13

The work carried in the research tenure has been compiled in the form of a thesis

entitled “Synthesis and Biological Evaluation of New Hybrids of Pyrrolo[2,1-

c][1,4]benzodiazepines and Quinazolinones as Potential Anticancer Agents”. The

main aim of this work has been to design and synthesize biologically active

quinazolinone-pyrrolobenzodiazepine conjugates and anthranilamide-

pyrrolobenzodiazepine conjugates, which exhibited DNA binding ability and

significant anticancer activity through mitochondrial mediated apoptosis.

Furthermore, the biologically active 2,3-dihydroquinazolinone-3,5-diaryl

isoxazoline/isoxazole conjugates and 2-styrylquinazolinone-3,5-diarylisoxazoline

/isoxazole conjugates have been prepared and evaluated for their anticancer activity

and cell cycle effects on MCF7 cell lines. The thesis has been divided into four

chapters.

CCHHAAPPTTEERR II:: This chapter gives the general introduction about cancer

chemotherapy, covalent interactions of drug-DNA, particularly of pyrrolo[2,1-c]

[1,4]benzodiazepine (PBD) antitumour antibiotics, and the objectives of the

present work.

CCHHAAPPTTEERR IIII:: This chapter describes the synthesis of a series of quinazolinone-PBD

conjugates connected through simple alkane spacers and these conjugates have

been evaluated for their biological activity. This chapter is mainly focused on the

mitochondrial mediated apoptotic pathway responsible for the significant

anticancer activity.

CCHHAAPPTTEERR IIIIII:: This chapter deals with the synthesis of new PBD hybrids by

linking anthranilamides through 4-piperazinyl alkane spacer to the C8-position

of the A ring of PBD scaffold. The present chapter is mostly focused on the DNA-

binding affinity and anticancer activity of the newly synthesized PBD hybrids.

SYNOPSIS

14

CCHHAAPPTTEERR IIVV:: This chapter comprises of two sections; section A consists of design,

synthesis and biological evaluation of 2,3-dihydroquinazolinone-3,5-diaryl

isoxazoline/isoxazole conjugates as potent anticancer agents. Further these

conjugates areevaluated for cell cycle effects on MCF 7 cell lines.. Section B

consists of design, synthesis anticancer activity and cellcycle effects (MCF7 cell

lines) of 2-styryl quinazolinone-3,5-diarylisoxazoline/isoxazole conjugates.

IINNTTRROODDUUCCTTIIOONN

This chapter describes the general introduction about pyrrolobenzodiazepines

and the objectives of the present work..

PPYYRRRROOLLOO[[22,,11--cc]][[11,,44]]BBEENNZZOODDIIAAZZEEPPIINNEESS

Cancer is a diseases characterized by uncontrolled growth or spread of

abnormal cells. It involves the conversion of any normal cell 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 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

CHAPTER-I

15

Leimgruber et. al., in 1963, and since then a number of compounds have been

developed on the PBD ring system leading to some efficient DNA binding ligands.

H3COH

N

HN

O

OCH3

CONH2

Anthramycin

12

453

67

8 910

11

N

N

O

HO

H3CO

Tomaymycin

H

N

NO

H3COO

HON

N

O

H

OCH3

SJG-136

H11a

Figure 1

PBD’s 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. A number of naturally occurring and synthetic

compounds based on PBD ring system, such as anthramycin, tomaymycin, DC-81

and its dimers (presently, one of the dimer SJG-136 is under clinical evaluation),

have shown varying degrees of DNA binding affinity and anticancer activity.

