Synthesis, Characterization and Biological
Studies of Ferrocenyl Ureas & Thioureas
Islamabad
A dissertation submitted to the Department of Chemistry, Quaid-i-Azam University,
Islamabad, in partial fulfillment of requirements for the degree of
Doctor of Philosophy
in
Inorganic/Analytical Chemistry
by
Ataf Ali Altaf
Department of Chemistry
Quaid-i-Azam University
Islamabad
2012
Synthesis, Characterization and Biological
Studies of Ferrocenyl Ureas & Thioureas
Islamabad
by
Ataf Ali Altaf
Department of Chemistry
Quaid-i-Azam University
Islamabad
2012
Declaration
This is to certify that this dissertation entitled “Synthesis, Characterization and Biological
Studies of Ferrocenyl Ureas & Thioureas” submitted by Mr. Ataf Ali Altaf is accepted in its
present form by the Department of Chemistry, Quaid-i-Azam University, Islamabad,
Pakistan, as satisfying the partial fulfillment for the degree of Doctor of Philosophy in
Inorganic/ Analytical Chemistry.
List of Foreign Refrees
This dissertation entitled “Synthesis, Characterization and Biological Studies of Ferrocenyl
Ureas and Thiourears” submitted by Mr. Ataf Ali Altaf, Department of Chemistry,
Quaid-i-Azam University, Islamabad, for the degree of Doctor of Philosophy in
Inorganic/Analytical Chemisty has been evaluated by the following panel of Foreign
Referees.
1. Dr. Wen- Hua Sun
Professor of Catalysis and Polyolefins
Institute of Chemistry
Chinese Academy of Sciences
Beijing 100080, China
Tel: 86-10-62557955; Fax: 86-10-62618239
E-mail:[email protected]
2. Prof. Dr. Akira Yamauchi
Kyushu University
6-10-1 Hakozaki, Highashi-ku
Fukuoka 812-8561,
Japan,
E-mail: [email protected]
3. Dr. Michael Bolte
Institut fuer Anorganische Chemie
J. –W. –Goethe-Universitaet, Max-Von-Laue-Str. 7, D-60438
Frankfurt/Main, Germany.
Tel: +49-69-7982-9136, Fax: +49-69-7982-9239
E-mail: [email protected]
Turnitin Orignality Report
IN THE NAME OF ALLAH
THE COMPASSIONATE
THE MERCIFUL
Dedicated to
My Loving Parents
i
Contents
Page
Acknowledgement vi
List of Figures viii
List of Tables xi
Abstract xiii
Chapter 1 Introduction 1 – 18
1.1 Cancer 1
1.1.1 Causes of cancer 3
1.1.2 Cancer treatment 3
1.1.3 Chemotherapy 4
1.2 Thioureas 5
1.2.1 Synthesis of thioureas 5
(a) From Cyanamid’s 5
(b) From Isothiocynates 6
(c) From Thiophosgene 6
1.2.2 Importance of thioureas 7
1.3 Ureas 7
1.3.1 Synthesis of ureas 7
(a) From Isocynates 7
(b) From carbamates 8
(c) From thioureas 8
1.3.2 Importance of ureas 9
1.4 Thioureas and ureas as anticancer agents 9
1.5 Limitations of thioureas and ureas using as drugs 11
1.6 Ferrocene 11
1.7 Preliminary screening of anti-cancer potency 13
1.8 DNA binding with small molecules 14
1.9 Techniques to study non-covalent interactions 15
1.9.1 UV-visible spectrophotometry in DNA binding 15
ii
Page
1.9.2 Thermal Melting Assay of DNA 16
1.10 Aims of study 18
Chapter 2 Experimental and Characterization 19 – 51
2.1 Materials 19
2.2 Instrumentation 19
2.3 General description of the work 19
2.4 Synthesis of Nitrophenylferrocenes (a – e) 22
2.4.1 4-nitrophenylferrocene (a) 22
2.4.2 2-methyl-4-nitrophenylferrocene (b) 23
2.4.3 2-methoxy-4-nitrophenylferrocene (c) 23
2.4.4 2-Chloro-4-nitrophenylferrocene (d) 23
2.4.5 3-nitrophenylferrocene (e) 24
2.5 Synthesis of ferrocenylanilines (A – E) 24
2.5.1 4-Ferrocenylaniline (A) 24
2.5.2 3-methyl-4-Ferrocenylaniline (B) 26
2.5.3 3-methoxy-4-Ferrocenylaniline (C) 26
2.5.4 3-Chloro-4-Ferrocenylaniline (D) 26
2.5.5 3-Ferrocenylaniline (E) 27
2.6 Synthesis of ferrocene based thioureas (At1 – Et7) 28
2.6.01 1-(4-(ferrocenyl)phenyl)-3-methylthiourea (At1) 28
2.6.02 1-(4-(ferrocenyl)phenyl)-3-ethylthiourea (At2) 29
2.6.03 1-(4-(ferrocenyl)phenyl)-3-propylthiourea (At3) 29
2.6.04 1-(4-(ferrocenyl)phenyl)-3-allylthiourea (At4) 29
2.6.05 1-(4-(ferrocenyl)phenyl)-3-phenylthiourea (At5) 30
2.6.06 1-(4-(ferrocenyl)phenyl)-3-(4-nitrophenyl)thiourea (At6) 30
2.6.07 1-(4-(ferrocenyl)phenyl)-3-(2,4-dichlorophenyl)thiourea (At7) 31
2.6.08 1-(4-(ferrocenyl)-3-methylphenyl)-3-methylthiourea (Bt1) 31
2.6.09 1-(4-(ferrocenyl)-3-methylphenyl)-3-ethylthiourea (Bt2) 31
2.6.10 1-(4-(ferrocenyl)-3-methylphenyl)-3-propylthiourea (Bt3) 32
2.6.11 1-(4-(ferrocenyl)-3-methylphenyl)-3-allylthiourea (Bt4) 32
iii
Page
2.6.12 1-(4-(ferrocenyl)-3-methylphenyl)-3-phenylthiourea (Bt5) 33
2.6.13 1-(4-(ferrocenyl)-3-methylphenyl)-3-(4-nitrophenyl)thiourea (Bt6) 33
2.6.14 1-(4-(ferrocenyl)-3-methylphenyl)-3-(2,4-dichlorophenyl)thiourea (Bt7) 33
2.6.15 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-methylthiourea (Ct1) 34
2.6.16 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-ethylthiourea (Ct2) 34
2.6.17 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-propylthiourea (Ct3) 35
2.6.18 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-allylthiourea (Ct4) 35
2.6.19 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-phenylthiourea (Ct5) 35
2.6.20 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-(4-nitrophenyl)thiourea (Ct6) 36
2.6.21 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-(2,4-dichlorophenyl)thiourea (Ct7) 36
2.6.22 1-(3-chloro-4-(ferrocenyl)phenyl)-3-methylthiourea (Dt1) 37
2.6.23 1-(3-chloro-4-(ferrocenyl)phenyl)-3-ethylthiourea (Dt2) 37
2.6.24 1-(3-chloro-4-(ferrocenyl)phenyl)-3-propylthiourea (Dt3) 37
2.6.25 1-(3-chloro-4-(ferrocenyl)phenyl)-3-allylthiourea (Dt4) 38
2.6.26 1-(3-chloro-4-(ferrocenyl)phenyl)-3-phenylthiourea (Dt5) 38
2.6.27 1-(3-chloro-4-(ferrocenyl)phenyl)-3-(4-nitrophenyl)thiourea (Dt6) 39
2.6.28 1-(3-chloro-4-(ferrocenyl)phenyl)-3-(2,4-dichlorophenyl)thiourea (Dt7) 39
2.6.29 1-(3-(ferrocenyl)phenyl)-3-methylthiourea (Et1) 39
2.6.30 1-(3-(ferrocenyl)phenyl)-3-ethylthiourea (Et2) 40
2.6.31 1-(3-(ferrocenyl)phenyl)-3-propylthiourea (Et3) 40
2.6.32 1-(3-(ferrocenyl)phenyl)-3-allylthiourea (Et4) 41
2.6.33 1-(3-(ferrocenyl)phenyl)-3-phenylthiourea (Et5) 41
2.6.34 1-(3-(ferrocenyl)phenyl)-3-(4-nitrophenyl)thiourea (Et6) 41
2.6.35 1-(3-(ferrocenyl)phenyl)-3-(2,4-dichlorophenyl)thiourea (Et7) 42
2.7 Synthesis of ferrocene based ureas (Au3 – Eu7) 43
2.7.01 1-(4-(ferrocenyl)phenyl)-3-propylurea (Au3) 43
2.7.02 1-(4-(ferrocenyl)phenyl)-3-allylurea (Au4) 43
2.7.03 1-(4-(ferrocenyl)phenyl)-3-phenylurea (Au5) 44
2.7.04 1-(4-(ferrocenyl)phenyl)-3-(4-nitrophenyl)urea (Au6) 44
2.7.05 1-(4-(ferrocenyl)-3-methylphenyl)-3-propylurea (Bu3) 45
iv
Page
2.7.06 1-(4-(ferrocenyl)-3-methylphenyl)-3-(4-nitrophenyl)urea (Bu6) 45
2.7.07 1-(4-(ferrocenyl)-3-methylphenyl)-3-(2,4-dichlorophenyl)urea (Bu7) 45
2.7.08 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-ethylurea (Cu2) 46
2.7.09 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-propylurea (Cu3) 46
2.7.10 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-phenylurea (Cu5) 47
2.7.11 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-(4-nitrophenyl)urea (Cu6) 47
2.7.12 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-(2,4-dichlorophenyl)urea (Cu7) 47
2.7.13 1-(3-chloro-4-(ferrocenyl)phenyl)-3-phenylurea (Du5) 48
2.7.14 1-(3-chloro-4-(ferrocenyl)phenyl)-3-(4-nitrophenyl)urea (Du6) 48
2.7.15 1-(3-chloro-4-(ferrocenyl)phenyl)-3-(2,4-dichlorophenyl)urea (Du7) 49
2.7.16 1-(3-(ferrocenyl)phenyl)-3-phenylurea (Eu5) 49
2.7.17 1-(3-(ferrocenyl)phenyl)-3-(4-nitrophenyl)urea (Eu6) 49
2.7.18 1-(3-(ferrocenyl)phenyl)-3-(2,4-dichlorophenyl)urea (Eu7) 50
2.8 Methods for activity studies 50
2.8.1 DNA binding Studies 50
2.8.2 Anti-Oxidant Studies (DPPH scavenging assay) 51
2.8.3 Modal Membrane Interaction Studies 51
Chapter 3 Results and Discussion 52 – 82
3.1 Synthesis 52
3.2 Elemental Analysis 53
3.3 Infra Red Spectroscopic Characterization 54
3.4 Nuclear Magnetic Resonance Spectroscopic Characterization 56
The 1HNMR 56
The 13
CNMR 60
3.5 Single crystal X-ray diffraction analysis 66
3.5.1 Crystal structures of nitrophenylferrocenes 66
3.5.1.1 2-methoxy-4-nitrophenyl ferrocene (c) 69
3.5.1.2 2-chloro-4-nitrophenyl ferrocene (d) 70
3.5.1.3 3-nitrophenyl ferrocene (e) 71
3.5.2 Crystal structure of 3-Ferrocenyl aniline (E) 74
v
Page
3.5.3 Crystal structures of Ferrocenyl thioureas 77
3.5.3.1 1-(4-(ferrocenyl)phenyl)-3-allylthiourea (At4) 77
3.5.3.2 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-phenylthiourea (Ct5) 80
Chapter 4 Biological Studies 83 – 106
4 Biological Screening 83
4.1 Modal membrane interaction studies 83
Nitrophenylferrocenes 84
Ferrocenyl anilines 87
Ferrocenyl ureas and thioureas 90
4.2 Antioxidant Activity (DPPH Free Radical Scavenging Assay) 95
4.3 DNA binding studies 98
4.3.1 UV-Visible Spectroscopic titration 99
4.3.2 Thermal Denaturing Assay 103
Conclusions 105
Future plans 106
Reference 107
List of Publications and Presentation 117
Publications 119
vi
Acknowledgements
All praises to Almighty Allah, Creator of the universe, most beneficent and Merciful.
He, Who blessed me with determination, potential and the ability to complete this research
work. Peace and blessing of Allah be upon the Holy Prophet Muhammad (PBUH) and his
pious progeny, who is the source of knowledge and guidance for the entire world forever.
I wish to express the vehement sense of thankfulness to my affectionate Mother
Zahida Altaf, father Altaf Mohu Din and my supervisor, Prof. Dr. Amin Badshah,
Chairman, Department of Chemistry, Quaid-i-Azam University, Islamabad, for their
enthusiastic interest and keen guidance. I admire my supervisor for his dedication to his
group; his strive for perfection and his genuine concern for the well-being of his students.
I am incredibly thankful to Prof. Dr. Saqib Ali, Head of Inorganic/Analytical Section,
Department of Chemistry, Quaid-i-Azam University, Islamabad, for providing Lab. facilities
during research work and his friendly behavior, caring attitude and fruitful discussions.
No calculated formula can properly express my feelings of indebtedness to my
friendly Teachers at Pak Junior Modal School, Green Market Sahiwal (1988 – 1991); Govt.
M. C. Junior Modal School, Green Market Sahiwal (1991 – 1994); Govt. Mahmoodia High
School, Sahiwal (1994 – 1999); Govt. Immamia Degree College, Sahiwal (1999 – 2001);
Chemistry Department, Govt. College Sahiwal (2001 – 2005) and Chemistry Department,
Quaid-i-Azam University Islamabad (2006 – 2012); and my dearest teachers Sir Talib
Hussain and Dr. Maliha Asma. Valuable co-operation of all of them will forever remain
alive in my memory.
I highly appreciate Higher Education Commission (HEC) of Pakistan, Quaid-i-Azam
University Islamabad Pakistan and Prof. Dr. Azim Khan Khattak at Gomal University, Dera
Ismail Khan and Prof. Dr. Debbie C. Cranes at Colorado State University USA for their
valuable support.
My cordial thanks and gratitude are due to my parents and family; my brothers Asif
Altaf, Ahsan Ali and Mohsan Ali; sisters Saima Saeed and Bushra Basharat; Brothers in
Law Muhammad Saeed, Basharat Hussain, Muhammad Asghar Sohail and Dr. Asad Ali
Sohail and my daughter Zahra Ataf. Their prayers love and support over the years has
enabled me to cross the finish line. Without their prayers, encouragement, excessive
generosity and patience, I would have not been able to complete this task. Special thanks to
my wife Lubna Ataf for her love and caring attitude.
vii
I cannot forget to aknowlege; the happy memories of my home are the kids; Munahil
Asif, Muhammad Bilal Asif, Marium Fatima, Muhammad Talha, Gulnaz Anum, Noor
Fatima, Muhammad Aans Saeed, Muhammad Awais Saeed, Mubashra Basharat, Muhammad
Ahmad and Ali Ahmer.
I am amazingly thankful to my great lab fellows. During my stay at Chemistry
Department, Quaid-i-Azam University Islamabad, I enjoyed working with the company of
Shafiqullah Marwat, Dr. Ghulam Murtaza, Dr. Muhammad Khawar Rauf, Bhajan Lal Bhatia,
Nasir Khan, Shafqat Ali, Muhammad Irshad Ali, Muhammad Ayaz, Shabeeb Hussain, Samad
Yaseen, Kamal Badshah, Muhammad Said, Dr. Hizbullah Khan, Raja Azadar Hussain,
Fatima Javed and Rukhsana Gul.
I express my gratitude to Dr. Pabitra B. Chatterjee, Dr. Ernestas Gaidamauskas, Dr.
Diganta Kalita, Mrs. Dolly Phattak, Michelle Romanishan, Ali Kamal and Maria Ali Kamal,
Syed Atta Muhammad Shah and Fayyaz Ul Amir Afsar for their care and help during my stay
at Colorado State University Fort Collins USA.
Friends, I believe are a vital part of one’s life. Their simply being with one is
sometimes enough for his survival. My sincere thanks and love to my most treasured
personalities of my life. Some of them are Taimoor Ali Noor, Sadaqat Ali, Nasir Majeed,
Shaikh Irfan, Rana Kashif Javad, Muhammad Hafeez, Muhammad Ayub, Muhammad Azam,
Muhammad Qasim, Muhammad Saeed Anjum, Hafiz Muhammad Ajmal Saeed, Muhammad
Qurban, Atif Iqbal, Muhammad Zeshan (Shani), Muhammad Nadeem Badshah, Ghulam
Muhammad Mujtaba Hashmi, Kamran Akber, Hafiz Muhammad Abdul Qayyum, Khuram
Shahzad Munawer, Muhammad Zaheer Yousaf, Adeel Asghar, Muhammad Siraj ud-Din and
many more.
From the bottom of my heart I salute to the gratefulness of all those souls that were
praying for the completion of this venture.
Ataf Ali Altaf
viii
List of Figures
Page
Figure 1.01: The cell cycle, in different types of cells 2
Figure 1.02: Thermal melting curve and Tm calculation. 17
Figure 2.01; The overall scheme of synthesis, L is the linker between ferrocene
and the functional moieties and R are different alkyl and aryl groups.
21
Figure 3.01: General framework of the entire synthesis. 52
Figure 3.02: Suggested mechanism for the conversion of ferrocenyl thioureas
into ureas (Step 4)
53
Figure 3.03: Comparative FT-IR spectra showing the spectral change of
conversion (d D Dt7 Du7)
55
Figure 3.04: Selective series of compounds discussed & characterized (d D
Dt7 Du7) using FTIR
55
Figure 3.05: 1HNMR spectra showing comparative chemical shifts on the
variation of R group in the series (A & At1 – At4)
57
Figure 3.06: 1HNMR spectra showing comparative chemical shifts on the
variation of the L group in (At4, Bt4 and Ct4) the spectra also show a well
splitting pattern.
58
Figure 3.07: 1HNMR spectra showing the spectral change of conversion (b B
Bt6 Bu6)
59
Figure 3.08: Selective series discussed for 1HNMR characterization (b B
Bt6 Bu6)
60
Figure 3.09: HSQCAD NMR spectrum of a and A indicates that the more acidic
protons are attached with more shielded carbons.
61
Figure 3.10: 13
CNMR spectra showing comparative chemical shifts on variation
of R group in series (C & Ct1 – Ct7)
63
Figure 3.11: 13
CNMR spectra showing comparative chemical shifts on the
variation of the L group in a series (At3, Bt3, Ct3, Dt3 and Et3)
64
Figure 3.12: 13
CNMR spectra showing the spectral change of conversion (e E
Et5 Eu5)
65
Figure 3.13: Selective series discussed for 13
CNMR characterization (e E
Et5 Eu5)
66
ix
Page
Figure 3.14: X) ORTEP diagram of c with atomic numbering scheme. Y) The
intra-molecular H-bonding represented by dotted lines. Z) The supramolecular
arrangement of compound c mediated by intermolecular H-bonding represented
by dotted lines
70
Figure 3.15: X) Molecular diagram of d with atomic numbering scheme. Y) The
intra-molecular H-bonding represented by dotted lines. Z) The supramolecular
arrangement of compound d mediated by intermolecular H-bonding represented
by dotted lines.
71
Figure 3.16: X) ORTEP diagram of e with atomic numbering scheme. Y) The
supramolecular arrangement of compound e along crystallographic axis-b. Z)
Molecules helices in the form of a screw due to NO---HC type hydrogen bonding
and Cδ+
---δ-
ON type interactions along crystallographic axis-a.
72
Figure 3.17: X) ORTEP diagram of the E with the atomic numbering scheme. Y)
The interaction of entrapped oxygen by H-bonding and NO type coordination
is represented by dotted lines, it shows that oxygen is nearly perpendicular to the
nitro plane. Z) The supramolecular arrangement of compound E helices due to
screw symmetry along the crystallographic axis-b
76
Figure 3.18: Y) ORTEP diagram of At4 with the atomic numbering scheme. Z)
The supramolecular arrangement of compound At4 along crystallographic axis-b,
presenting stairs like structure.
78
Figure 3.19: X) ORTEP diagram of Ct5 with the atomic numbering scheme. Y)
The intermolecular H-bonding represented by dotted lines enabling the acetone
molecule attachment and dimmer formation. Z) The supramolecular arrangement
of compound Ct5 mediated by intermolecular H---H Van der Waals interactions.
81
Figure 4.01: General micelle structure and surfactants used for modal membrane
formation
83
Figure 4.02: UV-Visible spectrum of nitrophenylferrocenes in different micelle
membranes and water after 12 hour sonication. a) for compound a, b) for d, c) for
b, d) for c, e) for e and f) is the HNMR spectra of a – e in TTAB micelles made
in D2O, 1 – 5 for a – e respectively
85
Figure 4.03: HNMR spectra of A, B and E in micelles made in D2O 87
Figure 4.04: Crystal packing of trapping atomic oxygen that is stabilized by the 89
x
Page
coordination of aniline nitrogen (N O) and N – H---O type hydrogen bonding
Figure 4.05: UV-Visible spectrum of selective ferrocenyl thiourea At4 (left) and
urea Au5 (right) in (–) DMSO, different micelle membranes (–) TTAB, (–)
SDS and (–) water after 12 hour sonication
90
Figure 4.06: HNMR spectrum of ferrocenyl urea Au5 in TTAB micelle
membranes and DMSO-d6 (upper)
91
Figure 4.07: HNMR spectra of selective series ferrocenyl thioureas in TTAB
micelle membranes
91
Figure 4.08: NOESY 2D NMR spectrum of ferrocenyl thiourea (At5) in TTAB
micelle membranes in D2O
92
Figure 4.09: Mechanism of ROS induced cell injury 96
Figure 4.10: Antioxidant activity data of selective ferrocenyl thioureas 97
Figure 4.11: Antioxidant activity data of selective ferrocenyl ureas 97
Figure 4.12: Comparative antioxidant activity of ferrocenyl ureas and thioureas 98
Figure 4.13: Most active ferrocenyl ureas and thioureas in comparison to
ascorbic acid (Standard drug)
98
Figure 4.14: UV–Visible absorption spectra of 20 µM compound At6 (left) and
60 µM compound Bt6 (right) in the absence of DNA and the presence of 10–80
µM DNA in 20% aqueous DMSO buffered at pH 6.8. (An increase of absorbance
at 260nm shows the continuous increase of DNA concentration.)
101
Figure 4.15: UV–Visible absorption spectra of 50 µM compound Ct5 (left) and
40 µM compound Ct6 in the absence of DNA and the presence of 10–80 µM
DNA in 20% aqueous DMSO buffered at pH 6.8, compounds At2, At3, At4, At5
and Dt5 behave like Ct5 when studied.
102
Figure 4.16: UV–Visible absorption spectra of 25µM compound Dt6 (left) and
60µM of Et6 (right) in the absence of DNA and the presence of 00 – 70µM DNA
in 20% aqueous DMSO buffered at pH 6.8. (An increase of absorbance at 260nm
shows the increasing of concentration DNA)
102
Figure 4.17: Thermal melting profiles of DNA in 1mM phosphate buffer (pH =
6.8) 80% DMSO: (a) with 1mM NaCl, (b) 1mM NaCl & compound Et6, and (c)
2mM NaCl & compound Et6. [DNA] = 100μM and [Et6] = 50μM.
104
xi
List of Tables
Page
Table 1.01: Top ten causes of death world wild 1
Table 1.02: A concise list of cancer causes 3
Table 1.03: A brief listing of representative commercial chemotherapeutic
agents
5
Table 3.01: Comparative 1HNMR data for nitrophenylferrocenes (a – e) and
ferrocenylanilines (A – E)
56
Table 3.02: Comparative 1HNMR data of 4-ferrocenylaniline (A) and its
thioureas (At1 – At4)
57
Table 3.03: Comparative 13
CNMR data for nitrophenylferrocenes (a – e) and
ferrocenylanilines (A – E)
60
Table 3.04: Comparative data of 13
CNMR 4-ferrocenyl-3-methoxyaniline (C)
and its thioureas (Ct1 – Ct7)
62
Table 3.05: Comparative 13
CNMR data of propyl thioureas (At3, Bt3, Ct3, Dt3
& Et3)
64
Table 3.06: Crystal data and structure refinement parameters for c, d and e 67
Table 3.07: Selected bond lengths, bond angles and torsion angles for
nitrophenylferrocenes (c, d & e)
68
Table 3.08: The intra-molecular hydrogen bonds in the nitrophenyl ferrocenes
(c, d & e)
69
Table 3.09: The inter-molecular hydrogen bonds in the nitrophenyl ferrocenes
(c, d & e)
69
Table 3.10: literature comparative data for solvent dependent torsion angles to
support push – pull delocalization of the π electrons.
73
Table 3.11: Selected bond lengths, bond angles and torsion angles for 3-
ferrocenylaniline (E)
75
Table 3.12: Crystal data and structure refinement parameters for At4 and Ct5 79
Table 3.13: Selected bond lengths, bond angles and torsion angles for ferrocene
based thioureas (At4 & Ct5)
80
Table 3.14: The inter-molecular hydrogen bonds in ferrocene based thioureas
(At4 & Ct5)
80
Table 4.01: 1HNMR spectral data of a – e in different solvents, CTAB and
TTAB micelles
86
xii
Table 4.02: 1Hnmr spectral data of A, B & E in different solvents, CTAB and
TTAB micelles
88
Table 4.03: 1Hnmr spectral data of ferrocenyl thioureas and ureas in DMSO-d
6
and TTAB micelles
93
Table 4.04: Some ROS with their normal physiological concentrations and half
lives
95
Table 4.05: DNA binding and UV-Visible spectroscopic data of selected
compounds
100
xiii
Abstract
Series of ferrocene substituted organometallic thioureas and ureas of general formula
[C5H5FeC5H4C6H3(R)NHC(X)NHR`] where, R = H, CH3, OCH3 or Cl, R`
= alkyl/aryl groups
and X = O or S, have been synthesized and characterized by using elemental analysis, FT-IR,
multinuclear (1H and
13C) NMR spectroscopy and UV-Visible spectroscopy. Single crystal
XRD was used for structural elucidation of some of the synthesized intermediate and end
products. In all crystal structures free cyclopendienyl (Cp) ring found to be disordered over
two sets of atoms. Based on the single crystal X-ray analysis most of the synthesized
compounds were found to be stabilized by intermolecular as well as intramolecular hydrogen
bonding and secondary non-covalent interactions. These intermolecular interactions permeate
these molecules to form supera-molecular structures.
The synthesized intermediates and the end products were studied for the interaction
with modal bio-mimetic micelle membranes by probing with 1H-NMR and UV-Visible
spectroscopy. Membrane penetration studies have been carried out for some compounds with
model lipid membrane interfaces prepared from SDS and TTAB surfactants using 1H NMR
and UV-Vis spectroscopic techniques. Results show the presence of these molecules in the
interfacial regions of the self assembled systems.
These studies justify the lipophilic character of these compounds as their ability to
penetrate into the modal membranes. Primarily these compounds were screened for their
DNA binding behavior and antioxidant activity to evaluate their anti-cancer potency.
Ferrocenyl ureas exhibited better anti-oxidant activity than the respective thioureas in general
and compound Bu6 was found to be the most active with IC50 = 11.94 ± 0.05 μM (IC50 for
standard thiourea is 30.53 ± 0.1 μM). DNA binding studies based on UV-Visible
spectrophotometric titration shows the potential of these organometallic compounds as an
anti-cancer agents. In ferrocenyl thioureas, the induction of nitro group found to increase the
binding ability of these compounds with DNA in these titrations. And Et6 was found to be
the most interacting compound among the tested ones with binding constant of Kb = 10810
M-1
.
1
Chapter 1
Introduction
Day by day development of science have industrialized and modernized the society.
Where, the science has created so many facilities for life like computers; using computers we
can solve problems of years and months in minutes and hours, communication; IT and
telecommunication facilitate to communicate across world at distances of millions meters,
transport; now we can travel thousands of kilometers in hours instead of months and years,
electricity; one of the most important discovery of science enlighten our lives, medicines;
now we have solution of many diseases and no one expire today due to malaria and fever, and
the science has granted many many more facilities we cannot count on tips. There also, the
modern industrialization has created some problems like pollution and huge industrial waste
that have polluted the purity of life. Due to these troubles many diseases have been grown up
like heart and cardiovascular problems and cancer. (But here I should say “Solution of
problems generated by science is in science”)
According to world health organization (WHO) survey 2011, the percentage of
natural deaths and deaths by accident (including suicide) is less than 30% of the total deaths
in the world. It means more than 70% peoples die today due health harms. Table 1.01
presents the list of the top ten causes of death in the world.
Table 1.01: Top ten causes of death world wild[1]
Sr. No. Cause Sr. No. Cause
1 Ischaemic heart disease 6 Diarrhoeal diseases
2 Cancers 7 HIV / AIDS
3 Stroke and other cerebrovascular disease 8 Tuberculosis
4 Lower respiratory infections 9 Diabetes mellitus
5 Chronic obstructive 10 Road Traffic accidents
Table 1.01 shows that cancer is the most dangerous disease after Ischaemic heart
disease that causes unnatural deaths on earth.
1.1 Cancer
Cancer is a broad term for a large group of diseases that can influence any component
of the body. Other terms used are malignant, tumours and neoplasms. One important feature
of cancer is the uncontrolled growth of abnormal cells beyond their usual boundaries, and
which can then attack adjoining parts of the body and widen to other organs. This process is
known as metastasis. Metastases are the foremost source of death from cancer.[2-4]
To
recognize cancer it is better to understand the normal cell cycle. The human life initiates from
2
a single cell, which divide to produce numerous millions of cells. This presents an example of
the controlled and normal process. Figure 1.01 shows the normal cell cycle explained by
Rubin et. al. (1999).[5]
Figure 1.01: The cell cycle, in different types of cells is; (I) Labile cells (e.g., Intestinal crypt
cells) endure continuous reproduction and the gap between two successive mitoses is called
the cycle. After partition, the cells go into a gap (G1) during which deoxyribonucleic acid
(DNA) synthesis stops, while ribonucleic acid (RNA) and protein synthesis occurs as the cell
build up its own particular function. Cells that carry on in the cell cycle go by the restriction
point (R), which assigns them to a new round of cell replication and continuance to the
synthesis (S) phase during which each and every chromosome is replicated. Followed by The
S phase cell division process enter into a small gap (G2) during which DNA synthesis ceases
and protein synthesis carry on. The M phase is the time of mitosis. After each cycle, one
daughter cell will committed to differentiation and the other will continue cycling. (II) Some
cell types, such as hepatocytes, are stable. After cell mitosis, the cells take up their
specialized function (G0) and do not reenter the cell cycle unless stimulated by the loss of
other cells. (III) Permanent cells (neuron) become terminally differentiated after mitosis and
cannot reenter the cell cycle.[5]
Any kind of disturbance that influences the cell replication process (increase the cell
cycle speed) can cause the cancer.[6]
Most cancers grow from a mutation in a single cell,
representing a monoclonal population, several tumors are polyclonal, following more than
one mutation.[4]
However, when cancer have effect, the controlled process is disturbed and
the cells multiply everlastingly to produce an ever-larger mass of cancer cel..[7]
The whole
mass of cancerous cells is called a tumor. The tumors are categorized according to the nature
3
of cells where the cancer initiates; for example, carcinomas (start in the skin or tissues that
wrap up the internal organs), sarcomas (cancer of connective tissues like as muscle, fat,
cartilage or bone), lymphomas and leukemias (initiate in body fluid e.g., blood and bone
marrow diagnosed by blood tests).[2]
There are 200+ types of cancer corresponding to
identical cells in the body.[8]
In general, a tumor is one centimeter thick or made up of more
than 1 million cells when it becomes detectable.[4]
There are two major forms of tumors i.e.
benign (non cancerous) and malignant (cancerous). Benign tumors do not widen to other
parts of the body and normally are not life ominous, however malignant tumors build up,
attack other tissues in the body and destroying them.[9]
1.1.1 Causes of cancer
Cancer develops by disorders in the genetic makeup that control the cellular growth
and cell division. Numerous factors are responsible for the development of cancer. These
factors may be genetic or environmental;[10]
in Table 1.02 many factors are enlisted
accountable to originate cancer. Along with these causes various others are also reported by
different authors.
Table 1.02: A concise list of cancer causes[10]
Sr. No. Cause Cancer caused
Genetic causes
1 Acquired mutation Can cause any kind of cancer
2 Inherited mutation (Germline mutation) Some time causes cancer but not always
Environmental causes
3 Tobacco use Larynx, lung, pancreas, bladder
4 Inorganic arsenic Skin, bladder, lung
5 Water chlorination Numerous
6 Aromatic hydrocarbons Numerous
7 Heavy metals Numerous
8 Viruses Liver, cervix and others
9 Alcohol abuse Breast, liver, larynx, esophagus, oropharynx
10 Recreational and prescribed drugs Increase the risk of cancer cell development
11 Radiation Skin, ovary, leukemia, breast sarcoma,
prostate
1.1.2 Cancer treatment
Although cancer is a dreadful disease, a number of cancers can be treated
successfully, while in case of several others, the life duration of patients can be extended
significantly, if diagnosed in early stages. The followings are the available remedies for the
cancer treatment; surgery, organ transplantation, biotherapy, palliative care, radiation therapy
and chemotherapy. Among these commonly used methods are surgery, radiation therapy and
chemotherapy.[11]
These are frequently used in combination. The type of treatment depends
upon the nature of disease and the extent to which the disease has been progressed. All these
4
treatments have their own benefits and side effects as well. But the most commonly used
methods now are chemotherapy.[12]
1.1.3 Chemotherapy
One of the most common remedy to fight against an assorted variety of cancers is
chemotherapy. Chemotherapy is an approach to cure cancer, which used chemicals to kill
cancer or the origin of cancer. Surgery and radiotherapy can be used against tumors confined
to a small area, but the Radiation treatment can be practiced in two ways: internally and
externally. In internal, the success is undermined by metastasis. The success rate of
radiotherapy and surgery is forty percent (mostly with small tumors). The remaining sixty
percent is still dying due to metastasis of cancer cells.[13]
Both these radiotherapy and surgery
are highly costly in comparison to chemotherapy and the success rate is also not very
impressive in that case. Chemotherapy has an advantage over surgery and radiation treatment,
it can be effectively utilized against the treatment of metastasized tumors[14]
An ideal
anticancer drug will be that which only eliminate the cancerous cells without affecting the
normal cells. In actual practice, all anticancer drugs affect healthy cells as well, which result
in various side effects such as nausea, vomiting and fatigue. Many chemotherapy agents are
available in the market to deal with cancer. In chemotherapy different drugs are used to slow
down the cell cycle. Figure 1.01 represents the normal cell cycle, to slow down cell
replication (for cancer treatment) a drug should have to target at least one process in the cell
cycle. Based on these target chemotherapy drugs are classified into various groups. In Table
1.03 a brief list of chemotherapeutic agents with their classification is presented.
