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Chapter 2

Pharmacophore hybridization

CHAPTER: 2 PHARMACOPHORE HYBRIDIZATION

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Infectious diseases are influencing the world with their morbidity and

mortality, out of which tuberculosis and malaria are major infectious

diseases caused by Mycobacterium tuberculosis and Plasmodium falciparum

respectively, have become more virulent now a days by claiming about three

to four million lives and infecting over ten million people annually. The

current chemotherapy is based on age old drugs like Chloroquine,

Pyrimethamine-sulfadoxine for malaria and Pyrazinamide, Isoniazid and

Rifampin for tuberculosis. The efficacy of these drugs has been deteriorated

by the emerging resistant strains. More over the pathogenic synergy of HIV

to these diseases is alarming the world to develop new efficient

chemotherapy for infectious diseases.

Although the development of drug resistance motivates the pursuit of

innovation in anti-infective drug development, it does not deter the

exploration of existing and effective drug discovery tools such as

pharmacophore hybridization. Pharmacophore hybridization is believed to

be analogous to conventional combination therapy, with the exception that

the two drugs are covalently linked and available as a single entity. The

successful utilization of this approach relies, in part, on the judicious

selection of monomers.

In demonstration, as part of our drug discovery programme on novel

antiinfective agents the synthesis of an exploratory library of natural

product-like hybrids modelled on the isatins moiety was undertaken and is

reported herein this Chapter.

Pharmacophore hybridization: Tetrahydropyrimidine–i satin hybridized derivatives: Synthesis and In vitro evaluation of Antibacterial, Antifungal, Antitubercular and Antim alarial activities.


Page 44

Pharmacophore hybridization (PH):

Pharmacophore or Molecular hybridization (PH or MH) is a new concept in drug design

and development based on the combination of pharmacophoric moieties of different

bioactive substances to produce a new hybrid compound with improved affinity and

efficacy, when compared to the parent drugs. Additionally, this strategy can result in

compounds presenting modified selectivity profile, different and/or dual modes of action

and reduced undesired side effects. So, in this chapter, we described several examples of

different strategies for drug design, discovery and pharmacomodulation focused on new

innovative hybrid compounds presenting analgesic, anti-inflammatory, platelet anti-

aggregating, anti-infectious, anticancer, cardio- and neuroactive properties.

2.1 Introduction:

Over the last decades, the registration of pharmaceuticals for the treatment of new

pathologies or that represent therapeutic innovations on known illnesses, mostly

infectious and with high social-economical impact, such as neurodegenerative diseases

and cancer, has suffered a continuous decrease, contrasting with the growing of

technological and scientific advances pursuing the improvement of the quality of life.

Reappearing diseases such as tuberculosis, hanseniasis, smallpox, schistosomiasis,

infectious diseases associated to resistant microorganisms, such as malaria, still

incurable new virus and tropical diseases, besides cancer, neurodegenerative and

autoimmune diseases, still represent a big challenge for the pharmaceutical sector and

demand a continuous effort to the development of new therapeutic tools: more efficient,

selective and economically accessible.

In this paradoxical context, the Pharmaceutical Industry has invested heavily in the

development of new techniques of diagnosis, investigation and creation of chemical

libraries with high molecular diversity, based on:

1) Combinatorial chemistry,

2) Computer-aided drug design (CADD),

3) Simulation and prediction of physicochemical and structural properties

associated to drug-receptor interactions (QSAR),

4) Automatized processes of pharmacological screening (High Throughput

Screening – HTS),


5

5) New methods of in vitro and in vivo pharmacological evaluation, based on the

advances of the molecular biology, genomics and biotechnological approaches [1-

3].

Recently, the natural products chemistry has been returning to a prominent position in the

prospection of bioactive compounds, justifying the new investments in research from the

Pharmaceutical sector pursuing new pharmaceuticals, especially the ones whose origin is

vegetal, marine or from microorganisms. The enormous chemical diversity of the

secondary metabolites still challenges and inspires the synthetic and medicinal

chemistry for their molecular complexity and diversity, working as role templates for the

discovery of new drugs and the planning of new synthetic and semi synthetic derivatives

[3-8].

On the other hand, the result of all this effort was not yet able of promoting the quick

access to massive quantities of new bioactive chemical entities (BioNCEs), frustrating the

expectation of high efficiency and productivity, which has not happened yet, since the

number of new registered pharmaceuticals has been decreasing significantly year after

year [4-5].

The rational planning of new synthetic prototypes has been using a series of methods of

structural modification that aim, a priori, at the generation of new compounds presenting

optimized pharmacodynamic and pharmacokinetic properties, by:

1) Exploring bioactive substances’ fragments (Fragment-Based Drug Design) [9],

2) Active metabolites of drugs [10],

3) Bioisosterism [11],

4) Selective optimization of side effects of drugs [12] and

5) Drug latentiation [13].

Pharmacophore hybridization as a tool in the planning of new ligands and

prototypes:

The pharmacophore hybridization (PH) is a strategy of rational design of new ligands or

prototypes based on the recognition of pharmacophoric sub-unities in the molecular

structure of two or more known bioactive derivatives which, through the adequate fusion


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of these sub-unities, lead to the design of new hybrid architectures that maintain pre-

selected characteristics of the original templates.

Considering the use of known template substances, already evaluated concerning the

physicochemical and pharmacological features, toxicity and mechanism of action, it is

possible the generation of extensive chemical libraries, constituted by hundreds or even

thousands of homologous pharmacophore hybrids, bringing a high level of accumulated

information, e.g. structural requirements, ligand-protein interaction mode, site ligand -

receptor interactions and quantitative structure-activity relationships, which tends to

become faster and more efficient the development of new drugs [14]. On the other hand,

if the degree of template-hybrid homology is either low or inexistent, the discovery of

new lead-compounds should be made by massive screening of the generated chemical

library.

The therapeutic tools for the treatment of complex heterogeneous diseases can be very

restricted, considering that only one drug will usually not be able to control the illness in

an effective way, demanding the combination of pharmaceuticals with different

pharmacotherapeutic profiles. In this context, the therapy by medicamentous association

may be reached by the utilization of two or more pure drugs or by pharmaceuticals

presenting only one active ingredient that combined the activities of two or more drugs,

i.e. a hybrid drug [15]. Accordingly, we can describe the several examples of the

application of PH approach in the design of new ligands or prototypes belonging to

different therapeutic categories.

As Cardioactive Agents

The search for new antihypertensive agents led Breschi and co-workers to recently

develop another new class of cardioactive hybrids [16]. Until last decade, the more

efficient antihypertensive drugs were inhibitors of the angiotensin-converting enzyme

(ACE), which present some adverse effects resultant from their ability in promote

bradykinin accumulation. One alternative found for the therapeutic replacement of this

class of antihypertensive agents was the development of sartans [17], which act as

selective antagonists of angiotensin II receptors AT1, blocking the action of this

vasoconstrictor peptide through a more efficient way than the ACE inhibitors, besides the

fact that the preservation of this enzyme does not avoid the degradation of the bradykinin


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[16], circumventing the undesirable side effects resulting from its accumulation.

Additionally, the use of drugs from sartan class modulates the bradykinin-dependent NO

biosynthesis, which displays remarkable vasorelaxant effect [18, 19]. According to this

panorama, Breschi and co-workers explored the structural pattern of lead-compound

losartan 1 for the design and synthesis of new hybrids 2 and 3, presenting the hydroxyl

group esterified with a NO-releasing sub-unit (Fig. 1) [16].

The hybrid compounds 2 and 3 almost complete vasorelaxant effect with an efficacy of

92 and 95%, respectively, which was strongly inhibited by 1H-[1,2,4]oxadiazolo[3,4-

a]quinoxalin-1-one, an inhibitor of guanylate cyclase, as expected to a vasodilator effect

dependent on the NO production [16]. Besides that, compounds 2 and 3 showed to be

AT1 receptor antagonists, and can be compared with losartan. Additional studies revealed

that the magnitude of the vasorelaxant properties of compound 2 are dependent of the

structural framework of the losartan 1 as well as of the lateral chain, besides being able

of promoting the NO liberation [16]. These results indicated an evolution on the

therapeutic application of this new class of hybrid compounds in comparison to the

sartans, since the original antihypertensive properties were assured, added to the

benefits from the NO production in the cardiovascular system.

N

N

NH

NN

N

OH

Cl

H3C

PHN

N

NH

NN

N

OR

Cl

H3C

1

2ONO2

O

O

ONO2R=

R=

3

Fig 1

Cardiovascular accidents, such as arterial coronary occlusion and chronic myocardial

ischemia, can be treated by the use of thrombolytic agents or percutaneous transluminal


Page 48

Coronary angioplasty, which effectively re-establish the myocardial blood flow and

reduce mortality [20]. However, these therapeutic resources are not able of protecting the

heart from the damage caused by reactive oxygen species (ROS) produced by the re-

oxygenation of the blood after the reperfusion of the ischemic myocard. It is believed that

oxygen free radicals can react with myocardial phospholipids affecting the selective

permeability of the cellular membranes and leading to a development of ventricular

arrhythmias and/or fibrillation [21]. An attractive strategy to the discovery of new

derivatives able of acting on ischemia/reperfusion-related anomalies involves the design

of bifunctional agents that present both antioxidant and antiarrhythmic properties.

Exploring the therapeutic application of this concept, Koufaki and colleagues

synthesized the pharmacophore hybrids 7-16, planned from α-tocopherol (4), lidocaine

(5) and procainamide (6), able of acting as antioxidant/antiarrhythmic bifunctional

agents [22]. In spite of α-tocopherol (4) be an efficient free radicals scavenger agent its

efficacy in therapies of emergency reperfusion is limited by its lipidic nature. The target

hybrid compounds (7-16) were designed by the combination in one only molecule of the

6-hydroxychromane ring, responsible for the antioxidant activity of 4 and the

diethylamine-carboxamide sub-unity of 5 and 6 , two class I belonging antiarrhythmic

drugs (Fig. 2) [22].

