H-3. The 13

C-NMR spectra (BB and DEPT) displayed 30 carbon signals for seven methyl, eleven methylene, six methine and six quaternary carbons. The downfield signals at δ 151.1, 109.7 and

79.3 assigned to double bond and an oxymethine. The above data was completely overlapped with the data reported

for lupol17

. The colorless amorphous solid compound 3 having molecular formula C32H52O2. The

1H-NMR spectrum of 2

was almost similar to that for 1 except with the additional signal for acetyl group at δ 2.01 (3H, s) and downfield shift

of oxymethine at δ 4.44 (1H, dd, J = 10.8, 5.8 Hz) indicated the attachment of acetyl group at C-3 position. The 13

C-

NMR spectra (BB and DEPT) of 3 disclosed altogether 32 carbon signals for eight methyl, eleven methylene, six methine and seven quaternary carbons. The signal at δ 171.0 and 21.3 was due to acetyl group. The remaining signals

were almost similar to those for 2. This data was in complete agreement with the data reported for lupeol acetate18

.

Compound 4 having the molecular formula C32H50O3 was established through HR-EI-MS by the molecular ion peak at m/z 482.3748. The

1H-NMR spectrum of 3 displayed a pair of olefinic doublets at δ 6.50 (1H, d, J = 5.4

Hz) and 5.45 (1H, d, J = 5.4 Hz) two oxymethines at δ 4.53 (1H, dd, J = 12.0, 3.6 Hz, H-3) and 3.91 (1H, dd, J = 11.4,

43

Riaz et al, 2015

4.2 Hz, H-1). The larger coupling constants of these oxymethines indicated both of these are axial and α in orientation.

The same spectrum also displayed six methyl singlets at δ 1.25, 1.14, 0.87, 0.86, 0.85, 0.82 (3H each, s) and a pair of doublets resonating at δ 0.90 and 0.78 (3H each, d, J = 6.0 Hz), indicating 4 an ursane type triterpene

19. The same

spectrum also showed a singlet at δ 2.04 (3H, s). The 13

C-NMR spectra of 4 (BB and DEPT) displayed 32 carbon

signals including nine methyl, seven methylene, eight methine and eight quaternary carbon atoms. The signal

resonating at δ 170.8 and 21.2 were due to the presence of acetyl group while the signals resonating at δ 152.0, 141.6, 123.3, 117.6, were attributed to two double bonds and signals at δ 77.2 and 75.6 were due to the presence of two

oxymethines, respectively. This data was in complete agreement with the data already published for conrauidienol13

.

O O

OOHOH

O

O

3

HO

COOH

O

HO

O

OOH

OH

HO

OH

O

OH

O

O

H

HO

2

OO

HOHO

OH

OH

COOH

OH

COOCH3

OH

O

O

H

1

4

10

11 12

5 6

8 97

1

1

35

7

9

11 13

27

23 24

25 26

15

17

19 21

29

30

28

Fig.1: Structures of compounds (1-12) isolated from F. bengalensis

Compound 5 having molecular C29H50O deduced through HR-EI-MS at m/z 414.3851. The 1H-NMR spectrum of 5

displayed six methyl signals: two tertiary methyl resonating at δ 1.00, 0.68, (3H each, s), three secondary at δ 0.92, 0.83, 0.80 (3H each, d, J = 6.2, 6.0 and 6.0 Hz, respectively) and a primary at δ 0.86 (3H, t, J = 6.0 Hz) and two

downfield signals at δ 5.13 (1H, br s, H-5) and 3.42 (1H, m, H-3). The 13

C-NMR spectra (BB and DEPT) of the 5

disclosed total 29 carbon signals for six methyl, eleven methylene, nine methine and three quaternary carbon atoms. The signals at δ 140.7 and 121.9 were due to presence of double bond and a signal at δ 71.6 was due an oxygenated

methine. All the physical and spectral data completely matched with the data reported for β-sitosterol20,21

.

