ABSTRACT

The work carried out during my research tenure has been compiled in the form of a thesis entitled ‘’Synthesis and biological activity of novel pyrrolobenzodiazepine hybrids and combinatorial synthesis of biologically active nitrogen heterocycles.’’ The main aim of this work is to design and synthesis of biologically active molecules like pyrrolobenozodiazepines, which are known to be

DNA-binding and potentially anticancer molecules. Nitrogen containing biologically active heterocyclics has been synthesiszed by solid-phase and polymer- assisted solution-phase combinatorial methods. The thesis has been divided into three chapters.

CHAPTER I: This chapter has been divided into three sections. Section-A gives the general introduction about cancer chemotheraphy, covalent interactions of drug-

DNA, particularly of pyrrolo[2,1-c][1,4]benzodiazepine (PBD) antitumour antibiotics and objectives of the present work. Section-B consists of the synthesis and DNA- binding affinity of novel fluoroaryl pyrimidine linked pyrrolo[2,1-c]

[1,4]benzodiazepine hybrids and their antitumour activity against sixty human tumour cell lines. Section-C consists of the design, synthesis and DNA-binding affinity of novel fluoroquinolone linked pyrrolo[2,1-c][1,4]benzodiazepine hybrids.

CHAPTER II: II: This chapter has been divided into two sections. Section-A gives the general introduction about drug discovery, solid-phase combinatorial chemistry, introduction of imidazo[1,2-a]pyridines. Section-B consists of the generation of imidazo[1,2-a]pyridine 225 compounds library on solid-phase.

CHAPTER III: This chapter has been divided into two sections. Section-A comprises general introduction of polymer-assisted solution phase combinatorial chemistry and utilized this strategy for the synthesis of pyrrolo[2,1-c][1,4]benzodiazepine anticancer agents, including naturally occurring DC-81 antibiotic. Section-B

I ABSTRACT

comprises general introduction pyrrolo[2,1-b]quinazoline alkaloids and synthesis of these alkaloids by polymer-assisted solution phase strategy. Chapter-I (Sec-A)

GENERAL IINTRODUCTION Cancer is a diseases characterized by uncontrolled growth or spread of abnormal cells. Since it involves the conversion of any normal cells to a cancerous cell showing tandem replication and cell division at much faster rate in comparison to the normal cells and thus provides a potential target area for the development of chemotherapeutic agents. It is now clear that chemotherapy’s most effective role in solid tumours is as an adjuvant to initial therapy by surgical or radiotherapeutic procedures. Chemotherapy becomes critical to effective treatment because only systemic therapy can attack micrometastases. These agents can be categorized into functional subgroups like alkylating agents, antimetabolites, antibiotics, and antimitotics. The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) belonging to the class of

DNA-interactive antitumour antibiotics have the potential as regulators of gene expression with possible therapeutic application in the treatment of genetic disorders including cancer. The first PBD antitumour antibiotic anthramycin has been described by Leimgruber et. al. in 1963, and since then a number of compounds have been developed on PBD ring system leading to DNA binding ligands.

II ABSTRACT

Figure-1

OH H OCH3 9 11 Me 8 N HO N N 10 H H HO H 11a 1 7 N 2 MeO N N 6 5 4 CONH2 MeO O 3 O O

Anthramycin Tomaymycin DC-81

H N O O N H

N OMe MeO N O O SJG-136

Pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a family of potent naturally occurring low molecular weight antitumour antibiotics originally isolated from various Streptomyces species. Their common interaction with DNA has been extensively investigated and it is considered unique since they bind within the minor groove of DNA forming a covalent aminal bond between the C11-position of the central B-ring and the N2 amino group of guanine base (Figure-2). A number of naturally occurring and synthetic compounds based on PBD ring system, such as anthramycin, tomaymycin, DC-81 (Figure-1) and its dimmers (presently, SJG-136 is under clinical evaluation), have shown varying degrees of DNA binding affinity and anti-cancer activity.

O O N HN N NH H N N 2HN N H H N N N DNA N DNA H H

N N O O N10-C11 imine 11R/S aminal

Figure-2: PBD-DNA interaction

Chapter-I (Sec-B)

III ABSTRACT

SYNTHESIS AND BIOLOGICAL ACTIVITY OF C-8C-8 FFLUOROARYL SUBSTITUTED PYRIMIDINE

LINKED-PYRROLOBENZODIAZEPINE CCONJUGATES

It has been of considerable interest in the past few years to design and synthesize symmetrical cross-linking agents, particularly based on pyrrolobenzodiazepines (PBDs). Pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are of current interest due to their ability to recognize and subsequently form covalent bonds to specific base sequences of double stranded DNA. Pyrrolo[2,1-c]

[1,4]benzodiazepine (PBD) antitumour antibiotics bind covalently to the N2 of guanine at purine-guanine-purine sites in the minor groove of DNA. Cancer has long been recognized as a disease of aberrant cellular proliferation. It is now known that proteins that regulate proliferation are frequently mutated, deleted or over- expressed in cancer cell lines, and that this leads to the deregulated growth of the tumour cell. Such proteins therefore represent potential targets for therapeutic intervention. The majority of cancers exhibit direct or indirect deregulation of cyclin- dependent kinase (CDK) function, members of the CDK family are attractive targets for the development of anticancer agents.