Six hybrid compounds analog to the procainamide, i.e. compounds 7-12 , and four

analogs based on the lidocaine, i.e. compounds 13-16 were synthesized, and in both

series the contribution of the R alkyl side chain was evaluated, varying from 1 to 12

carbon atoms. The evaluation of the antioxidant properties demonstrated that the

lidocaine analog 15 was the most active one, inhibiting by 72.4 % the lipid per-

oxidation in the concentration of 5 µM, followed by the compounds 7 and 10, with 95.2

% and 38.4% inhibition, respectively, in the concentration of 10 µM. These results also

reveal that the increase of the length of the side chain contributed to decrease of the anti-

oxidant activity in each series. The evaluation of the antiarrhythmic effects demonstrated

that all hybrid compounds, in concentrations of 30 and 100 µM, increased the post-

ischemic recovery without induction of ventricular fibrillation. Among the tested

compounds, the procainamide analog 40 was the one responsible for the greater decrease

concerning the premature beating. The validity of the bifunctional strategy in the therapy


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of the myocardial hyperfusion injury was demonstrated by the compounds 7, 14 and 16

(Fig. 2), which showed to be strong inhibitors of lipid per-oxidation and were also

effective in inhibiting reperfusion-derived arrhythmias, when compared to the

antiarrhythmic profile of lidocaine (5) and procainamide (6). The comparison of the

results obtained for these new hybrid compounds evidenced that its antioxidant properties

did not present parallelism with the antiarrhythmic effects, as it was observed for 16,

which proved to be a less potent antioxidant agent than 15, but induced clearer

antiarrhythmic effects. Likewise, the compound 13 presented antiarrhythmic effects, but

minimum antioxidant activity [22].

Anti-tumoral Agents

In 1999, Kuduk and collaborators [23] proposed the synthesis of hybrids of the

geldanamycine (GDM, 17 ) with estradiol (18 , Fig. 3), aiming at obtaining molecules

capable of causing the specific degradation of proteins, such as ligands of estradiol

receptors (ER) and of the transaminase of the HER2 membrane, which are highly

expressed in several types of breast cancer. The appropriate intervention on these proteins

could lead to the delay of the cellular growing and/or apoptosis [24].

GDM (17) is a macrocyclic antibiotic isolated from Streptomycis hygroscopicus, which

belongs to the ansamicine class. The fusion between the structures of the estradiol (18)


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and GDM (17) should lead to new hybrid ligands that maintain the activities of both

original ligands. In order to do that, a way of gathering these two structural templates in

the hybrid targets 19-22 (Fig. 3) was investigated, exploring the C-16 position of the

estradiol, whose relative stereochemistry should be α to avoid steric effects over the

pharmacoforic hydroxyl group at C-17. In relation to the GDM (17), prior studies

revealed that the methoxy attached carbon of benzoquinone system could act as Michael

acceptor when facing amines or other bionucleophilic species, supporting the structural

design that considered the connection of a spacer unit presenting a terminal primary

amino group, stereoselectively placed at C-16 of the estradiol (18), with this electrophilic

site of GDM (17) (Fig. 3) [23].

Other anti-tumoral antibiotics produced by species of Streptomyces species, such as the

pyrrolobenzodiazepine derivatives (PBDs), antramycin (23) and DC-81 (54, Fig. 4), and


Page 51

the pyrrolocarbamoylic oligopeptide distamicine A (30, Fig. 5), can act as selective

ligands in minor groove of the DNA and have been used as prototypes for the design of

NCE’s.


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Analgesic, Anti-inflammatory and Antithrombotic agents

Hybrid drugs have been proposed as new alternatives for the regulation of dependency

and tolerance to opioid pharmaceuticals [25]. An interesting strategy consists of the

combination, in only one molecule, of the pharmacophorical sub-unities of opioid agents

and of ligands of the subtype I2 of the imidazoline receptor (IBS), aiming at a synergic

and controlled antinociceptive effect, considering the recent evidences demonstrating

that some I2-IBS ligands can attenuate the opioid tolerance [26].

Dardonville and colleagues [25] planned the synthesis of new opioid pharmacophore

hybrids with high affinity to I2-IBS, conceived from the structures of fentanyl (38), an

opioid agent, and agmatine (39), a ligand of I2-IBS receptor. The evaluation of the

bioprofile of this series lead to the initial identification of the prototype 40 (Fig. 6), which

presented high affinity to the µ-opioid receptor, but low affinity to I2-IBS receptor [26].

Previous SAR study involving a series of aliphatic bis-guanidine alkaloids demonstrated

that the increase of the methylenic bridge between the two basic sub-unities leads to an

increase in the I2-IBS affinity [25]. The rationalization of the spacer unit size between the

fentanyl framework and the guanidine moiety (Fig. 6), anticipated an eventual

improvement of the affinity of some homologues of derivative 40 for I2-IBS, generating

the series of fentanyl-guanidine hybrids (41-43).


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The binding affinity to the I2-IBS and µ-opioid receptors was comparatively evaluated for

the derivatives of the new series. The hybrid derivative 43 presenting the longer spacer

unit (n = 12) between the propanamide and guanidine moieties showed a significantly

increase in the affinity to I2-IBS receptors, compared to fentanyl and idazoxan,

confirming the correlation between selectivity and the increase in the spacer size. All new

ligands showed nanomolar affinities to µ-opioid receptor, being the compound 42

almost equipotent, while 43 showed the smaller affinities. The compound 41 (Fig. 6)

showed to be an excellent ligand of the µ-opioid receptor, with affinity comparable to the

fentanyl [25]. These results demonstrated that the incorporation of the pharmacophoric

fentanyl framework and the alkyl-guanidine unit, in the same molecule, bound by an

appropriate alkyl spacer led to a synergic effect for target receptors, I2-IBS and µ-opioid

[25].

Studies undertaken by Carlos Alberto Manssour Fraga research group led to the

identification of N-aryl-(A) and N-acylhydrazone-(B) (Fig. 7) sub-unities as important

pharmacophoric groups for the design of new analgesic, anti-inflammatory and anti-

thrombotic agents [27-31]. It is believed that the biological activities of these compounds

are associated to their relative acidity and to their capacity of stabilizing free radicals,

mimicking the bis-allyl fragment (C), (Fig. 7) present in some fatty acids (e.g.

arachidonic acid (44), (Fig. 7) and contributing to the inhibition of the active site of

oxidative catabolic enzymes, such as cyclooxygenases (COX) and 5-lipoxigenase (5-

LO), responsible for the biosynthesis of prostaglandins, thromboxanes and leukotrienes

[27, 28]. The synthesis of new chemical entities presenting different structural patterns

and including acyl- and aryl-hydrazone pharmacophoric sub-unities led to the discovery


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of several new prototypes, which have been utilized in the development of drug

candidates acting over enzymes involved in the modulation of the biosynthesis of

arachidonic acid (44) cascade metabolites [32].

The nitropyrazolyl-hydrazone derivative (45) was designed as a hybrid isoster of BW-

755c (46) and CBS-1108 (47), two dual inhibitors of COX and 5-LO. Structural

simplification of 45 generated a new series of N-phenylpyrazole-4-acylhydrazone

prototypes (48-49, Fig. 8), amongst which the derivative 49 demonstrated analgesic

activity 11.0 and 1.2 times superior to dipyrone (51), in acetylcholine and acetic acid

induced abdominal contortions assays, respectively. It is interesting to highlight that the

compounds 45, 46 and 48-50 are structurally related to dipyrone (51) [29].


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More recently, Carlos Alberto Manssour Fraga research group described another

successful example of application of the pharmacophore hybridization as a tool in the

designing of new modified N-acylhydrazone prototypes with powerful analgesic

properties [33]. In this work, Bezerra-Netto and colleagues related a new series of

nitrophenoxyacetylhydrazone derivatives 65-72 (Fig. 9) planned through the

pharmacophore hybridization of a previously described safrole-derived phenylpropionic

N-acylhydrazone series 62-64 [33] with nimesulide (61), an anti-inflammatory drug that

present selective COX-2 inhibition properties and radical scavenger behavior [34]. In this

context, the nitro group of 61 was introduced at C-6 position of the 1,3-benzodioxole

ring of 62-64 (Fig. 9) in order to investigate its internal ‘catalytic’ effect in the formation

of free radicals on the N-acylhydrazone moiety, intending to improve the radical

scavenger profile in these new compounds, aiming the optimization of its

pharmacological profile through the selective modulation of its redox properties. The

evaluation of the antinociceptive properties of this series led us to discover two new

potent prototypes of analgesic and antipyretic agents, represented by the N-

acylhydrazone derivatives 65 and 67, which showed to be more potent than dipyrone

used as standard for both investigated activities [33,35].

Anti-infectious Agents


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After some years of lack of concern, the pharmaceutical industries have been showing

some interest again for the development of new anti-infectious agents. This strategic

change can be quantified by the great investments in research that culminated in more

than 50 new antibacterial drugs recently approved in the USA. Besides that, the

appearing of new diseases and the growing consumer market in countries of young

population has caused the recovery of research on anti-infectious agents, as well as

cancer, diabetes, arthritis, cardiovascular diseases and neuropathies [36]. Currently, one

of the biggest challenges in the anti-infectious therapy, especially the bacterial and

parasitary ones is the speed in which the microorganisms (MOs) suffer genetic mutations,

modifying their defense system and generating new resistant strains to the drugs

clinically used. Moreover, bacterial infections are responsible for multiple complications

in immune-suppressed patients with chronic diseases, cancer, transplanted and AIDS

(Acquired Immunodeficiency Syndrome) [36].

In this context, pharmaceutical companies have concluded that the best way to fight

against bacterial infections would be through new therapeutic targets and that, in the

case of a crisis in public health because of the development of multi-resistant bacteria,

the regulation agencies, such as Food & Drug Administration (FDA), should be faster

in the approval of new drugs,

H3C

H2C

CH2

H2C

CCH3

CH2

H3C

H2C

CH2

H2C

C

H2C

CH2

CH3NH2-peptiden n

fatty acidn = 2, X = AcNH-, NH2n = 7, X = CH3

73

Fig. 10

Contributing to the decrease of the development costs. The intensification of the efforts in

expanding the therapeutic arsenal has led many pharmaceutical industries to the

improvement of known chemotherapeutic classes such as penicillins, tetracyclines,

cyclosporines and glycopeptides, as well as the discovery of completely novel classes

such as the oxazolidinones. Another strategy adopted has been the investigation of new


Page 57

mechanisms of action on the metabolism and proliferation of MOs, since the antibacterial

agents currently in use act interfering in the protein biosynthesis, in DNA or in the

structure of the cellular wall [36].

In this scenario, in which the search for new therapeutic classes has become urgent and

continuous, demanding innovation, agility in the development and great sums of

investment from the industries and research centers, the pharmacophore hybridization can

be an important tool in the conception of new molecular patterns able of generating new

efficient and selective anti-infectious drugs.

This strategy was utilized by Oh and co-workers [37] in the development of new lipid-

peptide hybrid compounds (73) planned from natural peptides and fatty acids (Fig. 11).

The obtained results with this class of derivatives were not conclusive, indicating only


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that the introduction of long chains from fatty acids led to the loss of antibacterial

activity, when these chains did not present a NH2 terminal substituent [37].

In another work, Zhi and colleagues [38] explored the skeleton of 3-substituted 6-

anilinouracils, such as HB-EMAU (74), for the design of inhibitors of DNA-polymerase

IIIC in gram-positive bacteria. HB-EMAU was later hybridized with a series of

fluoroquinolones (FQs) with recognized activity against gram positive bacteria, including

norfloxacin (75), ciprofloxacin (76), sparfloxacin (77) and temafloxacin (78) (Fig. 11),

generating the new hybrid molecules AU-FQ (74-A).

