14.139.121.10614.139.121.106/pdf/2013/may13/5.pdf14.139.121.106

DESIGN AND DEVELOPMENT OF PROTEASE INHIBITORS AS POTENTIAL

ANTIMALARIAL AGENTS

A THESIS SUBMITTED TO

THE MAHARAJA SAYAJIRAO UNIVERSITY OF BARODA

FOR THE AWARD OF THE DEGREE OF

Doctor of Philosophy

in

Pharmacy

BY

UTTAM R. MANE

UNDER THE GUIDANCE OF

Prof. M. R. YADAV

Pharmacy Department,

Faculty of Technology and Engineering,

The M. S. University of Baroda,

Vadodara-390 001.

NOVEMBER 2012

Date:

CERTIFICATE

This is to certify that the thesis entitled “DESIGN AND DEVELOPMENT

OF PROTEASE INHIBITORS AS POTENTIAL ANTIMALARIAL AGENTS”

submitted for the award of Ph. D. Degree in Pharmacy by Mr. UTTAM RAJARAM

MANE incorporates the original research work carried out by him under my

supervision.

Supervisor

(Prof. M. R. Yadav)

HEAD DEAN

Pharmacy Department Faculty of Technology & Engineering,

The M.S. University of Baroda,

Vadodara -390 001

DECLARATION

I hereby declare that the topic entitled “DESIGN AND DEVELOPMENT

OF PROTEASE INHIBITORS AS POTENTIAL ANTIMALARIAL AGENTS”

submitted herewith to The Maharaja Sayajirao University of Baroda, Vadodara, for

the award of the degree of DOCTOR OF PHILOSOPHY IN PHARMACY is the

result of the work carried out by me in Pharmacy Department, Faculty of

Technology and Engineering, The M. S. University of Baroda, Vadodara.

The result of this work has not been previously submitted for any

degree/fellowship.

Date:

Place: Vadodara UTTAM R. MANE

Dedicated to my FAMILY

ACKNOWLEDGEMENT

I am deeply indebted to my supervisor Prof. M. R. Yadav, Head, Department of Pharmacy,

Faculty of Technology & Engineering, The M. S. University of Baroda, Vadodara, for

guiding me to accomplish this dissertation work. It was my privilege and pleasure to work

under his able guidance. A person who strongly believes in the principle of perfection and

enriches the true researchers’ spirit with intuition, logical reasoning and his well versed

knowledge has made him an oasis of ideas. I am indeed grateful to him for providing helpful

suggestions, from time to time. Due to his constant encouragement and inspiration, I am able

to present this thesis work.

I would like to thank Prof. A. N. Mishra, Dean, Faculty of Technology and Engineering, The

M. S. University of Baroda, for providing the facilities for research.

I convey my deepest gratitude to Prof. Rajani Giridhar, Coordinator, Q. I. P. Cell,

Pharmacy Department, for her moral support during the course of the study.

I gratefully acknowledge Dr. C. Dutt, Director and Dr. S. S. Nadkarni, ED, Dr. V.

Chauthaywale, VP, Torrent Research Centre for their enduring support to carry out this

dissertation work.

I sincerely acknowledge Dr. R. C. Gupta, GM, Torrent Research Centre for his never ending

support and fruitful suggestions at the hours of need.

My special thank go to Prof. Li Huang and Prof. Honglin Li, East China University of

Science and Technology, for carrying out biological studies of this research work.

I am also thankful to Dr. R. Shah, GM, Dr. Shailesh, GM, Dr. A. Mandhare, AGM and Dr.

Sanjay, AGM, my Seniors and Colleagues at Torrent Research Centre for their moral

support.

I am thankful to A. Shinde, Prakash and Anurag for their contributions.

I convey my thanks to Prashant, P. Bhaver, Suhas, Amit and Mahesh for giving me

valuable support.

I express my sincere thanks to teaching and non-teaching staff of Pharmacy Department for

their co-operation during the course of the work.

My parents deserve special mention for their support and prayers. I convey my deepest

gratitude to my late mother, who inculcated the fundamentals of learning and in true sense she

is the only source of my inspiration. My father is an ideal person in my life whom I follow

every step of my life. I owe thanks to my ever supporting wife Archana and my cute little

baby Anvi. I also owe so much to my sister, brother and Vahini and in laws, my late uncle

and aunty and other family members, friends and relatives for their encouragement and

support throughout my life. I express my deepest sentiments of affection and regards to them

for whatever I am today.

Finally, I would like to thank everybody who directly or indirectly involved in the successful

completion of this thesis, as well as expressing my apology that I could not mention

personally one by one.

Finally, Thanks to almighty for his blessings on me forever……………

Uttam R. Mane

ABBREVIATIONS

__________________________________________________________

Aq. : Aqueous

DMF : Dimethyl formamide

DMSO : Dimethyl sulphoxide

EtOAc : Ethyl acetate

DIEA : N.N-diisopropyl ethyl amine

TEA : Triethylamine

EMME : Diethyl ethoxymethylenemalonate

DPE : Diphenyl ether

conc. HCl : Concentrated hydrochloric acid

ECF : Ethyl chloroformate

HOBT : 1-Hydroxy benzotriazole hydrate

EDC : 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide

KBr : Potassium bromide

NaN3 : Sodium azide

RT : room temperature

ppt. : precipitate

Anal. : Analytical

TLC : Thin layer chromatography

Rf : Retention factor

ppm : Parts per million

M. P. : Melting point

Mass : Mass spectroscopy

M+

: Molecular ion

FP : Falcipain

FP-2 : Falcipain-2

FP-3 : Falcipain-3

IR : Infrared spectroscopy 1HNMR (PMR) : Proton nuclear magnetic resonance spectroscopy

INDEX

Certificate i

Declaration ii

Dedication iii

Acknowledgement iv

Abbreviations v

1. Introduction 1-57

2. Research envisaged 58-59

3. Results and Discussion 60-99

3.1 Chemical studies 60-96

3.1.1 Synthesis of 4H-pyrido[1,2-a]pyrimidin-4-one intermediates (4a-c & 6a-c) 61-66

Synthesis of 4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (4a) 61

Synthesis of 3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one (6a) 63

Synthesis of 8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (4b) 63

Synthesis of 3-isocyanato-8-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (6b) 64

Synthesis of 7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (4c) 65

Synthesis of 7-chloro-3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one (6c) 66

3.1.2 Synthesis of (4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea derivatives (Series I) 66

Synthesis of (4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea derivatives 66

Synthesis of 8-methyl-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea derivatives 74

Synthesis of 7-chloro-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea derivative 76

3.1.3 Synthesis of N-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-(pyridin-2-

yl)piperazine-1-carboxamide derivatives (Series II)

79

Synthesis of N-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-(pyridin-2-yl)-piperazine-1-

carboxamide derivatives

79

Synthesis of N-(8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-(pyridin-2-

yl)piperazine-1-carboxamide derivatives

80

Synthesis of N-(7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-(pyridin-2-


81

3.1.4 Synthesis of 4-oxo-4H-pyrido[1,2-a]pyrimidin-3-ylcarbamate derivatives (Series III)

83

Synthesis of 4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carbamate derivatives 83

Synthesis of 8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carbamate derivatives 85

Synthesis of 7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carbamate derivatives 85

3.1.5 Synthesis of 4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carboxamide derivatives (Series IV)

86

Synthesis of 4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carboxamide derivatives 87

Synthesis of 8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carboxamide derivatives 92

Synthesis of 7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carboxamide derivatives 93

Synthesis of 7-(4-methoxyphenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxamide 94

intermediates

Synthesis of 7-(4-methoxyphenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxamides

derivatives

94

3.2. Biological evaluation 96-99

4. Experimental work 100-143

4.1 Chemical studies 100-143

4.1.1 Synthesis of 4H-pyrido[1,2-a]pyrimidin-4-one intermediates (4a-c & 6a-c) 100

Synthesis of 4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (4a) 100

Synthesis of 3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one (6a) 101

Synthesis of 8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (4b) 102

Synthesis of 3-isocyanato-8-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (6b) 103

Synthesis of 7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (4c) 104

Synthesis of 7-chloro-3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one (6c) 105

4.1.2 Synthesis of (4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea derivatives (Series I) 106

Synthesis of (4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea derivatives 106

Synthesis of 8-methyl-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea derivatives 114

Synthesis of 7-chloro-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea derivative 116


yl)piperazine-1- carboxamide derivatives (Series II) 121

Synthesis of N-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-(pyridin-2-yl)-piperazine-1-

carboxamide derivatives

121

Synthesis of N-(8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-(pyridin-2-


123

Synthesis of N-(7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-(pyridin-2-


124

4.1.4 Synthesis of 4-oxo-4H-pyrido[1,2-a]pyrimidin-3-ylcarbamate derivatives

(Series III)

126

Synthesis of 4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carbamate derivatives 126

Synthesis of 8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carbamate derivatives 128

Synthesis of 7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carbamate derivatives 129

4.1.5 Synthesis of 4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carboxamide derivatives (Series IV)

130

Synthesis of 4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carboxamide derivatives 130

Synthesis of 8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carboxamide derivatives 137

Synthesis of 7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carboxamide derivatives 138

Synthesis of 7-(4-methoxyphenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxamide

intermediates

140

Synthesis of 7-(4-methoxyphenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxamides

derivatives

141

4.2. Biological work 143

5. Summary 144-151

6. References 152-165

1. Introduction

Chapter-1 Introduction

1

Parasite-borne diseases are having devastating impact on human

civilizations. However, none of the parasitic diseases has had as enormous an

impact on humans as that of malaria, which is one of the major causes of

morbidity and mortality of human civilizations in the past, and even today it

remains one of the deadliest diseases on the planet. It displays full explosive

power of vector-borne infections, erupting suddenly and with intensity that can

overwhelm vulnerable communities.1 Even today malaria, particularly the one

caused by Plasmodium falciparum, remains a serious health problem in Africa,

South America and many parts of Asia.2 As per the recent WHO report malaria is

having devastating impact on the lives of more than 3 billion people of the world,

about half of the humanity.3

An estimated 600 million people are at risk of contracting malaria

infection and the disease kills more than 1 million children and a large number of

pregnant women every year. About 90% of these casualties occur in tropical

Africa and majority of them are children under the age of five. Exacerbated by

poverty, malaria exerts a high toll on populations that can least afford a cure.4

Despite a heavy toll of tropical parasitic diseases on human life in the developing

world, present antimalarial therapy still rely on drugs developed decades ago.

There are several reasons for re-emergence and spread of the infection across the

globe as a major public health burden; the most common ones are complex life

cycle of the parasite, resistance developed by parasite against common

antimalarial drugs, inadequate control of the mosquito vector, and despite

tremendous impetus and thrust instituted legitimately, the dream of developing an

effective, safe and economical vaccine is a big illusion till date. The other major

contributors to the re-emergence of the disease and its spread in the new locations

and populations are increased population density, global warming, continuing

poverty and political instability. The business travels to malaria endemic areas

further added the spread of the disease to the developed countries where it has

been thought to be totally eradicated a long time ago. The prophylaxis of malaria

is also complex because of resistance, safety and tolerability issues of the existing

drugs, so there are very limited antimalarial drug options available.5

WHO aims for the global roll back of malaria with intensified efforts in

providing access to safe, affordable and effective antimalarial combination

treatments worldwide along with preventive measures.6 Science still has no magic


2

bullet for malaria and doubt remains whether such a single solution will ever exist.

In many cases, the clinical usefulness of new compounds is limited due to poor

efficacy, toxicity and constant attrition rate of drugs due to development of

resistance. The absence of profit incentives has resulted in the lack of investment

by the pharmaceutical industry in developing new chemotherapies7 because the

disease is more prevalent among the people of the developing countries who

hardly have any purchasing power. Efforts in the direction of vaccine

development and control/eradication of the mosquito vectors have either failed or

met with limited success. Development of new antimalarial drugs only remains an

economically and environmentally viable alternative.

Overall, there is an alarming situation as the parasite has developed

resistance to all of the available drugs except artemisinins, and to prevent

resistance development against artemisinins, these are generally used in

combination with other antimalarials. But some recent reports have challenged the

effectiveness and use of artemisinins alone or in combination due to reports on

their reduced efficacy on Thai-Combodian border, historically a site of emerging

antimalarial drug resistance and also because vaccine development has not yet

yielded any fruitful results.6 Hence there is an urgent need for identification and

characterization of new targets for antimalarial chemotherapy. Fortunately the P.

falciparum genome sequencing has revealed a number of new targets for drug and

vaccine development. But only the development of newer antimalarial drugs

remains an economically and environmentally viable alternative to fight out the

menace of the disease.8

1.1 Replicative life cycle of malaria

Malaria is caused by four species of protozoan parasites of the

Plasmodium genus. These are Plasmodium falciparum, P. vivax, P. ovale and P.

malariae, each of which presents slightly different clinical symptoms.

Geographically P. falciparum is the most widespread and the most pernicious of

the four species causing the major malaria related morbidity and mortality.9

In 1897, Ronald Ross first reported that the parasite Plasmodium could

infect the female anopheles mosquito, parasites are transmitted from one person to

another by this insect vector, thus completing parasite cycle. The malaria parasite

exhibits a complex life cycle (Fig. 1) involving an insect vector (mosquito) and a


3

vertebrate host (human). The major phases of the life cycle are: liver stage, blood

stage, sexual stage and sporogony.9

Figure 1: The life cycle of malaria parasite

Malaria infection is initiated when sporozoites are injected into the blood

of human host along with the saliva of a feeding mosquito. Sporozoites are carried

by the circulatory system to the liver where they invade parenchymal cells of the

hepatocytes (1), a process involving receptor ligand mediated adhesion. The

intracellular parasite undergoes an asexual replication known as exoerythrocytic

schizogony within the hepatocyte (2-4). Exoerythrocytic schizogony culminates in

the production of merozoites, which are released into the bloodstream (5). During

development and multiplication in the liver the host is asymptomatic. In

nonrelapsing malaria (falciparum, malariae) no parasites are left in the liver as

merozoites infection moves entirely in the blood. In relapsing malaria, a

proportion of the liver-stage parasites from P. vivax and P. ovale go through a non

dividing dormant stage known as hypnozoite (not shown in Fig. 1) instead of

immediately going through the asexual replication (i.e., temporarily stay at step

2). These hypnozoites are reservoir of parasites which will reactivate after several

weeks to months (or years) after the primary infection and are responsible for

relapses. Merozoites invade erythrocytes (6) and undergo a trophic period in


4

which the parasite enlarges (7-8). The early trophozoite is often referred to as 'ring

form' because of its morphology. Trophozoite enlargement is accompanied by an

active metabolism including the ingestion of host cytoplasm and the proteolysis of

hemoglobin into constituent amino acids. Multiple rounds of nuclear division

manifest the end of the trophic period without cytokinesis resulting in a schizont

(9). Merozoites bud from the mature schizont, also called a segmenter (10) and the

merozoites are released following rupture of the infected erythrocyte (11).

Invasion of erythrocytes reinitiates another round of the blood-stage replicative

cycle (6-11). The time intervals between the intermittent fever and chills often

associated with malaria is due to the synchronous rupture of infected erythrocytes

and release of merozoites, parasitic waste and the cell debris. The debris is the

cause of the episodes of fever and chills. Trophozoite and schizont infected

erythrocytes are rarely found in the peripheral circulation during P. falciparum

infections. Erythrocytes infected with these stages adhere to endothelial cells and

sequester in the microvasculature of vital organs, especially brain, heart and lungs.

Sequestration in the brain is a contributing factor in cerebral malaria. As an

alternative to the asexual replicative cycle, the parasite can differentiate into

sexual forms known as macro or microgametocytes (12). The gametocytes are

large parasites, which fill up the erythrocyte, but only contain one nucleus.

Ingestion of gametocytes by the mosquito vector induces gametogenesis (i.e., the

production of gametes) and their escape from the host erythrocyte. Factors which

participate in the induction of gametogenesis include a drop in temperature, an

increase in carbon dioxide concentration and mosquito metabolites. Microgametes

formed by a process known as exflagellation (13) are flagellated forms, which will

fertilize the macrogamete (14) leading to a zygote (15). The zygote develops into

a motile ookinete (16), which penetrates the gut epithelial cells and develops into

an oocyst (17). The oocyst undergoes multiple rounds of asexual replication (18)

resulting in the production of sporozoites (19). Rupture of the mature oocyst

releases the sporozoites into the hemocoel (body cavity) of the mosquito (20). The

sporozoites migrate to and invade the salivary glands (not shown in Fig. 1), thus

completing the life cycle. In summary, the malaria parasite undergoes three

distinct asexual replicative stages exoerythrocytic schizogony, blood stage

schizogony and sporogony, resulting in the production of invasive forms

(merozoites and sporozoites). Sexual reproduction occurs with the switch from


5

vertebrate to invertebrate host and leads to the invasive ookinete. All invasive

stages are characterized by the apical organelles typical of apicomplexan species.

The invasive stages differ in regard to the types of cells or tissues they invade and

their motility.

1.2. Currently available antimalarial drugs

A systemic malaria control program began after discovery of the malaria

parasite by Laveran (Nobel Prize for medicine, 1907) in 1880 and the

demonstration by Ross in 1897 that the female anopheles mosquito was the vector

of malaria. The mosquito control combined with therapeutic and

chemoprophylatic approaches in parallel are used in the successful disease

control. The efforts of complete eradication of the disease were limited by the

resistance developed by the parasite against common antimalarial drugs and also

by the mosquito vector.9 Mostly, antimalarial drugs are targeted against the

asexual erythrocytic stage of the parasite. The parasite degrades hemoglobin in its

acidic food vacuole,10

producing free heme, capable of reacting with molecular

oxygen and thus generating reactive oxygen species as toxic by-products. A major

pathway of detoxification of heme moieties is polymerization as malarial

pigment.11

Majority of antimalarial drugs act by disturbing the polymerization

(and/or the detoxification by any other way) of heme, thus killing the parasite with

its own metabolic waste.12

The main classes of active schizonticides are 8-

aminoquinolines, 4-aminoquinolines, arylalcohols including quinoline alcohols

and antifolate compounds which inhibit the synthesis of parasitic pyrimidines. A

newer class of antimalarials is based on the natural endoperoxide artemisinin, its

semisynthetic derivatives and synthetic analogs. Some antibiotics are also used,

generally in association with quinoline-alcohols.13

Very few compounds are active

against gametocytes and also against the intra-hepatic stages of the parasite.

1.2.1 8-Aminoquinolines

This is the only class of gametocytocides available to the therapeutic

purposes. Primaquine has been widely used for the treatment of the hypnozoites

(liver reservoirs) responsible for the relapsing forms of P. vivax and P. ovale.

However, primaquine was recently reconsidered for malaria chemoprophylaxis to

eliminate P. falciparum at the early stage of infection, when parasite develops in


6

the liver, thus preventing the clinical disease. Despite its good oral absorption, this

molecule has a short half life and needs to be administered daily. Serious toxicity

can be a major problem in patients with glucose-6-phosphate dehydrogenase

deficiency. Primaquine is interfering with the mitochondrial function of

Plasmodium. Tafenoquine (WR 238605) is a primaquine analog with a longer

elimination half-life (14 days compared to 4 hours for primaquine). It also has a

higher therapeutic index than primaquine. This molecule may be useful for

chemoprophylaxis of P. falciparum and for prevention of relapses of vivax

malaria.

1.2.2 4-Aminoquinolines

The main antimalarials are the 4-aminoquinolines because they have proven to be

a highly successful class of compounds for the treatment and prophylaxis of

malaria. They are easy to synthesize, cheap and generally well tolerated. These

compounds, as well as the quinoline alcohols, are active against the intra-

erythrocytic stages of the parasite. The 4-aminoquinolines are able to accumulate

in high concentrations within the acid food vacuole of Plasmodium, to kill the

parasite.14

Chloroquine (CQ) was introduced in 1944-1945 and soon became the

mainstay of therapy and prevention, as this drug was cheap, nontoxic and active

against all strains of malaria parasites. In 1994, CQ was the third most widely

consumed drug in the world after aspirin and paracetamol.15

The precise mode of

action of the quinoline antimalarials and the mechanism of parasite resistance are

still not completely understood. On the mode of action of CQ, one can cite 14

many hypotheses like (i) direct-heme binding, (ii) inhibition of an unidentified

heme ferriprotoporphyrin- IX “polymerase”, (iii) inhibition of vacuolar

phospholipase, (iv) inhibition of protein synthesis and (v) interaction with DNA.

However, the main mode of action of CQ seems to be related to the accumulation

of this weak base in the acidic lysosome and binding to ferriprotoporphyrin-IX

polymerase, thereby preventing the detoxification of ferriprotoporphyrin-IX by

polymerization and thus killing the parasite.16

CQ resistance was observed in

Southeast Asia and South America at the end of the 1950s and in Africa in the late

1970s. Resistant parasites accumulate CQ less avidly than do sensitive ones.

Resistance can be reversed in vitro using drugs known to reverse drug resistance

in tumor cells, such as verapamil.17

The observation that CQ resistance appeared


7

rather late, about 10 years after its widespread use, has been considered as an

argument to support the hypothesis that CQ resistance has genetic basis.18

NH

CH3

N

NH2

CH3

O F3C

NH

CH3

N

NH2

N

NH

CH3

N

CH3

CH3

Cl

N

OH

N

CH2

ClCl

CF3 OH

N CH3

CH3

N

NH

Cl

OH

N CH3

CH3

O

CH3

OR

CH3

HCH

3

H

O

O

N

OHNH

CF3

CF3

N

N

NH2

CH3

Cl

NH2

O

OH

O

Cl

NN

NH

S

NH2

O O

S

NH2

NH2

OO

O

O

CH3

CH3

OH CH3

CH3

OH

O

O

CH3

CH3

CF3

CF3

OOOCH

3

CH3

CH3

CH3

OH

Tafenoquine (WR 238605) Primaquine ChloroquineQuinine

HalofantrineAmodiaquine

Quinoline class of antimalarials

R = H DihydroartemisininR = CH3 Artmether

R = Et Arteether

Artemisinin antimalarials

Mefloquine

Quinoline class of antimalarials

Pyrimethamine

Atovaquone

Arylalcohol class of antimalarials

Folate antagonist class of antimalarials

Other antimalarials

Sulfadoxine Dapsone

Yingzhaosu A ArtefleneYingzhaosu C

OCH3

OCH3

OCH3

H3CO

OCH3

H3CO

Figure 2: Examples of some common antimalarial drugs

In spite of its reduced efficacy, CQ is still the most widely used

antimalarial drug in Africa, both for the reason of cost and because of the

widespread prevalence of resistance among people infected with the parasite

towards a large number of other antimalarial drugs. Moreover, tumor necrosis

factor (TNF), a cytokine responsible for some cerebral damages, which is


8

produced by immune system during the malaria crisis, has been proven to have a

synergistic effect with chloroquine, thus enhancing the effect of the drug.19

Amodiaquine is chemically related to CQ, but is more effective than CQ for

clearing parasitemia in cases of uncomplicated malaria, even against some

chloroquine-resistant strains.20

However, drug resistance and potential hepatic

toxicity limit its use. Amodiaquine has been shown to bind to heme and to inhibit

heme polymerization in vitro, with a similar efficiency as CQ.21

Furthermore,

amodiaquine exhibits cross-resistance with CQ suggesting that it exerts its activity

by a similar mechanism.22

1.2.3 Quinolinemethanols

Quinine, the active ingredient of cinchona bark, introduced into Europe

from South America in the 17th

century, had the longest period of effective use

and relieved more human suffering than any other drug for almost 3 centuries. But

now there is an overall decline in its clinical response for P. falciparum in some

areas. As on today quinine is not a first line treatment drug because of its small

therapeutic index, longer period of treatment and the toxicity associated with it.

Nevertheless, it remains an essential antimalarial drug for severe falciparum

malaria and intravenous infusion is, in this case, the preferred route of its

administration. The addition of a single dose of artemisinin enhances the parasite

elimination rate and thus increases the cure rate.23

Quinine interacts weakly with

heme, but has been shown to inhibit heme polymerization in vitro. The

mechanism of resistance to quinine is unknown, but a similar one as that of

mefloquine has been suggested.21

Combination of quinine and clindamycin24

or

quinine and allopurinol25

significantly shorten the duration of treatment with

respect to quinine used alone.

Mefloquine is structurally related to quinine and its long half-life (14-21

days) has probably contributed to the rapid development of resistance towards this

drug. For this reason, mefloquine should be used in combination with other

antimalarial agents. It binds with high affinity to membranes, causes

morphological changes in the food vacuole of Plasmodium and interacts relatively

weakly with free heme. The plasmodial P-glycoprotein 1 (Pgh 1) plays a role in

mefloquine resistance and it may also be the target of this drug.26,27

Lumefantrine

was synthesized by the Academy of Military Medical Sciences in Beijing. It


9

belongs to the quinoline alcohol group that includes quinine and mefloquine. Its

mechanism of action involves the interaction of the drug with heme and it is active

against CQ resistant P. falciparum infection. The antibiotics like tetracycline are

used in combination with quinine for multidrug resistant P. falciparum infection,28

and doxycycline and azithromycin29

are used for the purpose of prophylaxis.

Fluoroquinolone antibiotics showed activity only in the in vitro tests.

1.2.4 Other arylalcohols

Halofantrine is effective against chloroquine-resistant malaria.30

Despite

this, cardiotoxicity has limited its use as a therapeutic agent.31

Mefloquine usage

appears to lead to selection of parasites resistant to halofantrine.32

Furthermore, it

is an expensive drug without availability of parenteral formulations. Pyronaridine,

an acridine derivative, is a synthetic drug widely used in China that may have

utility for multiresistant falciparum malaria.33

Its current Chinese oral formulation

is reported to be effective and well tolerated, but its oral bioavailability is low and

this contributes to an unacceptably high cost of the treatment. It seems likely that

drug resistance would emerge rapidly if pyronaridine was used in monotherapy.

As reported above, resistance to a lot of antimalarial drugs has been observed in

clinical isolates, but resistance to mefloquine, quinine and halofantrine appears to

be inversely correlated with resistance to chloroquine and amodiaquine,

suggesting that the development of a high level of resistance to chloroquine makes

the parasite more sensitive to the arylmethanols.34

1.2.5 Folate antagonists

These compounds inhibit the synthesis of parasitic pyrimidines and thus

of parasitic DNA. There are two groups of antifolates (i) the dihydrofolate

reductase (DHFR) inhibitors like the antimetabolite antimalarial drugs,

pyrimethamine and proguanil and (ii) the dihydropteroate synthase (DHPS)

inhibitors having sulfones and sulphonamides like sulfadoxine and dapsone.

Due to a marked synergistic effect, a drug of the first group is usually

used in combination with a drug of the second one. Unfortunately, resistance is

widespread in Asia and now in Africa.35

Pyrimethamine-sulfadoxine (SP,

Fansidar) is the most widely used combination. It is cheap, practicable (only one

dose is needed because of the slow elimination of the drugs from the body) and


10

currently efficient in many parts of Africa. However, it is poorly active against

highly chloroquine-resistant strains. SP is also particularly prone to rapid

emergence of resistance. The mechanism of resistance for this combination has

been shown to be due to mutations in the genes of DHFR and DHPS.36

Proguanil

has also been combined with CQ in some oral formulations. It should be

mentioned that proguanil is a prodrug, its P-450 metabolite cycloguanil is the

active compound. Chlorproguanil is a chlorinated analog of proguanil that is also

metabolized as an active triazine compound. It is more efficient and has a larger

therapeutic index than proguanil and its combination with dapsone is eliminated

more rapidly than SP, offering the possibility of lowering the selection pressure

for resistance.37

Finally, the development of a combination of proguanil and

atovaquone (Malarone) would provide another useful way for the treatment of

malaria.38

Atovaquone, a hydroxynaphthoquinone derivative, is an analog of

ubiquinone, a parasite mitochondrial electron-carrier which is a cofactor of the

dihydroorotate dehydrogenase enzyme. Atovaquone acts by inhibiting parasite

mitochondrial electron transport. However, the mechanism of synergy of

proguanil with atovaquone is complex. This combination is well tolerated and

more effective than CQ alone, CQ-SP 39

or mefloquine,40

against acute

uncomplicated multidrug resistant P. falciparum. It is also effective in regions

where proguanil alone is ineffective due to resistance. Unfortunately, atovaquone

is expensive and not easily affordable in most of the African countries.

1.2.6 Artemisinin derivatives

To date, resistance has emerged to all classes of antimalarial drugs

except artemisinins41,42

and therefore, most of the African countries have adopted

artemisinin-based combination therapy (ACT) as the first-line treatment for

uncomplicated malaria but some recent reports have challenged credibility and use

of ACTs due to reduced efficacy of certain artemisinin-based combination

therapies.6 Artemisinin derivatives are the fastest acting antimalarial drugs.

43 Four

compounds that have been used are the parent artemisinin extracted from Chinese

herb ‘qinghao’ (Artemisia annua.)44

and three derivatives that are actually more

active than artemisinin itself.43

One of them is a water-soluble hemisuccinate

artesunate and the other two are oil-soluble ethers, artemether and arteether. All of

them are readily metabolized to the biologically active metabolite,


11

dihydroartemisinin. Artemisinin is active in nanomolar concentrations in vitro on

both CQ-sensitive and resistant P. falciparum strains. These drugs are fast acting

and act against gametocytes, the sexual forms of the parasite that infect

mosquitoes. The treatment of several million patients with artemisinin derivatives

for acute malaria failed to detect any significant toxicity,45

even for pregnant

women,46

despite the fact that neurotoxicity was observed in animals with higher

doses than used clinically. Artemisinin and its derivatives appear to be the best

alternative for the treatment of severe malaria47

and arteether has been included in

the WHO List of Essential Drugs for the treatment of severe multidrug resistant P.

falciparum infection. Arteether is having advantage over artemether as it is more

lipophilic and gives less toxic metabolite (ethanol versus methanol). In this

family, The Walter Reed Institute of Research has patented a stable, water-soluble

derivative called artelinic acid that is now being tested in animals. When

compared with other artemisinin derivatives, artelinic acid had the highest plasma

concentration, the highest oral bioavailability, the longest half-life, the lowest

metabolism rate and the lowest toxicity at equivalent doses.48

A key advantage of

these endoperoxide containing antimalarial agents, which have been used for

nearly two decades is the absence of any drug resistance. When several strains of

P. berghei or P. yoelii were exposed to a selection pressure by artemisinin or

synthetic analogs within infected mice, resistance proved very hard to induce. A

low level of resistance has been observed which disappeared as soon as the drug

selection pressure was withdrawn. Furthermore, with a synthetic analog of

artemisinin, BO7, resistance to the drug was lost when drug pressure was

removed; it was not regained once drug pressure was re-applied.49

Remarkably,

the introduction of artemisinin derivatives in routine treatment in some areas of

Southeast Asia has been associated with a significant reduction of incidences of

falciparum malaria. In fact, artemisinin derivatives prevent gametocyte

development and therefore reduce the transmission.

The major drawback of artemisinin derivatives is their short half-life (3-

5 hours). When used in monotherapy, a treatment as long as 5 days is required for

complete elimination of the parasites. They are then preferentially used in

combination with other antimalarial agents such as sulfadoxine-pyrimethamine,50

benflumetol,45

mefloquine51

or chlorproguanil-dapsone to increase cure rates and

to shorten the duration of therapy in order to minimize the emergence of resistant


12

parasites. Combination of artemether or artesunate and mefloquine has been used

in areas of multidrug resistance in Southeast Asia. When associated with

lumefantrine (benflumetol, a slow eliminated oral drug,) artemether is as effective

as the artesunate-mefloquine combination and better tolerated. Artemether clears

most of the infection and the lumefantrine concentration that remains at the end of

the 3 to 5-day treatment course is responsible for eliminating the residual

parasites. This combination is safe in patients with uncomplicated falciparum

malaria and even in children. It clears parasites rapidly and results in fewer

gametocytes carriers. The only limiting factor of artemisinins in comparison to

common antimalarials is their treatment cost and duration of course in

monotherapy, which make them least affordable in case of the low income

countries sharing world’s major malaria episodes and its associated deaths.52

The

relatively high cost and erratic supply of the natural parent compound artemisinin

make it necessary to develop new synthetic and cheap endoperoxide-based

antimalarials.53,54

1.2.7 Other antimalarials

Yingzhaosu A and Yingzhaosu C, two naturally occurring peroxides

with bisabolene skeleton have been isolated from the roots of the plant yingzhao

(Artabotrys uncinatus), both showing significant antimalarial activity in vitro.54-56

Arteflene a synthetic analog of Yingzhaosu A is under clinical developement. The

compound has low human toxicity as it interacts with specific parasite proteins,

fast acting and equipotent to artemisinin and has better chemical stability. Its

single dose can lasts for longer period of time.57-59

1.3 New antimalarial drug development approaches

Due to complex life cycle and different causative strains of the parasite

and high mutability of the genome of Plasmodium falciparum, development of

malaria vaccine has met with failures time and again hence, identification of new

targets have been explored for the development of new drugs acting via different

mechanisms in order to avoid fast development of resistance.60

The P. falciparum

genome sequencing has revealed a number of new targets for drug and vaccine

development.61,62

But discovery of new targets, the developmental time and costs

are also likely to be too high for such an approach. Inhibition of the proteases that


13

specifically degrade hemoglobin in the parasite food vacuole, the plasmepsins,

falcipains and falcilysins, is an interesting area of research.8 Also very promising

are targets associated with pathways specific to the apicoplast, such as the

shikimate pathway and the 2-deoxy-D-xylulose-5-phosphate (DOXP) pathway.63

Cellular and biochemical targets in the mosquito vector are worthy of

consideration as well.64

One group of workers has demonstrated the viability of

using a molecular connectivity QSAR approach to identify new active antimalarial

agents.65

1.3.1 Old targets new compounds

The main challenges associated with antimalarial drug development

program are high costs for developing new targets, development of resistance to

the available drugs by the parasite and general lack of interest among the major

pharma players, as the disease belongs to the poor third world countries. Other

challenges associated with it are the cost, efficacy, target specificity, oral-

bioavailability and safety. A new antimalarial drug must meet all standard drug

development criteria of a new molecule so, an alternative strategy is the

exploitation of the existing targets.66

As the major target population is from Africa

and other developing countries, any new drug development program must address

the challenge of cost and drug resistance along with all standard drug development

criteria. The free heme liberation in the parasite food vacuole is also an “old” but

always an attractive pharmacological target. It could be the most specific target

that can be exploited since it comes from the hemoglobin digestion by the parasite

that occurs only in infected erythrocytes.

Many chemical entities are directed toward this well known target, such

as CQ and artemisinin derivatives. CQ is a cheap and easy-to-prepare molecule

that has proved its credibility as it is highly effective, safe and well-tolerated drug

for treatment and prophylaxis. However, the spread of resistance has resulted in a

huge reduction in the utility of CQ. Many chemical modifications have been

attempted to obtain a molecule as affordable as CQ and equally active even on

resistant strains. These modifications carried out are substitutions in the quinoline

nucleus, variations in the side chain, synthesis of bisquinolines and more recently,

the introduction of a ferrocenyl moiety.67

A little modification could provide a


14

new compound that would be active on resistant strains; but the wait for a safe and

effective chloroquine alternative still continues.

Artemisinin and its derivatives (artemether, arteether and artesunate) are

increasingly used in Asia and Africa where multidrug-resistant P. falciparum is

prevalent. They are rapidly effective and well-tolerated treatments, but the total

synthesis is too complex to be exploited and the yield of extraction from the plant

is too low, despite research efforts toward the enhancement of artemisinin or one

of its precursor’s productions in A. annua. As a result, they remain expensive

treatments that are hardly accessible to people in endemic areas. The cost will also

be limitative for sophisticated artemisinin derivatives.68

Synthetic trioxanes,

simplified analogs of artemisinin retaining the crucial endoperoxide bridge have

been developed, but none of them could enter into clinical trials successfully.69

1.3.2 Proteases-general introduction

Proteases or proteolytic enzymes found in plants, bacteria, virus,

protozoa, fungi and mammals are essential for replication and are involved in a

series of essential metabolic and catabolic processes, such as protein turnover,

Table I: Proteases in disease propagation and their functions70

Protease Function Involved in disease

HIV-1 Protease HIV replication AIDS

Renin Generation of angiotensin I Hypertension

Thrombin Blood coagulation Stroke, vascular clots

Tryptase Phagocytosis Asthma

Cathepsin K Bone resorption Osteoporosis

ACE Generation of angiotensin II Hypertension

Neutral endoprotease Release of ANP Hypertension

Rhinovirus 3C protease Viral replication Common cold

Falcipain Proteolysis Malaria

Plasmepsin I & II Proteolysis Malaria

Cruzain Proteolysis Chagas’ disease


15

digestion, blood coagulation, apoptosis, fertilization, cell differentiation and the

immune response system.71

Proteases have enormous commercial importance in

industrial enzyme applications, mainly in detergent, food and leather industries.72

Proteases selectively catalyze hydrolysis of large polypeptides into smaller

peptides and further into amino acids thus facilitating their absorption by the

cells.73

Their virtual control over protein synthesis in human beings and other

microorganisms enable them to regulate physiological processes.74

They have

been implicated in many health problems (Table-I) involving the circulatory

system, immune system, allergies, sports injuries and infectious diseases. They

catalyze important proteolytic steps in tumor invasion or in infection cycle of a

number of pathogenic microorganisms and viruses. This makes proteases a

valuable target for the development of newer drugs.

1.3.3 Protease inhibitors as drugs

Proteases are involved in a number of diseased states and thus have

gained tremendous attention as therapeutic targets with regard to viral and

parasitic infections, stroke, cancer, Alzheimer’s disease, neuronal cell death and

arthritis. They are designated either as endo or exopeptidases that cleave peptide

bonds within a protein or peptide and remove amino acids from the N or the C-

terminus. Depending on the catalytic residue responsible for peptide hydrolysis,

the proteases have been divided into classes like serine, cysteine, aspartic and

metalloproteases.71,75

NNH

NH

O

O

OH

N

O CH3

N

O

NH2

CH3

CH3 S

N

N NH

NH

NHS

N

CH3

CH3

CH3

O

CH3

CH3

O OH

O

O

NSH

O

CH3

COOH

NNH

O

CH3

COOH

Saquinavir Ritonavir

Captopril Enalapril

EtOOC

Figure 3: Protease inhibitor drugs available in market

Proteases are crucial for propagation of certain diseases and are

emerging as promising therapeutic targets to be used in the treatment of these


16

diseases. These diseases include inflammation, tumor progression, parasitic,

fungal and viral infections (e.g. malaria, schistosomiasis, C. albicans, HIV, herpes

and hepatitis), immunological, respiratory, neurodegenerative and cardiovascular

disorders. Protease inhibitors thus, have considerable potential utility for

therapeutic intervention in a variety of disease states.73,76

There are many

examples of protease inhibitors as highly successful drugs.

The human immunodeficiency virus protease (HIV-1 protease) has

proved to be an attractive target due to its essential role in the replicative cycle of

HIV. Several low molecular weight HIV-1 protease inhibitors (MW < 1000 Da)

are now used as drugs, including saquinavir, ritonavir, indinavir, nelfinavir and

amprenavir. These are among the first successful examples of receptor/structure

based designer drugs. Keeping in mind the prior knowledge of inhibition of other

aspartic proteases (eg. renin) these compounds were designed and developed by

docking these structures on the active site of HIV-1 protease.70

Captopril and

enalapril are the examples of inhibitors of a metalloprotease, angiotensin-

converting enzyme (ACE) used successfully for the control of hypertension.77

1.3.4 Cysteine proteases and related aspects

Cysteine (thiol) proteases exist in three structurally distinct classes,75

which are papain-like (e.g. cathepsins), ICE-like (caspases) and picorna-viral type.

Several members of the cysteine protease family of enzymes have been implicated

as possible causative agents in a variety of diseases. More notable examples

include cathepsin K in degradation of bone matrix, cathepsin-L and S for MHC-II

antigen presentation, the caspases in programmed cell death, rhinovirus 3C

protease for viral processing, falcipain and cruzain in parasitic infections and the

possible role played by the gingipains in periodontal diseases. The biggest

problem in designing inhibitors for cysteine proteases is the similarity in their

substrate affinities and proteolytic mechanisms with the serine proteases.

Although, the spatial configurations of the catalytic triads of serine and cysteine

proteases are quite similar, it appears that the oxyanion hole and the negatively

charged tetrahedral intermediate are the central features of the catalytic

mechanisms of serine proteases, while cysteine proteases stabilize the later more

neutral acyl intermediate with the help of imidazole ring of histidine. This


17

mechanistic difference could be exploited in the development of potent, reversible

and selective transition state analogs as potential drugs.