OH

N

HN

O

NH2

O

HOH

HN

N N

N

H2N

O

DNA

carbinolomine

1110 OH

N

HN

O

NH2

O

NHH

HNN

N

NO

DNA

1110

Anthramycin

Figure 2

16

In search for new potential anticancer drugs, we designed and synthesized

combilexins, in which DNA intercalator is linked to a minor groove binding

component. A new type of DNA intercalator comprising of quinazolinone moiety

has been tethered by ether linkage to the PBD ring system and these have been

evaluated for their biological activity. An attempt, based on the in vitro cytotoxic

activity exhibited in these compounds made to rationalize their mechanism of action

through cell cycle analysis and DNA interaction studies. Cell cycle phase

distribution and apoptosis is measured using flow cytometry. The DNA binding

characteristics of these conjugates have been evaluated by thermal denaturation

studies. A molecular modeling study has been carried out for a set of compounds

22a-f and 25a-f and calculated their binding score. Further MD simulations have also

been carried out for the compounds 22b, 25b and 25c. One of the representative

compounds 25c of this series has been tested against a panel of 57 human cancer cell

lines. Further, compounds 22b and 25c induce marked influence on the cell cycle

phase distributions with significant G1 arrest in A-375 cells, followed by the

induction of apoptosis through mitochondrial mediated pathway along with

suppression of NF-B.

SYNTHESIS OF QUINAZOLINONE LINKED PYRROLOBENZODIAZEPINE (PBD)CONJUGATES

Synthesis of quinazolinone -pyrrolobenzodiazepine conjugates (22a-f and 25a-

f) has been carried out by employing the commercially available vanillin (1) as the

starting material. Oxidation of vanillin to form the corresponding carboxylic acid

followed by acid-catalyzed esterification with methanol provided methyl benzoate

in quantitative yield. This is followed by benzylation and nitration by employing the

literature method provides 4-benzyloxy-5-methoxy-2-nitrobenzoic acid (6). This has

been further coupled to L-proline methyl ester to afford the intermediate 7, which

upon reduction with DIBAL-H produces the corresponding aldehyde 8. The

CHAPTER-II

17

aldehyde group of this compound has been protected with EtSH/TMSCl to give 9,

which upon debenzylation provides the key intermediate 10 (Scheme 1).

MeO

HO

MeO

HO

MeO

HO

MeO

BnO

MeO

BnO

H OH OMe

OMeOMe

O O O

OO

NO2

MeO

BnO

MeO

HO

N

O

CH(SEt)2NO2

MeO

BnO

OH

O

NO2

(i) (ii)

(iii)

(iv)(v)

(vi)

(vii)

(viii)

(ix)

1 2 3

456

7 8

910Scheme 1. Reagents and conditions: (i) NH2SO3H, NaClO2, H2O, rt, 2 h, 90%; (ii) H2SO4, MeOH,reflux, 4 h, 85%; (iii) benzylbromide, K2CO3, acetone, reflux, 24 h, 92%; (iv) SnCl4, fumingHNO3, CH2Cl2, 5 min, -25 oc, 78%; (v) 2N LiOH, MeOH, H2O, THF (1:1:3), rt, 12 h, 83%; (vi)SOCl2, C6H6, L-proline methylester hydrochloride, THF- H2O, 1-2 h, rt, 85%; (vii) DIBAL-H,CH2Cl2, 1-1.30 h, -78 oC, 65%; (viii) EtSH, TMSCl, CH2Cl2, 8-12 h, rt, 72%; (ix) BF3

.OEt2, EtSH,CHCl3, rt, 8 h, 75%.

N

NO2

O

COOMe

MeO

BnO

N

NO2

O

CHO

MeO

BnO

N

NO2

O

CH(SEt)2

The quinazolinone precursors required for the preparation of the desired PBD-

conjugates have been prepared as shown in Scheme 2. This synthetic strategy is

involves to the cyclization of anthranilic acid to benzoxazinone followed by

coupling with aromatic amines. Starting materials 12 and 16 has been obtained by

condensation of commercially available 5-hydroxy anthranilic acid (11) and

anthranilic acid (15) with acetic anhydride respectively. The compound 12 on

coupling with substituted anilines in glacial acetic acid gives the compounds 13ae.

18

These compounds on subsequent etherification with dibromo alkanes afford

quinazolinone precursors (14af). The other types of quinazolinones have been

obtained by reaction of compound 16 with 4-aminophenol to give compound 17,

which upon etherification by employing dibromo alkanes give compounds 18a–c.