In this family, with 18 new hybrids containing a fluoroquinolone sub-unity as substituent

in the N-3 position, all the synthesized compounds showed to be potent inhibitors of

polymerase IIIC (pol IIIC) of Bacillus subtilis. The hybrids AU-FQ also showed

significant values of minimum inhibitory concentration (MIC= 0.16-1.25 µg/mL) after

screening against naive and resistant strains of S. aureus, Enterococcus faecalis and E.

Faecium [38]. Hybrid AU-FQ derivatives (Fig. 11) were ca. 2-4 times more potent than

the prototype 74, but less potent than the fluoroquinolone derivatives 75-78. In vivo

essays evaluating S. aureus-infected mice lethally demonstrated the complete protective

effect of the hybrids AU-FQ (74-A) in sub-toxic doses. Particularly the compound 81

also demonstrated the ability to face other gram-positive strains, motivating its selection

for further complementary studies [38,39].

Tropical diseases viz. tuberculosis and malaria also represent an important challenge

under the public health point of view, since, generally, they are endemic pathologies of

either poor or developing countries, which are consequently neglected by the global

pharmaceutical companies. Among these, Chagas’ disease affects about 17 million

people worldwide and it is caused by the hemoflagellate protozoan Trypanosoma cruzi

[39]. In 1968, Berkelhammer and Asato [40] synthesized a 5-nitroimidazole derivative

named megazol (84, Fig. 12) as an antimicrobial agent, which was later characterized by

the Brener group [41] as a potent trypanocide agent (IC50 = 9.9 µM), despite present

important toxicity [42]. Based on these data, Carvalho and co-workers [43] synthesized

two series of hybrids 86 and 87 designed through pharmacophore hybridization of the


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prototype megazol (84) and the guanylhydrazone derivative 86, which also presented

trypanocide activity [44].

The hybrid compound 86 showed to be the most active trypanocide (IC50 = 5.3 µM) of

the two hydrazone series. The simplified analogue 87 (Fig. 12) did not present significant

activity (IC50 = 63.4 µM), demonstrating the pharmacophoric contribution of the

nitroimidazole group in the mechanism of action against the T. cruzi [43].


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2.2 Present Work

Drug discovery and development is a very laborious and costly process involving

synthesis and screening of diverse organic compounds. In this regard, multicomponent

reactions (MCRs) are of increasing importance in the field of medicinal chemistry.

Currently, attention is put on speed, diversity, and efficiency in the drug discovery

process. MCRs can provide products with the diversity needed for the discovery of new

lead compounds or lead optimization employing combinatorial chemistry techniques. The

search and discovery for new MCRs on one hand, and the full exploitation of already

known MCRs on the other hand, are therefore of considerable current interest. The scope

of this reaction was gradually extended by the variation of all three building blocks,

allowing access to a large number of multi-functionalized di or tetrahydropyrimidines of

medicinal use.

Di- or tetra-hydropyrimidines showed a diverse range of biological activities. They are

known to possess activities such as antibacterial, antiviral, anticancer, analgesic and anti-

inflammatory as well as efficacy as calcium channel modulators and α1a-antagonists.

Furthermore, certain compounds bearing 1H-indole-2,3-dione nucleus is used as a

versatile lead molecule for designing potential antiviral, antitubercular, anticonvulsant

and anti-tumor therapeutic activities. While, imine bases of isatin (indoline-2,3-dione)

and its derivatives were reported for antibacterial, antifungal, anti-HIV, anticonvulsant

activities and GAL3 receptor antagonists.

Prompted by the biological properties of dihydropyrimidines and 1,3-dihydro-2H-indol-

2-ones nucleus, they were incorporated in one single molecule using principle of

pharmacophore hybridization and schiff bases were synthesized. This type of effort, in

the design of new drug entity, i.e. the development of hybrid molecules through the

combination of different pharmacophores in one frame may lead to compounds with

interesting dual biological profiles, which is being reflected in present work.

Thus, this section deals with design, synthesis of Tetrahydropyrimidine–isatin hybridized

derivatives and In-vitro evaluation of Antibacterial, Antifungal, Antitubercular and

Antimalarial activities.


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Section I:Section I:Section I:Section I:

Pharmacophore hybridization: Tetrahydropyrimidine–i satin

hybridized derivatives: Synthesis and In vitro evaluation of

Antibacterial, Antifungal, Antitubercular and Antim alarial

activities.

Dihydropyrimidin-2-one (DHPM)

In 1893, Pietro Biginelli has reported on the acid-catalyzed cyclocondensation reaction of

ethylacetoacetate, benzaldehyde and urea. The reaction was carried out by simply heating

a mixture of the three components dissolved in ethanol with a catalytic amount of Cons.

HCl at reflux temperature. The product of this novel one-pot, three-component synthesis

that precipitated on cooling the reaction mixture was identified correctly by Biginelli as

dihydropyrimidin-2-one (DHPM) [45a]. The scope of this reaction was gradually

extended by the variation of all three building blocks, allowing access to a large number

of multi-functionalized dihydropyrimidines of medicinal use [45b, 46-49].

Lewis acid Protic acid Ionic liquids Boron compounds TMSCl Tangstophosphoric acid Zeolite Conc. HCl

CaCl2 Montmorillonite Ion-exchange resins silica sulfuric acid PPE L-proline

Tetrahydropyrimidinone

(THPM)

Fig. 13 Effective catalysts for synthesis of tetrahydropyrimidinone heterocycle


Page 62

Two different approaches have been employed in recent years to synthesize DHPM

derivatives. The first method relies on the traditional Biginelli three component protocol

and involves the acid catalyzed cyclocondensation of a β-carbonyl component, with an

aromatic aldehyde and urea or thiourea derivative (Fig. 13). A major drawback of the

original Biginelli protocols, using ethanol and catalytic HCl as reaction medium, has been

the low yields that were frequently encountered when using sterically more demanding

aldehydes or thioureas [50]. In recent years, these problems have been largely overcome

by the development of improved and more robust reaction conditions, involving e.g.

Lewis acid catalyst as well as protic acid under classical reflux [51]. Other studies have

focused on the use of ionic liquids [52], microwave irradiation [53], combinatorial

techniques [54], use of boron compounds [55], TMSCl [56] and heterogeneous catalysts

viz. as tangstophosphoric acid [57], zeolite [58], montmorillonite [59], ion-exchange

resins [60], and the also use of silica sulfuric acid [61], PPE [62], L-proline [63] etc.

These, Di or tetrahydropyrimidines show a diverse range of biological activities. They are

known to possess activities such as antiviral [64], anticancer [65], antibacterial [66],

analgesic and antiinflammatory [66], antioxidant [67], HIV-1 replication [68], as well as

efficacy as calcium channel modulators and α1a-antagonists [64] (Fig. 14). Thus


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development of methodologies for efficient lead structure identification and for

pharmacophore variation of dihydropyrimidines motif has always attracted the attention

of pharmaceutical industry [69].

Furthermore, certain compounds bearing 1H-indole-2,3-dione nucleus is used as a

versatile lead molecule for designing potential antivirals [70], antimalarial [71],

antituberculars [72], anticonvulsants [73], progesterone receptor modulators [74] and

anti-tumor therapeutic activities [75]. While, Schiff bases of 1H-indole-2,3-dione and its

derivatives were reported for antibacterial [76], antifungal [76], anti-HIV [77],

anticonvulsant activities [78], anti-poxvirus agents [78] and GAL3 receptor antagonists

[79] (Fig. 15). Prompted by the biological properties of dihydropyrimidines and 1H-

indole-2,3-dione nucleus, they were incorporated with oxadiazole/thiadiazoles [80] and

Schiff bases were synthesized.

Thus, using the principle of pharmcophore hybridization, we have design of

hybrid molecules “5-substituted-2-({5-(6-methyl-2-oxo/thioxo-4-(phenyl/furan-2-yl)-

1,2,3,4-tetrahydro pyrimidin-5-yl)-(1,3,4-thiadiazol/1,3,4-oxadiazol)-2-yl}imino)-1,2-

dihydro-3H indol-3-ones, by combining tetrahydropyrimidinones nucleus with 1H-

indole-2,3-dione in one frame, which may lead to compounds with interesting dual


Page 64

biological profiles. The results of such studies are discussed in this Chapter and synthetic

pathway leading to the title compounds 5a-l, 6a-l and 7a-l is given in Scheme 1.

In first step, we report an efficient, practical, environmentally benign and high yielding

method for Biginelli’s three component, one–pot synthesis of tetrahydropyrimidinones

using CaCl2 [81] as catalyst for preparation of basic pharmacophores (Fig. 16). And

thereafter, via heterocyclization and condensation reactions respectively, the desired

products are obtained as follows (Scheme 1).

2.3 Experimental:

� All the reagents were obtained commercially and used with further purification.

Solvents used were of analytical grade.

� All melting points were taken in open capillaries and are uncorrected.

� Elemental analysis (% C, H, N) was carried out by Perkin-Elmer 2400 series-II

elemental analyzer and all compounds are within ±0.4% of theory specified.

� The IR spectra were recorded on a Shimadzu FTIR 8401 spectrophotometer using

KBr discs and only the characteristic peaks are reported in cm-1.

� 1H NMR and 13C NMR spectra were recorded in DMSO and CDCl3 on a Bruker

Avance 400 MHz spectrometer using solvent peak as internal standard. Chemical

shifts are reported in parts per million (ppm).


Page 65

� Mass spectra were scanned on a Shimadzu LCMS 2010 spectrometer. Mode of

ionization employed was ESI (electrospray ionization).

Experimental Scheme:

Entry X= R’= Entry X= R’=

1a, 2a, 3b, 3d, 5a-5f, 7g-7l.

S

4b, 6g-6l S

1b, 2b, 3a, 3c, 5g-5l, 7a-7f.

O

4a, 6a-6f O


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Chemistry:

Synthesis of ethyl 6-methyl-2-(oxo/thioxo)-4-(phenyl/furan-2yl)-1,2,3,4-

tetrahydropyrimidine-5-carboxylate (1a-b):

As per reported procedure [81] Urea/thiourea (0.5 mol), ethylacetoacetate (0.75 mol) and

benzaldehyde/furfural (0.75 mol) were mixed in ethanol (25 ml). Catalytic amount of

CaCl2 (0.020 mol) was added to the reaction mixture and refluxed for 2 hr. Completion of

reaction mass monitored TLC. White/yellowish precipitates were obtained. For workup,

reaction mass was slowly poured on crushed ice and product obtained as white/cream

precipitates, which was filtered under vacuum and washed with cold water, dried in hot

air oven. Pure product (single spot on TLC) obtained as white/cream solid powder, was

crystallized by methanol: water (60:40). Analytical data was compared with reported

literature [82].

