1

1. INTRODUCTION

Medicinal chemistry or Pharmaceutical chemistry is a discipline at the

intersection of chemistry and pharmacology involved with designing,

synthesizing and developing pharmaceutical drugs. It involves the identification,

synthesis and development of new chemical entities suitable for therapeutic use. It

is also includes the study of existing drugs, their biological properties, and their

quantitative structure activity relationships (QSAR). Pharmaceutical chemistry is

focused on quality aspects of medicines and aims to assure fitness for the purpose

of medicinal products1.

During the early stages of medicinal chemistry development, scientists were

primarily concerned with the isolation of medicinal agents found in plants. Today,

scientists in this field are also equally concerned with the creation of new

synthetic compounds as drugs. Medicinal chemistry is almost always geared

toward drug discovery and development 2. The approach to the practice of

medicinal chemistry has developed from an empirical one involving organic

synthesis of new compounds, based largely on modification of structures of

known activity, by a more logical approach. Now computers have been pressed

into the service of medicinal chemists. Computers augment the scientific process

in drug discovery by assisting the chemist with collecting, storing, manipulating,

analyzing and viewing the data. Further, computers provide a link to theoretical

chemistry and graphic modeling, providing calculated estimates of molecular

properties, models of molecules, models of biological sites and sometimes even

models of drug-receptor interactions 3.

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1. 1. Chemistry of Benzimidazoles:

The benzimidazole nucleus is an important pharmacophore in medicinal

chemistry. The synthesis of novel benzimidazole derivatives remains a main focus

of modern drug discovery. The versatility of new generation benzimidazole would

represent a fruitful pharmacophore for further development of better medicinal

agents. Since now, researchers have been attracted toward designing more potent

benzimidazole derivatives having wide range of biological activity.

Several benzimidazoles are commercially available as pharmaceuticals.

Benzimidazoles are most widely studied drugs as antihelmintics. Studies have

established that benzimidazole carbamates such as albendazole (1), mebendazole

(2), flubendazole (3), and fenbendazole (4) inhibit the in vitro growth of

Trichomonas vaginalis4 and G. lamblia5,6 and have a broad antiparasitic spectrum

of activity, low toxicity and have been used successfully to treat gastrointestinal

helmintic infections.

NH

N

NHCOOCH3

S

CH3

Albendazole

NH

N

NHCOOCH3

O

Mebendazole

(1) (2)

3

NH

N

NHCOOCH3

O

F

Flubendazole

NH

N

NHCOOCH3

S

Fenbenazole

(3) (4)

Another area of important use of benzimidazole has recently been as proton pump

inhibitor. Omeprazole (5) is appeared for the treatment and reduction of risk of

recurrence of duodenal ulcer, gastric ulcer and pathological hypersecretory

conditions. Lansoprazole (6) is used for the treatment of duodenal ulcer, and

Zollinger-Ellison syndrome.

NH

N

S

NCH3

CH3

H3CO

CH3

O

NH

N

S

NCH3

F3CH2CO

O

Lansoprazole

Omeprazole (5) (6)

The other therapeutic agents such as H1 antihistaminic agent clemizole (7), a

potent opioid analgesic etonitazene (8), non-nucleoside antiviral compound

enviroxime (9), for promotion of excretion of uric acid irtemazole (10), non

4

sedating antihistaminic agent astemizole (11), antinematodal agent nocodazole

(12) are based on benzimidazole heterocyclic nucleus.

N

N N

Cl

N

N

N

O2N

H5C2

C2H5

OEt

(7) (8)

N

N

SO2

NH2

CH3

CH3

N

NH

CH3

N

N

(9) (10)

N

N

NH

N

F

O

CH3

N

NH

NH

O

S O

CH3O

(11) (12)

5

1.1.1. Synthetic routes of benzimidazole nucles:

A variety of methods have been developed for the preparation of substituted

benzimidazoles. The traditional synthesis (13) of benzimidazoles involves the

reaction between a phenylenediamine and a carboxylic acid or its derivatives

under harsh dehydrating reaction conditions7-10.

NH

NR

NH2

NH2

RCOOH4N HCl

(13)

Subsequently, several improved protocols have been developed for the synthesis

of benzimidazoles via the condensation of o-phenylenediamines with aldehydes in

the presence of acid catalysts under various reaction conditions.

