Phytochemical Investigation on the Chemical Constituents of Zygophyllum eurypterum and Synthesis with Biological Evaluation of Schiff Bases of Substituted Phenyl Hydrazine and Benzimidazole Derivatives
Thesis submitted For the partial fulfillment of the degree of
DOCTOR OF PHILOSOPHY
BY
Mr. ZARBAD SHAH
H.E.J. RESEARCH INSTITUTE OF CHEMISTRY INTERNATIONAL CENTER FOR CHEMICAL AND BIOLOGICAL SCIENCES, UNIVERSITY OF KARACHI KARACHI-75270, PAKISTAN
2011
i,
Dedicated to parents especially my mother (late)
&
My beloved teachers,
Prof. Dr. Viqar Uddin Ahmad, H.I., S.I.
&
Prof. Dr. Khalid Mohammed Khan, T.I., S.I.
ii, iii,
iv, LIST OF CONTENTS
Acknowledgements xiii Personal Introduction xiv Summary xvi Khulasa xix Abbreviations xxi
Part- A
CHAPTER-1
1. Family Zygophyllaceae 2 1.1. The genus Zygophyllum 2 1.1.1. Zygophyllum eurypterum 2 1. 1.2. Zygophyllum megacarpum 2 1.1.3. Zygophyllum simplex 3 1.1.4. Zygophyllum propinquum 3 1.1.5. Zygophyllum fabago 3 1.2. Medicinal importance of the genus Zygophyllum 4 1.3. Literature review of the genus Zygophyllum 6
CHAPTER-2
2. Stigmasterol (1) 28 2.1. β-Sitosterol (2) 29 2.2. β-Sitosterol glucoside (3) 30 2.3. Oleanolic acid (4) 31 2.4. Harmine (5) 33 2.5. 7,3',4'-Trimethoxyflavone (6) 34 2.6. 5-Methylflavanone (7) 35
v, CHAPTER-3
3. Stigmasterol (1) 37 3.1. β-Sitosterol (2) 38 3.2. β-Sitosterol glucoside (3) 39 3.3. Oleanolic acid (4) 40 3.4. Harmine (5) 41 3.5. 7,3',4' -Trimethoxyflavone (6) 42 3.6. 5-Methylflavanone (7) 43 3.7. General notes 44 3.8. Instrumentation 44 3.9. Chromatography 44 3.10. Spray reagents 44 3.10.1. Ceric sulphate 45 3.10.2. Aniline phathalate 45 3.11. Zygophyllum eurypterum (Z. atriplicoides) 45 3.11.1. Plant collection 45 3.11.2. Isolation procedure 45 3.12. Isolation scheme of Zygophyllum eurypterum 47
CHAPTER-4
4. References 49
Part- B
CHAPTER-5
5. Introduction to Schiff bases 55 5.1.1. Synthetic approaches to Schiff bases 58 5.1.2. Synthesis of hydrazones by using ethanol and triethylamine 58 5.1.3. Synthesis of azines using solvent free condition 58
vi, 5.1.4. Synthesis of hydrazones by using methanol and sulphuric acid 59 5.1.5. Synthesis of hydrazones by using DMF 59 5.1.6. Synthesis of aryl-hydrazones using ultrasound irradiation 60 5.2. Results and Discussion 61 5.2.1 Chemistry 61 5.3. General structure elucidation of compounds by spectroscopic techniques 64 5.4. Biological Studies 66 5.5. Conclusion 85 5.6. General instrumentation 86 5.6.1. Synthetic procedure of compounds 28-62 87 5.6.1.1. 3, 4, 5-Trimethoxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (28) 87 5.6.1.2. 2-Hydroxy-3-methoxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone: (29) 87 5.6.1.3. Benzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (30) 87 5.6.1.4. 2-Fluorobenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (31) 88 5.6.1.5. 4-(Methylsulfanyl)benzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (32) 88 5.6.1.6. 4-(Dimethylamino)benzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (33) 88 5.6.1.7. 2-Hydroxy-5-methylbenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (34) 88 5.6.1.8. 2, 5-Dihydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (35) 88 5.6.1.9. 2, 4-Dichlorobenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (36) 89 5.6.1.10. 4-Chlorobenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (37) 89 5.6.1.11. 2-Chlorobenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (38) 89
vii, 5.6.1.12. 3, 5-Dichloro-2-hydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (39) 89 5.6.1.13. 3, 4-Dichlorobenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (40) 89 5.6.1.14. 3, 4-Dihydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (41) 90 5.6.1.15. 4-Hydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (42) 90 5.6.1.16. 3, 4-Dimethoxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (43) 90 5.6.1.17. 2, 3, 4-Trihydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (44) 90 5.6.1.18. 2, 3-Dihydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (45) 91 5.6.1.19. 3-Thiophenecarbaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (46) 91 5.6.1.20. Isonicotinaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (47) 91 5.6.1.21. 2-Bromobenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (48) 91 5.6.1.22. 2-Hydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (49) 92 5.6.1.23. 3-Chlorobenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (50) 92 5.6.1.24. 1-Phenanthrenecarbaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (51) 92 5.6.1.25. Nicotinaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (52) 92 5.6.1.26. 2-Naphthaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (53) 93 5.6.1.27. 2-Methylbenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (54) 93
viii, 5.6.1.28 2, 4, 6-Trihydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (55) 93 5.6.1.29. 2, 4-Dihydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (56) 93 5.6.1.30. 4-Methylbenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (57) 93 5.6.1.31. 3, 5-Dibromo-2-hydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (58) 94 5.6.1.32. 2-chloro-5-nitrobenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (59) 94 5.6.1.33. 4-Nitrobenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (60) 94 5.6.1.34. 5-Bromo-2-hydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (61) 94 5.6.1.35. 2-Nitrobenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (62) 95 5.7. References 96
CHAPTER-6
6. Introduction to Benzimidazole 110 6.1. Synthetic approaches to Benzimidazoles 115 6.1.1. Synthesis of benzimidazole from triethoxy methane and o-alkylated oximes 115 6.1.2. Photolysis of protected Benzimidazole 115 6.1.3. Synthesis of 2-substituted benzimidazole by using Lewis acids 116 6.1.4. Synthesis of 2-substituted benzimidazole via clay and IR 116 6.1.5. Microwave assisted synthesis of substituted benzimidazoles 116 6.1.6.. Synthesis of substituted benzimidazoles by using esters 117 6.1.7. Synthesis of substituted benzimidazoles via reductive cyclization 117 6.1.8. Synthesis of substituted benzimidazoles by using polyphosphoric acids 117
ix, 6.1.9. Synthesis of benzimidazoles by using 12-tungstophosphoricacid as a catalyst 118 6.2. Results and discussion 119 6.2.1 Chemistry 119
6.3. General structure elucidation of compound by spectroscopic techniques 122
6.4. Biological studies 124 6.5. Conclusion 137 6.6. General instrumentation 138 6.6.1 Synthetic procedure of compounds 37-66 138 6.6.1.1. 4-(6-Nitro-1H-benzimidazol-2-yl)phenol (37) 139 6.6.1.2. 2, 4-Dibromo-6-(6-nitro-1H-benzimidazol-2-yl)phenol (38) 139 6.6.1.3. 2-(3, 4-Dimethoxyphenyl)-6-nitro-1H-benzimidazole (39) 139 6.6.1.4. 2-(2-Chlorophenyl)-6-nitro-1H-benzimidazole (40) 139 6.6.1.5. 2, 4-Dichloro-6-(6-nitro-1H-benzimidazol-2-yl)phenol (41) 140 6.6.1.6. 6-Nitro-2-(4-nitrophenyl)-1H-benzimidazole (42) 140 6.6.1.7. 2-(4-Methylphenyl)-6-nitro-1H-benzimidazole (43) 140 6.6.1.8. 2-(3, 4-Dichlorophenyl)-6-nitro-1H-benzimidazole (44) 140 6.6.1.9. 4-(6-Nitro-1H-benzimidazol-2-yl)-1, 3-benzenediol (45) 141 6.6.1.10. 2-(4-Chlorophenyl)-6-nitro-1H-benzimidazole (46) 141 6.6.1.11. 4-(6-Nitro-1H-benzimidazol-2-yl)-1, 2-benzenediol (47) 141 6.6.1.12. 2-Methoxy-6-(6-nitro-1H-benzimidazol-2-yl)phenol (48) 141 6.6.1.13. 2-(6-Nitro-1H-benzimidazol-2-yl)-1, 3, 5-benzenetriol (49) 142 6.6.1.14. 4-(6-Nitro-1H-benzimidazol-2-yl)-1, 2, 3-benzenetriol (50) 142 6.6.1.15. N, N-dimethyl-4-(6-nitro-1H-benzimidazol-2-yl)aniline (51) 142 6.6.1.16. 2-Ethoxy-6-(6-nitro-1H-benzimidazol-2-yl)phenol (52) 142 6.6.1.17. 4-Chloro-2-(6-nitro-1H-benzimidazol-2-yl)phenol (53) 143 6.6.1.18. 2-(6-Nitro-1H-benzimidazol-2-yl)phenol (54) 143 6.6.1.19. 2-(2, 4-Dichlorophenyl)-6-nitro-1H-benzimidazole (55) 143 6.6.1.20. 2-(2-Chloro-5-nitrophenyl)-6-nitro-1H-benzimidazole (56) 143 6.6.1.21. 2-(2-Fluorophenyl)-6-nitro-1H-benzimidazole (57) 144
x, 6.6.1.22. 2-(4-Bromophenyl)-6-nitro-1H-benzimidazole (58) 144 6.6.1.23. 6-Nitro-2-(3-thienyl)-1H-benzimidazole (59) 144 6.6.1.24. 6-Nitro-2-(1-phenanthryl)-1H-benzimidazole (60) 144 6.6.1.25. 6-Nitro-2-phenyl-1H-benzimidazole (61) 145 6.6.1.26. Methyl 4-(6-nitro-1H-benzimidazol-2-yl)phenyl sulfide (62) 145 6.6.1.27. 6-Nitro-2-(4-pyridinyl)-1H-benzimidazole (63) 145 6.6.1.28. 2-(6-Nitro-1H-benzimidazol-2-yl)-1, 4-benzenediol (64) 145 6.6.1.29. 2-(9-Anthryl)-6-nitro-1H-benzimidazole (65) 146 6.6.1.30. 3-(6-Nitro-1H-benzimidazol-2-yl)-1, 2-benzenediol (66) 146 6.7. References 147
CHAPTER-7
7.1 In vitro antiglycation bioassay 159 7.2. In vitro carbonic anhydrase bioassay 159 7.3. In vitro α-chymotrypsin bioassay 160 7.4. In vitro urease bioassay 160 7.5. Antioxidant (DPPH and superoxide) bioassay 161
7.5.1. In vitro DPPH (1, 1-Diphenyl-2-picryl hydrazyl) free radical
scavenging bioassay 161
7.5.2. In vitro superoxide anion scavenging bioassay 162 7.6. In vitro phosphodiesterase inhibition bioassay 162 7.7. References 164 8. List of publications 166
xi, ACKNOWLEDGEMENTS First and foremost, I am thankful to Almighty Allah, the most beneficent and the most merciful, who enabled me to complete my Ph.D. research work. All respects for his last Holy prophet Muhammad (peace be upon him), whose teaching inspired me to widen my thoughts and deliberate upon nature deeply. I wish to express my gratitude to a number of people who were involved in this work, in one or another way. In this regard, first of all I am thankful to the founding Director of the
Institute, (late) Prof. Dr. Salimuzzaman Siddiqui, F.R.S. H.I., S.I., T.I. for building such a wonderful and world class institute of chemistry in this part of the world. Prof. Dr. Atta-ur-
Rahman, F.R.S., N.I., H.I., S.I., T.I. Ex-Chairman Higher Education Commission, chief patron of International Center for Chemical and Biological Sciences, University of Karachi, for providing us a nice environment for research, where I carried out all of my research work.
I am also thankful to Prof. Dr. M. Iqbal Choudhary, H.I., S.I., T.I. Director, H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, for his academic leadership, keen interest and co-operation. The work presented in this dissertation would never be accomplished without the keen interest, precious attention, and continuous encouragement of my research supervisor
Prof. Dr. Viqar Uddin Ahmad, H.I., S.I.,. I am grateful to him for his guidance and especially for his very kind behavior. I have great pleasure in expressing my deep gratitude to Prof. Dr. Khalid Mohammed
Khan, H.I., S.I. University of Karachi for allowing me to do synthetic work in his lab. and also his valuable suggestions, expert guidance, and active co-operation to produce this dissertation. Without him, this work would not be completed in its present form. I greatly appreciate his ever-ready helping attitude, which was a constant motivating factor for me to complete this study. Thanks also to all the faculty members of the institute that I had the privilege to learn from them the advance courses of M. Phil. and Ph.D. studies, especially Prof. Dr. Bina Shaheen
Siddiqui, S.I., T.I., Prof. Dr. Abdul Malik, S.I. ,, Prof. Dr. Shaheen Faizi, Prof. Dr. Sbira
Begum P.P. , Prof. Dr. M. Shaiq Ali, Dr. M. Raza Shah and Dr. Zaheer-ul-Haq Qasmi. I would like to express my heartiest gratitude and regards to my mother (late), father, brothers i.e. Abdul Wadud, Ahmad Jan, Abdul Majeed, Fida Muhammad, Muhammad
xii, Ibrahim, Khan Badshah, my sisters, my cousin S. Mutalib Shah and aunt for their love, prayers, encouragement and continuous support throughout my studies. My heartiest thanks are extended to my other best friends i.e. Mr. Momin Khan and Mr. Muhammad Taha whose guidance, moral support and continued support enabled me to complete my Ph.D. studies. I am also highly thankful to my senior colleagues, Dr. Saleha Suleman Khan and Dr. Nida Ambreen, Assistant Professor, Federal Urdu University, Karachi, whose continuous encouragement and vast experience in isolation/organic synthesis helped me to complete my Ph.D. work. I would like to express my sincere appreciation and thanks to my group fellows, Dr. S. S. Khan, Dr. Shazia Iqbal, Aqib Zahoor, Imran Nafees Siddiquii, Dr. Faryal Vali Muhammad, Mr. Viqar Hyder and Mr. Muhammad Ramzan Siddiqui. This dissertation could never be completed in its present form without an extensive bioassay work and I am therefore thankful to my colleagues Ms. Humera Jahan for the antiglycation bioassay, Mr. Ajmal Khan for urease inhibition, Ms. Shagufta for phosphodiesterase and β- glucoronidase inhibitory activities, Mr. Sajjad Ali for antioxidant activities, Mr. Bishnu for α-chymotrypsin activity, Ms. Shahida for cytoxicity, and Mr. Salim for carbonic anhydrase bioassays. My special thanks are extended to my friends especially Mr. M. Rabnawaz, Mr. Behramand, Mr. Nazimuddin, Mr. Farhatullah, Mr. Nizam Abbasi, Mr. Fazal Rahim, Mr. Naveed Iqbal, Mr. Muhammad Ateeq, my all course mates, hostel fellows and H .E. J. fellows. I am also thankful to all technical and non-technical staff members of the institute for their assistance and help, especially of Mr. Sohail, Mr. Rasees, Mr. Kamran of NMR section, Mr. Zubair, Mr. Arshad of analytical section, Mr. Abdul Wajid, technical officer, Mr. Mazoor and Mr. Itikhab of works and engineering section, Mr. Rafat Ali, Mr. Shamim Khan and Mr. Rahat Muhammad Khan.
Zarbad Shah H.E.J. Research Institute of Chemistry, University of Karachi, Karachi, Pakistan.
xiii, Personal Introduction of Mr. Zarbad Shah
ACADEMIC QUALIFICATION
SSC. A Grade/1st Div.
F.Sc. B Grade/1st Div.
B.Sc. B Grade/1st Div.
M.Sc. B Grade/1st Div.
M.Phil. 3.60 GPA/1st Div.
Chemistry GRE 68 Percentile
It was 18th January 1980 that I was born in the home of a farmer in a remote village of
Peshawar i.e Chaghar Matti Askhab Ba Ba. Our village which is about 5 km away from
Warsak dam, is very much green and fertile with a number of schools and colleges.
In 1994, I passed my middle board examination with 3rd position in a local school. For matriculation, i shifted to Govt. higher secondary school No.1, Peshawar city. I passed my matric board examination from this school with “A” grade. In 1998 and 2000, I passed F.Sc. and B.Sc. with “B” grades from Govt. Degree College, Peshawar. For Master studies, I was shifted to Gomal University, D. I. Khan and in 2004; I passed M.Sc. organic chemistry from this university. The thirst for knowledge was still not satisfied, and in October 8th 2005, I decided to join the H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi for Ph.D. studies. I have been fortunate to carry out my PhD research studies under the kind supervision of Prof. Dr. Viqar Uddin Ahmad at one of the best chemistry research institute of the world. I have worked on the isolation, structure elucidation, and biological screenings of the natural products. To continue further, I decided to change the research project and started working in the Prof. Dr. Khalid Mohammed Khan laboratory on synthesis of natural products and their derivatives. I feel proud that I
xiv, worked in this group. I am very much impressed from the cooperative behavior of Prof. Dr. Khalid Mohammed Khan and his group. At the H. E. J. Research Institute of Chemistry, I received generous guidance by highly experienced and dedicated faculty members who were always there to help me to broaden my scientific skill and knowledge. I learnt different ways of tackling complex situation, which not only enhanced my self-confidence but also improved my capacity of critical thinking. The areas of research remain phytochemical studies and small natural products synthesis. In future, I would like to take part in the development of phytochemical studies and will try to correlate it with the natural products synthesis for the search of biologically active molecules against diseases such as cancer, diabetes, tuberculosis, etc. I have dream of serving the humanity through relevant science all over the world generally and Pakistan particularly by contributing in the development of field laboratories to promote the scientific knowledge and ultimately contribute towards national development.
xv, Summary
This dissertation is divided into two sections; sections A and B. Section A describes the isolation and structure elucidation of seven known compounds [1-7] i.e. stigmasterol (1), β- sitosterol (2), β-sitosterol glucoside (3), oleanolic acid (4), harmine (5), 7,3',4'-
trimethoxyflavone (6), and 5-methylflavanone (7). Compounds [5-7] were for the first time reported from this plant. Section B describes the synthesis and biological evaluation of 2, 4,
6-trichlorophenyl hydrazones and 6-nitrobenzimidazole derivatives.
Section-A
This section consists of four chapters. Chapter 1 describes a brief introduction of the family
Zygophyllaceae and the genus Zygophyllum. Besides a short overview of the genus
Zygophyllum, some medicinal importance of the plants of this genus is also described. At the
end of this chapter, a detailed survey of the compounds isolated from the plants of the genus
Zygophyllum are summarized in the form of a table. The structure elucidation of the isolated compounds is given in chapter 2 whereas chapter 3 provides detailed spectroscopic data (i.e.
IR, Mass, 1H-NMR and 13C-NMR) of the seven compounds [1-7] which were isolated from
the aerial part of the Zygophyllum eurypterum. References are given at the end of the section.
Section-B
Section B consists of three chapters i.e. chapter 5, 6 and 7. Chapter 5 consists of a brief introduction of Schiff bases and their synthetic approaches. In this chapter we also described the synthesis of thirty five 2, 4, 6-trichlorophenyl hydrazone derivatives and their detailed bioactivities. During the detailed biological studies, we have screened all the synthetic compounds [28-62] for the glycation of protein, α-chymotrypsin, carbonic anhydrase, urease,
xvi, antioxidant (DPPH and superoxide anion scavenging assays) and phosphodiesterase
activities.
In the antiglycation bioassay, out of thirty five compounds, total eight compounds showed
activity in which two compounds showed much better activity than the rutin standard. In the
α-chymotrypsin assay, twelve compounds were active while in the carbonic anhydrase
inhibitory assay, eleven compounds showed activity. In the urease assay, total seven
compounds exhibited activity. All the thirty compounds were active in the DPPH radical
scavenging bioassay while thirteen compounds were active in the superoxide anion radical
scavenging assay. Finally, in the phosphodiesterase inhibitory assay, none of the compounds
were active.
Chapter 6 provide a brief introduction of benzimidazole and synthetic approaches.
Proceedingly, we also described the synthesis of thirty derivatives of 6-nitrobenzimidazole
along with their detailed bioactivities. During detailed biological activity studies, all the
synthetic compounds 37-66 were tested for the urease, α-chymotrypsin, carbonic anhydrase,
glycation of protein, phosphodiesterase and radical scavenging assays (DPPH & superoxide
anion). A total of thirty compounds were screened in urease bioassay, in which six compounds were active in this assay. In the α-chymotrypsin inhibitory assay, total nine
compounds showed activity while none of the compounds showed inhibition on the carbonic
anhydrase activity. Total fourteen compounds were active in the glycation of protein. In the
phosphodiesterase assay, total ten compounds showed activity. In the DPPH radical
scavenging assay, total six compounds were active while in the superoxide anion radical
scavenger assay, only four compounds exhibited activity.
xvii, The last chapter describes the bioassay techniques which were employed for the bioactivities for all the synthetic compounds.
xviii,
xix,
xx, ABBREVIATIONS
AGE Advance glycation end product (s)
Ac2O Acetic anhydride ATP Adenosine triphosphate BuOH Butanol CA Carbonic anhydrase CAN Ceric ammonium nitrate DMF N, N-Dimethylformamide DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DPPH 2, 2-Diphenyl-1-picrylhydrazyl EDTA Ethylenediaminetetraacetic acid EI-MS Electron impact mass spectrometry EtOH Ethanol FAD Flavin adenine dinucleotide HR-EI-MS High resolution electron impact mass spectrometry HP-TLC High performance thin layer chromatography h Hour(s) Me Methyl MeOH Methanol NAD Nicotinamide adenine dinucleotide
NEt3 Triethylamine NMR Nuclear magnetic resonance NPPs Nucleotide pyrophosphatases/phosphodiesterases Ph Phenyl PPA Phenylpropanolamine RNA Ribonucleic acid THF Tetrahydrofuran
xxi, TFA Trifluoroacetic acid TLC Thin layer chromatography TMP Thymidine monophosphate
xxii,
CHAPTER-1 INTRODUCTION
1, 1. Family Zygophyllaceae
Family Zygophyllaceae consists of 25 genera and approximately 240 species. Warm temperate
as well as subtropical and tropical areas are the origin of the plants of this family. About 22 species, which belong to about eight different genera, are found in Pakistan. The plants of this family are either perennial herbs or under shrubs. Trees are less common and usually the plants of this family require very less quantity of water [1].
1.1. The genus Zygophyllum
The Zygophyllum is the leading genus in the family Zygophyllaceae. The plants of this genus are
commonly found in Afganistan, Pakistan, South Africa, Australia, Iran and central Asia.
In Sindh and Baluchistan provinces of Pakistan, some species of this genus are found. So far six
species have been reported for Zygophyllum genus growing in Pakistan. Their names are,
Zygophyllum eurypterum (syn. Zygophyllum atriplicoides), Zygophyllum megacarpum,
Zygophyllum simplex, Zygophyllum propinquum and Zygophyllum fabago [1].
1.1.1. Zygophyllum eurypterum
Zygophyllum eurypterum is an erect shrub. It is 1-1.2 m tall. Stem and branches are whitish grey.
Leaves are 1.5-3 cm long. Flowers are creamy white. Seeds are brown and 6-7 mm long. It is widely distributed in Iran, Afghanistan, Russia and Pakistan [2].
1.1.2. Zygophyllum megacarpum
Zygophyllum megacarpum is a shrub. It is up to 3 m long. Leaves hight are about 1.6-2.8 cm tall.
Seeds are reuniform. It is widely distributed in Iran, Pakistan (Baluchistan) and Russia. This
species is closely related to Zygophyllum eurypterum, but differs in having bright yellow flowers
[2].