Synthesis of 6-methyl-2-(oxo/thioxo)-4-(phenyl/furan-2yl)-1,2,3,4-

tetrahydropyrimidine-5-carbohydrazide (2a-d):

1a/1b (0.01 mol), ethanol (20 ml), hydrazine hydrate (0.01 mol) was added followed by

the addition of a catalytic amount of conc. H2SO4 (4-5 drops) and allowed to stir for 3 hr

at 75°C. Yellowish precipitates were obtained during reflux, (monitored by TLC) In

workup process, reaction mass was allowed to cool to room temperature and poured on

crushed ice, product obtained as yellowish precipitates was filtered under vacuums and

dried in hot air oven. Product obtained as yellowish solid powder, crystallized by ethanol.

Characterization of selected compound is given below:

6-methyl-2-thioxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide (2a):

Yield 85%, M.p., 195 ºC. MS: m/z [263.45]+; 1H NMR : 1.89, 4.87, 8.93, 11.96 (4H, s, -

NH-), 2.30 (3H, s, -CH3), 5.46 (1H, s, -CH=), 7.20-7.42 (5H, m, aromatic). 13C NMR:

176.29, 167.42, 162.12, 110.34-148.13 (Ar-C), 60.40, 17.60. Anal. Calcd. for

C12H14N4OS : C, 54.94; H, 5.38; N, 21.36. Found: C, 54.71; H, 5.01; N, 21.00%.

6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide (2b):

Yield 82%, M.p., 190 ºC. MS: m/z [247.05]+; 1H NMR: 2.37 (3H, s, -CH3), 4.80 (2H, s, -

NH2), 5.40 (1H, s,-CH=), 6.51, 8.90 (2H, s, -NH-), 7.11-7.39 (5H, m, aromatic). 13C

NMR: 167.56, 154.73, 152.62, 115.10-146.90 (Ar-C), 58.87, 17.90. Anal. Calcd. for

C12H14N4O2 : C, 58.53; H, 5.73; N, 22.75. Found: C, 58.21; H, 5.52; N, 22.39%.


Page 67

Synthesis of 5-(5-amino-1,3,4-thiadiazol-2-yl)-4-(phenyl/furan-2-yl)-6-methyl-3,4-

dihydropyrimidin-2(1H)-one/thione (3a-d; 4a-b):

Title compounds 3a-d, 4a-b are obtained using reported procedure using 1N HCl,

ammonium thiocynate and conc. H2SO4 [83] and Title compound 3a-d, 4a-b are obtained

using reported procedure using CNBr [84].

Characterization of selected compound is given below:

5-(5-amino-1,3,4-thiadiazol-2-yl)-4-(phenyl)-6-methyl-3,4-dihydropyrimidin-2(1H)-one

(3a):

Yield 72%, M.p., 165-167 ºC. MS: m/z [288.05]+; 1H NMR: 1.98 (3H, s, -CH3), 5.65

(1H, s, -CH=), 5.90, 7.56, 8.90 (4H, s, -NH-), 6.99-7.36 (4H, m, aromatic). 13C NMR:

170.16, 162.28, 157.70, 109.12-145.06 (Ar-C), 17.20. Anal. Calcd. for C13H13N5OS : C,

54.34; H, 4.56; N, 24.37; Found: C, 54.28; H, 4.21; N, 24.07%.

5-(5-amino-1,3,4-thiadiazol-2-yl)-6-methyl-4-phenyl-3,4-dihydropyrimidine-2(1H)-

thione (3b):

Yield 65%, M.p., 150-152 ºC. MS: m/z [304.15]+; 1H NMR: 1.90, 6.96, 11.50 (4H, s, -

NH-), 2.16 (3H, s, -CH3), 5.25 (1H, s,-CH=), 7.05-7.40 (4H, m, aromatic). 13C NMR:

173.52, 160.17, 157.30, 110.56-141.08 (Ar-C), 16.80. Anal. Calcd. for C13H13N5S2 : C,

51.46; H, 4.32; N, 23.08; Found: C, 51.12; H, 3.91; N, 22.74%.

5-(5-amino-1,3,4-thiadiazol-2-yl)-4-(furan-2-yl)-6-methyl-3,4-dihydropyrimidine-

2(1H)-thione (4a):

Yield 72%, M.p., 210-211 oC, MS: m/z [272.36]+, 1H NMR: δ 8.36, 6.52 (s, 2H, -NH-),

7.42-7.94 (m, 5H, Ar-H), 5.27 (s, 1H, -CH=), 2.37 (s, 3H, -CH3). 13C NMR: δ 161.45,

154.13, 152.74, 142.43, 109.21-136.82 (Ar-C), 111.17, 54.25, 13.98. Anal. Calcd. for

C13H13N5O2: C, 57.56; H, 4.83; N, 25.82. Found: C, 57.17; H, 4.48; N, 25.58%.

5-(5-amino-1,3,4-thiadiazol-2-yl)-4-(furan-2-yl)-6-methyl-3,4-dihydropyrimidin-2(1H)-

one (4b):

Yield 79%, Mp., 222-223 oC, MS: m/z [288.57]+, 1H NMR: δ 8.36 (s, 2H, -NH-), 7.12-

7.57 (m, 5H, Ar-H), 5.46 (s, 1H, -CH=), 2.18 (s, 3H, -CH3). 13C NMR: δ 173.17, 155.23,

153.87, 142.68, 110.29-139.13 (Ar-C), 113.37, 55.76, 14.39. Anal. Calcd. for

C13H13N5OS: C, 54.34; H, 4.56; N, 24.37. Found: C, 53.89; H, 3.93; N, 23.76%.


Page 68

Synthesis of 5-substituted-2-({5-(6-methyl-2-oxo/thioxo-4-(phenyl/furan-2-yl)-1,2,3,4-

tetrahydro pyrimidin-5-yl)-1,3,4-thiadiazol-2-yl}imino)-1,2-dihydro-3H indol-3-one

(5a-l; 6a-l; 7a-l):

Compound 3a-d, 4a-b (0.01 mol) and 5-substituted indoline-2,3-dione (0.01 mol) were

dissolved in methanol (10 mL) in presence of catalytic amount of glacial acetic acid (2-3

drops) and reflux for 45 mins. Final product was obtained was filtered it under vacuum,

washed with cold ether (20-30ml), dried in hot air oven and purified by crystallization in

THF (tetrahydrofuran)-Water (60:40). The compounds 5a-l; 6a-l; 7a-l were prepared in

the same fashion using appropriate 5-substituted indoline-2,3-dione and 3a-d, 4a-b.

Characterization data is given below:

5a: 3-{[5-(6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-1,3-dihydro-2H-indol-2-one.

Yield: 70 % Mol. Wt.: 432.08

M.p.: 210-212 °C MS: (M+) 432.52

Elemental Analysis: Calcd: C, 58.31; H, 3.73; N, 19.43% Found: C, 58.03; H, 3.22; N, 18.90%.

Mol. For.: C21H16N6OS2

1H NMR: 400MHz, δ, ppm 13.76, 8.0, 2.0 (1H×3, s, NH), 7.86-7.23 (9H, m, Ar-H), 4.59 (1H, s, -CH=), 2.26 (3H, s, CH3).

13C NMR: 100MHz, δ, ppm 173.70, 164.90, 158.27, 156.60, 150.30, 143.22, 140.60, 119.20-132.15 (Ar-C), 117.11, 62.92, 16.74.

IR: νmax, cm-1, KBr 3331, 3167 (NH), 1724 (C=O), 1633 (C=N, iminebase), 1660 (C=S), 1377 (C=N), 1273, 1185 (C-S-C).


Page 69

5b: 5-bromo-3-{[5-(6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-1,3-dihydro-2H-indol-2-one

Yield: 72 % Mol. Wt.: 511.42

M.p.: 199-200 °C MS: (M+) 511.99

Elemental Analysis: Calcd: C, 49.32; H, 2.96; N, 16.43. Found: C, 49.12; H, 3.74; N, 16.08%.

Mol. For.: C21H15BrN6OS2


13C NMR: 100MHz, δ, ppm 173.65, 162.30, 158.35, 156.90, 149.90, 143.72, 140.10, 117.60-133.15 (Ar-C), 63.92, 16.94.

IR: νmax, cm-1, KBr 3344, 3167 (NH), 1739 (C=O), 1366 (C=N), 1283, 1185 (C-S-C), 1658 (C=S), 1643 (C=N, iminebase), 610 (C-Br)

5c: 3-{[5-(6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-5-nitro-1,3-dihydro-2H-indol-2-one

Yield: 74 % Mol. Wt.: 477.52

M.p.: 202-204 °C MS: (M+) 477.07


Mol. For.: C21H15N7O3S2


13C NMR: 100MHz, δ, ppm 174.15, 164.20, 159.12, 155.63, 150.55, 147.95, 122.50-144.15 (Ar-C), 143.34, 117.95, 65.35, 16.80.

IR: νmax, cm-1, KBr 3334, 3177 (NH), 1729 (C=O), 1668 (C=S), 1623 (C=N, iminebase), 1574 (N=O), 1283, 1195 (C-S-C), 1371 (C=N).


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5d: 5-fluoro-3-{[5-(6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-1,3-dihydro-2H-indol-2-one

Yield: 78 % Mol. Wt.: 550.51

M.p.: 205-206 °C MS: (M+) 550.07


Mol. For.: C21H15FN6OS2


13C NMR: 100MHz, δ, ppm 174.15, 164.25, 157.98, 155.23, 153.95, 150.13, 145.33, 111.06-142.68 (Ar-C), 65.05, 16.30.

IR: νmax, cm-1, KBr 3345, 3187 (NH), 1734 (C=O), 1658 (C=S), 1623 (C=N, iminebase),1371 (C=N), 1321 (C-F), 1268, 1205(C-S-C).

5e: 5-iodo-3-{[5-(6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-1,3-dihydro-2H-indol-2-one

Yield: 70 % Mol. Wt.: 558.42

M.p.: 215-218 °C MS: (M+) 557.98


Mol. For.: C21H15IN6OS2


13C NMR: 100MHz, δ, ppm 173.85, 163.35, 158.46, 155.30, 150.53, 143.33, 140.95, 91.66-138.18 (Ar-C), 59.95, 15.60.

IR: νmax, cm-1, KBr 3334, 3163 (NH), 1739 (C=O), 1668 (C=S), 1633 (C=N, iminebase), 1370 (C=N), 1264, 1185 (C-S-C), 550 (C-I).