Byeong Hyo Kim et al11 described indium-mediated reductive inter-molecular

coupling reaction of 2-nitroaniline with aromatic aldehydes to benzimidazoles

(14).

NH

NAr

NH2

NO2

BNP, InArCHO

MeOH, H2O, RT N

NAr

Ar

Major

(14)

Takashi Itoh et al12 synthesized 2-arylbenzothiazoles and imidazoles using

scandium triflate as a catalyst for both a ring closing and an oxidation steps (15).

6

NH

XAr

XH

NO2

ArCHO RT, under O2

Sc(OTf)3 (cat.)

X=S, NH X=S: Y.97-99%X=NH: Y.72-97%

(15)

Donglai Yang et al 13 reported a highly efficient and versatile method for the

synthesis of benzimidazoles in one step via the Na2S2O4 reduction of o-

nitroanilines by heating a solution of o-nitro aniline and an aldehyde in EtOH or

another appropriate solvent, in the presence of aqueous or solid Na2S2O4, provided

facile access to a series of 2-substituted benzimidazoles containing a wide range

of functional groups not always compatible with the existing synthetic methods

(16).

NO2

NHR'F

NO2

1 eq. R'NH2

DMSO, 1000C, 10 h

R R

N

NR"

R'

1 eq. R"CHO3 eq. Na2S2O4

EtOH/DMSO (4:1)800C, 12h

R

(16)

Khodabakhsh Niknam et al14 developed a highly selective synthesis of 2-aryl-1-

arylmethyl-1H-1,3-benzimidazoles from the reaction of o-phenylenediamines and

aromatic aldehydes in the presence of metal hydrogen sulfates [M(HSO4)n] in

water and also under solvent-free conditions in good to excellent yields (17).

NH

NAr

NH2

NH2

M(HSO4)nArCHO

(17)

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1.1.2. Reactions of benzimidazoles

Benzimidazoles undergoes following types of reactions:

1.1.2.1. Reactions with electrophilic reagents:

Preferential position of attack by electrophil is 5th position of unsubstituted

benzimidazole, and 2nd preferential position is 6th in absence of influence by the

attached substituent but if the 5-substituent is powerfully electron releasing the

second substituent enters at 4th position15. While an electron withdrawing

substituent at 5th position directs the entering electrophils to 6th position and to a

lesser extent the 7th position. Examples of electrophilic aromatic substitution

reaction are

Nitration:

N

NH

CF3

F

N

NH

CF3

F

NO2

HNO3/H2SO4

(18)

N

NH

R

O2NN

NH

R

O2N

O2N

HNO3/H2SO4

+N

NH

R

O2N

NO2

R = H, Ar

(19)

8

Sulfonation:

N

NH

RN

NH

R

HO3SH2SO4

R = H, Ar (20)

1.1.2.2. Substitution in the Imidazole ring:

Electrophilic substitution does not occur in the imidazole ring of benzimidazole.

Benzimidazole is quantitatively iodinated on treatment with iodine in aqueous

sodium hydroxide solution to give product that was believed to be 2-

iodobenzimidazole16.

Benzimidazole and 1-methyl benzimidazole react with bromine in chloroform at

room temperature to give dicoordinate complex and n- donor complexes that are

formulated17

N

NH

N NHBrH

+

n

Br3- N

N+

CH3

Br

Br-

(21)

1.1.2.3. Electrophilic attack at the 1-(or 3-) position: Alkylation and Related

Reactions:

There are four possible mechanisms for the alkylation depending on the alkylation

of substrate i.e. whether alkylated by base, an anion or conjugate acid. Alkylation

9

of neutral imidazoles and benzimidazoles by alkyl halides usually occurs by SE2

mechanism in which electrophilic attack is directed at the pyridine like nitrogen.

Under neutral conditions, the benzimidazolium intermediate reacts with

unchanged benzimidazoles18.

