“SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL EVALUATION OF SUBSTITUTED PYRAZOLE DERIVATIVES”

Transcription

“SYNTHESIS, CHARACTERIZATION AND
BIOLOGICAL EVALUATION OF SUBSTITUTED
PYRAZOLE DERIVATIVES”
By
SIDDIQUI SHAKEEL AHMED
Reg. No. 09PC251
Dissertation submitted to the
Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore.
In partial fulfilment of the requirements for the award of degree of
MASTER OF PHARMACY
IN
PHARMACEUTICALCHEMISTRY
Under the guidance of
Dr. SADATH ALI
M.Pharm. Ph.D
DEPARTMENT OF PHARMACEUTICAL CHEMISTRY
LUQMAN COLLEGE OF PHARMACY
GULBARGA- 585102 – KARNATAKA - INDIA
2010-2011
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE
DECLARATION BY THE CANDIDATE
I hereby declare that the matter embodied in the dissertation entitled
“SYNTHESIS,
CHARACTERIZATION
AND
BIOLOGICAL
EVALUATION OF SUBSTITUTED PYRAZOLE DERIVATIVES” is
a bonafide and genuine research work carried out by me under the
guidance of Dr. SADATH ALI M.Pharm. Ph.D , Luqman College of
Pharmacy, Gulbarga. The work embodied in this thesis is original and
has not been submitted the basis for the award of degree, diploma,
associate ship (or) fellowship of any other university (or) institution.
Date:
CERTIFICATE BY THE GUIDE
This is to certify that the dissertation entitled is “SYNTHESIS,
CHARACTERIZATION AND BIOLOGICAL EVALUATION OF
SUBSTITUTED PYRAZOLE DERIVATIVES” is a bonafide research
work carried out by Mr. SIDDIQUI SHAKEEL AHMED submitted in
partial fulfillment for the award of the degree of “Master of Pharmacy”
in Pharmaceutical chemistry by the Rajiv Gandhi University of Health
Sciences, Karnataka, Bangalore. He carried out this work in the library
and laboratories of Luqman College of pharmacy, Gulbarga, under my
guidance and direct supervision.
Date:
Place: Gulbarga
Dr. SADATH ALI
M.Pharm. Ph.D
PROFESSOR
Luqman College of Pharmacy,
Gulbarga- 585 102
ENDORSEMENT BY THE PRINCIPAL/ HEAD
OF THE INSTITUTION
This is to certify that the dissertation entitled “SYNTHESIS,
CHARACTERIZATION AND BIOLOGICAL EVALUATION OF
SUBSTITUTED PYRAZOLE DERIVATIVES” is a bonafide research
work carried out by Mr. SIDDIQUI SHAKEEL AHMED. Submitted in
partial fulfillment for the award of the degree of “Master of Pharmacy”
in Pharmaceutical chemistry by the Rajiv Gandhi University of Health
Sciences, Karnataka Bangalore. This work was carried out by him in
the library and laboratories of Luqman College of pharmacy, Gulbarga,
under the guidance of Dr. SADATH ALI M.Pharm. Ph.D Professor,
Luqman College of Pharmacy, Gulbarga.
Prof. Sudheendra
M.Pharm. (Ph.D)
Head
Deparment of Pharmaceutical Chemistry,
Luqman College of pharmacy,
Gulbarga -585 102
Principal
Luqman College of Pharmacy,
Gulbarga- 585 102
Karnataka
Date:
Date:
Place: Gulbarga
Place: Gulbarga
COPYRIGHT
DECLARATION BY THE CANDIDATE
I here by declare that the Rajiv Gandhi University of Health
Sciences, Karnataka, Bangalore, shall have the rights to preserve, use
and disseminate this dissertation / thesis in print or electronic format for
academic / research purpose.
Date:
SIDDIQUI SAHKEEL AHMED
Place: Gulbarga
© Rajiv Gandhi University of Health Science, Karnataka, Bangalore.
DEDICATED TO
Almighty,
Late grand parents,
Parents
And
Brother-Sister
ACKNOWLEDGEMENT
“Read in the name of your lord, who has created man from a clott.
Read! And your lord is the most generous, who has taught (the writting), by
the pen, has taught man which he knew not” (surah Aaq 1-5)
In the name of Allah almighty who is the most Merciful and the most
Knowledgeable, peace and blessings be upon the holy prophet Muhammed
Sallallah Alaihi Wasallam (PUBH) who was sent for the guidance of the
whole world. I am most thankfull to almighty Allah who guided me and
enabled me to pursue and accomplished this work.
With these words of Quran, I would like to take the privilege to thank the
selfless people from the core of my heart who with their constant support,
affection, inspiration and encouragement made me feel comfortable to
successfully complete this venture.
I would like to express my gratitude and indebtedness
Firstly, to my parents Mr. Siddiqui Khaleel Ahmed and Mrs. Shamim
Banu, whose full-hearted co-operation, love and moral support and by the
blessing of my peer Mr. Sayyed Shah Murutaza Hussaini Chisty made this
day possible in my life.
I consider myself most lucky to work under the guidance of Dr. Sadath
Ali, Professor, Luqman College of Pharmacy, Gulbarga. I take this opportunity
to express my heartfelt gratitude to my reverend guide. I am very much grateful
to him for his invaluable guidance and ever-lasting encouragement throughout
my course.
Very special thanks to Dr. M.G Purohit sir, for their constant support in
analytical work.
I am immensely thankful to Dr. Syed Rehmatullah, Founder Secretary
and Mr. Abdul Majeed, President Vocational Educational Society, Gulbarga
and Dr. Abdul Mujeeb, Chairman, College Governing Council, Luqman
college of Pharmacy, Gulbarga, for providing me all facilities for the successful
completion of this project.
I take this opportunity to express my deep sense of gratitude to my
esteemed master Mr. Sudheendra, Professor, HOD of Pharmaceutical
Chemistry under whose advice this work has materialized. I am highly indebted
to him for his valuable guidance, suggestions and keen interest throughout the
course of this research work. His discipline, principles, simplicity, caring
attitude and provision of fearless work environment will be cherished in all
walks of my life.
I owe my warmest and humble thanks to Mr. D.K Suresh, Mr. Kapse,
Mr. Syed Shakeel, Mrs. Nikhat Farhana, Mr. Liyaqat, Mr. M. S. Khalid,
Mrs. Syeda Humaira,
and other staff members of Luqman College of
Pharmacy, Gulbarga, for their timely help, encouragement, boosting my
confidence in the progress of my academics.
I solicit my deep sense of appreciation and love to my MOTHER &
FATHER and consider my self-privilege to have seen an entity of almighty in
them
and my strong source of inspiration .I feel deep sense of gratitude for my
sister Mrs. Zareen Fatima, Farheen Fatima and brother- in- law Mr. Habeeb
Ur Raheman, Muqthar Ahmed, Irsahd Ahmed Siddiqui also I would like to
express my affection to my beloved sister Afreen Fatima, Nasreen Fatima and
my brother Siddiqui Mustafa for their constant encouragement, moral support
and everlasting love that have served me as a source of inspiration, strength and
determination at each and every front of my life.
I also express my affection to my niece Tuba Hoorain and my nephews
.
Najmus Saqib, and Md. Kashif for their love throughout my life.
I express my deepest and very special thanks to my batch mates,
Md. laeeq Ahmed, Abdul Razzaq, Sushil Tiwari, Vikas Kakde, Neeraj Rai,
Abhishek Raj Pachouri, Moid Ansari, Md. Wasim Akram, Ramij Mulla for
their kind co-operation, help and encouragement throughout my course.
I heartly thank to my best friends Sarfaraz, Faisal Siddiqui, Moiz, Abdul
Khader, Baliq, Sameer, Asif, Taher, Anwar, Ilyas, Shaker who were with me
whenever I needed them, I wish them all great successes in life.
I express my heartful thanks to my colleagues Azhar Hussain, Ahnaf
Umair, Jjinesh, Jaspal Parmar sinh, Sheikh Salman, Wahid Mansoori,
Hakeemuddin khan, Raju, Hafeez for their nice co-operation during the course
of study.
I convey my thanks and well wishes to all my juniors and others who have
contributed directly or indirectly during my dissertation.
.
My sincere thankful to Mr. Maski, Mr. Ameen, Mr.Mallana, Mr.Maheboob
and other non teaching staff and Librarian of Luqman college of pharmacy,
Gulbarga, for their co-operation.
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.
Yes! My thesis is the sensible team effort of all these people, mentioned or notmentioned here, still, it is too less to express my deep sense of thanks to them.
Thankful I ever remain………
Date:
Place: Gulbarga
LIST OF ABBREVIATIONS
COX
Cyclo Oxygenase
DMF
Dimethyl Formamide
EIMS
Electron Impact Mass Spectroscopy
IR
Infra Red
LD50
Lethal Dose
MHZ
Mega Hertz
NMR
Nuclear Magnetic Resonance
LCMS
Liquid Chromatography Mass Spectrophotometer
Rf
Retardation factor
TLC
Thin Layer Chromatography
TMS
Tetra Methyl Silane
UV
Ultra-Violet
MP
Melting Point
NSAID’S
Non-Steroidal Anti-Inflammatory Drugs
ROV
Reduction in paw oedema volume
SEM
Standard Error of Mean
ABSTRACT
OBJECTIVE:The work presented in this thesis consists of synthesis, characterization and biological
evaluation of substituted pyrazole derivatives. Pyrazole derivatives have been shown
to have wide variety of pharmacological activities like antimicrobial, antiinflammatory, antidepressant and anticonvulsant. As combination of biologically
active moieties into one molecule and synthesis of totally newer moieties have been
the methods of research, we present here in the synthesis of some novel pyrazole
derivatives incorporating various biologically active aryl/aryloxy acid derivatives
such as ibuprofen, diclofenac, aceclofenac as well as potent antibacterial quinolones,
norfloxacin and ciprofloxacin. All the compounds synthesized were evaluated for
their antibacterial, antifungal (Cup-Plate method) and anti-inflammatory (Carrageenan
induced paw oedema method) activities.
METHODOLOGY:The mixture of aryl/aryloxy acid (0.1mol) and ethanol were refluxed for 6 hours in the
presence of sulphuric acid. The reaction mixture was concentrated and washed with
saturated sodium bicarbonate solution. The ester (0.1 mol) thus formed was dissolved
in appropriate quantity of ethanol and hydrazine hydrate (0.1 mol) was added. The
mixture was refluxed for 12-18 hours. Excess of ethanol was distilled off and poured
onto ice cold water and solid obtained was filtered, dried and recrystallized from
suitable solvents.
The equimolar quantities of hydrazides (Ia-h) and acetyl acetone were refluxed in
methanol (25ml) containing few drops of concentrated HCl for 5-6 hours on water
bath. The reaction mixture was cooled to room temperature and the solid separated
was filtered, washed with petroleum ether, dried and recrystallized from suitable
solvents. The derivatives were characterized by FT-IR, 1HNMR and Mass spectral
data.
CHARACTERIZATION:Melting Points were determined by using Toshniwal apparatus in open capillaries and
are corrected. The purity of the compounds were checked by TLC on silica gel G
plates using n-butanol, ethyl acetate (1:3) solvent system and UV lamp was used as a
visualizing agent.
IR spectra were recorded using KBr pellets on a Jasco FT/IR 5300 series
spectrophotometer. 1H NMR Spectra on an Avance 300MHZ spectrophotometer using
DMSO d6 as solvents and TMS as internal standard (chemical shift values are
expressed in δ ppm). Mass Spectra were recorded by LCMS technique on a liquid
chromatography mass spectrophotometer.
ANTI-INFLAMMATORY ACTIVITY:Anti-inflammatory activity is carried on albino rats of either sex, using carrageenan
induced rat paw oedema model. The potency of the synthesized compounds was
determined against standard drug ibuprofen.
ANTIMICROBIAL ACTIVITY:Antibacterial and antifungal activities is carried out by cup-plate method, using
Pseudomonous
aeruginosa
(ATCC-27853),
Escherichia
coli(ATCC-25923),
Enterococcus Fecalis(ATCC-29212) and Bacillus substilis organisms for antibacterial
activity using Amoxycillin as a standard drug and Aspergillus niger, Aspergillus
flavus organism for antifungal activity using clotrimazole as a standard drug. The
antimicrobial potency of the synthesized compounds was determined against standard
drug by measuring the zone of inhibition.
RESULT:SPECTRAL DATA:
IIb- Aromatic C-H was absorbed in the form of intense peak at 3100 cm-1, Aliphatic
C-H peaks are also obtained from 3032 cm-1 to 2843 cm-1. The C=O absorption peak
was seen at 1607 cm-1. The 1HNMR spectrum recorded in DMSO D6 exhibited two
identical peaks in the form of singlet at 2.3δ and CH2 protons absorption has merged
with DMSO protons at 3.5δ. The methyl proton and aromatic together have shown
multiplet from 7.1δ to 8.3δ. The base peak is observed by Mass spectra is m/z 91.
IIf-The N-H group present in the molecule sandwich between two phenyl molecules,
exhibited a sharp peak at 3323 cm-1, the aromatic and aliphatic C-H have exhibited an
absorbance peak from 2854 cm-1 to 3078 cm-1. The C=O group present in the
molecule in the form of imine exhibited a peak at 1694 cm-1. The 1HNMR spectra of
these molecules exhibites a broad peak at 3.3δ due to the presence of two CH3 protons
present in the molecule. The aromatic protons present in the molecule exhibited
aromatic cluster from 6.8δ to 7.3δ in the form of a multiplet. The C-H peakof
methylene appears to have merged with the aromatic cluster and the methylene
protons sandwich between carbonyl group as well as phenyl moiety have been
dishilded and give a peak at 6.8δ. The H of N-H protons as resonated at 7.1δ. These
measurement recorded are in concerns with proposed structure of the molecules. The
base peak is observed by Mass spectra is m/z 214.
BIOLOGICAL ACTIVITY:-
Compound IIc, IId and IIf shown significant anti- inflammatory activity where as
compound IIb, IIc, IIe and IIh found to possess good antimicrobial activity against
all the organisms used for the study and the compounds IIf and IIg was found to
exhibit moderate activities and the compounds IIa and IId were shown poor activity
compared to respective standard drugs by the maximum zone of inhibition.