Compound 6 was isolated as white amorphous solid. The molecular formula C20H16O5 was established by HR-

EI-MS by a peak at m/z 336.0987. The 1H-NMR spectrum of 5 displayed a pair of doublets at δ 6.72 (1H, d, J = 10.6

Hz) and 5.62 (1H, d, J = 10.6 Hz). A sharp singlet at δ 7.81 (1H, s) was the characteristic of C-2 of isoflavone

nucleus22

. An A2B

2 system type doublets at δ 7.40 (2H, d, J = 7.5 Hz) and 6.74 (2H, d, J = 7.5 Hz) were due to the

44


presence of p-substituted benzene. A singlet at δ 1.47 (6H, s) showing the presence of two magnetically equivalent

methyls. The 13

C-NMR spectra (BB and DEPT) of 6 showed eighteen signals for twenty carbons, for two methyl, eight methine and ten quaternary carbon atoms at δ 180.0, 159.0, 157.1, 155.7, 152.4, 141.2, 130.3, 128.1, 127.9,

123.4, 123.0, 115.5, 115.4, 94.8, 77.2 and 28.3. This data was in complete agreement with the data reported for

alpinum isoflavone23

.

Compound 7 having the molecular formula C8H8O3 was based on HR-EI-MS measurements at m/z 152.0462. Its

1H-NMR spectrum showed two aromatic doublets at δ 7.96 (2H, d, J = 8.8 Hz), 6.86 (2H, d, J = 8.8 Hz) and a

singlet at δ 3.90 (3H, s). The 13

C-NMR spectra (BB and DEPT) of 7 showed total six signals for eight carbons with

one methyl, four methine and three quaternary carbons. The downfield signals at δ 167.3 and 163.6 were due to an ester carbonyl and an oxygenated aromatic quaternary carbons. The signal appeared at δ 52.3 was due to the presence

of methoxy group. This data were in complete agreement with the data reported for the methyl 4-hydroxybenzoate25

.

The molecular formula C10H10O4 of 8 was established through HR-EI-MS due to a molecular ion peak at m/z 194.0568. The

1H-NMR spectrum displayed a pair of doublets at δ 7.03 (1H, d, J = 7.45 Hz), 6.96 (1H, d, J = 7.5 Hz)

and a triplet at δ 7.50 (1H, t, J = 6.0 Hz) in the aromatic region indicating the presence of ortho-trisubstituted benzene

ring. It also showed two oxygenated methines at δ 4.50 (1H, m), 4.52 (1H, d, J = 6.0 Hz) and a methyl at δ 1.44 (3H,

d, J = 6.5 Hz). The 13

C-NMR spectra of 8 (BB and DEPT) displayed 10 carbon signals for one methyl, five methine and four quaternary carbons. This data was in complete agreement with that reported for 4-hydroxymellein

25.

Compound 9 was obtained as crystalline solid having molecular formula C7H6O3. The 1H-NMR of 9 was

similar to that for 7 except the absence of methoxy group. The 13

C-NMR spectrum of 9 showed five carbon signals for seven carbons with similar resonance like that for 7 except the missing of methoxy signals. This information

completely matched with values reported for 4-hydroxybenzoic acid26

.

Compound 10 was obtained as white amorphous powder. Its molecular formula C9H8O3 was determined by

HR-EI-MS through a molecular ion peak at m/z 164.0461. Its 1H-NMR spectrum showed four doublets at δ 7.57 (1H,

d, J = 16.0 Hz), 7.43 (2H, d, J = 8.5 Hz), 6.79 (2H, d, J = 8.5 Hz) and 6.27 (1H, d, J = 16.0 Hz). The larger coupling

constants (16.0 Hz) of two doublets is a clear indication for the presence of a trans double bond. The 13

C-NMR

spectrum of 10 showed seven signals for nine carbon atoms. The signal at δ 172.0 was due to the presence of conjugated carboxyl acid where as four methines at δ 146.2, 130.2, 116.9 and 162.0 indicated the presence of p-

substituted cinnamic acid. The above data completely overlapped with the data reported for p-coumaric acid27

.

