In the Name of Allah, the Most Gracious, the Most Merciful

Bismillah Hirrahman Nirraheem In the Name of Allah, the Most Gracious, the Most Merciful

All praises for Almighty Allah, the most kind and cordial. He is ample and the best disposer of all affairs (for us). I am showing my humble submission to the heart and soul after The Holy Prophet Hazrat Muhammad (sallallahualayhewassallum) whose life is an ideal pattern for all of us .

Phytochemical Studies on the Chemical Constituents of Xanthium strumarium Linn., Synthesis in addition Bioactivities of 2, 3-Diaminonaphthalenimidazole Derivatives and Amides of Piperic Acid

Thesis submitted

for

The partial fulfillment of the Degree

DOCTOR OF PHILOSOPHY

Chemistry

AMINA SULTANA

Department of Chemistry Federal Urdu University of Arts, Science and Technology Karachi – 75260, Pakistan 2014

CERTIFICATE

Certified that Ms. Amina Sultana has carried out her research work on the topic entitled Phytochemical Studies on the Chemical Constituents of Xanthium strumarium Linn., Synthesis in addition Bioactivities of 2, 3- Diaminonaphtalenimidazole Derivatives and Amides of Piperic Acid at Department of Chemistry, Federal Urdu University of Arts, Science, and Technology, Karachi, Pakistan, under the supervision of Dr. Aneela Wahab. Her research work is original that has not been submitted to any other university. Some publications have been earned from the course of this research work and the all are acknowledged.

Dr. Aneela Wahab ------Assistant Professor Department of Chemistry Federal Urdu University, Karachi, Pakistan Research Supervisor

Dr. Iffat Mahmood ------Chairperson Department of Chemistry Federal Urdu University, Karachi, Pakistan

Dedicated to my loving and caring Parents whose prayers have always been a great source of Strength to me

Contents

Acknowledgement ------1 Summary ------3 KHULASA ------6

CHAPTER -1 Phytochemical Studies on the Chemical Constituents of Xanthium strumarium Linn. ------10

1.0 Introduction ------11 1.1. General Introduction ------12 1.1.1. Classification ------17 1.1.2. Introduction About Family Compositeae ------18 1.1.3. Introduction About Genus Xanthium ------20 1.1.4. General Description of Xanthium strumarium Linn. ------21 1.1.4.1. Chemical Constituents of Xanthium strumarium Linn.------21 1.1.4.2. Medicinal Importance of Xanthium strumarium Linn.------22 1.1.4.3. Literature Review ------24 1.2. Present Work ------53 1.3. Results and Discussion ------55 1.3.1. Lupenyl acetate (1) ------56 1.3.2. Stigmasterol (2) ------59 1.3.3. β-Sitosterol (3) ------60 1.3.4. Palmitic acid (4) ------61 1.3.5. β-Amyrin (5) ------62 1.3.6. Oleanolic acid (6) ------63 1.3.7. β-Sitosterol-3-O-β-D-Glucopyranoside (7) ------64 1.3.8. Ferulic acid (8) ------65 1.3.9. Biological Activities ------66 1.3.9.1. In vitro Anti-bacterial Activity ------66 1.3.9.2. In vitro Anti-fungal Activity ------68 1.3.9.3. In vitro Anti-oxidant Activity ------70 1.4. Experimental ------71 1.4.1. General Experimental ------72 1.4.2. Collection of Plant Material ------72 1.4.3. Extraction and Isolation ------72 1.4.4. Characterization of Compounds ------79 1.4.4.1. Characterization of Lupenyl acetate (1) ------79 1.4.4.2. Characterization of Stigmasterol (2) ------80 1.4.4.3. Characterization of β-Sitosterol (3) ------81 1.4.4.4. Characterization of Palmitic acid (4) ------82 1.4.4.5. Characterization of β-Amyrin (5) ------83 1.4.4.6. Characterization of Oleanolic acid (6) ------84 1.4.4.7. Characterization of β-Sitosterol-3-O-β-D- Glucopyranoside (7)------85 1.4.4.8. Characterization of Ferulic acid (8) ------86

1.5. References ------87

CHAPTER-2 Synthesis in addition Bioactivities of 2, 3-Diaminonaphthalenimidazole Derivatives------97 2.0. General Introduction For Chapter-2 and 3 ------99 2.1. Introduction of Benzimidazole ------104 2.1.1 Biological Importance ------104 2.2. Synthetic Approaches Towards Benzimidazole ------112 2.3. Results and Discussion ------123 2.3.1. Chemistry ------124 2.3.2. General Method for the Synthesis of Compounds (65-99) ------124 2.3.3. General Stucture Elucidation ------129 2.3.4. Biological Evaluation of Compounds (65-99) ------131 2.3.4.1. In Vitro Tyrosinase Inhibitory Activity ------131 2.3.4.2. In Vitro Acetylcholinesterase and Butrylcholinesterase. Inhibitory Activity ------133 2.3.4.3. In Vitro Urease Inhibitory Activity ------136 2.3.4.4. In Vitro Anti-bacterial Activity ------138 2.3.4.5. In Vitro Anti-fungal Activity ------146 2.3.4.6. In Vitro Anti-oxidant Activity ------149 2.3.5. Conclusion ------151

2.4. Experimental ------152 2.4.1. General Experimental ------153 2.4.2. General Method for the Synthesis of Compounds (65-99) ------153 2.4.2.1. 2-(1H-indol-3-yl)-1H-naphtho[2,3-d]imidazole (65) ------154 2.4.2.2. 2-(4-ethoxy-3-methoxyphenyl)-1H-naphtho[2,3-d]imidazole (66)-154 2.4.2.3. 4-(1H-naphtho[2,3-d]imidazol-2-yl)phenol (67) ------154 2.4.2.4. 2-methoxy-4-(1H-naphtho[2,3-d]imidazol-2-yl) phenyl acetate (68) ------154 2.4.2.5. 2-(5-bromo-2-methoxyphenyl)-1H-naphtho[2,3-d]imidazole (69)- 155 2.4.2.6. 2-(2, 5-dimethoxyphenyl)-1H-naphtho[2,3-d]imidazole (70) ----- 155 2.4.2.7. 4-chloro-2-(1H-naphtho[2,3-d]imidazol-2-yl)phenol (71) ------156 2.4.2.8. 2-(4-trifluoromethylphenyl)-1H-naphtho[2,3-d]imidazole (72) --- 156 2.4.2.9. 2-(4-nitrophenyl)-1H-naphtho[2,3-d]imidazole (73) ------156 2.4.2.10. 2, 6-dimethoxy-4-(1H-naphtho[2,3-d]imidazol-2-yl)phenol (74) -157 2.4.2.11. 2-(3-benzyloxyphenyl)-1H-naphtho[2,3-d]imidazole (75) ------157 2.4.2.12. 2-(2-fluoro-4-methoxyphenyl)-1H-naphtho[2,3-d] imidazole (76)------157 2.4.2.13. 2-methoxy-5-(1H-naphtho[2,3-d]imidazol-2-yl)phenol (77) ----- 157 2.4.2.14. 2-(4-benzyloxyphenyl)-1H-naphtho[2,3-d]imidazole (78) ------158 2.4.2.15. 2-(3-ethoxy-4-methoxyphenyl)-1H-naphtho[2,3-d] imidazole (79)------158 2.4.2.16. 4-(1H-naphtho[2,3-d]imidazol-2-yl)-3-nitrophenol (80) ------158 2.4.2.17. 2-(thiophen-2-yl)-1H-naphtho[2,3-d]imidazole (81) ------159 2.4.2.18. 2-(3, 4-dimethoxyphenyl)-1H-naphtho[2,3-d]imidazole (82) ---- 159 2.4.2.19. 4-(1H-naphtho[2,3-d]imidazol-2-yl)benzene-1,3-diol (83) ------159

2.4.2.20. 2-(2, 3, 4-trimethoxyphenyl)-1H-naphtho[2,3-d]imidazole (84)-- 160 2.4.2.21. 4-(1H-naphtho[2,3-d]imidazol-2-yl)benzene-1,2,3-triol (85) ----- 160 2.4.2.22. 2-(4-methylthiophenyl)-1H-naphtho[2,3-d]imidazole (86) ------160 2.4.2.23. 2-(2-nitrophenyl)-1H-naphtho[2,3-d]imidazole (87) ------161 2.4.2.24. 2-(naphthalen-2-yl)-1H-naphtho[2,3-d]imidazole (88) ------161 2.4.2.25. N, N-dimethyl-4-(1H-naphtho[2,3-d]imidazol-2-yl)aniline (89)-- 161 2.4.2.26. 3-bromo-6-methoxy-2-(1H-naphtho[2,3-d] imidazol-2-yl) phenol (90) ------162 2.4.2.27. 2-(2-bromo-4, 5-dimethoxyphenyl)-1H-naphtho [2,3-d] imidazole (91) ------162 2.4.2.28. 2-phenyl-1H-naphtho[2,3-d]imidazole (92) ------162 2.4.2.29. 2-(2-ethoxyphenyl)-1H-naphtho[2,3-d]imidazole (93) ------163 2.4.2.30. 2-(2, 3-dimethoxyphenyl)-1H-naphtho[2,3-d]imidazole (94) ---- 163 2.4.2.31. 2-(4-bromo-2, 5-dimethoxyphenyl)-1H-naphtho[2,3-d] imidazole (95) ------163 2.4.2.32. 2-(3-bromo-4-methoxyphenyl)-1H-naphtho[2,3-d] imidazole (96) ------164 2.4.2.33. 2-(4-bromo-2-fluorophenyl)-1H-naphtho[2,3-d] imidazole (97) ------164 2.4.2.34. 2-(2-chloro-3-methoxyphenyl)-1H-naphtho[2,3-d] imidazole (98) ------164 2.4.2.35. 2-(3-bromophenyl)-1H-naphtho[2,3-d]imidazole (99) ------165 2.5 References ------166

CHAPTER-3 Synthesis and Bioactivities of Amides of Piperic Acid------173

3.1. Introduction of Amides ------175 3.1.1. Importance of Amides ------177 3.2. Synthetic Approaches Towards Amides of Piperic Acid ------182 3.3. Results and Discussion ------186 3.3.1. Chemistry ------187 3.3.2. General Method for the Synthesis of Compounds (42-56) ------187 3.3.3. General Stucture Elucidation ------192 3.3.4. Biological Evaluation of Compounds (42-56) ------194 3.3.4.1. In Vitro Anti-bacterial Activity ------194 3.3.4.2. In Vitro Anti-fungal Activity ------196 3.3.4.3. In Vitro Nematicidal Activity------198 3.3.4.4. In Vitro Anti-oxidant Activity------200 3.3.5. Conclusion------202

3.4. Experimental------203 3.4.1. General Experimental------204 3.4.2. General Method for the Synthesis of Compounds (42-56)------204 3.4.3. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)penta-2,4-dienoic acid (Piperic acid, 28) ------205 3.4.3.1. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-1-(piperidin-1- yl)penta-2,4-dien-1-one (42)------206

3.4.3.2. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-1-morpholinopenta- 2,4-dien-1-one (43)------206 3.4.3.3. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-1-(4-methylpiperazin- 1-yl)penta-2,4-dien-1-one (44)------206 3.4.3.4. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)penta-2,4- dienamide (45)------207 3.4.3.5. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-phenylpenta- 2,4-dienamide (46)------207 3.4.3.6. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-p-tolylpenta- 2,4-dienamide (47)------207 3.4.3.7. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(4-chlorophenyl) penta-2,4-dienamide (48)------208 3.4.3.8. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(4-methoxyphenyl) penta-2,4-dienamide (49)------208 3.4.3.9. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-propylpenta- 2,4-dienamide (50)------208 3.4.3.10. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-mesitylpenta- 2,4-dienamide (51)------209 3.4.3.11. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(4-methylpiperazin- 1-yl)penta-2,4-dienamide (52)------209 3.4.3.12. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-1-(pyrrolidine-1-yl) penta-2,4-dien-1-one (53)------209 3.4.3.13. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(4-iodophenyl) penta-2,4-dienamide (54)------210 3.4.3.14. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-benzylpenta- 2,4-dienamide (55)------210 3.4.3.15. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(3-methoxy-4- methylphenyl)penta-2,4-dienamide (56)------210 3.5. References ------212

CHAPTER–4

Biological Activities Assays------216

4.0. Introduction about Biological Activities------218 4.1. Enzyme Inhibition Activity------218 4.2. Anti-microbial Activity------220 4.3. Anti-oxidant Activity------221 4.4. Nematicidal Activity------222

4.5. Protocols------223 4.5.1. Enzyme Inhibition Assay------224 4.5.1.1. In Vitro Tyrosinase Inhibitory Assay------224 4.5.1.2. In Vitro Acetylcholinesterase and Butrylcholinesterase Inhibitory Assay------224 4.5.1.3. In Vitro Urease Inhibitory Assay------225 4.5.2. In Vitro Anti-microbial Assay------226 4.5.2.1. In Vitro Anti-bacterial Assay------226

4.5.2.2. In Vitro Anti-fungal Assay------227 4.5.3. In Vitro Anti-oxidant Assay ------228 4.5.4. In Vitro Nematicidal Assay------228

4.6. References ------230

* Research Publications------234

Acknowledgement

At very first and foremost, I am much thankful to Almighty Allah, the most beneficient and merciful who bestow me to accomplish my Ph.D. research work. Limitless all respect to our beloved last Holy Prophet Hazrat Muhammad (sallallahoalaihiwasallum) who shower his blessing during the whole research period in deliberating upon things deeply. I would like to express my heartfelt and profound acknowledgements to all those people who helped me out to complete this research work.

I wish to express my high tribute to (Late) Prof.Dr.Salimuzzaman Siddiqui F.R.S.,

H.I. the founding director of H.E.J. Research Institute of Chemistry, for establishing a renowned institute of chemistry. I am also grateful to Prof.Dr.Atta- ur-Rahman F.R.S., N.I., S.I., T.I., the chief Patron, International Center for Chemical and Biological Sciences, University of Karachi for his dedication to the developments in the field of chemical Sciences. I am thankful to the director of H.E.J Research

Institute, Prof. Dr. M. Iqbal Choudhary S.I., T.I., for providing research facilities in this institute. I would like to express my special thanks to my co-supervisor, Prof. Dr. Khalid

Muhammad Khan T.I., H.E.J. Research Institute of Chemistry, University of Karachi, for his active co-operation, prompt and tactful suggestion and the most valuable, his ever ready helping attitude that was a motivating factor in finalizing this study. I have great pleasure to express my sincere and affectionate gratitude to my supervisor Dr. AneelaWahab, Assistant Professor, for her skilful guidance, keen interest and great co-operation at each and every step of this arduous task. Her encouraging attitude made me self –confident and kept me on the right track. I am thankful to Dr.Iffat Mahmood (chairperson), Head of the Department of Chemistry (Federal Urdu University of Arts, Science, and Technology, Karachi) for providing collaboration with H.E.J. Institute.

I am very grateful to Prof. Dr. Abdul Malik S.I., for his expert guidance to materialize this work.

It is my pleasure to thanks and my sincere appreciation to my colleagues, Dr. Ghulam Farid, scientific officer, PCSIR Laboratories Complex, Karachi, Mr. Shafqat Hussain and Dr. Farzana Naz for their valuable, kind and fruitful suggestions throughout the course of present tenure. I am pleased to express my sincere regards and appreciation to my dear friend Miss Shahla Noureen for her bonafide and whole hearted help, prayers, moral support and nice company. This dissertation required an extensive bioassay work. I thankfully acknowledge Dr. Mehrin Lateef, scientific officer, PCSIR Laboratories Complex, Karachi, for enzyme inhibition screening. Mr. Sikandar Khan Sherwani for nematicidal and anti-oxidant activities and Dr. Ayesha Irshad, Dr. Saima Faraz and Dr. Zeba Parveen for the determination of anti- bacterial and anti- fungal activities. I am also thankful to all technical and non-technical staff members of the institute for their help and sincere co-operation during this research studies. My extreme gratitude is also for all my teachers from primary school to university whom privilege me to submit a Ph.D. dissertation. Words are restrain to express my immense gratitude to my respected , gracious parents , my beloved sisters (especially Nasima Nasim for her sincere co- operation and sacrifice) and brothers for their encouragement , motivation , prayers and humble co-operation throughout my this research work.

Amina Sultana

SUMMARY

This dissertation has been divided into four chapters. Each chapter has its own numbering of compounds and references. The general introduction describes the importance of natural products and the drugs based on them.

The chapter 1 deals with the phytochemical studies on the chemical constituents of Xanthium strumarium Linn. The introduction provides a review of the earlier contributions made in the chemistry and pharmacology of the genus Xanthium and a brief account of the present work.

Studies undertaken on different fractions of methanolic extract (XS-HX, XS-DC, XS-EA, XS-BU and XS-ME) of the air dried aerial parts of X. Strumarium Linn. showed weak to moderate antibacterial and weak antioxidant activity except ethyl acetate fraction (XS-EA) which exhibited moderate to high antibacterial and antifungal activity (Table-2, 3) while significant antioxidant activity (Table-4) was observed among all fractions. Studies undertaken on the bioactive ethylacetate fractions led to the isolation and structure elucidation of eight known compounds. The known compound (1) is reported for the first time from X. strumarium Linn. The constituents obtained are listed below. I. Lupenyl acetate (1)

II. Stigmasterol (2)

III. β-Sitosterol (3)

IV. Palmitic acid (4)

V. β-Amyrin (5)

VI. Oleanolic acid (6)

VII. β-Sitosterol-3-O-β-D- Glucopyranoside (7)

VIII. Ferulic acid (8)

The structure of all the isolated compounds have been determined through various spectroscopic techniques such as, IR, EIMS, HR-EIMS, 1H-NMR, 13C-NMR, 2D- NMR and also by comparison of their spectral data with those reported in literature.

Chapter 2 is about characterization and bioassay screening of thirty five (35) synthesized derivatives of 2, 3-diaminonaphthalenimidazole (65-99). Out of these thirty five naphthalenimidazoles, twenty six (26) (65, 66, 68, 69, 70, 72, 74, 75, 76, 77, 78, 79, 80, 82, 84, 85, 86, 88, 90, 91, 93, 94, 95, 96, 97, 98) are newly synthesized compounds. All synthesized derivatives showed interesting in vitro enzyme inhibitory (urease, tyrosinase, acetylcholinesterase and butrylcholinesterase inhibitory), antimicrobial and antioxidant activities. Two compounds 81 and 85 revealed potent in vitro tyrosinase inhibitory activity. On the other hand compounds 65, 66, 68, 69, 71, 79, 88 and 94 were found moderately active for this activity. When tested for their in vitro butrylcholinesterase inhibitory activity, three compounds 65, 66 and 79 exhibited good activity while compounds 67, 81, 82 and 89 showed moderate butrylcholinesterase inhibitory activity but all synthesized compounds were found inactive for acetylcholinesterase inhibitory activity. In urease inhibitory activity two compounds 71 and 90 revealed good activity while moderate activity was observed in compounds 65, 66, 68, 81 and 82. All synthesized derivatives when screened for their anti-microbial activity, only two compounds 90 and 92 were found exhibiting remarkable activity against bacterial strains B. cereus, B. subtilis, S. epidermidis, S. paratyphi A, Enterobacter and S.dysenteriae. Significant activity against Enterobacter and S.dysenteriae was displayed by 99 and moderate activity was found in compounds 65, 74, 81, 82, 85 and 94 against various tested bacterial strains. All synthesized compounds showed weak antifungal activity. For in vitro antioxidant activity, the compounds 65, 68, 77, 90, and 99 revealed promising whereas compounds 79, 82, 85 and 95 showed good antioxidant activity.

Chapter 3 describes the synthesis, structure elucidation and biological activity of fifteen (15) amides of piperic acid (42-56). Five compounds (47, 51, 52, 54, 56) are new amides. All the synthesized derivatives were evaluated for their in vitro anti-microbial, nematicidal and anti-oxidant activity. In the case of antimicrobial activity compound 54 was found the most active against all applied bacterial strains except S. pneumoniae, compound 49 showed excellent activity for P. vulgaris and 53 was good against P. stutzeri where as compound 44, 46, 47 and 48 showed good activity against P. aeruginosa. It was determined that compound 50 was active against S. aureus, P. stutzeri and P. aeruginosa. Compound 52 showed good activity against P. aeruginosa and E. coli whereas compound 56 exhibited activity against E. coli only. It was observed that among all the synthesized amides only compound 54 showed antifungal activity against all applied fungal strains. When screened for nematicidal activity compounds 42, 43, 45, 47, 52 and 56 were found possessing excellent nematicidal activity against root-knot nematode, Meloidogyne incognita, where as compounds 44, 50 and 54 have significant mortality rate. During antioxidant testing three compounds 44, 49 and 51 showed significant and two compounds 46 and 54 showed moderate DPPH radical scavenging property.

Chapter 4 deals about introduction of biological activities and all the protocols used to determine the inhibitory potential of all fractions and synthesized compounds.

Urdu Kholasa

CHAPTER-1 Phytochemical Studies on the Chemical Constituents of Xanthium strumarium Linn.

1.0. Introduction

General Introduction The chemical compounds which derived from living organisms (plants, animals, insects, and microorganisms) are known as natural products. From the early history, human beings have been attracted in natural products for their basic needs (food, shelter, clothing and not least medicines). Plants have formed the basis of traditional medicine system for thousands of years [1] Our holy religion ISLAM has superb contribution to the development of therapy based on the doctrines of AL QURAN and SUNNAH. These two define the rules for hygiene and sound diet as TIBB AL NABI. The medicinal properties of date and honey have been described in HOLY QURAN where as our beloved prophet HAZART MUHAMMAD (sallallahu alaiyhi wassallam) quoted along with other plants the advantages of nigella sativa and crotalaria juncea [2]. The compounds isolated from plants and even insects had been used in traditional medicine as hypolipidemic, antiplatelet, antitumor, or immune- stimulating agents or for the treatment of cardiovascular diseases and cancers, for thousand of years in China, India, Egypt, and Greece. Many phytochemists have been formulated healing creams and liniments from plant extracts also. Several isolated phytochemicals have antimicrobial activity and can also either inhibit or stimulate the activity of certain enzymes. The volatile essential oils isolated from herbs and spices suppress cholesterol synthesis and tumor growth [3-14]. This plant-based medication is continuously playing an essential role in health care. We cannot ignore the importance of plant-derived drugs such as artemisinin (a sesquiterpene), plant derived alkaloids like morphine, quinine, digitalis, atropine, reserpine, vincristine, vinblastine, ajmaline and taxol (a diterpene) for the treatment of malaria, typhoid, hypertension, heart problems, and certain kinds of cancer [3-14]. As herbal medicine is based on the principle that plants contain natural substances that can uphold health and lighten illness with no or less side effects, people are usually interest in the use of herbal remedies over life- threatening medicines. Due to excess applications, the natural products have gained major importance in various aspects of human venture especially in health care [3-14].

It has been predicted by the World Health Organization that about 80% of the world population trust on traditional medicines for their basic health care. It is apparent in different countries of Asia where phytomedicine is generally under practice. In short, plants have a significant role in caring human health, improving their quality of life and also serving humans well with valuable components of seasonings, beverages, cosmetics and dyes [15]. The physiologically active constituents of medicinal plants have been studying right through from the development of organic chemistry. It has been estimated that about 40% of drugs have natural origin. A number of screening protocols are applied for bioactivity of compounds to led new drugs. Natural compounds also show an ecological role in maintaining interaction between plants, microorganisms, insects and animals. They may be defensive, attractants and pheromones. Chemotaxonomy is another motive for examining the plant constituents. Phytochemical assessment can reveal natural products as a “markers” for botanical and evolutionary relationships [16]. Despite the fact that thousands of natural compounds have been isolated, only less than 100 of them with defined structure are in use all over the world. Actually the plant material comprises of many chemical components that have therapeutic effects. These compounds are used for remedy from various ailments. In view of the above mentioned deliberations, organized efforts have been done by many researchers in subcontinent Indo-Pakistan to study pharmaco- chemical properties of physiologically active plant components. Purity of isolated compounds is highly necessary to investigate their structures, formation, uses and purpose. Researchers in this respect have been fully facilitated by modern physical methods of isolation and spectroscopic techniques for structural studies. Particular concentration is given to determine structure and activity correlation in physiologically active compounds. These studies have served two purposes, first to bring new medicines to facilitate theraputic leads and secondly to synthesise drugs via viable high yield route. Moreover the functional variations in the basic skeleton of drug have brought to enhance its bioactivity [17]. Taking these facts into account, the work presented in chapter-1 of the present doctoral dissertation entitled “Phytochemical Studies on the Chemical

Constituents of Xanthium strumarium Linn” is related to the brief introduction about family and genus, review of earlier researchers on the chemical constituents, their pharmacological significance and uses in indigenous system of medicine. This is followed by a brief discussion of present work with reference to the isolation and structure elucidation of isolated 8 known compounds, one of which was isolated for the first time from this genus. Further the anti-bacterial, anti- fungal activity along with their MIC values and anti-oxidant activity with EC50 of different fractions of methanolic extract is also included.

References

[1] K. T. Farrell, “Spices, condiments and seasonings”, C. T. Westport, AVI Publishing Company, p. 17 (1985). [2] Imam Shamsuddin Muhammad Bin Abi Bkr Ibnulqasim Aljozia, “Tib-e- Nabwi (S.A.W.W.)” Hakim Aziz ur Rehman Aazami (translator), Muktaba Muhammadia, Pakistan. www.kitabosunnat.com. [3] T. Larkin, FDA Consum, 17, 4 (1983). [4] T. G. Saxe, Am. Fam. Physician, 35, 135 (1987). [5] I. Nielsen and R. S. Pederson, Lancet, 1, 1305, (1984). [6] N. Mostefa-Kara, A. Pauwels, E. Pines, M. Biour and V. G. Levy, Lancet, 340, 674 (1992). [7] T. Y. K.Chan, J.C.N. Chan, B. Tanlinson and J.A. Critchley, Lancet, 342, 1532 (1993). [8] Encyclopedia Britannica, Halen Hemingway Benton, 9, 1043 (1974) . [9] J. Bruneton, in “Pharmacognosy, phytochemistry, medicinal plants”. Hatton, C. K., translator. Paris: Lavoisier Publishers (1995). [10] G. M.Cragg, S. A. Schepartz, M.Suffness and M. R. Grever, J. Nat. Prod., 56, 1657 (1993). [11] W.A. Niering and N. C. Olmstead, in “The Audubon Society field guide to North American wildflowers, eastern region”. Knopf, A. A., eds., New York (1979). [12] J. Mann, “Murder, Magic and Medicine”, Oxford University Press (1994). [13] K. McNutt, Nutr. Today, 30, 218 (1995). [14] M.J. Dew, B.K. Evans and J. Rhodes, Br. J. Clin. Pract, 38, 394 (1984). [15] G. Bodeker and F. Kronenberg, Am J Public Health, 92(10), 1582 (2002) [16] D.M. Eisenberg, R.C. Kessler, C. Foster, F.E. Norlock, D.R. Calkins and T. L. Delbanco, N. Engl. J. Med., 328, 246 (1993).

[17] http://www.fas.org/nuke/guide/pakistan/contractor/hejric.htm

Xanthium strumarium Linn.

1.1.1. Classification*

Kingdom------Plantae (plants) Subkingdom ------Tracheobionta (vascular plants) Super division ------Spermatophyta (seed plants) Division ------Magnoliophyta (flowering plants) Class ------Magnoliopsida (dicotyledons) Subclass ------Asteridae Order ------Asterales Family ------Asteraceae (aster family) or Compositeae Genus ------Xanthium L. (cocklebur) Species ------Xanthium strumarium Linn Common name ------Chotagokhru (rough cocklebur) Trade name ------Cocklebur or Burweed

* Natural Resources Conservation Service United State Department of Agriculture (USDA)

1.1.2. Introduction About Family Compositeae

The plant Xanthium strumarium Linn, commonly known as Chotagokhru or Chotadhatura in Hindi, the trade name is Cocklebur or Bur-weed, belongs to the sunflower family, commonly known as Compositeae. It is so called due to the composite flowers on a capitulum, which is their main feature [1]. The compositeae family members resemble to ‘stars’ in appearance, so these are also known as Asteraceae, the name comes from Greek term means ‘star’ [2]. The compositeae is the largest family of the angiosperms (flowering plants). It comprises of about 950 genera and more than 20,000 species. The members of this family are distributed worldwide in each and every possible habitat. They are usually found in tropical and in cold arctic alpine area in waste places and along river banks. They show every form of body known for plants, in Indo-pak the family is represented by many genera such as chrysanthemum, helianthus, xanthium, zinnia and others [3]. The plants in compositeae are herbs, shrubs and few of the stature of trees. Climbers are also reported. Some of the members are xerophytes and hydrophytes. The root is tapped, branched, sometimes tuberously thickened to store reserved food (e.g., Helianthus tuberosus). Stem is soft, erect, woody, very rarely climbing. Most of the plants possess milky or watery, bitter, resinous juice. The leaves are radical or alternate, simple or compound, of various shapes, hairy, rough surface exstipulate, some of them contain oil ducts and latex (e.g., sonchus). The inflorescence racemose comprises of a capitulum with many sessile flowers, gathered on a common raceptical surrounded by an involucres of bracts. The florates may be bisexual, unisexual or asexual. The flowers head may be ligulate, zygomorphic, actinomorphic and epigamous. Disc floret is usually unisexual while ray floret is either female or sterile. Calyx rudimentary, modified to scaly, hairy pappus which help in dispersal of fruit. Corolla five, gamopetalous, of various colours, sometime swollen due to presence of nectar. Androecium composed of five stamens, epipetalous, free and separate filaments, anthers are united to a tube (syngenecious), two- celled and superior. Gynoecium two- carpelled, inferior ovary with one anatropus basal ovule, style simple, stigma two,

bifid with many hairs. Fruit is cypsela (single seed fruit). Seed is exalbuminous with straight embryo [1, 3]. Phytochemical studies of its members have revealed the presence of a number of class of compounds including terpenoids, flavonoids, alkaloids, steroids, coumarines, quinones, saponines, glycosides, volatile oils and amino acids [4, 5, 6]. The extracts as well as compounds isolated from the members of compositae possess pharmacological activities such as anti-HIV, hepatoprotective, cytotoxic [4], hypoglycemic [5], analgesic, anti-inflammatory [7], anti-bacterial [8], antioxidant [9], anti-cancer, anti-malarial and insecticidal activities [10], cytotoxicity towards human cancer line cells [11], antifungal activity [12].

1.1.2.1. Economic Value of Family Compositeae The family Asteraceae has wide spread use in society. Many members of the family are ornamental plants for their flowers such as chicory, chrysanthemum, dandelion, daisy, dahlia, marigold, santolina, solidago and zinnia. Some plants are used as food like artichoke, endive, lettuce, sasifi in addition of safflower, sunflower and niger seeds which are good sources of oil. Most of the plants of compositeae are medically important, for example, artemisia (anti-malarial), calendula (wound healing, anti-spasmodic), echinacea (medicinal tea), chamomile (herbal tea). The family members of this family have been used for industrial use like pulicaria and tanacetum species (insecticides), tagetes (orange dye), parthenium (rubber), bertoni (sweetner), marigold oil (alcoholic beverage flavoring and cigarette) and wild silver oak and wild camphor ( building material) [13, 14].