FUTURE ACTION PLAN:Since the synthesized compounds were reported to posses several other
pharmacological actions, the substituted pyrazole derivatives can be screened for
other pharmacological actions.
KEYWORS:Pyrazoles, Anti-inflammatory, Anti-microbial.
TABLE OF CONTENTS
Chapter
No.
Title
Page No.
1
Introduction
1-20
2
Objectives
3
Review of Literature
22-46
4
Methodology
47-69
5
Results
70-95
6
Discussion
96-97
7
Conclusion
98
8
Summary
99
9
Bibliography
10
Annexure
21
100-107
108
LIST OF TABLES
Table No.
Table 1
Title
Physical data of intermediates (I a-h)
Page No.
52
Physical data of synthesized pyrazole derivatives
Table 2
57
( II a-h)
Table 3
In-vitro anti-inflammatory activity of substituted
pyrazole derivatives. ( II a-h)
91
Table 4
Anti-Microbial activity of substituted pyrazole
derivatives.( II a-h)
94
LIST OF FIGURES
Fig No.
Contents
Page
No.
71
1.1
I.R. of Compound IIb
1.2
N.M.R of Compound IIb
72
1.3
N.M.R of Compound IIb
73
1.4
MASS of Compound IIb
74
2.1
I.R. of Compound IIf
77
2.2
N.M.R of Compound IIf
78
2.3
N.M.R of Compound IIf
79
2.4
MASS of Compound IIf
80
3
I.R. of Compound IIa
84
4
I.R. of Compound IIc
85
5
I.R. of Compound IId
86
6
I.R. of Compound IIe
87
7
I.R. of Compound IIg
88
8
I.R. of Compound IIh
89
9
Anti-inflammatory activity of newly synthesized
Pyrazole derivatives
92
CHAPTER-O1
INTRODUCTION
01. INTRODUCTION
INFLAMMATION
History, Causes and Types
Throughout the centuries, inflammation has been considered as a disease in itself. The
misconception arose from the inability to distinguish between inflammatory changes
and the injuries which induce them. The understanding of the distinction between the
genesis of inflammation and the tissue reaction that follow is attributed to John
Hunter who at the end of the 18th century, substantially contributed to the analysis of
inflammation in objective terms1.
The structural and functional changes occurring in the inflamed tissue is still under
study. There are drugs which modulate these signs, but without a detailed knowledge
of the basic physio-pathological events, it is impossible to understand their
mechanism of action. The first coherent description of the phenomenon was presented
by Celsus, a Roman physician of the 1st century A.D. He described the classic signs of
inflammation: rubor (redness), tumor (swelling) with calor (heat) and dolor (pain).
The cardinal signs of inflammation as described by Celsus remain unchallenged even
now.
Our body is protected from the many injurious micro-organisms ever present in our
environment by the epithelia-skin and mucous membrane, which serve as a
mechanical barrier. Any injury to this barrier, usually by trauma or any other
destructive mechanism, there will be an immediate response in the form of
inflammation2. Although various definition were given to inflammation through the
centuries by different people, the earliest of the modern definitions was given by
Zeigler3 in 1889. According to him notion “inflammation” comprises a series of
DEPT. OF PHARMACEUTICAL CHEMISTRY, LUQMAN COLLEGE OF PHARMACY, GULBARGA
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INTRODUCTION
phenomena occurring partly in the circulatory apparatus, partly in the tissues, in
various proportions. As the phenomenon is not unique a brief and precise definition of
inflammation is altogether impossible.
At the beginning of the century, an interesting view of the phenomena of
inflammation was developed by immunologists, who have introduced the concept of
antibody production phagocytic stimulation (opsonization) which renders the noxious
agents (germs or foreign bodies) more vulnerable to the englobing and digestion by
phagocyties. Furthermore, from the work of physiologist and immunologists and
entirely new class of events (specific inflammation) became the object of
experimental works4. The introduction of the notion of “auto pharmacology” by Sir
Henry Dale5 in 1933 which described the phenomena to depend upon the formation,
synthesis or release of endogenous active substance, the so called mediator’s of
physio- pathological phenomena. According to this view, most of the physiological
phenomena of synaptic transmission, as well as many pathological events such as
anaphylaxis, allergy and some kind of shock and inflammatory reaction are mediated
by the release of acetylcholine, catecholamine’s, histamine and so forth.
Inflammation was defined by Houck6 as “vital response of a tissue to injury”. In
ordinary condition the inflammatory and reparative processes progress smoothly from
injury to healing and here the whole process is beneficial. Under special
circumstances eosinophils or basophils may concentrate in large number in tissues as
part of inflammatory response 2. Once the inflammatory stimulus has been eliminated,
the process leading to restoration begins as in the case of acute inflammation. In
contrast, there are many chronic inflammatory conditions of unknown etiology which
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INTRODUCTION
affect organ systems in the body and produce tissue destruction rather than
restoration.
Chronic inflammatory reactions can be divided into two groups, those with, and those
without the formation of granulomas, depending on the agent (infectious or noninfectious) responsible for inflammation. When the irritant cannot be easily dispose
by the mononuclear phagocytes (indigestible materials, e.g. mycobacteria,
streptococcal cell walls, carrageenin, paraffin oil etc.,) granuloma formation usually
occurs7. Such inflammation may become much more complex and inflammatory
response try to isolate from the rest of the organism by forming granuloma or pus.
This is the case we see in pulmonary tuberculosis and in syphilis. Apart from this,
there are various chronic inflammatory conditions of unknown etiology, like
rheumatoid arthritis, ankylosing spondylitis, gout etc.
Arthritis and rheumatism are two of the commonly used terms, the former to denote a
disease a inflammation of a joint while the later implies pain of various parts of the
body, usually referring to the soft tissues such as ligament, tendons and muscles8.
As mentioned earlier, inflammation is one of the most important mechanisms of host
defence, since it marshals the attack on injurious agents and leads to repair the
affected tissue. An acute inflammation is a cyclical reaction of the tissue in response
to many diverse types of injury.
ANTI-INFLAMMATORY AGENTS
In its widest sense, the designation anti-inflammatory implies that a drug inhibits any
facet of inflammation, whether this be part of an experimentally induced reaction or
observed as a clinical manifestation in disease. This definition includes the antiDEPT. OF PHARMACEUTICAL CHEMISTRY, LUQMAN COLLEGE OF PHARMACY, GULBARGA
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CHAPTER-O1
INTRODUCTION
rheumatic drugs which may be described as substances which suppress the overt signs
and symptoms of connective tissue inflammation in the rheumatic diseases.
Anti-inflammatory modify the inflammatory response to diseases but are not curative
and do not remove the underlined cause of the disease. Since inflammation is the host
defence mechanism to external injuries an ideal anti-inflammatory should only affect
the aberrant, uncontrolled inflammation and should not interfere with the normal
inflammatory response.
The future trend in the design of new anti-inflammatory agents useful in therapy lies
in:
a) Agent capable of minimizing the side effect of presently exicting drugs.
b) Development of smilar anti-inflammatory agents devoids of side effects.
c) Agents which can block the evolaution of chronic inflammatory disease
either by blocking effect of trauma or by acting some step of inflammatory
reaction which is responsible for irreversible lesions, such as the release or
action of the enzymes responsible for the deterioration of tissues structure.
The present day the anti-inflammatory durgs are of two types, namely the
steroidal drugs (e.g. Cortisone, Dexamethasone) and the non steroidal
drugs (e.g. Aspirin, Phenyl butazone)
STEROIDS AS ANTI-INFLAMMATORY AGENTS
Corticosteroids are important and widely used therapeutics in the treatment of a large
number of inflammatory and immunologically mediated diseases. Gluco corticoids
have a multiple of effects on many of stages in inflammatory in immune processes.
4
CHAPTER-O1
INTRODUCTION
They have attained important place especially in the treatment chronic inflammatory
conditions including rheumatic diseases.
In short, gluco corticoids exert their anti-inflammatory activity stabilizing the
lysosomal membrane, reducing the permeability of the capillaries which in turn
decreases migration of white cells out of the blood stream in to the surrounding the
tissue together with reduced lysosomal enzymes. The net result is in reduced joint
inflammation.
After the introduction of cortisone (1948) and later hydrocortisone (cortisol) for the
treatment of rheumatoid arthritis, many investigators began to search for superior
agents with pure side effects.
CH2OH
CH2OH
O
O
O
HO
OH
OH
O
O
Cortisone
Cortisole
Among the synthetic analogs the most potent anti-inflammatory agents are
Dexamethasone, Betamethasone, Triamcinolone, Prednisolone and 9α-flurocortisol.
5
CHAPTER-O1
INTRODUCTION
CH2OH
O
CH 2 OH
OH
HO
O
OH
HO
CH3
F
O
O
Prednisolone
D exam ethaso ne
These steroids drugs are used mainly in the treatment of chronic inflammatory
conditions like rheumatoid arthritis, ankylosing spondylitis etc. the adverse reaction
include moon faces, hirsutism GIT symptoms with or without ulceritis, oedema,
emotional disturbances etc.
NON-STEROIDAL ANTI- INFLAMMATORY AGENTS
Non-
steroidal
anti-inflammatory
agents
(NSAIA)
have
as
their
major
pharmacological effect, the reduction of oedema, erythema and resulting tissues
damage associated with the inflammatory condition. Besides, they also share the
actions of analgesia and antipyretic.
They can be broadly categorized as the classical NSAIA (e.g. aspirin, indomethacin)
as well as the immune suppressants, gold compounds, antimalarials, antipyretics and
penicillamine. The first category consists of drugs commonly called aspirin like and
they share anti-inflammatory, analgesic and antipyretic actions. They act mainly by
the inhibition of prostaglandins and other mediators of inflammation. The second
category consists of agents that are capable of modifying immune responsiveness and
are used for the treatment of rheumatic arthritis and other chronic inflammatory
conditions. The basis for their use is that such diseases may have an underlying
immunological abnormality and selective immune suppression may be beneficial.
6
CHAPTER-O1
INTRODUCTION
SALICYCLICACID DERIVATIVES
The drugs most widely used in the treatment of rheumatic diseases are derivatives of
salicylic acid9.
COOH
OH
OCOCH3
COOH
Aspirin
Salicyclic acid
Acetyl salicylic acid, commonly known as Aspirin is the drug of choice in the
treatment of rheumatoid arthritis10,11. It is also the drug of choice, along with
antibiotics in the treatment of rheumatic fever12.
Today, among the salicylic acid derivatives aspirin still remains the most important
drugs in the treatment of rheumatic diseases.
5-PYRAZOLONE DERIVATIVES
Antipyrine was one of the first synthetic compounds used in medicine and was
followed by aminopyrine. They showed anti inflammatory activity with analgesic and
antipyretic activities13.
CH 3
R1
R2
N
N
O
Antipyrine – R1 = CH3 : R2 = H
Aminopyrine- R1 = H : R2 = N(CH2)2
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INTRODUCTION
3,5-PYRAZOLIDINIDIONE DERIVATIVES
After the discovery of 5-pyrazolone derivatives as anti-inflammatory agents,
modifications were made on the basic structure and these resulted in the synthesis of
new compounds. Phenyl butazone was found to be a potent inhibitor of inflammation.
Its anti- rheumatic effect in human was demonstrated by White house14.
A metabolic product of phenyl butazone was found to be less toxic and equally potent
as the parent compound. This resulted in the synthesis of oxyphenbutazone15.
O
R1
R2
N
N
O
Phenyl butazone –
R1 = C6H6
R2 = n- C4H9
Oxyphen butazone - R1 = p-OHC6H5 R2 = n- C4H9
Sulfin pyrazone -
R1 = C6H5
R2 = CH2CH2-S-C6H6
Modification of the parent compound to increase the acidity of the molecule resulted
in the synthesis of a new derivative, Sulfin pyrazone16. It has enhanced uricosuric
activity and is potent against gout.
INDOLE DERIVATIVES
The possible role of serotonin in the medication of inflammation initiated the study of
indole derivatives as possible anti-inflammatory agents17. Several indole derivatives
were synthesized like Indo methacin18 Indoxole19 and tested in animals against
inflammation.
8
CHAPTER-O1
INTRODUCTION
CH 2 COOH
O CH
3
CH3
OCH 3
N
O
N
H
Indomethacin
OCH 3
Indoxole
PYRROLE ACETIC ACID DERIVATIVES
Tolmetin was introduced in 1976 as a anti-inflammatory, analgesic drug. It inhibits
PG synthesis and also capillary permeability. It has been proved for the treatment of
rheumatoid arthritis and osteoarthritis20.
O
H3C
N
CH2COOH
H3C
Tolmetin
N-ARYL ANTHRANILIC ACIDS
The discovery of the anti-inflammatory activity of N-aryl anthranilic acids led to the
synthesis of Mefenamic acid21 and Flufenamic acid22. They showed good antiinflammatory and analgesic properties. A dichloro derivatives Meclofenamic aid was
shown to be 15 time potent as phenyl butazone23.
9
CHAPTER-O1
INTRODUCTION
Cl
CH3
H 3C
COOH
CH3
COOH
NH
NH
Cl
M eclofenamic acid
M efenam ic acid
CF3
COOH
NH
Flufenamic acid
Similarly other agent like Benzydomine24 oxicam derivative like Piroxicam25, Napthy
pramide26,27, Chlorthenoxazine28,29, Hydroxamic acid30, Indoprofen31, etc. are found to
posses anti-inflammatory activity.
PHENYL ACETIC ACID DERIVATIVES
They include Ibuprofen, Flubriprofen, Fenoprofen and Naproxen.
Naproxen is naphthalene acetic acid derivatives with potent anti-inflammatory,
analgesic and antipyretic properties. It is useful in the treatment of rheumatoid
arthritis and also osteoarthritis, ankylosing spondylitis and acute gout.
