SYNTHESIS AND BIOLOGICAL INVESTIGATIONS OF SUBSTITUTED FLUORO CHLORO BENZPYRIDINE DERIVATIVES BY KANCHAM PRASANTHI Dissertation submitted to the KLE Academy of Higher Education & Research – Deemed University, Belgaum, Karnataka in partial fulfilment of the requirements for the degree of MASTER OF PHARMACY IN PHARMACEUTICAL CHEMISTRY Under the guidance of Dr. S M HIPPARAGI Department of Pharmaceutical Chemistry, KLE University’s College of Pharmacy, Bangalore-560010 2012 KLE ACADEMY OF HIGHER EDUCATION AND RESEARCH – DEEMED UNIVERSITY, BELGAUM, KARNATAKA DECLARATION BY THE CANDIDATE I hereby declare that this dissertation entitled SYNTHESIS AND BIOLOGICAL INVESTIGATIONS OF SUBSTITUTED FLUORO CHLORO BENZPYRIDINE DERIVATIVES is a bonafide and genuine research work carried out by me under the guidance of Dr. S.M.Hipparagi, Professor and HOD, Department of Pharmaceutical Chemistry, KLE University’s College of Pharmacy, Rajajinagar, Bangalore. Date: Place: Bangalore KANCHAM PRASANTHI KLE UNIVERSITY’S COLLEGE OF PHARMACY, BANGALORE-560010 (A constituent unit of KLE Academy of Higher Education and Research – Deemed University) CERTIFICATE BY THE GUIDE This is to certify that the dissertation entitled SYNTHESIS AND BIOLOGICAL INVESTIGATIONS OF SUBSTITUTED FLUORO CHLORO BENZPYRIDINE DERIVATIVES is a bonafide research work done by KANCHAM PRASANTHI under my supervision and guidance, in partial fulfilment of the requirements for the award of degree of MASTER OF PHARMACY IN PHARMACEUTICAL CHEMISTRY DATE: PLACE: Bangalore Dr. S.M.HIPPARAGI Professor and HOD Pharmaceutical Chemistry KLE University’s college of Pharmacy, Bangalore-560010 KLE UNIVERSITY’S COLLEGE OF PHARMACY, BANGALORE-560010 (A constituent unit of KLE Academy of Higher Education and Research – Deemed University) ENDORSEMENT BY THE HEAD OF THE DEPARTMENT AND THE PRINCIPAL/HEAD OF THE INSTITUTION This is to certify that the dissertation entitled SYNTHESIS AND BIOLOGICAL INVESTIGATIONS OF SUBSTITUTED FLUORO CHLORO BENZPYRIDINE DERIVATIVES is a bonafide research work done by KANCHAM PRASANTHI Under the guidance of Dr. S.M.Hipparagi Dr.S.M.HIPPARGI Head of the Department Dept. of pharmaceutical Chemistry DATE: PLACE: Bangalore Dr.B.G.DESAI Principal COPYRIGHT DECLARATION BY THE CANDIDATE I hereby declare that the KLE Academy of Higher Education & Research have the rights to preserve, use and disseminate this dissertation/thesis in print or electronic format for academic / research purpose. Date: Place: Bangalore KANCHAM PRASANTHI © KLE Academy of Higher education and Research-Deemed University Acknowledgement I have worked with a great number of people whose contribution in assorted ways to the research and the making of the thesis deserved special mention. It is a pleasure to convey my gratitude to them all in my humble acknowledgement. I would like to express my gratitude & indebtedness Firstly, to my Mother and Father, Smt. Sridevi and Sri Rajasekhar Reddy whose blessings, full-hearted cooperation, love and moral support made this day possible in my life. I would like to record my gratitude to my gracious mentor, Dr. S.M. Hippargi professor, Head of the department, Department of Pharmaceutical Chemistry, for his supervision, advices and guidance. Above all the encouragement and support in various ways. I thank his for the freedom of thought, trust and expression, which he bestowed upon me. Overall, it’s my fortune and so I am proud to have his as my guide. I wish to express my sincere thanks, with a deep sense of gratitude, to my Principal Prof. B.G. Desai, KLE University’s College of Pharmacy, Bangalore, for their generous consideration and facilities. I express my heartful thanks to Prof. Y.D. Satyanarayana, Vice principal, Dr. S.S. Karki, Mrs. Vanitha and other teaching staff of KLE University’s College of Pharmacy, Bangalore, for their valuable help and guidance during the course of my research work. I thank to Mrs. G.B Preethi for her guidance and for rendering me the permission to use the microbiology laboratory in carrying out anti-microbial activity. I would like to thank Mr. Sujeet Kumar for his constant help in taking IR. I thank to the Mr. Biradar, Mr. Sathish and other Non-teaching staff of the KLE University’s College of Pharmacy who helped me in various capacities. I especially thank to Indian Institute of Science and AstraZeneca Pharma India Ltd. Bengaluru for providing NMR analysis facility and Mr. suresh (G7 Pvt Ltd) for providing Mas spectra. i I take an opportunity to express my special thanks for my brother Pradeep and my husband Sreenivas for their love and cooperation towards me. My personal and sincere thanks to my cousins Neeraja, Kiran, Jagan and Narmada who stood behind me in every shade of my life. I express my special thanks to my uncle and aunty Sivareddy and Sujatha for their affection and being source of help whenever I needed. I am very much gratified by broad base of support and help that I received from my classmates Jayasree, Pavani, Reshma, Radha, Sravanthi, Sarala, Roshan, Rohit, Ruchika, Swetha, Anjali, Manjeera, Luv, Alok and Ramesh in completing this work and for making good friendly environment. With pleasure, I thank my seniors Viswa prakash, Parmesh, and all other for their support and valuable suggestions. With pleasure I thank my friends in other departments Anil, Shruti, Mounika, Usha, Rama, Deepti, Vamshi and all others who have confidence in me and supported my research. I would like to give special thanks to my best friend Chandralekha who had always supported and offered me helping hand whenever necessary. I thank my sweet roommates cum sisters Swetha, Sushma, Shilpa and Swetha reddy for there love and affection towards me. Memorable thanks to all who have bestowed love and help on me in timely completion of my thesis. I am grateful to all my Gurus from my kinder garden to my post graduation and specially to the teachers who preached me Chemistry and Inspired me to take up and be a master in pharmaceutical chemistry. Above all “Thank you” to the Almighty, who has given me this opportunity to extend my gratitude to all those people who have helped me and guided me throughout my life. I bow my head in complete submission before him for the blessings poured on me. Kancham Prasanthi ii LIST OF ABBREVIATIONS AIDS Acquired immuno deficiency syndrome ATCC American Type Culture Collection ACQ Amino chloroquinolines °C Degree centigrade -1 cm per centimeter (Wave number) CQ Chloroquine CDCl3 Deuteriated chloroform DNA Deoxy-ribonucleic acid DKA Diketo acids DMF Dimethyl formamide DMSO Dimethyl sulphoxide FT-IR Fourier transform infrared gm Gram HIV Human immunodeficiency virus HBV Anti-Hepatitis B Virus IC Inhibitory concentration IN Integrase enzyme IR Infrared spectroscopy K2CO3 Potassium carbonate KBr Potassium bromide LC-MS Liquid chromatography mass spectroscopy MIC Minimum inhibitory concentration m.p Melting point M.F Molecular formula M.wt Molecular weight μg Micro gram μM Micro Mole NMR Nuclear magnetic resonance POCl3 Phosphorous oxy chloride Ppm Parts per million % Percentage iii RTI Reverse transcriptase inhibitor RBF Round bottomed flask RNA Ribonucleic acid TLC Thin layer chromatography WHO World Health Organisation iv ABSTRACT Literature review shows that some substituted benzpyridine derivatives are known to exhibit diverse bioactivities such as antimicrobial, antifungal, antitubercular, anticancer, antitumour, antidepression, antianxiety, and antiviral. The main objective of this research project was to synthesize, characterize and biologically evaluate various benzpyridine derivatives for their antimicrobial activities. In the present work 7-chloro-6-fluoro-2-substituted quinoline-4-carboxylic acid derivatives were synthesized from 3-chloro 4-fluoro aniline, substituted benzaldehydes and pyruvic acid. Attempts were made to synthesize various derivatives from above compounds by using methyl chloro acetate, ethyl chloro acetate and thiosemicarbazide. All these newly synthesized derivatives were confirmed by IR, 1H-NMR and mass spectra. Antimicrobial study of these compounds against Gram +ve and Gram -ve show moderate activity by some derivatives but not as comparable to the standard (Ciprofloxacin). Keywords: Substituted benzpyridine, antimicrobial. v TABLE OF CONTENTS S. No. CONTENTS PAGES 1 Introduction 1-9 2 Objectives 10 3 Review of literature 11-29 4 Methodology 30-41 4.1 Scheme for synthesis 30-32 4.2 Experimental 33-41 Results and Discussion 42-55 Spectral studies 56-68 5 5.1 6 Conclusion 69 7 Summary 70 8 References 71-76 v LIST OF TABLES S.No. Title of the table Pages 1 Codes and Respective R groups of different derivatives. 30 2 Physical constants and Structures of the synthesized compounds. 44 3 Thin Layer Chromatography. 47 4 Infra-Red spectral study of the synthesized compounds. 48 5 1 51 6 Mass spectral data of synthesized compound. 52 7 Antibacterial activity of the synthesized compounds against S.aureus and P.auregenosa. 