1.1.2.4. Electrophilic Attack at Side-Chain Substituents:

Reactions in this category include substitution and addition processes. Some of

the reactions in this category are shown19

N

N

R

OH

Ph2CHClN

N

R

O

R= alkyl, aryl (22)N

NH

NH2

N

NH

N=CHR

RCHO

R= alkyl, aryl (23)

1.1.2.5. Reactions with Nucleophilic Reagents:

Substitution in the imidazole Ring:

In this type of reaction, the benzimidazole is heated in xylene with sodium amide.

For the compounds of this type, formation of anion of hetrocyclic prohibits the

nucleophilic attack at 2- position. Various other derivatives such as 5-alkyl and

thioalkyl derivatives are formed20.

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N

N

R

N

N

R

NH2

NaNH2/Xylene

R=alkyl, aryl (24)

1.2. Pyrazolones and Isoxazolinone:

1.2.1. Chemistry of Pyrazolones

The oxo derivatives of pyrazolines, known as pyrazolones, are best classified as

follows: 5-pyrazolone, also called 2-pyrazolin-5-one (25); 4-pyrazolone, also

called 2-pyrazolin-4-one (26); and 3-pyrazolone, also called 3-pyrazolin-5-one

(27). Within each class of pyrazolones many tautomeric forms are possible; for

simplicity only one form is shown.

NH

NO

NH

N

O

NH

NO H

(25) (26) (27)

NN

R

OH

NNH

R

O

NN

R

OH

NN

R

O

Enol Keto Enol Keto

(28) (29)

Substitution at N1 decreases the possible number of tautomers: for 3-pyrazolones,

two tautomeric forms are possible, (28) and (29), which in nonpolar solvents are

both present in about the same ratio. 5-Pyrazolones exhibit similar behavior.

11

In 4-pyrazolones, the enol form predominates, although the keto form has also

been observed. The tautomeric character of the pyrazolones is also illustrated by

the mixture of products isolated aftercertain reactions. Thus alkylation normally

takes place at C4, but on occasion it is accompanied by alkylation on O and N.

Similar problems can arise during acylation and carbamoylation reactions, which

also favor C4. Pyrazolones react with aldehydes and ketones at C4 to form a

carbon–carbon double bond, eg (30). Coupling takes place when pyrazolones

react with diazonium salts to produce azo compounds, eg (31).

Compounds of type (31) are widely used in the dye industry. The Mannich

reaction also takes place at C4, as does halogenation and nitration. The important

analgesic aminoantipyrine (32) on photolysis in methanol undergoes ring fission

to yield (33)21.

NN

R

O

R

R

NN

R

O

NN

R

12

(30) (31)

NN

O

NH2 CH3

CH3

CH3 OH

hv NH

O

OH

O

NHCH3

CH3

(32) (33)

1.2.2. Synthesis of pyrazolone derivatives

The pyrazolone-3-carboxylic acid (35) has been isolated by reaction of oxazolone

(34) with hydrazonyl chloride22.

O

N

Ar2

H

OAr1

+ NH N

COOR

Ar3

Cl

(C4H9)4N+Br

-

Na2CO3

NN

Ar1

O

CH3

COORNH

Ar2

O

(35)

(34)

NN

CH2

C(CH3)3(H3C)3C

+ NC

CN

Na H NN

C(CH3)3

ONC

NH2 C(CH3)3

13

(36)

The preferred synthetic method for the title compounds utilizes the reaction of

hydrazines with bifunctional compounds, such as β-diketones and esters, and β-

keto acetylenic compounds. In an alternative procedure, diazo compounds replace

hydrazines and ring formation takes place via 1,3-dipolar cycloaddition. Pyrazoles

and pyrazolones are widely used in the pharmaceutical industry to alleviate

inflammation, fever, pain, and infections. To a lesser extent, they are also used as

insecticides and herbicides. Pyrazolones linked to azo compounds are extensively

used in the dye industry; some pyrazolines display insecticidal activity23.

Pyrazolones with a free NH group are easily nitrosated and give rise to

nitrosamines, which cause tumors in the liver of test animals. The analgesics

antipyrine (37) and aminopyrine (38), if admixed with nitrites, are mutagenic

when tested in vitro; however, when tested in the absence of nitrites, negative

results are obtained24.