CH3
COOH
O CH3
10
CHAPTER-O1
INTRODUCTION
IBUPROFEN
In the recent tmes, phenyl- propionic acid derivatives, a large family of compounds
with similar biological properties have been found to be very effective as antiinflammatory and analgesic agents and have attained great importance. The interest in
these compounds developed after the discovery that isobutyl phenyl acetic acid
possessed anti-inflammatory properties.
CH3
H3C
COOH
Though it was found to be effective clinically, its use was limited due to the
hepatotoxicity.
Besides potent anti-inflammatory activity, ibuprofen showed considerable analgesic
and antipyretic activities. The analgesic activity was found to be peripherally induced.
The analgesic effect of ibuprofen is several times more than that of phenylbutazone.
Hence its use in chronic arthritis as well as acute inflammatory conditions is well
justified.
Ibuprofen was well absorbed after oral administration. Absorption was mainly from
the intestine and to some extent from the stomach. It was found in a state well bound
to plasma two metabolites were also found to some extent namely.
(1) 2-(4-(2-hydroxy-2-methyl propyl)phenyl ) propionic acid and
(2) 2-(4-(2-carboxy propyl)phenyl ) propionic acid.
The most common toxicity of ibuprofen was found to be gastrointestinal disturbances.
Long term therapy was found cause gastrointestinal lesions.
11
CHAPTER-O1
INTRODUCTION
IBUPROFEN DERIVATIVES
Ibuprofen is a useful anti-inflammatory agent with potent analgesic properties. Much
of the work done on this drug is with a view to reduce its gastrointestinal toxicities
and to enhance the anti-inflammatory potency.
An ester of ibuprofen with salicylic acid prepared and was found to be useful as antiinflammatory. Another interesting derivative which was found to be a useful anitinflammatory agent is an ester with phenylbutazone.
CH3
H 3C
CH
COO
C H3
HOOC
CH3
H9C4
H3C
CH
O
COO
C H3
N
Compounds of the following type were prepared and were found to be having antiinflammatory properties.
CH3
H3C
CH
CHNR1R2
C H3
R1 = H, R2 = CHMeCH2CH2CH2Net2
The taste of ibuprofen could be improved by converting it into the aluminium salt.
An ester of ibuprofen with 4-AcNHC6H4OH was found be useful as analgesic, antiinflammatory and antipyretic.
12
CHAPTER-O1
INTRODUCTION
CH3
H 3C
CH
NHAc
COO
C H3
Amide derivatives of ibuprofen of the following type was following type was found to
be anti-inflammatory and with much better taste.
CH3
H3C
CH
CO
NHR
C H3
R = 3-(F3C) C6H4
PURPOSE OF THE WORK
During the last decade considerable interest has arisen in the field of antiinflammatory agents. Inflammation although known in certain disease to affect the
connective tissues of the joints, tendons, bones and heart, the etiology of the diseases
and the mechanism is still eluding. A number of anti-inflammatory agents have been
discovered and many of them have disappeared from the market. The reasons are
mainly their side effects and lack of specificity moreover, the types of inflammation
also vary, and further the inflammations with regard to individuals also vary, which
can be explained to some extent by the involvement of immunological factors in the
medication of inflammation. All the anti-inflammatory agents discovered are not
effective in all types of inflammation. However it is very clear that till now, potent
inhibitors of inflammation does not really exist and an intensive investigation seems
to be definitely necessary.
After the advance of prostaglandin a new lease of life has been given to the field of
anti-inflammatory agents. The hope that is kindled in prostaglandins as a drug of
13
CHAPTER-O1
INTRODUCTION
tomorrow is also dwindling on account of the fact that the time duration of the release
of PG’s and their metabolism happens to be too short.
To review the situation so far, the steroidal anti-inflammatory agents have the extreme
drawback that they lower the immunity level in man. Also the undesirable’s effects
are more. So the choice is left with the non steroidal agents.
The compounds like ibuprofen have taken the market mainly due to the reasons of
fewer side effects compared to other. Indomethacin is taken off from the market in
Germany on account of its, highest incidence of side effects. Both ibuprofen and
indomethacin are acid derivatives. The present work is undertaken with a view to
synthesize some new pyrazoles with potent anti-inflammatory and anti-microbial
activities.
To synthesize the acid derivatives particularly with the reasons are many of the antiinflammatory compounds like Mefenamic acid, fluphenamic acid, flurbiprofen,
diclofenac, ketoprofen and naproxen have proved to effective in the symptomatic
treatment of rheumatic arthritis, juvenile rheumatic arthritis, oesto arthritic,
ankylosing spondylitis and related conditions. Besides being anti-inflammatory these
compound have also been proved as potent analgesic and antipyretic.
In the forgoing survey of literature, it is seen that the drug design by molecular
manipulation is a productive source of new drugs. Synthesis of compounds to explore
the potential biologically active agents still draws continued interest. Molecular
manipulation, combination of biologically active moieties into one molecule and
synthesis of totally newer moieties have been the methods of approach. Hence, we
present here in the synthesis of some novel pyrazole derivatives incorporating various
14
CHAPTER-O1
INTRODUCTION
biologically active aryl/aryloxy acid derivatives such as ibuprofen, diclofenac,
aceclofenac as well as potent antibacterial quinolones, norfloxacin and ciprofloxacin.
PYRAZOLES:
Pyrazole refers both to the class of simple aromatic ring organic compounds of the
heterocyclic series characterized by a 5-membered ring structure composed of three
carbon atoms and two nitrogen atoms in adjacent positions and to the unsubstituted
parent compound. Being so composed and having pharmacological effects on
humans, they are classified as alkaloids although they are not known to occur in
nature. IUPAC nomenclature is a system of naming chemical compounds and of
describing the science of chemistry in general.
Pyrazoles are produced synthetically through the reaction of α, β-unsaturated
aldehydes with hydrazine and subsequent dehydrogenation. Hydrogenation is a
chemical reaction in which unsaturated bonds between carbon atoms are reduced by
attachment of a hydrogen atom to each carbon.
H
N
H 2C
N
O
It may also be prepared by the union of diazomethane with acetylene and by warming
the acetal of propargyl aldehyde with an aqueous solution of hydrazine sulphate. It
crystallizes in colourless needles, is very stable and behaves as a weak base. It does
not combine with the alkyl iodides. On oxidation with potassium permanganate the Calkyl-derivatives give carboxylic acids, whilst the N-phenyl derivatives frequently
split off the phenyl group (especially if it is to be amidated) and have it replaced by
hydrogen. On reduction, the pyrazoles with a free NH group are scarcely affected,
15
CHAPTER-O1
INTRODUCTION
whilst the N-phenyl derivatives give pyrazolines, or by the use of very strong
reducing agents the ring is ruptured and trimethylenediamine derivatives are formed.
They yield substituted derivatives with the halogens, bromine being the most
effective. The chloro-derivatives are most readily prepared from the pyrazolones by
the action of phosphorus oxychloride.
The pyrazole carboxylic acids may also be obtained by condensing 1, 3-diketone or
oxymethylene ketone carboxylic esters with hydrazines, or the diazo fatty esters with
acetylene dicarboxylic esters. The dihydro pyrazoles or pyrazolines are less stable
than the pyrazoles and are more like unsaturated compounds. They may be obtained
by the reduction of pyrazoles (especially N-phenyl derivatives) with sodium in
alcoholic solution; by condensing diazo-acetic ester or diazomethane with ethylenic
compounds and by rearrangement of the hydrazones of α-olefine aldehydes or ketones
on warming or on distillation. They are weak bases, which are only soluble in
concentrated acids. On reduction they yield pyrazolidines, or the ring is broken; and
when oxidized they form blue or red colouring matters. The carboxylic acids show a
remarkable behavior on heating, the nitrogen is entirely eliminated, and trimethylene
carboxylic acids are obtained. Pyrazoline is a colourless liquid, which boils at 144° C.
It may be prepared by the action of diazomethane on ethylene.
The pyrazolones (ketodihydropyrazoles) first prepared from the elimination of the
elements of alcohol from the hydrazones of o-ketonic acids; or on the oxidation of the
pyrazolidones with ferric chloride. They form salts with both acids and bases, and
yield benzylidine and isonitroso derivatives. Pyrazolone is obtained by the
condensation of hydrazine with formyl acetic ester. It is a colourless crystalline solid,
which melts at 164°C. 1-Phenyl-3-methyl pyrazol-5-one is antipyrine. The isomeric
16
CHAPTER-O1
INTRODUCTION
1-phenyl-5-methyl pyrazol-3-one is formed by condensing aceto acetic ester with
aceto phenyl hydrazine in the presence of phosphorus oxychloride, or by the action of
ferric chloride on the corresponding pyrazolidone, which is produced by condensing
phenyl hydrazine with o-halogen butyric acid. When methylated it yields
isoantipyrine, an isomer of antipyrine, which is more poisonous.
Pyrazolidines are tetra hydro pyrazoles. The N-phenyl derivative, from sodium phenyl
hydrazine and trimethylene bromide, is oil, which readily oxidizes to phenyl
pyrazoline on exposure. The corresponding keto derivatives, or pyrazolidones, are
produced by the action of hydrazines on the 1, 3-halide acids or ß-olefine dicarboxylic
acids. Isomeric compounds may arise here when phenyl hydrazine is used, the ketogroup taking either the 3 or 5 position; thus with ß-iodopropionic acid 1-phenyl-5pyrazolidone is formed, whilst potassium 1, 3-iodopropionate gives the 3-compound.
Isomers of this type may be distinguished by the fact that the 5-pyrazolidone
compounds are basic, whilst the 3-compounds are acidic. The simplest member of the
series, 5- pyrazolidone, is a liquid that is formed by the action of hydrazine on acrylic
acid. The 3, 5-pyrazolidones are the cyclic hydrazides of the malonic acid series.
Thiopyrazoles have been obtained by the action of an aqueous or alcoholic solution of
the methyl chloride or iodide of phenyl methyl chloro pyrazole on a solution of an
alkaline hydrosulphide into which carbon bisulphide has been passed; or by the action
of sodium thiosulphate on antipyrine hydrochloride or a similar compound. Pyrazoles
are used for their analgesic, anti-inflammatory, antipyretic, antiarrhythmic,
tranquilizing, muscle relaxing, psychoanaleptic, anticonvulsant, monoamineoxidase
inhibiting, antidiabetic, antimicrobial32 and antibacterial activities.
17
CHAPTER-O1
INTRODUCTION
Structurally related compounds are pyrazoline and pyrazolidine.
H
N
H
N
N
N
N
NH
The above three represent heterocyclic nomenclature to pyrazolines require that
nitrogen atoms to be numbered one and two in each structure. Substituted 1pyrazolines are numbered to produce the lower locations, or in the case of
complicated structures produce the simplest name consistent with clarity of meaning.
Numbering of the 2-pyrazolines begins with the amino nitrogen and pyrazolines are
numbered to obtain for the double bond the lower of the two possible numbers. Thus,
here this structure will be referred as:
1
N
5
N2
4
3
The pyrazoles, the pyrazolines and the pyrazolidines form an interesting class of
compounds showing diverse biological activities. The pyrazoline and pyrazolidines
can be considered as the hydrogenated, compounds of pyrazoles. Many pyrazoles and
pyrazolines surveyed in the literature have been screened against various Microorganism and their pharmacological activities also compared.
18
CHAPTER-O1
INTRODUCTION
SOME REACTIONS OF PYRAZOLE:
Protonation:
The higher basicity of Pyrazole reflects the symmetry of the cation with its two
equivalent contributing resonance structure. Clearly, again oxygen has a larger
electron withdrawing effect than sulfur.
OH
H
N
N
NaH, (PhCO2 )2 O
N
N
H2O
Acylation at nitrogen:
The introduction of an acyl33 or phenyl sulfonyl34 group into Pyrazole, nitrogen is
usually achieved in the presence of weak base such as pyridine. Such a process
proceeds via imine nitrogen Acylation, then N+ -H- deprotonation. Since Acylation,
unlike alkylation is reversible the more stable product is obtained.
Ac
H
N
N
N
ACCl
N
H2O
(1)
Substitution at carbon:
Nitration:
Pyrazoles35 undergo straight nitration at C-4, it gives 1-nitro pyrazole but this can be
rearranged to 4-nitro pyrazole (2) in acid at low temperature36.
OH
H
N
N
Conc. HNO
3,
N
Ac 2 O
N
NO 2
H
N
Conc.H 2 SO 4
N
O 2N
(2)
19
CHAPTER-O1
INTRODUCTION
Halogenation:
Halogenation of pyrazole gives 4-mono halo pyrazoles, eg: 4-iodo37 or 4-bromo
pyrazole38 under controlled conditions. Poor yields are obtained on reaction of
isothiazole39 and isoxazole40. Bromine will attack at C-4, but with activating groups
present halogenation proceeds better. 3,4,5-tribromo pyrazole is formed efficiently in
alkaline solution; presumably the pyrazole anion is the reacting species .
Deprotonation of pyrazole N-hydrogen:
The PKa for loss of the N-hydrogen of pyrazole is 14.2 compared with 17.5 for
imidazole, though they are two equally contributing resonance forms.
H
N
Na
N
+
N
+ N
NaH
N
+
N
(3)
During the survey of literature it is found that much less study was carried on the
following molecule (10).
CH3
H3 C
N
N
R
(10)
The present work has been designed to synthesize a number of pyrazoles derivatives
containing above said moiety.
20
CHAPTER-02
OBJECTIVES
02. OBJECTIVES
A considerable amount of research activity is directed towards the synthesis of
potent, more specific and less toxic compounds. Substituted pyrazoles have
received considerable attention during last few decades as they are endowed with
variety of biological activities and have wide range of therapeutic properties. A
Literature
survey
indicates
that
pyrazole
derivatives
possess
different
pharmacological and biological activities; which of most potent activity are antiinflammatory and anti-microbial activities. Pyrazole derivatives have been shown
to have very interesting pharmacological activities, like antibacterial, antiinflammatory and antifungal. When one biologically active molecule is linked to
another, the resultant molecule generally has increased potency.
In the present investigation, we planned to synthesize substituted pyrazole
derivatives and evaluate for their anti-inflammatory and anti-microbial activity.
