Friday Probate/Guardianship Calendar

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]
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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]
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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]
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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]
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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 25C 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 25C 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 100g/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. Wilson and Gisvold’s Text book of Organic Medicinal and
Pharmaceutical Chemistry. 11th ed. Philadelphia. Lippincott Williams & Wilkins. P.
247-248.
4. Benard C, Zovhin F. Linker modified quinoline derivatives targeting HIV-1integrase:
synthesis and biological activity. Bioorganic and Medicinal Chemistry Letters 2004;
14:2473-76.
5. Mehanna SA, Abraham DJ. Rationale of design of anti HIV Drugs, In Burgers
medicinal chemistry and drug discovery. vol 5. 6th ed. Virginia. John Weiley and
Sons; 2003. P. 458-79.
6. Divo A, Sartorelli AC, Patton CL, Bia FJ. Activity of fluoroquinolines antibiotics
against plasmodium falciparum in vitro. Antimicrobial Agents Chemotherapy 1998;
32(8):1182-86.
7. Gorlitzer K, Gabriel B, Jomaa H, Wiesner J. Thieno(3,2-c)quinoline -4yl-amines:
Synthesis and investigations of activity against malaria. (article in German)
Pharmazie 2006: 61(4):278-84.
8. Khanfaruk MO, Levi SM, Takwani LB, Wilson HN, Borne FR. Synthesis of
isoquincilidine analogs of chloroquine: anti-malarial and anti-leishmanial activity.
Bioorganic and Medicinal Chemistry 2007; 15:3919-25. doi:10.1016/j.bmc.
2006.11.024.
9. Zhao YL, Chen YL, Tzeng CC, Chen IL, Wang TC, Han CH. Synthesis and
cytotoxicity evaluation of certain 4-(phenylamino)-furo-(2,3-b)quinoline and 2(furan-2-yl)-4-(phenylamino)quinoline
derivatives.
Bioorganic
and
Medicinal
Chemistry 2007; 17(4):942-45.
10. V.N.Ramachandran, W.F.Symth, F.O’Donnell, T.J.P.Symth. A study of the
antimicrobial activity of selected synthetic and naturally occuring quinolines.
International
Journal
of
Anti-microbial
agents
2010;
35(1):30-38.
doi:10.1016/j.ijantimicag.2009.06.031.
KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY]
Page 71
REFERENCES
11. Sun XY, Jin YZ, Li FN, Chai KY, Quan ZS. Synthesis of 8-alkoxy-4,5dihydro(1,2,4)triazole(4,3-a)quinoline-1-ones
and
evaluation
of
their
anticonvulsant
properties. Arch Pharm Res 2006; 29(12):1080-85.
12. Xie ZF, Chai KY, Piao HR, Kwak KC, Quan ZS. Synthesis and anticonvulsant
activity of 7-alkoxyl-4,5dihydro-(1,2,4)triazole(4,3-a) quinoline. Bioorganic and
Medicinal Chemistry 2005; 4803-05.
13. Aubry A, Pan XS, Fisher M, Jarlier V, Cambau E. Mycobacterium tuberculosis DNA
gyrase: Integrase with quinolones and correlation with antimycobacterial drug
Activity. Antimicrobial Agents Chemotherapy 2004; 48(4):1281-88.
14. Nayyar A, Monga V, Malde A, Coutinho E, Jain R. Synthesis and anti-tubercular
activity and 3D-QSAR study of 4-(admantan-1-yl)-2- subustituted quinolines.
Bioorganic and Medicinal Chemistry 2007; 15(2):626-40.
15. Zhu XY, Mardenborough LG, Li S, Khan A. Synthesis and evaluation of isosters of
N-methyl indole[3,2-b]-quinoline (cryptolepine) as new antiinfective agents.
Bioorganic and Medicinal Letters 2007; 15:686-95.