Compounds 19a–c have been prepared by employing benzaldehyde and 18a–c

(Scheme 3).

NH2

OH

O

(i)

N

O

O

(ii)

N

N

O

(iii)

N

N

OZ

n

11 12 13ae

14af

HO HO

ZXY

OBr ( )

HO

YX

14a: X,Y = H, Z = F, n = 214b: X = H, Y = Cl, Z = F, n = 314c: X, Z = Cl, Y =H, n = 214d: X = CH3, Y = H, Z = iodo, n = 214e: X = CH3, Y = H, Z = iodo, n = 314f: X,Y = H, Z = OCH3, n = 2

13a: X,Y = H, Z = F13b: X = H, Y = Cl, Z = F13c: X, Z = Cl, Y =H13d: X = CH3, Y = H, Z = iodo13e: X,Y = H, Z = OCH3

Scheme 2. Reagents and conditions: (i) (CH3CO)2O, 160-180 oC, 1 h; (ii) R-NH2, 180 oC, 2 h;(iii) dibromoalkanes, K2CO3, acetone, reflux, 24 h.

Scheme 3. Reagents and conditions: (i) (CH3CO)2O, 160-180 oC, 1 h; (ii) 4-amino phenol, 180 oC,2 h; (iii) dibromoalkanes, K2CO3, acetone, reflux, 24 h; (iv) benzaldehyde, glacial acetic acid,120 oC, 12 h.

NH2

OH

O

(i)

N

O

O

(ii)

N

N

OOH

(iii)

N

N

OO Br( )

n

N

N

OO Br( )

n

15 16 17

18ac19ac

(iv)

n = 13

19

PBD subunit precursors required for the preparation of these conjugates have

been synthesized by employing the procedure reported in the literature. PBD

subunit precursors required for the preparation of these conjugates have been

synthesized by employing the procedure reported in the literature. Accordingly

(2S)-N-[4-(hydroxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carb-oxaldehyde

diethyl thioacetal (10) has been prepared and linked to quinazolinone precursors

14af, 18ac and 19ac by etherification in the presence of K2CO3 and acetone to

provide intermediates 20af and 23af. To produce 22af and 25af the nitro

thioacetals 20af and 23af have been reduced to the amino thioacetals 21af and

24af with SnCl2.2H2O in refluxing MeOH and then cyclized by treatment with

HgCl2 and CaCO3 in MeCN-H2O (Schemes 4 and 5).

22a: X = H, Y = H, Z = F, n = 322b: X = H, Y = Cl, Z = F, n = 322c: X = Cl, Y = H, Z = Cl, n = 222d: X = CH3, Y = H, Z = iodo, n = 222e: X = CH3, Y = H, Z = iodo, n = 322f: X = H, Y = H, Z = OCH3, n = 3

N

NO2HO

H3CO

CH(SEt)2

10

N

NO2

O

O

H3CO

CH(SEt)2(i)

(ii)

22af

20af

N

N

OO

Z

YX

( )n

O

H3CO

N

N

OO

Z

YX

( )n

N

N H

Scheme 4. Reagents and conditions: (i) 14a-f, K2CO3, dry acetone, reflux, 24 h, 80-85%; (ii) SnCl2.2H2O,CH3OH, reflux, 6 h; (iii) HgCl2, CaCO3, CH3CN-H2O, (4:1), 12 h, 55-58%.

N

NH2

O

O

H3CO

CH(SEt)2

21af

N

N

OO

Z

YX

( )n

(iii)

O

O

20

N

NO2HO

H3CO

CH(SEt)2

10

(i)

N

N

O

R

O ( )n

N

NO2

O

O

H3CO

CH(SEt)2

25af

23af

25a: R= methyl, n = 125b: R= methyl, n = 225c: R= methyl, n = 325d: R= styryl, n = 125e: R= styryl, n = 225f: R= styryl, n = 3

N

N

O

R

O ( )n

O

H3CO N

N

O

H

(ii)

Scheme 5. Reagents and conditions: (i) 18ac and 19ac, K2CO3, dry acetone, reflux, 24 h, 8088%; (ii)SnCl2.2H2O, CH3OH, reflux, 6 h; (iii) HgCl2, CaCO3, CH3CN-H2O, (4:1), 12 h, 5558%.