1.1.3. Introduction About Genus Xanthium

The genus Xanthium belongs to tribe Ambrosieae of family Compositeae [15]. The members of this genus are annual, tall, branched, coarse and rough herbs. Their leaves are alternate, rough, palmately dentate. Head monoecious, axillary, male more apical, sterile, numerous flowers with tubular, 5-toothed corollas, cylindrical scaly receptical, short involucres, style unarmed and dilated slightly at the apex, achenes rudimentary; female fetile, apetalous, 2-flowered, involucral bracts, herbaceous, 2-celled utricle, corolla absent. Achenes enclosed in enlarged involucres, style branched, pappus absent, anthers and filament free and distinct [16, 17, 18, 19]. The members of this genus are distributed in nearly all tropical and temperate regions of the world [20]. The genus Xanthium is represented by 25 speceis, all are of American origin [21]. Out of these X. macrocarpum, X. strumarium, X. spinosum, and X. sibiricum are also found in Pakistan and India [16, 17, 22]. The species of Xanthium have been used as traditional herbal medicine in oriental countries for the treatment of nasal sinusitis, headache, urticaria, arthritis [23, 24], fever, scrofula, herpes and cancer [25, 26]. X. spinosum Linn. and X. strumarium Linn. are of medicinal use in Europe, North America and Brazil, X. canadense Mill is used in North America and Brazil, X. strumarium is medicinally important in China, India and Malaysia [27, 28].

1.1.4. General Description of Xanthium strumarium Linn.

Xanthium strumarium Linn, a member of Compositeae family, is an annual, erect, rough, and coarse herb or under shrub, near about 1.5 m tall [29]. Xanthium is the derivative of Greek word “xanthos” which means yellow and strumarium refers “cushion like swelling” because the seedpods when ripen turn from green to yellow. The reason behind its common name chotagokhru is the shape of its fruit that resemble the cow’s toe (chota- small; go-cow; khuru-toe) [28]. The weed probably of American origin and found in almost all of the hotter parts of Eurasia including up to the height of 5000 ft. in the Himalayas region [30]. It is a common roadside weed found abundantly in plain to 8000 ft. in almost all the four provinces of Pakistan and in Kashmir [31, 32]. The stem is hispid, slightly branched, erect, stout, rough and hairy. The leaves are three lobed, toothed, triangular heart shaped, broad, glandular with a long leaf stalk. Capitulum in terminal and axillary recemes, monoeciou, sessile having numerous white or green, male and female flowers on separate heads of the same plant. Sterile (Male) heads uppermost on stem with many flowers which are globose, hooked bristle, 5-toothed, tubular corolla. Fertile (Female) heads axillary, 2-flowered having no pappus and corolla. Fruits are involucres, ovoid or oblong, covered with hooked bristles, diverging ending, two strong and hooked beaks, hard and 2-celled. Achenes ovoid enclosed in a thick, tough and hard bracts covered with hooked spines [29, 32, 33, 34]. Flowering and fruiting period ranges from July to October i.e. after rainy season [33, 35]. The weed is propagated by seed [29, 26, 36].

1.1.4.1. Chemical Constituents of Xanthium strumarium Linn. The herb analysis reveals beneficial values for crude protein 17.6% ; carbohydrate 31.6% ; cellulose 12.3% ; lignin 12.5% ; nitrogen 2.82% ; phosphorous 1.13% ; potassium 2.42% ; calcium 3.15%; magnesium 1.40% ; sodium 0.47% and sulphur 0.61% [37]. The weed is cooked as vegetable in China while in Assam the floral tops and the leaves just below it are edible after boiling [28, 37, 38]. The aerial parts contain a mixture of alkaloids xanthinin, xanthatin [36], xanthumin,

xanthalol and isoxanthalol [37]. Stem is a source of fuel [37]. Leaves possesses compounds from class of phytosterols, sesquiterpenes, sesquiterpene lactones like β-sitosterol, isoxanthanol along with xanthinin and xanthumin, d-limonene, d- carveol, l-α-ionone, ρ-cymene, β-caryophyllene, isohexacosane, chlorobutanol, stearyl alcohol, palmitic acid. Leaves are also rich in ascorbic acid and iodine [39]. Leaves contain a substance due to which they are applied to dye yellow [40]. Fruits have glucose, fructose, sucrose, β-sitosterol, γ-sitosterol, stigmasterol, strumaroside (glucoside of β-sitosterol), phosphatides, KNO3 and is a good source of vitamin C [37, 39]. The seeds contain oxalic acid, xanthostrumarine and its glucoside, iodine [33, 37], toxic agents hydroquinone and choline due to which they cause poisoning to livestock [37]. The seeds comprises of a semidrying oil of the same taste like different vegetable oils, it is not only edible but also of industrial application for soap, paints and as a drying agents [36, 41]. The seed oil is a combination of saturate fatty acids such as capric, lauric, myristic, palmitic acids and unsaturated fatty acids oleic, linoleic, stearic acids, behenic [37]. The cake obtained after extraction of oil is rapidly nitrifying hence may be good fertilizer [37, 41]. The powdered shell is utilized to make activated carbon [41]. The roots comprises of phytosterols, n-heptacosanol, 3, 4-dihydroxycinnamic acid [39]. The whole plant can accumulate trace elements (Na, K, Ca, Cl) [42] and heavy metals (Cd, Cr, Cu, Mn, Ni, Pd) in different concentrations in plant body parts [43, 44].

1.1.4.2. Medicinal Importance of Xanthium strumarium Linn. The whole plant is of great importance in medicinal uses, the roots, leaves, fruits and seed oil all parts are utilized in herbal medications in China, India, Malaysia [36]. Even though the weed is supposed to be poisonous, the harmful and toxic substances are washed off by washing and boiling [37]. The herb is astringent, diaphoretic, diuretic, emollient, sialogogue, sudorific [33]. It is used to treat mouth ulcer and toothache [32]. The aerial parts composed of substances that exhibit anti-malarial activity [29], xanthinin is an anti-bacterial agent, xanthumin is a CNS depressant and also shows anti-bacterial activity [29, 37], xanthatin isolated from resin is a cytotoxic compound [39] and a hypoglycemic agent,

carboxyatractyloside exists in the weed [29]. The leaves exhibit characteristic properties of astringent, antisyphilitic, diuretic [37]. Their decoction is useful in scrofula, herpes and venereal sores [39, 41], an extract of leaves with honey is recommended in cough and fever [29], pounded leaves are applied on ulcer and for the remedy of snake bite [40, 45], their black pepper and sugar juice is suggested to cure blood dysentery [45], cocklebur tea has been used to cure fever [40]. The fruit contains glucoside of β-sitosterol which shows anti-inflammatory activity. It is given for the relief from kidney pain, scurvy, malaria, small-pox, recommended for hormonal regulation, for the treatment of urinogenital problems [41, 45]. The fruit is demulcent and cooling, its ash is applied on sores on lips and mucous membrane of mouth [33, 37]. The seed composed of anti-inflammatory agents hence are used to treat inflammatory swelling [37], seed oil is used to treat bladder infections, herpes, erysipelas, its massage beneficial in rheumatism, seed paste is useful on wounds [40, 45]. The root gives a bitter tonic which is used against cancer and scrofula [29]. It possesses anti-tumor and abortifacient properties [39], their extract is applied for the treatment of ulcer, boils, abscesses, pulmonary disorders [41], a decoction prepared with sugar is better for diarrhea [45].

1.1.4.3. Literature Review

In the year 1920, B.R. Leland observed the cold-pressed oil do not rancid after 6 months when keep in a cold dark place, he also determined 30.69% of kernel of burs of X. strumarium Linn [46]. A. Sado in 1937, found the aqueous extract of stem and leaves of X. struarium L. contained a substance which is weakly toxic to nerves and muscles [47]. I. C. Chopra and co-workers reported the physiologically inactivity of soluble glucoside of said species in 1946 [48]. Isamu Numato in 1951 obtained 115 – 131 mg % ascorbic acid (vit. C ) from leaves of X. strumarium L. in addition of other weeds [49]. In the same year, C.V.N. Rao reported pet.ether extract analysis of seeds of X. strumarium L. composed of oil, 31.5%; crude protein, 29.5%; crude fiber, 10.4%; carbohydrate, 22.8% and ash, 5.8% [50]. In 1953 R.C. Shrivastava suggested a method to remove hard covering of X. strumarium L. seed, also calculated its cake value , water, 7.93% ; minerals, 7.06 %; fiber, 2.46 % ; and carbohydrate, 22.11% [51]. In 1954 C.V.N. Rao with his fellow P.D. Kebra analysed seeds composition of the specie by Mc Cool Pulveriser, found occurance of oleic acid, linoleic acid and isolinoleic acid [52]. E.F. Leonova and co-workers in 1957 determined the presence of iodine in good amount in all parts of the said plant [53]. J.R. Plourde and J.A. Mockle in 1960 reported the pet.ether fraction of the fruit and leaves of Cocklebur composed of viscous oil, lactone and a flavones derivative. The lactone, C17H22O5, m.p. 121-122º, hydrogenolysed to C15H24O3 and upon deacetylation gives C15H18O3 [54]. D.S. Bhakuni and co-workers in 1961 investigate pet. ether fraction of fruits of Cocklebur for fattyacids. They found palmitic, stearic, linoleic and oleic acids whereas β-sitosterol, γ-sitosterol, ϵ-sitosterol and ceryl alcohol from unsaponified portion. From acetone fraction isolated strumaroside, m.p. 290-292º in addition of glucose, fructose, amino acid and aqueous extract contained sucrose, aspartic, fumaric, succinic and tartaric acids [55].

Itsuo Nishioka identified stigmasterol by gas chromatography of methanol extract of fruit of X. strumarium L. in 1965 [56]. In the year 1966, T. Takeo and his group identified a mixture of phytosterols, stigmasterol, campesterol and β-sitosterol in the methanolic extract of X. strumarium seeds [57]. In the same year Hitoshi Minato and Isao Horibe found a stereoisomer of xanthinin, which is a sesquiterpene lactone from the aerial parts of X. strumarium L. They suggested it xanthumin after elucidating the structure by NMR spectroscopy and derivatisation to iodoform , m.p. 100- 101º with molecular formula C17H22O5 [58]. M.M. Pashchenko and G.P. Pivenko, in the same year, isolated xanthatin from chloroform extract of aerial parts of Burweed, in addition to this, they also obtained a sesquiterpene lactone, xanthinin, m.p. 110- 120°, turned red in HCl and yellow in base from Et2O extract of powdered cocklebur [59, 60]. The same author in the year 1967 reported a flavonoid, m.p. 209-211° as 8-(Δ3- isopentenyl)- 5, 7, 3', 4'- tetrahydroxy flavone from ethanol extract of Bur weed [61]. Within the same year, Hitoshi Minato identified, xanthumin from acetone fraction of aerial parts of cocklebur [62]. S.M. Khafagy and fellows isolated xanthinin, choline and two new lactones, xanthanol and isoxanthanol from X. strumarium L. in 1974 [63]. In 1975 C. Mc Millan and group determined variant combinations of sesquiterpene lactones including xanthinin, xanthanol, xanthatin, xanthumin, xanthumanol, deactoxyxanthumin, xanthinosin and tomentosin in a wild population of X. strumarium [64]. A.S. Chawla and fellows in 1976 studied pet.ether soluble component of fruit of bur weed contained n-alkanes, n-alkanols and a mixture of sitosterol, stigmasterol and campesterol [65]. In the same year, S.M. Khafagy and group studied different fractions of the same specie they found xanthinin, from non- saponifible portion of petroleum extract a triterpene alcohol, m. p. 211- 213°, C30H50O2, a crystalline triterpene alcohol,

C30H50O5, m. p. 274- 276° and a phytosterol C28H48O2, m.p. 146-148° [66].

Xanthatin

N.P.S. Bisht and R. Singh in 1977 isolated from the leaves of X. strumarium palmitic acid, β-sitosterol, ϵ- sitosterol, stearyl acohol, isohexacosane in addition of chlorobutanol which was obtained first time from a natural source [67]. In 1978 same authors found oleic acid and a C-24 epimer of stigmasterol by the techniques of IR, NMR and Mass spectroscopy as poriferasterol from pet.ether extract. They also reported 3, 4-dihydroxycinnamic acid and β- sitosterol- D- glucoside from its leaves extract [68]. In the same year R.G. Patel and V.S. Patel observed the seed oil of the burweed on heating at 280 for 8 hr under CO2 polymerised and became so viscous that can be used as drying oil suitable for coating [69]. P. Debetto in 1978 discussed with 12 references the presence of atractyloside in X. strumarium along with in other plants [70]. N.P.S. Bisht and R. Singh in the same year isolated β-sitosterol, β-sitosterol-D- glucose, stigmasterol, heptacosanol, 3, 4-dihydroxycinnamic acid, KNO3 and

K2SO4 from the extract of stem of cocklebur [71]. The group of J.R. Cole in 1980 isolated and identified carboxyatractyloside. The structure of the compound was determined by spectroscopic analysis and a chemical identity with an authentic sample [72]. In 1982 E. Naidenova and companions isolated xanthatin from aqueous extract of leaves of X. strumarium in addition of two sesquiterpene lactones [73]. In the same year H.G. Cutler and J.P. Cole identified carboxyatractyloside as a plant growth inhibitor, isolated from the said specie [74]. In 1985 A. Harada and co-workers isolated xanthatin from burweed and found it as an anti-attaching repellent against blue mussel [75].

J. Molina-Torres and fellows observed in 1991 the accumulation of α-tocopherol is associated with the life of leaf while γ-tocopherol present throughout the age of leaf of X. strumarium Linn [76]. Agata Isao and co-workers in 1993 from fruit of X. strumarium 3,5-di- O- caffeoylquinic acid accompanied by a new polyphenol, 1,3,5- tri-O-caffeoylquinic acid [77]. M. S. Malik and fellows in same year reported a new sesquitrpene lactone from the extract of aerial parts of the same plant as 2-hydroxytomentosin -1β, 5β – epoxide [78]. In 1994 R. P. Rao analyzed the seed of X. strumarium as a good source of protein and fat [79].

V.K. Saxena and fellows in 1994 isolated β–sitosterol, stigmasterol, β-amyrin and octacosanol and a new xanthanolide, 6β,9β,-dihydroxy-8-epixanthatin from leaves extract of cocklebur [80]. J.A. Marco and companions reported xanthanol, isoxanthanol and their C-4 epimer from burweed extract within the same year [81]. V.K. Saxena and M. Mishra in 1995 identified a new xanthanolide, chloroxanthanolide in addition to 8-epi-xanthatin-1β, 5β-epoxide, 2- hydroxytomentosin and xanthumanol from the extract of same species [82]. P.S Joshi and co-workers in 1997 reported 4 active xanthanolides from acetone extract of X. strumarium, tomentosin, xanthumin, 8-epi-xanthatin and 8-epi- xanthatin-1β, 5β-epoxide [83]. A. Ahmed Mahmood et al. in 1999 reported xanthanolid and xanthane type sesquiterpenoid from cocklebur, identified by high resolution 1D and 2D NMR and NOE experiments as 11α, 13-dihydroxanthatin, 4β, 5β-epoxy xanthatin-1α, 4α-endoperoxide and 1β, 4β, 4α, 5α-diepoxyxanth-11(13)-en-12-oic acid. Also identified 11α, 13-dihydroxyxanthatin, a new xanthanolide diol using COSY NMR analysis along with other techniques [84, 85]. M. Kanauchi et. al in the same year with the help of IR, UV and 1H-NMR determined the structure of xanthatin, M.W. 246, m.p. 60º. The compound was acetone soluble and possesses anti-bacterial activity [86]. UI chenko, N.T. in 2000 using first time chromatography and spectral methods, analysed neutral lipids, normal and hydroxylated fatty acids and lipophilic components from seeds of X. strumarium linn. [87]. In 2003 Kim Young and co-workers obtained 8-epi-xanthatin and 8-epi-xanthatin epoxide from the leaves of cocklebur [88]. In the same year S. Shuenn-Jyi and fellows by using HPLC and electrophoretic methods reported the presence of Potassium 3-O- caffeoylqduinate and 7- hydoxymethyl-8, 8-dimethyl-4, 8-dihydrobenzo [1, 4] thiazine-3, 5-dione in X. strumarium linn. [89]. In 2003 B.B.S. Kapoor and companions isolated kaempferol and quercetin as a major flavonoid from ethanol fraction of extract of leaves and flower of cocklebur [90].

M.S. Kumar and co-workers in 2006 found stigmasterol, they also isolated very first time stigmasterol-3-O-β-D-glucopyranoside and 2-methylanthraquinone from methanolic extract of roots of the plant. Their structure was determined by detailed spectral studies [91].

O HO O O

1,4,4,5-diepoxyxanth-11(13)-en-12oic acid

In the same year W. Zwolan and fellows isolated phenolic acids from vegetative and generative parts of X. strumarium via HPLC technique. They identified them as caffeic, gallic, protocatechuic, vanillic, syringic and ferulic acid [92]. Han-Ting and co- workers in 2006 obtained xanthiazone, chlorogenic acid, ferulic acid, fermononetin and ononin from fruit extract of cocklebur along with two new thiazinediones whose structures were determined to be 7-hydroxymethyl-8,8- dimethyl-4,8-dihydrobenzol[1,4]thiazine-3,5-dione-11-O-β-D-glucopyranoside and 2-hydroxy-7-hydroxymethyl-8,8-dimethyl-4,8-dihydrobenzol[1,4]thiazine- 3,5-dione-11-β-D- glucopyranoside [93]. R.N. Yadava and group in same year isolated from leaves of X. strumarium a novel triterpenoidsaponin, 3-O-[α-L-rhamnopyranosyl-(1-3)-O-β-D-xylopyranosyl] manitadiol. The structural determination was done by various spectral analysis and chemical degradations [94]. In the same year E. Akbar and group analyzed essential oil obtained through hydrodistillation of X. strumarium stem and leaves via GC and GC/MS contained bornyl acetate, limonene and β-selinene [95]. R.E. Irving and fellows in 2008 reported first time xanthatin and xanthinosin from the burs of cocklebur [96]. T. Han and group in same year found caffeoylquinic acid by HPLC method from n- butanol fraction of the plant [97]. C-L Lin and fellows in 2008 found xanthialdehyde and (-)-xanthienopyrane from stirr fried seed extract of X. strumarium. The structure was elucidated by spectroscopic methods [98]. In same year a review study by D. Ying-Hui and others for chemical constituents found xanthanolide, kaurene glycoside and essential oils are prime components of xanthium species [99]. V. Kumar and G.S. Rawat in 2008 estimated nitrogen and protein in great amount in X. strumarium [100]. D.P. Pandey and M.A. Rather in 2012 found in ethyl acetate and methanolic extracts of air dried, powdered plant over silica gel and sephadex LH20, caffeic acid, xanthiazone and xanthiazone-(2-O-caffeoyl)-β-D-glucopyranoside. They

used 1D and 2D- NMR, Mass, UV and IR spectroscopy and chemical methods for structural elucidation [22]. Guleryuz and fellows determined heavy metals Cr, Cu, Mn, Ni and Zn accumulation in different concentrations in X. strumarium Linn. in 2008 and considered suitable for growing in industrially polluted regions as potential plant species for cleaning heavy metals from contaminated soil and remediation of polluted areas [101].

Table – 1: Constituents of Xanthium strumarium Linn.

S. M.P Name Parts References No ºC M.F. 1 β-amyrin L 80 2 2-methylanthraquinone R 91

3 Ascorbic acid 190 C6H8O6 L 100 4 Aspargine F 55 5 Aspartic acid F 55

6 Bornyl acetate C12H20O2 L 95 7 Borneol L,St 95

8 3,4-dihydroxybenzalde- C7H6O3 F 113,114 hyde 9 4-oxo-bedfordia acid A.P 84 10 Caffeoylquinic acid B 24

11 Caffeic acid 223 C9H8O4 F 22,24,109 12 Chlorogenic acid F 23,92 13 Pot.-3-O- F 24,89 Caffeoylquinate 14 1,5-di-O- F 24 Caffeoylquinic acid

15 1,3,5-tri-O- C34H30O15 F 24 Caffeoylquinic acid

16 Carboxyatractyloside C31H44O8S2K2 B 70,72,74,103

17 Chalcone derivatives 170 C16H14O4 St 68 18 Campesterol F 57,65

19 Chlorobutanol 96-97 C4H7Cl3O L 67

20 3,4-dihydroxy cinnamic 196-197 C9H9O4 L 68,71,92,109 acid

21 Ceryl alcohol F 55 22 Cephalins F 55 23 Fumaric acid F 55 24 Fructose F 55 25 8-(Δ3-isopentenyl) 5,7, 61 3’, 4’-tetrahydroxy-

flavone 26 Formononetin F 23 27 Ferulic acid F 23,92 28 Gallic acid A.P 92 29 Glycine F 55

30 Glucose 148 C6H12O6 F 55 31 Heptacosanol 74 St 71

32 Isohexacosane 60-61 C26H54 L 67

33 4-O-dihydroinusonio- C15H22O3 A.P 81 lide 34 Kaempferol 90

35 Limonene C10H16 L, St 95

36 Linoleic acid 229(BP) C18H32O2 F,S 52,55 37 Lecithins F 55 38 Leucine F 55

39 Linolenic acid 137(BP) C18H32O2 S 52 40 Malic acid F 55 41 Ononin F 23 42 Octacosanol L 80

43 Oleic acid 11-12 C18H34O2 F 52,55

44 Palmitic acid 61-62 C16H32O2 F,L 55,67 45 Protocatechuic acid 92 46 Phenylalanine F 55

47 Quercetin 90 48 3-O[α-L-rhamnopyrano- L 94 syl-O-β-D- xylopyranosyl] maniladiol

49 Є-Sitosterol 144 C29H50O F,L 67

50 β- Sitosterol 137 C29H50O S,F,L 57,65,67,71,80

51 γ- Sitosterol 158 C28H48O F 55,56

52 Stigmasterol 167 C29H48O S,F,L 57,65,71,80,91

53 Strumaroside 290-2 C35H60O5.H2O F 55 54 Sucrose F 55 55 Succinic acid F 55

56 Stearic acid 71-72 C18H36O2 F 55

57 Stearyl alcohol 59-60 C18H38O L 67

58 β- Sitosterol-D- 290 C35H66O6 St,L 68,71 glucoside 59 Stigmasterol-3-O-β-D- R 91 glucopyranoside 60 β-Selinene L,St 95 61 Syringic acid 92 62 Tomentosin A.P 83

63 Triterpene alcohol 211 C30H50O2 66

64 Triterpene alcohol 274 C30H50O5 66 65 2-hydroxy tomentosin- A.P 78 1β,5β-epoxide 66 Vanillic acid 92

67 Thiazenedione 186 C26H30O11NS F 93 68 8-epi-tomentosin L 108

69 2-Hydroxytomentosin C15H20O4 L 82

70 Xanthialdehyde 156-158 C11H11NO3S S 98

71 (-)-Xanthienopyran 218-220 C17H16O4S S 98 72 Xanthatin 113-115 B,L,F 86,96,108,75, 73,60,59 73 Xanthinosin B 96 74 epi-xanthatin R,L 88

75 8-epi-xanthatin epoxide C15H20O4 L,A.P 82,83,88

76 11α-13-dihydroxy C15H20O3 A.P 84 xanthatin

77 4β,5β-epoxy-xanthatin- C15H18O5 A.P 84 1α,4α-endoperoxide

78 1β,4β,4α,5α-diepoxy C15H22O4 A.P 84 xanth-11(13)-en-12-oic acid

79 Xanthiazone 159 C11H13O3SN A.P 23,24 80 8-epi-xanthatin 71-72 A.P,L 83,108

81 Xanthumin 100-101 C17H22O5 A.P,L 58,62,83,104 82 Xanthanol L 15,81,85,105 83 Iso-xanthanol A.P,L 81,15 84 6β,9β-dihydroxy-8-epi- L 80 xanthatin 86 Xanthumanol A.P 82

87 Xanthinin 123 C17H22O5 A.P,L 58,62

88 15-Chloro-2-epi- C17H23O5Cl A.P 82 xanthanol

89 K2SO4 St 71 90 KCl L 71

91 KNO3 A.P 55,71

92 CaSO4 A.P 55

A.P= Aerial part, B=Burs, F=Fruit, L=Leaves, S=Seed, St=Stem, R=Root, M.P=Melting point

1.1.4.3.1 Pharmacological activity

The toxicity of Xanthium strumarium Linn. took attention of many researcher, hence A. Sado in 1937, analyzed a substance weakly toxic to nerves and muscles, from the aqueous extract of stem and leaves of X. strumarium Linn [47]. Presence of physiologically inactive soluble glucoside in Xanthium strumarium Linn was found by I.C. Chopra and co-workers in 1946 [48]. In 1957, E.F. Leonova studied pharmacology on the basis of infusion results of leaves, stalk and seed tincture injection of X. strumarium, the leaves infusion increases peristalsis in rabbit intestine, caused cardiac blockade in frog heart, dilation of blood vessels of rabbit ear, results dilation following constriction of frog hind leg and a reduction of blood pressure by 20-40 mmHg, depressed stimulation of spinal cord when intravenously injected to cats whereas respiratory movements intensified in frog by the tincture of seed [53].

H. Minato in 1967 reported a CNS depressant agent, xanthumin, M.F C17H22O5, m.p.100.5-101º from acetone extract of aerial parts of cocklebur [62]. In 1980 the group of J.R. Cole identified carboxyatractyloside as a highly toxic substance, responsible for the poisoning character of cocklebur [72]. In 1982 E. Naidenova with companions communicated pharmacology of derivatised components. They isolated sesquiterpene lactone after treating aqueous extract of leaves and root of cocklebur at 80º for 30 min. following precipitation with lead acetate, chloroform washing, drying organic layer with sodium sulphate and removal of solvent under vacuum distillation. The residue was dissolved in aliquot of water. Under 284 nm of spectrophotometer and other techniques the compound was identified as xanthatin, its derivatives with hexamethyleneimine and morpholine revealed anti-tumor activity when tested with leukosis L- R 10 [73]. A. Harada et al. (1985) determined an anti- attaching repellent, xanthatin, from cocklebur extract, the isolated compound revealed strong repellent property and weak toxicity against Mytilus edulis (blue mussel) [75]. In 2007 R.N. Yadava and J. Jitendra obtained a novel anti-inflammatory active triterpene saponin from leaves of X. strumarium. The structure elucidation by

different spectral analysis identified it as, 3-O[α-L-rhamnopyranosyl-(1-3)-O-β-D- xylopyranosyl] manitadiol [94]. T. Han with his group in 2008 studied the polarity based ethanol extract fractions of X. strumarium amongst them the n-butanol was the most polar, exhibiting highest anti-inflammatory property in the test of croton oil induced edema and also possesses strong analgesic effect as it decreases writhing in mice. They also obtained 10 caffeoylquinic acid and decided them the cause of polarity of plant extract [21]. C-L Lee and coworkers in 2008 isolated xanthaialdehyde and (-) - xanthienopyrane from chloroform extract of seed of cocklebur. They found later one as a superoxide anion generation inhibitor produced by activated neutrophils showing 1.72µg/ml, IC50 value [98]. Y.H. Dai and group in 2007, research on pharmacological properties of xanthium species. They analyzed xanthanolide, kaurene glycoside and essential oils as active principals for antibacterial, antioxidant, anti-inflammatory and antitumor activities of many species of xanthium [99]. A crystalline hypoglycemic agent was isolated by P.F. Kupiecki and fellows in 1974, from the seeds of cocklebur by boiling mashed seeds in water for 30 min. following acetone and ethanol fractionation, column chromatography and crystallization. They analyzed the compound composed of C, H, O and S, it was active towards rats at a given i.p or s.c [102]. In 1976 J.C. Craige and fellows identified a glycoside from the burs of cocklebur M.F. C31H48O24S2, m.p. 278- 279o, exhibited hypoglycemic activity when given in 1-5 mg/kg i.v. in lab animals. The compound was purified by producing its potassium salt, recrystallisation in water and confirmed by comparing TLC, IR, and 1H-NMR with a pure and authentic sample as a potassium carboxyatractylate [103]. In the year 2000 M. Kanauchi and fellows isolated antibacterial agent, a white crystalline compound mp. 60º, MW 246, from leaves extract of X. strumarium. They determined its structure as xanthatin by IR, UV and 1H-NMR techniques. The compound exhibited MIC value 25- 100 µg/ml against Candida species, Pichia species, Sacchromycopsis species and Torulaspora species whereas 12.5- 100 µg/ml MIC against Bacillus species, contaminator of Koji used in manufacturing of alcoholic beverages. They concluded xanthatin as an anti-

bacterial agent that can be used to prevent contamination of Koji while preparing beverages [86]. Z. Cui et al. (2007) reported antibacterial activity of xanthium species in their review research about chemical constituents in xanthium species [99]. C.K. Gupta and D.R. Gupta in 1975 analysed xanthumin as an antibacterial agent from bioactive guided fraction of pet.ether extract of leaves of Xanthium strumarium, it was active towards gram positive. The structure was elucidated by comparison of IR spectrum and Rf values with a reliable sample as C17H22O5, m.p. 100- 101º [104]. In 1988 A.I.M. Jawad determined the anti-microbial activity of MeOH extract of cocklebur against Proteus vulgaris, Staphylococcus aureus, Basccilus subtilis, Candila albicans and C. pseudotropicallis. They further reported the property was due to a sesquiterpene xanthanol isolated from ethyl acetate extract by PTLC

(CHCl3/ EtOAc, 1:1) exhibited similar antimicrobial activity as methanol extract [105]. V.K. Saxena assessed the medicinally important bactericidal activity of lipids of cocklebur in the year of 1990 [106]. H.S. Kim and co-workers in the year 1997 isolated compounds A and B from EtOAc extracts of same species. Both compounds were purified through reverse phase HPLC, stable at 120º but not in acidic and alkaline medium. When tested with bacteria, yeast and fungi exhibited growth inhibition against both gram +ve and gram –ve bacteria in agar diffusion method while in FDA method within esterase compound B inhibited the growth of bacteria and yeast whereas compound A inhibited the growth of bacteria only [107]. J-W Ahn and coworkers in 1995 observed cytotoxicity of MeOH extract of leaves of X. strumarium against human tumor cell line. They realized the property was due to the presence of α-methylene containing sesquiterpene xanthatin, 8-epi- xanthatin and 8-epi-tomentosin. They also determined the greater cytotoxicity of 8-epixanthatin in comparison of 8-epi- tomentosin was the result of conjugated enone moiety in 8-epi-xanthatin [108]. Taking cytotoxicity under consideration Y.S. Kim and co-workers in 2003 isolated two xanthanolide sesquiterpene lactones, 8-epi-xanthatin and 8-epi-xanthatinepoxide from leaves extract of cocklebur and investigated for their cytotoxic effect on A549(non-small lungs), SK-OV-3(ovary), SK-MEL-2(melanoma), XF498(CNS) and HCT-15(colon).

They found the compounds were inhibitors of proliferation of above cultured human tumor cells and also inhibit fernesylation of human L-amine-B by fernesyltransferase (FTase) in high dose [88]. Showing keen interest for in vitro cytotoxicity of medicinally important plants R.E. Irving and group in 2008 recognized two sesquiterpene lactones, xanthatin and xanthinosine from the chloroform extract of X. strumarium leaves as potent cytotoxic agents having IC50 values 0.1 to 6.2µg/ml for human cancer cell line ATCC (colon), MDA-MB-231 ATCC (breast) and NCI-417 (lungs). These compounds were reported for the first time in the burs of this specie [96]. In the year 1999 F.L. Hsu and group isolated caffeic acid from fruit extract of cocklebur. They analyzed the compound as an anti-hyperglycemic after a decrease in plasma glucose when applying on diabetic rates of streptozotocin induced and insulin- resistant model through intravenous injection of it. On the other hand the caffeic acid was inactive on non diabetic, normal specimen [109]. The essential oils of X. strumarium possess strong anthelmintic property. It was reported by A.K. Gharia and fellows in 2002 when they observed superior anthelmintic activity of essential oils of said plant against earthworm, tapeworm, hookworm and nodular worm [110]. Y-H Dai et al. reported in 2007 xanthanolides and kaurene glycosides as prime substances of X. strumarium that possesses antioxidant activity [99]. Next year (2008) R. Scherer and fellows by applying three concentrations of DPPH suggested a new antioxidant and index (AAI) for different organic acids, clove essential oil, eugenol and X. strumarium extract. They observed among them X. strumarium exhibited strong antioxidant activity with AAI= 1.6 [111]. In 2008 Y. Xu and companions identified X. strumarium Linn as a good source of preparing environment friendly and green pesticides because the specie contains such active substances which prevent pests, weed and pathogens growth on plants [112]. Y.S. Bae along with his group in 2009 recognised 3,4-dihydroxybenzaldehyde as an active component in X. strumarium extract which induces apoptosis of blood cancer cells by inhibiting the activity of casein kinase 2 [113].