Like serine proteases, cysteine proteases tend to have relatively shallow

solvent exposed active sites that can accommodate short substrate/inhibitor

segments of protein loops or strands. Most of the inhibitors developed to date tend

to be 2-4 amino acids or their equivalents in chain length, interacting with the

nonprime subsites of the enzymes and terminating with various electrophilic

isosteres. Recently it has been convincingly demonstrated, for a wide range of

proteases including cysteine protease, that they universally bind their

inhibitors/substrates in extended or β-strand conformations.

1.4 Malarial cysteine proteases: new target for chemotherapy

1.4.1 Malarial cysteine proteases’ role in hemoglobin degradation

Erythrocytic stage of the malaria parasite P. falciparum has developed

complex proteolytic machinery for haemoglobin digestion.78

Malaria parasite

ingests and degrades most of the host cell hemoglobin during the morphologically

separate phases inside the erythrocyte (ring, trophozoite and schizont stages).79

Hemoglobin degradation is essential to the survival of the parasite, as interruption

of this process with a variety of protease inhibitors leads to death.80

The amino

acids derived from hemoglobin degradation are incorporated into parasite proteins

or utilized for energy metabolism.81,82

It has also been suggested that removal of

hemoglobin through degradation provides space in the red blood cell for the

growing parasite and prevents premature erythrocyte lysis.83

Massive degradation of hemoglobin generates a large amount of toxic

heme, which causes membrane damage due to its peroxidative properties.84

The

released hematin is detoxified to a cyclic dimer, -hematin. These dimers

crystallize to give hemozoin (insoluble malaria pigment).11

This massive catabolic

feat is carried out in a specialized organelle, the food vacuole and is most

pronounced during the trophozoite stage of development.10

More recent studies

indicate that the crystallization process from -hematin to hemozoin takes place in

or closely associated with, neutral lipid nanospheres in the aqueous content of the

vacuole.85

A major function of the food vacuole is to degrade the host red cell

hemoglobin sequestered through the cytostome machinery and provide amino


18

acids to the parasite. This is brought about by a variety of proteases and the

inhibition of this process is well investigated as a drug target.86,87

These enzymes

act at an optimum pH in the range of 4.5-5.0, i.e. pH of the digestive vacuole.78

A

low digestive vacuolar pH further promotes biomineralization of heme to

hemozoin.88

Endocytosis of host cell cytosol

Oxyhemoglobin

Plasmepsins I, II

(>20AA) Peptides

Falcipains (2, 3)

Small (6-8AA) Peptides

Falcilysins

DPAP

Amino Acids

Ferroproto-

porphyrinHeme

Polymerization

Hemazoin

Food Vacuole

2O-2H2O2

H+O2

H2O+1/2O2

Amino peptidaseSmall Peptides

Globin

Plasmepsin (IV & HAP)

Figure 4: Role of cysteine proteases in hemoglobin digestion89,90

In P. falciparum, proteases involved in hemoglobin degradation have

been proposed to act in a semi-ordered fashion as outlined in Fig. 4. Ten aspartic

proteases named plasmepsins (PMs) were identified in the genome of P.

falciparum and four of them, namely palsmepsin I, II and IV and histo-aspartic

protease (HAP) participate in hemoglobin degradation.89,90

Plasmepsin I and II

have been postulated to initiate hemoglobin degradation, by hydrolyzing the

peptide bond between residues Phe33 and Leu34 of native hemoglobin. 90,91

This

initial cleavage is thought to promote unfolding and release of the heme moiety

followed by further degradation by the plasmepsins,87

the cysteine proteases

(named falcipain-2 and falcipain-3),92-94

a metalloprotease (named falcilysin)95

and the lately discovered dipeptidyl aminopeptidase (DPAP).96

However, the

precise sequence of events particularly whether a plasmepsin or a falcipain

catalyzes the initial degradation step is still under debate.80

Disruption of each plasmepsin gene was achieved and these studies

confirmed that all these four vacuolar enzymes play important roles for parasite


19

development though not an essential one, as PMs knockouts possess the ability to

compensate for the loss of individual plasmepsin function.97

On the other hand, it

is known that inhibition of cysteine proteases turns out to be lethal for parasites.

Cysteine protease inhibitors irreversibly block the rupture of host cell membrane,

which prevent parasites to invade fresh erythrocytes.98

The metalloprotease,

falcilysin is only able to cleave small polypeptides, up to 20 amino acids,

producing even shorter oligopeptides.95

DPAP1 cleaves off dipeptides from

hemoglobin-derived oligopeptides in the food vacuole.96

Finally, small peptides

may be pumped out of the food vacuole into the cytoplasm and an amino

peptidase activity provides amino acids essential for parasite’s survival.99,100

1.4.2 Falcipains’ role in proteolysis

The best characterized P. falciparum cysteine proteases initially known

as trophozoite cysteine protease (TCP) and later termed as falcipains share

sequence identity and other features with papain family cysteine proteases.

Falcipain-1 (FP-1),92

nearly identical copies of two falcipain-2 (FP-2) (falcipain-2

and falcipain-2’, also known as falcipain-2A and falcipain-2B, respectively)93,101

and falcipain-3 (FP-3)94

are all expressed during the erythrocytic stage of the

Plasmodium life cycle.102

Falcipain-2’ is not fully understood and the knock out

studies have revealed that there is no significant change in its absence in parasite’s

function in erythrocytic stage.101

FP-1, located on chromosome 14, shares approximately 38-40%

sequence identity with FP-2 and FP-3. The function of FP-1 remains uncertain

because of its low abundance and inadequate systems for the production of

recombinant enzyme. FP-1 has been proposed to play a role in parasite invasion as

a relatively specific inhibitor of FP-1 blocked parasite invasion of host

erythrocytes independent of hemoglobin degradation.103

However, a subsequent

study showed that FP-1 knockout parasites developed normally in erythrocytes,

suggesting that this protease alone is neither required for parasite invasion nor

during the intracellular development within erythrocytes.92

The gene disruption

studies clearly demonstrated an important role for FP-1 in oocyst production

during parasite development in the mosquito midgut, but not an essential role in

asexual or gametocyte/gamete development. FP-1 could be directly involved in

the actual transition from gamete to oocyst by activating proteins by proteolytic


20

processing or, if secreted, it could degrade the peritrophic matrix or midgut

endothelium facilitating the migration of the ookinete.104

In contrast, the well-characterized function of FP-2 and FP-3 includes

degradation of host hemoglobin required for sustaining the metabolic needs of

rapidly growing parasite.93

They show a high level of amino acid sequence

homology (65%) but a different substrate specificity,105

although both enzymes

favor substrates with Leu at P2.93,94

FP-2 and FP-3 are primarily localized in the

food vacuole, consistent with their roles in hemoglobin digestion. Both falcipains

are synthesized over fairly long interval during the erythrocytic cycle as

membrane-bound pro-forms that are processed to soluble mature forms. However,

FP-2 is synthesized earlier, in the beginning of early trophozoites and is processed

more quickly than FP-3 which showed peak expression in late trophozoites/early

schizonts stage. Cysteine protease inhibitors and the lactone antibiotic brefeldin-

A, blocked the processing of both enzymes, suggesting that FP-2 and FP-3 are

trafficked to the food vacuole via the endoplasmic reticulum/Golgi network and

that they are processed by autohydrolysis (which occurs at neutral pH) before

arrival at the food vacuole.106

Genetic ablation of FP-2 gene, located on chromosome 11 underscored

the critical importance of this protease in hemoglobin degradation,107

which is

actually considered the prime target for discovery of novel antimalarial drugs.

During the late trophozoite and schizont stages, FP-2 is involved in the

degradation of erythrocyte membrane skeletal proteins, including ankyrin and the

band 4.1 protein.108

This activity displays a pH optimum in the range of 7.0-7.5

and is thought to contribute to destabilization of the erythrocyte membrane,

leading to host cell rupture and release of the mature merozoites, hence FP-2

shows stage specific activity i. e. in early trophozoite stage it degrades

hemoglobin in acidic pH and in neutral pH it cleaves the host erythrocytic skeletal

membrane proteins ankirin and protein 4.1, vital for the stability of RBC

membrane. 93,109

The disruption of FP-2 gene revealed that there is transient block in

hemoglobin hydrolysis but the loss of this enzyme alone is not sufficient to cause

parasite lethality, the parasite recovered from the defects later presumably due to

expression of FP-3 in the later stage of life cycle of the parasite, thus suggesting

the participation of additional cysteine proteases for parasite invasion and growth


21

in human erythrocytes. Alternatively, the development of parasite lethality might

require concomitant deletion of multiple cysteine proteases. Unlike other

falcipains, the FP-3 gene could not be disrupted, but its replacement with a tagged

functional copy has been recently achieved.110

These studies revealed that FP-3 is

essential to erythrocytic parasites, suggesting that efforts to develop cysteine

protease inhibitors as antimalarial drugs should probably be focused on FP-2 and

FP-3.

FP-2’ was the last cysteine protease to be identified through analysis of

genetic variability among isolates of P. falciparum. These studies revealed that P.

falciparum contains a nearly identical copy of the FP-2 gene, also located on

chromosome 11, encoding a distinct enzyme. These two genes are actually

paralogs, which share 96% identity at the nucleotide level and 93% identity at the

amino acid level. The products of these paralogs were designated as FP-2 and FP-

2’.111

Their amino acid sequences differ at seven positions within their mature

forms, including three amino acid replacements localized close to residues that are

predicted to interact with substrate and consequently may affect its affinity or

specificity.112

Recombinant FP-2’ protein expressed in bacteria exhibits protease

activity as FP-2 in erythrocytic parasites but importantly, the recombinant FP-2’

cleaves host ankyrin but not protein 4.1. Functional analysis revealed similar

biochemical properties to those of FP-2, including pH optima (pH 5.5-7.0),

reducing requirements and substrate preference. Notwithstanding its predicted

hemoglobinase function, the P. falciparum FP-2’ may contribute and orchestrate

selective proteolytic events during the exit of malaria parasite from human red

blood cells.113

However, gene disruption studies demonstred that FP-2’ knockout

parasites showed no differences in morphology neither in sensitivities to cysteine

or aspartic proteases compared to the wild-type parasites.110

These results indicate

a subordinate role for FP-2’ in parasite development.

All falcipains except FP-1 are involved in the conversion of

proplasmepsins into their active forms. FP-2’ and FP-3 are expressed later in the

erythrocytic cycle of the parasite. This would suggest that FP-2 might serve as the

dominant proplasmepsin maturase, although the contribution of FP-2’ and FP-3

cannot be ruled out.114


22

1.4.3 General structural features of falcipain-2

The mature FP-2 is a single polypeptide chain of 241 amino acids,

synthesized during the trophozoite stage as a membrane-bound proenzyme

comprising 484 amino-acid residues.93

The proenzyme is transported to the food

vacuole through the endoplasmatic reticulum/Golgi system and during this

process the N-terminal 243 residues containing the membrane anchor are

proteolytically removed. An autoproteolytic processing mechanism was suggested

on the basis of inhibitor studies110

and from the observation that the recombinant

proenzyme undergoes spontaneous processing during in vitro refolding.93

The crystal structure of the free FP-2 (inactivated by iodoacetamide, pdb

code 2GHU)115

and in complexation with cystatin (1YVB pdb code)116

(Fig. 5)

has been deposited in the Protein Data Bank. More recently, the crystal structures

of FP-2 in complexation with epoxysuccinate E-64 (3BPF) and FP-3 with

aldehyde leupeptin have also been reported.117

FP-2 generally adopts a classic papain-like fold in which the protease is

divided into L (left) and R (right) domains. The catalytic residues Cys42, His174

and Asn204 (Cys25, His159 and Asn175 following papain convention) are located

in a cleft at the junction between the two domains (Fig. 6). Like papain and the

majority of C1A proteases, FP-2 also has the disulfide bond Cys168-Cys229 to fix

the upper loops defining the S2 and S1’ substrate-binding sites. An additional

Cys99-Cys119 disulfide bond, predicted on the basis of homology modeling

studies,118

appears to play an important role together with the Cys73-Cys114

disulfide in stabilizing the long 4/ 2 loop (~30 residues) that blankets the lateral

surface of the L-domain.

The most extraordinary features of FP-2 are two unique structural

elements: a ‘‘noselike’’ projection (at the N-terminus) connecting the L and R

domains and an ‘‘arm-like’’ structure (near the C-terminus of the mature protein)

extending away from the protease surface.116

FP-2 nose consists of 17 amino acids

and deletional analysis of this sequence119

confirms that FP-2 nose is fundamental

for proper folding; generally it possesses a short significant element of secondary

structure and the amino acid Glu138 forms a buried hydrogen bond with Tyr4 and

a salt bridge with Arg5. This clearly indicates that FP-2 nose, even if shorter than

the standard papain family prodomain, may play a critical role as chaperone for

protease folding. Furthermore, in a different way from the other members of the


23

a

b

Figure 5: a) Crystal structure of FP-2 and cystatin complex. b) Overall structure of FP-2-cystatin is

depicted in stereo in a conversational orientation for papain-like cysteine proteases: with

the L domain (Left), the R domain (Right) and the active site on the top facing

forward.116

papain family, interactions of a residue in the protein core (Glu138) with the FP-2

nose may provide the additional binding energy essential to stabilize initial

interactions for folding.


24

FP-2 arm is composed of 14 amino acids located at the distal end of the

R domain (residues 183-196). It is a protruding arm-like structure of two extended

strands connected by an abrupt turn and generally it does not establish any

contact with the protease core. Mutagenesis studies suggest that FP-2 arm may

interact with the natural substrate of FP-2, hemoglobin. However, because FP-2

arm is > 25A˚ away from the protease active site, FP-2 arm probably functions in

hemoglobin hydrolysis through distal-site binding rather than direct participation

in substrate cleavage.

Figure 6: Crystal structure of FP-2 and FP-3 bound to small molecule inhibitors; implications

for substrate specificity 117

The 14-residue hemoglobin-binding -hairpin exhibits a high degree of

conformational flexibility when freely exposed to solvent bulk. The large

positional deviations exhibited by the -hairpin, while comparing the structure of

the free FP-2 and that of the FP-2-cystatin complex further underscores an

inherent flexibility and plasticity of this element that may have functional

implications in hemoglobin binding.

Recently, the co-crystallized FP-3-leupeptin structure (3BPM) has been

reported; FP-3 also possesses the two unique insertions discussed above, that

distinguishes plasmodial cysteine proteases from all other structurally

characterized papain family enzymes: the noselike domain located at the N-

http://pubs.acs.org/doi/abs/10.1021/jm8013663?prevSearch=falcipain&searchHistoryKey=

http://pubs.acs.org/doi/abs/10.1021/jm8013663?prevSearch=falcipain&searchHistoryKey=


25

terminus (AA 1-25) and the flexible 14-residue -hairpin at the C-terminus

outside of the two unique motifs discussed above. The rest of FP-2/3 is

structurally similar to homologous proteases in the C1A family. The active site is

located in a cleft between the structurally distinct domains of the papain-like fold.

The S2 pocket is the major determinant of specificity for most cysteine proteases

and its predominantly hydrophobic nature in FP-2 is confirmed by the fact that

FP-2 has a strong preference for substrates with a hydrophobic residue,

particularly Leu, at the P2 position.93

The crystal structure reveals that although

the S2 subsite is generally hydrophobic, the electronegative side chain of Asp234

is positioned very deep in the S2 pocket. Therefore, the presence of basic side

chain in the P2 position could form a salt-bridge interaction with Asp234, adding

further insights useful to explore the structural determinants of FP-2 specificity.

1.5 Falcipain-2 inhibitors

To date, many chemotypes have been identified as inhibitors of FP-2

such as peptides, peptidomimetics, isoquinolines, thiosemicarbazones, nonpeptidic

chalcones, pyrimidinenitriles and others which are able to inactivate the enzyme in

a reversible or irreversible manner. The majority of malarial cysteine protease

inhibitors are peptidic or peptidomimetic compounds in which the hydrolysable

amide bond is replaced by an electrophilic functionality. In this way, the catalytic

thiol of the enzyme reacts with the inhibitor to form a covalent complex. Until

recently, most potent cysteine protease inhibitors were irreversible inhibitors, in

which the electrophilic “warhead” would alkylate the enzyme, through

nucleophilic displacement or conjugate addition. Examples of this approach

include fluoromethyl ketones, acyloxymethyl ketones, vinyl sulfones, or

epoxysuccinates.

Alternatively, potent reversible inhibition can be achieved through

highly electrophilic warheads like aldehydes, ketoamides and nitriles, which form

reversible, covalent bonds to the thiol active site. As synthetic peptide inhibitor of

the P. falciparum schizont cysteine protease, Pf 68 inhibited erythrocyte invasion

by cultured parasites120

. The most effective peptide, GlcA-Val-Leu-Gly-Lys-

NHC2H5 inhibited the protease and blocked parasite development at high

micromolar concentrations (IC50 = 900 μM)120

. The natural triterpene betulinic

acid and its analogs (betulinic aldehyde, lupeol, betulin, methyl betulinate and


26

betulinic acid amide) caused concentration dependent alterations of erythrocyte

membrane shape towards stomatocytes or echinocytes according to their hydrogen

bonding properties.121

Thus, analogs with a functional group having a capacity of

donating a hydrogen bond (COOH, CH2OH and CONH2) caused formation of

echinocytes, whereas those lacking this ability (CH3, CHO, COOCH3) induced

formation of stomatocytes. Both kinds of erythrocyte alterations were prohibitive

with respect to P. falciparum invasion and growth; all compounds were inhibitory

with IC50 values in the range of 7-28 M and the growth inhibition correlated well

with the extent of membrane curvature changes assessed by transmission electron

microscopy. Although these results do not demonstrate the levels of inhibition

expected to be therapeutically relevant, they suggest that a specific protease

activity is required for erythrocyte invasion by malaria parasites and thus is a

potential target for antimalarial drugs.

The active site of papain and related proteases is large and can be

divided into pockets at each side of the catalytic site, S1-S4 and S1’-S3’, with each

pocket accommodating one amino acid residue of the peptide substrate.122

The

general structure of a cysteine protease inhibitor (Fig. 8) should comprise an

electrophilic moiety for interaction with the cysteine residue of the enzyme and

one or two series of substituents, P1-P4 and/or P1’-P3’, responsible for interactions

with the different binding pockets of the protease.

The irreversible inhibitors are better characterized by means of kinetic

constants: the dissociation constant Ki measures the affinity of inhibitor for the

enzyme, the kinac describes the velocity of formation of covalent enzyme inhibitor

complex and the second-order rate constant of inhibition k2nd that determines their

potency.

1.5.1 Peptide-based falcipain-2 inhibitors

1.5.1.1 Peptidyl fluoromethyl ketones

Fluoromethyl ketones are in general, highly reactive and selective

irreversible inhibitors of cysteine proteases.123

A number of peptidyl fluoromethyl

ketones were tested against FP-2,124

previously named as P. falciparum

trophozoite cysteine proteinase (TCP). In this context CBZ-Phe-Arg-CH2F (1)

(Fig. 7) proved to be the most potent inhibitor of FP-2 at picomolar concentration

(IC50 = 0.36 nM), to block the hemoglobin degradation (IC50 = 0.10 mM) and to


27

inhibit the development of cultured malaria parasites in the nanomolar range (IC50

= 64 nM).125

CBZ-Phe-Arg-CH2F (1) was effective at nanomolar concentrations

on four different strains of P. falciparum (Itg2, FCR3, W2, D6) and proved to be

nontoxic to four human cell lines.

A number of fluoromethyl ketones characterized by the presence of a

morpholineurea (Mu) group at P3 position was tested against FP-2; the most

effective inhibitors were Mu-Phe-HomoPhe-CH2F (2) (IC50 = 3 nM) and Mu-Leu-

HomoPhe-CH2F (3) (IC50 = 0.42 nM). Mu-Phe-HomoPhe-CH2F (2), which proved

to be the most effective inhibitor against P. vinckei cysteine protease (IC50 = 5.1

nM against P. vinckei protease) is biochemically similar in terms of molecular

mass, pH optimum, substrate specificity and inhibitor sensitivity to FP-2. It was

also tested in vivo in a murine malaria model.126

When administered parenterally

N NH

NH

CH2F

O

O

O

O

O NH

NH

CH2F

O

O

NH

O

NH2

NH

N NH

NH

CH2F

O

O

O

O

CH3

CH3

(1)(2)

(3)

Figure 7: Structures of peptidyl fluoromethyl ketones against FP-2

four times a day for 4 days to P. vinckei infected mice, the inhibitor (2) cured 80%

of the treated mice. However, the use of peptidyl fluromethyl ketones in therapy

had an important limitation due to their proteolysis by serum, tissue and gut

proteinases. These in vivo assays clearly evidenced that the serum half-life of 2

was short and that the efficacy of the inhibitor could be improved only by its

frequent administrations

1.5.1.2 Peptidyl vinyl sulfones

Vinyl sulfones (VS) well known covalent inhibitors of FP-2 are able to

inactivate the enzyme by irreversible addition of the thiol group of active site

Cys42 to the electrophilic vinyl sulfone moiety, which behaves as a Michael

acceptor.127

It is proposed that a hydrogen bond from the protonated active site

His174 to one of the sulfone oxygens might polarize the vinyl group, thus further

activating the -carbon proposed that a hydrogen bond from the protonated active

site His174 to one of the sulfone oxygens might polarize the vinyl group, thus


28

further activating the -carbon proposed that a hydrogen bond from the protonated

active site His174 to one of the sulfone oxygens might polarize the vinyl group,

thus further activating the -carbon toward nucleophilic attack (Fig. 9).123

NH

NH

O

O

SR

1'

OO

R3

R1

R2

P1'

P1

P2

P3

R1' = Phenyl, O-R, NH-R R1 = Ala, homoPhe, Phe, Arg, ValR2 = Phe, LeuR3 = Morpholine, N-Me-piperazine

Figure 8: General structure of vinyl sulfones, vinyl sulfonate esters and vinyl sulfonamides

The obtained negative charge at the -carbon is eliminated via protonation by the

histidinium residue.128

RNH

S

R

O

O

R

H-His174

H-Asp204

S

Cys42

S

Cys42

RNH

S

R

O

O

R

H-His174

H-Asp204

His174

S

Cys42

RNH

S

R

O

O

R

+ +

-

Figure 9: Mechanism of FP-2 inhibition by vinyl sulfones

Furthermore vinyl sulfones proved to be unreactive toward serine

proteases, metalloproteases, aspartyl proteases, nonactive-site cysteines and

circulating thiols such as glutathione; moreover, substituted vinyl sulfones proved

to be less reactive toward nucleophiles than the analogous vinyl ketones or esters

and sufficiently inert without the target.127

N NH

NH

O

OO

SO

O

N NH

NH

O

OO

CH3

CH3

SO

O

N NH

NH

O

ON

CH3

CH3

SR

OO

CH3

(4) (5) (6) R = Phenyl

(7) R = 2-Naphthyl

Figure 10: Structures of vinyl sulfones containing morpholineurea

A series of vinyl sulfones as novel peptide-based FP-2 cysteine

proteinase inhibitors were reported in P. vinckei.129

For most of the compounds,

inhibitory effects against FP-2 and P. vinckei proteases were similar; however, in

some cases efficacy against the two related enzymes proved to be different and

this clearly indicated a critical role of the peptide portion of the inhibitors in their


29

specificity toward similar enzymes. In this context, Mu-Phe-HomoPhe-VSPh (4)

(Fig. 10) has been proven to be a mid-nanomolar inhibitor of both proteinases

(IC50 = 0.08 M against FP-2 and IC50 = 0.1 M against P. vinckei protease); on

the contrary, the structurally similar peptide Mu-Leu-HomoPhe-VSPh (5)

containing a Leu residue at P2 site improved effectiveness against FP-2, while

activity against the P. vinckei enzyme decreased (IC50 = 0.003 M against FP-2

and IC50 = 0.2 M against P. vinckei protease).

An improved in vivo effectiveness resulted when the morpholineurea

group was replaced with the N-methylpiperazineurea (N-Pipu) group in

compound (6) due to increased aqueous solubility and bioavailability. The phenyl

vinyl moiety was also replaced with a 2-naphthalene group.130

Both of the

compounds (6 and 7) (Fig. 10) showed antimalarial effects correlating with

potency against FP-2. Both of them strongly inhibited hemoglobin degradation,

development and metabolic activity in cultured P. falciparum parasites in the

nanomolar range. The compounds (6 and 7) displayed better activity than the

parent molecule (5) in the inhibition of FP-2 and this clearly showed positive

effect of the replacement of morpholine nucleus with the N-methylpiperazine.

Also the extension of the aromatic area at the P1’ site (i.e. 7) resulted more

fruitful. When the most active compound (7) was administered orally to mice, it

markedly delayed the progression of murine malaria and cured about 40% of the

treated animals. However, many problems common to peptide-based inhibitors

still remained, like: (i) poor pharmacological profile, (ii) reduced absorption

through cell membranes, (iii) susceptibility to protease degradation and (iv)

limited bioavailability.

The structure-activity relationships of a new series of peptidyl vinyl

sulfones, vinyl sulfonate esters and vinyl sulfonamides (Table-II, III) for

inhibition of the protease FP-2 and of P. falciparum development has been

reported.131

Compounds containing the core sequence Phe-homoPhe showed

modest activity against FP-2 and substitutions at position P3 (Fig. 11) in these

compounds had relatively little impact on activity. Compounds with the core

sequence Phe-O-(phenyl)Ser had similar activities, with IC50 for the inhibition of

FP-2 in the mid-to-high nanomolar range. The Leu-homoPhe compounds revealed

to be very active against FP-2, with IC50 values generally in the high picomolar to

low nanomolar range. Identical compounds, except for the P1’ substituent


30

displayed the general rank order of activity: vinyl sulfonate esters > vinyl

sulfonamides > phenyl vinyl sulfones (Tables-II, III). The vinyl sulfones (8) were

potent inhibitors with IC50 in the low nanomolar range.

NNH

ON

OH

N

N

CH3

N

N

OH

N

O

N

O

OCH

3

N

OH

N

N

R NH

NH

O

O

CH3

CH3

SO

O

R NH

NH

O

O

CH3

CH3

SNH

OO

a b c d

e f g h

(9)(8)R =

Figure 11: P3 (R) substituents of peptidyl vinyl sulfones (8) and vinyl sulfonamides (9)

Sulfonates (10) and sulfonamides (9), despite their greater activity

against FP-2, were generally less active against the parasite. In this case, for

compounds with identical structures and with variable P1’ portion, the

antiparasitic activity order was generally (8) > (9) > (10); an order which was

opposite with respect to the activity against FP-2 (Table-I, II). In the vinyl

sulfonates (10) antiparasitic order was observed with ‘X’ group as OCH3 > F > H,

opposite to that reported for the enzymatic inhibition (Table-III).

It is noteworthy that P3 substituents (Fig. 11) showed a marked impact

on the activity against parasite cultures. Three of the six vinyl sulfonate esters (10)

with the highest antiparasitic activities contained an isonipecotic ester moiety (8f,

9f, 10f), while the other ones contained a tertiary amine (8c, 8h and 9c). It is

interesting to observe that compounds containing the isonipecotic ester moiety

proved to be active both against the enzymes as well as the cultured parasites. On

the contrary, those compounds containing a prolinol substituent at P3 (8b, 9b and

10b) were typically potent against the enzymes but showed relatively poor

antiplasmodial activity. The measured second-order rate constants (kassM-1

sec-1

)


31

Table II: Effects of R substituents (P3 Unit) on the activity of peptidyl vinyl sulfones (8) and vinyl

sulfonamides (9) toward FP-2 development of P. falciparum

R

Compound 8 Compound 9

FP-2

IC50

(nM)

P. falciparum

IC50 (nM)

FP-2

IC50

(nM)

P. falciparum

IC50 (nM)

a 35 200 14 1500

b 27 1800 3.6 220

c 8.7 4.5 2.3 4.4

d 9.2 15 2.5 22

e 3 22 2.2 46

f 6.9 3.9 2.2 1.6

g 9.9 21 - -

h 6.7 1.6 - -

Note : Compounds (8e) and (8c) correspond to compounds (5) and (6), respectively.

R NH

NH

SO

O

O

OOX

CH3

CH3

(10)

Table III: Effects of R substituents (P3 Unit) on the activity of vinyl sulfonate esters (10) toward

FP-2 and development of P. falciparum

R X FP-2 IC50 (nM) P. falciparum IC50 (nM)

b H 0.8 390

b F 0.8 394

b CH3O 0.9 220

e H 0.7 49

e F 0.7 84

e CH3O 0.7 14

f H 0.7 42

f F 0.7 120

f CH3O 0.9 9.7

for the inhibition of FP-2 and FP-3 of the most potent vinyl sulfone (8),

sulfonamide (9) and sulfonate (10) inhibitors were generally similar against both


32

of the recombinant enzymes. As seen with IC50 determinations, the vinyl sulfonate

esters were the most potent inhibitors, with kass values against both the proteases

being >105M

-1 sec

-1 for all of the tested compounds. The second-order rate

constants for the phenyl vinyl sulfones and vinyl sulfonamides were lower but

they were consistently >104 M

-1 sec

-1.

1.5.1.3 Peptidyl aldehyde and -ketoamide derivatives

It was found that peptidyl aldehydes and -ketoamide derivatives were

effective to inhibit the enzymatic activity of FP-2 at very low nanomolar range.

The best two inhibitors were revealed to be the aldehyde (11) and the ketoamide

(12) with an IC50 value of 1 nM. The other representative inhibitor was Z-Leu-

homoPhe-al (13) exhibiting an IC50 value of 2 nM.132

Compounds (11-13) (Table-

IV) proved to be active against multiple strains of P. falciparum with the best

activity shown by compound (11) (Table-IV).132

In agreement with older studies of vinyl sulfone inhibitors,129

compounds containing Leu at the P2 position were about an order of magnitude

more potent than those with Phe at this position. The key difference between the

plasmodial cysteine proteases and many other papain family cysteine proteases,

including the abundantly available host proteases cathepsin B and cathepsin L

suggests the potential for specific inhibition of plasmodial enzymes.133

The lead inhibitors (11-12) (Table-IV) when evaluated against

recombinant FP-2 and FP-3 proved to be similarly active against both of the

enzymes. Cysteine proteases of rodent malaria parasites that appear to be

orthologs of FP-2 and FP-3 were also characterized. A single ortholog of the two

P. falciparum proteases appeared to be present in P. vinckei and in three other

rodent malaria parasites.134

Interestingly, all the rodent parasite cysteine proteases

contain an unusual active site substitution that, in the case of the P. vinckei

protease vinckepain-2, mediates unusual kinetics.135

Taking into consideration that

assays in mouse models are generally a key step of malaria drug discovery, it was

of interest to check the activity of peptidyl aldehydes against P. falciparum and P.

vinckei cysteine proteases. The obtained results revealed that vinckepain-2 was

substantially less inhibited by the best inhibitors by about an order of magnitude;

inhibition was particularly poor with a compound containing Phe at P2 consistent

with literature data.


33

N NH

NH

H

O

O

O

O

CH3

CH3

N NH

NH

NH

O

O

O

O O

NH2

O

CH3

CH3

O NH

NH

H

O

O

O

CH3

CH3

(11)(12) (13)

Table IV: Falcipain-2 inhibition and activity against different strains of P. falciparum of peptidyl

aldehyde and a-ketoamide derivatives. (11-13)

Compound FP-2 IC50

(nM)

IC50 (nM)

W2 ItG D6 Dd2 HB3

11 1 1.1 0.96 1.4 1.9 2.6

12 1 2.9 5.3 2.5 2.4 3.1

13 2 8.2 19 12 19 16

Although activity against vinckepain-2 was weak, the in vivo

antimalarial activity of the lead compound Mu-Leu-homoPhe-al (11) was checked

against P. vinckei malaria and these studies demonstrated for this inhibitor: (i)

very poor pharmacokinetic profile, (ii) problems with its formulation and (iii)

need of subcutaneous infusion pumps for administration. This clearly emphasizes

that differences between P. falciparum and P. vinckei targets may contribute to the

limited in vivo efficacies of some cysteine protease inhibitors, if it is considered

that antimalarial drug discovery is based on validation with rodent models before

advancement to a full scale development.

1.5.1.4 E-64

The N-(L-3-trans carboxyoxiran-2-carbonyl)-L-leucyl)-amido(4-

guanido) butane (E-64, Fig. 12), was the first epoxysuccinyl peptide isolated from

a culture of Asperigillus japonicus,136

and its structure was determinated by

Hanada in 1978.137

E-64 is an irreversible inhibitor of papain-like cysteine

proteases (clan CA, family C1), including papain, bromelain, ficin,136

cathepsin

B,138

cathepsin L,139

calpain II 140,141

and cruzain,142

but it does not inhibit clan CD

proteases such as caspases,143

legumains,144

and gingipains,145

and inhibits

clostripain very slowly.146

It shows a second-order rate constant (k2nd) of FP-2

inhibition of 1.16x103

M-1

sec-1

and IC50 values of 0.015 M and of 0.075 M


34

toward FP-2 and FP-3, respectively. Additionally, when tested against P.

falciparum strain FCBR, it revealed an IC50 value of 5.3 M.147

O

NH

NH

NH

O

CH3

CH3

O

H

H

HOOCNH

NH2

E-64

Figure 12: Structure of E-64

E-64 contains a trans (2S,3S) configured epoxide ring, whereas the

amino acid residues of the peptidyl part of the inhibitor have the L-configuration.

On the epoxide ring the substituents at C-2 and C-3 are in trans position to one

another.148,149

Cis configuration leads to total loss of inhibition activity. E-64

inhibits cysteine proteases by S-alkylation of the active site cysteine, which results

in the opening of the epoxide ring.150,151

Its leucine side chain mimics the P2

amino acid of the substrate, occupying the target’s S2 binding pocket, while the

agmatine moiety binds in the S3 position.152

O

NH

NH

O

R

O

HH

X

O

S

Cys

H- His

S

Cys

OH

NH

NH

O

R

O

H

X

O

S

Cys

OH

NH

NH

O

R

O

H

X

O

R1

2 3

+

R1

2 3

R1

2 3

C2 attack C3 attack

Figure 13: Mechanism of inhibition of cysteine protease by Epoxysuccinates

Peptidyl epoxysuccinates can inhibit the enzyme by forming a thioether

bond via a nucleophilic attack at C-2 or C-3 of the epoxide ring by the active site

cysteine residue.153

Attack occurs at either C-2 or C-3 depending on the

orientation of the epoxysuccinate in the active site (Fig. 13). In case of E-64, the

site of attack was identified only at C-2 from NMR experiments.150

The

stereochemistry of the enzyme- inhibitor adduct has undergone an inversion of

configuration at the reaction site due to a nucleophilic attack by the active site


35

thiolate in an SN2 reaction. For instance, E-64, which has the 2S,3S configuration

before the nucleophilic attack, will become 2R,3R after the covalent bond between

the cysteine residue and C-2 of the oxirane ring is formed.

Initially, it was postulated that when E-64 inhibited papain, the oxirane

ring would be protonated by His159. This suggestion was rejected by Varughese

and co-workers,154,155

from the crystal structure of papain inhibited by E-64. They

argued that the epoxide was more likely protonated by water (Fig. 14) because of

the distance (5.5A˚) of the resulting hydroxyl group from His159.156

1.5.1.5 Peptidyl aziridines

Aziridines are aza-analogs of epoxides, that are equally susceptible to

ring opening by nucleophiles.123,157

This class of inhibitors has been tested against

several types of proteases, including serine proteases, aspartyl proteases and

metalloproteases, but aziridines are irreversible and selective inhibitors of cysteine

proteases.158

Further, aziridines such as aziridine-2-carboxylates and aziridine-2,3-

dicarboxylates, are hydrolyzed by serine proteases.159,160

There are three types of

aziridinyl peptides (Table-V) that are classified according to their structural

differences. 123

Type I are N-acylated aziridines with the aziridine moiety located

on the C-terminus of the peptides or amino acids. Type II are aza-analogs of

epoxysuccinyl peptides that have the aziridine-2,3-dicarboxylic acid moiety at the

N-terminus of the amino acid. Type III are N-acylated aziridines such as type I,

but the aziridine moiety is located in the middle of the peptide chain. Besides the

common inhibition mechanism, several differences can be found between the

epoxide and the aziridine heterocycles. These include reactivity, the influence of

the pH of medium, selectivity and stereospecificity of inhibition.161

The stereospecificity of the inhibition is one major difference between

aziridines and epoxides. Aziridines have been shown to be more reactive at low

pH,162

with maximum activity at pH 4, while the epoxides are more reactive at pH

6-7 due to protonation of aziridine nitrogen atom, which translates into the

increased reactivity of the inhibitor toward papain. Another difference between

aziridines and epoxides is the hydrogen-bonding abilities.163

Aziridines (Type-II)

are H-bond donors, whereas the epoxides are H-bond acceptors. These differences

suggest that the two classes of inhibitors may have different binding modes and

possibly show variable interactions with cysteine proteases. The aziridine ring can


36

N

NHNH

OH

HO

H

O

NH

NH

-O

O

O

O

NH

S

O

NH

NH

NH

-O

O

O

O

O

H

S

O

N

NHNH

OH

+

His 159

Cys 25

R1

R2

S2

S3

R1

R2+

Figure 14: Proposed inhibition mechanism of papain-like cysteine protease inhibitors

containing three-membered heterocycles

be opened by nucleophiles in two different ways-besides C-N bond cleavage, C-C

bond cleavage is also possible.164

Unlike epoxides, the R,R configuration of the

COOR2

N

NHR4

R1OOC

O

COOR2

NH

R1OOC

COOR1

N

O

EtOOC

NHR3

R3

R2

(I) (II) (III)

Table V: Aziridinyl peptide compounds.

Type (I) Type (II) Type (III)

R1= Et, H R1= Et, H R1= amino acid, peptide

R2= Et, Bn R2= amino acid R2= amino acid, side chain

R3= amino acid side chain - - R4= protecting group,

amino acid, dipeptide

- R3= protecting group, amino

acid

aziridine ring is preferred for inhibition in both types II and III aziridine inhibitors,

whereas the type I aziridine inhibitors with the S,S configuration are better

inhibitors.165

More recently,166

several aziridines were screened against FP-2, FP-3

and cultured P. falciparum. They can be divided in three groups (i) aziridine-2-

carboxylic acid derivatives, (ii) N-acylated aziridine-2,3-dicarboxylic acid


37

derivatives and (iii) aziridine-2-carboxylates containing a lysine residue. Within

the series of aziridine-2-carboxylic acid derivatives with free 3-position, inhibitor

(14) (Fig. 15) is the most active one with IC50 value of 2.2 M against FP-2.

Among the N-acylated aziridine-2,3-dicarboxylic acid derivatives, 15 and 16

R

O

N

O

N

OR

O

O

O

O

NO

(14) R = (S)-PheOBn

(16) R = Boc-(S)-Leu-(S)-Pro

(15) R =

Boc-(S)-Leu

OCH3

Figure 15: Aziridine-2-carboxylic and 2,3-dicarboxylic acid derivatives.

offered the most potent inhibitors of FP-2 (IC50 = 0.079 and 5.4 M, respectively)

and FP-3 (IC50 = 0.25 and 39.8 M, respectively).

1.5.1.6 The 1,2,4-trioxolane compounds

In a prodrug approach, 1,2,4-trioxolane carbonyl protected compounds

were reported to act by their cleavage/decomposition by iron chelation. The

peptidic carbonyl protected (aldehyde) morpholineurea (17) (Fig. 17) was reported

OO

ONH

HO

CH3

CH3

NH

R

R1

(17) R = Morphlineurea, R1 = CH2Ph

Figure 16: 1,2,4-Trioxolane compounds

to be weak falcipain inhibitor than parent aldehyde but it showed good

antimalarial activity with IC50 value of 9.3 nM in P. falciparum 3D7 strain.167

1.5.1.7 The γ-lactam or pyrrolidinone isosteres

The γ-lactam or pyrrolidinone isostere has proven to be an effective

conformational constraint for inhibitors of other classes of proteases.168

A series of

conformationally constrained pyrrolidinone inhibitors attached with electrophilic


38

aldehyde169

were reported as inhibitors of falcipain. Compound (18) (Fig. 17)

exhibited excellent binding affinity (50 nM) for falcipain.