NN

CH3O

CH3

NN

CH3

ON

CH3

CH3

CH3

(37) (38)

Pyrazolone-type drugs, such as phenylbutazone and sulfinpyrazone, are

metabolized in the liver by micro-somal enzymes, forming glucuronide

metabolites that are easily excreted because of enhanced water solubility.

14

The pyrazolone derivatives, which include dipyrone (39), antipyrine (37),

aminopyrine (38) and propyphenazone, are widely used analgesics. Dipyrone, the

most widely used pyrazolone, has been the most studied. Dipyrone is an inhibitor

of cyclo-oxygenase but, unlike aspirin, its effect is rapidly reversible. The

inhibition of prostaglandin biosynthesis contributes to the analgesic activity of the

pyrazolone derivatives. Unlike the Non-steroidal anti-inflammatory agents

(NSAIDs) generally, the pyrazolone derivatives antipyrine, aminopyrine and

propyphenazone are minimally bound to plasma proteins. The pyrazolones

undergo extensive biotransformation, aminopyrine and dipyrone being converted

to active metabolites. The most frequently reported side effects of the pyrazolone

derivatives are skin rashes. Gastrointestinal side effects are rare.

NN

CH3

ON

CH3

CH3

NaO3S

OH2

(39)

1.2.3. Important Pyrazolone and isoxazoline derivative in pharmaceuticals:

Some of the pharmaceuticals that incorporate the pyrazole nucleus are given

below. Their main uses are as antipyretic, anti-inflammatory, and analgesic

agents. To a lesser extent, they have shown efficacy as antibacterial/antimicrobial,

antipsychotic, anti-emetic, and diuretic agents. The analgesic aminopyrine, the

antipyretic dipyrone, and the anti-inflammatory phenylbutazone (40), though once

15

widely prescribed, are rarely used in the 1990s on account of their tendency to

cause agranulocytosis. Pyrazolone and pyrazolidinedione derivatives as like

benzimidazole derivatives have been found to possess some interesting

pharmacological activities. eg. Antipyrin, Ampyrone, edaravone, etc.

NN

O

O(CH2)3CH3

(40)

NN

O

O(CH2)3CH3

S

S

NH2O

O

NH2

OO

;

NN

O

O(H2C)3

CH3

O

O

Cl

(41) butaglyon, an antidiabetic; (42) feclobuzo, an antiinflammatory;

NN

O

OCH3

O

NN

O

OS

O

(43) kebuzone, an antirheumatic; (44) sulfinpyrazone, an anti gout

16

NN

NH2

O

CH3

Cl

Cl

NN

O CH3

CH3

(45) muzolimin, a diuretic (46) phenazobz, an antiasthmatic

NN

CH3

O

NN

CH3

O

NH2

CH3

Edaravone (47) Amprone (48)

Pyrazolones react with diazonium salts, an important process in the dye industry.

The majority of dyes are having pyrazolone nucleus with an azo linkage attached

at C4, eg, (49) and (50).

17

NN

O

R2

R1

CH3

R''

NN

NaO3S

NN

O

R1

NN

NN

NN

O

R1

Cl

Cl

(49) (50)

The survey of the pertinent literature reveals that isoxazolidine have been found to

possess a wide range of biological activity such as anti bacterial25, anti HIV26,

anti-inflammatory27, anticancer28, etc. Some isoxazole derivatives (51) have been

reported as anti-tubercular, anti bacterial and antifungal agets29.

18

O

NO

R

(51)

Azopyrazoles (52) and azoisoxazoles (53) are possessing good antifungal

activity30. Similarly, 2-alkyl isoaxazolidine derivatives have been as antifungal

agents31.

N

N

N

NH

R1

N

N

N

O

R1

(52) (53)

N

O

CH3

ClN

N

Cl

N

O

CH3

ClN

N

Cl

(54) (55)

1.3. IN-SILICO DRUG DESIGN

Drug discovery and development is an essential, intense, lengthy and an

interdisciplinary endeavor. Drug discovery is mostly portrayed as a linear,

consecutive process that starts with target and lead discovery, followed by lead

19

optimization and pre-clinical in vitro and in vivo studies to determine if such

compounds satisfy a number of pre-set criteria for initiating clinical development.