In the same year B.H. Lee with his fellows isolated a CKII inhibitory compound from X. strumarium fruit. The structure elucidation via different spectroscopy techniques identified as 3,4-dihydroxybenzaldehyde. It inhibited CKII phosphotransferase activity of 783µM, IC50. They determined that 50% growth of human cancer cell U937 by the isolated compound was the result of breaking of poly (ADP- ribose) polymerase and procarpase-3. The inhibitor also showed triggered apoptosis by fragmentation of DNA. They used flow cytometry analysis to confirm all these apoptosis and further concluded that as the CKII takes part in cell proliferation and oncogenesis, 3, 4 dihydroxybenzaldehyde may inhibit these diseases by inhibiting the CKII activity [114]. S.P. Joshi and fellows in 1997 isolated four anti-malarial active xanthanolides from bioactive direct fraction of acetone extract of aerial parts of X. strumarium. They observed all compounds possesses anti-malarial activity against chloroquine resistant Plasmodium falciparum strain under IC50 values 7.8, 7.8, 31 and 125 µg/ml for tomentosin, 8-epi-xanthati-1β,5β- epoxide xanthumin and 8-epi- xanthatin respectively [83]. To evaluate this property, S. Chandel and coworkers in 2012 determined malarial remedy property of X. strumarium Linn. by testing its ethanolic extract of leaves for antiplasmodial activity in Plasmodium berghei infected BALB/c micr. A dose dependent oral administration(500 mg/kg/day) showed 88.6% chemosuppression during early days of infection that was greater than that of standard chloroquine drug where as 90.40% chemosuppression was observed upon taken 350 mg/kg/day concentration during repository infection comparable to pyrimethamine (92.91% chemosuppression). They found the survival of mice enhanced from 21 to 26 days when concentration used 250 and 350 mg/kg/day. On the other hand upon 150 mg/kg/day concentration sustain all mice to 29 days. In the last they concluded that the plant can be used for malarial remedy [115]. In 2012 F. Khuda and Z. Iqbal with their companions studied solvent fractions of X. strumarium for bioactivities. They found chloroform extract revealed insecticidal activity due to which the plant can be a good source of natural insecticide in comparison of permethrin. They also determined maximum

cytotoxicity for brine shrimps with mortality rate 93% at highest dose of n-butanol fraction hence can apply safely in traditional medicine [116]. H.N. Yoon and coworkers in 2013 determined bioactive components from fruit of X. strumarium Linn. which are inhibitor of aldose reductase (AR) and galactitol formation in rat lenses with high glucose level. Methyl -3, 5-di-O-caffeoylquinate is the most potent inhibitor having IC50 value 0.30 and 0.67 µM for rAR and recombinant human AR (rh AR) respectively. They further analysed that it is galactitol formation inhibitor in rat lens and in erythrocytes, as well as the effectiveness of weed in diabetic applications. They also isolated neochlorogenic acid methyl ester, 3-hydroxy-1-(4-hydroxy phenyl)-propan-1-one and raffinose from ethyl acetate fraction by reverse phase C-18 chromatography for the first time from X. strumarium [117].

1.2. Present Work

In view of pharmacological characteristics and medicinal uses attributed to Xanthium strumarium Linn., the present studies were done on the chemical constituents of air dried aerial parts of this plant. The methanolic extrac (XS-Me) was subjected to classical methods of separation followed by various chromatographic techniques such as column chromatography (CC), pencil CC, preparative thin layer chromatography (TLC) (detail in experimental). The investigation of this extract led to the isolation of eight known constituents, (1) lupenyl acetate, (2) stigmasterol, (3) β-sitosterol, (4) palmitic acid, (5) β-amyrin, (6) oleanolic acid, (7) β-sitosterol-3-O-β-D-glucopyranoside and (8) ferulic acid among these, compound (1) is reported for the first time from this plant. The structure of (1) has been determined with detailed spectral studies including IR, EIMS, HR-EIMS, 1H-NMR, 13C-NMR and 2D-NMR analysis where as other constituents were recognized by the comparison of their spectral data with those of literature reported values. The anti-bacterial, anti-fungal and anti-oxidant activities of crude methanolic extract (XS-Me) and its fractions (XS-HX, XS-DC, XS-EA and XS-BU) were analyzed in collaboration with Department of Microbiology, Federal Urdu University of Arts, Science and Technology, Karachi, Pakistan. All the fractions possess significant biological activities, however the ethyl acetate fraction (XS- EA) is the most active fraction as it exhibited moderate to high anti-bacterial and anti-fungal activity along with this it also showed good anti-oxidant activity.

1.3. Results and Discussion

1.3.1. Lupenyl acetate (1)

Compound (1) appeared as colourless needles. It showed a molecular ion peak at m/z 468 [M]+ in EIMS and 468.3970 in HR-EIMS spectrum respectively corresponding to the molecular formula C32 H52O2. Other peaks in EIMS spectrum at m/z 453 and 408 were obtained by the loss of CH3 and CH3COOH where as peaks at m/z 249, 218 along with their counter ions at m/z 393 and 204 revealed the basic fragmentation pattern of pentacyclic tritrpenes. In IR spectrum absorption peaks at 1735, 1200 and 1630 cm-1 displayed the presence of ester and olefinic groups in the molecule. 1H-NMR spectrum of (1) exhibited resonances of seven methyl singlets at δ 1.68, 1.05, 0.98 (Me- 30, 26, 27), 0.85 (9H, Me- 25, 24, 23) and 0.81 (Me- 28). Another singlet at δ 2.02 identified methyl of ester linkage. However two doublets at δ 4.67 and 4.55 (J = 2.3 Hz) justified two olefinic protons (H-29), double doublets at δ 4.46 (J = 10.0, 5.7 Hz) attributing a proton with α (alpha) stereo (H- 3α) and H-19 resonate as multiplet of one proton at δ 2.36. This discussion agreed lup- 20(29)-ene skeleton that was further justified through HMBC (Heteronuclear Multiple Bond Coherence) assignment of (1) in which the spectrum showed connectivity of H-19 with C-29 (δ 109.3) and of H-3α with C=O (δ 171.0) of acetoxy group.

The above spectral data was in full agreement of literature values [118]. Hence the structure of (1) was established as 3β – acetoxylup-20(29) – ene.



H3C 

Fig. 1: Significant HMBC (H C) interactions of compound (1)

1.3.2. Stigmasterol (2)

29 28

22 21 26 24 18 20 25 23

12 17 27 19 11 13 H 16 9 14 15 1 2 10 8 H H 3 5 7 4 6 HO

+ The molecular formula C29H48O of (2) was obtained through HR-EIMS (M 412.3742). The EIMS spectrum of (2) showed peaks at m/z 397 and 394 due to + loss of CH3 and H2O, other remarkable peaks were observed at m/z 379 [C28H43] , + + + 273 [C20H33] , 255 [C19H27] and 229 [C18H13] . The IR spectrum showed the presence of hydroxyl group at 3380 cm-1 and olefinic group at 1660 cm-1 in the moiety. The 1H-NMR of compound (2) exhibited resonance as a broad singlet at δ 5.34 (H-6) and a multiplet at δ 3.50 (H- 3α) whereas multiplets at δ 5.17 and 5.03 were assigned to olefinic H-22 and H-23. It also showed two singlets at δ 0.99 and 0.67 (Me-19, Me-18) two doublets at δ 0.83 (J = 6.7 Hz, Me-27) and δ 0.78 (J = 6.7 Hz, Me-26) and a triplet at δ 0.81 (J = 7.0 Hz, Me-29). The above spectral data of stigmasterol (2) was in accordance with the reported values [119, 120]. It could be deduced as stigmasta-5, 22-diene-3β-ol.

1.3.3. β-Sitosterol (3)

29 28

22 21 26 24 18 20 25 23

12 17 27 19 11 13 H 16 9 14 15 1 2 10 8 H H 3 5 7 4 6 HO

The molecular ion [M]+ peak for (3) was observed at m/z 414.3862 in HR-EIMS spectrum corresponding to the molecular formula C29H50O. In the EIMS spectrum + compound (3) displayed characteristic fragment ions at m/z 396 [M-H2O] , 381 + + + [M-H2O-CH3] , 273 [M-side chain] and 255 [M-side chain- H2O] . The IR spectrum showed the hydroxyl band at 3400 cm-1 and band at 1640 cm-1 due to (C=C) double bond. The 1H-NMR spectrum of (3) was characteristic of a steroidal molecule. It displayed a signal of broad singlet at δ 5.33 for olefinic proton H-6 and a multiplet at δ 3.40 for H-3α, two singlets appeared at δ 1.00 and 0.67 were due to the quaternary Me-18 and Me-19 respectively. 1H-NMR spectrum also displayed three doublets at δ 0.90 (J = 6.0 Hz), 0.80 (J = 6.5 Hz) and 0.78 (J = 6.5Hz) of secondary Me-21, Me-26 and Me-27, respectively, whereas a triplet resonating at δ 0.82 (J = 7.0 Hz) was due to the primary Me-29. The comparative spectral study with the reported data [119, 121] led to the assignment of (3) as stigmasta-5-en-3-ol.

1.3.4. Palmitic acid (4)

O 3 5 7 9 11 13 15 HO 1 2 4 6 8 10 12 14 16

+ The molecular formula C16H32O2 of (4) was obtained through HR-EIMS (M , 256.2389). Its EIMS spectrum showed peak at m/z 45 indicating the presence of

(COOH) group whereas presence of (CH2) groups were exhibited by peaks at m/z 227, 213, and 199. The IR absorption bands at 3450-2610 cm-1 (COOH) and 1700 cm-1 (C=O) also confirm the presence of carboxylic group in the molecule. The 1H-NMR spectrum showed a triplet at δ 0.85 (J = 6.6 Hz) for terminal methyl

(Me- 16) and a triplet of (CH2) located next to COOH at δ 2.25 (J = 7.5 Hz, H-2) while a broad long singlet at δ 1.26 confirmed the presence of a long chain of

(CH2). In view of above spectral studies the structure of (2) was assigned as palmitic acid (Hexadecanoic acid). Its spectral data was in complete agreement with the reported values [122].

1.3.5. β-Amyrin (5)

29 30

20 19 21

12 18 22

11 13 17 25 26 H 9 14 16 28 1 15 2 10 8 H 3 5 7 27 HO 4 6 H 23 24

Compound (5) showed molecular ion peak [M]+ in HR-EIMS at m/z 426.0100 having molecular formula C30H50O. In EIMS spectrum the base peak at m/z 218 and 207 were due to retro Diels Alder fragmentation where as fragments at m/z

411 and 393 were obtained by the loss of CH3 and then H2O from the molecule. The fragment at m/z 189 indicates that -OH group is present either in ring A or B. It was placed at C-3 on biogenetic ground. The fragment at m/z 203 located the

CH3 group at C-17. This fragmentation identifies pentacyclic triterpene skeleton of oleanane and ursane series. In IR spectrum absorption bands at 3352 and 1648 cm-1 mentioned (OH) and (C=C) groups. The 1H-NMR spectrum determined Δ12 β–amyrin series of the compound as it displayed signals of eight methyl as singlets in upfield region at δ 1.09, 1.00, 0.99, 0.94, 0.87, 0.86, 0.85 and 0.79 (Me- 27, 26, 23, 25, 29, 30, 28 and 24). Similarly a signal at δ 3.09 (1H, dd, J = 11.0, 4.0 Hz) was assigned to H-3α and olefinic proton H-12 exhibited resonance as a triplet at δ 5.15 (J = 4.5 Hz). All above studies were in full agreement of literature values [123, 124], therefore structure of (5) was assigned as 3β-hydroxyolean-12-ene.

1.3.6. Oleanolic acid (6)

29 30

20 19 21

12 18 22

11 13 17 25 26 H COOH 9 14 16 28 1 15 2 10 8 H 3 5 7 27 4 HO 6 H 23 24

The compound (6) displayed molecular ion peak [M]+ at m/z 456 in EIMS and

456.3410 in HR-EIMS corresponding to the molecular formula C30H48O3. The retro Diels Alder fragments at m/z 248, 207, 203, 133 and 189 placed the (- COOH) group at C-17 and –OH group in ring A or B. The later was placed at C-3 on biogenetic ground. The IR absorption bands at 3410, 2789, 1710 and 1625 cm-1 justified the presence of hydroxyl, carboxylic and olefinic group in the molecule. The 1H-NMR spectrum displayed seven tertiary methyl singlets at δ1.20, 1.15, 0.97, 0.94, 0.86, 0.85 and 0.75 (Me-23, 27, 25, 24, 30, 29 and 26), a triplet resonated at δ 5.28 (J = 3.5 Hz) for olefinic proton (H-12) and δ 2.80 (dd, J= 14.1, 4.6 Hz) for H-18. These data attributed that compound (6 ) belongs to Δ12β – amyrin series of pentacyclic triterpenoids and its β-orientation was decided on the basis of δ value and coupling constant of H-3 (δ 3.40, dd, J = 14.0, 4.0 Hz). Structure of compound (6) was confirmed by comparing its spectral data with those reported in literature [125] as oleanolic acid or 3β-hydroxyolean-12-en-28- oic acid.

1.3.7. β-Sitosterol-3-O-β-D-glucopyranoside (7)

29 28

21 22 26 24 18 20 23 25

12 27 11 13 17 19 H 16 9 14 15 1 2 10 8 H H 6' OH 3 5 7 4 6 5' O O 4' OH 2' 1' OH 3' OH

The molecular formula C35H60O6 of Compound (7) was assigned by HR-FAB-MS (+ve) appeared at m/z 577.2877 [M+H]+ whereas in EIMS spectrum the characteristic peaks appeared at m/z 414 [M-gluc]+ and at m/z 396 [M- gluc- + H2O] . IR spectrum of (7) displayed absorption bands for (OH), (C=C), and (C-O) at 3410, 1638 and 1248 cm-1. The glycosidic moiety was identified in 1H-NMR by showing a signal of anomeric proton at δ 4.80 (d, J= 7.5 Hz, H-1') whereas other glucose protons appeared as multiplet in the range of δ 3.25-4.45. 1H-NMR spectrum showed resonance of olefinic proton at δ 5.32 (br. s, H-6) and oxymethine proton at δ 3.43 (m, H-3α). The 1H-NMR spectrum also showed the signals of six methyl groups out of which two were tertiary (δ 1.08, CH3-19 and δ 0.66, CH3-18), three secondary, CH3-21,

26 and 27 (δ 0.90, δ 0.81 and δ 0.79) and one primary methyl, CH3-29 at δ 0.82. The above spectral data was in full agreement of reported values [126] to decide the structure of (7) as β-sitosterol-3-O-β-D-glucopyranoside.

1.3.8. Ferulic acid (8)

In EIMS spectrum of compound (8) the peak at m/z 194 indicated the molecular ion [M]+ and 194.0136 in HR-EIMS corresponding to the molecular formula

C10H10O4. The mass spectrum also showed the presence of COOH, OCH3, and olefinic group in the molecule by showing the peaks at m/z 149, 163 and 123. The compound displayed 3345-2578cm-1 (OH and COOH), 1675(C=O), 1619 (C=C) and 1439(aromatic C=C) absorption peaks in IR spectrum. 1H-NMR spectrum showed signals of olefinic protons as two doublet at δ 7.48 and 6.01 (J= 14.5 Hz, H-7, H-8) large coupling constant indicated there trans substitution. Whereas resonance in downfield defined aromatic protons at δ 7.15 (br. s, H-2) and two doublets at δ 7.21 (J =7.8 Hz, H-6) and 6.60 (J = 7.8 Hz, H-

5). It further gave a singlet at δ 3.89 for methoxy (OCH3) moiety attached to aromatic ring. On the basis of above spectral studies that coincided with the literature values [23, 127] compound (8) was characterized as ferulic acid.

1.3.9. Biological activities

All fractions of methanolic extract of air dried aerial parts of X. strumarium Linn. (XS- HX, XS-DC, XS-EA, XS-BU and XS-ME) were analysed for their antimicrobial and anti-oxidant activity by the methods as described in chapter 4.

1.3.9.1. In Vitro Anti-bacterial activity The anti-bacterial activity of XS-HX, XS-DC, XS-BU and XS-ME fractions was determined by disc diffusion method whereas agar-well method was applied for fraction XS-EA. The anti-bacterial activity of all fractions were determined against Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, Corynebacterium xerosis, Mycobacterium smegmatis, Staphylococcus auereus, Staphylococcus epidermidis, streptococcus saprophyticus, streptococcus faecalis, streptococcus pyogenes gram positive bacterial strains and Campylobacter coli, Enterobacter aerugenus, Escherichia coli, Klebsiella pneumonieae, Proteus mirabilis, Pseudomonas aeruginosa ATCC 9027, Salmonella typhi , Salmonella typhi A , Salmonella typhi B, Shigella dysenteriae and Vibrio choleraeae gram negative bacterial strains. The fraction XS-EA was found to be the most active fraction. It showed remarkable anti- bacterial activity against Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis and Klebsiella pneumonieae having zone of inhibition 26, 34, 30 and 22 mm with MIC = 88, 120, 102 and 56 mg/ml. It further showed good activity against Campylobacter coli, Enterobacter aerugenus, Salmonella typhi , Salmonella typhi A , Salmonella typhi B and Vibrio choleraeae creating 19, 17, 18, 19, 20 and 19 mm zone of inhibition respectively (MIC = 200, 200, 89, 90, 52 and 160 mg/ml). Gentamicin was used as standard. All other fractions exhibited weak to moderate anti-bacterial activity displaying zone of inhibition in the range of 8-13 mm in comparison of standard Ampicillin. Results are displayed in Table-2.

Table-2: In vitro anti-bacterial activity of different fractions of methanolic extract of X. strumarium Linn

Organisms XS-HX XS-DC XS-BU XS-ME Ampicillin XS-EA Gentamicin MIC mm mm mm mm mm mm mm mg/ml

Gram positive bacteria Bacillus cereus - 09 - - >25 26 >15 88 Bacillus subtillis - - - 34 >15 120 Bacillus thuringiensis 08 09 - - >45 30 >15 102 Corynebacterium xerosis ------Mycobacterium smegmatis ------Staphylococcus aureus 09 - - - >30 - - - Staphylococcus epidermidis ------Streptococcus saprophyticus - - 09 - >35 - - - Streptococcus faecalis - - - 13 >30 - - - Streptococcus pyogenes ------Gram negative bacteria Campylobacter coli - - - - - 19 >15 200 Enterobacter aerogenus - - - - - 17 >15 200 Escherichia coli ------Klebsiella pneumoniae - - - - - 22 >15 56 Proteus mirabilis - 08 - - >35 - - - P. aeruginosa ATCC 9027 10 08 - - >30 - - - Salmonella typhi - - - - >30 18 >15 89 Salmonella para typhi A - - - - - 19 >15 90 Salmonella para typhi B - - - - - 20 >15 52 Shigella dysenteriae ------Vibrio cholerae - - - - - 19 >15 160

Key: Values are zone of inhibition diameter (mm) and an average of triplicate. (-) indicates inactivity. Fraction : XS= Xanthium strumarium, HX=n-Hexane, DC= Dichloromethane, BU= n-Butanol, ME= Methanol and EA= Ethyl acetate. MIC = minimum inhibitory concentration

1.3.9.2. In Vitro Anti-fungal activity All the fractions were tested for their anti-fungal activity against Candida albicans, Candida albicans ATCC (0383), Saccharomyces cerevisiae, Microsporum canis, Microsporum gypseum, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Aspergillus flavus, Aspergillus niger, Fusarium species, Helminthosporium, Penicillium species and Rhizopus species. It was found that XS- EA was the only fraction that showed excellent anti-fungal activity against Microsporum canis, Aspergillus flavus, Aspergillus niger, Helminthosporium, Penicillium species and Rhizopus species showing zone of inhibition 12, 19, 15, 14, 12, and 16 mm (MIC = 312, 320, 100, 212, 149, and 300 mg /ml ) respectively. Gresiofulvin was used as standard. Results are presented in Table-3.

Table-3: In vitro anti-fungal activity of different fractions of methanolic extract of X. strumarium Linn. Fungi XS-HX XS-DC XS-EA XS-BU XS-ME Gresiofulvin MIC (mm) (mm) (mm) (mm) (mm) (mm) (mg/ml)

Yeast Candida albicans - - - - - >12 Candida albicans ATCC 0383 - - - - - >12 Saccharomyces cerevisiae - - - - - >12 Dermatophytes Microsporum canis - - 12 - - >12 312 Microsporum gypseum - - - - - >12 Trichophyton rubrum - - - - - >12 Trichophyton mentagrophytes - - - - - >12 Trichophyton tonsurans - - - - - >12 Saprophytes Aspergillus flavus - - 19 - - >12 320 Aspergillus niger - - 15 - - >12 100 Fusarium specie - - - - - >12 Helminthosporium - - 14 - - >12 212 Penicillium specie - - 12 - - >12 149 Rhizopus specie - - 16 - - >12 300 Key: Values are inhibition zones (mm) and an average of triplicate. (-) sign indicates no activity. MIC=minimum inhibitory conc. Fractions: XS=Xanthium strumarium, HX= n-Hexane, DC=Dichloromethane, EA= Ethylacetate, BU= n-Butanol, ME= Methanol

1.3.9.3. In Vitro Anti-oxidant activity All the methanolic fractions when tested for their anti-oxidant activity in comparison of ascorbic acid only EtOAc fraction (XS-EA) exhibited significant activity with

70% inhibition (EC50 = 937 µg/ml). The remaining fractions showed less than 50% inhibition hence were not further studied. Results are displayed in Table-4.

Table-4: In vitro anti-oxidant activity of methanolic fractions of X. strumarium

Fractions % inhibition EC50 (µg/ml) XS-HX -4.16 - XS-DC 41.66 - XS-EA 70 937 ± 0.5 XS-BU 30 - XS-ME 25 - Ascorbic acid 80 8.3

Key: Values are inhibition (%) and an average of triplicate. EC50 = effective concentration to scavenge 50 % of DPPH Fractions: XS=Xanthium strumarium, HX=n-Hexane, DC=Dichloromethane, EA= Ethylacetate, BU= n-Butanol, ME= Methanol.

1.4. Experimental

1.4.1. General Experimental

Melting points were determined in glass capillary tubes on a Gallenkamp melting point apparatus and were incorrected. Column chromatography was carried out on silica gel (70-230 mesh, Merck). TLC was performed on pre-coated silica gel GF- 254. IR spectra were recorded on a Jasco-302-A spectrophotometer. The mass spectra were scanned on a Jeol-JMS HX-110 mass spectrometer. The 1H and 13C-NMR spectra were recorded on a Bruker spectrometer operating at 500, 300 and 75 MHz.

The chemical shift values are reported in δ (ppm) relative to SiMe4 (TMS) as an internal standard. The coupling constant (J) are given in Hz.

1.4.2. Plant Material Aerial parts of Xanthium strumarium Linn. was collected by Mohammad Farman Ali from Lakimarwat (Khyber Pakhtun Khawa) Pakistan and identified by Dr. Sahar. A voucher specimen (G.H. No.86398, No. 01) has been deposited in the herbarium at Department of Botany, Faculty of Science, University of Karachi, Karachi, Sind, Pakistan.

1.4.3. Extraction and Isolation The air dried aerial parts of Xanthium strumarium Linn (10 kg) were ground to fine powder and extracted repeatedly with methanol at room temperature. The solvent was evaporated under vacuum to obtain 250g crude extract (XS-Me). The resultant dark greenish brown gummy mass was partitioned between EtOAc and H2O (Scheme I).

Aqueous phase was neglected while the EtOAc phase was treated with aq. Na2CO3 solution (4%) to remove acidic portion from neutral fractions. This EtOAc portion that composed of neutral fractions was again washed with distilled water, dried with

Na2SO4 treated with activated charcoal to remove green colored chlorophyll part and filtered. The filtrate was concentrated under reduced pressure to obtain 150g neutral fraction (XS-N). The charcoal bed was eluted with MeOH–C6H6 (1:1) repeatedly. The total extract was combined and solvent was removed by vaccum distillation.

Both fractions were combined after comparison of their TLC on silica gel GF-254 using mobile phase n-Hexane-EtOAc (9.5:0.5) to give total neutral fraction 154g (150g+4g), (XS-N). The resultant total neutral fraction thus obtained was partitioned into n-Hexane soluble (XS-HX; 13g) and n-Hexane insoluble portions (Scheme II). The n-Hexane insoluble fraction was again divided into dichloromethane (DCM) soluble (XS-DC; 14g) and DCM insoluble portions. The DCM insoluble portions was again partitioned into EtOAc soluble (XS-EA; 97g) and EtOAc insoluble portion. The EtOAc insoluble portion was again divided into n-butanol soluble (XS-BU; 21g) and n-butonal insoluble portion. All fractions (XS-HX, XS-DC, XS-EA, XS-BU and XS-Me) were analyzed for their anti-microbial and anti-oxidant activities. Among all these fractions XS-EA was found to be the most active fraction as it showed high anti-bacterial, moderate anti- fungal and good anti-oxidant activity whereas all other fractions displayed weak to moderate anti-bacterial activity (Table-2, 3 and 4). The EtOAc soluble fraction was subjected to column chromatography (CC) over silica gel with successive elution with n-hexane and n-hexane- EtOAc in increasing order of polarity. It ultimately furnished sixteen fractions (FR-1 to FR-16) (Scheme III). The fraction FR-2 which eluted with n-hexane- EtOAc (9.5 : 0.5) provided white crystalline solid on slow evaporation which was filtered and recrystallized from same solvent system to obtain colorless needles of lupenyl acetate (1 ; 40 mg). The mother liquor (2.0 g) of FR-2 was subjected to pencil CC over silica gel and eluted with n- hexane, n-hexane –EtOAc in increasing order of polarity to gave 38 fractions (FR-2-1 to FR-2-38). The fraction FR-2-5 obtained with n-hexane–EtOAc (9.5: 0.5) afforded stigmasterol (2; 20 mg) and FR-2-11 eluted with n-hexane-EtOAc (9:1) gave β- sitosterol (3; 25 mg). The fraction FR-3 of the main EtOAc fraction which eluted with n-hexane-EtOAc (9:1) showed two major spots on TLC. Purification through preparative TLC in the same solvent system provided palmitic acid (4; 22 mg) and β- amyrin (5; 6.0 mg). The fraction FR-6 was subjected to CC over silica gel (scheme IV) eluting with n-hexane, n-hexane–EtOAc in increasing order of polarity furnished 10 fractions (FR-6-1 to FR-6-10). The fraction FR-6-3 obtained with n-hexane-

EtOAc (8:2) was purified through preparative TLC using mobile phase CHCl3– MeOH (1:1) to obtain a colorless crystalline compound which was characterized as oleanolic acid (6; 4.0 mg). On the other hand, FR-6-7 obtained with n-hexane- EtOAc

(1:1) crystallized from CHCl3-MeOH (1:1) gave β-sitosterol-3-O- β–D- glucopyranoside (7; 5.5 mg). The fraction FR-9 of the main EtOAc fraction was further subjected to CC over silica gel using CHCl3, CHCl3-MeOH in increasing order of polarity to obtain 6 fractions (FR-9-1 to FR-9-6). The fraction FR-9-3 obtained with CHCl3-MeOH (9:1) gave colorless solid which was recrystallized from the same solvent system to afford ferulic acid (8; 3.0 mg).

Extraction and Isolation

Aerial Parts of Xanthium strumarium Linn + Methanol(5 times repeatedly; R.T.)

Methanolic Extract Solvent removed under vaccum

Crude Extract (XS- ME)* +EtOAc + H2O

EtOAc Phase Aqueous Phase (Neglected) +Aqueous Na2CO3 (4%)

EtOAc Phase Aqueous Na2CO3 phase 1) General work up (Not worked up in the present studies) 2) Charcoal

EtOAc eluate Charcoal bed

solvent removed under MeOH-C H (1:1) reduced pressure 6 6

EtOAc residue MeOH-C6H6 eluate solvent removed under vaccum

MeOH-C6H6 eluate residue

combined

Residue (XS- N) Scheme-I

Residue (XS-N) + n-Hexane

n-Hexane n-Hexane insoluble fraction solublefraction (XS- HX)* + Dichloromethane (DCM)

DCM DCM insoluble fraction * soluble fraction (XS- DC) +EtOAc

EtOAc insoluble fraction EtOAc soluble fraction + n- Butanol solvent removed under reduced pressure n- Butanol n- Butanol insoluble fraction soluble fraction (XS- BU)* (neglected)

Residue (XS- EA)*

* attributes the fractions exhibiting antimicrobial and antioxidant activities

Scheme-II

XS-EA* CC (n-Hexane, n-Hexane-EtOAc in order ofincreasing polarity)

FR-1 FR-2 FR-3 FR-4 FR-5 FR-6 FR-7 FR-8 FR-9 FR-10 FR-11 FR-12 FR-13 FR-14 FR-15 FR-16 (scheme IV) Preparative TLC CC n-Hexane- EtOAc(9:1) (CHCl3,CHCl3-MeOH; in order of increasing polarity)

FR-9-1 FR-9-2 FR-9-3 FR-9-4 FR-9-5 FR-9-6 Palmitic acid(4) CHCl3- MeOH(9:1)

Crystals amyrin (5)

Recrystallization from Kept at room temperature CHCl - MeOH(1:1) over night in n-Hexane-EtOAc(9.5:0.5), 3 filter

Ferulic acid (8)

Lupenyl acetate (1) Mother Liquor

concentrated in vaccum CC (n-Hexane, n-Hexane-EtOAc; in order of increasing polarity)

FR-2-1 FR-2-2 FR-2-3 FR-2-4 FR-2-5 FR-2-6 FR-2-7 FR-2-8 FR-2-9 FR-2-10 FR-2-11 FR-2-12 -38 n-Hexane- n-Hexane-EtOAc(9.5:0.5) EtOAc (9:1)

Stigmasterol (2) -sitosterol (3)

Scheme-III

FR-6

CC (n-Hexane, n-Hexane-EtOAc in order of increasing polarity)

FR-6-1 FR-6-2 FR-6-3 FR-6-4 FR-6-5 FR-6-6 FR-6-7 FR-6-8 FR-6-9 FR-6-10

Preperative TLC Crystallized from CHCl3 -MeOH (I:1) CHCl3- MeOH (1:1)

Oleanolic acid (6)

sitosterol-3-O-D-glucopyranoside (7)

Scheme-IV

1.4.4 Characterization of compounds

1.4.4.1. Characterization of Lupenyl acetate (1)

Colourless needles (40 mg). M.P: 214-215°C. -1 IR (KBr) νmaxcm : 1735 (C=O), 1630 (C=C), 1200(C-O). EIMS: m/z (rel. int., %), 468 (M +, 51), 453 (17), 408 (13), 249 (18), 218 (91) and 204 (44). + HR-EIMS: m/z 468.3970 (calcd for C32H52O2, 468.3969); 453.3770 [C31H49O2] , + + + 408.3751 [C30H48] , 249.1850 [C16H25O2] , 218.2030 [C16H26] and + 204.1871[C15H24] . 1 H-NMR: (CDCl3, 300 MHz): δ 4.67 and 4.55 (each1H, d, J =2.3 Hz, H-29), 4.46

(1H, dd, J=10.0, 5.7 Hz,H-3α), 2.36 (1H, m, H-19), 2.02 (3H, s, COCH3), 1.68 (3H, s, H-30), 1.05 (3H, s, H-26), 0.98 (3H, s, H-27), 0.85 (9H, s, H-25, 24, 23), 0.81 (3H, s, H-28). 13 C-NMR: (CDCl3, 75 MHz): δ 38.3 (C-1), 23.8 (C-2), 81.0 (C-3), 37.8 (C-4), 55.3 (C-5), 18.2 (C-6), 34.2 (C-7), 40.8 (C-8), 50.3 (C-9), 37.1 (C-10), 20.9 (C-11), 25.1 (C-12), 38.0 (C-13), 42.8 (C-14), 27.4 (C-15), 35.5 (C-16), 43.0 (C-17), 48.0 (C-18), 48.3 (C-19), 150.9 (C-20), 29.8 (C-21), 40.0 (C-22), 27.9 (C-23), 16.5 (C-24), 16.1 (C-25), 15.9 (C-26), 14.5 (C-27), 18.0 (C-28), 109.3 (C-29), 19.2 (C-30), 171.0

(C=O) and 21.3 (COCH3).