2, 1.1.3. Zygophyllum simplex
Zygophyllum simplex represents a very prominent species of the genus Zygophyllum growing in
Egypt. It is also common in North America as well as in Arabian regions. It is a perennial, 50-
250 cm tall shrub. Leaves flat, oblong-obovate-spathulate or subrotundate to almost rotundate.
Capsule is large and 4-5 winged [1].
1.1.4. Zygophyllum propinquum
Zygophyllum propinquum is a perennial herb. It is 20-50 cm tall, diffusely branched dull green or
pinkish purple herbs. Young shoots are grayish. Leaves are 5-10 mm long, flowers are yellowish
white. Seeds are compressed and are considered antihelmenthic. It is widely distributed in
Pakistan, Afghanistan, Iran, Iraq, Syria, Palestine, Egypt (Sinai), Jordan, Kuwait and Saudi
Arabia [3].
1.1.5. Zygophyllum fabago
Zygophyllum fabago commonly known as Syrian bean-caper, is distributed in Pakistan, Iran,
Afghanistan, Iraq, Arabia, North Africa, Spain, France and Italy. Zygophyllum fabago is also found in Turkey.
It is a perennial glabrous erect herb. Branches divaricate, striate, basal shoots spreading, upper ones ascending. Leaves pinnately bifoliolate, petiole 12-15 mm long, somewhat flattened; and stipules are ovate-elliptic which is about 5-10 mm long [1].
3, 1.2. Medicinal importance of the genus Zygophyllum
The plants belonging to the genus Zygophyllum are medicinally important and hence can be used to cure different types of diseases. Some medicinal properties associated with these plants are given below,
1. Zygophyllum cornutum can be used as a local medicine due to its sugar reducing
properties. Some compounds of this plant can be used to lower cholesterol and lipid
contents in the blood [4].
2. The leaves of Zygophyllum simplex have skin cleaning properties. In gulf and African
countries, it was also useful against horny patches on the skin [5]. Some compounds of
this plant are used in antibiotics as well as in laxative problems [6].
3. Some compounds isolated from Zygophyllum geslini are used to treat diabetic disease.
One compound of this species is usefull against KB cells [7].
4. In Egypt, Zygophyllum decumbens extracts are useful against asthma, hypertension,
gout and rheumatism [8]. The extracts of this species are also used in many diseases
like diuretic, spasmolytic, antipyretic, hypotensive, etc. [9].
5. In the folk medicine, Zygophyllum gaetulum Emb. Marie may be used as an
antieczema, antispasmodic, and in stomach and in liver diseases. This plant also
shows antidiabetic properties [10].
6. The compounds so far isolated from Zygophyllum fabago L. have a great medicinal
value and are used for different activities such as anti-inflammatory, expectorant,
antitussive, pain killers, anti-asthmatic, anti-rheumatic and in anthelminthic [12, 13].
4, This plant shows strong antifungal activity but the pollens of this plant can cause
allergic symptoms to the body [13-15].
7. Zygophyllum coccineum L. extracts are used in Kuwait as a local medicine for the
anthelminthic and diuretic agent. Some compounds of this plant have medicinal value
and can be used to cure various diseases like diabetes, asthma, gout, rheumatism, and
hypertension [16, 17].
5, 1.3. Literature review of the genus Zygophyllum
Phytochemical investigation of the genus Zygophyllum L. shows that this genus is very rich in
saponins. Beside saponins, several other compounds such as flavonoids, alkaloids and tannins
are also reported from this genus. A brief review of these compounds are given in the following
table 1:
Table-1. Literature review of the genus Zygophyllum No. Structure of compound M. F Source Rf & M. Wt 1
C30H45O6 Z. coccineum 18 H COOH 486.335
H COOH
HO H 2
C H N Z. fabago N 12 10 2 19 N H 182.221 CH3
3
N H3CO N H C13H12N2O Z. fabago 20 CH3 212.247
6, 4 CH3 OH
HO O
C16H12O5 Zygophyllaceae 21
OH 300.063 OH O
5 OH OH
C15H10O7 Zygophyllaceae HO O 22 302.043
OH
OH O 6 OH OCH3
HO O C16H12O7 Zygophyllaceae 22 316.058 OH
OH O
7
COOH C30H48O3 Z. fabago 23 456.700
HO
7, 8
O C30H46O3 Z. fabago 23 454.354
O
HO
9
C30H48O4 Z. obliquum 24 COOH 472.700
HO
CH2OH 10
H C H O Z. obliquum 24 COOH 29 46 3 456.700
HO H
8, 11 OCH3 OH
HO O
O
C22H22O13 Z. cornutum 4 OH O 494.402
OH
O OH OH OH 12
H
H O
OH H CH 3 C41H66O11 Z. propinquum 25 O O 734.956 OH
OH
OH O O OH
OH
9, 13
COOH
COOH CH3 O O OH C41H64O13 Z .propinquum 25
OH 764.939 O OH O OH
OH 14
COOH
CH C O 3 C47H74O18 Z. propinquum 26 O O O 927.080 OH CH2OH
OH O
OH O O OH OH OH
OH OH 15
HO OH
C39H56O5 Z. propinquum 26
CH2OH 604.859
O O
10, 16
O OH OH
C16H12O7 Z. simplex 5 OCH3 HO O 316.262
OH
17
H COOH C36H56O9 Z. simplex 5
H COOH 632.824
O O
CH3
OH
OH OH 18
H COOH C36H56O12S Z. propinquum 27 712.889 H CH3 COOH
O O OH
OH
OSO3H
11, 19
O HO O C H O Z. propinquum 27 COOH 41 62 16 CH2OH O O 810.921 O OH OH OH OH OH OH OH 20
H COOH
H CH3 C41H66O11 Z. album 28 O O 734.46 OH CH3
O O OH OH
OH OH
12, 21
H O
O COOH CH OH 2 HO O C H O Z. album 28 OH 42 66 15 810.964 OH
CH2OH O
O OH
OH OH
22
H COOH
C42H68O12 Z. album 28 H CO 764.982 HO CH2OH O O OH
OH CH2OH
O O OH
OH OH
13, 23
H COOH
H COOH C42H66O11 Z. album 28 CH2OH 794.965 O O OH
OH
OH O O
CH3
OH OH 24
H COOH
H C O CH3 C42H66O17S Z. album, 29 O O 875.029 Z. coccineum, OH Z. dumosum CH2OH OH O O OSO3H OH
OH OH
14, 25
H COOH
CH H C O 3 C42H66O14 Z. album, 29 O O 794.965 Z. coccineum, OH Z. dumosum OH CH2OH O OH O OH
OH OH 26
H COOH
C36H56O9 Z. album, 29 H COOH CH3 632.824 Z. coccineum, O O Z. dumosum OH
OH OH
15, 27 OH
HO O
OCH3
O OH O
O C28H32O19S Z. dumosum 30 O 704.608 OSO3H CH3 OH O OH OH
OH OH 28
O C42H66O18S Zygophyllum 31 O COOH 891.029 Sp. CH2OH
O O CH2OH OH O
OH OH
OSO3H OH
OH 29
O C42H66O15 Zygophyllum 31 COOH O 810.964 Sp. CH2OH O CH2OH O OH O OH OH
OH OH OH
16, 30
H O
O C43H68O17S Zygophyllum 31 H COOH CH2OH 889.056 Sp. O
CH2OH OH O OH OH
OSO3H OH OH 31
H O
O C47H74O18 Zygophyllum 31 H COOH CH 3 CH2OH 927.080 Sp. O O O
OH OH
OH OH
OH O O OH OH
OH
17, 32
O
OH COOH CH3 C41H64O13 Zygophyllum 31 O O 764.939 Sp. OH
OH
OH O O OH
OH 33
O C42H66O14 Zygophyllum 31 O 794.965 Sp. CH3 COOH
O O CH2OH OH O OH OH OH OH OH 34
O C H O Z. decumbens 32 HO 42 66 16 O 826.964 COOH CH2OH
O O O OH OH OH OH OH OH OH
18, 35
O C41H62O16 Z. decumbens 32 HO O 810.921 COOH CH OH 2 O O O OH OH OH OH OH OH OH 36 HO
O C41H66O15 Z. decumbens 33 H O 810.964 COOH CH2OH O O O OH OH
OH OH OH OH
19, 37
O
O
CH2OH C55H88O22S Z. gaetulum 33 O O CH2OH OH 1132.549 O OH OH OH O OH OH OSO3H
CH3
O
OH
OH OH 38
O
O C43H70O11 Z. gaetulum 33
CH3 763.009 O O CH2OH OH O OH OH
OH OH OH
20, 39
O
O CH 3 C55H88O11 Z. gaetulum 34 O O CH2OH OH 1036.438 O OH OH
OH O OH OH OH
CH3 O
OH
OH OH
40 OH
O
O C43H70O16S Z. eichwaldii 34 CH2OH 874.438 OH O
OH CH2OH
O
OSO3H OH
OH OH
41 OH
C35H56O8 Z. eichwaldii 35 COOH 604.814
OH O O OH
OH
21, 42 OH
COOH C35H56O11S Z. eichwaldii 36 684.354
OH O O OH
OSO3H 43
CH2OH
C39H56O5 Z. geslini 37 O
O 604.413
OH HO 44
O OH
O O C41H66O13 Z. eurypterum 38 OH 766.450 (Z atriplicoides.) OH OH
O CH2OH
O OH
OH OH
22, 45 CH3 OO H C19H18O4 Z. atriplicoides 38 O H 310.121 (Z. eurypterum) OH
46
CH2OH MeO O C18H18O7 Z .atriplicoides 38 H 346.105 (Z. eurypterum) HO H OH O OMe 47
OCH3 OO
H C19H18O7 Z. atriplicoides 38 O H 358.105 (Z. eurypterum) O OH OCH3
48
HOOC O H C18H16O6 Z. atriplicoides 38
H 328.095 (Z. eurypterum) OCH O 3 OCH3
23, 49 O
O CH3 C H O Z .atriplicoides 38 H 23 26 5 382.178 (Z. eurypterum) H3CO H O OCH 3 50
H O
O H CH3 CH2OH O O OH O O C53H84O25S Z. geslini 39 OH OH 1152.502 OH OH O OSO3H OH
COOH
O O OH
OH OH 51
H O C42H66O20S Z. geslini 39 O 954.359 COOH H
O O CH2OH
OH O OH OH
OSO3H OH
OSO3H
24, 52
H COOH C36H56O13S Z. geslini 39 728.344 H COOH CH2OH
O O OH
OH
OSO3H 53
H O O H COOH C47H74O19 Z. geslini 39 CH2OH 942.482 O O CH2OH OH O OH OH
O O OH OH OH
OH OH 54
H COOH C35H56O8 Z. fabago 40
H 604.398 CH2OH
O O OH
OH OH
25, 55
C35H54O12S Z. fabago 12 H COOH 698.344 H COOH O O OH
OH
OSO3H 56
H O
O C41H64O17S Z .fabago 12 H COOH
CH2OH 860.386 O O OH O OH OH OH OSO3H OH
HOH2C 57 O O
OH OH OH
H CH2OH C41H68O16S Z. fabago 41
H 848.423 O O OH
OSO H OH 3 OH
26,
CHAPTER-2
RESULTS AND DISCUSSION
27, 2. Stigmasterol (1)
Compound 1 was isolated from the chloroform fraction of the methanolic extract as colorless
crystals.
H
H H
HO Stigmasterol (1)
The molecular formula of compound 1 was obtained as C29H48O through high resolution electron
mass ionization (HR-EI-MS) spectrometric studies which indicated [M]+ peak at m/z 412.3711
(calcd for C29H48O; 412.3709) showing six degrees of unsaturation.
Proton NMR of compound 1 revealed a doublet for olefinic proton at δ 5.42 (1H, d, J = 4.2 Hz,
H-6) and a multiplet for one proton at δ 3.84 (1H, s, H-3). The two olefinic protons appeared as double doublet at δ 5.21 (1H, dd, J = 8.71 Hz, J = 6.7 Hz, H-22) and δ 5.06 (1H, dd, J = 8.72 Hz,
J = 6.2 Hz, H-23). The secondary methyl groups at Me-26 and Me-27 resonated as a doublet at δ
0.85 ( 3H, d, J = 6.2 Hz, H-26) and δ 0.90 (3H, d, J = 6.3 Hz), the Me-21 signal appeared as a doublet at δ 1.06 (3H, d, J = 6.6 Hz, ) and Me-29 peak appeared as a multiplet peak at δ 0.85
(3H, multiplet). The Me-18 and Me-19 protons showed singlets at δ 0.68 and 1.1 respectively.
By comparing the spectral data of compound 1 with the known data [41], compound 1 was identified as stigmasterol. 28, 2.1. β-Sitosterol (2)
The chloroform fraction of the methanolic extract yielded compound 2 as white powder.
H
H H HO β-Sitosterol (2)
Based on HR-EI-MS, the molecular formula of compound 2 was deduced as C29H50O (calcd for
C29H50O; 414.3861) corresponding to five degrees of unsaturation.
IR spectrum of compound 2 showed the absorption band at 3450 cm-1 for hydroxyl group at 1650 cm-1 and 815 cm-1 due to trisubstituted double bond.
By the comparison study of EI-MS and 1HNMR spectrum of compound 2 with literature data [41], compound 2 was characterized as β-sitosterol
29, 2.2. β-Sitosterol glucoside (3)
β-Sitosterol glucoside 3 was isolated from the EtOAc soluble part of the methanolic extract of
Zygophyllum eurypterum as amorphous solid. The HR-FAB-MS [M+1] + of compound 3 exhibited the molecular ion peak at m/z 577.4386 (calcd for C35H61O6; 577.4389).
H
CH2OH H H O O OH β-Sitosterol glucoside (3) HO OH
The 13C-NMR spectrum of compound 3 revealed 35 signals showing six methyl, twelve methylene, fourteen methine and three quaternary carbons. The detailed spectroscopic data is given in the experimental section.
The comparative study of proton NMR and 13C-NMR data with known data [42], revealed compound 3 as 3–O–β–D–-glucopyranosyl β–sitosterol.
30, 2.3. Oleanolic acid (4)
Oleanolic acid is also reported from the genus Zygophyllum and its molecular formula is
C30H48O3.
H COOH
H
HO Oleanolic acid (4) H
The EI-MS revealed the molecular ion peak at m/z 456 and HR–EI-MS showed the exact mass of at m/z 456.3601. A peak at m/z 248 demonstrates the base peak which is characteristic for pentacyclic triterpenes of β–amyrin series with a double bond at C–12. Beside molecular ion peak, the other fragment ions obtained are justified in the following scheme.
31, H RDA H COOH H COOH H A m/z = 248 HO H CCO2H + H2 m/z = 456
CH2
m/z = 133 m/z = 203 B m/z = 189
Scheme 2.1 Mass fragmentation of oleanolic acid (4)
The 1H-NMR spectrum of compound 4 showed the presence of an olefinic proton resonating at δ 5.51 (1H, t, J= 3.5 Hz), corresponding to H-12. Another downfield signal at δ 3.45 (1H, dd, J= 10.0, 5.0 Hz) was assigned to H-3α. The 1H-NMR also showed signals for seven methyls. The tertiary status of these methyls was evident from their sharp singlets in the 1H-NMR spectrum. The 13C-NMR spectrum of compound 4 indicated thirty carbons comprising of seven methyl, ten methylene, five methine and eight quaternary carbons.
The spectral data of compound 4 was in complete agreement with those of reported literature data [43], and hence the compound 4 was identified as oleanolic acid.
32, 2.4. Harmine (5)
Harmine (7–Methoxy–1–methyl–β–carboline) was first isolated in 1967 from the extract of
Zygophyllum fabago by Jerzy Lutomski, Krystyne Drost and Krystyne Schmidt [20]. However, this compound was isolated from this plant for the first time.
N H CO N 3 H
Harmine (5) CH3
The compound 5 indicated characteristic orange color on TLC with Dragendroff’s reagent which proved its alkaloid nature. The EI-MS revealed the [M] + at m/z 212.1.
The proton NMR (in DMSO) of compound 5 indicated a doublet at δ 8.14 (1H, d, J = 5.4 Hz, H-
5) and a doublet at δ 7.79 (1H, d, J = 5.4 Hz, H-6) which is ortho (o-coupled) with H-5. The vicinally coupled C-9 and C-10 protons appeared as a doublet and double doublet at δ 8.05 (1H, d, J = 8.4 Hz, H-9) and δ 6.85 (1H, dd, J = 8.7 Hz, J = 2.4 Hz, H-10). Furthermore, C-12 proton appeared as a doublet at δ 7.0 (1H, d, J = 2.1, H-12).
The 13C-NMR spectrum of compound 5 indicated two methyl, five methine and six quaternary carbon signals.
The spectral data of compound 5 were in complete agreement with those of reported literature data [44] and hence the compound 5 was identified as 7–methoxy–1–methyl–β–carboline.
33, 2.5. 7,3',4'-Trimethoxyflavone (6)
Some flavonoids are also reported from the genus Zygophyllum [47]. The present flavonoid compound (6) is the first report from this plant.
OCH3
OCH3
H CO 3 O
O 7,3',4'-Trimethoxyflavone (6)
The molecular formula of the compound 6 was established through EI-MS indicating the [M] + at m/z 312.1.
The proton NMR of compound 6 revealed a downfield doublet signal at δ 8.12 (1H, d, J = 8.72
Hz, H-5). The double doublet signal at δ 7.52 (1H, dd, J = 8.4 Hz, J = 2.1 Hz, H-6') was assigned to be C-6' proton. A doublet representing C-2' proton appeared at δ 7.35 (1H, J = 2.1 Hz, H-2') and another doublet indicating the meta coupled C-6 and C-8 protons at δ 6.94 (2H, d, J = 2.1
Hz, H-6 and H-8). Furthermore, H-5' signal appeared as a multiplet at δ 6.94 (1H, m, H-5') and a singlet at δ 6.66 (1H, s, H-3) was assigned to be C-3 proton. An upfield signal at δ 3.94 (9H, s, 3-
OMe) is represented by three methoxy protons which are attached to C-7, C-3' and C-4'.
The 13C-NMR spectrum of compound 6 indicated three methyl, seven methine and eight quaternary carbon signals. The detailed spectroscopic data are given in the experimental section.
The physical and spectral data of compound 6 are in complete agreement with those of reported literature [45], and hence compound 6 was identified as 7,3',4'-trimethoxyflavone.
34, 2.6. 5-Methylflavone (7)
5-Methylflavone (7) is another isolated compound from this plant for the first time. The EI-MS of compound 7 showed the molecular ion peak [M]+ at m/z 236.1.
O
CH3 O 5-methylflavone (7)
The proton NMR of compound 7 indicated a downfield multiplet signal at δ 7.89 (2H, m, H-
2'and H-6') indicating the meta coupled C-2' and C-6' protons and another multiplet signal showing four protons which are attached to C-3', C-4', C-5' and C-8 at δ 7.48 (4H, m, H-3', H-4',
H-5' and H-8) respectively. The two broad doublets for C-7 and C-6 protons at δ 7.39 (1H, br. d,
J = 8.1 Hz, H-7) and δ 7.13 (1H, br. d, J = 7.2 Hz, H-6). A singlet at δ 6.71 (1H, s, H-3) is assigned to be C-3 proton and another singlet at δ 2.88 (3H, s, H-14) is assigned to be the methyl protons.
The 13C-NMR spectrum of compound 7 indicated one methyl, nine methine and six quaternary carbon signals.
The physical and spectral data of compound 7 were in complete agreement with those of reported literature data [46], and hence the compound 7 was identified as 5-methylflavone.
35,
CHAPTER-3 EXPERIMENTAL
36, 3. Stigmasterol (1)
H
H H
HO Stigmasterol (1)
Physical Data
Crystallized from chloroform as colorless crystals (8.0 mg)
-1 IR (CHCl3) νmax cm : 3443 (OH), 1648 (C=C).
EI-MS m/z (rel. int. %): 412 [M] +.
1 H-NMR (pyridine, 300 MHz) : δH 5.42 (1H, d, J = 4.2 Hz, H-6), 3.84 (1H, m, H-3),
2.62 (2H, br. d, J = 6.9 Hz, H-4), 1.1 (3H, s, H-19), 0.68 (3H, s, H-18), 1.06 (3H, d, J =
6 Hz, H-21), 5.21 ( 1H, dd, J = 8.7 Hz, J = 6.7 Hz, H-22), 5.06 (1H, dd, J = 8.7, J= 6.3
Hz, H-23), 0.85 (3H, d, J = 6.3 Hz, H-26), 0.90 ( 3H, d, J = 6.3 Hz, H-27), 0.86 (3H, m,
H-29).
37, 3.1. β-Sitosterol (2)
H
H H HO β-Sitosterol (2)
Physical Data
Crystallized from chloroform as white powder (8.2 mg)
-1 IR (KBr) νmax cm : 3600-3400 (OH), 1570 (C=C).
EI-MS m/z (rel. int. %): 414 [M] +.
1 H-NMR (CDCl3+CD3OD, 300 MHz): δH 0.66 (3H, s, H-18), 0.77 (3H, d, J= 6.2 Hz, H-
27), 0.80 (3H, d, J= 6.3 Hz, H-26), 0.79 (3H, t, J= 7.1 Hz, H-29), 0.89 (3H, d, J= 6.5 Hz,
H-21), 0.99 (3H, s, H-19), 5.32 (1H, br. s, H-6), 4.35 (H1, d, J=7.7 Hz, H-1′).
38, 3.2. β-Sitosterol glucoside (3)
H
CH2OH H H O O OH β-Sitosterol glucoside (3) HO OH
Physical Data
Amorphous solid (10 mg)
-1 IR (KBr) νmax cm : 3600-3400 (O-H), 1570 (C=C).
HR-FAB-MS (+ve) m/z : 577.4386 (calcd for C35H61O6; 577.4389).
1 H-NMR (Pyridine, 300 MHz) : δH 5.33 ( 1H, br. s, H-6), 4.56 (1H, m, H-3), 4.42-4.03
(6H, m, glucosidal protons), 2.71 (1H, br. dd, J = 4.5 Hz, J = 2.7 Hz, H-4) 0.64 (3H, s, H-
18), 0.92 (3H, d, J = 6.4 Hz, H-21), 0.88 (3H, s, H-29), 0.86 (3H, s, H-26), 0.84 (3H, s,
H-27), 1.01 (3H, s, H-19).
13 C-NMR (Pyridine, 75 MHz) : δc 37.5 (C-1), 30.3 (C-2), 78.5 (C-3), 29.8 (C-4), 140.9
(C-5), 121.9 (C-6), 32.2 (C-7), 32.1 (C-8), 50.4 (C-9), 36.6 (C-10), 21.3 (C-11), 39.9 (C-
12), 42.5 (C-13), 56.9 (C-14), 24.5 (C-15), 28.7 (C-16), 56.3 (C-17), 19.5 (C-18), 12.0
(C-19), 36.4 (C-20), 19.0 (C-21), 34.3 (C-22), 26.4 (C-23), 46.1 (C-24), 29.5 (C-25), 20.0
(C-26), 19.2 (C-27), 23.4 (C-28), 12.2 (C-29), 102.6 (C-1', 71.8 (C-2'), 78.2 (C-3'), 75.4
(C-4'), 78.6 (C-5'), 62.9 (C-6').
39, 3.3. Oleanolic acid (4)
H COOH
H
HO Oleanolic acid (4) H
Physical Data
White powder (10.1 mg)
-1 IR νmax (CHCl3) cm : 3450 (OH), 1630 (C=C).
EI-MS m/z (rel. int. %): 456 [M] +.
HR– EI-MS: m/z 456.3601 (calcd for C30 H48 O3; 456.3601).