Compound 11 was obtained as white powder. The molecular formula C30H48O3 was established by HR-EI-MS through its molecular ion peak at m/z 456.3593. The

1H-NMR spectrum of 11 showed a tertiary methine at δ 5.49 (1H,

t, J = 3.5 Hz), an oxymethine at δ 3.44 (1H, dd, J = 9.8, 4.2 Hz) and seven tertiary methyls at δ 1.30, 1.24, 1.04, 1.02,

1.01, 0.97 and 0.93. The 13

C-NMR spectra of 11 showed 30 carbon signals, for seven methyls, ten methylenes, five

methines and eight quaternary carbons. The signals resonating at δ 144.8 and 122.6 were attributed to double bond where as the signal at δ 78.2 was due to the presence of oxygenated methine. The above spectral data resembles

completely with the data reported for the oleanolic acid28,29

.

The molecular formula C35H61O6 compound 12 was established by HR-FAB-MS through a molecular ion peak at m/z 577.4455 [M+H]

+. The

1H-NMR spectrum displayed same signals as observed for 5 with the additional signals

for glucose moiety whose anomeric proton was observd at δ 4.38 (1H, d, J = 6.5 Hz) with multiplets observed between

δ 3.01-3.73. The 13

C-NMR spectrum of 12 displayed 35 carbon signals for six methyl, twelve methylene, fourteen methine and three quaternary carbon atoms. The signals at δ 140.7 and 121.9 were due to the presence of double bond.

The signal for sugar moiety were appeared at δ 101.0, 74.4, 79.1, 70.1, 78.2 and 61.9. The downfield shift of C-

3 (δ 76.2) is an indication for the glycosidation at this position. This data was in complete agreement with that

reported for β-sitosterol 3-O-β-D-glucopyranoside30

.

Table 2: α-Glucosidase inhibition of compounds 1-12

Compound Percentage Inhibition IC50

1 95.36 ± 0.04 169.97 ± 0.07

2 98.74 ± 0.03 51.29 ± 0.003

3 99.09 ± 0.01 26.31 ± 0.003

4 97.16 ± 0.02 288.2 ± 0.003

5 42.37 ± 0.04 277.7 ± 0.003

6 97.12 ± 0.09 75.89 ± 0.008

7 47.21 ± 0.03 277.7 ± 0.003

8 87.68 ± 0.02 202.82 ± 0.09

9 41.12 ± 0.04 297.7 ± 0.002

10 99.59 ± 0.05 54.15 ± 0.005

11 98.35 ± 0.04 231.33 ± 0.05

12 87.15 ± 0.09 258.71 ± 0.07

Acarbose 92.23 ± 0.14 38.25 ± 0.12

All the samples were dissolved in methanol and experiments were performed in triplicate (mean±sem, n=3).

45

Riaz et al, 2015

2.1 α-Glucosidase Inhibition Studies of compounds 1-12 The compounds 1-12 were subjected for α-glucosidase inhibitory activity and the results are shown in Table 2.

Compound 3 with IC50 value 26.3 µM showed promising activity followed by compounds 2, 10 & 6 with IC50 value

51.2, 54.1 & 75.8 µM, respectively.

3. EXPERIMENTAL

3.1 General experimental procedures Melting points were recorded by using apparatus of Buchi 434. UV spectra were measuerd (in methanol) on

spectrophotometer of Schimadazu UV-240. Optical rotations were measured by polarimeter (JASCO DIP-360). IR spectra were performed on IR spectrometer (IR-460 Shimadzu).

1H- and

13C-NMR, HMQC, COSY and HMBC

spectra were recorded using spectrometer of Bruker, operating at 500 MHz for 1H- and 125 MHz for

13C-NMR,

respectively. Chemical shift values (δ) are reported in ppm and the coupling constants (J) measured in Hz. Mass spectra (EI-MS, HR-EI-MS) were measured on mass (JMS HX 110) instrument and HR-FAB-MS were recorded on

mass spectrometers (JMS-DA 500) and unit is shown in m/z. Thin layer chromatography (TLC) were carried out by

using aluminum sheets coated with silica gel 60 F254 (20 × 20 cm, 0.2 mm thick; E. Merck) and column

chromatography (CC) using silica gel (230-400 mesh). TLC plates were visualized under UV at 254 and 366 nm and sprayed with ceric sulfate solution (1% in 10% H2SO4) with heating.