53 8 Antifungal activity of the synthesized compounds against C.albicans 55 H NMR spectral data of synthesized compounds. viii LIST OF FIGURES S.No. Title of the figure Pages 1 Schematic representation of mechanism of action shown by antibacterial agents. 5 2 Antibacterial activity for the synthesized compounds against S. aureus. 54 3 Antibacterial activity for the synthesized compounds against P.auregenosa. 54 4 Infrared spectra of 7-chloro-6-fluoro-2-phenyl quinoline4-carboxylic acid. 56 5 Infrared spectra of 7-chloro-6-fluoro-2-(4-methyl phenyl) quinoline-4-carboxylic acid. 57 6 Infrared spectra of 7-chloro-6-fluoro-2-(3-nitro phenyl) quinoline-4-carboxylic acid. 58 7 Infrared spectra of 7-chloro-6-fluoro-2-(4-methoxy phenyl) quinoline-4-carboxylic acid. 59 8 Infrared spectra of 2-methoxy-2-oxoethyl-7-chloro-6fluoro-2-phenyl quinoline-4-carboxylate. 60 9 Infrared spectra of 2-methoxy-2-oxoethyl-7-chloro-6fluoro-2-(4-methyl phenyl) quinoline-4-carboxylate. 61 10 Infrared spectra of 5-(7-chloro-6-fluoro-2-phenyl quinolin-4-yl)-1,3,4-thiadiazol-2- amine. 62 11 Infrared spectra of 5-(2-(4-methoxy phenyl)-7-chloro-6fluoro quinolin-4-yl)-1,3,4-thiadiazol-2- amine. 63 12 1 H NMR spectra of 7-chloro-6-fluoro-2-phenylquinoline4-carboxylic acid. 64 13 1 H NMR spectra of 7-chloro-6-fluoro-2-(4-methyl phenyl) quinoline-4-carboxylic acid. 65 14 1 66 H NMR spectra of 7-chloro-6-fluoro-2-(4-methoxy phenyl) quinoline-4-carboxylic acid. vi 15 Mass spectra of 7-chloro-6-fluoro-2-(4-methyl phenyl) quinoline-4-carboxylic acid. 67 16 Mass spectra of 5-(2-(4-methyl phenyl)-7-chloro-6fluoro quinolin-4-yl)-1,3,4-thiadiazol-2- amine. 68 vii INTRODUCTION INTRODUCTION Benzpyridine Also more commonly known as quinoline (1) or 1-azanaphthalene, is a heterocyclic aromatic compound characterized by a double ring structure containing a benzene fused to pyridine at two adjacent carbon atoms. Quinoline itself is the simplest member of quinoline. It is hygroscopic yellowish oily liquid; slightly soluble in water, soluble in alcohol, ether, carbon disulfide and readily in many organic solvents. It has the formula C9H7N.1 Quinoline structure is known ever since 1908 and proved by total synthesis by Woodward and Doering in 1945. It was first isolated by Runge in 1834 from coal tar bases and subsequently, Gerhardt in 1842 obtained it from alkaline pyrolysis of cinchonine, an alkaloid to quinoline. (1) Isoquinoline differs from quinoline in nitrogen position at 2. Quinoline family compounds are widely used as a parent compound to make drugs, fungicides, biocides, alkaloids, dye, rubber chemicals and flavouring agents. It is used in manufacturing oil soluble dyes, food, colorants, pharmaceuticals, pH indicator and other organic compounds.2 The quinolone was introduced for the treatment of urinary tract infections in 1963.The drugs containing quinolone nucleus includes oxolinic acid, norfloxacin, ciprofloxacin etc.3 Since then, this nucleus of quinoline has been explored widely and its derivatives have been found to posses various activities ranging from anti-HIV,4,5 antimalarial,6,7,8 anticancer,9 antimicrobial,10 anticonvulsant,11,12 antitubercular,13,14 KLEUCOP Bangalore (DEPT.OF.PHARM CHEMISTRY) anti-infective,15 Page 1 INTRODUCTION melanin concentrating hormone antagonists16 etc. This nucleus still holds a broad potential. Quinine may be claimed without exaggerations the drug to have relieved more human suffering than any other in history.For 300 years, it was the only known effective treatment for a life- threatening infectious disease. Only a handful of other treatments like emetine for amoebic dysentery, mercury for syphilis, chaulmoogra oil for leprosy, and herbal anthelmintics, were effective as specific anti-infective agents until this century. The fanciful story of the miraculous cure of the Countess of Cinchon, wife of the Viceroy of Peru, by administration of a native remedy produced from tree bark is charming but very far from fact, for a scholarly discussion of the early history of cinchona.17 Tree bark from the cinchona tree like Cinchona officinals and other Cinchona species, a native plant from South America, was the source for an effective treatment of recurrent fevers. In the 19th century, the active principles, the cinchona alkaloids, quinine (2), and cinchonidine (3), were isolated and purified. The formal synthesis of quinine by Woodward and Doering in 1944-1945 was a landmark in modern synthetic chemistry.18The first stereoselective total synthesis of quinine was recently reported by Stork and co-workers in their paper which also includes a brief history of synthetic efforts toward quinine. The principal areas producing cinchona are central Africa, India, and Indonesia. Commercial formulations of quinine have approximately 10% dihydroquinine as an impurity. The preparation Quinimax is a mixture of cinchona alkaloids, predominately quinine, is reported to be more effective than quinine alone. The stereochemical differences among the cinchona alkaloids result in differences in potency, and the stereo electronic features have been examined. Conformational differences between the diastereomers apparently lead to differing ability to form critical hydrogen bonds. Quinidine is two- to threefold more active than quinine in both chloroquine-sensitive and chloroquine-resistant strains of P. falciparum.19 Likewise, cinchona isomer are active than cinchonidine in vitro. The differences in activity based on stereochemistry is greater for those compounds like quinine and quinidine, which bear a piperidine ring.20 KLEUCOP Bangalore (DEPT.OF.PHARM CHEMISTRY) Page 2 INTRODUCTION CH=CH2 H HO H CH=CH2 H N HO H3CO H N H N N (2) (3) ANTIMICROBIAL ACTIVTY: Medicinal microbiology is the study of microbes that infect humans, the diseases they cause, their diagnosis, prevention and treatment. The 4-quinolones have a number of advantages over other classes of antibacterial agents. They are effective against many organisms, well-absorbed orally, well-distributed in tissues, and they have relatively long serum half-lives and minimal toxicity. Because of deep-tissue and cell penetration, they are useful for urinary tract infections, prostatitis, infections of the skin and bones, and penicillan-resistant sexually transmitted diseases. Some well known quinolones antibiotics like ciprofloxacin (4) and lomefloxacin (5). O NH2 COOH F O COOH F H3C N N N N N N H OCH3 H3C (4) KLEUCOP Bangalore (DEPT.OF.PHARM CHEMISTRY) (5) Page 3 INTRODUCTION Among the compounds that currently are available for clinical use in the United States are quinolones containing a carboxylic acid moiety at position 3 of the primary ring structure.22 Many of the newer fluoroquinolones also contain a fluorine substituent at position 6 and a piperazine moiety at position 7. Bacteria are one among the different types of microbes which are capable of causing infections among the human beings eg:- Bacillus subtilis, Pseudomonas Aeruginosa, Escherichia coli etc. The agents which fight against these microbes are generally termed as antibacterial agents. A few examples of antibacterial agents are βlactam antibiotics, aminoglycosides, sulphonamides etc. The rational uses of these agents depend on understanding their mechanism of action, pharmacokinetics, pharmacodynamics, toxicities, and interactions. Antibacterial agents like all other antimicrobial agents are directed against unique targets not present in mammalian cells.21 The main aim of these agents is to limit toxicity to the host and maximize chemotherapeutic (a term coined by Ehlrich) activity against invading organisms. Mechanism of action: The quinoline antibiotics target bacterial DNA gyrase and topoisomerase IV. For many Gram-positive bacteria (such as S.aureus),topoisomerase IV is the primary activity inhibited by quinolones. In contrast, for many Gram-negative bacteria (such as E.coli), DNA gyrase is the primar y quinolone target.The individual strands of double helical DNA must be separated to permit DNA replication or transcription.22 In the place of DNA gyrase or topoisomerase IV, the mammalian cells possess an enzyme topoisomerasse II which has low affinity for fluoroquinolones- hence the low toxicity to host cells.21 KLEUCOP Bangalore (DEPT.OF.PHARM CHEMISTRY) Page 4 INTRODUCTION Fig 1- Schematic representation of mechanism of action shown by antibacterial agents. Fungi are plant-like, nonphotosynthetic eukaryotes growing either in colonies of single cells (yeasts) or in filamentous multicellular aggregates (molds).eg:-Aspergillus nidulans,Candida albicans. Most fungi live as saprophytes in soil or on dead plant material and are important in the mineralization of organic matter. Unfortunately some species are parasites of terrestrial plants and can cause serious crop damage. A small number cause disease in humans and animals. Mycotic illnesses in humans are dividing into three groups: contagious skin and hair infections; noncontagious soil borne or air borne systemic infections; and noncontagious food borne toxemias. The responsible organisms and methods of prevention and treatment differ with each other. The agents which fight