1 H-NMR (CDCl3, 500 MHz) : δH 5.51 (1H, t, J= 3.5 Hz, H-12), 3.45 (1H, dd, J= 10.0, J= 5.0 Hz, H-3), 3.32 (1H, dd, J= 14.0, J= 4.0 Hz, H-18), 1.12, 0.96, 0.91, 0.90, 0.89, 0.76 and 0.74 (each 3H, s). 13 C-NMR (pyridine, 75 MHz) : δc 180.7 (C-28), 143.7 (C-13), 122.1 (C-12), 78.6 (C-3),
55.1 (C-5), 47.5 (C-9), 46.2 (C-17), 45.8 (C-19), 41.5 (C-14), 41.1 (C-18), 39.1 (C-8),
38.5 (C-4), 38.3 (C-1), 36.8 (C-10), 33.7 (C-21), 32.8 (C-29), 32.5 (C-7), 32.4 (C-22),
30.5 (C-20), 28.0 (C-23), 27.5 (C-15), 26.6 (C-2), 25.6 (C-27), 23.3 (C-30), 23.2 (C-16),
18.1 (C-6), 16.6 (C-26), 15.3 (C-24), 15.1 (C-25).
40, 3.4. Harmine (5)
N H CO N 3 H
Harmine (5) CH3
Physical Data
Amorphous solid (30 mg)
-1 IR (KBr) νmax cm : 3421 (N-H), 1624-1417(Aromatic C=C).
EI-MS m/z (rel. int. %): 212.
HR–EI-MS m/z: 212.2473.
1 H-NMR (DMSO-d6, 300 MHz) : δH 8.14 (1H, d, J = 5.4 Hz, H-5), 7.79 (1H, d, J = 5.4 Hz,
H-6), 8.05 (1H, d, J = 8.4 Hz, H-9), 6.85 (1H, dd, J = 8.7 Hz, J = 2.4 Hz, , H-10), 7.0 (1H, d, J
= 2.1 Hz, H-12), 2.71 (3H, s, H-14), 3.86 (3H, s, OMe-11).
13 C-NMR (DMSO-d6, 100 MHz) : δc 134.5 (C-2), 141.2 (C-3), 137.7 (C-5), 111.9 (C-6),
114.8 (C-7), 127.2 (C-8), 122.5 (C-9), 109.0 (C-10), 160.1 (C-11), 94.6 (C-12), 20.3 (C-
13), 141.9 (C-14), 55.3 (OMe-11).
41, 3.5. 7,3',4'-Trimethoxyflavone (6)
OCH3
OCH3
H CO 3 O
O 7,3',4'-Trimethoxyflavone (6)
Physical Data
White crystals (10.65 mg)
EI-MS m/z (rel. int. %): 312.1
-1 IR (KBr) νmax cm : 1627 (C=O), 1601-1425 (Aromatic C=C).
1 H-NMR (CDCl3, 300 MHz) : δH 8.12 (1H, d, J = 8.7 Hz, H-5), 7.52 (1H, dd, J = 8.4 Hz, J =
2.1 Hz, H-6'), 7.34 (1H, d, J = 2.1 Hz, H-2'), 6.94 (2H, m, H-6 and H-8), 6.94 (1H, m, H-5'), 6.66
( 1H, s, H-3), 3.94 ( 9H, s, 3-OMe).
13 C-NMR (CDCl3, 75 MHz) : δc 164.1 (C-2), 106.4 (C-3), 177.9 (C-4), 127.0 (C-5),
114.2 (C-6), 163.0 (C-7), 100.4 (C-8), 117.7.0 (C-9) 157.8 (C-10), 124.3 (C-1'), 111.1 (C-
2'), 151.9 (C-3'), 149.2 (C-4'), 108.7 (C-5'), 119.8 (C-6'), 56.1 (OMe).
42, 3.6. 5-Methylflavone (7)
O
CH3 O 5-methylflavone (7)
Physical Data
White crystals (10.4 mg)
EI-MS m/z (rel. int. %): 236.1
-1 IR (KBr) νmax cm : 1651 (C=O), 1602-1413 (Aromatic C=C).
1 H-NMR (CDCl3, 300 MHz): δH 7.89 (2H, m, H-2' and H-6'), 7.48 (4H, m, H-3', H-4', H-5' and H-8), 7.39 (1H, br. d, J = 8.1 Hz, H-7), 7.13 (1H, br. d, J = 7.2 Hz, H-6), 6.71 (1H, s, H-3),
2.88 ( 3H, s, H-11).
13 C-NMR (CDCl3, 75 MHz): δc 161.6 (C-2), 108.8 (C-3), 180.6 (C-4), 141.0 (C-5),
127.7 (C-6), 132.6 (C-7), 116.1 (C-8), 122.3 (C-9), 157.7 (C-10), 22.7 (C-11), 131.7 (C-
1'), 126.1 (C-2'), 128.9 (C-3'), 131.3 (C-4'), 128.9 (C-5'), 126.1 (C-6'').
43, 3.7. General notes
3.8. Instrumentation
Melting points of all the compounds were determined on Büchi 434 apparatus. Proton NMR spectra were recorded on Bruker AM 500, 400, 300 MHz. instruments. The 13C-NMR spectra were run on 125, 100 and 75 MHz on Bruker AM 500, 400, and 300 Fourier transform NMR spectrometers, respectively.
The mass spectra were recorded on Varian-MAT 112s and Finnigan MAT-112 and 312A double focusing mass spectrometers connected to DEC PDP 11/34 and IBM-AT compatible PC based system, respectively. FAB-MS were run in glycerol-water (1:1) matrix in the presence of potassium iodide. HR-EI-MS were recorded on a Jeol-JMS H X-110 mass spectrometer.
The UV spectra were recorded on a Shimadzu UV-240 spectrophotometer. The IR spectra were recorded on a Shimadzu IR-460 (Shimadzu Corporation, Tokyo, Japan) instrument.
3.9. Chromatography
Column chromatography was carried out on silica gel (type 60, 70-230 mesh, E.Merck,
Germany). TLC was done on silica gel GF-254 precoated plates. The commercially available solvents were distillated and used for thin layer and column chromatographic techniques.
3.10. Spray reagents
Ceric sulphate reagent was used for the detection of triterpenoids and steroids aniline phthalate reagent for sugar’s detection and dragondroff reagent for alkaloids.
44, 3.10.1. Ceric sulphate
Ceric sulphate (0.1 g) and trichloroacetic acid (1 gm) were mixed in 4-5 ml distilled water. This mixed solution was boiled and conc. H2SO4 was added drop-wise until the disappearance of turbidity.
3.10.2. Aniline phthalate
Aniline (0.93 g) and o-phthalic acid (1.66 gm) were dissolved in 100 ml n-butanol saturated with
H2O.
3.11. Zygophyllum eurypterum (Z. atriplicoides)
3.11.1. Plant collection
The plant Zygophyllum eurypterum (Zygophyllaceae) was collected from Quetta, Balochistan,
(Pakistan), in 2002, and was identified by Rasool Bukhsh Tareen. A voucher specimen no.1408 was submitted to the Botany Department, Balochistan University, Quetta.
3.11.2. Isolation procedure
The plant Zygophyllum eurypterum was dried under shade for 18 days. The whole plant (20 kilogram) was crushed and concentrated with methanol at room temperature. This methanolic extract was kept in fuming hood for some days which finally resulted in a gummy residue (100.5 g). Four fractions i.e chloroform, ethyl acetate, n-butanol and water were obtained starting re- extaction from this gummy residue. The chloroform (20.2 g) and ethyl acetate fractions (60.7 g) were passed to extensive column chromatography on silica gel using solvent systems of hexane, chloroform and ethyl acetate.
45, The chloroform fraction resulted the isolation of two known compounds, stigmasterol (1), β- sitosterol (2) while the ethyl acetate fraction yielded the isolation of five known compounds i.e
β-sitosterol glucoside (3), oleanolic acid (4), harmine (5), 7,3',4'-trimethoxyflavone (6) and 5- methylflavone (7). The compounds (5-7) were reported from this plant for the first time.
Purity of compounds was finally checked on HP-TLC plates, and spots were visualized by spraying with ceric ammonium sulfate reagent followed by heating.
46, 3.12. Isolation scheme of Zygophyllum eurypterum
Zygophyllum eurypterum (20 kg)
MeOH
Methanolic Extract (100g)
Butanol Chloroform Ethyl Acetate (10.3 g) (27.4 g) (60 g)
Stigmasterol (1) β-Sitosterol (2)
Oleanolic acid (4) Harmine (5) 3`,4`,7-trimethoxyflavone (6) 5-methylflavone (7)
47,
CHAPTER-4 REFERENCES
48, 4.1. References
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53,
CHAPTER-5
SYNTHESIS OF 2, 4, 6- TRICHLOROPHENYL HYDRAZONE DERIVATIVES
54, 5. Introduction to Schiff bases Schiff bases constitute very important class of organic compounds with a large number of biological activities [1-4]. Investigation of new class of biologically active Schiff bases has been attracting the interest of medicinal chemists [5]. A number of papers and reviews have been published regarding the biological activities of Schiff bases such as antibacterial [6-9], anticancer
[10], antifungal [11-12], and herbicidal activities [13]. Heterocyclic Schiff base derivatives are reported to have anticonvulsant [14-15], antiproliferative activities [16]. A wide range of biological activities are reported for the Schiff bases of acylhyrazide such as antifungal, analgesic, antimalarial, antiplatelets, antibacterial, anticonvulsant, antituberculosis, anticancer activities [17-25], adriamycin immunoconjugates, proteinase inhibition for antiparasitic activity against Trypanosoma brucei, antilieshmanial, antimyco-bacterial, insecticidal, [26-30] and N- cyanoethylhydrazide derivatives also showed activity against β-glucuronidase enzyme [31].
The derivative of 5-nitro-2-furylhydrazone 1 [32], 4-arylhydrazono-2-pyrazoline-5-ones derivatives 2 [33], 1, 2, 4-triazoline-3(2H)-thione derivatives containing thiourea moiety 3 was found to have antitubercular active.
H C O 3 O2N N Me O N N N O N HN NH N H N Me N S O O H 1 O 2
N N Cl S N Cl HN Me 3 N S H
Figure 1. Structures of 5-nitro-2-furylhydrazone (1), 4-arylhydrazono-2-pyrazoline-5-ones (2) and 1, 2, 4-triazoline-3(2H)-thione (3).
55, Three aroylhydrazone Fe chelators, 2-hydroxy-1-naphthylaldehyde m-fluorobenzoyl hydrazone
4, salicylaldehyde isonicotinoyl hydrazone 5 and pyridoxal isonicotinoyl hydrazone 6 showed excellent antimalarial activity [34,35]. The antimalarial activity was mainly due to highest chelating behavior of iron with these three compounds.
O N F O N N OH H N 4 OH H N 5 O
HN NN
HO OH
6
Figure 2. Structures of m-fluorobenzoyl hydrazone (4), salicylaldehyde isonicotinoyl hydrazone (5), and pyridoxal isonicotinoyl hydrazone (6).
The four compounds i.e 7, 8, 9a and 9b showing strong analgesic activity may only be changed by changing aryl group or substitution on aryl group [36]. In fact, derivative 8 shows potency 11- fold greater than dipyrone as an analgesic agent [37]. Ionized and hydrazones derivatives [38-
42], 4-aminobenzoic acid hydrazide-hydrazones derivatives [43] and the final products with indole [44], p-aminosalicylic acid hydrazide derivatives [45], pyrazolone derivatives [46], 1- methyl-1H- 2-imidazo pyridine carboxylic acid hydrazide-hydrazones [47] triazole derivatives
[48] are known to have excellent antitubercular activity against Mycobacterium tuberculosis
H37Rv.
56, O O
O O N O N HN N Me H3C C N N N H H Me 7 H R CF3 8
CH3
N O O N NH R N N HN N N CH C Me N 9a H CF3 9b
Figure 3. Structures of some analgesic hydrazones.
57, 5.1. Synthetic approaches to Schiff bases
There are a large number of methods reported in literature for the synthesis of Schiff bases hydrazones [49]. Some common methods are given as follows;
5.1.1. Synthesis of hydrazones by using ethanol and triethylamine
This method is most commonly used for the synthesis of Schiff base hydrazones. For example, for the synthesis of hydrazone 12, 50 mg of compound 10 was dissolved in 5 mL ethanol, the resultant solution was treated with hydrazine hydrate 11 to get the desired hydrazone 12. In this reaction, NEt3 is used as a catalyst.The overall yield of this reaction was 61% [50] [Scheme-1].
R EtOH HN NH 2 N 11 NEt3 O NH 10 R 12 O NH2 R= H, S NH O
[Scheme-1]
5.1.2. Synthesis of azines using solvent free condition
This method involves solvent free conditions. An appropriate aldehyde like benzaldehyde, hydrazine monohydrochloride and FeCl3.6H2O were mixed thoroughly in a motar for 5 minutes.
When the reaction was completed, DCM was mixed to the reaction and thus the catalyst was taken out by filteration [51] [Scheme-2].
58, O H
FeCl3. 6H2O NH2NH2. HCl CH N N 5 min. grinding 14 13 15 Scheme-2
5.1.3. Synthesis of hydrazones by using methanol and sulphuric acid
In this case, the substrate like 16 has carboxlic acid (i.e 2, 4-diflouro-4-hydroxybipheny-3- carboxylic acid) is first esterfied and then the resultant product 17 was converted into hydrazine- hydrate 18 which is finally treated with a suitable aldehyde and thus resulting hyrazone 19 [52]
[Scheme-3].
Ar O OH C O OCH3 NH2 N C OH O NH O NH OH C C OH MeOH NH NH .H O OH 2 2 2 ArCHO H2SO4 F F F F 16 17 F F F F 18 19 [Scheme-3]
5.1.4. Synthesis of hydrazones by using DMF
In the literature, different types of solvents are used for the synthesis of Shciff base hydrazones and each method involves its own reaction time. For example, 50µL of hydrazide was mixed with 100 µL of aldehyde in DMF in the deepwell plates. Stirring the above reaction mixture at room temperatre for 24 hrs, resulted hydrazone 22 in high yield [22] [Scheme-4].
59, OH O OH DMF O R O `R NH N r.t. overnight R NH R` NH2 20H 21 22
R = H, naphthyl, 3-OH, 3-OMe, 4-OH, 4-OMe, 4-NEt2, 5-OH, 5-Br, 5-tBu, 5-Nitro R`= aryl, hetrocyclic [Scheme-4]
5.1.5. Synthesis of aryl-hydrazones using ultrasound irradiation
A very good and fast aryl-hydrazone synthesis involves ultrasound irradiation in water. The reactions was run by taking equal ratio of hydrazides 24 (such as semicarbazones 24a, guanyl hydrazide 24b or thiosemicarbazones 24c) and aryl aldehyde or ketone 23 in minium amount of water and irradiated with low intensity for about 20-30 min and then the product 25 was washed with water or ethanol [53] [Scheme-5].
O X NH2 R2 R2 H2N N NH NH X 2 ))) H2O N H R 23 24 R 25
24a: X = O Semicarbazide, 24b: X = NH Aminoguanidine, 24c: X = S Thiosemicarbazide
[Scheme-5]
60, 5.2. Results and Discussion
5.2.1. Chemistry
Drug designing in the exploration of biologically active compounds is an ongoing research of our group and for this purpose our group has synthesized a number Schiff bases of different classes of compounds. Hydrazone-hydrazide is an attractive moiety for the synthesis of different classes of organic compounds. Previouly we have reported Schiff base hydrazone derivatives and other class of compounds showing very good antiglycation activities as well as anti-leishmanial, thymidine phosphorylase and β-glucorinodase activities [1, 4, 31, 54-56]. In the light of these results, we designed our project for the synthesis of 2, 4, 6-trichlorophenyl hydrazone Schiff bases. In the present work, we have synthesized and studied some vital biological properties of 2,
4, 6-trichlorophenyl hydrazone derivatives. In our present study, we have synthesized 2, 4, 6- trichlorophenyl hydrazones 28-62 derivatives.
In a typical reaction, the commercially available 2, 4, 6-trichlorophenylhydrazine was refluxed with a variety of aromatic aldehydes in 1:1 ratio in methanol for 2 h [Scheme-6]. The reaction progress was checked by TLC. After cooling, the resultant compounds were washed with methanol and then it were dried which yielded 2, 4, 6-trichlorophenyl hydrazones 28-62 in high yields.
These compounds were finally recrystallized from hexane/ethyl acetate solvents. The structures of of these synthesized compounds were elucidated by using proton NMR, EI-MS spectroscopy and elemental analysis.
61, NH Cl HN 2 Cl Cl RCHO, MeOH Cl HN N Reflux, 2h Cl R Cl 28-62 26, 27
Scheme 6. Synthesis of 2, 4, 6-trichlorophenyl hydrazones 28-62. Compounds R Compounds R
1' 2' 6' 2' 28 46 S1' MeO OMe 4' 5' OMe
1' OH 6' 5' 3' 29 47 6' 2' 5' OMe N 4' 1'
1' 1' 6' 2' 6' Br 30 48 5' 3' 5' 3' 4' 4'
1' F 1' OH 6' 6' 31 49 5' 3' 5' 3' 4' 4'
1' 6' 2' 1' 6' 2' 32 50 5' 3' 5' Cl SMe 4'
10' 1' 2' 9' 6' 2'
33 51 3' 8' 5' 3' 4' 5' 7' NMe2 6'
62, 4' 1' OH 5' 6' 34 52 6' 2' Me 3' N 4' 1'
1' 8' 1' 7' 6' OH 35 53 3' 6' HO 3' 4' 4' 5'
1' 6' Cl 1' 6' Me 36 54 5' 3' 5' 3' Cl 4'
1' 1' 6' 2' HO OH 37 55 5' 3' 5' 3' Cl OH
1' 1' OH 6' Cl 6' 38 56 5' 3' 5' 3' 4' OH
1' 1' 6' 2' 6' OH 39 57 5' 3' Cl Cl 4' Me
1' 6' 2' 1' 6' OH 40 58 5' Cl Br Br Cl 4'
1' 6' 2' 1' 6' Cl 41 59 5' OH 3' O2N OH 4'
63, 1' 1' 6' 2' 6' 2' 42 60 5' 3' 5' 3'
OH NO2
1' 2' 1' 6' 6' OH 43 61 5' OMe Br 3' OMe 4'
1' 6' OH 1' NO 6' 2 44 62 5' OH 5' 3' OH 4'
1' 6' OH 45 5' OH 4'
5.3. General structure elucidation of compounds by spectroscopic techniques
The structure of the 2, 4, 6-trichlorophenyl hydrazone 28 was established by spectroscopic techniques. The proton NMR was carried out in deuterated methanol on 500 MHz instrument. A singlet at δ 7.83 (s, 1H, =CH) shows the imine proton and a singlet at δ 7.45 (s, 2H, H-3/5) represents the integral of two protons which are attached to the C-3 and C-5 atoms. Similarly, another singlet at δ 6.95 (s, 2H, H-2'/6') indicates the integral of two protons which are attached to C-2' and C-6' atoms. Finally, two singlets appeared for the three methoxy protons, one singlet at δ 3.86 (s, 6H, -OCH3) showing an integral of six protons and another singlet at δ 3.76 (s, 3H,
OCH3) shows the integral of the trimethoxy protons.
64, 3` OMe N 4` Cl OMe 2 NH 5` OMe 6 Cl 4 Cl
Figure-4. 1H-NMR analysis of compound 28.
The synthetic compound 28 was also proved by EI-MS showing the molecular ion peak at m/z =
389 which lead to molecular formula C16H15Cl3N2O3. Ion at m/z 374 appeared due to the loss of one chlorine atom and another two chlorine atoms corresponds to the ion at m/z 283. Finally, the loss of 3, 4, 5-trimethoxybezene lead to the most stable ion at m/z 115.
. . OMe OMe . OMe MeO OMe OMe MeO OMe MeO . N NH -Cl -2Cl 3 Cl N Cl N N NH NH NH m/z = 115 Cl Cl Cl m/z = 354 m/z = 283 m/z = 389
OMe 3 = - OMe OMe
Figure-5. Fragmentation pattern of compound 28.
65, 5.4. Biological Studies
Antiglycation activity
In our present study, in vitro antiglycation activity of thirty five compounds [28-62] of 2, 4, 6- trichlorophenyl hydrazone derivatives have been studied in an extension of our preceding study
[54-56]. In diabetes mellitus, elevated blood sugar level leads to chronic complications such as, retinopathy, micro and macro-vascular diseases, nephropathy and peripheral neuropathy [57-58].
Diabetic individuals are prone to oxidative stress, which causes oxidative damage of proteins.
Several hypotheses are involved in the pathogenesis of oxidative stress in diabetic individuals that include glycation.
Glycation is a non-enzymatic modification of proteins, which occurs following synthesis of proteins. It comprises of two phases, early and advanced phases. In the early phase of glycation, reducing sugar reacts with epsilon amino group of lysine and terminal amino groups of proteins to form reversible Schiff base, which rearranges itself into an Amadori product. Glycated proteins in the presence of transition metals and molecular oxygen can undergo further modifications and form dicarbonyl intermediates such as methylglyoxal (MG), glyoxal (GO) and
3-deoxyglucosones (3-DG), which accelerate the process of glycation and form poorly characterized structures called advanced glycation end products (AGEs) [59].
Several inhibitors, for instance aminoguanidine, vitamin B6, aspirin and flavonoids have been reported in this regard against glycation and the process is gaining increasingly much interest as a potential therapeutic target [60].
In our present study, we investigated the therapeutic potential of Schiff bases hydrazones against glycation. Presently diabetic disease is more spreading, so further study on new antiglycation
66, compouds is more demonding. Since currently number of valuable antiglycating compouds is very small, the need of new antiglycating compouds are still required [61].
In vitro anti-glycation activity
The compounds 28-62 were studied for the antiglycation activity. These compounds exhibited
IC50 values ranging between 27.2–949.019 µM. Compounds 41 (IC50 = 27.2 ± 0.00 µM), and 45
(IC50 = 55.7 ± 0.00 µM) revealed potent antiglycation activity than the rutin standard (IC50 = 70
± 0.5 µM). Compounds 35 (IC50 = 95 ± 0.004 µM), 55 (IC50 = 175 ± 0.004 µM), 56 (IC50 = 178
± 0.005 µM), and 42 (IC50 = 276 ± 0.014 µM) exhibited less antiglycation potential activity than the above two compounds. Nevertheless, two compounds 44 (IC50 = 354 ± 0.008 µM) and 51
(IC50= 949 ± 0.019 µM) were found least active among the series. Rest of the compounds which have shown inhibitions under 50%, were not further studied.
Compounds 41 and 45, having dihydroxyl groups are the highest active analogues among the active compounds with IC50 values of 27.2 ± 0.00 and 55.7 ± 0.00 µM, respectively. A relatively high antiglycation potential of compounds 41 (3, 4-dihydroxyl analogue) and 45 (2, 3-dihydroxy analog) might be due to the acetal formation of hydroxyl groups with carbonyl group of methylglyoxal. Our hypothesis is confirmed when we compared compound 41 with its methylated derivative 43 which is completely inactive, similarly compound 29 was found to be inactive as compared to its free dihydroxyl analogue 45. Furthermore, we found that the position of hydroxyl group alter the antiglycation potential as shown in compounds 35, and 56 with IC50 values 95 ± 0.004, and 178 ± 0.005 µM, respectively. This may be due to the formation of hemiacetal instead of acetal as in case of compounds 41 and 45.
67, The sharp decrease in activity of trihydroxyl analogues of compounds 55 and 44 is presumably caused by the steric hindrance in the formation of acetal. Nevertheless, compound 51 showed very weak antiglycation activity with IC50 = 949 ± 0.019 µM.