NSNH

O

O

O

H

O

(18)

Figure 17: The γ-lactam or pyrrolidinone derivative

1.5.1.8 Peptidic fumaric acids and other peptidic analogs

Fumaric acid derived oligopeptide compounds library was successfully

identified from a high-throughput screening of a solid phase-bound combinatorial

library with the glycine, asparagine or phenylalanine in P1 and phenylglycine and

cyclohexylglycine in P2 site. These fumaric acid derivatives from the library were

found to be the most potent.170

This information was used for synthesis of more

potent vinylogous Michael-type peptidic cysteine protease inhibitors, displaying

different inhibition mechanisms depending on the configuration of the double

bond and the peptide sequence. It has been reported that the E configured

vinylogous Gln-tert-esters are time-dependent inhibitors and on the other hand, Z

or E configured maleic and fumaric acid derivaites containing two phenyl residues

are reversible inhibitors. Some of the compounds show preferential selectivity to

FP-2 over FP-3.171

1.5.1.9 Azapeptide nitriles derivatives

Azadipeptide nitrile cysteine protease inhibitors display structure

dependent antimalarial activity against both chloroquine sensitive and chloroquine

O NH

NN N

O

O

CH3

R2

(19a) R1 = -i.Pr, R2 = -Methyl

(19b) R1 = -CH2Ph, R2 = -Pentyl

(19c) R1 = -CH2Ph, R2 = -(CH2)2Ph

R1

Figure 18: Azapeptide nitrile derivatives

resistant P. falciparum malaria parasites. Inhibition of parasite’s hemoglobin

degrading cysteine proteases was also investigated, revealing the azadipeptide


39

nitriles as potent inhibitors of FP-2 and 3. A correlation between the cysteine

protease inhibiting activity and the antimalarial potential of the compounds was

observed. The FP-2, IC50 for the compound derivatives are more potent than

phenylalanine peptides suggesting that the S2 pocket of the enzyme is deep rather

than shallow.172

1.5.2 Peptidomimetic falcipain-2 inhibitors

1.5.2.1 Peptidomimetics based on a 1,4-benzodiazepine scaffold

Even though several peptide-based FP-2 inhibitors have been identified,

their utility as therapeutic agents is limited due to their susceptibility to protease

degradation and poor absorption through cell membranes; a common strategy to

improve pharmacokinetic and pharmacodynamic parameters is to lock a defined

conformation of the peptide into a rigid scaffold, which could mimic protein’s

secondary structure. In this context the -turn is a structural motif that has been

postulated in many cases for the biologically active form of linear peptides and in

recent years several scaffolds mimicking -turns have been reported.173-176

The benzodiazepine (BZD) nucleus with its similar molecular

dimensions has been proven to be a good -turn mimetic.177,178

Coupled with the

fact that BZDs possess good oral bioavailability as a drug class that are certainly

well tolerated; some novel peptidomimetic FP-2 inhibitors have been designed.

They are based on a 1,4-BZD scaffold 179

as -turn mimetics introduced internally

to a peptide sequence which mimics the dipeptide D-Ser-Gly, and on a C-terminal

aspartyl aldehyde building block, which inhibits the enzyme by forming a

reversible covalent bond with the active site cysteine. The serine hydroxyl group

was functionalized with various aryl isocyanates.

All new peptidomimetics displayed a significant inhibition of both FP-

2A and FP-2B with IC50 values in the range of 8-26 M. In this context compound

(20) (Fig. 19) proved to be the most active inhibitor with an IC50 = 8.2 M for the

FP-2A and of 11.2 M for FP-2B. Trying to define a structure-activity

relationship, in this test series the phenyl derivative proved to be the least potent

inhibitor (IC50 about 20 M in both the enzymes); the introduction of an electron-

donating or an electron-withdrawing group at position 4 of the phenyl ring of the

carbamoyl moiety increased the inhibitory activity in both the cases. The best


40

results were obtained when a methyl (20) or a trifluoromethyl (21) group was

introduced at position 2 of 4-chloro derivative. Also the extension of the arom-

NNH

ON

ONH

OO

R

Cl

O

O

OH

FP-2A FP-2B

(IC50 in mM)

(20) R = CH3 8.2 11.2

(21) R = CF3 8.7 25.9

Figure 19: Inhibition of FP-2A and FP-2B by peptidomimetic 1,4-benzodiazepine

compounds.

atic area seemed to be fruitful. It is interesting to see that all new peptidomimetics

inhibit both FP-2A and FP-2B at a similar level and this is a key step in the

development of a FP-2 inhibitors, as it is well known that the disruption of FP-2A

gene and in this case a decrease of the expression of this paralog seems to be

compensated by the increased expression of FP-2B.86

Furthermore, all compounds

proved to possess good selectivity when tested against other cysteine proteases;

such a panel of active recombinant human caspases (i.e., caspases 1-9) did not

show any inhibitory activity up to 50 M. The peptidomimetics bearing a

protected aspartyl aldehyde warhead led to the thioacylals (22) (Fig. 20) and the

acylals (23) with increased antiplasmodial activity in comparison to the parent

molecule (21).180

NNH

ON

ONH

OO

CF3

Cl

O

O

R

FP-2 FP-2

Ki ( M) IC50 in M

(22) R = S-t-Bu 8.2 28.5

(23) R = OBn 8.9 49.5

Figure 20: Thioacylal and acylal compounds

In order to further investigate the structural requirements of this class of

inhibitors, the peptidomimetics were modified by replacing the P1 aspartyl


41

aldehyde in the lead compound (20) with a vinyl sulfone moiety which was able to

interact as classical Michael acceptor with the active site cysteine leading to an

irreversible inactivation of the target enzyme as described above. In particular,

two different series of vinyl sulfones were synthesized,181

first one containing, at

NNH

S

O O

ON

ONH

OO

CF3

Cl

(24) R1 = H

(25) R1 = CH2CH2Ph

R1

R2

Table VI: Inhibition of FP-2 and antiplasmodial activity of compounds (25)

Compound R1 k2nd

(M-1

min-1

)

kinacM-1

/kinac M

P.

falciparum

IC50 (nM)

25a Methyl 432,000 0.16/0.39 55.4

25b Ethyl 307000 0.1/0.32 9.1

25c Phenyl 175000 0.11/0.60 9.2

25d 4-OCH3-C6H4 634000 0.034/0.053 59.6

the P1 site, a homoPhe residue (e.g. 25, Table-VI), an amino acid known to

strongly increase the potency of FP-2 inhibitors and second one containing in the

same position, a glycine (e.g. 24, Table-VI), useful to evaluate the relevance of the

amino acid side chain in the process of recognition of the ligand by the enzyme.130

It was observed that all of the compounds (24-25) exhibited high second-order

rate constants in the range of 161,000-634,000M-1

min-1

, in particular compounds

(25) (Table-VI) generally showed higher second order rate constants with respect

to analogs (24). Furthermore, it was possible to observe that the introduction, at

the P1’ site, of a methoxy group in position 4 of the phenyl ring, strongly

stabilized the enzyme-inhibitor interaction, probably acting as hydrogen bond

acceptor, as shown in the most active compound (25d) with the highest value of

the second order rate constants of inhibition (K2nd = 634,000M-1

min-1

) and the

lowest dissociation constant value (Kinac = 53 nM). Although there was no clear

correlation between the antiplasmodial activity and the inhibitory potency,

compounds (25) proved to be more active than their glycine analogs (24). It is

interesting to point out that compound (25c), one of the lesser potent inhibitors

against the enzyme, displayed the highest antiplasmodial activity, while the


42

presence of the methoxy group para to the phenyl ring (i.e. 25d), basic for the

enzyme inhibitor interaction, decreased the activity against the parasite, probably

reducing the penetration through cell membranes.

Selectivity assays were also performed against papain family human

cysteine proteases cathepsins B and L by demonstrating good selectivity of all

vinyl sulfones toward FP-2. Other efforts were made to further optimize the

pharmacophore portion by replacing the vinyl sulfone moiety with a vinyl

phosphonate one, generating unsatisfactory results; a decrease of potency and

selectivity was obtained.182

NNH

ON

ONH

OO O

NH

CF3

Cl

NNH

ON

O

OO O

R1

(27)

R2

R3

(26)

OCH3

OCH3

Table VII: Inhibition of falcipain-2 and antiplasmodial activity of compounds (27).

Comp. R1 R2 R3 k2nd (M-

1 min

-1)

kinacM-1

/kinac M

P. falciparum

IC50 (nM)

27a H 4-Cl, 2-CF3-C6H4 Br 24,500 0.038/1.56 6.33

27b H 4-Cl, 2-CF3-C6H4 H 3570000 0.06/0.017 12

27c H 1-Adamantyl H 1160000 0.013/0.014 10.7

27d H 1-Naphthyl H * * 17.6

* Time independent inhibition.

Based on the constrained peptidomimetic vinyl ester (26) (IC50 12

M)183

an irreversible inhibitor of FP-2, the constrained vinyl ester

peptidomimetics were evaluated; compound (27a) (Table-VII) was found to be the

most potent one having an IC50 of 10.7 nM. Any further modification in the parent

compound leads to decrease in FP-2 inhibition.184

1.5.2.2 Peptidomimetics based on a pyridine/pyrimidone ring scaffold

Recently, another nonpeptidic scaffold was incorporated on a peptide

sequence suitable for FP-2 recognition, in a way to lock the amino acid backbone.

The pyridone ring core was selected as a replacement for the peptidic leucine

residue, at the P2 position, within the inhibitor’s framework. Aldehyde, vinyl

sulfone or vinyl ester moieties were the chosen pharmacophores.185

The new


43

synthesized peptidomimetics showed in vitro antimalarial activity against the 3D7

parasite strain in the range of 5-40 M, with the vinyl sulfone (28) (Fig. 21) as the

best inhibitor in the series (IC50 = 5.7 M).

Consistently, with binding features previously reported, compounds

bearing a homoPhe residue at P2 site proved to be more active than those bearing a

phenylalanine in the same position. Some of the synthesized peptidomimetics

were also evaluated on recombinant FP-2 and FP-3. In this evaluation they proved

to be weak inhibitors of FP-2 (IC50 10-20 M) and inactive toward FP-3 (IC50 >

25 M). The best inhibitory profile against FP-2 was shown by compound (29)

(IC50 = 10.9 M), whereas it was not possible to test the most active compound

(28) because of its low solubility.

N R

O

O NH

O

N

N

NH

NH

O CH3

OCOCH3

OO

Ph

(28) R = CH=CHSO2Ph

(29) R = CHO

(30)

Z OCH3

Figure 21: Pyridone and pyrimidone compounds

A new class of pyrimidinyl peptidomimetics (30) was reported as

malarial cysteine protease inhibitors. The core structure of the new agents consists

of a substituted 5-aminopyrimidone ring and a Michael acceptor side chain methyl

2-hydroxymethyl-but-2-enoate. The most effective compound (30) of the series

(IC50 = 9 ng/ml) showed comparable efficacy to that of CQ (IC50 = 6 ng/ml). In

general, this class of compounds exhibited weak to moderate in vitro cytotoxicity

against neuronal and macrophage cells and less toxicity in colon cell line.

Preliminary results indicated that compound (30) is active against P. bergehi

prolonging the life span of the parasite-bearing mice from 6 days for untreated

control to 16-24 days for drug treated animals.186

1.5.3 Nonpeptidic falcipain-2 inhibitors

1.5.3.1 Chalcones

Modeling studies on acylhydrazones have been made on a model

structure of FP-2, designed on the basis of the X-ray structures of papain and

actinidin. This investigation on the most active compound of this class, the oxalic


44

bis[(2-hydroxy-1-naphthylmethylene)hydrazide], clearly revealed that one

naphthyl group filled the hydrophobic S2 site while the other one interacted with

the Trp177 of the S1’ site. On the basis of these results, the length of the backbone

was shortened; the aromatic rings being replaced with heteroaromatic nucleus in

order to improve the water solubility and to enhance interaction with His67 of the

S2 pocket. Finally, to improve the metabolic stability, the hydrazide linker was

replaced with an , -unsaturated ketone backbone leading to calchones.162

Some

chalcones were thought to inhibit malarial cysteine proteases and today their

ability to inhibit the protease of P. falciparum FP-2 is well known. Chalcones are

biosynthetic precursors of flavonoids.

Licochalcone A (Fig. 22), a natural product isolated from Chinese

liquorice roots, was the first compound of this family to exhibit in vivo and in

vitro antimalarial activity.187,188

Chalcones-artemisinins combination therapy have

been reported to have synergistic or additive effect in the treatment of malaria.189

O

OH

CH3

CH3

CH2

OH

OCH3

Figure 22: Licochalcone

First study on synthetic chalcones was performed by Li and co-workers

in 1995.190

The most active chalcone derivative, 1-(2,5-dichlorophenyl)-3(4-

quinolinyl)-2-propen-1-one (31) (Fig. 23) showed an IC50 value of 200 nM against

both CQ-resistant (W2) and CQ-sensitive (D6) strain of P. falciparum. The main

conclusions of this work were: (i) the C2-C3 double bond was revealed to be

essential for the inhibitory properties, because it kept the molecular structure in

extended conformation leading to a better interaction with the active site, (ii)

substitutions on the bridge portion caused a pronounced decrease in the inhibitory

activity, probably due to steric interactions, (iii) introduction of halogens on ring

A (fluorine and chlorine at 2,3 or 2,4-positions e.g. 33) and electron-donating

groups on the B ring increased the antimalarial activity and (iv) the presence of

quinolinyl group in ring A resulted in increased activity. Under acidic conditions,

nitrogen-containing heterocycles can be protonated within the acidic food vacuole

of the parasite facilitating the interaction with His67 of FP-2. Additionally, this


45

chemical entity which is common in established antimalarials may interfere with

heme detoxification affecting the whole process.

O F

FNH

NH

OCl

O

N

Cl

Cl

O

N

(31) (32)

(33)

B A

OCH3H3CO

OCH3

Figure 23: Chalcone compounds.

Later, a broad series of alkoxylated and hydroxylated chalcones have

been synthesized by Liu and co-workers in 2001,191

with the most active

compound 1-(2’,3’,4’-trimethoxyphenyl)-3-(3-quinolinyl)-2-propan-1-one (32)

(Fig. 23) showing an in vitro IC50 value of 2 M against a strain of CQ-resistant

human malaria parasite, P. falciparum (K1). The whole series was not tested

directly against FP-2. Its FP-2 inhibitory activity was presumed. The results

indicated that (i) the substitution pattern on the B ring could influence the

antimalarial activity, (ii) hydrophobicity and size factors, respectively, for the A

and B rings were important parameters for activity, (iii) presence of halogens on A

ring did not necessarily increase the antimalarial activity while the influence of the

latter groups on A ring seemed to be influenced by substitution pattern on B ring

and (iv) an interesting observation was the association of good antimalarial

activity with the presence of a 3-quinolinyl A ring. This was observed with 3-

quinolinyl A ring and several B ring series of compounds except 4’-

hydroxylchalcone. The 4-quinolinyl A ring derivatives were significantly less

active than their 3-quinolinyl counterparts indicating that a steric element is also

involved in the interaction with the target.

Dominguez et al. reported phenylurenyl chalcones to possess good

inhibitory properties against FP-2.192

The best inhibitor of this series, was 1-[4’-N-

(N’-p-chlorophenylurenyl)phenyl]-3-(3,4,5-trimethoxyphenyl)-2-propen-1-one

disp-laying an IC50 value of 1.76 M toward the protease, coupled with a

satisfactory inhibition of cultured malaria parasite development (IC50 = 3 M).


46

Quantum chemical QSAR study on the chalcone derivatives confirmed

that their activity against W2 and D6 P. falciparum strains depended upon steric

and hydrophobic factors. Generally, a width limiting chemical substituent on A

ring is optimal for activity on W2 and D6 strains. Molecular weight, which is

related to molecular volume, appears to influence only the activity of D6 strain.

This study also indicates that chalcones are capable of taking resonance structures

along unsaturated chain, thus they can be good Michael acceptors. This would

result in irreversible inhibition. The oxygen of the carbonyl is subject to the

protonation in the internal acidic environment of the digestive vacuole of

plasmodium, intensifying the positive partial charges on carbons C-1 and C-3 and

favoring the nucleophilic addition at these carbons. However, these studies were

performed in the absence of crystal structure of the parasitic cysteine protease,

giving a rough evaluation for enzyme-inhibitor interaction mechanism.193

O

O

N

NN

OH

R

S CH3

CH3

O

O

N

O

O

Cl

R1 = 4-methoxy, 2,4-dimethoxy, 2,3,4-trimethoxy

R1

ThiolactonesIsatins

R =

Figure 24: -Amino alcohol thiolactone-chalcone and isatin-chalcone derivatives

Hans et al. reported two types of chalcone, thiolactone and isatin

scaffold- containing compounds (Fig. 24). A 36-member -aminoalcohol triazole

library showed that the thiolactone-chalcones, with IC50 ranging from 0.68 to 6.08

M, were more active against W2 strain of P. falciparum than the isatin-chalcones

with IC50 values of 14.9 M or less. Interestingly, isatin-chalcones diaplayed FP-2

inhibitory activity whereas the thiolactone-chalcones lacked enzyme inhibitory

activity. No correlation was observed between the antiplasmodial activity and the

FP-2 enzyme inhibition suggesting that it may not be the primary mode of action

of the chalcones.194


47

1.5.3.2 The -pyranochalcones and pyrazoline derivatives

Wanare G et al. reported the -pyranochalcones and pyrazoline

derivatives as falcipain inhibitors. Docking studies (GLIDE) on (E)-3-(3-(2,3,4-

O O

O

O

O

O

OH

O O

N N

O

O

CH3

CH3

R R

O O

N NCH

3

O

O

O

CH3

CH3

(34) (35)

(36)(37)

Figure 25: Chalcone compounds

Table VIII: -Pyranochalcones and pyrazoline falcipain inhibitors

Compound R

Antimalarial activity P. falciparum (mg/ml)

3D7

IC50

3D7

IC80

RKL9

IC50

Resistance index

(IC50 D7/IC50 RKL9)

34a 3-OCH3- 11 24 4.9 2.2

34b 3,4-di OCH3- 26.7 100 - -

34c 2,3,4-tri OCH3- 3.1 6.2 1.1 2.8

35a 2-Cl 20 25 13.5 1.5

35b 2,5-di OCH3- >100 - - -

36 - 10 10 9 1.1

37 - 10 44 7.6 1.3

trimethoxyphenyl)-acryloyl)-2H-chromen-2-one with active site residue Cys42 of

falcipain enzyme revealed the requirement of hydrogen bond acceptor in ring B.

The compound (34c) with trimethoxy substituent (Table-VIII) was found to be the

most potent compound.195

1.5.3.3 Isoquinoline derivatives

Isoquinolines derivatives (Fig. 26) were designed as FP-2 inhibitors

according to homology modeling studies and subsequent validation by docking. It

was found that isoquinoline framework was not essential for the activity against

FP-2.196

The planarity of the isoquinolines was unimportant as both the

dihydroisoquinoline and isoquinoline cores were found to have IC50 values in the


48

same range. Moreover, all of the compounds bearing a p-methoxyphenyl

substituent at position 1 of the isoquinoline core were almost equipotent to the

N

R

N

O

O

O

(38) R= OMe, IC50 3 M

(39) R= OBu, IC50 3 M

(40) IC50 3 M

Figure 26: Isoquinoline derivatives

earlier reported ligands.197

On the other hand, compounds with p-benzyloxyphenyl

group (38-40, Fig. 26) docked well into the active pocket of the homology-

modeled FP-2 and were found to be more potent than the corresponding hydroxyl

analogs suggesting that S2 pocket of the enzyme prefered to accommodate the

hydrophobic groups.196

1.5.3.4 Thiosemicarbazones

1.5.3.4.1 2-Acetylpyridine-4-phenyl-3-thiosemicarbazone

The first thiosemicarbazone synthesized as potential antimalarial agent

was 2-acetylpyridine-4-phenyl-3-thiosemicarbazone (41, Fig. 27). Klayman and

co-workers198

prepared a series of thiosemicarbazone derivatives from the parent

compound (41) and tested them against P. berghei in mice. The main conclusions

of this work were (i) the 2-pyridylethylidene group was important for antimalarial

NN

NH

NH

S

CH3

(41)

Figure 27: 2-Acetylpyridine-4-phenyl-3-thiosemicarbazone

activity and (ii) at N-4 position the presence of unsubstituted phenyl, benzyl,

phenethyl or cycloalkyl groups such as adamantyl and cyclohexyl could also

contribute to antimalarial activity.


49

1.5.3.4.2 Isatin thiosemicarbazones and other thiosemicarbazone compounds

Chiyanzu et al. designed a new class of thiosemicarbazones as FP-2

inhibitors bearing the isatin scaffold (Fig. 28).199

It was evident that the

commercially available isatin was inactive, while the corresponding

thiosemicarbazones showed certain degree of activity. The most promising FP-2

inhibitor of this series was revealed to be compound (42) with an IC50 value of 4.4

M.

NH

N

O

NH

NH2S

R1

R2

(42) R1 = NO2 R2 = H

Figure 28: Isatin thiosemicarbazone derivatives

Recently, some thiosemicarbazones have been prepared and evaluated

against FP-2, rhodesain and cruzain. Additionally all of the compounds were

screened against the respective parasites. The obtained data revealed significant

differences between the structural requirements essential for the inhibition of the

NNH

NH2

CH3

S

NNH

NH2

OHS

R

OH

N N N N

N

Cl

(43)

(44) (45) (46)

(44) FP-2 IC50 5.8 M: P. falciparum W2 ED50 >10 M

(45) FP-2 IC50 3.8 M: P. falciparum W2 ED50 >10 M

(46) FP-2 IC50 2.25 M: P. falciparum W2 ED >3.75 M

R =

Figure 29: Thiosemicarbazone compounds.

enzyme and the parasite. Compound (43) (Fig. 29) was the only one active against

FP-2 (IC50 = 10 M) but it was found to be almost inactive against the parasite


50

(ED50 20 M). On the contrary, all other compounds (44-46) (Fig. 29) showed

good activity against FP-2. As the corresponding aldehyde precursors were

practically inactive against FP-2, it was reasonable to assume that the (thio)

semicarbazide moiety played an important role in the inhibition of FP-2; but

compounds without mannich bases i. e. phenolic aldehydes and

(thio)semicarbazides independently were found inactive against both FP-2 and the

parasite. This clearly suggested that the activity of the discussed compounds was

not due to the thiosemicarbazide side chain alone, but it was due to the combined

effects of both, the mannich base of aldehyde and thiosemicarbazide

components.200

More recently, new mannich bases of phenolic benzaldehyde and

4-aminoquinoline-(thio)semicarbazide were synthesized. These small compounds

showed good activity against CQ-resistant P. falciparum W2 strain but were very

weak FP-2 inhibitors hence they primarily act some by some mechanism other

than FP-2 inhibition.201

1.5.3.4.3 Gold thiosemicarbazones

The gold (I) thiosemicarbazone derivatives were evaluated for their FP-2

inhibiton. There was no correlation of antiplasmodial activity and FP-2 inhibition

of the parent ligands hence these compounds were assumed to exhibit the

antiplasmodial activity by inhibition of multiple targets.202

1.5.3.5 Acridinediones

Acridinediones like compound (47) (Fig. 30), have been shown to have

antimalarial activity, but their mechanism of action remains unknown. From a

N

OO

Cl

OHCF

3

NH

S

OO

Cl

O

(47) (48)H3CO

OCH3

Figure 30: Acridione and isostere analog

series of chalcones, previously identified as inhibitors of falcipain, a

conformationally constrained series was synthesized. This conformationally

constrained series utilized a sulfur isostere of acridindione to afford the


51

phenothiazine (48) that blocked parasite metabolism and development at low

micromolar concentrations.203

1.5.3.6 2-Cyanopyrimidines

Coteron et al. have reported a detailed study of optimization of the FP-2

and FP-3 activity on 5-chloro/bromo-2-cyanopyrimidine, (Fig. 31) an active

scaffold. Based on initial studies on 4-chloro/bromo-2-cyanopyrimidines, it has

been identified as an active scaffold at micromolar concentrations which was

N

N

NNH

X

N

O

N

Nalkyl

O

NN

CH3

S

N

NCH

3

O

X = Br, Cl

P2

P3

P2 =

P3 =

Figure 31: The 2-cyanopyrimidine derivatives

further subjected to optimization studies by varying the groups at X, P2 and P3

positions (Fig. 33) to obtain potent FP-2/FP-3 inhibitors. The FP-2/FP-3 inhibition

concentrations were found to be in low nanomolar to picomolar range (IC50 0.2

nM & 1nM in Pf W2 strain).204

1.5.3.7 Cryptolepine derivatives

Cryptolepine derivatives (Fig. 32) containing basic side chains at the C-

11 position containing propyl, butyl and cycloalkyl diamine groups significantly

increased activity against chloroquine-resistant P. falciparum strains with less

cytotoxicity, compared to chloroquine. Cryptolepine derivatives containing linear

alkyl (eg. ethyl, propyl) or cyclic groups with terminal secondary or tertiary

amines were found to be potent compounds with IC50 values of 20-90 nM. Any

branching led to decrease in FP-2 inhibitory activity.205


52

N

NH

CH3

R2

R3

R1+

R1, R2 = H/Cl

R3 = NH-Linker-NR4R5

R4, R5 = H, Alkyl, Cycloalkyl

. Cl

Figure 32: Cryptolepine derivatives

1.5.3.8 Indolin-2-one derivatives

The 3-methyleneindolin-2-one derivatives (Fig. 33) showed moderate

antimalarial activity (IC50 = 10 M). The recognition moiety interaction of the

Leu-iso-amyl sequence with the S2 pocket plays important role in activity.

Furthermore, the antiplasmodial activity could be significantly improved when a

NH

O

NH

CH3

CH3

N

O

R1

R2

R3

R1 = H, methyl

R2 = H, Cl

R3 = H, isobutyl, phenyl ethyl/methyl

Figure 33: 3-Methyleneindolin-2-one derivatives

4-aminoquinoline moiety was coupled to the 3-methylene moiety of the indolin-2-

one scaffold (IC50 = 140 nM).206

1.5.3.9 Allicin derivatives

Allicin (S-allyl-2-propenylthiosulfinate) and its derivatives were studied

for FP-2 inhibition (Fig. 34). The mode of action is through the attack of active-

site Cys residue on the primary carbon atom in vicinity to the thiosulfinate sulfur.

Compounds 49 (allicin, Ki = 1.04 M, IC50 = 5.21 M), 55 (Ki = 1.8 M, IC50 =

10.9 M) and 51 (Ki = 4.5 M, IC50 = 30.3 M) showed good FP-2 inhibition.207

RS

SR

O

SS

O

CH3

CH3

CH3

(49-55) (56)

Figure 34: Allicin derivatives


53

Table IX: Allicin derivatives and their antimalarial activity

Compound R FP-2 Ki ( M) P. falciparum IC50 ( M)

49 allyl 1.04 5.21

50 n-propyl 26.4 78.3

51 benzyl 4.5 30.3

52 tert-butyl >100 72.5

53 cyclohexyl >100 34.4

54 iso-pentyl 3.04 54.7

55 n-hexyl 1.8 10.9

56 - >100 52.9

1.5.4 Molecular modeling studies

1.5.4.1 Virtual screening of ChemBridge database

Virtual screening of ChemBridge database (consisting of approximately

2,41,000 compounds) was performed in an attempt to identify nonpeptide

inhibitors of parasitic cysteine proteases as novel drugs.208

The compounds were

screened against homology models of falcipain-2 and falcipain-3 in three

consecutive stages of docking. A total of 24 diverse inhibitors were identified, out

NH

N

NNH

COOH

N

Cl

N

OH

OHOH

OH

NH

O

NNH

O

CN

FO

(57)

HOOC (58)

(59)

Figure 35: FP-2 and FP-3 dual inhibitors identified by virtual screening of

ChemBridge database

of which 12 compounds appeared to be dual inhibitors of FP-2 and FP-3 (Fig. 35).

Some of them showed preferential selectivity for FP-2 over FP-3. 203

1.5.4.2 Docking studies on compounds of SPECS database

In another virtual screening study, the SPECS database (2,87,000) compounds

were screened and reduced to about 80,000 using the drug likeness filter and

subsequently docked by two methods Glide and GAs Dock, with different scoring


54

functions to identify leads for novel FP-2 inhibitors. The top 1000 compounds

were selected using Glide with XP docking used for further accurate docking. The

NN

N

OH

SN

CH3

O

Cl

ON

NN

ON

NH

CH3

ClNH

N

CH3

Cl

N

CH2

NS

O

SNH

O

S

ONH

2(60)

(61)

(62)

(60) 53.1 2.4

(61) 71.4 5.8

(62) 56.5 6.4

Inhibition rate(%) IC50 M

Figure 36: Potent compounds identified by docking studies on of SPECS database

[Note: Inhibition rate (%) calculated with respect to percent inhibition of control (E64)]

top 200 compounds were subjected to visual inspection of the docking geometry

by (1) complementarity between the ligand and the hydrophobic S2 pocket of the

protein and (2) formation of hydrogen bonds between the ligand and residues near

the catalytic cysteine (Cys42), resulting into identification of 53 compounds. In

GAs Dock method top druglike 1000 compounds were selected by its energy

score. The compounds were further evaluated and ranked using the CSCORE

module of the Sybyl package and 154 compounds were then selected with the

consensus score of 5. By visual analysis of the 154 docked poses by elimination

criteria, 56 compounds were selected. Total 81 compounds were then screened for

in vitro evaluation of FP-2 inhi bition. The compounds (60-62) (Fig. 36) were

found to be the most potent ones.209

1.5.4.3 2-Amido-3-(1H-indol-3-yl)-N-substitued propanamides

2-Amido-3-(1H-indol-3-yl)-N-substitued-propanamides were designed

and synthesized based on structure-based virtual screening in conjunction with


55

surface plasmon resonance (SPR) based binding assays. The compounds showed

moderate FP-2 inhibition activity, with IC50 values ranging from 10.0-39.4 μM.210

NH

NHNH

O

R1

R2

Figure 37: 2-Amido-3-(1H-indol-3-yl)-N-substitued propanamides

1.5.4.4 2-(3,4-Dihydro-4-oxothieno[2,3-d]pyrimidin-2-ylthio)acetamides

A series of novel small molecule FP-2 inhibitors have been designed and

synthesized on the basis of structure-based virtual screening in conjunction with

an enzyme inhibition assay. All of the 2-(3,4-dihydro-4-oxothieno[2,3-

d]pyrimidin-2-ylthio)acetamide derivatives showed high inhibitory activity

against FP-2 with IC50 values ranging from 1.46-11.38 μM.211

N

N

S

O

SNH

O

R4

R1

R2

R3

Figure 38: 2-(3,4-Dihydro-4-oxothieno[2,3-d]pyrimidin-2-ylthio)acetamides

1.5.5 Combination or multi-target/hybrid structural approaches

Combination therapies are generally used to combat malaria that can

minimize the chances of developing dug resistance. Certain combinations of

falcipain inhibiting scaffolds and other antimalarial drugs or multi-target/hybrid

therapy approaches have been studied in recent papers.

Aspartic and cysteine protease inhibitors act synergistically to degrade

hemoglobin in vitro212

therefore inhibitors of these enzymes would possess

synergistic effects in inhibiting the growth of cultured parasites. So combination

therapy of inhibitors of malarial cysteine and aspartic proteases may provide the

most effective chemotherapeutic regimen and could possibly limit the

development of parasite resistance to protease inhibitors in the best possible way.

Hans et al. reported two types of chalcones, thiolactone and isatin

scaffolds containing hybrid compounds (Fig. 24) as antimalarial agents. 194

Chalcone-azole derivatives and artemisinins combination therapy have been

reported to have synergistic or additive effect in the treatment of malaria. The


56

synergistic combinations decrease hemozoin formation in parasitized erythrocytes.

These combinations do not affect new permeation pathways induced in the host

cells. The combinations showed very potent FP-2 inhibition with IC50 values

ranging from 0.29 to 10.63 M. The binding affinities and interaction modes were

studied by molecular docking studies using AutoDock 3.05 for the potent

compounds.189

O O

O

O

O N

O

N SO O

R3

R2

R1

R1 = (CH2)2Ph, CH2Ph

R2 = CH2CHMe2, CH2Ph, H

R3 = Ph, Me

(63)

H3CCH3

CH3

H3C

Figure 39: Artemisinin-dipeptidyl vinylsulfone hybrids

Peptidomimetic-thioacylals and acylals bearing protected aspartyl

protease aldehyde warhead were studied for inhibiton of protozoal cysteine

proteases falcipain-2 and rhodesain. Compounds showed potent anti-tripanosomal

activity.180

Artemisinin-dipeptidyl vinylsulfone hybrid molecules (63) were acting

in the parasites food vacuole via endoperoxide activation and falcipain inhibition.

Compounds showed FP-2 inhibition in micromolar range but almost all the

compounds displayed IC50 values ranging from 2-6 nM in P. falciparum W2

strain.213

In another combination strategy through a single prodrug approach,

1,2,4-trioxolane carbonyl protected compounds were reported to act by their

cleavage/decomposition by iron chelation. Peptidic aldehydes and esters were

used to get hybrid compounds. The aldehyde endoperoxide hybrids showed

activity in nanomolar concentration in 3D7 strain of P. falciparum but were

observed to be not good falcipain inhibitors when compared with the parent

peptidic aldehydes.167


57

SNH

NH

O

F

SN

N

CH3

O O

IC50 FP-2 = 7 M, DHFR = 6.3 M

Figure 40: FP-2 and DHFR duel inhibitor

In a multitarget approach a series of small molecules were designed and

synthesized. The compounds showed dual inhibition of falcipain-2 and

dihydrofolate reductase as antimalarial agent and their potency increased 6 to 8

times in comparison to the individual inhibitors. The IC50 values for FP-2 ranged

from 2.7–13.2 μM and for DHFR IC50 values ranged from 1.8–19.8 μM. The most

potent compound (Fig. 40) exhibited moderate in vivo antimalarial activity in a

dose dependent fashion. The molecular docking studies have been performed to

identify the key structural information to attain dual inhibitory activity214

The existing armamentarium of antimalarial drugs is not sufficient to

combat malaria, primarily because of resistance developed by the parasite. P.

falciparum genome sequencing has revealed a number of drug targets. Although

the enzymes of lethal malaria parasite species still has not drawn much attention

of medicinal chemists, biologist and clinicians’ worldwide to the extent it is

required for the development of new drug. Ultimately, a better understanding of

the biochemical and biological roles played by malarial proteases will foster the

development of protease inhibitors that would specifically inhibit malaria parasite

enzymes. There exist a great potential in the exploration of cysteine proteases

falcipain inhibitor and thus might prove to be the most suitable drug target for the

chemotherapy of malaria. This thesis work has been directed towards design and

synthesis of FP-2 inhibitors. The details about design strategy, synthetic

methodologies and biological screening results are described in the Chapter-2

Research Envisaged, Chapter-3 Result and discussion and Chapter-4 Experimental

work sections.

Introduction 2. Research Envisaged

Chapter-2 Research envisaged

58

As discussed earlier, majority of the antimalarial drugs were discovered

during the Second World War when the soldiers of the western countries while

fighting in the topical countries started contracting malaria in mosquito infested areas.

Over a long period of their usage against malaria caused development of resistance in

the parasite against these drugs. The problem got further compounded due to non-

enrichment of antimalarial drug inventory as the cash rich western countries were not

interested in finding new therapeutics for a disease which was considered to be the

disease of the poor third world tropical countries. Unfortunately for these poor

malaria infested countries an effective antimalarial vaccine also could not be

developed due to fast and constant mutations in the parasite genome. All these factors

led to a woefully thin antimalarial drug development pipeline with little chemical

diversity. There exist a great potential in the exploration of cysteine proteases

falcipain inhibitor and thus might prove to be the most suitable drug target for the

chemotherapy of malaria.

Genome mapping of the malaria parasite offered a number of attractive

drug targets including plasmepsins and falcipains, the enzymes involved in parasite

metabolism and in providing nutrients to the parasite to meet energy requirements.

Falcipains 2 and 3 especially were more attractive targets as these enzymes degrade

haemoglobin to small sized peptides in the acidic environment of the vacuole and

degrade the membrane proteins of the host cells and help the parasite rupture the host

cells in the alkaline environment. So, inhibition of FP-2/3 enzymes could effectively

contain the growth of the parasite and control their progression in the host system.

A large number of peptidic and non-peptidic motifs like chalcones, (thio)

semicarbozones, hydrazides, heterocycles, oxiranes and succinates have been reported

in the literature to exhibit FP-2/3 inhibitory activity. Unfortunately none of such

compounds have

N

N

X

O

AX = Urea, carbamate, amide

A = Alkyl/aryl/heteroaryl

R = H, CH3, Cl, 4-C6H4OCH3

R

(M)

crossed the clinical phase of evaluation as antimalarial agents. There are some reports

of some heterocyclic215

and polycyclic216

pyridopyrimidines exhibiting antimalarial

activity. Taking the basic information from literature about the enzyme inhibitors into

consideration, it was planned to synthesize pyrido[1,2-a]pyrimidin-4-one class of

Chapter-2 Research envisaged

59

compounds (M). It is worth noting that the circled pharmacophore may act as Michael

acceptor for reversible/irreversible binding to the thiol nucleophile present in the FP-

2/3 enzymes. While the groups, X and R were selected in such a way that compounds

could exhibit better binding profile in the P2 and P3 binding pockets of the falcipain

enzyme that could translate in to enhanced potency of the compounds.

It was planned to synthesize the given four (Series-I to IV) of

compounds and to evaluate them against FP-2 enzyme in the in vitro tests. FP-3

enzyme having a high degree of homology to FP-2 was presumed to react with all

such compounds which would prove to be powerful inhibitors of FP-2.

N

N

N

O

O

NRR N N

N

N

N

O

O

N

N

O

N

O

AR

N

N

N

O

O

OR

I II

AA

R1

IV

R1

III

A

Where: R = H, 8-CH3, 7-Cl, 4-C6H4OCH3

R1 = H, Alkyl

A = Alkyl/heteroaryl or alkyl

3. Results and Discussion

Envisaged

Chapter-3 Results and discussion

60

The research work carried out towards achieving the proposed plan has

been discussed under the following two main headings:

3.1 Chemical studies

3.2 Biological evaluation

3.1 Chemical studies

3.1.1 Synthesis of 4H-pyrido[1,2-a]pyrimidin-4-one intermediates (4a-c & 6a-c)

Synthesis of 4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (4a)

Synthesis of 3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one (6a)

Synthesis of 8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid

(4b)

Synthesis of 3-isocyanato-8-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (6b)

Synthesis of 7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid

(4c)

Synthesis of 7-chloro-3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one (6c)

3.1.2 Synthesis of (4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea derivatives

(Series I)

Synthesis of (4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea derivatives

Synthesis of 8-methyl-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea

derivatives

Synthesis of 7-chloro-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea

derivative


yl)piperazine-1-carboxamide derivatives (Series II)

Synthesis of N-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-(pyridin-2-yl)-

piperazine-1-carboxamide derivatives

Synthesis of N-(8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-

(pyridin-2-yl)piperazine-1-carboxamide derivatives

Synthesis of N-(7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-(pyridin-

2-yl)piperazine-1-carboxamide derivatives

3.1.4 Synthesis of 4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carbamate derivatives

(Series III)

Synthesis of 4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carbamate derivatives


61

Synthesis of 8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carbamate

derivatives

Synthesis of 7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carbamate

derivatives

3.1.5 Synthesis of 4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carboxamide derivatives

(Series IV)

Synthesis of 4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carboxamide derivatives

Synthesis of 8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carboxamide

derivatives

Synthesis of 7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carboxamide

derivatives

Synthesis of 7-(4-methoxyphenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-

carbox-amide intermediates

Synthesis of 7-(4-methoxyphenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-

carbox-amides derivatives

Compounds belonging to these individual categories have been

synthesized by following six major schemes as described below:

3.1.1 Synthesis of 4H-pyrido[1,2-a]pyrimidin-4-one intermediates

(4a-c & 6a-c)

The acids (4a-c) and the isocyanates (6a-c) are the key intermediates in

the preparation of the targeted compounds. Their synthesis was accomplished by

following the key steps i) Gould and Jacob reaction ii) cyclization, iii) hydrolysis

and iv) Curtius rearrangement reaction. Synthesis of theses intermediates is

accomplished by following the reaction sequence as outlined in the Scheme-1.