3.2 Plant material The aerial roots of F. bengalensis was collected from Sahiwal District (Pakistan) in July 2010 and was identified by

Dr. Muhammad Arshad (late), Plant Taxonomist, Cholistan Institute of Desert Studies (CIDS), The Islamia University

of Bahawalpur, where a plant specimen is deposited (0046-FB/CIDS/10).

3.3 Extraction and isolation The shade dried ground aerial roots was extracted in metanol. The methanolic extract was evaporated on rotary evaporator to obtain black gummy material (750 g) which suspended in water and extracted with n-hexane, ethyl

acetate, and n-butanol. The ethyl acetate fraction (50 g) was subjected to the column chromatography over flash silica

gel and eluted with dichloromethane (DCM), DCM-ethyl acetate, ethyl acetate, ethyl acetate-methanol, and methanol in increasing order of polarity to get eight sub-frations. These sub-fractions were further purified by gradient elution

using n-hexane and DCM (10% DCM in n-hexane) to afford 1, (12% DCM in n-hexane), lupol (2), (15% DCM in n-

hexane), lupeol acetate (3), (20% DCM in n-hexane), conrauidienol (4), (20% DCM in n-hexane), β-sitosterol (5), (28% DCM in n-hexane), alpinum isoflavone (6), (35% DCM in n-hexane), methyl 4-hydroxybenzoate (7), (50%

DCM in n-hexane), 4-hydroxymellein (8), (45% DCM in n-hexane), 4-hydroxybenzoic acid (9), (55% DCM in n-

hexane), p-coumaric acid (10), (70% DCM in n-hexane) oleanolic acid (11), (1% MeOH in DCM) and β-sitosteryl 3-

O-β-D-glucopyranoside (12).

3.4 Chracterization of isolated compounds (1-12)

3.4.1 3β-acetoxyurs-9(11),12-diene (1)

Colorless amorphous powder (20 mg); [α]D24

+ 24.7 (c 0.16, MeOH); IR (KBr, max, cm-1

): 2966, 1735, 1650; 1H- and

13C-NMR spectral data, see Table 1; HR-EI-MS: m/z 466.3800 [M]

+ (calcd for C32H50O2, 466.3810).

3.4.2 Lupeol (2)

White amorphous solid (12mg); m.p 213 ˚C; [α]D24

+ 25.7 (c = 0.70 in CHCl3); IR (KBr, max, cm-1

): 3326, 2931, 1631, 1450, 1377, 1035, 874;

1H-NMR (CDCl3, 400 MHz, δ/ppm): 4.69 (br s, 1H, H-29a), 4.57 (br s, 1H, H-29b),

3.19 (dd, J = 11.6, 4.8 Hz, 1H, H-3), 2.39 (dd, J = 9.6, 4.0 Hz, 1H, H-19), 1.70 (s, 3H, H-30), 1.03 (s, 3H, H-28), 0.97 (s, 3H, H-27), 0.94 (s, 3H, H-26), 0.83 (s, 3H, H-25), 0.81 (s, 3H, H-24), 0.76 (s, 3H, H-23);

13C-NMR (CDCl3, 100

MHz, δ/ppm): δ 151.1 (C-20), 109.7 (C-29), 79.3 (C-3), 55.6 (C-5), 50.7 (C-9), 48.8 (C-19), 48.3 (C-18), 43.4 (C-17),

43.2 (C-14), 41.2 (C-8), 40.4 (C-22), 39.2 (C-4), 39.1 (C-1), 38.4 (C-13), 37.5 (C-10), 35.9 (C-16), 34.6 (C-7), 30.2 (C-21), 28.4 (C-23), 27.9 (C-2), 27.8 (C-15), 25.5 (C-12), 21.3 (C-11), 19.8 (C-30), 18.7 (C-6), 18.4 (C-28), 16.5 (C-

25), 16.3 (C-26), 15.8 (C-24), 14.9 (C-27); HR-EI-MS m/z: 426.3851 (calcd for C30H50O, 426.3861).