Table-1. Results of antiglycation assay of 2, 4, 6-trichlorophenyl hydrazones 28-62. a a Compound No. IC50 (µM ± SEM Compound No. IC50 (µM ± SEM ) 28 NAb 46 NAb 29 NAb 47 NAb 30 NAb 48 NAb 31 NAb 49 NAb 32 NAb 50 NAb 33 NAb 51 949 ± 0.019 34 NAb 52 NAb 35 95 ± 0.004 53 NAb 36 NAb 54 NAb 37 NAb 55 175 ± 0.004 38 NAb 56 178 ± 0.005 39 NAb 57 NAb 40 NAb 58 NAb 41 27.2 ± 0.00 59 NAb 42 276 ± 0.014 60 NAb 43 NAb 61 NAb 44 354 ± 0.008 62 NAb 45 55.7 ± 0.00 Rutin c 70 ± 0.5 SEMa is the standard error of the mean, NAb Not active, Rutin,c standard inhibitor for antiglycation activity.
68, α-Chymotrypsin inhibitory activity
Natural substrates of enzymes are interceded in their conversion particularly by the enzyme inhibitors. Investigation of the choice of drugs in the pharmaceutical research rewards to enzyme inhibition study of the reported compounds. Therefore, the enzyme inhibitors are urgent need in case of numerous physiological abnormalities which debilitates enzymes hyperactivity [62].
In the enzyme mundane, proteases are not only decimating cellular proteins and peptides but also help in replication of some viruses. Proteases of HCV (NS3 protease) and HIV are important enzymes that accelerate in the replication of virus are the target for anti-HCV and anti-HIV drugs
[63].
Serine proteases are also the target for plant pathogens if inhibitors are declared in genetically engineered plants. So far, genetically engineered tobacco plants signifying protease inhibitors have shown as insect/pest resistant breed [64, 65]. Bowman-Birk-type protease inhibitors demonstrated as tumor suppressor in vitro as well as in vivo [66].
Kalikreins are in fact serine proteases concerned in skin desquamation, plasticity, psoriasis, prostate cancer, and neural breast [67]. Serine protease cleaves CD14 into soluble sCD14 which is bio-marker of pediatric pneumonia and enhances the death rate [68]. The majority of the serine protease inhibitors obstruct more than one type of serine protease [69]. Both chymotrypsin and cathepsin are responsible for the breakage of interleukin 1-β (IL-1β) precursor into IL-1β which causes inflammatory arthritis [70]. Chymotrypsin mobilizes epithelial sodium channel (EnaC) by proteolytic cleavage and consequences in cystic fibrosis [71].
69, In vitro α-chymotrypsin inhibitory activity
The compounds 28-62 were screened for the in vitro the α-chymotrypsin enzyme. These compounds showed a range of inhibition between 44.45-447.57 µM. The compounds 42 (IC50 =
43.81 ± 5.88 µM), 41 (IC50 = 44.45 ± 1.25 µM), 54 (IC50 = 55.23 ± 1.68 µM), 45 (IC50 = 57.43 ±
0.52 µM), 43 (IC50 = 68.71 ± 2.29 µM) and 57 (IC50 = 70.27 ± 4.45 µM), showed comparatively activity relative to the chymostatin standard (IC50 = 5.7 ± 0.130). The compounds 46 (IC50 =
111.94 ± 2.26 µM), 33 (IC50 = 166.38 ± 0.78 µM), 48 (IC50 = 224.59 ± 4.12 µM), 30 (IC50 =
240.03 ± 5.75 µM) and 29 (IC50 = 279.46 ± 5.34 µM) showed moderate activity. Nevertheless, compound 50 (IC50 = 447.57 ± 1.08 µM) is the least active among the active compounds in the series. The compounds 28, 31, 32 34-40, 44, 47, 49, 51-53, 55, 56, and 58-62 revealed inhibitions under 50%, so therefore, these compounds were not further studied.
The compound 42 is p-hydroxyl substituted and its highest activity is presumably caused by its greater chelation capacity with the enzyme. On the other hand, compound 54, an o-methylated analogue is the second most active; however, its p- methylated analogue i.e compound 57 is less active which is probably due to the position of methyl group. Compounds 41 and 45 having 3, 4- dihydroxyl and 2, 3-dihydroxyl groups are less active relative to compound 42 due to the above reason. The less activity of compound 45 than compound 41 is probably due to its less chelating behavior with the enzyme related to the later compound. Surprisingly, the compound 35 that is 2,
5-dihydroxyl substituted found to be inactive due to the same reason. The compound 43 showed less activity then its free dihydroxyl analogue. The compound 46 is active due to the thiophene ring. The compound 33 showed moderate activity due to N, N-dimethylamine substitution. The compound 48 shows activity presumably by the nitro group which has capability to chelate with enzyme. The compound 30 is more active than compound 29 which means enzyme have specific
70, behavior towards different compounds. The compound 50 showed least activity having meta chloro substitution even less active than unsubstituted analogue.
Table-2. Results of α-chymotrypsin inhibitory assay of 2, 4, 6-trichlorophenylhydrazones 28-62. a a Compound No. IC50 (µM ± SEM Compound No. IC50 (µM ± SEM ) 28 NAb 46 111.94 ± 2.26 29 279.46 ± 5.34 47 NAb 30 240.03 ± 5.75 48 224.59 ± 4.12 31 NAb 49 NAb 32 NAb 50 447.57 ± 1.08 33 166.38 ± 0.78 51 NAb 34 NAb 52 NAb 35 NAb 53 NAb 36 NAb 54 55.23 ± 1.68 37 NAb 55 NAb 38 NAb 56 NAb 39 NAb 57 70.27 ± 4.45 40 NAb 58 NAb 41 44.45 ± 1.25 59 NAb 42 43.81 ± 5.88 60 NAb 43 68.71 ± 2.29 61 NAb 44 NAb 62 NAb 45 57.43 ± 0.52 Chymostatinc 5.7 ± 0.13 SEMa is the standard error of the mean, NAb Not active, chymostatinc, standard inhibitor for α- chymotrypsin activity.
71, Carbonic anhydrase inhibitory activity
In living system, the enzyme inhibition activity is a vital means by which metabolic pathways are regulated. Various enzyme inhibitors have discovered effective chemotherapeutic agents since they can block or reduce the activity of exact enzymes [72].
Carbonic anhydrases (CA) are ubiquitous zinc containing enzymes present in archaea prokaryotes and eukaryotes and in higher animals, like human. Fourteen different carbonic anhydrases isoenzymes are recognized having varied tissue distribution and subcellular localization. Generally, there are some enzymes which are classified in this group, such as cytosolic types (CA-I, CA-II, CA-III and, CA-IV), 4 membrane-bound isoenzymes (CA-IV, CA-
IX, CA-XII and CA-XIV), one mitochondrial type (CA-V), and secreted CA-isozyme CA-IV
[73].
The interconversion between the carbon dioxide and bicarbonate ion, CA (EC 4.2.1.1,. CA-II) catalyses a very simple physiological reaction. It is also concerned in certain physiological process associated with oxygen intake and transfer of CO2/HCO3 between metabolizing lungs as well as tissues. CA also play a vital role in pH and CO2 homeostasis, electrolyte secretion, biosynthetic reactions like lipogenesis, glucoenogenesis, bone resorption, ureogenesis, tumorgenicity, calcification, and several other pathological or physiological processes. Cancer cells have an elevated replication rate than the normal cells. This requires a high flux of bicarbonate in the metabolic pathway [74], hence the expression of CA’s is enhanced in many tumors, where they act to acidify the extra cellular milieu and provide tumors a growth advantage over normal tissues
[75]. It was reported that expression of CA-I and CA-II correlate well with the aggressiveness of colorectal cancer and synchronous distant metastasis.
72, Carbonic anhydrase CO2 + H2O H2CO3
Figure-6. Action of carbonic anhydrase.
The majority of the inhibitors of carbonic anhydrase belong to the sulfonamide or sulfamate classes of compounds [76]. These inhibitors are clinically used for the treatment of different diseases like epilepsy, anticancer, glaucoma, acid-base disequilibria, and other minor neuromuscular disorders. Some other classes of compounds are in clinical investigation as antiobesity agents [77]. Since with passage of time, many advancements have been done to discover successful CAIs which can be used to cure glaucoma, with two available drugs, brinzolamide (AZOPTTM, Alcon Laboratories), and dorzolamide (TRUSOPTTM, Merck &
Co.). These drugs solved many problems which were earlier noted, previously observed with the systemically used CAIs for the treatment of glaucoma. Both brinzolamide and dorzolamide are helpful antiglaucoma compounds but still they cause some problems in many patients.
In vitro carbonic anhydrase inhibitory activity
The compounds 28-62 were screened for the in vitro carbonic anhydrase activity. These compounds showed a range of inhibition between 75.95-227.96 µM. The compounds 38 (IC50 =
75.95 ± 3.93 µM), 43 (IC50 = 82.64 ± 0.73 µM), 51 (IC50 = 91.75 ± 4.21 µM), 60 (IC50 = 94.31 ±
2.34 µM) and 29 (IC50 = 97.60 ± 1.31 µM) were relatively less active compared to the acetazolamide standard (IC50 = 0.12 ± 0.03) while the compounds 59 (IC50 = 113.43 ± 1.70 µM),
34 (IC50 = 125.32 ± 3.00 µM), 30 (IC50 = 154.0 ± 2.72 µM) showed moderate activity. On the other hand, the compounds 46 (IC50 = 201.70 ± 1.43 µM), 36 (IC50 = 217.90 ± 1.06 µM), and 53
(IC50 = 227.96 ± 3.95 µM) are the least active compounds among the active compounds. The compounds 28, 31-33, 35, 37, 39-42, 44, 45, 47-50, 52, 54-58, 61 and 62 showed inhibitions under 50%, so, therefore, these compounds were not further studied. 73, The compound 38 which is 2-chloro substituted showed higher activity which is presumably caused by its greater binding capacity with the enzyme. On the other hand, the compound 37 which is p-chloro substituted is not allowed active and hence we assume that p-cholro substituted analogue of 37 is not allowed to bind with the enzyme. Compound 43, a 3,4-dimethoxy analogue is another good active compound and its activity is probably caused by the electronic effects of the two methoxy groups. Similarly, compound 29 which is 2-hydroxy-3-methoxy substituted, is less active than compound 43, because of the presence of one methoxy group. We assume that as the number of hydroxyl groups increases, the the activity of compound will decreases. Therefore, compounds 41, 44, 45 and 55 are inactive because of the above reason. However, to compound
43, compound 28 which is 3, 4, 5-trimethoxy substituted is inactive which is because of the steric bulk of the three methoxy groups.
The activity of of compound 51 is most probably due to the presence phenanthrene ring which might show hydrophobic interaction with the enzyme. Conversely to compound 51, compound
30 which has only one benzene ring is less active which is possibly due to less hydrophobic interaction with the enzyme. On the other hand, compound 53 is not active and hence from this we assume that enzymes have different behavior towards different molecules. The compound 34, a 2-hydroxyl-5-methyl substituted analogue is active while compound 35 which is 2, 5- dihydroxyl substituted is inactive, the main reason of this is that the methyl sustituent in the former compound has electron donating effect while the two hydroxyl groups in the later compound has electron withdrawing effects which probably diminishes the activity of this compound. The activity of compound 60 is due to p-nitro substituted group. On the other hand, compound 59 which is 2-chloro-5-nitro substituted is less active than compound 60 and from this we assume that the former compound is not as effectively chelated with the enzyme as the later compound does, so that is why, it show less activity than the later compound. The
74, compound 46 is active due to the presence of thiophene ring. Finally, the weaker activity of compound 37 is presumably caused by 2, 4-dichloro atoms.
Table-3. Results of carbonic anhydrase inhibitory assay of 2, 4, 6-trichlorophenyl hydrazones 28-62. a a Compound No. IC50 (µM ± SEM Compound No. IC50 (µM ± SEM ) 28 NAb 46 201.70 ± 1.43 29 97.60 ± 1.31 47 NAb 30 154.0 ± 2.72 48 NAb 31 NAb 49 NAb 32 NAb 50 NAb 33 NAb 51 91.75 ± 4.21 34 125.32 ± 3.00 52 NAb 35 NAb 53 227.96 ± 3.95 36 217.90 ± 1.06 54 NAb 37 NAb 55 NAb 38 75.95 ± 3.93 56 NAb 39 NAb 57 NAb 40 NAb 58 NAb 41 NAb 59 113.43 ± 1.70 42 NAb 60 94.31 ± 2.34 43 82.64 ± 0.73 61 NAb 44 NAb 62 NAb 45 NAb Acetazolamidec 0.12 ± 0.03 SEMa is the standard error of the mean, NAb : Not active, acetazolamide c standard inhibitor for carbonic anhydrase activity.
75, Urease inhibitory activity
The hydrolysis of urea to ammonia and carbon dioxide is catalyzed by the urease (urea amidohydrolase EC 3.5.15) [78]. For an animal to utilize urea as an alternative source of nitrogen, urease enzyme plays a very important role [79]. In plants, urease can serve as a guard protein in systemic nitrogen transport pathways [80]. Urease is responsible for the problems of pathologies induced by Helicobacter pylori, thus permit them to live at low pH of the stomach and, therefore, play a vital role in the pathogenesis of gastric and peptic ulcer, apart from cancer as well [81]. Urease is directly concerned in the formation of infection stones and furnishes to the pathogensis of pyelnephritis, urolithiasis, ammonia and hepatic encephalopathy, urinary catheter encrustation and hepatic coma [82].
In vitro urease inhibitory activity
The compounds 28-62 were screened against the urease enzyme. These compounds showed a range of inhibition between 44.1-328.06 µM. Compared to the thiourea standard (IC50 = 21 ±
0.011), compounds 44 (IC50 = 44.1 ± 0.44 µM) and 55 (IC50 = 83.13 ± 0.78 µM) were less active while compounds 41 (IC50 = 167.1 ± 1.23 µM) and 56 (IC50 = 171.53 ± 0.95 µM) showed moderate activity. On the other hand, the compounds 43 (IC50 = 290.1 ± 2.07 µM), 35 (IC50 =
312.2 ± 4.97 µM), and 53 (IC50 = 328.06 ± 3.4 µM), are the least active compounds among the active compounds. The compounds 28-34, 36-40, 46-54, and 59-62 indicated inhibitions under
50%, so, therefore, these compounds were not further studied.
The compound 44 which is 2, 3, 4-trihydroxyl substituted show highest activity and it might be due to its higher hydrogen chelation capacity with the enzyme. Compared to compound 44, compound 55 which is 2, 4, 5-trihydroxyl substituted shows less activity which is possibily due to steric reasons. On the other hand, the compounds 41 and 56 having 3,4-dihydroxyl and 2,4-
76, dihydroxyl groups showed very less activity in comparison to its trisubstituted analogues and hence it proves that higher number of hydroxyl groups in the molecule is enhancing the activity of the molecule. If compound 41 is compared with compound 56 then even though both these compounds have very close IC50 values but slight higher IC50 value of former compound than the later compound is probably due to the adjacent hydroxyl groups which are in better position to chelate with the enzyme. If we assume that in dihydroxyl substituted analogues, as the position of the two hydroxyl groups is changing, then the enzyme chelation behaviour to those molecules will decreases. This hypothesis is proved in compound 35 which is 2, 5-dihydroxyl substituted, showed very least activity in comparison to compounds 41 and 56 because of the same reasons.
On the other hand, if the two hydroxyl groups in compound 41 are substituted by the two methoxy groups as like in compound 43, then the later compound activity is sharply declined.
Finally, compound 58 show very leas activity due to the presence of one hydroxyl group.
Table-4. Results of urease assay inhibitory of 2, 4, 6-trichlorophenyl hydrazones 28-62. a a Compound No. IC50 (µM ± SEM Compound No. IC50 (µM ± SEM ) 28 NAb 46 NAb 29 NAb 47 NAb 30 NAb 48 NAb 31 NAb 49 NAb 32 NAb 50 NAb 33 NAb 51 NAb 34 NAb 52 NAb 35 312.2 ± 4.97 53 NAb 36 NAb 54 NAb 37 NAb 55 83.13 ± 0.78 38 NAb 56 171.53 ± 0.95 39 NAb 57 NAb
77, 40 NAb 58 328.06 ± 3.4 41 167.1 ± 1.23 59 NAb 42 NAb 60 NAb 43 290.1 ± 2.07 61 NAb 44 44.1 ± 0.44 62 NAb 45 NAb Thioureac 21 ± 0.011
SEMa is the standard error of the mean, NAb Not active, Thioureac, standard inhibitor for urease activity.
Antioxidant studies
In ordinary physiological circumstances, free radicals are formed to protect the body from external agents, and living system has a balance system of their production and elimanation.
Thus variation between the production and elimanation of radicals may results the oxidative damage of biomolecules. Reactive oxygen species (ROS) [83, 84] such as singlet oxygen [85], hydroxyl [86], peroxyl [87], superoxide [88], and peroxynitrite [89] radicals represents the most harmful species. Antioxidant compounds can impede free radicals damage and thus protect the body from various dangerous effects [90]. An imbalance between reactive free radicals and the antioxidant may results an oxidative pressure which finally results cell injury. Oxidative strain can demage the balance between oxidants and reductants and thus consequently lead to numerous diseases like cancer, stroke, and immunodeficiency syndrome, coronary artery and tumor diseases [91-92].
Superoxide anion radical is produced by the living organisms in an array of various metabolic processes like glucose metabolism, inflammation, and fats oxidation in peroxisomes [93]. Free radicals spoil proteins, nucleic acids, lipids, and other biomolecules, for example, the extra cellular matrix glycosaminoglycans e.g. hyaluronic acid, sulfur-containing amino acids and the polyunsaturated fatty acids are generally susceptible. 78, In vitro DPPH radical scavenging activity
In search of a novel class of DPPH radical scavenger compounds, the compounds 28-57 were screened for the in vitro DPPH radical scavenging activity. These compounds showed a varying degree of IC50 values ranging between 4.05-369.30 µM. The compounds 44 (IC50 = 4.05 ± 0.06
µM), 56 (IC50 = 4.23 ± 0. 09 µM), 45 (IC50 = 4.41 ± 0.21 µM), 41 (IC50 = 4.49 ± 0.04 µM), 35
(IC50 = 5.85 ± 0.24 µM), 42 (IC50 = 6.30 ± 0.29 µM), 39 (IC50 = 6.32 ± 0.072 µM), 29 (IC50 =
7.21 ± 9.12 µM), 55 (IC50 = 20.09 ± 0.30), 34 (IC50 = 24.42 ± 0.86 µM) and 49 (IC50 = 30.25 ±
2.53 µM) showed IC50 values which were much better than the standard value of n-propylgallate
(IC50 = 30.12 ± 0.27 µM). The compounds 32 (IC50 = 95.20 ± 4.94 µM), 33 (IC50 = 115.54 ±
3.07), 47 (IC50 = 125.27 ± 3.09), 36 (IC50 = 136.26 ± 3.56 µM) and 30 (IC50 = 185.25 ± 5.81
µM) showed moderate IC50 values. On the other hand, the compounds 31 (IC50 = 231.58 ±
2.79µM), 40 (IC50 = 240.39 ± 1. 94 µM), 52 (IC50 = 251.51 ± 1. 60 µM), 54 (IC50 = 253.80 ±
4.82 µM), 28 (IC50 = 255.40 ± 3.85 µM), 50 (IC50 = 278.73 ± 7.67µM), 46 (IC50 = 291.43 ± 6.16
µM), 38 (IC50 = 295.85 ± 6.29 µM), 51 (324.65 ± 8.35 µM), 48 (329. 94 ± 6.66 µM), 53 (330.66
± 4.82 µM), 37 (IC50 = 353.90 ± 1.15µM), 43 (IC50 = 364.85 ± 5.21µM) and 57 (IC50 = 369.30 ±
1. 52µM) revealed least activity among the active compounds.
The highest activity of compound 44 which is a 2, 3, 4-trihydroxyl analogue is presumably caused by the trihydroxyl groups which can stabilizes resonatively the free radical in a better way. It can be understood by the following resonance structures,
R R R R R O O O O O
O O O O O O O O O O
Figure-7. Different resonance forms of compound 44
79, On the other hand, compound 56, having 2, 4, 6-trihydroxyl groups which is a closest counterpart of compound 44 is not fully stabilized and hence show slight lower activity as compared to this compound. This can also be shown by the following resonance structures,
R R R R O O O O (Z)
(E) (E) (E) O O O O
Figure-8. Different resonance forms of compound 56.
Similarly, compounds 45, a 2, 3-dihydroxyl analogue, 41, a 3, 4-dihydroxyl analogue, 35, a 2, 5- dihydroxyl analogue, and 42, a 4-hydroxyl analogue is less active than compounds 44 and 56 which probably have less resonance free radical stabilities as compared to its trihydroxyl counterparts. Analogously, if we compare the IC50 value of compound 42 with compounds 34 and 49, then it proves that resonance stability is mainly responsible for the enhanced activity of compound 42 relative to these compounds.
R R R R
O O O O
Figure-9. Resonance stabilization of compound 42.
Alternatively, compound 39 which is a 3, 5-dichloro-2-hydroxyl analogue showed higher activity as compared to compounds 29, and 55. This is possibily due to the presence of one hydroxyl and two chlorine atoms which can stabilizes the free radicals in a much better manner in comparison to these compounds. Compound 32 shows slight higher activity than compound 33, which is presumably caused by the presence of methylated sulfur which exert less mesomeric electron
80, donating effect as compared to N, N-dimethylamino group. The compound 47 shows moderate activity which may be due to the presence of nitrogen atom of the pyridine ring. The compound
36, a 2, 4-dichloroanalogue, show higher activity as compared to compounds 30 and 31 which is probably due to electron withdrawing effects of the two chlorine atoms. Analogously, compound
40 shows higher activity relative to compounds 52, 54, and 28, due to the same reason. Similarly, compounds 50, 38 and 48 are less active than compound 40 due to the presence of one halogen atom in each of these compounds. Finally, compounds 46, 51, 43, 53, 37 and 57 show least activities in the series which is presumably caused by the formation of an unstabilized or not fully stabilized free radical in these compounds.
Table-5. Results of DPPH radical scavenging assay of 2, 4, 6-trichlorophenylhydrazones 28-62. a a Compound # IC50 (µM ± SEM ) Compound #. IC50 (µM ± SEM ) 28 255.40 ± 3.85 44 4.05 ± 0.06 29 7.21 ± 9.12 45 4.41 ± 0.21 30 185.25 ± 5.81 46 291.43 ± 6.16 31 231.58 ± 2.79 47 125.27 ± 3.09 32 95.20 ± 4.94 48 329. 94 ± 6.66 33 115.54 ± 3.07 49 30.25 ± 2.53 34 24.42 ± 0.86 50 278.73 ± 7.67 35 5.85 ± 0.24 51 324.65 ± 8.35 36 136.26 ± 3.56 52 251.51 ± 1. 60 37 353.90 ± 1.15 53 330.66 ± 4.82 38 295.85 ± 6.29 54 253.80 ± 4.82 39 6.32 ± 0.072 55 20.09 ± 0.30 40 240.39 ± 1.94 56 4.23 ± 0. 09 369.30 ± 1. 52 41 4.49 ± 0.04 57
42 6.30 ± 0.29 n-Propylgallatec 30.12 ± 0.27 43 364.85 ± 5.21 SEMa is the standard error of the mean, NAb Not active, n-propylgallatec c, standard inhibitor for DPPH radical scavenging activity
81, Superoxide anion radical scavenger activity
All the synthesized compounds (28-57) were screened for the superoxide anion radical scavenging activity which exhibit IC50 values ranging between 91.23-406.90 µM. The compounds 55 (IC50 = 91.23 ± 1.2 µM), 35 (IC50 = 95.5 ± 1.53 µM), 44 (IC50 = 97.5 ± 1.65 µM),
42 (IC50 = 103.72 ± 1.78 µM), 41 (IC50 = 105.31 ± 2.29 µM), showed IC50 values which were much better than the standard value of n-propylgallate (IC50 = 106.34 ± 1.6 µM). Similarly, compounds 45 (IC50 = 110.01 ± 2.1 µM), 39 (IC50 = 111.15 ± 1.57 µM) and 29 (IC50 = 113.38 ±
2.56 µM) showed IC50 values which were comparable the standard value of n-propylgallate. The compounds 34 (IC50 = 144.92 ± 2.03 µM), 47 (IC50 = 209.57 ± 2.3 µM) showed moderate IC50 values relative to the standard value. On the other hand, the compounds 43 (IC50 = 258.8 ± 1.92
µM), 40 (IC50 = 268.9 ± 1.66 µM) and 31 (IC50 = 406.90 ± 2.4 µM), revealed least activity among the active compounds. The compounds 28, 32, 33, 36-38, 43, 46, 48-54 and 56-62 indicated inhibitions under 50%, so, therefore, these compounds were not further studied.