Page 71

5f: 5-chloro-3-{[5-(6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-1,3-dihydro-2H-indol-2-one

Yield: 70 % Mol. Wt.: 466.97

M.p.: 210-212 °C MS: (M+) 466.04


Mol. For.: C21H15ClN6OS2


13C NMR: 100MHz, δ, ppm 174.10, 164.60, 159.10, 155.48, 150.40, 143.10, 141.30, 92.00-137.05 (Ar-C), 59.90, 16.55.

IR: νmax, cm-1, KBr 3350, 3177 (NH), 1735 (C=O), 1643 (C=N, iminebase), 1659 (C=S), 1378 (C=N), 1267, 1205 (C-S-C), 715 (C-Cl).

5g: 3-{[5-(6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-1,3-dihydro-2H-indol-2-one

Yield: 74 % Mol. Wt.: 416.46

M.p.: 214-216 °C MS: (M+) 416.11


Mol. For.: C21H16N6O2S

1H NMR: 400MHz, δ, ppm 8.0, 6.1 (1H×3, s, NH), 7.25-7.89 (9H, m, Ar-H), 4.57 (1H, s, -CH=), 2.28 (3H, s, CH3).

13C NMR: 100MHz, δ, ppm 162.90, 157.85, 155.20, 152.05, 151.30, 142.72, 141.60, 120.10-131.15 (Ar-C), 60.92, 15.74.

IR: νmax, cm-1, KBr 3334, 2912 (NH), 1701, 1729 (C=O), 1623 (C=N, iminebase), 1371 (C=N), 1283, 1195 (C-S-C).


Page 72

5h: 5-bromo-3-{[5-(6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-1,3-dihydro-2H-indol-2-one

Yield: 79 % Mol. Wt.: 495.35

M.p.: 250-252 °C MS: (M+) 494.02


Mol. For.: C21H15BrN6O2S


13C NMR: 100MHz, δ, ppm 163.40, 158.52, 155.80, 152.90, 151.45, 142.68, 141.22, 118.40-133.95 (Ar-C), 60.15, 15.10.

IR: νmax, cm-1, KBr 1712 , 1724 (C=O), 1633 (C=N, iminebase), 1263, 1205 (C-S-C), 1377 (C=N), 630 (C-Br).

5i: 3-{[5-(6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-5-nitro-1,3-dihydro-2H-indol-2-one

Yield: 75 % Mol. Wt.: 461.45

M.p.: 253-254 °C MS: (M+) 461.09




13C NMR: 100MHz, δ, ppm 163.50, 158.68, 155.90, 151.35, 150.05, 148.95, 123.76-144.58 (Ar-C), 117.60, 58.95, 15.00.

IR: νmax, cm-1, KBr 3318, 2945 (NH), 1720, 1749 (C=O), 1630 (C=N, iminebase), 1574 (N=O), 1368 (C=N), 1273, 1175 (C-S-C).


Page 73

5j: 5-fluoro-3-{[5-(6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-1,3-dihydro-2H-indol-2-one

Yield: 78 % Mol. Wt.: 434.35

M.p.: 214-216 °C MS: (M+) 435.10

Elemental Analysis: Calcd: C, 58.06; H, 3.48; N, 19.34. Found: 57.86; H, 3.26; N, 19.03%.

Mol. For.: C21H15FN6O2S


13C NMR: 100MHz, δ, ppm 163.58, 158.48, 155.37, 154.60, 151.43, 150.28, 110.18-146.88 (Ar-C), 57.05, 15.85.

IR: νmax, cm-1, KBr 3244, 2922 (NH), 1724, 1701 (C=O), 1647 (C=N, iminebase), 1368 (C=N), 1313 (C-F), 1290, 1222 (C-S-C).

5k: 5-iodo-3-{[5-(6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-1,3-dihydro-2H-indol-2-one

Yield: 70 % Mol. Wt.: 542.35

M.p.: 218-220 °C MS: (M+) 542.45


Mol. For.: C21H15IN6O2S

1H NMR: 400MHz, δ, ppm 8.1, 6.1 (1H×3, s, NH), 7.68-8.08 (3H, m, aromatic fused ring), 7.24-7.37 (5H, m, Ar-H), 4.55 (1H, s, -CH=), 2.26 (3H, s, CH3).

13C NMR: 100MHz, δ, ppm 173.85, 163.55, 158.90, 155.28, 150.10, 143.52, 141.42, 91.80-137.58 (Ar-C), 58.70, 16.15

IR: νmax, cm-1, KBr 3332, 2922 (NH), 1729, 1749 (C=O), 1643 (C=N, iminebase), 1371 (C=N), 1293, 1176 (C-S-C), 556 (C-I).


Page 74

5l: 5-chloro-3-{[5-(6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-1,3-dihydro-2H-indol-2-one

Yield: 71 % Mol. Wt.: 542.35

M.p.: 225-227 °C MS: (M+) 543.07


Mol. For.: C21H15ClN6O2S


13C NMR: 100MHz, δ, ppm 164.88, 157.98, 155.28, 151.90, 150.70, 143.30, 141.10, 91.80-137.18 (Ar-C), 59.05, 15.05.

IR: νmax, cm-1, KBr 3334, 2936 (NH), 1730, 1749 (C=O), 1619 (C=N, iminebase), 1359 (C=N), 1276, 1195 (C-S-C),710 (C-Cl).

6a: 3-(5-(4-(furan-2-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-ylimino)-1,3-dihydro-indol-2-one

Yield: 75 % Mol. Wt.: 406.12

M.p.: 202-203 °C MS: (M+) 407.37



1H NMR: 400MHz, δ, ppm 8.42, 6.58 (1H×3, s, -NH-), 7.17-7.83 (m, 7H, Ar-H), 2.32 (s, 3H, -CH3), 4.41 (s, 1H, -CH=).

13C NMR: 100MHz, δ, ppm 170.52, 167.41, 158.74, 153.42, 151.63, 95.72-146.41 (Ar-C), 58.79, 15.82.

IR: νmax, cm-1, KBr 3358, 3173 (N-H), 1725 (C=O), 1634 (C=N, Iminebase), 1372 (C=N), 1256, 1217 (C-S-C), 1011 (C-O-C).


Page 75

6b: 5-bromo-3-(5-(4-(furan-2-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-ylimino)-1,3-dihydro-indol-2-one

Yield: 78 % Mol. Wt.: 485.05

M.p.: 231-232 °C MS: (M+) 486.14




13C NMR: 100MHz, δ, ppm 170.52, 167.41, 158.74, 153.42, 151.63, 95.72-146.41 (Ar-C), 58.79, 15.82.

IR: νmax, cm-1, KBr 3362, 3161 (N-H), 1724 (C=O), 1638 (C=N, Iminebase), 1376 (C=N), 1252, 1211(C-S-C), 1027(C-O-C), 617 (C-Br).

6c: 5-nitro-3-(5-(4-(furan-2-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-ylimino)-1,3-dihydro-indol-2-one

Yield: 73 % Mol. Wt.: 451.45

M.p.: 205-206 °C MS: (M+) 452.10




13C NMR: 100MHz, δ, ppm 171.21, 168.42, 158.31, 153.73, 152.46, 96.05-145.32 (Ar-C), 57.99, 15.37.

IR: νmax, cm-1, KBr 3344, 3165 (N-H), 1725 (C=O), 1633 (C=N, Iminebase), 1572 (N=O), 1379 (C=N), 1243, 1223 (C-S-C), 1029 (C-O-C).


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6d: 5-fluoro-3-(5-(4-(furan-2-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-ylimino)-1,3-dihydro-indol-2-one

Yield: 80 % Mol. Wt.: 424.41

M.p.: 261-262 °C MS: (M+) 425.65




13C NMR: 100MHz, δ, ppm 169.26, 166.10, 157.51, 152.68, 150.81, 96.79-146.89 (Ar-C), 59.16, 16.26.

IR: νmax, cm-1, KBr 3365, 3127 (N-H), 1715 (C=O), 1630 (C=N, Iminebase), 1319 (C-F), 1348 (C=N), 1256, 1222 (C-S-C), 1031 (C-O-C).

6e: 5-iodo-3-(5-(4-(furan-2-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-ylimino)-1,3-dihydro-indol-2-one

Yield: 69 % Mol. Wt.: 532.31

M.p.: 199-201 °C MS: (M+) 533.03




13C NMR: 100MHz, δ, ppm 172.48, 169.42, 159.52, 153.58, 151.43, 95.58-148.32 (Ar-C), 59.47, 17.36.

IR: νmax, cm-1, KBr 3338, 3139 (N-H), 1736 (C=O), 1629 (C=N, Iminebase), 1349 (C=N), 1248, 1228 (C-S-C), 1022 (C-O-C), 545 (C-I).


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6f: 5-chloro-3-(5-(4-(furan-2-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-ylimino)-1,3-dihydro-indol-2-one

Yield: 87 % Mol. Wt.: 440.58

M.p.: 217-219 °C MS: (M+) 441.72




13C NMR: 100MHz, δ, ppm 170.14, 167.82, 159.14, 153.56, 151.37, 95.60-147.75 (Ar-C), 58.62, 15.36.

IR: νmax, cm-1, KBr 3371, 3189 (N-H), 1712 (C=O), 1631 (C=N, Iminebase), 1375 (C=N), 1252, 1216 (C-S-C), 1024 (C-O-C), 710 (C-Cl).

6g: 3-(5-(4-(furan-2-yl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-ylimino)indolin-2-one

Yield: 66 % Mol. Wt.: 422.48

M.p.: 189-190 °C MS: (M+) 423.62

Elemental Analysis: Calcd: C, 54.01; H, 3.34; N, 19.89. Found: C, 53.61; H, 2.83; N, 19.69 %.


1H NMR: 400MHz, δ, ppm 11.83, 8.42, 6.58 (1H×3, s, -NH-), 7.50-8.21 (m, 7H, Ar-H), 2.46 (s, 3H, -CH3), 4.33 (s, 1H, -CH=).

13C NMR: 100MHz, δ, ppm 172.71, 167.83, 158.46, 154.25, 152.59, 96.31-147.12 (Ar-C), 59.80, 15.50.

IR: νmax, cm-1, KBr 3351, 3158 (N-H), 1722 (C=O), 1661 (C=S), 1637 (C=N, Iminebase), 1361 (C=N), 1258, 1219 (C-S-C), 1015 (C-O-C).


Page 78

6h: 5-bromo-3-(5-(4-(furan-2-yl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-ylimino)-1,3-dihydro-indol-2-one

Yield: 78 % Mol. Wt.: 501.38

M.p.: 231-232 °C MS: (M+) 502.27

Elemental Analysis: Calcd: C, 45.52; H, 2.61; N, 16.76, Found: C, 45.18; H, 2.24; N, 16.03 %.

Mol. For.: C19H13BrN6O2S2


13C NMR: 100MHz, δ, ppm 172.41, 167.43, 158.51, 153.18, 151.95, 95.92-146.78 (Ar-C), 59.18, 15.69.