Pyrimidine ring systems are reported to have shown a variety of biological activities particularly CDK inhibitors.

The objective of the present work is to combine the feature of CDK inhibitor property in the pyrimidine molecule. Therefore, it has been considered of interest to couple to C8-position of PBD through alkyl spacer. In the present chapter Sec-B the synthesis and biological activity of C-8 fluoroaryl substituted pyrimidine linked- pyrrolobenzodiazepine conjugates has been described.

IV ABSTRACT

Scheme-1 CH3

O O O N N

CH3 i OEt ii OH

F F F 1 2 3

iii

CH3 N N

O n Br

F n = 1,2,3 4a-c

o Reagents and conditions: (i) CO(OEt)2, NaH, THF, 0 C, 1 h, 90%; (ii) acetamidine hydrochloride, o K2CO3, EtOH, 70 C, 16 h, 70%; (iii) dibromo alkanes, K2CO3, dry acetone, reflux, 48 h, 93%.

Synthesis of bromoalkyl-6-(4-fluorophenyl)-2-methyl-4-pyrimidylether 4a-c has been carried by monoalkylation of 6-(4-fluorophenyl)-2-methyl-4-pyrimidinol 3 with dibromoalkanes and whereas compound 3 has been prepared by condensation of ethyl-3-(4-fluorophenyl)-3-oxopropanoate 2 wtih acetamidinehydrochloride. The ethyl-3-(4-fluorophenyl)-3-oxopropanoate 2 has been prepared from 4-fluoro acetophenone 1 by using NaH/diethylcarbonate (Scheme-1).

Synthesis of C-8 fluoroaryl substituted pyrimidine-PBD hybrids has been carried out by employing the commercially available vanillin. Oxidation of vanillin followed by benzylation and nitration by literature methods provides the 4- benzyloxy-5-methoxy-2-nitrobenzoic acid (5). L-Proline methyl ester has been coupled to 4-benzyloxy-5-methoxy-2-nitrobenzoic acid to afford compound 6 followed by reduction of ester group with DIABAL-H to produce the corresponding aldehyde, which upon protection with EtSH/TMSCl gives 7. This upon debenzylation gives (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2- carboxaldehyde diethylthioacetal (9). The main precursors 10a-c have been obtained

V ABSTRACT

by coupling of 9 and 4-(4-fluorophenyl)-6-bromoalkyloxy-2-methylpyrimidines (4a- c). These upon nitro reduction followed by deprotection of thioacetal group afford the target compounds 12a-c (Scheme-2).

Compounds 12a-c have been evaluated for in vitro antitumour screening programme of the National Cancer Institute against sixty human tumour cell lines derived from nine cancer types, leukemia, non-small lung, colon, CNS, melanoma, ovarian, renal, prostate and breast cancer. For each compound, dose response curves for each cell line were measured at a minimum of five concentrations at 10 fold dilutions in protocol of 48 h continuous drug exposure, and a sulphorodhamine B

(SRB) protein assay is used to estimate cell viability or growth. The concentration causing 50% cell growth inhibition (GI50), total cell growth inhibition (TGI, 0% growth), and 50% cell death (LC50 -50% growth) concentration for killing 50% of the cells) for compounds 12a-c against the 60 different cell lines are shown good activity.

VI ABSTRACT

Scheme-2

BnO NO BnO NO2 CO Me 2 i 2 BnO NO2 ii CHO OH N H3CO H3CO H3CO N O O O 5 6 7

CH3 iii N N HO NO2 CH(SEt) BnO NO2 CH(SEt) 2 iv 2 O n Br + N H3CO N H3CO F n =1-3 O O 4a-c 9 8 v

CH3 N N NO O n O 2 CH(SEt)2

F H3CO 10a-c O vi CH3 N N NH O n O 2 CH(SEt)2

F H3CO 11a-c O

vii CH3 N N

N O n O H F H CO N n = 1-3 3 12a-c O

Reagents and conditions: (i) SOCl2, C6H6, L-prolinemethylester hydrochloride, THF-H2O, 2 h, rt, 85%; (ii) o DIBAL-H, CH2Cl2,1 h, -78 C, 71%; (iii) EtSH, TMSCl, CH2Cl2, 8 h, rt; (iv) EtSH-BF3OEt2, CH2Cl2, 12 h, rt ;

(v) K2CO3, dry acetone, 48 h, reflux, 94-96%; (vi) SnCl2.2H2O, MeOH, 2 h, reflux, 85-87%; (vii) HgCl2-CaCO3,

CH3CN-H2O (4:1), 12 h, rt, 68-71%.