2-Aminopyridine (1a) was reacted with diethyl ethoxymethylene-

malonate affording the diethyl 2-pyridylaminomethylenemalonate (2a).

Compound (2a) offered characteristic peaks at 3274 (N-H str.), 1686 (C=O, str.)

and 1248 cm-1

(C-O, str.) in its IR spectrum. The compound (2a) displayed


62

characteristic signals at 1.22-1.29 (m, 6H, CH3(8/10)), 4.12-4.18 (q, 2H, CH2(7/9)),

4.20-4.25 (q, 2H, CH2(7/9)), 7.14-7.17

N

NH2

N

N

O

O

CH3

O

N

N

O

OH

O

N

N

N

O

ON

N

O O

N

N+

N

R

R R

R

R

N

NH

O

O

O

CH3

O

CH3

R

(1a-c)(2a-c)

(3a-c) (4a-c)

(5a-c)(6a-c)

a. R = H, b. R = 8-CH3, c. R = 7-Cl

i. ECF, TEA, DMF, 0OC

ii. NaN3, H2O,

EMME, Reflux

DPE,Reflux

HCl, Reflux

Toluene, Reflux

a. R = H, b. R = 4-CH3, c. R = 5-Cl

Scheme 1

(m, 1H, Ar-H(2)), 7.39-7.41 (d, 1H, Ar-H(4)), 7.79-7.83 (m, 1H, Ar-H(3)), 8.36-

8.37 (d, 1Ar-H(1)), 9.05-9.08 (d, 1H, Ar-H(6)) and 10.76-10.80 (d, 1H, NH(5)) in its

NMR spectrum. The (M++1) peak was observed at (m/z) 265 in its mass spectrum.

N

1

4

2

3 6NH

5

O

7

O

O

9

CH3

10

O

CH3

8

N

1

4

2

3 5N

O

O

O6

CH3

7N

1

4

2

3 5N

O

OH

O

(2a) (3a) (4a)

The diester (2a) was refluxed in diphenyl ether to get the cyclized

product ethyl 4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (3a). Its IR

spectrum displayed characteristic peaks at 1730 (C=O str.) and 1103 cm-1

(C-O

str.). The cyclized product showed characteristic signals at 1.29-1.32 (3H,

CH3(7)) and 4.25-4.30 (m, 2H, CH2(6)), 7.56-7.59 (m, 1H, Ar-H(2)), 7.84-7.87 (d,


63

1H, Ar-H(4)), 8.19-8.23 (m, 1H, Ar-H(3)), 8.87 (s, 1H, Ar-H(5)) and 9.16-9.18 (d,

1H, Ar-H(1)) in its NMR spectrum. The (M++1) peak was obtained at (m/z) 219 in

its mass spectrum.

4-Oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (4a) was prepared

by acid catalyzed ester hydrolysis of the compound (3a) using concentrated

hydrochloric acid. Characteristic peaks were displayed at 3091 (O-H str.) and

1757 cm-1

(C=O str.) in its IR spectrum. The 1H-NMR displayed characteristic

signals at 7.67-7.71 (m, 1H, Ar-H(2)), 7.97-8.00 (d, 1H, Ar-H(4)), 8.31-8.35 (m,

1H, Ar-H(3)), 8.96 (s, 1H, Ar-H(5)) and 9.22-9.23 (d, 1H, Ar-H(1)). The (M++1)

peak was observed in its mass spectrum at (m/z) 191.


The carbonyl azide (5a) was prepared by reacting the acid (4a) with

ethyl chloroformate to get the mixed anhydride which was treated in situ with

aqueous sodium azide offering 4-oxo-4H-pyrido[1,2-a]pyrimidine-3-

carbonylazide (5a). Characteristic absorption bands were observed at 2140 for the

azide and 1724 cm-1

(C=O, str.) in its IR spectrum. The 1H-NMR showed peaks

at 7.61-7.64 (m, 1H, Ar-H(2)), 7.87-7.91 (d, 1H, Ar-H(4)), 8.24-8.35 (m, 1H, Ar-

H(3)), 8.94 (s, 1H, Ar-H(5)) and 9.10-9.28 (d, 1H, Ar-H(1)). The mass spectrum of

the compound (5a) showed (M+-1) peak at (m/z) 214.

N

1

4

2

3 5N

O

N

O

N+

N

N

1

4

2

3 5N

O

N=C=O

(5a) (6a)

The azide (5a) was refluxed in toluene to get the Curtius rearrangement

product 3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one (6a). Characteristic peaks

were observed at 2140 (N=C=O str.) and 1687 cm-1

(C=O str.) in its IR spectrum.

The mass spectrum displayed (M++1) peak at (m/z) 188.

Synthesis of 8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic

acid (4b)

Similarly, diethyl (4-methyl-2-pyridylamino)methylenemalonate (2b)

was prepared by reacting compound (1b) with diethyl ethoxymethylenemalonate


64

as described above for compound (2a). It gave strong characteristic absorption

bands at 1682 (C=O str.) and 1218 cm-1

(C-O str.) in its IR spectrum. The mass

spectrum displayed (M++1) peak at (m/z) 279.

N

1

4

2

6NH

5

O

7

O

O

9

CH3

10

O

CH3

8

CH3

3

N

1

4

2

5N

O

O

O6

CH3

7

CH3

3

N

1

4

2

5N

O

OH

O

CH3

3

(2b) (3b) (4b)

Ethyl 8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (3b)

was prepared from compound (2b) as described above for compound (3a). Strong

characteristic peaks were observed at 1689, (C=O str.) and 1143 cm-1

(C-O str.) in

its IR spectrum. Its 1H-NMR displayed signals of the ethyl ester groups at 1.30

(t, 3H, CH3(7)), 2.51 (s, 3H, CH3(3)), 4.23-4.29 (m, 2H, CH2(6)), 7.42-7.44 (m, 1H,

Ar-H(2)), 7.67 (s, 1H, Ar-H(4)), 8.82 (s, 1H, Ar-H(5)), 9.04-9.05 (d, 1H, Ar-H(1)).

The mass spectrum showed (M++1) peak at (m/z) 233.

8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (4b) was

obtained on acidic hydrolysis of the ester (3b). It gave characteristic absorption

bands at 2645 (O-H str.) and 1764 cm-1

(C=O str.) in its IR spectrum. 1H-NMR

displayed characteristic signals for methyl group at 2.62 (s, 3H, CH3(3)) and acid

at 10.90 (b, 1H, COOH). The aromatic protons were observed at 7.62-7.64 (m,

1H, Ar-H(2)), 7.89 (s, 1H, Ar-H(4)), 8.88-8.90 (d, 1H, Ar-H(5)) and 9.14-9.16 (d,

1H, Ar-H(1)). The compound gave (M+-1) peak at (m/z) 203 in its mass spectrum.

Synthesis of 3-isocyanato-8-methyl-4H-pyrido[1,2-a]pyrimidin-4-one

(6b)

The synthesis of 8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-

carbonylazide (5b) was accomplished by treating (4b) with ethyl chloroformate

and sodium azide as outlined in (Scheme 1). Characteristic IR peaks were

observed at 2142 (azide str.) and 1712 cm-1

(C=O str.) in its IR spectrum. Its 1H-

NMR signals appeared at 2.56 (s, 3H, CH3(3)), 7.49-7.51 (d, 1H, Ar-H(2)), 7.75

(s, 1H, Ar-H(4)), 8.84 (s, 1H, Ar-H(5)) and 9.09-9.13 (d, 1H, Ar-H(1)). The mass

spectrum showed (M++1) signal at (m/z) 230.


65

N

1

4

2

5N

O

N

O

N+

NCH

3

3

N

1

4

2

5N

O

CH3

3

N=C=O

(5b) (6b)

The acid azide (5b) was refluxed in toluene as described above for (6a)

yielding 3-isocyanato-8-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (6b). Strong

absorption bands were observed at 2223 (N=C=O str.) and 1670 cm-1

(C=O str.) in

its IR spectrum. The mass showed (M++1) peak at (m/z) 201.

Synthesis of 7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic

acid (4c)

Diethyl (5-chloro-2-pyridylamino)methylenemalonate (2c) was

synthesized by reacting compound (1c) with diethyl ethoxymethylenemalonate as

described above for compound (2a). Its IR spectrum showed strong absorption

bands at 1676 (C=O

N

1

4

2

3 6NH

5

O

7

O

O

9

CH3

10

O

CH3

8

ClN

1

4

2

3 5N

O

O

O6

ClCH

37

N

1

4

2

3 5N

O

OH

O

Cl

(2c) (3c) (4c)

str.) and 1219 cm-1

(C-O str.). The characteristic NMR signals were displayed at

1.11-1.29 (m, 6H, CH3(8/10)), 4.13-4.26 (m, 4H, CH2(7/9)), 7.47-7.49 (d, 1H, Ar-

H(3)), 7.91-7.94 (m, 1H, Ar-H(4)), 8.41 (s, 1H, Ar-H(1)), 8.94-8.97 (d, 1H, Ar-H(6))

and 10.79-10.82 (d, 1H, NH(5)). Molecular ion peak (M++1) was observed at (m/z)

299 in its mass spectrum.

Ethyl 7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (3c)

was prepared from compound (2c) as described above for compound (3a). Strong

peaks were observed in its IR spectrum at 1681 (C=O str.) and 1222 cm-1

(C-O str.).

The mass spectrum displayed a prominent (M++1) peak at (m/z) 253.


66

7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (4c) was

prepared from compound (3c) as described above for compound (4a).

Characteristic IR bands were observed at 3063 (O-H str.), 1756 cm-1

(C=O str.) in

its IR spectrum. The mass spectrum showed (M++1) peak at (m/z) 225.6.

Synthesis of 7-chloro-3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one

(6c)

Reaction of compound (4c) with ethyl chloroformate and sodium azide

was done as described above for compound (5a) to get 7-chloro-4-oxo-4H-

pyrido[1,2-a]pyrimidine-3-carbonylazide (5c). Its IR spectrum showed absorption

bands at 2158 (azide str.) and 1732 cm-1

(C=O str.). A prominent (M++1) peak

was observed at (m/z) 250.6 in its mass spectrum.

N

1

4

2

3 5N

O

N

O

N+

N

Cl

N

1

4

2

3 5N

O

Cl N=C=O

(5c) (6c)

7-Chloro-3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one (6c) was

synthesized as described above for compound (6a). It displayed characteristics

peaks at 2243 (N=C=O str.) and 1668 cm-1

(C=O str.) in its IR spectrum. The

mass spectrum showed (M++1) peak at 222.6.

3.1.2 Synthesis of substituted (4-oxo-4H-pyrido[1,2-a]pyrimidin-3-

yl)urea derivatives (Series I)

The ureas Series-I (I-1 to I-34) were prepared by coupling of suitable

isocyanate (6a-c) with amine derivatives of type R1NHA in toluene under reflux

conditions following the procedure as depicted in Scheme 2 or Scheme 3.


The synthesis of 1-(4-chlorobenzyl)-3-(4-oxo-4H-pyrido[1,2-

a]pyrimidin-3-yl)urea (I-1) has been carried out by refluxing the isocyanate

derivative (6a) (Scheme 1) and 4-chlorobenzylamine in toluene as depicted in

Scheme 2 to get the compound (I-1). Its IR spectrum showed characteristic peaks


67

at 3318 (N-H str.) and 1693 cm-1

(C=O str.). The 1H-NMR displayed

characteristic signals at 4.39-4.41 (d, 2H, CH2(8)), 7.26-7.28 (m, 1H, Ar-H(2)),

N

N

NH

O

O

NR R N NN

N

NH

O

O

NH N A,

N

N

O

N

CF3

CF3

NH

CH3

O

N N

O

CF3

FF

NO

CH3

CH3

N

N

ClN

ONN

O

CH3

CH3

O

OCH

3

N

N

O

O

O

CH3

NH2

O

O

OSS

O

OCH

3

CH3

CH3

6 (a-c)

Toluene, reflux

A A

R1NHA, X=

R1

Toluene, reflux

(I-1 to I-34)

N=C=O

OCH3

A =

R1 = H, i.Pr, Cycloproyl, Benzyl

OCH3

OCH3

a. R = H, b. R = 8-CH3, c. R = 7-Cl

(II-1 to II-11)

Scheme 2

7.30-7.35 (m, 2H, Ar-H(9,10)), 7.41-7.47 (m, 2H, Ar-H(11,12)), 7.56 (t, 1H, NH(7)),

7.61-7.64 (d, 1H, Ar-H(4)), 7.68-7.71 (m, 1H, Ar-H(3)), 8.55 (s, 1H, NH(6)), 8.85-

8.87 (d, 1H, Ar-H(1)) and 9.16 (s, 1H, Ar-H(5)). The mass spectrum displayed

molecular ion peak (M+) at 328.9 and (M

++2) at (m/z) 330.8 characteristic of the

chloro compound.

1-(4-Methoxybenzyl)-3-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea (I-

2) was prepared by reacting the isocyanate (6a) with 2-methoxyethylamine as


68

described above for compound (I-1). Characteristic signals appeared at 3361 (N-H

str.), 1639 (C=O str.) and 1180 cm-1

(C-O str.) in its IR spectrum. 1H-NMR

spectrum showed peaks at 3.73 (s, 3H, OCH3(13)), 4.24–4.25 (d, 2H, CH2(8)),

6.89–6.91 (d, 2H, Ar-H(9,10)), 7.22–7.27 (m, 3H, Ar-H(11,12) & Ar-H(2)), 7.40–7.43

(t, 1H, NH(7)), 7.61–7.63 (d, 1H, Ar-H(4)), 7.68–7.72 (m, 1H, Ar-H(3)), 8.39 (s, 1H,

NH(6)), 8.84–8.86 (d, 1H, Ar-H(1)) and 9.18 (s, 1H, Ar-H(5)). A molecular ion peak

(M++1) was observed at (m/z) 325 in its mass spectrum.

N

1

4

2

3 5N

O

NH

6NH

7

O8 9

11

12

10 Cl

N

1

4

2

3 5N

O

NH

6NH

7

O8 9

11

12

10 OCH3

(I-1) (I-2)

The synthesis of 1-(2-methoxyethyl)-3-(4-oxo-4H-pyrido[1,2-a]pyri-

midin-3-yl)urea (I-3) was accomplished by reacting the isocyanate derivative (6a)

with 2-methoxyethylamine as described above for compound (I-1). It gave

characteristic IR absorption bands at 3362 (N-H str.), 1639 (C=O str.) and 1099

cm-1

(C-O str.). Its 1H-NMR showed characteristic signals at 3.25-3.28 (m, 5H,

OCH3(10) & CH2(9)), 3.70-3.39 (t, 2H, CH2(8)), 7.16-7.17 (m, 1H, NH(7)), 7.23-7.27,

(m, 1H, Ar-H(2)), 7.60-7.63 (d, H, Ar-H(4)), 7.67-7.72 (m, 1H, Ar-H(3)), 8.41 (s,

1H, NH(6)), 8.45-8.86 (d, 1H, Ar-H(1)) and 9.16 (s, 1H, Ar-H(5)). The mass

spectrum displayed (M++1) peak at (m/z) 263.

N

1

4

2

3 5N

O

NH

6NH

7

O8

9

OCH3

10

N

1

4

2

3 5N

O

NH

6NH

7

O8

9

NH

10

O

CH3

11

(I-3) (I-4)

The isocyanate (6a) was reacted with 2-acetamidoethylamine as

described above for compound (I-1) affording N-{2-[3-(4-Oxo-4H-pyrido[1,2-

a]pyrimidin-3-yl)ureido]ethyl}acetamide (I-4). It showed characteristic peaks at

3317 (N-H str.), 1633 cm-1

(C=O str.) in its IR spectrum. The characteristic peaks

were observed at 1.81 (s, 3H, CH3(11)), 3.11-3.16 (m, 4H, CH2(8,9)), 7.09-7.11 (m,

1H, NH(7)), 7.23-7.27 (m, 1H, Ar-H(2)), 7.61-7.63 (d, 1H, Ar-H(4)), 7.68-7.72 (m,

1H, Ar-H(3)), 7.94 (b, 1H, NH(10)), 8.35 (s, 1H, NH(6)), 8.84-8.86 (d, 1H, Ar-H(1))


69

and 9.17 (s, 1H, Ar-H(5)) in its NMR spectrum. The compound (I-4) offered

(M++1) peak at (m/z) 290 in its mass spectrum.

1-(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-(2-trifluoromethylbenzyl)-

urea (I-5) was prepared by reacting the isocyanate (6a) with 2-trifluoromethyl-

benzylamine as described above for compound (I-1). The compound showed

strong peaks at 3315 (N-H str.) and 1667 cm-1

(C=O str.) in its IR spectrum. The

characteristic signals were displayed at 4.51-4.52 (d, 2H, CH2(8)), 7.25-7.28 (m,

1H, Ar-H(2)), 7.47-7.51 (m, 1H, NH(7)), 7.56-7.64 (m, 3H, Ar-H(4,11,12)), 7.68-7.74

(m, 3H, Ar-H(3,9,10)), 8.59 (s, 1H, NH(6)), 8.86-8.88 (d, 1H, Ar-H(1)) and 9.17 (s,

1H, Ar-H(5)) in its NMR spectrum. The compound gave (M

++1) peak at (m/z) 363

in its mass spectrum.

N

1

4

2

3 5N

O

NH

6NH

7

O8

10

12

11

9

CF3

N

1

4

2

3 5N

O

NH

6NH

7

O8 9

11

12

10 CF3

(I-5) (I-6)

1-(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-(4-trifluoromethylbenzyl)-

urea (I-6) displayed characteristic peaks at 3328 (N-H str.), 3048 (Ar-H str.) and

1670 cm-1

(C=O str.) in its IR spectrum. The NMR spectrum displayed

characteristic signals at 4.42-4.44 (d, 2H, CH2(8)), 7.25-7.28 (m, 1H, Ar-H(2)),

7.51-7.54 (d, 2H, Ar-H(9,10)), 7.59-7.64 (m, 2H, Ar-H(3,4)), 7.69-7.73 (m, 3H, Ar-

H(11,12) & NH(7)), 8.50 (s, 1H, NH(6)), 8.86-8.87 (d, 1H, Ar-H(1)) and 9.16 (s, 1H,

Ar-H(5)). The (M+-1) peak was observed at (m/z) 361 in its mass spectrum.

The compound 1-(2,4-difluorobenzyl)-3-(4-oxo-4H-pyrido[1,2-

a]pyrimidin-3-yl)urea (I-7) showed characteristic IR signals at 3328 (N-H str.)

and 1676 cm-1

(C=O str.). The 1H-NMR displayed characteristic peaks at 4.35-

N

1

4

2

3 5N

O

NH

6NH

7

O8

10

11

9F

F

10 11

12S9

N

1

4

2

3 5N

O

NH

6NH

7

O8

(I-7) (I-8)

4.37 (d, 2H, CH2(8)), 7.14-7.20 (m, 2H, Ar-H(9,11)), 7.24-7.28 (m, 2H, Ar-H(2,10)),

7.52-7.55 (t, 1H, NH(7)), 7.62-7.64 (d, 1H, Ar-H(4)), 7.69-7.74 (m, 1H , Ar-H(3)),


70

8.50 (s, 1H, NH(6)), 8.85-8.87 (d, 1H, Ar-H(1)) and 9.14 (s, 1H, , Ar-H(5)). The

molecular ion (M++1) peak was observed at (m/z) 331 in its mass spectrum.

1-(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-[2-(thiophen-2-yl)ethyl]-

urea (I-8) showed characteristic IR signals at 3213 of urea (N-H str.), 3033 (Ar-H

str.) and 1656 cm-1

(C=O str.). The compound gave the signals at 2.97-3.01 (t,

2H, CH2(9)), 3.37-3.4 (t, 2H, CH2(8)), 6.92-6.93 (b, 1H, Ar-H(10)), 6.97-6.99 (m,

1H, Ar-H(11)), 7.15-7.18 (t, 1H, NH(7)), 7.23-7.27 (m, 1H, Ar-H(2)), 7.35-7.37 (d,

1H, Ar-H(12)), 7.61-7.63 (d, 1H, Ar-H(4)), 7.68-7.72 (m, 1H, Ar-H(3)), 8.41 (s, 1H,

NH(6)), 8.84-8.85 (d, 1H, Ar-H(1)) and 9.19 (s, 1H, Ar-H(5)) in its NMR spectrum.

The mass spectrum showed (M++1) peak at (m/z) 315.

The compound 1-[3-(morpholin-4-yl)propyl]-3-(4-oxo-4H-pyrido[1,2-

a]pyrimidin-3-yl)urea (I-9) displayed characteristic peaks at 3297 (N-H str.) and

3072 (Ar-H str.), 1663 (C=O str.) and 1118 cm-1

(C-O str.) in its IR spectrum. The

signals were displayed at 1.55–1.62 (m, 2H, CH2(9)), 2.28–2.32 (m, 2H, CH2(10)),

2.34 (b, 4H, CH2(11/12)), 3.10–3.15 (m, 2H, CH2(8)), 3.56–3.58 (t, 4H, CH2(13/14)),

7.01–7.04 (m, 1H, NH(7)), 7.23–7.27 (m, 1H, Ar-H(2)), 7.60–7.63 (d, 1H, Ar-H(4)),

7.67–7.71 (t, 1H, Ar-H(3)), 8.29 (s, 1H, NH(6)), 8.84–8.86 (d, 1H, Ar-H(1)) and 9.17

(s, 1H, Ar-H(5)) in its NMR spectrum. The compound gave (M++1) peak at (m/z)


N

1

4

2

3 5N

O

NH

6NH

7

O8

9

10

N

11 13

O

1412

N

1

4

2

3 5N

O

NH

6NH

7

O8

9

10

1211

1413

15

17

16

19

18

20

(I-9) (I-10)

1-(3,3-Diphenyl)propyl-3-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea

(I-10) displayed strong IR signals at 3324 (N-H str.) and 1664 cm-1

(C=O Str.).

The signals were observed at 2.18-2.23 (m, 2H, CH2(9)), 2.97-3.05 (m, 2H,

CH2(8)), 4.01-4.05 (t, 1H CH(10)), 7.11-7.34 (m, 12H, Ar-H(2,11-20) & NH(7)), 7.60-

7.63 (d, 1H, Ar-H(4)), 7.67-7.74 (m, 1H, Ar-H(3)), 8.32 (s, 1H, NH(6)), 8.85-8.92

(d, 1H, Ar-H(1)) and 9.15 (s, 1H, Ar-H(5)) in its 1H-NMR spectrum. The (M

++1)

peak was displayed at (m/z) 399 in the mass spectrum.


71

The IR spectrum of the compound 1-(4-oxo-4H-pyrido[1,2-a]pyrimidin-

3-yl)-3-[3-(n.pentyloxy)pyridin-2-yl]urea (I-11) displayed characteristic bands at

3350 (N-H str.), 1665 (C=O str.) and 1220 cm-1

(C-O Str.). The 1H-NMR signals

were displayed at 0.90-0.94 (t, 3H, CH3(15)), 1.32-1.47 (m, 4H, CH2(13,14)), 1.78-

1.85 (m, 2H, CH2(12)), 4.07-4.11 (t, 2H, CH2(11)), 7.05-7.08 (m, 1H, Ar-H(9)), 7.30-

7.34 (m, 1H, Ar-H(2)), 7.44-7.46 (d, 1H, Ar-H(8)), 7.67-7.69 (d, 1H, Ar-H(4)), 7.76-

7.81 (m, 1H, Ar-H(3)), 7.89-7.91 (dd, 1H, Ar-H(10)), 8.35 (s, 1H, NH(6)), 8.92-8.94

(d, 1H, Ar-H(1)), 9.29 (s, 1H, Ar-H(5)) and 12.15 (s, 1H, NH(7)). The compound

gave (M++1) peak at (m/z) 368 in its mass spectrum.

Characteristic peaks were displayed in the IR spectrum of 1-[3-

(benzyloxy)pyridin-2-yl]-3-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea (I-12) at


(Ar-O str.). 1H-NMR signals

appeared at 5.29 (s, 2H, CH2(11)), 7.07 (m, 1H, Ar-H(9)), 7.33-7.42 (m, 3H, Ar-

H(14-16)), 7.51-7.57 (m, 3H, Ar-H(2,12,13)), 7.70-7.76 (m, 1H, Ar-H(4)), 7.77-7.79 (m,

1H, Ar-H(3)), 7.91-7.92 (d, 1H, Ar-H(8)), 8.46 (s, 1H, NH(6)), 8.90-8.94 (m, 1H, Ar-

H(10)), 9.22-9.28 (d, 1H, Ar-H(1)), 9.52 (s, 1H, Ar-H(5)) and 12.06 (s, 1H, NH(7)).

The compound offered (M++1) peak at (m/z) 388 in its mass spectrum.

N

1

4

2

3 5N

O

NH

6NH

7

O N10

9

8

O

11

12

13

14

CH3

15

N

1

4

2

3 5N

O

NH

6NH

7

O N

8

10

9

O

11

12 14

16

1513

(I-11) (I-12)

N

1

4

2

3 5N

O

NH

6NH

7

O

N

8

9

10

N

O

14

13

12

11

(I-13)

1-[6-(Morpholin-4-yl)pyridin-2-yl]-3-(4-oxo-4H-pyrido[1,2-a]pyrimi-

din-3-yl)urea (I-13) displayed characteristic Ibands at 3118 (N-H str.), 1688

(C=O str.) and 1229 cm-1

(C-O str.). Its NMR spectrum showed characteristic

signals at 3.66-3.68 (b, 4H, Ar-H(11,12)), 3.95-3.97 (b, 4H, Ar-H(13,14)), 6.17-6.20

(d, 1H, Ar-H(10)), 6.29-6.31 (d, 1H, Ar-H(8)) and 11.48 (s, 1H, NH(7)). The mass

spectrum showed (M++1) peak at (m/z) 367.


72

To study the binding pattern in the P1 and P2 pocket, some additional

compounds bearing esters (I-14), (I-19), amides (I-15), (I-17) and nitriles (I-18),

(I-19) were also synthesized. The esters, amide and nitriles have been reported to

show good binding affinity for FPs. Hence compounds (I-14 to I-19) were

synthesized by following the reaction sequence as outlined in the Scheme 3.

NH2

R'

O

O CH3

n

NH

R'

O

O

N

N

NH

O

O

CH3

n

NH

R'

O

NH2

N

N

NH

O

O

CH3

CH3

NH

R'

N

N

NH

O

O

N

N

N

N

Cl

Cl

Cl

liq. ammonia

(6a,b) +

Toluene Reflux

(I-14, I-16)

R' =

n = 0, 1

DMF, 0OC

(I-15, I-17) (I-18, I-19)

Scheme 3

Ethyl (2S)-2-{[(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)carbamoyl]ami-

no}-3-phenylpropanoate (I-14) was prepared by reacting compound (6a) with L-

phenylalanine ethyl ester, afforded compound (I-14). The IR spectrum showed

characteristic peaks at 3338 (N-H str.), 3032 (Ar-H str.) and 1733, 1629 cm-1

(C=O str.). The mass spectrum displayed the (M++1) at (m/z) 381.

N

1

4

2

3 5N

O

NH

6NH

7

O

O8

9

O

10

12

14

13

11

CH3

N

1

4

2

3 5N

O

NH

6NH

7

O

NH2

158

9

O

10

12

14

13

11

(I-14) (I-15)

(2S)-2-{[(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)carbamoyl]amino}-3-

phenylpro-panamide (I-15) was prepared by reacting compound (I-14) with


73

ammonia solution yielding the compound (I-15). Its IR spectrum showed peaks at

3329 (N-H str,) and 1634 cm-1

(C=O str.). The mass spectrum displayed (M++1)

signal at (m/z) 352.

Methyl (2S)-4-methyl-2-{[(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)carba-

moyl]amino}n.pentanoate (I-16) was obtained by reacting the compound (6a)

with L-leucine methyl ester. Its IR spectrum showed characteristic peaks at 3325

(N-H str.) and 1745, 1657 cm-1

(C=O str.). The peak (M++1) was observed at

(m/z) 333 in its mass spectrum.

N

1

4

2

3 5N

O

NH

6NH

7

O

8

9 10

O

CH3

12

CH3

11

OCH313

N

1

4

2

3 5N

O

NH

6NH

7

O

NH2

138

9 10

O

CH3

12

CH3

11

(I-16) (I-17)

(2S)-4-Methyl-2-{[(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)carbamoyl]-

amino}n.pentanamide (I-17) was prepared by treating the compound (I-16) with

ammonia solution affording the compound (I-17). Its IR spectrum displayed

characteristic peaks at 3361 (N-H str.) and 1672, 1628 (C=O str.). The compound

offered (M++1) peak at (m/z) 318 in its mass spectrum.

Dehydration of the amide compound (I-16) was done using cyanuric

chloride in dimethylformamamide to get the nitrile product 1-[(1S)-1-(cyano-2-

phenyl)ethyl]-3-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea (I-18). Characteristic

peaks at 3.09-3.19 (m, 2H, CH2(9)), 4.92-4.98 (m, 1H, CH(8)), 7.26-7.34 (m, 2H,

Ar-H(2,14)), 7.36-7.37 (m, 4H, Ar-H(10-13)), 7.63-7.67 (m, 2H, Ar-H(4) & NH(7)),

7.72-7.77 (t, 1H, Ar-H(3)), 8.58 (s, 1H, NH(6)), 8.85-8.87 (d, 1H, Ar-H(1)) and 9.10

(s, 1H, Ar-H(5)) were observed in its PMR. The mass spectrum showed (M++1)

peak at (m/z) 334.

N

1

4

2

3 5N

O

NH

6NH

7

O

8

910

12

14

13

11

N

N

1

4

2

3 5N

O

NH

6NH

7

O

8

9 10 CH3

12

CH3

11

N

(I-18) (I-19)


74

1-[(1S) 1-(Cyano-3-methyl)butyl]-3-(4-oxo-4H-pyrido[1,2-a]pyrimidin-

3-yl)urea (I-19) was obtained by the dehydration of the compound (I-17) using

cyanuric chloride affording the compound (I-19). The 1H-NMR gave peaks at

0.91-0.96 (m, 6H, CH3(11,12)), 1.64-1.80 (m, 3H, CH2(9) & CH(10)), 4.65-4.71 (m,

1H, CH(8)), 7.27-7.31 (m, 1H, NH(2)), 7.65-7.67 (d, 2H, Ar-H(4) & NH(7)), 7.73-

7.77 (m, 1H, Ar-H(3)), 8.49 (s, 1H, NH(6)), 8.86-8.88 (d, 1H, Ar-H(1)) and 9.12 (s,

1H, Ar-H(5)). The mass spectrum gave (M++1) peak at (m/z) 300.

9

11

13

12

10

N

1

4

2

3 5N

O

NH

6N7

O8

14

CH3

16

CH3

15

(I-20)

1-Benzyl-1-isopropyl-3-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea (I-

20) showed strong signals at 3373 (N-H str.) and 1641 cm-1

(C=O str.) in its IR

spectrum. Characteristic NMR peaks appeared at 1.14-1.16 (d, 6H, CH3(15,16)),

4.51-4.52 (m, 1H, CH(14)), 4.55 (s, 2H, CH2(8)), 7.25-7.29 (m, 2H, Ar-H(2,13)), 7.36-

7.37 (d, 4H, Ar-H(9-12)), 7.50 (s, 1H, NH(6)), 7.63-7.65 (d, 1H, Ar-H(4)), 7.73-7.77

(m, 1H, Ar-H(3)), 8.78-8.80 (d, 1H, Ar-H(1)) and 8.96 (s, 1H, Ar-H(5)). The

compound offered (M++1) peak at (m/z) 337 in its mass spectrum.


derivatives

1-(2-Methoxyethyl)-3-(8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-

yl)urea (I-21) showed characteristic peaks at 3305 (N-H str.), 1633 (C=O str.) and

1228 cm-1

(C-O str.) in its IR spectrum. Characteristic signals appeared at 2.39

(s, 3H, CH3(3)), 3.22-3.36 (m, 7H, CH2(8,9) & OCH3(10)), 7.09-7.7.11 (m, 2H, Ar-

H(2) & , NH(7)), 7.41(s, 1H, Ar-H(4)), 8.30 (s, 1H, NH(6)), 8.73-8.75 (d, 1H, Ar-H(1))

and 9.06 (s, 1H, Ar-H(5)) in its 1HNMR. The compound displayed (M

+-1) peak at


N-{2-[3-(8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)ureido]ethyl}-

acetamide (I-22) gave strong IR signals at 3356 (N-H str.) and 1633 cm-1

(C=O

str) in its IR spectrum. The characteristic signals were displayed at 1.81 (s, 3H,

CH3(11)), 2.41 (s, 3H, CH3(3)), 3.1-3.15 (m, 4H, CH2(8,9)), 7.03-7.06 (t, 1H, NH(7)),


75

7.11-7.13 (dd, 1H, Ar-H(2)), 7.43 (s, 1H, Ar-H(4)), 7.93-7.94 (t, 1H, NH(10)), 8.26

(s, 1H, NH(6)), 8.76-8.78 (d, 1H, Ar-H(1)) and 9.09 (s, 1H, Ar-H(5)) in its NMR.

The mass showed the (M++1) peak at (m/z) 304.

NH

7

9

8N

1

4

2

5N

O

NH

6

OCH

3

3

OCH3

10

N

1

4

2

5N

O

NH

6NH

7

O8

9

NH

10

O

CH3

11

CH3

3

(I-21) (I-22)

The characteristic IR signals of 1-(8-methyl-4-oxo-4H-pyrido[1,2-

a]pyrimidin-3-yl)-3-[2-(thiophen-2-yl)ethyl]urea (I-23) appeared at 3278 (N-H

str) and 1634 cm-1

(C=O str.). The characteristic peaks at 2.41 (s, 3H, CH3(3)),

2.95-2.98 (m, 4H, CH2(8,9)), 6.92 (s, 1H, Ar-H(10)), 6.98 (b, 1H, Ar-H(11)), 7.11-

7.12 (m, 2H, Ar-H(2) & NH(7)), 7.35-7.37 (d, 1H, Ar-H(12)), 7.43 (s, 1H, Ar-H(4)),

8.32 (s, 1H, NH(6)), 8.75-8.77 (d, 1H, Ar-H(1)) and 9.11 (s, 1H, Ar-H(5)) in its

NMR spectrum. The mass spectrum displayed the (M++1) peak at (m/z) 329.

10 11

12S9

N

1

4

2

5N

O

NH

6NH

7

O8

CH3

3

N

10 12

O

1311

N

1

4

2

5N

O

NH

6NH

7

O8

9

CH3

3

(I-23) (I-24)

The IR spectrum of 1-(8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-

3-[2-(morpholin-4-yl)ethyl]urea (I-24) showed characteristic peaks at 3334 (N-H

str.), 1632, (C=O str.) and 1145 cm-1

(C-O str.). The 1H-NMR displayed peaks at

2.37-2.41 (m, 9H, CH3(3) & CH2(9,10,11)), 3.21-3.24 (m, 2H, CH2(8)), 3.58-3.59 (b,

4H, CH2(12,13)), 6.98-6.99 (m, 1H NH(7)), 7.11-7.12 (d, 1H Ar-H(2)), 7.42 (s, 1H

Ar-H(4)), 8.37 (s, 1H NH(6)), 8.76-8.78 (d, 1H Ar-H(1)) and 9.08-9.09 (d, 1H Ar-

H(5)). The mass spectrum displayed (M++1) peak at (m/z) 332.

1-Benzyl-1-isopropyl-3-(8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-

yl)urea (I-25) gave characteristic bands at 3353 (N-H str.) and 1642 cm-1

(C=O

str.) in its IR spectrum. The characteristic

1HNMR peaks were observed at 1.13-

1.15 (d, 6H, CH3(15,16)), 2.41 (s, 3H, CH3(3)), 4.49-4,51 (m, 1H, CH(14)), 4.54 (s,

2H, CH2(8)), 7.13-7.15 (d, 1H, Ar-H(2)), 7.25- 7.26 (m, 1H, Ar-H(13)), 7.35-7.36 (d,

4H, Ar-H(9-12)), 7.45 (b, 2H, NH(6) & Ar-H(4)), 8.70-8.72 (d, 1H, Ar-H(1)) and 8.87


76

(s, 1H, Ar-H(5)). The compound displayed (M++1) peak at (m/z) 351 in its mass

spectrum.

9

11

13

12

10

N

1

4

2

5N

O

NH

6N7

O8

14

CH3

16

CH3

15

CH3

3

(I-25)


derivatives

IR spectrum of 1-(7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-[2-

(morpholin-4-yl)ethyl] urea (I-26) gave characteristic peaks at 3285 (N-H str.),

1729, 1646 (C=O str.) and 1284 cm-1

(C-O str.). The signals appeared at 2.39 (b,

6H, CH2(9,10,11)), 3.23-3.24 (m, 2H, CH2(8)), 3.60 (b, 4H, CH2(12,13)), 7.08 (b, 1H,

NH(7)), 7.63-7.65 (d, 1H, Ar-H(4)), 7.70-7.73 (dd, 1H, Ar-H(3)), 8.57 (s, 1H, NH(6)),

8.84 (s, 1H, Ar-H(1)) and 9.17 (s, 1H, Ar-H(5)) in its 1H-NMR. The compound

showed (M+) peak at 352.1 and (M

++2) at (m/z) 354.1 in its mass spectrum.

N

10 12

O

1311

N

1

4

2

3 5N

O

NH

6NH

7

O8

9Cl

10 11

12S9

N

1

4

2

3 5N

O

NH

6NH

7

O8

Cl

(I-26) (I-27)

1-(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-[2-(thiophen-2-

yl)ethyl]urea (I-27) in its IR spectrum showed characteristic signals at 3387 (N-H

str.) and 1651 cm-1

(C=O str.). 1H-NMR displayed the peaks at 2.97-3.00 (t, 2H,

CH2(9)), 3.37-3.40 (t, 2H, CH2(8)), 6.92-6.93 (d, 1H, Ar-H(10)), 6.97-6.99 (m, 1H,

Ar-H(11)), 7.18-7.20 (m, 1H, NH(7)), 7.35-7.37 (m, 1H, Ar-H(12)), 7.63-7.66 (d, 1H,

Ar-H(4)), 7.71-7.74 (dd, 1H, Ar-H(3)), 8.51 (s, 1H, Ar-H(6)), 8.83-8.84 (d, 1H, Ar-

H(1)) and 9.19 (s, 1H, Ar-H(5)). The mass spectrum showed the molecular ion peak

(M+) at 349 and (M

++2) at (m/z) 350.9.

The IR spectrum of 1-(7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-

3-(thiophen-2-yl)methylurea (I-28) gave characteristics IR signals at 1702, 3272

(N-H str.) and 1652 cm-1

(C=O str.). The compound showed peaks at 4.49-4.50


77

(d, 2H, CH2(8)), 6.97-6.98 (m, 1H, Ar-H(10)), 7.00 (b, 1H, Ar-H(9)), 7.40-7.41 (d,

1H, Ar-H(11)), 7.56 (b, 1H, NH(7)), 7.64-7.66 (d, 1H, Ar-H(4)), 7.72-7.74 (d, 1H,

Ar-H(3)), 8.51 (s, 1H, NH(6)), 8.89 (s, 1H, Ar-H(1)) and 9.18 (s, 1H, Ar-H(5)) in its

NMR spectrum. The compound showed (M+) peak at (m/z) 335.34 and (M

++2) at

(m/z) 337.38 in its mass spectrum.

S11

109

N

1

4

2

3 5N

O

NH

6NH

7

O8

Cl

N

1

4

2

3 5N

O

NH

6NH

7

O8

9Cl

OCH3

10

(I-28) (I-29)

1-(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-(2-methoxyeth-

yl)urea (I-29) displayed characteristic peaks 3370 (N-H str.), 1649 (C=O str.) and

1229 cm-1

(C-O str.) in its IR spectrum.

NMR spectrum displayed characteristic

peaks at 3.24-3.39 (m, 7H, CH2(8,9) & CH3(10)), 7.19-7.21 (t, 1H, NH(7)), 7.63-

7.65 (d, 1H, Ar-H(4)), 7.70-7.73 (dd, 1H, Ar-H(3)), 8.51 (s, 1H, NH(6)), 8.83-8.84

(b, 1H, Ar-H(1)) and 9.16 (s, 1H, Ar-H(5)). The mass spectrum gave (M+) peak at

(m/z) 297 and (M++2) at (m/z) 299.