Table 1.03 shows urea and thiourea derivatives belong to many classes of
chemotherapeutics. This demonstrates the importance of ureas and thioureas. It is one of the
driving forces for the synthesis of ureas and thioureas in this specific research project.
5
Table 1.03: A brief listing of representative commercial chemotherapeutic agents [15-24]
Sr. No. Chemotherapeutics Cancer against which practiced
Alkylating agents Interact with DNA to inhibit the cell replication process
1 Nitrogen Mustards
(Mechlorethamine)
Hodgkin’s disease, non-Hodgkin’s lymphoma breast and
lung
2 Nitrosoureas
(Carmustine)
Brain tumors, Hodgkin’s disease, non-Hodgkin’s
lymphoma, melanoma, lung cancer, colon cancer
3 Alkyl Sulfonates
(Busulfan)
Chronic myelogenous leukemia
4 Ethylenimines
(Thiotepa)
Breast cancer, ovarian cancer, Hodgkin’s disease, and non-
Hodgkin’s lymphoma
Anti-metabolites
Induce cell death during the S phase of cell growth, incorporated into RNA, DNA or inhibit
enzymes
5 Pyrimidines
(Flurouracil)
Breast, head, neck, adrenal, pancreatic, gastric, colon,
rectal, esophageal, liver
6 Purines
(6-Mercaptopurine )
Acute lymphocytic leukemia
7 Folate antagonists
(Pemetrexed)
Mesothelioma, non-small cell lung cancer
8 Hydroxyurea Melanoma, chronic myelogenous leukemia, squamous cell
carcinomas
Topoisomerase inhibitors
Makes the enzyme nonfunctional by blocking the ability of the topoisomerase to bind the DNA
9 Doxorubicin
Hodgkin's lymphoma, bladder, breast, stomach, lung,
ovaries, thyroid, soft tissue sarcoma, multiple myeloma
10 Mitoxantrone
Breast cancer, acute myeloid leukemia, non-Hodgkin's
lymphoma.
Mitotic inhibitors
Arrest the division of cells and cause cell death, By binding to the building blocks of a tubulin
protein
11 Vincristine Acute leukemia, rhabdomyosarcoma, neuroblastoma,
Wilm’s tumor, Hodgkin’s disease
12 Vindesine Melanoma, lung cancers, uterine cancers
Kinase Inhibitors
Blocks a kinase gene from binding to ATP, preventing the phosphorylation that would benefit
the cancerous cell and promote cell division.
13 Sorafenib Renal cancer, Liver cancer
1.2 Thioureas
Thioureas are the organosulfur compounds with the general formula
(R1R2N)(R3R4N)C=S, It is structurally similar to urea, except that the oxygen atom is
replaced by a sulfur atom, the properties of urea and thiourea vary appreciably.
1.2.1 Synthesis of thioureas
(a) From Cyanamid’s
6
Cyanamid is the compounds with general formula R1R2NCN, reacts with "LiAlHSH"
in the presence of 1N HCl in anhydrous diethyl ether to yield corresponding N, N-substituted
thioureas. (Scheme 1.01) In this reaction scheme one or two substitutions take place only on
single nitrogen. The "LiAlHSH" is synthesized by reacting elemental sulfur with lithium
aluminium hydride.[25]
N,N' disubstituted thioureas can not be prepared by method.
Scheme 1.01: Synthesis of thioureas from Cyanamid’s. R1 and R2 may be same or different
alkyl and aryl groups
(b) From Isothiocynates
Isothiocynates are the compounds with general formula R1NCS, on condensation with
primary (RNH2) or secondary (R2R3NH) amine yields corresponding N, N`-substituted
thioureas. (Scheme 1.02) Different acyl, aroyl, alkyl and aryl substituted thioureas can be
prepared by this method.[26, 27]
This type of condensations is reported under different
conditions like: in two different immiscible solvents using phase transfer catalyst[28, 29]
,
solvent free microwave synthesis.[30, 31]
But N,N,N`,N`-tetra substituted thioureas cannot be
synthesized in this way.
Scheme 1.02: Synthesis of thioureas from isothiocyanates. R1, R2 and R3 may be same or
different alkyl and aryl groups, R3 may also be alkoyl or aroyl group
(c) From Thiophosgene
Any kind of thiourea can be prepared by coupling two amine (primary or secondary)
molecules with thiophosgene (CSCl2) in the presence of pyridine.[32]
(Scheme 1.03) On using
two different amines mixture of thioureas formed those can be separated by different
chromatographic techniques.
Scheme 1.03: Synthesis of thioureas from thiophosgene. R and R1 may be same or different
alkyl and aryl groups.
7
1.2.2 Importance of thioureas
The importance of substituted thioureas is established mostly in heterocyclic
chemistry of 1, 3-Thiazoles, 1H-1, 2, 4-Triazoles, Imidazolidin-2-thiones, 1, 2, 4-
Oxathiazoles, 1, 2 ,4-Thiadiazolidines, 1, 3-thiazin-4-ones and 1, 3, 5-oxadiazinium, 1, 2, 4-
dithiazolium, 1, 3, 5-thiadiazinium 2-Aryl-4, 6-diamino-1, 3, 5-thiadiazinium salts and
Tetrazoles,. Thioureas have power over various biological activities, such as antiviral,[33, 34]
antibacterial,[35]
fungicidal,[36, 37]
herbicidal, [38, 39]
plant growth regulating, [40]
antitumor
agents inhibiting c-Met/VEGFR2 tyrosine kinase [41]
and potent and selective inhibitors of the
platelet derived growth factor (PDGF) receptor autophosphorylation[42]
. Thioureas have
antiarrythmic, analgesic, antihyperlipidemic, and local anesthetic activities [43]
. Recently
some thioureas were reported to demonstrate drug sensitive human oral carcinoma (KB) cells
and nasopharyngeal carcinoma (CNE2) cells [44]
. Thioureas coordination compounds show
more activity against several cancer cells when compared with well known anticancer agent
cis-platin [45]
. Thioureas have also been established to comprise applications in molecular
electronics and metal complexes. Thioureas have been effectively used for the extraction and
purification of Nickel, Palladium and Platinum metals.[46-50]
These compounds are a rich
source of materials for the development of pharmaceuticals, industrial products and
agrochemicals.
1.3 Ureas
Ureas are the organic compounds with the general formula (R1R2N)(R3R4N)C=O,
these have two nitrogen atoms attached to carbonyl carbon. These are the derivatives of urea
that is known to be first natural organic compound synthesized in laboratory.[51]
Urea and its
derivatives are used in various fields such as fertilizer, environmental protection, food
industry, pharmaceutical, and the most important applications are the biomedical and clinical
analysis.
1.3.1 Synthesis of ureas
Large numbers of protocols are reported in literature for the synthesis of urea
derivatives strating with simple compounds like amines, carbon monoxide, carbon dioxide,
organic acid chlorides, alkyl or aryl isocyanates, thioureas and many more. In this dissertation
some of these methods are summarized as below.
(a) From Isocynates
Isocynates are the compounds with general formula R1NCO, on condensation with
primary (RNH2) or secondary (R2R3NH) amine yields corresponding N, N`-substituted ureas.
(Scheme 1.04) Different acyl, aroyl, alkyl and aryl substituted ureas can be prepared by this
method.[52, 53]
this type of condensation is also reported using microwave radiations and
8
ultrasonic waves for synthesis.[54, 55]
But N,N,N`,N`-tetra substituted ureas cannot be
synthesized in this way.
Scheme 1.04: Synthesis of ureas from isocyanates. R, R1 and R2 may be same or different
alkyl and aryl groups
(b) From carbamates
Alkyl or aryl substituted carbamates, are the ester derivatives of the carbamic acid
(NH2COOH) obtained by the replacement of hydrogen’s, on reaction with primary or
secondary amines generate urea derivatives (Scheme 1.05). Many authors have reported this
protocol for the synthesis of tetra substituted ureas. Using this method we can make any
either symmetrical or unsymmetrical urea.[55, 56]
Scheme 1.05: Synthesis of ureas from carbamates. R1, R2, R3 and R4 may be same or different
alkyl and aryl groups
(c) From thioureas
Thioureas can be converted into respective ureas by the replacement of sulfur with
oxygen. Many reagents are known to replace sulfur with oxygen in such reactions like;
NaIO4[57]
, Br2/H2O[58]
, NaOH/H2O2[59]
, cetyltrimethylammonium dichromate.[60]
Use of these
oxidizing agents for the synthesis of urea’s in not appropriate because of low yield. We used
mercury (Hg-II) for the conversion of thioureas into urea derivatives in the analogues passion
of guanylation reaction (Scheme 1.06). Detailed synthesis protocol is reported in the
experimental section (Chapter 2) of the dissertation.
Scheme 1.06: Synthesis of ureas from thioureas using HgCl2. R1, R2, R3 and R4 may be same
or different alkyl and aryl groups
9
1.3.2 Importance of ureas
Urea derivatives are used in various fields such as fertilizer, environmental protection,
food industry, pharmaceutical, and the most important applications are the biomedical and
clinical analysis.[61]
Urea is a popular Nitrogen fertilizer, while certain substituted urea acts as
herbicides, fungicides, insecticides, bactericides and pesticides in agriculture applications.[62-
64] Urea derivatives have important applications; in polarography
[65], as solvents in industry,
catalyst [66]
, as raw materials in polymer industry[67]
, in green chemistry NOx control[68]
,
explosives, in food industry[69]
. Biological applications of urea and its derivatives are found;
as anticancer drugs for different carcinomas[70-72]
and obesity control[73, 74]
, potential in
biosensor[75]
, in modrn drug delivery systems.[76, 77]
1.4 Thioureas and ureas as anticancer agents
The urea analogs like as thioureas, aroylthioureas, N-nitrosoureas,
diarylsulphonylureas, and benzoylureas symbolize one of the most valuable classes of
anticancer agents, with broad spectrum activities against different leukemias and solid
tumors.[78-80]
In current era; several studies have been published about the mechanistic and
synthetic aspects of the urea and thiourea derivatives, the evaluation of their anticancer
activities, structure- activity relationships, and their interactions with biological systems, like
DNA and proteins;[81-83]
and diarylsulfonylurea derivatives have been studied to have broad
spectrum anticancer activities[84]
; the benzoylureas as new drugs with powerful anticancer
activities and the pyrazolylureas were explored as P38 kinase inhibitors.[85]
(I) An antineoplastic drug
used in myeloproliferative
disorders, polycythemia
vera, thrombocythemia
(II) Used as broad
spectrum anti-
cancer drug
(III) Used for several types of brain
cancer
(IV) Highly active against Human Oral Carcinoma and Nasopharyngeal Carcenoma
10
(V) Used for metastatic cancer of the pancreatic
islet cells.
(VI) Used for advanced stage prostate
cancer
(VII) Active against Lung Cancer
(VIII) Highly active against different
types of metastatic pancreatic cancer and
brain tumor cancer cells
Hydroxyurea has been recently investigated for the treatment of a wide range of solid
tumors as well as acute and chronic leukemia.[86]
Studies on rodent and human models of
cancer showed diarylsulphonylureas (DSU's) correspond to a new class of antitumor agents
with significant therapeutic-activity. The proposed mechanism of action for DSU's
outstanding activities is so for the inhibition of a drug-responsive NADH oxidase activity
located on the external surface of the plasma membrane of cancer cells.[80, 87]
Compounds (I –
XI) are the selective literature reported examples of anticancer ureas and thioureas having
well repute as anticancer drugs or potential drugs. Thus the design of new generations of urea
and thiourea derivatives as drugs can be benefited from chemotherapy.
(IX) Used for brain tumors
(X) Highly active against different types of
metastatic pancreatic cancer and brain tumor
cancer cells
11
(XI) Active against Brain cancer
1.5 Limitations of thioureas and ureas using as drugs
One of the major issues with using of urea and thiourea derivatives as the drug is their
least lipophilic character which limits their efficacy and impart lots of fetal side effects.[88]
As
compounds (I – III, V, VI, IX) are used as drugs in the market. Reported side effects due to
the use of high dose are; In vitro, nilutamide (VI) has been shown to inhibit the activity of
liver cytochrome P-450 isoenzymes and, therefore, may reduce the metabolism of
compounds requiring these systems. Administration of nilutamide to rats for 18 months at
doses of 0, 5, 15, or 45 mg/kg/day produced benign Leydig cell tumors in 35% of the high-
dose male rats.[89]
Long-term hydroxyurea (I) therapy develops multiple solar kera- toses and
skin tumours, both squamous cell carcinomas and basal cell carcinomas, leg ulcer, lichenoid
eruption and widespread skin changes.[90-92]
Long-term carmustine (III) therapy develops
telangiectasia, Glioma, erythema and Mild leukopenia.[15, 93, 94]
Numbers of reports are
available to describe less lipophilic character of urea and thiourea derivatives in the field of
chemotherapy, for the reason their high doses are required for chemotherapy.[88, 94, 95]
To
overcomee problems of less lipophilic nature ferrocene come into action. Ferrocene is well
known molecule for its lipid uptake characteristics.
1.6 Ferrocene
Ferrocene was reported as highly stable compound in
1951.[96]
Its correct sandwich structure was suggested
independently by Wilkinson and Fischer[97, 98]
, Woodward
suggests its name owing to the resemblance of its reactivity with
benzene. The ferrocene discovery was arguably the opening of
modern organometallic chemistry. In recent years, a rapidly
maturing and growing area, which links classical organometallic
chemistry to medicine, biology and molecular biotechnology, has
been developed as bioorganometallic chemistry.[99]
12
Since its discovery after 60 years, ferrocene has remained a molecule of interest, due
to the rich chemistry of the iron (II) center and the variety of available methods for
functionalizing the cyclopentadienyl (Cp) rings.[100, 101]
The ability to tailor the chemical
reactivity and chemical behavior of ferrocene derivatives are enhanced by the outstanding air,
heat and photochemical-stability. Ferrocene derivatives have a variety of applications in
material sciences, industry, biotechnology and medicine.[102]
Transition metal complexes
predominantly those containing redox-active species like ferrocenyl group are increasingly
being investigated for a variety of potential uses such as in the development of novel
materials including; materials for non-linear optics[103]
, building blocks in polymers, redox
sensors for molecular recognition, mediators in amperometric biosensors, coatings to modify
electrode surfaces and variety of ferrocenyl surfactants have been reported for colloidal
chemistry.[104-108]
Ferrocenyl derivatives with low oxidation potentials are attracting attention due to
their ability to catalyze the production of reactive oxygenated species (ROS), under
physiological conditions that generates cytotoxic effects.[109]
Some ferrocene derivatives have
shown cytotoxicity against lung tumours, breast cancer, antiproliferative effect in vitro, DNA
detection, antimalarial activity.[110]
The search for metal-based antitumor drugs results from
the discovery by Rosenberg in 1965 that cisplatin could effectively inhibit tumor growth and
cisplatin is used in 50-70% of all cancer patients showing good activity.[111]
Its toxicity is
high, leading to side effects which limit administered dose and some tumors are resistant to
cisplatin.[112]
Apart from the development of other platinum drugs, other metal-based
anticancer agents have been developed which appear to exhibit fewer side effects. The iron
(ferrocene) based drugs are better alternatives to platinum drug and show good cytoxic
effects with reduced general toxicity and side effects.[110]
Scheme 1.07: Ferrocifen (right one) is more active and efficient than simple organic analog
(existing drug Temoxifen at left of the equation). It is in Phase-II clinical trails for the
treatment of Breast Cancer[101, 113]
13
Scheme 1.08: Ferrochloroquine (right one) is more active and efficient than simple organic
analog (existing drug Temoxifen at left of the equation) for the treatment of malaria.[101, 114]
Scheme 1.09: Ferrocene analog (right one) of Cytosine (a nucleoside) has high apoptosis-
inducing activity against tumor cells[115]
One of the most interesting properties of ferrocene is its lipophilic character, which
attracted the bioorganometallic chemists. And it also induces lipophilicity to its
derivatives.[100]
Many commercial drugs are going to substitute by their ferrocene analogs.
Schemes 1.06 – 1.08 show the selective examples in this regard. Studies show that the
insertion of ferrocene in existing drugs enhances their activities many fold. Insertion of
ferrocene in peptide structures increased their anticancer activity, ferrocenyl-cisplatin also
been reported, chloroquien is going to replaced by frequent (ferrocene analog) and a one of
the best examples is the discovery of ferrociefen, a ferrocene analogue of tamoxifen that is in
clinical trials for breast cancer treatment, and give very impressive results.[116]
1.7 Preliminary screening of anti-cancer potency
Before moving towards expensive cell line studies it is better to know the potency of
compound to act as an anti cancer agent using simple cheap assays. To decide simple assays
it is helpful to understand, cancer hazardous as discussed earlier, the harms for normal cell
cycle can be the potentials of cancers. Highly rash free radicals and reactive oxygen species
(ROS) are being found in biological systems from different sources. These free radicals are
liable for the oxidation of nucleic acids, proteins, lipids or DNA and can initiate degenerative
disease like cancer. So we decide to do antioxidant screening of target compounds. The mode
of action of existing anticancer drugs can also be helpful to choose preliminary tests.[117-119]
As discussed earlier in the chemotherapy (Section 1.1.3); many of the chemotherapeutic
agents interact with DNA or the DNA synthesis process to stop the cell proliferation process.
Literature reports provide the evidence that majority of the drugs containing ureas and
thioureas interact with DNA to control cancer. Helleday et al described, “The control over
DNA can control different cancerous cell growth”.[120]
Keeping in mind the importance of
14
DNA binding with different compounds we decide to screen the target compounds for their
DNA binding potency. Detail of preliminary tests will also be discus in chapter 4.
1.8 DNA binding with small molecules
Different drugs interact with DNA in three different ways. First, through control of
transcription factors and polymerases; here the drug interacts with the proteins that bind to
DNA. Secondly through RNA binding to DNA double helix to form nucleic acid triple
helical structure or RNA hybridization (sequence specific binding), to expose DNA single
strand regions forming DNA-RNA hybrids that may interfere with transcriptional activity.
Third, small molecules that bind to DNA double helical structures directly. The third one
mode is the most important and common for anti-cancer agents in this way two kinds of
interactions are known in literature. These are (a) covalent binding and (b) non-covalent
binding. In covalent binding small molecules make covalent or coordinate covalent bond with
different components of DNA. For example a famous covalent binder is cis-platin (used as an
anticancer drug) which makes an intra and inter strand cross-link through the replacement of
chlorides with the nitrogen's on the DNA bases. Covalent binding is irreversible and
invariably proceeds to complete inhibition of DNA and as a result cell death will take place.
In a non-covalent interactionn there is no permanent affiliation of small molecules
with DNA. In this way small molecules cause temporary conformational changes in DNA.
This kind of binding is reversible and is preferred over a covalent interaction because of toxic
side effects and drug metabolism in covalent binding. However there is the advantage of high
binding strength in covalent binding.
The non-covalent interactions are further classified into three categories (i)
intercalation (ii) groove binding and (iii) electrostatic interaction.[121, 122]
To understand these
interactions lets have a look at the
DNA structure
(I) In intercalation planer groups in
molecules penetrate between base
pairs of DNA double strand and
introduce strong structural
disturbance in DNA. The classical
examples of intercalators are
Nogalamycin and ethidium bromide.
(II) The groove binding is the attachment of small drug molecules bind with DNA by
promoting Van der Waals interactions and hydrogen bonds to the bases. Groove binders are
usually crescent shaped. Mostly groove binders bind at the AT (adenine and thymine) base
pair of the DNA i.e., Netropsin, Distamycin etc. Some exceptional groove binders have
15
specificity for G-C and C-G regions in the grooves for example few synthetic polyamides
such as lextropsins and amadazole-pyrrole polyamides.
(III) The electrostatic interaction is the ionic type of attraction between the negative charge of
DNA phosphates and positively charged molecules those can not intercalate into the DNA
helical structure due to structural make up.
1.9 Techniques to study non-covalent interactions
The manifestation of the reality of non-covalent bonding interactions among DNA
and drugs or potentially mutagenic agents involves sensitive analytical techniques.
Technological advances in key techniques, together with the efficiency in surface plasmons
resonance or fluorescence techniques have permitted to obtain information about the nature,
the strength and the localization of drug-DNA interactions. Variety of techniques has been
devoted to evidence drug-DNA interactions and growing continuously. For example UV-
visible spectrophotometry, Vibrational spectroscopy, NMR spectroscopy, Circular and linear
dichroism, Fluorescence emission spectroscopy, Metal enhanced fluorescence (MEF),
Enzimatic methods: footprinting, competition dialysis assay, gel mobility electrophoresis
assay, HPLC-MS, capillary electrophoresis-mass spectrometry; Genosensors:
Electrochemical genosensors, Optical genosensors, are the techniques available at the time.
Keeping into consideration the available facilities we tried to explore the DNA binding
behavior of synthesized compounds using UV-visible spectrophotometry.[123]
1.9.1 UV-visible spectrophotometry in DNA binding
The UV-visible spectrophotometric method is highly sensitive, efficient, fast, and low
cost technique for studying of the interaction between small molecules (optically active i.e.
absorbing in UV-visible spectrum) and DNA. This approach could contribute a lot in order to
speed up the drug screening process. The evidence for the interaction mechanism, the nature
of the complex formed, binding constant, binding site size and the role of free radicals gene
generated during interaction in drug action can be provided by UV-visible
spectrophotometric signal related to DNA interactions. The interaction can be detected by
measuring the changes in the absorption properties of the interacting compound or the DNA
molecules. The UV-Vis absorption spectrum of DNA exhibits a broad band (200-350 nm) in
the UV region with a maximum placed at 260 nm. This maximum is a consequence of the
chromophoric groups in purine and pyrimidine moieties responsible for the electronic
transitions. The probability of these transitions is high and thus the molar absorptivity (ε) is
of the order of 6600 M-1
cm-1
. So the compounds absorbing in other than this region can
easily be studied for DNA interaction by UV-visible spectrophotometeric titration. By
keeping compound concentration constant and varying the DNA concentration.[124]
16
The different kind of changes occurs in the spectrum of compound after addition of
DNA aliquots. These changes are; the maximum absorption can shift up-to 70nm towards red
or blue wavelengths upon DNA interaction. Hyperchromic or hypochromic effects usually
escort these shifts. In the case of poor associations, only hyperchromic or hypochromic effects
are observed without noteworthy changes of shifts in the spectra. When a compound interacts
with DNA; its electronic structure changes those results into these changes.[124]
The
hypochromic effect is due to the interaction between the electronic states of the intercalating
chromophore and those of the DNA bases. The strength of this electronic interaction would
decrease as the cube of the distance of separation between the chromophore and the DNA
bases. The bathochromic effect or red shift is associated with the decrease in the energy gap
between the highest occupied (HOMO) and the lowest unoccupied (LUMO) molecular
orbitals after the interaction of compounds with DNA. Hypochromic effect along with red
shift in UV-visible spectra is a typical characteristic of an intercalating mode. The
compactness in the structure of either the compound alone and/or DNA after the formation of
the complex may result in hypochromism. The blue shift is associated with the increase in the
energy gap between the highest occupied (HOMO) and the lowest unoccupied (LUMO)
molecular orbitals after the interaction of compounds with DNA. Hypochromic effect along
with blue shift in UV-visible spectra is a typical characteristic of an ionic or electrostatic
mode of interaction.[125]
The quantitative association of the compound with DNA can be achieved on the basis
of changes absorption on increasing amounts of DNA. The equilibrium constants (binding
constant) can be calculated with data fitting in the Benesi-Hildebrand equation (equation
1.1).[126]
(1.1)
Where Ao and A are the absorbance of free compound and compound–DNA complex
respectively, εG and εH-G is the molar extinction coefficients of free compound and
compound–DNA complex respectively.
1.9.2 Thermal melting assay of DNA
The coupling of thermal treatment of DNA with UV-visible spectroscopy yields
interesting phenomenon to understand the binding behavior of a compound to the DNA
helical structure. The study of this phenomenon is known as thermal melting assay of DNA.
The use of this flexible and easy technique permits to calculate the molar concentration of
DNA by measuring the absorbance value at 260 nm. The ε values (at λmax= 260 nm) of free
oligonucleotides are higher than the ones matching to the same oligonucleotides in double
strand DNA (ds-DNA) and single strand DNA (ss-DNA) because base-base stacking
17
consequences in a hypochromic effect. This performance can be subjected to prove
denaturalization of DNA by measuring its absorbance before and after denaturing treatment.
The denaturalization of DNA can be done by temperature treatment. The temperature at
which the absorbance of DNA at 260nm increase abruptly is called melting temperature (Tm)
of DNA. It can also be defined as Melting temperature (Tm) is the temperature value
corresponding to the transfer of 50 % of the double strands into single strands of DNA,
according to the equilibrium shown in equation (1.2).[123]
(1.2)
The changes in the Tm value can be followed by techniques such as circular
dichroism, calorimetry, NMR or fluorescence, but UV-Vis absorption spectrometry is the
most frequently employed methods due to its good sensitivity, reproducibility, simplicity and
versatility. Tm value is calculated from the inflexion point in the absorbance-temperature plot
(Figure 1.02).
Figure 1.02: Thermal melting curve and Tm calculation. (Left) A thermal denaturation curve
for a parallel bimolecular duplex at pH 6.5. (Right) The dotted line represents the median.
The Tm matches to the intersection between the median and the experimental curve. [127]
When a compound–DNA interaction exists, Tm shifts to values different from native
ds-DNA. Direction of shifting decides the nature of covalent or non-covalent binding.
Decrease in the Tm value is characteristic of covalent binding that is actually the
destabilizing interaction while the increase in Tm value shows the existence of non-covalent
stabilizing interaction. The magnitude of the shift depends on the type of interaction. Thus,
for intercalating agents the increase observed in the Tm value is higher than in the case of
agents interacting through the DNA minor or major grooves or through electrostatic
interaction. Tm value is highly ionic strength dependent for the compounds interacts via
electrostatic interaction.[127]
18
1.10 Aims of study
Thioureas and ureas are well known for their role in biological applications specifically in
cancer treatment. Keeping in mind their importance and the problem of their less lipophilic
character as discussed earlier, we decide to synthesize thiourea and urea derivatives with
better lipid soluble qualities. Ferrocene was selected to insert lipophilic properties of these
molecules due to its non-toxic, redox active and important biological activity enhancing
properties. Along with these it is highly stable and varieties of methods are available to
functionalize it. Keeping these significances in view the following aims were set for the
present study
To synthesize some novel ferrocenyl substituted thioureas in good yield.
To synthesize the ferrocenyl substituted ureas of these synthesized thioureas.
To fully characterize these compounds by using UV-visible spectroscopic, elemental
analysis, NMR, FT-IR measurement and single crystal X-ray diffraction analysis.
On the basis of biological activities of related compounds reported in literature and
available facilities, it was decided to preliminary investigate the synthesized
compounds for their anticancer potency.
The preliminary anticancer potency investigation was decided by the screening of
synthesized compounds for their antioxidant activity and DNA binding potency.
To find out the lipophilic properties of synthesized compounds micelle membrane
interaction studies was decided to perform.
To find out the structure activity relationship among the designed compounds and
their determined activities.
19
Chapter 2
Experimental and Characterization
2.1 Materials
Ferrocene, 4-nitroaniline, 2-methyl-4-nitroaniline, 2-methoxy-4-nitroaniline, 2-
chloro-4-nitroaniline, 3-nitroaniline, methylisothiocynate, ethylisothiocynate,
propylisothiocynate, alleylisothiocynate, phenylisothiocynate, 4-nitrophenylisothiocynate,
2,4-dichlorophenylisothiocynate, sodium hydroxide and mercuric chloride were purchased
from Acros Organics (Geel, Belgium), sodium nitrite, cetyltrimethylammonium bromide
(CTAB), tetradecyltrimethylammonium bromide (TTAB), sodium dodecylsulphate (SDS),
hydrochloric acid, Zinc dust, formic acid, ammonium formate, diethyl ether, chloroform
(CHCl3), dimethylsulfoxide (DMSO) and other solvents were purchased from Aldrich. All
the chemicals purchased were of analytical reagent grade and were used as such. Doubly
distilled and deionized water (17.5 MΩ cm Barnstead E-pure) was used without purification.
D2O (99.9 %), CDCl3, DMSO-d6 and Acetone-d
6 were acquired from Cambridge Isotope
Laboratories Andover, MA. USA.
2.2 Instrumentation
Melting points were determined using melting point apparatus, model Bio Cote
SMP10- UK, all the values are uncorrected. Infrared absorption spectra were recorded in the
range of 4000-400 cm-1
as KBr discs on Thermo Scientific Nicolet-6700 FTIR
spectrophotometer. UV-visible spectrometric measurements were recorded on Perkins
Erlenmeyer lambda-25 UV-visible spectrometer.
NMR spectra were recorded on Varian MR NMR spectrometer using deuterated
solvents. 1H NMR (400 MHz) internal standard solvent DMSO-d
6 (2.50 ppm from TMS
(tetramethyl silane - Si(CH3)4)); 13
C NMR (101.0 MHz) internal standard solvent DMSO-d6
(39.5 ppm from TMS)[128]
. The splitting of proton resonance in the 1H NMR spectra is
defined as s = singlets, d = doublet, t = triplet, q = quartet and m = multiplet etc.; Coupling
constants are reported in Hz.
Elemental analysis (CHNS analysis) was performed by using an in house instrument
of Leco CHNS-932 Elemental analyzer USA in Quaid-i-Azam University Islamabad and
some of the samples were analyzed on payment from Columbia Analytical Services, Tucson,
AZ. USA. The crystallographic data of the synthesized ferrocene derivatives were collected
on a Bruker kappa APEXII CCD diffractometer.
2.3 General description of the work
Ferrocene based thioureas and ureas were the target compounds of the synthetic work.
The targeted syntheses were achieved in four steps. In the first step; substituted
20
nitrophenylferrocenes (a - e) were made by the coupling of ferrocene with diazonium salts of
different nitroanilines using phase transfer catalyst.[129]
In the second step; these nitro
derivatives were reduced into ferrocenylanilines (A - E) using different reducing agents.[130]
In the third step; one of the targeted series of compounds, ferrocenylthioureas (At1 – Et7),
were achieved by the coupling of ferrocenyl anilines with different isothiocynates in
acetone.[131]
In the fourth step; selected thioureas were converted into ferrocenylureas (Au3 –
Eu7) by the replacement of sulfur with oxygen using mercuric chloride in DMF.[132]
The overall scheme of synthesis is shown in figure 2.01. All the synthesized
compounds were characterized by elemental analysis, FTIR, 1H &
13C NMR, and some were
characterized by single crystal XRD. These compounds were then screened for antioxidant
activity, DNA binding ability by using UV-visible spectroscopy & DNA thermal denaturing
assay, and modal membrane interactions using UV-visible and 1HNMR spectroscopy.
[133]
21
Figure 2.01; The overall scheme of synthesis, L is the linker between ferrocene and the
functional moieties and R are different alkyl and aryl groups.
Code Linker (L) Number R group
A, a
1 2
B, b
3
4
C, c
5
D, d
6
E, e
7
22
2.4 Synthesis of Nitrophenylferrocenes (a – e)
Five different nitrophenylferrocenes were synthesized in an ether / water mixture using phase
transfer catalysis, by the coupling of diazonium salts of nitroanilines as shown in scheme
2.01.
Scheme 2.01; Synthesis of Nitrophenylferrocenes (a – e), X is H for a and e, CH3 for b,
OCH3 for c and Cl for d. Nitro group is substituted at para position in a – d and meta in e,
from ferrocene.
*PTC = Phase Transfer Catalyst i.e. cetyltrimethylammonium bromide (CTAB).
2.4.1 4-nitrophenylferrocene (a)
Compound (a) was synthesized using the reported method with some modifications[129]
. 4-
Nitroaniline (14 g, 100 mmol), 30 ml of water and 30 ml of concentrated hydrochloric acid
were mixed together and cooled to 0-5 °C using salt water-ice bath. A solution of sodium
nitrite (7 g, 100 mmol) in 50 ml of water was added drop wise under stirring. After complete
addition, the resulting solution was stirred for 30 min and kept below 4 °C. Ferrocene (9.5 g,
50 mmol), and hexadecyltrimethylammonium bromide (CTAB) (0.5 g, 1.37 mmol) were
added to 150 ml diethyl ether and cooled to 0-5 °C. The diazonium salt solution was added
drop wise to CTAB containing ferrocene solution under constant stirring, keeping the
temperature below 5 °C. After complete addition, the reaction mixture was stirred overnight
at room temperature. The mixture was concentrated by rotary evaporation and the residue
was washed with water. The crude solid was then steam distilled to recover unreacted
ferrocene. The residual crude product was recrystallized from n-heptane to give 4-
nitrophenylferrocene (a) as violet plates (14.2 g, 73.6 %), Elemental analysis calculated for
C16H13FeNO2 C, 62.57; H, 4.27; N, 4.56 found C, 62.59; H, 4.31; N, 4.59 %; 1H NMR (400
MHz, DMSO-d6, ppm) δ 8.14 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 8.8 Hz, 2H), 4.74 (t, J = 1.8
Hz, 2H), 4.48 (t, J = 1.8 Hz, 2H), 4.06 (s, 5H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ
148.2, 145.5, 125.9(2C), 123.8(2C), 81.7, 70.5(2C), 70.0(5C), 67.2(2C); FTIR (KBr, υ cm-1
):
2990, 1596, 1506, 1340, 1286, 1105, 1080, 847, 756, 696, 505, 472.