VII ABSTRACT

Compounds 12a-c exhibit cytotoxic potency against many cell lines.

Compound 12b exhibits a wide spectrum of activity against 60 human cancer cell lines in nine-cell line panel with GI50 value of <1 μM. The average GI50 value of compound 12b against leukemia cancer RFMI-8226, CCRFN CEM and MOLT-4 are

0.01, 0.08 and 0.08 μM respectively. In repeated testing of compound 12b the cytotoxic activity is further confirmed in most of the cell lines particularly GI 50 value is 0.002 μM in case of colon cancer HCC-2998. Compounds 12a and 12c exhibit cytotoxic potency against number of cell lines in the nine panels, with the GI50 value range of <20 μM. Compound 12c exhibits cytotoxic potency in the leukemia which

CCRF-CEM, EPMI-8226 and SR cell lines are 2.86, 2.21 and 2.98 μM respectively. In the non-small cell lung cancer, the growth of HOP-92 cell line is affected by compound 12c with GI50 value 0.37 μM. The in vitro cytotoxicity (IC50) for the naturally occurring DC-81 is 0.38, 0.33 and 0.1 μM in L1210, PC6 and CH1 cell lines respectively.

The DNA binding ability of these compounds has also been investigated by thermal denaturation studies using calf thymus (CT) DNA at pH 7.0, incubated at 37

°C. It is observed that compound 12b elevates the helix melting temperature of the

CT-DNA to 1.4 °C after incubation of 18 h while compounds 12a and 12c have not exhibited any significant ∆Tm value. In the same experiment the naturally occurring

DC-81 exhibits a ∆Tm of 0.7 °C

A quantitative restriction enzyme digest (RED100) assay has been carried out to determine the ability of 12a-c, which inhibits the DNA linearization by BamH1. It is observed that for the compound 12b the inhibition of BamH1 cleavage is in agreement with the DNA binding affinity as determined by thermal denaturation.

(Letters in Drug Design & Discovery, 2005, 1, 55-61).

VIII ABSTRACT

Chapter-I (Sec-C)

SYNTHESIS AND BIOLOGICAL ACTIVITY OF FFLUOROQUINOLONE-PYRROLO[2,1-c][1,4]

BENZODIAZEPINE CONJUGATES The development of conjugates or hybrid molecules between two types of cytotoxic moieties represent a new approach in the discovery of new antitumour agents, as they could posses not only high potency but also different alkylation sites, both the aspects are useful for tumour treatment. Recent efforts in the structural modification of the PBD-ring system have led to the synthesis of a variety of PBD hybrids that exhibited not only good DNA-binding affinity but also promising in vitro anticancer activity.

Topoisomerase II is one of the most important protein targets currently available for the treatment of human cancers. In literature, quinolone ring system has been extensively exploited for the development of broad spectrum of antimicrobial drugs. The compounds based on quinolones skelton particularly fluoroquinolones are the only direct inhibitors of the DNA synthesis by binding to the enzyme DNA complex, they stabilize DNA strand breaks created by DNA gyrase and topoisomerase II and IV. Further, in literature a number of antiviral and antitumour agents have been developed in which fluorine substitution has played a key role in their biological activity.

The objective of the present work is to combine the feature of inhibition of topoisomerase II property in the fluoroquinolone molecule. Therefore, it has been considered of interest to couple to C8-position of PBD through alkyl spacer. In the

IX ABSTRACT

present chapter Sec-C, the synthesis and biological activity of C-8 fluoroquinolone linked-pyrrolobenzodiazepine conjugates has been described.

Synthesis of fluoroquinolone-PBD conjugates 19a-c has been carried out by employing the corresponding N-alkylbromo-6,7-difluoro-4-hydroxyquinolone-3- ethylcarboxylate (16a-c). The precursors ethyl-1-(4-bromoalkyl)-6,7-difluoro-4-oxo-

1,4-dihydro-3-carboxlate 16a-c have been prepared by the monoalkylation of compound 15 with dibromoalkanes. The compound ethyl-6,7-difluoro-4-hydroxy-3- quinoline carboxylate 15 has been prepared by the cyclization of diethyl-2-(3,4- difluoroanilinomethylene) malonate (14), which has been obtained by the condensation of 3,4-difluoroaniline (13) with diethyl ethoxymethylenemalonate

(Scheme-3).