IR: νmax, cm-1, KBr 3355, 3139 (N-H), 1728 (C=O), 1668 (C=S), 1641 (C=N, Iminebase), 1389 (C=N), 1247, 1219 (C-S-C), 1028 (C-O-C), 610 (C-Br).

6i: 5-nitro-3-(5-(4-(furan-2-yl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-ylimino)-1,3-dihydro-indol-2-one

Yield: 70 % Mol. Wt.: 467.48

M.p.: 225-226 °C MS: (M+) 468.51

Elemental Analysis: Calcd: C, 48.82; H, 2.80; N, 20.97, Found: C, 48.51; H, 2.69; N, 20.72 %.



13C NMR: 100MHz, δ, ppm 171.68, 168.22, 158.39, 154.59, 152.15, 95.65-145.39 (Ar-C), 58.05, 16.88.

IR: νmax, cm-1, KBr 3345, 3161 (N-H), 1752 (C=O), 1657 (C=S), 1638 (C=N, Iminebase), 1582 (N=O), 1372 (C=N), 1259, 1231 (C-S-C), 1021 (C-O-C).


Page 79

6j: 5-fluoro-3-(5-(4-(furan-2-yl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-ylimino)-1,3-dihydro-indol-2-one

Yield: 64 % Mol. Wt.: 440.05

M.p.: 276-277 °C MS: (M+) 441.69


Mol. For.: C19H13FN6O2S2


13C NMR: 100MHz, δ, ppm 169.47, 166.42, 157.58, 151.57, 149.34, 95.87-146.12 (Ar-C), 59.15, 17.21.

IR: νmax, cm-1, KBr 3365, 3127 (N-H), 1720 (C=O), 1660 (C=S), 1635 (C=N, Iminebase), 1321 (C-F), 1343 (C=N), 1257, 1221 (C-S-C), 1039 (C-O-C).

6k: 5-iodo-3-(5-(4-(furan-2-yl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-ylimino)-1,3-dihydro-indol-2-one

Yield: 58 % Mol. Wt.: 548.38

M.p.: 182-183 °C MS: (M+) 549.48


Mol. For.: C19H13IN6O2S2


13C NMR: 100MHz, δ, ppm 171.23, 169.64, 158.71, 153.12, 151.63, 95.42-149.14 (Ar-C), 60.14, 16.89.

IR: νmax, cm-1, KBr 3342, 3111 (N-H), 1724 (C=O),1658 (C=S), 1636 (C=N, Iminebase), 1347 (C=N), 1256, 1224 (C-S-C), 1021 (C-O-C), 540 (C-I).


Page 80

6l: 5-chloro-3-(5-(4-(furan-2-yl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-ylimino)-1,3-dihydro-indol-2-one

Yield: 78 % Mol. Wt.: 456.93

M.p.: 231-232 °C MS: (M+) 458.03


Mol. For.: C19H13ClN6O2S2


13C NMR: 100MHz, δ, ppm 172.10, 161.51, 158.31, 153.58, 152.91, 95.81-148.13 (Ar-C), 58.62, 15.10.

IR: νmax, cm-1, KBr 3362, 3175 (N-H), 1721 (C=O), 1635 (C=N, Iminebase), 1646 (C=S), 1362 (C=N), 1266, 1223 (C-S-C), 1018 (C-O-C), 718 (C-Cl).

7a: 3-(5-(6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-oxadiazol-2-ylimino)indolin-2-one

Yield: 81 % Mol. Wt.: 400.39

M.p.: 267-268 °C MS: (M+) 401.43


Mol. For.: C21H16N6O3

1H NMR: 400MHz, δ, ppm 8.87, 6.31 (1H×3, s, -NH-), 7.00-7.87 (m, 9H, Ar-H), 5.10 (s, 1H, -CH=), 2.03 (s, 3H, -CH3).

13C NMR: 100MHz, δ, ppm 169.24, 167.89, 155.41, 152.58, 151.87, 95.45-147.08 (Ar-C), 59.12, 13.65.

IR: νmax, cm-1, KBr 3356, 3192 (N-H), 1719 (C=O), 1621 (C=N, Iminebase), 1375 (C=N), 1251, 1012 (C-O-C).


Page 81

7b: 5-bromo-3-(5-(6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-oxadiazol-2-ylimino)indolin-2-one

Yield: 86 % Mol. Wt.: 479.29

M.p.: 279-280 °C MS: (M+) 480.34


Mol. For.: C21H15BrN6O3


13C NMR: 100MHz, δ, ppm 170.21, 169.67, 155.10, 153.71, 152.83, 96.19-148.43 (Ar-C), 57.44, 14.70.

IR: νmax, cm-1, KBr 3336, 3193 (N-H), 1738 (C=O), 1620 (C=N, Iminebase), 1366 (C=N), 1248, 1021 (C-O-C), 636 (C-Br).

7c: 3-(5-(6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-oxadiazol-2-ylimino)-5-nitroindolin-2-one

Yield: 90 % Mol. Wt.: 446.27

M.p.: 251-253 °C MS: (M+) 447.17


Mol. For.: C21H15N7O5


13C NMR: 100MHz, δ, ppm 171.29, 168.34, 155.45, 153.72, 151.65, 97.12-149.32 (Ar-C), 57.43, 14.78.

IR: νmax, cm-1, KBr 3371, 3189 (N-H), 1712 (C=O), 1631 (C=N, Iminebase), 1565 (N=O), 1375 (C=N), 1252, 1024 (C-O-C).


Page 82

7d: 5-fluoro-3-(5-(6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-oxadiazol-2-ylimino)indolin-2-one

Yield: 92 % Mol. Wt.: 418.38

M.p.: 273-274 °C MS: (M+) 419.47


Mol. For.: C21H15FN6O3


13C NMR: 100MHz, δ, ppm 170.67, 168.58, 155.16, 153.18, 151.61, 96.19-147.89 (Ar-C), 61.36, 14.89.

IR: νmax, cm-1, KBr 3357, 3190 (N-H), 1726 (C=O), 1630 (C=N, Iminebase), 1349 (C=N), 1321 (C-F), 1233, 1022 (C-O-C).

7e: 5-iodo-3-(5-(6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-oxadiazol-2-ylimino)indolin-2-one

Yield: 79 % Mol. Wt.: 526.29

M.p.: 217-219 °C MS: (M+) 527.10


Mol. For.: C21H15IN6O3


13C NMR: 100MHz, δ, ppm 169.92, 167.49, 155.87, 152.78, 151.27, 96.54-146.83 (Ar-C), 61.57, 15.37.

IR: νmax, cm-1, KBr 3372, 3187 (N-H), 1739 (C=O), 1616 (C=N, Iminebase), 1333 (C=N), 1231, 1039 (C-O-C), 555 (C-I).


Page 83

7f: 5-chloro-3-(5-(6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-oxadiazol-2-ylimino)indolin-2-one

Yield: 77 % Mol. Wt.: 434.84

M.p.: 212-213 °C MS: (M+) 436.02


Mol. For.: C21H15ClN6O3


13C NMR: 100MHz, δ, ppm 170.47, 167.98, 155.43, 152.55, 151.09, 95.41-147.07 (Ar-C), 60.70, 15.33.

IR: νmax, cm-1, KBr 3350, 3163 (N-H), 1746 (C=O), 1619 (C=N, Iminebase), 1348 (C=N), 1230, 1046 (C-O-C), 721 (C-Cl).

7g: 3-(5-(6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-oxadiazol-2-ylimino)indolin-2-one

Yield: 80 % Mol. Wt.: 416.46

M.p.: 190-191 °C MS: (M+) 417.69



1H NMR: 400MHz, δ, ppm 11.87, 9.09, 2.89 (1H×3, s, -NH-), 7.16-7.84 (m, 9H, Ar-H), 4.98 (s, 1H, -CH=), 1.98 (s, 3H, -CH3).

13C NMR: 100MHz, δ, ppm 170.53, 166.58, 154.87, 152.19, 151.54, 96.89-148.93 (Ar-C), 55.96, 14.77.

IR: νmax, cm-1, KBr 3369, 3161 (N-H), 1710 (C=O), 1649 (C=N, Iminebase), 1367 (C=N), 1243, 1040 (C-O-C).


Page 84

7h: 5-bromo-3-(5-(6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-oxadiazol-2-ylimino)indolin-2-one

Yield: 78 % Mol. Wt.: 495.35

M.p.: 239-240 °C MS: (M+) 496.79




13C NMR: 100MHz, δ, ppm 173.11, 168.65, 154.71, 152.28, 151.89, 97.99-147.06 (Ar-C), 55.66, 15.06.

IR: νmax, cm-1, KBr 3334, 3171 (N-H), 1725 (C=O), 1650 (C=N, Iminebase), 1372 (C=N), 1241, 1048 (C-O-C), 616 (C-Br).

7i: 3-(5-(6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-oxadiazol-2-ylimino)-5-nitroindolin-2-one

Yield: 88 % Mol. Wt.: 461.45

M.p.: 244-245 °C MS: (M+) 462.57




13C NMR: 100MHz, δ, ppm 172.43, 168.49, 155.41, 153.55, 151.88, 97.72-146.12 (Ar-C), 55.71, 14.78.

IR: νmax, cm-1, KBr 3350, 3165 (N-H), 1718 (C=O), 1638 (C=N, Iminebase), 1571 (N=O), 1379 (C=N), 1247, 1052 (C-O-C).


Page 85

7j: 5-fluoro-3-(5-(6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-oxadiazol-2-ylimino)indolin-2-one

Yield: 90 % Mol. Wt.: 434.45

M.p.: 186-187 °C MS: (M+) 435.18




13C NMR: 100MHz, δ, ppm 170.69, 168.38, 154.19, 154.28, 151.69, 94.80-144.56 (Ar-C), 54.98, 16.08.

IR: νmax, cm-1, KBr 3326, 3148 (N-H), 1743 (C=O), 1645 (C=N, Iminebase), 1331 (C-F), 1363 (C=N), 1228, 1050 (C-O-C).

7k: 5-iodo-3-(5-(6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-oxadiazol-2-ylimino)indolin-2-one

Yield: 83 % Mol. Wt.: 542.35

M.p.: 189-190 °C MS: (M+) 543.16




13C NMR: 100MHz, δ, ppm 173.78, 168.32, 154.86, 152.34, 150.95, 95.37-145.19 (Ar-C), 54.67, 16.00.

IR: νmax, cm-1, KBr 3329, 3161 (N-H), 1719 (C=O), 1657 (C=N, Iminebase), 1379 (C=N), 1249, 1037 (C-O-C), 561 (C-I).