1-(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-(4-trifluorome-

thyl)benzylurea (I-30) gave characteristic peaks at 3397 (N-H str.) and 1692, 1656

cm-1

(C=O str.) in its IR spectrum. Characteristic signals appeared at 4.42-4.44

(d, 2H, CH2(8)), 7.57-7.66 (m, 6H, NH(7) & Ar-H(4,9-12)), 7.72- 7.75 (dd, 1H, Ar-

H(3)), 8.58 (s, 1H, NH(6)), 8.84-8.85 (d, 1H, Ar-H(1)) and 9.16 (s, 1H, Ar-H(5)) in its

NMR spectrum. The compound offered (M+) at (m/z) 397.34 and (M

++1) at (m/z)

399.38.

CF3

9

11

12

10

2

ClN

1

4

3 5N

O

NH

6NH

7

O8

N

1

4

2

3 5N

O

NH

6NH

7

O8 9

12

11

10

ClCF

3

(I-30) (I-31)

1-(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-(3-trifluorometh-

yl)benzylurea (I-31) displayed characteristic peaks at 3312 (N-H str.) and 1677,

1646 cm-1

(C=O str.) in its IR spectrum. 1

HNMR spectrum signals appeared at

4.42-4.43 (d, 2H, CH2(8)), 7.51-7.53 (d, 2H, Ar-H(4,10)), 7.61-7.63 (m, 2H, NH(7)

& Ar-H(11)), 7.70-7.75 (m, 3H, Ar-H(3,9,12)), 8.60 (s, 1H, NH(6)), 8.84-8.85 (d, 1H,


78

Ar-H(1)) and 9.17 (s, 1H, Ar-H(5)). The compound gave (M+) peak at (m/z) 397.34

and (M++2) at (m/z) 399.32.

Characteristic IR signals of 1-(7-Chloro-4-oxo-4H-pyrido[1,2-

a]pyrimidin-3-yl)-3-(2-trifluoromethylbenzyl)urea (I-32) appeared at 3362 (N-H

str.) and 1707, 1622 cm-1

(C=O str.). NMR spectrum showed peaks at 4.51-4.52

(d, 2H, CH2(8)), 7.47-7.51 (m, 1H, Ar-H(11)), 7.59-7.75 (m, 6H, Ar-H(3,4,9-12) &

NH(7)), 8.68 (s, 1H, NH(6)), 8.85 (b, 1H, Ar-H(1)) and 9.17 (s, 1H, Ar-H(5)). The

compound showed (M+) peak at (m/z) 397.34 and (M

++2) at (m/z) 399.32 in its

mass spectrum.

N

1

4

2

3 5N

O

NH

6NH

7

O8

10

12

11

9

CF3

Cl

N

1

4

2

3 5N

O

NH

6N7

O8

14

CH3

16

CH3

15

Cl

9

11

13

12

10

(I-32) (I-33)

N

1

4

2

3 5N

O

NH

6N7

O

Cl14

15

8

16

18

20

19

179

11

13

12

10

(I-34)

1-Benzyl-3-(7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-1-isoprop-

ylurea (I-33) displayed IR bands at 3447 (N-H str.) and 1663, 1630 cm-1

(C=O

str.). Its mass spectrum showed (M+) peak at (m/z) 371.42 and (M

++2) at (m/z)

373.41.

1-Benzyl-3-(7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-1-pheneth-

ylurea (I-34) showed characteristic IR signals at 3345 (N-H str.) 1647 cm-1

(C=O

str.). NMR peaks were displayed at 2.85-2.89 (t, 2H, CH2(15)), 3.35-3.59 (t, 2H,

CH2(14)),

4.58 (s, 2H, CH2(8)), 7.14-7.38 (m, 10H, Ar-H(9-13,16-20)), 7.68-7.71 (d,

1H, Ar-H(4)), 7.81-82 (m, 2H, Ar-H(3), NH(6)), 8.88 (b, 1H, Ar-H(1)) and 8.92 (s,

1H, Ar-H(5)). The compound showed (M+) peak at (m/z) 433.42 and (M

++2) at

(m/z) 435.41.


79

3.1.3 Synthesis of N-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-


(Series II)

Synthesis of N-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-(pyridin-2-

yl)piperazine-1-carboxamide derivatives (piperazine urea analogs) of the Series-II

(II-1 to II-11) were synthesized by coupling of isocyanate (6a-c) with suitably

substituted piperazine derivatives under reflux conditions in toluene as depicted in

Scheme 2.



The synthesis of compound N-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-

4-(pyridin-2-yl)piperazine-1-carboxamide (II-1) was done by reacting compound

(6a) with 4-(2-pyridyl)piperazine as described for compound (I-1). Characteristic

IR peaks were observed at 3393 (N-H str.) and 1659 cm-1

(C=O) str.). The NMR

spectrum showed characteristic peaks at 3.57-3.58 (b, 8H, CH2(7-10)), 6.65-6.69

(m, 1H, Ar-H(14)), 6.86-6.88 (d, 1H, Ar-H(11)), 7.31-7.35 (m, 1H, Ar-H(2)), 7.54-

7.58 (m, H, Ar-H(13)), 7.67-7.69 (d, 1H, Ar-H(4)), 7.80-7.84 (m, 1H, Ar-H(3)), 8.08

(s, 1H, NH(6)), 8.13-8.14 (d, 1H, Ar-H(12)), 8.77 (s, 1H, Ar-H(5)) and 8.90-8.93 (d,

1H, Ar-H(1)). The compound displayed (M++1) peak at (m/z) 351 in its mass

spectrum.

N

7 9

N

108

N12

14

1311

N

1

4

2

3 5N

O

NH

6

O

N

7 9

N

108

11 13

1412

N

1

4

2

3 5N

O

NH

6

O

OCH3

15

(II-1) (II-2)

The IR spectrum of 4-(4-methoxyphenyl)-N-(4-oxo-4H-pyrido[1,2-

a]pyrimidin-3-yl)piperazine-1-carboxamide (II-2) showed characteristic bands at


(C-O str.). The characteristic

signals appeared at 2.99 (b, 4H CH2(9,10)), 3.61(b, 4H CH2(7,8)), 3.81 (s, 3H,

OCH3(15)), 6.87-7.01 (m, 4H, Ar-H(11-14)), 7.32-7.35 (m, 1H, Ar-H(2)), 7.67-7.69 (d,

1H, Ar-H(4)), 7.80-7.84 (t, 1H, Ar-H(3)), 8.06 (s, 1H, NH(6)), 8.77 (s, 1H, Ar-H(5))

and 8.91-8.93 (d, 1H, Ar-H(1)) in its NMR spectrum. The (M++1) peak was

observed at (m/z) 380 in its mass spectrum.


80

N-(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-[3-(N-cyclopropylcarbo-

xamido)pyridine-2-yl]piperazinecarboxamide (II-3) offered the peaks at 3296 (N-

H str.) and 1654 cm-1

(C=O str.). The 1H-NMR gave characteristic signals at

0.55-0.58 (m, 2H, CH2(16/17)), 0.69-0.72 (m, 2H, CH2(16/17)), 2.82-2.83 (m, 1H,

CH(15)), 3.31-3.36 (t, 4H, CH2(9,10)), 3.57-3.59 (t, 4H, CH2(7,8)), 6.89-6.92 (m, 1H,

Ar-H(12)), 7.32-7.36 (m, 1H, Ar-H(2)), 7.65-7.67 (m, 2H, Ar-H(4,11)), 7.81-7.85 (m,

1H, Ar-H(3)), 8.08 (s, 1H, NH(6)), 8.23-8.25 (dd, 1H, Ar-H(13)), 8.51-8.53 (d, 1H,

NH(14)), 8.76 (s, 1H, Ar-H(5)) and 8.91-8.93 (d, 1H, Ar-H(1)). The (M++1) peak

was observed at (m/z) 434 in its mass spectrum.

N

1

4

2

3 5N

O

NH

6N

O

N

10

9

8

7

N13

12

11

NH14

O

15

16 17

N

1

4

2

3 5N

O

NH

6N

O

11

13

12

15

14

16

18

17 20

19 21

N

9

10

7

8

(II-3) (II-4)

4-Benzhydryl-1-[N-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)]carboxami-

dopiperazine (II-4) displayed characteristic signals at 3403 (N-H str.) and 1657

cm-1

(C=O Str.). The 1H-NMR displayed characteristic peaks at 2.33-2.36 (b,

4H, CH2(9,10)), 3.48-3.49 (b, 4H, CH2(7,8)), 4.35 (s, 1H, CH(11)), 7.19-7.22 (m, 2H,

Ar-H(16,21)), 7.30-7.34 (m, 5H, Ar-H(2,14,15,19,20)), 7.45-7.47 (d, 4H, Ar-

H(14,15,19,20)), 7.66-7.68 (d, 1H, Ar-H(4)), 7.79-7.83 (m, 1H, Ar-H(3)), 7.93 (s, 1H,

NH(6)), 8.74 (s, 1H, Ar-H(5)) and 8.88-8.90 (d, 1H, Ar-H(1)). The compound gave




N-(8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-(pyridin-2-yl)-

piperazine-1-carboxamide (II-5) offered characteristic bands at 3395 (N-H str.)

and 1654 cm-1

(C=O str.) in its IR spectrum. Characteristic peaks appeared at

2.45 (3H, CH3(3)), 3.57 (b, 8H, CH2(7-10)), 6.65-6.68 (t, 1H, Ar-H(14)), 6.86-6.88 (d,

1H, Ar-H(11)), 7.20-7.22 (d, 1H, Ar-H(2)), 7.50 (s, 1H, Ar-H(4)), 7.54-7.58 (m, 1H,

Ar-H(13)), 8.05 (s, 1H, NH(6)), 8.13-8.14 (d, 1H, Ar-H(12)), 8.66 (s, 1H, Ar-H(5)) and


81

8.82-8.84 (d, 1H, Ar-H(1)) in its NMR spectrum. The mass spectrum displayed the

(M++1) peak at (m/z) 365.

N

7 9

N

108

N12

14

1311

N

1

4

2

3 5N

O

NH

6

OCH

3

N

7 9

N

108

11 13

1412

N

1

4

2

3 5N

O

NH

6

OCH

3

OCH3

15

(II-5) (II-6)

N

1

4

2

3 5N

O

NH

6N

O

11

13

12

15

14

16

18

17 20

19 21

N

9

10

7

8

CH3

(II-7)

N-(8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-[(4-methoxy-

phenyl)]piperazine-1-carboxamide (II-6) gave characteristic IR signals at 3393

(N-H str.), 1648 cm-1

(C=O str.) and 1241 (C-O str.). Its PMR spectra displayed

characteristic peaks at 2.44 (s, 3H, CH3(3)), 2.97-2.99 (b, 4H, CH2(9,10)), 3.58-

3.61 (b, 4H, CH2(7,8)), 3.80 (s, 3H, OCH3(15)), 6.87-7.00 (m, 4H, Ar-H(11-14)), 7.19-

7.22 (dd, 1H, Ar-H(2)), 7.50 (s, 1H, Ar-H(4)), 8.00 (s, 1H, NH(6)), 8.66 (s, 1H, Ar-

H(5)), 8.82-8.84 (d, 1H, Ar-H(1)) in its NMR spectrum. The mass spectrum

displayed (M++1) peak at (m/z) 394.

N-(8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-benzhydrylpip-

erazine-1-carboxamide (II-7) displayed characteristic IR bands at 3390 (N-H str.),

1644 cm-1

(C=O str.). The compound displayed characteristic signals at 2.32-

2.34 (b, 4H, CH2(9,10)), 2.43 (s, 3H, CH3(3) ), 3.46-3.48 (b, 4H, CH2(7,8)), 4.34 (s,

1H, CH(11)), 7.18-7.22 (m, 3H, Ar-H(2 & diphenyl)), 7.29-7.33 (m, 4H, Ar-H(diphenyl)),

7.44-7.48 (m, 5H, Ar-H(4 & diphenyl), 7.87 (s, 1H, NH(6)), 8.63 (s, 1H, Ar-H(5)) and

8.79-8.81 (d, 1H, Ar-H(1)) in its NMR spectrum. The (M++1) peak was observed at


Synthesis of N-(7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-


N-[(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-(pyridin-2-

yl)piperazine]-1-carboxamide (II-8) was prepared by refluxing compound (6c)


82

with 4-(pyridin-2-yl)piperazine as depicted in Scheme 1. Characteristic peaks

were observed in its IR spectrum at 3397 (N-H str.) and 1729, 1643 cm-1

(C=O

str.). Characteristic signals were observed at 3.57-3.58 (m, 8H, CH2(7-10)), 6.65-

6.68 (m, 1H, Ar-H(14)), 6.86-6.88 (d, 1H, Ar-H(11)), 7.54-7.58 (m, 1H, Ar-H(13)),

7.69-7.71 (d, 1H, Ar-H(4)), 7.83-7.86 (dd, 1H, Ar-H(3)), 8.13-8.14 (d, 1H, Ar-

H(12)), 8.16 (s, 1H, NH(6)), 8.82 (s, 1H, Ar-H(5)) and 8.89-8.90 (d, 1H, Ar-H(1)) in

its NMR spectrum. The compound gave (M+) peak at (m/z) 384.8 and (M

++2) at

(m/z) 386.5.

N

7 9

N

108

N12

14

1311

N

1

4

2

3 5N

O

NH

6

O

Cl

N

7 9

N

108

11 13

1412

N

1

4

2

3 5N

O

NH

6

O

ClOCH3

15

(II-8) (II-9)

N-[(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-(4-methoxyphe-

nyl) piperazine]-1-carboxamide (II-9) was synthesized by reacting (6c) with 4-(4-

methoxyphenyl)piperazine. The compound offered characteristic IR peaks at 3393

(N-H str.), 1665 (C=O str.) and 1236 cm-1

(C-O str.). Characteristic signals were

observed at 2.97-2.99 (b, 4H, CH2(9,10)), 3.60-3.61 (b, 4H, CH2(7,8)), 3.80 (s, 3H,

OCH3(15)), 6.87-7.00 (m, 4H, Ar-H(11,14)), 7.69-7.71 (d, 1H, Ar-H(4)), 7.83-7.86 (m,

1H, Ar-H(3)), 8.13 (s, 1H, NH(6)), 8.89 (s, 1H, Ar-H(5)) and 8.90 (s, 1H, Ar-H(1)) in

its NMR spectrum. The mass spectrum displayed (M+) peak at 414.4 and (M

++1)

at (m/z) 416.2 in its mass spectrum.

N-[(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-(4-tert-butyloxy-

carbonyl)piperazine]-1-carboxamide (II-10) gave strong absorption bands at 3393

(N-H str.), 1691, 1650 cm-1

(C=O str.). NMR spectrum displayed characteristic

peaks at 1.42 (s, 9H, CH3(11)), 3.39 (b, 4H, CH2(9,10)), 3.44-3.45 (b, 4H, CH2(7,8)),

7.68-7.71 (d, 1H, Ar-H(4)), 7.83-7.85 (d, 1H, Ar-H(3)), 8.13 (s, 1H, NH(6)), 8.78 (s,

1H, Ar-H(5)) and 8.88-8.89 (d, 1H, Ar-H(1)). The compound gave (M+) peak at

(m/z) 408.41 and (M++2) at (m/z) 410.39 in its mass spectrum.

N

7 9

N

108

N

1

4

2

3 5N

O

NH

6

O

O 11 CH3

O CH3

CH3

Cl

O13

1211

N

7 9

N

108

N

1

4

2

3 5N

O

NH

6

O O

Cl

(II-10) (II-11)


83

N-[(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-(furan-2-carbo-

nyl)-piperazine]-1-carboxamide (II-11) displayed peaks at 3446 (N-H str.) and

1626 cm-1

(C=O str.) in its IR spectrum. 1HNMR peaks were displayed at 3.57-

3.58 (b, 4H, CH2(9,10)), 3.74 (b, 4H, CH2(7,8)), 6.65 (b, 1H, Ar-H(11)), 7.04-7.05 (m,

1H, Ar-H(12)), 7.69-7.71 (d, 1H, Ar-H(4)), 7.83-7.86 (m, 1H, Ar-H(3)), 7.87 (s, 1H,

Ar-H(13)), 8.18 (s, 1H, NH(6)), 8.80 (s, 1H, Ar-H(5)) and 8.89 (b, 1H, Ar-H(1)). The

compound gave molecular ion (M+) peak at (m/z) 402.37 in its mass spectrum.

3.1.4 Synthesis of 4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-

carbamate derivatives (Series III)

Synthesis of the carbamate derivatives of Series-III (III-1 to III-9) have

been accomplished by reaction of the isocyanate (6a-c) and suitable alcohol

derivative (A-HO) in toluene under reflux conditions following the procedure as

outlined in Scheme 4.

N

N

O

R N

N

NH

O

O

OR

N F

O

O

N

6 (a-c)

A HO-A

Toluene, reflux

N=C=O

(III-1) - (III-9)

a. R = H, b. R = 8-CH3, c. R = 7-Cl

A =

Scheme 4

Synthesis of 4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carbamate

derivatives

The isocyanate derivative (6a) and benzyl alcohol were refluxed in

toluene offering the compound benzyl (4-oxo-4H-pyrido[1,2-a]pyrimidin-3-

yl)carbamate (III-1). The compound (III-1) displayed characteristic peaks at 3239

(N-H str.), 1718, 1650 cm-1



The 1H-NMR showed characteristic peaks at 5.17 (s, 2H CH2(7)), 7.32-7.44 (m,

6H, Ar-H(2,8-12)), 7.69-7.71 (d, 1H Ar-H(4)), 7.84-7.89

(m, 1H Ar-H(3)), 8.70 (b,

1H, Ar-H(5)), 8.92-8.94 (d, 1H Ar-H(1)) and 9.05 (b, 1H, NH(6)). The mass

spectrum showed (M++1) peak at (m/z) 296.


84

12

8

10

11

9

N

1

4

2

3 5N

O

NH

6O

O7

11

N9

10

8

N

1

4

2

3 5N

O

NH

6O

O7

(III-1) (III-2)

The compound pyridin-2-ylmethyl (4-oxo-4H-pyrido[1,2-a]pyrimidin-3-

yl)carbamate (II-2) displayed characteristic bands at 3245 (N-H str.), 1713, 1666


(C-O str.) in its IR spectrum. The 1H-NMR showed

characteristic signals at 5.22 (s, 2H, CH2(7)), 7.32-7.37 (m, 2H, Ar-H(2,11)), 7.51-

7.53 (d, 1H, Ar-H(4)), 7.69-7.72 (d, 1H, Ar-H(8)) , 7.83-7.89 (m, 2H, Ar-H(3,10)),

8.55-8.56 (d, 1H, Ar-H(9)), 8.72 (s, 1H, Ar-H(5)), 8.94-8.95 (d, 1H, Ar-H(1)) and

9.21 (b, 1H, NH(6)) in its NMR spectrum. The (M++1) peak was observed in the

mass spectrum at (m/z) 297.

3-Fluorobenzyl (4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)carbamate (III-

3) was obtained by reacting isocyanate derivative (6a) with 3-fluorobenzyl alcohol

as described above for compound (III-1). Characteristic absorption bands were

observed at 3416 (N-H str.), 1716, 1659 (C=O str.) and 1234 cm-1

(C-O str.) in its

IR spectrum. The NMR signals appeared at 5.19 (s, 2H, CH2(7)), 7.14-7.19 (m,

1H, Ar-H(10)), 7.27-7.37 (m, 3H, Ar-H(2,9,11)), 7.41-7.47 (m, 1H, Ar-H(8)), 7.69-

7.71 (d, 1H, Ar-H(4)), 7.85-7.89 (m, 1H, Ar-H(3)), 8.72 (b, 1H, Ar-H(5)), 8.93-8.95

(d, 1H, Ar-H(1)) and 9.16 (b, 1H, NH(6)). The mass spectrum gave (M++1) signal

at (m/z) 314.

11

8

10

9

N

1

4

2

3 5N

O

NH

6O

O7 F

8

10

9

N

1

4

2

3 5N

O

NH

6O

O7

O

11O

(III-3) (III-4)

The IR spectrum of 3,4-methylenedioxybenzyl(4-oxo-4H-pyrido[1,2-

a]pyrimdin-3-yl)carbamate (III-4) showed characteristic peaks at 3242 (N-H str.),

1717, 1666 (C=O str.) and 1235 cm-1

(C-O str.). The compound (III-4) displayed

characteristic signals at 5.06 (s, 2H, CH2(7)), 6.02 (s, 2H, CH2(11)), 6.92 (s, 2H,

Ar-H(8,10)), 7.02 (s, 1H, Ar-H(9)), 7.33-7.37 (m, 1H, Ar-H(2)), 7.69-7.71 (d, 1H, Ar-


85

H(4)), 7.84-7.89 (m, 1H, Ar-H(3)), 8.70 (b, 1H, Ar-H(5)), 8.92-8.94 (d, 1H, Ar-H(1))

and 9.01 (b, 1H, NH(6)) in its NMR spectrum. The mass spectrum showed (M++1)

peak at (m/z) 340.

Synthesis of 8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carbam-

ate derivatives

Benzyl (8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)carbamate (III-

5) gave characteristic signals at 3242 (N-H str.), 1717, 1635 (C=O str.) and 1224

cm-1

(C-O str). The

compound (III-5) offered characteristic peaks at 2.45 (s, 3H,

CH3(3)), 5.15 (s, 2H, CH2(7)), 7.21-7.23 (d, 1H, Ar-H(2)), 7.31-7.44 (m, 5H, Ar-H(8-

11)), 7.52 (s, 1H, Ar-H(4)), 8.60 (b, 1H, Ar-H(5)), 8.83-8.85 (d, 1H, Ar-H(1)) and

8.97 (b, 1H, NH(6)) in its NMR spectrum. The (M++1) peak was observed at (m/z)


12

8

10

11

9

N

1

4

2

5N

O

NH

6O

O7

CH3

3

11

N9

10

8

N

1

4

2

5N

O

NH

6O

O7

CH3

3

(III-5) (III-6)

Pyridin-2-ylmethyl (8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-

yl)carbamate (III-6) displayed characteristic peaks at 3247 (N-H str.), 1721, 1667


(C-O str) in its IR spectrum. The 1H-NMR displayed

characteristic peaks at 2.45 (s, 3H, CH3(3)), 5.20 (s, 2H, CH2(7)), 7.22-7.24 (d,

1H, Ar-H(2)), 7.33-7.36 (m, 1H, Ar-H(11)), 7.49-7.51 (m, 1H, Ar-H(8)), 7.53 (b, 1H,

Ar-H(4)), 7.83-7.87 (m, 1H, Ar-H(10)), 8.55-8.56 (d, 1H, Ar-H(9)), 8.62 (b, 1H, Ar-

H(5)), 8.85-8.87 (d, 1H, Ar-H(1)) and 9.14 (b, 1H, NH(6)). The mass spectrum

showed (M++1) peak at (m/z) 311.

Synthesis of 7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carba-

mate derivatives

2-Pyridinylmethyl (7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-

yl)carbamate (III-7) gave characteristic peaks at 3451 (N-H str.), 1724, 1652


(C-O str) in its IR spectrum. Characteristic signals were

appeared at 5.23 (s, 2H, CH2(7)), 7.33-7.36 (t, 1H, Ar-H(11)), 7.51-7.53 (d, 1H,


86

Ar-H(8)), 7.71-7.73 (d, 1H, Ar-H(4)), 7.83-7.87 (t, 1H, Ar-H(10)), 7.88-7.91 (dd, 1H,

Ar-H(3)), 8.55-8.56 (d, 1H, Ar-H(9)), 8.76 (s, 1H, Ar-H(5)), 8.92-8.93 (d, 1H, Ar-

H(1)) and 9.32 (s, 1H, NH(6)) in its NMR spectrum. The compound showed (M+)

peak at (m/z) 331.37 and (M++1) at (m/z) 333.35 in its mass spectrum.

3-Pyridinylmethyl (7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-

carbamate (III-8) displayed characteristic IR absorption bands at 3431 (N-H str.),

1722, 1664 (C=O str.) and 1232 cm-1

(C-O str) in its IR spectrum. The PMR gave

peaks at 5.22 (s, 2H, CH2(7)), 7.41-7.45 (m, 1H, Ar-H(10)), 7.70-7.73 (d, 1H, Ar-

H(4)), 7.86-7.91 (m, 2H, Ar-H(3,9)), 8.54-8.55 (d, 1H, Ar-H(11)), 8.66 (b, 1H, Ar-

H(8)), 8.74 (b, 1H, Ar-H(5)), 8.91- 8.92 (d, 1H, Ar-H(1)) and 9.16 (s, 1H, NH(6)).

The signals, (M+) at (m/z) 331.37 and (M

++2) at (m/z) 333.35 displayed in its mass

spectrum.

11

N9

10

8

N

1

4

2

3 5N

O

NH

6O

O7

Cl

11

8

N

10

9

N

1

4

2

3 5N

O

NH

6O

O7

Cl

(III-7) (III-8)

11

8

10

9

N

1

4

2

3 5N

O

NH

6O

O7 F

Cl

(III-9)

3-Fluorobenzyl (7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)carba-

mate (III-9) displayed characteristic bands at 3276 (N-H str.), 1726, 1660 (C=O

str.) and 1229 cm-1

(C-O str) in its IR spectrum. Its NMR signals were observed

at 5.15 (s, 2H, CH2(7)), 7.20-7.24 (m, 2H, Ar-H(9,10)), 7.48-7.51 (m, 2H, Ar-

H(8,11)), 7.70-7.72 (d, 1H, Ar-H(4)), 7.88-7.90 (d, 1H, Ar-H(3)), 8.74 (s, 1H, Ar-

H(5)), 8.91 (s, 1H, Ar-H(1)), 9.18 (s, 1H, NH(6)). The compound showed (M+) peak

at (m/z) 348 and (m/z) 350 (M++2) in its mass spectrum.

3.1.5 Synthesis of 4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carboxa-

mide derivatives (Series IV)


87

Synthesis of the amide derivatives of Series IV (IV-1 to IV-26) have

been accomplished by reacting the acid (4a-c) with suitable amine (R’-NHA)

using EDC as a coupling agent as outlined in Scheme 5 and Scheme 6.

N

N

O

OH

O

N

N

O

N

O

ARR

CF3

CF3

N

O

CF3

FF

N

ON

NO

CH3

N O

O

CH3

O

CH3

N

O

O

CH3

N

O

CH3

F

FOCH

3

N

O

4 (a-c) (IV-1) - (IV-20)

EDC, HOBT, DIEA, 0OC/ RT

R'-NHA

R'

a. R = H, b. R = 8-CH3, c. R = 7-Cl

OCH3

A =

OCH3

R' = H, i.Pr, Cyclopropyl, Benzyl

Scheme 5

Synthesis of 4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carboxamide

derivatives

N-(4-Methoxybenzyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxam-

ide (IV-1) was prepared by the carbodimide coupling reaction. The acid derivative

(4a) was coupled with 4-methoxybenzylamine using 1-(3-dimethylaminopropyl)-

3-ethylcarbodiimide affording the compound (IV-1). Its IR spectrum showed

characteristic peaks at 3336 (N-H str.) and 1679 cm-1

(C=O str.). The NMR

spectrum of the compound (IV-1) displayed characteristic peaks at 3.80 (3H,

OCH3(12)), 4.62-4.64 (m, 2H, CH2(7)), 6.86-6.90 (d, 2H, Ar-H(8,9)) 7.31-7.33 (d,

2H, Ar-H(10,11)) 7.34-7.38 (m, 1H, Ar-H(2)) 7.84-7.86 (d, 1H, Ar-H(4)) 7.92-7.97

(m, 1H, Ar-H(3)) 9.19-9.21 (d, 1H, Ar-H(1)), 9.30 (b, 1H, NH(6)) and 9.38 (s,

1H,Ar-H(5)). The mass spectrum displayed (M++1) peak at (m/z) 310.


88

N-(3-Fluorobenzyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-carboxamide

(IV-2) in its IR spectrum displayed peaks at 3327 (N-H str.), 1662 cm-1

(C=O

str.). The 1H-NMR showed characteristic peaks at 4.59-4.61 (d, 2H, CH2(7)),

7.06-7.11 (m, 1H, Ar-H(10)), 7.15-7.21 (m, 2H, Ar-H(9,11)), 7.36-7.41 (m, 1H, Ar-

H(8)), 7.59-7.63 (m, 1H, Ar-H(2)), 7.90-7.92 (d, 1H, Ar-H(4)), 8.19-8.23 (m, 1H,

Ar-H(3)), 9.05 (s, H, Ar-H(5)), 9.19-9.21 (d, 1H, Ar-H(1)) and 9.43-9.46 (t, 1H,

NH(6)). The mass spectrum gave (M++1) peak at (m/z) 298.

8

10

11

97

N

1

4

2

3 5N

O

NH

6

O

OCH3

12

118

10

97

F

N

1

4

2

3 5N

O

NH

6

O

(IV-1) (IV-2)

108

11

9

7

CF3

N

1

4

2

3 5N

O

NH

6

O

118

10

97CF

3N

1

4

2

3 5N

O

NH

6

O

(IV-3) (IV-4)

4-Oxo-N-[2-(trifluoromethyl)benzyl]-4H-pyrido[1,2-a]pyrimidin-3-

carboxamide (IV-3) displayed characteristic peaks at 3299 (N-H str.), 1695 cm-1

(C=O str.) in its IR spectrum. 1H-NMR showed characteristic signals at 4.76-

4.78 (d, 2H, CH2(7)), 7.48-7.52 (m, 1H Ar-H(2)), 7.59-7.69 (m, 3H, Ar-H(8,10,11)),

7.75-7.77 (d, 1H, Ar-H(9)), 7.91-7.93 (d, 1H, Ar-H(4)), 8.20-8.24 (m, 1H, Ar-H(3)),

9.05 (s, 1H, Ar-H(5)), 9.21-9.23 (d, 1H, Ar-H(1)) and 9.48-9.51 (t, 1H, NH(6)). The

mass spectrum displayed the (M++1) peak at (m/z) 348.

The compound 4-oxo-N-[3-(trifluoromethyl)benzyl]-4H-pyrido[1,2-

a]pyrimidin-3-carboxamide (IV-4) displayed characteristic peaks at 3328 (N-H

str.), 1691, 1645 and 1490 cm-1

(C=O str.). The NMR spectrum of the compound

(IV-4) displayed characteristic peaks at 4.66-4.67 (d, 2H, CH2(7)), 7.56-7.63 (m,

3H, Ar-H(2,8,10)), 7.66-7.68 (d, 1H, Ar-H(11)), 7.72 (s, 1H, Ar-H(9)), 7.90-7.92 (d,

1H, Ar-H(4)), 8.18-8.23 (m, 1H, Ar-H(3)), 9.05 (s, 1H, Ar-H(5)), 9.20-9.21 (d, 1H,

Ar-H(1)) and 9.50-9.53 (t, 1H, NH(6)). The mass spectrum showed (M++1) peak at

(m/z) 348.

4-Oxo-N-[4-(trifluoromethyl)benzyl]-4H-pyrido[1,2-a]pyrimidin-3-

carboxamide (IV-5) displayed characteristic IR signals at 3336 (N-H str.), 1663


89


(C-O str.). The compound showed characteristic peaks at

4.75-4.76 (d, 2H, CH2(7)), 7.38-7.41 (m, 1H, Ar-H(2)), 7.49-7.51 (d, 2H, Ar-

H(8,9)), 7.58-7.60 (d, 2H, Ar-H(10,11)), 7.86-7.88 (d, 1H, Ar-H(4)), 7.96-8.00 (m, 1H,

Ar-H(3)), 9.21-9.23 (d, 1H, Ar-H(1)), 9.37-9.38 (s, 1H, Ar-H(5)) and 9.47 (b, 1H,

NH(6)) in its NMR spectrum. Its mass spectrum displayed the (M++1) peak at (m/z)

348.

8

10

11

97

CF3

N

1

4

2

3 5N

O

NH

6

O

8

10

9

7

F

F

N

1

4

2

3 5N

O

NH

6

O

(IV-5) (IV-6)

The compound N-(2,4-Difluorobenzyl)-4-oxo-4H-pyrido[1,2-

a]pyrimidin-3-carboxamide (IV-6) displayed characteristic IR peaks at 3328 (N-H

str.) and 1664 cm-1

(C=O str.) in its IR spectrum. The NMR signals appeared at

4.60-4.62 (d, 2H, CH2(7)), 7.14-7.21 (m, 2H, Ar-H(8,10)), 7.24-7.29 (m, 1H, Ar-

H(9)), 7.59-7.63 (m, 1H, Ar-H(2)), 7.89-7.92 (d, 1H, Ar-H(4)), 8.19-8.23 (m, 1H,

Ar-H(3)), 9.03 (s, 1H, Ar-H(5)), 9.20-9.23 (d, 1H, Ar-H(1)) and 9.42-9.45 (t, 1H,

NH(6)). The mass spectrum showed (M++1) peak at (m/z) 316.

N

10

9

12

11

ON

1

4

2

3 5N

O

NH

6

O7

8

N

1

4

2

3 5N

O

NH

6

O

7

8

9

N

O10

11

12

13

CH3

14

(IV-7) (IV-8)

N-[2-(Morpholin-4-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-

carboxamide (IV-7) displayed characteristic IR peaks at 3423 (N-H str.), 1690


(C-O str.). Characteristic 1H-NMR signals were

observed at 2.43 (b, 4H, CH2(9,10)), 2.50-2.51 (m, 2H, CH2(8)), 3.45-3.50 (m, 2H,

CH2(7)), 3.59-3.61 (t, 4H, CH2(11,12)), 7.57-7.61 (m, 1H, Ar-H(2)), 7.84-7.90 (d, 1H,

Ar-H(4)), 8.17-8.21 (m, 1H, Ar-H(3)), 9.02 (s, 1H, Ar-H(5)), 9.15-9.16 (t, 1H, NH(6))

and 9.20-9.22 (d, 1H, Ar-H(1)). The mass spectrum showed (M++1) peak at (m/z)

303.

4-Oxo-N-[3-(n.pentyloxy)pyridin-2-yl]-4H-pyrido[1,2-a]pyrimidin-3-

carboxamide (IV-8) displayed characteristic peaks at 3297 (N-H str.) 1693 (C=O

str.) and 1218 cm-1

(C-O str.) in its IR spectrum. The 1H-NMR spectrum


90

displayed characteristic signals at 0.89-0.93 (t, 3H, CH3(14)), 1.36-1.43 (m, 2H,

CH2(13)), 1.49-1.57 (m, 2H, CH2(12)), 1.78-1.84 (m, 2H, CH2(11)), 4.09-4.13 (t, 2H,

CH2(10)), 7.95-7.97 (d, 2H, Ar-H(4,9)), 8.24-8.28 (m, 1H, Ar-H(3)), 9.13 (s, 1H, Ar-

H(5)), 9.21-9.24 (d, 1H, Ar-H(1)) and 11.51 (s, 1H, NH(6)). The (M++1) peak

appeared in mass spectrum at (m/z) 353.

N-[3-(Benzyloxy)pyridin-2-yl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-

carboxamide (IV-9) IR spectrum showed characteristic peaks at 3480 (N-H str.),

1699 (C=O str.) and 1285 cm-1

(Ar-O str.). The characteristic signals were

appeared at 5.28 (s, 2H, CH2(10)), 7.00-7.04 (m, 1H, Ar-H(8)), 7.58-7.60 (d, 2H,

Ar-H(11,12)), 8.16-8.18 (m, 1H, Ar-H(7)), 9.27-9.31 (m, 1H, Ar-H(1)), 9.49 (s, 1H,

Ar-H(5)), 11.76 (s, 1H, NH(6)) in its NMR spectrum. (M++1) peak was observed in

the mass spectrum at (m/z) 373 in its mass spectrum.

11

13

15

14

12

N

1

4

2

3 5N

O

NH

6

O

7

8

9

N

O10

7

8

N

9

10

12

11

O

O

13

CH3

14

O

15

CH3

16

NH

6

N

1

4

2

3 5N

O O

(IV-9) (IV-10)

The IR spectrum of N-[(2,5-diethoxy)-4-(morpholin-4-yl)phenyl]-4-oxo-

4H-pyrido[1,2-a]pyrimidine-3-carboxamide (IV-10) displayed characteristic

peaks at 3263 (N-H str.), 1685 (C=O str.) and 1199 cm-1

(C=O str.). Characteristic

signals were observed at 1.44-1.47 (t, 3H, CH3(14/16)) and 1.54-1.58 (3H,

CH3(14/16)), 3.09-3.10 (t, 4H, CH2(9,10)), 3.89-3.91 (t, 4H, CH2(11/12)), 6.60 (s, 1H,

Ar-H(8)), 7.37-7.41 (m, 1H, Ar-H(2)), 7.85-7.87 (d, 1H, Ar-H(4)), 7.95-7.99 (m, 1H,

Ar-H(3)), 8.40 (s, 1H, Ar-H(7)), 9.33-9.34 (d, 1H, Ar-H(1)), 9.42 (s, 1H, Ar-H(5)) and

11.49 (s, 1H, NH(6)) in its NMR spectrum. The mass showed (M++1) peak at (m/z)

439.

The IR spectrum of N-(3,5-Dimethyl-1,2-oxazol-4-yl)-4-oxo-4H-

pyrido[1,2-a]pyrimidin-3-carboxamide (IV-11) displayed characteristic peaks at

3253 (N-H str.) and 1694 cm-1

(C=O str.). The characteristic peaks were displayed

at 2.15 (s, 3H, CH3(8)), 2.33 (s, 3H, CH3(7)), 7.65-7.69 (m, 1H, Ar-H(2)), 7.95-

7.97 (d, 1H, Ar-H(4)), 8.24-8.29 (m, 1H, Ar-H(3)), 9.09 (s, 1H, Ar-H(5)), 9.26-9.27


91

(d, 1H, Ar-H(1)) and 10.20 (s, 1H, NH(6)) in its NMR spectrum. The (M++1) peak

at (m/z) 285 was present in its mass spectrum.

CH3

8

N

O

CH3

7

NH

6

N

1

4

2

3 5N

O O

N

1

4

2

3 5N

O

NH

6

O

7

9

8

11

10

CH3

12

OCH3

(IV-11) (IV-12)

N-[1-(4-Methoxyphenyl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-

carboxamide (IV-12) gave peaks at 3315 (N-H str.), 1667 (C=O str.), 1240 (C-O

str.) and 1025 cm-1

in its IR spectrum. Characteristic peaks were displayed 1.61-

1.66 (m, 3H, CH3(12)), 3.79 (s, 3H, OCH3(13)) and 5.28-5.35 (m, 1H, CH(7)), 6.87-

6.91(d, 2H, Ar-H(8,9)), 7.34-7.38 (m, 3H, Ar-H(2,10,11)), 7.83-7.86 (d, 1H, Ar-H(4)),

7.92-7.97 (m, 1H, Ar-H(3)), 9.20-9.22 (d, 1H, Ar-H(1)) and 9.36 (s, 1H, Ar-H(5),

NH(6)) in its NMR spectrum. The mass spectrum showed (M++1) peak at (m/z)

324.

The IR spectrum of N-(1-benzylpiperidin-4-yl)-4-oxo-4H-pyrido[1,2-

a]pyrimidin-3-carboxamide (IV-13) showed characteristic peaks at 3288 (N-H

str.) and 1695 cm-1

(C=O str.). Characteristic signals appeared at 1.52-1.58 (m,

2H, CH2(8,9)), 1.89-1.92 (b, 2H, CH2(8,9)), 2.18-2.22 (t, 2H, CH2(10,11)), 2.70 (b, 2H,

CH2(10,11)), 3.50 (s, 2H, CH2(12)), 3.88 (b, 1H, CH(7)) and 9.20-9.22 (d, 1H, NH(6))

in its NMR. The (M++1) signal was displayed at (m/z) 363 in mass spectrum.

N-(3,4-Methylenedioxybenzyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-

carboxamide (IV-14) IR spectrum displayed 3260 (N-H str.), 1666 (C=O str.) and

1224 cm-1

(C-O str.). Characteristic peaks were observed at 4.46-4.48 (d, 2H,

CH2(7)), 5.98 (s, 2H, CH2(11)), 6.82-6.88 (m, 2H, Ar-H(8,10)), 6.93 (s, 1H, Ar-H(9)),

7.58-7.62 (m, 1H, Ar-H(2)), 7.89-7.91 (d, 1H, Ar-H(4)), 8.17-8.22 (m, 1H, Ar-H(3)),

9.05 (s, 1H, Ar-H(5)), 9.17-9.19 (d, 1H, Ar-H(1)) and 9.27-9.33 (t, 1H, NH(6)). in its

NMR spectrum. Its mass spectrum showed (M++1) peak at (m/z) 324.