against these microbes are generally termed as antifungal agents.21 A few examples of antifungal agents are fluconazole,ketoconazole,itraconazole,griseofulvin etc. KLEUCOP Bangalore (DEPT.OF.PHARM CHEMISTRY) Page 5 INTRODUCTION Antimalarial activity Malaria is one of the major killer diseases of the world. According to WHO, malaria is a significant public health problem in more than 90 countries. Malaria causes up to 2.7 million deaths per year with the vast majority of these among young children in Africa. There are an estimated 300-500 million clinical causes each year with more than 90% of these occurring in sub-Saharan Africa.23 The name malaria derives from the Italian word which literally means bad air, for it was thought that the disease resulted from effluvia from the marshes. The bark of Cinchona tree, growing in Peru, was introduced in Europe in the early 17th century as a cure for fevers. Later it was realized to be a specific remedy for malaria. Quinine, isolated from cinchona bark in1820, replaced the crude preparation and continued to be the major antimalarial drug till 1942. Mepacrine was produced in Germany in 1926. Chloroquine(6) was produced in USA soon after as a less toxic alternative to mepacrine.24 Pamaquine was the first 8-aminoquinoline to be tested in Germany in 1920s.21 However no attention was paid to it because of its poor schizontozide action and Primaquine emerged as the most desirable drug. (6) Quinine is an erythrocytic schizontozide for all species of plasmodia; less effective and more toxic than chloroquine. Resurgence of interest in quinine is due to the fact that most chloroquine and multidrug resistant strains of Plasmodium falciparum are still KLEUCOP Bangalore (DEPT.OF.PHARM CHEMISTRY) Page 6 INTRODUCTION sensitive to it. However even quinine resistance has been described in certain parts south east Asia and Brazil where qunine + tetracycline has been the standard treatment of complicated malaria. Quinine resistance has not been encountered in India. Quinine has no effect on pre-erythrocytic stage and on hypnozoites of relapsing malaria, but kills vivax gamates.21 Malaria is a parasitic disease caused by protozoa of the genus Plasmodium which is introduced through the bite of the female Anopheles mosquito.23 Anti-malarial drugs are classified in terms of the action against the different stages of the life cycle of the parasite. The chief species of human malaria parasites are as follows: 25 P. falciparum, which has an erythrocytic cycle of 48 hours in humans, produces malignant tertian malaria. The plasmodium induces, on the infected red cell’s membrane, receptors for the adhesion molecules on vascular endothelial cells. These parasitized red cells then stick to uninfected red cells forming clusters. They also adhere to and pack the vessels of the microcirculation, interfering with tissue blood flow and causing organ dysfunction. P. vivax produces benign tertian malaria. Exoerythrocytic forms may persist for years and cause relapses. P. ovale, which has a 48 hour cycle and an exoerythrocytic stage, is the cause of a rare form of malaria. P. malariae has a 72 hour cycle, causes quartan malaria and has no exoerythrocytic cycle. Mechanism of Action Chloroquine is considered as the prototypical structure that succeeded quinine. Its main site of action appears to involve the lysosome of the parasite-infected erythrocyte. By accumulating in the acidic vesicles of the parasite and because of its weakly basic nature it raises the vesicular pH and thereby interferes with degradation of haemoglobin by parasitic lysosomes. Polymerization of toxic heme to nontoxic parasite pigment hemozoin is inhibited by formation of chloroquine-heme complex.21 Heme itself or its complex with chloroquine then damages the plasmodial membranes. KLEUCOP Bangalore (DEPT.OF.PHARM CHEMISTRY) Page 7 INTRODUCTION IMPORTANCE OF FLUORINE The small size and higher electro negativity of fluorine are among the special properties that contribute to the well-recognized importance of this element in the field of medicinal chemistry. Sometimes predictable effects of fluorine substitution on the biological behaviour of biologically active molecules have been used extensively in drug design. E.g.:- Anticancer agent 5- Fluorouracil. Increasing interest in fluorinated pharmaceutical and medicinal agents helped in development of new fluorinating agents that in turn produced yet more applications in medicinal chemistry. The biological consequences of fluorine substitution now often become rationalized. Interpretation of such data in turn has added to our understanding how fluorine interacts with macro molecular recognition sites and this has aided further drug design. Selective aromatic fluorine substitution can increase the affinity of a molecule for macromolecular recognition site through non covalent interactions. These effects are evaluated most accurately by direct comparison of binding affinities of selectively fluorinated compounds with their corresponding hydrocarbons. Since the mid 1950 the progress in organic fluorine chemistry has been rapidly translated into useful applications in medicinal and biochemistry. Advance in the area has been accelerated by the development of new technique and reagents for the site selective introduction of fluorine for the development of drugs likes anticancer, antiviral agents, anti-inflammatory, antiparasitic agents, antibiotics and general anesthetics. Fluoroquinolones are known to have two enzyme targets, DNA gyrase and topoisomerase IV in the bacterial cell.26 Both of these targets are essential for bacterial DNA replication.eg:- sparfloxacin (7) KLEUCOP Bangalore (DEPT.OF.PHARM CHEMISTRY) Page 8 INTRODUCTION NH2 O O F OH N N NH F (7) Fluorine in Bioactive Molecules:The incorporation of fluorine in drug molecule as a mean of increasing therapeutic efficacy is based on several considerations. 1. Fluorine, the second smallest substituent, closely mimics hydrogen with respect to steric requirement at enzyme receptor sites. (Vanderwaals radii F=1.35 A0 , H=1.2 A0) 2. The strong electron withdrawing inductive effect of fluorine can significantly influence reactivity and stability of functional groups and the reactivity of neighboring reaction centers. 3. The substitution of hydrogen by fluorine or near a reactive site frequently causes inhibition of metabolism because of the high C-F bond energy. 4. The replacement hydrogen by fluorine usually increases lipid solubility, thereby enhancing the rate of absorption and transport of drugs in vivo. 5. Sometimes the presence of fluorine instead of hydrogen actually blocks in essential biochemical reaction. Eg:-5-Fluorouracil, fluoroquinolones. KLEUCOP Bangalore (DEPT.OF.PHARM CHEMISTRY) Page 9 OBJECTIVES OBJECTIVES Benzpyridine ring holds a broad potential for various activities. In this project we have aimed to synthesized various derivatives of benzpyridine and evaluate them for certain pharmacodynamic attributes. To achieve these following objectives were set: To carry out literature survey of various benzpyridine derivatives. To establish the method of synthesis for the proposed compounds. To carry out the preliminary tests such as physical constant determination, solubility, TLC. To confirm the structure of the synthesized compounds by IR, 1H NMR, Mass analysis. To evaluate the synthesized compounds for their antimicrobial activity. KLEUCOP Bangalore [DEPT.OF.PHARM CHEMISTRY] Page 10 REVIEW OF LITERATURE REVIEW OF LITERATURE Quinoline nucleus has been explored widely and its derivatives have been found to posses various activities ranging from antimalarial, antimicrobial, anticancer, anti-HIV etc. Stated below is the review of quinoline moiety possessing various pharmacological activities. Antimicrobial activity The first quinoline of commercial importance was a nalidixic acid derivative (8) prepared in 1962 by Lesher.27 Norfloxacin (9) is a fluoroquinolone with a broad spectrum of antibacterial activity. Chemical modifications based on their structures have since led to thousands of new analogs, some of which have significantly improved effectiveness. (8) (9) Monsouri et al.,28 synthesized a series of N-[5(chlorobenzylthio)-1,3,4-thiadiazol-2yl]piperazinyl quinoline derivatives (10) by reaction of piperazinyl quinolones with 5chloro-2-(chlorobenzylthio)-1,3,4-thiadiazoles. These compounds found to be a good antibacterial activity against Gram +ve and Gram –ve bacteria. Among these compounds with a 2-chlorobenzylthio moiety is (11) which is a ciprofloxacin derivative, exhibited high activity against Staphylococcus aureus and S. epidermidis (MIC=0.06µg/ml). KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 11 REVIEW OF LITERATURE (10) (11) A series of 7-[4-(5-amino-1,3-thiadiazole-2-sulfonyl)]-1-piperazinyl fluoroquinolonic derivatives (12) and (13) were synthesized by Talath et al.29 These compounds were evaluated for their preliminary in vitro antibacterial activity against some Gram-positive and Gram-negative bacteria and selected compounds (13) were