3.4.3 Lupeol acetate (3)

Colorless amorphous solid (14 mg); IR IR (KBr, max, cm-1

): 2942, 2866, 1735, 1451, 1379, 1243, 1027 cm−1

; 1H-

NMR (CDCl3, 400 MHz, δ/ppm): 4.69 (br s, 1H, H-29a), 4.57 (br s, 1H, H-29b), 4.44 (dd, J = 10.8, 5.8 Hz, 1H, H-3),

2.01 (s, 3H, OAc), 1.70 (s, 3H, H-30), 1.03 (s, 3H, H-28) 0.94 (s, 3H, H-26), 0.91 (s, 3H, H-27), 0.81 (s, 3H, H-

23,24), 0.83 (s, 3H, H-25), 0.76 (s, 3H, H-23); 13

C NMR (CDCl3, 100 MHz, δ/ppm): 171.0 (OAc), 150.9 (C-20), 109.3 (C-29), 80.9 (C-3), 55.3 (C-5), 50.3 (C-9), 48.2 (C-19), 48.0 (C-18), 42.9 (C-17), 42.8 (C-14), 40.8 (C-8), 39.9 (C-22),

38.3 (C-1), 38.0 (C-13), 37.3 (C-4), 37.0 (C-10), 35.5 (C-16), 34.7 (C-7), 29.1 (C-21), 28.2 (C-23), 27.4 (C-15), 25.0

46


(C-12), 23.2 (C-2), 21.3 (OAc), 20.9 (C-11), 19.0 (C-30), 18.1 (C-6), 17.9 (C-28), 16.4 (C-26), 16.1 (C-25), 15.9 (C-

24), 14.5 (C-27); HR-EI-MS at m/z 442.3801 (calcd for C32H52O2, 442.3811)

3.4.4 Conrauidienol (4)

White amorphous powder; [α]D24

+ 12.4 (c 0.0014, acetone); UV (MeOH, max, log ε, nm): 198 (3.4), 203 (3.45), 215 (3.36), 220 (3.32);

1H-NMR (CDCl3, 400 MHz, δ/ppm): 6.50 (d, J = 5.4 Hz, 1H, H-11), 5.45 (d, J = 5.4 Hz, 1H, H-

12), 4.53 (dd, J = 12.0, 3.6 Hz, 1H, H-3), 3.91 (dd, J = 11.4, 4.2 Hz, 1H, H-1), 2.04 (s, 3H, OAc), 1.25 (s, 3H, H-25), 1.14 (s, 3H, H-26), 0.90 (d, J = 6.0 Hz, 3H, H-30), 0.87 (s, 3H, H-27), 0.86 (s, 3H, H-24), 0.85 (s, 3H, H-23), 0.82 (s,

3H, H-28), 0.78 (d, J = 6.0 Hz, 3H, H-29), 13

C-NMR (CDCl3, 125 MHz, δ/ppm): 170.8 (OAc), 152.0 (C-9), 141.6 (C-

13), 123.3 (C-12), 117.6 (C-11), 77.2 (C-3), 76.5 (C-1), 57.2 (C-18), 48.7 (C-5), 44,5 (C-10), 43.1 (C-8), 41.2 (C-16), 40.7 (C-14), 39.3 (C-20), 39.0 (C-19), 37.9 (C-4), 34.4 (C-2), 33.6 (C-17), 31.1(C-21), 30.9 (C-7), 28.6 (C-28), 28.2

(C-22), 27.6 (C-23), 26.1 (C-15), 22.9 (C-26), 21.5 (C-30), 21.2 (OAc), 18.6 (C-25), 18.2 (C-6), 17.7 (C-27), 17.3 (C-

29), 16.1 (C-24); HR-EI-MS m/z: 482.3748 (calcd for C32H50O3, 482.3759).