The present work was undertaken with the following objectives:
1. To achieve the synthesis of the new title compounds adapting unambiguous
synthetic routes.
2. To establish the structure of the newly synthesized substituted pyrazole
derivatives based on chemical data and spectral analysis.
3. To evaluate the derivatives for their anti-inflammatory and anti-microbial
activity using literature methods.
21
CHAPTER-03
REVIEW OF LITERATURE
03. REVIEW OF LITERATURE
PYRAZOLES AND THEIR BIOLOGICAL ACTIVITIES:
Franco Chimenti et al.,41 synthesized N1-thiocarbamoyl-3,5-di(hetero)aryl-4,5dihydro-(1H)-pyrazole derivatives (11) and reported their activity against human
monoamine oxidase.
Ar
N
N
H2N
Ar'
S
(11)
Ar = Ph, 4’-CH3-Ph, 4’-Cl-Ph, fur-2’-yl; Ar’= Ph, 4’-CH3-Ph, 4’-F-Ph
Mehdi Bakavoli et al.,42 synthesized new pyrazolo [3,4-d] pyrimidine derivatives
(12) and reported their anti bacterial activity.
O
NH
N
O2N
N
N
X
O2 N
(12)
X= H, 4-OCH3, 3-OCH3, 4-Br, 3-NO2, 3-OH, 2-OH, 4-Cl
22
CHAPTER-03
Flora
F
Barsoum
al.,43
et
synthesized
bis(4,5-dihydro-1H-pyrazole-1-
carboxamides) (13) and their thio-analogues and reported their potential PGE2
inhibitory properties.
A
R
R
N
N
N
O
NH2
O
N
NH2
(13)
A= 2-O(CH2)2O-2’, 4-O(CH2)2O-4’; R= Ph, 2-thienyl, 4-ClC6H5
Babasaheb P Bandgar et al.,44 synthesized and evaluated novel series of pyrazole
chalcones
like
1-(2,4-dimethoxy-phenyl)-3
substituted-1H
pyrazole-4-yl-
propenone (14) as anti-inflammatory, antioxidant and antimicrobial agents.
R
O
N
H3 C
O
O
CH3
N
(14)
R= 4-H, 4-CH3, 4-OCH3, 4-Cl, 4-Br, 4-F, 3-Cl, 2-Cl, 2,4-Cl, 2,4-OCH3
23
CHAPTER-03
El-Shimaa MN Abdel-Hafez et al.,45 synthesized and evaluated certain pyrazole 3-carboxylic acid derivatives (15) as novel carriers for nitric oxide.
O
O
ONO2
N
N
Ph
N
Ph
O
N
Ph
(15)
Olga Bruno et al.,46 synthesized 1-methyl and 1-(2-hydroxyalkyl)-5-(3-alkyl/
cycloalkyl/ phenyl/ naphthylureido)- 1H- pyrazole -4- carboxylic
acid ethyl
ester (16) and evaluated as potent human neutrophil chemotaxis inhibitor.
CH3
O
O
NH
NR'
N
O
N
R
(16)
R= CH2-CHOH-C6H5, CH2-CHOH-CH3, CH2-CHOH-C2H5, CH3
NR’= α-naphthylamino, isopropylamino, benzylamino, N-benzylpiperazino,
p-F-anilino, o-F-anilino, m-F-anilino.
Sureshbabu Dadiboyena et al.,47 synthesized pyrazole derivatives (17) and
reported their anti bacterial and anti inflammatory activity.
24
CHAPTER-03
N
N
NH
CH3
R
(17)
John Porter et al.,48 synthesized Tetra hydro isoquinoline amide (18) substituted
phenyl pyrazoles as selective Bcl-2 inhibitors.
O
N
Cl
N
N
O
N
NH2
(18)
Abid et al.,49 synthesized and evaluated antimicrobial activity of 1-(benzofuran-2yl)-4-nitro-3-arylbutan-1-ones and 3-(benzofuran-2-yl)-4,5-dihydro-5-aryl-1- [4(aryl)-1,3-thiazol-2-yl]-1H-pyrazoles (19), (20), (21).
Ar
NO2
O
O
Ar
N N
NH2
O
N N
N
S
(19)
(20)
S
Ar
(21)
25
CHAPTER-03
Guiping Ouyang et al.,50 synthesized and evaluated antiviral activity of novel
pyrazole derivatives containing oxime esters group(22).
3
H3 C
CHNOCOR
N
S
N
R2
R1
(22)
R1= H, 4-Cl, 4-Me; R2 = Me, F, OMe; R3= Me, Ph, -(CH2)4-CH3
Nada M Abunada et al.,51 synthesized some new pyrazoline and pyrrolo [3, 4-c] –
pyrazole-4, 6-dione derivatives (23) and reported their antifungal, antidepressant,
anticonvulsant, anti-inflammatory, antibacterial and anti-tumor activity.
H
Ar
O
N
N
C6 H 4 R
N
H
Ar'
O
(23)
Rahat Khan et al.,52 synthesized brominated 5-methyl -2,4-dihydropyrazol-3-one
(24) and its derivatives as cytotoxic agents.
26
CHAPTER-03
R1
H 3C
R2
N
O
N
R
(24)
Adnan A Behkit et al.,53 synthesized and evaluated some thiazolyl and
thiadiazolyl derivatives of 1H-pyrazole (25) as anti-inflammatory, antimicrobial
agents.
R
N
N
H
Br
N
S
H 3C
N
N
N
N
CH3
H
S
O
O
(25)
R= C6H5, 4-CH3C6H4, 4Cl-C6H4
Om Prakash et al.,54 synthesized and evaluated antibacterial activity of some new
2,3-dimethoxy-3-hydroxy-2- (1-phenyl-3-aryl-4-pyrazolyl) chromanones (26).
Ph
Ph
N
N
O
OMe
IBD
MeOH
Ar
O
OH
O
N
N
Ar
OH
O OMe
(26)
27
CHAPTER-03
Sanjay Gupta et al.,55 synthesized N-aryl-5-amino-4-cyanopyrazole derivatives
(27) as potent xanthine oxidase inhibitors.
R
R
HC(OEt)3,Ac2O
N
N
NH2
NH3
N
N
N
N
CN
NH2
(27)
David Kralj et al.,56 have given a simple synthesis of 4-(2-aminoethyl)-5-hydroxy1H-pyrazole (28).
COPh
COPh
N
N
O
N
O
OH
N
NH2
NMe2
(28)
Nesrin Gokhan-Kelekci et al.,57 synthesized some novel pyrazole derivatives (29)
as dual MAO-B inhibitors and anti-inflammatory analgesic.
R
N
N
N
H
S
NH R1
(29)
28
CHAPTER-03
Yong Xia et al.,58 synthesized novel 1-arylmethyl-3aryl-1H-pyrazole-5carbohydrazide derivative (30) and reported their activity against A 549 lung
cancer cell.
H 2 NHNOC
N
N
R1
X
R2
(30)
R1 = H, Cl, OMe; R2 = H, Cl, t-Bu, OMe; X= C, N
Olga Bruno al.,59 synthesized 2-phenyl-2,3-dihydro-1H-imidazo[1,2-b] pyrazole
derivatives (31) and reported their potent inhibitors of fMLP- induced neutrophil
chemotaxis activity.
N
N
H 5 C6
NR2
N
H
O
(31)
Pande PS et al.,60 synthesized substituted 1,3(a),4,5-tetrahydro pyrazoles[3,4-c]
pyrazoles and benzo [4,5] imidazo-5H-thiazolo[5,4-c] 2,3-dihydropyrazoles (32)
and reported their fungicidal and insecticidal activity.
29
CHAPTER-03
O
HO
CH3
N
N
N
N
H
R
(32)
R= H, OCH3, Cl
Jagdhani SG et al.61
demonstrated the synthesis of pyrazolyl chromones by
conventional and non conventional methods. They report the synthesis of different
pyrazole derivatives by using traditional method, microwave method and
Ultrasound method.
R
N
N
N
N
O
(33)
R= phenyl, 4-chlorophenyl, 4-bromophenyl, 4-methylphenyl, 2-naphthyl,biphenyl
Subas M Sakya et al.,62 synthesized some 1,4-unsubstituted and substituted 5alkyl ether and 5-alkyl thioether pyrazole (34) and evaluated their COX-2
inhibitor activity.
H 3C
H 3 CO 2 S
N
N
N
CF 3
(34)
30
CHAPTER-03
Subas M Sakya et al.,63 synthesized and studied Structure-activity relationship of
5-alkylamino pyrazoles as potent, selective and orally active analog of some 3difluoromethyl-5-(cis-2,6-imethylmorpholin-4-yl)-1-(5-methyanesulfonyl-pyridin2-yl) -1 H-pyrazole-4-carbonitrile(35) as selective canine COX-2 inhibitors.
NR 2 R3
SO 2 R1
CN
N
N
N
CF2 R
(35)
N
R= H Me, R1= H, Me, R2= R3=
Wageeh S El-Hamouly et al.,64 synthesized new 4-new aryl-isoxazolyl [5,4-d]
pyrimidine-6-one\thione and 4-aryl-pyrazolol[3,4-d]-pyrimidine-6-one derivatives
(36) and reported their antihypertensive activity.
X
HN
NH
Me
H
Ar
NH
H
Me
N
(36)
Vartale SP et al.,65 synthesized 6/7/8-substituted-1- [aryl/ 6’-substituted-2’benzothiazolyl]- pyrazolo [4,5-b] quinolines (37) and reported their antimicrobial
activity.
31
CHAPTER-03
H
R1
N
R2
S
N
N
R3
Y
N
(37)
R1= OCH3, H; R2= OCH3, H; R3= OCH3, H; Y= OCH3, H, CH3, NO2,
Mohd Amir et al.,66 synthesized new 3-chloro-4-substituted pyrazolyl -1benzenesulphonamide and N-[4-(substituted pyrazolyl)-3-chlorophenyl] methane
sulphonamide (38) and reported their anti-inflammatory activity.
Cl
N
R1
N
R
H3 C
(38)
R= SO2NH2; NHSO2CH3; R1= CH3, C6H5
Suthakaran
R
et
al.,67
synthesized
8-(methylene-2’’,3’’-disubstituted
benzoquinazolo -4’’-one)- 9 ,2 - (4’) - disubstituted benzopyrano pyrazoles (39)
and reported their antimicrobial activity.
32
CHAPTER-03
R3
R2
N
R1
O
R4
N
O
N
N
(39)
R1= H, OCH3; R2= H, OCH3; R3= CH3, C6H5; R4= 3,5 dibromo-4chloro aniline, 4-morpholino aniline, 4’-phenyl-4-piperazino aniline
Wadhal SA et al.,68 synthesized N-aroyl-3, 5-diaryl pyrazoles (40) and reported
their biological activity.
X
Z
Y
N
N
O
R2
R1
R3
(40)
R1= CH3, H; R2= H, CH3, OCH3; R3= H, CH3, OCH3, Cl; X= CH, N; Y, Z=
OH, H
33
CHAPTER-03
Ezawa M et al.,69 synthesized 1,5–disubstituted pyrazoles (41) as cyclooxygenase
-2 (COX-2) selective inhibitor.
N
N
N
O
O
S
ONO 2
CH3
(41)
Rossella Fioravani et al.,70 synthesized some pyrazole derivatives (42) and carried
out preliminary investigation of their affinity binding to P-glycoprotein.
R
N
N
H3C
O
(42)
R= H
Akihiko Tanitame et al.,71 synthesized novel series of DNA gyrase inhibitors: 5[(E)-2-arylvinyl] pyrazoles (43) and antibacterial activity.
34
CHAPTER-03
Cl
Cl
N
N
O
Cl
HN
(43)
Kee-In Lee et al.,72 synthesized some of the pyrazole oxime ethers (44) and
carried out its antitumor activity.
O
N
R1
R4
H
N
O
R3
N
R2
(44)
R1=R2= H; R3= Me; R4= Ph-3-OMe
Dhar et al.,73 reported a series of substituted chalcones and corresponding
pyrazoles were synthesized and evaluated for in vitro cytotoxic activity against a
panel of human cancer cell lines.
Ar
Ar
N
N
Ac
(45)
35
CHAPTER-03
El-Sied A Aly et al., 74 synthesized novel pyrazole derivatives (46). They reported
the building of various heterocyclic systems on the pyrazole nucleus.
Ar
N
COOEt
N
N
H
Ph
(46)
Ar= Ph, p-Tol
Solanki PR et al.,75 demonstrated the conventional heating and microwave
promoted synthesis of some substituted pyrazole and isoxazoles and reported their
anti-inflammatory, antimicrobial activity.
R
N
N
Me
Ph
(47)
Giuseppe Daidone et al.,76 synthesized 1-methyl-5-[substituted-4(3H)-oxo- 1, 2,
3- benzotriazin-3-yl-1H-pyrazole-4-acetic acid derivative (48) and evaluated their
anti-inflammatory activity.
36
CHAPTER-03
HOOCH 2 C
O
N
R3
R2
N
N
N
CH3
N
R1
(48)
R1 =H, CH3; R2= R3= H, Cl
Pyrazoline as antimicrobial and anti bacterial agent:
Sachchar SP and Singh AK77 synthesized several 1-phenyl-3(substituted fluoro
phenyl)-5-heteroayl-2-pyrazolines (49) and noted their antimicrobial as well as
anti bacterial actions especially against bacteria B. anthrasis.
R
R1
N
N
(49)
R = furyl thienyl and pyridyl; R1 = fluoro, chloro, hydroxyl and methyl.
Substituted pyrazolines have been reported for their antibacterial activity.
Stirrewiberg WE
78
and co-workers reported antibacterial as well as insecticidal
action of pyrazoline derivatives (50).
N N CO
R2
NH
R
R3
R1
(50)
37
CHAPTER-03
Desai NC et al.,79 synthesized several pyrazoline derivatives of phenothiazines
(51) and showed moderate to good anti bacterial activity. These compounds
exhibited anti-tubercular activity.
X
S
X
N
N
N
X
CH 2 CO
R1
R
(51)
R= 2-OH-C6H5, 3-OH-C6H5, 4-OH-C6H5; R1= H, Cl; X= Br.