23
2.4.2 2-methyl-4-nitrophenylferrocene (b)
Quantities used were 15.21 g (100 mmol) 2-methyl-4-nitroaniline, 6.89 g (100 mmol) sodium
nitrite, 50 ml concentrated HCl, (0.5 g, 1.37 mmol) hexadecyltrimethylammonium bromide
(CTAB) and 13.95 g (75 mmol) Ferrocene. Yield 17.6 g (73.2 %), Calc. for C17H15FeNO2 C,
63.58; H, 4.71; N, 4.36 found C, 63.52; H, 4.75; N, 4.39 %; 1H NMR (400 MHz, DMSO-d
6,
ppm) δ 7.95 – 8.00 (m, 2H), 7.63 (d, J = 6.1 Hz, 1H), 4.69 (t, J = 1.8 Hz, 2H), 4.51 (t, J = 1.8
Hz, 2H), 4.23 (s, 5H), 2.52 (s, 3H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 146.3, 136.6,
132.7, 130.7, 125.4, 120.5, 80.3, 70.8(2C), 70.6(5C), 69.8(2C), 21.6; FTIR (KBr, υ cm-1
):
3091, 2964, 2927, 1606, 1581, 1510, 1346, 1132, 1105, 1070, 1002, 876, 829, 802, 750, 717,
557, 472.
2.4.3 2-methoxy-4-nitrophenylferrocene (c)
Quantities used were 16.81 g (100 mmol) 2-methoxy-4-nitroaniline, 6.89 g (100 mmol)
sodium nitrite, 50 ml concentrated HCl, (0.5 g, 1.37 mmol) hexadecyltrimethylammonium
bromide (CTAB) and 13.95 g (75 mmol) Ferrocene. Yield 17.3 g (68.4 %), Elemental
analysis calculated for C17H15FeNO3 C, 60.56; H, 4.48; N, 4.15 found C, 60.62; H, 4.44; N,
4.38 %; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 7.79 – 7.81 (m, 2H), 7.61 (d, J = 8.5 Hz,
1H), 4.87 (t, J = 1.9 Hz, 2H), 4.42 (t, J = 1.9 Hz, 2H), 4.06 (s, 5H), 4.01 (s, 3H); 13
C NMR
(101 MHz, DMSO-d6, ppm) δ 156.5, 146.2, 136.6, 128.6, 116.0, 105.9, 79.6, 69.82(2C),
69.8(5C), 69.5(2C), 55.8; FTIR (KBr, υ cm-1
): 3112, 3081, 3016, 2956, 2933, 2916, 1585,
1517, 1334, 1242, 1103, 1078, 1035, 860, 814, 800, 746, 732, 497, 484, 462.
2.4.4 2-Chloro-4-nitrophenylferrocene (d)
Quantities used were 17.2 g (100 mmol) 2-chloro-4-nitroaniline, 6.89 g (100 mmol) sodium
nitrite, 50 ml concentrated HCl, (0.5 g, 1.37 mmol) hexadecyltrimethylammonium bromide
(CTAB) and 9.3 g (50 mmol) Ferrocene. Yield 10.82 g (63.5 %), Elemental analysis
calculated For C16H12FeNClO2 C, 56.26; H, 4.54; N, 4.10 found C, 56.10; H, 4.69; N, 4.79
24
%; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 8.21 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.73 (d, J =
8.0 Hz, 1H), 4.99 (bs, 2H), 4.59 (bs, 2H), 4.27 (s, 5H). 13
C NMR (101 MHz, DMSO-d6,
ppm) δ 145.8, 145.5, 132.3, 131.1, 125.8, 121.2, 81.3, 70.3(2C), 70.1(5C), 68.9(2C); FTIR
(KBr, υ cm-1
): 3104, 3074, 2923, 1592, 1585, 1514, 1336, 1105, 885, 864, 821, 759, 744,
715, 511, 469.
2.4.5 3-nitrophenylferrocene (e)
Quantities used were 13.8 g (100 mmol) 3-nitroaniline, 6.89 g (100 mmol) sodium nitrite, 50
ml concentrated HCl, (0.5 g, 1.37 mmol) hexadecyltrimethylammonium bromide (CTAB)
and 13.95 g (75 mmol) Ferrocene. Yield 16.2 g (70.2 %), Elemental analysis calculated For
C16H13FeNO2 C, 62.57; H, 4.27; N, 4.56 found C, 6.58; H, 4.21; N, 4.57 %; 1H NMR (400
MHz, DMSO-d6, ppm) δ 8.28 (s, 1H), 8.03 (d, J = 9.3 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.44
(t, J = 7.9 Hz, 1H), 4.73 (bs, 2H), 4.42 (bs, 2H), 4.07 (s,5H); 13
C NMR (101 MHz, DMSO-
d6, ppm) δ 145.1, 139.6, 131.6, 129.2, 120.5, 120.4, 81.1, 70.6(2C), 69.8(5C), 66.8(2C);
FTIR (KBr, υ cm-1
): 3084, 2931, 1527, 1346, 1105, 742, 723, 675, 478, 462.
2.5 Synthesis of ferrocenylanilines (A – E)
All the synthesized nitrophenylferrocenes (a – e) were reduced to respective anilines as
shown in scheme 2.02. The reduction was first optimized for the synthesis of A and then the
optimized protocol was used for the synthesis of B – E.
Scheme 2.02; Synthesis of ferrocenylanilines (A – E).
2.5.1 4-Ferrocenylaniline (A)
4-Ferrocenylaniline (A) was synthesized in four different ways to improve the yield and the
results were published.[130]
Following are the different procedures.
Procedure (I) Conc. HCl (30 mL) was added to the suspension of Zn dust (2.62 g, 40.0
mmol) in solution of 4-nitrophenylferrocene (a) (2.0 g, 7.0 mmol) in ethanol (100 mL). The
reaction mixture was refluxed for 8 hours and monitored on TLC. The reaction completed
with the change of color from violet to orange. The hot reaction mixture was filtered off and
25
volume of the filtrate was reduced to 30 ml using rotary evaporator and further diluted with
water NaOH solution by adjusting pH at 14. Ferrocenylaniline (A) was then extracted from
that mixture with CH2Cl2 (6 x 50 ml). The solvent was evaporated and the product was re-
crystallized from the pet ether of boiling range (60-80 ºC) to give 4-ferrocenylaniline (A) as a
red-orange solid. (1.17 g, 65 %)
Procedure (II) Conc. HCl (30 ml) was added to the suspension of Sn powder (4.50 g, 40.0
mmol) in a solution of 4-nitrophenylferrocene (a) (2.0 g, 7.0 mmol) in 100 ml of ethanol. The
reaction mixture was refluxed for 8 hours and monitored on TLC. The reaction completed
with the change of color from violet to orange. The hot reaction mixture was filtered off and
volume of filtrate was reduced to 30 ml using rotary evaporator and further diluted with water
NaOH solution by adjusting pH at 14. Ferrocenylaniline (A) was then extracted from that
mixture with CH2Cl2 (6 x 50 ml). The solvent was evaporated and the product was re-
crystallized from pet-ether of boiling range (60-80 ºC) to give 4-ferrocenylaniline (A) as a
red-orange solid. Yield 1.42 g (79 %)
Procedure (III) Suspension of Zn dust (2.62 g, 40.0 mmol) was made in solution of 4-
nitrophenylferrocene (a) (2.0g, 7.0 mmol) in 100 ml of methanol. Formic acid (3.82 ml, 100
mmol) was added drop wise to the suspension, stirred for 3 hours and monitored on TLC. On
completion, the reaction mixture was rotary evaporated, orange yellow crude solid was
dissolved in 100 ml of CH2Cl2 and washed with water (4 x 100 ml). The solvent was
evaporated and the product was re-crystallized from the pet ether of boiling range (60-80 ºC)
to give 4-ferrocenylaniline (A) as a red-orange solid. Yield 1.62 g (90%)
Procedure (IV) Zn dust (2.62 g, 40.0 mmol) was added to the solution of 4-
nitrophenylferrocene (a) (2.0 g, 7.0 mmol) in 50 ml of Formic acid, the mixture was stirred
vigorously for 5 min and monitored on TLC. On completion, the reaction mixture was
filtered and the filtrate was rotary evaporated and the orange yellow crude solid was
dissolved in 100 ml of CH2Cl2 and washed with 2.0% NaOH solution (3 x 100 ml) and the
water (4 x 100 ml). The solvent was rotary evaporated and product was re-crystallized from
the pet ether of boiling range (60-80 ºC) to give 4-ferrocenylaniline (A) as a red orange solid
(1.69 g, 94 %). The melting point of the product of all entries was in the range of 156-159 ºC.
Elemental analysis calculated For C16H15FeN; C, 69.34; H, 5.46; N, 5.05 found C, 69.29; H,
5.51; N, 5.05 %; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 7.33 (d, J = 8.4 Hz, 2H), 6.67 (d, J
= 8.4 Hz, 2H), 4.56 (t, J = 1.8 Hz. 2H), 4.26 (t, J = 1.8 Hz. 2H), 4.06 (s, 5H), 3.66 (bs, 2H);
13C NMR (101 MHz, DMSO-d
6, ppm) δ 144.5, 129.0, 127.2(2C), 115.2(2C), 86.6, 69.4(5C),
68.2(2C), 65.8(2C); FT-IR (KBr, υ cm-1
): 3435, 3372, 3354, 1616, 1528, 1455, 1103, 999,
814.
26
2.5.2 3-methyl-4-Ferrocenylaniline (B)
Quantities used were 16.05 g (50 mmol) 2-methyl-4-nitrophenylferrocene (b), 13.0 g, (200.0
mmol) Zn dust and 50 ml formic acid. Yield 13.56 g (93.2 %), Calc. for C17H17FeN; C,
70.12; H, 5.88; N, 4.81 found C 70.10; H, 5.87; N, 4.84 %; 1H NMR (400 MHz, DMSO-d
6,
ppm) δ 7.50 (d, J = 7.9 Hz, 1H), 6.54 (d, J = 7.9 Hz, 1H), 6.49 (s, 1H), 4.39 (bs, 2H), 4.23
(bs, 2H), 4.12 (s, 5H), 3.61 (bs, 2H), 2.30 (s, 3H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ
138.5, 135.1, 132.8, 124.6, 120.3, 117.0, 84.1, 69.4(5C), 69.3(2C), 67.4(2C), 21.2; FT-IR
(KBr, υ cm-1
): 3425, 3327, 3254, 2930, 1616, 1532, 1451, 1110, 993, 804, 540.
2.5.3 3-methoxy-4-Ferrocenylaniline (C)
Quantities used were 16.85 g (50 mmol) 2-methoxy-4-nitrophenylferrocene(c), 13.0 g, (200.0
mmol) Zn dust and 50 ml formic acid. Yield 13.57 g (88.4 %), Elemental analysis calculated
for C17H17FeNO; C, 66.47; H, 5.58; N, 4.56; found C, 66.44; H, 5.60; N, 4.58 %; 1H NMR
(400 MHz, DMSO-d6, ppm) δ 7.29 – 7.32 (m, 2H), 6.34 (d, J = 6.5 Hz, 1H), 4.39 (t, J = 1.8
Hz, 2H), 4.22 (t, J = 1.7 Hz, 2H), 4.06 (s, 5H), 4.01 (s, 3H). 3.90 (bs, 2H); 13
C NMR (101
MHz, DMSO-d6, ppm) δ 141.5, 127.2, 118.6, 116.0, 112.6, 105.9, 82.1, 69.8(5C), 67.8(2C),
64.5(2C), 55.0; FTIR (KBr, υ cm-1
): 3377, 3302, 3112, 3081, 3016, 2956, 2933, 2916, 1585,
1517, 1334, 1242, 1103, 1078, 1035, 860, 814, 800, 746, 732, 497, 484, 462.
2.5.4 3-Chloro-4-Ferrocenylaniline (D)
Quantities used were 17.1 g (50 mmol) 2-chloro-4-nitrophenylferrocene(d), 13.0 g, (200.0
mmol) Zn dust and 50 ml formic acid. Yield 14.56 g (93.5 %), Elemental analysis calculated
For C16H14FeNCl; C, 61.67; H, 4.53; N, 4.50 found C, 61.64; H, 4.55; N, 4.53 %; 1H NMR
(400 MHz, DMSO-d6, ppm) δ 7.51 (s, 1H), 7.21 (d, J = 7.3 Hz, 1H), 7.03 (d, J = 7.4 Hz, 1H),
4.61 (bs, 2H), 4.36 (bs, 2H), 4.07 (s, 5H), 3.71 (bs, 2H); 13
C NMR (101 MHz, DMSO-d6,
ppm) δ 144.0, 142.4, 130.3, 128.1, 123.8, 120.2, 76.3, 70.1(5C), 68.5(2C), 64.9(2C); FTIR
27
(KBr, υ cm-1
): 3358, 3285, 3104, 3074, 2923, 1592, 1585, 1514, 1336, 1105, 885, 864, 821,
759, 744, 715, 511, 469.
2.5.5 3-Ferrocenylaniline (E)
Quantities used were 15.35 g (50 mmol) 3-nitrophenylferrocene (e), 13.0 g, (200.0 mmol) Zn
dust and 50 ml formic acid. Yield 12.5 g (90.2 %), Elemental analysis calculated For
C16H15FeN; C, 69.34; H, 5.46; N, 5.05 found C, 69.39; H, 5.50; N, 5.08 %; 1H NMR (400
MHz, DMSO-d6, ppm) δ 7.09 (t, J = 7.3 Hz, 1H), 6.92 (d, J = 6.9 Hz, 1H), 6.82 (s, 1H), 6.54
(d, J = 6.8 Hz, 1H), 4.59 (bs, 2H), 4.28 (bs, 2H), 4.05 (s, 5H), 3.67 (bs, 2H); 13
C NMR (101
MHz, DMSO-d6, ppm) δ 142.1, 131.6, 129.2, 123.1, 120.5, 120.3, 82.4, 69.8(5C), 69.8(2C),
66.8(2C); FT-IR (KBr, υ cm-1
): 3395, 3307, 3250, 2925, 1595, 1527, 1431, 1115, 986, 811,
541.
28
2.6 Synthesis of ferrocene based thioureas (At1 – Et7)
Five series of ferrocenylthioureas (At1 – At7, Bt1 – Bt7, Ct1 – Ct7, Dt1 – Dt7 & Et1 – Et7)
were accomplished by the coupling of ferrocenylanilines (A – E) with different
isothiocynates, having R groups (1 – 7) in acetone. The general reaction is shown in scheme
2.03.
Scheme 2.03; Synthesis of ferrocenylthioureas (At1 – Et7).
Number R group
1 2 3 4
5
6
7
2.6.01 1-(4-(ferrocenyl)phenyl)-3-methylthiourea (At1)
To the solution of 2.77 g (10 mmol) 4-ferrocenylaniline (A) in acetone, 0.73 g (10 mmol)
methylisothiocynate was added, The reaction mixture was refluxed for 8 hours and the extent
of the reaction was monitored on TLC. On completion of the reaction solvent was rotary
evaporated. Crude product was washed with n-hexane and recrystallized from methanol by
slow evaporation. 1-(4-(ferrocenyl)phenyl)-3-methylthiourea (At1) was obtained as orange
solid crystals. Yield 2.22 g (63.5%), Elemental analysis calculated For C18H18FeN2S; C,
61.72; H, 5.18; N, 8.00; S, 9.15 found C, 61.78; H, 5.08; N, 8.01; S, 9.17 %; 1H NMR (400
MHz, DMSO-d6, ppm) δ 9.32 (bs, 1H), 7.50 (bs, 1H), 7.40 (d, J = 7.4 Hz, 2H), 6.95 (d, J =
7.3 Hz, 2H), 4.73 (t, J = 1.7 Hz, 2H), 4.32 (t, J = 1.7 Hz, 2H), 4.02 (s, 5H), 2.67 (s, 3H); 13
C
NMR (101 MHz, DMSO-d6, ppm) δ 179.5, 137.0, 134.1, 125.1(2C), 122.0(2C), 83.9,
68.2(5C), 67.6(2C), 66.0(2C), 21.6; FTIR (KBr, υ cm-1
): 3467, 3377, 3095, 2964, 2928,
29
2868, 1515, 1476, 1449, 1409, 1300, 1256, 1240, 1190, 1129, 1106, 1046, 1000, 906, 854,
816, 787, 689, 491.
2.6.02 1-(4-(ferrocenyl)phenyl)-3-ethylthiourea (At2)
Quantities used were 2.77 g (10 mmol) 4-ferrocenylaniline (A) and 0.874 ml (10 mmol)
ethylisothiocynate. Yield 2.25 g (62.0%), Elemental analysis calculated For C19H20FeN2S; C,
62.64; H, 5.53; N, 7.69; S, 8.80 found C, 62.63; H, 5.53; N, 7.67; S, 8.81 %; 1H NMR (400
MHz, DMSO-d6, ppm) δ 9.39 (bs, 1H), 7.70 (bs, 1H), 7.51 – 7.43 (m, 2H), 7.31 (d, J = 8.5
Hz, 2H), 4.74 (t, J = 1.9 Hz, 2H), 4.32 (t, J = 1.9 Hz, 2H), 4.03 (s, 5H), 3.44 – 3.55 (m, 2H),
1.12 (t, J = 7.2 Hz, 3H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 179.7, 136.9, 134.6,
125.9(2C), 123.0(2C), 84.7, 69.2(5C), 68.6(2C), 66.0(2C), 38.6, 14.1; FTIR (KBr, υ cm-1
):
3374, 3261, 3187, 3056, 2973, 2925, 1620, 1558, 1527, 1456, 1425, 1407, 1367, 1346, 1326,
1292, 1270, 1236, 1203, 1186, 1143, 1103, 1087, 1053, 1028, 1002, 943, 887, 844, 821, 806,
721, 692, 6446, 628, 611, 574, 526, 514, 503, 484, 433, 418, 412.
2.6.03 1-(4-(ferrocenyl)phenyl)-3-propylthiourea (At3)
Quantities used were 2.77 g (10 mmol) 4-ferrocenylaniline (A) and 1.03 ml (10 mmol)
propylisothiocynate. Yield 2.45 g (65.0%), Elemental analysis calculated For C20H22FeN2S;
C, 63.50; H, 5.86; N, 7.40; S, 8.48 found C, 63.52; H, 5.83; N, 7.41; S, 8.47 %; 1H NMR
(400 MHz, DMSO-d6, ppm) δ 9.39 (bs, 1H), 7.71 (bs, 1H), 7.47 (d, J = 8.6 Hz, 2H), 7.32 (d,
J = 8.6 Hz, 2H), 4.74 (t, J = 1.9 Hz, 2H), 4.33 (t, J = 1.9 Hz, 2H), 4.03 (s, 5H), 3.42 (dd, J =
12.0, 5.8 Hz, 2H), 1.63 – 1.49 (m, 2H), 0.89 (t, J = 7.4 Hz, 3H); 13
C NMR (101 MHz,
DMSO-d6, ppm) δ 180.0, 134.5, 126.5(2C), 125.9, 122.8(2C), 84.7, 69.2(5C), 68.6(2C),
66.0(2C), 45.5, 21.7, 11.3; FTIR (KBr, υ cm-1
): 3259, 3031, 2958, 2923, 2860, 1602, 1556,
1525, 1454, 1334, 1317, 1263, 1103, 1078, 998, 885, 823, 651, 734, 524, 482, 434.
2.6.04 1-(4-(ferrocenyl)phenyl)-3-allylthiourea (At4)
Quantities used were 2.77 g (10 mmol) 4-ferrocenylaniline (A) and 0.98 ml (10 mmol)
allylisothiocynate. Yield 2.24 g (59.5%), Elemental analysis calculated For C20H20FeN2S; C,
63.84; H, 5.36; N, 7.44; S, 8.52 found C, 63.82; H, 5.37; N, 7.43; S, 8.52 %; 1H NMR (400
30
MHz, DMSO-d6, ppm) δ 9.50 (bs, 1H), 7.81 (bs, 1H), 7.51 – 7.46 (m, 2H), 7.36 – 7.31 (m,
2H), 5.90 (ddt, J = 17.2, 10.5, 5.4 Hz, 1H), 5.15 (ddq, J = 28.6, 10.3, 1.6 Hz, 2H), 4.74 (t, J =
1.9 Hz, 2H), 4.33 (t, J = 1.9 Hz, 2H), 4.15 (dd, J = 7.3, 3.0 Hz, 2H), 4.03 (s, 5H); 13
C NMR
(101 MHz, DMSO-d6, ppm) δ 180.2, 136.8, 134.8, 134.7, 125.9(2C), 123.1(2C), 115.6, 84.6,
69.2(5C), 68.6(2C), 66.0(2C), 46.0; FTIR (KBr, υ cm-1
): 3246, 3081, 3074, 2947, 2917,
2846, 1616, 1555, 1527, 1456, 1407, 1346, 1317, 1103, 1081, 1025, 1000, 922, 889, 831,
821, 684, 622, 528, 512, 503, 482, 433, 412.
2.6.05 1-(4-(ferrocenyl)phenyl)-3-phenylthiourea (At5)
Quantities used were 2.77 g (10 mmol) 4-ferrocenylaniline (A) and 1.19 ml (10 mmol)
phenylisothiocynate. Yield 3.03 g (73.5%), Elemental analysis calculated For C23H20FeN2S-
MeOH; C, 64.87; H, 5.44; N, 6.30; S, 7.22 found C, 64.85; H, 5.49; N, 6.32; S, 7.28 %; 1H
NMR (400 MHz, DMSO-d6, ppm) δ 9.76 (bs, 1H), 7.53 – 7.29 (m, 8H), 7.13 (d, J = 7.4 Hz,
2H), 4.75 (t, J = 1.9 Hz, 2H), 4.33 (t, J = 1.9 Hz, 2H), 4.03 (s, 5H); 13
C NMR (101 MHz,
DMSO-d6, ppm) δ 179.2, 139.4, 137.1, 134.9, 128.3(2C), 125.7(2C), 124.3(2C), 123.5(2C),
123.4, 84.6, 69.2(5C), 68.6(2C), 66.0(2C); FTIR (KBr, υ cm-1
): 3191, 3087, 3037, 3006,
2998, 1598, 1540, 1492, 1448, 1419, 1328, 1103, 813, 765, 701, 653, 549, 505, 476, 441.
2.6.06 1-(4-(ferrocenyl)phenyl)-3-(4-nitrophenyl)thiourea (At6)
Quantities used were 2.77 g (10 mmol) 4-ferrocenylaniline (A) and 1.81 g (10 mmol) 4-
nitrophenyllisothiocynate. Yield 3.58 g (78.5%), Elemental analysis calculated For
C23H19FeN3O2S-EtOAc; C, 59.46; H, 4.99; N, 7.70; S, 5.88 found C, 59.51; H, 4.92; N, 7.69;
S, 5.83 %; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 10.36 (bs, 1H), 10.25 (bs, 1H), 8.20 (d, J
= 9.3 Hz, 2H), 7.82 (d, J = 9.3 Hz, 2H), 7.52 (d, J = 8.7 Hz, 2H), 7.42 (d, J = 8.6 Hz, 2H),
4.76 (t, J = 1.9 Hz, 2H), 4.34 (t, J = 1.9 Hz, 2H), 4.02 (s, 5H); 13
C NMR (101 MHz, DMSO-
d6, ppm) δ 179.3, 146.7, 142.6, 137.1, 136.2, 126.4(2C), 124.8(2C), 123.9(2C), 121.9(2C),
84.9, 69.8(5C), 69.3(2C), 66.6(2C); FTIR (KBr, υ cm-1
): 3344, 3224, 3091, 3023, 3012,
1594, 1567, 1521, 1456, 1411, 1385, 1340, 1333, 1303, 1282, 1255, 1201, 1176, 1105, 1081,
1054, 1029, 1014, 1000, 944, 929, 887, 850, 837, 749, 723, 682, 528, 509, 495, 484, 458,
440, 412.
31
2.6.07 1-(4-(ferrocenyl)phenyl)-3-(2,4-dichlorophenyl)thiourea (At7)
Quantities used were 2.77 g (10 mmol) 4-ferrocenylaniline (A) and 2.04 g (10 mmol) 2,4-
dichlorophenylisothiocynate. Yield 3.95 g (82.0%), Elemental analysis calculated For
C23H18Cl2FeN2S-EtOAc; C, 56.96; H, 4.60; N, 4.92; S, 5.63 found C, 56.87; H, 4.67; N, 4.85;
S, 5.66 %; 1
H NMR (400 MHz, DMSO-d6, ppm) δ 10.05 (bs, 1H), 9.40 (bs, 1H), 7.93 – 7.22
(m, 7H), 4.76 (t, J = 1.8 Hz, 2H), 4.34 (t, J = 1.8 Hz, 2H), 4.03 (s, 5H); 13
C NMR (101 MHz,
DMSO-d6, ppm) δ 180.0, 137.2, 137.1, 133.0, 127.6, 126.2(2C), 125.3, 124.7, 122.9(2C),
121.9, 121.4, 74.9, 72.6(5C), 69.3(2C), 63.2(2C); FTIR (KBr, υ cm-1
): 3450, 3158, 2954,
2923, 2852, 1624, 1594, 1558, 1506, 1456, 1398, 1385, 1307, 1263, 1230, 1182, 1101, 1070,
1012, 833, 725, 628, 609, 584, 569, 543, 520, 505, 476, 447, 433, 412, 403.
2.6.08 1-(4-(ferrocenyl)-3-methylphenyl)-3-methylthiourea (Bt1)
Quantities used were 2.91 g (10 mmol) 3-methyl-4-ferrocenylaniline (B) and 0.73 g (10
mmol) methylisothiocynate. Yield 2.31 g (63.5%), Elemental analysis calculated For
C19H20FeN2S C; 62.64; H, 5.53; N, 7.69; S, 8.80 found C; 62.62; H, 5.54; N, 7.69; S, 8.82 %;
1H NMR (400 MHz, DMSO-d
6, ppm) δ 9.67 (bs, 1H), 7.51 (bs, 1H), 7.39 – 7.46 (m, 2H),
7.12 (d, J = 7.3 Hz, 1H), 4.51 (t, J = 1.7 Hz, 2H), 4.32 (t, J = 1.7 Hz, 2H), 4.12 (s, 5H), 2.69
(s, 3H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 179.5, 136.8, 135.2, 132.8, 130.3, 124.7,
120.3, 86.4, 69.1(5C), 69.0(2C), 67.7(2C), 21.4, 20.9; FTIR (KBr, υ cm-1
): 3415, 3118, 3095,
3072, 2915, 2850, 1587, 1583, 1513, 1436, 1409, 1380, 1336, 1305, 1270, 1249, 1137, 1122,
1105, 1054, 1049, 1039, 1008, 998, 885, 873, 863, 819, 760, 745, 715, 663, 592, 560, 532,
509, 487, 478, 468.
2.6.09 1-(4-(ferrocenyl)-3-methylphenyl)-3-ethylthiourea (Bt2)
Quantities used were 2.91 g (10 mmol) 3-methyl-4-ferrocenylaniline (B) and 0.874 ml (10
mmol) ethylisothiocynate. Yield 2.53 g (67.0%), Elemental analysis calculated For
C20H22FeN2S; C, 63.50; H, 5.86; N, 7.40; S, 8.48 found C, 63.52; H, 5.85; N, 7.41; S, 8.45 %;
1H NMR (400 MHz, DMSO-d
6, ppm) δ 9.71 (bs, 1H), 7.50 (bs, 1H), 7.22 – 7.37 (m, 2H),
7.11 (d, J = 7.4 Hz, 1H), 4.52 (t, J = 1.8 Hz, 2H), 4.32 (t, J = 1.8 Hz, 2H), 4.13 (s, 5H), 3.49
32
(q, J = 6.9 Hz, 2H), 2.33 (s, 3H), 1.20 (t, J = 6.9 Hz, 3H); 13
C NMR (101 MHz, DMSO-d6,
ppm) δ 179.9, 136.8, 135.2, 132.8, 130.3, 124.7, 120.3, 86.4, 69.2(5C), 69.1(2C), 67.7(2C),
39.3, 20.9, 15.3; FTIR (KBr, υ cm-1
): 3442, 3360, 3209, 3089, 2927, 2852, 1560, 1525, 1452,
1403, 1385, 1361, 1346, 1313, 1280, 1259, 1245, 1205, 1182, 1147, 1105, 1081, 1051, 1029,
1016, 1000, 981, 889, 819, 777, 721, 646, 628, 613, 540, 513, 499, 440, 418, 412.
2.6.10 1-(4-(ferrocenyl)-3-methylphenyl)-3-propylthiourea (Bt3)
Quantities used were 2.91 g (10 mmol) 3-methyl-4-ferrocenylaniline (B) and 1.03 ml (10
mmol) propylisothiocynate. Yield 2.76 g (70.5%), Elemental analysis calculated For
C21H24FeN2S; C, 64.29; H, 6.17; N, 7.14; S, 8.17 found C, 64.25; H, 6.15; N, 7.18; S, 8.16 %;
1H NMR (400 MHz, DMSO-d
6, ppm) δ 9.35 (bs, 1H), 7.70 (bs, 1H), 7.61 (d, J = 8.3 Hz,
1H), 7.21 (dd, J = 8.4, 2.2 Hz, 1H), 7.13 (d, J = 2.1 Hz, 1H), 4.52 (t, J = 1.8 Hz, 2H), 4.32 (t,
J = 1.8 Hz, 2H), 4.13 (s, 5H), 3.42 (dd, J = 12.6, 6.2 Hz, 2H), 2.32 (s, 3H), 1.61 – 1.50 (m,
2H), 0.89 (t, J = 7.4 Hz, 3H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 180.0, 136.8, 135.2,
132.8, 130.3, 124.7, 120.3, 86.4, 69.1(5C), 69.0(2C), 67.7(2C), 45.5, 40.1, 21.7, 11.2; FTIR
(KBr, υ cm-1
): 3483, 3379, 3243, 3066, 2959, 2926, 2868, 1593, 1545, 1515, 1455, 1417,
1378, 1336, 1307, 1256, 1131, 1104, 1071, 1025, 1002, 956, 881, 816, 739, 689, 616, 518,
501, 486.
2.6.11 1-(4-(ferrocenyl)-3-methylphenyl)-3-allylthiourea (Bt4)
Quantities used were 2.91 g (10 mmol) 3-methyl-4-ferrocenylaniline (B) and 0.98 ml (10
mmol) allylisothiocynate. Yield 2.36 g (60.5%), Elemental analysis calculated For
C21H22FeN2S; C, 64.62; H, 5.68; N, 7.18; S, 8.21 found C, 64.57; H, 5.69; N, 7.17; S, 8.23 %;
1H NMR (400 MHz, DMSO-d
6, ppm) δ 9.46 (bs, 1H), 7.79 (bs, 1H), 7.62 (d, J = 8.4 Hz,
1H), 7.23 (dd, J = 8.3, 2.2 Hz, 1H), 7.14 (d, J = 2.1 Hz, 1H), 5.90 (ddt, J = 17.2, 10.5, 5.4 Hz,
1H), 5.15 (ddq, J = 27.7, 10.3, 1.5 Hz, 2H), 4.52 (t, J = 1.8 Hz, 2H), 4.33 (t, J = 1.8 Hz, 2H),
4.14 – 4.17 (m, 2H), 4.13 (s, 5H), 2.33 (s, 3H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ
180.2, 136.7, 135.3, 134.8, 133.1, 130.3, 124.9, 120.6, 115.5, 86.4, 69.1(5C), 69.0(2C),
67.7(2C), 46.1, 20.9; FTIR (KBr, υ cm-1
): 3232, 3101, 3058, 2969, 2923, 2856, 1641, 1591,
1556, 1544, 1513, 1456, 1411, 1384, 1338, 1305, 1270, 1220, 1199, 1168, 1128, 1105, 1070,
1024, 1001, 956, 910, 879, 866, 837, 831, 815, 750, 738, 694, 613, 521, 501, 486, 476, 451,
412.
33
2.6.12 1-(4-(ferrocenyl)-3-methylphenyl)-3-phenylthiourea (Bt5)
Quantities used were 2.91 g (10 mmol) 3-methyl-4-ferrocenylaniline (B) and 1.19 ml (10
mmol) phenylisothiocynate. Yield 3.13 g (73.5%), Elemental analysis calculated For
C24H22FeN2S-Acetone; C, 66.94; H, 5.83; N, 5.78; S, 6.62 found C, 66.91; H, 5.82; N, 5.72;
S, 6.52 %; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 9.72 (bs, 1H), 7.66 – 7.07 (m, 9H), 4.53
(bs, 2H), 4.33 (bs, 2H), 4.13 (s, 5H), 2.33 (s, 3H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ
179.4, 139.3, 138.2, 133.4, 128.4, 128.3, 124.3, 123.6, 122.5(2C), 120.1(2C), 115.1, 86.2,
69.2(5C), 69.1(2C), 67.8(2C), 20.9; FTIR (KBr, υ cm-1
): 3430, 3097, 3039, 2975, 2958,
2919, 2829, 1606, 1581, 1510, 1436, 1407, 1346, 1307, 127, 1130, 1105, 1068, 1029, 1002,
927, 887, 875, 829, 802, 750, 715, 590, 555, 532, 512, 498, 484, 472, 455, 437, 428, 404.