16. Souers A, Wodka D, Gao J, Lewis JC, Vasudevan A, Gentles R. Synthesis and
evaluation of 2-amino-8-alkoxy quinolines as MCHr1 antagonist. Part 1. Bioorganic
and Medicinal Letters 2004; 14:4873-77. doi:10.1016/j.bmcl. 2004.07.032.
17. 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.
211.
18. Woodward, R.B, Doering W.E. Synthesis of quinoline analogues. J. Am. Chem. Soc,
1945; 67:860-874.
19. Andrejus K. Essentials of Medicinal chemistry. 2nd ed. New York. John Wiley and
Sons; 2008. P. 629-631.
20. Karle J. M., Bhattachq'ee, A. K. Gerena, L Milhous. Comparision of quinine and
quinidine on chloroquine resistant strains. J. Bioorg. Med. Chem., 1999; 7:1769-1774.
21. Tripathi KD. Antimicrobial Drugs. Essentials of Medical Pharmacology. 5th ed. New
Delhi, Jaypee brothers Medical Publisher; 2004. P.735-736.
22. Brunton LL, Lazo JS, Parker KL. Goodman and Gilman’s. The pharmacological basis
of therapeutics. 11th ed. New York. McGraw-Hill medical publishing division. P.
1119.
KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY]
Page 72
REFERENCES
23. Casteel DA. Antimalarial agents. Burgers Medicinal Chemistry and drug discovery.
6th ed. Virginia.A John Wiley and Sons, Inc. Publication; 1991. P.920.
24. Tripathi KD. Antimalarial Drugs. Essentials of Medical Pharmacology. 5th ed. New
Delhi. Jaypee brothers Medical Publisher; 2004. P.735-736.
25. Rang HP, Dale MM, Ritter JM, Moore PK. Pharmacology. 5th ed. Edinburgh.
Churchill Livingstone; 2003. P. 673-74.
26. Hooper DC. Bacterial topoisomerases, anti-topoisomerases, and anti-topoisomerase
resistance. Clin Infect Dis 1998; 27:S54-S63.
27. Lesher GY. Froelich ED, Froelich ED, Grant MD, Bailey JH, Brundage RP. J. Med.
Pharm. Chem. 1962; 5:1063.
28. Monsouri E, Firoozpour F, Asadipour E, Ammi M. Synthesis and antibacterial
activity
of
N-[5(chlorobenzylthio)-1,3,4-thiadiazol-2-yl]piperzinyl
quinoline
derivatives. Arch. Pharm. Res. 2007; 30(2):138-45.
29. Talath S, Gadad AK. Synthesis, antibacterial and antitubercular activities of some 7[4-5-amino-(1,
3,
4)
thiadiazole-2-sulfonyl]-piperazin-1-yl]
fluoro-quinolonic
derivatives. European Journal of Medicinal Chemistry 2006; 41(8):918-24.
30. De Souza M, Pais K, Kaiser C, Peralta M. Synthesis and in vitro antitubercular
activity of a quinoline derivatives. Bioorganic and Medicinal Chemistry 2009;
17(4):1474-80.
31. Wang Y, Lv J, Qian J, Liu T. Synthesis and evaluation of ampiphilic cationic quinine
derived for antibacterial activity against methicillin resistant staphylococcus aureus.
Bioorganic and Medicinal chemistry Letters 2007; 17(2):4102-4106.
32. Zhu X, Mardenborough L, Li S, Khan A, Zhang W, Fan P. Synthesis and evaluation
of N-methyl indolo(3,2-b)quinoline(cryptolepine) as new anti
-infective
agents 2007; 15: 686-695.
33. John H.Block, John M.Beale, In Antimalarials, Wilson and Gisvold’s Textbook of
Organic Medicinal and Pharmaceutical chemistry, eleventh edition, Lippincott
Company, United States of America, 2004. P. 282.
34. Sato M, Motomura T, Aramaki H, Matsuda T, Yamshita M. Novel HIV integrase
inhibitors derived from quinolone antibiotics. J. Med Chem. 2006; 49:1506-08.
KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY]
Page 73
REFERENCES
35. Ohnmacht CJ, Patel AR, Lutz ER. Antimalarials.7-bis (trifluoromethyl)-alpha-(2piperidyl-4-quinolinemethanols).
J.
Med.
Chem.
1971;
14(10):926-28.
doi:
10.1021/jm00292a008.
36. Schmidt HL, Alexender S, Allen L, Rasco J. Synthesis of 8-quinolinamines, N1-{4[2-(tert-butyl)-6-methoxy-8-quinolinamino]pentyl}-(2S/2R)-2-aminosubstituted
amides as anti-malarial activity. J. Antimicrob. Agents Chemother. 1977; 12:51-66.
37. Kaur K, Patel RS, Patil P, Jain M, Khan IS, Jacob R. Synthesis, antimalarial,
antileshmanial, antimicrobial, cytotoxicity and methemoglobin formation, activities of
new 8-quinolinamines. Bioorganic and Medicinal Chemistry 2007; 15(2):915-30.
38. Watkins HM, Nevill CG, Lury JD, Mosobo KM. Synthesis of 2-[3-(7-chloroquinolin4-yl-amino)alkyl]-1-(substituted phenyl)-2,3,4,9-tetrahydro-1H-β-carbolines as antimalarial against chloroquine sensitive strains of P.falciparum. Soc. Trop. Med. Hyg.
1994; 10:319-20.
39. Black M, Beagley C, Kelly C, Clarck C. Synthesis and antimalarial activity in vitro of
new hetero-bimetallic complexes: Rh and Au derivatives of chloroquine and a series
of ferrocenyl-4-amino-7-chloroquinolines. Bioorganic and Medicinal Chemistry
2007; 15(20):6510-16.
40. Cochin DS, Grellier P, Maes L, Mouray E. Synthesis and anti-malarial activity of
carbamate and amide derivatives of 4-anilinoquinoline. European Journal of
Medicinal Chemistry 2008; 43(10):2045-55.
41. Ashok kumar, Kumkum Srivastava, S. Raja kumar, S. K. Puri, M. S Chauhan.
Synthesis of new 4-amino quinolines and quinoline-acridine hybrids as antimalarial
agents. Bio organic and Medicinal Chemistry Letters 2010; 20:7059-63.
doi:10.1016/j.bmcl.2010.09.107.
42. Chen YL, Hung HM, Lu CM, Li KC, Tzeng CC. Synthesis and anticancer evaluation
of certain indolo(2,3-b)quinoline derivatives. Bioorganic and Medicinal Chemistry
2004; 12(24):6539-46.
43. Shi A, Ngugen TA, Battina SK, Rana S, Takemoto DJ, Chiang PK, Hua DH.
Synthesis and anticancer activities of substituted quinolines. Bioorganic and
Medicinal Chemistry Letters 2008; 18(11):3364-3368.
44. Mostafa M. Ghorab, Mansour S. Al-Said, Mohammed S. Al-Dosari, Mostafa M.
Hamed.
Synthesis
and
invitro
anticancer
KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY]
evaluation
of
some
novel
Page 74
REFERENCES
hexahydroquinoline derivatives having a benzene sulfonamide moiety. Eur J Med
Chem. 2011; 46(1):201-207. doi:10.1016/j.ejmech.2010.11.002.
45. Ferlin MG, Marzano C, Via LD, Chilin A, Zagotto G, Guitto A, Maro S. New water
soluble pyrroloquinoline derivatives as new potential anticancer agents. Bioorganic
and Medicinal Chemistry 2005; 13(15):4733-39.
46. Guo LJ, Nei CX, Jia JH, Zhao LM, Quan ZS. Design and synthesis of 5-alkoxy[1,2,4]triazolo[4,3-a]quinoline derivatives with anti-convulsant activity. Eur J Med
Chem. 2009; 44(3):954-8. doi:10.1016/j.ejmech.2008.07.010.