1.4.4.2. Characterization of Stigmasterol (2)

Colourless needles (20 mg). M.P: 169-170°C. -1 IR (KBr) νmax cm : 3380 (OH), 1660 (C=C). EIMS: m/z (rel. int., %), 412 (M + , 22), 397 (45), 394 (38), 379 (17), 367 (18), 273 (24), 255 (76), 229 (32), 211 (38), 199 (27), 173 (29), 145 (50) and 119 (40). + HR-EIMS: m/z 412.3742 (calcd for C29H48O, 412.3707); 397.3353 [C28H45O] , + + + + 394.3570 [C29H46] , 379.3372 [C28H43] , 367.3006 [C26H39O] , 273.2219 [C20H33] , + + + + 255.2093 [C19H27] , 229.1617 [C18H13] , 211.1462 [C16H19] , 199.1464 [C15H19] , + + + 173.1317 [C13H17] , 145.1019 [C11H13] and 119.0860 [C9H11] . 1 H-NMR: (CDCl3, 300 MHz): δ 5.34 (1H, br.s, H-6), 5.17 (1H, m, H-22), 5.03 (1H, m, H-23), 3.50 (1H, m, H-3), 0.99 (3H, s, Me-19), 0.83 (3H, d, J = 6.7 Hz, Me-27), 0.81 (3H, t, J =7.0 Hz, Me-29), 0.78 (3H, d, J = 6.7 Hz, Me-26) and 0.67 (3H, s, Me- 18).

1.4.4.3. Characterization of β-Sitosterol (3)

Colourless solid (25 mg). M.P: 134-135°C. -1 IR (KBr) νmax cm : 3400 (OH), 1640 (C=C). EIMS: m/z (rel. int., %), 414 (M +, 32), 396 (43), 381 (17), 273 (26), 255 (74), 213 (35), 133 (12) and 55 (36). + HR-EIMS: m/z 414.3862 (calcd. for C29H50O, 414.3864); 396.3773 [C29H48] , + + + + 381.3563 [C28H45] , 273.2563 [C20H33] , 255.2160 [C19H27] , 213.1625 [C16H21] , + + 133.1021 [C10H13] and 55.6955 [C4H7] . 1 H-NMR: (CDCl3, 500 MHz): δ 5.33 (1H, br. s, H-6), 3.40 (1H, m, H-3), 1.00 (3H, s, Me-19), 0.90 (3H, d, J = 6.0 Hz, Me-21), 0.82 (3H, t, J = 7.0 Hz, Me-29), 0.80 (3H, d, J = 6.5 Hz, Me-26), 0.78 (3H, d, J = 6.5 Hz, Me-27) and 0.67 (3H, s, Me-18).

1.4.4.4. Characterization of Palmitic acid (4)

White crystals (22 mg). M.P: 61-62ºC. -1 IR (KBr) νmax cm : 3450-2610 (COOH), 2925 and 2850 (CH), 1700 (C=O) and 1120 (C-O). EIMS: m/z (rel. int., %), 256 (M +, 12), 227 (17), 213 (34), 199 (16), 171 (27), 129 (61), 73 (90) and 45 (22). + HR-EIMS m/z: 256.2389 (calcd for C16H32O2, 256.2403); 227.2026 [C14H27O2] , + + + 213.1840 [C13H25O2] , 199.1705 [C12H23O2] , 171.1368 [C10H19O2] , 129.0925 + + + [C7H13O2] , 73.0302 [C3H5O2] , 45.0120 [CO2H] . 1H-NMR: (MeOD, 300 MHz): δ 2.25 (2H, t, J = 7.5 Hz, H-2), 1.26 (26H, br. s, 13 х

CH2 chain), 0.85 (3H, t, J = 6.6 Hz, Me-16).

1.4.4.5. Characterization of β–Amyrin (5)

Colourless solid (6.0 mg). M.P: 195- 196ºC. -1 IR (KBr) νmax cm : 3352 (OH), 1648 (C=C), 2720, 2801 (CH). EIMS: m/z (rel. int., %), 426 (M+, 49), 411 (15), 408 (10), 393 (10), 218 (100), 207 (42), 203 (53), 189 (60), 175 (24), 147 (25), 135 (45), 119 (27), 109 (41) and 69 (55). + HR-EIMS: m/z 426.0100 (calcd. for C30H50O, 426.3862); 411.3412 [C29H47O] , + + + + 408.1502 [C30H48] , 393.223 [C29H45] , 218.0420 [C16H26] , 207.3301 [C14H23O] , + + + + 203.1354 [C15H23] , 189.3010 [C14H21] , 175.0132 [C13H19] , 147.1945 [C11H15] , + + + + 135.1093 [C10H15] , 119.2203 [C9H11] , 109.0110 [C8H13] and 69.2015 [C5H9] . 1 H- NMR: (CDCl3, 500 MHz): δ 5.15 (1H, t, J = 4.5 Hz, H- 12), 3.09 (1H, dd, J = 11.0, 4.0 Hz, H-3α), 1.09, 1.00, 0.99, 0.94, 0.87, 0.86, 0.85 and 0.79 (each 3H, s, 8×Me).

1.4.4.6. Characterization of Oleanolic acid (6)

Colourless needles (4.0 mg). M.P: 195-197C. -1 IR (CHCl3) νmax cm : 3410-2789 (COOH), 2768 (C-H), 1710 (C=O), 1625 (C=C), and 1066 (C-O). EIMS: m/z (rel. int.), 456 (M +, 4), 248 (100), 207 (23), 203 (31), 189 (10) and 133 (21). + HR-EIMS: m/z 456.3410 (calcd for C30H48O3, 456.3530), 248.1730 [C16H24O2] , + + + 207.1643 [C14H23O] , 203.2735 [C15H23] , 189.1549 [C14H21] and 133.1081 + [C10H13] . 1 H-NMR: (CDCl3, 300 MHz):  5.28 (t, J = 3.5 Hz, H-12), 3.40 (1H, dd, J = 14.0, 4.0 Hz, H-3α), 2.80 (1H, dd, J = 14.1, 4.6 Hz, H-18) 1.20, 1.15, 0.97, 0.94, 0.86, 0.85 and 0.75 (3H each, s, 7×Me).

1.4.4.7. Characterization of β-Sitosterol-3-O-β-D-glucopyranoside (7)

Colourless crystals (5.5 mg ). M.P: 284º- 285ºC -1 IR (KBr) νmax cm : 3410 (OH), 1638 (C=C), 1248 (C-O). EIMS: m/z (rel. int. %), 414 (19), 396 (100), 255 (36), 135 (20), 120 (15). HRFAB-MS (+ve): m/z 577.2877 [M + 1]+ . 1 H-NMR: (CDCl3, 500 MHz): δ 5.32 (1H, br. s, H-6), 4.48 (1H, d, J= 7.5 Hz, H-1'), 3.43 (1H, m, H-3), 3.25 – 4.45 (glucose protons) (5H, m, H-2',3',4',5' and 6'), 1.02 (3H, s, Me-19), 0.90 (3H, d, J= 6.3 Hz, Me-21), 0.82 (3H, t, J = 6.9 Hz, Me-29), 0.81 (3H, d, J = 6.4 Hz, Me- 26), 0.79 (3H, d, J = 7.0 Hz, Me-27) and 0.66 (3H, s, Me-18).

1.4.4.8. Characterization of Ferulic acid (8)

Colourless solid (3.0 mg ). M.P: 168-172 ºC. -1 IR (KBr) νmax cm : 3345 – 2578 (COOH), 1675 (C=O), and 1619 (C=C). EIMS: m/z (rel. int. %), 194 (M +, 10), 163 (32), 149 (24), 123 (19) and 92 (5). + HR-EIMS: m/z 194.0136 (calcd. for C10H10O4, 194.2110), 163 [C9H7O3] , 149 + + + [C9H9O2] , 123 [C7H7O2] and 92 [C6H4O] . 1 H-NMR: (CDCl3, 500 MHz): δ 7.48 (1H, d, J = 14.5 Hz, H-7), 7.21 (1H, d, J = 7.8 Hz, H-6), 7.15 (1H, br. s, H-2), 6.60 (1H, d, J = 7.8 Hz, H-5). 6.01 (1H, d, J = 14.5

Hz, H-8) and 3.89 (3H, s, OCH3).

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CHAPTER-2 Synthesis in addition Bioactivities of 2,3-Diaminonaphthalenimidazole Derivatives

Introduction

2.0. General Introduction For Chapter 2 and 3

General Introduction

Organic chemistry is the study of carbon compounds. The carbon has unique property of combining with itself and with other atoms (H, O, N, S, P and halogens) in a number of ways producing acyclic or cyclic compounds. The cyclic organic compounds are of two types, homocyclic compounds (composed of carbon and hydrogen only) such as benzene and heterocyclic compounds (containing at least one atom other than carbon and hydrogen in their cyclic skeleton) like pyridine [1-4]. The heterocyclic compounds are more prominent because of their variable properties. They play a pivotal role and have great importance in medicinal and pharmaceutical field. They are abundantly distributed in nature and many of them are essential for life because these compounds perform significant and vital role in all physiological functions. For instance, DNA which is fundamental for existence of life, is genetic code material comprises of heteronuclei i.e. purine and pyrimidine bases. Proteins that build the external and internal skeleton of a living organism is actually a polymer of amino acids. Enzymes which are natural biological catalyst assist in many physiological processes in every second with high level of substrate specificity thus, maintain, control and speed up body functions. Likewise vitamins such as B12, A, E, riboflavin, biotin and pyridoxine etc., are essential constituents of daily diet. These are necessary for better body performance. Hemoglobin transport oxygen to each and every cell of the body. Similarly, hormones like thyroids balance and regulate body functions. ATP and ADP (Adenosintri and di phosphate) are energy packets. Chlorophyll, a green color pigment found in plant is photosynthesizing compound [5- 8]. A vast number of natural and synthetic heterocyclic compounds possesses valuable pharmacological properties hence, are of clinical uses. For example penicillin is a potent antibiotic. Chloramphenicol is a protein synthesis inhibitor. Quinine is used to treat malaria. Caffeine and benzodiazepines are psychopharmacological agents. It is noticed that nearly all synthetic drugs such as diazepam, barbiturates and azidothymidine are heterocyclic compounds [9-16].

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Besides the field of medicine these compounds have found extensive uses in applied chemistry like dyeing, polymer, rubber, paints, solvents, photographic sensitizers, valuable synthetics intermediates. In addition, insecticides, pesticides and rodenticides all are different composition of organic compounds [17-22]. The medicinal chemist takes keen interest in infectious diseases because these infections are continuously going to rise as the resistance against human pathogens is increasing. There is a vast range of synthetic organic compounds which possesses valuable bioactivities due to which they play a crucial role in the development of medicines. Now there is a list of analgesics, sedatives, anti-aging, anti-inflammatory, CNS depressants, anti- diabetics etc [23-27]. Organic synthesis has been appeared as an indispensable, influential and valuable tool for the development of drugs. It is difficult to understand the basics biological phenomenon without the fundamental knowledge of organic chemistry. Generally organic synthesis can be divided in to two main groups: (a) Target oriented synthesis. This is the synthetic route for a natural product, defined or designed molecule. (b) Method oriented synthesis. In this group usually an already existing scheme is used preferably to designed methods in order to get high quality and pure products [28-30]. The aim of this study was to synthesize different derivatives of 2, 3- diaminonaphthalenimidaozle and amides of piperic acid then subsequent screening of their biological activity. The synthetic portion therefore, comprises of synthesis, in addition of biological screening of derivatives of 2, 3-diaminonaphthalenimidazole in chapter-2 and of amides of piperic acid dealt in chapter-3 of this dissertation.

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References

[1] J. Claydon, N. Greeves and S. Warren, “Organic Chemistry”, Oxford University Press, Inc., 2nd Ed., Chap-42 (2012). [2] J. McMurry, “Organic Chemistry”, 6th Ed., Thomson, Brooks/Cole (USA), (2004). [3] T. Eicher, S. Hanptmann and A. Speicher, “The Chemistry of Heterocycles, Structure, Reaction, Synthesis and Application”, 3rd Ed., Wiley-VCH Verlag and CO., p. 5-15 (2012). [4] J. A. Joule and K. Mills, “Heterocyclic Chemistry”, 4th Ed., Blackwell Publishing, UK, p. 53-61 (2008) [5] D. L. Nelson and M. M. Cox “Lehninger, Principles of Biochemistry” L. Schultz (Editor), 6th Ed., W. H. Freeman and Company, New York, p.75-400 (2013). [6] R. K. Murray , D. K. Granner and V. W. Rodwell, “Harper’s Illustrated Biochemistry”, 27th Ed., The McGraw-Hill company, Singapore, p. 14-450 (2003). [7] A. M. Lesk, “Introduction to Protien Science, Architecture, Function and Genomics”, 2nd Ed., Oxford University Press, New York (2010). [8] B. Albert, “Molecular Biology of the Cell”, 4th Ed., Garland Science, New York, p. 129-235, 767-831(2002). [9] L. P. Garrod, Brit. Med. J., 1(5172), 527 (1960). [10] R. Holt, Lancet, 289(7502), 1259 (1967). [11] O. Jardetzky, The J Biol. Chem., 238(7), 2498 (1963). [12] H. Barennes, E. Pussard and M. Sani, Brit. J Clin. Pharmacol., 41(5), 389 (1996). [13] A. Dorndorp and F. Nosten, Lancet, 366(9487), 717 (2005). [14] Y. Shiraishi, K. Kamamoto and A. A. Sandberg, Mutant Research, 62(1), 139 (1979). [15] A. Nehlig, J. L. Daval and G.Debry, Brain Res. Rev., 17(2), 139 (1992).

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[16] A. D. Fraser, Ther. Drug Monit., 20(5), 481 (1998). [17] I. L. Finar, “Organic Chemistry”, 6th Ed., Pearson Education Pvt. Ltd., Singapore, p. 801 (2003). [18] J. Claydon, N. Greeves and S. Warren, “Organic Chemistry”, Oxford University Press Inc., New York, p.1 (2012). [19] J. G. Kim and D. O. Jang, Synlett, 8, 1231 (2010). [20] J. D. Robert and M. C. Caserio, “Basic Principles of Organic Chemistry”, 2nd Ed., W. A. Benjamin Inc., Philipines, p. 561, 1328-1492 (1984). [21] M. H. Sarvari and H. Sharghi, J. Org. Chem., 71, 6652 (2006). [22] A. E. Akelah, R. Kenawy and D. C. Sherrington, European Polymer J., 29(8), 1041 (1993). [23] L. D. Luca, Current Medicinal Chemistry, 13, 1 (2006). [24] J. C. Lee, S. Kumar and D. C. Underword, Immunopharmacology, 47, 185 (2000). [25] B. Narasimhan, D. Sharma and P. Kumar, Med. Chem. Res., DOI 10.1007/s 00044-0109472-5. [26] A. R. Todeschini, A. L. Miranda and C.M. Silva, Europ. J. Med. Chem., 33, 189 (1998). [27] P. C. Lima, L. M. Lima and P. H. Leda, Europ. J. Med. Chem., 35, 187 (2000). [28] K. C. Nicolaou and E. J. Sorensen, “Classics in Total Synthesis, Target, Strategies, method”, VCH Publishers, Inc., New York, p. 1-18 (1996). [29] L. F. Tietze and T. Eicher, “Reaction and Synthesis in the Organic Chemistry Laboratory”, D. Ringe (Translator), University Science Books, Mill Valley, California, p.4 (1998). [30] W. A. Smit, A. F. Bochkov and R. Caple, “Organic Synthesis, the Science behind the Art”, The Royal Society of Chemistry, UK, p.232-350 (1998).

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2.1 Introduction of Benzimidazole

The condensation product of primary amines with an aldehyde or ketone is known as Schiff base (1). The term is named after Hugo Schiff. The compound contains a carbon–nitrogen double bond where nitrogen atom connected to an alkyl or aryl group. The Schiff base which is comprised of a five membered planar aromatic ring having two nitrogen atoms, one bonded with a hydrogen atom while other shows pyridine like character is termed as Imidazole (2). When imidazole fused with benzene ring is called as Benzimidazole (3). It is bicyclic, aromatic heterocycle compound. The word ‘azole’ represents nitrogen atoms. The protonated nitrogen is assigned as 1 and tertiary nitrogen as 3.

R1, R2, R3 = alkyl or aryl Figure 1: Structure of Schiff base (1), Imidazole (2) and Benzimidazole (3)

Imidazoles have much strong chemical stability towards bases, acids and catalytic hydrogenation. It is observed that benzimidazoles undergo hydrogenation in the benzene nucleus while imidazole moiety remains unaffected. Benzimidazole which is an elaborated imidazole exists in natural products possessing biological importance [1-4].

2.1.1. Biological Importance Benzimidazole moiety exist as a part of highly significant biomolecule found in nature such as fifty percent of bases of nucleic acid which are genetic code

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material, is comprised of benzimidazole moiety i.e adenine (4) and guanine (5) [5].

Vitamin B12, a naturally occurring compound, is essential for blood cells formation and better function of central nervous system, has cobalt in co-ordination with N- riboysl-dimethyl benzimidazole [6-9]. Kealiiquinone (6) is a natural benzimidazole alkaloid which was isolated from Lucetta. It exhibited anti-cancer activity [10].

Figure 2: Structure of Adenine (4), Guanine (5) and Kealiiquinone (6)

The compounds containing benzimidazole moiety attached to a heterocyclic nucleus revealed a broad spectrum of biological activities. A wide range of synthetic benzimidazole derivatives have been reported for their physiological and pharmacological properties hence their scope in remedying several diseases like epilepsy, influenza, herpes, obesity, infections and depression etc. [11-13].

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It is also reported by many researchers that the bio-activities of benzimidazole and its analogues are due to modification in the nucleus by insertion of different heterocyclic system to exhibit broad spectrum of biological activities [14] such as anti-bacterial (7) [15-17], anti-fungal (8) [18,19], anti-tubercular (9) [20], anti- inflammatory (10), analgesic [21], anti-cancer towards human breast (11) and human colon carcinoma [22], anti-viral (12) [23,24], diuretic, cytotoxic [25], anti-asthmatic, anti-diabetic [26], anti-oxidant (13) and anthelmintic activities [27].

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Figure 3: Structures of some biologically active benzimidazoles

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A wide range of benzimidazoles are in general use like thiabendazole (14) (anthelmintic), lansoprazole (15) (anti-ulcerative), astemizole (anti-histamine), albendazole and oxytetracycline (veterinary medicine), as well as (5-substituted benzimidazole) alkane (16) possesses good anti-leishmanial activity, on the other hand 2-mercaptobenzimidazole derivatives (17) exhibited analgesic property [28-30].

H3C OCH3

CH3

NH N H3CO NH S S N N O (14) (15)

N N NH2

N N

(16) N

N S N

(17)

Figure 4: Structures of generally used benzimidazoles

A series of 2-substituted N-hydroxyacrylamide benzimidazoles were found to be inhibitor of human histone deacetylase towards A 549, HL 60 and PC 3 cells [31] where as aminoacridine derived benzimidazoles showed moderate to significant kinase inhibition against CDK-5, 3 and 1 [32]. Additionally, 1-substituted

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benzimidazole displayed potential for inhibitory activity in platelet derived growth factor receptors (PDGFRs) [33].

N-substituted bis-benzimidazole derivatives have been appeared good for anti-HIV activity as well as cytotoxicity against human erythrocyte kidney cell line (HET-293T) and human lungs cancer cell line (NCI-H 23) [34]. In addition, a series of benzylvanilline benzimidazole compound (18) has shown anti-proliferative property in leukemia cancer cell HL 60 line. [35].

The derivatives of bis-benzimidazole with malonic acid appeared as an anti- cancer agent because of their DNA topoisomerase I and cytotoxic character against A 431 and MCF 7 cells [36].

It was also observed that 2-amino benzimidazoles which are attached to an imidazole moiety (19) were active H3-antagonist [37].

O N OCH3

H (18) H N NH H N N (19) N OCH3

Figure 5: Structures of anti-leukemic (18) and H3-antagonist (19) benzimidazoles

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Recently many researchers have elucidated naphthoimidazoles for their biological activities. The derivatives are found as an effective anti-tumor agent- against human cervical carcinoma (He La), human hepatoma (SMMC -7721) (20) and some solid tumors [38-40]. Novel naphthoimidazoles (21) have been developed that reveal considerable potential against Mycobacterium tuberculosis with MIC value ≤ 0.78 g / ml. The property can be modified against H374 RV (ATCC 27294) showing MIC values 9.12 and 4.2 M on introducing p.toluyl and indolyl groups of chalcone [41, 42]. A series of compounds based upon naphthoimidazole derivatives of natural quinones proved to possess trypanosomal activity against blood stream form of trypanosoma cruzi, some show EC50 value 15.5 M (22) [43-48].

N CF3 N O NH

H3CO N N

H O N O (20) (21) O

O N NH

(22)

Figure 6: Structures of biologically active naphthoimidazole containing compounds

Both Benzimidazole and Naphthoimidazoles also have important and appreciable industrial as well as analytical applications. It is found that aldo-

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naphthimidazoles can efficiently be used to determine compositional and absolute configuration in natural polysaccharides through HPLC techniques and by capillary electrophoresis (CE) methods [49, 50].

Naphthalene based polyimides display high stability and selectivity with strong electrode surface co-herence due to which they can be used as glucose immobilization oxidase [51]. These are also analysed to display sensor properties towards heavy metals captions [52]. A bilayer heterojunction solar cell has been developed through conjugation of naphthalene-bis-imidazole and zinc phthalocyanin (23). It serve as an electron acceptor, V = 0.50 V [53]. Some derivatives of naphthalene benzimidazoles (24) were applied as useful substance in organic dye sensitized solar cell [54].

O O N

N N N

N N n O (24) (23)

Figure 7: Structure of naphthoimidazoles used in solar cell

Many compounds with benzimidazole moiety are used in electronics, photography, as fire retardant and as transition metal corrosion inhibitor [55].

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2.2 Synthetic Approaches Towards Benzimidazoles

A variety of different methods are reported in literature for the synthesis of Benzimidazoles and its analogues. Some of them are given below:

2.2.1. Environment friendly synthesis of Benzimidazoles

A library of benzimidazole (26) has been synthesized via copper mediated intramolecular N-arylation of substituted imides (25). The use of water as a solvent rendered the method exclusively economical and green (Scheme 1) [56].

X CU O, K CO H NH 2 2 3 N o R' H2O, 100 C N R' N 30h H (25) (26) X = Br, I R' = Me, Ar

Scheme-1

2.2.2. Infra red radiation and clay mediated synthesis of Benzimidazole

A new simple and inexpensive methodology was reported for the synthesis of benzimidazole (29) by reaction of o-phenylenediamine (27) and carboxylic acid (28) using clay and infra red radiations in absence of solvent (Scheme-2) [57].

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O NH2 Bentonite N + ClCH OH Cl 2 IR, 20 min N NH2 H (27) (28) (29)

Scheme-2

2.2.3. Synthesis of Benzimidazole via recyclable catalyst

The condensation of o-phenylenediamine (27) and aromatic addehyde (30) was carried out successfully via oxidation through sulfonic acid functionalized silica catalyst to afford benzimidazole (31). The recovery and reusing ability of the catalyst for three reaction cycle with similar reactivity is the advantage of the reaction (Scheme-3) [58].

H O Sulfonic acid NH2 functionalised N + H Ar silica Ar N NH2 CH2Cl2, r. t., 1-2 hr

(27) (30) (31)

Scheme-3

2.2.4 Synthesis of 2-substituted Benzimidazoles

o-phenylenediamine (27) and o-anisaldehyde (32) were allowed to couple effectively in presence of benzoquinone to afford benzimidazole (33) in appreciable yield (Scheme-4) [59].

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H H3CO NH2 CHO N + Benzoquinone

NH2 OCH3 Ethanol N

(27) (32) (33)

Scheme-4

2.2.5 Microwave-assisted synthesis of Benzimidazole

Microwave-assisted condensation of o-phenylenediamine (27) and aldehyde (30) is a fast and efficient technology to obtain benzimidazole (31) in solvent free condition (Scheme-5) [60].

H O NH2 N Na2S2O5 Ar + H Ar microwave N NH2 60 sec. (27) (30) (31)

Scheme-5

2.2.6. Chemoselective synthesis of 2-Arylbenzimidazoles

Ortho substituted aniline (34) and arylaldehyde (30) undergo condensation through hydrogenperoxide and cericammonium nitrate (CAN) in solvent free condition to give chemoselective 2-arylbenzimidazole (35) in high yield (Scheme-6) [61].

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O NH2 Y CAN, H2O2 Ar + Ar H o YH 50 C, 70 min. N solvent free Y = S, NH (34) (30) (35)

Scheme-6

2.2.7 Copper catalysed synthesis of Benzimidazoles

One pot condensation of three components 2-halo aniline (36), aryl aldehyde (30) and sodium azide (37) was efficiently done by using catalytic amount of CuCl in dimethylsulfoxide (DMSO) to obtain benzimidazole (31) in good yield (Scheme-7) [62].

H NH O 2 N CUCl, DMSO Ar + Ar H + NaN3 o X 120 C, 12 hr. N X = Br, I

(36) (30) (37) (31)

Scheme-7

2.2.8 Synthesis of Benzimidazole through functionalized orthoesters

An efficient method for preparation of benzimidazole (40) has been developed by condensation of ortho-substituted aniline (38) with functionalized orthoester (39) using BF3.OEt2 at room temperature (Scheme-8) [63].

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NH 2 Y R'O OR' BF3. OEt2 + R" YH R'' OMe DCM N r.t., 4h Y = O, S, NH R', R'' = alkyl (38) (39) (40)

Scheme-8

2.2.9 Synthesis of Benzimidazole by using lodine as an oxidant

Hypervalent iodine can be used as an oxidant for the condensation of phenylenediamine (27) and aldehyde (30) in presence of dioxane to obtain benzimidazole (31) in high yield at ambient temperature in very short time (Scheme- 9) [64].

Scheme-9 2.2.10 Synthesis of Benzimidazole via heterogeneous base catalyst

KF/Al2O3 was found as a favorable solid heterogeneous base catalyst in one- pot coupling of o-phenylenediamine (27) and carbonyl compound (41) for the synthesis of benzimidazole (42) at room temperature (Scheme-10) [65].

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NH O 2 N KF-Al2O3 + R X R CH2Cl2, r.t. NH2 NH

(27) (41) (42) R = Alkyl, Aryl

X = Cl, -OR, -O2CR

Scheme-10

2.2.11 Synthesis of Benzimidazoles from esters

o-phenylenediamine (27) undergo condensation with esters (43) in microwave (mw) irradiation mediated method to produce benzimidazoles (42) with high yield in very short time (Scheme-11) [66].

NH2 O (CH2OH)2 N + R C OEt R mw, 1.5 min. NH2 NH R = alkyl, aryl (27) (43) (42)

Scheme-11 2.2.12 Phtolysis of protected Benzimidazole

Protected benzimidazole (44) under longer photolysis in presence of dioxane got deprotected to give benzimidazole (3) (Scheme-12) [67].

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N N Dioxane N h NH

(44) NO2 (3)

Scheme-12

2.2.13 Synthesis of Benzimidazole from a common intermediate

Benzimidazole derivatives (46) have been prepared from cyclization of a common intermediate i-e arylaminoxime (45) by using triethylamine (TEA) in dichloromethane. The product obtained in good yield (Scheme-13) [68].

OH N N TEA, CH2Cl2

o NH 23 C, 6h N

Ar Ar (45) (46)

Scheme-13

2.2.14 Synthesis of 2-substituted Benzimidazole in presence of H2O2

Preparation of 2-substituted benzimidazoles (31) has been carried out in excellent yield through an efficient one–pot coupling of o-phenylenediamine (27) and aromatic aldehydes (30) by the use of hydrogen peroxide and HCl in acetonitrile at room temperature (Scheme-14) [69].

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NH2 H O 30% O 2 2 N HCl 37% + Ar H Ar MeCN, r.t. NH2 NH 30-50 min (27) (30) (31)

Scheme-14

2.2.15 Synthesis of Benzimidazoles through reductive cyclisation

The reductive cyclisation of aromatic nitro aniline (47) with formic acid (48) has been achieved in presence of iron powder and 2-propanol to produce benzimidazole (49) in appreciable yield. (Scheme-15) [70].

NO2 O Fe, NH Cl N + H C OH 4 2-propanol NH N 80oC, 1-3 hr R R R = H, Ph, Et (47) (48) (49)

Scheme-15

2.2.16 Solution-phase synthesis of Benzimidazole

Solution phase synthesis of benzimidazoles (52) was carried out by condensation of 1,2-phenylenediamine (50) and aldehyde (51) in wet dimethyl formamide (DMF) and oxone. Products of high purity were obtained by simple aqueous precipitation (Scheme-16) [71].

119

NH2 O Oxone N + C R' R' H DMF/H O NH 2 N r.t. 22h R R R, R' = alkyl, Aryl (50) (51) (52)

Scheme-16

2.2.17 Synthesis of naphtho [1,2-d] imidazoles

1,2-diaminonaphthalene (53) undergoes condensation with furfural (54) in presence of organic solvent at room temperature to produce naphtho [1,2-d] imidazole (55) (Scheme-17) [72].

O X NH2 HN

NH2 N DCM + r.t OHC O X

(53) (54) (55) X = H, Br, NO2

Scheme-17 2.2.18 Synthesis of naphtho [2,3-d] imidazoles

Naphtho [2,3-d] imidazoles (58) was synthesized by simple condensation of 2,3-diamino naphthalene (56) with formazylglyoxylic acid (57) in presence of ethanol (Scheme-18) [73].

120

NH2 HOOC N-NH-Ar + NH2 O N=NH-Ar (56) (57) N N-NH-Ar EtOH reflux N N=NH-Ar

(58) H

Scheme-18

2.2.19 A simple and efficient synthesis of naphtho [2,3-d] imidazoles

2,3-Diaminonaphthalene (56) was effectively condensed with 1-(9-alkyl-9H- carbazol-3-yl)-4-carboxy-2-pyrrolidinone (59) in DMF at 170-230oC to obtain 2- substituted naphtho [2,3-d] imidazole (60) in good yield (Scheme-19) [74].

N COOH NH2

+ NH2 N O C H N (56) (59) 2 5 N DMF N 170o - 230oC H 3hr N (60) C2H5

Scheme-19

121

2.2.20 Iodine-catalysed synthesis of aldo-naphthimidazole

Aldo-naphthimidazoles (62) were obtained by iodine-catalysed oxidative condensation of 2,3-diamine naphthalene (56) with different aldoses (61) in presence of acetic acid at room temperature (Scheme-20) [75].

NH2 O I2, CH3COOH HO + HO r.t. reflux NH2 OH OH OH (56) (61) OH N HO HO OH N (62) H

Scheme-20

122

2.3. Results and Discussion

123

2.3.1 Chemistry

The above literature study shows that the nitrogen containing heterocyclic compounds are associated with a number of biological properties, same for benzimidazole system which is nitrogen containing five–membered heterocycles of immense biological and clinical uses. Their high therapeutic character switched our interest towards synthesis of naphthalenimidazole derivatives. With a little difference, it is almost similar in structure to that of benzimidazole. We assumed it might possesses biological properties and uses similar to that of benzimidazole moiety. Very little work is reported on its synthesis. So we aimed to synthesize 2,3-diamino naphthalenimidazole derivatives first time from conventional method that is generally used for preparation of imidazoles and to screen them for their biological activities.