3.4.5 β-Sitosterol (5)

White crystalline solid (20 mg); m.p 135˚C; IR (KBr, max, cm-1

): 3446, 3050, 1650; 1H-NMR (CDCl3, 400 MHz,

δ/ppm): 5.13 (m, 1H, H-5), 3.42 (m, 1H, H-3), 1.00 (s, 3H, H-19), 0.92 (d, J = 6.2 Hz, 3H, H-21), 0.86 (t, J = 7.0 Hz,

3H, H-29), 0.83 (d, J = 6.0 Hz, 3H, H-26), 0.80 (d, J = 6.0 Hz, 3H, H-27), 0.68 (s, 3H, H-18); 13

C-NMR (CDCl3, 100 MHz, δ/ppm): 140.7 (C-5), 121.9 (C-6), 71.6 (C-3), 56.6 (C-14), 55.9 (C-17), 51.3 (C-9), 48.9 (C-24), 42.8 (C-4), 42.2

(C-13), 40.3 (C-12), 36.9 (C-1), 36.6 (C-10), 36.1 (C-20), 34.4 (C-22), 32.7 (C-7), 32.2 (C-16), 32.0 (C-8), 31.4 (C-2),

28.9 (C-23), 26.2 (C-25), 25.5 (C-15), 23.2 (C-28), 21.1 (C-11), 19.8 (C-27), 19.6 (C-19), 19.0 (C-21), 18.7 (C-26), 12.1 (C-29), 11.9 (C-18); HR-EI-MS m/z 414.3851 (calcd for C29H50O, 414.3862).

3.4.6 Alpinum isoflavone (6)

White amorphous solid (10 mg); m.p 213-214˚C; UV (MeOH, max, log ε, nm): 284 (4.7); IR (KBr, max, cm-1

): 3570, 3300, 1650;

1H-NMR (CDCl3, 400 MHz, δ/ppm): 7.81 (s, 1H, H-2), 6.32 (s, 1H, H-8), 7.40 (d, J = 7.5 Hz, 2H, H-2,6),

6.74 (d, J = 7.5 Hz, 2H, H-3,5), 6.72 (d, J = 10.0 Hz, 1H, H-4''), 5.62 (d, J = 10.0 Hz, 1H, H-3''), 1.47 (s, 6H, H-9,10); 13

C-NMR (CDCl3, 100 MHz, δ/ppm): 180.0 (C-4), 159.0 (C-5), 157.1 (C-8a), 155.7 (C-4'), 152.4 (C-2), 141.2 (C-6),

130.3 (C-2',5'), 128.1 (C-3''), 127.9 (C-3), 123.4 (C-1'), 123.0 (C-7), 115.5 (C-4''), 115.4 (C-3',5'), 94.8 (C-8), 77.2 (C-2''), 28.3 (Me-9,10); HR-EI-MS m/z: 336.0987 (calcd for C20H16O5, 336.0998).

3.4.7 Methyl 4-hydroxybenzoate (7)

Crystalline solid (13 mg); m.p 128˚C; UV (MeOH, max, log ε, nm): 220 (3.80), 308 (3.91); IR (KBr, max, cm

-1): 3515,

1696; 1H-NMR (CD3OD, 400 MHz, δ/ppm): 7.96 (d, J = 8.8 Hz, 2H, H-2,6), 6.86 (d, J = 8.8 Hz, 2H, H-3,5), 3.90 (s,

3H, OCH3); 13

C-NMR (CD3OD, 100 MHz, δ/ppm): 167.3 (C-7), 163.6, (C-4), 132.7, (C-2,6), 122.6 (C-1), 116.1, (C-

3,5), 52.3 (OCH3); HR-EI-MS m/z 152.0462 (calcd for C8H8O3, 152.0473).