The highest activity of compound 55 is presumably caused by the presence of 2, 4, 6-trihydroxyl groups which may stabilises the phenoxide ion radicals formed in the bioassay. Similarly, compound 35, a 2, 5-dihydroxyl analogue is less active than compound 55 which is possibily due to comparatively less stabilization of the phenoxide ion radicals relative to its highest active counterpart. Analogously, due to highest resonance stability of the phenoxide ions of the 2, 5- dihyroxyl groups of compound 35 compared to the 2, 3, 4-trihydroxyl analogue of compound 44, the former compound is more active than the later compound. On the other hand, compound 42 which is a 4-hydroxyl analogue shows higher activity than compounds 41 and 45 due to the same reasons. Alternatively, compounds 39, 29 and 34 are less active relative to their dihydroxyl analogues which might be due to the electron withdrawing and electron donating effects of the substituents in these compounds. Finally, compounds 47, 43, 40 and 54 are the least active 82, compounds which are possibly caused by the electronic effects of the substituents in these compounds.
Table-6. Results of superoxide anion radical scavenging assay of 2, 4, 6-trichlorophenyl hydrazones 28-62. a a Compound # IC50 (µM ± SEM ) Compound # IC50 (µM ± SEM ) 28 NAb 44 97.5 ± 1.65 29 113.38 ± 2.56 45 110.01 ± 2.1 30 NAb 46 NAb 31 NAb 47 209.57 ± 2.3 32 NAb 48 NAb 33 NAb 49 NAb 34 144.92 ± 2.03 50 NAb 35 95.5 ± 1.53 51 NAb 36 NAb 52 NAb 37 NAb 53 NAb 38 NAb 54 406.90 ± 2.4 39 111.15 ± 1.57 55 91.23 ± 1.2 40 268.9 ± 1.66 56 NAb 41 105.31 ± 2.29 57 NAb 42 103.72 ± 1.78 n-Propylgallatec 106.34 ± 1.6 43 258.8 ± 1.92 SEMa is the standard error of the mean, NAb Not active, n-Propylgallatec c, standard inhibitor for superoxide anion radical scavenging.
Phosphodiesterase inhibitory activity
Phosphodiesterases/Nucleotide pyrophosphatases (E.C. 3.1.4.1) are hydrolases that act on diesters of phosphoric acid. They catalyses the liberation of nucleoside-5´-monophosphates from a variety of pyrophosphate bonds (e.g. nucleoside diphosphates and triphosphates, NAD, FAD, and UDP-glucose) and phosphodiester bonds (in oligonucleotides and exogenous substrates like di-p-nitrophenyl phosphate, and p-nitrophenyl ester of TMP) [94]. The NPP family consists of three members which are NPP-1, NPP-2 and NPP-3. They are commonly dispersed in
83, mammalian intestinal mucosa, liver cells, and serum, snake venom and in different plants. They have specific patterns of circulation in different cells types and even within the same types of cells [95]. NPP-1 has been restricted on cells of the distal convoluted tubule of the kidney, chondrocytes, osteoblasts, epididymic and hepatocytes. NPP1 or PC1 (plasma cell membrane glycoprotein) is a key controller of calcification of bone and other tissues. Over-expression of
NPP1 is related with chondrocalcirosis [96], while under expression causes severe periarticular calcification in mice [97] and the syndrome of idiopathic infantile arterial calcification in human
[98].
In vitro phosphodiesterase inhibitory activity
The synthetic compounds 28-57 were screened for the phosphodiesterase assay. All the synthesized compounds were inactive in this assay.
Table-7. Results of phosphodiesterase inhibitory assay of 2, 4, 6-trichlorophenyl hydrazones 28-62. a a Compound No. IC50 (µM ± SEM Compound No. IC50 (µM ± SEM ) 28 NAb 46 NAb 29 NAb 47 NAb 30 NAb 48 NAb 31 NAb 49 NAb 32 NAb 50 NAb 33 NAb 51 NAb 34 NAb 52 NAb 35 NAb 53 NAb 36 NAb 54 NAb 37 NAb 55 NAb 38 NAb 56 NAb 39 NAb 57 NAb
84, 40 NAb 58 NAb 41 NAb 59 NAb 42 NAb 60 NAb 43 NAb 61 NAb 44 NAb 62 NAb
45 NAb EDTAc 274 ± 0.007
SEMa is the standard error of the mean, NAb Not active, EDTAc standard inhibitor for phosphodiesterease activity.
Cytotoxic activity
All the synthetic compounds 28-62 exhibited no cytotoxicity against Artemia salina.
5.5. Conclusion
All of the synthetic compounds 28-62 were randomly screened for the in vitro antiglycation, α- chymotrypsin, carbonic anhydrase, urease, antioxidant (DPPH & superoxide anion scavenging assays) and phosphodiesterase activities.
In the antiglycation assay, out of 35 compounds, a total of eight compounds showed activity in which two compounds 41 and 45 were much better than the standard drug, rutin, four compounds 35, 55, 56 and 42 were good active and the rest of two compounds 44 and 51 were weakly active.
In the α-chymotrypsin inhibition assay, total twelve compounds exhibited activity in which six compounds 42, 41, 54, 45, 43 and 57 showed good activity, five compounds 46 , 33 , 48 , 30 and
29 showed moderate activity and the remaining one compound 50 was least active in this assay.
In the carbonic anhydrase assay, total eleven compounds showed activity, in which five compounds 38, 43, 51, 60 and 29 were good active, three compounds 59, 34 and 30 were moderately active and the remaining three compounds 46, 36 and 53 were weakly active.
85, In the urease assay, total seven compounds exhibited activity in which two compounds 44 and 55 were good active, two compounds 41 and 56 were moderately active and the rest of three compounds 43, 35 and 53 were weakly active.
In the DPPH radical scavenging assay, all the thirty compounds showed activity in which eleven compounds 44, 55, 45, 41, 35, 42, 39, 29, 56, 34 and 49 were much better than the standard n- propylgallate, five compounds 32, 33, 47, 36 and 30 were moderately active while the remaining fourteen compounds 31, 40, 52, 54, 28, 50, 46, 38, 51, 48, 53, 37, 43 and 57 were weak active.
In the superoxide anion radical scavenging assay, out of 30 compounds, total thirteen compounds exhibited activity in which five compounds 55, 35, 44, 42, 41 showed much better three compounds 45, 39 and 29 have comparable values to the standard n-propylgallate, two compounds 34 and 47 were moderately active and the remaining three compounds 43, 40 and 31 were weakly active.
Finally, in the phosphodiesterase assay, all the compounds showed no activity.
5.6. General Instrumentation
Melting points of all the compounds were recorded on a Büchi 434 apparatus. NMR spectra were done on Bruker AM 500, 400, 300 MHz. instruments. For elemental analysis, A Carlo Erba
Strumentazion-Mod-1106, Italy was used. Electron impact mass spectra (EI-MS) spectra were recorded on Finnigan MAT-311A, Germany. TLC (thin layer chromatography) was carried out on precoated silica gel glass plates (Kieselgel 60, 254, E. Merck, Germany). UV visualized chromatograms at 254 and 365 nm.
86, 5.6.1. Synthetic procedure of compounds 28-62
2, 4, 6-Trichlorophenylhydrazones (28-62) were prepared by refluxing the commercially available
2, 4, 6-trichlorophenylhydrazine with different aromatic aldehydes in 1:1 ratio in methanol for 2 hrs (Scheme-6). The reaction progress of was checked by TLC. After cooling, the resultant compounds were washed with methanol and then it were dried to afford 2, 4, 6-trichlorophenyl hydrazones 28-62. These compounds were finally recrystallized from hexane/ethyl acetate solvents. The structures of of these synthesized compounds were elucidated by using proton NMR,
CHN analysis and EI-MS spectroscopy.
5.6.1.1. 3, 4, 5-Trimethoxybenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone: (28)
1 Yield: 0.77 g (98%); H-NMR: (500 MHz, MeOD): δH 7.83 (s, 1H, =CH), 7.45 (s, 2H, H-3/5),
6.95 (s, 2H, H-2'/6'), 3.86 (s, 6H, -OCH3), 3.76 (s, 3H, OCH3); MS: m/z (rel. abund. %), 289
(M+, 100), 273 (48), 194 (20).
5.6.1.2. 2-Hydroxy-3-methoxybenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone: (29)
1 Yield: 0.63 g (91%); H-NMR: (400 MHz, DMSO-d6): δH 10.07 (s, 1H, -OH), 9.65
(s, 1H, -NH), 8.30 (s, 1H, =CH), 7.65 (s, 2H, H-3/5), 7.02 (dd, 1H, J6',5' = 7.5 Hz, , J6',4' = 0.8
Hz, H-6'), 6.92 (dd, 1H, J4',5' = 7.5 Hz, J4',6' = 0.8 Hz, H-4'), 6.79 (t, 1H, J5'/4',6' = 7.5 Hz, H-5'),
3.77 (s, 3H, -OCH3); MS: m/z (rel. abund. %), 344 (M+, 84), 195 (100), 135 (64).
5.6.1.3. Benzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone: (30)
1 Yield: 0.577 g (96%); H-NMR: (400 MHz, DMSO-d6): δH 9.57 (s, 1H, -NH), 8.02 (s, 1H,
=CH), 7.61 (s, 2H, H-3/5), 7.56 (d, 2H, J2',3'/6',5' = 7.2 Hz, H-2'/6'), 7.36 (t, 2H, J3'/2',4' and 5'/4',6'
= 7.2 Hz, H-3'/5'), 7.30 (t, 1H, J4'/3',5' = 7.2 Hz, H-4'); MS: m/z (rel. abund. %), 298 (M+, 79), 194
(100), 169 (30), 77 (50).
87, 5.6.1.4. 2-Fluorobenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone: (31)
1 Yield: 0.588 g (92%); H-NMR: (400 MHz, DMSO-d6): δH 9.88 (s, 1H, -NH), 8.25 (s, 1H,
=CH), 7.80 (td, 1H, J 4',6' = 1.6 Hz, J4'/3', 5' = 7.5 Hz, H-4'), 7.61 (s, 2H, H-3/5), 7.33 (m, 1H, H-
3'), 7.20 (m, 2H , H-5'/6'); MS: m/z (rel. abund. %), 316 (M+, 76), 194 (100), 169 (24).
5.6.1.5. 4-(Methylsulfanyl)benzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (32)
1 Yield: 0.64 g (93%); H-NMR: (500 MHz, DMSO-d6): δH 9.52 (s, 1H, -NH), 7.98 (s, 1H, =CH),
7.59 (s, 2H, H-3/5), 7.50 (d, 2H, J3',2'/5',6' = 8.4 Hz, H-3'/5'), 7.24 (d, 2H, J2',3'/6',5' = 8.4 Hz, H-
2'/6'), 2.49 (s, 3H, -SCH3); MS: m/z (rel. abund. %), 344 (M+, 100), 195 (40), 150 (44).
5.6.1.6. 4-(Dimethylamino)benzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (33)
1 Yield: 0.63 g (92%); H-NMR: (500 MHz, DMSO-d6): δH 9.10 (s, 1H, -NH), 7.95 (s, 1H, =CH),
7.57 (s, 2H, H-3/5), 7.39 (d, 2H, J3',2'/5',6' = 8.8 Hz, H-3'/5'), 7.24 (d, 2H, J2',3'/6',5' = 8.8 Hz, H-
2'/6'), 2.49 (s, 6H, -N(CH3)2); MS: m/z (rel. abund. %), 341 (M+, 100), 195 (13), 147 (58).
5.6.1.7. 2-Hydroxy-5-methylbenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (34)
1 Yield: 0.61 g (94%); H-NMR: (400 MHz, DMSO-d6): δH 10.17 (s, 1H, -NH), 9.62
(s, 1H, -OH), 8.25 (s, 1H, =CH), 7.65 (s, 2H, H-3/5), 7.19 (d, 1H, J6',4' = 2.0 Hz, H-6'), 6.98
(dd, 1H, J4',3' = 8.0 Hz, J4',6' = 2.0 Hz, H-4'), 6.74 (d, 1H, J3',4' = 8.0 Hz, H-3'), 2.20 (s, 3H, 5'-
CH3); MS: m/z (rel. abund. %), 328 (M+, 57), 195 (100), 83 (50).
5.6.1. 8. 2, 5-Dihydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (35)
1 Yield: 0.62 g (94%); H-NMR: (400 MHz, DMSO-d6): δH 9.62 (s, 1H, -NH), 9.56 (s, 1H, -OH),
8.81 (s, 1H, -OH), 8.23 (s, 1H, =CH), 7.63 (s, 2H, H-3/5), 6.83 (d, 1H, J6',4' = 2.8 Hz, H-6'),
6.66 (d, 1H, J3',4' = 8.8 Hz, H-3'), 6.60 (dd, 1H, J4',3' = 8.8 Hz, J4',6' = 2.8 Hz, H-4'); MS: m/z (rel. abund. %), 330 (M+, 43), 195 (90), 161 (100). 88, 5.6.1. 9. 2, 4-Dichlorobenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (36)
1 Yield: 0.68 g (93%); H-NMR: (400 MHz, DMSO-d6): δH 10.11 (s, 1H, -NH), 8.23 (s, 1H,
=CH), 7.88 (d, 1H, J6',5' = 8.4 Hz, H-6'), 7.64 (s, 2H, H-3/5), 7.61 (d, 1H, J3',5' = 2.0 Hz, H-3'),
7.41 (dd, 1H, J5',6' = 8.4 Hz, J5',3' = 2.0 Hz, H-5'); MS: m/z (rel. abund. %), 368 (M+, 100), 196
(80), 167 (16).
5.6.1.10. 4-Chlorobenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (37)
1 Yield: 0.62 g (91%); H-NMR: (500 MHz, DMSO-d6): δH 9.69 (s, 1H, -NH), 7.99 (s, 1H, =CH),
7.62 (s, 2H, H-3/5), 7.58 (d, 2H, J3',2'/5',6' = 8.4 Hz, H-3'/5'), 7.41 (d, 2H, J2',3'/6',5' = 8.4 Hz, H-
2'/6'); MS: m/z (rel. abund. %), 334 (M+, 56), 195 (100), 167 (22).
5.6.1.11. 2-Chlorobenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (38)
1 Yield: 0.63 g (95%); H-NMR: (400 MHz, DMSO-d6): δH 10.03 (s, 1H, -NH), 8.41 (s, 1H,
=CH), 7.89 (m, 1H, H-3'), 7.63 (s, 2H, H-3/5), 7.44 (m, 1H, H-4'), 7.31 (m, 2H, H-5'/6'); MS: m/z (rel. abund. %), 334 (M+, 71), 196 (100), 167 (26).
5.6.1.12. 3, 5-Dichloro-2-hydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (39)
1 Yield: 0.72 g (94%); H-NMR: (400 MHz, DMSO-d6): δH 11.28 (s, 1H, -OH), 10.10 (s, 1H, -
NH), 8.18 (s, 1H, =CH), 7.70 (s, 2H, H-4'/6'), 7.47 (s, 2H, H-3/5); MS: m/z (rel. abund. %),
284 (M+, 100), 349 (21), 195 (66).
5.6.1.13. 3, 4-Dichlorobenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (40)
1 Yield: 0.70 g (96%); H-NMR: (400 MHz, DMSO-d6): δH 9.88 (s, 1H, -NH), 7.92 (s, 1H, =CH),
7.77 (d, 1H, J2',6' = 2.0 Hz, H-2'), 7.65 (s, 2H, H-3/5), 7.61 (d, 1H, J5',6' = 8.4 Hz, H-5'), 7.55
(dd, 1H, J6',5'm = 8.4 Hz, J6',2' = 2.0 Hz,, H-6'); MS: m/z (rel. abund. %), 368 (M+, 58), 196 (100),
194 (100). 89, 5.6.1.14. 3, 4-Dihydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (41)
1 Yield: 0.62 g (94%); H-NMR: (400 MHz, DMSO-d6): δH 9.18 (s, 1H, -NH), 9.05 (br. s, 2H, -
OH) 7.90 (s, 1H, =CH), 7.58 (s, 2H, H-3/5), 7.07 (d, 1H, J2',6' = 2.0 Hz, H-2'), 6.78 (dd, 1H, J6',5'
= 8.0 Hz, J6',2' = 2.0 Hz, H-6'), 6.70 (d, 1H, J5',6' = 8.0 Hz, H-5'),. MS: m/z (rel. abund. %), 330
(M+, 100), 195 (74), 137 (49).
5.6.1.15. 4-Hydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (42)
1 Yield: 0.60 g (95%); H-NMR: (500 MHz, MeOD): δH 7.87 (s, 1H, =CH), 7.44 (d, 2H, J2',3'/6',5' =
8.4 Hz, H-2'/6'), 7.41 (s, 2H, H-3/5), 6.76 (d, 2H, J3',2'/5',6' = 8.4 Hz, H-3'/5'); MS: m/z (rel. abund.
%), 314 (M+, 100), 196 (47), 194 (47).
5.6.1.16. 3, 4-Dimethoxybenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (43)
1 Yield: 0.68 g (95%); H-NMR: (400 MHz, DMSO-d6): δH 9.38 (s, 1H, -NH), 7.94 (s, 1H, =CH),
7.61 (s, 2H, H-3/5), 7.22 (d, 1H, J2',6' = 1.6 Hz, H-2'), 7.04 (dd, 1H, J6',5' = 8.0 Hz, J6',2' = 1.6 Hz,
H-6') 6.94 (d, 1H, J5',6' = 8.0 Hz, H-5'), 3.76 (s, 3H, -OCH3), 3.75 (s, 3H, OCH3); MS: m/z (rel. abund. %), 358 (M+, 100), 195 (29), 137 (42).
5.6.1.17. 2, 3, 4-Trihydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (44)
1 Yield: 0.65 g (93%); H-NMR: (400 MHz, DMSO-d6): δH 10.47 (br. s, 1H, -OH), 9.34 (s, 1H, -
NH), 9.25 (br. s, 1H, -OH), 8.34 (br. s, 1H, -OH), 8.22 (s, 1H, =CH), 7.63 (s, 2H, H-3/5), 6.64
(d, 1H, J6',5' = 8.4 Hz, H-6'), 6.34 (d, 1H, J5',6' = 8.4 Hz, H-5'); MS: m/z (rel. abund. %), 346 (M+,
51), 195 (100), 83 (62).
90, 5.6.1.18. 2, 3-Dihydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (45)
1 Yield: 0.64 g (97%); H-NMR: (400 MHz, DMSO-d6): δH 10.12 (s, 1H, -OH), 9.62 (s, 1H, -
NH), 9.07 (s, 1H, -OH), 8.28 (s, 1H, =CH), 7.66 (s, 2H, H-3/5), 6.83 (dd, 1H, J4',5' = 7.6 Hz, J4',6'
= 1.2 Hz, H-4'), 6.75 (dd, 1H, J6',5' = 7.6 Hz, J6',4' = 1.2 Hz, H-6'), 6.67 (t, 1H, J5'/4',6' = 7.6 Hz, H-
5'); MS: m/z (rel. abund. %), 332 (M+, 65), 195 (100), 161 (22).
5.6.1.19. 3-Thiophenecarbaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (46)
1 Yield: .57 g (93%); H-NMR: (400 MHz, DMSO-d6): δH 9.38 (s, 1H, -NH), 8.03 (s, 1H, =CH),
7.63 (d, 1H, J2',4' = 2.4 Hz, H-2'), 7.61 (s, 2H, H-3/5), 7.53 (dd, 1H, J4',5' = 4.8 Hz, J4',2' = 2.8
Hz, H-4'), 7.34 (d, 1H, J5',4' = 4.8 Hz, H-5'); MS: m/z (rel. abund. %), 304 (M+, 100), 194 (100),
83 (35).
5.6.1.20. Isonicotinaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (47)
1 Yield: 0.55 g (92%); H-NMR: (400 MHz, DMSO-d6): δH 10.07 (s, 1H, -NH), 8.51 (d, 2H,
J2',3'/6',5' = 5.6 Hz, H-2'/6'), 7.89 (s, 1H, =CH), 7.66 (s, 2H, H-3/5), 7.49 (d, 2H, J3',2'/5',6' = 5.6 Hz,
H-3'/5'); MS: m/z (rel. abund. %), 299 (M+, 75), 194 (100), 167 (25).
5.6.1.21. 2-Bromobenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (48)
1 Yield: 0.73 g (97%); H-NMR: (400 MHz, DMSO-d6): δH 9.71 (s, 1H, -NH), 7.97 (s, 1H, =CH),
7.63 (s, 2H, H-3/5), 7.53 (m, 4H, H-3'/4'/5'/6'); MS: m/z (rel. abund. %), 378 (M+, 72), 194
(100), 83 (51).
91, 5.6.1.22. 2-Hydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (49)
1 Yield: 0.59 g (93%); H-NMR: (400 MHz, DMSO-d6): δH 10.41 (s, 1H, -OH), 9.65 (s, 1H, -NH),
8.31 (s, 1H, =CH), 7.64 (s, 2H, H-3/5), 7.40 (dd, 1H, J6',5' = 8.4 Hz, J6',4' = 1.6 Hz,, H-6'), 7.18
(td, 1H, J5'/4',6' = 8.4 Hz, J5',3' = 1.6 Hz, H-5'), 6.84 (m, 2H, H-3'/4'); MS: m/z (rel. abund. %), 314
(M+, 90), 279 (25), 195 (100).
5.6.1.23. 3-Chlorobenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (50)
1 Yield: 0.64 g ( 95%); H-NMR: (400 MHz, DMSO-d6): δH 9.79 (s, 1H, -NH), 7.95 (s, 1H, =CH),
7.65 (s, 2H, H-3/5), 7.60 (s, 1H, H-2'), 7.40 (d, 1H, J4',5' = 7.6 Hz, H-4'), 7.38 (t, 1H, J5'/4',6' =
7.6 Hz, H-5'), 6.34 (d, 1H, J6',5' = 7.6 Hz, H-6'); MS: m/z (rel. abund. %), 334 (M+, 72), 194
(100), 167 (25).
5.6.1.24. 1-Phenanthrenecarbaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (51)
1 Yield: 0.78 g (98%); H-NMR: (400 MHz, DMSO-d6): δH 9.75 (s, 1H, -NH), 8.91 (m, 2H, H-
3'/7'), 8.80 (m, 2H, H-2'/4'), 8.08 (s, 1H, =CH), 8.03 (d, 1H, J5',6' = 9.6 Hz, H-5'), 7.69 (m, 6H,
H-3/5/6'/8'/9'/10'); MS: m/z (rel. abund. %), 398 (M+, 100), 204 (88), 177 (69).