N

1

4

2

3 5N

O

NH

6

O

7

8 10

N

119

12

13

15 17

16

14N

1

4

2

3 5N

O

NH

6

O7 9

8

10O

11

O

(IV-13) (IV-14)


92

N

1

4

2

3 5N

O

N

O6

7

10

8

911

12 13

F

(IV-15)

N-Cyclopropyl-N-(2-fluorobenzyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-

carboxamide (IV-15) displayed characteristic peaks at 1669, 1636 cm-1

(C=O, str.)

in its IR spectrum. The 1H-NMR at 0.58-0.61 (d, 4H, CH2(12,13)), 3.06 (b, 1H,

CH(11)) and 4.93 (s, 2H, CH2(6)). The mass spectrum showed (M++1) peak at (m/z)

338.

Synthesis of 8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxa-

mide derivatives

N-(4-Fluorobenzyl)-8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-

carboxamide (IV-16) was prepared by treating (4b) with 4-fluorobenzylamine as

described above for compound (IV-1) yielding (IV-16). IR spectrum of the

compound displayed characteristic peaks at 3287 (N-H str.) and 1677 cm-1

(C=O

str.). The 1H-NMR displayed characteristic peaks at 2.58 (s, 3H, CH3(3)), 4.65-

4.66 (d, 2H, CH2(7)), 7.00-7.04 (t, 2H, Ar-H(8,9)), 7.18-7.21 (m, 1H, Ar-H(2)), 7.34-

7.37 (m, 2H, Ar-H(10,11)), 7.63 (s, 1H, Ar-H(4)), 9.08-9.09 (d, 1H, Ar-H(1)), 9.33 (s,

1H, Ar-H(5)) and 9.35 (b, 1H, NH(6)). The mass spectrum showed the (M++1) peak

at (m/z) 312.

N

1

4

2

5N

O

NH

6

O7 9

8

11

10FCH

3

3

N

1

4

2

5N

O

NH

6

O7 9

8

10

11

CH3

3

CF3

(IV-16) (IV-17)

The IR spectrum of N-(4-trifluorobenzyl)-8-methyl-4-oxo-4H-

pyrido[1,2-a]pyrimidin-3-carboxamide (IV-17) displayed characteristic peaks at

3414 (N-H str.), 1695 (C=O str.) cm-1

. Its 1H-NMR showed characteristics peaks

at 2.55 (s, 3H, CH(3)), 4.65-4.67 (d, 2H, CH(7)), 7.46-7.49 (dd, 1H, Ar-H(2)),

7.55-7.57 (d, 2H, Ar-H(8,9)), 7.69-7.73 (m, 3H, Ar-H(4,10,11)), 9.00 (s, 1H, Ar-H(5)),

9.08-9.10 (d, 1H, Ar-H(1)) and 9.45-9.48 (t, 1H, NH(6)) The compound gave



93

Synthesis of 7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxami-

de derivatives

7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (4c) was

reacted with 3,4-methylenedioxy benzylamine) as described above for compound

(IV-1) yielding the compound (IV-18) Characteristic IR peaks were observed at

3300 (N-H str.) and 1672 cm-1

(C=O str.). Characteristic NMR signals were

present at 4.46-4.48 (d, 2H, CH2(7)), 5.98 (s, 2H, CH2(11)), 6.81-6.87 (m, 2H, Ar-

H(8,10)), 6.92 (s, 1H, Ar-H(9)), 7.90-7.93 (d, 1H, Ar-H(4)), 8.24-8.26 (dd, 1H, Ar-

H(3)), 9.04 (s, 1H, Ar-H(5)), 9.15-9.16 (d, 1H, Ar-H(1)) and 9.23-9.26 (t, 1H, NH(6)).

The compound gave molecular ion peak (M+) at (m/z) 358.3 and (M

++2) peak at

(m/z) 360.4.

N

1

4

2

3 5N

O

NH

6

O7 9

8

10O

11

OCl

N

1

4

2

3 5N

O

NH

6

O7 9

8

11

10CF

3

Cl

(IV-18) (IV-19)

7-Chloro-4-oxo-N-[4-(trifluoromethyl)benzyl]-4H-pyrido[1,2-a]pyrimid-

ine-3-carboxamide (IV-19) gave characteristic IR signals at 3315 (N-H str.) and

1674 cm-1

. The compound (IV-19) gave characteristic peaks at 4.67-4.68 (d,

2H, CH2(7)), 755-7.57 (d, 2H, CH2(8,9)), 7.70-7.72 (d, 2H, Ar-H(10,11)), 7.92-7.95 (d,

1H, Ar-H(4)), 8.26 -8.29 (dd, 1H, Ar-H(3)), 9.04 (s, 1H, Ar-H(5)), 9.17-9.18 (d, 1H,

Ar-H(1)) and 9.43-9.46 (t, 1H, NH(6)) in its NMR spectrum. The mass spectrum

displayed (M+) at (m/z) 382.2 and (M

++2) (m/z) 384.2.

N

1

4

2

3 5N

O

N

O6

7

10

8

911

12 13

F

Cl

(IV-20)

The characteristic signals were displayed in IR spectrum of 7-chloro-N-

cyclopropyl-N-(2-fluorobenzyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-carboxamide

(IV-20) at 3102 (N-H str.) and 1630 cm-1

(C=O str.). The characteristic signals

were displayed at 0.51-0.55 (d, 4H, CH2(12,13)), 2.87 (b, 1H, CH(11)), 4.75 (b, 2H,

CH2(6)), 7.21-7.24 (m, 2H, Ar-H(7,8)), 7.33-7.36 (m, 1H, Ar-H(9)), 7.63 (b, 1H, Ar-

H(4)), 7.84-7.87 (d, 1H, Ar-H(3)), 8.14-8.17 (dd, 1H, Ar-H(1)), 8.47 (s, 1H, Ar-H(5))


94

and 9.14 (s, 1H, NH(6)) in its NMR spectrum. The mass spectrum displayed

(M++1) peak at (m/z) 372.4.

7-(4-Methoxyphenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic

acid intermediate

The 7-chloro ester (3c) was reacted with p-methoxyphenylboronic acid

through Suzuki coupling offering the ester (7) which on acid hydrolysis yielded

the acid (8). The acid derivative (8) was coupled with suitable amines to get the

targeted compounds (IV23 - IV24) as depicted in Scheme 6. Ethyl 7-(4-

methoxyphenyl)-4-

ON

1

4

3 5N

O O

CH3

H3CO

N

1

4

2

3 5N

O

OH

OH3CO

(7) (8)

oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (7) displayed characteristic IR

signals at 1744 (C=O str.) and 1113 cm-1

(C-O str.) in its IR spectrum. The

compound gave (M++1) peak at (m/z) 325 in its mass spectrum.

The ester (7) on acidic hydrolysis furnished the free acid (8) 7-(4-

methoxyphenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (8). Its IR

spectrum displayed characteristic bands at 3087 (COO-H str.) and 1772 (C=O str.)

cm-1

. The (M

++1) peak at (m/z) 297 was observed in its mass spectrum.

Synthesis of 7-(4-methoxyphenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-

3-carboxamide derivatives

In order to study the hydrophobic binding interactions by increasing the

steric bulk of the group attached to the B-ring (i.e. pyrido) of the pyridopyrimidine

ring, some additional compounds (IV23 - IV24) were synthesized. Synthesis of 7-

(4-methoxyphenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxamide derivatives

of (Series IV) has been was accomplished by using the intermediates of Scheme 1

and by following the reaction sequence outlined in Scheme 6.


95

N

N

O

O

O

ClCH

3 N

N

O

O

O

CH3

N

N

O

OH

O

N

N

O

NH

O

A

B(OH)2

CF3

CF3

F O

(3c) (7)

DME, P(Ph3)4, K2CO3,

Reflux

HCl, Reflux

(8) (IV-21) - (IV-24)

EDC, HOBT, DIEA,

A-NH2, 0OC/ RT

H3COH3CO

H3CO

A =

H3CO

Scheme 6

N-(3-Fluorobenzyl)-7-(4-methoxyphenyl)-4-oxo-4H-pyrido[1,2-a]pyri-

midine-3-carboxamide (IV-21) was synthesized by reacting compound (8) with 3-

fluorobenzylamine as described for compound (IV-1). Its IR spectrum displayed

characteristic peaks at 3319 (N-H str.), 1688 (C=O str.) and 1264 cm-1

(C-O str.).

The compound gave characteristic signals at 3.89 (s, 3H, OCH3(16)), 4.60-4.62

(d, 2H, CH2(7)), 7.07-7.16 (m, 3H, Ar-H(9-11)), 7.20-7.22 (m, 2H, Ar-H(14,15)), 7.37-

7.42 (m, 1H, Ar-H(8)), 7.79-7.81 (d, 2H, Ar-H(12,13)), 7.95-7.98 (d, 1H, Ar-H(4)),

8.54-8.57 (dd, 1H, Ar-H(3)), 9.04 (s, 1H, Ar-H(5)), 9.27-9.28 (d, 1H, Ar-H(1)) and

9.45-9.48 (t, 1H, NH(6)) in its NMR spectrum. A strong peak (M+-1) peak was

observed at (m/z) 403 in its mass spectrum.

13

15

14

12N

1

4

3 5N

O

NH

6

O7 9

8

10

11

F

13

15

14

12N

1

4

3 5N

O

NH

6

O7 9

8

10

11

CF3

H3CO16

H3CO16

(IV-21) (IV-22)

Characteristic bands were observed for the compound N-(3-

trifluoromethylbenzyl)-7-(4-methoxyphenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-

3-carboxamides (IV-22) in its IR spectrum displayed characteristic bands at 3414

(N-H str.), 1692 (C=O str.) and 1163 (C-O str.) cm-1

. The PMR spectrum gave


96

characteristic peaks at 3.83 (s, 3H, OCH3(16)), 4.67-4.68 (d, 2H, CH2(7)), 7.11-

7.14 (d, 2H, Ar-H(14,15)), 7.57-7.64 (m, 2H, Ar-H(9,10)), 7.78-7.81 (d, 2H, Ar-

H(12,13)), 7.67-7.69 (m, 1H, Ar-H(11)), 7.72 (b, 1H, Ar-H(8)), 7.95-7.97 (d, 1H, Ar-

H(4)), 8.53-8.56 (dd, 1H, Ar-H(3)), 9.04 (s, 1H, Ar-H(5)), 9.26 (s, 1H, Ar-H(1)) and

9.51-9.54 (t, 1H, NH(6)). Its mass spectrum gave (M++1) peak at (m/z) 454.

13

15

14

12N

1

4

3 5N

O

NH

6

O7 9

8

11

10CF

3

H3CO16

13

15

14

12

O

8

9 10

11N

1

4

3 5N

O

NH

6

O7

H3CO16

(IV-23) (IV-24)

N-(4-Trifluoromethylbenzyl)-7-(4-methoxyphenyl)-4-oxo-4H-pyrido-

[1,2-a]pyrimidine-3-carboxamide (IV-23) gave strong peaks at 3425 (N-H str.),

1689 (C=O str.) and 1263 cm-1

(C-O str.) in its IR spectrum. Characteristic peaks

were observed in its NMR at 3.84 (s, 3H, OCH3(16)), 4.68-4.69 (d, 2H, CH2(7)),

7.13-7.15 (d, 2H, Ar-H(14,15)), 7.57-7.59 (d, 2H, Ar-H(8,9)), 7.71-7.73 (d, 2H, Ar-

H(10,11)), 7.80-7.82 (d, 2H, Ar-H(12,13)), 7.96- 7.99 (d, 1H, Ar-H(4)), 8.55-8.58 (dd,

1H, Ar-H(3)), 9.04 (s, 1H, Ar-H(5)), 9.28-9.29 (d, 1H, Ar-H(1)) and 9.52-9.55 (t, 1H,

NH(6)). Its mass spectrum gave (M++1) signal at (m/z) 454.

N-(Tetrahydrofuran-2-ylmethyl)-7-(4-methoxyphenyl)-4-oxo-4H-

pyrido [1,2-a]pyrimidine-3-carboxamide (IV24) displayed IR bands at 3453 (N-H

str.), 1689 (C=O str.) and 1180 cm-1


Characteristic

peaks were appeared at 3.84 (s, 3H, OCH3(16)), 3.96-3.99 (m, 1H, CH(8)), 7.12-

7.15 (d, 2H, Ar-H(14,15)) and 7.79-7.84 (d, 2H, Ar-H(12,13)), 7.91-7.97 (d, 1H, Ar-

H(4)), 8.54-8.56 (dd, 1H, Ar-H(3)), 9.03 (s, 1H, Ar-H(5)), 9.17-9.19 (t, 1H, NH(6))

and 9.29-9.30 (d, 1H, Ar-H(1)) in its NMR spectrum. The compound gave (M++1)

signal at (m/z) 380 in its mass spectrum.

3.2 Biological evaluation

The aim of the current study was to develop new antimalarial agents,

ideally directed against the targets which were not previously exploited in the

antimalarial chemotherapy. Malarial cysteine protease falcipain-2 is such a target.

The synthesized compounds were tested for falcipain inhibition-2 (in vitro

inhibition assay) following the reported procedure.93


97

Compounds were evaluated for their inhibitory activity against

recombinant FP-2 using Cbz-Phe-Arg-AMC as a fluorogenic substrate.

Preliminary screening was performed at 10 M concentration. An equivalent

concentration of DMSO was used as negative control and the irreversible standard

inhibitor of clan CA family C1 cysteine proteases (papain family), namely E-64

was used as positive control. Assays were performed to determine the percentage

inhibition of the enzyme at a concentration of 10 M. FP-2 activity is assessed by

cleavage of the fluorogenic substrate Cbz-Phe-Arg-AMC releasing the fluorescent

AMC group. Hence decrease in fluorescence intensity in a sample represents

inhibition of enzyme activity. The IC50 values were determined for those

compounds only which showed more than 40 % inhibition at 10 M

concentration.

3.2.1 Assay of falcipain-2 inhibition

The diluted soluble Plasmodium falciparum FP-2 (30 nM) was incubated

for 10 min at room temperature in 100 mM sodium acetate buffer of pH 5.5, 10

mM dithiothreitol (DTT), with a fixed/different concentration of the compounds

to be tested or the standard (E64). Compound solutions were prepared from a

stock in DMSO. After 10 min incubation, the substrate Z-Phe-Arg-AMC was

added to a final concentration of 25 µM. The fluorescence intensity was

monitored (excitation 355 nm; emission 460 nm) for 10 min at room temperature

with a SynergyTM

4 Multi-Mode Microplate Reader (BioTek). The inhibition rate

(%) is calculated using the given equation:

% Inhibition = [1 - (F test / F stand.)] × 100

Where (F test is the fluorescence intensity of the test compound, F

standard is the fluorescence intensity of the standard compound (E64). All values

are the means of three independent determinations and the deviations are <10 %

of the mean value. Fluorescence was monitored for 15 min at room temperature

in a Labsystems Fluoroskan Ascent spectrofluorometer. IC50 values were

determined from plots of percents of activity over the compound concentration

using Graphpad Prism Software. . The IC50 values were determined for those

compounds only which showed 40 % or more of enzyme inhibition at 10 μM. The

results of the study are summarized in Table 1.


98

Table 1: In vitro falcipain-2 inhibition activity data of the test compounds

Comp. No.

Falcipain-2

Inhibition

Ratio (%)

IC50

(μM） Comp. No.

Falcipain-2

Inhibition

Ratio (%)

IC50

(μM）

I-1 38.83 NP II-5 37 NP

I-2 18.35 NP II-6 46.14 18.87

I-3 31.11 NP II-7 23.2 NP

I-4 46.37 16.08 II-8 41.34 12.16

I-8 44.43 14.27 II-9 36.01 NP

I-9 25.12 NP III-1 11.52 NP

I-21 62.51 6.36 III-2 17.53 NP

I- 22 15.28 NP III-5 30.71 NP

I-23 45.09 21.16 III-6 41.91 18.32

I-24 27.21 NP IV-14 7.4 NP

I-26 67.97 6.93 IV-16 5.56 NP

I-27 41.81 23.35 IV-18 23.65 NP

II-1 46.13 14.84 E-64 92 (2 M) 0.02

II-2 42.79 NP DMSO 0

NP- not performed

The enzyme inhibition data given in Table 1 shows that the pyrido[1,2-

a]pyrimidin-4-ones show FP-2 inhibition in micromolar range. Based on the result

of evaluated compounds a broad structure activity relationship could be deduced

for this series of the compounds. Urea moiety has the potential to provide potent

FP-2 inhibitors as the carbamate and carboxamide derivatives are much less active

in comparison to the substituted ureas.

Ethylmorpholine (I-26, IC50 6.93 M) and methoxyethyl (I-21, IC50 6.36

M) urea derivatives proved to be the most potent compounds. 2-Thiophenethyl

(I-8, IC50 14.27 M; IV-23, IC50 21.16 M; I-27, IC50 23.35 M), acetamidoethyl

(I-4, IC50 16.08 M) and 4-methoxyphenylpiperazine (II-5, IC50 18.87 M) were

the other groups which gave active compounds when attached to the other end of

the urea moiety. 2-Pyridylpiperazine is another moiety which offered quite active

compounds (II-8, IC50 12.16 M; II-1, IC50 14.84 M).

In general 8-methyl and 7-chloro substituted derivatives offered more

potent compounds indicating that increasing the steric bulk of the group attached

to the B-ring (i.e. pyrido) is likely to provide more potent FP-2 inhibitors. An


99

electron rich environment at the end of the carbon chain attached to the urea

nitrogen also seems to be conducive for better activity as indicated by the

compounds having morpholine, 2-thiophenenyl, 2-pyridyl, 4-methoxyphenyl,

acetamidoethyl and 2-methoxyethyl groupings. But bulky groupings (II-7,

benzhydrylpiperazine and I-9, 4-morpholinopropyl) may not offer potent

derivatives.

The above given SAR points have been framed on the basis of available

biological data for a limited number of compounds. For the remaining compounds

the biological data is yet to be obtained. A detailed structure activity relationship

would be framed once the biological data of all the synthesized compounds is

available at hand.

With the availability of biological activity for compounds the current

work has proved the potential of pyrido[1,2-a]pyrimidin-4-one derivatives as

potent FP-2 inhibitors. Based on the activity data of the synthesized compounds it

could be claimed that pyrido[1,2-a]pyrimidin-4-one can serve as a lead series for

future investigations. By appropriate structural optimizations the potency of this

series of compounds can be improved substantially against FP-2 enzyme which in

turn can serve to be potential antimalarial agents. Further optimization of the lead

structures and biological screening is in progress in the laboratory.

4. Experimental

Chapter-4 Experimental work

100

All the reagents and solvents required for synthesis were purified by

general laboratory techniques before use. Compounds were purified by passing

them through silica gel H (100-200 mesh) purifying column using mixture of ethyl

acetate and hexane or methanol as eluent. Melting points were determined using a

Labindia make melting point apparatus (heating block type) and are uncorrected.

Purity of the compounds and completion of reactions were monitored by thin layer

chromatography (TLC) on silica gel plates (60 F254; Merck), visualizing with

ultraviolet light or iodine vapors. The yields reported here are un-optimized. The

IR spectra were recorded using KBr disc method on a Bruker FT-IR, model alpha.

The 1H-NMR spectra (on a Bruker 400 MHz spectrometer) were recorded in

DMSO-d6 (chemical shifts in δ ppm). The assignments of exchangeable protons

were confirmed by the D2O exchange studies wherever required. Mass spectral

data was obtained on a Waters Micromass ESCi spectrometer.

4.1. Chemical studies

4.1.1 Synthesis of 4H-pyrido[1,2-a]pyrimidin-4-one intermediates

(4a-c & 6a-c)


Diethyl 2-pyridylaminomethylenemalonate (2a)

A mixture of 2-aminopyridine (1a) (10 g, 10.6 mmole) and diethyl

ethoxymethylenemalonate (24.47 g, 10.6 mmole) was refluxed for 2 h. The

reaction mixture was cooled to RT to get (2a) as a light brown solid (14 g, 50%),

m. p. 68-69 °C (Lit. 217,218

67.5-68 °C).

Anal.:

TLC : Rf 0.85 (EtOAc)

IR : 3274, 1686, 1649, 1601, 1558, 1414, 1248 and 1147 cm-1

1H-NMR : 1.22-1.29 (m, 6H), 4.12-4.18 (q, 2H), 4.20-4.25 (q,

2H), 7.14-

7.17 (m, 1H), 7.39-7.41 (d, 1H), 7.79-7.83 (m, 1H), 8.36-8.37

(d, 1H), 9.05-9.08 (d, 1H), 10.79-10.82 (d, 1H)

MS : (m/z) 265 (M++1)


101

Ethyl 4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (3a)

The pyridine derivative (2a) (13 g, 5.9 mmole) was refluxed in diphenyl

ether (100 ml) using air condenser for 8 h in a two-neck round-bottom flask (250

ml). The reaction mixture was cooled to RT and hexane (500 ml) was added in to

it to get a solid precipitate. The precipitated solid was filtered, washed with

hexane and dried to obtain the compound (3a) (10 g, 93.1%), m. p. 112-13 °C

(Lit.217

110-11 °C).

Anal.:


IR : 1730, 1482, 1288, 1227 and 1103 cm-1

1H-NMR : 1.29-1.32 (t, 3H), 4.25-4.30 (m, 2H), 7.56-7.59 (m, 1H), 7.84-

7.87 (d, 1H), 8.19-8.23 (m, 1H), 8.87 (s, 1H), 9.16-9.18 (d,

1H)

MS : (m/z) 219 (M++1)

4-Oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (4a)

A solution of the ester (3a) (10 g, 5.2 mmole) in conc. HCl (15 ml) was

refluxed for 6 h. The reaction mixture was cooled to RT to get a solid precipitate.

The solid precipitate was filtered, washed with ether and dried to get compound

(4a) as a dark brown solid (8 g, 85.5%), m. p. 268 °C (decomp.) [Lit.217

265 °C

(decomp.)].

Anal.:


IR : 3221, 3091, 1757, 1677, 1636, 1519, 1483, 1275, 1247 cm-1

1H-NMR : 7.67-7.71 (m, 1H), 7.97-8.00 (d, 1H), 8.31-8.35 (m, 1H), 8.96

(s, 1H), 9.22-9.23 (d, 1H)

MS : (m/z) 191 (M++1)


4-Oxo-4H-pyrido[1,2-a]pyrimidine-3-carbonylazide (5a)

In a two-neck round-bottom flask (250 ml), compound (4a) (8 g, 4.2

mmole) and triethylamine (8.5 g, 8.4 mmole) were dissolved in DMF (25 ml). The

reaction mixture was cooled to 0 oC. Ethyl chloroformate (5.03 g, 4.6 mmole) was

added to the reaction mixture and stirred for 15 min followed by addition of


102

aqueous solution of sodium azide (10.95 g, 16.8 mmole) resulting into a yellow

solid precipitate. The solid precipitate was filtered, washed with cold water,

hexane and then air dried affording the compound (5a) as brown solid (6.8 g,

75.1%) m. p. 130-31 °C.

Anal.:


IR : 2310, 1725, 1472, 1185 and 1012 cm-1

1H-NMR : 7.61-7.64 (m, 1H), 7.87-7.91 (d, 1H), 8.24-8.35

(m, 1H), 8.94

(s, 1H), 9.10-9.28 (d, 1H)

MS : (m/z) 214 (M+-1)

3-Isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one (6a)

The acid azide (5a) (4 g, 1.8 mmole) in toluene was refluxed for 2 h.

Work up of the reaction mixture was done by removing toluene under vacuum to

get the compound (6a) (3.2 g, 40.6%), m. p. 180-81 °C.

Anal.:


IR : 2140, 1687, 1650, 1575, 1550, 1490 and 1190 cm-1

MS : (m/z) 188 (M++1)

Synthesis of 8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic

acid (4b)

Diethyl (4-methyl-2-pyridylamino)methylenemalonate (2b)

4-Methyl-2-aminopyridine (10 g, 9.25 mmole) was reacted with diethyl

ethoxymethylenemalonate (21.29 g, 9.25 mmole) as described above for

compound (2a), offering compound (2b) (15 g, 58.2%), m. p. 73-74 °C (Lit.217

72-

73 °C).

Anal.:


IR : 1731, 1682, 1648, 1605, 1548, 1374 and 1218 cm-1

MS : (m/z) 279 (M++1)


103

Ethyl 8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (3b)

In analogy to compound (3a), 2b (10 g, 4.31 mmole) was refluxed in

diphenyl ether to obtain compound (3b) (10 g, 88.4%), m. p. 174-75 °C (Lit.217

171-72 °C).

Anal.:


IR : 1689, 1639, 1556, 1501, 1284 and 1143 cm-1

1H-NMR : 1.30 (t, 3H), 2.51 (s, 3H), 4.23-4.29 (m, 2H), 7.42-7.44 (m,

1H), 7.67 (s, 1H), 8.82 (s, 1H), 9.04-9.05 (d, 1H)

MS : (m/z) 233 (M++1)

8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (4b)

A solution of the ester (3b) (10 g, 4.3 mmole) in conc. HCl (15 ml) was

refluxed for 2 h. The reaction mixture was cooled to RT to get a solid precipitate.

The solid so obtained was filtered, washed with ether (100 ml) and dried to offer

8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-carboxylic acid (4b) (8.5 g, 96.7%),

m. p. 225 °C [Lit.219

223 °C].

Anal.:


IR : 2645, 1764, 1696, 1620, 1522, 1268, 1204 and 1152 cm-1

1H-NMR : 2.62 (s, 3H), 7.62-7.64 (m, 1H), 7.89 (s, 1H), 8.88-8.90 (d,

1H), 9.14-9.16 (d, 1H), 10.9 (b, 1H)

MS : (m/z) 203 (M+-1)

Synthesis of 3-isocyanato-8-methyl-4H-pyrido[1,2-a]pyrimidin-4-one

(6b)

8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbonylazide (5b)

Compound (5b) was prepared by reacting the acid (4b) (8 g, 3.9 mmole)

with ethyl chloroformate (4.68 g, 4.3 mmole) and sodium azide (10.19 g, 15.7

mmole) as described above for compound (5a) resulting in to compound (5b) (7 g,

77.9%), m. p. 148-50 °C.

Anal.:


IR : 2142, 1712, 1640, 1569, 1485, 1285 and 1172 cm-1


104

1H-NMR : 2.56 (s, 3H), 7.49-7.51 (d, 1H), 7.75 (s, 1H), 8.84 (s, 1H),

9.09-9.13 (d, 1H)

MS : (m/z) 230 (M++1)

3-Isocyanato-8-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (6b)

The azide (5b) (4 g) was refluxed in toluene for 2 h. The reaction

mixture was cooled to RT and the solvent was removed under vacuo to get 3-

isocyanato-8-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (6b) (3.34 g, 95.1%), m. p.

234-35 °C.

Anal.:


IR : 2353, 2316, 2223, 1670, 1639 and 1482 cm-1

MS : (m/z) 201 (M++1)

Synthesis of 7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic

acid (4c)

Diethyl (5-chloro-2-pyridylamino)methylenemalonate (2c)

Compound (2c) was prepared by reacting compound (1c) (10 g, 7.8

mmole) with diethyl ethoxymethylenemalonate (17.96 g, 7.8 mmole) as described

above for compound (2a), affording the compound (2c) (15 g, 42.9%), m. p. 133-

34 °C (Lit.220

131-32 °C).

Anal.:


IR : 3270, 1676, 1644, 1588, 1552, 1395, 1219 and 1007 cm-1

1H-NMR : 1.11-1.29 (m, 6H), 4.13-4.26 (m, 4H), 7.47-7.49 (d, 1H),

7.91-7.94 (m, 1H), 8.41 (s, 1H), 8.94-8.97 (d, 1H), 10.79-

10.82 (d, 1H)

MS : (m/z) 299 (M++1)

Ethyl 7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (3c)

With a procedure similar to that adopted for compound (3a) compound

(3c) was prepared from compound (2c) (15 g, 5.03 mmole) providing the titled

compound (3c) (10 g, 78.8%), m. p. 146-47 °C (Lit.220

147-48 °C).

Anal.:


105


IR : 1681, 1645, 1591, 1552, 1466, 1366, 1222 and 1091 cm-1

MS : (m/z) 253 (M++1)

7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (4c)

In analogy to compound (4a), compound (4c) was obtained by

employing the same procedure using 3c (10 g, 3.9 mmole) to get 7-chloro-4-oxo-

4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (4c) (8 g, 89.9%), m. p. 256-57 °C.

Anal.:


IR : 3063, 1756, 1661, 1612, 1510, 1418, 1263 and 1163 cm-1

MS : (m/z) 225.6 (M++1)

Synthesis of 7-chloro-3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one

(6c)

7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbonylazide (5c)

Compound (5c) was prepared by reacting compound (4c) (8 g, 6.25

mmole) with ethyl chloroformate (6.78 g, 6.87 mmole) and sodium azide (16.25 g,

25 mmole) as described above for compound (5a) producing the compound (5c)

(6.5 g, 74.2%), m. p. 159-60 °C.

Anal.:


IR : 2158, 1732, 1563, 1472, 1296, 1201 and 1023 cm-1

MS : (m/z) 250.6 (M++1)

7-Chloro-3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one (6c)

5c (4 g, 1.8 mmole) was refluxed in toluene for 2 h. The reaction mixture

was concentrated to obtain 7-chloro-3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-

one (6c) (3.34 g, 98.5%), m. p. 312-13 °C.

Anal.:


IR : 2317, 2243, 1668, 1625, 1547, 1525, 1475 and 1182 cm-1

MS : (m/z) 222.6 (M++1)


106

4.1.2 Synthesis of substituted (4-oxo-4H-pyrido[1,2-a]pyrimidin-3-

yl)urea derivatives (Series I)


1-(4-Chlorobenzyl)-3-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea (I-1)

The isocyanate (6a) (0.5 g, 0.27 mmole) and 4-chlorobenzylamine

(0.227 g, 0.16 mmole) in toluene (25 ml) were refluxed together for 6 h. The

reaction mixture was cooled to RT to get a solid precipitate. The solid precipitate

was filtered and washed with hexane. The product was purified by column

chromatography using hexane (20%) in ethyl acetate as eluent to get the titled

compound (I-1) (0.2 g, 37.9%), m. p. 250-51 °C.

Anal.:


IR : 3318, 3051, 1693, 1637, 1536, 1479, 1220 and 1136 cm-1

1H-NMR : 4.39-4.41 (d, 2H), 7.26-7.35 (m, 3H), 7.41-7.47 (m, 2H), 7.56

(t, 1H), 7.61-7.64 (d, 1H), 7.68-7.71 (m, 1H), 8.55 (s, 1H),

8.85-8.87 (d, 1H), 9.16 (s, 1H)

MS : (m/z) 328.9 (M+), 330.8 (M

++2)

1-(4-Methoxybenzyl)-3-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea (I-2)

Compound (I-2) was prepared by treating the isocyanate (6a) (0.3 g,

0.16 mmole) with 4-methoxybenzylamine (0.172 g, 0.16 mmole) as described

above for compound (I-1). The crude product was purified by column

chromatography using methanol (1%) in ethyl acetate as eluent offering the

compound (I-2) (0.15 g, 51.9%), m. p. 229-30 °C.

Anal.:

TLC : Rf 0.68 (1% MeOH in EtOAc)

IR : 3361, 3277, 3038, 1639, 1242, 1220, 1180 and 1122 cm-1

1H-NMR : 3.73 (s, 3H), 4.24-4.25 (d, 2H), 6.89-6.91 (d, 2H), 7.22-7.27

(m, 3H), 7.40-7.43 (t, 1H), 7.61-7.63 (d, 1H), 7.68-7.72 (m,

1H), 8.39 (s, 1H), 8.84-8.86 (d, 1H), 9.18 (s, 1H)

MS : (m/z) 325 (M++1)


107

1-(2-Methoxyethyl)-3-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea (I-3)

Compound (I-3) was prepared by reacting the isocyanate (6a) (0.3 g,

0.16 mmole) with 2-methoxyethylamine (0.119 g, 0.16 mmole) as described

above for compound (I-1). The crude product after chromatographic purification

using methanol (0.9%) in ethyl acetate as eluent provided compound (I-3) (0.5 g,

61.1%), m. p. 196-97 °C.

Anal.:


IR : 3362, 1696, 1639, 1548, 1463, 1226, 1192 and 1099 cm-1

1H-NMR : 3.25-3.28 (m, 5H), 3.70-3.39 (t, 2H), 7.16-7.17 (m, 1H), 7.23-

7.27 (m, 1H), 7.60-7.63 (d, 1H), 7.67-7.72 (m, 1H), 8.41 (s,

1H), 8.45-8.86 (d, 1H), 9.16(s, 1H)

MS : (m/z) 263 (M++1)

N-{2-[3-(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)ureido]ethyl}acetamide (I-4)

The isocyanate (6a) (0.3 g, 0.16 mmole) was reacted with 2-

acetamidoethylamine (0.62 g, 0.16 mmole) as described above for compound (I-

1). Chromatographic purification of the crude product using methanol (0.9%) in

ethyl acetate as eluent furnished compound (I-4) (0.5 g, 61.1%), m. p. 247-49 °C.

Anal.:


IR : 3317, 3251, 1726, 1633, 1536, 1467, 1219 and 1120 cm-1

1H-NMR : 1.81 (s, 3H), 3.11-3.16 (m, 4H), 7.09-7.11 (m, 1H), 7.23-7.27

(m, 1H), 7.61-7.63 (d, 1H), 7.68-7.72 (m, 1H), 7.94 (b, 1H),

8.35 (s, 1H), 8.84-8.86 (d, 1H), 9.17 (s, 1H)

MS : (m/z) 290 (M++1)

1-(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-(2-trifluoromethylbenzyl)urea

(I-5)

Compound (I-5) was prepared by reacting the isocyanate (6a) (0.3 g,

0.16 mmole) with 2-trifluoromethylbenzylamine (0.281 g, 0.16 mmole) as

described above for compound (I-1). Work-up of the reaction mixture followed by

chromatographic purification of the crude product using hexane (10%) in ethyl


108

acetate as eluent earned the titled compound (I-5) (0.22 g, 37.8%), m. p. 243-45

°C.

Anal.:


IR : 3315, 3047, 1667, 1638, 1548, 1478, 1328 and 1224 cm-1

1H-NMR : 4.51-4.52 (d, 2H), 7.25-7.28 (t, 1H), 7.47-7.51 (m, 1H), 7.56-

7.64 (m, 3H), 7.68-7.74 (m, 3H), 8.59 (s, 1H), 8.86-8.88 (d,

1H), 9.17 (s, 1H)

MS : (m/z) 361 (M+-1)

1-(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-(4-trifluoromethylbenzyl)urea

(I-6)

The isocyanate (6a) (0.3 g, 0.16 mmole) was treated with 4-

trifluoromethylbenzylamine (0.281 g, 0.16 mmole) as described above for

compound (I-1). The crude product after chromatographic purification using

hexane (10%) in ethyl acetate as eluent offered the titled compound (I-6) (0.21 g,

34.4%), m. p. 256-57 °C.

Anal.:


IR : 3328, 3048, 1670, 1636, 1478, 1332, 1222 and 1107 cm-1

1H-NMR : 4.42-4.44 (d, 2H), 7.25-7.28 (m, 1H), 7.51-7.54 (d, 2H), 7.59-

7.64 (m, 2H), 7.69-7.73 (m, 3H), 8.50 (s, 1H), 8.86-8.87 (d,

1H), 9.16 (s, 1H)

MS : (m/z) 363 (M++1)

1-(2,4-Difluorobenzyl)-3-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea (I-7)

The coupling reaction of the isocynate (6a) (0.3 g, 0.16 mmole) was

done with 2,4-difluorobenzylamine (0.229 g, 0.16 mmole) as described above for

compound (I-1). Work-up of the reaction mixture followed by chromatographic

purification of the crude product using hexane (5%) in ethyl acetate as eluent

yielded compound (I-7) (0.1 g, 18.8%), m. p. 170-71 °C.

Anal.:


IR : 3328, 1676, 1558, 1483, 1236 and 1127 cm-1


109

1H-NMR : 4.35-4.37 (d, 2H), 7.14-7.20 (m, 2H), 7.24-7.28 (m, 2H),

7.52-7.55 (t, 1H), 7.62-7.64 (d, 1H), 7.69-7.74 (m, 1H), 8.50

(s, 1H), 8.85-8.87 (d, 1H), 9.14 (s, 1H)

MS : (m/z) 331 (M++1)

1-(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-[2-(thiophen-2-yl)ethyl]urea (I-8)

A mixture of isocyanate (6a) (0.3 g, 0.16 mmole) and 2-(2-

thiophenyl)ethylamine (0.308 g, 0.16 mmole) were reacted together as described

above for compound (I-1). The crude product was purified by column

chromatography using methanol (2%) in ethyl acetate as eluent to obtain the

compound (I-8) (0.2 g, 39.6%), m. p. 216-17 °C.

Anal.:


IR : 3213, 3033, 1656, 1525, 1469, 1218, 1182 and 1127 cm-1

1H-NMR : 2.97-3.01 (t, 2H), 3.37-3.4 (t, 2H), 6.92-6.93 (b, 1H), 6.97-

6.99 (m, 1H), 7.15-7.18 (t, 1H), 7.23-7.27 (m, 1H), 7.35-7.37

(d, 1H), 7.61-7.63 (d, 1H), 7.68-7.72 (m, 1H), 8.41 (s, 1H),

8.84-8.85 (d, 1H), 9.19 (s, 1H)

MS : (m/z) 315 (M++1)

1-[3-(Morpholin-4-yl)propyl]-3-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea

(I-9)

6a (0.5 g, 0.27 mmole) and 3-(4-morpholino)propylamine (0.227 g,

0.16 mmole) were reacted together as described above for compound (I-1). The

crude product after chromatographic purification using hexane (10%) in ethyl

acetate as eluent offered the titled compound (I-9) (0.18 g, 40.6%), m. p. 189-90

°C.

Anal.:


IR : 3297, 3072, 1663, 1549, 1479, 1228 and 1118 cm-1

1H-NMR : 1.55-1.62 (m, 2H), 2.28-2.32 (m, 2H), 2.34 (b, 4H), 3.10-3.15

(m, 2H), 3.56-3.58 (t, 4H), 7.01-7.04 (m, 1H), 7.23-7.27 (m,

1H), 7.60-7.63 (d, 1H), 7.67-7.71 (m, 1H), 8.29 (s, 1H), 8.84-

8.86 (d, 1H), 9.17 (s, 1H)


110

MS : (m/z) 332 (M++1)

1-(3, 3-Diphenyl)propyl-3-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea (I-10)

The isocyanate (6a) (0.25 g, 0.13 mmole) was reacted with 3,3-

diphenylpropylamine (0.282 g, 0.13 mmole) as described above for compound (I-

1). The chromatographic purification of the crude product using hexane (10%) in

ethyl acetate as eluent afforded the urea derivative (I-10) (0.21 g, 39.4%), m. p.

259-60 °C.

Anal.:


IR : 3324, 3133, 3024, 1664, 1631, 1559, 1476 and 1241cm-1

1H-NMR : 2.18-2.23 (m, 2H), 2.97-3.05 (m, 2H), 4.01-4.05 (t, 1H), 7.11-

7.34 (m, 12H), 7.60-7.63 (d, 1H), 7.67-7.74 (m, 1H), 8.32 (s,

1H), 8.85-8.92 (dd, 1H), 9.15(s, 1H)

MS : (m/z) 399 (M++1)

1-(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-[3-(n.pentyloxy)pyridin-2-yl]-

urea (I-11)

Compound (I-11) was prepared by reacting the isocynate (6a) (0.3 g,

0.16 mmole) with 3-n.pentyloxy-2-aminopyridine (0.289 g, 0.16 mmole) as

described above for compound (I-1). The crude product after chromatographic

purification using methanol (1%) in ethyl acetate as eluent offered the desired

product (I-11) (0.2 g, 33.96%), m. p. 178-80 °C.