3.4.8 4-Hydroxymellein (8)

White amporphous solid (20 mg); m.p 118-119˚C; UV (MeOH, max, log ε, nm): 246 (2.9), 314 (3.6); IR (KBr, max, cm

-1): 3300, 1680;

1H-NMR (CDCl3, 400 MHz, δ/ppm): 7.03 (d, J = 7.5 Hz, 1H, H-7), 6.96 (d, J = 7.5 Hz, 1H, H-5),

7.53 (t, J = 7.5 Hz, 1H, H-6), 4.52 (d, J = 6.0 Hz, 1H, H-4), 4.50 (m, 1H, H-3), 1.44 (d, J = 6.5 Hz, 3H, C-9); 13

C-

NMR (CDCl3, 100 MHz, δ/ppm): 168.5 (C-1), 161.9 (C-8), 141.1 (C-4a), 136.8 (C-6), 117.7 (C-7), 116.2 (C-5), 106.5

(C-1a), 79.9 (C-4), 69.0 (C-3), 17.8 (C-9); HR-EI-MS m/z 194.0568 (calcd for C10H10O4, 194.0579).

3.4.9 4-Hydroxybenzoic acid (9)

Crystalline solid (8 mg); m.p 213-214°C; UV (MeOH, max, log ε, nm): 222 (3.80), 310 (3.89); IR (KBr, max, cm

-1):

3515, 3335-2730, 1710; 1H-NMR (CD3OD, 400 MHz, δ/ppm): 7.86 (d, J = 8.4 Hz, 2H, H-2,6), 6.80 (d, J = 8.4 Hz,

2H, H-3,5); 13

C-NMR (CD3OD, 100 MHz, δ/ppm): 170.9 (C-7), 163.3 (C-4), 132.9 (C-2,6), 122.9 (C-1), 116.0 (C-3,5); HR-EI-MS m/z: 138.0307 (calcd for C7H6O3, 138.0317).

3.4.10 p-Coumaric acid (10)

White powder (14 mg); m.p 211-213°C; UV (MeOH, max, log ε, nm): 290 (4.4), 306 (4.6) nm; IR (KBr, max, cm

-1):

3400, 3350-2250, 1685, 1625, 1425, 1380; 1H-NMR (CD3OD, 400 MHz, δ/ppm): 7.57 (d, J = 16.0 Hz, 1H, H-1 ), 7.43

(d, J = 8.5 Hz, 2H, H-2,6), 6.79 (d, J = 8.5 Hz, 2H, H-3,5), 6.27 (d, J = 16.0 Hz, 1H, H-2 ); 13

C-NMR (CD3OD, 100 MHz, δ/ppm): 172.0 (C-7), 162.0 (C-4), 146.2 (C-7), 130.2 (C-2,6), 127.5 (C-1), 116.9 (C-3,5), 116.0 (C-8); HR-EI-MS m/z: 164.0461 (calcd. for C9H8O3, 164.0473).

3.4.11 Oleanolic acid (11)

White powder (15 mg); m.p 305-306°C; [ ]D

25 + 78.9

° (c 0.015, CHCl3); IR

47

Riaz et al, 2015

(KBr, max, cm-1): 3410-2650, 1710, 1660, 820;

1H-NMR (CDCl3, 400 MHz, δ/ppm): 5.49 (t, J = 3.5 Hz, 1H, H-12),

3.44 (dd, J = 9.8, 4.2 Hz, 1H, H-3), 1.30 (s, 3H, H-27), 1.24 (s, 3H, H-23), 1.04 (s, 3H, H-26), 1.02 (s, 3H, H-24), 1.01

(s, 3H, H-30), 0.97 (s, 3H, H-29), 0.93 (s, 3H, H-25); 13

C-NMR (CDCl3, 100 MHz, δ/ppm): 180.0 (C-28), 144.8 (C-

13), 122.6 (C-12), 78.2 (C-3), 55.9 (C-5), 48.2 (C-9), 46.7 (C-17), 42.2 (C-19), 41.7 (C-14), 40.7 (C-18), 39.2 (C-8), 38.7 (C-4), 38.5 (C-1), 37.2 (C-10), 33.8 (C-21), 32.9 (C-29), 32.5 (C-7), 32.4 (C-22), 30.6 (C-20), 28.2 (C-23), 27.7

(C-15), 27.8 (C-2), 25.9 (C-27), 23.5 (C-30), 23.4 (C-11), 23.3 (C-30), 23.2 (C-16), 18.3 (C-6), 17.20 (C-26), 15.5 (C-

24), 15.3 (C-25); HR-EI-MS m/z 456.3593 (calcd for C30H48O3, 456.3603).