2.3.2. General method for the synthesis of compounds (65-99).

2,3-diaminonaphthalenimidazoles (65-99) were synthesized by reacting commercially available 2,3-diaminonaphthalene with different aromatic aldehydes in N, N-dimethyl formamide (DMF). The resulted products were obtained in good yield (Scheme-21).

In a typical reaction, sodium metabisulfite (Na2S2O5) was mixed to stirring solution of 2,3-diaminonaphthalene (3.12 mmol) and substituted aromatic aldehyde (3.16 mmol) in DMF (15 ml). The reaction mixture was refluxed at 110oC for 4 hr. The reaction progress was monitored by TLC. After completion of reaction, the reaction contents were cooled at room temperature. Then cold distilled water was added with vigorous shaking till the precipitates of solid residue. It was kept aside on an ice-bath to settle down the precipitates. The solid was filtered and washed with distilled water to obtain product in good yield. Recrystallization from ethanol afforded pure 2,3-diaminonaphthalenimidazole derivatives (65-99).

124

The structures of all synthesized compounds were elucidated with the help of 1H-NMR and EI spectroscopy techniques. All compounds also give satisfactory elemental (CHN) analysis.

H O 10 NH 6 1 2 11 N Na2S2O5 + H R R o 2 DMF, 110 C 12 NH N3 2 Refluxed 13 9 (63) (64) (65 - 99)

Scheme-21 Synthesis of 2,3-diaminonaphthalenimidazole derivatives (65-99)

125

Table-1: Synthesis of 2,3-diaminonaphthalenimidazole derivatives (65-99) Comp. R Comp. R 65 72 2' 3' 5'

6' CF3 2' 7' 6' 5' N1' 8' H 2' 66 OCH3 73 3' 2'

NO2 OC2H5 6' 5' 6' 5' 3' 67 2' 74 OCH3 2' OH OH 6' 5' 6' OCH3

68 OCH3 75 OCH2C6H5 2' 2' 3'

OCOCH3 4'

6' 5' 6' 5'

69 H3CO 76 F 3' 3'

4' OCH3

6' 5' 6' Br

70 H3CO 77 OH 3' 2'

OCH3 4'

6' 6' 5' OCH3 71 HO 78 2' 3' 3' OCH2C6H5

4' 6' 5' 6' Cl

126

2' 3' 79 OC2H5 86 2' SCH3 OCH3 6' 5' 6' 5'

80 O2N 87 O2N 3' 3'

OH 4'

6' 5' 6' 5' 81 3' 4' 88 1' 8' 7' 5' 6' S 3' 4' 5' 2' 3' OCH3 82 89 CH3 2' N OCH3 CH 6' 5' 3 6' 5'

83 HO 90 HO OCH3 3'

OH 4'

6' 5' 5' Br

84 H3CO OCH3 91 Br 3'

OCH 3 OCH3

6' 5' 6' OCH3 85 HO OH 92 2' 3'

4' OH 6' 5' 6' 5'

127

93 C2H5O 94 H3CO OCH3 3'

4' 4'

6' 5' 6' 5'

95 H3CO 96 Br 3' 2'

Br OCH3

6' 6' 5' OCH3

97 F 98 Cl OCH3 3'

Br 4'

6' 5' 6' 5' 99 Br 2'

6' 5'

128

2.3.3. General structure elucidation of compounds (65-99)

H 10 6 2' 3' N1 11 2 NO2 12 N 13 9 3 6' 5'

Figure-8: Structure of compound (73)

The structure of compound (73), taken as a representative example, was established through spectroscopic techniques. The 1H-NMR was carried out in

DMSO-d6 on 300 MHz. The signal of a singlet at  13.31 confirmed a proton of –NH and a singlet of two protons, H-6 and 9 was appeared at  8.12. Signals of doublet of doublet at  8.04 (J = 6.4, 3.2 Hz) and at  7.41 (J = 6.4, 3.2 Hz) identified H-10/13 and H-11/12 respectively of naphthalene moiety where as presence of two doublets one at  8.44 and other on  8.55 with same coupling constant (J = 8.7 Hz) indicates two protons H-2/ 6 and next two protons H-3/ 5 of phenyl ring respectively. The structure of synthetic compound (73) was also determined by fragmentation patterns, through EI-MS spectra where the molecular ion peak at m/z =

289 leads to molecular formula C17H11N3O2. A peak at m/z = 259 is due to the loss of NO groups, subsequent loss of CO group from this fragment give a peak at m/z = 231.

Where as the peak appeared due to loss of nitro group (–NO2 ) at m/z = 243. The EI- MS spectrum also displayed a peak at m/z = 288, obtained by the loss of Hydrogen from the molecular ion. The loss of nitro phenyl group was determined by the peak at m/z = 167. The peak at m/z = 141 was obtained by the subsequent loss of nitril group (CN) from it where as the further loss of hydrogen was indicated by the peak at m/z = 140. A peak at m/z = 122 is due to nitro phenyl ion (Figure-9). From the above discussion the compound (73) assigned as 2-(4-nitrophenyl)-1H-naphtho[2,3- d]imidazole. The structures of other synthetic compounds were elucidated in the same way.

129

NH +

N m/z = 243 N +

NO2 N -NO m/z = 288 2 -H

NH +

NO2 -NO N m/z = 289 NH + NH O N N NO2 m/z = 259 - + - CO NO2

+ NH m/z = 122 NH + N m/z = 231 N m/z = 167 -CN

N H

m/z = 141

Figure-9: Fragmentation patterns of compound (73)

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2.3.4. Biological Evaluation of Compounds (65-99)

2.3.4.1. In Vitro Tyrosinase Inhibitory Activity

The synthetic compounds (65-99) were screened for their in vitro tyrosinase inhibitory activity (for method see chapter 4). These compounds showed inhibition in the range of 52.3-136.2 µM. The compound 81 was found as the most active among the series exhibiting IC50 = 52.3±0.196 µM. The compound 85 appeared as a second highest active compound having IC50 = 66.5±0.350 µM. Moreover the compounds 65, 66, 68, 69, 71, 79, 88 and 94 showed moderate tyrosinase inhibitory activity with

IC50 = 106.8 ±0.269, 107.2 ± 0.159, 103.5± 0.210, 102.0± 0.524, 133.6± 0.215, 132.0± 0.125, 136.2± 0.159 and 125.2± 0.212 µM respectively. The activity exhibited by compounds 81 and 85 might be due to presence of thiophene nucleus and trihydroxyl groups in phenyl nucleus. The rest of compounds revealed inhibition under 50%, therefore, were not studied further for IC50. The results are depicted in Table-2.

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Table- 2 In vitro tyrosinase inhibitory activity of compounds 65-99

Compounds IC50 ± SEM Compounds IC50± SEM (µM) (µM) 65 106.8±0.269 83 NA 66 107.2±0.159 84 NA 67 NA 85 66.5±0.350 68 103.5±0.210 86 NA 69 102.0±0.524 87 NA 70 NA 88 136.2±0.159 71 133.6±0.215 89 NA 72 NA 90 NA 73 NA 91 NA 74 NA 92 NA 75 NA 93 NA 76 NA 94 125.2±0.212 77 NA 95 NA 78 NA 96 NA 79 132.0±0.125 97 NA 80 NA 98 NA 81 52.3±0.196 99 NA 82 NA Kojic acid 16.67±0.06

Key: SEM = standard error of the mean, IC50 = concentration of compounds that inhibit 50% enzyme activity, NA= not active

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2.3.4.2. In Vitro Acetylcholinesterase and Butrylcholinesterase Inhibitory Activity

All the synthesized compounds (65-99) were screened for their in vitro acetylcholinesterase and butrylcholinesterase inhibitory activity (for protocol see chater 4). These compounds were observed possessing IC50 values in the range of 22.7-85.9 µM for butrylcholinesterase inhibitory activity. Among these, compounds 65, 66 and 79 were found to be the most active for butrylcholinesterase inhibitory activity with IC50= 22.7±0.09, 35.2±0.213 and 29.3±0.121µM respectively. Their activity may be due to the presence of indole and alkoxy moiety whereas moderate activity with no structure correlation was observed for compounds 67, 81 82 and 89

(IC50= 85.9 ±0.125, 55.4± 0.121, 58.2 ±0.192 and 52.1± 0.22 µM respectively). The rest of compounds showed inhibition under 50% therefore, were not further studied. Unfortunately all compounds were found inactive for acetylcholinesterase activity. The results are displayed in Table-3 and 4.

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Table – 3 In vitro butrylcholinesterase inhibitory activity of compounds 65-99

Compounds IC50± SEM Compounds IC50± SEM (µM) (µM) 65 22.7±0.09 83 NA 66 35.2±0.213 84 NA 67 85.9±0.125 85 NA 68 NA 86 NA 69 NA 87 NA 70 NA 88 NA 71 NA 89 52.1±0.22 72 NA 90 NA 73 NA 91 NA 74 NA 92 NA 75 NA 93 NA 76 NA 94 NA 77 NA 95 NA 78 NA 96 NA 79 29.3±0.121 97 NA 80 NA 98 NA 81 55.4±0.121 99 NA 82 58.2±0.192 Galanthamine 0.5±0.001

Key: SEM = standard error of the mean, IC50 = concentration of compounds that inhibit 50% enzyme activity, NA= not active

134

Table – 4 In vitro acetylcholinesterase inhibitory activity of compounds 65-99

Compounds IC50± SEM Compounds IC50 ± SEM (µM) (µM) 65 NA 83 NA 66 NA 84 NA 67 NA 85 NA 68 NA 86 NA 69 NA 87 NA 70 NA 88 NA 71 NA 89 NA 72 NA 90 NA 73 NA 91 NA 74 NA 92 NA 75 NA 93 NA 76 NA 94 NA 77 NA 95 NA 78 NA 96 NA 79 NA 97 NA 80 NA 98 NA 81 NA 99 NA 82 NA Galanthamine 0.5±0.001

Key: SEM = standard error of the mean, IC50 = concentration of compounds that inhibit 50% enzyme activity, NA= not active

135

2.3.4.3. In Vitro Urease Inhibitory Activity

The synthesized compounds (65-99) were screened for their urease inhibitory activity according to the method described in chapter 4. Only seven compounds exhibited varying degree of urease inhibition. Compound 71 appeared as the most active and compound 90 as a second most active compound with IC50 values 34.2±0.72 and 42.43±0.65 µM. The highest activity of these compounds is probably due to the presence of hydroxyl group at position-2 in phenyl nucleus. Other compounds 65, 66,

68, 81 and 82 showed moderate urease inhibitory activity with IC50 = 92.3±0.20, 73.5±0.65, 56.3±0.78, 86.4±0.78 and 67.14±0.67µM respectively. Remaining compounds showed inhibition less than 50%, so, were not further studied. Results are displayed in Table -5.

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Table- 5 In vitro urease inhibitory activity of compounds 65-99

Compounds IC50± SEM Compounds IC50± SEM (µM) (µM) 65 92.3±0.20 83 NA

66 73.5±0.65 84 NA 67 NA 85 NA 68 56.3±0.78 86 NA

69 NA 87 NA 70 NA 88 NA 71 34.2±0.72 89 NA

NA 42.43±0.65 72 90 73 NA 91 NA 74 NA 92 NA

75 NA 93 NA

76 NA 94 NA 77 NA 95 NA 78 NA 96 NA

79 NA 97 NA 80 NA 98 NA 81 86.4±0.78 99 NA

82 67.14±0.67 Thiourea 21.7±0.12

Key: SEM = standard error of the mean, IC50 = concentration of compounds that inhibit 50% enzyme activity, NA= not active

137

2.3.4.4. In Vitro Anti-bacterial Activity

All synthetic compounds (65-99) were tested for their in vitro anti-bacterial activity by using disc diffusion method (described in chapter 4). The anti-bacterial activity was determined in comparison of streptomycine as standard, against twelve gram positive bacterial strains i.e. Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, Corynebacterium diphtheriae, Corynebacterium hoffmanii, Mycobacterium luteus ATCC 9341, Staphylococcus aureus, Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus faecalis and Streptococcus pyogenes whereas the nine gram negative bacterial strained used were Enterobacter, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, Salmonella paratyphi A, Salmonella paratyphi B, Shigella flexeneri and Shigella dysenteriae. The results were reported on the basis of diameter of zone of inhibition in mm that was appeared around the disc (7mm). All of the synthesized compounds showed varying degree of anti-bacterial activity. Only two compounds 90 and 92 showed promising activity against B. cereus creating zone of inhibition 15mm (MIC= 25 µg/ml) and 11mm zone of inhibition (MIC= 100 µg/ml) was displayed by the compound 81 against the same bacterial strain. Compounds 82, 90, 92 and 94 were found moderately active against B. subtilis showing zone of inhibition 11, 13, 13and 11mm respectively (MIC=100, 50, 50 and 100 µg/ml). Only compound 65 showed weak activity (zone of inhibition =10 mm, MIC= 100 µg/ml) against B. thuringiensis. Compound 74 was found moderately active for C. diphtheriae creating zone of inhibition=12mm (MIC= 100 µg/ml) and also exhibited activity with 11mm zone of inhibition against S. aureus. 12 mm zone of inhibition and MIC= 100 µg/ml was observed by compound 82 against C. hoffmanii. Furthermore compounds 82, 90 and 92 displayed moderate activity against S. epidermidis creating 12, 12 and 11mm zone of inhibition respectively ( MIC= 100 µg/ml each). Only compound 65 showed moderate activity for S. saprophyticus with zone of inhibition=11mm and MIC= 100 µg/ml. Two compounds 72 and 82 showed moderate activity by creating zone of inhibition 11 and 13mm against S. feacalis with

138

MIC values 100 and 50 µg/ml respectively. The rest of compounds were found weakly active against tested gram positive bacterial strains. For the activity against gram negative bacteria compounds 90, 92 and 99 showed significant activity against Enterobacter , exhibiting zone of inhibition 16, 14 and 15 mm with MIC= 25, 50 25µg/ml respectively. Compound 82 and 85 were moderately active against E. coli showing zone of inhibition 12 and 10 mm (MIC= 35 and 25µg/ml respectively). Only compound 65 was active against K. pneumoniae showing zone of inhibition 12mm and MIC=100 µg/ml. Compound 90 was moderately active against S. paratyphi A with 13 mm zone of inhibition (MIC= 100 µg/ml). Compounds 90, 92 and 99 exhibited good activity with 12, 15 and 13 mm zone of inhibition against S. dysenteriae (MIC= 100, 25 50 µg/ml). Compound 77 was the only one that was found inactive in this anti-bacterial assay. The rest of compounds were weakly active, they showed less than 50% anti-bacterial activity, hence, were not further studied. The results of anti-bacterial assay are displayed in Table- 6 and 7.

139

Table – 6 In vitro anti-bacterial activity of compounds 65- 99

Microorganisms 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 Gram positive bacteria Bacillus cereus 9 - 8 - - - 7 9 - 9 7 7 - - 9 - 11 7 - Bacillus subtilis 8 - 7 9 - 7 9 9 - 9 9 8 - - 10 - 7 11 7 Bacillus thuringiensis 10 7 7 7 7 - 8 9 - 8 ------8 - Corynebacterium diphtheriae 8 7 8 7 7 9 7 9 7 12 7 8 - - - - - 9 9 Corynebacterium hoffmanii 10 - - - 9 7 7 8 - 10 7 8 - 7 7 8 8 12 - Mycobacterium luteus ATCC 9341 8 - 8 8 - - 7 8 - 9 - - - 7 7 8 - 7 9 Staphylococcus aureus 7 7 7 8 7 - 7 9 7 11 7 8 ------Staphylococcus aureus (MRSA) 8 8 9 8 7 7 7 7 - 9 8 - - - - - 8 7 - Staphylococcus epidermidis 8 - 8 - - - 7 - - 8 7 7 - - 8 7 8 12 - Staphylococcus saprophyticus 11 7 8 - - 7 8 9 7 9 7 8 - 7 7 8 8 7 9 Streptococcus feacalis 9 7 10 7 8 - 7 11 - 10 9 8 - 9 7 8 - 13 - Streptococcus pyogenes ------8 - 10 10 10

140

Continue Table-6 Microorganisms 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 Streptomycin Bacillus cereus 7 - - - - - 15 8 15 - - 8 - - - 9 18 Bacillus subtilis - - - - - 9 13 9 13 10 11 9 7 7 9 10 18 Bacillus thuringiensis - 7 - 7 7 7 8 7 9 8 8 7 - - - 9 18 Corynebacterium diphtheriae - - - - 10 7 7 7 10 ------22 Corynebacterium hoffmanii 8 - - - - 7 10 7 9 7 7 7 7 7 7 7 22 Mycobacterium luteus ATCC 9341 7 - 7 10 - - 10 - 10 ------10 22 Staphylococcus aureus - - 7 8 9 - 10 10 - - 7 - - - - - 25 Staphylococcus aureus (MRSA) - - - 7 7 9 10 8 10 - 8 7 7 7 8 9 25 Staphylococcus epidermidis - 8 - - - 8 12 7 11 8 10 9 9 7 8 10 18 Staphylococcus saprophyticus 9 - 7 7 7 - - - - - 10 - - - - - 25 Streptococcus feacalis 7 - - - - 8 7 7 8 8 10 8 7 7 7 7 * Streptococcus pyogenes 8 9 8 7 7 - - - - 8 9 8 7 8 7 7 18

141

Continue Table-6

Microorganisms 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 Gram negative bacteria Enterobacter 9 - 10 7 - - 7 9 7 9 9 7 - - - 8 8 10 - Escherichia coli 7 7 8 - - - 7 ------8 - 12 - Klebsiella pneumoniae 12 7 11 8 7 8 8 8 7 10 8 8 - - 8 - - 9 - Pseudomonas aeruginosa ------10 - - - - - 8 7 7 7 7 - Salmonella typhi - 7 ------7 7 8 - 8 8 Salmonella paratyphi A 7 - 8 ------7 7 Salmonella paratyphi B ------8 -

Shigella flexeneri 7 ------Shigella dysenteriae 7 7 8 8 7 7 7 7 7 7 8 8 - - - 8 10 10 9

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Continue Table-6

Gram negative bacteria 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 Streptomycin Enterobacter - - - 7 - - 16 - 14 ------15 22 Escherichia coli 7 10 - 9 8 8 ------10 Klebsiella pneumoniae - - - 7 ------18 Pseudomonas aeruginosa ------8 7 - - 7 7 - 7 - - 16 Salmonella typhi - 9 7 7 7 - 8 7 - - 7 7 7 - 7 - 15 Salmonella paratyphi A ------13 7 9 - 7 - - - - - * Salmonella paratyphi B ------17 Shigella flexeneri ------7 8 8 ------* Shigella dysenteriae - - - 8 - 7 12 7 15 8 10 8 10 10 9 13 10 key: values are zone of inhibition (mm) and an average of triplicate, (-) indicates resistance. * Means not applied.

143

Table-7 MIC (minimum inhibitory concentration) values of compounds

Microorganism MIC values of compounds (µg/ml) 65 67 72 74 79 81 82 83 85 87 88 90 92 93 94 96 97 99 Gram positive bacteria Bacillus cereus - - - - - 100 - - - - - 25 25 - - - - - Bacillus subtilis - - - - 100 - 100 - - - - 50 50 100 100 - - - Bacillus 100 ------thuringiensis Corynebacterium - - - 100 ------100 - 100 - - - - - diphtheriae Corynebacterium 100 - - 100 - - 100 - - - - 100 ------hoffmanii Mycobacterium ------100 - 100 100 - - - - - luteus ATCC 9341 Staphylococcus - - - 100 ------100 100 - - - - - aureus Staphylococcus ------100 - - - - 100 100 - 100 - - - epidermidis Staphylococcus 100 ------100 - - - saprophyticus Streptococcus - 100 100 100 - - 50 ------100 - - - feacalis Streptococcus ------100 100 ------pyogenes

144

Microorganism MIC values of compounds (µg/ml) 65 67 72 74 79 81 82 83 85 87 88 90 92 93 94 96 97 99 Gram negative ------bacteria Enterobacter - 100 ------25 50 - - - - 25 Escherichia coli ------35 - 25 ------Klebsiella pneumoniae 100 100 - 100 ------Pseudomonas - - 100 ------aeruginosa Salmonella paratyphi ------100 ------A Shigella dysenteriae ------100 25 - 100 100 100 50

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2.3.4.5. In Vitro Anti-fungal Activity

All the synthetic compounds (65-99) were also tested for their in vitro anti-fungal activity in comparison of ketoconazole as standard (for methods see chapter 4), against Aspergillus flavis, Aspergillus nigar, Penicillium species, Rhizopus species, Candida albicans, Candida albicans ATCC, Sachromyces cerevisiae, Fusarium species, Helminthosporum, Microsporum canis, Microsporum gypsium, Trichophyton rubrum, Trichophyton tonsurans and Trichophyton mentagrophytes. The compounds 65, 66, 67, 68, 74, 80 and 82 were found exhibiting moderate activity against C. albicans with zone of inhibition in the range of 9-11mm. Compound 74 also showed moderate activity against Fusarium species, M. canis, and T. rubrum with 9, 9 and 11 mm zone of inhibition. Whereas the compound 92 showed moderate activity with zone of inhibition 10, 10 and 9 mm against S. cerevisiae, Fusarium species and T. tonsurans. All the rest of synthesized compounds showed less than 50% zone of inhibition against all the applied fungal strains. Therefore, they were not further studied. Anti-fungal results are presented in Table-8.

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Table – 8 In vitro anti-fungal activity of compounds 65- 99

Microorganisms 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 Aspergillus flavis ------Aspergillus nigar ------Penicillium species ------Rhizopus species ------Candida albicans 9 11 10 10 7 8 - 7 - 10 - - - - 7 9 8 10 8 Candida albicans ATCC ------Sachromyces cerevisiae 7 - 7 ------7 ------Fusarium species 8 7 ------9 ------Helminthosporum 7 8 ------8 ------Microsporum canis - 7 ------9 - - - - 7 7 - 8 - Microsporum gypsium 7 ------Trichophyton rubrum - 7 7 - - 7 - 8 - 11 - - - 7 - - 7 8 - Trichophyton tonsurans ------Trichophyton ------mentagrophytes

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Continue Table-8

Microorganisms 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 Ketoconazole Aspergillus nigar ------24 Penicillium species ------24 Rhizopus species ------22 Candida albicans - 8 - 7 7 8 ------7 22 Candida albicans ATCC ------22 Sachromyces cerevisiae - - - - - 7 7 - 10 - 8 - - - - - 24 Fusarium species ------10 ------22 Helminthosporum ------22 Microsporum canis ------22 Microsporum gypsium ------24 Trichophyton rubrum - - - 8 ------24 Trichophyton tonsurans ------9 ------22 Trichophyton ------22 mentagrophytes key: values are zone of inhibition (mm) and an average of triplicate, (-) indicates resistance.

148

2.3.4.6. In Vitro Anti-oxidant Activity

All the synthesized compounds (65-99) were screened for their in vitro antioxidant activity against 1, 1-diphenyl-2-picryl hydrazil (DPPH) radical along with Ascorbic acid as a standard by using protocol described in chapter 4. The compounds 65, 68, 77, 90 and 99 showed significant anti-oxidant activity having

% inhibition 71.1, 67.4, 71, 71, and 69.3% with EC50 = 37.5, 75, 37.5, 37.5, and 75 µg/ml respectively. Whereas compounds 79, 82, 85 and 95 exhibited moderate anti-oxidant activity with % inhibition of 57.8, 57.8, 57.9 and 57.7 having EC50 = 100 µg/ml for each compound. The rest of compounds revealed % inhibition less than 50%, therefore, were not further studied. The results are shown in Table-9.

149

Table–9 In vitro anti-oxidant activity of compounds 65-99

Compds % Inhibition + SD EC50 Compds % Inhibition + SD EC50 (µg/ml) (µg/ml) 65 71.1 + 0.01 37.5 83 43 + 0.01 - 66 43 + 0.01 - 84 13 + 0.01 - 67 27 + 0.01 - 85 57.9 + 0.02 100 68 67.4 + 0.01 75 86 17 + 0.01 - 69 43 + 0.01 - 87 32 + 0.01 - 70 43 + 0.01 88 22 + 0.01 - 71 29 + 0.01 - 89 33 + 0.01 - 72 13 + 0.01 - 90 71.1 + 0.01 37.5 73 47 + 0.01 - 91 45 + 0.01 - 74 12 + 0.01 - 92 43 + 0.01 - 75 19 + 0.01 - 93 29 + 0.01 - 76 29 + 0.01 - 94 43 + 0.01 - 77 71 + 0.01 37.5 95 57.7 + 0.01 100 78 47 + 0.01 - 96 49 + 0.01 - 79 57.8 + 0.01 100 97 23 + 0.01 - 80 47 + 0.01 - 98 27 + 0.01 - 81 32 + 0.01 - 99 69.3 + 0.01 75 82 57.8 + 0.02 100 Ascorbic 80 8.3 acid

Key: SD = standard deviation, EC50 = effective concentration of compounds that scavange 50% radical, (-) indicates resistance.

150

2.3.5. Conclusion

All the synthesized derivatives of 2, 3-diaminonaphthalenimidazole (65-99) were screened for their various biological activities. Only ten compounds showed tyrosinase inhibitory activity in which two compounds 81 and 85 were found most active and the rest of eight compounds 65, 66, 68, 69, 71, 79, 88 and 94 were moderately active. In acetyl and butrylcholinesterase inhibitory assay, total seven compounds exhibited butrylcholinesterase activity in which three compounds 65, 66 and 79 showed good activity while four compounds 67, 81, 82 and 89 were moderately active. Unfortunately all candidates were found inactive towards acetylcholinesterase inhibition activity. Total seven compounds were found active in urease inhibitory assay. Two compounds 71 and 90 exhibited good activity and rest of five 60, 65, 66, 81 and 82 were moderately active. The antimicrobial activity of all the synthesized compounds was also determined. The compounds 90 and 92 were found significantly active against nearly all applied bacterial strains, whereas 65, 72, 74, 77, 81, 82, 85, 94 and 99 have moderate antibacterial activity. It is found that 65, 66, 67, 68, 74, 80, 82 and 92 were the candidates having moderate antifungal activity. The synthesized compounds were also tested for antioxidant activity. They displayed varying % inhibition, among them 65, 68, 77, 90 and 99 exhibited remarkable antioxidant activity. In view of these biological assays we can conclude that the above cited candidates may serve as lead compounds for further studies.

151

2.4. Experimental

152

2.4.1 General Experimental

Dimethylsulphoxide (DMSO-d6) with trimethylsilane (TMS) as an internal standard. Thin layer chromatography (TLC) was carried out on precoated silica gel glass plates (Kieselgel 60, 254, E. Merck, Germany), UV visualized chromatograms at 254 and 365 nm. The elemental (CHN) analysis was done on a Carlo Erba Strumentazion-Mod-1106, Italy.

2.4.2 General method for the synthesis of compounds (65-99)

In a typical reaction, sodium metabisulfite (Na2S2O5) was mixed to a stirring solution of 2,3-diaminonaphthalene (3.12 mmol) and substituted aromatic aldehyde (3.16 mmol) in DMF (15 ml). The reaction mixture was refluxed at 110oC for 4hr. The reaction progress was monitored by TLC. After completion of reaction, the reaction contents were cooled at room temperature. Then cold distilled water was added with vigorous shaking till the precipitated of solid residue. It was kept aside on an ice-bath to settle down the precipitates. The solid was filtered and washed with distilled water to obtain product in good yield. Recrystallisation from ethanol afford pure 2,3-diaminonaphthalenimidizole derivatives.

All compounds were synthesized applying same methodology. The structures of all synthesized compounds were determined with the help of 1H- NMR and EI-MS spectroscopy techniques. All compounds also gave satisfactory elemental (CHN) analysis.

153

2.4.2.1. 2-(1H-indol-3-yl)-1H-naphtho[2,3-d]imidazole (65)*

1 Yield: 0.2g (70%); M.P: 289-291ºC; H-NMR: (500 MHz, DMSO-d6):  13.71

(1H, bs, -NH), 12.21 (1H, s, H-1′), 8.45 (1H, br.d , J5’,6’ = 6.0 Hz, H-5′), 8.44 (1H, s, H-2′), 8.13 (2H, s, H-6/9), 8.08 (2H, dd, J10/11,12= 6.4, 3.2 Hz, H-10/13), 7.60

(1H, br.d, J8’,7’ = 6.0 Hz, H-8′), 7.44 (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12), 7.30 (2H, m, H-6′/7′); EI-MS: m/z (rel. abund. %), 283 (M+, 44), 282 (10), 149 (15),

141 (10), 140 (11), 135 (38), 115 (9), 71 (34), 44 (100); Anal. Calcd for C19H13N3 (283. 11): C, 80.54; H, 4.62; N, 14.83; Found: C, 80.52, H, 4.61; N, 14.85

2.4.2.2 2-(4-ethoxy-3-methoxyphenyl)-1H-naphtho[2,3-d]imidazole (66)*

1 Yield: 0.18g (58 %); M.P: 265-267ºC; H-NMR (400 MHz, DMSO-d6):  13.22

(1H, bs, -NH), 8.16 (2H, s, H-6/9), 8.07 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13),

7.87 (2H, dd, J6’/5’,2’ = 8.4, 2.0 Hz, H-2′/6′), 7.43 (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-

11/12), 7.23 (1H, d, J5’,6’ = 8.4 Hz, H-5′), 4.14 (2H, q, J = 7.2 H2, - OCH2), 3.92

(3H, s, -OCH3), 1.37 (3H, t, J = 7.2 Hz, - CH3); EI-MS: m/z (rel. abund. %), 318 (M+, 100 %), 303 (6), 289 (57), 288 (15), 275 (13), 273 (7), 261 (31), 260 (13),

168 (5), 141 (7), 140 (18); Anal. Cald for C20H18N2O2 (318.14): C, 75.45; H, 5.70; N, 8.80; Found: C, 75.43; H, 5.69; N, 8.78.

2.4.2.3 4-(1H-naphtho[2,3-d]imidazole-2-yl)phenol (67) 1 Yield: 0.15g (47 %): M.P: 268–269ºC; H-NMR (400 MHz, DMSO-d6):  10.51

(1H, s, -NH), 8.15 (2H, d, J2’,3’ = 8.4 Hz, H-2′/6′) 8.12 (2H, s, H-6/9), 8.07 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13), 7.44 (2H, dd, J11/10,13= 6.4, 3.2 Hz, H-11/12), + 7.04 (2H, d, J3’,2’ = 8.4 Hz, H-3′/5′); EI-MS: m/z (rel. abund %), 260 (M , 100),

231 (11), 141 (10), 140 (27), 130 (19), 120 (6); Anal. Calcd for C17H12N2O (260.09): C, 78.44; H, 4.65; N, 10.76; Found: C, 78.42; H, 4.64; N, 10.78.

2.4.2.4 2-methoxy-4-(1H-naphtho[2,3-d]imidazol-2-yl)phenyl acetate (68)* o 1 Yield: 0.17g (56 %): M.P: 244-245 C; H-NMR (400 MHz, DMSO-d6):  10.81

(1H, s, -NH), 8.22 (2H, s, H-6/9), 8.08 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13),

154

8.02 (1H, d, J2’/6’ = 2.0 Hz, H-2′), 7.89 (1H, dd, J6’/5’,2’ = 8.4, 2.0 Hz, H-6′), 7.44

(2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12), 7.40 (1H, d, J5’/6’ = 8.4 Hz, H-5′), 3.94 + (3H, s, OCH3), 2.31 (3H, s, -COCH3); EI-MS: m/z (rel. abund. %), 332 (M , 87), 298 (6), 291 (56), 290 (100), 289 (54), 275 (23), 192 (5), 141 (8), 140 (17); Anal.