Scheme-3 OH O EtO2C CO2Et F i F ii F OEt

F NH2 F N F N H 13 14 15

iii

O O F OEt F N Br n 16a-c n = 1-3

Reagents and conditions: (i) diethylethoxymethylenemalonate, 110 oC, 1.5 h, 98%; (ii) o diphenyl ether, 250 C, 1 h, 81%; (iii) dibromoalkanes, K2CO3, acetone, reflux, 48 h, 95-98%.

Where as (2S)-N-(4-benzyloxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2- carboxaldehyde diethylthioacetal has been debenzylated to give (2S)-N-(4-hydroxy-

5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carboxaldehydediethylthioacetal (9). The

X ABSTRACT

main precursors 17a-c have been obtained by coupling of compound 9 and N- alkylbromo-6,7-difluoro-4-hydroxyquinolone-3-ethylcarboxylate (16a-c). These nitrothioacetals have been reduced with SnCl2.2H2O to give 18a-c. The deprotection of these amino thioacetal by employing HgCl2/CaCO3 affords the target imine compounds 19a-c (Scheme-4).

Scheme- 4

F F F F

HO NO2 CH(SEt)2 i NO O N O 2 CH(SEt)2 + O N n Br n H3CO N N O O H3CO O n = 1-3 OEt OEt O 9 16a-c 17a-c

ii

F F F F

iii O N O NH2 CH(SEt)2 O N n H O N n

O H3CO N O H3CO N OEt O OEt O

19a-c 18a-c

Reagents and conditions: (i) K2CO3, dry acetone, reflux, 48 h, 72-76%; (ii) SnCl2.2H2O, MeOH, reflux, 6-8 h, 80-85%; (iii) HgCl2, CaCO3, CH3CN-H2O (4:1), rt, 12 h, 55-61%.

As representative members, compounds 19b-c have been evaluated for their in vitro cytotoxicity in selected human cancer cell lines of colon (HT-29, HCT-15), lung (A-549, HOP-62), cervix (SiHa) origin by using SRB method. Usually, when the concentration of the solution is 10-6 mol/L, the inhibition of the solution is more than

50%, and then compound is considered as an effective agent. According to this standard, it has been observed that both 19b and 19c exhibit a strong effect to HCT-

XI ABSTRACT

15, A-549 cell lines. However, the in vitro cytotoxicity (IC50) for naturally occurring

DC-81 is 0.38 and 0.33 M in L1210 and PC6 cell lines.

The DNA binding ability for these C8-linked PBD conjugates has been examined by thermal denaturation studies using calf thymus (CT)-DNA. Melting studies show that these compounds stabilize the thermal helix®coil or melting

o stabilization (∆Tm) for the CT-DNA duplex at pH 7.0, incubated for 18 h at 37 C, where PBD/DNA molar ratio is 1:5. Interestingly, in this study PBD conjugates have shown moderate melting temperature values (1.6-3.9 oC). It is interesting, to observe that both the fluoroquinolone PBD-conjugates 19b and 19c the ∆Tm values are higher when the length of alkyl chain spacer is four or five. In the same experiment the

o naturally occurring DC-81 exhibits a ∆Tm of 0.7 C.

A quantitative restriction enzyme digest (RED100) assay study has been carried out to determine the ability of 19a-c, which inhibits the DNA linearization by

BamH1. The results of this experiment for compounds 19a, 19b and 19c are suggest that the fluoroquinolone-PBDs inhibit BamH1. There are differences in the inhibitory activity exhibited by PBDs evaluated in this assay. It is observed that the ranking order is 19c>19b>19a for inhibition of BamH1 cleavage and is in agreement with the

DNA-binding affinity as determined by thermal denaturation. These results clearly demonstrate that as the linker chain increases from three to five carbon spacer as in case of 19c there is an enhancement in the inhibitory activity. (Bio. org. Med. Chem.

2005, 13, 2021-2029).

CHAPTER-II

COMBINATORIAL CCHEMISTRY

Combinatorial chemistry is a new methodology developed by researchers in the pharmaceutical industry to reduce the time and costs associated with producing

XII ABSTRACT

effective and competitive new drugs. By accelerating the process of chemical synthesis, this method is having a profound effect on all branches of chemistry, especially on drug discovery. Through the rapidly evolving technology of combinatorial chemistry, it is now possible to produce libraries of small molecules to screen for novel bioactivities. This powerful new technology has begun to help pharmaceutical companies to find new drug candidates quickly, save significant money in preclinical development costs and ultimately change their fundamental approach to drug discovery.