Traditionally drugs were discovered by synthesizing compounds in a time

consuming multi-step processes against battery in-vivo biological screens and

further investigating the promising candidates for the pharmacokinetic properties,

metabolism and potential toxicity. Such a development processes has resulted in

high attrition rates with failures attributed to poor pharmacokinetics (39%), lack

of efficacy (30%), animal toxicity (11%), adverse effects in humans (10%) and

various commercial and miscellaneous factors. Today, the processing of drug

discovery has been revolutionized with the advent of genomics, proteomics,

bioinformatics and efficient technologies like, combinatorial chemistry, high

throughput screening (HTS), virtual screening, de novo design in vitro, in silico

ADMET screening and structure- based drug design.

Computer aided drug design is an interdisciplinary of bioinformatics, medicine

and biophysics. Bioinformatics and computational methods recently were used to

design new drug candidates that could potentially bind with target proteins, thus

producing drug molecules for many disease. They also promise to speedup drug

research by predicting potential effectiveness of designed compounds prior to

experimental studies and preclinical trials.

In-silico methods can help in identifying the drug targets via bioinformatics tools.

They can also be used to analyze the target structure for possible binding/ active

sites, generate candidate molecules, check for their drug likeness, dock these

20

molecules with the target, rank them according to their biding affinities, further

optimize the molecules to improve binding characteristics. The use of computers

and computational methods permeates all aspects of drug discovery today which

is essential core of structure-based drug design. The use of in-silico drug design

techniques increases the chance of success in many stages of the drug discovery

process, from the identification of novel targets and elucidation of their function

to the discovery and development of lead compounds with desired properties.

Computational tools provide the advantage of delivering the new drug candidates

more quickly and at lower cost32.

1.3.1. RATIONAL DRUG DESIGN

In-silico techniques save great amounts of time and money in R&D projects. A

good modeling support is often what makes the difference between a successful

drug design project and one that fails. With a strong background in the fields of

molecular modeling, molecular biology and computational chemistry, we are able

to offer full in-silico support for projects of drug design, protein engineering and

intermolecular recognition. The possibility of developing software to tailor the in-

silico approach to different problems is what makes us unique.

1.3.2. TECHNIQUES

• Molecular Docking and Virtual Screening: Docking studies are

computational techniques for the exploration of the possible binding modes of a

substrate to a given receptor, enzyme or other binding site. Docking is the process

by which two molecules fit together in 3D space. Docking studies may help to

21

increase ligand specificity; and also better therapeutic index can be achieved if the

drug produces undesirable side effects due to its binding with another site, the

affinity for that competing site can be diminished. Different types of docking

include- flexible protein-ligand docking, flexible protein-protein docking and

hydrophobic docking. Docking may play an important role in the QSAR studies

and homology modeling very useful in structure based drug design. Various

docking programs are available DOCK, FLOG, ADAM, and UGIN.

• Molecular Dynamics: The prediction of the evolution of molecular

systems over time, the study of protein conformation, protein-protein interactions,

the simulation of biological membranes.

• Quantum Mechanics: The study of chemical reactions, the effects of

substitutions on electronic properties and reactivity of molecules.

• QSAR: Quantitative structure-activity relationship. The ability of

predicting biological properties of molecules without even the need of knowing

their target.

• Homology Modelling: Predicting the structures of proteins that has not

been yet crystallized.

1 . 3.3. DOCKING STUDIES:

The ability to propose reasonable binding modes of a designed structure to a

known receptor site called docking studies, which is crucial to the success of

structure based design. One approach is to dock or position ligand or receptor

molecules together in many different possible ways and then scores each

orientation according to an evaluation function of some kind. These studies can

22

predict binding confirmations and affinities of millions of molecules without the

need of a single synthetic step. These rational drug design methods accelerate the

process by speeding up the discovery of new chemical substances that may

become a new drug.

1.3.4. DRUG-LIKENESS AND LEAD-LIKENESS

Christopher A. Lipinski33 defined the Drug likeness as the compounds those have

sufficiently acceptable absorption, distribution, metabolism and elimination

properties to get successful entry in to human Phase 1 clinical trials. For the drug

development, drug properties are important prominent component. A chemically

synthesized compound library can contain many non-drug-like compounds.