2.6.13 1-(4-(ferrocenyl)-3-methylphenyl)-3-(4-nitrophenyl)thiourea (Bt6)
Quantities used were 2.91 g (10 mmol) 3-methyl-4-ferrocenylaniline (B) and 1.81 g (10
mmol) 4-nitrophenyllisothiocynate. Yield 3.93 g (83.5%), Elemental analysis calculated For
C24H21FeN3O2S-Acetone; C, 61.25; H, 5.14; N, 7.94; S, 6.06 found C, 61.16; H, 4.19; N,
7.91; S, 6.20 %; 1
H NMR (400 MHz, DMSO-d6, ppm) δ 10.33 (bs, 1H), 10.21 (bs, 1H), 8.20
(d, J = 9.2 Hz, 2H), 7.84 (d, J = 9.2 Hz, 2H), 7.66 (d, J = 8.4 Hz, 1H), 7.31 (dd, J = 8.3, 2.2
Hz, 1H), 7.23 (d, J = 2.1 Hz, 1H), 4.55 (t, J = 1.8 Hz, 2H), 4.34 (t, J = 1.8 Hz, 2H), 4.13 (s,
5H), 2.34 (s, 3H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 178.9, 146.3, 142.1, 136.5, 135.3,
133.9, 130.3, 125.1, 124.2(2C), 121.5(2C), 120.8, 86.2, 70.0(5C), 69.1(2C), 67.8(2C), 21.0;
FTIR (KBr, υ cm-1
): 3344, 3220, 3189, 3141, 3077, 3002, 1614, 1600, 1581, 1561, 1519,
1450, 1437, 1412, 1379, 1346, 1332, 1301, 1255, 1240, 1211, 1176, 1105, 1072, 1029, 1002,
970, 956, 893, 873, 852, 823, 809, 748, 723, 711, 678, 661, 590, 576, 555, 546, 518, 505,
487, 451, 418.
2.6.14 1-(4-(ferrocenyl)-3-methylphenyl)-3-(2,4-dichlorophenyl)thiourea (Bt7)
Quantities used were 2.91 g (10 mmol) 3-methyl-4-ferrocenylaniline (B) and 2.04 g (10
mmol) 2,4-dichlorophenylisothiocynate. Yield 3.91 g (79%), Elemental analysis calculated
For C24H20Cl2FeN2S-Acetone; C, 58.61; H, 4.74; N, 5.06; S, 5.79 found C, 58.52; H, 4.71; N,
34
5.06; S, 5.70 %; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 10.00 (bs, 1H), 9.40 (bs, 1H), 7.70 –
7.59 (m, 3H), 7.42 (dd, J = 8.6, 2.4 Hz, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.24 (d, J = 2.3 Hz,
1H), 4.54 (t, J = 1.8 Hz, 2H), 4.34 (t, J = 1.8 Hz, 2H), 4.13 (s, 5H), 2.34 (s, 3H); 13
C NMR
(101 MHz, DMSO-d6, ppm) δ 179.9, 136.5, 135.8, 135.2, 133.7, 131.1, 130.9, 130.7, 130.3,
128.7, 127.2, 125.4, 121.0, 86.2, 69.2(5C), 69.1(2C), 67.8(2C), 20.9; FTIR (KBr, υ cm-1
):
3220, 3097, 3074, 3010, 2925, 2850, 1581, 1531, 1473, 1454, 1394, 1390, 1332, 1300, 1245,
1207, 1144, 1105, 1097, 1074, 1057, 1028, 1005, 968, 941, 885, 864, 848, 812, 776, 756,
725, 719, 666, 611, 588, 577, 520, 501, 484, 436, 417.
2.6.15 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-methylthiourea (Ct1)
Quantities used were 3.07 g (10 mmol) 3-methoxy-4-ferrocenylaniline (C) and 0.73 g (10
mmol) methylisothiocynate. Yield 2.45 g (64.5%), Elemental analysis calculated For
C19H20FeN2OS; C, 60.01; H, 5.30; N, 7.37; S, 8.43 found C, 60.05; H, 5.29; N, 7.35; S, 8.46
%; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 9.45 (bs, 1H), 7.64 (bs, 1H), 7.00 – 7.15 (m, 2H),
6.91 (d, J = 7.4 Hz, 1H), 4.74 (t, J = 1.7 Hz, 2H), 4.26 (t, J = 1.7 Hz, 2H), 4.03 (s, 5H), 3.82
(s, 3H), 2.64 (s, 3H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 180.7, 156.1, 137.9, 128.6,
121.9, 114.2, 110.5, 82.2, 69.0(5C), 68.2(2C), 67.7(2C), 55.3. 21.7; FTIR (KBr, υ cm-1
):
3432, 3077, 2960, 2917, 1525, 1496, 1344, 1305, 1287, 1267, 1205, 1157, 1105, 1066, 1033,
1016, 999, 910, 892, 854, 825, 806, 742, 723, 674, 534, 491, 478, 462, 437, 428, 404.
2.6.16 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-ethylthiourea (Ct2)
Quantities used were 3.07 g (10 mmol) 3-methoxy-4-ferrocenylaniline (C) and 0.874 ml (10
mmol) ethylisothiocynate. Yield 2.42 g (61.5%), Elemental analysis calculated For
C20H22FeN2OS; C, 60.92; H, 5.62; N, 7.10; S, 8.13 found C, 60.90; H, 5.65; N, 7.13; S, 8.10
%; 1
H NMR (400 MHz, DMSO-d6, ppm) δ 9.45 (bs, 1H), 7.77 (bs, 1H), 7.46 (d, J = 8.3 Hz,
1H), 7.15 (s, 1H), 6.87 (d, J = 8.2 Hz, 1H), 4.72 (t, J = 1.8 Hz, 2H), 4.27 (t, J = 1.8 Hz, 2H),
4.03 (s, 5H), 3.83 (s, 3H), 3.50 (m, 2H), 1.13 (t, J = 7.1 Hz, 3H); 13
C NMR (101 MHz,
DMSO-d6, ppm) δ 180.1, 156.1, 138.0, 128.6, 121.9, 114.2, 109.4, 82.3, 69.0(5C), 68.1(2C),
67.8(2C), 55.3. 38.5, 14.1; FTIR (KBr, υ cm-1
): 3421, 3377, 3329, 3139, 2980, 2932, 2870,
2834, 1603, 1576, 1530, 1453, 1415, 1382, 1332, 1313, 1269, 1236, 1169, 1146, 1131, 1104,
1058, 1035, 1004, 960, 933, 881, 852, 824, 747, 687, 645, 605, 564, 488.
35
2.6.17 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-propylthiourea (Ct3)
Quantities used were 3.07 g (10 mmol) 3-methoxy-4-ferrocenylaniline (C) and 1.03 ml (10
mmol) propylisothiocynate. Yield 2.59 g (63.5%), Elemental analysis calculated For
C21H24FeN2OS; C, 61.77; H, 5.92; N, 6.86; S, 7.85 found C, 61.74; H, 5.94; N, 6.90; S, 7.81
%; 1
H NMR (400 MHz, DMSO-d6, ppm) δ 9.44 (bs, 1H), 7.77 (bs, 1H), 7.46 (d, J = 8.3 Hz,
1H), 7.18 (d, J = 2.1 Hz, 1H), 6.85 – 6.93 (m, 1H), 4.72 (t, J = 1.9 Hz, 2H), 4.27 (t, J = 1.9
Hz, 2H), 4.03 (s, 5H), 3.83 (s, 3H), 3.44 (dd, J = 12.9, 6.3 Hz, 2H), 1.62 – 1.51 (m, 2H), 0.90
(t, J = 7.4 Hz, 3H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 179.7, 156.1, 138.0, 128.7,
122.1, 114.3, 106.2, 82.3, 69.0(5C), 68.1(2C), 67.8(2C), 55.2, 45.5, 21.7, 11.3; FTIR (KBr, υ
cm-1
): 3433, 3392, 3164, 3026, 2962, 2936, 2874, 2828, 1576, 1530, 1457, 1440, 1417, 1384,
1315, 1256, 1223, 1171, 1125, 1077, 1033, 1002, 966, 881, 860, 818, 751, 662, 633, 614,
489.
2.6.18 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-allylthiourea (Ct4)
Quantities used were 3.07 g (10 mmol) 3-methoxy-4-ferrocenylaniline (C) and 0.98 ml (10
mmol) allylisothiocynate. Yield 2.92 g (72.0%), Elemental analysis calculated For
C21H22FeN2OS; C, 62.08; H, 5.46; N, 6.89; S, 7.89 found C, 62.10; H, 5.43; N, 6.90; S, 7.85
%; 1
H NMR (400 MHz, DMSO-d6, ppm) δ 9.55 (bs, 1H), 7.86 (bs, 1H), 7.47 (d, J = 8.3 Hz,
1H), 7.20 (d, J = 2.0 Hz, 1H), 6.91 (dd, J = 8.3, 2.0 Hz, 1H), 5.91 (ddt, J = 17.2, 10.5, 5.4 Hz,
1H), 5.16 (ddq, J = 29.7, 10.3, 1.6 Hz, 2H), 4.72 (t, J = 1.9 Hz, 2H), 4.27 (t, J = 1.9 Hz, 2H),
4.16 (t, J = 5.3 Hz, 2H), 4.03 (s, 5H), 3.83 (s, 3H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ
180.5, 156.6, 138.5, 135.3, 129.2, 122.9, 116.1, 115.2, 106.9, 82.7, 69.5(5C), 68.7(2C),
68.3(2C), 55.8, 46.6; FTIR (KBr, υ cm-1
): 3387, 3166, 3081, 3028, 2974, 2959, 2936, 2868,
2830, 1603, 1576, 1520, 1440, 1415, 1384, 1302, 1256, 1219, 1169, 1125, 1077, 1035, 1000,
969, 933, 881, 862, 820, 751, 662, 633, 608, 562, 489.
2.6.19 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-phenylthiourea (Ct5)
36
Quantities used were 3.07 g (10 mmol) 3-methoxy-4-ferrocenylaniline (C) and 1.19 ml (10
mmol) phenylisothiocynate. Yield 3.09 g (70.0%), Elemental analysis calculated For
C24H22FeN2OS-Acetone; C, 64.80; H, 5.64; N, 5.60; S, 6.41 found C, 64.86; H, 5.61; N, 5.63;
S, 6.35 %; 1
H NMR (400 MHz, DMSO-d6, ppm) δ 9.79 (bs, 1H), 9.77 (bs, 1H), 7.01 – 7.51
(m, 8H), 4.73 (t, J = 1.9 Hz, 2H), 4.28 (t, J = 1.9 Hz, 2H), 4.03 (s, 5H), 3.83 (s, 3H); 13
C
NMR (101 MHz, DMSO-d6, ppm) δ 179.0, 155.9, 139.3, 138.2, 128.5, 128.3(2C), 124.3,
123.6(2C), 122.5, 115.1, 106.7, 82.2, 69.0(5C), 68.2(2C), 67.8(2C), 55.3; FTIR (KBr, υ cm-
1): 3354, 3266, 3093, 3058, 3012, 2960, 2932, 2903, 2857, 2832, 1595, 1547, 1517, 1497,
1449, 1411, 1359, 1300, 1279, 1256, 1225, 1198, 1129, 1077, 1031, 1002, 956, 927, 883,
848, 820, 754, 739, 691, 662, 635, 612, 578, 532, 505, 489, 463, 424.
2.6.20 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-(4-nitrophenyl)thiourea (Ct6)
Quantities used were 3.07 g (10 mmol) 3-methoxy-4-ferrocenylaniline (C) and 1.81 g (10
mmol) 4-nitrophenyllisothiocynate. Yield 4.11 g (84.5%), Elemental analysis calculated For
C24H21FeN3O3S-Acetone; C, 59.46; H, 4.99; N, 7.70; S, 5.88 found C, 59.45; H, 4.94; N,
7.62; S, 5.88 %; 1
H NMR (400 MHz, DMSO-d6, ppm) δ 10.36 (bs, 1H), 10.29 (bs, 1H), 8.16
– 8.24 (m, 2H), 7.80 – 7.86 (m, 2H), 7.51 (d, J = 8.3 Hz, 1H), 7.26 (d, J = 1.9 Hz, 1H), 7.04
(dd, J = 8.3, 2.0 Hz, 1H), 4.75 (t, J = 1.9 Hz, 2H), 4.29 (t, J = 1.9 Hz, 2H), 4.03 (s, 5H), 3.84
(s, 3H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 178.6, 156.0, 146.1, 142.2, 137.7, 128.6,
124.3(2C), 123.3, 121.5(2C), 115.1, 106.7, 82.0, 69.0(5C), 68.3(2C), 67.9(2C), 55.3; FTIR
(KBr, υ cm-1
): 3446, 3339, 3245, 3204, 3156, 3101, 3014, 2962, 2937, 2870, 2834, 1595,
1574, 1536, 1515, 1453, 1415, 1334, 1302, 1259, 1217, 1177, 1133, 1104, 1077, 1035, 1000,
893, 845, 814, 747, 695, 670, 645, 562, 488, 415.
2.6.21 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-(2,4-dichlorophenyl)thiourea (Ct7)
Quantities used were 3.07 g (10 mmol) 3-methoxy-4-ferrocenylaniline (C) and 2.04 g (10
mmol) 2,4-dichlorophenylisothiocynate. Yield 4.06 g (79.5%), Elemental analysis calculated
For C24H20Cl2FeN2OS-Acetone; C, 56.96; H, 4.60; N, 4.92; S, 5.63 found C, 56.98; H, 4.64;
N, 4.98; S, 5.67 %; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 10.10 (bs, 1H), 9.43 (bs, 1H),
7.68 (d, J = 2.4 Hz, 1H), 7.62 (d, J = 8.6 Hz, 1H), 7.51 (d, J = 8.3 Hz, 1H), 7.43 (dd, J = 8.6,
2.4 Hz, 1H), 7.27 (d, J = 2.0 Hz, 1H), 7.03 (dd, J = 8.3, 2.0 Hz, 1H), 4.75 (t, J = 1.9 Hz, 2H),
37
4.28 (t, J = 1.9 Hz, 2H), 4.03 (s, 5H), 3.85 (s, 3H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ
180.1, 138.2, 136.4, 134.2, 133.0, 131.5, 130.2, 128.7, 125.3, 124.0, 123.7, 122.5, 121.3,
82.2, 69.0(5C), 68.1(2C), 67.9(2C), 55.3; FTIR (KBr, υ cm-1
): 3421, 3335, 3231, 3176, 3097,
3014, 2955, 2934, 2855, 1605, 1576, 1528, 1465, 1419, 1388, 1342, 1296, 1265, 1223, 1167,
1144, 1104, 1079, 1056, 1039, 1004, 935, 893, 806, 735, 670, 608, 566, 493.
2.6.22 1-(3-chloro-4-(ferrocenyl)phenyl)-3-methylthiourea (Dt1)
Quantities used were 3.12 g (10 mmol) 3-Chloro-4-ferrocenylaniline (D) and 0.73 g (10
mmol) methylisothiocynate. Yield 2.74 g (71.2%), Elemental analysis calculated For
C18H17ClFeN2S; C, 56.20; H, 4.45; N, 7.28; S, 8. found C, 56.24; H, 4.47; N, 7.25; S, 8.38 %;
1H NMR (400 MHz, DMSO-d
6, ppm) δ 9.83 (bs, 1H), 7.59 (bs, 1H), 7.41 (s, 1H), 7.28 (d, J
= 7.4 Hz, 1H), 6.89 (d, J = 7.4 Hz, 1H), 4.75 (t, J = 1.9 Hz, 2H), 4.37 (t, J = 1.9 Hz, 2H), 4.14
(s, 5H), 2.71 (s, 3H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 180.2, 138.4, 133.0, 130.3,
128.4, 124.4, 123.4, 83.2, 69.4(5C), 69.2(2C), 67.9(2C), 23.1; FTIR (KBr, υ cm-1
): 3432,
3093, 2956, 2925, 2871, 1612, 1585, 1529, 1508, 1458, 1415, 1313, 1234, 1209, 1182, 1151,
1012, 835, 553, 470, 435.
2.6.23 1-(3-chloro-4-(ferrocenyl)phenyl)-3-ethylthiourea (Dt2)
Quantities used were 3.12 g (10 mmol) 3-Chloro-4-ferrocenylaniline (D) and 0.874 ml (10
mmol) ethylisothiocynate. Yield 2.81 g (70.5%), Elemental analysis calculated For
C19H19ClFeN2S; C, 57.23; H, 4.80; N, 7.03; S, 8.04 found C, 57.25; H, 4.81; N, 7.05; S, 8.02
%; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 9.93 (bs, 1H), 7.85 (bs, 1H), 7.45 (s, 1H), 7.24 (d,
J = 7.4 Hz, 1H), 6.84 (d, J = 7.4 Hz, 1H), 4.73 (t, J = 1.8 Hz, 2H), 4.37 (t, J = 1.8 Hz, 2H),
4.14 (s, 5H), 3.49 (q, J = 8.1 Hz, 2H), 1.20 (t, J = 8.1 Hz, 3H); 13
C NMR (101 MHz, DMSO-
d6, ppm) δ 179.7, 137.9, 133.6, 130.3, 128.4, 124.4, 123.5, 83.2, 69.4(5C), 69.2(2C),
68.3(2C), 38.6, 14.3; FTIR (KBr, υ cm-1
): 3411, 3338, 3087, 2944, 2917, 2850, 1621, 1558,
1539, 1516, 1454, 1402, 1382, 1309, 1253, 1197, 1105, 925, 910, 881, 836, 814, 738, 709,
682, 651, 611, 578, 555, 526, 513, 497, 485, 470, 457, 443, 428, 412.
2.6.24 1-(3-chloro-4-(ferrocenyl)phenyl)-3-propylthiourea (D t3)
38
Quantities used were 3.12 g (10 mmol) 3-Chloro-4-ferrocenylaniline (D) and 1.03 ml (10
mmol) propylisothiocynate. Yield 2.62 g (63.5%), Elemental analysis calculated For
C20H21ClFeN2S; C, 58.20; H, 5.13; N, 6.79; S, 7.77 found C, 58.21; H, 5.12; N, 6.75; S, 7.77
%; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 10.07 (bs, 1H), 9.50 (bs, 1H), 7.46 (s, 1H), 7.23
(d, J = 7.4 Hz, 1H), 6.93 (d, J = 7.4 Hz, 1H), 4.77 (t, J = 1.9 Hz, 2H), 4.38 (t, J = 1.9 Hz, 2H),
4.15 (s, 5H), 3.43 (t, J = 4.8 Hz, 2H), 1.74 – 1.41 (m, 2H), 0.92 (t, J = 12.4 Hz, 3H); 13
C
NMR (101 MHz, DMSO-d6, ppm) δ 179.9, 139.0, 134.0, 130.3, 128.4, 124.5, 123.6, 83.2,
69.4(5C), 69.2(2C), 68.0(2C), 45.5, 21.7, 11.3; FTIR (KBr, υ cm-1
): 3327, 3295, 3195, 3126,
3091, 3062, 3032, 1597, 1551, 1499, 1442, 1417, 1350, 1313, 1286, 1234, 1104, 1085, 1050,
1035, 998, 939, 887, 841, 816, 785, 751, 695, 668, 641, 528, 495, 443, 420.
2.6.25 1-(3-chloro-4-(ferrocenyl)phenyl)-3-allylthiourea (D t4)
Quantities used were 3.12 g (10 mmol) 3-Chloro-4-ferrocenylaniline (D) and 0.98 ml (10
mmol) allylisothiocynate. Yield 2.73 g (66.5%), Elemental analysis calculated For
C20H19ClFeN2S; C, 58.48; H, 4.66; N, 6.82; S, 7.81 found C, 58.45; H, 4.63; N, 6.85; S, 7.82
%; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 9.97 (bs, 1H), 7.80 (bs, 1H), 7.49 (s, 1H), 7.31 (d,
J = 7.4 Hz, 1H), 6.93 (d, J = 7.4 Hz, 1H), 5.89 (ddt, J = 12.1, 10.2, 6.1 Hz, 1H), 5.21 (dd, J =
13.3, 8.1 Hz, 2H), 4.73 (t, J = 1.9 Hz, 2H), 4.38 (t, J = 1.9 Hz, 2H), 4.15 (s, 5H), 4.12 (d, J =
6.1 Hz, 2H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 180.5, 138.2, 132.9, 130.2, 128.4
124.5, 123.5 121.7, 115.4, 83.2, 69.4(5C), 69.2(2C), 68.3(2C), 46.6; FTIR (KBr, υ cm-1
):
3433, 3357, 3101, 2917, 2839, 1616, 1600, 1581, 1508, 1467, 1438, 1404, 1385, 1307, 1284,
1232, 1166, 1103, 1072, 1047, 1028, 1018, 999, 862, 814, 806, 781, 694, 590, 555, 516, 503,
486, 451, 436, 422.
2.6.26 1-(3-chloro-4-(ferrocenyl)phenyl)-3-phenylthiourea (D t5)
Quantities used were 3.12 g (10 mmol) 3-Chloro-4-ferrocenylaniline (D) and 1.19 ml (10
mmol) phenylisothiocynate. Yield 3.13 g (70.2%), Elemental analysis calculated For
C23H19ClFeN2S-Acetone; C, 61.86; H, 4.99; N, 5.55; S, 6.35 found C, 61.83; H, 4.99; N,
5.57; S, 6.28 %; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 9.92 (bs, 1H), 9.88 (bs, 1H), 7.73 (d,
J = 8.5 Hz, 1H), 7.65 (d, J = 2.2 Hz, 1H), 7.10 – 7.43 (m, 6H), 4.74 (t, J = 1.9 Hz, 2H), 4.38
(t, J = 1.9 Hz, 2H), 4.15 (s, 5H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 179.3, 139.2,
138.4, 132.0, 131.2, 130.2, 128.4, 124.5(2C), 123.9, 123.6(2C), 121.7, 83.3, 69.4(5C),
39
69.2(2C), 68.2(2C); FTIR (KBr, υ cm-1
): 3356, 3220, 3089, 3024, 1595, 1522, 1448, 1401,
1384, 1334, 1311, 1273, 1236, 1188, 1131, 1106, 1050, 1002, 958, 875, 822, 731, 693, 670,
647, 580, 486.
2.6.27 1-(3-chloro-4-(ferrocenyl)phenyl)-3-(4-nitrophenyl)thiourea (D t6)
Quantities used were 3.12 g (10 mmol) 3-Chloro-4-ferrocenylaniline (D) and 1.81 g (10
mmol) 4-nitrophenyllisothiocynate. Yield 4.11 g (83.5%), Elemental analysis calculated For
C23H18ClFeN3O2S-Acetone; C, 56.79; H, 4.40; N, 7.64; S, 5.83 found C, 56.70; H, 4.49; N,
7.65; S, 5.82 %; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 10.49 (bs, 1H), 10.34 (bs, 1H), 7.72
(d, J = 9.2 Hz, 2H), 7.65 (d, J = 2.1 Hz, 1H), 7.30 - 7.43 (m, 4H), 4.75 (t, J = 1.9 Hz, 2H),
4.39 (t, J = 1.9 Hz, 2H), 4.15 (s, 5H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 179.0, 156.1,
146.4, 142.4, 138.6, 133.7, 129.7(2C), 126.3, 124.6, 122.5(2C), 118.3, 113.0, 83.2, 69.5(5C),
69.3(2C), 68.4(2C); FTIR (KBr, υ cm-1
): 3333, 3256, 3208, 3151, 3114, 3085, 1593, 1547,
1496, 1426, 1409, 1328, 1300, 1246, 1179, 1113, 1006, 962, 931, 848, 751, 693, 607, 564,
493.
2.6.28 1-(3-chloro-4-(ferrocenyl)phenyl)-3-(2,4-dichlorophenyl)thiourea (D t7)
Quantities used were 3.12 g (10 mmol) 3-Chloro-4-ferrocenylaniline (D) and 2.04 g (10
mmol) 2,4-dichlorophenylisothiocynate. Yield 4.23 g (82.0%), Elemental analysis calculated
For C23H17Cl3FeN2S-Acetone; C, 54.43; H, 4.04; N, 4.88; S, 5.59 found C, 54.49; H, 4.02; N,
4.83; S, 5.52 %; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 10.13 (bs, 1H), 9.62 (bs, 1H), 7.40 –
7.75 (m, 6H), 4.75 (t, J = 1.9 Hz, 2H), 4.38 (t, J = 1.9 Hz, 2H), 4.15 (s, 5H); 13
C NMR (101
MHz, DMSO-d6, ppm) δ 180.1, 137.9, 135.5, 132.5, 132.5, 131.3, 131.0, 130.9, 130.3, 128.9,
127.3, 124.2, 121.9, 83.2, 69.4(5C), 69.2(2C), 68.3(2C); FTIR (KBr, υ cm-1
): 3335, 3206,
3101, 3078, 3003, 1699, 1578, 1528, 1474, 1448, 1384, 1332, 1300, 1242, 1221, 1144, 1104,
1052, 1029, 1004, 952, 912, 862, 818, 777, 749, 726, 680, 524, 505, 440.
2.6.29 1-(3-(ferrocenyl)phenyl)-3-methylthiourea (E t1)
Quantities used were 2.77 g (10 mmol) 3-ferrocenylaniline (E) and 0.73 g (10 mmol)
methylisothiocynate. Yield 2.04 g (58.2%), Elemental analysis calculated For C18H18FeN2S;
40
C, 61.72; H, 5.18; N, 8.00; S, 9.15 found C, 61.69; H, 5.13; N, 8.07; S, 9.11 %; 1H NMR
(400 MHz, DMSO-d6, ppm) δ 10.10 (bs, 1H), 7.50 (bs, 1H), 7.33 (t, J = 7.4 Hz, 1H), 6.96 –
7.15 (m, 2H), 6.74 (d, J = 7.4 Hz, 1H), 4.50 (t, J = 1.8 Hz, 2H), 4.22 (t, J = 1.8 Hz, 2H), 4.00
(s, 5H), 2.67 (s, 3H); 13
C NMR (101 MHz, DMSO-d6, ppm) 180.1, 139.4, 128.3, 126.7,
124.1, 123.2, 121.2, 84.4, 69.2(5C), 68.5(2C), 66.2(2C), 22.4; FTIR (KBr, υ cm-1
): 3360,
3312, 3156, 3089, 3033, 2928, 1651, 1595, 1540, 1501, 1457, 1415, 1388, 1328, 1275, 1242,
1200, 1177, 1110, 1060, 1031, 1002, 885, 850, 818, 751, 691, 664, 532, 497, 440.
2.6.30 1-(3-(ferrocenyl)phenyl)-3-ethylthiourea (E t2)
Quantities used were 2.77 g (10 mmol) 3-ferrocenylaniline (E) and 0.874 ml (10 mmol)
ethylisothiocynate. Yield 2.37 g (65.2%), Elemental analysis calculated For C19H20FeN2S; C,
62.64; H, 5.53; N, 7.69; S, 8.80 found C, 62.66; H, 5.59; N, 7.61; S, 8.85 %; 1H NMR (400
MHz, DMSO-d6, ppm) δ 10.10 (bs, 1H), 7.50 (bs, 1H), 7.33 (t, J = 7.4 Hz, 1H), 6.95 – 7.15
(m, 2H), 6.74 (d, J = 7.4 Hz, 1H), 4.50 (t, J = 1.7 Hz, 2H), 4.22 (t, J = 1.7 Hz, 2H), 4.00 (s,
5H), 3.49 (q, J = 6.2 Hz, 2H), 1.20 (t, J = 6.1 Hz, 3H); 13
C NMR (101 MHz, DMSO-d6, ppm)
179.5, 138.9, 128.3, 126.9, 124.2, 123.3, 121.6, 84.5, 69.3(5C), 68.3(2C), 66.2(2C), 38.9,
15.1; FTIR (KBr, υ cm-1
): 3383, 3093, 2930, 1580, 1511, 1471, 1448, 1384, 1294, 1229,
1202, 1142, 1098, 1052, 1004, 908, 864, 820, 760, 691, 662, 555, 491.
2.6.31 1-(3-(ferrocenyl)phenyl)-3-propylthiourea (E t3)
Quantities used were 2.77 g (10 mmol) 3-ferrocenylaniline (E) and 1.03 ml (10 mmol)
propylisothiocynate. Yield 2.28 g (60.2%), Elemental analysis calculated For C20H22FeN2S;
C, 63.50; H, 5.86; N, 7.40; S, 8.48 found C, 63.52; H, 5.83; N, 7.42; S, 8.41 %; 1H NMR
(400 MHz, DMSO-d6, ppm) δ 10.10 (bs, 1H), 7.50 (bs, 1H), 7.33 (t, J = 7.4 Hz, 1H), 6.90 –
7.10 (m, 2H), 6.74 (d, J = 7.4 Hz, 1H), 4.50 (t, J = 1.6 Hz, 2H), 4.22 (t, J = 1.6 Hz, 2H), 4.00
(s, 5H), 3.43 (t, J = 6.8 Hz, 2H), 1.41 – 1.74 (m, 2H), 0.96 (t, J = 12.4 Hz, 3H); 13
C NMR
(101 MHz, DMSO-d6, ppm) 179.9, 139.6, 128.3, 126.7, 124.1, 123.2, 121.8, 84.4, 69.3(5C),
68.4(2C), 66.8(2C), 45.5, 21.8, 11.2; FTIR (KBr, υ cm-1
): 3272, 3095, 2999, 2937, 2857,
2838, 1568, 1519, 1453, 1413, 1388, 1311, 1279, 1252, 1227, 1169, 1104, 1033, 966, 927,
881, 850, 818, 745, 703, 622, 489.
41
2.6.32 1-(3-(ferrocenyl)phenyl)-3-allylthiourea (E t4)
Quantities used were 2.77 g (10 mmol) 3-ferrocenylaniline (E) and 0.98 ml (10 mmol)
allylisothiocynate. Yield 2.38 g (63.5%), Elemental analysis calculated For C20H20FeN2S; C,
63.84; H, 5.36; N, 7.44; S, 8.52 found C, 63.85; H, 5.31; N, 7.40; S, 8.56 %; 1H NMR (400
MHz, DMSO-d6, ppm) δ 9.93 (bs, 1H), 9.19 (bs, 1H), 7.48 (t, J = 7.4 Hz, 1H), 6.89 – 7.15
(m, 2H), 6.64 (d, J = 7.4 Hz, 1H), 5.74 (ddt, J = 12.1, 10.2, 6.1 Hz, 2H), 5.12 (dd, J = 13.3,
8.1 Hz, 1H), 4.74 (t, J = 1.8 Hz, 2H), 4.34 (t, J = 1.8 Hz, 2H), 4.15 (d, J = 6.1 Hz, 2H), 4.05
(s, 5H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 180.1, 139.1, 128.3, 126.6, 124.1, 123.4,
122.0, 121.4, 115.5, 84.5, 69.4(5C), 68.4(2C), 66.3(2C), 46.3; FTIR (KBr, υ cm-1
): 3421,
3118, 3085, 3004, 2950, 2917, 2823, 1583, 1515, 1465, 1448, 1394, 1333, 1243, 1102, 1078,
1033, 877, 860, 800, 730, 497, 482, 453, 422.
2.6.33 1-(3-(ferrocenyl)phenyl)-3-phenylthiourea (E t5)
Quantities used were 2.77 g (10 mmol) 3-ferrocenylaniline (E) and 1.19 ml (10 mmol)
phenylisothiocynate. Yield 2.89 g (70.2%), Elemental analysis calculated For C23H20FeN2S-
MeOH; C, 64.87; H, 5.44; N, 6.30; S, 7.22 found C, 64.91; H, 5.41; N, 6.32; S, 7.17 %; 1
H
NMR (400 MHz, DMSO-d6, ppm) δ 9.78 (bs, 1H), 9.77 (bs, 1H), 7.68 (s, 1H), 7.21 – 7.54
(m, 6H), 7.13 (t, J = 7.4 Hz, 1H), 4.71 (t, J = 1.8 Hz, 2H), 4.34 (t, J = 1.8 Hz, 2H), 4.06 (s,
5H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 179.5, 139.3, 129.8, 128.3 128.2(2C), 125.8,
124.3, 123.5(2C), 122.1, 121.4, 121.1, 84.6, 69.3(5C), 68.7(2C), 66.3(2C); FTIR (KBr, υ cm-
1): 3356, 3172, 3103, 3055, 3026, 1593, 1553, 1522, 1496, 1424, 1367, 1313, 1294, 1242,
1183, 1104, 1073, 1019, 998, 954, 925, 902, 837, 812, 779, 749, 714, 691, 645, 626, 576,
505, 484, 453, 416.
2.6.34 1-(3-(ferrocenyl)phenyl)-3-(4-nitrophenyl)thiourea (E t6)
Quantities used were 2.77 g (10 mmol) 3-ferrocenylaniline (E) and 1.81 g (10 mmol) 4-
nitrophenyllisothiocynate. Yield 3.61 g (79%), Elemental analysis calculated For
C23H19FeN3O2S-Acetone; C, 60.59; H, 4.89; N, 8.15; S, 6.22 found C, 60.60; H, 4.90; N,
8.19; S, 6.31 %; 1
H NMR (400 MHz, DMSO-d6, ppm) δ 10.38 (bs, 1H), 10.26 (bs, 1H), 8.21
42
(d, J = 9.1 Hz, 2H), 7.86 (d, J = 9.1 Hz, 2H), 7.68 (s, 1H), 7.19 – 7.36 (m, 3H), 4.72 (bs, 2H),
4.35 (bs, 2H), 4.05 (s, 5H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 179.8, 156.1, 146.7,
142.7, 140.0, 139.4, 129.0, 126.8(2C), 124.8, 122.0(2C), 112.8, 85.0, 69.8(5C), 69.4(2C),
66.9(2C); FTIR (KBr, υ cm-1
): 3378, 3293, 3216, 3135, 3108, 3099, 2996, 2958, 1699, 1598,
1583, 1560, 1504, 1494, 1463, 1425, 1417, 1386, 1365, 1346, 1331, 1305, 1288, 1272, 1257,
1232, 1182, 1110, 1103, 1089, 1076, 1051, 1031, 1020, 999, 953, 894, 848, 833, 811, 804,
794, 729, 696, 686, 654, 638, 611, 555, 526, 503, 485, 460, 443, 410, 403.