Calcd for C20H16N2O3 (332.12); C, 72.28; H, 4.85; N, 8.43; Found: C, 72.23; H, 4.84; N, 8.44

2.4.2.5 2-(5-bromo-2-methoxyphenyl)-1H-naphtho[2,3-d]imidazole (69)*

1 Yield: 0.19g (57 %); M.P: 230-232ºC; H-NMR (300 MHz, DMSO-d6):  12.27

(1H, s, -NH), 8.49 (1H, d, J6’,4’ = 2.4 Hz, H-6′), 8.19 (1H, s, H-6), 8.05 (1H, s, H-

9), 8.01 (2H, m, H-10/13), 7.96 (1H, dd, J4’/3’,6’ = 8.0, 2.4 Hz, H-4′), 7.35 (2H, m,

H-11/12), 7.28 (1H, d, J3’,4’ = 8.0 Hz, H-3′), 4.05 (3H, s, -OCH3); EI-MS: m/z (rel. abund. %), 352 (M+, 100), 351 (38), 337 (18), 322 (23), 308 (6), 272 (9), 243 (26), 170 (10), 168 (18), 154 (8), 141 (14), 140 (50), 107 (11), 75 (6); Anal. Calcd for

C18H13BrN2O (352.02); C, 61.21; H, 3.71; N, 7.93; Found: C, 61.23; H, 3.70; N, 7.91

2.4.2.6 2-(2, 5-dimethoxyphenyl)-1H-naphtho[2,3-d]imidazole (70)*

1 Yield: 0.18g (61%); M.P: 225-226ºC; H-NMR (300 MHz, DMSO-d6):  12.22

(1H, s, -NH) 8.18 (2H, s, H-6/9), 8.05 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13),

7.92 (1H, d, J6’,4’ = 2.4 Hz, H-6′), 7.42 (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12),

7.28 (1H, d, J3’,4’ = 8.0 Hz, H-3′), 7.20 (1H, dd, J4’/3’,6’ = 8.0, 2.4 Hz, H-4′), 4.02 + (3H, s, -OCH3), 3.83 (3H, s, -OCH3); EI-MS: m/z (rel. abund. %), 304 (M , 98), 303 (20), 289 (13), 286 (100), 274 (42), 246 (8), 144 (14), 140 (11), 107 (6), 91

(8); Anal. Calcd for C19H16N2O2 (304.12); C, 74.98; H, 5.30; N, 9.20; Found: C, 74.99; H, 5.30; N, 9.21

155

2.4.2.7 4-Chloro-2-(1H-naphtho[2,3-d]imidazol-2yl)phenol (71)

1 Yield: 0.19g (67 %); M.P: 289-290ºC; H-NMR (400 MHz, DMSO-d6):  13.30

(1H, s, -NH), 8.25 (1H, d, J6’,4’ = 2.4 Hz, H-6′), 8.09 (2H, s, H-6/9), 8.06 (2H, m,

H-10 /13), 7.49 (2H, m, H-11/12), 7.48 (1H, dd, J4’/3’,6’ = 8.8, 2.4 Hz, H-4′), 7.10 + (1H, d, J3’,4’ = 8.8 Hz, H-3′); EI-MS: m/z (rel. abund. %), 294 (M , 100), 258 (10),

140 (26), 111 (8), 76 (9); Anal. Calcd for C17H11ClN2O (294.06); C, 69.28; H, 3.76; N, 9.50; Found: C, 69.26; H, 3.77; N, 9.49

2.4.2.8 2-(4-trifluoromethylphenyl)-1H-naphtho[2,3-d]imidazole (72)*

1 Yield: 0.17g (60 %): M.P: 255-257ºC; H-NMR (300 MHz, DMSO-d6):  13.29

(1H, s, -NH), 8.50 (2H, d, J2’,3’ = 8.4 Hz, H-2′/6′), 8.24 (2H, s, H-6/9), 8.10 (2H, m, H-10/13), 8.06 (2H, d, J3’,2’ = 8.4 Hz, H-3′/ 5′), 7.46 (2H, m, H-11/12); EI-MS: m/z (rel. abund. %), 312 (M+, 100), 293 (7), 274 (5), 243 (6), 173 (15), 171 (7), 157 (13), 141 (10), 140 (28), 127 (6), 114 (23), 77 (3); Anal. Calcd for

C18H11F3N2 (312.09); C, 69.23; H, 3.55; N, 8.97; Found: C, 69.22; H, 3.53; N, 8.96

2.4.2.9 2-(4-nitrophenyl)-1H-naphtho[2,3-d]imidazole (73)

1 Yield: 0.19g (67.8%); M.P: 283-285ºC; H-NMR (300 MHz, DMSO-d6):  13.31

(1H, s, -NH), 8.55 (2H, d, J3’,2’ = 8.7 Hz, H-3′/5′), 8.44 (2H, d, J2’,3’ = 8.7 Hz, H-

2′/6′), 8.12 (2H, s, H-6/9), 8.04 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13), 7.41 (2H, + dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12); EI-MS: m/z (rel. abund. %), 289 (M , 100), 288 (5), 259 (36), 243 (73), 231 (11), 167 (9), 141 (5), 140 (15), 122 (9); Anal.

Calcd for C17H11N3O2 (289.09); C, 70.58; H, 3.83; N, 14.53; Found: C, 70.56; H, 3.82; N, 14.54

156

2.4.2.10 2, 6-dimethoxy-4-(1H-naphtho[2,3-d]imidazol-2-yl)phenol (74)*

1 Yield: 0.21 g (64 %); M.P: 282-284 ºC; H-NMR (400 MHz, DMSO-d6): 10.99

(1H, s, -NH), 8.17 (2H, s, H-6/9), 8.07 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13),

7.63 (2H, s, H-2′/6′), 7.44 (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12), 3.92 (6H, s, 2× + –OCH3); EI-MS: m/z (rel. abund. %), 320 (M , 100), 319 (15), 304 (7), 289 (10),

273 (9), 140 (11), 115 (8); Anal. Calcd for C19H16N2O3 (320.12); C, 71.24; H, 5.03; N, 8.74; Found: 71.22; H, 5.02; N, 8.75

2.4.2.11 2-(3-Benzyloxyphenyl)-1H-naphtho[2,3-d]imidazole (75)*

1 Yield: 0.19g (52 %); M.P: 232 – 234ºC; H-NMR (400 MHz, DMSO-d6):  9.96

(1H, s, -NH), 8.17 (2H, s, H-6/9), 8.04 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13),

7.96 (1H, br, s, H-2′), 7.87 (1H, d, J6’,5’ = 8.0 Hz, H-6′), 7.56 (1H, m, H-5′), 7.47

(5H, m, Ar-H), 7.41 (2H, m, H-11/12) 7.27 (1H, dd, J4’/5’,6’ = 8.0, 2.0, Hz, H-4′), + 5.25 (2H, s, -OCH2); EI-MS: m/z (rel. abund. %), 350 (M , 60), 349 (6), 259

(10), 231 (12), 140 (11), 92 (10), 91 (100), 65 (8); Anal. Calcd for C24H18N2O (350.14); C, 82.26; H, 5.18; N, 7.99; Found: C, 82.27; H, 5.19; N, 7.98

2.4.2.12 2-(2-fluoro-4-methoxyphenyl)-1H-naphtho[2,3-d]imidazole (76)*

1 Yield: 0.08g (56 %); M.P: 178–179ºC; H-NMR (300 MHz, DMSO-d6):  12.46

(1H, s, -NH), 8.24 (1H, d, J6’,5’ =8.2 Hz, H-6′), 8.17 (2H, s, H-6/9), 8.00 (1H, s, H-

3′), 7.98 (2H, m, H-10/13), 7.37 (2H, m, H-11 /12), 7.08 (1H, d, J5’,6’= 8.2 Hz, H- + 5′), 3.88 (3H, s, -OCH3); EI-MS: m/z (rel. abund. %), 292 (M , 100), 291 (6), 277

(45), 249 (27), 152 (5), 141 (7), 140 (18), 125 (9); Anal. Calcd for C18H13FN2O (292.10); C, 73.96; H, 4.48; N, 9.58; Found: C, 73.95; H, 4.46; N, 9.57

2.4.2.13 2-methoxy-5-(1H-Naphtho[2,3-d]imidazol-2-yl) phenol (77)*

1 Yield: 0.2g (85 %); M.P: 287–288ºC; H-NMR (400 MHz, DMSO-d6):  10.55

(1H, s, -NH), 8.12 (2H, s, H-6/9), 8.05 (2H, dd, J10/11,12 = 6.4, Hz, 3.2, H-10/13),

157

7.73 (2H, m, H-2′/6′), 7.43 (2H, dd, J11/10,13 = 6.4, 3.2, Hz, H-11/12), 7.21 (1H, d, + J5’,6’ = 8.4 Hz, H-5′), 3.89 (3H, s, -OCH3); EI-MS: m/z (rel. abund. %), 290 (M , 100), 289 (5), 275 (8), 274 (35), 247 (19), 141 (7), 140 (10), 123 (11), 108 (9);

Anal. Calcd. for C18H14N2O2 (290.11); C, 74.47; H, 4.86; N, 9.65; Found: C, 74.45; H, 4.87; N, 9.63

2.4.2.14. 2-(4-benzyloxyphenyl)-1H-naphtho[2,3-d]imidazole (78)*

1 Yield: 0.91g (52 %); M.P: 289–290ºC; H-NMR (400 MHz, DMSO-d6):  9.85

(1H, s, -NH), 8.23 (2H, d, J2’,3’ = 8.8 Hz, H-2′/6′), 8.14 (2H, s, H-6/9), 8.06 (2H, dd, J10/11,12 = 6.4, 3.2, Hz, H-10/13), 7.44 (7H, m, Ar-H), 7.35 (2H, d, J3’,2’ = 8.8 + Hz, H-3′/ 5′), 5.25 (2H, s, -OCH2); EI-MS: m/z (rel. abund. %), 350 (M , 48), 270 (6), 259 (41), 231 (28), 141 (7), 140 (26), 92 (15), 91 (100), 65 (32); Anal. Calcd for Anal. Calcd for C24H18N2O (350.14); C, 82.26; H, 5.18; N, 7.99; Found: C, 82.29; H, 5.20; N, 8.00

2.4.2.15. 2-(3-ethoxy-4-methoxyphenyl)-1H-naphtho[2,3-d]imidazole (79)*

1 Yield: 0.09 g (55 %); M.P: 274–275ºC; H-NMR (300 MHz, DMSO-d6):  9.99

(1H, s, -NH), 8.16 (2H, s, H-6/9), 8.05 (2H, dd, J10/11,12 = 6.4, 3.2, Hz, H-10/13),

7.86 (2H, br. s, H-2′/6′), 7.45 (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12), 7.25 (1H, d, J5’,6’ = 8.8 Hz, H-5′), 4.14 (2H, q, J = 6.9 Hz, -OCH2), 4.19 (3H, s, -OCH3), + 1.37 (3H, t, J = 6.9 Hz, -CH3); EI-MS: m/z (rel. abund. %), 318 (M , 100), 317 (5), 287 (5), 273 (7), 258 (12), 243 (9), 230 (10), 168 (6), 152 (11), 141 (9), 140

(43), 123 (11), 115 (13); Anal. Calcd for C20H18N2O2 (318.14); C, 75.45; H, 5.70; N, 8.80; Found: C, 75.44; H, 5.71; N, 8.79

2.4.2.16. 4-(1H-naphtho[2,3-d]imidazol-2-yl)-3-nitrophenol (80)*

1 Yield: 0.09 g (60 %); M.P: 274–276ºC; H-NMR (300 MHz, DMSO-d6):  11. 26

(1H, s, -NH), 8.14 (1H, s, H-3′), 8.10 (2H, s, H-6/9), 8.04 (2H, dd, J10/11,12 = 6.4,

3.2 Hz, H-10/13), 7.42 (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12), 7.23 (1H, d, J5’,6’

158

= 8.0, Hz, H-5′), 7.12 (1H, d, J6’,5’ = 8.0, Hz, H-6′); EI-MS: m/z (rel. abund. %), 305 (M+, 55), 304 (52), 288 (19), 259 (8), 257 (7), 141 (13), 140 (100), 138 (5),

120 (9), 114 (24), 107 (10), 92 (12), 74 (20); Anal. Calcd. For C17H11N3O3 (305. 08); C, 66.88; H, 3.63; N, 13.76; Found: C, 66.86; H, 3.65; N, 13.77

2.4.2.17. 2-(thiophen-2-yl)-1H-naphtho[2,3-d]imidazole (81)

1 Yield: 0.13g (65 %); M.P: 264 – 265ºC; H-NMR (300 MHz, DMSO-d6):  11. 06

(1H, s, -NH), 8.04 (2H, s, H-6/9), 8.00 (3H, m, Ar-H) 7.84 (1H, d, J5’,4’= 4.2 Hz,

H-5), 7.38 (2H, dd, J11/10.13 = 6.4, 3.2, Hz, H-11/12), 7.28 (1H, t, J4’,5’ = 4.2 Hz, H-4); EI-MS: m/z (rel. abund. %), 250 (M+, 100), 249 (5), 141 (18), 140 (22), 114

(43), 109 (15), 97 (13), 83 (14), 64 (57); Anal. Calcd for C15H10N2S (250.06); C, 71.97; H, 4.03; N, 11.19; Found: C, 71.96; H, 4.04; N, 11.20

2.4.2.18. 2-(3, 4-dimethoxyphenyl)-1H-naphtho[2,3-d]imidazole (82)*

1 Yield: 0.12g (61 %); M.P: 184 – 185ºC; H-NMR (300 MHz, DMSO-d6): 

10.61(1H, s, -NH), 8.06 (2H, s, H-6/9), 8.01 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-

10/13), 7.85 (1H, br. s, H-2), 7.82 (1H, d, J6’,5’ = 8.0 Hz, H-6), 7.38 (2H, dd,

J11/10,13 = 6.4, 3.2 Hz, H-11/12), 7.20 (1H, d, J5’,6’ = 8.0 Hz, H-5), 3.90, (3H, s, - + OCH3), 3.86 (3H, s, -OCH3); EI-MS: m/z (rel. abund. %), 304 (M , 100), 303 (12), 289 (15), 273 (9), 261 (32), 242 (7), 218 (31), 192 (15), 168 (10), 163 (11), 141 (21) 140 (95), 138 (12), 121 (8), 113 (40), 91 (5), 75 (13); Anal. Calcd for

C19H16N2O2 (304.12); C, 74.98; H, 5.30; N, 9.20; Found: C, 74.96; H, 5.31; N, 9.22

2.4.2.19. 4-(1H-naphtho[2,3-d]imidazol-2-yl)benzene-1, 3-diol (83)

1 Yield: 0.15 g (52 %); M.P: 262 – 264ºC; H-NMR (300 MHz, DMSO-d6):  13.10

(1H, br. s, -NH ), 8.06 (2H, s, H-6/9), 8.01 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10

/13), 7.92 (1H, d, J6,5 = 8.0, H-6), 7.40 (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12),

6.50 (1H, dd, J5’/6’,3’ = 8.0, 2.0 Hz, H-5), 6.42 (1H, d, J3’,5’ = 2.0 Hz, H-3); EI-

159

MS: m/z (rel. abund. %), 276 (M+, 100), 275 (6), 219 (61), 168 (10), 141 (12), 140

(27), 115 (25), 109 (16), 79 (22), 75 (8); Anal. Calcd. for C17H12N2O2 (276. 09); C, 73.90; H, 4.38; N, 10.14; Found: C, 73.91; H, 4.37; N, 10.13.

2.4.2.20. 2-(2, 3, 4-trimethoxyphenyl)-1H-naphtho[2,3-d]imidazole (84)*

o 1 Yield: 0.17 (72 %); M.P: 166–167 C; HNMR (300 MHz, DMSO-d6):

s, -NH8.20 (2H, s, H-6/9), 8.07 (2H, dd, J10/11,12 = 6.3, 3.1 Hz, H–

10/13), 8.04 (1H, d, J6’,5’ = 8.0 Hz, H– 6), 7.44 (2H, dd, J11/10,13 = 6.3, 3.1 Hz, H–

11/12), 7.12 (1H, d, J5’,6’ = 8.0 Hz, H-5), 4.01 (3H, s, -OCH3), 3.93 (3H, s, - + OCH3), 3.86 (3H, s, -OCH3); EI-MS: m/z (rel. abund. %), 334 (M , 100), 330 (30), 319 (77), 303 (21), 289 (22), 193 (7), 181 (11), 167 (15), 141 (7), 140 (33);

Anal. Calcd for C20H18N2O3 (334.13); C, 71.84; H, 5.43; N, 8.38; Found: C, 71.83; H, 5.40; N, 8.40

2.4.2.21. 4-(1H-naphtho[2,3-d]imidazol-2-yl)benzene-1, 2, 3-triol (85)*

o 1 Yield: 0.12 g (53 %); M.P: 284–285 C; H-NMR (400 MHz, DMSO-d6):  10.82 (1H, s, -OH), 10.65 (1H, s, -OH), 10.27 (1H, s, -OH), 8.70 (2H, s, H-6/9), 8.03

(2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13), 7.42 (2H, dd, J11/10,13 =6.4, 3.2 H-11/12),

7.09 (1H, d, J65 = 8.4 Hz, H-6) Hz), 6.51 (1H, d, J5,6 = 8.4 Hz, H-5); EI-MS: m/z (rel. abund %), 292 (M+, 100) 262 (12), 168 (9), 151 (5), 140 (14), 126 (13),

115 (23), 79 (12); Anal. Calcd for C17H12N2O3 (292.08); C, 69.86; H, 4.14; N, 9.58; Found: C, 69.85; H, 4.14; N, 9.57

2.4.2.22. 2-(4-methylthiophenyl)-1H-naphtho[2,3-d]imidazole (86)*

o 1 Yield: 0.16 g (83 %); M.P: 290–291 C; H-NMR (400 MHz, DMSO-d6):

br.s.,-NH), 8.20 (2H, d, J2’,3’ = 8.4 Hz, H-2/6), 8.17 (2H, s, H–

6/9), 8.05 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H–10/13), 7.52 (2H, d, J3,2 = 8.4 Hz, H–

3/5), 7.44 (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H–11/12), 2.58 (3H, s, -CH3); EI-MS: m/z (rel. abund. %), 290 (M+, 100), 275 (8), 243 (20), 141 (9), 140 (42), 113 (16),

160

75 (5); Anal. Calcd for C18H14N2S (290.09); C, 74.45; H, 4.86; N, 9.65; Found: C, 74.44; H, 4.84; N, 9.66

2.4.2.23. 2-(2-nitrophenyl)-1H-naphtho[2,3-d]imidazole (87)

o 1 Yield: 0.12 g (57 %): M.P: 250–252 C; H-NMR (400 MHz, DMSO-d6):  13.07 (1H, s, -NH), 8.09 (2H, m, H-3/6), 8.06 (2H, s, H-6/9), 8.01 (2H, m, H-10/13),

7.90 (1H, t, J4/5,6 = 7.6 Hz, H-4), 7.81 (1H, t, J5/4,6 = 7.6 Hz, H-5), 7.40 (2H, m, H-11/12); EI-MS: m/z (rel. abund. %), 289 (M+, 100), 273 (6), 259 (15), 243 (18),

141 (12), 140 (93), 107 (5), 90 (9), 77 (13), 63 (48); Anal. Calcd for C17H11N3O2 (289.09); C, 70.58; H, 3.83; N, 14.53; Found: C, 70.59; H, 3.82; N, 14.54

2.4.2.24. 2-(naphthalen-2-yl)-1H-naphtho[2,3-d]imidazole (88)*

o 1 Yield: 0.15 g (78 %); M.P: 241–243 C; H-NMR (400 MHz, DMSO-d6):  br s, -NH8.89 (1H, br. s, H-1), 8.36 (1H, m, H-6), 8.22 (2H, s, H- 6/9), 8.18 (2H, m, H-7/8), 8.09 (2H, m, H-10/13), 7.69 (3H, m, H-3/4/5), 7.55 (2H, m, H-11/12); EI-MS: m/z (rel. abund. %), 294 (M+, 100), 293 (41), 153 (12),

140 (42), 127 (22), 114 (31); Anal. Calcd for C21H14N2 (294.12): C, 85.69; H, 4.79; N, 9.52; Found: C, 85.67; H, 4.80; N, 9.50

2.4.2.25. N, N-dimethyl-4-(1H-naphtho[2,3-d]imidazol-2-yl)aniline (89)

o 1 Yield: 0.15g (85 %); M.P: 298–299 C; H-NMR (400 MHz, DMSO-d6):

s, -NH 8.12 (2H, d, J2,3 = 8.0 Hz, H-2/6), 8.07 (2H, s, H-6/9),

8.04 (2H, dd, J10/11,12 = 6.5, 3.1 Hz, H-10/13), 7.44 (2H, dd, J11/10,13 = 6.5, 3.1 Hz,

H-11/12), 6.91 (2H, d, J3, 2 = 8.0 Hz, H-3/5), 3.00 (6H, s, -N (CH3)2); EI-MS: m/z (rel. abund. %), 287 (M+, 100), 286 (20), 272 (15), 271 (21), 244 (13), 146 (12), 141 (8), 140 (31), 121 (10), 113 (23), 105 (8), 90 (6), 76 (7); Anal.Calcd for

C19H17N3 (287.14); C, 79.41; H, 5.96; N, 14.62; Found: C, 80.00; H, 5.90; N, 14.60

161

2.4.2.26. 3-bromo-6-methoxy-2-(1H-naphtho[2,3-d]imidazol-2-yl)phenol (90)*

o 1 Yield: 0.17 g (77 %); M.P: 209–210 C; H-NMR (400 MHz, DMSO–d6):

s, -NH 8.12 (2H, s, H-6/9), 8.03 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-

10/13), 7.40 (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12), 7.21 (1H, d, J5’,4’ = 8.0 Hz,

H-5′), 7.09 (1H, d, J4’,5’ = 8.0 Hz, H-4′), 3.86 (3H, s, -OCH3); EI-MS: m/z (rel. abund. %), 368 (M+, 50), 367 (20), 289 (5), 259 (7), 243 (15), 229 (8), 169 (6),

152 (13), 141 (9), 140 (47), 76 (10); Anal. Calcd. for C18H13Br N2O2 (368.02); C, 58.56; H, 3.55; N, 7.59; Found: C, 57.69 ; H, 3.57; N, 7.58.

2.4.2.27. 2-(2-bromo-4, 5-dimethoxyphenyl)-1H-naphtho[2,3-d]imidazole (91)*

o 1 Yield: 0.14 g (58 %): M.P: 241–242 C; H-NMR (400 MHz, DMSO–d6):

s, -NH 8.20 (2H, s, H-6/9), 8.08 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-

10/13), 7.48 (1H, s, H-6′), 7.44 (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12), 7.39 (1H, s, H-3′), 3.89 (3H, s, -OCH3), 3.84 (3H, s, -OCH3); EI-MS: m/z (rel. abund. %), 382 (M+, 100), 381 (21), 367 (15), 351 (12), 339 (10), 322 (7), 303 (6), 242 (6), 217 (17), 216 (16), 168 (8), 141 (13), 140 (92), 115 (26), 74 (5); Anal. Calcd for

C19H15Br N2O2 (382.03); C, 59.55; H, 3.95; N, 7.31; Found: C, 59.99; H, 3.97; N, 7.29

2.4.2.28. 2-phenyl-1H-naphtho[2,3-d]imidazole (92)

o 1 Yield: 0.14 g (95 %); M.P: 280–281 C; H-NMR (400 MHz, DMSO-d6):

s, -NH 8.29 (2H, dd, J2’/3’,4’ = 7.2, 3.6 Hz, H-2′/6′), 8.21 (2H, s, H-

6/9), 8.08 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13), 7.68 (3H, m, H-3′/4′/5′), 7.44 + (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12);EI-MS: m/z (rel. abund. %), 244 (M , 72), 243 (6), 141 (10), 140 (28), 114 (29), 104 (5), 89 (6), 77 (99); Anal Calcd for

162

C17H12N2 (244.10); C, 83.58; H, 4.95; N, 11.47; Found: C, 83.61; H, 4.91; N, 11.45

2.4.2.29. 2-(2-ethoxyphenyl)-1H-naphtho[2,3-d]imidazole (93)*

o 1 Yield: 0.13 g (73 %); M.P: 196–197 C; H-NMR (400 MHz, DMSO–d6): 

s, -NH8.26 (1H, m, H-6′), 8.12 (2H, s, H-6/9), 8.01 (2H, dd, J10/11,12

= 6.3, 3.3 Hz, H-10/13), 7.50 (1H, m, H-5′), 7.38 (2H, dd, J11/10,13 = 6.3, 3.3 Hz,

H-11/12), 7.29 (1H, d, J3’,4’ = 8.0 Hz, H-3′), 7.14 (1H, t, J4’,3’ = 8.0 Hz, H-4′),

4.37 (2H, q, J = 6.8 Hz, -OCH2), 1.47 (3H, t, J = 6.8 Hz,-CH3); EI-MS: m/z (rel. abund. %), 288 (M+, 69), 287 (14), 273 (83), 259 (4), 244 (100), 243 (7), 231 (17),

168 (7), 141 (17), 140 (82), 107 (5), 91 (9), 76, (6); Anal. Calcd for C19H16N2O (288.13); C, 79.14; H, 5.59; N, 9.72; Found: C, 79.12; H, 5.63; N, 9.69

2.4.2.30. 2-(2, 3-dimethoxyphenyl)-1H-naphtho[2,3-d]imidazole (94)*

o 1 Yield: 0.18 g (96 %); M.P: 247–248 C; H-NMR (400 MHz, DMSO-d6):  12.26 (1H, s, -NH), 8.12 (2H, s, H-6/9), 7.99 (2H, m, H-10/13), 7.90 (1H, m, H-6′), 7.37

(2H, dd, J11/10,13 = 6.4, 3.2 Hz, H-11/12), 7.26 (2H, m, H-4′/5′), 3.90 (3H, s, - + OCH3), 3.89 (3H, s, -OCH3); EI-MS: m/z (rel. abund. %), 304 (M , 100), 303 (16), 289 (25), 274 (6), 273 (19), 258 (10), 243 (11), 169 (8), 140 (9); Anal. Calcd for C19H16N2O2 (304.12); C, 74.98; H, 5.30; N, 9.20; Found: C, 75.00; H, 5.30; N, 9.19

2.4.2.31. 2-(4-bromo-2, 5-dimethoxyphenyl)-1H-naphtho[2,3-d]imidazole (95)*

o 1 Yield: 0.18 g (94 %); M.P: 246–248 C; H-NMR (400 MHz, DMSO-d6):  12.15 (1H, s, -NH), 8.17 (2H, s, H-6/9), 8.04 (3H, m, H-10/13/6′), 7.58 (1H, s, H-3′),

7.42 (2H, m, H-11/12), 4.05 (3H, s, -OCH3), 3.94 (3H, s, -OCH3); EI-MS: m/z (rel. abund. %), 384 (M+ 2, 100), 381 (23), 382 (96), 367 (20), 352 (38), 335 (6),

163

242 (9), 217 (31), 168 (5), 141 (6), 140 (26), 74 (7); Anal. Calcd for C19H15 Br

N2O2 (382.03); C, 59.55; H, 3.95; N, 7.31; Found: C, 59.59; H, 3.97; N, 7.35

2.4.2.32. 2-(3-bromo-4-methoxyphenyl)-1H-naphtho[2,3-d]imidazole (96)*

o 1 Yield; 0.20g (95 %); M.P: 218–220 C; H-NMR (400 MHz, DMSO-d6):  12.47

(1H, s, -NH), 8.51 (1H, d, J2’,6’ = 2.0 Hz, H-2′), 8.29 (1H, dd, J6’/5’,2’ = 8.4, 2.0 Hz,

H-6′), 8.14 (2H, s, H-6/9), 8.06 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13), 7.43 (2H, m, H-11/12), 7.39 (1H, d, J5’,6’= 8.4 Hz, H-5′), 3.97 (3H, s, -OCH3); EI-MS: m/z (rel. abund. %), 352 (M+, 100), 351 (5), 337 (12), 309 (6), 273 (4), 258 (13), 154

(6), 141 (7), 140 (21), 74 (3); Anal. Calcd for C18H13BrN2O (352.02); C, 61.21; H, 3.71; N, 7.93; Found: C, 61.18; H, 3.69; N, 7.96

2.4.2.33. 2-(4-bromo-2-fluorophenyl)-1H-naphtho[2,3-d]imidazole (97)*

o 1 Yield: 0.15 g (72 %); M.P: 211–213 C; H-NMR (300 MHz, DMSO–d6): 10.49

(1H, br s, -NH), 8.28 (1H, d, J6’,5’ = 8.4 Hz, H-6′), 8.22 (2H, s, H-6/9), 8.02 (2H, m, H-10/13), 7.88 (1H, d, J3’,5’ = 2.1 Hz, H-3′), 7.68 (1H, dd, J5’/6’,3’ = 8.4, 2.1 Hz, H-5′), 7.39 (2H, m, H-11/12); EI-MS: m/z (rel. abund. %), 340 (M+, 90), 187 (5), 172 (7), 156 (6), 141 (8), 140 (23), 80 (4), 78 (72), 63 (70); Anal. Calcd for

C17H10Br F N2 (340.00); C, 59.85; H, 2.95; N, 8.21; Found: C, 59.83; H, 2.99; N, 8.18

2.4.2.34. 2-(2-Chloro-3-methoxyphenyl)-1H-naphtho[2,3-d]imidazole (98)*

o 1 Yield: 0.15 g (75 %). M.P: 148-149 C; H-NMR (400 MHz, DMSO-d6):  10.98

(1H, s, -NH), 8.16 (2H, s, H-6/9), 8.04 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13),

7.52 (2H, m, H-5′/6′), 7.42 (3H, m, H-11/12/4′), 3.95 (3H, s, -OCH3); EI-MS: m/z (rel. abund. %), 308 (M+, 100), 307 (5), 293 (4), 276 (3), 265 (19), 242 (5), 169 (3), 155 (6), 141 (7), 140 (18), 114 (7), 113 (7), 78 (27); Anal. Calcd for

C18H13ClN2O (308.07); C, 70.02; H, 4.24; N, 9.07; Found: C, 70.00; H, 4.21; N, 9.09

164

2.4.2.35. 2-(3-bromophenyl)-1H-naphtho[2,3-d]imidazole (99)

o 1 Yield: 0.16 g (80 %); M.P: 180–181 C; H-NMR (300 MHz, DMSO-d6): 11.88

(1H, s, -NH), 8.48 (1H, br. s, H-2′), 8.29 (1H, d, J6’/5’ = 7.8 Hz, H-6′), 8.21 (2H, s,

H-6/9), 8.08 (2H, dd, J10/11,12 = 6.4, 3.2 Hz, H-10/13), 7.83 (1H, d, J4’,5’ = 7.8 Hz,

H-4′), 7.62 (1H, t, J5’/4’,6’ = 7.8 Hz, H-5′), 7.44 (2H, dd, J11/10,13 = 6.4, 3.2 Hz, H- 11/12); EI-MS: m/z (rel. abund. %), 322 (M+, 100), 321 (3), 243 (20), 214 (10); 183 (9), 162 (18), 155 (8), 141 (7), 140 (31), 121 (19), 114 (12), 75 (5); Anal.

Calcd for C17H11BrN2 (322.01); c, 63.18; H, 3.43; N, 8.67; Found: C, 63.20; H, 3.45; N, 8.65

* indicates newly synthesized compounds.

165

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172

CHAPTER-3 Synthesis and Bioactivities of Amides of Piperic Acid

173

Introduction

174

3.1 INTRODUCTION OF AMIDES

An amide is an organic compound that comprises of a trivalent nitrogen atom attached to a carbonyl group. The nitrogen may bear substitution [1].