3.4.12 β-Sitosterol 3-O-β-D-glucopyranoside (12) Colorless amorphous powder (25mg); [α]D

25: ˚, (c 0.003, MeOH); IR (KBr, max, cm

-1): 3452. 3044, 1646, 1618,

1559, 1550; 1H-NMR (CDCl3+ CD3OD, 400 MHz, δ/ppm): 5.13 (br s, 1H, H-5), 4.38 (d, J = 6.5 Hz, 1H, H-1'), 3.01

(m, 1H, H-2'), 3.32 (m, 1H, H-3'), 3.2 (m, 1H, H-4'), 3.39 (m, 1H, H-5 ), 3.73, 3.65 (br s, 2H, H-6'), 3.45 (m, 1H, H-3), 1.00 (s, 3H, H-19), 0.92 (d, J = 6.2 Hz, 3H, H-21), 0.86 (t, J = 7.0 Hz, 3H, H-29), 0.83 (d, J = 6.0 Hz, 3H, H-26),

0.80 (d, J = 6.0 Hz, 3H, H-27), 0.68 (s, 3H, H-18); 13

C-NMR (CDCl3+CD3OD, 100 MHz, δ/ppm): 140.7 (C-5), 121.9

(C-6), 101.0 (C-1'), 78.2 (C-5 ), 76.2 (C-3), 74.4 (C-2'), 79.1 (C-3'), 70.1 (C-4'), 61.9 (C-6'), 56.6 (C-14), 55.9 (C-17),

51.3 (C-9), 48.9 (C-24), 42.8 (C-4), 42.2 (C-13), 40.3 (C-12), 36.9 (C-1), 36.6 (C-10), 36.1 (C-20), 34.4 (C-22), 32.7 (C-7), 32.2 (C-16), 32.0 (C-8), 31.4 (C-2), 28.9 (C-23), 26.2 (C-25), 25.5 (C-15), 23.2 (C-28), 21.1 (C-11), 19.8 (C-

27), 19.6 (C-19), 19.0 (C-21), 18.7 (C-26), 12.1 (C-29), 11.9 (C-18); HR-FAB-MS m/z 577.4455 [M+H]+ (calcd for

C35H61O6, 577.4468).

3.4.13 α-Glucosidase Inhibition Assay The method used to perform α-glucosidase inhibition assay was similar but with slight modifications as done by Pierre

et al31

. 100 µL of total volume of reaction mixture having 70 µL (50 mM) phosphate buffer, pH 6.8, 10 µL (0.5 mM) test compound, continued with the addition of 10 µL (0.0234 units, Sigma Inc.) enzyme. These reagents were mixed,

preincubated for 10 min at 37˚C and pre-read at 400 nm. This was further initiated by adding 10 µL (0.5 mM)

substrate (p-nitrophenyl glucopyranoside, Sigma Inc.). The incubation was done for 30 min at 37˚C, yellow color

showed absorbance due to the formation of p-nitrophenol which was measured at 400 nm using Synergy HT (BioTek, USA) using 96-well microplate reader. For positive control, acarbose was used. The inhibition percentage was

calculated by using the equation below

Inhibition (%) = (abs of control – abs of test / abs of control) × 100

IC50 values were calculated using EZ-Fit Enzyme Kinetics Software (Perrella Scientific Inc. Amherst, USA).

4. ACKNOWLEDGEMENT The authors are thankful to Higher Education Commission (HEC) of Pakistan and Alexander von Humboldt (AvH) Foundation, Germany for financial support.

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