2.6.35 1-(3-(ferrocenyl)phenyl)-3-(2,4-dichlorophenyl)thiourea (E t7)
Quantities used were 2.77 g (10 mmol) 3-ferrocenylaniline (E) and 2.04 g (10 mmol) 2,4-
dichlorophenylisothiocynate. Yield 3.90 g (81.0%), Elemental analysis calculated For
C23H18Cl2FeN2S-Acetone; C, 57.90; H, 4.49; N, 5.19; S, 5.95 found C, 57.91; H, 4.44; N,
5.12; S, 5.96 %; 1
H NMR (400 MHz, DMSO-d6, ppm) δ 10.07 (bs, 1H), 9.46 (bs, 1H), 7.62 –
7.73 (m, 3H), 7.43 (dd, J = 8.6, 2.4 Hz, 1H), 7.23 – 7.34 (m, 3H), 4.73 (t, J = 1.7 Hz, 2H),
4.35 (t, J = 1.7 Hz, 2H), 4.06 (s, 4H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 180.2, 139.5,
138.9, 135.7, 131.0, 130.8, 130.7, 128.8, 128.4, 127.2, 122.5, 121.4, 121.3, 84.4, 69.3(5C),
68.8(2C), 66.3(2C); FTIR (KBr, υ cm-1
): 3371, 3155, 3110, 3093, 3052, 3029, 3006, 2993,
2848, 1602, 1585, 1552, 1508, 1490, 1458, 1394, 1380, 1344, 1330, 1298, 1284, 1265, 1236,
1220, 1191, 1099, 1081, 1054, 1034, 1018, 998, 919, 883, 867, 846, 812, 791, 711, 698, 678,
630, 613, 588, 553, 513, 501, 484, 445, 413, 403.
43
2.7 Synthesis of ferrocene based ureas (Au3 – Eu7)
Selected eighteen ferrocenylthioureas (At3 – At6, Bt3, Bt6, Bt7, Ct2, Ct3, Ct5 – Ct7, Dt5 –
Dt7 & Et5 – Et7) were treated with alkaline mercuric chloride in DMF to yield respective
ureas. The reaction conditions are shown in scheme 2.04.
Scheme 2.04; Synthesis of ferrocenylureas (Au3 – Eu7).
2.7.01 1-(4-(ferrocenyl)phenyl)-3-propylurea (Au3)
To the solution of 1.89 g (5 mmol) 1-(4-(ferrocenyl)phenyl)-3-propylthiourea (At3) in 95%
DMF 5% H2O, 1.36 g (5 mmol) HgCl2 was added. The reaction mixture was stirred for an
hour, afterward 1ml of 100 mM NaOH(aq) was added to the mixture and refluxed for 4 hours.
Completion of the reaction was monitored on TLC. On completion of the reaction black
precipitates of HgS were filtered off and the filtrate was then poured into the ice cold water
and stirred well. Solid product separated by filtration, washed with deionized water and
recrystallized from ethyl acetate. 1-(4-(ferrocenyl)phenyl)-3-propylurea (Au3) was obtained
as orange colored crystals. Yield 1.18 g (65.0%), Elemental analysis calculated For
C20H22FeN2O; C, 66.31; H, 6.12; N, 7.73 found C, 66.35; H, 6.15; N, 7.81 %; 1H NMR (400
MHz, DMSO-d6, ppm) δ 9.44 (bs, 1H), 7.96 (bs, 1H), 7.63 (d, J = 8.6 Hz, 2H), 7.32 (d, J =
8.6 Hz, 2H), 4.72 (t, J = 1.9 Hz, 2H), 4.27 (t, J = 1.9 Hz, 2H), 4.03 (s, 5H), 3.44 (dd, J = 12.9,
6.3 Hz, 2H), 1.62 – 1.51 (m, 2H), 0.90 (t, J = 7.4 Hz, 3H); 13
C NMR (101 MHz, DMSO-d6,
ppm) δ 164.7, 138.0, 128.7(2C), 122.1(2C), 114.4, 82.3, 69.0(5C), 68.1(2C), 67.8(2C), 45.5,
21.7, 11.3; FTIR (KBr, υ cm-1
): 3320, 3177, 3095, 3035, 2960, 2930, 2872, 1647, 1601,
1553, 1530, 1455, 1415, 1384, 1315, 1280, 1234, 1184, 1158, 1131, 1104, 1083, 1054, 1031,
1004, 941, 889, 820, 756, 683, 530, 440.
2.7.02 1-(4-(ferrocenyl)phenyl)-3-allylurea (Au4)
Quantities used were 1.88 g (5 mmol) 1-(4-(ferrocenyl)phenyl)-3-allylthiourea (At4), 1.36 g
(5 mmol) HgCl2 and 1ml of 100mM NaOH(aq). Yield 1.43 g (79.5%), Elemental analysis
calculated For C20H20FeN2O; C, 66.68; H, 5.60; N, 7.78 found C, 66.82; H, 5.63; N, 7.73 %;
44
1H NMR (400 MHz, DMSO-d
6, ppm) δ 9.61 (bs, 1H), 7.75 (bs, 1H), 7.35 – 7.58 (m, 4H),
5.96 (ddt, J = 17.2, 10.5, 5.4 Hz, 1H), 5.01 (ddq, J = 28.6, 10.3, 1.6 Hz, 2H), 4.79 (t, J = 1.9
Hz, 2H), 4.36 (t, J = 1.9 Hz, 2H), 4.12 (dd, J = 7.3, 3.0 Hz, 2H), 4.03 (s, 5H); 13
C NMR (101
MHz, DMSO-d6, ppm) δ 165.2, 137.1, 134.9, 134.1, 127.9(2C), 124.1(2C), 117.6, 84.8,
70.2(5C), 68.6(2C), 65.7(2C), 47.1; FTIR (KBr, υ cm-1
): 3383, 3093, 2930, 2851, 1611,
1576, 1544, 1522, 1448, 1411, 1357, 1294, 1234, 1184, 1158, 1131, 1104, 1083, 1054, 1031,
1004, 941, 889, 820, 756, 683, 530, 440.
2.7.03 1-(4-(ferrocenyl)phenyl)-3-phenylurea (Au5)
Quantities used were 2.06 g (5 mmol) 1-(4-(ferrocenyl)phenyl)-3-phenylthiourea (At5), 1.36
g (5 mmol) HgCl2 and 1ml of 100mM NaOH(aq). Yield 1.45 g (73.5%), Elemental analysis
calculated For C23H20FeN2O-MeOH; C, 67.30; H, 5.65; N, 6.54 found C, 67.35; H, 5.69; N,
6.52 %; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 9.78 (bs, 1H), 8.77 (bs, 1H), 7.68 (s, 1H),
7.21 – 7.54 (m, 6H), 7.13 (t, J = 7.4 Hz, 2H), 4.71 (t, J = 1.8 Hz, 2H), 4.34 (t, J = 1.8 Hz,
2H), 4.06 (s, 5H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 164.5, 139.3, 129.8, 128.3(2C),
125.8(2C), 124.3, 123.5(2C), 122.1(2C), 121.4, 84.6, 69.3(5C), 68.7(2C), 66.3(2C); FTIR
(KBr, υ cm-1
): 3356, 3172, 3103, 3055, 3026, 1593, 1553, 1522, 1496, 1424, 1367, 1313,
1294, 1242, 1183, 1104, 1073, 1019, 998, 954, 925, 902, 837, 812, 779, 749, 714, 691, 645,
626, 576, 505, 484, 453, 416.
2.7.04 1-(4-(ferrocenyl)phenyl)-3-(4-nitrophenyl)urea (Au6)
Quantities used were 2.28 g (5 mmol) 1-(4-(ferrocenyl)phenyl)-3-(4-nitrophenyl)thiourea
(At6), 1.36 g (5 mmol) HgCl2 and 1ml of 100mM NaOH(aq). Yield 1.73 g (78.5%), Elemental
analysis calculated For C23H19FeN3O3; C, 62.60; H, 4.34; N, 9.52 found C, 62.51; H, 4.32; N,
9.59 %; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 10.09 (bs, 1H), 9.48 (bs, 1H), 8.19 (d, J =
9.2 Hz, 2H), 7.72 (d, J = 9.3 Hz, 2H), 7.40 – 7.47 (m, 4H), 4.73 (t, J = 1.8 Hz, 2H), 4.31 (t, J
= 1.8 Hz, 2H), 4.02 (s, 5H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 162.2, 152.0, 140.7,
132.7, 126.5(2C), 126.1(2C), 125.1(2C), 118.4(2C), 117.1, 84.9, 69.1(5C), 68.4(2C),
65.7(2C); FTIR (KBr, υ cm-1
): 3360, 3312, 3156, 3089, 3033, 2928, 1651, 1595, 1540, 1501,
1457, 1415, 1388, 1328, 1275, 1242, 1200, 1177, 1110, 1060, 1031, 1002, 885, 850, 818,
751, 691, 664, 532, 497, 440.
45
2.7.05 1-(4-(ferrocenyl)-3-methylphenyl)-3-propylurea (Bu3)
Quantities used were 1.96 g (5 mmol) 1-(4-(ferrocenyl)-3-methylphenyl)-3-propylthiourea
(Bt3), 1.36 g (5 mmol) HgCl2 and 1ml of 100mM NaOH(aq). Yield 1.32 g (70.5%), Elemental
analysis calculated For C21H24FeN2O; C, 67.03; H, 6.43; N, 7.44 found C, 67.25; H, 6.35; N,
7.46 %; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 9.39 (bs, 1H), 8.13 (bs, 1H), 7.61 (d, J = 8.4
Hz, 1H), 7.21 (dd, J = 8.4, 2.2 Hz, 1H), 7.13 (d, J = 2.2 Hz, 1H), 4.74 (t, J = 1.9 Hz, 2H),
4.33 (t, J = 1.9 Hz, 2H), 4.03 (s, 5H), 3.19 (dd, J = 12.0, 5.8 Hz, 2H), 2.32 (s, 3H), 1.49 –
1.63 (m, 2H), 0.89 (t, J = 7.4 Hz, 3H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 166.0, 136.9,
134.5, 130.3, 126.5, 125.9, 122.8, 84.7, 69.2(5C), 68.6(2C), 66.0(2C), 45.5, 40.1, 21.7, 11.3;
FTIR (KBr, υ cm-1
): 3360, 3312, 3156, 3089, 3033, 2926, 2864, 1615, 1559, 1499, 1386,
1323, 1275, 1242, 1200, 1177, 1110, 1060, 1031, 1002, 885, 850, 818, 751, 691, 664, 532,
497, 440.
2.7.06 1-(4-(ferrocenyl)-3-methylphenyl)-3-(4-nitrophenyl)urea (Bu6)
Quantities used were 2.35 g (5 mmol) 1-(4-(ferrocenyl)-3-methylphenyl)-3-(4-
nitrophenyl)thiourea (Bt6), 1.36 g (5 mmol) HgCl2 and 1ml of 100mM NaOH(aq). Yield 1.90
g (83.5%), Elemental analysis calculated For C24H21FeN3O3; C, 63.31; H, 4.65; N, 9.23 found
C, 63.36; H, 4.69; N, 9.29 %; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 10.38 (bs, 1H), 9.26
(bs, 1H), 8.21 (d, J = 9.1 Hz, 2H), 7.86 (d, J = 9.1 Hz, 2H), 7.68 (s, 1H), 7.25 – 7.36 (m, 2H),
4.72 (bs, 2H), 4.35 (bs, 2H), 4.05 (s, 5H); 2.34 (s, 3H); 13
C NMR (101 MHz, DMSO-d6,
ppm) δ 162.6, 142.2, 137.7, 129.1,128.6, 126.0(2C), 124.3, 123.3(2C), 121.5, 115.1, 106.7,
82.0, 69.0(5C), 68.2(2C), 67.9(2C), 21.1; FTIR (KBr, υ cm-1
): 3379, 3093, 3016, 2960, 2926,
2864, 1615, 1559, 1499, 1449, 1407, 1386, 1323, 1261, 1223, 1171, 1142, 1108, 1073, 1025,
1002, 944, 860, 818, 754, 701, 668, 580, 488, 453.
2.7.07 1-(4-(ferrocenyl)-3-methylphenyl)-3-(2,4-dichlorophenyl)urea (Bu7)
Quantities used were 2.48 g (5 mmol) 1-(4-(ferrocenyl)-3-methylphenyl)-3-(2,4-
dichlorophenyl)thiourea (Bu7), 1.36 g (5 mmol) HgCl2 and 1ml of 100mM NaOH(aq). Yield
1.89 g (79.0%), Elemental analysis calculated For C24H20Cl2FeN2O; C, 60.16; H, 4.21; N,
46
5.85 found C, 60.12; H, 4.27; N, 5.56 %; 1H NMR (400 MHz, DMSO-d
6, ppm) , ppm) δ
10.07 (bs, 1H), 8.46 (bs, 1H), 7.62 – 7.73 (m, 2H), 7.43 (dd, J = 8.6, 2.4 Hz, 1H), 7.23 – 7.34
(m, 3H), 4.73 (t, J = 1.7 Hz, 2H), 4.35 (t, J = 1.7 Hz, 2H), 4.06 (s, 5H); 2.34 (s, 3H); 13
C
NMR (101 MHz, DMSO-d6, ppm) δ 164.9, 146.5, 143.8, 139.2, 137.7, 134.1, 132.9, 131.7,
130.3, 129.2, 128.7, 126.8, 125.4, 86.6, 69.7(5C), 69.1(2C), 67.1(2C), 21.2; FTIR (KBr, υ
cm-1
): 3379, 3093, 3016, 2960, 2930, 2872, 1647, 1601, 1553, 1530, 1449, 1407, 1386, 1323,
1261, 1223, 1171, 1142, 1125, 1077, 1033, 1002, 966, 881, 860, 818, 751, 662, 633, 614,
489.
2.7.08 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-ethylurea (Cu2)
Quantities used were 1.97 g (5 mmol) 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-ethylthiourea
(Ct2), 1.36 g (5 mmol) HgCl2 and 1ml of 100mM NaOH(aq). Yield 1.16 g (61.5%), Elemental
analysis calculated For C20H22FeN2O2; C, 63.51; H, 5.86; N, 7.41 found C, 63.51; H, 5.85; N,
7.43 %; 1
H NMR (400 MHz, DMSO-d6, ppm) δ 9.56 (bs, 1H), 7.86 (bs, 1H), 7.56 (d, J = 8.3
Hz, 1H), 7.25 (s, 1H), 6.97 (d, J = 8.2 Hz, 1H), 4.77 (t, J = 1.8 Hz, 2H), 4.32 (t, J = 1.8 Hz,
2H), 4.03 (s, 5H), 3.88 (s, 3H), 3.21 (q, J = 7.1 Hz, 2H), 1.18 (t, J = 7.1 Hz, 3H); 13
C NMR
(101 MHz, DMSO-d6, ppm) δ 166.1, 155.1, 139.2, 132.6, 129.4, 121.9, 110.2, 82.5, 69.3(5C),
68.1(2C), 66.8(2C), 55.3, 38.5, 15.1; FTIR (KBr, υ cm-1
): 3354, 3266, 3093, 3012, 2960,
2903, 2857, 1651, 1595, 1501, 1457, 1388, 1328, 1300, 1279, 1256, 1225, 1198, 1129, 1077,
1031, 1002, 956, 883, 820, 754, 691, 662, 612, 578, 505, 489, 424.
2.7.09 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-propylurea (Cu3)
Quantities used were 2.04 g (5 mmol) 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-propylthiourea
(Ct3), 1.36 g (5 mmol) HgCl2 and 1ml of 100mM NaOH(aq). Yield 1.24 g (63.5%), Elemental
analysis calculated For C21H24FeN2O2; C, 64.30; H, 6.17; N, 7.14 found C, 64.37; H, 6.14; N,
7.19 %; 1
H NMR (400 MHz, DMSO-d6, ppm) δ 9.35 (bs, 1H), 7.96 (bs, 1H), 7.61 (d, J = 8.3
Hz, 1H), 7.21 (dd, J = 8.4, 2.2 Hz, 1H), 7.13 (d, J = 2.1 Hz, 1H), 4.52 (t, J = 1.8 Hz, 2H),
4.32 (t, J = 1.8 Hz, 2H), 4.13 (s, 5H), 3.93 (s, 3H), 3.42 (dd, J = 12.6, 6.2 Hz, 2H), 1.61 –
1.50 (m, 2H), 0.89 (t, J = 7.4 Hz, 3H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 164.0, 156.1,
138.8, 135.2, 132.8, 130.3, 124.7, 86.4, 69.1(5C), 69.0(2C), 67.7(2C), 55.2, 45.5, 21.7, 11.2;
FTIR (KBr, υ cm-1
): 3433, 3392, 3164, 3026, 2962, 2936, 2874, 2828, 1595, 1547, 1517,
47
1497, 1449, 1411, 1359, 1256, 1223, 1171, 1125, 1077, 1033, 1002, 966, 881, 860, 818, 751,
662, 633, 614, 489, 419.
2.7.10 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-phenylurea (Cu5)
Quantities used were 2.21 g (5 mmol) 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-phenylthiourea
(Ct5), 1.36 g (5 mmol) HgCl2 and 1ml of 100mM NaOH(aq). Yield 1.49 g (70.0%), Elemental
analysis calculated For C24H22FeN2O2; C, 67.62; H, 5.20; N, 6.57 found C, 67.86; H, 5.21; N,
6.63 %; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 10.15 (bs, 1H), 9.55 (bs, 1H), 7.03 – 7.91
(m, 8H), 4.78 (t, J = 1.9 Hz, 2H), 4.29 (t, J = 1.9 Hz, 2H), 4.03 (s, 5H), 3.85 (s, 3H); 13
C
NMR (101 MHz, DMSO-d6, ppm) δ 165.3, 139.2, 138.4, 132.0, 131.2, 130.2, 128.4(2C),
124.5, 123.9, 123.6(2C), 121.7, 83.3, 69.4(5C), 69.2(2C), 68.2(2C), 55.4; FTIR (KBr, υ cm-
1): 3446, 3392, 3279, 3197, 3093, 3008, 2934, 2851, 1611, 1576, 1530, 1457, 1440, 1417,
1384, 1315, 1254, 1217, 1198, 1169, 1146, 1106, 1073, 1031, 1002, 946, 883, 810, 749, 662,
612, 582, 545, 493, 428.
2.7.11 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-(4-nitrophenyl)urea (Cu6)
Quantities used were 2.44 g (5 mmol) 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-(4-
nitrophenyl)thiourea (Ct6), 1.36 g (5 mmol) HgCl2 and 1ml of 100mM NaOH(aq). Yield 1.99
g (84.5%), Elemental analysis calculated For C24H21FeN3O4; C, 61.16; H, 4.49; N, 8.92 found
C, 61.14; H, 4.44; N, 8.90 %; 1
H NMR (400 MHz, DMSO-d6, ppm) δ 10.49 (bs, 1H), 9.87
(bs, 1H), 8.22 (d, J = 9.2 Hz, 2H), 7.85 (d, J = 2.1 Hz, 1H), 7.40 - 7.49 (m, 4H), 4.75 (t, J =
1.9 Hz, 2H), 4.39 (t, J = 1.9 Hz, 2H), 4.15 (s, 5H) 3.84 (s, 3H); 13
C NMR (101 MHz, DMSO-
d6, ppm) δ 165.6, 146.3, 142.1, 136.5, 135.3, 133.9, 130.3(2C), 125.1, 124.2(2C), 121.5,
120.8, 86.2, 70.0(5C), 69.2(2C), 67.8(2C), 55.9; FTIR (KBr, υ cm-1
): 3446, 3339, 3245,
3204, 3156, 3101, 3014, 2962, 2937, 2870, 2834, 1576, 1544, 1522, 1448, 1411, 1357, 1300,
1277, 1217, 1177, 1104, 1077, 1035, 1000, 893, 845, 814, 747, 695, 645, 562, 488, 415.
2.7.12 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-(2,4-dichlorophenyl)urea (Cu7)
48
Quantities used were 2.56 g (5 mmol) 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-(2,4-
dichlorophenyl)thiourea (Ct7), 1.36 g (5 mmol) HgCl2 and 1ml of 100mM NaOH(aq). Yield
1.97 g (79.5%), Elemental analysis calculated For C24H20Cl2FeN2O2; C, 58.21; H, 4.07; N,
5.66 found C, 58.22; H, 4.04; N, 5.71 %; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 10.18 (bs,
1H), 9.15 (bs, 1H), 7.24 – 8.12 (m, 6H), 4.79 (t, J = 1.9 Hz, 2H), 4.29 (t, J = 1.9 Hz, 2H),
4.03 (s, 5H), 3.89 (s, 3H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 166.1, 137.9, 135.5,
132.5, 132.5, 131.3, 131.0, 130.9, 130.3, 128.9, 127.3, 124.2, 121.9, 83.2, 69.4(5C),
69.2(2C), 68.3(2C), 55.4; FTIR (KBr, υ cm-1
): 3371, 3272, 3091, 2995, 2932, 2834, 1668,
1607, 1570, 1519, 1449, 1409, 1384, 1307, 1252, 1196, 1171, 1135, 1104, 1077, 1054, 1035,
1017, 973, 946, 883, 862, 814, 752, 718, 699, 660, 612, 562, 489.
2.7.13 1-(3-chloro-4-(ferrocenyl)phenyl)-3-phenylurea (Du5)
Quantities used were 2.23 g (5 mmol) 1-(3-chloro-4-(ferrocenyl)phenyl)-3-phenylthiourea
(Dt5), 1.36 g (5 mmol) HgCl2 and 1ml of 100mM NaOH(aq). Yield 1.51 g (70.2%), Elemental
analysis calculated For C23H19ClFeN2O; C, 64.14; H, 4.45; N, 6.50 found C, 64.18; H, 4.49;
N, 6.57 %; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 9.60 (bs, 1H), 8.43 (bs, 1H), 7.71 (d, J =
5.4 Hz, 2H), 7.37 – 7.53 (m, 5H), 7.03 (dd, J = 8.6, 2.5 Hz, 1H), 4.72 (t, J = 1.9 Hz, 2H), 4.35
(t, J = 1.9 Hz, 2H), 4.14 (s, 5H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 165.0, 155.9,
139.3, 138.2, 128.5(2C), 128.3, 124.3, 123.6(2C), 122.5, 115.1, 106.7, 82.2, 69.0(5C),
68.2(2C), 67.8(2C); FTIR (KBr, υ cm-1
): 3356, 3220, 3089, 3024, 1595, 1522, 1448, 1401,
1384, 1334, 1311, 1273, 1236, 1188, 1131, 1106, 1050, 1002, 958, 875, 822, 731, 693, 670,
647, 580, 486.
2.7.14 1-(3-chloro-4-(ferrocenyl)phenyl)-3-(4-nitrophenyl)urea (Du6)
Quantities used were 2.46 g (5 mmol) 1-(3-chloro-4-(ferrocenyl)phenyl)-3-(4-
nitrophenyl)thiourea (Dt6), 1.36 g (5 mmol) HgCl2 and 1ml of 100mM NaOH(aq). Yield 1.98
g (83.5%), Elemental analysis calculated For C23H18ClFeN3O3; C, 58.07; H, 3.81; N, 8.83
found C, 57.90; H, 3.85; N, 8.76 %; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 10.36 (bs, 1H),
9.13 (bs, 1H), 7.75 – 7.90 (m, 4H), 7.38 - 7.49 (m, 3H), 4.75 (t, J = 1.9 Hz, 2H), 4.29 (t, J =
1.9 Hz, 2H), 4.03 (s, 5H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 165.8, 156.1, 146.7,
142.7, 140.0, 139.4, 130.0(2C), 126.8, 124.8, 122.0(2C), 112.8, 84.9, 69.8(5C), 69.3(2C),
66.8(2C); FTIR (KBr, υ cm-1
): 3320, 3177, 3095, 3035, 2960, 2930, 2872, 1647, 1601, 1553,
49
1530, 1455, 1415, 1384, 1315, 1280, 1223, 1171, 1142, 1108, 1073, 1025, 1002, 944, 860,
818, 754, 701, 668, 580, 488, 453.
2.7.15 1-(3-chloro-4-(ferrocenyl)phenyl)-3-(2,4-dichlorophenyl)urea (Du7)
Quantities used were 2.56 g (5 mmol) -(3-chloro-4-(ferrocenyl)phenyl)-3-(2,4-
dichlorophenyl)thiourea (Dt7), 1.36 g (5 mmol) HgCl2 and 1ml of 100mM NaOH(aq). Yield
2.04 g (82.0%), Elemental analysis calculated For C23H17Cl3FeN2O; C, 55.29; H, 3.43; N,
5.61 found C, 55.49; H, 4.42; N, 5.63 %; 1H NMR (400 MHz, DMSO-d
6, ppm) δ 9.60 (bs,
1H), 8.43 (bs, 1H), 7.47 – 7.92 (m, 5H), 7.03 (dd, J = 8.6, 2.5 Hz, 1H), 4.72 (t, J = 1.9 Hz,
2H), 4.35 (t, J = 1.9 Hz, 2H), 4.14 (s, 5H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 165.1,
138.2, 136.4, 134.2, 133.0, 131.5, 130.2, 128.7, 125.3, 124.0, 123.7, 122.5, 121.3, 82.2,
69.0(5C), 68.1(2C), 67.9(2C); FTIR (KBr, υ cm-1
): 3383, 3093, 2930, 1580, 1511, 1471,
1448, 1384, 1294, 1229, 1202, 1142, 1098, 1052, 1004, 908, 864, 820, 760, 691, 662, 555,
491.
2.7.16 1-(3-(ferrocenyl)phenyl)-3-phenylurea (Eu5)
Quantities used were 2.06 g (5 mmol) 1-(3-(ferrocenyl)phenyl)-3-phenylthiourea (Et5), 1.36
g (5 mmol) HgCl2 and 1ml of 100mM NaOH(aq). Yield 1.39 g (70.2%), Elemental analysis
calculated For C23H20FeN2O; C, 69.71; H, 5.09; N, 7.07; O, 4.04 found C, 69.70; H, 5.01; N,
7.12 %; 1
H NMR (400 MHz, DMSO-d6, ppm) δ 9.76 (bs, 1H), 7.29 – 7.53 (m, 8H), 7.13 (d, J
= 7.4 Hz, 2H), 4.75 (t, J = 1.9 Hz, 2H), 4.33 (t, J = 1.9 Hz, 2H), 4.03 (s, 5H); 13
C NMR (101
MHz, DMSO-d6, ppm) δ 164.3, 139.4, 137.1, 134.9, 128.3, 127.2, 125.7(2C), 124.3, 123.5,
123.4(2C), 121.1, 84.6, 69.2(5C), 68.6(2C), 66.0(2C); FTIR (KBr, υ cm-1
): 3346, 3191,
3087, 3037, 3006, 2998, 1598, 1540, 1492, 1448, 1419, 1328, 1103, 813, 765, 701, 653, 549,
505, 476, 441.
2.7.17 1-(3-(ferrocenyl)phenyl)-3-(4-nitrophenyl)urea (Eu6)
Quantities used were 2.28 g (5 mmol) 1-(3-(ferrocenyl)phenyl)-3-(4-nitrophenyl)thiourea
(Et6), 1.36 g (5 mmol) HgCl2 and 1ml of 100mM NaOH(aq). Yield 1.74 g (79.0%), Elemental
analysis calculated For C23H19FeN3O3; C, 62.60; H, 4.34; N, 9.52 found C, 62.60; H, 4.40; N,
50
9.59 %; 1
H NMR (400 MHz, DMSO-d6, ppm) δ 10.33 (bs, 1H), 8.51 (bs, 1H), 8.20 (d, J =
9.2 Hz, 2H), 7.84 (d, J = 9.2 Hz, 2H), 7.66 (s, 1H), 7.19 – 7.36 (m, 3H), 4.55 (t, J = 1.8 Hz,
2H), 4.34 (t, J = 1.8 Hz, 2H), 4.13 (s, 5H); 13
C NMR (101 MHz, DMSO-d6, ppm) δ 164.7,
146.3, 142.1, 136.5, 135.3, 133.9(2C), 130.3, 125.1(2C), 124.2, 121.5, 120.8, 86.2, 70.0(5C),
69.1(2C), 67.8(2C); FTIR (KBr, υ cm-1
): 3360, 3312, 3156, 3089, 3033, 2928, 1651, 1595,
1540, 1453, 1415, 1382, 1332, 1313, 1269, 1236, 1169, 1146, 1131, 1104, 1033, 966, 927,
881, 850, 818, 745, 703, 622, 489.
2.7.18 1-(3-(ferrocenyl)phenyl)-3-(2,4-dichlorophenyl)urea (Eu7)
Quantities used were 2.40 g (5 mmol) 1-(3-(ferrocenyl)phenyl)-3-(2,4-
dichlorophenyl)thiourea (Eu7), 1.36 g (5 mmol) HgCl2 and 1ml of 100mM NaOH(aq). Yield
1.88 g (81.0%), Elemental analysis calculated For C23H18Cl2FeN2O; C, 59.39; H, 3.90; N,
6.02 found C, 59.91; H, 3.94; N, 6.03 %; 1
H NMR (400 MHz, DMSO-d6, ppm) δ 10.00 (bs,
1H), 8.40 (bs, 1H), 7.42 – 7.70 (m, 5H), 7.32 (dd, J = 8.3, 2.2 Hz, 1H), 7.24 (d, J = 2.1 Hz,
1H), 4.54 (t, J = 1.8 Hz, 2H), 4.34 (t, J = 1.8 Hz, 2H), 4.13 (s, 5H); 13
C NMR (101 MHz,
DMSO-d6, ppm) δ 165.2, 140.5, 138.9, 136.7, 131.6, 130.8, 130.1, 128.7, 128.4, 125.1,
122.5, 121.4, 121.3, 84.4, 69.3(5C), 68.8(2C), 66.3(2C); FTIR (KBr, υ cm-1
): 3320, 3177,
3095, 3035, 2960, 2930, 2872, 1647, 1601, 1559, 1499, 1449, 1407, 1386, 1323, 1261, 1223,
1171, 1142, 1077, 1035, 1000, 969, 933, 881, 862, 820, 751, 662, 633, 608, 562, 489.
2.8 Methods for activity studies
2.8.1 DNA binding Studies
Solutions of DNA in 10 mM phosphate buffer (pH 6.8) gave a ratio of UV absorbance
at 260 and 280 nm, A260 /A280, of 1.8-1.9 indicating the purity of DNA. Concentrated stock
solution of DNA was prepared in 10 mM phosphate buffer (pH 6.8) in water and the
concentration of DNA was determined by UV absorbance at 260 nm after 1:100 dilutions.
The molar absorption coefficient was taken as 6600 M-1
cm-1
. Stock solutions were stored
below 4 °C and were used within 4 days. All DNA binding studies were carried out with the
water: DMSO (1:4) buffered at pH = 6.8 with 10 mM phosphate buffer[134]
.
UV-Visible Spectrometry; Absorption spectra were measured on Perkins Erlenmeyer
lambda-25 UV–Visible spectrometer at constant temperature of 25 ± 1 °C. The electronic
spectrum of a known concentration of the complexes was obtained without DNA. The
spectroscopic response to the same amount of the complex was then monitored by the
addition of small aliquots of DNA solution. All the samples were sonicated to degas and
placed for 30 min to equilibrate before the spectral measurements. The DNA binding constant
51
of the compounds was calculated by UV-visible spectroscopy using the equation (2.1).
Intercept to slope ratio of the plot 1/[DNA] vs Ao/A-Ao gives the value of binding constant
K[135]
.
Ao / (A-Ao) = εG / (εH-G - εG) + εG / (εH-G – εG) . 1/ Kb [DNA] ……… (2.1)
Thermal Denaturation; Melting curves were recorded in media containing 1 mM phosphate
buffer (pH = 6.8) 80% DMSO(aq).[127]
The absorbance at 260 nm was monitored for sonicated
solutions of DNA (100 μM) with a solution of the compound under study for increasing
temperature. Equimolar solution of the test compound was placed in the reference cell during
the experiment. The temperature was increased by 1°C/min on the average between 30 and
98 °C.
2.8.2 Anti-Oxidant Studies (DPPH scavenging assay)
1,1-Diphenyl-2-picryl-hydrazyl (DPPH) and DMSO were purchased from Sigma Aldrich and
were used as such. DPPH solution and test samples were prepared in DMSO and buffered at
pH 6.8 by phosphate buffer. All the samples, in triplicates, were incubated at 37 C for 30-35
minutes and the absorbance at 519 nm was measured by (Perkins Erlenmeyer lambda-25 UV-
visible spectrometer) under dim light[136]
.
2.8.3 Modal Membrane Interaction Studies
Micelle Preparation; A 0.1M (Conc. >> CMC) stock solution of each CTAB, TTAB and
SDS were prepared, dissolving in doubly distilled deionized water, and solution made for
nmr studies were in D2O, under ambient conditions and CTAB micelles solutions stored at
37°C. All the compounds 1-5 are water insoluble, these solid compounds were sonicated with
micelles solutions for 2 hours. (CMC = critical micelle concentration that is for CTAB = 0.79
mM, TTAB = 3.79 mM,[137]
SDS = 5.7 mM).[133]
UV-Visible Spectroscopy; Absorption spectra were recorded at constant temperature of 26°C
for TTAB and SDS micelle systems, and at 37°C for CTAB micelles. The electronic
spectrum of known concentrations of the compounds in different solvents was obtained at
room temperature. All the readings were referenced to air by placing empty cell in the path of
the reference light beam.
H NMR Spectroscopy; 1HNMR spectra for CTAB micelles were recorded using a Varian
INOVA-300 spectrometer at 37°C. All the other 1HNMR spectral measures were taken at
26°C temperature using Varian MR-400 spectrometer. All the proton chemical shifts were
referenced against an external sample of DSS in D2O, also equilibrated by internal refrencing
in some cases.