Combinatorial chemistry is used to synthesize large number of chemical compounds by combining sets of building blocks. Each newly synthesized compound’s composition is slightly different from the previous one. In this way the bench chemists can single handedly prepare many hundreds or thousands of compounds in the time usually taken to prepare only a few by orthodox methodologies. Over the last few years, the combinatorial chemistry has emerged as an exciting new paradigm for the drug discovery. In a very short time the topic has become the focus of considerable scientific interests and research efforts.

IMIDAZO[1,2-a]PYRIDINE Nitrogen-bridgehead fused heterocycles containing an imidazole ring are common structural motifs in pharmacologically important molecules, with activities spanning a diverse range of targets. Probably the most widely used heterocyclic system from this group is imidazo[1,2-a]pyridine, which is contained in marketed drugs such as the benzodiazepine agonist Zolpidem and the PDE 3 inhibitor

Olprinone, as well as other experimental molecules. However, alternative derivatives, such as the closely related imidazo[1,2-a]pyrimidine Divaplon, are also prevalent (Figure-3). The imidazo[1,2-a]pyridine units appear as important building

XIII ABSTRACT

blocks in both natural and synthetic bioactive compounds. Such alterations in structure offer the potential to change the base pair recognition on DNA-binding and to yield different pharmacokinetic profiles.

Figure-3

N N Me N N O N CN N Me N Et O O N Me Me N H

Zolpidem Olprinone Divaplon

Imidazo[1,2-a]pyridines are cyclin-dependent kinases (CDK) inhibitors.

Evidence from in vitro studies suggests that inhibition of CDK2 selectively kills tumour cells with deregulated E2F-1 activity. CDK inhibitors are therefore particularly attractive as potential therapeutic agents and may offer new opportunities for selective and tolerable therapy for human cancer. Imidazo[1,2- a]pyridine moieties have been shown to possess diverse therapeutic activities, antiulcer, antibacterial, antifungal, herbicidal, calcium channel blockers, GABAA receptor modulator and also has been explored more recently 2-arylimidazo[1,2- a]pyridines were described as ligands for detecting β-Amyloid (Aβ) plaques in the brain, which production is a pivotal event in the pathology of Alzheimer’s disease

SOLID-PHASE SYNTHESIS OF IMADAZO[1,2-a]PYRIDINE-8-CARBOXAMIDES

The imidazo[1,2-a]pyridines moiety has gained much attention in the synthetic community, mainly because for its representation as a member of the family of ‘privileged scaffolds.’ In the literature previous reports has been described

XIV ABSTRACT

for the solid-phase synthesis of imidazo[1,2-a]pyridines first one involved a multi- component reaction (MCR) with 2-amino pyridine, aldehyde and isonitrile. Second method used benzene sulfinate as a traceless linker. Third method involved α- bromoketone bound to solid-support condensation with various 2-aminopyridines.

However, not much effort has been made for the development of solid-phase synthesis of such 8-substituted imidazo[1,2-a]pyridines and utilizing the solid- supported liquid-liquid extraction (SLE) extraction method for the final product purification.

imidazo[1,2-a]pyridines (Scheme-5) by directly attachment of the Boc- protected 2-aminonicotincacid 21 to 4-hydroxyphenyl-sulfide resin 20 was performed by a carbodiimde mediated esterification reaction using EDC and DMAP in a mixture of dichloromethane and DMF to afforded the polymer-bound Boc- protected 2-aminonicotincester 22. Deprotection of the Boc-group in acidic condition to gave the 2-aminonicotinicester resin. This resin condensation with various α- haloketones 23{1-5} (Figure-4) to results the formation of imidazo[1,2-a]pyridine moiety (24). Selective halogenation’s at the 3-position of 24 was achieved by means of N-chlorosuccinamide (NCS) or N-bromosuccinamide (NBS) in THF to gave 25 and 26. The desired amides 28, 29 and 30 were liberated from the resins (24, 25, and

26) using excess of an amine 27{1-15} (Figure-5) in pyridine. Removal of the excess amine was accomplished using solid-supported liquid-liquid extraction (SLE) with diatomaceous earth as support.