Ead HA, et al.,80 have reported, the synthesis of two novel series of pyrazoles and
2-pyrazolines, and their antibacterial activity. The tested compounds showed
significant activity.
Pyrazolines as antifungal:
Sachchar SP81 extended their studies on several pyrazoline derivatives and has
reported them as antifungal agents. Aspergillus niger and Helmithosporium
sativum were employed as fungi to test the fungicidal activity.
Ritcha S and Horsfall JC82 synthesized 3,5-dimethyl-4-nitro pyrazole and 1,3,5
trimethyl-4-nitrosopyrazole compounds and have reported these as anti-fungal
agents. Their fungicidal activity increased by increasing the size of hydrocarbon
side group.
38
CHAPTER-03
Mitra P and Nayak A83 reported that 5-pyrazolone and its derivatives 4-acetyl-2pyrazoline-5-one and 2-pyrazoline-5-thione (52) was associated with significant
fungicidal activity against the rice blast pathogen Pyricularia oryzae and brown
leaf spot pathogen Helminthosporium oryzae.
S
CH2
N
N
N
O
S
R
(52)
Parvati Mitra and Mitra AG84 have synthesized 4-N (aryl) amino methyl-2pyrazoline-5-one by mannich reaction of 2-pyrazoline-5-one derivative. The same
authors also reported cobalt II complex with 2-pyrazoline-5-one derivative as
having anti fungal activity.
Sadasiva Shankar M85 synthesized several hydroxyl aryl-pyrazole (53) and tested
them for antibacterial as well as anti-fungal activity where none of the compounds
were found to possess anti-bacterial activity. But all compounds were found to
possess anti-microbial activity. Antifungal activity has been assessed by
employing Drechslera prostrate (Drechs) and Alternatia alternate (Keissler). All
compounds tested could inhibit the spore germination at 30-mcg/ml upto a
maximum level.
OH
R
N
N
R1
(53)
R= Alkyl, Aryl; R1= H, Phenyl
39
CHAPTER-03
Nayak A and Mitra AS86 synthesized several 4,4’,bis-5-pyrazoline and 4,
4’,unsaturated products and found fungicidal activity, against rice blast pathogen
Pyricularia oryazae and brown leaf spot pathogen, Helmithosporium oryzae.
Tiwari N, Dwevedi B and Nizamuddin87 synthesized several 1-acetyl/aroyl-3methyl-4-substituted anilido -5-aryl pyrazolines and 3-methyl-4-substituted
anilido-5-aryl pyrazolines and 3-methyl-4-substituted anilido-5-aryl isoxazolines,
and tested against Cephalosporium sacchari, Helmithosporium oryzne, and
Saprolegina parasitica, Acellya orion all the compounds showed remarkable
activity.
Mohanthy SK88 reported that 1-phenyl-5-aryl-1-2-pyrazoline 3-4-thiazolidine-2one derivative showing antifungal properties.
The fungicidal activity of the compounds was determined by poisoned food
technique at various concentrations. It was also reported that all compounds
inhibit the growth of the fungus Aspergillus flavours.
Mazahir Kidwai89 synthesized the substituted pyrazole (54) showing antifungal
activity.
O
O
O
NH
H3C
N
H2 C
Ar
N
H
CH3
(54)
40
CHAPTER-03
Pyrazoline as antiviral:
Sachchar SP90 extended their study on several phenyl-1-3-(substituted fluoro
phenyl)-5-heteroaryl-2-pyrazoline derivatives and found antiviral activity against
Sunn hemp rosette virus (SRV). These compounds were evaluated by the method
of Verma and Awasthi.
Pyrazoline as local anesthetic, C.N.S depressant and anti convulsant:
Bheemasankara Rao CH and co-workers synthesized many 3-aryl, 4-aryl, 2pyrazoline (55) and found them to be active as local anaesthetic, C.N.S.
deprassant and anticonvulsant.
R1
R
O
C
N
R2
R3
N
H
(55)
Where R = R1 = R2 = R3 = H
R = R1 = R2 = R3 = OCH3
Anti-inflammatory activity with pyrazole nucleus:
Pyrazolone derivatives as anti inflammatory:
Antipyrine was one of the first synthetic compound used in medicine and was
followed by aminopyrine. They showed anti-inflammatory activity along with
analgesic and antipyretic activities.
41
CHAPTER-03
R2
N
R1
R3
N
O
R4
(56)
R1
R2
R3
R4
Antipyrin
-C6H5
-CH3
-CH3
-H
Aminopyrin
-C6H5
-CH3
-CH3
-N-CH3
Dipyrone
-C6H5
-CH3
-CH3
-N-CH2SO3Na
Another derivative dipyrone was prepared which showed strong antiserotonin and
anti-oedema activities in rats but found to be clinically disappointing in the
treatment of rheumatic arthritis. Other compounds like 4-(N-Nicotinyl amino) was
also found to be less toxic and clinically effective in various inflammation
diseases.
Frangnly AM et al.,91 have reported anti-inflammatory activity of some new
pyrazoles, pyrazolines and 4(3H)-quinazolines.
Derivative of 3, 5-pyrazolindinedione as anti inflammatory:
After the discovery of the 5-pyrazoline derivatives as anti-inflammatory agents,
modifications were made on the basic structure and this resulted in the synthesis
of a new compound, phenyl butazone, which was found to be a potent inhibitor of
inflammation.
42
CHAPTER-03
A metabolic product of phenyl butazone was found to be less toxic and equally
potent as the parent compound. The result was the synthesis of oxyphenbutazone.
Modification of the parent compound to increase the acidity of the molecule
resulted in the synthesis of a new derivative sulfin pyrazone (57). It also enhances
uricosuric activity and is potent against gout.
R1
N
C6H5
O
N
O
R2
(57)
R1
R2
Phenyl butazone
-C6H5
-C4H9
Oxyphen butazone
-C6H4-OH (P)
-C4H9
Sulfin pyrazone
-C6H5
-CH2-CH2-S-C6H5
Pyrazole derivatives as anti-inflammatory agent:
Coli B92 reported that pyrazole derivative itself has anti-inflammatory activity.
e.g. Benzylame (Tantum) (58).
OCH2R
N
R = -CH2-CH2-N(CH3)2
N
R = -COCH3
CH2Ph
(58)
43
CHAPTER-03
Sarangam S93 have synthesized number of derivatives of pyrazole (3, 4-d)
pyrimidine-4-6 diones (59) and reported the screening for C.N.S. depression
properties and anti-inflammatory activity. It was also reported that some
derivatives showed anti-inflammatory properties equivalent to aspirin.
O
R2
N
N
O
N
N
R1
CH3
(59)
R1= Phenyl, O-Tolyl
R2= Phenyl, O-Tolyl, O-Anisyl
Pyrazole derivatives as antidiabetic:
Froesch EE94 has reported antidiabetic activity in the 5-methyl pyrazole-3carboxilic acid (60).
COOH
H3C
N
N
H
(60)
Pyrazole derivative as vasodilator:
Burner HR et al.,95 have reported vasodilator activity in pyrazole derivative.
N
N
N
N
CH3
(61)
44
CHAPTER-03
Pyrazole derivative as anti-hypoglycemic and anti-hypotensive agent:
Smith DL96 reported that 3, 5-dimethyl pyrazole (63) and 3-methyl pyrazole-5carboxilic acid (64) exhibited anti-hypoglycemic activity.
Arya VP et al.,97 synthesized several pyrazolidine derivatives and reported the
anti-hypotensive activity.
OCH2R
N
R = -CH2-CH2-N(CH3)2,
N
-COCH3
CH2Ph
(62)
CH3
H3 C
N
CH3
N
N
HOOC
N
H
(63)
(64)
Pyrazolidone derivatives as inhibitor:
Sweeny MJ98 reported that pyrazopurine which is a natural antibiotic was very
effective in inhibiting pyrimidine biosynthesis.
ChasinM et al.,99 discovered a potent new compound pyrazole pyridine (65) that
was 60 times more potent as an inhibitor of rat brain PDE than theophyllin.
N
N
N
C2H5OOC
NH-N=C-(CH3)
(65)
45
CHAPTER-03
Novinson T100 reported a series of pyrazole pyrimidine derivatives (66), (67) and
showed them to be inhibitor of PDE from rabbit lung and beef heart.
H
R
N
N
N
N
R1
N
EtOOC
N
N
R
R1
(66)
(67)
The presence of the following nucleus in the molecule may be the cause of high
activity of such compounds. Many compounds containing this nucleus have been
found to show biological activity 101-104.
X
N
N
Y
(68)
Pyrazolines as diuretics:
Varanayan BA et al., has shown that 1,3,4,5 tetra-substituted pyrazoline-5-ones
could change the diuretic activity compared to furosemide pyrazoline for the
treatment of hormone dependant breast tumor and ovulary infertility.
Edward Philip Neil et al., 105 had found that certain Pyrazoline derivatives could
be used for the treatment of breast tumors and ovulary infertility.
Pyrazolines as anti-depressants:
Bilgin AA and Sunal R106 have reported synthesis of 1-thiocarbamyl-3, 5diphenyl-2-pyrazoline derivatives. Some of the compounds have shown
equivalent or higher activity than paragyline hydrochloride and tranyl
cyproomiline sulphate as an antidepressant.
46
CHAPTER-04
METHODOLOGY
04. METHODOLOGY
The importance of pyrazole moiety has been discussed in the previous chapter.
Among the many methods available for the synthesis of pyrazole derivatives,
in the
present chapter a convenient and versatile methodology has been
adopted for the synthesis of pyrazole derivatives. The hydroxyl substitution in
the molecule is always favorable for the bioactivity of various heterocyclic
molecules, because of their partial ionic structure.
The organic chemist is frequently faced with the problem of characterizing and
ultimately elucidating the structure of organic compounds. The worker in the field
of natural products
has the prospects of isolating such compounds from their
sources in a pure state and then of determining the structure. On the other hand,
the synthetic organic chemist encounters new or unexpected compounds in the
course of investigations in to the applicability of new reagents or techniques or as
by products of established reactions. All the reactions were carried out under
prescribed laboratory conditions. The products were purified by recrystallization.
Melting points were determined by capillary method and were uncorrected. All
the aldehydes were obtained commercially.
4.1
Materials and methods:
a) The entire chemicals used were procured from Qualingens, Himedia and Lobachemicals. Purity of starting materials used for reaction was confirmed by
checking their melting point or boiling point and by thin layer chromatography.
47
CHAPTER-04
METHODOLOGY
b) Melting points were determined in open capillary tube using precision melting
point apparatus and uncorrected.
c) The FT-IR a spectrum of the synthesized compounds has been obtained from
Karnataka University, university science instrument centre Dharwaad.
The IR spectra were carried out by SHIMADZU PERKIN EKMER 8201 PC
IR SPECTROMETER using a thin film on potassium bromide pellets.
d) The 1HNMR of the selected compounds has been obtained from Karnataka
University, university science instrument centre Dharwaad.The PMR
spectra
were
recorded
on
BRUKER
AVANCE
II
300
NMR
SPECTROMETER in a mixture of CDCl3. Chemical shift values are reported
as values in ppm relative to TMS (d=0) as internal standard.
e) The Mass spectrum of the selected synthesized compounds has been
performed in IICT Hyderabad. The FAB mass spectra were recorded on
JEOL SX-102/DA-6000 Mass Spectrometer using Argon/Xenon (6Kv,
10Ma) as the FAB gas.
f) Purity of compounds was checked on “Silica Gel G” coated on laboratory
micro slides prepared by dipping method or precoated plates, eluent was the
mixture of different polar and non-polar solvents in varying proportions and
detection was done either by observing in UV (ultra-violet) light or exposure to
iodine vapours as required. The absence of TLC spots for starting materials and
appearance of new TLC spot at different Rf value ensured the completion of
reaction.
48
CHAPTER-04
METHODOLOGY
SCHEME
R-COOH
C2H5OH
H2SO4
IIa -(2-hydrophenyl)methanone
R-COOC2H5
IIb -2-phenyl-1-ethanone
NH2-NH2.H2O
IIc -2-phenoxy-1-ethanone
IId -2-(4-isobutylphenyl)-1-ethanone
R-CONHNH2
I (a-h)
IIe -3-carbonyl-1ethyl-6-fluro-7-piperazino-1,4-dihydro-4quinolinone.
IIf -2-[2-(3,5-dichloroanilino)phenyl]-1-ethanone.
CH3
H3C
N
N
IIg
-2-oxoethyl-1-{2-[2-(2,6-dichloroanilino)phenyl]}acetate.
IIh -[(1-cyclopropyl-6-fluro-7-piperazino)-3-carbonyl]1,4-dihydro-4-quinolinone.
R
II (a-h)
49
CHAPTER-04
4.2
METHODOLOGY
EXPERIMENTAL
General method of preapararion of hydrazide I (a-h):
The mixture of aryl/aryloxy acid (0.1mol) and ethanol (50ml) were taken with a
few drop of concentrated sulphuric acid and it was refluxed for 6 hours. The
reaction mixture was concentrated by distilling off the excess of ethanol under
reduced pressure and treated with a saturated solution of sodium bicarbonate. The
ester obtained was used for the preparation of hydrazides directly.
The ester (0.1 mole) was dissolved in appropriate quantity of ethanol and to this
hydrazine hydrate (0.1 mole) was added. The reaction mixture was taken in a
round bottomed flask and refluxed for a period of 12-18 hours. Excess of ethanol
was distilled off under reduced pressure. It was then poured into ice cold water
and the solid obtained was filtered. It was recrystallised from suitable solvent.
The following hydrazides were prepared.
1. 2-hydroxy-1-benzenecarbohydrazide
2. 2-phenylethanohydrazide
3. 2-Pheoxyethano hydrazide
4. 2-(4-isobutylphenyl)propanohydrazide
5. 1-ethyl-6-fluoro-4-oxo-7-piperazino-1,4-dihydro -3-quinoline
carbohydrazide
6.