screened for antitubercular activity against Mycobacterium tuberculosis H37Rv strain by broth dilution assay method. The antibacterial data of the tested N-sulfonylfluoroquinolones (12) indicated that all the synthesized compounds showed better activity against Grampositive bacteria S. aureus, E. faecelis and Bacillus sp. (MIC = 1–5 μg ml–1, respectively) compared to reference drugs. The in vitro antitubercular activity reports of selected compounds (13) against M. tuberculosis strain H37Rv showed moderate activity at MIC of 10 μg ml.29 KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 12 REVIEW OF LITERATURE (12) R1= -H, -NH2, R2= -H, -F, -OCH3 R3= -H, -CH3, -C2H5, NO2 (13) R1=- H, -NH2 R2= -H, - F, -OCH3 R3= -H, -CH3, -C2H5, NO2 De Souza et al.,30 synthesized a series of 33 quinoline derivatives and they were evaluated for their in vitro antibacterial activity against Mycobacterium tuberculosis H37Rv using the alamar Blue susceptibility test and the activity expressed as the minimum inhibitory concentration (MIC) in μg/mL. KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 13 REVIEW OF LITERATURE Compounds (14) and (15) exhibited a significant activity at 6.25µg/ml and 3.12 μg/ml respectively, when compared with first line drugs such as ethambutol and could be a good starting point to develop new lead compounds in the fight against multidrug resistant tuberculosis. (14) (15) Wang et al.,31 carried out the synthesis of ampiphilic cationic quinine-derivatives and evaluated for antibacterial activity against methicillin resistant Staphylococcus aureus. The in vitro MIC of the compound (16) and (17) ranged from 0.4 to 1.6 µg/ml. (16) (17) The isosters of cryptolepine were synthesized and evaluated for their anti-infective activities.Among these derivatives of 5-methyl-11H-indeno [3, 2-b]quinolinium iodide compound (18) found to possess similar potency as cryptolepine against C. albicans and C. neoformans. These compounds also showed significant effect against leishmania as compared to pentamidine and moderate activities on P. falciparum.32 KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 14 REVIEW OF LITERATURE (18) Antimalarial activity Quinine has been in use for hundreds of years. Several derivatives of it were discovered and being approved recently.33 Some find uses in other infectious diseases while many are specifically used in malaria. Tree bark from the cinchona tree, Cinchona officinalis and other Cinchona species, a native plant from South America, was the source for an effective treatment of recurrent fevers. Racemic mefloquine WR 142 and its derivatives (19, 20 and 21) were synthesized and evaluate for their antimalarial activity.34 The isomers that differ in their position of trifluoromethyl groups around the quinoline system were proved to be most active in this series. HO N H HO CF3 N H N . N (19) KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] CF3 CF3 (20) Page 15 REVIEW OF LITERATURE HO N H N CF3 (21) Ohnmacht et al.,35 synthesized Primaquine CWR 2975 (22), and found to be extensively used as the anti-relapse drug of choice against P. cynomolg. d and l primaquine have essentially identical curative properties for P. cynomolgi. NH2 HN N H3CO (22) Schmidt et al.,36 synthesized the two series of 8-quinolinamines, N1-{4-[2-(tert-butyl)-6methoxy-8-quinolylamino] pentyl}-(2S/2R)-2-amino substituted amides (23) and N1-[4(4-ethyl-6-methoxy-5-pentyloxy-8-quinolylamino)pentyl]-(2S/2R)-2-amino substituted amides (24) in six steps from 6-methoxy-8-nitroquinoline and 4-methoxy-2-nitro-5pentyloxyaniline, respectively. OC5H11 C2H5 H3CO H3CO N H3C R3 N H N N NH2 H3C R3 H N N (23) KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] NH2 (24) Page 16 REVIEW OF LITERATURE Lapudrine (25) was synthesized by Kaur et al.,37 and used as a good alternative to proguanil in areas of drug resistance. Cl NH N H NH N H N H (25) Watkins et al.,38 synthesized a series of hybrid molecules 2-[3-(7-chloro-quinolin-4-ylamino)alkyl]-1-(substituted phenyl)-2,3,4,9-tetrahydro-1H-β-carbolines and screened for their in vitro antimalarial activity against chloroquine-sensitive strains of Plasmodium falciparum. Compounds 26, 27 and 28 have shown MIC in the range of 0.05–0.11 μM and are in vitro several folds more active than chloroquine. COOCH3 H N N N H CH3 N Cl (26) COOCH3 N H N N H C2H5 N Cl (27) KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 17 REVIEW OF LITERATURE COOCH3 H N N N H N Cl (28) A new series of 4-aminoquinoline isatin derivatives, 29, 30 were synthesized and screened for in vitro antiplasmodial activity and inhibition of falcipain-2. These compounds showed IC50 values in the range of 0.05–2 μM against drug resistant and sensitive strains of P. falciparum.39 S S C2H5 H3C O O N N C2H5 CH3 NH NH N N Cl (29) Cl (30) A total of 80 new 2-methyl-6-ureido-4-quinolinamides were synthesized and evaluated for their antimalarial activity by Cochin and his coworkers.40 Several analogs elicited the antimalarial effect at MIC of 0.25 mg/mL against the chloroquine-sensitive P. falciparum strain. The IC50 values of the active compounds like 31, 32 were observed to be in ng/mL range and these two analogs have better IC50 value than the standard chloroquine. KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 18 REVIEW OF LITERATURE CH3 N N F3C HN H N H N O N (31) CH3 N N Cl HN H N H N O N Cl (32) Kumar et al.,41 carried out the synthesis of new 4-aminoquinoline derivatives and quinoline–acridine hybrids. All the synthesized compounds were evaluated in vitro for their antimalarial activity against NF 54 strain of P. falciparum. Among the evaluated compounds, compound 33 (MIC = 0.125 lg/mL) was equipotent to standard drug CQ (MIC = 0.125 lg/mL) and compound 34 (MIC = 0.031 lg/mL) was four times more potent than CQ. N HN N N Cl N (33) KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 19 REVIEW OF LITERATURE N HN Cl N (34) Anticancer activity Y.L.Chen and co-workers42 reported the synthesis and anticancer activity of certain 11substituted 6H-indolo [2, 3b] quinolines (35, 36, and 37) and their methylated derivatives. (35) (36) (37) KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 20 REVIEW OF LITERATURE In 2008, Aibin et al.,28 synthesized and reported anti-breast cancer activities of some substituted quinolines (38) bearing biologically active sulfonamide moiety as a new class of antitumor agents. (38) Mostafa et al.,44 reported the synthesis of some novel hexahydroquinoline derivatives having a benzenesulfonamide moiety. All the newly synthesized compounds were evaluated for their in vitro anticancer activity. Compounds 39 and 40 showed significant activity compared to the reference drug doxorubicin. O Ar O Ar H H O CN N NH N NHCOC6H5 N Br SO2NH2 (39) SO2NH2 (40) KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 21 REVIEW OF LITERATURE Some pyrroloquinoline (41, 42) derivatives were synthesized by Maria et al.,45 as potential anticancer agents. (41) (42) Anticonvulsant activity Guo et al.,46 synthesized a series of 5-alkoxy-[1,2,4]triazolo[4,3-a]quinoline derivatives using 4-hydroxyquinolin-2(1H)-one as the starting material. Their anticonvulsant activities were evaluated by the maximal electroshock test (MES) and their neurotoxicities were measured by the rotarod test. The results of these tests demonstrated that 5-hexyloxy-[1,2,4]triazolo[4,3-a]quinoline (43) was the most potent anticonvulsant and the compound 5-benzyloxy-[1,2,4]triazolo[4,3-a]quinoline (44), exhibited a little weaker activity than compound (43) in controlling the seizure, but it possessed lower neurotoxicity which was safer than marketed drug carbamazepine. O CH2 O N N N (43) KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] N N N (44) Page 22 REVIEW OF LITERATURE Quan et al.,47 synthesized a series of 1-substituted 7-benzloxy-4,5-dihydro[1,2,4] triazolo[4,3-a] quinolines from 6-hydroxy-3,4-dihydro-1H-quinoline-2-one and the compounds were evaluated for anti-convulsant activity. Among them, the most active compound was 7-benzyloxy-4,5-dihydro-[1,2,4]triazolo[4,3-a]quinoline (45) and the safest compound was 1-phenyl-7-benzyloxy-4,5-dihydro-[1,2,4]triazolo[4,3-a]quinoline (45).The protective index of the compound (46) was better than that of most marketed drugs. O N N N (45) O N N N (46) Anti-HIV activity Sato and his co-workers48 reported that the 4-quinolone carboxylic acid antibiotics can be used as an alternative scaffold to diketo acids (DKA) in order to identify new integrase inhibitors. These novel quinolone integrase inhibitors, such as (47, 48) exhibit potent inhibitory activity against IN-catalyzed DNA strand transfer (IC50= 7.2 nM) and antiviral activity in vitro (EC50= 0.9 nM). KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 23 REVIEW OF LITERATURE (47) (48) Bailing et al.,49 reported the synthesis and biological evaluation of N4-(hetero) arylsulfonylquinoxalines (49, 50, 51) as HIV-1 reverse transcriptase inhibitors. (49) KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] (50) Page 24 REVIEW OF LITERATURE (51) A new series of HIV-1 integrase inhibitors (52, 53, 54, 55) were synthesized by Mouscadet et al.,47 and tested in both in vitro and ex vivo assays. These inhibitors featured quinoline subunit and ancillary aromatic ring linked by functionalized spacers such as amide, hydrazide, urea and 1-hydroxyprop-1-en-3-one moiety.50 Among these derivatives the amide group containing derivatives were the most promising ones. (52) KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 25 REVIEW OF LITERATURE (53) (54) (55) Immunosupressive activity Qiang et al.,51 synthesized a series of quinoline derivatives and their immunosuppressive activity and cytotoxicity were evaluated with a T-cell functional assay and MTT method, respectively. Most of 5,7-dimethoxyquinolin-4-yl ortho-substituted benzoate derivatives showed a quite stronger inhibitory activity. Among the synthesized compounds, 5,7dimethoxyquinolin-4-yl 2,6-dichlorobenzoate (56) and 5,7-dimethoxyquinolin-4-yl 4methylbenzenesulfonate (57) exhibited a potent inhibitory activity without significant cytotoxicity at 10 µM concentration. KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 26 REVIEW OF LITERATURE CH3 O Cl Cl S OCH3 H3CO O OCH3 O H3CO N O O N (56) (57) Anti-Hepatitis B Virus activity A series of novel 6-chloro-4-(2-chlorophenyl)-3-(2-hydroxyethyl) quinolin-2(1H)-one derivatives were synthesized by Zhang et al.,52 and evaluated for their anti-hepatitis B virus (anti-HBV) activities. Most of the synthesized compounds possessed potent antiHBV activity of which the promising compound (58) exhibited significantly inhibitory potency against the secretion of hepatitis surface antigen (HBsAg), hepatitis e antigen (HBeAg) and the replication of HBV DNA. H N O O Cl O Cl OCH3 (58) KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 27 REVIEW OF LITERATURE Cyclooxygenase-2 inhibitors A group of 4-carboxyl quinoline derivatives possessing a methylsulfonyl COX-2 pharmacophore at the para position of the C-2 phenyl ring were designed and synthesized as selective COX-2 inhibitors by Ebrahim and his coworkers.53 Among the 4-carboxyl quinolines, 7,8,9,10-tetrahydro-2-(4-(methyl sulfonyl) phenyl)benzo[h]quinoline-4- carboxylic acid (59) was identified as potent and high selective COX-2 inhibitor, that was more potent than the reference drug celecoxib. A molecular modeling study where 9e was docked in the binding site of COX-2 showed that the p-MeSO2 substituent on the C-2 phenyl ring is oriented in the vicinity of the COX-2 secondary pocket (Arg513, Phe518 and Val523) and the carboxyl group can interact with Arg120. COOH N SO2CH3 (59) Leishmanicidal and enzyme inhibitory activities A series of quinoline-4-carboxylic acids was synthesized by Mohammed et al.,54 and screened for their leishmanicidal, phosphodiesterase, β-glucuronidase and urease inhibitory properties. Among all the tested compounds, only the compounds (60) and (61) were found to be active against leishmaniasis and the compound (62) showed maximum percentage inhibition against both phosphodiesterase and urease enzymes. Compound (63) showed activity against β-glucuronidase enzyme. KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 28 REVIEW OF LITERATURE COOH COOH OH N N CH3 (60) (61) COOH COOH OCH3 N N OCH3 OCH3 (62) KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] (63) Page 29 METHODOLOGY SCHEME FOR SYNTHESIS STEP-1 COOH F RCHO Cl CH3COCOOH NH2 F C2H5OH reflux for 3 hrs Cl Substituted Pyruvic acid 3-chloro 4-fluoro aniline aldehyde N R TABLE-1 CODE R KP-1 CH3 KP-2 NO2 KP-3 KP-4 OCH3 KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 30 METHODOLOGY HO KP-5 O2N KP-6 STEP-2 COOCH2COOCH3 COOH F F DMF Cl N + ClCH2COOCH3 R Methyl chloro acetate reflux, K2CO3 72 hrs Cl N R STEP-3 COOCH2COOC2H5 COOH F F DMF Cl N + ClCH2COOC2H5 R Ethyl chloro acetate reflux, K2CO3 72 hrs KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Cl N R Page 31 METHODOLOGY STEP-4 COOH S F POCl3 Cl N R H2N N H NH2 700C 7 hrs Thiosemicarbazide NH2 N N S F Cl KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] N R Page 32 METHODOLOGY EXPERIMENTAL The compounds synthesized were identified and characterized by following methods Melting point determination Thin layer chromatography Infra red spectroscopy Nuclear magnetic resonance spectroscopy Mass spectroscopy Melting point determination: The melting point of an organic compound was determined by Thiel’s melting point apparatus by capillary tube method. The determination of melting point is the most important and easy way of differentiating one compound from the other. Thin layer chromatography (TLC): The TLC of the compounds are determined by using pre-coated TLC plates. It is an important method for synthetic chemistry to confirm the completion or progress of the reaction and the purity of compounds based on the Rf values. Infra red spectroscopy (IR): IR is the most important tools for determining the various functional groups and the possible chemical structure. The important advantage of IR over other technique is that it gives fingerprints (1300-650 cm-1) information about the structure (functional group, bonding with each other) of molecules easily. No two compounds have identical fingerprint region. This technique is based upon the molecular vibration of the compound such that each and every bond will vibrate at the different frequency and this vibration frequency correspond to the IR frequency. Thus IR spectra of each and every bond will be formed. The IR spectra were recorded in KBr on a Jasco FTIR 460 plus spectrometer by diffuse reflectance technique. Nuclear magnetic resonance spectroscopy (NMR): The introduction between matter and electromagnetic forces can be observed by subjecting a substance simultaneously to KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 33 METHODOLOGY two magnetic forces, one stationary and other varying at some radio frequency. At a particular combination of fields, energy is observed by the sample and absorption can be observed as a change in signal developed by a radio frequency detector and amplifier. This energy of absorption can be related to a magnetic dipolar nature of a spinning nucleus. This technique is known as Nuclear Magnetic Resonance. This technique is useful in assuming the structure of the molecule. 1H NMR spectra were measured in CDCl3 and DMSO on a Bruker Ultraspec 500MHz/ AMX400MHz spectrometer. Mass spectroscopy (MS): The mass spectrometer is an instrument in which the substance in gaseous (or) vapor state is bombarded with a beam of electrons, to form positively charged ions (cations) which are further sorted according to their mass to charge ratio to record their masses and relative abundances. Both positive and negative ions can be studied using mass spectrometer but usually positive ions are analyzed since they are produced in large amounts as compared to negative ions. Negative ion spectra although less commonly used than positive ion spectra, can also be obtained. The mass spectra of compounds were recorded on LCMS-2010 SHIMADZU mass spectrometer in G7 Synergon, Bangalore. A) METHOD OF SYNTHESIS: 1. Synthesis of 7-chloro-6-fluoro-2-phenyl quinoline-4-carboxylic acid, (KP-1):55 In a 500 ml round bottom flask, equipped with a reflux condenser, 12.5 g (12 ml, 0.118 mol) of purified benzaldehyde, 11 g (8.66 ml, 0.125 mol) of freshly distilled pyruvic acid and 100 ml of absolute ethanol was placed. The mixture was heated to the boiling point on a water bath and a solution of 11.5 g (0.079 mol) of pure 3-chloro 4fluoro aniline in 100 ml of absolute ethanol was added slowly to it, with frequent shaking. The addition occupied about one hour. The mixture was then refluxed on a water bath for 3 hr and then allowed to stand overnight. The crude chlorofluoroquinoline-4carboxylic acid (KP-1) was filtered off at the pump and crystals were washed with a little KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 34 METHODOLOGY ether. The crude product was recrystallised from ethanol to give a cream color crystals whose yield was 12.6 g (52.83%). 2. Synthesis of 7-chloro-6-fluoro-2-(4-methyl phenyl) quinoline-4carboxylic acid, (KP-2): In a 500 ml round bottom flask, equipped with a reflux condenser, 12.5 g (12.3 ml, 0.104 mol) of purified 4-methyl benzaldehyde (p-tolualdehyde), 11 g (8.66 ml, 0.125 mol) of freshly distilled pyruvic acid and 100 ml of absolute ethanol was placed. The mixture was heated to the boiling point on a water bath and a solution of 11.5 g (0.079 mol) of pure 3-chloro 4-fluoro aniline in 100 ml of absolute ethanol was added slowly to it, with frequent shaking. The addition occupied about one hour. The mixture was then refluxed on a water bath for 3 hr and then allowed to stand overnight. The crude chlorofluoroquinoline-4-carboxylic acid (KP-2) was filtered off at the pump and crystals were washed with a little ether. The crude product was recrystallised from ethanol to give a white color crystals whose yield was 14.5 g (58.08%). 