XV ABSTRACT

Scheme-5 N N Boc x H N O OH N b N c R OH 21 N Boc R H N N a O O R = 23{1-5} O O O O 20 22 x = Br, 25{23[1-5]} OH 24{23[1-5]} x= Cl, 26{23[1-5]} OH S

1 d,e d,e R1 = 27{1-15}

x N R N N R N 1 R 1 O N R H O N H 28{1-15} 29{1-15} 30{1-15}

Reagents and conditions: a) EDC, DMAP, CH2Cl2-DMF (1:1), 48 h; b) i) 4 N HCl in 1,4-dioxane; ii) xCH2COR 23{1-5} EtOH, reflux for 5 h; c) NBS or NCS, EtOH, reflux for 2 h; d) R1R2NH (27), py, 48 h; e) amine extraction.

Br Br Br Br Cl

O O O O O CH3 CH Cl 3 OCH3

1 2 3 4 5

Figure-4: A set of a-haloketones 23{1-5} for the library

XVI ABSTRACT

HN N H N NH2 HN 2 H2N F O H3CO F F 1 2 3 4 5

Cl O H C HN H N 3 N H N 2 H2N 2 H

6 7 8 9 10

H H N H3C N H2N H2N HN H3C CH3

11 12 13 14 15

Figure-5: A set of amines 27{1-15} for library

A versatile approach for the solid-phase synthesis of imidazo[1,2-a]pyridines has been developed. The diversity at C(8)-position can be created in a facile manner by employing this methodology. Moreover, halogination at C(3)-position has been achieved by employing this synthetic sequence. The reaction conditions used in this protocol are mild and compounds are obtained in good yields. This method can be potentially used for the generation of large number of imidazo[1,2-a]pyridines-based compounds using automated synthesizer. (J. Com. Chem. 2006, manuscript under preparation)

XVII ABSTRACT

CHAPTER-III: SEC-A

POLYMER ASSISTED SSOLUTION PPHASE STRATEGY FOR THE SSYNTHESIS OF PYRROLO[2,1-

C]BENZODIAZEPINES AS ANTICANCER AGENTS

In the last few years, the use of solution-phase methods has received a considerable amount of attention for the parallel synthesis of low molecular weight compound libraries. Moreover, in the field of solution phase library generation, the use of polymer-supported reagents is emerging as a leading strategy that not only gives the advantage of product isolation and purification of solid phase chemistry but also provides the benefits of the traditional solution-phase reactions.

Thus, by choosing an appropriate combination of scavenger resins and immobilized reagents, it is possible to perform multi-step synthesis without the classical purification of intermediates. In this way the advantages of solid phase synthesis, i.e., the ability to use an excess of reagents to drive reactions to completion and the ease of product isolation, become applicable to solution synthesis. Polymer- supported reagents and scavengers have now been successfully applied to multi- step synthesis to yield complex products in high purities and yields.

Polymer-supported reagents and scavengers represent a versatile addition to solid-phase organic synthesis and parallel solution-phase chemistry. The combination of these reagents offers exciting possibilities (Table-1).

The imine or carbinolamine-containing pyrrolo[2,1-c][1,4]benzodiazepines are a family of low molecular weight natural products originally isolated from

Streptomyces species, that are known to exhibit antitumour activity. These antibiotics bind selectively in the minor groove of DNA while a covalent aminal bond is formed between the electrophilic C11-position of the PBD and the nucleophilic N2-amino

XVIII ABSTRACT

group of a guanine base, resulting in biological activity. The S-configuration at the chiral C11a-position provides the PBD structure with the necessary right handed twist to fit snugly within the minor groove. In conjunction with these efforts and to

Table-1. Advantages of combinatorial chemistry

PASP Solid-phase Solution-phase Use of excess √ √

reagent Easy of work up √ √ Minimal √ √

purification Easy of reaction √ √

monitoring Simple chemistry √ √

set up Large libraries √ High quantities √ √ the best of my knowledge, for the first time polymer-supported reagents has been used for the synthesis of pyrrolo[2,1-c][1,4]benzodiazepines.Further, dilactams are known to exhibit different type of biological properties such as antiphage activity, analgesic antagonist, anti-inflammatory, psychomotor depressant activity and herbicidal properties.

The oxidation of amine to the corresponding imine functionality is a very useful transformation particularly in the field of natural products. In the literature, some procedures have been developed only, which allow the biologically and synthetically relevant transition-metal catalyzed transformation of amines into imines. The present work described for the conversion of amine to imine functionality by using polymer-supported reagents (Figure-6, Scheme-6). This

XIX ABSTRACT

process has also been extended towards the synthesis of pyrrolo[2,1-c]

[1,4]benzodiazepine antibiotics and further the use of orchestrated multi-step PS- reagents has also been demonstrated in its total synthesis. Figure-6 O O S N C N O N+Me RuO - 5 3 4

PS-carodiimide (31) PS-sulfoxide (32) PS-perruthenate (33)

Polymer-supported reagents.