2-[2-(3,5-dichloroanilino)phenyl]ethanohydrazide
7. 2-hydrazino-2-oxoethyl2-[2-(2,6-dichloroanilino)phenyl]acetate
50
CHAPTER-04
METHODOLOGY
8. 1-cyclopropyl-6-fluoro-4-oxo-7-piperazino-1,4-dihydro-3quinolinecarbohydrazide.
The physical data of the intermediates synthesized are listed in Table 1.
Preparation of 3, 5-dimethyl-1H-1-substituted pyrazoles II (a-h): The
equimolar quantities of hydrazides I (a-h) and acetyl acetone was refluxed in
methanol (25ml) containing few drops of concentrated HCl for 5-6 hours on water
bath. The reaction mixture was cooled to room temperature and the solid
separated was filtered, washed with petroleum ether, dried and recrystallized from
suitable solvent. The physical data of the substituted pyrazoles synthesized are
listed in Table 2.
51
CHAPTER-04
METHODOLOGY
TABLE 1: Physical characteristic data of intermediats I(a-h):
Sr.No.
1
Compound
code
Salicyclic acid
Ia
R
Molecular
formula
C17H8N202
152.152
Melting
point
1780C
MOL.WT
Yield
70
2
Ib
Phenyl acetic acid
C8H10N20
150.179
1210C
73
3
Ic
Phenoxy acetic acid
C8H10N202
166.178
1100C
74
4
Id
2(4-isobutyl phenyl)Propionic acid
C13H20N20
220.313
720C
68
5
Ie
335.380
2220C
65
6
If
1-Ethyl-6-fluro-1,4
dihydro-4-oxo-7-(1- C16H22FN502
piperazinyl)-3-quinolinecarboxylic acid
[o-(2,6-dichloroanilino)phenyl]acetate
C14H13 Cl2N30
310.182
1040C
60
7
Ig
C16H15Cl2N303
368.218
1450C
76
8
Ih
2-({2-[(2,6-dichloroanilino)phenyl]acetyl}oxy)acetic acid
1-cyclopropyl-6-fluro-1,4dihydro-4-oxo-7(1-piprazinyl)-3-quinolinecarboxylic acid
C17H22FN502
347.391
2650C
70
52
CHAPTER-04
METHODOLOGY
PHYSICAL DATA OF THE SYNTHESIZED COMPOUNDS (II a-h):
COMPOUND II a
O
CH3
N
N
OH
CH3
(3,5-dimethyl-1H-1-pyrazolyl)(2-hydroxyphenyl)methanone
Molecular Formula : C12H12N202
Molecular weight
: 216.238
Melting point
: 1540C
COMPOUND II b
CH3
H3C
N
N
O
1-(3,5-dimethyl-1H-1-pyrazolyl)-2-phenyl-1-ethanone
Molecular weight
: 214.268
Melting point
: 2270C
53
CHAPTER-04
METHODOLOGY
COMPOUND II c
CH3
N
H3C
O
N
O
1-(3,5-dimethyl-1H-1-pyrazolyl)-2-phenoxy-1-ethanone
Molecular weight
: 230.265
Melting point
: 1350C
COMPOUND II d
CH3
H3C
O
N
N
CH3
CH3
1-(3,5-dimethyl-1H-1-pyrazolyl)-2-(4-isobutylphenyl)-1-ethanone
Molecular weight
: 270.373
Melting point
: 2200C
54
CHAPTER-04
METHODOLOGY
COMPOUND II e
O
O
CH3
F
N
N
N
N
CH3
C2 H5
N
H
3 -[(3,5-dimethyl-1H-1-pyrazolyl)carbonyl]
-1-ethyl-6-fluoro-7-piperazino-1,4-dihydro-4-quinolinone
Molecular Formula : C21H24 F N502
Molecular weight
: 397.451
Melting point
: 2450C
COMPOUND II f
CH3
H3C
N
N
O
Cl
NH
Cl
2-[2-(3,5-dichloroanilino)phenyl]-1-(3,5-dimethyl-1H-1-pyrazolyl)-1-ethanone
Molecular Formula : C19H17 Cl2N30
Molecular weight
: 374.268
Melting point
: 1770C
55
CHAPTER-04
METHODOLOGY
COMPOUND II g
Cl
NH
O
Cl
O
N
N
CH3
O
H3C
2-(3,5-dimethyl-1H-1-pyrazolyl)-2-oxoethyl 2-[2-(2,6-dichloroanilino)phenyl]acetate
Molecular Formula : C21H19 Cl2N303
Molecular weight
: 432.304
Melting point
: 1450C
COMPOUND II h
O
O
F
N
N
N
CH3
N
H3C
N
H
1
-cyclopropyl-3-[(3,5-dimethyl-1H-1-pyrazolyl)carbonyl]-6-fluoro-7-piperazino-1,4-dihy
dro-4-quinolinone
Molecular Formula : C22H24 F N502
Molecular weight
: 409.462
Melting point
: 2690C
56
CHAPTER-04
METHODOLOGY
TABLE 2: Physical characteristic data of synthesized compounds II (ah):
CH3
H3 C
N
N
R
Sr.
No.
Compound
1
Code
IIa
2
IIb
R
Molecular
MOL.W
T
Melting
point
Yield
-(2-hydrophenyl)methanone
Formula
C12H12N202
216.238
1540C
75
-2-phenyl-1-ethanone
C13H14N20
214.268
2270C
72
0
3
IIc
-2-phenoxy-1-ethanone
C13H14N202
230.265
135 C
67
4
IId
-2-(4-isobutylphenyl)-1-ethanone
C17H22N20
270.373
2200C
77
5
IIe
6
IIf
7
8
IIg
IIh
-3-carbonyl-1ethyl-6-fluro-7-piperazino-1,4dihydro-4-quinolinone.
-2-[2-(3,5-dichloroanilino)phenyl]-1-ethanone.
-2-oxoethyl-1-{2-[2-(2,6-dichloroanilino)
phenyl]}acetate.
-[(1-cyclopropyl-6-fluro-7-piperazino)-3carbonyl]-1,4-dihydro-4-quinolinone.
0
C21H24F N502
397.451
245 C
80
C19H17 Cl2N30
374.268
1770C
70
C21H19 Cl2N303
C22H24 F N502
432.304
409.462
0
145 C
0
269 C
74
72
Rf
0.51
0.63
0.58
0.49
0.53
0.51
0.76
0.47
57
CHAPTER-04
4.3
METHODOLOGY
BIOLOGICAL ACTIVITIES:
Determination of acute Toxicity:
Acute oral toxicity studies for the formulations were carried out using OECD
guideline 420 (modified, adopted 23rd march 2006). The test procedure minimizes
the number of animals required to estimate the oral acute toxicity of a chemical
and in addition estimation of LD50, confidence intervals. The test also allows the
observation of signs of toxicity and can also be used to identify chemicals that are
likely to have low toxicity.
Principle of the FDP:
The fixed dose procedure (FDP) is method for assessing acute oral toxicity that
involve the identification of a dose level that cause evidence of non-lethal toxicity
(termed evident toxicity) rather than a dose level that cause lethality. The stumuli
for the development of the FDP were a combination of ethical and scientific
concerns regarding the traditional methods that use lethality as the key end point.
Evident toxicity is a general term describing clear signs of toxicity following
administration of test substance, such that an increase to the next highest fixed
dose would result in the development of severe toxic signs and probably mortality.
Procedure: As suggested, after acclimatization of animals for 4-5 days, study was
carried out as follows:
Healthy, young adult Albino Swiss female mice (18-25gm), nulliporous and
non pregnant were used for this study. Food, but not water was withheld for 3-4
hours and further 1-2 hours post administration of sample under study.
Fixed dose level of 5, 50, 300, 500 mg/kg were initially chosen as dose level that
would be expected to allow the identification of dose producing evident toxicity.
58
CHAPTER-04
METHODOLOGY
During the validation procedure, a fixed dose of 2000 mg/kg was added to
provide more information on substance of low acute toxicity.
Dosed one animal at the test dose by oral route.
Since, this first test animal survived, four other animals were dosed (orally) as
subsequent days, so that a total of five animals were tested.
Observation:
After the administration of synthesized pyrazole derivatives(II a-h), animals were
observed individually during the first 30 min and periodically during 24 hours
with special attention during the first four hours and daily thereafter for a period
of 14 days. Once daily animals were observed principally in relation to changes in
skin, fur, eyes and mucous membrane (nasal) and also autonomic symptoms like
sedation, lacrimation, perspiration, piloerection, urinary incontinence and control
nervous system (ptosis, drawsiness, gait tremors and convulsion).
a. ANTI-INFLAMMATORY ACTIVITY
Method for determination of anti-inflammatory activity107,108:
Carrageenan induced rat paw oedema model:
11 groups of albino rats of either sex (each comprising of six animals) weighing
between 160-200 gms were deprived of food and water for 18 hours prior to the
experiment.
59
CHAPTER-04
METHODOLOGY
Groups
Treatment protocol
Group I
Control (5% acacia gum)
Group II
Standard drug (Ibuprofen 100 mg/kg .In distilled water p.o)
Group III
Toxicant
Group IV
IIa H
(100 mg/kg p.o) 5% acacia gum suspension
Group IV
IIa L
Group V
IIa H
Group V
IIa L
Group VI
IIa H
Group VI
IIa L
Group VII
IIa H
Group VII
IIa L
Group VIII IIa H
Group VIII IIa L
Group IX
IIa H
Group IX
IIa L
Group X
IIa H
Group X
IIa L
Group XI
IIa H
Group XI
IIa L
60
CHAPTER-04
METHODOLOGY
The standard ibuprofen and synthesized compounds under study i.e. IIa to IIh
were administered orally to all rats. After 30 minutes 0.1 ml of 1% carrageenan
suspension in normal saline was injected into the sub planar region of the paw of
each rat. The edema volumes of the injected paw were measured at 1st, 2st, 3rd and
4th hour. The difference between the paw volumes of treated animals were
compared with that of the control group and the mean edema volume was
calculated.
From the data obtained mean volume of Oedema, ± SEM and percentage
reduction in Oedema were calculated. Percentage reduction or inhibition in edema
volume was calculated by using the formula.
Percentage reduction
=
V0 - Vt
V0
X 100
where
V0 = Volume of the paw of control at time t
Vt = Volume of the paw of drug treated at time t
From the data obtained the mean edema volume and percentage reduction in
edema volume were calculated and are summarized in Table 3.
b. ANTI- MICROBIAL ACTIVITY:
An anti-microbial activity is anything that can kill or inhibit the growth of
bacteria, such as high heat or a chemical. Antibacterial chemicals can be grouped
into three broad categories like antibacterial drigs, antiseptic and disinfectant.
61
CHAPTER-04
METHODOLOGY
Bacteria are single celled organism that lives in and around as. Bacteria may he
helpful, but in certain condition may cause illness like throat, most ear infection
and bacterial pneumonia.
Antibacterial drugs are used in relatively low concentration in or upon the bodies
of organisms to prevent or treat specific bacterial disease without harming the host
organism. Unlie antibacterial drugs, antiseptic and disinfectants are usually nonspecfic with respect to their targets, i.e. they can kill or inhibit a variety of
microbes. Antiseptic are used topically in or on living tissue, where as
disinfectant, are used on object or in water.
Antimicrobial chemotherapy plays an important role in the treatment of many
infectious diseases. However repeated use of some antibiotics results in resistance
i.e. ineffectiveness of drug against the microorganism. In the recent past, the
emergence of drug resistance to antibiotics is more. This situation stimulated us to
prepare new series of antimicrobials.
Drug resistances can arise as a consequence of the following.
1. Reduced drug delivery.
2. Increased drug efflux.
3. Decreased drug intake.
4. Increased deactivation of crug.
5. Structural alteriation in target site.
6. Duplication of function of the target site.
62
CHAPTER-04
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Antimicrobial resistance can develop in any type of microbe. Microbes can
develop resistance to specific medicines. A common misconception is that a
person’s body become resistant to specific drugs however, it is microbes, not
people, which become resistant to drugs.
Drug resistance happens when microbes develop ways to survive the use of drugs
meant to kill or weaken thme. If a microbe is resistant to many drugs, treating hte
infections it causes can become difficult or even impossible. Someone with an
infection that is resistant to a certain medicines can pass that resistant infection to
another person. In this way, a hard to teat illness can be spread from person to
person. In some cases, the illness leads to serious diability or even death.
The development of resistancce to an antimicrobial is complex. Susceptible
bacteria can become resistant by accquring resistant genes from other bacteria or
through mutation in their own genetic material (DNA). Once acquiring, the
resistance characteristic is passed on to the future generation and sometimes to
other bacterial species.
While antibacterial are major factors in the development of resistance many other
factors involved are as follows.
1. Nature of the specific bacteria
2. Antibacterial involved i.e. the way the antimicrobial is used.
3. Characteristic of the host.
4. Environmental factors.
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Therefore the use of antibacterials does not always lead to resistance.
The synthetic antibacterial agents are comprised of two major classes of
compounds, those effective systematically and used topically.
The important factors to be controlled in the testing of antimicrobial activity are as
follows:
1. Type of test organism.
2. Temperarure and time of incubation.
3. Composition and PH of the culture.
4. Inoculun concentration.
4.3.1 EVALUATION OF ANTIBACTERIAL ACTIVITY:
Antibacterial activity is determined based on the in vitro activity in pure cultures.
In vitro susceptibility tests are done by the cup-plate method. The antibacterial
activity of pyrazole derivatives was evaluated by cup-plate method against the
strains of common pathogens; gram negative organisms Escherichia coli,
Pseudomonas auriginosa and Gram positive organisms Basillus subtilus,
Enterococcus fecalis. Amoxycillin is used as standard drug at the concentration of
50µg/ml and 100µg/ml.
Antibacterial are of two types of drugs, those are
bactericidal and bacteriostatics. Bacteriocidals are the drugs used to destroy the
growth of bacteria and bacteriostatics are the drugs which are used to inhibit the
multiplication of bacteria.In view of these facts a attept has been made to carry out
the antimicrobial activity of synthesized compounds.