3. Synthesis of 7-chloro-6-fluoro-2-(3-nitro phenyl) quinoline-4- carboxylic acid, (KP-3): In a 500 ml round bottom flask, equipped with a reflux condenser, 12.5 g (0.082 mol) of purified 3-nitro benzaldehyde, 11 g (8.66 ml, 0.125 mol) of freshly distilled pyruvic acid and 100 ml of absolute ethanol was placed. The mixture was heated to the boiling point on a water bath and a solution of 11.5 g (0.079 mol) of pure 3-chloro 4fluoro aniline in 100 ml of absolute ethanol was added slowly to it, with frequent shaking. The addition occupied about one hour. The mixture was then refluxed on a water bath for 3 hr and then allowed to stand overnight. The crude chlorofluoroquinoline-4carboxylic acid (KP-3) was filtered off at the pump and crystals were washed with a little ether. The crude product was recrystallised from ethanol to give a light greenish color powder whose yield was 12.4 g (45.3%). 4. Synthesis of 7-chloro-6-fluoro-2-(4-methoxy phenyl) quinoline-4carboxylic acid, (KP-4): In a 500 ml round bottom flask, equipped with a reflux condenser, 12.5 g (11.17 ml, 0.092 mol) of purified 4-methoxy benzaldehyde (anisaldehyde), 11 g (8.66 ml, 0.125 KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 35 METHODOLOGY mol) of freshly distilled pyruvic acid and 100 ml of absolute ethanol was placed. The mixture was heated to the boiling point on a water bath and a solution of 11.5 g (0.079 mol) of pure 3-chloro 4-fluoro aniline in 100 ml of absolute ethanol was added slowly to it, with frequent shaking. The addition occupied about one hour. The mixture was then refluxed on a water bath for 3 hr and then allowed to stand overnight. The crude chlorofluoroquinoline-4-carboxylic acid (KP-4) was filtered off at the pump and crystals were washed with a little ether. The crude product was recrystallised from ethanol to give a light yelow color crystals whose yield was 16.5 g (62.9%). 5. Synthesis of 7-chloro-6-fluoro-2-(2-hydroxy phenyl) quinoline-4carboxylic acid, (KP-5): In a 500 ml round bottom flask, equipped with a reflux condenser, 12.5 g (10.74 ml, 0.102 mol) of purified 2-hydroxy benzaldehyde (salicylaldehyde), 11 g (8.66 ml, 0.125 mol) of freshly distilled pyruvic acid and 100 ml of absolute ethanol was placed. The mixture was heated to the boiling point on a water bath and a solution of 11.5 g (0.079 mol) of pure 3-chloro 4-fluoro aniline in 100 ml of absolute ethanol was added slowly to it, with frequent shaking. The addition occupied about one hour. The mixture was then refluxed on a water bath for 3 hr and then allowed to stand overnight. The crude chlorofluoroquinoline-4-carboxylic acid (KP-5) was filtered off at the pump and crystals were washed with a little ether. The crude product was recrystallised from ethanol to give a yelow color crystals whose yield was 10.5 g (41.7%). 6. Synthesis of 7-chloro-6-fluoro-2-(2-nitro phenyl) quinoline-4- carboxylic acid, (KP-6): In a 500 ml round bottom flask, equipped with a reflux condenser, 12.5 g (0.083 mol) of purified 2-nitro benzaldehyde , 11 g (8.66 ml, 0.125 mol) of freshly distilled pyruvic acid and 100 ml of absolute ethanol was placed. The mixture was heated to the boiling point on a water bath and a solution of 11.5 g (0.079 mol) of pure 3-chloro 4fluoro aniline in 100 ml of absolute ethanol was added slowly to it, with frequent shaking. The addition occupied about one hour. The mixture was then refluxed on a water bath for 3 hr and then allowed to stand overnight. The crude chlorofluoroquinoline-4carboxylic acid (KP-6) was filtered off at the pump and crystals were washed with a little KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 36 METHODOLOGY ether. The crude product was recrystallised from ethanol to give a reddish-orange color powder whose yield was 9.7 g (35.43%). 7. Synthesis of 2-methoxy-2-oxoethyl-7-chloro-6-fluoro-2-phenyl quinoline-4-carboxylate, (KP-1A): To a solution of 7-chloro-6-fluoro-2-phenyl quinoline-4-carboxylic acid (KP-1, 1 g, 0.0034 mol) in dry DMF (10 ml), methyl chloroacetate 0.36 ml (0.0034 mol) and anhydrous potassium carbonate 0.457 g (0.0033 mol) were added and the reaction was refluxed for 72 h. The reaction mixture was poured into ice-cold water; the solid was separated, collected and dried. Recrystallization was carried out with ethanol:water(1:4) to give a dark yellow color compound (KP-1A) with yield 0.8 g (64.51%). 8. Synthesis of 2-methoxy-2-oxoethyl-7-chloro-6-fluoro-2-(4-methyl phenyl) quinoline-4-carboxylate, (KP-2A): To a solution of 7-chloro-6-fluoro-2-(4-methyl phenyl) quinoline-4-carboxylic acid (KP3, 1 g, 0.0032 mol) in dry DMF (10 ml), methyl chloroacetate 0.34 ml (0.0032 mol) and anhydrous potassium carbonate 0.437 g (0.0032 mol) were added and the reaction was refluxed for 72 h. The reaction mixture was poured into ice-cold water; the solid was separated, collected and dried. Recrystallization was carried out with ethanol:water(1:4) to give a creamish-yellow color compound (KP-2A) with yield 0.48 g (40.37%). 9. Synthesis of 2-ethoxy-2-oxoethyl-7-chloro-6-fluoro-2-phenyl quinoline-4-carboxylate, (KP-1C): To a solution of 7-chloro-6-fluoro-2-phenyl quinoline-4-carboxylic acid (KP-1, 1 g, 0.0034 mol) in dry DMF (10 ml), ethyl chloroacetate 0.34 ml (0.0033 mol) and anhydrous potassium carbonate 0.457 g (0.0033 mol) were added and the reaction was refluxed for 72 h. The reaction mixture was poured into ice-cold water; the solid was separated, collected and dried. Recrystallization was carried out with ethanol:water(1:4) to give a brown color compound (KP-1C) with yield 0.46 g (37.03%). 10. Synthesis of 5-(7-chloro-6-fluoro-2-phenylquinolin-4-yl)-1,3,4thiadiazol-2- amine, (KP-1B):56 The mixture of 7-chloro-6-fluoro-2-phenyl quinoline-4-carboxylic acid (KP-1, 1 g, 0.0034 mol) and thiosemicarbazide (0.301 g, 0.0033 mol) was added slowly to the RBF KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 37 METHODOLOGY containing POCl3 (1 ml) with constant stirring in ice bath. After completion of addition ice bath was replaced by water bath and slowly heated to 70-800C. Temperature was maintained for 7-8 hrs. It was cooled and then poured into ice water and made alkaline with ammonia. The product was filtered, washed with water and recrystallized from ethanol to obtain the brown coloured compound (KP-1B) and the yield was 0.7 g (58.9%). 11. Synthesis of 5-(2-(4-methyl phenyl)-7-chloro-6-fluoro quinolin-4-yl)1,3,4-thiadiazol-2- amine, (KP-2B): The mixture of 7-chloro-6-fluoro-2-(4-methyl phenyl) quinoline-4-carboxylic acid (KP3, 1 g, 0.0032 mol) and thiosemicarbazide (0.288 g, 0.0032 mol) was added slowly to the RBF containing POCl3 (1 ml) with constant stirring in ice bath. After completion of addition ice bath was replaced by water bath and slowly heated to 70-800C. Temperature was maintained for 7-8 hrs. It was cooled and then poured into ice water and made alkaline with ammonia. The product was filtered, washed with water and recrystallized from ethanol to obtain the reddish-brown coloured compound (KP-2B) and the yield was 0.77 g (65.58%). 12. Synthesis of 5-(2-(4-methoxy phenyl)-7-chloro-6-fluoro quinolin-4yl)-1,3,4-thiadiazol-2- amine, (KP-4B): The mixture of 7-chloro-6-fluoro-2-(4-methoxy phenyl) quinoline-4-carboxylic acid (KP-4B, 1 g, 0.003 mol) and thiosemicarbazide (0.274 g, 0.003 mol) was added slowly to the RBF containing POCl3 (1 ml) with constant stirring in ice bath. After completion of addition ice bath was replaced by water bath and slowly heated to 70-800C. Temperature was maintained for 7-8 hrs. It was cooled and then poured into ice water and made alkaline with ammonia. The product was filtered, washed with water and recrystallized from ethanol to obtain the dark reddish-brown coloured compound (KP-4B) and the yield was 0.42 g (36.03%). KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 38 METHODOLOGY B] BIOLOGICAL EVALUATION Antimicrobial Activity Biological evaluation involves testing the microbial susceptibility to chemotherapeutic agents. After the development of desired new drug molecules, with different structure, an antimicrobial screening programme is necessary to uncover the interesting activity of the compounds. The inhibition of the microbial growth under standardization may be utilized for demonstrating the therapeutic efficacy of the synthesized compounds. The method adopted for screening of the antimicrobial substances was 1. Disc diffusion method.57 Anti bacterial activity Preparation of stock solution Stock solutions of the synthesized compounds used were prepared in dimethyl sulfoxide in the concentration of 100 µg/ml. The stock solution of standard drug (ciprofloxacin) was prepared using distilled water in the concentration of 100 µg/ml. Preparation of pure culture The pour plate method of obtaining pure culture is used which involved serial dilution, transferring to melted agar, a specific volume of the dilution containing a few organisms and picking up cells from colony of agar. Culture media Enriched media that provide a nutrient that fastens the growth of organisms was used as the growth media. Most routine laboratory culture make use of peptones (general purpose media) or digested meat or fish proteins.58 Other cultures used are yeast extracts, casein, hydrolysate, serum, whole blood or heated whole blood (enriched media). KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 39 METHODOLOGY Composition of nutrient broth used for bacteria S. No. 1. 