Page 86

7l: 5-chloro-3-(5-(6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,4-oxadiazol-2-ylimino)indolin-2-one

Yield: 79 % Mol. Wt.: 450.90

M.p.: 245-246 °C MS: (M+) 452.0




13C NMR: 100MHz, δ, ppm 173.67, 166.75, 155.65, 153.68, 152.05, 97.12-149.19 (Ar-C), 56.96, 16.19.

IR: νmax, cm-1, KBr 3365, 3183 (N-H), 1718 (C=O), 1643 (C=N, Iminebase), 1367 (C=N), 1246, 1029 (C-O-C), 710 (C-Cl).


Page 87

1H-NMR of 5g

13C-NMR of 5g

CHAPTER: 2

PHARMACOPHORE HYBRIDIZATION

Mass Spectra of 5g

IR Spectra of 5g


Page 88


Page 89

1H-NMR of 6c

13C-NMR of 6c

CHAPTER: 2


Mass Spectra of 6c

IR Spectra of 6c


Page 90

CHAPTER: 2


NH

O

NO

NN

HN

NH

S CH3

Cl

7l

1H-NMR of 7l

13C-NMR of 7l


Page 91

CHAPTER: 2


Mass Spectra of 7l

IR Spectra of 7l


Page 92


Page 93

2.4 Biological Evolution

1) Antibacterial Activity:

Man is closely influenced by the activities if microorganisms. Microorganisms are a part

of our lives in more ways than most of us understand. They have shaped our present

environment and their activities will greatly influence our future. Microorganisms should

not be considered separate from human beings, but profound beneficial influence as a

part of our life. They are employed in the manufacture of dairy products, certain foods,

min processing of certain medicines and therapeutic agents, in manufacture of certain

chemicals and in numerous other ways.

Despite the established useful functions and potentially valuable activities of

microorganism, these microscopic dorms of life may be best known as agents of food

spoilage and causal agents of human beings viz. Acquired Immune Deficiency Syndrome

(AIDS), Herpes, Legionnaires disease, Influenza, Jaundice, Tuberculosis, Typhoid,

Dermatomycoses, Dysentery, Malaria etc.... in human being. Animals (infected with

Brucellosis, Tularemia etc...) and Plants (infected with Mildews, Rusts, Smuts, Cankers,

Leaf spots, etc...) have also been known to be victims of microbial pathogens. So far as is

known, all primitive and civilized societies have experienced diseases caused by

Microbes, frequently with disastrous results. Moreover, microorganisms have played

profound roles in warfare, religion and the migration of populations.

Control of microbial population is necessary to prevent transmission of disease, infection,

decomposition; contamination and spoilage caused by them, man’s personal comforts and

convenience depend to a large extent on the control of microbial population.

In 1928, a german scientist C. E. Chrenberg first used the term “Bacterium” to denote

small Microscopic organism with a relatively simple and primitive form of the cellular

organization known as “Prokaryotic”.


Page 94

Danish physician, gram in peculiarity, bacteria are generally unicellular e.g. Cocci,

Bacilli, etc… filamentous, eg. Actinomycetes, some being sheathed having certain cells

specialized for reproduction. The microorganisms are capable of producing diseases in

host are known as ‘Pathogenic’. Most of the microorganisms present on the skin and

mucous membrane are non pathogenic and are often referred to as “Commensals” or if

they live on food residues as in intestine, they may be called “Saprophytes”. Generally,

the pathogenic Cocci and Bacilli are gram positive and the pathogenic Coco Bacilli are

gram negative.

2) Antifungal Activity

It has been estimated that the life expectancy of humans has increased by atleast 10 years

since the discovery of antimicrobial agents for the treatment of microbial infections. A

consequence of our success with antimicrobial agents and improved medical care is the

number of fungal infections.

The incidence of fungal infections has increased dramatically in the past 20 years partly

because of the increase in the number of people whose immune systems are compromised

by wither AIDS, aging, organ transplantation or cancer therapy. Accordingly, the

increase in rates of morbidity and mortality because of fungal infections has been now

recognized as a major problem. In response to the increased incidence of fungal

infections, the pharmaceutical industry has developed a number of newer less toxic

antifungal for clinical use. The increased use of antifungal, often for prolonged periods,

has lead to recognition of the phenomenon of acquired antifungal resistance to one or

more of the available antifungal.

Fungi are non photosynthetic eukaryotes growing either as colonies of single cells

(yeasts) or as filamentous multi-cellular aggregate (molds). Most fungi live as saprophytes

in soil or on dead plant material and are important in the mineralization of organic matter.

A smaller number produce disease in human and animals. The in vitro methods used for

detections of antifungal potency are similar to those used in antibacterial screening. As


Page 95

with bacteria, it is easy to discover several synthetic and natural compounds that, in small

quantity, can retard or prevent growth of fungi in culture media.

� Minimal Inhibition Concentration (MIC)

1. Serial dilutions were prepared in primary and secondary screening.

2. The control tube containing no antibiotic is immediately sub cultured [before

inoculation] by spreading a loopful evenly over a quarter of plate of medium suitable

for the growth of the test organism and put for incubation at 37 oC OVERNIGHT.

The tubes are then incubated overnight.

3. The MIC of the control organism is read to check the accuracy of the drug

concentrations.

4. The lowest concentration inhibiting growth of the organism is recorded as the MIC.

5. The amount of growth from the control tube before incubation (which represents the

original inoculums) is compared.

� Methods Used For Primary And Secondary Screening

Each synthesized drug was diluted obtaining 2000 µg/ml concentration, as a stock

solution.

Primary screen:

In primary screening 1000 µg/ml, 500 µg/ml and 250 µg/ml concentrations of the

synthesized drugs were taken. The active synthesized drugs found in this primary

screening were further tested in a second set of dilution against all microorganisms.

Secondary screen:

The drugs found active in primary screening were similarly diluted to obtain 200 µg/ml

100 µg/ml, 50 µg/ml, 25 µg/ml, 12.5 µg/ml, 6.250 µg/ml, and concentrations.

Reading Result:

The highest dilution showing at least 99 % inhibition zone is taken as MIC. The result of

this is much affected by the size of the inoculums. The test mixture should contain 108

organism/ml.


Page 96

The MICs of synthesized compounds were carried out by broth microdilution method as

described by Rattan [85]. Antibacterial activity was screened against E. Coli (MTCC-

443), P. Aeruginosa (MTCC-1688), Kl. Pneumoniae (MTCC-109), S. Typhi (MTCC-98),

S. Aureus (MTCC-96), S. Pyogenus (MTCC-442) and B. Subtilis (MTCC-441).

Gentamycin, Ampicillin, Chloramphenicol, Ciprofloxacin, Norfloxacin was used as a

standard antibacterial agent. Antifungal activity was screened against three fungal species

C. Albicans (MTCC 227), A. Niger (MTCC 282) and A. Clavatus (MTCC 1323).

Nystatin and Griseofulvin was used as a standard antifungal agent. The antimicrobial

screening data are shown in Table 1 & 2.

All MTCC cultures were collected from Institute of Microbial Technology, Chandigarh

and tested against known drugs. Mueller–Hinton broth was used as nutrient medium to

grow and dilute the drug suspension for the test. Inoculums size for test strain was

adjusted to 108 CFU (Colony Forming Unit) per millilitre by comparing the turbidity.

DMSO was used as diluents to get desired concentration of drugs to test upon standard

bacterial strains.


Page 97

Table 1: Antibacterial Activity (Minimal Inhibition Concentration, MIC, µg/mL) Entry R X E.c. P.a. Kl.pn. S.ty S.a. S.py. B.s.

5a H S 500 200 200 200 200 200 200 5b Br S 200 200 200 250 100 100 100 5c NO2 S 62.5 100 100 100 100 200 100 5d F S 62.5 62.5 100 100 62.5 100 100 5e I S 250 250 200 250 200 200 200 5f Cl S 200 100 200 200 200 200 100 5g H O 500 250 250 250 500 500 500 5h Br O 250 250 200 250 100 100 100 5i NO2 O 62.5 100 200 200 200 250 200 5j F O 100 62.5 100 100 62.5 100 100 5k I O 250 250 250 250 250 200 250 5l Cl O 100 200 200 200 200 200 200 6a H O 500 200 200 200 200 200 200 6b Br O 100 200 200 200 200 200 200 6c NO2 O 500 200 250 200 250 250 200 6d F O 200 250 200 200 250 200 250 6e I O 250 250 500 250 200 200 200 6f Cl O 100 100 62.5 100 62.5 100 62.5 6g H S 500 250 250 250 500 500 500 6h Br S 250 250 200 250 100 100 100 6i NO2 S 500 250 250 250 500 500 500 6j F S 100 100 100 62.5 62.5 100 100 6k I S 250 250 250 500 250 200 250 6l Cl S 62.5 100 100 62.5 100 62.5 100

Gentamycin 0.05 1 0.05 1 0.25 0.5 0.5 Ampicillin 100 100 100 100 250 100 100 Chloramphenicol 50 50 50 50 50 50 50 Ciprofloxacin 25 25 25 25 50 50 50 Norfloxacin 10 10 10 10 10 10 10 E.c.=E. coli (MTCC-443); P.a.=P. aeruginosa (MTCC-1688); Kl.pn.=Kl. pneumoniae (MTCC-109);S.ty.=S. typhi (MTCC-98);S.a.=S. aureus (MTCC-96);S.py.=S. pyogenus (MTCC-442) ;B.s.=B. subtilis (MTCC-441).


Page 98

Table 2: Antifungal Activity (Minimal Inhibition Concentration, MIC, µg/mL)

Entry C. albicans A. niger A. clavatus 5a >1000 500 500 5b 500 500 500 5c 250 100 250 5d 100 100 100 5e >1000 >1000 >1000 5f 500 500 500 5g 250 250 250 5h 500 500 >1000 5i 250 250 500 5j 100 100 200 5k >1000 >1000 >1000 5l >1000 500 500 6a >1000 500 500 6b 250 250 250 6c >1000 100 250 6d 500 200 500 6e >1000 500 >1000 6f 100 100 100 6g 250 >1000 250 6h 500 500 >1000 6i >1000 500 500 6j 250 100 250 6k >1000 >1000 500 6l 200 100 100

Nystatin 100 100 100 Greseofulvin 500 100 100 C. albicans (MTCC 227); A. niger (MTCC 282); A. clavatus (MTCC 1323)


Page 99

3) Antitubercular Activity

Hansen (1868) discovered the first member (lepra bacilli), Robert Koch (1882) isolated

mammalian tubercle bacilli and Johne (1895) described Mycoparatuberculosis (Johne’s

bacillu). Mycobacteria are slender bacilli and sometimes exhibit filamentous forms

resembling fungal mycelium (from Greek, Myces meaning fungus) and hence they are so

named. They are difficult to stain by ordinary stains because of the presence of waxy

materials in their cell walls. Although they are gram positive, they stain poorly, if at all,

by gram’s technique. They are better stained by hot carbol fuchsin, and once stained; they

resist decolourisation by dilute minerals acids and are, therefore, referred to as Acid Fast

Bacilli or AFB. These organisms are non motile, non capsulated, non-sporing and mostly

very slow growing. The genus includes obligate parasites pathogenic to man, mammals;

birds and reptiles, opportunistic pathogens and saprophytic varieties.