5.6.1.25. Nicotinaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (52)
1 Yield: 0.55 g (91%); H-NMR: (400 MHz, DMSO-d6): δH 9.82 (s, 1H, -NH), 8.71 (d, 1H, J2',4' =
1.6 Hz, H-2'), 8.47 (dd, 1H, J6',5' = 4.8 Hz, J6',4' = 1.6 Hz, H-6'), 8.00 (s, 1H, =CH), 7.95 (dd, 1H,
J4',5' = 8.0 Hz, J4',6' = 2.0 Hz, H-4'), 7.63 (s, 2H, H-3/5), 7.37 (m, 1H, H-5'); MS: m/z (rel. abund.
%), 300 (M+, 89), 194 (100), 167 (26).
92, 5.6.1.26. 2-Naphthaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (53)
1 Yield: 0.67 g (97%); H-NMR: (300 MHz, DMSO-d6): δH 9.72 (s, 1H, -NH), 8.18 (s, 1H, H-2'),
7.89 (m, 5H, =CH/H-3'/4'/6'/8'), 7.63 (s, 2H, H-3/5), 7.49 (m, 2H, H-5'/7'); MS: m/z (rel. abund. %), 348 (M+, 88), 194 (39), 127 (100).
5.6.1.27. 2-Methylbenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (54)
1 Yield: 0.60 g (97%); H-NMR: (300 MHz, DMSO-d6): δH 9.60 (s, 1H, -NH), 8.37 (s, 1H, =CH),
7.70 (m, 1H, H-6'), 7.60 (s, 2H, H-3/5), 7.17 (m, 3H, H-3'/4'/5'), 2.34 (s, 3H, 2'-CH3); MS: m/z
(rel. abund. %), 312 (M+, 100), 194 (23), 118 (95).
5.6.1.28. 2, 4, 6-Trihydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (55)
1 Yield: 0.65 g (94%); H-NMR: (300 MHz, DMSO-d6): δH 10.49 (s, 2H, -OH), 10.41 (s, 1H, -
OH), 9.60 (s, 1H, -OH), 9.29 (s, 1H, -NH), 8.56 (s, 1H, =CH), 7.61 (s, 2H, H-3/5), 5.79 (s, 2H,
H-3'/5'), MS: m/z (rel. abund. %), 346 (M+, 82), 195 (100), 161 (28).
5.6.1.29. 2, 4-Dihydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (56)
1 Yield: 0.61 g (92%); H-NMR: (400 MHz, DMSO-d6): δH 10.55 (br. s, 1H, -OH), 10.72 (br. s,
1H, -OH), 9.32 (s, 1H, -NH), 8.23 (s, 1H, =CH), 7.62 (s, 2H, H-3/5), 7.16 (d, 1H, J6',5' = 8.8
Hz, H-6'), 6.30 (dd, 1H, J5',6' = 8.8 Hz, J5',3' = 2.0 Hz, H-5'), 6.25 (d, 1H, J3',5' = 2.0 Hz, H-3');
MS: m/z (rel. abund. %), 329 (M+, 100), 195 (86), 167 (20).
5.6.1.30. 4-Methylbenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (57)
1 Yield: 0.59 g (95%); H-NMR: (400 MHz, DMSO-d6): δH 9.46 (s, 1H, -NH), 8.00 (s, 1H, =CH),
7.61 (s, 2H, H-3/5), 7.47 (d, 2H, J3',2'/5',6' = 8.0 Hz, H-3'/5'), 7.18 (d, 2H, J2',3'/6',5' = 8.0 Hz, H-
2'/6'), 2.29 (s, 3H, 4'-CH3); MS: m/z (rel. abund. %), 312 (M+, 100), 194 (75), 91 (30).
93, 5.6.1.31. 3, 5-Dibromo-2-hydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (58)
1 Yield: 0.88 g (93%); H-NMR: (300 MHz, DMSO-d6): δH 11.50 (s, 1H, -OH), 10.13 (s, 1H, -
NH), 8.14 (s, 1H, =CH), 7.71 (s, 2H, H-3/5), 7.69 (d, 1H, J4',6' = 2.4 Hz, H-4'), 6.61 (d, 1H, J6',4'
= 2.4 Hz, H-6') ; MS: m/z (rel. abund. %), 474 (M+, 66), 195 (100), 167 (21).
5.6.1.32. 2-Chloro-5-nitrobenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (59)
1 Yield: 0.73g (96%); H-NMR: (300 MHz, DMSO-d6): δH 10.45 (s, 1H, -NH), 8.62 (d, 1H, J6',4' =
2.7 Hz, H-6'), 8.40 (s, 1H, =CH), 8.10 (dd, 1H, J4',3' = 8.7 Hz, J4',6' = 2.7 Hz, H-4'), 7.76 (d, 1H,
J3',4' = 8.8 Hz, H-3'), 7.70 (s, 2H, H-3/5); MS: m/z (rel. abund. %), 379 (M+, 67), 194 (100), 169
(22).
5.6.1.33. 4-Nitrobenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (60)
1 Yield: 0.65g (95%); H-NMR: (400 MHz, DMSO-d6): δH 10.19 (s, 1H, -NH), 8.21 (d, 2H,
J3',2'/5',6' = 8.7 Hz, H-3'/5'), 8.04 (s, 1H, =CH), 7.80 (d, 2H, J2',3'/6',5' = 8.7 Hz, H-2'/6'), 7.68 (s, 2H,
H-3/5); MS: m/z (rel. abund. %), 343 (M+, 77), 194 (100), 167 (21).
5.6.1.34. 5-Bromo-2-hydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (61)
1 Yield: 0.766g (97%); H-NMR: (300 MHz, DMSO-d6): δH 10.48 (s, 1H, -NH), 9.82 (s, 1H,
-OH), 8.22 (s, 1H, =CH), 7.64 (s, 2H, H-3/5), 7.62 (s, 1H, H-6'), 7.30 (dd, 1H, J4',3' = 8.7 Hz,
J4',6' = 2.4, H-4'), 6.82 (d, 1H, J3',4' = 8.7 Hz, H-3'),; MS: m/z (rel. abund. %), 394 (M+, 94), 197
(100), 167 (19).
94, 5.6.1.35. 2-Nitrobenzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (62)
1 Yield: 0.64g (97%); H-NMR: (300 MHz, DMSO-d6): δH 10.24 (s, 1H, -NH), 8.42 (s, 1H,
=CH), 8.04 (d, 1H, J3',4' = 7.8 Hz, H-3'), 7.98 (d, 1H, J6',5' = 7.8 Hz, H-6'), 7.69 (t, 1H, J4'/3',5' = 7.5
Hz, H-4'), 7.66 (s, 2H, H-3/5), 7.52 (t, 1H, J5'/4',6' =7.5 Hz, H-5'); MS: m/z (rel. abund. %), 343
(M+, 24), 207 (100), 179 (40).
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108,
CHAPTER-6
SYNTHESIS OF 6-NITROBENZIMIDAZOLE DERIVATIVES
109, 6. Introduction to Benzimidazole
Benzimidazole is an aromatic, bicyclic heterocyclic compound in which the benzene ring is fused with imidazole (Figure 1).
4 3 5 N 2 6 N 1 7 H 1
Figure 1. Structure of Benzimidazole 1.
In nature, benzimidazole moiety exists in different natural products as well as in other living organisms. For example, granulatimide 2 is a natural product which was isolated from the
Didemnum granulatum [1]. 5, 6-dimethylbenzimidazole 3 which is a derivative of benzimidazole is biosynthesized from riboflavin by aerobic microorganisms and by another pathway in anaerobic microorganisms. It is also used in the biosynthesis of ribazole [2].
Similarly, 2-butyl-1H-benzimidazole 4 is another benzimidazole derivative which is constituent of chicken eggs and shows antifungal activity [3, 4].
H N O O N H3C N NH (CH2)3Me N H3C N N N H 4 H H 3 2
Fig. 2. Structures of granulatimide 2, dimethylbenzimidazole 3 and 2-butyl-1H-benzimidazole 4.
110, There are some other natural products which have benzimidazole moiety like compound 5, isolated from the sponge Leucetta species [5], compound 6, isolated from the marine sponge of the genus Xestospongia [6], and compound 7 which is isolated from the sardines [7].
O CH3 OMe NH2 MeO N N N N CH3 MeO N N O N N 7 6 5 OMe
Figure 3. Structures of compounds 5, 6 and 7 having benzimidazole moiety.
Vitamin B12 is another naturally occurring compound having benzimidazole moiety [8]. Several derivatives of benzimidazole have been synthesized which are found to be pharmacologically and physiologically active and thus used to cure different diseases like diabetes, anti-fertility, epilepsy, etc. [9, 10]. Very recently several papers have reported that the biological and pharmacological properties of the benzimidazole analogues can be modified by the introduction of different types heterocyclic moieties to display a wide range of biological activities e.g anthelmintic (compound 8) [11,12], anti-ulcer agents (compound 9) [13], anti-cancer (compound
11), anti-fungal [14-17], antiviral [18-21], antibacterial [22-28], anti-inflammatory [29], antihistaminic [30], proton pump inhibitors [31,32], antioxidant [33-36], antihypertensive [37], anticoagulant [38], and antileukaemia [39].
111, O Me MeO N O Me OMe N O S NH N Me S N H N H 9 8
OH S N NH O CO2CH3 N NH N N H O 10 N H 11
Figure 4. Structures of some biligically active benzimidazole derivatives.
Benzimidazoles are some time used as herbicides as well as in veterinary problems. Some benzimidazoles like, cambendazole 12 and thiabendazole 13 are used effectively as fungicides, herbicides and antiheliminthies. 2-aminoimidazolines 14 is a well known fungicide [40].
O H H H R N N N N 2 O N R N NH N S N 1 R3 O 14 12 13 CH3
R1= an aromatic or cycloaliphatic radical, R2 = H, R3 = H or CH3 OR R1 and R2 = some alkyl radical having 1-4 carbon atoms, R3 = H or CH3
Figure 5. Structures of cambendazole 12, thiabendazole 13 and 2-aminoimidazolines 14.
A series of substituted 2-aminobenzimidazole compounds were found to be inhibitors of p38 map kinase [41]. It was found that Z-Hymenialdisine 15 was a nanomolar inhibitor of nitrogen activated protein kinase. Addionally, Z-2-debromohymenialdisine has inhibitory activity of the
G2DNA damage checkpoint. A considerable number of patents claiming the pharmacological
112, activities of these compounds for the prevention and treatment of neurodegenerative disorders, inflammatory pathologies, cancer, diabetes, osteoarthritis and ocular disorders are recently appeared [42-45].
NH N 2 O NH
Br HN N H O 15
Figure 6. Structures of Z-Hymenialdisine 15.
Benzimidazole shows important activity against numerous viruses like HIV, RNA influenza, human cytomegalovirus (HCMV) and herpes (HSV-1) [46-57]. A significant number of benzimidazole compounds showed good antitumor activities [58-71]. The nitroimidazoles, particularly metronidazole 16, is the most commonly used drug for the chemotherapy of anaerobic bacteria and protozoal disease as well as for the radiosensization of hypoxic tumor
[72-74].
N N NO2 N F N OCH3 HN N OH 17 16
Figure 7. Structures of metronidazole 16 and astemizole 17.
Naturally occurring vitamin B12 recently showed important biological activities, i.e. antiviral activities [75]. Introducing a small substituent at 2- or 5-position is distinguishing benzimidazole
113, antihelminthics; rather than bulkier substituents at position 2 which are characteristic drug used in the treatment of peptic ulcer. Benzimidazole nucleus has established widespread attention of medicinal chemists, particularly after the commercialization of the antiulcerative omeprazole 9 and the antihistamine astemizole 17 [76, 77].
114, 6.1. Synthetic approaches to Benzimidazoles
In the literature, different methods are available for the synthesis of benzimidazoles and its substituted analogues. Some methods are described here;
6.1.1. Synthesis of benzimidazole from triethoxymethane and o-alkylated oximes. o-Phenylenediamine 19 undergoes ring closure with triethoxymethane and o-alkylated oximes 19 in the presence of DMF and thus yields benzimidazole. o-Alkylated oximes 19 acts as building blocks in the creation of other rings [78] [Scheme-1].
HON N OH NH2 N reflux, 13h CH(OEt) 3 DMF Me Me NH2 N H 18 19 1
Scheme-1
6.1.2. Photolysis of protected benzimidazole
Protected benzimidazole 20 undergoes photolysis yielding deprotected benzimidazole in reasonable yields. Some other functional groups such as ester, activated chloride, aldehyde and nitrile show compatibility with the mildness of this reaction [79] [Scheme-2].
N N N Dioxane hv N H O2N 1 20 Scheme-2
115, 6.1.3. Synthesis of 2-substituted benzimidazole by using Lewis acids
Employing Lewis acids and room temperature conditions, benzimidazole derivative 24 can be synthesized by the coupling of o-phenyldiamines 22 with orthoesters 23 in reasonable yields [80]
[Scheme-3].
NH2 R O 2 OR N R 2 Lewis acids R R2 R1 EtOH, rt NH2 OR2 N H 22 23 24
R= H, Me, Cl, NO2, R1= H, Me, Et, CH3(CH2)3, R2= Me, Et
Scheme-3
6.1.4. Synthesis of 2-substituted benzimidazole via clay and IR
Using clay and infrared irradiations, reaction of o-phenylenediamine 19 with various carboxylic acids 25 gives 2-alkylbenzimidazoles 26. A great advantage of this procedure is that no solvent is used and this method is simple and inexpensive [81] [Scheme-4].
NH2 N RCO2H IR, bentonite R NH2 N 25 H 19 26 Scheme-4
6.1.5. Microwave assisted synthesis of substituted benzimidazoles
Microwave is a very important technology which is commonly used in organic synthesis. The following are some methods in which microwave radiations are used for the benzimidazoles synthesis; 116, 6.1.6. Synthesis of substituted benzimidazoles by using esters
2-Substituted benzimidazoles 28 are prepared via one pot method by treating o- phenylenediamine 19 with esters 27 under microwave irradiations [82] [Scheme-5].
NH2 N (CH OH) RCO Et 2 2 R 2 1.5 min, mw NH N 2 27 H 19 28 Scheme-5
6.1.7. Synthesis of substituted benzimidazoles via reductive cyclization
In this method, 2-nitroanilines 29 is first reduced followed by cyclization with a variety of carboxylic acids 30 under microwave irradiation and thus giving substituted benzimidazoles 31 in higher yields [83] [Scheme-6].
NO2 N SnCl2. 2H2O R R CO H R R 1 2 2 5 min, mw, 130 C 1 2 NH2 N 30 H 29 31 Scheme-6
6.1.8. Synthesis of substituted benzimidazoles by using polyphosphoric acid
A mixture of organic acid 32 and o-phenyldiamine 19 were stirred and irradiated in microwave oven in the presence of polyphosphonic acid (PPA) which yielded 2-substituted benzimidazoles
33 [84] [Scheme-7].
NH 2 O N PPA R ROH M.W NH2 N 32 H 19 33 Scheme-7
117, 6.1.9. Synthesis of benzimidazoles by using 12-tungstonphosphoricacid as a catalyst.
Benzimidazole derivatives 34c were more efficiently synthesized by the condensation of substituted o-phenylenediamine 34a with a variety of different aromatic aldehydes 34b in the presence of monoammonium salt, 12-tungstonphosphoric acid as catalyst [85] [Scheme-8].
H R NH R 1 2 O 1 N (NH4)H2PW12O40 R DCE, Reflux N R2 NH2 RH R2 R3 R3 34b 34a 34c
R-Ph; R1-H, CH3, CF3; R2-H, CH3; R3- H, NO2
Scheme-8
118, 6.2. Results and Discussion
6.2.1. Chemistry
In our previous investigations, we have reported benzothiazole as β-glucoronidase inhibitors
[86]. In the light of that report we synthesized 6-nitrobenzimidazole derivatives 37-66 due to the functional groups similarity with benzothiazole with this assumption that the 6- nitrobenzimidazole might have some β-glucoronidase inhibition properties. The antiglycation, phosphodiesterases inhibitory activities of the nitrobenzimidazole derivatives 37-66 have proven our initial hypothesis. We designed 6-nitrobenzimidazole derivatives project in such a way that the R substituent should remain close to benzyl substitution.
6-Nitrobenzimidazoles 37-66 were synthesized by treating commercially available
4-nitrophenylenediamine with different aromatic aldehydes in DMSO. The resulted products were formed in high yields [Scheme-9].
In a typical reaction, sodium metabisulfite (Na2S2O5) was mixed to a stirring solution of
4-nitrophenylenediamine (3.12 equivalent) and substituted aromatic aldehydes (3.16 equivalent) in DMSO. The above reaction was heated for 4 hrs and the reaction progress was checked by
TLC. When the reaction was completed, the reaction contents were then cooled at normal temperature. Addition of water (30 ml) resulted in precipitation of a crude solid residues, which were then passed through several coloumn chromatography to afford pure 6-nitrobenzimidazole derivatives 37-66 in high yields. The structures of these compounds were elucidated with the help of proton NMR and EI spectroscopy. All compounds also give satisfactory CHN analysis.
119, NH 2 N R RCHO, Na2S2O5 O N NH DMSO, reflux 4h O N N 2 2 2 H 37-66 35, 36
Scheme-9. Synthesis of 6-nitrobenzimidazole derivatives 37-66
Compounds R Compounds R
1' 37 6' 2' 1' OH 52 3' 5' 3' 5' OEt OH 4'
1' 1' 6' OH 6' OH 38 53 Br Br Cl 3' 4' 4'
1' 6' 2' 1' OH 6' 39 54 5' OMe 5' 3' 4' OMe
1' 6' Cl 1' 6' Cl 40 5' 3' 55 4' 5' 3' Cl
1' 6' OH 1' 6' Cl 41 Cl Cl 56 4' O N 3' 2 4'
1' 6' 2' 1' F 6' 42 57 5' 3' 5' 3' 4' NO2
120, 1' 1' 6' 2' 6' 2' 43 58 5' 3' 5' 3' Me Br
1' 2' 6' 2' 44 59 S1' 5' 4' Cl 5' Cl
10' 1' OH 2' 9' 6' 45 60 3' 8' 5' 3' 4' 5' 7' OH 6'
1' 6' 1' 2' 6' 2' 46 61 5' 3' 5' 3' Cl 4'
1' 1' 6' 2' 6' 2' 47 62 3' 5' OH 5' OH SMe
1' OH 6' 5' 2' 48 63 6' 3' 5' OMe N 4' 1'
1' HO OH 1' 6' OH 49 64 5' 3' HO 3' OH 4'
121, 1' OH 10' 9' 3' 50 65 2' 8' 5' OH 3' 7' 4' 5' 6' OH
5' 1' 3' 6' OH 51 6' 2' 66 5' OH 4' NMe2
6.3. General structure elucidation of compound by spectroscopic techniques
The structure of the 2-(6-nitro-1H-benzimidazol-2-yl)-1, 3, 5-benzenetriol 49 was established through spectroscopic techniques. The 1H-NMR was carried out in deuterated methanol on 300
MHz instrument. A doublet at δ 8.37 (d, 1H, J7,5 = 2.0 Hz, H-7) and a double doublet at δ 8.14
(dd, 1H, J5,4 = 9.0 Hz, J5,7 = 2.0 Hz, H-5) shows the meta coupled C-7 and C-5 protons respectively. Similarly another doublet at δ 7.68 (d, 1H, J4,5 = 9.0 Hz, H-4) reveals the C-4 proton which is ortho coupled with C-5 proton. Finally, a sharp singlet at δ 6.00 (s, 2H, H-3'/5') demonstrates the integral of two protons which are attached to C-3' and C-5' atoms.
HO OH N
OH O2N N H
Figure-2. 1H-NMR analysis of compound 49.
The synthetic compound 49 was also confirmed by EI-MS showing the molecular ion peak at m/z = 287 which lead to molecular formula C13H9N3O5. Ion at m/z 242 appeared due to the loss of nitro group. Similary, a peak at 118 corresponds to the loss 2, 4, 6-trihydroxy benzene.
122, + . HO OH +. _ HO OH HO OH N +. N -NO2 N OH N OH OH O2N N N H H H m/z = 118 m/z = 242 m/z = 287
Figure 3. Fragmentation patterns of compound 49.
123, 6.4. Biological Studies
In vitro urease inhibitory assay
The synthetic compounds 37-66 were screened for the in vitro urease activity.These compounds showed a range of inhibition between 41.9-424.4 µM. The compound 49 (IC50 = 41.9 ± 0.44 µM) exhibited good activity relative to the thiourea standard (IC50 = 21 ± 0.011). On the other hand, compounds 50 (IC50 = 161.63 ± 4.35 µM), 66 (IC50 = 283.73 ± 4.83 µM) and 47 (IC50 = 307.20 ±
3.67 µM) showed moderate activity. Nevertheless, compounds 64 (IC50 = 447.57 ± 1.08 µM) and
54 (IC50 = 424.4 ± 6.67 µM) are the least active among the active compounds in the series. The compounds 37-46, 48, 51-53, 55-63 and 65 indicated inhibitions under 50%, so, therefore, these compounds were not further studied.
Compound 49 shows highest activity which is probably due to the presence of 2, 4, 6-trihydroxyl groups. On the other hand, compound 50 which is another trihydroxyl analogue is less active and the lesser activity of this compound is probably due to the presence of 2, 3, 4-trihydroxyl groups which are not better position to chelate with the enzyme as like in the fomer compound.
Similarly, compound 66 which is 2, 3-dihydroxyl analogue show less activity relative to compounds 49 and 50 which are presumably caused by the the presence of two hydroxyl groups.
Therefore, it is assumed that the presence of higher number of hydroxyl groups in a compound is responsible for the enhanced urease activity. Similary, the less activity of compounds 47 and 64 which have 3,4- and 2,5-dihydroxyl groups relative to compound 66 possibly due to different position of hydroxyl groups. Finally, compound 72 show least activity due to the presence of one hydroxyl group.
124,
Table-1. Results of urease inhibitory assay of 6-nitrobenzimidazole derivatives 37-66
a a Compound No. IC50 (µM ± SEM Compound No. IC50 (µM ± SEM ) 37 53 NAb NAb
38 54 NAb 424.4 ± 6.67
39 55 NAb NAb
40 56 NAb NAb
41 57 NAb NAb
42 58 NAb NAb
59 43 NAb NAb
44 60 NAb NAb
45 61 NAb NAb
46 62 NAb NAb
47 63 307.20 ± 3.67 NAb
48 64 NAb 394.76 ± 4.66
65 49 41.9 ± 0.44 NAb
50 66 161.63 ± 4.35 283.73 ± 4.83
51 NAb Thioureac 21 ± 0.011
52 NAb
SEMa is the standard error of the mean, NAb Not active, Thioureac, standard inhibitor for urease activity.
125, In vitro α-chymotrypsin inhibitory assay
All the synthetic compounds 37-66 were screened for in vitro α-chymotrypsin activity. These compounds showed a range of inhibition between 21.25-430.47 µM. The compound 52 (IC50 =
21.25 ± 0.72 µM) revealed comparatively less activity relative to the chymostatin standard (IC50
= 5.7 ± 0.13). On the other hand, compounds 64 (IC50 = 150.34 ± 4.86 µM), 47 (IC50 = 213.66 ±
1.7 µM) 66 (IC50 = 302.61 ± 4.23 µM), 54 (IC50 = 321.48 ± 6.63 µM), 49 (IC50 = 333.77 ± 7.03
µM) and 50 (IC50 = 347.41 ± 0.83 µM) are moderately active. Nevertheless, compounds 41 (IC50
= 412.49 ± 3.85 µM) and 58 (IC50 = 430.47 ± 4.27 µM) are the least active among the active compounds in the series. The compounds 37-40, 42-46, 48, 51, 53, 55-57, 59-63 and 65 showed inhibitions under 50%, so, therefore, these compounds were not further studied.