O O O

R NH2 R NHR' R NR'R''

1 2 3

Where: R,R and R′′ = alkyl groups

Figure-1: General structure of amides

An amide is named after the name of its related carboxylic acid by attaching the word “amide” at the end in place of -oic or -ic acid. Where as the alkyl substituent attached to nitrogen atom is placed preceded by N- alkyl.

O O CH3

H3C NH2 H3C N H IUPAC: ethanamide N-methyl ethanamide Trivial: acetamide N-methyl acetamide 4 5

Figure-2: Naming of amides

Cyclic amide is known as Lactams. The ring size is designated by Greek letters (3), (4), (5),  (6) and  (7) [2].

N O H 6

Figure-3: Structure of Butyrolactam

175

Followings are classes of compounds related to amides.

Class of Basic structure Compounds O

Urea N N

O O Imide N H O Carbamate O N

Sulfonamide S N O

Figure-4: Classes of compounds related to amides

Urea (7) which is excreted by higher animals is the excess nitrogenous product of metabolism of protein. Lower animals excrete ammonia (8) where as birds and reptiles excrete guanidine (9). These excreted substances are good and chief nitrogen fertilizers [1-3].

Figure-5 Structures of Urea, Ammonia and Guanidine

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3.1.1 Importance of Amides 3.1.1.1 Biological importance of Amides

Amides are very important in biological system. They play a vital role in protein synthesis which are polyamides and build the body structure as well as perform various functions in the organism. Glutamine (10) that enhances immune system is a common amino acid found in proteins. Niacinamide (11) is vitamin

B3, it helps in production of natural emollient to keep skin hydrated [4]. Most of amides of naturally occurring endogenous fatty acid serve as chemical messengers. Anandamide (12) is a cannabinoid receptor in mammalian brain and display analgesic effects [5]. Oleamide (13) that induces physiological sleep is brain lipid, accumulated in cerebrospinal fluid [6]. Another endogenous fatty acid amide, erucamide (14) stimulates new blood vessel formation [7].

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Figure-6: Structures of natural amides

Thyrotropic–hormone releasing factor (TRF) (15) is a tripeptide. It was isolated from the hypothalamus glands of cattle. It stimulates thyroid hormone secretion. Another brain peptide is enkephalins (16). It is body natural painkiller, comprises of at least five amino acid residues [8].

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O H N O N N NH2

H O O NH

15 Tyr-gly-gly-phe-met

Where: Tyr = tyrosine, gly = glycine, phe = phenylalanine, met = methionine

Figure-7: Structure of TRF and Enkephalins

3.1.1.2 Medicinal importance of Amides Amide bond and amide derivatives are widely involve in both naturally occurring and synthetic biologically active compounds. Several researchers elucidated their biological activities like antimicrobial [9], antitumor [10], antituberculosis [11], insecticidal [12], anti-inflammatory [13], anticonvulsant [14] and anti-platelet [15] activities, hence are pharmacologically active in the treatment of many diseases, for examples, meprobamate (17) is a tranquillizer, ampicillin (18) has been used as an antibiotics and sulfathiazole (19) is a potent bacteriostatic sulfadrug [1]. A naturally occurring alkaloid, piperine (20) is the chief component of Piper nigrum Linn., and is reported to possessed valuable therapeutic actions like anti-depressent [16], antioxidant [17], anti-inflammatory, anti-carcinogenic, antimicrobial [18] functions. Some researches also evaluated its property as bioavailability enhancer for many drugs [19, 20].

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Figure-8: Structures of biologically active compounds containing amide bond

Piperine (20) also exhibited phytotoxic effect on many vegetables [21]. Not only piperine but also its synthetic analogues have been evaluated for their biological properties. Some researchers examined isobutylamide containing analogues exhibited potency towards insecticidal activity [22, 23]. Synthetic analogue (21) examined for trypanocidal activity against Trypanosoma cruzi which causes incurable disease in humans ‘chagas’ [24]. A novel synthesized piperamide (22) is an antioxidant and antidepressant agent [25]. Its derivative piperlonguminine (23) has shown inhibitory activity against histone deacetylase and human colon cancerline HCT-116, where as a hydroxamic moiety containing analogue (24) exhibited in vitro cytotoxicity and HDAC activity [26].

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Figure-9: Structures of synthetic piperamides

3.1.1.3 Industrial application of Amides

Amides are also utilized in industry for photographic materials, as polymer stabilizers, pigments, foaming materials, photo developers [27, 28] and for conformational switching [29].

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3.2 Synthetic Approaches Towards Amides of Piperic acid 3.2.1 Design and synthesis of bioconjugates of piperic acid–glycine

Bioconjugates of piperoyl glycine were designed and synthesized by condensation of an activated ester of piperic acid (25) with glycine (26) in presence of dicyclohexylcarbodiimide (DCC) and dimethylaminopropionitrile (DMAP). The reaction mixture stirred for 3 hr. at room temp. to get product (27). All conjugates exhibited good antibacterial and antifungal activity than cefepime and fluconazole (Scheme-1) [30].

O OH O DCC/DMAP + H2N r.t., 3hr. O O

26 25 O

O OH N H O O 27

Scheme-1

3.2.2 Synthesis and insecticidal activity of piperamides

Amide derivatives of piperine were synthesized from piperic acid (28) coupling with amine (29) in presence of oxalyl chloride (COCl)2 in dry tetrahydrofuran (THF). The reaction mixture was stirred for 6 hr. at room temperature. The pure product (30) was obtained after purification by flash column chromatography on silica gel (diethyl ether – Hexane; 1: 2). All the synthetic amides were tested for their insecticidal property. The results revealed mortality range from 0 to 97.5% depending on compound and insects (Scheme-2) [31].

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O (COCl)2, THF O o OH + RR'NH 25 C, 6h

O 28 29 O

O R N

R' O 30 R = H, Et, i-pr R' = aliphatic or alicyclic alkyl group

Scheme-2

3.2.3 Synthesis and anti-leishmanial property of bioconjugates of piperoyl- amino acid

Piperoyl amino acid conjugates were synthesized by reaction of piperic o acid (28) with amino acid methyl ester (31) in dry CH2Cl2 at -15 C with drop wise addition of triethylamine (TEA) following methanesulfonyl chloride and then stirred at 0oC for 4 hr. After aqueous work up crude product was subjected to column chromatography on silica gel (CHCl3 – MeOH with increasing polarity) to afford pure product as piperoyl–amino acid conjugate (32). Evaluation of synthetic compounds for their anti-leishmanial activity exhibited better activity than either amino acid methyl ester and piperine (Scheme-3) [32].

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O COOMe CH Cl , OoC O 2 2 OH + H2N R CH3SO2Cl, TEA

O 28 31 O COOMe

O N R H O 32

R = CH2C6H5, CH(CH3)2 H2C-C6H4-p-OH

Scheme-3

3.2.4 Synthesis of piperine analogues and study of their structure activity relationship An attempt was made to obtain piperine analogues from piperic acid (28) and an appropriate amine (33) using SOCl2 as coupling agent in CH2Cl2 (DCM) with stirring of 1hr. The crude product (34) that was obtained after aqueous workup was purified by CC over SiO2 with mobile phase pet.ether–EtOAc (4:1). The synthesized analogues were related with modification in parent molecule for inhibition of cytochrome P 450 activities. It was found that modification in parent molecule results lost of inhibitory potential while saturation in side chain enhanced the property (Scheme-4) [33].

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O SOCl , DCM O 2 OH 1 2 + R R NH Refluxed, 1hr. O O 28 33 O NR1R2

O 34 R1R2NH = Piperidine, pyrrolidine, n-butyl amine

Scheme-4

3.2.5 Synthesis of piperamides analogues from natural safrole

Piperamide analogues were synthesized from natural safrole (35) by applying following scheme (Scheme-5) [34].

O O OH 1- BF3, THF R. T., 2Hr. O O 2- NaOH aq PCC, DCM 35 Reflux 36 1 hr

COH O CO2Et O

KH, DME, O 38 O triethylphos- 37 Aq. LiOH, THF phonoacetate 4 hr O

O O CO2H N

1- SOCl2, Reflux O 40 O 1 hr X X = CH2, O and S 39 2- RNH2, DCM R.T., 30 min

Scheme-5

185

3.3. Results and Discussion

186

3.3.1 Chemistry

The importance and applications of amides of piperic acid have been revealed from above literature discussion. One of naturally occurring piperidine amide of piperic acid, commonly known as piperine is well known for various pharmacological uses. Many researchers have synthesized its analogues and evaluated them for their application in medicinally and industrially valuable fields. In view of this literature survey we assumed the possibility of compounds which are similar in structure to that of piperine and may possesses activities like it. Here in this research work we aim to synthesise amides of piperic acid by aliphatic and aromatic amines in order to get compounds which are more potent in their antimicrobial, antioxidant and nematicidal activities.

3.3.2 General method for the synthesis of compounds (42-56) The amides (42-56) were synthesized from piperic acid (28) (obtained by basic hydrolysis of commercially available piperine (20) through the formation of an active intermediate acid chloride (41) and its subsequent condensation with appropriate amine. The resulted products were afforded in good yields (Scheme- 6).

Generally the preparation of amides comprises of two steps: (i) conversion of acid to acid chloride and (ii) condensation of acid chloride with an appropriate amines.

3.3.2.1 Preparation of piperic acid

Piperine (20) (5g, 0.0175 mol) was refluxed with 300 ml of 20% KOH in methanol at 150oC for 120 hr. Completion of reaction was monitored on TLC. After complete hydrolysis excess of methanol was removed under reduced pressure. The reaction worked up by suspending the residue in hot distilled water followed by acidification with 2N HCl. The resulted precipitates were filtered, washed with ice cold water and dried at room temperature. The crude product was

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purified through recrystallisation from ethanol to afford pale yellow crystals of pure piperic acid (28), m.p. 215-216 oC, [Lit. 215 oC] yield 94% [32].

3.3.2.2 Conversion of piperic acid to acid chloride

Piperic acid (28), 0.5g (0.00229 mol) was dissolved in dry dichloromethane (DCM) 15 ml kept over an ice-bath under nitrogen atmosphere, stirred for 10 min. After that freshly distilled thionyl chloride (SOCl2) 2ml (0.27 mol) was added dropwise and stirred the resultant solution for 2 hr till the reaction mixture indicated orange-brown colouration. Completion of reaction was confirmed by TLC. The excess thionyl chloride was then removed under vaccuo leaving acid chloride (41) as an orange-brown residue.

3.3.2.3 Synthesis of amides of piperic acid by condensation of acid chloride with an amine

To the resultant solution of crude acid chloride (41) in dry DCM 10 ml, appropriate amine 0.7 ml (0.585 g, 0.00688 mol) dissolved in 5 ml DCM was added. The resultant reaction mixture was refluxed at 60oC for 2-2.5 hr. Completion of reaction was monitored on TLC (n-Hexane-EtOAc, 7:3). The content was diluted with distilled water. The organic layer was separated out through separating funnel. Washed with distilled water (2x25 ml), dried with anhydrous Na2SO4 and concentrated to crude product. The crude product was subjected to column chromatography (CC) over silica gel using mobile phase n- Hexane – Ethyl acetate (with increasing order of polarity) to get pure product (42- 56) in good yield. The structures of synthesized compounds (42-56) were determined by IR, 1H-NMR and EIMS spectroscopy. All compounds give satisfactory elemental (CHN) analysis.

188

O O

O 20 % KOH O N OH 150oC, 120 hr O O refluxed 28 20 N2 atmosphere, Dry DCM

SOCl2 2hr, stirr over ice bath O O 2 Amine 7 5 3 R O O 8 6 Dry DCM Cl 1 N 4 o 2 1 60 , 2-2.5hr R O 11 O 9 refluxed 41 10 42-56

Scheme-6

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Table-1: Synthesis of amides of piperic acid (42-56)

Compd - NR1R2 M.P. M.P. Yield No. oC oC [lit] % 2' 3'

42 N 4' 130-131 132 [35] 74 6' 5'

2' 3'

43 N O 159-160 162 [36] 89 6' 5'

2' 3'

44 N N CH3 178-180 185 [36] 61 6' 5'

H N 45 102-105  81 H

H 2' 3'

46 N 4' 173-175 174 [36] 87

6' 5'

H 2' 3'

47 N CH3 190-191  81

6' 5'

H 2' 3'

48 N Cl 118-120  80

6' 5'

H 2' 3'

49 N OCH3 163-165 167 [36] 51

6' 5'

190

2' 4' H N 50 3' 128-129 151 [36] 76

H3C 3' 51 265-269  92 H N CH3

5' H3C

2' 3'

52 H N N N CH3 70-71  72

6' 5'

2' 3' 53 N 141-143 142 [33] 67 4' 5'

54 115-117  75

2' 3'

55 H N 4' 170-173 177 [36] 36

6' 5'

OCH3 2' 56 201-203  41 H N CH3

6' 5'

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3.3.3. General Structure Elucidation of Compounds (42-56)

O 2' 3' 7 5 3 O 8 6 NH N N CH3 4 2 6' 5' O 9 11 10

Figure-10: Structure of compound (52)

The structure of compound (52) (as a representative example) was established through spectroscopic techniques. The 1H-NMR was carried out in deutarated methanol on 300 MHz. A singlet at  5.97 indicates two protons of methylene dioxy group (O-CH2-O). A doublet and a double doublet at  6.10 (J = 15.0 Hz) and 7.38 (J = 15.0, 10.0 Hz) show trans protons H-2 and H-3. Where as a multiplet in range of 6.79-7.09 indicates olefinic (H-4, H-5) and aromatic protons (H-7, 10, 11). A singlet at  2.91 indicates three protons of methyl (N-

CH3). It also shows signals at  3.50 and 3.35 as two broad singlets of –N (CH2)2

(H-2′, 6′) and (CH2)2 N (H-3′, 5′) respectively. The synthetic compound (52) was also confirmed by EIMS showing a molecular ion peak at m/z = 315 which leads to molecular formula C17H21N3O3. Ion at m/z = 300 appeared due to loss of methyl group similarly peak at m/z 201 appeared due to loss of amine linkage. It further gives a peak at m/z 173 by the loss of CO group. In addition a peak at m/z = 114 corresponds to piprazine moiety, subsequent loss of methyl give peak at m/z = 99. The IR spectrum displayed peaks at 3330 (N-H), 1628-1700 (C=C, C=O) and 1194 (C-O). The structures of other compounds were elucidated in the same way.

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Figure-11: Fragmentation patterns of compound (52)

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3.3.4. Biological Evaluation of Compounds (42-56)

3.3.4.1 In Vitro Anti-bacterial Activity

The anti-bacterial activity of all the synthesized compounds 42-56 was determined by using disc diffusion method (see chapter 4). Susceptibility test in vitro was performed against bacterial strain i.e. Streptococcus pneumoniae, Proteus vulgaris, Streptococcus aureus, Pseudomonas stutzeri, Pseudomonas aeruginosa and Escherichia coli. Streptomycine was taken as standard for comparison of antibacterial activity under similar conditions. The results of anti- bacterial activity of synthesized compounds were reported on the basis of diameter of zone of inhibition in mm that was appeared around the disc. All of the synthesized compounds showed varying degree of antibacterial activity. None of the synthesized compounds showed antibacterial activity against S. pneumoniae. Compound 54 showed significant anti-bacterial activity against all remaining strains creating highest zone of inhibition 30 mm against S. aureus, second highest inhibition zone 29 mm against P. stutzeri and 25 mm against P. vulgaris, 20 mm against E. coli and moderate inhibition zone 19 mm against P. aeruginosa. Compound 49 did not showed anti-bacterial activity against all strains except P. vulgaris where it produced significant zone of inhibition 30 mm. Compound 50 showed moderate anti-bacterial activity against S. aureus with zone of inhibition 14 mm. It also showed moderate anti-bacterial activity with zone of inhibition 16 mm against P. stutzeri and P. aeruginosa. Compound 53 was found active against P. stutzeri (zone of inhibition = 16mm). Compound 44 showed anti-bacterial activity with zone of inhibition 10 mm against P. stutzeri and 14 mm against P. aeruginosa. Compound 52 showed excellent zone of inhibition against P. aeruginosa and E. coli (18 and 14 mm). On the other hand compounds 46, 47 and 48 exhibited good anti-bacterial activity against P. aeruginosa with zone of inhibition in the range of 12-16 mm. Compound 56 showed moderate anti- bacterial activity with 16 mm zone of inhibition against E. coli. The results of anti-bacterial activity are given in Table-2.

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Tabel-2: In vitro anti-bacterial activity of compounds 42-56

Compound No.

Microorganisms 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Streptomycin Streptococcus pneumoniae ------35 Proteus vulgaris ------30 - - - - 25 - - 30 Streptococcus aureus ------14 - - - 30 - - 30 Pseudomonas stutzeri - - 10 - - - - - 16 - - 16 29 - - 15 Pseudomonas aeruginosa - 7 14 - 16 16 12 - 16 - 18 - 19 - - 15 Escherichia coli ------14 - 20 - 16 20

Key: Values are diameter of zone of inhibition (mm) and an average of triplicate, (-) indicates resistant.

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3.3.4.2 In Vitro Anti-fungal Activity

All the synthesized compounds (42-56) were evaluated for their anti- fungal activity against saprophytic fungi (Rhizopus, Aspergillus niger, Aspergillus flavus) and yeast (Candida albican). The anti-fungal activity was determined by disc diffusion method as in chapter 4, using ketoconazol as standard. Only compound 54 showed weak anti-fungal activity while the rest of the compounds found inactive. So, were not further studied. The results of anti-fungal activity are displayed in Table-3.

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Tabel-3: In vitro anti-fungal activity of compounds 42-56

Compound No.

Microorganisms 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Ketoconazole Rhizopus ------7 - - 22 Aspergillus niger ------5 - - 24 Aspergillus flavus ------5 - - 24 Candida albican ------6 - - 22

Key: values are diameter of zone of inhibition (mm) and an average of triplicate, (-) indicates resistant.

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3.3.4.3 In Vitro Nematicidal Activity

The nematicidal activity of synthesized compounds (42-56) was determined by applying the method as described in chapter 4. The nematicidal activity was tested against second stage juveniles of root-knot nematode Meloidogyne incognita while the toxicity was found by the determination of their lethal concentration (LC) at LC20,

LC50 and LC90 mg/ml (i.e. the lethal concentration at which 20, 50 and 90% nematodes become immobile). Compound 42 showed highest mortality (98 ± 0.02%) with good LC50 value 4.2 mg/ml. where as compounds 52 and 56 showed excellent mortality rate (96 ± 0.03 and 95 ± 0.01 % respectively) having LC50= 3.4 and 3.5mg/ml. The compounds 43, 47 and 45 also exhibited good mortality rate (92 ±

0.03, 92 ± 0.04 and 90 ± 0.02%) with LC50= 5.8, 4.4 and 3.0 respectively. Beside these the compounds 44, 54 and 50 showed mortality rate 83 ± 0.02, 82 ± 0.02 and

80 ± 0.02 % respectively, with favorable LC50 value 5.8, 4.5 and 3.8 mg/ml respectively . Results are shown in Table-4.

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Table-4: In vitro nematicidal activity of compounds 42-56.

Compounds Mortality LC20 LC50 LC90 No. Rate % mg/ml mg/ml mg/ml 42 98 ± 0.02 3.6 4.2 5.2 43 92 ± 0.03 4.1 5.8 6.0 44 83 ± 0.02 4.2 5.8 6.6 45 90 ± 0.02 2.6 3.0 4.4 46 20 ± 0.02 20.2 26.7 31.0 47 92 ± 0.04 3.2 4.4 5.8 48 12 ± 0.02 22.4 34.3 37.6 49 14 ± 0.06 19.3 22.6 34.2 50 80 ± 0.02 3.3 3.8 4.4 51 21 ± 0.04 18.7 22.5 31.8 52 96 ± 0.03 2.5 3.4 4.1 53 13 ± 0.02 28.6 39.0 40.1 54 82 ± 0.02 3.3 4.5 4.9 55 21 ± 0.03 20.1 31.3 39.2 56 95 ± 0.01 2.2 3.5 4.3 Furadan 100

Key: values are mortality rate (%), LC = Lethal concentration (mg/ml)

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3.3.4.4 In Vitro Anti-oxidant Activity

The compounds (42-56) were screened for their in vitro anti-oxidant activity by the method used in chapter 4. Free radical solution of DPPH (1,1-diphenyl-2- picryl hydrazyl) was used to study radical scavenging activity, along with Ascorbic acid as standard, varying degree of DPPH radical scavenging activity was shown by these compounds. Compound 49, 51 and 44 exhibited significant % inhibition (80 ±

0.01, 72 ± 0.01 and 70 ± 0.02 respectively). The EC50 value of these compounds is 625, 937.5 and 937 g/ml. Compound 46 and 54 revealed % inhibition (58 ± 0.02 and

57.9 ± 0.01 respectively) showing EC50 value 100 µg/ml hence recognized as moderate antioxidant. While the rest of compounds revealed inhibition under 50%, so these compounds were not further studied. The results of anti-oxidant-activity are given in Table-5.

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Table-5: In vitro anti-oxidant activity of compounds 42-56.

Compound No. % Inhibition ± SD EC50 g/ml

42 12 ± 0.01 - 43 - - 44 70 ± 0.02 937 45 - - 46 58 ± 0.02 100 47 44 ± 0.02 - 48 - - 49 80 ± 0.01 625 50 15 ± 0.01 - 51 72 ± 0.02 937.5 52 47 ± 0.01 - 53 44 ± 0.01 - 54 57.9 ± 0.01 100 55 - - 56 40 ± 0.02 - Ascorbic acid 80 8.3

Key: Values are zone of inhibition (%) and an average of triplicate. EC50 = Effective concentration to scavange 50% of DPPH (-) indicates inactivity

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3.3.5. Conclusion

All the synthesized amide derivatives of piperic acid 42-56 were evaluated for their biological activity. Compounds 44, 46, 47, 48, 49, 50, 52, 53 and 56 exhibited significant anti-bacterial activity. On the other hand compound 54 showed marvelous anti-bacterial activity and it was the only compound that showed anti-fungal activity against all the applied strains. When tested for their nematicidal activity compounds

42, 43, 45, 47, 52 and 56 showed significant mortality rate and LC50 values against root-knot nematode i.e. Meloidogyne incognita. Hence, these candidates may serve as a fruitful nematicidal agent. Only three compounds i.e. 44, 49 and 51 showed excellent and two compounds 46 and 54 showed moderate anti-oxidant activity.

In view of these biological assays we can conclude that the above candidates may serve as a lead compounds for the further studies.

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3.4. Experimental

203

3.4.1 General Experimental

All the reagents and solvents used for synthesis were purchased from E. Merck, Germany. Melting points were determined in glass capillary using Gallen Kamp melting point apparatus and are uncorrected. EIMS spectra were recorded on JEOL JMS-600H. 1H-NMR spectra were performed on Avance AV-300, 400, 500 and 600 NMR spectrometers operating at 300, 400, 500 and 600 MHz. in deuterated methanol (MeOD) with trimethylsilane (TMS) as an internal standard. IR spectra were done on a JASCO–302-A spectrophotometer. A Carlo Erba Strumentazion- Mod-1106, Italy was used for elemental (CHN) analysis. TLC (Thin Layer Chromatography) was carried out on precoated silica gel glass plates (Kieselgel 60, 254, E. Merck. Germany). UV visualized chromatogram at 254 and 365 nm.

3.4.1 General method for the synthesis of compounds (42-56) The general synthetic procedure of amides of piperic acid comprises of following steps:

3.4.2.1 Preparation of pipeic acid

Piperine (20) (5g, 0.0175 mol) was refluxed with 300 ml of 20% KOH in methanol at 150oC for 120 hr. Completion of reaction was monitored on TLC. After complete hydrolysis excess of methanol was removed under reduced pressure. The reaction worked up by suspending the residue in hot water followed by acidification with 2N HCl. The resulted precipitates were filtered, washed with ice-cold water and dried at room temperature. The crude product was purified through recrystallisation from ethanol to afford pale yellow crystals of pure piperic acid (28), M. P. 215-216oC [Lit. 215oC], yield 94% [32].

3.4.2.2 Conversion of piperic acid to acid chloride Piperic acid (28) (0.5g, 0.00229 mol) was dissolved in dry Dichloromethane (DCM) 15 ml, kept over an ice-bath under nitrogen atmosphere, stirred for 10 min.

204

After that freshly distilled thionyl chloride (SOCl2) 2ml (0.27 mol) was added drop wise and stirred the resultant solution for 2 hr. till the reaction mixture indicated organge-brown colouration. Completion of reaction was confirmed by TLC. The excess SOCl2 was then removed under vaccuo leaving acid chloride (41) as an orange-brown residue.

3.4.2.3 Synthesis of amides of piperic acid by condensation of acid chloride with an amine

To the resultant solution of crude acid chloride (41) in 10 ml dry DCM, appropriate amine 0.7 ml (0.585 g, 0.00688 mol) dissolved in 5 ml DCM, was added. The resultant reaction mixture was refluxed at 60oC for 2-2.5 hr. Completion of reaction was monitored on TLC (n-Hexane-EtOAc, 7:3). The content was diluted with distilled water. The organic layer was separated out through separating funnel, washed with distilled water (2x25 ml), dried with anhydrous Na2SO4 and concentrated to crude product. It was subjected to column chromatography over silica gel using n-Hexane–EtOAc as mobile phase with increasing order of polarity to get pure product (42-56) in good yield.

All other compounds were synthesized applying same methodology. The structures of the synthesized compounds (42-56) were determined by IR, 1H-NMR and EIMS spectroscopy. All synthesized compounds gave satisfactory elemental (CHN) analysis.

3.4.3. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)penta-2,4-dienoic acid (Piperic acid, 28)

-1 1 Yield: 94% ; m.p: 215-216ºC; IR (KBr) νmax cm : 2940, 1673, 1597, 1447; H-NMR:

(300 MHz, DMSO-d6): δ 12.17 (1H, s, -COOH), 7.01 (1H, dd, J3/2,4 = 15.6, 8.1 Hz,

H-3), 6.89-7.32 (5H, m, olefinic and Ar-H), 6.04 (2H, s, O-CH2-O), 6.01 (1H, d, J2,3

205

= 15.6 Hz, H-2); EI-MS: m/z (rel.abund.%), 218 (M+ , 82), 201 (6), 173 (100), 143

(31), 121(2), 115 (63); Anal. Calcd for C12H10O4 (218.06): C, 66.05; H, 4.62; Found: C, 66.00; H, 4.63.

3.4.3.1. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-1-(piperidin-1-yl)penta-2,4-dien-1- one (42)

-1 1 Yield: 74% ; IR (KBr) νmax cm : 1631, 1490, 844; H-NMR: (500 MHz, MeOD); δ

7.28 (1H, dd, J3/2,4 = 15.0, 14.0 Hz, H-3), 6.94 (2H, m, H-4/5), 6.89 (2H, m, H-

10/11), 6.82 (1H, s, H-7), 6.59 (1H, d, J2,3 = 15.0 Hz, H-2), 5.95 (2H, s, O-CH2-O),

3.60 (4H, m, -N(CH2)2), 1.69 (2H, m, H-4′), 1.59 (4H, bs, H-3′/5′); EI-MS: m/z (rel. abund. %), 285 (M+, 36), 201 (71), 173 (60), 159 (13), 143 (36), 115 (100), 84 (18);

Anal. Calcd for C17H19NO3 (285.14): C, 71.56; H, 6.71; N, 4.91; Found: C, 17.54; H, 6.73; N, 4.90.

3.4.3.2. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-1-morpholinopenta-2,4-dien-1-one (43)

-1 1 Yield: 89% ; IR (KBr) νmax cm : 2941, 1633, 1491, 997; H-NMR: (500 MHz,

MeOD): δ 7.33 (1H, dd, J3/2,4 = 15.0, 14.0 Hz, H-3), 6.95 (2H, m, H-4/5), 6.89 (2H, m, H-10/11), 6.86 (1H, s, H-7), 6.58 (1H, d, J2,3 = 15.0 Hz, H-2), 5.96 (2H, s, O-CH2-

O), 3.70 (4H, m, N(CH2)2), 3.61 (4H, m, O (CH2)2); EI-MS: m/z (rel. abund. %), 287 + (M , 94), 201 (100), 173 (60), 115 (68), 86 (6); Anal. Calcd for C16H17NO4 (287.12): C, 66.89; H, 5.96; N, 4.88; Found: C, 66.87; H, 5.97; N, 4.86.

3.4.3.3. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-1-(4-methylpiperazin-1-yl)penta-2,4- dien-1-one (44)

-1 1 Yield: 61% ; IR (KBr) νmax cm : 2954, 1630, 845; H-NMR: (500 MHz, MeOD): δ

7.31 (1H, dd, J3/2,4 = 15.0, 14.0 Hz, H-3), 6.78- 7.08 (5H, m, olefinic and Ar-H), 6.59

206

(1H, d, J2,3 = 15.0 Hz, H-2), 5.95 (2H, s, O-CH2-O), 3.29 (4H, m, H-2′/6′), 2.48 (4H, + bs, H-3′/5′), 2.33 (3H, s, N-CH3): EI-MS: m/z (rel. abund. %), 300 (M , 28), 201 (23),

173 (32), 143 (16), 115 (46), 99 (48), 70 (100); Anal. Calcd for C17H20N2O3 (300.15): C, 67.98; H, 6.71; N, 9.33; Found: C, 67.97; H; 6.72; N, 9.35.

3.4.3.4. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)penta-2,4-dienamide (45)

-1 1 Yield: 81% ; IR (KBr) νmax cm : 3021, 1644, 970; H-NMR (300 MHz, MeOD): δ

7.24 (1H, dd, J3/2,4 = 15.0, 10.0 Hz, H-3), 6.77-7.08 (5H, m, olefinic and Ar-H), 6.12

(1H, d, J2,3 = 15.0 Hz, H-2), 5.95 (2H, s, O-CH2-O), 5.47 (2H, s, -NH2); EI-MS: m/z (rel. abund. %), 217 (M+, 68), 173 (100), 143 (31), 115 (80), 96 (14), 44 (8); Anal.

Calcd for C12H11NO3 (217.07): C, 66.35; H, 5.10: N, 6.45; Found: C, 66.33; H, 5.12; N, 6.44.

3.4.3.5. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-phenylpenta-2,4-dienamide (46)

-1 1 Yield: 87% ; IR (KBr) νmax cm : 3215, 1639, 890; H-NMR: (500 MHz, MeOD): δ

7.61 (2H, d, J2’,3’ = 7.5 Hz, H-2′/6′), 7.40 (1H, dd, J3/2,4 = 15.0, 10.0 Hz, H-3), 7.29-

7.08 (3H, m, H-3′/4′/5′), 6.79-7.10 (5H, m, olefinic and Ar-H), 6.25 (1H, d, J2,3 = 15.0 + Hz, H-2), 5.96 (2H, s, O-CH2O-); EI-MS: m/z (rel. abund, %), 293 (M , 12) 201 (92),

173 (11), 115 (100), 92 (3), 77 (10); Anal. Calcd for C18H15NO3 (293.11): C, 73.71; H, 5.15; N, 4.78; Found: C, 73.69; H, 5.13: N, 4.79.

3.4.3.6. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-p-tolylpenta-2,4-dienamide (47)*

-1 1 Yield: 81% ; IR (KBr) νmax cm : 3198, 1647, 929; H-NMR: (500 MHz, MeOD): δ

7.88 (1H, s, -CONH), 7.60 (2H, d, J2’,3’ = 8.5 Hz, H-2′/6′), 7.41 (1H, dd, J3/2,4 = 15.0,

10.0 Hz, H-3), 7.22 (2H, d, J3’/2’ = 8.5 Hz, H-3′/5′), 6.79-7.10 (5H, m, olefinic and

Ar-H), 6.01 (1H, d, J2,3 = 15.0 Hz, H-2), 5.96 (2H, s, O-CH2-O), 2.21 (3H, s, Ar- + CH3); EI-MS: m/z (rel. abund, %), 307 (M , 76), 217 (13), 201 (45), 187 (6), 173

207

(100), 143 (60), 115 (79); Anal. Calcd for C19H17NO3 (307.12): C, 74.25; H, 5.58; N, 4.56; Found: C, 74.24; H, 5.59; N, 4.54.