O

N

N

O

R

O ( )n

N

NH2

O

O

H3CO

CH(SEt)2

24af(iii)

The thermal denaturation studies show that hybrid agents (22a-f and 25a-f)

possess good DNA binding ability compared to DC-81. Our finding suggested that

these hybrid agents bind to DNA more efficiently than DC-81. In addition,

molecular modeling study has been carried out for a set of quinazolinone-PBDs

(22a-f and 25a-f) in order to rank them through their complementarity to the

CGCGATCGCG, according to their interaction energies with B-DNA. The optimal

number of methylene linkers calculated from the GOLD docking and the

experimental numbers are in reasonably good agreement.

Compounds 22a–f, 25a, 25b, and 25d–f have exhibited significant anticancer

activity against eleven cell lines with GI50 values ranging from <0.1 to 26.2 M in

comparison to adriamycin (GI50 from 0.1–7.25 M). According to the in vitro

screening data, compound 25c has significant cytotoxicity against all the cancer cell

lines with GI50 values ranging from 0.06 to 0.43 M and the mean GI50 value for the

compound 25c is 0.27 M, which exhibits an interesting profile of activity for various

21

cell lines, indicating that this compound 25c has the potent broad-spectrum

anticancer activity. Further compounds 22b and 25c induced strong sub G1/G0

arrest of the cell cycle followed by apoptosis, in A-375 cells. Two promising

compounds 22b and 25c induced the release of cytochrome c, activation of caspase-3,

cleavage of PARP and subsequent cell death. Further, these compounds when

treated with A375 cells show the characteristic features of apoptosis like

enhancement in the levels of p53, p21 and p27 inhibition of CDK2 and suppression

of NF-B. Also these compounds 22b and 25c control cell proliferation by regulating

anti apoptotic genes like Bcl-2. Hence the data obtained suggests that the ability of

these PBD conjugates to activate p53 and inhibit NF-B could be useful for the

development of anticancer agents with a greater therapeutic potential for tumour

suppression (Bioorg. Med. Chem. 2010, 18, 526-542, Patent filed 2598/DEL/08 (India),

PCT/IN2009/000400).

In an effort to establish new candidates with improved anticancer activity,

pyrrolobenzodiazepine derivatives bearing anthranilamide moiety synthesized and

their DNA interaction has been evaluated by thermal denaturation studies. The

cytotoxic studies of the hybrid agents on human cancer cell lines indicate most of the

hybrids induced higher cytotoxicity.

SYNTHESIS OF C8-LINKED PYRROLO[2,1-C][1,4]BENZODIAZEPINE HYBRIDS WITHANTHRANILAMIDE

The synthetic strategies adopted to obtain the target compounds are depicted

in Schemes 12. The precursors in the present study is the 28 anthranilamides of

formula 4 and (2S)-N-[4-(n-bromoalkyl)oxy-5-methoxy-2-nitro-benzoyl]pyrrolidine-

2-carboxaldehyde dieth-ylthioacetal51 of formula 5 have been prepared by literature

methods.

CHAPTER-III

22

R =

MeOOMe

OMe N O

O

NH

O

O

O

X

NH2

N

O

NBoc

X

NH

N

R

O

NBoc

X

1a: X = H1b: X = Cl

2a, b 3af

NH

N

R

O

NH

X

(i) (ii)

(iii)

1a

a: R = 3,4,5-trimethoxyphenyl, X = Hb: R = 4 -pyridinyl, X = Hc: R = 2- napthyl, X = Hd: R = 3-chromenyl, X = He: R = 2-napthyl, X = Clf: R = 3-chromenyl, X = Cl

4af

Scheme 1. Reagents and conditions: (i) N-boc piperazine, 1,4-dioxane, reflux, 4 h; (ii) R-CHO,NaCNBH3, MeOH, CH3COOH, rt, 22 h; (iii) TFA, CH2Cl2, rt, 5 h

The precursors 4af have been prepared as shown in Scheme 1.