The highest activity of compound 52 which has one hydroxyl and ethoxy groups is probably caused by the electron donating effect of the ethoxy group. Alternatively, compound 64 which has 2, 5-dihydoxyl groups is less active relative to compound 52 which is possibly due to the electron withdrawing effects of the two hydoxyl groups which decreases the activity. Similarly, compounds 47 and 66 are less active relative to compound 64 which might be due to less chelation behavior of these compounds with the enzyme. The less activity of compound 72 which has only one hydoxyl group is presumably due to the same reason. Analogously, compounds 49 and 50 are less active due to the electron withdrawing effects of the three hydroxyl groups and hence minimises the activity of these compounds. Finally, a slight higher activity of compound 41 relative to compound 58 might be due to some what higher chelation capacity of the former compound with the enzyme than the later one.
126, Table-2. Results of α-chymotrypsin inhibitory assay of 6-nitrobenzimidazole derivatives 37-66. a a Compound # IC50 (µM ± SEM Compound # IC50 (µM ± SEM ) 37 53 NAb NAb
38 54 NAb 321.48 ± 6.63
39 55 NAb NAb
40 56 NAb NAb
41 57 412.49 ± 3.85 NAb
42 58 NAb 430.47 ± 4.27
59 43 NAb NAb
44 60 NAb NAb
45 61 NAb NAb
46 62 NAb NAb
47 63 213.66 ± 1.7 NAb
48 64 NAb 150.34± 4.86
65 49 333.77 ± 7.03 NAb
50 66 347.41 ± 0.83 302.61 ± 4.23
51 NAb Chymostatinc 5.7 ± 0.13
52 21.25 ± 0.72
SEMa is the standard error of the mean, NAb Not active, chymostatinc, standard inhibitor for α- chymotrypsin activity
127, In vitro carbonic anhydrase inhibitory activity
The synthetic compounds 37-66 were also tested for the carbonic anhydrase activity. All the synthesized compounds were inactive in this assay.
Table-3. Results of carbonic anhydrase inhibitory assay of 6-nitrobenzimidazole derivatives 37- 66.
a a Compound No. IC50 (µM ± SEM Compound No. IC50 (µM ± SEM )
37 53 NAb NAb
38 54 NAb NAb
39 55 NAb NAb
40 56 NAb NAb
41 57 NAb NAb
42 58 NAb NAb
59 43 NAb NAb
44 60 NAb NAb
45 61 NAb NAb
46 62 NAb NAb
47 63 NAb NAb
48 64 NAb NAb
65 49 NAb NAb
50 66 NAb NAb
128, 51 NAb Acetazolamidec 0.12 ± 0.03
52 NAb
SEMa is the standard error of the mean, NAb Not active, acetazolamidec, standard inhibitor for carbonic anhydrase activity.
In vitro antiglycation inhibitory activity
The synthetic compounds 37-66 were screened for antiglycation activity. These compounds showed a range of inhibition between 17.7-340.0 µM. The compounds 66 (IC50 = 17.7 ± 0.001
µM), 47 (IC50 = 25.5 ± 0.00 µM), 65 (IC50 = 37.5 ± 0.033 µM), 64 (IC50 = 48.7 ± 0.00 µM), 45
(IC50 = 52.4 ± 0.001 µM) and 38 (IC50 = 63.0 ± 0.02 µM) showed IC50 values which much better than the rutin standard (IC50 = 70 ± 0.5 µM). Compounds 50 (IC50 = 71.7 ± 0.008 µM) and 41
(IC50 = 89.2 ± 0.001 µM) showed IC50 values which were near to the standard value. On the other hand, compounds 49 (IC50 = 109.0 ± 0.00 µM), 42 (IC50 = 114.0 ± 0.005 µM) and 59 (IC50
= 142.0 ± 0.014 µM) showed moderate IC50 values in comparison to the standard value.
Nevertheless, compounds 43 (IC50 = 192.0 ± 0.017 µM), 37 (IC50 = 194.0 ± 0.039 µM) and 54
(IC50 = 340.0 ± 0.00 µM) were considered weak active among the series. The compounds 39, 40,
44, 46, 48, 51, 52, 53, 55-58, 60-63 revealed inhibitions under 50%, so, therefore, these compounds were not further studied.
The highest activity of compounds 66 and 47 are most probably caused by the presence of 2, 3- and 3, 4-dihydroxyl groups which are in better position to form acetal with the carbonyl group of the methylglyoxal. However, compounds 64 and 45 which are 2, 5- and 2, 4- dihydroxyl analogues are less active in comparison to the parent compounds due to the position of hydroxyl groups. On the other hand, compound 65 which has anthracene ring is unusually the third most active which probably show hydrophobic interaction with the enzyme. Compound 38 show higher activity as compared to compound 41 which is possibly due to the presence of bromine
129, atoms at 3, 5-positions which can better interact with the enzyme relative to the chlorine atoms at
3,5-positions in the later compound. Compound 50 which have one more hydroxyl group at p- position in comparison to the highest active compound showed sharp decline in activity.
Interestingly, if the position of the 2, 3, 4-trihydroxyl groups in compound 50 are interchanged with 2, 4, 6- positions as in the case of compound 49, then the activity of this compound is further declined. Compound 42 and 59 showed moderate activity due to the presence of nitro and thiophene rings. The activity of compound 43 is due to electron donating effect of the methyl group. Finally, compounds 37 and 54 are the least active among the series due to one hydroxyl groups in each of the above compounds.
Table-4. Results of antiglycation inhibitory assay of 6-nitrobenzimidazole derivatives 37-66. a a Compound # IC50 (µM ± SEM Compound # IC50 (µM ± SEM ) 37 53 194.0 ± 0.039 NAb
38 54 63.0 ± 0.02 340.0 ± 0.000
39 NAb 55 NAb
40 56 NAb NAb
41 57 89.2 ± 0.001 NAb
42 58 114.0 ± 0.005 NAb
59 43 192.0 ± 0.017 142.0 ± 0.014
44 60 NAb NAb
45 61 52.4 ± 0.01 NAb
46 62 NAb NAb
130, 47 63 25.5 ± 0.000 NAb
48 64 NAb 48.7 ± 0.000
65 49 109.0 ± 0.000 37.5 ± 0.033
50 66 71.7 ± 0.008 17.7 ± 0.001
51 NAb Rutin c 70 ± 0.5
52 NAb
SEMa is the standard error of the mean, NAb Not active, rutin,c standard inhibitor for antiglycation activity.
In vitro phosphodiesterase inhibitory activity
All the synthetic compounds 37-66 were screened for the in vitro phosphodiesterase activity.
These compounds showed a range of inhibition between 1.5-294.0 µM. The compounds 66 (IC50
= 1.5 ± 0.043 µM), 37 (IC50 = 2.4 ± 0.049 µM), 47 (IC50 = 5.7 ± 0.113 µM), 49 (IC50 = 6.4 ±
0.148 µM), 50 (IC50 = 10.5 ± 0.51 µM), 45 (IC50 = 11.49 ± 0.08 µM), 39 (IC50 = 63.1 ± 1.48
µM), 46 (IC50 = 120.0 ± 4.47 µM) and 42 (IC50 = 153.2 ± 5.6 µM) showed IC50 values which much better than the EDTA standard (IC50 = 274 ± 0.007 µM) and thus were considered excellent inhibitors against the thymidine phosphorylase enzyme. Only one compound 41 (IC50 =
294.0 ± 16.7 µM) showed IC50 value which was slightly higher than the standard value. The compounds 38, 40, 43, 44, 48 and 51-65 showed inhibitions under 50%, so, therefore, these compounds were not further studied.
Compounds 66 showed remarkably highest activity which is presumably caused by the presence of two hydroxyl groups at 2, 3-positions. On the other hand, compounds 47 and 45 which are 3,
4- and 2, 4-dihydroxyl analogues are less active relative to the parent compound due to the position of hydroxyl groups. Compound 37, a p-hydroxyl analogue is the second most active 131, compound; however, the o-hydroxyl analogue of compound 54 is completely inactive. Our hypothesis about this behavior is that the hydroxyl group at p-position in the former compound is in better position to interact with the enzyme relative to its o-hydroxyl analogue. Compound 49, a 2, 4, 6- trihydroxyl analogue, is the fourth most active compound, however, the 2, 3, 4- trihydroxyl analogue of compound 50 is slightly less active which might be due to the above reason. If the 3, 4-dihydroxyl groups in compound 47 are replaced by two methoxy groups as in the case of compound 39, then the activity of this compound is sharply declined. The activity of compounds 46 and 41 are most probably caused by the presence of chloro and nitro groups at p- positions. Finally, least activity of compound 46 is might be due to two chloro groups at 3, position.
Table-5. Results of phosphodiasterase inhibitory assay of 6-nitrobenzimidazole derivatives 37-66. a a Compound # IC50 (µM ± SEM Compound # IC50 (µM ± SEM ) 37 53 2.4 ± 0.049 NAb
38 54 NAb NAb
39 55 63.1 ± 1.48 NAb
40 56 NAb NAb
41 57 294.0 ± 16.7 NAb
42 58 153.2 ± 5.6 NAb
59 43 NAb NAb
44 60 NAb NAb
45 61 11.49 ± 0.08 NAb
132, 46 62 120.0 ± 4.47 NAb
47 63 5.7 ± 0.113 NAb
48 64 NAb NAb
65 49 6.4 ± 0.148 NAb
50 66 10.5 ± 0.51 1.5 ± 0.043
51 c NAb EDTA 274 ± 0.007
52 NAb
SEMa is the standard error of the mean, NAb Not active, EDTAc standard inhibitor for phosphodiesterease activity
Antioxidant Studies
In vitro DPPH inhibitory acivity
In search of a potential class of DPPH radical scavenging compounds, the compounds 37-66 were screened for the in vitro DPPH radical scavenging activity. These compounds have shown varying degree of DPPH radical scavenging activy and their IC50 values ranging between 9. 3-
417.5 µM. The compounds 47 (IC50 = 9.3 ± 1.11 µM), 64 (IC50 = 11.17 ± 0.42 µM), 66 (IC50 =
11.58 ± 0.06 µM), and 50 (IC50 = 15.6 ± 3.68 µM), showed IC50 values which were much better than the n-propylgallate standard (IC50 = 106.34 ± 1.6 µM). The compound 49 (IC50 = 73.06 ±
0.06 µM) showed moderate IC50 values relative to the standard value. On the other hand, compound 52 (IC50 = 417.5 ± 2.28 µM), was least active among the active compounds. The compounds 37-46, 48, 51, 53-63 and 84 revealed inhibitions under 50%, so, therefore, these compounds were not further studied.
133, The compounds 47, 64, 66 and 50 showed higher activity which are presumably caused by the presence of dihydroxyl groups and thus stabilizing the phenoxide ion radicals. On the other hand, compounds 50, a 2, 3, 4-trihydroxyl analogue and 49, a 2, 4, 6-trihydroxyl analogues showed less activity relative to their dihydroxyl analogues which are probably due to less stabilization of the phenoxide ion radicals in comparison to these compounds. Finally, compound 52 is least active which might be due to the electron donating effect of the ethoxy group.
Table-6. Results of DPPH radical scavenging inhibitory assay 6-nitrobenzimidazole derivatives 37-66. a a Compound # IC50 (µM ± SEM ) Compound # IC50 (µM ± SEM )
37 NAb 53 NAb
38 NAb 54 NAb
39 NAb 55 NAb
40 NAb 56 NAb
41 NAb 57 NAb
42 NAb 58 NAb
43 NAb 59 NAb
44 NAb 60 NAb
45 NAb 61 NAb
46 NAb 62 NAb
47 9.3 ± 1.11 63 NAb
48 NAb 64 11.17 ± 0.42
49 73.06 ± 0.60 65 NAb
134, 50 15.6 ± 3.68 66 11.58 ± 0.06
51 NAb n-Propylgallatec 30.12 ± 0.27
52 417.5 ± 2.28
SEMa is the standard error of the mean, NAb Not active, n-propylgallatec c, standard inhibitor for DPPH radical scavenging activity.
In vitro superoxide inhibitory activity
In search of a potential class of superoxide anion radical scavenging compounds, the compounds
37-66 were screened for the in vitro superoxide anion radical scavenger activity. These compounds have shown varying degree of superoxide anion radical scavenging activity and their
IC50 values ranging between 89.4- 455.0 µM. The compounds 47 (IC50 = 89.4 ± 2.72 µM) and 50
(IC50 = 104.0 ± 1.4 µM) showed IC50 values which were much better than the n-propylgallate standard (IC50 = 106.34 ± 1.6 µM). The compounds 49 (IC50 = 121.78 ± 2.25µM), showed moderate IC50 values relative to the standard value. On the other hand, the compound 52 (IC50 =
455.0 ± 1.8 µM) was least active among the active compounds. The compounds 37-46, 48, 51,
53-66 revealed inhibitions under 50%, so, therefore, these compounds were not further studied.
The compounds 47 and 50 showed highest activity which are presumably caused by the the presence of 3, 4-dihydroxyl and 2, 3, 4-trihydroxyl groups which can better stabilises the phenoxide ion radicals. On the other hand, compound 49, a 2, 4, 6-trihydroxyl analogue showed less activity relative to their di- and trihydroxyl analogues which is probably caused by less stabilization of the phenoxide ion radicals in comparison to these compounds. Finally, compound
52 is least active which is prossibly due to the electronic effects of the ethoxy group.
135, Table-7. Results of superoxide anion radical scavenging inhibitory assay of 6-nitrobenzimidazole derivatives 37-66. a a Compound # IC50 (µM ± SEM ) Compound # IC50 (µM ± SEM )
37 NAb 53 NAb
38 NAb 54 NAb
39 NAb 55 NAb
40 NAb 56 NAb
41 NAb 57 NAb
42 NAb 58 NAb
43 NAb 59 NAb
44 NAb 60 NAb
45 NAb 61 NAb
46 NAb 62 NAb
47 89.4 ± 2.72 63 NAb
48 NAb 64 NAb
49 121.78 ± 2.25 65 NAb
50 104.0 ± 1.4 66 NAb
51 NAb n-Propylgallatec 106.34 ± 1.6
52 455.0 ± 1.8
SEMa is the standard error of the mean, NAb Not active, n-propylgallatec c, standard inhibitor for superoxide anion radical scavenging.
136, Cytotoxic activity
All the synthetic compounds 37-66 exhibited no cytotoxicity against Artemia salina.
6.5. Conclusion
All The synthetic compounds 37-66 were randomly screened for the in vitro urease, α- chymotrypsin, carbonic anhydrase, antiglycation, phosphodiesterase and antioxidant activities
(DPPH & superoxide anion scavenging assays).
In the urease assay, out of 30 compounds, total six compounds exhibited activity in which only one compound 49 showed good activity, three compounds 50, 66 and 47 were moderately active and the rest of two compounds 64 and 54 were least active.
In the α-chymotrypsin assay, total nine compounds showed activity in which only one compound
52 was good active, six compounds 64, 47, 66, 54, 49 and 50 were moderately active and the remaining two compounds 41 and 58 were least active.
In the carbonic anhydrase assay, none of the compounds showed activity in this assay.
In the antiglycation assay, total fourteen compounds showed activity in which six compounds 66,
47, 65, 64, 45 and 38 were much better than the standard rutin, two compounds 50 and 41 exhibited IC50 values near the standard rutin, three compounds 49, 42 and 59 showed moderately active and the remaining three compounds 43, 37 and 54 were weakly active.
In the phosphodiesterase assay, total ten compounds showed activity in which nine compounds
66, 37, 47, 49, 50, 45, 39, 46 and 42 were much better than the standard EDTA while one compound 41 showed IC50 value which was slightly higher than the standard value.
In the DPPH radical scavenging assay, total six compounds showed activity in which four compounds 47, 64, 66, and 50 were much better than the standard n-propylgallate, one
137, compound 49 showed moderate activity while the remaining one compound 52 was the least active in this assay.
In the superoxide anion radical scavenging assay, total four compounds revealed activity two of them (47 and 50)were much better than the standard n-propylgallate, while compound 49 showed moderate activity and the remaining one compound 52 was least active.
6.6. General Instrumentation
Melting points of all the compounds were recorded on a Büchi 434 apparatus. NMR spectra were done on Bruker AM 500, 400, 300 MHz. instruments. For elemental analysis, A Carlo Erba
Strumentazion-Mod-1106, Italy was used. EI-MS spectra were recorded on a Finnigan MAT-
311A, Germany. TLC (thin layer chromatography) was carried out on precoated silica gel glass plates (Kieselgel 60, 254, E. Merck, Germany). UV visualized chromatograms at 254 and 365 nm.
6.6.1. Synthetic procedure of compounds 37-66
6-Nitrobenzimidazole derivatives were synthesized by taking a solution of 4- nitrophenylenediamine (3.12 eq.) and substituted aromatic aldehydes (3.16 eq.) in 15 ml DMSO in a round bottom flask (100 ml). Sodium metabisulfite (Na2S2O5) was added to the stirred solution of 4-nitrophenylenediamine and 30 different substituted aromatic aldehydes. The reaction contents were heated for 4h and the reaction progress was checked by TLC. When the reaction was completed then it was cooled at room temperature. Addition of water (30 ml) resulted in the precipitation of crude solid residues, which were then passed through extensive coloumn chromatography and hence it resulted the 6-nitrobenzimidazole derivatives 37-66 in high yields. The structures of the synthesized compounds were elucidated with the help of proton
NMR, CHN, and EI spectroscopy. All compounds give satisfactory CHN analysis.
138, 6.6.1.1. 4-(6-Nitro-1H-benzimidazol-2-yl)phenol (37)
1 Yield: 0.65 g (81%); H-NMR: (300 MHz, DMSO-d6): δH 8.38 (d, 1H, J7,5 = 2.0 Hz, H-7), 8.10
(dd, 1H, J5,4 = 9.0 Hz, J5,7 = 2.0 Hz, H-5), 8.04 (d, 2H, J2',3'/6',5' = 8.7 Hz, H-2'/6'), 7.69 (d, 1H, J4,5
+ = 9.0 Hz, H-4), 6.94 (d, 2H, J3',2'/5',6' = 8.7 Hz, H-3'/5'); MS: m/z (rel. abund. %), 255 (M , 100),
209 (37), 182 (25).
6.6.1.2. 2, 4-Dibromo-6-(6-nitro-1H-benzimidazol-2-yl)phenol (38)
1 Yield: 0.82 g (82%); H-NMR: (300 MHz, DMSO-d6): δH 8.59 (br. s, 1H, H-7), 8.34 (d, 1H, J4',6'
= 2.5 Hz, H-4'), 8.21 (dd, 1H, J5,4 = 9.0 Hz, J5,7 = 2.5 Hz, H-5), 7.98 (d, 1H, J6',4' = 2.5 Hz, H-6'),
+ 7.88 (d, 1H, J4,5 = 9.0 Hz, H-4); MS: m/z (rel. abund. %), 412 (M , 100), 367 (36), 169 (10).
6.6.1.3. 2-(3, 4-Dimethoxyphenyl)-6-nitro-1H-benzimidazole (39)
1 Yield: 0.79 g (85%); H-NMR: (300 MHz, MeOD): δH 8.47 (d, 1H, J7,5 = 2.0 Hz, H-7), 8.18 (dd,
1H, J5,4 = 9.0 Hz, J5,7 = 2.5 Hz, H-5), 7.70 (m, 3H, H-4/2'/6'), 7.15 (d, 1H, J5',6' = 8.5 Hz, H-5'),
3.96 (s, 3H, -OMe), 3.92 (s, 3H, -OMe); MS: m/z (rel. abund. %), 299 (M+, 100), 253 (30), 210
(49).
6.6.1.4. 2-(2-Chlorophenyl)-6-nitro-1H-benzimidazole (40)
1 Yield: 0.688 g (80%); H-NMR: (400 MHz, DMSO-d6): δH 8.54 (br. s, 1H, H-7), 8.16 (d, 1H,
J5,4 = 8.5 Hz, H-5), 7.94 (d, 1H, J6',5' = 7.0 Hz, H-6'), 7.81 (d, 1H, J4,5 = 8.5 Hz, H-4), 7.70 (d, 1H,
+ J3',4' = 7.0 Hz, H-3'), 7.70 (m, 2H, H-4'/5'); MS: m/z (rel. abund. %), 273 (M , 100), 243 (28), 227
(29).
139, 6.6.1.5. 2, 4-Dichloro-6-(6-nitro-1H-benzimidazol-2-yl)phenol (41)
1 Yield: 0.83 g (83%); H-NMR: (300 MHz, DMSO-d6): δH 8.58 (d, 1H, J7,5 = 1.5 Hz, H-7), 8.18
(m, 2H, H-5/6'), 7.84 (d, 1H, J4,5 = 9.0 Hz, H-4), 7.72 (d, 1H, J4',6' = 2.0 Hz, H-4'); MS: m/z (rel. abund. %), 323 (M+, 100), 293 (23), 277 (47).
6.6.1.6. 6-Nitro-2-(4-nitrophenyl)-1H-benzimidazole (42)
1 Yield: 0.71 g (80%); H-NMR: (300 MHz, MeOD): δH 8.59 (d, 1H, J7,5 = 2.0 Hz, H-7), 8.44 (d,
2H, J3',2'/5',6' = 9.0 Hz, H-3'/5'), 8.38, (d, 2H, J2',3'/6',5' = 9.0 Hz, H-2'/6'), 8.25 (dd, 1H, J5,4 = 9.0 Hz,
+ J5,7 = 2.0 Hz, H-5), 7.79 (d, 1H, J4,5 = 9.0 Hz, H-4); MS: m/z (rel. abund. %), 284 (M , 100), 238
(15), 192 (15).
6.6.1.7. 2-(4-Methylphenyl)-6-nitro-1H-benzimidazole (43)
1 Yield: 0.61 g (81%); H-NMR: (300 MHz, DMSO-d6): δH 8.44 (d, 1H, J7,5 = 2.0 Hz, H-7), 8.12
(m, 3H, H-5/2'/6'), 7.72 (d, 1H, J4,5 = 9.0 Hz, H-4), 7.40 (d, 2H, J3',2'/5',6' = 8.0 Hz, H-3'/5'); MS: m/z (rel. abund. %), 253 (M+, 100), 209 (23), 180 (13).
6.6.1.8. 2-(3, 4-Dichlorophenyl)-6-nitro-1H-benzimidazole (44)
1 Yield: 0.76 g (79%); H-NMR: (300 MHz, MeOD): δH 8.58 (d, 1H, J7,5 = 2.0 Hz, H-7), 8.25 (dd,
1H, J5,4 = 9.0 Hz, J5,7 = 2.5 Hz, H-5), 7.90 (d, 1H, J5',6' = 8.4 Hz, H-5'), 7.78 (d, 1H, J4,5 = 9.0 Hz,
H-4), 7.74 (d, 1H, J2',6' = 2.0 Hz, H-2'), 7.56 (dd, 1H, J6',5' = 8.4 Hz, J6',2' = 2.0 Hz, H-6'); MS: m/z
(rel. abund. %), 307 (M+, 100), 277 (33), 261 (33).
140, 6.6.1.9. 4-(6-Nitro-1H-benzimidazol-2-yl)-1, 3-benzenediol (45)
1 Yield: 0.69 g (81%); H-NMR: (300 MHz, DMSO-d6): δH 8.44 (br. s, 1H, H-7), 8.12 (dd, 1H, J5,4
= 9.0 Hz, J5,7 = 2.0 Hz, H-5), 7.92 (d, 1H, J6',5' = 8.7 Hz, H-6'), 7.74 (d, 1H, J4,5 = 9.0 Hz, H-4),
6.45 (m, 2H, H-3'/5'), 7.56 (dd, 1H, J6',5' = 8.4 Hz, J6',2' = 2.0 Hz, H-6'); MS: m/z (rel. abund. %),
271 (M+, 100), 225 (88).