Therefore, recent technologies helped to develop recognized drug-like compounds

from a diverse compound library34-39. These drug-like measuring and filtering

technologies have partly solved the screening problems. However, they have not

been good enough to completely solve these problems. It has been observed that

many drug-like compounds, which should be potential candidates; do not come up

as hits when they are screened against biological targets. Drug-likeness is the

descriptors of all important pharmacological properties such as potency,

selectivity toward receptor, absorption, distribution, metabolism and toxicity. In

the past, these parameters were optimized sequentially. Now, it is mandatory that

these parameters should be optimized simultaneously. Properties that have been

associated with oral drug-likeness include:

• Oral bioavailability

23

• Appropriate toxicity to pass phase I clinical trials.

• Aqueous solubility

• Synthetics accessibility

• Pharmacokinetic viability

• Blood-brain barrier permeability.

Lipophilicity is a key property for pharmacological activity in drug discovery and

used to estimate the permeability of a drug molecule in the cell membrane. It is

measured as logP value that distribution coefficient of compounds between n-

octanol and water.

When logP value is very low or very high, the permeability of drug components

get dropped due to the inability of weakly lipophilic compounds to penetrate the

lipid portion of the membrane and the excessive partitioning of strongly lipophilic

compounds into the lipid portion of the membrane and their subsequent inability

to pass through the aqueous portion of the membrane.

Lipinski's rule helps to predict the poor absorption and permeability of potential

drug candidates. It will occur if,

• A molecular weight less than 500.

• An octanol-water partition coefficient log P of less than 5.

• Molar refractivity not more than 150

• Not more than 5 hydrogen bond donors (nitrogen or oxygen atoms with

one or more hydrogen atoms)

• Not more than 10 hydrogen bond acceptors (nitrogen or oxygen atoms).

24

1.4. NON-STEROIDAL ANTI-INFLAMMATORY DRUGS

Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most widely used

therapeutics, primarily for the treatment of inflammation, especially arthritis40. In

addition to their anti-inflammatory effects, agents belonging to the NSAID class

possess both analgesic and antipyretic activities. Hence, NSAIDs are sometimes

referred to as non-narcotic analgesics or as aspirin-like drugs41. They provide

symptomatic relief from pain and swelling in chronic joint disease such as occurs

in osteo- and rheumatoid arthritis, and in more acute inflammatory conditions

such as sports injuries, fractures, sprains and other soft tissue injuries. They also

provide relief from postoperative, dental and menstrual pain, and from the pain of

headaches and migraine.

1.4.1. Pharmacological Actions:

All the NSAIDs have actions very similar to those of aspirin. The three main

therapeutic effects are an anti-inflammatory effect: modification of the

inflammatory reaction, an analgesic effect: reduction of certain types of

(especially inflammatory) pain and an antipyretic effect: lowering of body

temperature when this is raised in disease (i.e. fever).

In addition, all the NSAIDs share, to a greater or lesser degree, the same types of

mechanism-based side effects. These include:

• gastric irritation, which may range from simple discomfort to ulcer

formation

• an effect on renal blood flow in the compromised kidney

25

• a tendency to prolong bleeding through inhibition of platelet function.

Controversially, it is argued that they may also all-but especially COX-2 selective

drugs-increase the likelihood of thrombotic events such as myocardial infarction

by inhibiting prostaglandin (PG I2) synthesis.

A number of aryl and heteroaryl substituted compounds such as Diclofenac42 (1),

Lumiracoxib43 (2), Lonazolac (3), Etodolac42 (4) have been commercialized as

non-steroidal anti-inflammatory drugs (NSAIDS).

Important Non-Steroidal Anti-inflammatory Drugs:

26

COOH

CH3

F

SCH3 O

Diclofenac (56) Indomethacin (57) Sulindac (58)

PROFEN DERIVATIVES:

CH3

CH3

COOH

CH3

COOH

CH3

F

Ibuprofen (59) Flubiprofen (60)

NH

COOH

ClCl

27

COOH

CH3

O

H3CO

COOH

CH3

Ketoprofen (61) Naproxen (62)

OXICAMES:

NS

NH N

O

OH

OOCH3

NS

NH N

O

OH

OOCH3

S

Piroxicam (63) Tenoxicam (64)

Others:

CH3

N

COOHCH3O

N

O

COOH

Tolmetin (65) Ketorolac (66)

Selective COX-2 Inhibitors:

SO2NH2

NN

CH3

CF3

SO2CH3

O

O

Celecoxib (67) Rofecoxib (68)

28

COOH

NH

Cl F

CH3

(69) Lumiracoxib

NN

COOHCl

NH

O

CH3

CH3

COOH

Lonazolac (70) Etodolac (71)

While there are differences between individual drugs, all these effects are

generally thought to be related to the primary action of the drugs-inhibition of the

fatty acid COX enzyme, and thus inhibition of the production of prostaglandins

and thromboxanes. There are three known isoforms-COX-1, COX-2 and COX-3-

as well as some non-catalytic species. As it is not yet certain that COX-3 actually

occurs in humans in a functional form, we will confine the discussion mainly to a

consideration of COX-1 and COX-2. While they are closely related (> 60%

sequence identity) and catalyse the same reaction, it is clear that there are

important differences between the expression and role of these two isoforms.

COX-1 is a constitutive enzyme expressed in most tissues, including blood

platelets. It has a 'housekeeping' role in the body, being involved in tissue

homeostasis, and is responsible for the production of prostaglandins involved in,

for example, gastric cytoprotection, platelet aggregation, renal blood flow

autoregulation and the initiation of parturition).

29

In contrast, COX-2 is induced in inflammatory cells when they are activated, and

the primary inflammatory cytokines-interleukin (IL)-1 and tumour necrosis factor

(TNF)-α are important in this regard. Thus the COX-2 isoform is responsible for

the production of the prostanoid mediators of inflammation, although there are

some significant exceptions. For example, there is a considerable pool of

'constitutive' COX-2 present in the central nervous system (CNS) and some other

tissues, although its function is not yet completely clear.

Although, non-steroidal anti-inflammatory drugs (NSAIDS) have been used in the

treatment of various inflammatory diseases, their usage is limited by the side

effects produced by them, thereby necessitating the need for searching new

molecular entities42.

1.5. Anti bacterial activity:

The emergence of resistance to the major classes of antibacterial agent is

recognized as a significant medical crisis and serious health concern. Particularly,

the emergence of multi drug-resistance strains of Gram-positive bacterial

pathogens is a problem of ever increasing significance. As the limited number of

antimicrobial classes and the common occurrence of resistance within and

between classes, the search for antibacterial agents with novel mechanism of

actions is always remains an important and challenging task.

The control of microorganism is critical for the prevention and treatment of

disease. Microorganisms also grow on and within other organism, and microbial

colonization can lead to disease, disability, and death. Thus the control or

30

destruction of microorganisms residing within the bodies of humans and other

animals is great importance.

Modern medicine is dependant on chemotherapeutic agents, chemical agents that

are used to treat disease. Chemotherapeutic agents destroy pathogenic

microorganisms or inhibit their growth at concentrations low enough to avoid

undesirable damage to the host. Most of these agents are antibiotics, microbial

products or their derivatives that can kill susceptible microorganisms or inhibit

their growth. Drugs such as the sulfonamides are sometimes called antibiotics

although they are synthetic chemotherapeutic agents, not microbially synthesized.

Antibiotics are chemical substances excreted by some microorganism which

inhibit the growth and development of other microbes. Some of these drugs that

were obtained naturally were put to chemical modifications in attempts to

enhance beneficial effects while minimizing the toxic effects. The resultant

modified product is termed as semi synthetic antibiotics. Most antibiotic currently

used are semi synthetic. The chemist has synthesized many drugs that have got

the antibacterial property and less toxicity. These drugs are called synthetic

antibiotic drugs. Naturally occurring antibiotic, their semi synthetic derivatives

and synthetic antibiotics have got the same target. i.e., antimicrobial action. Hence

all these drugs were put together to be called antimicrobial agents.

1.5.1. Drug resistance:

The emergence of drug resistance bacteria is posing a major problem in

antimicrobial therapy. The frequency varies with the organism and the antibiotic

31

used. At first, there is an emergence of a small number of drug resistant bacteria

which sooner multiplies selectively in the presence of the drug at the cost of

sensitive bacteria.