3.4.3.7. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(4-chlorophenyl)penta- 2,4-dienamide (48)

-1 1 Yield: 80% ; IR (KBr) νmax cm : 3291, 1638, 997; H-NMR: (300 MHz, MeOD): δ

7.63 (2H, d, J2’,3’ = 8.7 Hz, H-2′/6′), 7.46 (2H, d, J3’,2’ = 8.7 Hz, H-3′/5′), 7.40-6.70

(6H, m, olefinic and Ar-H), 6.23 (1H, d, J2/3 = 15.0 Hz, H-2), 5.97 (2H, s, O-CH2-O); EI-MS: m/z (rel. abund. %), 327 (M+, 13), 201 (79), 173 (18), 143 (22), 121 (32);

Anal. Calcd for C18H14ClNO3 (327.07): C, 65.96; H, 4.31; N, 4.27; Found: C, 65.93; H, 4.30; N, 4.25.

3.4.3.8. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(4-methoxyphenyl)penta-2,4- dienamide (49)

-1 1 Yield: 51% ; IR (KBr) νmax cm : 3350, 1649; H-NMR: (300 MHz, MeOD): δ 8.17

(1H, s, -CONH-), 6.70-7.47 (10H, m, olefinic and Ar-H), 6.00 (1H, d, J2,3 = 15.0 Hz,

H-2), 5.93 (2H, s, O-CH2-O), 3.76 (3H, s, -OCH3); EI-MS: m/z (rel. abund %), 323 (M+, 64), 307 (8), 293 (6), 201 (14), 173 (6), 143 (10), 121 (13), 115 (17); Anal.

Calcd for C19H17NO4 (323.12): C, 70.58; H, 5.30; N, 4.33; Found: C, 70.57; H, 5.30; N, 4.34.

3.4.3.9. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-propylpenta-2,4-dienamide (50)

-1 1 Yield: 76% ; IR (KBr) νmax cm : 3263, 1634, 1539, 787; H-NMR: (300 MHz,

MeOD): δ 7.40 (1H, dd, J3/2,4 = 15.0, 10.0 Hz, H-3), 6.78-7.10 (5H, m, olefinic and

Ar-H), 6.09 (1H, d, J2,3 = 15.0 Hz, H-2), 5.96 (2H, s, O-CH2-O), 5.91 (1H, s, -CONH-

), 3.52 (2H, m, H-2′), 1.68 (2H, m, H-3′), 1.00 (3H, t, J4’,3’ = 7.2 Hz, H-4′); EI-MS: m/z (rel. abund %), 259 (M+, 13), 218 (83), 201 (30), 173 (100), 143 (45), 115 (69);

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Anal. Calcd for C15H17NO3 (259.12): C, 69.48; H, 6.61; N, 5.40; Found: C, 69.45; H, 6.60; N, 5.41.

3.4.3.10. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-mesitylpenta-2,4-dienamide (51)* -1 1 Yield: 92% ; IR (KBr) νmax cm : 3259, 1633, 845; H-NMR: (300 MHz, MeOD): δ

7.40 (1H, dd, J3/2,4 = 15.0, 10.0 Hz, H-3), 6.79-7.11 (7H, m, olefinic and Ar-H), 6.34

(1H, d, J2,3 = 15.0 Hz, H-2), 5.97 (2H, s, -O-CH2-O) 2.25 (3H, s, p-CH3), 2.16 (6H, s, + 2 x o-CH3); EI-MS: m/z (rel. abund %), 335 (M , 57), 201 (100), 171 (34), 143 (47),

135 (26), 115 (61), 89 (9); Anal. Calcd for C21H21NO3 (335.15): C, 75.20; H, 6.31; N, 4.18; Found: C, 75.19; H, 6.32; N, 4.20.

3.4.3.11. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(4-methylpiperazin-1-yl)penta- 2,4-dienamide (52)*

-1 1 Yield: 72% ; IR (KBr) νmax cm 3330, 1628, 1194; H-NMR: (300 MHz, MeOD): δ

7.38 (1H, dd, J3/2,4 = 15.0, 10.0 Hz, H-3), 6.79-7.09 (5H, m, olefinic and Ar-H), 6.10

(1H, d, J2,3 = 15.0 Hz, H-2), 5.97 (2H, s, O-CH2-O), 3.50 (4H, bs, N-N(CH2)2), 3.35 + (4H, bs, (CH2 )2N-), 2.91 (3H, s, N-CH3); EI-MS: m/z (rel. abund %), 315 (M , 15), 300 (4), 217 (19), 201 (96), 173 (14), 143 (9), 114 (28), 99 (85); Anal. Calcd. for

C17H21N3O3 (315.16): C, 64.74; H, 6.71; N, 13.32; Found: C, 64.72; H, 6.70; N, 13.33.

3.4.3.12. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-1-(pyrrolidine-1-yl)penta-2,4-dien- 1-one (53)

-1 1 Yield: 67% ; IR (KBr) νmax cm : 1649, 1448, 929; H-NMR: (300 MHz, MeOD): δ

7.37 (1H, dd, J3/2,4 = 15.0, 10.0 Hz, H-3), 6.78-7.09 (5H, m, olefinic and Ar-H), 6.44

(1H, d, J2,3 = 15.0 Hz, H-2), 5.96 (2H, s, O-CH2-O), 3.59 (4H, m, H-2′/5′), 1.98 (4H, m, H-3′/4′); EI-MS: m/z (rel. abund %), 271 (M+, 90), 201 (100), 173 (20), 143 (14),

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115 (53), 70 (6); Anal. Calcd for C16H17NO3 (271.12): C, 70.83; H, 6.32; N, 5.16; Found: C, 70.81; H, 6.33; N, 5.17.

3.4.3.13. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(4-iodophenyl)penta-2,4- dienamide (54)*

-1 1 Yield: 75% ; IR (KBr) νmax cm : 3321, 1627, 987; H-NMR: (600 MHz, MeOD): δ

7.72 (2H, d, J2’,3’ = 8.4 Hz, H-2′/6′), 6.90 (1H, dd, J3/2,4 = 15.0, 10.0 Hz, H-3), 6.80

(2H, d, J3’,2’ = 8.4 Hz, H-3′/5′), 6.83-7.54 (5H, m, olefinic and Ar-H), 5.96 (2H, s, O-

CH2-O), 5.94 (1H, d, J2,3 = 15.0 Hz, H-2), 3.63 (1H, s, -CONH-); EI-MS: m/z (rel. abund %), 419 (M+, 8), 418 (9), 218 (41), 173 (47), 143 (23), 115 (62), 43 (100);

Anal. Calcd for C18H14NO3I (419.00): C, 51.57; H, 3.37; N, 3.34; Found: C, 51.55; H, 3.39; N, 3.35.

3.4.3.14. (2E, 4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-benzylpenta-2,4-dienamide (55)

-1 1 Yield: 76% ; IR (KBr) νmax cm : 3239, 1623; H-NMR: (400 MHz, MeOD): δ 7.29

(1H, dd, J3/2,4 = 15.0, 10.0 Hz, H-3), 6.78- 7.23 (10H, m, olefinic and Ar-H), 6.13

(1H, d, J2,3 = 15.0 Hz, H-2), 5.95 (2H, s, O-CH2-O), 4.45 (2H, s, N-CH2-); EI-MS: m/z (rel. abund. %), 307(M+, 54), 218 (52), 201 (24), 173 (92), 143 (35), 115 (77),

106 (100), 91 (37), 77 (20), Anal.Calcd for C19H17NO3 (307.12): C, 74.25; H, 5.58; N, 4.56; Found: C, 74.21; H, 5.59; N, 4.58.

3.4.3.15. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(3-methoxy-4-methylphenyl) penta-2,4-dienamide (56)*

-1 1 Yield: 71% ; IR (KBr) νmax cm : 3257, 1647; H-NMR: (300 MHz, MeOD): δ 7.40

(1H, dd, J3/2,4 = 15.0, 10.0 Hz, H-3), 6.79-7.10 (8H, m, olefinic and Ar-H), 6.26 (1H, d, J2,3 = 15.0 Hz, H-2), 5.96 (2H, s, -O-CH2-O), 3.82 (3H, s, -OCH3), 2.13 (3H, s, - + CH3); EI-MS: m/z (rel. abund. %), 337 (M , 35), 215 (25), 201 (100), 143 (34), 115

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(78); Anal. Calcd for C20H19NO4 (337.13): C, 71.20; H, 5.68; N, 4.15; Found: C, 71.17; H, 5.69; N, 4.17. * indicates newly synthesized compounds.

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3.5. References

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[1] R. J. Fessenden and J. S. Fessenden, “Organic Chemistry”, 3rd edition, Brooks / Cole Publishing Company, California (U.S.A.), p. 657-64 (1986).

[2] J. B. Hendrickson, D. J. Cram and G. S. Hammond, “Organic chemistry”, 3rd edition, Mc Graw – Hill KogaKusha, Ltd., p. 137 (1970).

[3] T. W. Graham Solomons, “Fundamentals of Organic Chemistry”, 4th edition, John Willey and Sons, Inc. (Canada), p. 740 (1994). [4] J. Clayden, “Organic Chemistry”, Oxford University Press, p. 1356 (2001). [4a] Philip Newsholme, J. Nutr., 131(9), S2515(2001). [4b] W. Gehring, J Cosmet Dermatol., 3(2), 88 (2004).

[5] A. W. Devane, L. Hanus and A. Breuer, Science, 258, 1946 (1992).

[6] H–R Salvador, L. Gombart and F. C. Benjamin, Experimental Neurology, 172(1), 235 (2001).

[7] E. K. Farrell and D. J. Merkler, Drug Discov. Today, 13(13), 558 (2008).

[8] R. J. Fessenden and J. S. Fessenden, “Organic Chemistry”, 3rd edition, Brooks / Cole Publishing Company, California (U.S.A.), p. 903 (1986).

[9] M. Moise, V. Sunel, L. Profire and M. Popa, Farmacia, LVL(3), 283 (2008).

[10] A. Warnecke, I. Fichtner and G. Sab, Archiv der Pharmazia, Chemistry in Life Sciences, 340, 8 (2007).

[11] M. I. Hegab, M. Abdel-Samee and M. Yousef, Archiv der Pharmazia, Chemistry in Life Sciences, 340(8), 396 (2007).

[12] G. T. L. Gray bill, M. J. Ross and B. R. Gauvin, Bioorganic Medicinal Chemistry Letter, 1375 (1992).

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[13] K. G. Walesa and A. P. Stec, “Medical Academy in Lublin”, 9, 118 (2003).

[14] N. Siddiqui, M. S. Alam and W. Ahsan, Acta Pharma, 58, 445 (2008).

[15] F. G. Mann and B. C. Saunders, “Practical Organic Chemistry”, 4th edition, p. 96 (2003).

[16] W. Jintanaporn, C. Pennapa, M. Supaporn and P. Aroonsri, Food and Chemical Toxicology, 46(9), 3106 (2008).

[17] K. Shashi, P. Neelima and M. K. Singh, Toxicology International, 15(2), 85 (2008).

[18] D. P. Bezerra, D. Pereira, P. Claudia and D. Loiola, Recent Progress in Medicinal Plants, 24, 91 (2009).

[19] G. Sharma and B. Mishra, J. of Pharmaceutical Research, 6(3), 129 (2007).

[20] K. Janakiraman and R. Manavalan, African J. Traditional, Complementary and Alternative Medicines [Online Computer file], 5(3), 257 (2008).

[21] W. S. Tavares, I. Cruz and S. S. Freltas, J. Medicinal Plants Research, 5(21), 5301 (2011).

[22] M. Miyakado, I. Nakayama, A. Inoue, M. Hatakoshi and N. Ohno, J. Pest Sci., 10, 11 (1985).

[23] I–K Park, S-G Lee, S–C Shin and Y–J Ahn, J. Agric. Food Chem., 50, 1866 (2002).

[24] T. S. Ribiero, L. F. Lima and J. O. Previato, Bioorganic and Medicinal Chemistry Letters, 14(13), 3555 (2004).

[25] M. K. Prashanth, K. M. Lokanatha Rai and B. Veeresh, Bioorganic and Medicinal Chemistry Letters, 22(23), 7065 (2012).

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[26] Y. Luo, H–M Liu and M-B Su, Bioorganic and Medicinal Chemistry Letters, 21, 4844 (2011).

[27] Y. Terada, N. Leda, K. Komura and Y. Sugi, Synthesis, 15, 2318 (2008).

[28] E–S Lower, Pigm Resin Technol, 22, 19 (1993).

[29] I. Azumaya, T. Okamoto, F. Imabeppu and H. Takayanagi, Tetrarhedron, 59, 2325 (2003).

[30] S. Mishra, U. Narain, R. Mishra and K. Misra, Bioorganic and Medicinal Chemistry, 13, 1477 (2005).

[31] V. F de Paula, L. C de A Barbosa, A. J. Demuner and M. C. Picanco, Pest Management Science, 56, 168 (2000).

[32] I. P. Singh, S. K. Jain, A. Kaur, S. Singh and S. K. Arora, European J Medicinal Chemistry, 45, 3439 (2010).

[33] S. Koul, J. L. Kaul, S. C. Taneja, K. L. Dhar and J. Singh, Bio-organic and Medicinal Chemistry, 8, 251 (2000).

[34] J–X de Aranjo, E. J. Barreiro, J. S. Parente and C. A. Fraga, Synthetic communication: An International J. for Rapid communication of Synthetic Organic Chemistry, 29(2), 263 (1999).

[35] M. Sheeja, A. Mathew and J. Varkey, Asian J. Pharma. Hea. Sci., 2(1), 256 (2012).

[36] P. L. Sangwan, J. L. Koul, M. V. Reddy and G. N. Qazi, Bioorganic and Medicinal Chemistry, 16, 9847 (2008).

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CHAPTER-4 Biological Activities Assays

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Introduction

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4.0. Introduction About Biological Activities

4.1. Enzymes Inhibition Activity

The enzymes are biological catalyst. They perform many physiological functions without being altered in the reactions. They possess high degree of substrate specificity. They maintain and regulate body functions and also defend against severe disease [1, 2].

Enzymes inhibition is a prime therapeutic strategy for the treatment of various infections which are caused by extra ordinary activity of different enzymes via relevant enzymes inhibitor [1-3].

The enzyme inhibitor is the drug which after binding with the relevant enzyme inhibits or reduces it’s over activity that is generating ailments. These either kill a pathogen or regulate the metabolic imbalance. The enzyme inhibitors are valuable chemotherapeutic agent and are considered as an important tool for the treatment of diverse state diseases. Chymotrypsin and trypsin (the serine proteases) are digestive enzymes. They protect cellular protein destruction. Their imbalance may cause cirrhosis, pancreatic cancer or hepatitis C [1-3].

4.1.1. Urease Inhibition Activity Urease which is a nickel containing enzyme hydrolyses urea to carbon dioxide and ammonia. Urea is a basic soil fertilizer, urease help plants, bacteria and algae in nitrogen assimilation. Ureases are also significant virulence factor implicated in the pathogenesis of hepatic coma, urinary stones and other infections. Peptic ulcer (the small erosions in digestive track) is the infection caused by gram-negative bacterium, Helicobacter pylori (H. pylori). This bacterium can transfer from one to another person via food and water. Indigestion, abdominal pain, gastric obstruction and ulcer bleeding are its main symptoms. About 5-10% world population suffer this problem. This bacterial infection produces enzymes, urease, this increasing concentration

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causes ulcer. Therefore, the urease inhibitors are of great importance in medicinal chemistry [4,5].

4.1.2. Tyrosinase Inhibition Activity Tyrosinase is a copper containing enzyme, widely distributed in every plant and animal including human. It control and catalyzes the synthesis of melanin from tyrosine which is a pigment that provides colour to skin, hair and eye. The melanin protects skin from harmful effects of uv radiations and also plays a vital role in normal vision. Tyrosinase is further responsible of browning of fruits and vegetables especially when their tissues are ruptured. Any mutation or uncontrolled activity in tyrosine gene (TYR gene) may results in impaired production of tyrosinase. It may lead to over production of melanin (melanogenesis) where it get accumulated abnormally in different parts of skin to produce pigmented patches on the skin. This hyperpigmentation of skin is an esthetic problem and the browning of fruit and vegetables is undesirable as it reduces their commercial values. These problems have encouraged researchers to develop new effective tyrosinase inhibitors for anti- browning of fruits and vegetables and to treat hyperpigmention in skin. The tyrosinase inhibitors prevent over production of tyrosinase, which in turn block abnormal synthesis of melanin. This blocking of excess formation of melanin is helpful in controlling of hyperpigmentation and browning action. Now these days’ tyrosinase inhibitors have broad application in cosmetics and food industries as skin whitening and anti-browning agents [6-9].

4.1.3. Cholinesterase Inhibition Activity

Butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) are cholinesterase enzyme found in mammalian brain.The serum BuChE is produced in liver, lungs, heart and brain while AChE in brain, muscles and erythrocyte membrane. AChE is very important part of cholinergic brain synapses. It helps in termination of impulses through hydrolysis of neurotransmitter acetylcholine. BuChE is abundantly present in the nervous system and in blood plasma. BuChE (a glycoprotein) is a hydrolase

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that also catalyse choline hydrolysis. The BuChE participates in nerve conduction and in CNS function. It also has a role in neurodegenerative disorders. In human brain BuChE is present in neurons and neuritic plaque. It is found that BuChE takes the place of AChE after its degradation. BuChE have minor whereas AChE play a major role in regulating brain acetylcholine. It is found that the Alzheimer’s disease (AD) is the result of insufficient cholinergic function in the brain. It is observed that BuChE activity increases in patients of Alzheimer’s disease (AD) whereas AChE activity decelerated or remains unchanged. Therefore, it is accepted that the inhibition of brain BuChE may weaken neurodegeneration in AD. So it is suggested that BuChE inhibitors may be favored for the treatment of AD. Moreover the development of BuChE inhibitors may lead to an effective treatment and important therapeutic goal in AD. [10-13]

4.2. Anti-microbial Activity The anti-microbial resistance is the relative insensitivity of a microorganism (bacteria, fungi, viruses) to an antimicrobial agent. This resistance is increasing in various pathogens with the passage of time, allowing them to be able to reduce the effectiveness of conventional treatment and increases the risk of complications. This results in the wide spread use of antimicrobial medicines [14, 15].

The Anti-bacterial agents destroy, kill or inhibit the growth of bacteria. In the same way the drug which selectively removes fungal pathogens with minimal toxicity from a host is known as antifungal agent. The increased implication of these antimicrobial agents renders the resistant pathogens to grow and evolve continuously with acquired resistance such as salmonella species resistant to fluroquinolones, mycobacterium tuberculosis show resistance to rifampin and staphylococcus aureus to vancomycin. Moreover, vibrio cholera and shigella species have made difficult to control infections caused by them. It has been observed that klebsiella speices resistance to carapenum and enterococcal are resistant towards erythromycin. Furthermore, the resistant microorganisms diminishes the ability of an antimicrobial medicine to

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engage with its target like oxyiminocephalalosporin resistant pseudomonas aeruginosa and aminoglycoside resistant Acinetobacter baumanii, resistance to anti- malarial drug ( chloroquine) is worldwide problem [16-23].

Similarly some medicines that prepared from aspergillus and penicillum species possess intestinal ailments. Deep-seated mycosis is due to resistant filamentous fungi ( fusarium species). The plague causing agent yersinia pestis exihibit resistance to ampicillin, where as candida glabrate shows resistance in both azole and echinocandin anti-fungal agent. Pseudomonas species have multidrug resistance while E. coli is third generation (Cephalosporin) resistant. It is also found that the spoilage and deterioration of food stuff is usually associated with fungi and bacteria. The emergence of antimicrobial resistance in a variety of pathogens results infectious diseases as a leading cause of death. So, it is a serious health and economic problem. It can be controlled by the development and delivery of new antimicrobial agents [16- 23].

4.3. Anti-oxidant Activity The anti-oxidants are imperative compounds which either inhibit or delay the oxidation process that takes place in body under the influence of atmospheric oxygen or by reactive oxygen species. The anti-oxidant species control the generation of free radical by converting them to stable harmless substance after giving an electron to them. The free radical formed attack the normal healthy cells and react with lipids, proteins and other molecules. Hence, lead severe disorders, damages and diseases associated with them like aging, cancer, heart diseases, CNS disorders, alzheimer, immune system decline, hypertension, cataract, obesity etc. [24-26].

The antioxidant scavenges and deactivates free radicals to protect body from oxidative damage and repair damaged tissue and cells. Thus, antioxidants are essential to health. Moreover they act as an anti- cancer, anti-allergic, vasodilator agent and prevent body from many ailments. Many free radicals scavenging

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substances exist within the body like vitamin C and E, β-carotenes, flavonoides, carotenoids and certain enzymes. Some have dietary sources such as fruits and vegetables. Gallic acid ester is a synthetic antioxidant. Currently radical scavengers are used for stabilization of pharmaceuticals, food and polymers [24-26].

4.4. Nematicidal Activity The nematicide is a substance or chemical pesticide that is used to destroy or kill plant-parasitic nematodes. The nematodes are simple unsegmented organisms belong to phylum Nematoda which comrises of nearly 25000 species. They are found in almost every part of earth. They can adapt all ecosystems and can live in highly extreme atmosphere. Besides, they have tendency to live on or in a plant and animal (including humans). In addition, more than half of species are parasitic and are responsible of many ailments such as stunting shoot of alfa alfa by ditylenchus depsaci, galling of root by meloidogyne species and ascaris in humans. It has been found that carbondioxide acts as an attractant for many phyto-parasitic nematodes to locate their target in complex environment of soil. The phyto-parasitic nematodes affect worth and mass of crop that results drastic loss of cultivation and so, economy. These parasitic nematodes must be controlled by means of nematicides, natural bio control, rotation of plant and soil steaming [27-32].

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Protocols

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4.5.0. Biological activities Assays

4.5.1. Enzyme Inhibition Assay

4.5.1.1. In Vitro Tyrosinase Inhibition Assay Tyrosinase inhibition was determined spectrophotometrically following the method described in literature [33]. All synthesized compounds were dissolved in DMSO and screened for o-diphenolase inhibitory activity of tyrosinase. L-Dopa was used as substrate and kojic acid as a standard inhibitor for tyrosinase. At first 30 units of mushroom tyrosinase (28 mM) were preincubated with compounds at 25˚ C for 10 min in presence of 50mM sodiumphospahte buffer (pH 6.8). Then L-Dopa (0.5 mM) was added. The enzyme reaction thus started to produce L-DOPA chrome, was monitored by measuring absorbance at 475nm (at 37˚C) for 10 min in spectramax 340 microplate reader (Molecular Devices USA). It was compared to the curve of standard. The percent of inhibition was calculated by following formula:

% Inhibition = [B –S / B] × 100.

Here, B is the absorbance for the blank and S is the absorbance for the samples. The results were in an average of triplicate and represent means ± SEM (standard error of the mean).

4.5.1.2. In Vitro Acetylcholinesterase and Butrylcholinesterase Inhibition Assay

Acetylcholinesterase (AChE) and Butrylcholinesterase (BChE) inhibition was determined spectrophotometrically in a 96 well micro plate on Spectra Max 340 (Molecular device, U.S.A) by slight modification in the method described in literature [34]. Acetylcholine iodide and butrylcholine chloride were used as substrates for Acetylcholinesterase and Butrylcholinesterase activity, Electric eel acetylcholinesterase (EC 3.1.1.7) and horse serum butyrylcholinesterase (EC 3.1.1.8) were used for enzyme preparations, 5,5′-dithio-bis-nitrobenzoic acid (DTNB) were

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used to measure cholinesterase activity and galanthamine as standard. 140µL of sodium phosphate buffer (pH= 8), 20µL of AChE / BChE solutions and 20 µL of test compounds were mixed then incubated at 25oC for 15 minutes. Subsequent addition of 10µL of DTNB and 10 µL of Acetylcholine iodide or butrylcholine chloride, in that order, initiated the reaction. The enzymatic hydrolysis of substrate released thiocholine which react with DTBN to form yellow 5-thio-2-nitrobenzoate anion. The hydrolysis was monitored spectrophotometrically at a wavelength of 412 nm. The results are in average of triplicate. All reagents and chemicals were purchased from Sigma and of analytical grades.

4.5.1.3. In Vitro Urease Inhibition Assay Urease activity was determined by using the indophenol method [35]. The results were obtained after measuring the ammonia produced during the reaction. The reaction mixtures containing 25 μL of enzyme (Jack bean Urease) solution, 55 μL of buffers (0.01 M K2HPO4.3H2O, 1 mM EDTA and 0.01 M LiCl2, pH= 8.2) and 100 mM urea were incubated in 96-well plates with 5 μL of test compounds (1 mM) for 15 min at 30oC. Briefly, 45 μL each of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 70 μL of alkali reagent (0.5% w/v NaOH and 0.1 % active chloride NaOCl) were added to each well. After 50 minutes the increasing absorbance was measured at 630 nm by using a microplate reader (Molecular Device, USA). All reactions were performed in a final volume of 200 μL in triplicate. The results i.e. change in absorbance per min., were processed on a SoftMax Pro software (Molecular Device, USA). Thiourea was used as a standard for comparison. Percentage inhibition was calculated from the formula:

% inhibition = 100 – (ODtestwell / ODcontrol) x 100

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4.5.2. In Vitro Anti-microbial Assay

Collection of pathogenic organism

All the bacterial and fungal pathogens were obtained from the Department of Microbiology, Federal Urdu University of Arts, Science and Technology, Karachi, Pakistan.

Preparation of culture To culture bacterial strains Muller Hinton agar (Oxoid) and Muller Hinton broth (Oxoid) were used as a media and Sabourd dextrose agar (SDA) plates for fungal strains [36].

4.5.2.1. In Vitro Anti-bacterial Assay The anti-bacterial activity of fractions and all the synthesized compounds was determined by the disc diffusion method and agar-well method as described in literaure [37, 38].

Disc Diffusion Method The suspensions of the applied bacterial strains were prepared in accordance with 0.5McFarland scale and swabbed on to the surface of sterile Mueller Hinton agar plates. 100 mg/ml of stock solution was prepared by dissolving pure compounds in DMSO. The sterile filter paper discs (diameter = 7mm) were impregnated with every tested sample. The discs were allowed to dry at room temperature to evaporate remaining solvent and placed on the surface of the inoculated plates. The discs impregnated with DMSO served as the negative control and standards as positive control. The plates were incubated at 37 °C for 24 hours. The results were recorded in triplicate on the basis of measurement of the diameter of zone of inhibition appeared around the disc [37].

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Agar – well Method Autoclaved Muller Hinton broth was used to keep bacterial culture in log phase for 2 h with constant agitation then wells were dug onto Muller Hinton Agar. Then 10 µl of culture were poured into the wells. 10 mg/ml of the test sample was taken for activity and incubated at 28+ 2°C for 24- 48 hr. After incubation diameter of zone of inhibition was measured in triplicate to get results [38].

4.5.2.2. In Vitro Anti-fungal Activity Assay All the fungal strains were checked for purity and maintained on SDA at 4ºC in the refrigerator until required for use. Antifungal activity of fractions was screened using agar-well method [39]. Briefly, a little amount of culture was transferred to 2-3 ml distilled water or normal saline in a screw capped tube with few glass beads of 1 mm in diameter, vortexes for 5-10 minutes to get a homogeneous suspension of fungal culture. The fungal spore suspension that was prepared in autoclaved distilled water transferred aseptically into each SDA plates. Now, the test samples (10 mg/ml) was taken for activity. All plates were incubated at 28+ 2°C for 24 -48 h. After incubation, results were recorded in triplicate by measuring diameter of zone of inhibition in mm.

4.5.2.3. Determination of Minimum inhibitory concentration (MIC) MIC of test samples was determined by Micro broth dilution method using 96-well microtitre plate. The stock solution of samples (100 mg/ ml) was prepared in distilled water. Two fold serial dilutions of samples was made in 100 µl broth subsequently 10 µl of 2h refreshed culture matched with 0.5 Mac Farland index was added to each well. One well served as culture control while other served as antibiotic control. Microtitre plate was incubated at 37ºC for 24 h. The MIC was found when the well showing no visible growth. Results were recorded in an average of triplicate [40].

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4.5.3. In Vitro Antioxidant Assay

Antioxidant activity of the synthesized compounds was determined by using the procedure described in literature [41]. The stable radical solution of 1, 1-diphenyl-2- picrylhydrazyl (DPPH) was prepared in ethanol (300 µM). 10 µL of test samples and 90 μL solution of stable radical (DPPH) was added in 96-well microtiter plates and incubated at 37º C for 30 minutes. Absorbance was measured at 515 nm by means of a spectrophotometer. Percent inhibition of radicals by treatment of test sample was found out by comparison with DMSO as negative control. Ascorbic acid was used as standard control. % inhibition was calculated by following formula:

% Inhibition = (absorbance of the control-absorbance of the test sample) x 100

Absorbance of the control

The EC50 value calculated denotes the concentration (in µg/ml) of sample required to scavenge 50% of DPPH.

4.5.4. Nematicidal Assay

Harvesting of Meloidogyne incognita

The nematicidal activity of synthesized compounds was determined through the method reported in literature [42]. The tested nematode species, Meloidogyne incognita was harvested and the roots of about 3 month old tomato plants, which had been infected with the nematode, were washed in fresh tap water. After that the roots were cut into 1-2 cm length and put in a round filter container then gently placed in the funnel, which had been placed in a mist chamber. Active nematodes passed through the filter and sank to the bottom of the funnel stem. After 4 days nematodes could be harvested and used for the experiments.

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Mortality test and lethal concentration (LC) determination

The test concentrations were prepared by adding 40 µL of synthetic drug stocks to 460 µL of fresh tap water in a 12-well plate. The mixing-plate was gently shaken by hands around 2 min to allow the drug to mix well. After that, 150 ml of the solution was transferred into 24 well test plates. Next, 90 µL of the nematode suspension containing approximately 150 second stage juveniles was added into the wells and gently mix for another 2 min then kept standing overnight at 24ºC. After 24 h the dead and alive nematodes were counted under stereoscopic binocular microscope to evaluate the mortality rate. Nematodes were considered dead when no movement was observed even after mechanical prodding. The % mortality was calculated from an average of replicate. The lethal concentration was determined at LC20, LC50 and LC90 mg/ml (the concentration needs to kill 20, 50 and 90% nematodes).

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4.6. References

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Research Publications

1. Aneela Wahab, Amina Sultana, Khalid M. Khan, Ayesha Irshad, Nida Ambreen and M. Bilal, Chemical investigation of Xanthium strumarium Linn. and biological activity of its different fractions, J. Pharm. Res., 5(4), 1984 (2012).

2. Aneela Wahab, Amina Sultana, Khalid M. Khan, Sikandar Khan Sherwani and Sandaleen Kanwal, Chemical constituents from the bioactive ethyl acetate fraction of Xanthium strumarium Linn., Pak. J Pharm. Sci. (Submitted).

3. Aneela Wahab, Amina Sultana, Khalid M. Khan, Sikandar Khan Sherwani and Zeba Parveen, Synthesis, Antimicrobial, Antioxidant and Nematicidal activity of (2E, 4E) 5(benzo[d][1,3]dioxol-5yl)penta-2,4-dienamides, J Saud. Chem. Soc., (Submitted).

4. Aneela Wahab, Amina Sultana, Khalid M. Khan, Sikandar Khan Sherwani and Saima Faraz, Anti-microbial and antioxidant activity of conventionally synthesized 2, 3-Diaminonaphthalenimidazole Derivatives, (in Process).

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