Isatoicanhydride 1a or 6-chloroisatoicanhydride 1b has been treated with N-boc

piperazine in 1:1.2 M ratio, and refluxed in 1,4-dioxane to give the 4-(2-

aminobenzoyl)N-boc piperazine 2a and 4-(2-amino-5-chlorobenzoyl) N-boc

piperazine 2b respectively in almost quantitative yield. These compounds 2a and 2b

upon reductive amination with different aromatic aldehydes by using

sodiumcyanoborohydride (NaCNBH3) in methanol with catalytic amount of acetic

acid afford compounds 3af, which on further deprotection with triflouroaceticacid

give precursors 4af (Scheme 1). Synthesis of anthranilamide-PBD conjugates (8a-l)

has been carried out by employing (2S)-N-[4-(bromoalkoxy)-5-methoxy-2-

nitrobenzoyl]pyrrolidine-2-carboxaldehyde diethyl thioacetal (5a-c) as the starting

material. The nitro thioacetal intermediate (5a-c) was coupled with anthranilamide

precursors of formulae 4af to give nitrothioacetal intermediates 6al. The nitro

group of this compound has been efficiently reduced employing SnCl2.2H2O to

afford the corresponding aminothioacetal 7a-l. Deprotection of the thioacetal group

with HgCl2 and CaCO3 afford the desired PBD hybrids 8a-l (Scheme 2).

23

(i)N

NO2O

H3COO

CH(SEt)2

5ac

Br ( )n

N

NO2O

H3CO

CH(SEt)2( )

nNH

N

R

O

N

X

(iii)

n = 1, 3, 4

N

O

H3CO

( )nNH

N

R

O

N

X

N

O

H

4af

a: R = 3,4,5-trimethoxy phenyl, X = H, n = 1b: R = 3,4,5-trimethoxy phenyl, X = H, n = 3c: R = 3,4,5-trimethoxy phenyl, X = H, n = 4d: R = 4-pyridinyl, X = H, n = 1e: R = 4-pyridinyl, X = H, n = 3f: R = 4-pyridinyl, X = H, n = 4g: R = 2-napthyl, X = H; n = 1h: R = 2-napthyl, X = H, n = 3i: R = 2-napthyl, X = H, n = 4j: R = 3-chromenyl, X = H, n = 3k: R = 2-napthyl, X = Cl, n = 3l: R = 3-chromenyl, X = Cl, n = 3

6al

8al

(ii)

N

NH2O

H3CO

CH(SEt)2( )

nNH

N

R

O

N

X

7al

Scheme 2. Reagents and conditions: (i) dibromoalkane, K2CO3, acetone, reflux, 24 h; (ii) SnCl2.2H2O,MeOH, reflux, 6 h; (iii) HgCl2, CaCO3, CH3CN:H2O (4:1), rt, o/n

O

O

BIOLOGICAL ACTIVITY OF C8-LINKED PYRROLO[2,1-C][1,4]BENZODIAZEPINEHYBRIDS

Compounds 8a–l have been evaluated for their in vitro anticancer activity in

selected human cancer cell lines of MCF7 (breast), A2780 (ovarian), Colo205 (colon),

PC3 (prostate), SiHa (cervix), A-549 and Hop62 (lung) and KB (Leukemia) by

sulforhodamine B (SRB) assay. The compounds exhibiting GI50 ≤ 10-5M (10 M) are

considered to be active on the respective cell lines. A protocol of 48 h continuous

drug exposure was used and a sulforhodamine B (SRB) protein assay was used to

estimate cell viability or growth. The concentration causing 50% cell growth

inhibition (GI50) compared with the control was calculated. The results show that all

the new compounds are significantly cytotoxic, with the molar concentration of the

drug that inhibits 50% net cell growth inhibition (GI50) ranging from 0.13 to 29 M.