2. 3. 4. Weight (g) Ingredients Beef extract Peptic digest of animal tissue Yeast 1.50 5.00 1.50 Sodium chloride 5.00 Final pH at 25C 7.4 ± 0.2 Composition of nutrient media used for agar plate S. No. 1. 2. Weight (g) Ingredients Beef extract Peptic digest of animal tissue 1.50 5.00 3. Yeast 1.50 4. Sodium chloride 5.00 5. Agar 15.00 Final pH at 25C 7.4 ± 0.2 Cultures used Standard cultures of Staphylococcus aureus and Pseudomonas auregenosa species were obtained from from Microbiology laboratory, K.L.E University’s College of Pharmacy, Bangalore. KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 40 METHODOLOGY Strain No. Staphylococcus aureus (ATCC 6538) Pseudomonas auregenosa (ATCC 10145) Disc diffusion method The petri dishes were washed thoroughly and sterilized in hot air oven at 170˚C for one hour. Around 120 ml of sterile nutrient agar medium for bacteria was poured into sterile petri dishes and allowed to solidify. The petri dishes were incubated at 37˚C for 24 hr to check for sterility. The medium was seeded with the organism by spread plate method using sterile cotton swabs and then placed the disc of Whatmann filter paper, pre-saturated with different sterile dilutions of KP-1, KP-2, KP-3, KP-4, KP-5, KP-5, KP-1A, KP-2A, KP1B, KP-2B, KP-4B and a standard solution of ciprofloxacin at a concentration of 100 μg/ml was taken as standard reference. The petri plates were incubated for 24 hr at 37˚C and then the zones of inhibition were measured. Anti fungal activity The anti fungal activity of the derivatives was done in Sunrise hospitals, Hyderabad. The drug Fluconazole was taken as standard and the concentration of standard was taken as 100µg/ml. The sample concentrations were also taken as 100 µg/ml each. The method used was disc diffusion method.The procedure done for antifungal activity was same as above. Composition of Fungi media (Sabouand Dextrose Broth) S. No. Ingredients Weight (g) 1. Special Peptone 10gm 2. Dextrose 20gm KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 41 RESULTS AND DISCUSSION In this research project we have tried to further explore the quinoline nucleus by synthesizing some quinoline derivatives using the proposed scheme given in methodology section. As per scheme, in the first step, some substituted 7-chloro 6-fluoro quinoline-4carboxylic acids were synthesized by reacting substituted aniline and pyruvic acid with different aldehydes. All the synthesized derivatives were recyrstallized by using various solvents. Physical characterization was done by melting point and TLC. The structures of the compounds were confirmed by IR, 1H NMR and mass spectral studies. The synthesis of 2-substituted 7-chloro 6-fluoro quinoline-4-carboxylic acid (KP 1-6) was accomplished by reacting the substituted aniline and pyruvic acid with appropriate aldehyde. The IR in these compounds showed C=O stretch peak at 1689 cm-1, O-H stretch appeared at 3331 cm-1, C-H stretch aromatic at 3058 cm-1, C-F stretch at 1223 cm1 and C-Cl stretch 649.89 cm-1. In 1H NMR, there are well resolved resonance peaks at δ 6.58-7.01 for CH aromatic, 9.98 for –COOH ppm. In the second step, substituted 7-chloro 6-fluoro quinoline-4-carboxylic acids were made to react with methylchloro acetate in the presence of DMF and K2CO3 to give substituted methyl esters. e.g. in 2-methoxy-2-oxoethyl-7-chloro-6-fluoro-2-phenyl quinoline-4carboxylate (KP-1A) IR vibrations were seen at 3065 cm-1 for aromatic C-H, 2924 cm-1 for aliphatic C-H stretch, 1675 cm-1 for C=O, 1259 cm-1 for C-O-C and 1603 cm-1 for C=N stretch in ring. The substituted 7-chloro 6-fluoro quinoline-4-carboxylic acids were treated with ethylchloro acetate in the presence of DMF and K2CO3 to give substituted ethyl esters in the third step. The IR spectra of the compound, 2-ethoxy-2-oxoethyl-7-chloro-6-fluoro-2phenyl quinoline-4-carboxylate (KP-1C) showed bands at 3058 cm-1 for C-H aromatic stretching, 2924 cm-1 for aliphatic C-H stretch, 1686 cm-1 for C=O, 1250 cm-1 for C-O-C, 1463 cm-1 for C=N stretch in ring. KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 42 RESULTS AND DISCUSSION Various thiadiazole derivatives were synthesized by treating equimolar mixture of substituted quinolinic-4-carboxylic acid and thiosemicarbazide with concentrated sulphuric acid. The compound 5-(2-(4-methyl phenyl)-7-chloro-6-fluoro quinolin-4-yl)1,3,4-thiadiazol-2- amine (KP-2B) shows IR vibrations at 3077 for aromatic C-H , 3117 for N-H, 2295 for C-S-C, 1254 for C-O-C, 1595 for C=N stretch in ring, C-F stretch at 1245 cm-1 and C-Cl stretch 709.89 cm-1. The mass analysis of the compound shows the molecular ion peak at m/z 371.1 by which we have confirmed its structure. All the synthesized compounds were characterized using TLC, NMR, IR and Mass analysis. Physical constants and characterized data for these compounds are given in table 1-10. KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 43 RESULTS AND DISCUSSION TABLE-2: Physical constants and Structures of the synthesized compounds Code Physical state Structure M.P ° C Molecular formula Mol. wt 170-172 C16H9ClFNO2 301.7 100-102 C17H11ClFNO2 316.05 160-162 C16H8ClFN2O4 346.7 110-112 C17H11ClFNO3 331.04 OH O Cream KP-1 crystals F Cl N O KP-2 White OH F crystals N Cl CH3 OH O Light F KP-3 green powder Cl O Light KP-4 NO2 N OH F yellow crystals Cl N OCH3 KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 44 RESULTS AND DISCUSSION O KP-5 Yellow OH F crystals 98-100 C16H9ClFNO3 317.7 190-192 C16H8ClFN2O4 346.7 80-82 C19H13ClFNO4 373.76 90-92 C21H17ClFNO4 401.82 N Cl OH O ReddishKP-6 orange OH F NO2 powder N Cl O O O CH3 O KP-1A Dark F yellow powder N Cl O O Creamish KP-2A -yellow powder O CH3 O F Cl N CH3 KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 45 RESULTS AND DISCUSSION O O O O Brown KP-1C CH3 F powder 74-76 C20H15ClFNO4 387.79 198-200 C17H10ClFN4S 356.8 N Cl NH2 N N S Brown KP-1B F powder N Cl NH2 N ReddishKP-2B brown N S 172-175 C18H12ClFN4S 370.83 F powder N Cl CH3 NH2 N Dark KP-4B reddish- S N 180-182 C18H12ClFN4OS 386.83 F brown powder Cl N OCH3 KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 46 RESULTS AND DISCUSSION TABLE-3: Thin Layer Chromatography Code Solubility Mobile phase KP-1 Chloroform Toluene:Ethyl acetate 0.84 KP-2 Chloroform Toluene:Ethyl acetate 0.78 KP-3 Chloroform Toluene:Ethyl acetate 0.70 KP-4 Chloroform Toluene:Ethyl acetate 0.73 KP-5 Chloroform Toluene:Ethyl acetate 0.8 KP-6 Chloroform Toluene:Ethyl acetate 0.64 KP-1A Chloroform Chloroform:Toluene 0.42 KP-2A Chloroform Chloroform:Toluene 0.71 KP-1C Chloroform Chloroform:Toluene 0.59 KP-1B Chloroform Chloroform:Ethanol 0.64 KP-2B Chloroform Chloroform:Ethanol 0.55 KP-4B Chloroform Chloroform:Ethanol 0.48 KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Rf value Page 47 RESULTS AND DISCUSSION SPECTRAL DATA TABLE-4: Infra Red spectral study of the synthesized compounds Code KP-1 KP-2 KP-3 KP-4 Spectral Peaks in cm-1 Functional Group 3114.47 3071.08 1696.09 1600.63 1223.61 649.893 -O-H stretching -C-H stretching aromatic -C=O stretching -C=N stretch in ring -C-F stretching -C-Cl stretching 3108.69 3023.84 1671.98 1599.66 2923.56 1230.36 663.393 -O-H stretching -C-H stretching aromatic -C=O stretching -C=N stretch in ring -C-H stretching aliphatic -C-F stretching -C-Cl stretching 3118.33 3085.55 1701.87 1602.56 1514.81 1240.00 685.57 -O-H stretching -C-H stretching aromatic -C=O stretching -C=N stretch in ring -N=O stretching -C-F stretching -C-Cl stretching 3308.28 3041.47 1694.84 1598.77 1192.76 1221.37 654.715 -O-H stretching -C-H stretching aromatic -C=O stretching -C=N stretch in ring -C-O stretching (methoxy) -C-F stretching -C-Cl stretching KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 48 RESULTS AND DISCUSSION 3303.46 3034.76 1710.56 1602.94 1187.94 654.715 -O-H stretching -C-H stretching aromatic -C=O stretching -C=N stretch in ring -C-F stretching -C-Cl stretching 3318.89 3070.12 1695.12 1603.52 1426.10 1237.11 736.674 -O-H stretching -C-H stretching aromatic -C=O stretching -C=N stretch in ring -N=O stretching -C-F stretching -C-Cl stretching KP-1A 3065.30 2923.56 1675.84 1603.52 1259.29 1062.59 701.962 -C-H stretching aromatic -C-H stretching aliphatic -C=O stretching -C=N stretch in ring -C-O stretching -C-F stretching -C-Cl stretching KP-2A 3109.25 2920.66 1671.02 1609.31 1217.14 1114.65 764.637 -C-H stretching aromatic -C-H stretching aliphatic -C=O stretching -C=N stretch in ring -C-O stretching -C-F stretching -C-Cl stretching KP-5 KP-6 KP-1B 3349.75 3059.51 2208.09 1600.63 1258.32 701.962 KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] -N-H stretching -C-H stretching aromatic -C-S-C stretching C=N stretch in ring -C-F stretching -C-Cl stretching Page 49 RESULTS AND DISCUSSION KP-4B 3364.21 3046.98 2360.44 1610.27 1256.43 