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MATERIAL AND METHODS:
Test Organisms (Bacteria)
Basillus subtilus
Gram positive bacteria
Enterococcus fecalis
Gram positive bacteria
Escherichia coli
Gram negative bacteria
Pseudomonas auriginosa
Gram negative bacteria
All the synthesized compounds were screened for antibacterial activity against the
above mentioned strains by cup-plate method109,110. The following materials were
used for the testing.
1. Nutrient agar.
2. Sterilized petridishes, pipettes and beakers.
3. Sterilized 6 mm cork borer and tuberculin syringes.
4. 18-24 h old growth culture in nutrient broth.
5. Sterilized test tubes containing solution of test compounds in desired
concentration
Preparation of Nutrient agar media
Nutrient agar (40g), bacteriological peptone (1g), beef extract (5g) and sodium
chloride (5g) were dissolved in distilled water (1000 ml). The pH of the solution
was adjusted to 7 to 7.4 by using sodium hydroxide solution (40%, approximately
0.25 ml for 100 ml of nutrient broth) and then sterilized for 30 min. at 15 lbs
pressure in an autoclave.
Preparation of sub culture
One day prior to test the microorganisms were inoculated into the sterilized
nutrient broth and incubated at 370C for 24 hr. On the day of testing the organisms
ware sub-cultured into sterile nutrient broth. After incubating for 3 hr, the growth
thus obtained was used as inoculums for the test.
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Sterilization of media and glass wares
The media used in the present study, nutrient agar and nutrient broth were
sterilized in a conical flask of suitable capacity by autoclaving the same at 15 lbs
pressure for 20 min. The cork borer, petridishes, test tubes and pipettes, were
sterilized by employing hot air oven at 1600C for 1 hr.
Preparation of solution of test compound
The test compound (10 mg each) was dissolved in freshly distilled DMF (10 ml)
in serially labeled sterile test tubes, thus giving a final concentration of
100µgm/0.1 ml; similarly 50µgm/0.1 ml concentrations were also prepared.
Preparation of standard solution
The standard compound amoxycillin (10 mg ) was dissolved in freshly distilled
DMF (10 ml) in serially labeled sterile test tubes, thus giving a final concentration
of 100µgm/0.1 ml; similarly 50µgm/0.1 ml concentration were also prepared.
METHOD OF TESTING
The method depends on the diffusion of an antibiotic from a cavity through the
solidified agar layer in a petridish to an extent such that growth of the added
microorganisms is prevented entirely in a circular area or zone around the cavity
containing a solution of test compounds. About 15-20 ml of molten nutrient agar
was poured into each of the sterile petridishes. The cups were made by scooping
out nutrient agar with a sterile cork borer. The agar plates so prepared were
divided into different set and each set of the plates were inoculated with the
suspension of particular organism by spread plate technique.
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The cups of inoculated plates were then filled with 0.1 ml of the test solution; the
plates were then incubated at 370C for 24 hours. The zone of inhibition (diameter
in mm) developed, if any, was then measured for the particular compound with
each organism. The solvent DMF was used as negative-control to know the
activity of the solvent. The results of antibacterial testing are summarized in the
following Table 4. The tested compounds are then compored with that of standard
drug used i.e Amoxycillin to measure the activity of the compounds
4.3.2 ANTIFUNGAL ACTIVITY:
The antifungal activity of pyrazole derivatives was carried out by cup and plate
method in comparison with that of standard antifungal drug clotrimazole. The
fungi cultures used were Aspergillus niger and Aspergillus flavous.
MATERIAL AND METHODS
Cup-plate diffusion method: Antifungal activity of the test compounds was
assessed against the above strains of fungi by cup plate diffusion method. The
following materials were used:
1. Sabourauds agar and tuberculin syringes with needles.
2. Sterilized Petri-dishes and pipettes of 0.1 ml and 0.2 ml
3. 16-18 hr old Cultures grown in Sabourauds broth
4. Sterilized test tubes for preparation of solution of the test compounds in
desired concentration.
Preparation of Sabourauds agar media
Bacteriological peptone (1 g) and glucose (4 g) were dissolved in distilled water
(100 ml) and filtered. Agar powder (2 g) was added and sterilized for 30 min at
15 lbs pressure.
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Preparation of sub cultures
One day prior to the test, inoculation of the microorganism (Aspergillus niger and
Aspergillus flavous) was made in sabourauds broth and incubated at 370C for 18hr.
Sterilization of media and glass wares
The media used in the present study was sterilized in conical flask of suitable
capacity by autoclaving at 15 lbs pressure for about 20 min. The cork borer,
petridishes, test tubes and pipettes were sterilized in hot air oven at 1600C for one
hour.
Preparation of solution
1. Clotrimazole: 10 mg of the clotimazole was dissolved in 10 ml, of DMF
(dimethyl formamide) to get a concentration of 100µg/0.1 ml; similarly
50µgm/0.1 ml concentration were also prepared.
2. Compounds: 10 mg of each test compounds was dissolved in 10 ml of DMF in
serially and suitably labeled in sterile test tubes thus giving a final concentration
of 100µg/0.1 ml; similarly 50µgm/0.1 ml concentrations were also prepared.
METHOD OF TESTING
Cup-plate method: This method depends on the diffusion of an antifungal agent
from a cavity through the solidified agar layer in a petridish to an extent such that
growth of the added microorganism is prevented in a circular area or zone around
the cavity containing a solution of antifungal agent. A previously liquefied
medium was inoculated appropriate to the assay with the requisite of the
68
CHAPTER-04
METHODOLOGY
suspension of the microorganisms between 40-500C and inoculated medium was
poured into petridishes to give a depth of 3 to 4 mm. Ensured that the layer of
medium were uniform in thickness by placing the dishes on a leveled surface.
With the help of a cork borer, scooped out the set agar from each petridish. Using
sterile pipettes, the standard and the sample solution (0.1 ml) of known
concentrations ware fed into the bored cups. The dishes ware left standing for 1 to
4 hrs at room temperature as a period of pre-incubation diffusion. These were then
incubated for 48 hr. at 370C. The zone of inhibition developed; if any was then
accurately measured in mm. growth of the added microorganism is prevented in a
circular area or zone around the cavity containing a solution of antifungal
agent.The results obtained are summarized in Table 4.
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05. RESULTS
5.1 Characterization of the synthesized compounds:
All the synthesized substituted Pyrazole derivatives remitted in products with good
yield. Purity of all the synthesized compounds was checked by their melting point as
well as TLC. The structure of synthesized compounds has been established and
confirmed by spectral and elemental data obtained viz, FT-IR, 1HNMR and Mass
spectroscopy. By analysis of spectral data of the representative compounds reveals the
successful information of the synthesized substituted Pyrazole derivatives.
The FT-IR a spectrum of the synthesized compounds has been obtained from
Karnataka University, university science instrument centre Dharwaad. The IR
spectra were carried out by SHIMADZU PERKIN EKMER 8201 PC IR
SPECTROMETER using a thin film on potassium bromide pellets.
The 1HNMR of the selected synthesized compounds has been obtained from
Karnataka University, university science instrument centre Dharwaad.The PMR
spectra were recorded on BRUKER AVANCE II 300 NMR SPECTROMETER in
a mixture of CDCl3. Chemical shift values are reported as values in ppm relative to
TMS (d=0) as internal standard.
The Mass spectrum of the selected synthesized compounds has been performed in
IICT, Hyderabad. The FAB mass spectra were recorded on JEOL SX-102/DA-6000
Mass Spectrometer using Argon/Xenon (6Kv, 10Ma) as the FAB gas.
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CHAPTER-05
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Result and discussion of spectral data::
Figure 1.1- I.R. of Compound IIb
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CHAPTER-05
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Figure 1.2- NMR. Of Compound IIb
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CHAPTER-05
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Figure 1.3- NMR. Of Compound IIb
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CHAPTER-05
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Figure 1.4- MASS of Compound IIb
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IR Spectra:
Types of Vibrations
Group frequency in
Wave number (cm-1)
3100cm-1
Aromatic CH stretching
3032-2843cm-1
Aliphatic CH
1607cm-1
C=O absorption
1
HNMR spectra:
Value (δ
δ)
Nature of segment
Type
2.3-3.5δ
Singlet
CH2 protons
7.1-8.3δ
Multiplet
Methyl and aromatic proton
Mass Spectra (m/z): Molecular ion peak at m/z 91.
IIb- Interpretation
The compound synthesized from the reaction carboxy hydrazide and diketones
subjected to I.R. measurement, expected no absorption due to NH or OH were not
absorbed. However aromatic C-H was absoebed in the form of intense peak at 3100cm-1
aliphatic C-H peaks are also obtained from 3032cm-1 to 2843cm-1. The C=O absorption
peak was seen at 1607cm-1.
The 1HNMR spectrum recorded in DMSO D6 exhibited two identical peaks in the form
of singlet at 2.3δ and CH2 protons absorption has merged with DMSO protons at 3.5δ.
The methyl proton and aromatic together have shown multiplet from 7.1δ to 8.3δ. All
these data support the structure assign to compound IIb {I-(3,5 dimethyl-1H-pyrazo-1yl)-2-phenyl-1-ethanone.}
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The Mass spectrum of the compound has not exhibited molecular ion peaks as expected
at m/z 214. However the fragements obtained due to the elimination of CH3-C-C-CH
{CH2=C-CH} to give rise to another fragment m/z 150. The base peak is obtained at
m/z 91 which may due to benzylic cation. The fragment ion at m/z 150 on the loss of
two nitrogen atom and C=O gives to benzyl cation at m/z 91.
The fragementation pattern discuss for the compound IIb {I-(3,5 dimethyl-1H-pyrazo-1yl)-2-phenyl-1-ethanone.} is in concerns with the structure assigned to the molecule.
IIb.
+.
CH3
H3C
H3C
N
N
m/z 37
C
+
N
N
. N
C=O
C=O
H 2C
CH2
M+ m/z 214
+
N .
-m/z 27
C=O
H2C
(M+ -37) m/z 177
[(M+ -37)-27]m/z 150
m/z 150
-m/z 37
+
. C=O
+
CH2 .
{[(M+ -37)-27]-32}-28
m/z 91
-m/z 28
CH2
{[(M+ -37)-27]-32}
m/z 118
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CHAPTER-05
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Figure 2.1- I.R. Of Compound IIf
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CHAPTER-05
RESULTS
Figure 2.2- N.M.R. Of Compound IIf
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CHAPTER-05
RESULTS
Figure 2.3- N.M.R. Of Compound IIf
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CHAPTER-05
RESULTS
Figure 2.4-MASS. Of Compound IIf
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CHAPTER-05
RESULTS
IR Spectra:
Types of Vibrations
Group frequency in
Wave number (cm-1)
N-H
3323cm-1
Aromatic and Aliphatic
2854- 3078cm-1
C-H
1694cm-1
C=O
1
HNMR spectra:
Value (δ
δ)
Nature of segment
Type
3.3δ
Singlet
CH3 protons
6.8δ-7.3δ
Multiplet
Aromatic proton
6.8δ
Disheild
C-H
7.1δ
Resonated
H of N-H protons
Mass Spectra (m/z): Molecular ion peak at m/z 242.
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IIf - Intereperation
Compound IIf{2-[2-(3,5-dichloroanilino)phenyl]-1-(3,5-dimethyl-1- H-1-pyrazolyl)-1ethanone}after purification was taken for I.R.measurement.The N-H group present in
the molecule sandwich between two phenyl molecules, exhibited a sharp peak at
3323cm-1,which the normal range of secondary imines present in the molecule. The
aromatic and aliphatic C-H has exhibited an absorbance peak from 2854cm-1 to
3078cm-1. The C=O group present in the molecule in the form of imine exhibited a peak
at 1694cm-1. This data are in concerns with the structure of the molecule.
The 1HNMR spectra of these molecules exhibites a broad peak at 3.3δ due to the
presence of two CH3 protons present in the molecule. The aromatic protons present in
the molecule exhibited aromatic cluster from 6.8δ to 7.3δ in the form of a multiplet.
The C-H peakof methylene appears to have merged with the aromatic cluster and the
methylene protons sandwich between carbonyl group as well as phenyl moiety have
been dishilded and give a peak at 6.8δ. The H of N-H protons as resonated at 7.1δ.
These measurement recorded are in concerns with proposed structure of the
molecules.
The Mass spectrum of these compounds when recorded has not shown any peak
concentration or ionic concentration nearing to the molecular weight of the compound
m/z 277 due to the loss of heterocyclic moiety present in the molecule. This molecule
contain two Cl atom, hence it applyes the two Cl cluster fragmentation of M+, M++2
M++2+2. These is also supported for the fact that fragment ion at m/z 277 corresponds
to the cleavage of heterocyclic moiety and presence of halogen cluster i.e M+2 and
M++2. Further fragmentation indicated the fragment ion peak at m/z 242 which is
obtained by the loss of m/z 214. These two fragments presence of halogen cluster is
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CHAPTER-05
RESULTS
distunguishly noted. Finally the conclusion can withdraw also these spectral data
present supports the proposed structure of the molecule.
+.
CH3
H3C
+.
N
N
C
-95
O
O
Cl
Cl
NH
NH
Cl
Cl
M+ m/z 373,M++2 m/z 375, M++2+2 m/z 377
M+ m/z 277, M++2 m/z 279, M++2+2 m/z 281
-35
+.
Cl
NH
Cl
M+ m/z 242,M++2 m/z 244,M++2+2 m/z 246
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RESULTS
Figure 3- I.R. of Compound IIa
The hydrogen bonded O-H peak was noticed at 3350cm-1 also it is varying in intensity.
Wherein partial O-H character is distributed, the similar observaton is made in case of
C=O carbonyl absorptiom peak at 1659cm-1, wherein the carbonyl absorption is
relatively less intense then as aspected. In addition to this absorption peak the aromatic
and aliphatic C-H peaks are noticed at 3000cm-1. These data confirms the structure
assign to molecule.