1.5.2. Types of drug resistance:

Drug resistance is of two types, primary and acquired.

1. Primary resistance: some bacteria possess an innate property of resistance to

certain drug, e.g. resistance of E.coli to penicillin.

2. Acquired resistance: it results either from mutation or gene transfer.

1.5.3. Recent targets for finding antibacterial agents

Beta-Ketoacyl-acyl carrier protein (KAS) synthase III encoded by the

fabH gene is thought to catalyze the first elongation reaction of type II fatty acid

synthesis in bacteria and plant plastids. Beta-ketoacyl-acyl carrier protein

synthase (KAS) I is important enzyme system for the construction of the

unsaturated fatty acid carbon skeletons characterizing E. coli membrane lipids.

Recent research reported that Type II fatty acid synthesis (FAS II) pathway is an

attractive target for their efficacy against infections caused by multi-resistant

Gram-positive bacteria and Gram-negative bacteria44. Among the related FAS II

enzymes, beta ketoacyl-acyl carrier protein synthase (KAS) is an essential target

for novel antibacterial drug design45-46.

The enzyme bacterial peptide deformylase (PDF) is another novel target for novel

antibacterial agents. The metalloproteases enzyme, Bacterial peptide deformylase

(PDF) deformylates the N-formyl methionine of newly synthesized

32

polypeptides through Fe2+-mediated catalytic reaction. PDF is essential in

prokaryotes and this enzyme is absent in mammalian cells and provides a unique

target for antimicrobial chemotherapy 47-50. Thus, it may be another target for new

chemotherapeutic agents.

Lipopolysaccharides constitute the outer leaflet of the outer membrane of Gram-

negative bacteria and are therefore essential for cell growth and viability. The

glycosyltransferase (GT) enzyme, heptosyltransferase WaaC involved in the

synthesis of the inner core region of lipopolysaccharides. It catalyzes the addition

of the first l-glycero-d-manno-heptose molecule to one molecule of 3-deoxy-d-

manno-oct-2-ulosonic acid (Kdo) residue of the Kdo2-lipid A molecule. These

heptose is an essential component of the Lipopolysaccharides core domain; its

absence results in a truncated lipopolysaccharide associated with the deep-rough

phenotype causing a greater susceptibility to antibiotic. Thus, WaaC represents a

promising target in antibacterial drug design51.

1.6. Anti fungal activity:

The object of antifungal drug discovery has become a subject of greater challenge

due to increasing incidences of fungal drug resistance. This appears due largely to

the extensive use of antifungal agents to treat fungal infections. In the past

decade, number of patients diagnosed with fungal infections have increased

drastically, whereas, relatively very few clinically useful drugs were discovered.

The azole derivatives such as such as clotrimazole, fluconazole, itraconazole,

ketoconazole, etc. have been widely used to treat a verity of fungal infections.

33

These azole derivatives inhibit the fungal enzyme 14-alpha demethylase which is

essential for the ergosterol synthesis pathway leads to the depletion of this

steroidal compound in the cell membrane and accumulation of toxic intermediate

sterols, leads increased membrane permeability and inhibition of fungal growth52-

54. But broad usage of these drugs led to development of acquired resistance

especially among Candida albicans. Thus, searching not only improved version

of existing drug but also for new drug targets has become an urgent need55.

Recent reports showed that 2-glutamine, D-fructose-6-phosphate

aminotransferase known as a new target for antifungals, it catalyzes a complex

reaction involving ammonia transfer from L-glutamine to fructose-6-phosphate,

followed by isomerisation of the formed fructosamine-6-phosphate to

glucosamine-6-phosphate56.

1.7. Antioxidant activity:

The knowledge of free radicals and reactive oxygen species is producing a

revolution in the field of medicine that promises a new age of health and disease

management. The formation and activity of a number of compounds, known as

reactive oxygen species, which have a tendency to donate oxygen to other

substances are producing various potential harmful effects. Evidences show that

free radical damage contributes to the etiology of many chronic health problems

such as cardiovascular and inflammatory disease, cataract and cancer.

Antioxidants can prevent free radical induced tissue damage by preventing the

formation of radicals, scavenging them, or by promoting their decomposition57-59.

34

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