Anal.:


IR : 3387, 3350, 3067, 1665, 1550, 1478 and 1220 cm-1

1H-NMR : 0.90-0.94 (t, 3H), 1.32-1.47 (m, 4H), 1.78-1.85 (m, 2H), 4.07-

4.11 (t, 2H), 7.05-7.08 (m, 1H), 7.30-7.34 (m, 1H), 7.44-7.46

(d, 1H), 7.67-7.69 (d, 1H), 7.76-7.81 (m, 1H), 7.89-7.91 (dd,

1H), 8.35 (s, 1H), 8.92-8.94 (d, 1H), 9.29 (s, 1H), 12.15 (s,

1H)

MS : (m/z) 368 (M++1)


111

1-[3-(Benzyloxy)pyridin-2-yl]-3-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea

(I-12)

Compound (6a) (0.3 g, 0.16 mmole) was treated with 2-amino-3-

benzyloxypyridine (0.319 g, 0.16 mmole) as described above for compound (I-1).

The crude product was purified by column chromatography using methanol (1%)

in ethyl acetate as eluent offering the titled compound (I-12) (0.19 g, 30.6%), m.

p. 249-50 °C.

Anal.:


IR : 3133, 1675, 1558, 1482, 1192 and 1118 cm-1

1H-NMR : 5.29 (s, 2H), 7.07 (m, 1H), 7.33-7.42 (m, 3H), 7.51-7.57 (m,

3H), 7.70-7.76 (m, 1H), 7.77-7.79 (m, 1H), 7.91-7.92 (d, 1H),

8.46 (s, 1H), 8.90-8.94 (m, 1H), 9.22-9.28 (d, 1H), 9.52 (s,

1H), 12.06 (s, 1H)

MS : (m/z) 388 (M++1)

1-[6-(Morpholin-4-yl)pyridin-2-yl]-3-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-

urea (I-13)

Compound (I-13) was prepared by treating the isocyanate (6a) (0.3 g,

0.16 mmole) with 2-amino-6-morpholinopyridine (0.287 g, 0.16 mmole) as

described above for compound (I-1). The chromatographic purification of the

crude product using methanol (1%) in ethyl acetate as eluent offered compound

(I-13) (0.25 g, 42.5%), m. p. 275-76 °C.

Anal.:


IR : 3118, 1688, 1558, 1484, 1440, 1229 and 1113 cm-1

1H-NMR : 3.66-3.68 (b, 4H), 3.95-3.97 (b, 4H), 6.17-6.20 (d, 1H), 6.29-

6.31 (d, 1H), 7.11-7.15 (m, 1H), 7.48-7.52 (m, 2H), 7.58-7.62

(m, 1H), 7.67-7.70 (d, 1H), 8.97-8.99 (d, 1H), 9.53 (s, 1H),

11.48 (s, 1H)

MS : (m/z) 367 (M++1)


112

Ethyl (2S)-2-{[(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)carbamoyl]amino}-3-

phenylpropanoate (I-14)

The isocyanate (6a) (1.94 g, 6.1 mmole) and L-phenylalanine ethyl ester

(2 g, 6.1 mmole) in toluene (25 ml) were refluxed together for 6 h. The reaction

mixture was cooled to RT to yield a solid precipitate. The solid precipitate was

filtered, washed with hexane and dried to get the titled compound (I-14) (2 g,

62.5%), m. p. 158-59 °C.

Anal.:


IR : 3338, 3032, 1733, 1688, 1629, 1132 and 1185 cm-1

MS : 381(M++1)

Methyl (2S)-4-methyl-2-{[(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)carbamoyl]-

amino}n.pentanoate (I-15)

Compound (I-15) was obtained by reacting compound (6a) (2.85 g, 1.37

mmole) with L-leucine methyl ester (2 g, 1.37 mmole) as described above for

compound (I-14). The crude product was recrystallized from methanol to yield

compound (I-15) (2.1 g, 45.8%), m. p. 129-30 °C.

Anal.:


IR : 3325, 3276, 1745, 1657, 1634, 1550, 1528 and 1191 cm-1

MS : (m/z) 333 (M++1)

(2S)-2-{[(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)carbamoyl]amino}-3-phenyl-

propanamide (I-16)

A mixture of compound (I-14) (2 g, 0.52 mmole) and ammonia solution

was stirred for 12 h. The solid precipitate so obtained was filtered, washed with

water and recrystallized from methanol to furnish compound (I-16) (1.8 g,

97.4%), m. p. 239-40 °C.

Anal.:


IR : 3329, 1634, 1519, 1477, 1244 and 1191 cm-1

MS : (m/z) 352 (M++1)


113

(2S)-4-Methyl-2-{[(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)carbamoyl]amino}-

n.pentanamide (I-17)

Compound (I-15) (2 g, 0.52 mmole) was stirred in ammonia solution for

12 h to get the solid precipitate. The precipitated recrystallized from methanol to

obtain compound (I-17) (1.6 g 83.7%), m. p. 243-44 °C.

Anal.:


IR : 3361, 3320, 1672, 1628, 1528, 1469, 1437 and 1234 cm-1

MS : (m/z) 318 (M++1)

1-[(1S)-1-(Cyano-2-phenyl)ethyl]-3-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-

urea (I-18)

The amide (I-16) (1.2 g, 0.34 mmole) was dissolved in

dimethylformamide (2.5 ml) and cooled to 0°C. Cyanuric chloride (1.26 g, 0.68

mmole) was added in small portions and stirred for 2 h. The reaction mixture was

quenched in ice-cold water (20 ml) to get solid precipitate. The solid precipitate

was filtered and dried. The crude product so obtained was purified by column

chromatography to earn the compound (I-18) (0.34 g, 29.8%), m. p. 223-25 °C.

Anal.:


IR : 3303, 2210, 1686, 1631, 1530, 1433, 1227 and 1133 cm-1

1H-NMR : 3.09-3.19 (m, 2H), 4.92-4.98 (m, 1H), 7.26-7.34 (m, 2H),

7.36-7.37 (m, 4H), 7.63-7.67 (m, 2H), 7.72-7.77 (m, 1H), 8.58

(s, 1H), 8.85-8.87 (d, 1H), 9.10 (s, 1H)

MS : (m/z) 334 (M++1)

1-[(1S) 1-(Cyano-3-methyl)butyl]-3-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-

urea (I-19)

Compound (I-19) was prepared by treating (I-17) (2.0 g, 0.63 mmole)

with cyanuric chloride (2.33 g, 1.26 mmole) as described above for compound (I-

18). The chromatographic purification of the crude product using hexane (25%) in

ethyl acetate as eluent offered compound (I-19) (0.35 g, 18%), m. p. 237-39 °C.

Anal.:



114

IR : 3342, 2210, 1642, 1557, 1510, 1465, 1434 and 1235 cm-1

1H-NMR : 0.91-0.96 (d, 6H), 1.64-1.80 (m, 3H), 4.65-4.71(m, 1H), 7.27-

7.31 (m, 1H), 7.65-7.67 (d, 2H), 7.73-7.77 (m, 1H), 8.49 (s,

1H), 8.86-8.88 (d, 1H), 9.12 (s, 1H)

MS : (m/z) 300 (M++1)

1-Benzyl-1-isopropyl-3-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea (I-20)

Isocyanate (6a) (0.25 g, 0.13 mmole) and N-isopropylbenzylamine

(0.199 g, 0.13 mmole) were reacted as described above for compound (I-1). The

crude product by chromatographic purification using hexane (10%) in ethyl

acetate offered the titled compound (I-20) (0.15 g, 30.6%), m. p. 145-46 °C.

Anal.:

TCL : Rf 0.61 (EtOAc)

IR : 3373, 1641, 1527, 1483, 1410, 1232, 1176, and 940 cm-1

1HNMR : 1.14-1.16 (d, 6H), 4.51-4.52 (m, 1H), 4.55 (s, 2H), 7.25-7.29

(m, 2H), 7.36-7.37 (d, 4H), 7.50 (s, 1H), 7.63-7.65 (d, 1H),

7.73-7.77 (m, 1H), 8.78-8.80 (d, 1H), 8.96 (s, 1H)

MS : (m/z) 337 (M+-1)


derivatives

1-(2-Methoxyethyl)-3-(8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea

(I-21)

Compound (I-21) was prepared by reacting compound (6b) (0.3 g, 0.149

mmole) with 2-methoxyethyl amine (0.111 g, 0.149 mmole) as described above

for compound (I-1). Work up of the reaction mixture followed by

chromatographic purification using hexane (5%) in ethyl acetate as eluent yielded

compound (I-21) (0.16 g, 38.84%), m. p. 233-34 °C.

Anal.:


IR : 3388, 3305, 2890, 2360, 1633, 1534, 1452, 1228, 1179,

1088, 1006, 935, 809 and 630 cm-1

1HNMR : 2.39 (s, 3H), 3.22-3.25 (t, 4H), 3.35 (s, 3H), 7.09-7.11 (m,

2H), 7.41 (s, 1H), 8.30 (s, 1H), 8.73-8.75 (d, 1H), 9.06 (s, 1H)


115

MS : (m/z) 277 (M+-1)

N-{2-[3-(8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)ureido]ethyl}aceta-

mide (I-22)

Compound (I-22) was prepared by reacting compound (6b) (0.3 g, 0.149

mmole) with 2-acetamidoethylamine (0.151 g, 0.149 mmole) as described above

for compound (I-1). Chromatographic purification of the crude product was done

using hexane (5%) in ethyl acetate as eluent to yield the desired porduct (I-22)

(0.2 g, 44.2%), m. p. 269-70 °C.

Anal.:


IR : 3356, 3289, 1633, 1524, 1451, 1239 and 1004 cm-1

1H-NMR : 1.81 (s, 3H), 2.41 (s, 3H), 3.1-3.15 (m, 4H), 7.03-7.06 (m,

1H) 7.11-7.13 (dd, 1H), 7.43 (s, 1H), 7.93-7.94 (m, 1H), 8.26

(s, 1H), 8.76-8.78 (d, 1H), 9.09 (s, 1H)

MS : (m/z) 304 (M++1)

1-(8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-[2-(thiophen-2-yl)ethyl]

urea (I-23)

Compound (6b) (0.3 g, 0.149 mmole) was treated with 2-(2-

thiophenyl)ethylamine (0.188 g, 0.149 mmole) as described above for compound

(I-1). Work-up of the reaction mixture followed by chromatographic purification

of the crude product using methanol (0.2%) in ethyl acetate as eluent produced 1-

(8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-[2-(thiophen-2-yl)-ethyl]urea

(I-23) (0.2 g, 40.8%), m. p. 249-50 °C

Anal.:


IR : 3278, 1634, 1518 and 1449 cm-1

1H-NMR : 2.41 (s, 3H), 2.95-2.98 (m, 4H), 6.92 (s, 1H), 6.98 (d, 1H),

7.11-7.12 (m, 2H), 7.35-7.37 (d, 1H), 7.43 (s, 1H), 8.32 (s,

1H), 8.75-8.77 (d, 1H), 9.11 (s, 1H)

MS : (m/z) 329 (M++1)


116

1-(8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-[2-(morpholin-4-yl)eth-

yl]urea (I-24)

The isocyanate derivative (6b) (0.3 g, 0.149 mmole) was reacted with 2-

(4-morpholinyl)ethylamine (0.163 g, 0.149 mmole) as described above for

compound (I-1). Chromatographic purification of the crude product using

methanol (0.5%) in ethyl acetate as eluent offered compound (I-24) (0.21 g,

42.5%), m. p. 225-26 °C.

Anal.:


IR : 3334, 1632, 1569, 1460, 1235, 1145, 1104 and 1002 cm-1

1H-NMR : 2.37-2.41 (m, 9H), 3.21-3.24 (m, 2H), 3.58-3.59 (b, 4H),

6.98-6.99 (m, 1H), 7.11-7.12 (d, 1H), 7.42 (s, 1H), 8.37 (s,

1H), 8.76-8.78 (d, 1H), 9.08-9.09 (d, 1H)

MS : (m/z) 332 (M++1)

1-Benzyl-1-isopropyl-3-(8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)urea

(I-25)

Isocyanate derivative (6b) (0.25 g, 0.12 mmole) in toluene (25 ml) was

refluxed with N-isopropylbenzylamine (0.185 g, 0.12 mmole) for 4 h to get a

solid. The solid so obtained was filtered and washed with hexane.

Chromatographic purification of the crude product using hexane (5%) in ethyl

acetate provided the titled compound (I-25) (0.16 g, 36.7%), m. p. 161-62 °C.

Anal.:


IR : 3353, 3031, 2971, 1642, 1546, 1486, 1403 and 1242 cm-1

1HNMR : 1.13-1.15 (d, 6H), 2.41 (s, 3H), 4.49-4,51 (m, 1H), 4.54 (s,

2H), 7.13-7.15 (d, 1H), 7.25- 7.26 (m, 1H), 7.35-7.36 (d, 4H),

7.45 (b, 2H), 8.70-8.72 (d, 1H), 8.87 (s, 1H)

MS : (m/z) 349 (M+), 350.9 (M

++2)


derivatives


117

1-(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-[2-(morpholin-4-

yl)ethyl]urea (I-26)

Compound (I-26) was prepared by reacting compound (6c) (0.3 g, 0.135

mmole) with 2-(4-morpholinyl)ethylamine (0.176 g, 0.135 mmole) as described

above for compound (I-1). Chromatographic purification of the crude product

using methanol (2%) in ethyl acetate as eluent furnished the titled compound (I-

26) (0.22 g, 46.2%), m. p. 226-27 °C.

Anal.:


IR : 3285, 1729, 1646, 1552, 1464, 1284, 1247 and 1117 cm-1

1H-NMR : 2.39 (b, 6H), 3.23-3.24 (m, 2H), 3.60 (b, 4H), 7.08 (b, 1H),

7.63-7.65 (d, 1H), 7.70-7.73 (dd, 1H), 8.57 (s, 1H), 8.84 (s,

1H), 9.17 (s, 1H)

MS : (m/z) 352.1 (M+), 354.1 (M

++2)

1-(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-[2-(thiophen-2-yl)eth-

yl]urea (I-27)

Compound (I-27) was obtained by treating compound (6c) (0.3 g, 0.135

mmole) with 2-(2-thiophenyl)ethylamine (0.17 g, 0.135 mmole) as described

above for compound (I-1). Work-up of the reaction mixture followed by


acetate as eluent offered compound (I-27) (0.24 g, 50.8%), m. p. 216-17 °C.

Anal.:


IR : 3387, 1651, 1544, 1510, 1470, 1224 and 1189 cm-1

1H-NMR : 2.97-3.00 (t, 2H), 3.37-3.40 (t, 2H), 6.92-6.93 (d, 1H), 6.97-

6.99 (m, 1H), 7.18-7.20 (m, 1H), 7.35-7.37 (m, 1H), 7.63-7.66

(d, 1H), 7.71-7.74 (dd, 1H), 8.51 (s, 1H), 8.83-8.84 (d, 1H),

9.19 (s, 1H)

MS : (m/z) 349 (M+), 350.9 (M

++2)

1-(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-(thiophen-2-yl)methyl-

urea (I-28)

Isocyanate derivative (6c) (0.25 g, 0.11 mmole) in toluene was refluxed


118

with (2-thiophenyl)methylamine (0.127 g, 0.11 mmole) to get a solid.

The solid so obtained was filtered and washed with hexane. The crude product

was purified by column chromatography using 1% methanol in ethyl acetate to

obtain the compound (I-28) (0.15 g, 39.76%), m. p. 285-87 °C.

Anal.:


IR : 3272, 3024, 1652, 1562, 1472, 1337, 1240, and 1155 cm-1

1HNMR : 4.49-4.50 (d, 2H), 6.97-6.98 (m, 1H), 7.00 (s, 1H), 7.40-7.41

(d, 1H), 7.56 (b, 1H), 7.64-7.66 (d, 1H), 7.72-7.74 (d, 1H),

8.51 (s, 1H), 8.89 (s, 1H), 9.18 (s, 1H)

MS : (m/z) 335.34(M++1), 337.38 (M

++2)

1-(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-(2-methoxyethyl)urea

(I-29)

A mixture of isocyanate derivative (6c) (0.25 g, 0.11 mmole), 2-

methoxyethylamine (0.162 g, 0.11 mmole) and toluene was refluxed for 4 h to get

solid precipitate. The solid so obtained was filtered and washed with hexane.

Chromatographic purification of the product using hexane (15%) in ethyl acetate

accomplished the desired compound (I-29) (0.22 g, 65.74%), m. p. 254-56 °C.

Anal.:


IR : 3370, 3116, 2972, 1702, 1649, 1557, 1528, 1434, 1354, 1229,

1199, 1106, 817, 757 and 593 cm-1

1HNMR : 3.24-3.39 (m, 7H,), 7.19-7.21 (m, 1H), 7.63-7.65 (d, 1H),

7.70-7.73 (dd, 1H), 8.51 (s, 1H), 8.83-8.84 (b, 1H), 9.16 (s,

1H)

MS : (m/z) 297.39 (M+), 299.38 (M

++2)

1-(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-(4-trifluoromethyl)ben-

zylurea (I-30)

A mixture of isocyanate derivative (6c) (0.25 g, 0.11 mmole) and 4-

trifluoromethylbenzylamine (0.197 g, 0.11 mmole) in toluene was refluxed for 4

h. Solid precipitate so obtained was filtered and washed with hexane.


119

Chromatographic purification of the crude product using 1% methanol in ethyl

acetate yielded the titled compound (I-30) (0.23 g, (51.4%), m. p. 287-89 °C.

Anal.:


IR : 3397, 3187, 1692, 1656, 1632, 1364 and 1226 cm-1

1HNMR : 4.42-4.44 (d, 2H), 7.57-7.66 (m, 6H), 7.72-7.75 (dd, 1H), 8.58

(s, 1H), 8.84-8.85 (d, 1H), 9.16 (s, 1H)

MS : (m/z) 397.34(M+), 399.38 (M

++2)


zylurea (I-31)

A mixture of isocyanate derivative (6c) (0.25 g, 0.11 mmole) and 3-

trifluoromethylbenzylamine (0.197 g, 0.11 mmole) and toluene (50 ml) was

refluxed for 4 h. Solid precipitate so obtained was filtered and washed with

hexane. The crude product was purified by column chromatography using hexane

(5%) in ethyl acetate to furnish the titled compound (I-31) (0.22 g, 49.1%), m. p.

260-62 °C.

Anal.:


IR : 3312, 3282, 3085, 1677, 1646, 1564, 1488, 1433, 1331, 1243,

1160, 1107, 1016, 821, 765 and 644 cm-1

1HNMR : 4.42-4.43 (d, 2H), 7.51-7.53 (d, 2H), 7.61-7.63 (m, 2H), 7.70-

7.75 (m, 3H), 8.60 (s, 1H), 8.84-8.85 (d, 1H), 9.17 (s, 1H)

MS : (m/z) 397.34(M+), 399.32 (M

++2)


zylurea (I-32)

Isocyanate derivative (6c) (0.25 g, 0.11 mmole) in toluene was refluxed

with 2-trifluoromethylbenzylamine (0.197 g, 0.11 mmole) to get solid precipitate.

The solid so obtained was filtered and washed with hexane. Chromatographic

purification of the crude product using hexane (5%) in ethyl acetate furnished the

titled compound (I-32) (0.23 g, 51.4%), m. p. 276-78 °C.

Anal.:


120


IR : 3362, 3126, 1707, 1622, 1558, 1461, 1311 and 1225 cm-1

1HNMR : 4.51-4.52 (d, 2H), 7.47-7.51 (m, 1H), 7.59-7.75 (m, 6H), 8.68

(s, 1H), 8.85 (b, 1H), 9.17 (s, 1H)

MS : (m/z) 397.34 (M+), 399.32, (M

++2)

1-Benzyl-3-(7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-1-isopropylurea

(I-33)

Isocyanate derivative (6c) (0.25 g, 0.11 mmole) in toluene (25 ml) was

refluxed with N-isopropylbenzylamine (0.168 g, 0.11 mmole) for 5 h to get solid

precipitate. Chromatographic purification of the crude product using hexane

(10%) in ethyl acetate offered the desired compound (I-33) (0.2 g, 47.8%), m. p.

158-60 °C.

Anal.:


IR : 3447, 1663, 1630, 1521, 1487, 1234, 1114 and 1070 cm-1

MS : (m/z) 371.42 (M+), 373.41(M

++2)

1-Benzyl-3-(7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-1-phenethylurea

(I-34)

Reaction of isocyanate derivative (6c) (0.25 g, 0.11 mmole) and N-

benzyl phenylethylamine (0.238 g, 0.11 mmole) was done as described above for

compound (I-1). The crude product was purified by column chromatography

using 2% methanol in ethyl acetate to obtain the titled compound (I-34) (0.18 g,

36.8%), m. p. 195-97 °C.

Anal.:


IR : 3345, 3028, 1647, 1550, 1493, 1435, 1366 and 1233 cm-1

1HNMR : 2.85-2.89 (t, 2H), 3.35-3.59 (t, 2H),

4.58 (s, 2H), 7.14-7.38

(m, 11H), 7.68-7.71 (d, 1H), 7.81-7.82 (b, 2H), 8.88 (b, 1H),

8.92 (s, 1H)

MS : (m/z) 433.42 (M+), 435.41 (M

++2)


121

3.1.3 Synthesis of N-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-

(pyridin-2-yl)piperazine-1-carboxamide derivatives (Series II)


yl)- piperazine-1-carboxamide derivatives

N-(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-(pyridin-2-yl)piperazine-1-

carboxamide (II-1)

Compound (II-1) was prepared by reacting compound (6a) (0.3 g, 0.16

mmole) with 4-(2-pyridyl)piperazine (0.262 g, 0.16 mmole) as described above

for compound (I-1). The crude product was purified by column chromatography

using hexane (10%) in ethyl acetate as eluent to afford the compound (II-1) (0.19

g, 33.8%), m. p. 194-95 °C.

Anal.:


IR : 3393, 3107, 1659, 1596, 1537, 1482 and 1238 cm-1

1H-NMR : 3.57-3.58 (b, 8H), 6.65-6.69 (m, 1H), 6.86-6.88 (d, 1H), 7.31-

7.35 (m, 1H), 7.54-7.58 (m, 1H), 7.67-7.69 (d, 1H), 7.80-7.84

(m, 1H), 8.08 (s, 1H), 8.13-8.14 (d, 1H), 8.77 (s, 1H) 8.90-

8.93 (d, 1H)

MS : (m/z) 351 (M++1)

4-(4-Methoxyphenyl)-N-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)piperazine-1-

carboxamide (II-2)

The urea derivative (II-2) was obtained by refluxing the isocyanate (6a)

(0.3 g, 0.16 mmole) with 1-(4-methoxyphenyl piperazine (0.308 g, 0.16 mmole) in

toluene for 6 h. The reaction mixture was cooled to RT and the solid precipitate

was filtered. The chromatographic purification of the crude product using hexane

(10%) in ethyl acetate as eluent yielded the titled compound (II-2) (0.19 g 31.2%),

m. p. 143-45 °C.

Anal.:


IR : 3396, 3031, 2926, 1649, 1479, 1437, 1229 and 1021 cm-1

1H-NMR : 2.99 (b, 4H), 3.61(b, 4H), 3.81 (s, 3H), 6.87-7.01 (m, 4H),


122

7.32-7.35 (m, 1H), 7.67-7.69 (d, 1H), 7.80-7.84 (m, 1H), 8.06

(s, 1H), 8.77 (s, 1H), 8.91-8.93 (d, 1H)

MS : (m/z) 380 (M++1)

N-(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-[3-(N-cyclopropylcarboxamido)

pyridine-2-yl]piperazinecarboxamide (II-3)

Compound (II-3) was prepared by reacting the isocyanate (6a) (0.3 g,

0.16 mmole) with N-cyclopropyl-2-(piperazine-1-yl)pyridine-3-carboxamide

(0.319 g, 0.16 mmole) as described above for compound (I-1). The crude

compound was purified by column chromatography using methanol (3%) in ethyl

acetate as eluent to get the desired compound (II-3) (0.22 g, 31.6%), m. p. 140-42

°C.

Anal.:


IR : 3296, 3035, 1654, 1530, 1484, 1434, 1234 and 1134 cm-1

1H-NMR : 0.55-0.58 (m, 2H), 0.69-0.72 (m, 2H), 2.82-2.83 (m, 1H),

3.31-3.36 (t, 4H), 3.57-3.59 (t, 4H), 6.89-6.92 (m, 1H), 7.32-

7.36 (m, 1H), 7.65-7.67 (m, 2H), 7.81-7.85 (m, 1H), 8.08 (s,

1H), 8.23-8.25 (dd, 1H), 8.51-8.53 (d, 1H), 8.76 (s, 1H), 8.91-

8.93 (d, 1H)

MS : (m/z) 434 (M++1)

4-Benzhydryl-1-[N-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)]carboxamido-

piperazine (II-4)

The isocyanate (6a) (0.3 g, 0.16 mmole) was reacted with

benzhydrylpiperazine (0.404 g, 0.16 mmole) as described above for compound (I-

1). The crude product was purified by column chromatography using hexane (5%)

in ethyl acetate as eluent offering the titled compound (II-4) (0.2 g, 42.5%), m. p.

253-55 °C.

Anal.:


IR : 3403, 3032, 1657, 1527, 1484, 1294, 1232 and 1110 cm-1

1H-NMR : 2.33-2.36 (t, 4H), 3.48-3.49 (t, 4H), 4.35 (s, 1H), 7.19-7.22


123

(m, 2H), 7.30-7.34 (m, 5H), 7.45-7.47 (d, 4H), 7.66-7.68 (d,

1H), 7.79-7.83 (m, 1H), 7.93 (s, 1H), 8.74 (s, 1H), 8.88-8.90

(d, 1H)

MS : (m/z) 440 (M++1)



N-(8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-(pyridin-2-yl)piperazi-

ne-1-carboxamide (II-5)

Compound (6b) (0.3 g, 0.149 mmole) was reacted with 4-(2-

pyridyl)piperazine (0.243 g, 0.149 mmole) as described above for compound (I-

1). The chromatographic purification of the crude product was done using

methanol (2%) in ethyl acetate as eluent to get the product (II-5) (0.18 g, 33.1%),

m. p. 160-61°C.

Anal.:


IR : 3395, 3045, 1654, 1596, 1531, 1484, 1245, and 1192 cm-1

1H-NMR : 2.45 (s, 3H), 3.57 (b, 8H), 6.65-6.68 (m, 1H), 6.86-6.88 (d,

1H), 7.20-7.22 (d, 1H), 7.50 (s, 1H), 7.54-7.58 (m, 1H), 8.05

(s, 1H), 8.13-8.14 (d, 1H), 8.66 (s, 1H), 8.82-8.84 (d, 1H)

MS : (m/z) 365 (M++1)

N-(8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-[(4-methoxyphenyl)]-

piperazine-1-carboxamide (II-6)

Compound (II-6) was prepared by reacting compound (6b) (0.3 g, 0.149

mmole) with 4-(4-methoxyphenyl)piperazine (0.287 g, 0.149 mmole) as described

above for compound (I-1). Chromatographic purification of the crude product

using methanol (2%) in ethyl acetate as eluent provided the titled compound (II-6)

(0.195 g, 33.2%), m. p. 134-35 °C.

Anal.:


IR : 3393, 1648, 1530, 1483, 1241, 1188 and 1153 cm-1

1H-NMR : 2.44 (s, 3H), 2.97-2.99 (b, 4H), 3.58-3.61 (b, 4H), 3.80 (s,

3H), 6.87-7.00 (m, 4H), 7.19-7.22 (dd, 1H), 7.50 (s, 1H), 8.00


124

(s, 1H), 8.66 (s, 1H), 8.82-8.84 (d, 1H)

MS : (m/z) 394 (M++1)

N-(8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-benzhydrylpiperazine-

1-carboxamide (II-7)

The isocyanate derivative (6b) (0.3 g, 0.149 mmole) and benzhydryl

piperazine (0.376 g, 0.147 mmole) were reacted together as described above for

compound (I-1). The crude product was purified by chromatographic purification

using methanol (0.5%) in ethyl acetate as eluent to obtain the compound (II-7)

(0.2 g, 29.5%), m. p. 233-35 °C.

Anal.:


IR : 3000, 1644, 1529, 1481, 1237 and 1186 cm-1

1H-NMR : 2.32-2.34 (b, 4H), 2.43 (s, 3H), 3.46-3.48 (b, 4H), 4.34 (s,1H),

7.18-7.22 (m, 3H), 7.29-7.33 (m, 4H), 7.44-7.48 (m, 5H),

7.87 (s, 1H), 8.63 (s, 1H), 8.79-8.81 (d, 1H)

MS : (m/z) 454 (M++1)

Synthesis of N-(7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-


N-[(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-(pyridin-2-yl)pipera-

zine]-1-carboxamide (II-8)

Compound (II-8) was prepared by reacting compound (6c) (0.3 g, 0.135

mmole) with 4-(2-pyridyl)piperazine (0.221 g, 0.135 mmole) as described above

for compound (I-1). The chromatographic purification of the crude product was

done using methanol (2%) in ethyl acetate as eluent to earn the compound (II-8)

(0.21 g, 40.3%), m. p. 189-90 °C.

Anal.:


IR : 3397, 3088, 1729, 1643, 1531, 1482, 1243 and 1124 cm-1

1H-NMR : 3.57-3.58 (m, 8H), 6.65-6.68 (m, 1H), 6.86-6.88 (d, 1H),

7.54-7.58 (m, 1H), 7.69-7.71 (d, 1H), 7.83-7.86 (dd, 1H),

8.13-8.14 (d, 1H), 8.16 (s, 1H), 8.82 (s, 1H), 8.89-8.90 (d,

1H)


125

MS : (m/z) 385 (M++1)

N-[(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-(4-methoxyphenyl)-

piperazine]-1-carboxamide (II-9)

Compound (6c) (0.3 g, 0.135 mmole) was reacted with 4-(4-

methoxyphenyl)piperazine (0.26 g, 0.135 mmole) as described above for

compound (I-1). Chromatographic purification of the crude product was done

using methanol (2%) in ethyl acetate as eluent yielding the titled compound (II-9)

(0.25 g, 44.72%), m. p. 163-64 °C.

Anal.:


IR : 3393, 1656, 1626, 1525, 1483, 1347, 1236 and 1065 cm-1

1H-NMR : 2.97-2.99 (b, 4H), 3.60-3.61(b, 4H), 3.80 (s, 3H), 6.87-7.00

(m, 4H), 7.69-7.71 (d, 1H), 7.83-7.86 (m, 1H), 8.13 (s, 1H),

8.89 (s, 1H), 8.90 (s, 1H)

MS : (m/z) 414 (M++1)

N-[(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-(4-tert-butyloxycarbony-

l)-piperazine]-1-carboxamide (II-10)

Isocyanate derivative (6c) (0.25 g, 11 mmole) in toluene (25 ml) was

refluxed with boc-piperazine (0.209 g, 0.11 mmole) to get a solid precipitate. The

solid so obtained was filtered and washed with hexane. Chromatographic

purification of the crude product using hexane (10%) in ethyl acetate provided the

desired compound (II-10) (0.2 g, 43.4%), m. p. 229-30 °C.

Anal.:


IR : 3393, 1691, 1650, 1629, 1490, 1422, 1233 and 1173 cm-1

1HNMR : 1.42 (s, 9H), 3.39 (b, 4H), 3.44-3.45 (b, 4H), 7.68-7.71 (d,

1H), 7.83-7.85 (d, 1H), 8.13 (s, 1H), 8.78 (s, 1H), 8.88-8.89

(d, 1H)

MS : (m/z) 408.41, 410.39 (M+-1)


126

N-[(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-(furan-2-carbonyl)-

piperazine]-1-carboxamide (II-11)

Isocyanate derivative (6c) (0.25 g, 0.11 mmole) was reacted with 1-

(furan-2-carbonyl)piperazine (0.203 g, 0.11 mmole) as described above for

compound (I-1). Chromatographic purification of the crude product using 1%

methanol in ethyl acetate offered the desired compound (II-11) (0.19 g, 41.9%),

m. p. 227-28 °C.

Anal.:


IR : 3446, 3086, 1680, 1626, 1427, 1290, 1242 and 1199 cm-1

1HNMR : 3.57-3.58 (b, 4H), 3.74 (b, 4H), 6.65 (s,

1H), 7.04-7.05 (d,

1H), 7.69-7.71 (d, 1H), 7.83-7.86 (m, 1H), 7.87 (s, 1H), 8.18

(s, 1H), 8.80 (s, 1H), 8.89 (b, 1H)

MS : (m/z) 402 (M+-1)


carbamate derivatives (Series III)

Synthesis of 4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carbamate

derivatives

Benzyl (4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)carbamate (III-1)

Compound (6a) (0.3 g, 16 mmole) and benzyl alcohol (0.173 g, 16

mmole) in toluene (25ml) were refluxed for 6 h to get a solid precipitate. The solid

precipitate was filtered and washed with hexane and the crude product so obtained

was purified by column chromatography using hexane (10%) in ethyl acetate as

eluent to offer the titled compound (III-1) (0.5 g, 61.1%), m. p. 158-60 °C (Lit.221

153-55 °C).

Anal.:


IR : 3239, 1718, 1650, 1461, 1274, 1228, 1180 and 1116 cm-1

1H-NMR : 5.17 (s, 2H), 7.32-7.44 (m, 6H), 7.69-7.71 (d, 1H), 7.84-7.89

(m, 1H), 8.70 (b, 1H), 8.92-8.94 (d, 1H), 9.05 (b, 1H)

MS : (m/z) 296 (M++1)


127

Pyridin-2-ylmethyl (4-oxo-4H-[1,2-a]pyrimidin-3-yl)carbamate (III-2)

Compound (III-2) was prepared by reacting (6a) (0.5 g, 0.26 mmole)

with pyridine-2-methanol (0.175 g, 0.16 mmole) as described above for

compound (III-1). The crude product was subjected to chromatographic

purification using hexane (10%) in ethyl acetate as eluent affording the titled

compound (III-2) (0.24 g, 50.5%), m. p. 178-80 °C.

Anal.:


IR : 3245, 3070, 1713, 1666, 1549, 1477, 1234 and 1125 cm-1

1H-NMR : 5.22 (s, 2H), 7.33-7.38 (m, 2H), 7.51-7.53 (d, 1H), 7.69-7.72

(d, 1H), 7.83-7.89 (m, 2H), 8.55-8.57 (d, 1H), 8.72 (s, 1H),

8.94-8.95 (d, 1H), 9.21 (b, 1H)

MS : (m/z) 297 (M++1)

3-Fluorobenzyl (4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)carbamate (III-3)

A mixture of isocyanate derivative (6a) (0.25 g, 0.13 mmole) and 3-

fluorobenzyl alcohol (0.169 g, 0.13 mmole) were reacted as described above for

compound (III-1). The crude product was purified by column chromatography

using hexane (20%) in ethyl acetate to attain the titled compound (III-3) (0.11 g,

26.2%), m. p. 158-60 °C.

Anal.:


IR : 3416, 3264, 1716, 1659, 1480, 1441, 1403, 1234, 1200, 1184,

1134, 796 and 773 cm-1

1HNMR : 5.19 (s, 2H), 7.14-7.19 (m, 1H), 7.27-7.37 (m, 3H), 7.41-7.47

(m, 1H), 7.69-7.71 (d, 1H), 7.85-7.89 (m, 1H), 8.72 (s, 1H),

8.93-8.95 (d, 1H), 9.16 (b, 1H)

MS : (m/z) 314, (M+-1)

3,4-Methylenedioxybenzyl(4-oxo-4H-pyrido[1,2-a]pyrimdin-3-yl)carbamate

(III-4)

6a (0.5 g, 0.26 mmole) was reacted with 3,4-methylenedioxybenzyl

alcohol (0.244 g, 0.106 mmole) as described above for compound (III-1). The



128

acetate as eluent offered the titled compound (III-4) (0.18 g, 33%), m. p. 175-76

°C.

Anal.:


IR : 3242, 3031, 1717, 1666, 1485, 1443, 1235 and 1137 cm-1

1H-NMR : 5.06 (s, 2H), 6.02 (s, 2H), 6.92 (s, 2H), 7.02 (s, 1H), 7.33-7.37

(m, 1H), 7.69-7.71 (d, 1H), 7.84-7.89 (m, 1H), 8.70 (b, 1H),

8.92-8.94 (d, 1H), 9.01 (b, 1H)

MS : (m/z) 340 (M++1)

Synthesis of 8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carbam-

ate derivatives

Benzyl (8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)carbamate (III-5)

Compound (III-5) was prepared by treating the isocyanate derivative

(6b) (0.5 g, 0.26 mmole) with benzyl alcohol (0.161 g, 0.149 mmole) as described

above for compound (III-1). Chromatographic purification of the crude product

using hexane (10%) in ethyl acetate as eluent yielded compound (III-5) (0.18 g,

39%), m. p. 168-70 °C (Lit.221

177-79 °C).

Anal.:


IR : 3242, 1717, 1635, 1546, 1465, 1224 and 1056 cm-1

1H-NMR : 2.45 (s, 3H), 5.15 (s, 2H), 7.21-7.23 (d, 1H), 7.31-7.44 (m,

5H), 7.52 (s, 1H), 8.60 (b, 1H), 8.83-8.85 (d, 1H), 8.97 (b, 1H)

MS : (m/z) 310 (M++1)

Pyridin-2-ylmethyl (8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)

carbamate (III-6)

Compound (6b) (0.5 g, 0.26 mmole) was reacted with pyridine-2-

methanol (0.163 g, 0.149 mmole) as described above for compound (III-1).


acetate as eluent earned the titled compound (III-6) (0.19 g, 41%), m. p. 180-82

°C.

Anal.:



129

IR : 3429, 3247, 1721, 1667, 1644, 1480, 1244 and 1117 cm-1

1H-NMR : 2.45 (s, 3H), 5.20 (s, 2H), 7.22-7.24 (d, 1H), 7.33-7.36 (m,

1H), 7.49-7.51 (d, 1H), 7.53 (s, 1H), 7.83-7.87 (m, 1H), 8.55-

8.56 (d, 1H), 8.62 (b, 1H), 8.85-8.87 (d, 1H), 9.14 (b, 1H)

MS : (m/z) 311 (M++1)

Synthesis of 7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carba-

mate derivatives

2-Pyridinylmethyl (7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)carba-

mate (III-7)

6c (0.25 g, 0.11 mmole) was reacted with pyridine-2-methanol (0.176 g,

0.135 mmole) as described above for compound (III-1). The crude product was

purified by column chromatography using 2% methanol in ethyl acetate to

accomplish the titled compound (III-7) (0.05 g, 13.4%), m. p. 196-97 °C.

Anal.:


IR : 3451, 3245, 1724, 1652, 1477, 1354, 1236 and 694 cm-1

1HNMR : 5.23 (s, 2H), 7.33-7.36 (d, 1H), 7.51-7.53 (d, 1H), 7.71-7.73

(d,

1H), 7.83-7.87 (t, 1H), 7.88-7.91 (dd, 1H), 8.55-8.56 (d,

1H), 8.76 (s, 1H), 8.92-8.93 (d, 1H), 9.32 (s, 1H)

MS : (m/z) 331.37, 333.35 (M+-1)

3-Pyridinylmethyl (7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)carba-

mate (III-8)

6c (0.25 g, 0.11 mmole) and pyridine-3-methanol (0.123 g, 0.11 mmole)

were reacted as described above for compound (III-1) to get solid precipitate. The

solid so obtained was filtered and washed with hexane. Chromatographic

purification of the crude product using hexane (5%) in ethyl acetate provided the

intended compound (III-8) (0.15 g, 40.2%), m. p. 293-95 °C.

Anal.:

TLC : Rf 0.65 (ETOAc)

IR : 3415, 3251, 1722, 1664, 1630, 1402 and 1232 cm-1

1HNMR : 5.22 (s, 2H), 7.41-7.45 (m, 1H), 7.70-7.73 (d, 1H), 7.86-7.91

(m, 2H), 8.54-8.55 (d, 1H), 8.66 (b, 1H), 8.74 (b, 1H), 8.91-


130

8.92 (d, 1H) 9.16 (s, 1H)

MS : (m/z) 331.37, 333.35, (M+-1)

3-Fluorobenzyl (7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)carbamate

(III-9)

Compound (III-9) was obtained by reaction of compound (6c) (0.25 g,

0.11 mmole) with 3-fluorobenzyl alcohol (0.142 g, 0.11 mmole) to get solid

precipitate. The solid so obtained was filtered and washed with hexane and

purified by column chromatography using hexane (10%) in ethyl acetate to give

the titled compound (III-9) (0.06 g, 15.3%), m. p. 240-42 °C.