52
Chapter 3
Results and Discussion
3.1 Synthesis
Compounds a – e were prepared from ferrocene and the diazonium salt of mono and
disubstituted anilines as shown in Figure 3.01. The reactions were carried out in the ether-
water mixtures using CTAB as a phase transfer catalyst in analogy with previous preparations
of many different substituted ferrocene derivatives.[129]
The presence of the phase transfer
catalyst increased the yields from about 15% to > 70% for compounds a – e, in agreement
with literature reports on other substituted ferrocene derivatives.[138, 139]
Different reduction procedures have been reported for the synthesis of
ferrocenylaniline (A) from 4-nitrophenylferrocene (a). Hu Ping, et al. (2001)[140]
has reported
the reduction of (a) with granular tin and HCl yielding 76% ferrocenylaniline. Zaheer M.
(2008)[141]
has reduced (a) using Pd-charcoal and hydrazine with 78% yield. But in the
present work (a) has been reduced with Zn / formic acid which showed much higher (94%)
yield than ever reported. In this study Sn-powder / HCl and Zn-dust / HCl have also been
tried for the reduction a. This work showed the better reducing properties of Zn / formic acid
system for the nitro functionality, which is in agreement with the work of D. Gowda et al.
(2001)[142]
After optimizing the reduction method, compounds A – E were synthesized using
the best method.
Figure 3.01: General framework of the entire synthesis.
L varies
R varies
Compounds At1 – Et7 were synthesized by the coupling of ferrocenylanilines (A – E)
with respective thiocyanates (1 – 7) in acetone. In analogous method, reported for simple
organic thioureas that followed the addition of N–H into C=N double bond of isothiocynate
(–N=C=S) group.[131]
The compounds Au3 – Eu7 were synthesized by the replacement of
sulfur with oxygen,[132]
in the presence of alkaline Hg2+
as sulfur capturing agent, that yielded
53
urea more than 70%. NaOH was used as alkali which provides OH` anion that attack on the
thio-carbon of thiourea. The proposed mechanism, similar to that of guanylation, is shown in
Figure 3.02.[143]
Figure 3.02: Suggested mechanism for the conversion of ferrocenyl thioureas into ureas
(Step 4)
All the synthesized compounds were characterized using CHNS elemental analysis,
FT-IR, 1H and
13C NMR and UV-Visible spectroscopy. Some of the compounds were also
characterized by single crystal X-rays diffraction analysis.
3.2 Elemental Analysis
CHNS analysis is an appropriate tool for the determination of the bulk purity of
compounds. It is helpful for the analysis of salvation as well as for the structural elucidation
of compounds. The percentage of carbon, hydrogen, nitrogen and sulfur in the case of
compounds At1 – Et7 were determined and reported in the experimental section Chapter 2.
Results indicate that the elemental composition of the compounds is in close
agreement with the calculated values of the supposed structures. It was observed from the
elemental analysis data that the compounds are sufficiently pure in bulk and, in the case of
some thioureas containing aromatic arms on both sides; the compounds have solvent
molecules in equal proportion. The thioureas containing one aliphatic arm, on the other hand,
did not have any solvent molecule. It might be due to the bulky size of aromatic groups.
Intermolecular distance increased which inhibited the intermolecular H-bonding. As a result,
the deficiency of H-bonding insisted the polar solvent molecule to come into action. This will
be further elaborated in X-rays crystallographic section.
54
3.3 Infra Red Spectroscopic Characterization
Infra red spectroscopy is an important tool for the solid phase characterization of
chemicals. All the synthesized compounds have been analyzed by Fourier transformation
infra red (FTIR) spectroscopy using Nicolet FTIR instrument. The analysis data have been
reported in the experimental section Chapter 2, along with the other characterization data.
Most significant bands have been tentatively assigned according to the literature.[144]
The presence of N-O stretching band in the range of 1350 – 1450 cm-1
in compounds
a – e and no characteristic band at about 3200 cm-1
and above shows the coupling of nitro
anilines with ferrocene. Other aromatic and aliphatic C-H and C-C bands were observed in
their normal regions of absorbance and have less importance regarding the progress of
reaction. In compounds A – E, the absence of N-O stretching bands and the appearance of N-
H stretching bands in the range of 3200 – 3400 cm-1
provide the authentication of the aniline
formation which confirms the reduction process.
After the coupling of compounds A – E with isothiocynates, the success of the
thioureas formation can be confirmed from the fact that there is no characteristic band for –
N=C=S in the range of 2000 cm-1
. And the presence of N-CS-N (thiourea C=S) stretching
band about 1200 – 1300 cm-1
also expresses the formation of thioureas At1 – Et7. The
distinguishing IR bands of N, N'-disubstituted thioureas are between/ near to 3120-3395 cm-1
(NH), 2960-3090 cm-1
Ph(CH), 1235-1300 cm-1
(C=S) and 1135-1180 cm-1
(C-S). Conversion
of the broad band into sharp bands in the region of 3200 – 3400 cm-1
also supports the
formation of thioureas from ferrocenyl anilines. Other characteristic bands for aliphatic and
aromatic C-H and C-C bands are found in their normal characteristic regions and are of less
importance.
In compounds Au3 – Eu7, the disappearance of C=S stretching band and the
appearance of urea carbonyl (C=O) stretching frequency in the region of 1550 – 1660 cm-1
provide evidence for the conversion of thioureas into the urea functionality. This conversion
is also supported by shifting of stretching bands, for the affiliated NH and CH groups,
towards higher frequency. A selective example is discussed in the succeeding paragraph that
presents the FTIR changes of the conversions (d D Dt7 Du7).
Figure 3.03 presents the spectral changes for the success of the formation of the
selective series of compounds as shown in the Figure 3.04 below. The reduction of the
compound d to the compound D is confirmed by the appearance of a broad band of NH at
3374 cm-1
and the disappearance of N – O band at 1403 cm-1
.
55
Figure 3.03: Comparative FT-IR spectra showing the spectral change of conversion (d D
Dt7 Du7)
Figure 3.04: Selective series of compounds discussed & characterized (d D Dt7
Du7) using FTIR
On the coupling of 2,4-dichlorophenylisothiocynate with amine (D), the broad NH
band converts into sharp signals (at 3178 and 3302 cm-1
) and a new band at 1292 cm-1
characteristic of C=S appears, which demonstrates the success of coupling between
compound D and respective isothiocynate, i.e., the formation of compound Dt7. In the latter
reaction, conversion of the C=S bond to the urea C=O group is proposed from the appearance
of new band at 1580 cm-1
for urea carbonyl. The shifting of NH band towards higher
frequency (3334 cm-1
) demonstrates the conversion of thiourea Dt7 into urea Du7, as the
higher electronegative oxygen induces more polarity to the NH bond which is shifted from
3302 cm-1
, the C–H and C=C bonds also show shifting on the conversion of Dt7 into Du7.
56
3.4 Nuclear Magnetic Resonance Spectroscopic Characterization
1H and
13C NMR spectra have been recorded on broad band Varian MR 400 MHz for
1HNMR and 101 MHz for
13CNMR and the data has been reported in the experimental
section of the dissertation along with the other characterization data in Chapter 2.
Table 3.01: Comparative 1HNMR data for nitrophenylferrocenes (a – e) and
ferrocenylanilines (A – E)
Comp Fc
(5H)
Fc
(2H)
Fc
(2H) Aromatic Protons
Other
group NH2
A 4.06 4.48 4.74 7.56 (2H) 8.14 (2H) -- -- --
A 4.06 4.26 4.56 6.67 (2H) 7.33 (2H) -- -- 3.66
B 4.23 4.51 4.69 7.63 (1H) 7.95 – 8.00 (2H) 2.52 (CH3) --
B 4.12 4.23 4.39 6.49 (1H) 6.54 (1H) 7.50 (1H) 2.30 (CH3) 3.61
C 4.06 4.42 4.87 7.61 (1H) 7.79 – 7.81 (2H) 4.01 (OCH3) --
C 4.06 4.22 4.39 6.34 (1H) 7.29 – 7.32 (2H) 4.01 (OCH3) 3.90
D 4.27 4.59 4.99 7.73 (1H) 8.01 (1H) 8.21 (1H) Cl --
D 4.07 4.36 4.61 7.03 (1H) 7.21 (1H) 7.51 (1H) Cl 3.71
E 4.07 4.42 4.73 7.44 (1H) 7.76 (1H) 8.03 (1H) 8.28 (1H) --
E 4.05 4.28 4.59 6.54 (1H) 6.82 (1H) 6.92 (1H) 7.09 (1H) 3.67
The 1HNMR data for the compounds a – e describe the substitution of nitrophenyl
group at one Cp ring of ferrocene. As one nmr signal of ferrocene split into three bands after
the reaction,[145]
the un-substituted C5H5 ring of ferrocene appears as a singlet in the range of
4.05 – 4.12 ppm for 5H, whereas the substitution on the other Cp ring revealed two bands at
about 4.7 and 4.4 ppm. Both these bands appeared, as a triplet (j = 1.8 Hz) which shows that
the coupling is due to the similar chemical environment. Aromatic protons appeared in the
pattern similar to that of nitroaniline with different chemical shift values is due to the
replacement of NH2 group with ferrocene. All the protons in these compounds have been
identified by intensity and multiplicity pattern. Total protons calculated from the integration
curve are in close agreement with the molecular structures.
In compounds A – E, the appearance of the NH protons signal provides the evidence
for the successful reduction of nitro derivative into the aniline derivatives. The data given in
table 3.01 shows chemical shift values of different protons, shifted up field as compared to
the respective nitro derivatives that is in accordance with the normal change of inductive and
resonance effects. The data also shows the change of 1HNMR signals of ferrocene with the
change of substituents on the phenyl ring. On the coupling of ferrocenyl anilines with
isothiocynates, the broad singlet of two NH protons split into two separate singlets which
provide evidence for the formation of thioureas.
57
Table 3.02: Comparative 1HNMR data of 4-ferrocenylaniline (A) and its thioureas (At1 –
At4)
Comp. Fc (5H) Fc (2H) Fc (2H) Aromatic Protons NH NH R group A 4.060 4.260 4.560 6.670 7.330 3.660 --- ---
At1 4.024 4.322 4.732 7.291 7.474 7.687 9.381 CH3
At2 4.028 4.323 4.735 7.306 7.476 7.699 9.389 C2H5
At3 4.026 4.322 4.737 7.323 7.475 7.712 9.393 C3H7
At4 4.027 4.325 4.741 7.339 7.484 7.805 9.501 C3H5
Figure 3.05: 1HNMR spectra showing comparative chemical shifts on the variation of R
group in the series (A & At1 – At4)
The formation of thioureas does effect slightly the chemical shift of ferrocenyl
protons as compared to the respective anilines. For example, a representative case is shown in
Figure 3.05. A general skeleton of the synthesized thioureas is presented in Figure 3.01.
It was observed in the 1HNMR spectra that the change of the R group of the thioureas
does not effect significantly the ferrocenyl signals on keeping the linker L the same. Signals
at 2.5 and 3.3 ppm appear for the solvent and moisture in solvent respectively.
But aromatic protons on L show minute disparity. A selective example is reported in
Table 3.02, and spectra’s are shown in Figure 3.05. Such examples are observable in cases of
aliphatic R substituents. The 1HNMR signals of the aliphatic R groups of molecules are well
split and appear at the characteristic values. As shown in Figure 3.06 for allyl substituted
derivatives, It is clear from the Figure 3.06 that the variation of the L group does not
influence significantly the chemical shift of allyl group.
58
Figure 3.06: 1HNMR spectra showing comparative chemical shifts on the variation of the L
group in (At4, Bt4 and Ct4) the spectra also show a well splitting pattern.
The data for the synthesized N, N`-disubstituted thioureas and ureas show that the NH
hydrogen resonates well down field from the other protons in the spectra. In compounds At1
– Eu7, the NH protons give signals in the region of 7.5 to 10.5 ppm, with reference to the
DMSO-d6 (Solvent) residual signal (observed at 2.5ppm). In most of the cases these NH
protons are well separated from each other. One of them appears in the range of 7.5 – 8.0
ppm, and the other at 9.0 – 10.0 ppm. In some cases, both the NH protons appear above 10.0
ppm (When R is nitrophenyl group) as (compound Bt6) in Figure 3.07. This Figure also
demonstrates the 1HNMR spectral change of a selective series of reactions which shows the
overall formation route of the ferrocenyl urea Bu6. Figure 3.08 demonstrate the scheme for
the synthesis of Bu6.
59
Figure 3.07: 1HNMR spectra showing the spectral change of conversion (b B Bt6
Bu6)
In this specific case, the formation of b has been probed in 1HNMR by the appearance
of three signals in the ferrocene region at 4.23 (5H), 4.51 (2H) and 4.69 (2H) ppm. These
three bands appear due to the substitution of one proton on one Cp ring of ferrocene by
nitrotoluyl group. The aromatic protons appear at 7.63 (1H) and 7.95 – 8.00 (2H) ppm. After
reduction of b, the appearance of a broad signal at 3.61 ppm for two protons confirms the
formation of B. The ferrocenyl protons shifted up-field (appeared at 4.12 (5H), 4.23 (2H) and
4.39 (2H) ppm), along with aromatic protons (appeared at 6.49 (1H), 6.54 (1H) and 7.50 (1H)
ppm), due to the reduction of electron withdrawing nitro group into the electron donating
amino group. The coupling of B with 4-nitrophenylisothiocynate has been confirmed by the
splitting of the NH2 signal along with down field shifting (appeared at 10.21 (1H) and 10.33
(1H) ppm). Aromatic (7.23 (1H), 7.31 (2H), 7.66 (1H), 7.84 (2H) and 8.20 (2H) ppm) as well
as ferrocenyl (4.13 (5H), 4.34 (2H), 4.55 (2H) ppm) proton signals reveal shifting on the
formation of thiourea Bt6. After the conversion of thiourea into urea, all the aromatic (7.25 –
7.36 (2H), 7.68 (1H), 7.86 (2H) and 8.21 (2H) ppm) and ferrocenyl (4.05 (5H), 4.35 (2H),
4.72 (2H) ppm) protons shifted slightly, NH proton signals were also shifted as well
(appeared at 9.26 (1H) and 10.38 (1H) ppm).
60
Figure 3.08: Selective series discussed for 1HNMR characterization (b B Bt6 Bu6)
The 13
CNMR data for the compounds a – e depict the replacement of hydrogen with
nitrophenyl assembly at one Cp ring of ferrocene as one 13
CNMR signal of ferrocene split
into four signals after the coupling of diazonium salt of nitro anilines.[145]
Table 3.03: Comparative 13
CNMR data for nitrophenylferrocenes (a – e) and
ferrocenylanilines (A – E)
Comp. Aromatic Carbons Ferrocenyl Carbons Branch a 148.2 145.5 125.9 123.8 81.7 70.5 70.0 67.2
A 144.5 129.0 127.2 115.2 86.6 69.4 68.2 65.8
b 146.3 136.6 132.7 130.7 125.4 120.5 80.3 70.8 70.6 69.8 21.6
B 138.5 135.1 132.8 124.6 120.3 117.0 84.1 69.4 69.3 67.4 21.2
c 156.5 146.2 136.6 128.6 116.0 105.9 79.6 69.82 69.8 69.5 55.8
C 155.9 137.6 127.2 121.6 115.0 105.9 82.1 69.8 67.8 64.5 55.0
d 145.8 145.5 132.3 131.1 125.8 121.2 81.3 70.3 70.1 68.9
D 144.0 142.4 130.3 128.1 123.8 120.2 76.3 70.1 68.5 64.9
e 145.1 139.6 131.6 129.2 120.5 120.4 81.1 70.6 69.8 66.8
E 142.1 131.6 129.2 123.1 120.45 120.34 82.4 69.84 69.78 66.8
One un-substituted C5H5 ring of ferrocene appears as a single band in the range of 69
– 71 ppm for five carbons, while the substitution on other Cp ring generates three bands, two
of which are in the range of 64 - 71 ppm and the third one is in the range of 79 – 82 ppm.
61
Figure 3.09: HSQCAD NMR spectrum of a and A indicates that the more acidic protons are
attached with more shielded carbons.
The signal intensities infer that the signals at about 64 – 71 ppm are for un-substituted
carbons, which appear in the form of pairs. The chemical shift and signal intensity value of
the signal at about 79 – 82 ppm reveal that it is the signal of tertiary carbon on which
branching takes place. Aromatic carbons appear in the pattern similar to that of nitroaniline
with different chemical shift values because of the replacement of NH2 group with ferrocene.
All magnetically different carbons appear at different chemical shift values; the intensities
were found in agreement with the molecular structures.
In compounds A – E, after the reduction of strong electron withdrawing nitro group
into an electron donating NH2 moiety effect the chemical shift values of all the carbons as
reported in the Table 3.03. Chemical shift values of different carbons shifted up field as
62
compared to the respective nitro derivatives that are in accordance with the normal change of
inductive and resonance effects. From the data given in Table 3.03, there exists an ambiguity,
as some of the aromatic carbons (underlined in Table 3.03) do not shift in accordance with
classical organic chemistry. In order to understand, the HSQCAD NMR spectrum of a and A
shown in Figure 3.09 was measured to assign exact chemical shift values to the carbons. The
careful assignment of chemical shifts shows that there is a downfield shifting of some
carbons in aniline. This might be due to the electron donating effect of both NH2 and
ferrocene groups. (But we cannot be for certain.)
Table 3.04: Comparative data of 13
CNMR 4-ferrocenyl-3-methoxyaniline (C) and its
thioureas (Ct1 – Ct7)
The most de-shielded signal appears at about 177 – 182 ppm in all the thioureas for
C=S group on the coupling of ferrocenyl anilines with isothiocynates. This provides evidence
for the formation of thioureas. The formation of thioureas does effect slightly the chemical
shift of ferrocenyl carbons as compared to the respective anilines. For example, a
representative case is shown in Figure 3.10. It was observed in the 13
CNMR spectra that the
change of the R group of the thioureas does not effect significantly the ferrocenyl signals on
keeping the linker L the same. But aromatic carbons on L show just slight shifting, A
selective example is reported in Table 3.04, and spectra’s are shown in Figure 3.10 that are
observable in the case of aliphatic R substituents.
Compound C Ct1 Ct2 Ct3 Ct4 Ct5 Ct6 Ct7
C=S 180.7 180.1 179.72 180.48 178.97 178.57 180.10
Aromatic 155.9
137.6
127.2
121.6
115.0
105.9
156.06
137.95
128.61
121.90
114.19
110.53
156.06
138.01
128.65
121.94
114.17
109.44
156.06
138.04
128.67
122.13
114.37
106.21
156.59
138.45
135.30
129.21
122.89
116.09
115.22
106.95
155.95
139.35
138.23
128.45
128.31
124.34
123.62
122.54
115.06
106.72
156.01
146.13
142.18
137.70
128.59
124.25
123.26
121.46
115.08
106.68
154.98
138.20
136.40
134.20
131.50
130.20
128.70
125.30
124.00
122.50
115.24
106.53
Ferrocene 82.1
69.8
67.8
64.5
82.22
69.00
68.17
67.73
82.26
68.99
68.13
67.78
82.27
68.99
68.14
67.78
82.75
69.54
68.69
68.34
82.21
69.02
68.19
67.84
82.00
69.03
68.25
67.93
82.17
68.98
68.11
67.97
Other
groups
55.0 55.30
21.66
55.32
38.50
14.12
55.21
45.54
21.66
11.27
55.78
46.56
55.30 55.34 55.31
63
Figure 3.10:
13CNMR spectra showing comparative chemical shifts on variation of R group
in series (C & Ct1 – Ct7)
The 13CNMR signals of the aliphatic R group of molecules show slight effect on the
variation of the L group as shown in Figure 3.11 and is demonstrated in the Table 3.05.
64
Figure 3.11: 13
CNMR spectra showing comparative chemical shifts on the variation of the L
group in a series (At3, Bt3, Ct3, Dt3 and Et3)
Table 3.05: Comparative 13
CNMR data of propyl thioureas (At3, Bt3, Ct3, Dt3 & Et3)
In the subsequent reactions, thioureas were converted into ureas, those are confirmed
by the conversion of the C=S band (at about 177 – 182 ppm) into the urea C=O band (at
about 161 – 167 ppm) with reference to the DMSO-d6 (solvent) residual signal (at 40.0 ppm).
Figure 3.12 demonstrates the 13
CNMR spectral change of a selective series (Figure 3.13) of
the reactions which shows the overall formation route of the ferrocenyl urea Eu5. (e E
Et5 Eu5).
Comp.
Code
At3 Bt3 Ct3 Dt3 Et3
C=S 180.0 180.0 179.7 179.9 179.9
Aromatic 134.5, 126.5
125.9, 122.8
136.8, 135.2
132.8, 130.3
124.7, 120.3
156.1, 138.0
128.7, 122.1
114.3, 106.2
139.0, 134.0
130.3, 128.4
124.5, 123.6
139.6, 128.3
126.7, 124.1
123.2, 121.8
Ferrocene 84.7, 69.2
68.6, 66.0
86.4, 69.1
69.0, 67.7
82.3, 69.0
68.1, 67.8
83.2, 69.4
69.2, 68.0
84.4, 69.3
68.4, 66.8
Propyl 45.5, 21.7
11.3
45.5, 21.7
11.2
45.5, 21.7
11.3
45.5, 21.7
11.3
45.5, 21.8
11.2
Others 40.1 55.2
65
Figure 3.12: 13
CNMR spectra showing the spectral change of conversion (e E Et5
Eu5)
In this specific case, the formation e has been probed in 13
CNMR by the appearance
of four signals in the ferrocene region at 81.3, 70.3, 70.1 and 68.9 ppm. These four bands
appear due to the substitution of one proton, on one Cp ring of ferrocene, with the nitrophenyl
group. The aromatic carbons appear at 145.8, 145.5, 132.3, 131.1, 125.8 and 121.2 ppm.
After the reduction of e into E, three ferrocenyl signals shifted up-field appeared (at 69.8,
69.8 and 66.8. ppm) along with aromatic carbons (at 142.1, 131.6, 129.2, 123.1, 120.5 and
120.3 ppm) due to the reduction of the electron withdrawing nitro group into the electron
donating amino group. But one ferrocenyl carbon signal shifted down field, which appears at
82.4 ppm. The reason of shifting is ambiguous. The coupling of E with phenylisothiocynate
was confirmed by the appearance of the thiourea C=S signal at 179.5 ppm. Aromatic carbon
signals (at 139.3, 129.8, 128.3 128.2, 125.8, 124.3, 123.5, 122.1, 121.4 and 121.1 ppm) as
well as ferrocenyl carbon signals (at 84.6, 69.3, 68.7, 66.3) reveal shifting on the formation of
thiourea Et5. After the conversion of thiourea Et5 into urea Eu5, all the aromatic carbons (at
139.4, 137.1, 134.9, 128.3, 127.2, 125.7, 124.3, 123.5, 123.4 and 121.1 ppm) and ferrocenyl
66
carbons (at 84.6, 69.2, 68.6 and 66.0 ppm) shifted slightly. The C=S signal disappears and
urea C=O signal come into sight at 164.3 ppm.
Figure 3.13: Selective series discussed for 13
CNMR characterization (e E Et5 Eu5)
3.5 Single crystal X-ray diffraction analysis
The single crystal X-ray diffraction has been practiced to bear out the structure of
synthesized compounds. The different diffractometers have been used for the data collection
and the structures were resolved by direct method using SHELXS-97and refined by the full-
matrix least squares on F2 using SHELXL-97.
[146, 147] The structural drawings were generated
by using different software’s like Mercury, Diamond, ENCIFER, Ortep and Platon etc.
3.5.1 Crystal structures of nitrophenylferrocenes
The crystals of nitrophenylferrocenes (c, d & e) were grown by recrystallization in
petroleum ether fractions. Suitable crystals were selected for solid state X-ray diffraction
studies. The ORTEP representations of molecular structure of compounds c, d and e are
given in Figures 3.14(X) – 3.16(X). The crystallographic parameters are given in Tables 3.06,
while the selected bond lengths, bond angles and torsion angles are presented in Table 3.07.
The intra-molecular hydrogen bonding (Table 3.08) and intermolecular interactions present in
these compounds are summarized in Table 3.09 and drawn in Figures 3.14(Y, Z) – 3.16(Y,
Z).
67
Table 3.06: Crystal data and structure refinement parameters for c, d and e
Crystal Parameters c d e
Empirical formula C17H15NO3Fe C16H12NO2ClFe C16H13NO2Fe
Formula weight 337.15 341.57 307.12
Temperature (K) 296(2) 296(2) 296(2)
Wavelength (Å) 0.71073 0.71073 0.71073
Crystal system Orthorhombic Monoclinic Orthorhombic
Space group Pbca P 21/c Pbca
Unit cell
dimensio
ns
a(Å) 11.7790(6) 9.5788 7.4635(3)
b(Å) 9.6666(5) 23.945(2) 11.8515(7)
c(Å) 25.3185(13) 11.8371(11) 30.3350(15)
(o) 90 90 90
(o) 90 90.096(4) 90
(o) 90 90 90
V(Å3), Z 2882.8(3), 8 2715.0(4),8 2683.2(2), 8
Density (g/cm3) 1.554 1.671 1.521
Crystal size (mm3) 0.40 x 0.39 x 0.08 0.52 x 0.44 x 0.26 0.35 x 0.14 x 0.08
Index ranges
-16<=h<=16
-13<=k<=13
-36<=l<=36
-14<=h<=14
-36<=k<=36
-17<=l<=18
-6<=h<=9
-14<=k<=9
-29<=l<=37
F(000) 1392 1392 1264
Total reflections 4497 10440 11492
Refinement method Full-matrix least-
squares on F2
Full-matrix least-
squares on F2
Full-matrix least-
squares on F2
Mu (mm-1
) 1.058 1.309 1.123
R indices (all data) R1 = 0. 0551,
wR2 = 0.2025
R1 = 0.0325,
wR2 = 0.1289
R1 = 0.0667,
wR2 = 0.0751
Final R indices
[I>2(I)]
R1 = 0.0480,
wR2 = 0.1919
R1 = 0.0271,
wR2 = 0.1202
R1 = 0.0343,
wR2 = 0.0651
Goodness-of-fit 1.713 1.073 1.014
θ range for data
collection (o)
2.84 to 30.82 1.70 to 33.27 2.69 to 26.00
68
Table 3.07: Selected bond lengths, bond angles and torsion angles for nitrophenylferrocenes
(c, d & e)
Bond lengths (Å) Bond angles (°) Torsion angles (°)
c
C1–Fe1 2.06 C10–C6–Fe1 69.51 C2–C1–C11–C12 -23.33
C3–C2 1.424 C6–C10–C9 107.83 C2–C1–C11–C16 156.93
C6–Fe1 2.051 C16–C11–C1 119.29 C1–C11–C12–C13 179.37
C12–O1 1.367 C10–Fe1–C1 122.51 C13–C14–N1–O2 -7.64
C12–C11 1.419 O1–C12–C11 115.96 C13–C14–C1–O3 73.19
C14–N 1 1.464 C15–C14 –N1 118.78 C13–C12–O1–C17 9.19
C15–C 14 1.383 O2–N1–O3 123.33 C11–C12–O1–C17 170.92
C17–O 1 1.428 O2–N1–C14 118.33 Fe1–C1–C11–C12 69.63
N1–O 2 1.22 C2–Fe1–C9 158.84 Fe1–C1–C11–C16 -110.11
d
Fe–C6 2.046 C4–Fe–C6 107.18 N–C14–C13–C12 -179.08
Fe–C4 2.048 Fe–C6–C11 128.35 N–C14–C15–C16 179.32
C6–C11 1.468 C12–C13–C14 118.42 O1–N–C14–C15 -176.54
C12–Cl 1.739 C11–C12–Cl 122.01 Cl–C12–C11–C6 1.480
C14–N 1.465 C13–C12–Cl 115.58 C12–C11–C6–C7 26.55
C13–C14 1.386 C13–C14–N 118.34 C12–C11–C6–C10 -156.61
N–O1 1.220 N–C14–C15 119.37 C10–C6–C11–C16 23.53
N–O2 1.235 O1–N–O2 123.65 Fe1–C6–C11–C12 -67.94
C1–C2 1.427 C14–N–O1 118.33 Fe1–C6–C11–C16 112.20
e
Fe1–C10 2.036 C4–Fe1–C6 106.70 N1–C15–C16–C11 -176.49
Fe1–C4 2.043 Fe1–C10–C11 125.30 N1–C15–C14–C13 176.69
C10–C11 1.478 C12–C13–C14 120.33 O1–N1–C15–C16 168.88
C12–C13 1.379 C10–C11–C12 121.42 O2–N1–C15–C16 -10.68
C15–N1 1.486 C13–C12–C11 121.90 C12–C11–C10–C6 17.37
C13–C14 1.378 C14–C15–N1 118.88 C12–C11–C10–C9 -163.96
N1–O1 1.222 N1–C15–C16 117.71 C10–C11–C16–C15 23.53
N1–O2 1.217 O1–N1–O2 124.05 Fe1–C10–C11–C12 106.81
C1–C2 1.382 C15–N1–O1 117.71 Fe1–C10–C11–C16 -72.80
C15–N1–O2 118.24
69
3.5.1.1 2-methoxy-4-nitrophenyl ferrocene (c)
The violet colored, slide shaped crystal was analyzed by Bruker kappa APEXII CCD
diffractometer equipped with a graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å).
Data collection used ω scans, and a multi-scan absorption correction was applied. The
molecular structure is given in Figure 3.14(X).
Table 3.08: The intra-molecular hydrogen bonds in the nitrophenyl ferrocenes (c, d & e)
Compound X H Y d(X–H)
(Å)
d(H--Y)
(Å)
d(X--Y)
(Å)
<(XHY)
(˚)
c C2 H2 O1 0.941(21) 2.386(24) 2.840(2) 109.29(168)
C13 H13 O2 1.012(33) 2.369(34) 2.707(3) 98.30(228)
d C7 H7 Cl 0.980(2) 2.656(1) 3.156(3) 111.99(6)
C16 H16 C10 0.930(2) 2.626(2) 2.964(2) 102.10(7)
C13 H13 O1 0.930(2) 2.418(3) 2.706(2) 97.85(8)
C15 H15 O2 0.931(1) 2.446(2) 2.727(2) 97.40(8)
e C16 H16 O2 0.930(2) 2.410(2) 2.712(3) 98.74(14)
C14 H14 O1 0.930(3) 2.451(2) 2.725(3) 96.94(18)
Table 3.09: The inter-molecular hydrogen bonds in the nitrophenyl ferrocenes (c, d & e)
Compound X H Y d(X–H)
(Å)
d(H--Y)
(Å)
d(X--Y)
(Å)
<(XHY)
(˚)
c C3 H3 O2 0.971 2.620 3.251 122.8
C16 H16 O3 0.994 2.524 3.504 167.47
d C16 H16 O2 0.931 2.609 3.522 167.15
e C13 H13 O2 0.930 2.630 3.441 146.11
C15 --- O2 Dipole interaction 3.204 Å
The single crystal X-ray results indicate, in c, the planes of cyclopentadienyl ring A
(C1–C5) and phenyl ring B (C11–C16) cross each other with the dihedral angle of 22.42°
whereas the nitro group is in plane with phenyl ring B (dihedral angle is 7.75°). The Fe atom
is at a distance of 1.651 Å from the centroid of ring A. Extensive conjugation is responsible
for planarity, along with intra-molecular H-bonding as shown in Figure 3.14(Y). The packing
diagram of c (Figure 3.14(Z)) shows a supramolecular chain structure mediated by NO --- H
secondary non-covalent interactions (a kind of H-bonding). In this supramolecular
arrangement, molecules make C32
(15) cavities.[148]
Such non-bonding secondary interactions
are important for the biological activities of a compound.[149]
70
X Y
Z
Figure 3.14: X) ORTEP diagram of c with atomic numbering scheme. Y) The intra-molecular
H-bonding represented by dotted lines. Z) The supramolecular arrangement of compound c
mediated by intermolecular H-bonding represented by dotted lines.
3.5.1.2 2-chloro-4-nitrophenyl ferrocene (d)
The violet colored, needle like crystal was analyzed by Bruker kappa APEXII CCD
diffractometer equipped with a graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å).
Data collection used ω scans, and a multi-scan absorption correction was applied. The
molecular structure is given in Figure 3.15(X).
The single crystal X-ray results indicate, in d, the planes of cyclopentadienyl ring A
(C6–C10) and phenyl ring B (C11–C16) cross each other with the dihedral angle of 24.88°;
however the nitro group is in-plane with phenyl ring B (dihedral angle is 2.95°). The Fe atom
is at a distance of 1.651 Å from the centroid of ring A. Widespread conjugation is
accountable for planarity, along with intra-molecular H-bonding, as shown in Figure 3.15(Y).
The packing diagram of d (Figure 3.15(Z)) shows that the molecule is stabilized by zigzag
chain, formed along the crystallographic axis-b, structure mediated by NO --- HC
intermolecular hydrogen bonding.
71
X Y
Z
Figure 3.15: X) Molecular diagram of d with atomic numbering scheme. Y) The intra-
molecular H-bonding represented by dotted lines. Z) The supramolecular arrangement of
compound d mediated by intermolecular H-bonding represented by dotted lines.
3.5.1.3 3-nitrophenyl ferrocene (e)
The orange colored, needle like crystal was analyzed by Bruker kappa APEXII CCD
diffractometer equipped with a graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å).
Data collection used ω scans, and a multi-scan absorption correction was applied. The crystal
structure of e is sketched out in Figure 3.16(X). The cyclopentadienyl ring A (C6–C10) and
phenyl ring B (C11–C16) is planar with r.m.s. deviations of 0.0024 and 0.0030 Å,
respectively. The dihedral angle between rings A and B is 17.46° which shows that phenyl
ring is almost planar with attached cyclopentadienyl. However, the nitro group is also almost
planer with phenyl ring B (dihedral angle is 10.52°). The Fe atom is at a distance of 1.645 Å
from the centroid of ring A.