47. Quan ZS, Cui LJ, Xie Zf, Paio HR, Chai KY. Synthesis and anticonvulsant activity of
1-substituted-7-benzyloxy-4,5-dihydro-(1,2,4)triazolo(4,3-a)quinoline. Biological and
Pharmaceutical Bulletin 2005; 28(7):1216-20.
48. Sato M, Motomura T, Aramaki H, Matsuda T, Yamshita M, Ito Y. Novel HIV
integrase inhibitors derived from quinolone antibiotics. J. Med. Chem. 2006; 49:15061508.
49. Xu B, sun Y, Gua Y, Cao Y, Yu T. Synthesis and biological evaluation of N4(hetero)arylsulfonylquinoxalines as HIV-1 reverse transcriptase inhibitors. Bioorganic
and Medicinal Chemistry 2009; 17(7):2767-74.
50. Benard C, Zouhiri F, Normand-Bayle DM, Desmaele D Leh H, Mouscadet JF.
Mbemba G. Linker modified quinoline derivatives targeting HIV-1 integrase:
synthesis and biological activity. Biorg. and Med. Chem. Lett. 2004; 14:2473-2476.
51. Qiang Xu, Guo-Biao Liu, Jian-Liang Xu, Cui-Cui He, Gong chen, Hong-Xixu, Jianxin Li. Synthesis and evaluation of a novel series of quinoline derivatives with
immunosuppressive activity. Bio organic & Medicinal Chemistry 2009; 17:5433-41.
doi:10.1016/j.bmc.2009.06.043.
52. Quaz Zhang, Rui-Hua Gua, Yun Bao Ma, Ji-Jun Chen, Jun Zhou, Zhi-Yong Jiang.
Structural activity relationship study of 6-chloro-4-(2-chlorophenyl)-3-(2-hydroxy
ethyl)quinolin-2-(1H)-one derivative as novel non-nucleoside anti-hepatitis B virus
agents. Eur J Med Chem 2011; 46(1):307-319. doi:10.1016/j.ejmech.2010.11.019.
53. Ebrahim Azizi, Afshin Zarghi, Razieh Ghodsi, Bahram Davaie, Mehdi Hedayati,
Orkideh G. Dadrass. Synthesis and biological evaluation of new 4-Carboxyl quinoline
derivatives as Cyclo oxygenase-2-inhibitors. Bio organic and Medicinal Chemistry
2009; 17:5312-17.
KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY]
Page 75
REFERENCES
54. Khalid Mohammed Khan, Sumayya Saied, Shahnaz Perveen, Marium Munawar,
Samreen.Ajmal Khan, Uzma Rasool Mughal. Synthesis, Leishmanicidal and Enzyme
Inhibitory Activities of Quinoline-4-Carboxylic Acids. J. Chem. Soc. Pak. 2009;
31(5):809-819.
55. Furniss BS, Hannaford AJ, Smith PWG, Tatchell AR. Vogel’s textbook of practical
organic chemistry. 5th ed. New York. John Willey & Sons; 1989. P. 1185-86.
56. Jadhav VB, Kulkarni MV, Rasal VP, Biradar SS, Vinay MD. Synthesis and antiinflammatory evaluation of methylene bridged benzofuranyl Imidazo[2,1-b]-1,3,4thiadiazoles. European journal of Medicinal Chemistry 2008; 43:1721-29.
57. Prescott LM, Harley JP, Klein DA. Microbial growth and metabolisim. Microbiology.
Second Edition; 2001. p.104-8.
58. Harry WS, Paul J, John J. Culturing microorganism microbes in action. A laboratory
manual of microbiology.4th ed. Philadelphia. J.B.Lippincott Company; 1991. P. 3761.
KLEUCOP Bangalore [DEPT.OF PHARM CHEMISTRY]
Page 76
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