Anal.:


IR : 3431, 3276, 1726, 1660, 1546, 1229 and 1127 cm-1

1HNMR : 5.15 (s, 2H), 7.20-7.24 (m, 2H), 7.48-7.51 (m, 2H), 7.70-7.72

(d, 1H), 7.88-7.90 (d, 1H), 8.74 (s, 1H), 8.91 (s, 1H), 9.18 (s,

1H)

MS : (m/z) 350 (M+-1), 348 (M

+-3)


carboxamide derivatives (Series IV)

Synthesis of 4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-carboxamide

derivatives

N-(4-Methoxybenzyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxamide

(IV-1)

In a two neck round bottom flask (100 ml) equipped with calcium

chloride guard tube, the acid (4a) (0.5 g, 0.26 mmole) was dissolved in

dichloromethane (50 ml) and the solution was cooled to 0 °C. 1-

Hydroxybenzotriazole hydrate (0.355 g, 0.26 mmole), 1-(3-

dimethylaminopropyl)-3-ethylcarbodiimide (0.603 g, 0.31 mmole) and N,N-

diisopropylethylamine (0.679 g, 0.52 mmole) were added to the above solution.

After 20 min 4-methoxybenzylamine (0.397 g, 0.26 mmole) was added and the

reaction mixture was stirred for 1 h at 0 °C and for another 8 h at room


131

temperature. The reaction mass was quenched by adding cold water (20 ml) and

the medium was neutralized using dilute hydrochloric acid, extracted using ethyl

acetate (3x100 ml), dried over anhydrous sodium sulfate and concentrated to get a

dark brown product. The crude product was purified by column chromatography

using methanol (2%) in ethyl acetate to offer the compound (IV-1) (0.24 g,

28.2%), m. p. 192-94 °C.

Anal.:


IR : 3336, 1679, 1489, 1334, 1297, 1243 and 1030 cm-1

1H-NMR : 3.80 (s, 3H), 4.62-4.64 (d, 2H), 6.86-6.90 (m, 2H), 7.31-7.33

(d, 2H), 7.34-7.38 (m, 1H), 7.84-7.86 (d, 1H), 7.92-7.97 (m,

1H), 9.19-9.21 (d, 1H), 9.30 (b, 1H), 9.38 (s, 1H)

MS : (m/z) 310 (M++1)

N-(3-Fluorobenzyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxamide (IV-2)

Compound (IV-1) was prepared by reacting compound (4a) (0.5 g, 0.26

mmole) with 3-fluorobenzylamine (0.329 g, 0.26 mmole) as described above for

compound (IV-1). The crude product was purified by column chromatography

using hexane (5%) in ethyl acetate as eluent offering the compound (IV-2) (0.32

g, 40.9%), m. p. 168-70 °C.

Anal.:


IR : 3327, 3122, 1662, 1623, 1569, 1533, 1500 and 1240 cm-1

1H-NMR : 4.59-4.61 (d, 2H), 7.06-7.11 (m, 1H), 7.15-7.21 (m, 2H),

7.36-7.41 (m, 1H), 7.59-7.63 (m, 1H), 7.90-7.92 (d, 1H), 8.19-

8.23 (m, 1H), 9.05 (s, 1H), 9.19-9.21 (d, 1H), 9.43-9.46 (t,

1H)

MS : (m/z) 298 (M++1)

4-Oxo-N-[2-(trifluoromethyl)benzyl]-4H-pyrido[1,2-a]pyrimidin-3-carbox-

amide (IV-3)

Compound (IV-3) was prepared by treating compound (4a) (0.5 g, 0.26

mmole) with 2-trifluoromethylbenzylamine (0.461 g, 0.26 mmole) as described

above for compound (IV-1). Chromatographic purification of the crude product


132

using hexane (5%) in ethyl acetate as eluent yielded compound (IV-3) (0.32 g,

35%), m. p. 158-60 °C.

Anal.:


IR : 3299, 3079, 1695, 1630, 1476, 1315, 1167 and 1094 cm-1

1H-NMR : 4.76-4.78 (d, 2H), 7.48-7.52 (m, 1H), 7.59-7.69 (m, 3H),

7.75-7.77 (d, 1H), 7.91-7.93 (d, 1H), 8.20-8.24 (m, 1H), 9.05

(s, 1H), 9.21-9.23 (d, 1H), 9.48-9.51 (t, 1H)

MS : (m/z) 348 (M++1)


amide (IV-4)

Compound (4a) (0.5 g, 0.26 mmole) was reacted with 3-


compound (IV-1). The chromatographic purification of the crude product using

hexane (5%) in ethyl acetate as eluent furnished the compound (IV-4) (0.33 g,

36.1%), m. p. 139-40 °C.

Anal.:


IR : 3328, 3113, 1691, 1645, 1532, 1490, 1415 and 1335 cm-1

1H-NMR : 4.66-4.67 (d, 2H), 7.56-7.63 (m, 3H), 7.66-7.68 (d, 1H), 7.72

(s, 1H), 7.90-7.92 (d, 1H), 8.18-8.23 (m, 1H), 9.05 (s, 1H),

9.20-9.21 (d, 1H), 9.50-9.53 (t, 1H)

MS : (m/z) 348 (M++1)


amide (IV-5)

Compound (IV-5) was prepared by reacting (4a) (0.5 g, 0.26 mmole)

with 4-trifluoromethylbenzylamine (0.461 g, 0.26 mmole) as described above for


hexane (5%) in ethyl acetate as eluent yielded compound (IV-5) (0.25 g, 26.2%),

m. p. 167-69 °C.

Anal.:



133

IR : 3336, 3115, 1663, 1600, 1570, 1500, 1325 and 1123 cm-1

1H-NMR : 4.75-4.76 (d, 2H), 7.38-7.41 (m, 1H), 7.49-7.51 (d, 2H), 7.58-

7.60 (d, 2H), 7.86-7.88 (d, 1H), 7.96-8.00 (m, 1H), 9.21-9.23

(d, 1H), 9.37-9.38 (d, 1H), 9.47 (b, 1H)

MS : (m/z) 348 (M++1)

N-(2,4-Difluorobenzyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-carboxamide

(IV-6)


with 2,4-difluoromethylbenzylamine (0.376 g, 0.26 mmole) as described above

for compound (IV-1). The chromatographic purification of the crude product

using hexane (20%) in ethyl acetate as eluent afforded the desired product (IV-6)

(0.21 g, 25.3%), m. p. 172-75 °C.

Anal.:


IR : 3328, 3125, 1664, 1500, 1430, 1184 and 1072 cm-1

1H-NMR : 4.60-4.62 (d, 2H), 7.14-7.21 (m, 2H), 7.24-7.29 (m, 1H),

7.59-7.63 (m, 1H), 7.89-7.92 (d, 1H), 8.19-8.23 (m, 1H), 9.03

(s, 1H), 9.20-9.23 (d, 1H), 9.42-9.45 (t, 1H)

MS : (m/z) 316 (M++1)

N-[2-(Morpholin-4-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-carboxamide

(IV-7)

Reaction of compound (4a) (0.5 g, 0.26 mmole) with 2-(4-

morpholinyl)ethylamine (0.342 g, 0.26 mmole) was effected as described above

for compound (IV-1). The chromatographic purification of the crude product

using methanol (0.2%) in ethyl acetate as eluent offered the titled compound (IV-

7) (0.18 g, 22.6%), m. p. 193-95 °C.

Anal.:


IR : 3123, 1690, 1631, 1483, 1400, 1251 and 1115 cm-1

1H-NMR : 2.43 (b, 4H), 2.50-2.51 (m, 2H), 3.45-3.50 (m, 2H), 3.59-3.61

(t, 4H), 7.57-7.61 (m, 1H), 7.84-7.90 (d, 1H), 8.17-8.21 (m,

1H), 9.02 (s, 1H), 9.15-9.16 (t, 1H), 9.20-9.22 (d, 1H)


134

MS : (m/z) 303 (M++1)

4-Oxo-N-[3-(n.pentyloxy)pyridin-2-yl]-4H-pyrido[1,2-a]pyrimidin-3-

carboxami-de (IV-8)


with 2-amino-3-n.pentyloxypyridine (0.474 g, 0.26 mmole) as described above for


methanol (2.5%) in ethyl acetate as eluent produced the titled product (IV-8) (0.3

g, 32.3%), m. p. 159-60 °C.

Anal.:


IR : 3292, 3237, 1693, 1595, 1470, 1333 and 1218 cm-1

1H-NMR : 0.89-0.93 (t, 3H), 1.36-1.43 (m, 2H), 1.49-1.57 (m, 2H), 1.78-

1.84 (m, 2H), 4.09-4.13 (t, 2H), 6.55 (s, 1H), 7.13-7.16 (m,

1H), 7.46-7.48 (d, 1H), 7.66-7.70 (m, 1H), 7.95-7.97 (d, 1H),

8.24-8.28 (m, 1H), 9.13 (s, 1 H), 9.21-9.24 (d, 1H), 11.51 (s,

1H)

MS : (m/z) 353 (M++1)

N-[3-(Benzyloxy)pyridin-2-yl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-carboxam-

ide (IV-9)

Compound (4a) (0.5 g, 0.26 mmole) and 2-amino-3-benzyloxypyridine

(0.526 g, 0.26 mmole) were reacted together as described above for compound

(IV-1). The chromatographic purification of the crude product using methanol

(1%) in ethyl acetate as eluent offered compound (IV-9) (0.23 g, 23.4%), m. p.

193-95 °C.

Anal.:


IR : 3480, 3106, 1699, 1598, 1482, 1285 and 1012 cm-1

1H-NMR : 5.28 (s, 2H), 7.00-7.04 (dd, 1H), 7.23-7.26 (dd, 1H), 7.34-

7.38 (m, 1H), 7.41-7.44 (m, 2H), 7.53-7.55 (m, 1H), 7.58-

7.60 (d, 2H), 7.88-7.91 (d, 1H), 7.98-8.02 (m, 1H), 8.10-8.14

(m, 1H), 8.16-8.18 (dd, 1H), 9.27-9.31 (m, 1H), 9.49 (s, 1H),

11.76 (s, 1H)


135

MS : (m/z) 373 (M++1)

N-[(2,5-Diethoxy)-4-(morpholin-4-yl)phenyl]-4-oxo-4H-pyrido[1,2-a]pyrimid-

ine-3-carboxamide (IV-10)

Compound (IV-10) was prepared by treating compound (4a) (0.5 g,

0.26 mmole) with 2,5-diethoxy-4-morpholin-4-yl-phenylamine (0.7 g, 0.26

mmole) as described above for compound (IV-1). The chromatographic

purification of the crude product using methanol (1%) in ethyl acetate as eluent

provided compound (IV-10) (0.35 g, 30.3%), m. p. 166-67 °C.

Anal.:


IR : 3263, 1685, 1487, 1260, 1199, 1113, 1043 cm-1

1H-NMR : 1.44-1.47 (t, 3H), 1.54-1.58 (t, 3H), 3.09-3.10 (t, 4H), 3.89-

3.91 (t, 4H), 4.12-4.17 (m, 4H), 6.60 (s, 1H), 7.37-7.41 (m,

1H), 7.85-7.87 (d, 1H), 7.95-7.99 (m, 1H), 8.40 (s, 1H), 9.33-

9.34 (d, 1H), 9.42 (s, 1H), 11.49 (s, 1H)

MS : (m/z) 439 (M++1)

N-(3,5-Dimethyl-1,2-oxazol-4-yl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-carbox-

amide (IV-11)


with 3,5-dimethyl-1,2-oxazol-4yl-amine (0.295 g, 0.26 mmole) as described

above for compound (IV-1). The chromatographic purification of the crude

product using hexane (10%) in ethyl acetate as eluent offered compound (IV-11)

(0.34 g, 45.4%), m. p. 243-45 °C.

Anal.:


IR : 3253, 3209, 1694, 1486, 1332, 1235 and 1067 cm-1

1H-NMR : 2.15 (s, 3H), 2.33 (s, 3H), 7.65-7.69 (m, 1H), 7.95-7.97 (d,

1H),

8.24-8.29 (m, 1H), 9.09 (s, 1H), 9.26-9.27 (d, 1H), 10.20

(s, 1H)

MS : (m/z) 285 (M++1)


136

N-[1-(4-Methoxyphenyl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-carboxa-

mide (IV-12)

Compound (4a) (0.5 g, 0.26 mmole) was reacted with -methyl-4-

methoxybenzylamine (0.397 g, 0.26 mmole) as described above for compound

(IV-1) Chromatographic purification of the crude product using methanol (0.2%)

in ethyl acetate as eluent afforded compound (IV-12) (0.24 g, 28.2%), m. p. 173-

75 °C.

Anal.:


IR : 3315, 3128, 1667, 1630, 1506, 1240 and 1025 cm-1

1H-NMR : 1.61-1.66 (d, 3H), 3.79 (s, 3H), 5.28-5.35 (m, 1H), 6.87-6.91

(d, 2H), 7.34-7.38 (m, 3H), 7.83-7.86 (d, 1H), 7.92-7.97 (m,

1H), 9.20-9.22 (d, 1H), 9.33-9.36 (m, 2H)

MS : (m/z) 324 (M++1)

N-(1-Benzylpiperidin-4-yl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-carboxamide

(IV-13)

Compound (IV-13) was obtained by reaction of compound (4a) (0.5 g,

0.26 mmole) with 4-amino-1-benzylpiperidine (0.5 g, 0.26 mmole) as described


product using hexane (10%) in ethyl acetate as eluent earned the titled compound

(IV-13) (0.25 g, 5%), m. p. 180-83 °C.

Anal.:


IR : 3288, 1695, 1544, 1479, 1335 and 1061 cm-1

1H-NMR : 1.52-1.58 (m, 2H), 1.89-1.92 (d, 2H), 2.18-2.22 (t, 2H), 2.70

(b, 2H), 3.50 (s, 2H), 3.88 (b, 1H), 7.26 (b, 1H), 7.33 (b, 4H),

7.60-7.64 (t, 1H), 7.90-7.92 (d, 1H), 8.19-8.23 (t, 1H), 9.04

(b, 2H), 9.20-9.22 (d, 1H)

MS : (m/z) 363 (M++1)


137

N-(3,4-Methylenedioxybenzyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbox-

amide (IV-14)

The reaction of compound (4a) (0.5 g, 0.26 mmole) with 3,4-

methylenedioxybenzyl amine (0.397 g, 0.26 mmole) was performed as described


product using hexane (20%) in ethyl acetate as eluent produced compound (IV-

14) (0.3 g, 35.2%), m. p. 193-95 °C.

Anal.:

TLC : Rf 0.6 (EtOAc)66

IR : 3260, 3071, 1665, 1627, 1490, 1434, 1246 and 1224 cm-1

1H-NMR : 4.46-4.48 (d, 2H), 5.98 (s, 2H), 6.82-6.88 (m, 2H), 6.93 (s,

1H), 7.58-7.62 (m, 1H), 7.89-7.91 (d, 1H), 8.17-8.22 (m, 1H),

9.05 (s, 1H), 9.17-9.19 (d, 1H), 9.27-9.33 (t, 1H)

MS : (m/z) 324 (M++1)

N-Cyclopropyl-N-(2-fluorobenzyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-

carboxamide (IV-15)

The reaction of (4a) (0.5 g, 0.26 mmole) with N-cyclopropyl-2-fluoro

benzylamine (0.434 g, 0.26 mmole) was performed as described above for


hexane (5%) in ethyl acetate as eluent yielded the desired compound (IV-15)

(0.24 g, 27%), m. p. 137-40 °C.

Anal.:


IR : 3110, 1669, 1492, 1404, 1090 and 1031cm-1

1H-NMR : 0.58-0.61 (d, 4H), 3.06 (b, 1H), 4.93 (s, 2H), 7.11-7.13 (m,

1H), 7.23-7.25 (m, 1H), 7.32-7.34 (m, 2H), 7.67 (b, 1H), 7.80-

7.82 (d, 1H), 7.90-7.94 (m, 1H), 8.60 (s, 1H), 9.21-9.23 (d,

1H)

MS : (m/z) 338 (M++1)

Synthesis of 8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbox-

amide derivatives


138

N-(4-Fluorobenzyl)-8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-carbox-

amide (IV-16)

Compound (IV-16) was prepared by coupling reaction of (4b) (0.5 g,

0.26 mmole) with 4-fluorobenzylamine (0.306 g, 0.24 mmole) as described above

for compound (IV-1). Chromatographic purification of the crude product using

hexane (10%) in ethyl acetate as eluent furnished the titled product (IV-16) (0.15

g, 51.9%), m. p. 185-87 °C.

Anal.:


IR : 3287, 3014, 1677, 1633, 1481, 1279, 1222 and 1146 cm-1

1H-NMR : 2.58 (s, 3H), 4.65-4.66 (d, 2H), 7.00-7.04 (m, 2H), 7.18-7.21

(m, 1H), 7.34-7.37 (m, 2H), 7.63 (s, 1H), 9.08-9.09 (d, 1H),

9.33 (s, 1H), 9.35 (b, 1H)

MS : (m/z) 312 (M++1)

N-(4-Trifluorobenzyl)-8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-carbox-

amide (IV-17)

Compound (IV-17) was prepared by reacting (4b) (0.4 g, 0.19 mmole)


compound (IV-1). Chromatographic purification of the crude product using

methanol (5%) in methylene chloride as eluent yielded the titled product (IV-17)

(0.15 g, 21.2%), m. p. 175-77 °C.

Anal.:


IR : 3414, 3294, 1695, 1634, 1545, 1330, 1117, 1015 and 793 cm-1

1H-NMR : 2.55 (s, 3H), 4.65-4.67 (d, 2H), 7.46-7.49 (dd, 1H), 7.55-7.57

(d, 2H), 7.69-7.73 (m, 3H), 9.00 (s, 1H), 9.08-9.10 (d, 1H),

9.45-9.48 (t, 1H)

MS : (m/z) 362 (M++1)

Synthesis of 7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbox-

amide derivatives


139

N-(3,4-Methylenedioxybenzyl)-7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-

carboxamide (IV-18)

Compound (4c) (0.5 g, 0.22 mmole) was coupled with 3,4-

methylenedioxybenzylamine (0.336 g, 0.22 mmole) as described above for


hexane (10%) in ethyl acetate as eluent yielded the desired product (IV-18) (0.4 g,

50.2%), m. p. 200-01 °C.

Anal.:


IR : 3500, 3300, 1672, 1490, 1434, 1248 and 1066 cm-1

1H-NMR : 4.46-4.48 (d, 2H), 5.98 (s, 2H), 6.81-6.87 (m, 2H), 6.92 (s,

1H), 7.90-7.93 (d, 1H), 8.24-8.26 (dd, 1H), 9.04 (s, 1H), 9.15-

9.16 (d, 1H), 9.23-9.26 (t, 1H)

MS : (m/z) 358.3 (M++1), 360.4 (M

++2)

7-Chloro-4-oxo-N-[4-(trifluoromethyl)benzyl]-4H-pyrido[1,2-a]pyrimidine-3-

carboxamide (IV-19)

Compound (IV-19) was prepared by reacting the acid (4c) (0.5 g, 0.22

mmole) with 4-trifluoromethylbenzylamine (0.39 g, 0.22 mmole) as described

above for compound (IV-1). Chromatographic purification of the crude product

using hexane (10%) in ethyl acetate as eluent offered compound (IV-19) (0.2 g,

23.5%), m. p. 212-14 °C.

Anal.:


IR : 3315, 3091, 1674, 1620, 1532, 1498, 1323 and 1134 cm-1

1H-NMR : 4.67-4.68 (d, 2H), 7.55-7.57 (d, 2H), 7.70-7.72 (d, 2H), 7.92-

7.95 (d, 1H), 8.26 -8.29 (dd, 1H), 9.04 (s, 1H), 9.17-9.18 (d,

1H), 9.43-9.46 (t, 1H)

MS : (m/z) 382.2 (M++1), 384.2 (M

++2)

7-Chloro-[N-cyclopropyl-N-(2-fluorobenzyl)]-4-oxo-4H-pyrido[1,2-a]pyrimi-

dine-3-carboxamide (IV-20)

Compound (IV-20) was prepared by reacting the acid (4c) (0.5 g, 0.22

mmole) with N-cyclopropyl-2-fluorobenzylamine (0.367 g, 0.22 mmole) as


140

described above for compound (IV-1). The chromatographic purification of the

crude product using hexane (10%) in ethyl acetate as eluent provided compound

(IV-20) (0.28 g, 33.8%), m. p. 209-10 °C.

Anal.:


IR : 3068, 3019, 1688, 1630, 1481, 1344, 1223 and 1030 cm-1

1H-NMR : 0.51-0.55 (d, 4H), 2.87 (b, 1H), 4.75 (b, 2H), 7.21-7.24 (m,

2H), 7.33-7.36 (m, 1H), 7.63 (b, 1H), 7.84-7.87 (d, 1H), 8.14-

8.17 (dd, 1H), 8.47 (s, 1H), 9.14 (s, 1H)

MS : (m/z) 372.4 (M++1), 374.3 (M

++2)

7-(4-Methoxyphenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic

acid intermediates

Ethyl 7-(4-methoxyphenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate

(7)

Ethyl 7-chloro-4-oxo-4H-pyrido[1,2a]pyrimidine-3-carboxylate (4c) (10

gm, 39.6 mmole) was dissolved in dimethoxyethane (300 ml) and 4-

methoxyphenylboronic acid (6 g, 39.6 mmole), potassium carbonate (8.194 gm,

59.4 mmole) and tetrakis(triphenylphosphene)palladium(0) (0.046 gm, 0.039

mmole) were added to the above reaction mixture under N2 atmosphere. The

reaction mixture was refluxed for 36 h at 90 °C. The reaction mass was filtered

and the filtrate was washed with water and extracted using methylene chloride (3

x100 ml), dried over anhydrous sodium sulfate and concentrated to get a dark

brown product. Chromatographic purification of the crude product using ethyl

acetate (15%) in hexane as eluent offered the titled compound (7) (3.5 g, 27.3%),

m. p. 166-67 °C.

Anal.:

TLC : Rf 0.7 (50% EtOAc in hexane)

IR : 3087, 1744, 1609, 1569, 1487, 1296, 1188, 1113, 1045,

826 and 795 cm-1

MS : (m/z) 325, (M+-1)

7-(4-Methoxyphenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (8)


141

A solution of 7 (5 g, 4.3 mmole) in conc. HCl (15 ml) was refluxed for 2

h. The reaction mixture was cooled to RT to get a solid precipitate. The solid so

obtained was filtered, washed with ether (100 ml) and dried to obtain 7-(4-

methoxyphenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (8) (3.1 g,

99.8%), m. p. 211-12 °C.

Anal.:


IR : 3317, 3087, 2474, 1791, 1772, 1606, 1289 and 1187 cm-1

MS : (m/z) 297, (M+-1)

N-(3-Fluorobenzyl)-7-(4-methoxyphenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-

3-carboxamide (IV-21)

Compound (8) (0.25 gm, 0.08 mmole) and 3-fluorobenzylamine (0.105

g, 0.08 mmole) were reacted as described above for compound (IV-1).


acetate as eluent afforded the titled compound (IV-21) (0.16 g, 47.0%), m. p. 171-

73 °C.

Anal.:


IR : 3445, 3319, 3072, 1688, 1611, 1533, 1482, 1336, 1290, 1264,

1184, 1143, 1060, 941, 832, 793, 691, 649 and 556 cm-1

1HNMR : 3.83 (s, 3H), 4.60-4.62 (d, 2H), 7.07-7.16

(m, 3H), 7.20-7.22

(d, 2H), 7.37-7.42 (d, 1H), 7.79-7.81(d, 2H), 7.95-7.98 (d,

1H), 8.54-8.57 (dd, 1H), 9.04 (s, 1H), 9.27-9.28 (d, 1H), 9.45-

9.48 (t, 1H)

MS : (m/z) 404, (M+-1)

Synthesis of 7-(4-methoxyphenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-

3-carboxamide derivatives

N-(3-Trifluoromethylbenzyl)-7-(4-methoxyphenyl)-4-oxo-4H-pyrido[1,2-

a]pyrimidine-3-carboxamide (IV-22)

Compound (8) (0.3 gm, 0.10 mmole) was reacted with 3-




142

hexane (5%) in ethyl acetate as eluent offered the desired compound (IV-22) (0.14

g, 30.49%), m. p. 197-98 °C.

Anal.:


IR : 3414, 3290, 3110, 1692, 1563, 1478, 1333, 1262, 1163, 1112,

1066, 828 and 704 cm-1

1HNMR : 3.84 (s, 3H), 4.68-4.69 (d, 2H), 7.13-7.15 (d, 2H), 7.57-7.59

(d, 2H), 7.71-7.73 (d, 2H), 7.80-7.82 (d, 2H), 7.96- 7.99 (d,

1H), 8.55-8.58 (dd, 1H), 9.04 (s, 1H), 9.28-9.29 (d, 1H), 9.52-

9.55 (t, 1H)

MS : (m/z) 454, (M+-1)

N-(4-Trifluoromethylbenzyl)-7-(4-methoxyphenyl)-4-oxo-4H-pyrido[1,2-


Compound (IV-23) was obtained by reacting (8) (0.3 gm, 0.10 mmole)



hexane (5%) in ethyl acetate as eluent yielded the titled compound (IV-23) (0.16

g, 45.9%), m. p. 209-10 °C.

Anal.:


IR : 3425, 3293, 1689, 1623, 1333, 1263, 792, 583 and 558 cm-1

1HNMR : 3.83 (s, 3H), 4.67-4.68 (d, 2H), 7.11-7.14 (d,

2H), 7.57-7.64

(m, 2H), 7.67-7.69 (m, 1H), 7.72 (b, 1H), 7.78-7.81 (d, 2H),

7.95-7.97 (d, 1H), 8.53-8.56 (dd, 1H), 9.04 (s, 1H), 9.26 (s,

1H), 9.51-9.54 (t, 1H)

MS : (m/z) 454, (M+-1)

N-(Tetrahydrofuran-2-ylmethyl)-7-(4-methoxyphenyl)-4-oxo-4H-pyrido[1,2-


Compound (IV-24) (0.25 gm, 0.08 mmole) was reacted with 2-

tetrahydrofuranyl methylamine (0.085 g, 0.08 mmole) as described above for



143

hexane (20%) in ethyl acetate as eluent provided compound (IV-24) (0.1 g,

31.2%), m. p. 176-77 °C.

Anal.:

TLC : Rf 0.61(1% MeOH in EtOAc)

IR : 3453, 3306, 1689, 1611, 1545, 1337, 1264 and 1180 cm-1

1HNMR : 1.54-1.61 (m, 1H), 1.80-1.89 (m, 2H), 1.90-1.99 (m, 1H),

3.38-3.39

(d, 1H), 3.52-3.57 (m, 1H), 3.66-3.69 (m, 1H),

3.80-3.81 (d, 1H), 3.84 (s, 3H), 3.96-3.99 (m, 1H), 7.12-7.15

(d, 2H), 7.79-7.84 (d, 2H), 7.91-7.97 (d, 1H), 8.54-8.56 (dd,

1H), 9.03 (s, 1H), 9.17-9.19 (t, 1H), 9.29-9.30 (d, 1H)

MS : (m/z) 380, (M+-1)

4.2 Biological work

4.2.1 Falcipain-2 enzyme inhibition assay

The diluted soluble Plasmodium falciparum FP-2 (30 nM) was incubated

for 10 min at room temperature in 100 mM sodium acetate, pH 5.5, 10 mM DTT,

with a fixed/different concentration of the compounds to be tested or the standard

(E64). Compound solutions were prepared from stock in DMSO. After 10 min

incubation, the substrate Z-Phe-Arg-AMC was added to a final concentration of

25 µM. The fluorescence intensity was monitored (excitation 355 nm; emission

460 nm) for 10 min at room temperature with a SynergyTM

4 Multi-Mode

Microplate Reader (BioTek). The inhibition rate (%) is calculated using the given

equation:

% Inhibition = [1 - (F test / F stand.)] × 100

where, F test is the fluorescence intensity of the test compound, F control is the

fluorescence intensity of the standard (E64). All values are the means of three

independent determinations and the deviations are <10 % of the mean value. The

IC50 values were determined for those compounds only which showed 40 % or

more of enzyme inhibition at 10 μM.

5. Summary

Summary

144

The existing armamentarium of antimalarial drugs is not sufficient to combat

malaria, primarily because of resistance developed by the parasite. The problem got

further compounded due to non-enrichment of antimalarial drug inventory.

Unfortunately an effective antimalarial vaccine also could not be developed due to

fast and constant mutations in the parasite genome. All these factors led to a woefully

thin antimalarial drug development pipeline with little chemical diversity.

Genome mapping of the malaria parasite offered a number of attractive drug

targets including plasmepsins and falcipains, the enzymes involved in parasite

metabolism and in providing nutrients to the parasite to meet its energy requirements.

There exist a great potential in the exploration of cysteine proteases falcipain

inhibitors. Falcipains 2 and 3 especially were more attractive targets as these enzymes

degrade haemoglobin to small sized peptides in the acidic environment of the vacuole

and degrade the membrane proteins of the host cells and help the parasite rupture the

host cells in the alkaline environment. So, inhibition of FP-2/3 enzymes could

effectively arrest the growth of the parasite and control their progression in the host

system.

A large number of peptidic and non-peptidic motifs like chalcones, (thio)

semicarbozones, hydrazides, heterocycles, oxiranes and succinates have been reported

in the literature to exhibit FP-2/3 inhibitory activity. Unfortunately none of such

compounds have crossed the clinical phase of evaluation as antimalarial agents. There

are some reports of some heterocyclic and polycyclic compounds exhibiting FP-2/3

inhibiting activities. Taking the basic information from literature about the enzyme

inhibitors into consideration, it was planned to synthesize pyrido[1,2-a]pyrimidin-4-

one class of compounds (M).

N

N

X

O

AX = Urea, carbamate, amide

A = Alkyl/aryl/heteroaryl

R = H, CH3, Cl, 4-C6H4OCH3

R

(M)

It is worth noting that the circled pharmacophore may act as Michael

acceptor for reversible/irreversible binding to the thiol nucleophile present in the FP-

2/3 enzymes in the P1 binding pocket. While the groups, X and R were selected in

such a way that compounds could exhibit better binding profile in the P2 and P3

Summary

145

binding pockets of the falcipain enzyme that could translate in to enhanced potency of

the compounds.

It was planned to synthesize the given four (Series-I to IV) of compounds

and to evaluate them against FP-2 enzyme in the in vitro tests. FP-3 enzyme having a

high degree of homology to FP-2 was presumed to react with all such compounds

which would prove to be powerful inhibitors of FP-2.

N

N

N

O

O

NRR N N

N

N

N

O

O

N

N

O

N

O

AR

N

N

N

O

O

OR

I II

AA

R1

IV

R1

III

A

Where: R = H, 8-CH3, 7-Cl, 4-C6H4OCH3

R1 = H, Alkyl

A = Alkyl/heteroaryl or alkyl

All of the derivatives of the given four (Series-I to IV) were synthesized

by following either of the Schemes 1-6 and the compounds were characterized on

the basis of their spectral data. Synthetic methods for preparing these new

chemical entities, spectral (IR, PMR and Mass spectrometry) data and biological

activities of these compounds have been discussed in detail in this thesis work.

The acids (4a-c) and the isocyanates (6a-c) are the key intermediates in

the preparation of the targeted compounds. Their synthesis was accomplished by

following the key steps i) Gould and Jacob reaction ii) cyclization, iii) hydrolysis

and iv) Curtius rearrangement reaction. Synthesis of theses intermediates is

accomplished by following the reaction sequence as outlined in the Scheme 1.

2-Aminopyridines (1a-c) by Gould and Jacob coupling reaction with

diethyl ethoxymethylenemalonate yielded (2a-c), which on cyclization by

Summary

146

refluxing in diphenyl ether resulted into pyrido[1,2-a]pyrimidin-4-one monoesters

(3a-c). The esters (3a-c) on acid catalyzed hydrolysis offered free acids (4a-c).

The acids (4a-c) when reacted with ethyl chloroformate in presence of

triethylamine yielded anhydrides which on further in situ reaction with sodium

azide yielded the acid azides (5a-c). The azides (5a-c) offered isocyanate

derivatives (6a-c) (Scheme 1) by refluxing them in toluene.

N

NH2

N

N

O

O

CH3

O

N

N

O

OH

O

N

N

N

O

ON

N

O O

N

N+

N

R

R R

R

R

N

NH

O

O

O

CH3

O

CH3

R

(1a-c)(2a-c)

(3a-c) (4a-c)

(5a-c)(6a-c)

a. R = H, b. R = 8-CH3, c. R = 7-Cl

i. ECF, TEA, DMF, 0OC

ii. NaN3, H2O,

EMME, Reflux

DPE,Reflux

HCl, Reflux

Toluene, Reflux

a. R = H, b. R = 4-CH3, c. R = 5-Cl

Scheme 1

The ureas Series-I (I-1 to I-34) were prepared by coupling of suitable

isocyanate (6a-c) with amine derivatives of type R1NHA in toluene under reflux

conditions following the procedure as depicted in Scheme 2 or Scheme 3.

While piperazine-urea analogs of the Series-II (II-1 to II-11) were

synthesized by coupling of isocyanate (6a-c) with suitably substituted piperazine

derivatives under reflux conditions in toluene as depicted in Scheme 2.

Summary

147

N

N

NH

O

O

NR R N NN

N

NH

O

O

NH N A,

N

N

O

N

CF3

CF3

NH

CH3

O

N N

O

CF3

FF

NO

CH3

CH3

N

N

ClN

ONN

O

CH3

CH3

O

OCH

3

N

N

O

O

O

CH3

NH2

O

O

OSS

O

OCH

3

CH3

CH3

6 (a-c)

Toluene, reflux

A A

R1NHA, X=

R1

Toluene, reflux

(I-1 to I-34)

N=C=O

OCH3

A =

R1 = H, i.Pr, Cycloproyl, Benzyl

OCH3

OCH3

a. R = H, b. R = 8-CH3, c. R = 7-Cl

(II-1 to II-11)

Scheme 2

To study the binding pattern in the P1 and P2 pocket, some additional

compounds bearing esters (I-14), (I-19), amides (I-15), (I-17) and nitriles (I-18),

(I-19) were also synthesized. The esters, amide and nitriles have been reported to

show good binding affinity for FPs. Hence compounds (I-14 to I-19) were

synthesized by following the reaction sequence as outlined in the Scheme 3. The

ester derivatives (I-14 to I-16) obtained by coupling of the isocyanate derivative

(6a) with esters of L-phenylalanine or L-leucine affording ester derivatives (I-14,

I-16). The esters on treatment with ammonia solution yielded amides (I-15, I-17).

The amides on dehydration reaction using cyanuric chloride in

dimethylformamamide offered the nitriles (I-18, I-19).

Summary

148

NH2

R'

O

O CH3

n

NH

R'

O

O

N

N

NH

O

O

CH3

n

NH

R'

O

NH2

N

N

NH

O

O

CH3

CH3

NH

R'

N

N

NH

O

O

N

N

N

N

Cl

Cl

Cl

liq. ammonia

(6a,b) +

Toluene Reflux

(I-14, I-16)

R' =

n = 0, 1

DMF, 0OC

(I-15, I-17) (I-18, I-19)

Scheme 3

Synthesis of the carbamate derivatives of Series-III (III-1 to III-9) have

been accomplished by reaction of the isocyanate (6a-c) and suitable alcohol

derivative (A-HO) in toluene under reflux conditions following the procedure as

outlined in Scheme 4.

N

N

O

R N

N

NH

O

O

OR

N F

O

O

N

6 (a-c)

A HO-A

Toluene, reflux

N=C=O

(III-1) - (III-9)

a. R = H, b. R = 8-CH3, c. R = 7-Cl

A =

Scheme 4

Synthesis of the amide derivatives of Series IV (IV-1 to IV-26) have

been accomplished by reacting the acid (4a-c) with suitable amine (R’-NHA)

using EDC as a coupling agent as outlined in Scheme 5.

Summary

149

N

N

O

OH

O

N

N

O

N

O

ARR

CF3

CF3

N

O

CF3

FF

N

ON

NO

CH3

N O

O

CH3

O

CH3

N

O

O

CH3

N

O

CH3

F

FOCH

3

N

O

4 (a-c) (IV-1) - (IV-20)

EDC, HOBT, DIEA, 0OC/ RT

R'-NHA

R'

a. R = H, b. R = 8-CH3, c. R = 7-Cl

OCH3

A =

OCH3

R' = H, i.Pr, Cyclopropyl, Benzyl

Scheme 5

In order to study the hydrophobic binding interactions by increasing the

steric bulk of the group attached to the B-ring (i.e. pyrido) of the pyridopyrimidine

ring, some additional compounds (IV23 - IV24) were synthesized. The 7-chloro

ester (3c) was reacted with p-methoxyphenylboronic acid through Suzuki

coupling offering the ester (7) which on acid hydrolysis yielded the acid (8). The

acid derivative (8) was coupled with suitable amines to get the targeted

compounds (IV23 - IV24) as depicted in Scheme 6.

Summary

150

N

N

O

O

O

ClCH

3 N

N

O

O

O

CH3

N

N

O

OH

O

N

N

O

NH

O

A

B(OH)2

CF3

CF3

F O

(3c) (7)

DME, P(Ph3)4, K2CO3,

Reflux

HCl, Reflux

(8) (IV-21) - (IV-24)

EDC, HOBT, DIEA,

A-NH2, 0OC/ RT

H3COH3CO

H3CO

A =

H3CO

Scheme 6

The synthesised compounds were submitted for in vitro FP-2 inhibition

evaluation. Compounds were evaluated for their inhibitory activity against

recombinant FP-2 using Cbz-Phe-Arg-AMC as a fluorogenic substrate.

Preliminary screening was performed at 10 M concentration. An equivalent

concentration of DMSO was used as negative control and the irreversible standard

inhibitor of clan CA family C1 cysteine proteases (papain family), namely E-64

was used as positive control. Assays were performed to determine the percentage

inhibition of the enzyme at a concentration of 10 M. FP-2 activity is assessed by

cleavage of the fluorogenic substrate Cbz-Phe-Arg-AMC releasing the fluorescent

AMC group. Hence decrease in fluorescence intensity in a sample represents

inhibition of enzyme activity. The IC50 values were determined for those

compounds only which showed more than 40 % inhibition at 10 M

concentration. The FP-2 enzyme inhibition data of the synthesised compounds

shows that the pyrido[1,2-a]pyrimidin-4-ones show FP-2 inhibition in micromolar

range. Based on the result of evaluated compounds a broad structure activity

relationship could be deduced for this series of the compounds. Urea moiety has

the potential to provide potent FP-2 inhibitors as the carbamate and carboxamide

derivatives are much less active in comparison to the substituted ureas.

Ethylmorpholine (I-26, IC50 6.93 M) and methoxyethyl (I-21, IC50 6.36

M) urea derivatives proved to be the most potent compounds. 2-Thiophenethyl

(I-8, IC50 14.27 M; IV-23, IC50 21.16 M; I-27, IC50 23.35 M), acetamidoethyl

Summary

151

(I-4, IC50 16.08 M) and 4-methoxyphenylpiperazine (II-5, IC50 18.87 M) were

the other groups which gave active compounds when attached to the other end of

the urea moiety. 2-Pyridylpiperazine is another moiety which offered quite active

compounds (II-8, IC50 12.16 M; II-1, IC50 14.84 M).

In general 8-methyl and 7-chloro substituted derivatives offered more

potent compounds indicating that increasing the steric bulk of the group attached

to the B-ring (i.e. pyrido) is likely to provide more potent FP-2 inhibitors. An

electron rich environment at the end of the carbon chain attached to the urea

nitrogen also seems to be conducive for better activity as indicated by the

compounds having morpholine, 2-thiophenenyl, 2-pyridyl, 4-methoxyphenyl,

acetamidoethyl and 2-methoxyethyl groupings. But bulky groupings (II-7,

benzhydrylpiperazine and I-9, 4-morpholinopropyl) may not offer potent

derivatives.

With the availability of biological activity for compounds the current

work has proved the potential of pyrido[1,2-a]pyrimidin-4-one derivatives as

potent FP-2 inhibitors. Based on the activity data of the synthesized compounds it

could be claimed that pyrido[1,2-a]pyrimidin-4-one can serve as a lead series for

future investigations. By appropriate structural optimizations the potency of this

series of compounds can be improved substantially against FP-2 enzyme which in

turn can serve to be potential antimalarial agents. Further optimization of the lead

structures and biological screening is in progress in the laboratory.

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