72
X Y
Z
Figure 3.16: X) ORTEP diagram of e with atomic numbering scheme. Y) The supramolecular
arrangement of compound e along crystallographic axis-b. Z) Molecules helices in the form of
a screw due to NO---HC type hydrogen bonding and Cδ+
---δ-
ON type interactions along
crystallographic axis-a.
The cyclopentadienyl ring of ferrocene not attached with phenyl is disordered over
two sets of sites. The intermolecular H-bonding of C—H···O type exist to stabilize the
molecule in the form of polymeric chains extending in two directions. Along the
crystallographic axis-b two molecules replicate and form a polymeric chain by NO---HC
hydrogen bonding, while along the crystallographic axis-a, molecule helices in the form of a
screw due to NO---HC type hydrogen bonding and Cδ+
---δ-
ON type interactions. The packing
diagram of e is shown in Figure 3.16(Y, Z) which present a superamolecular chain structure
mediated by CH---O type non-covalent interactions and chain molecules helices due to screw
symmetry. Robert et. al. (1988) also reports a crystal structure for the same compound with
the same crystal system and space group. But some of the bond distances and angles are
different as shown in Table 3.10.
73
The crystal structural study shows that the secondary interactions are more in the
compound under observation in comparison to the already reported para nitro isomer.[150, 151]
The capability of forming such secondary interactions is favorable for a compound to bind
with biological macro molecules like proteins and DNA.[135]
Table 3.10: literature comparative data for solvent dependent torsion angles to support push –
pull delocalization of the π electrons.
The conformation and rotation of the phenyl group out of plane of the ferrocene is
approximately 22.4° in c, 24.9° in d and 17.46° in e. The 4-nitrophenylferrocene (a) has been
characterized previously using X-ray crystallography, and hence three different structures
have been reported.[150, 151]
These structures differ in the torsion angle responsible for the
conjugation between the ferrocene and the phenyl group. In compound a, there is no ortho
substituent, and the observed effects are due to the most favorable conformation. The fact
those three different structures were observed documenting the flat energy surface of these
types of structures. The conjugation between the ferrocene and the phenyl group is not
important to the stability of the compound. In the case of structures c and d, the methoxy and
chloro groups respectively provide some steric hindrance and disturb the planarity of
ferrocene cyclopentadienyl group and phenyl ring. The conjugation between the phenyl and
the ferrocene is also reflected in the C-C bond length between the ferrocene and phenyl ring.
Since this bond length is shorter for the p-nitro substituted derivatives. This provides the
evidence of push – pull delocalization of the π-electrons. So, in non-conjugated systems and
the m-nitro substituted derivative e; the concerned bond is elongated in comparison to
electron withdrawing (a, c and d) and donating (p-ferrocenylaniline) conjugated systems.
Comp. #
Compound
substituent
Torsion angle
(o)
Ph–Cp
Bond
length
(Å)
Fe to ortho
carbon
distance
(Å)
Solvent Ref.
Down
ward
Up
ward
a H 15.46 18.30 1.469 3.599 EtOAc/DCM [150]
a H 11.27 13.32 1.466 3.704 EtOH/DCM [151]
a H 12.5 1.455 MeOH [152]
c OCH3 23.33 22.18 1.466 3.670 Iso octane This work
d Cl 26.55 23.53 1.468 3.890 Iso octane This work
e H 4.95 8.55 1.497 3.864 n-Hexane This work
e H 17.4 1.463 MeOH [152]
p-Fc
aniline
H 7.60 8.29 1.477 3.965 Hexane
/EtOAc
[153]
Non
conjugated
system
- - - 1.493 CH2Cl2/
MeOH
[154]
Fc - Fc - 49.9 55.7 1.477 EtOAc [155]
74
Therefore the observation of a near planar phenyl group and ferrocene group are not
anticipated.
The conformer observed is particularly surprising, because one could also have
anticipated that some electrostatic interaction between the oxygen atom and the iron atom
would exist. Since the distances between the ortho carbon atom of structure c and that of the
corresponding phenyl derivatives are very similar, there is no structural support for such
attraction, as it is expected that the torsion angle should be larger in the compound c than in d
due to the larger size of methoxy group.[156]
But, the observed values are opposite to the
expectation. One could also anticipate that it might be due to intra-molecular hydrogen
bonding (shown in Figure 3.14(Y) and 3.15(Y)), as oxygen is more electronegative than
chlorine.
3.5.2 Crystal structure of 3-Ferrocenyl aniline (E)
The orange colored, platelet like crystals were grown during modal membrane studies
(interaction with micelle) in D2O TTAB micelle solution. The selected suitable crystal was
analyzed by Bruker kappa APEXII CCD diffractometer equipped with a graphite-
monochromated Mo-Kα radiation (λ = 0.71073 Å) radiation. Data collection used ω scans,
and a multi-scan absorption correction was applied. The compound E was crystallized as
[2(C16H15NFe)O] (2E.O) in monoclinic crystal system with space group P-21, the unit cell
dimensions are a = 12.8048(19) Å, b = 6.0256(9) Å, c = 16.977(3) Å, = = 90o, =
91.990(4)o, volume = 1309.1(4) Å
3.
Other crystallographic parameters generated, using Full-matrix least-squares on F2
refinement method at 296K, are formula weight 570.28, Z = 2, density = 1.447 g/cm3, crystal
size = 0.33 x 0.11 x 0.05 (mm3), index ranges h, k, lmax (18, 8, 24) and h, k, lmin (-18, -8, -24),
F(000) = 592.0, total reflections = 7677, Mu = 1.136 mm-1
, R indices (all data) R1 = 0. 0923,
wR2 = 0.1647, final R indices [I>2(I)] R1 = 0.0537, wR2 = 0.1373, goodness-of-fit = 0.880,
and θ range for data collection = 2.03o – 30.52
o.
75
Table 3.11: Selected bond lengths, bond angles and torsion angles for 3-ferrocenylaniline (E)
Bond lengths (Å) Bond angles (°)
N1–C28 1.393(7) N2–C8–C7 120.07(5)
N1–H1B 0.860(4) C9–C4–C1 121.04(4)
N1–H1A 0.860(5) C4–C1–Fe1 125.53(3)
C25–C16 1.487(6) C11–Fe1–C13 111.05(3)
C16–Fe2 2.055(4) N1–C28–C29 120.01(4)
Fe2–C22 2.026(7) N1–C28–C24 121.18(4)
C18–C19 1.411(8) C25–C16–C17 127.62(4)
C31–C30 1.420(9) C25–C16–Fe2 126.75(2)
C24–C28 1.389(7) C18–Fe2–C31 109.07(2)
C4–C1 1.470(7) Torsion angles (°)
C8–N2 1.407(8) N1–C28–C24–C25 -177.30(4)
N2–H2A 0.860(5) C24–C25–C16–C17 -6.37(7)
N2–H2B 0.860(5) C24–C25–C16–C20 173.09(4)
C1–Fe1 2.043(5) C25–C16–Fe2–C22 -11.35(6)
C15–Fe1 2.001(9) C26–C25–C16–Fe2 -97.31(5)
C11–C10 1.435(9) N1–C28–C29–C27 177.91(4)
C9–C8 1.388(8) C30–Fe2–C16–C25 66.36(7)
C15–C14 1.310(9) C24–C25–C16–Fe2 83.97(5)
The crystal structure of 2E.O is outlined in Figure 3.17(X). The selected bond lengths,
bond angles and torsion angles are given in Table 3.11. The cyclopentadienyl ring A (C1 –
C3, C10 & C11) and phenyl ring B (C4 – C9) are planar with r.m.s. deviations of 0.0031 and
0.0027 Å, respectively. The dihedral angle between rings A and B is 6.94(23)° which shows
that, in comparison to the respective nitro derivative (e), the phenyl ring is more planar with
attached cyclopentadienyl ring in the aniline derivative (E). The Fe atom is at a distance of
1.648(17) Å from the centroid of ring A. Cyclopentadienyl ring of ferrocene not attached
with phenyl is disordered over two sets of sites. NH---O type hydrogen bonding exists with
O---H, N–H, and N---O bond distances are 2.221, 0.860 and 3.018 Å respectively. The N–H–
O angle is 154.08°.
76
X
Y Z
Figure 3.17: X) ORTEP diagram of the E with the atomic numbering scheme. Y) The
interaction of entrapped oxygen by H-bonding and NO type coordination is represented by
dotted lines, it shows that oxygen is nearly perpendicular to the nitro plane. Z) The
supramolecular arrangement of compound E helices due to screw symmetry along the
crystallographic axis-b.
In the packing of crystal an oxygen atom is entrapped due to NH---O type H-bonding
and NO type coordination of oxygen. Oxygen is almost perpendicular to NH2 planes of two
molecules and the perpendicular distances being 2.923 Å and 2.515 Å. The packing diagram
of E (Figure 3.17 (Z) shows a helical chain structure mediated by NH---O type H-bonding
and NO type coordination. Here chain molecules are helices due to screw symmetry along
the crystallographic axis-b.
77
3.5.3 Crystal structures of Ferrocenyl thioureas
The crystals of Ferrocenyl thioureas were developed by re-crystallization in different
solvents and suitable crystals were selected for solid state X-ray diffraction studies. The
ORTEP representations of molecular structure of compounds At4 and Ct5 are given in
Figures 3.18(Y) and 3.19(X). The intermolecular hydrogen bonding and Van der Waal
interactions present in these compounds are drawn in Figures 3.18(Z) and 3.19(Y, Z).
3.5.3.1 1-(4-(ferrocenyl)phenyl)-3-allylthiourea (At4)
The orange brown colored, needle like crystal was analyzed by Bruker kappa APEXII
CCD diffractometer equipped with a graphite-monochromated Mo-Kα radiation (λ = 0.71073
Å). Data collection used ω scans, and a multi-scan absorption correction was applied. The
molecular structure is given in Figure 3.18(Y). The crystallographic parameters are given in
Table 3.12; the selected bond lengths, bond angles and torsion angles, in Table 3.13. The
intermolecular hydrogen bonding interactions are summarized in Table 3.14.
The phenyl ring A (C5–C10) and cyclopentadienyl ring B (C11–C15) are planar with
r.m.s. deviations of 0.0017 and 0.0012 Å respectively. The dihedral angle between ring A and
B is 28.53° The Fe atom is at a distance of 1.654 Å from the centroid of ring B. The
intermolecular H-bonding of N—H···S type exist to stabilize the molecule in the form of
polymeric chains extending along the crystallographic axis-b. In these chains, molecules are
connected in stairs like structure as shown in Figure 3.18(Z). The capability of forming such
secondary interactions is favorable for a compound to bind with biological macro molecules
like proteins and DNA.[135]
78
Y
Z
Figure 3.18: Y) ORTEP diagram of At4 with the atomic numbering scheme. Z) The
supramolecular arrangement of compound At4 along crystallographic axis-b, presenting stairs
like structure.
79
Table 3.12: Crystal data and structure refinement parameters for At4 and Ct5
Crystal Parameters At4 Ct5
Empirical formula C20H20N2SFe C24H22N2OFeS .
C3H6O
Formula weight 376.30 500.43
Temperature (K) 296(2) 296(2)
Wavelength (Å) 0.71073 0.71073
Crystal system Monoclinic Monoclinic
Space group P 21/c P 21/c
Unit cell
dimensio
ns
a(Å) 21.060(3) 27.4038(10)
b(Å) b=8.0823(10) 7.7445(3)
c(Å) c=9.9972(12) c=22.0818(8)
(o) 90 90
(o) 95.374(3) 94.077(2)
(o) 90 90
V(Å3), Z 1694.2(4), 4 4674.5(3), 8
Density (g/cm3) 1.475 1.422
Crystal size (mm3) 0.23 x 0.15 x 0.02 0.46 x 0.28 x 0.16
Index ranges
-27<=h<=27
-10<=k<=10
-13<=l<=13
-42<=h<=42
-11<=k<=11
-25<=l<=33
F(000) 784.0 2096
Total reflections 3999 17633
Refinement method Full-matrix least-
squares on F2
Full-matrix least-
squares on F2
Mu (mm-1
) 1.016 0.762
R indices (all data) R1 = 0.1495,
wR2 = 0. 4380
R1 = 0.1051,
wR2 = 0.1650
Final R indices
[I>2(I)]
R1 = 0. 1374,
wR2 = 0.4344
R1 = 0.0480,
wR2 = 0.1244
Goodness-of-fit 3.492 0. 903
θ range for data
collection (o)
1.94 to 27.84 1.85 to 33.14
80
Table 3.13: Selected bond lengths, bond angles and torsion angles for ferrocene based
thioureas (At4 & Ct5)
Bond Lengths Bond Angles Torsion Angles
At4
Fe–C1 2.041 C1–Fe–C6 106.61 C7–C6–C11–C12 28.07
Fe–C6 2.053 Fe–C6–C11 127.05 C7–C6–C11–C16 152.20
C6–C11 1.488 C1–C2–C3 108.91 C6–C11–C12–C13 177.54
C19–C20 1.322 C7–C6–C10 108.53 C13–C14–N1–C17 152.85
C17–S 1.689 C6–C11–C12 121.93 C14–N1–C17–S 0.180
C14–N1 1.410 C14–N1–C17 130.92 C14–N1–C17–N2 179.81
N1–C17 1.361 N1–C17–S 125.89 N1–C17–N2–C18 169.97
C17–N2 1.359 S–C17–N2 121.98 S–C17–N2–C18 9.67
N2–C18 1.462 C17–N2–C18 124.53 C17–N2–C18–C19 179.75
Ct5
Fe – C1 2.049 C1 – Fe – C6 109.96 C7 – C6 – C11 – C12 24.29
Fe – C6 2.074 Fe – C6 – C11 128.71 C7 – C6 – C11 – C16 159.91
C6 – C11 1.483 C1 – C2 – C3 108.04 C6 – C11 – C12 – O1 5.79
C12 – O1 1.370 C7 – C6 – C10 107.01 C13 – C14 – N1 – C17 121.83
C17 – S1 1.690 C6 – C11 – C12 123.70 C14 – N1 – C17 – S1 173.62
C14 – N1 1.427 C14 – N1 – C17 125.95 C14 – N1 – C17 – N2 8.24
N1 – C17 1.350 N1 – C17 – S1 122.84 N1 – C17 – N2 – C18 171.66
C17 – N2 1.356 S1 – C17 – N2 124.75 S1 – C17 – N2 – C18 10.30
N2 – C18 1.417 C17 – N2 – C18 128.19 C17 – N2 – C18 – C19 43.35
Table 3.14: The inter-molecular hydrogen bonds in ferrocene based thioureas (At4 & Ct5)
Compound X H Y d(X–H)
(Å)
d(H--Y)
(Å)
d(X--Y)
(Å)
<(XHY)
(˚)
At4 N1 H1 S1 0.860 2.632 3.443 157.53
N2 H2 S1 0.860 2.523 3.371 168.80
Ct5 N1 H1 S1 0.859 2.696 3.480 152.27
N2 H2 O2 0.859 2.224 3.045 159.91
H --- H van der Waals interaction 2.096 Å
3.5.3.2 1-(4-(ferrocenyl)-3-methoxyphenyl)-3-phenylthiourea (Ct5)
The orange brown colored, leaf like crystal was analyzed by Bruker kappa APEXII CCD
diffractometer equipped with a graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å).
Data collection used ω scans, and a multi-scan absorption correction was applied. The
molecular structure is given in Figure 3.19(X). The crystallographic parameters are given in
Table 3.12; the selected bond lengths, bond angles and torsion angles, in Table 3.13. The
intermolecular interactions are reported in Table 3.14.
81
X Y
Z
Figure 3.19: X) ORTEP diagram of Ct5 with the atomic numbering scheme. Y) The
intermolecular H-bonding represented by dotted lines enabling the acetone molecule
attachment and dimer formation. Z) The supramolecular arrangement of compound Ct5
mediated by intermolecular H---H Van der Waals interactions.
The phenyl rings A (C18 – C23), B (C11 – C16) and cyclopentadienyl ring C (C6–
C10) are planar with r.m.s. deviations of 0.0017, 0.0013 and 0.0024 Å respectively. The
dihedral angle between rings B and C is 24.21°. The dihedral angle between rings B and C
increased in comparison to nitro intermediate (c) in which the dihedral angle is 22.42°, which
shows on conversion into thiourea, aromatic delocalization is decreased and planarity lost.
The Fe-atom is at a distance of 1.653 Å from the centroid of ring C. The intermolecular H-
bonding of N—H···S type exist to stabilize the molecule in the dimmer formation (Figure
3.19(Y)). H-bonding of NH --- O type also exists which traps acetone molecules during
crystallization. There also exists H---H non bonding Van der Waal interaction which
promotes the supra-molecular structure formation as shown in Figure 3.19(Z). The existence
of such interactions enables these molecules to bind with biological systems like proteins and
DNA.[149]
It was observed that molecule (Ct5) with aromatic groups of the both thiourea
nitrogen’s exhibit stronger secondary interaction forces in comparison to (At4) which has one
82
aromatic group and one aliphatic substituent. On the basis of such observations, stronger
DNA binding abilities of compounds with stronger secondary interaction forces are expected.
83
Chapter 4
Biological Studies
4.0 Biological Screening
The progress in metallopharmaceuticals is one of the very valuable strategies for the
acceleration of medicinal chemistry. Iron being an essential metal for animals and non-toxic
in controlled quantity is implicated in many diseases.[157]
Substituted thioureas are also well-
known physiologically active compounds having a broad spectrum of biological activity as
discussed in chapter 1. Some of the synthesized ferrocene based ureas and thioureas were
investigated for their anti-oxidant activity, DNA binding ability and modal membrane
interaction behavior to evaluate their anticancer potency before moving towards expensive
living cell line studies, taking into account the available facilities.
4.1 Modal membrane interaction studies
According to Fluid Mosaic model the cell membrane is made up of the zwitter ionic
surfactants (phospholipids), those have hydrophobic and hydrophilic regions.[158]
These
phospholipids align in the bi-layers form and make the living cell membrane in which
proteins and other membrane components are impregnated. The formation of such
membranes in the laboratory is not an easy job. However different surfactants having a
hydrophobic tail and hydrophilic head are used in the laboratory to make bio-mimetic
membranes. By using such surfactants negative, positive, neutral or even zwitterionic
membranes can be made.[137]
Figure 4.01: General micelle structure and surfactants used for modal membrane formation
We have used CTAB, TTAB and SDS surfactants in water. These surfactants make
micelle in water, these are polar sphere having a hydrophobic core as shown in Figure 4.01
these have resemblance with the cell membrane. CTAB and TTAB make positive membrane
where as SDS forms a negative membrane. Biomemtic modal membranes of CTAB, TTAB
and SDS have been made to check the ability of penetration of these potential drug
84
molecules. These potential drug molecules have been probed by using modern sophisticated
techniques like UV-Visible and 1HNMR spectroscopy.
UV-Visible spectroscopy is a useful technique for probing ferrocene and its
derivative. Ferrocene and its derivatives are highly sensitive to oxidation and shows a
remarkable change in the UV-Visible spectrum. On oxidation ferrocene gives entirely
different colored ferrocenium ions with one positive charge overall on the complexes with Fe
(III) oxidation state which have one unpaired electron that gives d-d transition in the region
of 650 – 750nm with a deep green color where as ferrocene itself has brown color. All the
synthesized compounds are insoluble in water and give no peak when scan for UV-Visible
spectrum in water. These compounds show uptake in the micelle membranes of CTAB,
TTAB and SDS. And give characteristic bands in 1HNMR and UV-Visible spectrum when
probed in CTAB and TTAB micelle. Initially CTAB have been used as a positive membrane
for selected compounds (a, A, At5 and Au5) and the probing were done at 37 °C (well above
the micelle formation temperature of CTAB). Afterward CTAB was replaced with TTAB to
overcome temperature complication and probing were done at 25 °C for TTAB and SDS.
Following four different positions available for a molecule in the micelle membrane solution
are shown in Figure 4.01
(1) Solvent (D2O) far outside the micelle
(2) Just outside micelle in the form of adduct
(3) Just on the interface inside the micelle
(4) At the core of the micelle non-polar region
Nitrophenylferrocenes (a – e); Lambda max shifting data is shown in Figure 4.02,
there appears a new band at about 650 – 715 nm for compounds a – d, when these are probed
in the SDS negative membrane. This new band is assumed for the oxidation of the ferrocene
into ferrocenium specie. Which is then proved by the UV-Visible spectral studies of
ferrocene and ferrocenium ion in accordance with the literature.[159]
It is interesting to see that
the oxidation band has been observed only for compounds a – d, but not for the compound e.
This band is the argument that the push pull systems support the oxidation of ferrocene into a
ferrocenium ion by the strong delocalization of pi electronic cloud due to the presence of
strong electron with-drawing nitro group at the para position to the ferrocene. It seems that
the delocalized π – electron cloud facilitate the un-pairing of paired electrons, which cannot
be possible in e (Meta substituted nitro derivative) that is in accordance with classical organic
chemistry.
85
Figure 4.02: UV-Visible spectrum of nitrophenylferrocenes in different micelle membranes
and water after 12 hour sonication. a) for compound a, b) for d, c) for b, d) for c, e) for e
and f) is the HNMR spectra of a – e in TTAB micelles made in D2O, 1 – 5 for a – e
respectively
The probing of these compounds has also been done by using HNMR spectroscopy
along with UV-Visible spectroscopy. NMR is highly sensitive technique in this regard. It
gives a lot of information about the probe qualitatively as well as quantitatively. In this
regard, qualitative information relates to the position of the probe in/or outside of micelle
membrane. The relative integration value reveals the number of molecules per micelle and
the extent of a molecule to penetrate into the micelle relative to the other molecules. In other
words, it means the lipophilic ability of the molecule in reference to the other molecules.
86
Table 4.01: 1HNMR spectral data of a – e in different solvents, CTAB and TTAB micelles
*
Molecule Ferroocene region Aromatic region R group No. of probe
Molecules
per micelle** Cp ring
(s, 5H)
Cp ring
(t, 2H)
Cp ring
(t, 2H)
a in CDCl3 4.047 4.465 4.734 7.549, 8.132 -
a in Acetone-d6 4.084 4.524 4.961 7.798, 8.153 -
a in iso-octane*** 4.034 4.417 4.699 7.530, 8.145 -
a in cyclohexane-d12
3.947 4.333 4.616 7.459, 8.060 -
a in DMSO-d6 4.063 4.482 4.739 8.141, 7.564
a in CTAB 4.128 ----- 4.928 7.751, 8.201 1.04
a in 75mM CTAB 4.127 ----- 4.926 7.748, 8.201 -
a in TTAB 4.135 4.614 4.937 7.759, 8.210 0.94
b in CDCl3 4.230 4.506 4.693 7.618, 7.954-7.999 CH3 (2.525) -
b in DMSO-d6 4.234 4.514 4.695 7.95 – 8.00, 7.63 CH3 (2.526)
b in TTAB 4.197 4.559 4.903 7.993, 8.033, 8.108 CH3 (2.592) 1.55
c in CDCl3 4.058 4.425 4.869 7.607, 7.741-7.814 OCH3 (4.013) -
c in DMSO-d6 4.063 4.425 4.874 7.790 – 7.814, 7.613 OCH3 (4.014)
c in TTAB 4.111 4.526 4.996 7.764, 7.842, 7.863 OCH3 (4.065) 1.21
d in CDCl3 4.273 4.585 4.994 7.730, 8.012, 8.208 Cl -
d in DMSO-d6 4.27 4.59 4.99 8.21, 8.01 7.73 Cl
d in TTAB 4.229 4.643 5.096 8.140, 8.195, 8.295 Cl 1.39
e in CDCl3 4.069 4.419 4.727 7.440, 7.763, 8.026,
8.279
- -
e in DMSO-d6 4.074 4.421 4.732 8.280, 8.033, 7.761,
7.444
e in TTAB 4.113 4.482 4.878 7.640, 8.069, 8.139,
8.258
- 2.86
*All the micelle solutions for NMR study were made in D2O with 100mM surfactant concentration otherwise mentioned.
**Number of probe molecules per micelle calculated by considering aggregation number for TTAB = 100[160]
***NMR spectra of iso-octane solution were taken by locking and shimming on cyclohexane-d12
The chemical shift values and the quantitative data of compounds are given in the
Table 4.01. The quantitative data shows that the compound e (having 2.86 molecules per
micelle) has the highest lipophilic nature among the five compounds. It might be due to the
presence of nitro group at the meta position which decreases the dipole moment (qualitatively
we can judge that e should have least dipole moment among these) and make the molecule
non-polar. The change of the substitution on the phenyl ring disturbs the planarity between
the phenyl and Cp ring of the molecule which results in the change in polarity and hence the
lipophilic behavior. On the basis of the change in the chemical shift value comment on the
position of the probe molecule in the micelle membrane system can be made.
Results (given in Table 4.01 and shown in Figure 4.02) infer that these molecules
might be present inside the micelle membrane but in the polar region (at position 3 in Figure
4.01), because of the downfield shifting of the proton resonating frequency in reference to the
chemical shift value of the probe in different solvents (for compound a); and in CDCl3 for all
the other compounds. It can be noticed that ferrocene protons show less downfield shifting as
compared to the aromatic protons which suggests the orientation of nitrophenyl group
towards the interface. On comparing CTAB and TTAB for compound (a) 1HNMR data reveal
87
that probe molecule feels more acidic environment in the TTAB micelle membrane. It might
be due to the smaller size of TTAB as compared to CTAB micelle and the positive membrane
near to the probe molecule. From this comparison it can also be concluded that these
molecules are inside the micelle and do not exist on the surface of the micelles as the adduct
form via ionic interaction between them. (Like position (2) in Figure 4.01; if such kind of
adduct exist then there must be no change in chemical shift of probe a on changing CTAB to
TTAB)
In SDS micelle membrane none of the probe molecules band appears in the NMR
spectrum. It provides supporting evidence for the proposed oxidation of the ferrocene into
ferrocenium specie which makes the probe paramagnetic and NMR inactive that result in the
absence of the probe band in NMR spectra. For SDS micelle, one can think about the
oxidation of the probe due to the protonation of the ferrocene which generates ferrocenium
due to the presence of normal RSO3H impurity in the SDS surfactant. But it must be the least
probable chance because the SDS purified using a reported protocol.
In the same way ferrocenyl anilines were studied to check their ability to penetrate in
biomemtic membrane, ferrocenyl anilines (A, B and E) have been probed by using 1HNMR.
The data for chemical shift and the measure of quantitative permeability of these molecules is
given in Table 4.02; and the spectral changes in the NMR spectra are pictured in Figure 4.03;
data reveal that the proton of free Cp-ring and one pair of protons in bounded Cp-ring in
these compounds resonate up-field in reference to their chemical shift DMSO and CDCl3 that
is the indication of their presence in non-polar region of the micelle.
Figure 4.03: HNMR spectra of A, B and E in micelles made in D2O
88
The quantity of these compounds moved into the micelles is in the similar order as for
nitro analogs but these molecules have much more quantity permeated. In case of anilines (A,
B and E) it was observed that large quantities get suspended in the colloidal solution.
Solutions were transparent and from these solutions it was observed that there might exist an
equilibrium among the solid in the container and the molecules moved into the micelles core.
It was evident from the fact that on long time (3-4 week) standing, E was crystallized in the
micelle solution. The crystals formed in this way were well shaped and suitable for single
crystal analysis. In this specific case, E was found crystallized by trapping atomic oxygen
that was stabilized by the coordination of aniline nitrogen (N O) and N – H---O type
hydrogen bonding as shown in Figure 4.04.
This oxygen might be the dissolved oxygen present in D2O used[161-163]
and entrapped
into aniline molecules during the crystallization process. It might be the oxygen radicals that
can harm the micelle membrane and entrapped in anilines during moment of E for
crystallization. In any case, it can be confidently comment that these molecules have ability
to capture the harmful reactive oxygen present near the modal membrane. So, in the same
way these molecules have potential to protect the lipid cell membrane that is similar to these
model micelle membranes.
Table 4.02: 1Hnmr spectral data of A, B & E in different solvents, CTAB and TTAB
micelles*
Molecule Ferroocene region Aromatic region NH2/R group No. of probe
Molecules
per micelle** Cp ring
(s, 5H)
Cp ring
(t, 2H)
Cp ring
(t, 2H)
A in CDCl3 4.036 4.242 4.544 6.639, 7.295 3.623 (NH2) --
A in DMSO-d6
4.056 4.257 4.559 6.671, 7.334 3.664 --
A in CTAB 4.044 4.204 -- 6.772, 7.332 -- 15.055
A in TTAB (Two sets of Signals)
4.044
3.698
4.205
4.101
4.629
4.363
6.776, 7.334
6.652, 7.010
-- 13.604
1.60
B in DMSO-d6 4.121 4.233 4.394 6.495, 6.543, 7.501 3.615 (NH2) /
2.304 (CH3)
--
B in CDCl3 4.117 4.229 4.392 6.491, 6.538, 7.497 3.611 (NH2) /
2.306 (CH3)
--
B in TTAB 4.090 4.181 4.449 6.599, 6.651, 7.478 2.341 (CH3) 16.55
E in DMSO-d6 4.052 4.285 4.595 6.543, 6.822, 6.924,
7.095
3.674 (NH2) --
E in CDCl3 4.051 4.281 4.595 6.539, 6.819, 6.918,
7.086
3.673 (NH2) --
E in TTAB 4.054 4.267 4.681 6.671, 6.739, 6.996,
7.104
16.33
*All the micelle solutions for NMR study were made in D2O with 100mM surfactant concentration otherwise mentioned.
**Number of probe molecules per micelle calculated by considering aggregation number for TTAB = 100[160]
1HNMR of the compound A in TTAB micelle shows there appears two groups of
signals for it. One group at (4.044, 4.205, 4.629, 6.776 and 7.334 ppm) and the other group of
signals for complete molecule appear at (3.698, 4.101, 4.363, 6.652 and 7.010 ppm). These
89
signals for the same compound at different positions in the same solution suggest the
presence of these molecules in the different environments. From the chemical shift values of
these groups it can be concluded that some of the molecules are at the core of the micelle and
some other molecules are at polar region of the micelle. These different positions exert
different environments on the molecules. The data show that more molecules are present in
the polar regions with high chemical shift values and less number of molecules present in the
non-polar region of the micelle. It is in agreement with the fact that the polar region (the
interfacial region) of the micelle has more area as compared to the non-polar region (core) of
the micelle (Figure 4.01). So location 3 and 4 are the potential positions for these compounds
and are justified from the NMR data.
For anilines in addition to these four locations there is another possibility which is the
formation of ammonium ion and become soluble in water. But this possibility can be rejected
on the basis of crystallographic studies which provide evidence that no ammonium salt is
formed in this case as aniline E is crystallized as such having trapped oxygen. In this case the
oxygen is trapped by H-bonding and coordination with nitrogen (N O). If the ammonium
ion forms the electronic charge density on nitrogen changes and behaves differently in
XRD.[164, 165]
Another possibility in this case is the ammonium salt would form with bromide
counter ion not the oxygen on the bases of statistical availability. The bromide ion
concentration (0.1M) is much higher than the atomic oxygen (7-10ppm) in these
solutions.[163]
Figure 4.04: Crystal packing of trapping atomic oxygen that is stabilized by the coordination
of aniline nitrogen (N O) and N – H---O type hydrogen bonding
90
Hence conclusively it can be argued that anilines penetrate in micelle membrane and
exist in non-polar regions of the micelle and also have a much higher ability of permeation
into micelles than the corresponding nitro compounds. This permeability may be due to the
less dipole creation in the aniline. Qualitatively we can judge the dipole moment of anilines
(A, B and E) must be less than the nitro analogue on the basis of classical organic chemistry
as nitro derivatives are the good examples of π-electronic push–pull system.
Ferrocenyl ureas and thioureas have also been studied to check their ability of
penetration into cell membrane using biomemtic model membrane and probed by UV-
Visible and proton NMR spectroscopy.
Figure 4.05: UV-Visible spectrum of selective ferrocenyl thiourea At4 (left) and urea Au5
(right) in (–) DMSO, different micelle membranes (–) TTAB, (–) SDS and (–) water after
12 hour sonication
These molecules have been tested on the positive membrane of TTAB as well as on
the negative membrane of SDS. UV-Visible probing shows that these compounds are
insoluble in water and behave differently in different membrane. Evidences from the λmax
shifting, in different solvent and micelle membranes indicate that ferrocenyl thioureas and
ureas have the ability of permeation into membrane and are not at the core of the micelle (the
similar region in both micelle). So these molecules present in the interfacial region inside the
micelle position (3) shown in Figure 4.01. These molecules have also been probed by
1HNMR. The
1HNMR data are summarized in Table 4.03 and for selected compounds the
spectra are drawn in the Figure 4.06 and 4.07.
The 1HNMR data support the position of probe molecules suggested by the UV-
Visible spectroscopy. Comments of similar kind can be made for the position of ferrocenyl
thioureas and ureas as for nitrophenylferrocenes and ferrocenylanilines. The ability of
permeation into the membrane (Number of probe molecules per micelle) of ferrocenyl
thioureas and ureas, it is argued that all the tested compounds show permeation into modal
membrane with no significant variation in the permeation number (number of probe
molecules per micelle).
91
Figure 4.06: HNMR spectrum of ferrocenyl urea Au5 in TTAB micelle membranes and
DMSO-d6 (upper)
Figure 4.07: HNMR spectra of selective series ferrocenyl thioureas in TTAB micelle
membranes
The data obtained show that thiourea molecules are more capable than ureas in
membrane penetration. The more penetration of thioureas is due to their less polar character
then the ureas. Oxygen in ureas increases the polarity of these molecules. The selective
thiourea (At5) in TTAB membrane has also been probed by 2D NMR NOESY experiment to
check the interaction between the thiourea molecule and the surfactant molecules. Result
pictured in Figure 4.08 shows no cross peak in the spectrum indicating that there is no
92
binding interaction between At5 and the surfactant molecules and hence it is the inherent
ability of these molecules to penetrate into the modal membrane.