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RESULTS
Figure 4- I.R. Of Compound IIc
The I.R spectrum of the synthesized compound indicated the sence of tatumeric
heteroxyic group at 3466 cm-1 which is characterstic property of amide molecule. The
N-H absorption is noticed at 3050 cm-1 and 3022 cm-1. The aliphatic part of the moiety
indicated the presence of C-H by absorption at 2914 cm-1 to 2852 cm-1. The C=O
carbonyl group indicated its presence by exhibiting absorption peak at 1703-1cm. All
these data are in regimen with structure assign to the molecule.
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Figure 5- I.R. Of Compound IId
The I.R spectrum of these compound indicatded broad peak at 3400 cm-1 due to the
absorption of O-H at N-H moieties. The aromatic and aliphatic C-H peaks are found by
exhibiting broad peak around 3000 cm-1. The presence of carbonyl group in the
molecule indicated by the absorption peak at 1700 cm-1. This are in regimen with
structure assign to the molecule.
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RESULTS
Figure 6- I.R. Of Compound IIe
A broad hump noticed around 3350 cm-1 which reperesent the presence of enolic keto
group along with N-H peaks, as aspected in previous spectrum, in this case also
aromatic and aliphatic C-H are recorded from 3019 cm-1 to 2724 cm-1. The presence of
conjugated diketones is reperesent by an absorption peak at 1721 cm-1. The I.R values
noticed in thse case are in regimen with structure of molecule
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RESULTS
Figure 7- I.R. Of Compound IIg
This molecule when subjected for I.R spectrum recording give broad peak at 3310cm-1
due to the C=O carboxylate residue present in the molecule. The N-H absorption peaks
found to be present at 3323cm-1, aromatic and aliphatic C-H absorption are noticed at
3100cm-1 to 2850cm-1. The carboxylate carbonyl residue present in the molecule
exhibited short peak 1696cm-1, with a shoulder at 1750cm-1. Hence the data observed,
confirm the structure of the molecule.
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RESULTS
Figure 8- I.R. Of Compound IIh
In case of next compound, we has exhibited a very broad band at 3400 cm-1 due to the
carboxylate carbonyl residue and N-H as expected from perivous data the aromatic
and aliphatic C-H peaks from 3100 cm-1 to 2922 cm-1. The carboxylate carbonyl
residue absorption peak is noticed at 1726 cm-1. This data supports the structure
assign to molecule.
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5.2 Biological Activity:
Antiinflammatory activity:
All the synthesized compounds were evaluated for their anti-inflammatory activity
using carrageenan induced rat hind paw oedema method at two dose levels, 50mg/kg
(low dose) and 100mg/kg (high dose). The reduction in paw oedema volume was
measured in mm using plethysmograph and the percent reduction in edema volume
was determined comparing with control. The anti-inflammatory drug Ibuprofen was
used as reference standard. The findings are summarized in Table 3 and graphically
depicted in Figure 9.
.
90
CHAPTER-05
RESULTS
TABLE 3: Anti-inflammatory activity of newly synthesized Pyrazole derivatives
Group
Treatment
Dose
Mg/kg
st
After 1 hr
Mean
%ROV
Paw Oedema volume
After 2 hr
After 3rd hr
Mean
%ROV
Mean
%ROV
nd
Control
0.5ml 1.18±0.005
1.22±0.002
1.27±0.001
Standard
100
0.96±0.005*** 18.64
1±0.001***
18.03
0.9±0.002***
29.13
Ibuprofen
1
IIa H
100
1.28±0.002*
8.47
1.13±0.003*
7.37
1.02±0.002***
19.68
IIa L
50
1.29±0.001**
9.32
1.11±0.006**
9.01
1.04±0.002***
18.11
2
IIb H
100
1.28±0.004*
8.47
1.18±0.005n.s 3.27
1.08±0.003***
14.96
IIb L
50
1.27±0.004*
7.62
1.17±0.003*
4.09
1.07±0.005***
15.74
3
IIc H
100
1.24±0.002*
5.08
1.14±0.001*
6.55
1.04±0.004***
18.11
IIc L
50
1.24±0.001*
5.08
1.12±0.002**
8.19
1.02±0.001***
19.68
4
IId H
100
1.24±0.002n.s 3.38
1.13±0.003*
7.37
1.04±0.005***
18.11
IId L
50
1.22±0.002*
5.03
1.11±0.003**
9.01
1.05±0.005***
17.32
5
IIe H
100
1.24±0.005n.s 4.23
1.16±0.005*
4.91
1.08±0.004***
14.96
IIe L
50
1.23±0.006*
5.96
1.16±0.006*
4.91
1.09±0.001***
14.17
6
IIf H
100
1.25±0.002*
5.96
1.11±0.007**
9.01
1.01±0.003***
20.47
IIf L
50
1.26±0.003*
6.77
1.12±0.006**
8.91
1.01±0.003***
20.47
7
IIg H
100
1.25±0.004*
5.93
1.15±0.007*
5.73
1.05±0.004***
17.32
IIg L
50
1.23±0.004*
4.23
1.13±0.006*
7.37
1.03±0.006***
18.69
8
IIh H
100
1.22±0.003*
7.62
1.18±0.004n.s 3.27
1.09±0.001***
14.17
IIh L
50
1.25±0.002*
5.93
1.18±0.004n.s 3.27
1.09±0.002***
14.17
Toxicant control compared with normal control. Standard and synthesized compounds compared with toxicant control.
After 4th hr
Mean
%ROV
1.4±0.0057
0.87±0.005***
37.85
0.99±0.005***
1±0.003***
0.93±0.004***
0.95±0.005***
0.91±0.002***
0.93±0.003***
0.92±0.002***
0.93±0.002***
1.01±0.006***
0.98±0.004***
0.91±0.005***
0.94±0.004***
0.94±0.002***
0.96±0.002***
1.01±0.003***
1.04±0.005***
29.28
28.57
33.57
32.14
35
33.57
34.28
33.27
27.85
30
35
32.85
32.85
31.42
27.85
25.71
ROV – Reduction in paw oedema volume.
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Fig No. 9: Anti-inflammatory activity of newly synthesized Pyrazole
derivatives
Anti-inflammatory activity:
All the synthesized compounds II (a-h) were tested at dose of 50 mg/kg as a low dose
and 100mg/kg as a high dose exhibited significant anti-inflammatory activity in acute
inflammatory models in rats, results are tabulated in Tabel 3. All the compounds at
4th hours show maximum reduction in oedema volume. Compounds IIc, IId and IIf
produced maximum inhibition i.e. 35%, 34.28% and 35% respectively is equipotent to
that of standard Ibuprofen employed for comparison i.e. 37.85% where as the
compounds IIb and IIg exhibited moderate inhibition of 33.57% and 32.87% and
compounds IIa, IIe and IIh exhibited poor inhibition of 29.28% 27.85% and 27.85%
respectively at high dose level. Where as compounds IIc, IId and IIf produced
maximum inhibition i.e 33.57%, 33.57% and 32.85% and ompounds IIb and IIg
exhibited moderate inhibition i.e. 32.14% and 31.42% and compounds IIa, IIe and
IIh exhibited poor inhibition.
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Antimicrobial activity:
All the synthesized derivatives were also evaluated for their antibacterial and
antifungal activities following cup and plate method at the concentration levels of
100µg/ml and 50µg/ml. The zone of inhibition was measured in mm using scale. The
antibacteril drug Amoxycillin and antifungal drug Clotrimazole was used as reference
standard for antibacterial and antifungal activities respectively. The findings are
summarized in Table4.
.
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TABLE 4 - Antimicrobial activity of newly synthesized Pyrazole derivatives.
Sample
Code
P. aeruginosa
50µ
µg
100µ
µg
Inhibition zone diameter in mm (Average triplicate ± Standard deviation)
E.coli
E.Fecalis
B.substilis
A.niger
50µg
100 µg
50µ
µg
100µ
µg
50µ
µg
100µ
µg
50µ
µg
100µ
µg
A.flavus
50µg
100 µg
IIa
9
13
11
16
9
11
8
12
7
9
8
10
IIb
13
17
14
17
15
18
14
18
12
15
12
14
IIc
15
18
14
17
15
18
14
18
12
17
14
17
IId
12
13
10
12
11
12
12
13
6
9
7
9
IIe
14
17
15
17
15
18
15
18
14
18
15
17
IIf
14
15
14
15
13
17
13
16
8
11
10
12
IIg
13
14
13
17
15
16
14
15
9
13
11
13
IIh
14
18
15
17
15
18
15
18
12
16
13
17
Amoxicillin
25
28
26
27
26
29
27
29
-
-
-
-
Clotrimazole
-
-
-
-
-
-
-
-
17
20
19
23
DMSO
-
-
-
-
-
-
-
-
-
-
-
-
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Antibacterial activity:
All the synthesized compounds were screened for antibacterial activity studies at a
concentration of 100 µg/ml and 50 µg/ml using DMSO as a control against
Pseudomonous aeruginosa, Escherichia coli, Enterococcus fecalis and Bacillus
substilis by cup- plate method on nutrient agar media, The standard drug used was
Amoxycillin 50 µg/ml and 100 µg/ml used for comparison against Gram positive
and Gram negative bacteria. The data in Table 4 indicates that most of the
synthesized compounds are active against bacteria. The compounds IIb, IIc, IIe
and IIh, has shown good antibacterial activity and ,IIf and IIg has shown
moderate activity and compounds IIa and IId were shown poor antibacterial
activity.
Antifungal activity: All the synthesized compounds were screened for antifungal
activity studies at a concentration of 100 µg/ml and 50 µg/ml using DMSO as
control against Aspergillus niger and Aspergillus flavus on potato dextrose agar
media. Clotrimazole were used as standard. The data in Table 4 indicates that
most of the synthesized agents are signifacantly active against fungal strains used
for the study. The compounds IIb, IIc, IIe and IIh, have shown good antifungal
activity and IIf and IIg have shown moderate activity and compounds IIa and
IId were shown to posess poor antifungal activity.
.
95
CHAPTER-06
DISCUSSION
06. DISCUSSION
All the reactions were monitored by TLC, structures and purity of the anticipated
compounds were characterized by physical constant and FTIR spectral studies
initially followed by 1H-NMR and Mass spectroscopy. Absence of TLC spots for
starting materials and appearance of new TLC single spot at different Rf value
were ensured to declare completion of reaction. The TLC plates were visualized
either by Iodine vapours or by viewing in UV-Visible chamber. The reaction
products of all the reactions were purified by different workup processes to
remove unreacted starting materials if any and then by recrystallisation using
suitable solvents. Most of the steps were optimized in order to achieve
quantitative yields i.e. more than 70% yields.
Anti-inflammatory activity:
From the anti-inflammatory screening it was found that the compounds have
shown significant activity in reducing edema volume. Compounds (IIc and IIf)
produced maximum inhibition i.e. 35% which is nearly equipotent to that of
standard Ibuprofen employed for comparison i.e. 37.85%; where as the compound
IId exhibited maximum inhibition of 34.28% at high dose and the compounds
IIc and IId produced maximum inhibition of 33.57% and compound IIf exhibited
significant inhibition of 32.85% at low dose. Few of the tested compounds were
shown to possess nearly equipotent to moderate activity to that of the standard
employed for comparison. Hence, these compounds appear to be promising antiinflammatory agents.
96
CHAPTER-06
DISCUSSION
Anti-microbial activity:
From the antimicrobial screening it was found that the few compounds showed to
possess significant antimicrobial activity.
The compounds from pyrazole
derivatives evaluated for antibacterial and antifungal activities, IIb, IIc, IIe and
IIh were found to possess good antibacterial and antifungal activities against all
the organisms used for the study and the compounds IIf and IIg were found to
exhibit moderate activities where as the compounds IIa and IId were shown poor
activities compared to standard. Since, most of the compounds have been shown
to possess good to moderate potency compared to that of the standards, these
compounds belonging to 1,2,4-trisubstituted pyrazoles, appear to be promising
anti-microbial agents.
.
97
CHAPTER-07
CONCLUSION
07. CONCLUSION
During the present investigation, the new pyrazole derivatives have been
successfully synthesized by linking two biologically active moieties such as antiinflammatory molecules, Ibupofen, Diclofenac, Acelofenac and synthetic
antibacterial agents, Ciprofloxacin and Norfloxacin with pyrazole moiety. This
was done based on the observation that combination of biologically active
moieties into one molecule and synthesis of totally newer moieties may result into
compounds with improved potency, selectively and reduced toxicity.
Even though the results obtained in anti-inflammtory and antimicrobial data
reveals that the above pyrazole derivatives possessed by the most of compounds
are inferior to that of the standard drugs employed, it is encouraging that few of
the compounds were shown to possess good anti-inflammatory and antimicrobial
activities.
All the above results establish the fact that the above pyrazole moiety can be a
rich source for further exploitation.
Hence, in search for new generation of drugs with high potency, selectively and
reduced toxicity, it may be worthwhile to explore the possibility in this area by
fusing different moieties. If suitably exploited it may results in better compounds.
98
CHAPTER-08
SUMMARY
08. SUMMARY
The object of the present work is to synthesize certain new derivatives of
Pyrazoles, which has been considered as active moiety and is a core structure in a
various synthetic pharmaceuticals displaying a wide spectrum of biological
activities.
The target molecules were successfully synthesized in which Pyrazole nucleus is
linked to other biologically active moieties such as anti-inflammatory molecules,
Ibupofen,
Diclofenac,
Acelofenac
and
synthetic
antibacterial
agents,
Ciprofloxacin and Norfloxacin. The purity of the compounds synthesized was
established by using TLC technique and the structures were confirmed by their
FT-IR, 1HNMR and MASS spectral data.
All the derivatives synthesized were screened for their anti-inflammatory,
antibacterial and antifungal activities.
The anti-inflammatory and antimicrobial activities screening of the compounds
suggest that these possessed significant anti-inflammatory and anti-microbial
activites that is comparable with the standard drug.
All these above results only showed that the pyrazole moiety could be rich source
for further exploitation. The Pyrazole moiety needs more attention and if it is
suitably exploited by molecular modification can still give better lead compounds.
99
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CHAPTER-10
ANNEXURE
10. ANNEXURE
108

“SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL EVALUATION OF SUBSTITUTED PYRAZOLE DERIVATIVES”

Transcription

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