1177.33 710.64 KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] -N-H stretching -C-H stretching aromatic -C-S-C stretching C=N stretch in ring -C-O stretching (methoxy) -C-F stretching -C-Cl stretching Page 50 RESULTS AND DISCUSSION TABLE-5: 1H NMR spectral data of synthesized compounds Code Chemical shift value (δ) Nature of proton (ppm) 9.826 KP-1 KP-2 KP-4 KP-1A s, 1H, -COOH 7.685-7.734 t, 2H, Ar-H 7.118-7.334 m, 3H, Ar-H 6.035-6.048 d, 2H, Ar-H 6.578 s, 1H, Ar-H 10.184 s, 1H, -COOH 7.683-7.715 d, 1H, Ar-H 7.259-7.331 t,2H, Ar-H 6.919-7.176 m, 3H, Ar-H 6.115-6.128 d, 1H, Ar-H 2.304 s, 3H, -CH3 9.984 s, 1H, -COOH 7.663-7.696 d, 1H, Ar-H 7.261-7.337 q, 3H, Ar-H 6.886-7.107 m, 2H, Ar-H 6.582 s, 1H, Ar-H 3.767 s, 3H, -OCH3 7.33-7.56 m, 4H, Ar-H 7.745 s, 2H, Ar-H 7.85-7.92 d, 2H, Ar-H 2.13 s, 3H, -CH3 3.03 s, 2H, -OCH2CO- KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 51 RESULTS AND DISCUSSION TABLE-6: Mass spectral data of synthesized compounds Code Molecular weight m/z (Relative intensity) (calculated) (observed) KP-2 316.05 315.3 KP-2B 370.83 371.1 KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 52 RESULTS AND DISCUSSION Anti microbial Studies Antibacterial activity: The synthesized compounds (KP 1, 2, 3, 4, 5, 1A, 2A, 1B, 2B, 4B) were subjected to antibacterial evaluation against pseudomonas auregenosa and staphylococcus aureus. The antibacterial activity of the compounds compared in term of zone of inhibition was summarized in the Table 7 and Fig. 2. TABLE-7: Antibacterial activity of the synthesized compounds. Code Dose (µg/ml) Zone of inhibition (mm) S.aureus P.auregenosa (gram+ve) (gram-ve) KP-1 100 µg/ml 15 mm 12 mm KP-2 100 µg/ml 10 mm None KP-3 100 µg/ml 11 mm None KP-4 100 µg/ml None None KP-5 100 µg/ml 12 mm 11 mm KP-1A 100 µg/ml 13 mm 12 mm KP-2A 100 µg/ml None None KP-1B 100 µg/ml 14 mm 11 mm KP-2B 100 µg/ml None None KP-4B 100 µg/ml 10 mm None 100 µg/ml 22 mm 19 mm Ciprofloxacin (standard) KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 53 RESULTS AND DISCUSSION As evident from the table the synthesized compounds have not shown good antimicrobial activity against both Gram-negative and Gram-positive bacteria. The compounds KP-1, KP-3, KP-5, KP-1A and KP-1B have shown activity against S.aureus (Gram+ve bacteria) and the compounds KP-1 and KP-1A have shown activity against P.auregenosa (Gramve bacteria). Some compounds have almost no activity against gram -ve bacteria. No compound had shown better activity than the standard ciprofloxacin. Fig-2: Antibacterial activity for the synthesized compounds against S. aureus Fig-3: Antibacterial activity for the synthesized compounds against P.auregenosa KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 54 RESULTS AND DISCUSSION Antifungal activity: The synthesized compounds (KP 1,2,4,5,1A,1B) were subjected to antifungal evaluation against C.albicans. The antifungal activity of the synthesized compounds were found to be showing mild activity. The compounds KP-1 and KP-1A showed mild activity against the organism and other compounds have almost no activity. Overall none of the compounds showed higher activity than the standard drug. Table-8: Antifungal activity of synthesized compounds against C.albicans S.No. Code Concentration(µg/ml) Zone of inhibition(mm) 1. KP-1 100µg/ml 12 2. KP-2 100µg/ml None 3. KP-4 100µg/ml None 4. KP-5 100µg/ml 10 5. KP-1A 100µg/ml 11 6. KP-1B 100µg/ml None 100 µg/ml 20 7. Fluconazole(standard) KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 55 SPECTRAL STUDIES Fig-4: Infrared spectra of 7-chloro-6-fluoro-2-phenyl quinoline-4carboxylic acid (KP-1) OH O F Cl N KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 56 SPECTRAL STUDIES Fig-5: Infrared spectra of 7-chloro-6-fluoro-2-(4-methyl phenyl) quinoline-4-carboxylic acid (KP-2) O OH F Cl N CH3 KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 57 SPECTRAL STUDIES Fig-6: Infrared spectra of 7-chloro-6-fluoro-2-(3-nitro phenyl) quinoline-4-carboxylic acid (KP-3) OH O F Cl N KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] NO2 Page 58 SPECTRAL STUDIES Fig-7: Infrared spectra of 7-chloro-6-fluoro-2-(4-methoxy phenyl) quinoline-4-carboxylic acid (KP-4) O OH F Cl N OCH3 KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 59 SPECTRAL STUDIES Fig-8: Infrared spectra of 2-methoxy-2-oxoethyl-7-chloro-6-fluoro-2phenyl quinoline-4-carboxylate (KP-1A) O O O CH3 O F Cl N KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 60 SPECTRAL STUDIES Fig-9: Infrared spectra of 2-methoxy-2-oxoethyl-7-chloro-6-fluoro-2-(4methyl phenyl) quinoline-4-carboxylate (KP-2A) O O O CH3 O F Cl N CH3 KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 61 SPECTRAL STUDIES Fig-10: Infrared spectra of 5-(7-chloro-6-fluoro-2-phenylquinolin-4-yl)1,3,4-thiadiazol-2- amine (KP-1B) NH2 N N S F Cl N KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 62 SPECTRAL STUDIES Fig-11: Infrared spectra of 5-(2-(4-methoxy phenyl)-7-chloro-6-fluoro quinolin-4-yl)-1,3,4-thiadiazol-2- amine (KP-4B) NH2 N S N F Cl N OCH3 KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 63 SPECTRAL STUDIES Fig-12: 1H NMR spectra of 7-chloro-6-fluoro-2-phenyl quinoline-4carboxylic acid (KP-1) OH O F Cl N KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 64 SPECTRAL STUDIES Fig-14: 1H NMR spectra of 7-chloro-6-fluoro-2-(4-methyl phenyl) quinoline-4-carboxylic acid (KP-2) O OH F Cl N CH3 KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 65 SPECTRAL STUDIES Fig-14: 1H NMR spectra of 7-chloro-6-fluoro-2-(4-methoxy phenyl) quinoline-4-carboxylic acid (KP-4) O OH F Cl N OCH3 KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 66 SPECTRAL STUDIES Fig-15: Mass spectra of 7-chloro-6-fluoro-2-(4-methyl phenyl) quinoline-4-carboxylic acid (KP-2) O OH F Cl N CH3 KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 67 SPECTRAL STUDIES Fig-16: Mass spectra of 5-(2-(4-methyl phenyl)-7-chloro-6-fluoro quinolin-4-yl)-1,3,4-thiadiazol-2- amine (KP-2B) NH2 N N S F Cl N CH3 KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 68 CONCLUSION CONCLUSION As evident from the literature survey, quinoline derivatives are potent anti-microbial, anti-malarial, anticancer and anti-HIV agents. Many quinoline compounds are undergoing clinical trials under different phases. Taking a lead from these evidences some quinoline derivatives were synthesized using pyruvic acid, substituted benzaldehydes, aniline, methyl chloroacetate and ethyl chloroacetate. Treatment of quinolinic acid with thiosemicarbazide was carried out to obtain thiadiazole derivatives of quinoline. The structures of the synthesized compounds were confirmed with the help of m.p, TLC, spectroscopic analysis like IR, 1HNMR, and Mass analysis. The synthesized compounds have been tested for antimicrobial activity. The antibacterial activity was carried out on Gram positive (staphylococcus aureus) and Gram negative (pseudomonas auregenosa) bacteria. Some of the compounds were showing moderate activity at concentration of 100 µg/ml. But none have found to be better than the standard (ciprofloxacin). Some synthesized derivatives showed mild antifungal activity against C. albicans at a concentration of 100g/ml. A further research in this direction holds promising aspect for the development of more effective and safer drugs. KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 69 SUMMARY SUMMARY Section I describes the necessity to synthesize substituted fluoro chloro benzpyrine derivatives Section II deals with the objectives of the entire research work of this dissertation by explaining the need to develop newer derivatives as antimicrobial agents. In section III review of literature has discussed with special reference to various benzpyridine derivatives for antimicrobial activity. In section IV a detailed method of synthesis of various fluoro chloro benzpyridine derivatives. Section V describes methodology of compounds along with their purification, physical constants has been given. All the compounds were synthesized in good yield and high purity. The derivatives were characterized by subjecting to various spectral studies such as FT-IR, 1HNMR, Mass spectroscopy and antimicrobial studies have also given here. Section VI deals with the result and discussion of fluoro chloro benzpyridine derivatives. Section VII deals with the conclusion of the synthesized fluoro chloro benzpyridine derivatives. Section VIII deals with the summary of the entire research work of this dissertation. Section IX deals with the various references that led to formation of this dissertation. KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 70 SUMMARY KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY] Page 71 REFERENCES REFERENCES 1. Delgado JN, Remers WA. Wilson and Gisvold’s Text book of organic medicinal and pharmaceutical chemistry. 9th ed. Philadelphia. J.B.Lippincott Company; 1991. P. 155-8. 2. Bansal RK. Text book of heterocyclic chemistry. 4th ed. New Delhi. New Age Publishers; 2005. P. 366. 3. Block JH, Beale JM. 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