Figure 4.08: NOESY 2D NMR spectrum of ferrocenyl thiourea (At5) in TTAB micelle
membranes in D2O
93
Table 4.03: 1Hnmr spectral data of ferrocenyl thioureas and ureas in DMSO-d
6 and TTAB
micelles*
Molecule Ferroocene region Aromatic region Other
group
No. of
probe
Molecules
Per micelle*
Cp ring
(s, 5H)
Cp ring
(t, 2H)
Cp ring
(t, 2H)
At1 in DMSO 4.025 4.322 4.734 6.947,7.396, 9.321, 7.502,
2.671
At1 in TTAB 4.026 4.160 6.593, 7.297 1.54
At2 in DMSO 4.033 4.324 4.741 7.312,7.510 – 7.434, 9.391, 7.702,
3.444 – 3.553,
1.121
At2 in TTAB 4.087 4.333 7.510, 7.663 3.606 1.95
At3 in DMSO 4.034 4.331 4.745 7.323,7.471, 9.391, 7.712,
3.422, 1.634 –
1.487, 0.891
At3 in TTAB 4.092 4.358 7.518, 7.653 1.15
At4 in DMSO 4.032 4.331 4.742 7.360 – 7.312,7.512 –
7.460,
9.501, 7.813,
5.901, 5.1544,
4.152
At4 in TTAB 4.091 4.348 7.521, 7.685 4.278, 5.287,
6.005
1.59
At5 in DMSO 4.032 4.331 4.754 7.134,7.531 – 7.290, 9.761
At5 in TTAB 4.109 4.384 4.784 7.266, 7.436, 7.561, 7.780 0.25
At6 in DMSO 4.022 4.341 4.763 7.424, 7.519,7.823, 8.202, 10.361, 10.250
At6 in TTAB 4.110 4.412 4.796 6.825, 7.575, 8.021, 8.293 -- 1.09
Bt3 in DMSO 4.130 4.324 4.523 7.132, 7.211, 7.610, 9.351, 7.702,
3.423, 2.321,
1.614 – 1.501,
0.890
Bt3 in TTAB 4.175 4.361 4.606 7.480, 7.531, 7.692 0.59
Bt4 in DMSO 4.132 4.330 4.523 7.141, 7.234, 7.620, 9.461, 7.793,
5.905, 5.150,
4.140– 4.168,
2.330
Bt4 in TTAB 4.169 4.346 4.599 7.513, 7.569, 7.692 4.278, 5.284,
6.004
1.28
Bt5 in DMSO 4.130 4.332 4.534 7.661 – 7.070 9.721, 2.33
Bt5 in TTAB 4.179 4.372 4.622 7.222, 7.414, 7.716, 7.782 0.41
Bt6 in DMSO 4.129 4.341 4.554 7.234, 7.312, 7.661, 7.840
, 8.201
10.330,
10.212, 2.345
Bt6 in TTAB 4.080 4.308 4.597 6.851, 6.893, 6.941, 7.956
– 8.105
1.58
Ct2 in DMSO 4.031 4.274 4.723 6.873, 7.150, 7.485, 9.451, 7.770,
3.834, 3.505,
1.132
Ct2 in TTAB 4.031 4.162 -- 6.404, 6.600, 7.303 3.893, -- 0.58
Ct3 in DMSO 4.034 4.273 4.720 6.346 – 6.930, 7.185,
7.461,
9.440, 7.771,
3.834, 3.445,
1.620 – 1.514,
0.904
Ct3 in TTAB 4.067 4.300 4.841 7.182, 7.514, 7.727 3.922 2.07
Continued . . .
94
Molecule Ferroocene region Aromatic region Other
group
No. of
probe
Molecules
Per micelle*
Cp ring
(s, 5H)
Cp ring
(t, 2H)
Cp ring
(t, 2H)
Ct4 in DMSO 4.035 4.274 4.723 6.907, 7.205, 7.471, 9.551, 7.862,
5.910, 5.164,
4.159, 3.833
Ct4 in TTAB 4.062 4.288 4.839 7.228, 7.516, 7.743 3.920, 4.276,
5.291, 6.007
2.68
Ct5 in DMSO 4.032 4.284 4.730 7.010 – 7.510 9.791, 9.770,
3.834
Ct5 in TTAB 4.032 4.267 4.824 7.209, 7.260, 7.385,
7.520, 7.760, 7.868
3.899 8.31
Ct6 in DMSO 4.033 4.292 4.751 7.044, 7.263, 7.510, 7.804
– 7.863 , 8.16 0– 8.241,
10.361,
10.294, 3.843
Ct6 in TTAB 4.071 4.332 4.878 6.806, 7.606, 8.005, 8.299 3.939 2.83
Et5 in DMSO 4.063 4.341 4.714 7.132, 7.210 – 7.544,
7.680,
9.781, 9.769
Et5 in TTAB 4.105 4.328 4.790 7.190 – 7.169, 7.749 –
7.806, 8.175
4.37
Et6 in DMSO 4.054 4.352 4.721 7.190 – 7.360,
7.683,7.861, 8.210,
10.381, 10.262
Et6 in TTAB 4.113 4.346 4.776 6.809, 7.373, 7.462,
8.009, 8.296
3.23
Au3 in DMSO 4.032 4.274 4.723 7.3217.321,7.632, 9.440, 7.964,
3.443, 1.620 –
1.512, 0.904
Au3 in TTAB 4.060 4.275 4.813 6.962, 7.229, 0.48
Au4 in DMSO 4.034 4.361 4.793 7.350 – 7.585 9.610, 7.749,
5.964, 5.010,
4.123
Au4 in TTAB 4.059 4.176 4.712 6.863, 7.127, 7.360 4.276, 5.291,
6.007
0.48
Au5 in DMSO 4.063 4.344 4.712 7.210 – 7.544,7.680, 9.783, 8.771
Au5 in TTAB 4.073 4.330 -- 7.045, 7.352, 7.501, 7.659 1.19
Au6 in DMSO 4.022 4.314 4.731 7.400 – 7.474, 7.721,
8.193,
10.091, 9.482
Au6 in TTAB 4.036 4.304 7.496, 7.634, 7.920, 8.246 3.85
Bu3 in DMSO 4.034 4.333 4.742 7.133, 7.211, 7.608, 9.391, 8.132,
3.191, 2.324,
1.491 – 1.634,
0.893
Bu3 in TTAB 4.041 4.296 4.760 6.909, 7.176, 7.565,
7.918, 8.057
0.13
Bu6 in DMSO 4.052 4.354 4.721 7.250 – 7.363, 7.681,
7.857, 8.210,
10.381, 9.263,
2.344
Bu6 in TTAB 4.044 4.254 4.476 6.630, 6.809, 6.881,
7.451, 7.963
2.58
Cu2 in DMSO 4.031 4.324 4.773 6.974, 7.252, 7.560, 9.561, 7.864,
3.213, 1.184
Cu2 in TTAB 4.068 4.284 4.840 7.225, 7.511, 7.709 3.926, 3.621 0.34
Continued . . .
95
Molecule Ferroocene region Aromatic region Other
group
No. of
probe
Molecules
Per micelle*
Cp ring
(s, 5H)
Cp ring
(t, 2H)
Cp ring
(t, 2H)
Cu3 in DMSO 4.134 4.320 4.520 7.133, 7.214, 7.610 9.350, 7.961,
3.934, 3.422,
1.610– 1.504,
0.888
Cu3 in TTAB 4.054 4.462 4.363 7.165, 7.531, 7.831 0.31
Cu5 in DMSO 4.034 4.291 4.780 7.034 – 7.912 10.150, 9.554,
3.853
Cu5 in TTAB 4.065 4.327 4.836 7.204, 7.371, 7.536,
7.675, 7.938, 8.013, 8.118
3.964 0.14
Cu6 in DMSO 4.154 4.393 4.750 7.400 - 7.491, 7.855,
8.221,
10.491, 9.868,
3.844
Cu6 in TTAB 4.036 4.541 -- 6.221, 7.084, 7.60, 8.121
Eu5 in DMSO 4.030 4.331 4.749 7.125,7.352, 7.409, 7.471
– 7.501
9.759
Eu5 in TTAB 4.106 4.381 -- 7.263, 7.434, 7.558, 7.780 0.80
Eu6 in DMSO 4.134 4.341 4.550 7.190 – 7.358, 7.663,
7.844, 8.201
10.331, 8.510
Eu6 in TTAB 4.036 4.304 -- 7.496, 7.634, 7.920, 8.246 3.85
4.2 Antioxidant Activity (DPPH Free Radical Scavenging Assay)
Antioxidants have the capability to capture free radicals. Highly rash free radicals and
reactive oxygen species (ROS) are being found in biological systems from different sources.
These free radicals are liable for the oxidation of nucleic acids, proteins, lipids or DNA and
can initiate degenerative disease like cancer. Table 4.04 contains the list of different ROS
with their normal physiological concentrations and half lives.
Table 4.04: Some ROS with their normal physiological concentrations and half lives[166-168]
Reactive Specie - ROS
Physiological half life (s) Physiological concentrations
(μM)
Hydroxyl radical (OH) 10
-9
Alcoxyl radical (RO) 10
-6
Peroxynitrite anion (ONOO-
)
0.05 – 1.0 ~ 10-3
Peroxyl radical (ROO) 7 10
-4 – 10
-2
Nitric oxide (NO) 1 – 10 10
-3
Hydrogen peroxide (H2O2) Hours–days (accelerated
by enzymes)
10-3
– 10-1
Superoxide anion (O2-
) Hours–days 10-6
– 10-5
Hypochlorous acid (HOCl) Depanding upon substrate
Under normal physiological conditions these free radicals are tightly controlled by
biological antioxidants. The biological antioxidants are enzymatic antioxidants like
superoxide dismutases (SOD), glutathione peroxidase (GPX) and chloramphenicol
96
acetyltransferase (CAT); and non-enzymatic antioxidants like vitamins (A, C & E), thiols,
uric acid, ceruloplasmin, transferrin, phenols and albumin etc. When biological antioxidants
fail, these harmful free radicals increase in conc. And on increasing, these radicals injure the
cells by damaging lipids, proteins, DNA and sugars in the way as shown in the mechanism.
Figure 4.09 shows the mechanism of the different degenerative activities of these risky free
radicals.
Figure 4.09: Mechanism of ROS induced cell injury
Thus it is important to capture ROS after malfunction of natural biological antioxidant
defense system. In this way synthetic antioxidants are used to protect cell injury and
ultimately to encumber the corridor of degenerative diseases.[168]
Several assays have been
repeatedly used to approximate antioxidant capacities of different natural and synthetic
compounds for clinical ram. Assays include the oxygen radical absorption capacity
(ORAC),[169, 170]
2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS),[171, 172]
2,2-
diphenyl-1-picrylhydrazyl (DPPH)[136, 173]
and ferric reducing antioxidant power (FRAP)[174]
methods. The ORAC assay is said to be more relevant because it utilizes a biologically
relevant radical source.[175]
These techniques have shown different results among different
compounds and across laboratories. Awika et al. (2003) and K. Thaipong et al. (2006) have
observed high correlation between ABTS, DPPH, and ORAC methods during the study of a
large number of compounds.[176, 177]
Many other researchers also provide the evidence that the
DPPH assayy can efficiently be used to estimate the antioxidant ability of synthetic
Cell Injury
LIPIDS PROTEINS DNA SUGARS
Lipid
Peroxidation
Oxidation of thiols
Carbonyl formation DNA
damage
Damage to Ca2+
and
other ion transport
systems
ROS potent to induce cell injury
Poly ADP
ribosylation
Altered
gene
expression
Membrane
damage
Instability to
maintain normal ion
gradients
Depletion of ATP
and NAD(P)(H)
Amadori
products
Activation/Deactivation of
various enzyme systems
AGEs (Advanced glycation end products)
97
compounds, because this assay is simple, easy to perform, inexpensive and provides high
degree of precision.[136, 173]
At4 At5 At6 Bt3 Bt6 Bt7 Ct2 Ct3 Ct5 Ct6 Ct7 Dt5 Dt6 Dt7 Et5 Et6 Et70
2
4
Compound Code
IC-5
0 va
lues
(mM
)
Figure 4.10: Antioxidant activity data of selective ferrocenyl thioureas
Antioxidant ability of selective ferrocenyl ureas and thioureas has been estimated
using DPPH free radical scavenging assay in DMSO. The experimental procedure is reported
in experimental section chapter 2. The estimated activities are reported as 50% inhibitory
concentration (IC50) values in graphical Figure 4.10 for ferrocenyl thioureas and Figure 4.11
for ferrocenyl ureas.
Au4 Au5 Au6 Bu3 Bu6 Bu7 Cu2 Cu3 Cu5 Cu6 Cu7 Du5 Du6 Du7 Eu5 Eu6 Eu70
0.7
1.4
Compound Code
IC-5
0 Va
lues
(mM
)
Figure 4.11: Antioxidant activity data of selective ferrocenyl ureas
Results show that among ferrocenyl ureas and thioureas, compounds having aromatic
substituent on both nitrogen show relatively higher activities than the compounds having
aliphatic group on one side. It has also been observed that the insertion of nitro group in the
structure enhances the antioxidant activity. It might be due to the high electron withdrawing
ability of the nitro group which enhances the stability of the end product formed from these
compounds as a result of DPPH reduction.
Figure 4.12 presents the comparison of antioxidant activity between ferrocenyl ureas
and thioureas: Results reveal that ureas have reasonably higher activities than the
corresponding thioureas. It might be due to the more acidic nature of NH proton in ureas
relative to thioureas.
98
A4 A5 A6 B3 B6 B7 C2 C3 C5 C6 C7 D5 D6 D7 E5 E6 E7
0
2
4
IC-5
0 Va
lues
(mM
)
Thioureas Ureas
Figure 4.12: Comparative antioxidant activity of ferrocenyl ureas and thioureas
The IC50 value of Ascorbic acid (a standard drug used) was found to be 30.53µM. The
free radical scavenging profile of compounds (At6, Bt6, Ct7, Dt6, Au6, Bu6, Bu7, Cu7 and
Du6) was found to be fairly impressive as IC50 is less than 60µM for these compounds which
is comparable to the standard drug used as shown in Figure 4.13. Conclusively we can say
that these compounds have low ability to capture free radicals (ROS). But compounds, with
IC50 less than 60µM, are suspected to be used as therapeutic agents.
21.99
13.58
59.57
48.6
17.3311.94
47.57
19.8616.2
30.53
0
30
60
Compound Code
IC-5
0 Va
lues
(µM
)
Figure 4.13: Most active ferrocenyl ureas and thioureas in comparison to ascorbic acid
(Standard drug)
4.3 DNA binding studies
The main cause of several diseases such as cancer, diabetes, hemophilia, etc., is
related to over or under production of proteins or mutated proteins. As DNA is the genetic
material that codes for proteins, therefore drug interactions with DNA which can affect the
replication processes are potential treatments for such diseases. DNA was a target of
anticancer drug discovery before its structure was even discovered.[178, 179]
Empirically
identified compounds with anti-cancer activity was later on shown to target DNA either
directly or through inhibition of enzymes that control DNA integrity or provide building
blocks for DNA. By the time the structure of DNA was revealed by Watson and Crick in
99
1953.[180]
There were several established therapeutic modalities targeting DNA;
antimetabolites, those trim down nucleotides; alkylating agents, those cause direct DNA
damage and intercalators such as actinomycins, which bind DNA and inhibit the activity of
many enzymes that use DNA as a substrate. Among the most widely and successfully used
anticancer agents today are nonspecific DNA targeting chemicals.[181]
The use of metal based compounds as biological probes represent one of the most
successful application of bioinorganic chemistry. The DNA binding properties of such
compounds play a vital role in their anticancer activity.[182, 183]
Thus before moving towards
expensive cell line studies it is valuable to study the ability of a compound to interact with
DNA. Binding of small molecules to DNA can be detected by a number of techniques. The
present report discusses DNA binding studies of new compounds using various techniques
like UV-Visible absorption spectroscopic and thermal denaturing assay of DNA. To avoid the
decomposition of the ferrocenium state if formed in basic or even neutral solution and
protonation of the ferrocenyl group in strongly acidic conditions these studies have been
carried out in phosphate buffer at pH = 6.8.[184]
4.3.1 UV-Visible Spectroscopic titration
The affinity of ferrocene derivatives for DNA was measured using a binding assay
based on UV-Visible spectroscopic changes. This assay is based on measuring an absorption
change as a direct measure for the quantification of the interaction of DNA with the metal
complexes. This method is suitable for the ferrocenes, because their absorption spectrum is
particularly sensitive to the environment and thus readily reports on changes such as
interaction with DNA.
UV-Visible spectroscopic technique is especially employed for the study of
ferrocenes owing to their intense color. It has been reported[185]
that the color of the
ferrocenes has strongly changed upon oxidation, making it easier to measure through
spectroscopy in the visible range. UV-Visible spectroscopy is an effective tool for the
quantification (using host guest equation 4.1) of binding strength of DNA with metal
complexes.[186, 187]
Ao/(A-Ao) = εF / (εB – εF) + εF / (εB – εF)*1/K [DNA] (4.1)
Where K is the binding constant, Ao and A are the observances of the free compound
and the apparent one after addition of DNA, εF and εB are their absorption coefficients
respectively. The slope to intercept ratio of the plot between Ao/(A−Ao) versus 1/[DNA]
yielded the binding constant K.
100
Table 4.05: DNA binding and UV-Visible spectroscopic data of selected compounds
Comp. # DNA binding constant
Kb (M-1
)
ΔG of binding
(kJ / K mol)
Wavelength
(nm)
Absorption coefficients
(ɛap, cm/mol)
At2 Ng -- 313, 448 12830, 460
At3 Ng -- 301, 451 10493, 298
At4 Ng -- 299, 450 16707, 486
At5 243 13.6 290, 449 8140, 222
At6 812 16.6 317, 363,
430s
48787, 46022, 8681
Bt5 U.A -- 448 (x) 413
Bt6 760 16.4 352, 455s 10439, 1067
Ct5 560 15.7 309, 449 19392, 2251
Ct6 1308 17.8 351, 440s 24199, 3974
Dt5 517 15.5 301, 447 19142, 566
Dt6 6421 21.7 364, 450s 34052, 6406
Et5 U.A -- 449 (x) 1049
Et6 10810 23.0 287, 354,
440s
13758, 15500, 1111
U.A; Unable to measure due to the peak appears in region lower than 290nm; (x) another band present in lower
than 290nm; Ng stands for Negligible value less than 100; s stands for the shoulder.
Most of the ferrocenyl thioureas have two to three characteristic bands in the UV-
Visible spectra. In these ferrocenyl thioureas complexes the π – π* transitions of the ligand
attributable to the aromatic rings and Cp rings of ferrocene is observed around 270 – 390 nm.
The d-d transition band appears around 400 – 500 nm. Unfortunately, the π – π* transition
appeared below 290 nm for compound Bt5 and Et5 prevent studies of these systems, because
DNA nucleotides absorbs in this region and absorbance of DNA nucleotides and/or
compound cannot readily be distinguished.[188, 189]
in nitro derivatives (At6, Bt6, Ct6, Dt6 and
Et6) the MLCT band appear in the range of 400 – 450 nm as a shoulder which overlap the d-
d transition band in the compounds. Importantly, the color of the ferrocenes has strongly
changed upon oxidation, and thus provides confirmation of the stability of the system during
the studies. The DNA binding constants (Kb) of selected compounds have been calculated
from absorption data using equation (1) and the results are listed in Table 4.05.
Addition of DNA aliquots to the constant concentration of compounds reveals
decrease in the absorbance in most of the compounds which are attributed to the consumption
of the compound by DNA along with another possibility. It may well be the pi-stacking of the
aromatic groups that ultimately reduces the probability of the π – π* transitions[190]
(decrease
in the number of electrons participating in π – π* transitions). This phenomenon makes us to
think about the electrostatic interaction of these compounds with DNA i.e., H- bonding π ---
H or N-H --- N type.
101
250 350 450 550
-0.2
0.2
0.6
1
1.4
00 µM DNA
10 µM DNA
20 µM DNA
30 µM DNA
40 µM DNA
50 µM DNA
60 µM DNA
70 µM DNA
80 µM DNA
Wave length
(nm)
Ab
s.
260 360 460 560
-0.2
0.2
0.6
1
1.400 µM DNA
10 µM DNA
20 µM DNA
30 µM DNA
40 µM DNA
50 µM DNA
60 µM DNA
70 µM DNA
Wave length
(nm)
Ab
s.
Figure 4.14: UV–Visible absorption spectra of 20 µM compound At6 (left) and 60 µM
compound Bt6 (right) in the absence of DNA and the presence of 10–80 µM DNA in 20%
aqueous DMSO buffered at pH 6.8. (An increase of absorbance at 260nm shows the
continuous increase of DNA concentration.)
Aliphatic thioureas (aliphatic group on one nitrogen of the thiourea At2 - At4) shows
negligible (or no) interaction with DNA which reveals slight change in intensity of the
absorbance band as compared to the aromatic thioureas (aromatic group on both nitrogen’s of
the thiourea), although the aromatic thioureas exhibit weak binding. However, aromatic
thioureas containing nitro substitution reveals interesting results and bind comparatively in
better way i.e., shows better affinity towards DNA, as binding constant justifies the
interactions tabulated and pictured in Table 4.05 and Figures 4.14 – 4.16.
Among the thioureas having aromatic arms on both sides, phenyl thioureas are found
to have less affinity towards the DNA as compared to nitro-phenyl thioureas. Electron-
withdrawing groups appear to be more favorable then electron-donators in enhancing binding
affinity36.[191]
In compounds (At6, Bt6, Ct6, Dt6, Et6), more suppression of the peak intensity
also supports our assumption of electrostatic interaction. (Through H-bonding) more
suppression of the peak reveals the more penetration of the compound into the DNA
helix.[192]
Isosebistic point (below 300nm) existing in all titration spectra’s shows a continuous
increase of DNA concentration. In compounds (At6, Bt6, Ct6, Dt6 and Et6) the presence of
an isosebistic point near 400nm, shows that these compounds interact with DNA either from
two different sites or make covalent interaction. In the current case it is not the covalent
interaction because of the fact that the binding constants measured from these points are
different. Binding constant measured from π – π* transition is different from the one
measured from d – d transition. Along with this, the binding affinities (binding constant K) of
these compounds are much less than the reported covalent binders like Mitomycins.[193]
The
same mode of binding is explored by thermal melting experiment.
102
260 360 460
0
0.4
0.8
1.2
00 µM DNA
10 µM DNA
20 µM DNA
30 µM DNA
40 µM DNA
50 µM DNA
60 µM DNA
70 µM DNA
80 µM DNA
Wave length
(nm)
Ab
s.
255 355 455 555
0
0.4
0.8
1.2
1.6
00 µM DNA
10 µM DNA
20 µM DNA
30 µM DNA
40 µM DNA
50 µM DNA
60 µM DNA
70 µM DNA
Wave length
(nm)
Ab
s.
Figure 4.15: UV–Visible absorption spectra of 50 µM compound Ct5 (left) and 40 µM
compound Ct6 in the absence of DNA and the presence of 10–80 µM DNA in 20% aqueous
DMSO buffered at pH 6.8, compounds At2, At3, At4, At5 and Dt5 behave like Ct5 when
studied. (An increase of absorbance at 260nm shows the continuous increase of DNA
concentration.)
The interaction from two sites can also be explained that the nitro group decreases the
electrons density from the aromatic groups which eventually polarizes the N-H group (in
thiourea) and thus enhances the ability of H-bonding the thioureas (similar as the urea’s have
more H-bonding ability then thioureas). Due to the same reasons compounds may start
penetrating deep into the DNA helix and show depressed π–π* transition. On the other hand
overall molecules have key like shape (L–shaped) which restricts the complete penetration
into the DNA helix. This competition may result in the slight tilting of the ferrocene
sandwich, which increases the probability of the d – d electronic transitions revealing in the
increase of peak height. In other words the slight tilting of the ferrocene Cp rings decreases
the back bonding ability and enhance the metal to ligand charge transfer increasing the molar
absorptivity.[194, 195]
250 350 450 550
-0.2
0.2
0.6
1
00 µM DNA
10 µM DNA
20 µM DNA
30 µM DNA
40 µM DNA
50 µM DNA
60 µM DNA
70 µM DNA
Wave length
(nm)
Ab
s.
260 360 460 560
0
0.4
0.8
1.2
00 µM DNA
06 µM DNA
12 µM DNA
18 µM DNA
24 µM DNA
30 µM DNA
36 µM DNA
42 µM DNA
48 µM DNA
Wave length
(nm)
Ab
s.
Figure 4.16: UV–Visible absorption spectra of 25µM compound Dt6 (left) and 60µM of Et6
(right) in the absence of DNA and the presence of 00 – 70µM DNA in 20% aqueous DMSO
103
buffered at pH 6.8. (An increase of absorbance at 260nm shows the increasing of
concentration DNA)
Among the nitro group containing compounds (At6, Bt6, Ct6, Dt6 and Et6),
Compounds having ferrocene at the para position (At6, Bt6, Ct6 and Dt6) show less
interaction as compared to compound having ferrocene at meta position Et6 because of its
structural arrangements. It is plausible on the basis of classical fact that ferrocene is electron
rich and can donate electrons effectively at the para position via resonance where as
resonance does not effect at meta position. So in case of para position it weakens the H-
bonding ability of the thiourea with DNA basis through pi-stacking or N – H --- N bonding.
One more substitution on the linking phenyl group of para ferrocenyl derivatives (At6, Bt6,
Ct6, and Dt6) reveal more affinity. The substitution on ortho to ferrocene group disturbs the
planarity between the linking phenyl and ferrocene Cp ring, which results in the suppression
of electron donation via resonance and thus enhances the interaction between these
compounds and DNA via the H-bonding.
4.3.2 Thermal Denaturing Assay
The melting point technique is a sensitive and easy tool to detect even slight DNA
conformational changes. It is known that a destabilizing interaction with the double helix
(typically covalent) is observed as a decrease in the melting point (Tm), while a stabilizing
interaction usually by intercalation or by electrostatic attraction induces an increase of Tm.[127]
The reactivity of the compound Et6 the most active among the compounds tested by
UV- Visible spectroscopy towards double-stranded DNA was assessed by measuring its
induced effect on the melting behavior (Tm) of DNA duplex.[196]
The results are summarized
in Figure 4.17. These results indicate that the tested compound has interactions with DNA as
demonstrated by a change in Tm. Et6 was found to shift the DNA melting curve (Tm =
64.3°C) significantly (indicating the stabilizing intercalation or electrostatic interaction)
compared to pure DNA (Tm = 61.2 °C) under similar conditions.[197]
Decrease in the slope of
the melting curve indicates stabilizing interaction of Et6 which restrict the opening of the
DNA duplex. To demonstrate, either the interaction is electrostatic or intercalation, the same
experiment was repeated with the higher Ionic strength of the test solution keeping the
remaining parameters constant, which indicates a decrease in the melting temperature (Tm =
62.1 °C) supporting the electrostatic interaction of the test sample which is in agreement with
UV-Visible spectroscopic titration results.
104
30 50 70 90
0.6
0.7
0.8
0.9
1
c
b
a
Temp. (°C)
Ab
s.
Figure 4.17: Thermal melting profiles of DNA in 1mM phosphate buffer (pH = 6.8)
80% DMSO: (a) with 1mM NaCl, (b) 1mM NaCl & compound Et6, and (c) 2mM NaCl &
compound Et6. [DNA] = 100μM and [Et6] = 50μM.
105
Conclusions
1. A series of novel ferrocene substituted thioureas and ureas were synthesized in three
and four steps respectively starting form ferrocene. At every step intermediate and
end products are fully characterized.
2. All the synthesized complexes (intermediate and end products) were successfully
characterized, using different instrumental techniques like elemental analysis, FT-IR,
multinuclear (1H,
13C) NMR, and single crystal XRD.
3. Elemental analysis results show the presence of salvation molecules in some of the
synthesized complexes.
4. Single crystal X-ray diffraction analyses exhibit that the studied compounds have
been stabilized in crystalline form by intermolecular as well as intramolecular H-
bonding.
5. Primarily some compounds were screened for their interaction with modal bio-
mimetic membranes, DNA binding behavior and antioxidant activity to evaluate their
anti-cancer potency.
6. The DNA binding abilities of some of the synthesized complexes were checked by
UV-Visible spectroscopy. The DNA interaction studies of the complexes indicate that
the induction of electron withdrawing groups in the structure increases the binding
abilities.
7. IC50 values for anti-oxidant studies revealed that the ferrocene substituted ureas have
much better activity than thioureas and some of the synthesized complexes can be
potential therapeutic agents. It was observed that the electron withdrawing effect of
the substituent plays a significant effect on the anti-oxidant properties.
8. Membrane penetration studies have been carried out for some compounds with model
lipid membrane interfaces prepared from SDS and TTAB surfactants using 1HNMR
and UV-Visible spectroscopic techniques. Results show the presence of these
molecules in the interfacial regions of the self assembled systems.
9. These studies justify the lipophilic character of these compounds as their ability of
these compounds to penetrate into the modal membranes.
106
Future plans
1. Since some of the test compounds show promising interaction with DNA, so the
future plan is to test these compounds against other biological molecules like proteins
and enzymes as well.
2. Based on the anti-oxidant and DNA binding results of some leading compounds it is
in the pipeline to carry out their anti-cancer cell line studies. And the synthesis of
more compounds with similar structures having more electron loving groups.
3. It is in the pipeline to find out the effects of the synthesized compounds on various
body tissues to take further steps to use it as a drug.
4. The DNA binding studies of ureas are also in the pipeline, along with the synthesis of
more compounds having both thiourea and urea functionalities keeping in mind the
better membrane penetration of thioureas and anti-oxidant properties of ureas.
107
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List of Publications and Presentations
1. R. A. Hussain; A. Badshah; B. Lal; A. A. Altaf; A. Shah (2012) “A Review on
Cadmium Selenide (CdSe) Nanostructures” Chemical Reviews (submitted manuscript
ID cr-2012-002802)
2. B. Lal, A.A. Altaf; A. Badshah; S. Marwat; N. Khan; F. Huq (2012) “Study of new
ferrocene incorporated N, N'-disubstituted thioureas as potential antitumour agents”
Journal of Inorganic Biochemistry (Submitted)
3. A.A. Altaf; A. Badshah; S. Marwat; N. Khan; B. Lal, Debbie C. Crans (2012)
“Synthesis, modal membrane interaction and anti-cancer cell line studies of
nitrophenyl ferrocenes” Inorganic Chemistry (submitted manuscript ID ic-2012-
01403w)
4. M. Jamil; A. A. Altaf; A. Badshah; Shafiqullah; I Ahmad; M. Zubair; S. Kemal; M. I.
Ali (2012) “Naked Eye DNA Detection: Synthesis, Characterization and DNA
binding studies of a novel Azo-Guanidine” Spectrochim. Acta. A. DOI:
10.1016/j.saa.2012.12.020
5. S. Ali; A. Badshah; A. A. Altaf; Imtiaz-ud-Din; B. Lal; K. M. Khan (2012)
“Synthesis of 3-ferrocenylaniline: DNA interaction, antibacterial and antifungal
activity” Medicinal Chem. Res. DOI: 10.1007/s00044-012-0311-8
6. B. Lal, A.A. Altaf; A. Badshah; S. Marwat; N. Khan; F. Huq (2012) “Synthesis,
Characterization and Antitumor activity of new Ferrocene incorporated N, N’-
disubstituted Thioureas” Dalton Trans. 41 14643 - 14650
7. A. A. Altaf; F. Javed; A. Badshah; B. Lal; M. Siddiq; Z.-U. Rehman; A. Shah;
Shafiqullah; M. N. Tahir (2012) “New supramolecular ferrocenyl amides: synthesis
characterization and preliminary DNA-binding studies” J. Coord. Chem. 65 969 –
979.
8. B. Lal, A. Badshah, A. A. Altaf, N. Khan, S. Ullah (2011) “Miscellaneous
applications of ferrocene-based peptides/amides” Appl. Organometal. Chem. 25 843 –
855.
9. S. Ullah; A. Badshah1; F. Ahmed; R. Raza; A. A. Altaf; R. Hussain,
(2011)“Electrodeposited Zinc Electrodes for High Current Zn/AgO Bipolar Batteries”
Int. J. Electrochem. Sci., 6 3801 – 3811.
10. A.A. Altaf; A. Badshah; S. Marwat; N. Khan; B. Lal, (2011) “Improved synthesis of
ferrocenyl aniline.” J. Chem. Soc. Pak. 33 691 – 693.
118
11. A.A. Altaf; A. Badshah; S. Marwat; N. Khan; S. Ali, (2011) “Zirconium complexes
in homogeneous ethylene polymerization” J. Coord. Chem. 64 1815 – 1836.
12. A. A. Altaf; A. Badshah; N. Khan; M. N. Tahir, (2010) “N-(4-
Ferrocenylphenyl)benzamide” Acta Cryst. E66, m831
13. M. Riaz, N. Rasool, I. H. Bukhari, K. Rizwan, A. A. Altaf, M. Zubair, H. M. A.
Qayyum, (2011) “An Ivestigation Of Biological Studies And Protective Effect On
H2O2 Induced Oxidative Damage On Pbr322 DNA By Verbascum Thapsus Extracts”
Abstract in 1st International Conference of Safe Food and Human Health -
January 10 2012, GCU Faisalabad, Pakistan.
14. A. A. Altaf; F. Javed; A. Badshah; M. N. Tahir, D. C. Crans, (2012) “Ferrocenyl
amides as potential anti-cancer agents: Synthesis, characterization and interaction
with lipid membrane interfaces” Abstract and presented in 242nd
ACS National
Meeting & Exposition - August 30, 2011, Denver, Colorado USA.
15. A. A. Altaf; A. Badshah; D. C. Crans; “Thiourea based ferrocene and its analog as
potential anti cancer agents: Synthesis, characterization, DNA binding, and model
lipid membrane interaction study” Abstract in 244th
ACS National Meeting &
Exposition - August 22, 2012, Philadelphia, Pennsylvania USA.
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