M. tuberculosis is non-motile, non-sporing and non-capsulated bacilli, arranged singly or

in groups. They are acid-fast due to the presence of mycolic acid in cell-wall and weakly

Gram positive. With Ziehl-Neelsen stain, M. tuberculosis look slender, straight or slightly

curved rod with beaded or barred appearance and M.bovis appear straighter, stouter and

shorter with uniform staining. Tubercle bacilli are aerobes, grow slowly (generation time

14-15 hours), optimum temperature 37 oC, pH 6.4-7.0. They grow only in specially

enriched media containing egg, asparagines, potatoes, serum and meat extracts. Colonies

appear in 2-6 weeks. M. tuberculosis grows more luxuriantly in culture (eugenic) than M.

bovis which grows sparsely (dysgenic).

The drug susceptibility test may be performed by either the direct or the indirect method.

The direct drug susceptibility test is performed by using a subculture from a primary

culture as the inoculums.


Page 100

Methods used for primary and secondary screening:

Each synthesized drug was diluted obtaining 2000 microgram /ml concentration, as a

stock solution.

Primary screen:

In primary screening 500 µg/ml, 250 µg/ml, and 125 µg/ml concentrations of the

synthesized drugs were taken. The active synthesized drugs found in this primary

screening were further tested in a second set of dilution against all microorganisms.

Secondary screen:

The drugs found active in primary screening were similarly diluted to obtain 100 µg/ml,

50 µg/ml, 25 µg/ml, 12.5 µg/ml, 6.250 µg/ml, 3.125 µg/ml and 1.5625 µg/ml

concentrations.

Reading Result:

The highest dilution showing at least 99 % inhibition is taken as MIC.The result of this is

much affected by the size of the inoculums. The test mixture should contain 108

organism/ml (CFU).

The Standard Drugs:

The Standard strain M. Tuberculosis H37RV is tested with each new batch of medium.

The recommended drug concentrations are 4 mg/l for Streptomycin, 0.2 mg/l for

Isoniazid, 40 mg/l for Rifampicin and 2 mg/ l for Ethambutol.

Antituberculosis activity against M. tuberculosis H37RV of synthesized compound is

mentioned in Table 3.


Page 101

4) Antimalarial Activity All the synthesized compounds were screened for antimalarial activity in the

Microcare laboratory & TRC, Surat (Gujarat) India. The in vitro antimalarial assay was

carried out against Plasmodium falciparum 3D7 chloroquine-sensitive strain in 96 well

microtitre plates according to the microassay protocol of Rieckmann and co-workers with

minor modifications [86, 87]. The test concentration which inhibited the complete

maturation into schizonts was recorded as the minimum inhibitory concentrations (MIC).

Chloroquine was used as the reference drug. Observations of the in vitro antimalarial

screening are presented in the Table 4.

Table 3: Antitubercular Activity (Minimal Inhibition Concentration, MIC, µg/mL)

Entry M. tuberculosis H37RV

% Inhibition Entry M.

tuberculosis H37RV

% Inhibition

5a 500 98 6g 1000 98 5b 250 98 6h 500 99 5c 62.5 99 6i 250 98 5d 25 99 6j 500 99 5e 1000 98 6k 1000 98 5f 62.5 99 6l 62.5 99 5g 1000 98 7a 200 92 5h 500 98 7b 500 74 5i 62.5 99 7c 3.10 99 5j 50 99 7d 250 84 5k 1000 98 7e 500 93 5l 100 98 7f 100 97 6a 1000 98 7g 500 94 6b 250 99 7h 500 75 6c 500 98 7i 100 97 6d 250 98 7j 200 89 6e 1000 98 7k 500 94 6f 100 99 7l 3.12 99

Isoniazid 40 98 Isoniazid 40 98 Rifampicin 0.20 99 Rifampicin 0.20 99 M. tuberculosis H37RV (MTCC-200)


Page 102

2.5 Result & Discussion

For compounds 5a-l:

The literature survey revealed that introduction of electron-withdrawing groups at

positions 5, 6, and 7 greatly increased activity from that of 1,3-dihydro-2H-indol-2-one,

with substitution at the 5th position being most favorable. This is not surprising, as C-5

substitution has previously been associated with increased biological activity for a range

of indole-based compounds [88, 89] and the presence of substituted aromatic ring at 3rd

position has been reported to be associated with antimicrobial properties [90, 91]. The

various substituent at 3rd position of the isatin which were reported, are various

substituted phenyl ring moieties [92, 93] heterocyclic rings [94-96] and aliphatic system

[97]. These observations led to the conception that a series of some different novel Schiff

bases of 5-(5-amino-1,3,4-thiadiazol-2-yl)-6-methyl-4-aryl-3,4-dihydropyrimidin-2(1H)-

one/thione using different 5-substituted indoline-2,3-diones

From in-vitro antibacterial activity data, it is confirmed that compounds containing strong

electron withdrawing (fluorine group) i.e. 5d & 5j exhibited excellent activity against all

microbial strains, while compounds 5b & 5h exhibited comparable activity against gram

positive strains, while compound 5c & 5i are found to be highly active against gram

negative strains as compared to standard antibiotic ampicillin.

From in-vitro antifungal activity data, It is found that compound 5d & 5f is displaying

highest activity against all fungal strains, while compounds 5c & 5i are showing

somewhat less activity compare to compounds 5d & 5i. But overall, all the compounds

Table 4: Antimalarial Activity* (Minimal Inhibition Concentration, IC50, µg/mL)

Entry IC50 #C log P Entry IC50 #C log P 7a 10 3.13 ± 0.83 7g 5 3.74 ± 0.85 7b 10 4.11 ± 0.88 7h 10 4.71 ± 0.89 7c 0.177 3.04 ± 0.84 7i 5 3.65 ± 0.86 7d 5 3.39 ± 0.88 7j 5 3.99 ± 0.89 7e 10 4.37 ± 0.88 7k 10 4.97 ± 0.89 7f 10 3.93 ± 0.84 7l 0.035 4.53 ± 0.86

Chloroquine 0.125 - Chloroquine 0.125 - *Plasmodium falciparum 3D7 chloroquine-sensitive strain #C log P value calculated using Chem bio office 2010


Page 103

have displayed significant antibacterial and antifungal activity. In general, the order of

antibacterial activity of the substituents at the 5th position of 1H-indole-2,3-dione is

F>NO2>Br>Cl>H=I and also due to presence of sulphur atom at position–2 in the

compounds 5a-5f is responsible for better activity compared to oxygen atom (at position–

2) in compounds 5g-5l. The in-vitro antibacterial and antifungal screening results are

summarized in Table 1 & 2.

The encouraging results from the antibacterial and antifungal studies impelled us to go

for preliminary screening of synthesized compounds against M .tuberculosis, which is

summarized in Table 3. Compound 5j containing 5-flouro substituent on indolone ring

with oxygen atom on tetrahydropyrimidine nucleus showed better activity (50 µg/ml) and

compounds 5c, 5f and 5j showed good activity (50-62.5 µg/ml) which is attributed due to

5- nitro, 5-chloro substituents of 2-thioxo-tetrahydropyrimidine indolone nucleus and 5-

nitro substituents of 2-oxo-tetrahydropyrimidine indolone nucleus respectively, where as

compound 5d which is having inductively electron withdrawing but mesomerically

electron releasing sulphur atom with 5-flouro substituent on indolone ring showed better

activity (25 µg/ml) compared to other analogs.

For compound 6a-l:

From in vitro antibacterial and antifungal activity data, It is confirmed that compounds

6b, 6e, 6j & 6l exhibited excellent activity against all tested microbial and fungal strains,

while compounds 6d & 6h displayed comparable activity against gram-positive strains,

while compounds 6e & 6k are found to be moderate active against gram-negative as

compared to standard antibiotics. The encouraging results from the antibacterial and

antifungal studies impelled us to go for preliminary screening of synthesized compounds

against M. tuberculosis H37RV, which is summarized in Table 3. In vitro

antituberculostic activity of all the newly synthesized compounds against Mycobacterium

tuberculosis H37RV strain was determined by using Lowenstein-Jensen medium

(conventional method). Majority of the compounds displayed moderate to good while

compounds 6f, 6l gives best activity against M. tuberculosis H37RV.


Page 104

For compound 7a-l:

The synthesized compound 7a-l were also evaluated in vitro for antimalarial assay

against Plasmodium falciparum 3D7 chloroquine-sensitive strain (Microcare laboratory

& TRC, Surat, Gujarat, India), in 96 well microtitre plates according to the microassay

protocol of Rieckmann and co-workers with minor modifications [98-101]. The test

concentration which inhibited the complete maturation into schizonts was recorded as the

minimum inhibitory concentrations (MIC). Chloroquine was used as the reference drug.

Observations of the in vitro antimalarial screening are presented in the Table 4. The

compounds 7a, 7b, 7d, 7e, 7j and 7k has no effect on antimalarial activity (MIC = 10

µg/mL) while compounds 7c, 7b, 7g, 7i, 7j & 7l has remarkable improvement in

antimalarial potency with MIC value in the range of 0.035-5.0 µg/mL. Presence of nitro

(7c, MIC=0.177 µg/mL) and presence of chloro (7l, MIC=0.035 µg/mL) displayed

excellent antimalarial potency. Overall, among the various substitution, the order of

highest antimalarial potency is NO2>F>Br>H. It is well known from the literature that the

presence of these groups imparts a variety of properties including steric, electronic

properties, enhanced binding interactions, metabolic stability, changes in physical

properties and selective reactivities [102, 103]. This promising antitubercular and

antimalarial activity may be due to sufficient hydrogen bonding capacity with desired

lipophilicity or with favorable stearic hindrance [104].

In Conclution, Tetrahydropyrimidinyl-1,3,4-(thia/oxa)diazolylimino-1,3-dihydro-2H-

indol-2-ones derivatives 5a-l; 6a-l; 7a-l were synthesized and characterized for their

structure elucidation. Various chemical and spectral data supported the structures of

newly synthesized compounds. The Biginelli’s reaction for preparation of

tetrahydropyrimidinones derivatives 1a-b, was efficiently carried out using CaCl2 as

catalyst. Some Compounds showed significant Antibacterial and Antifungal activity.

While some the compound displayed promising Antitubercular and Antimalarial activity

compared to standards. Thus, present library model can be used to design the new ligand

of this class for their Antimicrobial, Antitubercular and Antimalarial activities.


Page 105

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