6.6.1.10. 2-(4-Chlorophenyl)-6-nitro-1H-benzimidazole (46)
1 Yield: 0.73 g (85%); H-NMR: (300 MHz, DMSO-d6): δH 8.47 (br. s, 1H, H-7), 8.21 (d, 2H,
J3',2'/5',6' = 8.5 Hz, H-3'/5'), 8.13 (dd, 1H, J5,4 = 9.0 Hz, J5,7 = 2.5 Hz, H-5), 7.76 (d, 1H, J4,5 = 9.0
+ Hz, H-4), 7.69 (d, 2H, J2',3'/6',5' = 8.5 Hz, H-2'/6'); MS: m/z (rel. abund. %), 273 (M , 100), 227
(70), 200 (33).
6.6.1.11. 4-(6-Nitro-1H-benzimidazol-2-yl)-1, 2-benzenediol (47)
1 Yield: 0.69 g (81%); H-NMR: (300 MHz, DMSO-d6): δH 8.37 (d, 1H, J7,5 = 2.0 Hz, H-7), 8.08
(dd, 1H, J5,4 = 9.0 Hz, J5,7 = 2.0 Hz, H-5), 7.66 (d, 1H, J5',6' = 8.7 Hz, H-5'), 7.63 (d, 1H, J2',6' = 2.0
Hz, H-2'), 7.50 (dd, 1H, J6',5' = 8.0 Hz, J6',2' = 2.0 Hz, H-6'), 6.90 (d, 1H, J4,5 = 9.0 Hz, H-4); MS: m/z (rel. abund. %), 271 (M+, 100), 241 (38), 225 (83).
6.6.1.12. 2-Methoxy-6-(6-nitro-1H-benzimidazol-2-yl)phenol (48)
1 Yield: 0.68 g (77%); H-NMR: (300 MHz, DMSO-d6): δH 8.46 (d, 1H, J7,5 = 2.0 Hz, H-7), 8.05
(dd, 1H, J5,4 = 9.0 Hz, J5,7 = 2.0 Hz, H-5), 7.73 (d, 1H, J4',5' = 8.0 Hz, H-4'), 7.70 (d, 1H, J4,5 = 9.0
Hz, H-4), 7.06 (d, 1H, J6',5' = 8.0 Hz, H-6'), 7.50 (t, 1H, J5'/4',6' = 8.0 Hz, H-5'), 3.82 (s, 3H, -OMe);
MS: m/z (rel. abund. %), 285 (M+, 100), 267 (82), 196 (57).
141, 6.6.1.13. 2-(6-Nitro-1H-benzimidazol-2-yl)-1, 3, 5-benzenetriol (49)
1 Yield: 0.72 g (81%); H-NMR: (300 MHz, MeOD): δH 8.37 (d, 1H, J7,5 = 2.0 Hz, H-7), 8.14 (dd,
1H, J5,4 = 9.0 Hz, J5,7 = 2.0 Hz, H-5), 7.68 (d, 1H, J4,5 = 9.0 Hz, H-4), 6.00 (s, 2H, H-3'/5'); MS: m/z (rel. abund. %), 287 (M+, 100), 241 (26), 203 (30).
6.6.1.14. 4-(6-Nitro-1H-benzimidazol-2-yl)-1, 2, 3-benzenetriol (50)
1 Yield: 0.75 g (84%); H-NMR: (400 MHz, DMSO-d6): δH 8.45 (br. s, 1H, H-7), 8.13 (dd, 1H, J5,4
= 9.0 Hz, J5,7 = 2.5 Hz, H-5), 7.73 (d, 1H, J6',5' = 8.5 Hz, H-6'), 7.41 (d, 1H, J4,5 = 9.0 Hz, H-4),
+ 7.73 (d, 1H, J5',6' = 8.5 Hz, H-5'); MS: m/z (rel. abund. %), 287 (M , 100), 241 (69), 212 (13).
6.6.1.15. N, N-Dimethyl-4-(6-nitro-1H-benzimidazol-2-yl)aniline (51)
1 Yield: 0.37 g (82%); H-NMR: (400 MHz, DMSO-d6): δH 8.33 (br. s, 1H, H-7), 8.06 (dd, 1H,
J5,4 = 9.0 Hz, J5,7 = 2.0 Hz, H-5), 8.04 (d, 2H, J2',3'/6',5' = 8.8 Hz, H-2'/6'), 7.63 (d, 1H, J4,5 = 9.0
Hz, H-4), 6.94 (d, 2H, J3',2'/5',6' = 8.8 Hz, H-3'/5'), 3.26 (s, 3H, NCH3), 3.01 (s, 3H, NCH3); MS: m/z (rel. abund. %), 282 (M+, 100), 236 (73.
6.6.1.16. 2-Ethoxy-6-(6-nitro-1H-benzimidazol-2-yl)phenol (52)
1 Yield: 0.73 g (78%); H-NMR: (400 MHz, DMSO-d6): δH 8.53 (br. s, 1H, H-7), 8.16 (dd, 1H,
J5,4 = 9.0 Hz, J5,7 = 2.5 Hz, H-5), 7.81 (d, 1H, J4,5 = 9.0 Hz, H-4), 7.68 (d, 1H, J6',5' = 8.0 Hz, H-
6'),7.14 (d, 1H, J4',5' = 8.0 Hz, H-4'), 6.97 (t, 1H, J5'/4',6' = 8.0 Hz, H-5'), 4.10 (q, 2H, J = 7.0 Hz, -
+ OCH2), 1.37 (t, 3H, J = 7.0 Hz, -CH3); MS: m/z (rel. abund. %), 299 (M , 78), 284 (100), 196
(48).
142, 6.6.1.17. 4-Chloro-2-(6-nitro-1H-benzimidazol-2-yl)phenol (53)
1 Yield: 0.73 g (81%); H-NMR: (300 MHz, DMSO-d6): δH 8.53 (d, 1H, J7,5 = 2.0 Hz, H-7), 8.16
(m, 2H, H-5/6'), 7.82 (d, 1H, J4,5 = 9.0 Hz, H-4), 7.66 (dd, 1H, J4',5' = 8.7 Hz, J4',6' = 2.5 Hz, H-4'),
+ 7.11 (d, 1H, J5',4' = 8.7 Hz, H-5'); MS: m/z (rel. abund. %), 289 (M , 100), 243 (53), 179 (09).
6.6.1.18. 2-(6-Nitro-1H-benzimidazol-2-yl)phenol (54)
1 Yield: 0.66 g (82%); H-NMR: (300 MHz, DMSO-d6): δH 8.53 (br. s, 1H, H-7), 8.16 (m, 2H, H-
5/6'), 7.82 (d, 1H, J4,5 = 9.0 Hz, H-4), 7.43 (t, 1H, J4'/5',6' = 7.5 Hz, H-4'), 7.06 (m, 2H, H-3'/5');
MS: m/z (rel. abund. %), 255 (M+, 100), 209 (94), 182 (12).
6.6.1.19. 2-(2, 4-Dichlorophenyl)-6-nitro-1H-benzimidazole (55)
1 Yield: 0.78 g (81%); H-NMR: (300 MHz, DMSO-d6): δH 8.55 (d, 1H, J7,5 = 1.5 Hz, H-7), 8.18
(dd, 1H, J5,4 = 9.0 Hz, J5,7 = 2.5 Hz, H-5), 7.98 (d, 1H, J6',5' = 8.5 Hz, H-6'), 7.90 (d, 1H, J3',5' = 2.0
Hz, H-3'), 7.41 (d, 1H, J4,5 = 9.0 Hz, H-4), 7.73 (d, 1H, J5',3' = 2.0, J5',6' = 8.5 Hz, H-5'); MS: m/z
(rel. abund. %), 307 (M+, 100), 261 (49), 159 (42).
6.6.1.20. 2-(2-Chloro-5-nitrophenyl)-6-nitro-1H-benzimidazole (56)
1 Yield: 0.77 g (77%); H-NMR: (300 MHz, DMSO-d6): δH 8.79 (d, 1H, J6',4' = 2.5, H-6'), 8.60 (d,
1H, J7,5 = 1.5 Hz, H-7), 8.41 (dd, 1H, J5,4 = 9.0 Hz, J5,7 = 2.5 Hz, H-5), 7.73 (dd, 1H, J4',6' = 2.0,
J4',3' = 8.5 Hz, H-4'), 8.01 (d, 1H, J3',4' = 8.5 Hz, H-3'), 7.88 (d, 1H, J4,5 = 9.0 Hz, H-4); MS: m/z
(rel. abund. %), 318 (M+, 100), 272 (75), 226 (47).
143, 6.6.1.21. 2-(2-Fluorophenyl)-6-nitro-1H-benzimidazole (57)
1 Yield: 0.645 g (79%); H-NMR: (400 MHz, DMSO-d6): δH 8.52 (d, 1H, J7,5 = 2.0 Hz, H-7), 8.27
(td, 1H, J4',6' = 1.5, J4'/3',5' = 8.5 Hz, H-4'), 8.14 (dd, 1H, J5,4 = 9.0 Hz, J5,7 = 2.5 Hz, H-5), 7.81 (d,
1H, J4,5 = 9.0 Hz, H-4); 7.62 (m, 1H, H-3'), 7.48 (m, 2H, H-5'/6'); MS: m/z (rel. abund. %), 257
(M+, 100), 211 (57), 184 (34).
6.6.1.22. 2-(4-Bromophenyl)-6-nitro-1H-benzimidazole (58)
1 Yield: 0.79 g (80%); H-NMR: (300 MHz, DMSO-d6): δH 8.47 (br. s, 1H, H-7), 8.15 (d, 2H,
J3',2'/5',6' = 8.5 Hz, H-3'/5'), 8.13 (dd, 1H, J5,4 = 9.0 Hz, J5,7 = 2.5 Hz, H-5), 7.80 (d, 2H, J2',3'/6',5' =
+ 8.5 Hz, H-2'/6'), 7.76 (d, 1H, J4,5 = 9.0 Hz, H-4); MS: m/z (rel. abund. %), 317 (M , 100), 289
(29), 192 (21).
6.6.1.23. 6-Nitro-2-(3-thienyl)-1H-benzimidazole (59)
1 Yield: 0.65 g (84%); H-NMR: (300 MHz, DMSO-d6): δH 8.44 (br d, 1H, J7,5 = 2.1, H-7), 8.38
(m, 1H, H-2'), 8.12 (dd, 1H, J5,4 = 8.7 Hz, J5,7 = 2.1 Hz, H-5), 7.79 (m, 3H, H-4/4'/5');MS: m/z
(rel. abund. %), 245 (M+, 100), 199 (61), 172 (37).
6.6.1.24. 6-Nitro-2-(1-phenanthryl)-1H-benzimidazole (60)
1 Yield: 0.85 g (86%); H-NMR: (300 MHz, DMSO-d6): δH 8.98 (d, 2H, J4',3'/5',6' = 8.1, Hz, H-
4'/5'), 8.95 (d, 1H, J2',3' = 8.4 Hz, H-2'), 8.60 (br. s, 1H, H-7), 8.44 (m, 1H, H-10'), 8.20 (dd, 1H,
J5,4 = 8.7 Hz, J5,7 = 2.1 Hz, H-5), 8.15 (d, 1H, J9',10' = 8.2 Hz, H-9'), 7.78 (m, 5H, H-4/3'/6'/7'/8');
MS: m/z (rel. abund. %), 339 (M+, 100), 292 (86), 203 (25).
144, 6.6.1.25. 6-Nitro-2-phenyl-1H-benzimidazole (61)
1 Yield: 0.57 g (76%); H-NMR: (400 MHz, DMSO-d6): δH 8.46 (d, 1H, J7,5 = 2.0, H-7), 8.21 (dd,
2H, J5,4/2',3' = 8.0 Hz, J5,7/2',4' = 2.0 Hz, H-5/2'), 8.12 (dd, 1H, J6',5' = 9.0 Hz, J6',4' = 2.4 Hz, H-6'),
+ 7.75 (d, 1H, J4,5 = 9.0 Hz, H-4), 7.58 (m, 3H, H-3'/4'/5'); MS: m/z (rel. abund. %), 239 (M , 100),
209 (38), 166 (19).
6.6.1.26. Methyl 4-(6-nitro-1H-benzimidazol-2-yl)phenyl sulfide (62)
1 Yield: 0.74 g (74%); H-NMR: (400 MHz, DMSO-d6): δH 8.43 (d, 1H, J7,5 = 2.0 Hz, H-7),
8.13(d, 2H, J2',3'/6',5' = 8.5 Hz, H-2'/6'), 8.10 (dd, 1H, J5,4 = 9.0 Hz, J5,7 = 2.0 Hz, H-5), 7.74 (d, 1H,
+ J4,5 = 9.0 Hz, H-4), 7.46 (d, 2H, J3',2'/5',6' = 8.5 Hz, H-3'/5'); MS: m/z (rel. abund. %), 285 (M ,
100), 239 (80), 212 (41).
6.6.1.27. 6-Nitro-2-(4-pyridinyl)-1H-benzimidazole (63)
1 Yield: 0.61 g (81%); H-NMR: (400 MHz, DMSO-d6): δH 8.81 (d, 2H, J2',3'/6',5' = 8.5 Hz, H-2'/6'),
8.55 (br. s, 1H, H-7), 8.16 (dd, 1H, J5,4 = 8.8 Hz, J5,7 = 2.0 Hz, H-5), 8.12 (d, 2H, J3',2'/5',6' = 8.5
+ Hz, H-3'/5'), 7.83 (d, 1H, J4,5 = 8.8 Hz, H-4); MS: m/z (rel. abund. %), 240 (M , 100), 194 (48),
63 (24).
6.6.1.28. 2-(6-Nitro-1H-benzimidazol-2-yl)-1, 4-benzenediol (64)
1 Yield: 0.71 g (83%); H-NMR: (400 MHz, DMSO-d6): δH 9.17 (br. s, 1H, NH), 8.51 (br. s, 1H,
H-7), 8.13 (dd, 1H, J5,4 = 9.0 Hz, J5,7 = 2.5 Hz, H-5), 7.79 (d, 1H, J4,5 = 9.0 Hz, H-4), 7.53 (d, 1H,
+ J6',4' = 2.5 Hz, H-6'), 6.88 (m, 2H, H-3'/4'); MS: m/z (rel. abund. %), 271 (M , 100), 225 (65).
145, 6.6.1.29. 2-(9-Anthryl)-6-nitro-1H-benzimidazole (65)
1 Yield: 0.83 g (84%); H-NMR: (300 MHz, DMSO-d6): δH 8.90 (s, 1H, H-6'), 8.64 (br. s, 1H, H-
7), 8.24 (m, 3H, H-5/2'/10'), 7.88 (d, 1H, J4,5 = 8.7 Hz, H-4), 7.67 (d, 2H, J5',4'/7',8' = 8.7 Hz, H-
5'/7'), 7.57 (m, 4H, H-3'/4'/8'/9'); MS: m/z (rel. abund. %), 339 (M+, 100), 292 (57), 146 (10).
6.6.1.30. 3-(6-Nitro-1H-benzimidazol-2-yl)-1, 2-benzenediol (66)
1 Yield: 0.73 g (86%); H-NMR: (300 MHz, DMSO-d6): δH 8.53 (br. s, 1H, H-7), 8.17 (m, 1H, H-
5), 7.80 (d, 1H, J4,5 = 8.7 Hz, H-4), 7.53 (t, 1H, J5'/4',6' = 8.0 Hz, H-5'), 6.93 (m, 2H, H-4'/6'); MS: m/z (rel. abund. %), 271 (M+, 100), 225 (53), 196 (8).
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157,
CHAPTER-7
BIOASSAY TECHNIQUES
158, 7.1. In vitro antiglycation bioassay
This assay is used to inhibit the Methyl Glyoxal mediated development of fluorescence of BSA.
This assay was done according to the method described by et al., (2005) with minor changes.
Triplicate samples of Rahbar BSA 10 mg/ml, 14 mM MGO, 0.1 M phosphate buffer (pH 7.4) containing NaN3 (30 mM) was incubated under aseptic conditions (in such a way that each well of 96-well plate contain 50 µL BSA solution, 50 µL MGO, and 20 µL test sample) at 37 °C for 9 days in the presence or absence of a variety of concentrations of the test compounds. After 9 days of incubation, each compound was studied for the development of specific fluorescence
(excitation, 330 nm; emission, 440 nm), against sample blank. On a microtitre plate spectrophotometer (Spectra Max, Molecular Devices, CA USA). Rutin was used as a positive control (IC50 = 294 ± 1.50 µM) [1].
The percent inhibition of AGE formation in the test sample versus control was measured for each inhibiting compound by using the following formula:
% inhibition = (1- fluorescence of test sample/ Fluorescence of the control group) X 100
7.2. In vitro carbonic anhydrase bioassay
In this assay, 4-NPA is hydrolyzed to 4-nitrophenol and acetate and then this reaction is followed by observing the formation of 4-nitrophenol at a temperature of 25-28 °C [2].
The experiment was carried out in buffer solution having HEPES an acidic and Tris alkaline at a total concentration of 20mM and pH ranges from 7.2-7.9. For every test sample, the reaction tube consisted of 140 µl of the HEPES-Tris solution, 20 µl of freshly prepared aqueous solution of purified bovine erythrocyte CA-II (0.1-0.2mg/2000 µl of deionized water for 96-well plates), and sigma aldrick. 20 µl of test sample which was dissolved in DMSO. 20µl of substrate 4-
PNA at concentration of 0.7 mM diluted in ethanol. 159, 7.3. In vitro α-chymotrypsin bioassay
The α-chymotrypsin activity is performed in 50 mM Tris-HCl buffer pH 7.6 with 10 mM CaCl2 according to the known method [3].
The α-chymotrypsin enzyme; (12 Units/mL prepared in buffer mentioned above) with the test compound (0.5 mM) prepared in DMSO (final concentration 7%) is incubated at 30 °C for 25 min. The reaction is initiated by the addition of the substrate N-succinyl-L-phenylalanine-p- nitroanilide (SPpNA; 0.4 mM prepared in the buffer as above). The variation in absorbance by dischrging p-nitroanilide will be constantly checked at 410 nm. The positive control without test sample and the negative control without enzyme or with standard inhibitor run in parallel. The % of inhibition is based upon initial velocity and calculated as:
OD of test compound _ 100 % of Inhibition = 100 X OD of positive control
IC50 Calculation
IC50 (Inhibition of enzymatic hydrolysis of the substrate SPpNA by 50%) is calculated by observing the inhibition value upon the rising the concentration of the test samples. This IC50 value is measured using EZ-Fit enzyme kinetics program (Perellela Scientific, Inc., Amherst,
Mars, USA).
7.4. In vitro urease bioassay
Reaction mixtures consisting of 25µL of enzyme (jack bean urease) solution and 55 µL of buffers having 100 mM urea were incubated with 5 µL of test compounds (0.5 mM concentration) at 30
°C for 15 min in 96-well plates. Urease activity was determined by measuring ammonia 160, production using the indophenol method as stated by weather burn. Shortly, 45 µl each phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprussside) and 70 µL of alkali reagent
(0.5% w/v NaOH and 0.1% active chloride NaOCl) were mixed to each other. The rising of absorbance at 630 nm was determined after 50 min using a microplate reader (Molecular Device,
USA). All reactions were performed in triplicate in a final volume of 200 µL. The results (change in absorbance per min) were processed by using soft Max Pro software (molecular Device,
USA). All the assays were performed at pH 6.8. Percentage inhibitions were calculated from the formula 100-(ODtestwell/ODcontrol) ×100. For the urease enzyme, thiourea was selected as a standard inhibitor [4].
7.5. Antioxidant (DPPH and Superoxide)
7.5.1. In vitro DPPH (1, 1-Diphenyl-2-picryl hydrazyl) free radical scavenging bioassay The free radical scavenging activity was calculated by 1, 1-diphenyl-2-picrylhydrazil (DPPH) using literature protocols [5,6]. Reaction mixture contains 5 µL of test compound (1 mM in
DMSO) and 95 µL of DPPH (Sigma, 300 µM) in ethanol. The reaction mixture was taken into a
96-well microtiter plate and incubated at 37 º C for 30 min. The absorbance was measured at 515 nm on microtiter plate reader (Molecular Devices, USA). Percent radical scavenging activity was determined by comparison with DMSO containing control (Table-1). IC50 values indicates concentration of compounds to scavenge 50% of DPPH radicals. BHA (3-t-Butyl-4- hydroxyanisole) was used as a positive control. All the chemicals used were of analytical grade
(Sigma, USA).
161, 7.5.2. In vitro superoxide anion scavenging bioassay
Test compounds whether natural or synthetic origin was assayed by the technique used by
Gaulejac et al., in 1999. In aerobic reaction mixtures having NADH, phenazine methosulphate and Nitroblue tetrazolium, PMS is reduced by NADH and then yield O2-, which then reduced to
NBT. This PMS is habitually used to mediate O2-. The reaction mixture consist of 40 µL of 280
µM β-nicotinamide adenine dinucleotide reduced form (NADH), 40 µL of 80 µM, nitro blue tetrazolium (NBT), 20 µL 8 µM phenazine methosulphate (PMS) 10 µL of 1 mM sample and 90
µL of 0.1 M phosphate buffer (pH 7.4). The reagents were prepared in buffer and sample in
DMSO. The reaction is done in 96-well microtitre plate at room temperature and absorbance was studied at 560 nm. The superoxide formation was checked by analysing the formation of water soluble blue Formazan dye. A lesser absorbance of reaction mixture reveals a higher scavenging activity of sample.
% Radical scavenging activity (%RSA) of test compounds can be determined in comparison with a control.
% RSA= 100 – {(OD test compound / OD control) X 100}
7.6. In vitro phosphodiesterase inhibition bioassay
Activity against snake venom was done by taking 33 mM Tris-HC1 buffer pH 8.8, 30 mM
Mgacetate with 0.000742 U/well final concentration of enzyme using a microtiter plate assay and
0.33 mM bis-(p-nitropheny1) phosphate (Sigma N-3002) as substrate. From Merck Cystein and
EDTA were used as positive controls (1C50 = 748 ± 0.015 µM, 274 ± 0.007 µM, respectively).
After 30 min pre-incubation of the enzyme with the test compounds, enzyme activity was done by spectrophotometrically at 37 °C on a microtitre plate reader (SpectraMax, Molecular Devices)
162, by following the rate (change in O.D/min) of release of p-nitrophenol from p-nitrophenyl phosphate at 410 nm. All assays were conducted in triplicate [7-10].
163, 7.7. References
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165, 8. List of publications
1. Khalid M. Khan, Zarbad Shah, Viqar Uddin Ahmad, Momin Khan, Muhammad
Taha, Humera Jahan and Muhammad Iqbal Choudhary, Synthesis of 2, 4, 6-
Tricholorophenyl Hydrazones and their Antiglycation Protein Activity, Medicinal
Chemistry, 2011 ,7, 572-580.
2. Khalid M. Khan, Zarbad Shah, Viqar Uddin Ahmad, Momin Khan, Muhammad
Taha, Shagufta Noreen, and Muhammad Iqbal Choudhary, 6-Nitrobenzimidazole
Derivatives: Potential Phosphodiesterase Inhibitors: Synthesis and Structure-Activity
Relationship, Bioorganic and Medicinal Chemistry, 2011 (Accepted).
3. Khalid M. Khan, Zarbad Shah, Viqar Uddin Ahmad, Momin Khan, Muhammad
Taha, Sajjad Ali and Muhammad Iqbal Choudhary, 2,4,6-Trichlorophenylhydrazine
Schiff Bases as DPPH Radical and Super Oxide Anion Scavengers, Medicinal
Chemistry, 2011 (Submitted).
166,