Scheme-6

R2 i or ii R2 R1 N R1 N H 34 35

R1 = R2 = 10 examples

Reagents and conditions: i) 32, (COCl)2/Et3N, CH2Cl2, -50 o C to rt for 3-5 h, 62-78% ; ii) 33, NMO, CH2Cl2, rt, 8-10 h, 81-96%.

XX ABSTRACT

Scheme-7

NO2 NO CH OH NO i 2 2 ii or iii 2 CHO R R R OH N N O O O 36 37 38 iv

H N N H H R ii or iii R N N O O 40 39 Seven examples Polymer-assisted solution phase strategy for the synthesis of pyrrolo[2,1-c][1,4]benzodiazepines.

Reagents and conditions: i) L-prolinol, 31, CH2Cl2, rt,12 h, 94-96%; ii) 32, (COCl)2/Et3N, CH2Cl2, -50 o C to rt, 3-3.5 h; iii) 33, NMO, CH2Cl2, rt, 8-9 h; iv) 10% Pd-C, H2 (1.5 atm.), C2H5OH, rt, 6 h, 80%.

The synthetic route comprises of the coupling of L-prolinol with the corresponding 2-nitrobenzoic acids (36). Interestingly, in the coupling reaction of prolinol to the nitrobenzoic acid by using polymer-supported cyclohexylcarbodiimide (31) there is excess of acid and urea by-products that can be simply filtered from the N-(2-nitrobenzoyl)pyrrolidine-2-carbinols (37). The N-(2- nitrobenzoyl)pyrrolidine-2-carboxaldehyde (38) has been obtained by the oxidation of 37 using PS-sulfoxide (32) or PS-perruthenate (33). The reductive cyclization of the nitroaldehyde (38) employing Pd/C affords the secondary amines (39). These amines (39) have been treated with PS-sulfoxide (32) or alternatively by PS- perruthenate (33) to afford the desired imines 40 (Scheme-7) in good yield. Apart from the advantage of reuse of the PS-reagents there is absence of side-products no unpleasant smell of DMSO particularly when PS-sulfoxide is employed. Moreover the synthesis of pyrrolo[2,1-c][1,4]benzodiazepine antitumour antibiotics employing

PS-reagents is a rapid and efficient procedure.

XXI ABSTRACT

An efficient, clean process has been developed for the conversion of amine to imine functionality. This process has also been extended towards the synthesis of pyrrolo[2,1-c][1,4]benzodiazepine antibiotics and further the use of orchestrated multi-step PS-reagents has also been demonstrated in its total synthesis. This elegant preparation of imines from amines has potential for its use in combinatorial chemistry. Noteworthy aspect is that these polymer-supported reagents can be recovered and reused with negligible loss of activity and are enormously beneficial from the environmental and economic basis in the context of ‘green’ chemistry. (Adv.

Syn. Cat. 2006, 348, 249-254)

CHAPTER-III: SEC-B

POLYMER-ASSISTED SOLUTION-PHASE STRATEGY FOR THE SYNTHESIS OF FUSED[2,1- b]QUINAZOLINONES AND PREPARATION OF OOPTICALLY AACTIVE VASICINONE

Solid-phase synthesis has been instrumental in building compound libraries efficiently, however progress of the reaction and compound loading determination on the polymer usually require some specialized techniques and analytical equipments. Further, cleavage of the compound from the polymer at the final step is also a requirement. Therefore, an attractive alternative is the use of polymer- supported material as reagents instead of as anchors for the substrates. Polymer- assisted solution-phase (PASP) synthesis has many advantages over the conventional solution-phase and solid-phase chemistry. Some of these include, monitoring of the reaction in real time by conventional methods, easy reaction

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optimization, and no residual functionality from bead attachment in the final product.

FUSED [2,1-[2,1-b]QUINAZOLINONES AND VVASICINONE

Adathoda vasica Nees is an evergreen subherbaceous bush, and is extensively used in indigenous medicine for cold, cough, bronchitis and asthma. The leaves of this plant are rich in essential oil and quinazoline alkaloids viz, (Figure-5) (-)- vasicinone (41), (-)-vasicine (42), vasicinolone (43), deoxyvasicinone (44), vasicoline, adathaodine and anisotine. The roots contain vasicol, adhavasinone, pegamine (47) and 2-hydroxy-4-glucosyl chalcone. Vasicinone and related alkaloids have also been reported to be present in other plants namely, Peganum harmala and Sida cordifolia.

Fitzgerald et al., isolated mackinazoline (45) and 6,7,8,9-tetrahydro-11H-pyrido[2,1- b]quinazoline, analogues of vasicinone and vasicine analogues respectively from

Mackinlaya macrosciadia and Mackinlaya subulata of the family Araliaceae. The seven membered aliphatic ring analogue dihomo (C) vasicinone (46) has been synthesized but has not been reported to occurring naturally.

Figure-5

O O O HO N N N N

N N N N OH OH OH (-)-Vasicinone (41) (-)-Vasicine (42) Vasicinolone (43) Deoxyvasicinone (44)

O O O NH OH

N N N

Mackinazolinone (45) Dihomo (C) vasicinone (46) Pegamine (47)

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In continuation to our earlier efforts for the synthesis of biologically important compounds employing polymer-supported reagents. The present work described the synthesis of pyrrolo[2,1-b]quinazolinones and related analogues including vasicinone employing polymer-supported reagents for the first time to starting from 2-azidobenzoic acids (48) and various lactams. The synthetic route consists of the coupling of 2-Azidobenzoic acids 48 with different lactams (49) employing N-cyclohexylcarbodiimide, N’-methyl polystyrene (A) to give N-(2- azidobenzoyl)lactams (50), the excess of acid and urea by-products can be simply filtered from azido lactams 49. The intramolecular aza-Wittig reaction has been used extensively for the synthesis of five to seven membered nitrogen heterocyles including vasicinone. Cyclization with triphenyl phospine usually takes place in about 5 h under reflux condition in xylene. This has prompted us to envisage an azidoreductive cyclization process for azidobenzoyl lactams (50-52) to obtain the fused [2,1-b]quinazolinones (53-55) employing polymer-supported triphenyl phosphine (B) under mild conditions in quantitative yield (Scheme-8).

Bromination of deoxyvasicinone 53a by employing polymer-bound

- brominating agent (Amberlyst A-26 Br3 -form) (C) gives allylic monobromo derivatives (56a) in excellent yield. These bromo substituted deoxyvasicinone gave the acylated derivatives upon treatment with Amberlyst A-26 AcO--form. (D).

(±)-Vasicinone (57a) has been obtained by the treatment of these acylated

54e intermediate with borohydride exchange resin (E) in presence of Pd(OAc)2.

Oxidation of (±)-vasicinone by using poly(vinylpyridinium dichromate) (F) peovide the diones (58a) (Scheme-9). Enantioselective reduction of this dione 58a by using polymer-supported chiral sulfonamide (H) in the presence of NaBH4/Me3SiCl as a reducing agent32g to afford the optically active (l)-vasicinone (41a)S. Alternatively

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enantioselective reduction of dione by using polymer-supported chiral sulfonamide

(G) affords the (d)-vasicinone (41a)R in good yield (Scheme-9) with >94% of ee

(determined by chiral HPLC).33 These polymer-supported chiral reagents have been recovered and reused.

Scheme-8 O N C N O R R 1 OH A 1 N + HN n n R N CH2Cl2, r.t., 95-98% R N 2 3 O 2 3O n = 1, 2, 3 n = 1; 50 48 49 n = 2; 51 a R1 = R2 = H n = 3; 52 b R =H, R = Me 1 2 PPh c R = Cl, R = H 2 1 2 B

CH2Cl2, r.t., 96-98% O R 1 N n R2 N n = 1; 53 n = 2; 54 n = 3; 55

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Scheme-9

+ - NMe3 AcO O + - O i) O NMe3 Br3 D N C N MeOH, r.t., N THF, r.t., + - ii) NMe3 BH4 N >99% N E N Br OH 53a 56a Pd(OAc)2/MeOH 57a refulx, 1 h, 90%

+ -2 NH Cr2O7

2 o F DMF, 70 C, 18 h, 93%

O O O NaBH /Me SiCl NaBH /Me SiCl N 4 3 N 4 3 N

O O O O R N S S N N S N N OH O OH 58a H Ph Ph G Ph Ph (d)-Vasicinone (41a)R (l)-Vasicinone(41a)S HO HO THF, reflux, THF, reflux, 3 h, 93% 3 h, 95%

In conclusion the present work describes a clean preparation of fused [2,1- b]quinazolinones from substituted 2-azidobenzoic acids and various lactams with polymer-supported triphenylphosphine utilizing the intramolecular azido reductive process. This method has been extended towards the preparation of optically active

(d) and (l)-vasicinone by employing polymer-supported reagents. This synthetic strategy is readily amenable for designing and preparing a combinatorial library. It is noteworthy in the entire process, that the work-up has been simplified to filtration and evaporation for all the steps and all the reagents could be recovered and reused thus addressing the problems of environmental and economic sustainability.

(Synlett, 2006 in press)

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