24

Thermal denaturation studies showed that these compounds stabilized the

thermal helix coil or melting stabilization (ΔTm) for the CT-DNA duplex at pH 7.0,

where PBD/DNA molar ratio is 1:5. Interestingly, all the anthranilamide–PBD

conjugates (8a–l) elevate the helix melting temperature of CT-DNA in the range of

4.1–8.6 oC at 0 h and also examined after 18 h incubation at 37 o C. Compound 8f

showed the highest ΔTm of 8.6 oC at 0 h which increased up to 9.2 oC after 18 h

incubation. All the compounds 8a–l have shown better fit in the minor groove of

DNA, leading to enhanced binding affinity compared to DC-81. Moreover it is

interesting to note that the incorporation of a piperazine moiety in the linker spacer

in all the anthranilamide-PBD conjugates have exhibited potent DNA-binding

affinity with ΔTm values ranging from 4.1–8.6 oC.

Among all the PBD conjugates, three potent compounds 8g, 8h and 8j have

been tested for cell viability of A375 cell lines by MTT assay. The significant

inhibition of cell proliferation of A375 cell line showed by these PBD conjugates

prompted us to further study the effect of them on cell cycle distribution as well as

caspase-3 analysis (Bioorg. Med. Chem. Lett. 2010, in press).

In continuation to our efforts on the design of new anticancer agents, we became

interested in the development of new hybrid molecules that consists of two

phamacophores in a single molecule with an intention to enhance the efficacy of

such compounds.31 In this context, 3,5-diaryl isoxazoline/isoxazoles have been

linked to quinazolinones through different alkane spacers and as well as through a

piperazine side armed alkane spacer. These new hybrids have been evaluated for

their anticancer activity. In view of this promising activity, it has been considered of

interest to investigate their role in cell cycle effects of MCF7 cell lines. Further, some

CHAPTER-IV

25

detailed biological studies related to the mechanism aspects have been carried out

for some representative compounds. This chapter is divided into two sections.

SECTION A

SYNTHESIS OF 3,5-DIARYL ISOXAZOLINE/ISOXAZOLE LINKED 2,3-

DIHYDROQUINAZOLINONE HYBRIDS

The synthesis of 3,5-diaryl isoxazoline and isoxazole derivatives (5a,b and 10a,b)24,25 has been carried out from aldehydes 1a,b and 6 as the starting materials.

Reaction of these aldehydes with hydroxylamine in a MeOH/H2O (3:1) solution

produces the corresponding oximes 2a,b and 7 in high yields. Olefins 3a and 8a

have been obtained by the reaction of aldehydes 6 and 1b respectively with

methyltriphenyl phosphonium bromide, in the presence of sodium hydride. Then

these olefins 3a and 8a have been coupled to oximes 2a and 7 to provide the

corresponding tertiary butyl dimethyl silyl (TBDMS) protected isoxazolines 4a and

9a. Similarly, TBDMS protected isoxazoles 4b and 9b have been prepared by

employing alkynes 3b and 8b and oximes 2b and 7. Further, these compounds 4a,b

and 9a,b upon deprotection with tetrabutylammonium fluoride give the desired

precursors (5a,b and 10a,b). The compound 11 has been obtained by etherification of

compound 5a as shown in Scheme 1.

26

R2R3R1

CHO

(i)

(ii)

(iii)

1a,b

a: R1 = OMe, R2 = OTBDMS, R3 = OMeb: R1 = OTBDMS, R2 = OMe, R3 = H

3a: Z = CH23b: Z = CH

4a: Z = CH2, R1 = OMe, R2 = OTBDMS, R3 = OMe4b: Z = CH, R1 = OTBDMS, R2 = OMe, R3 = H

4a,b

5a: Z = CH2, R1 = OMe, R2 = OH, R3 = OMe5b: Z = CH, R1 = OH, R2 = OMe, R3 = H

R2R3R1

NOH

MeO

MeO

OMe

Z

MeO

MeOOMe

NO

ZR1

R2

R3

+

2a,b

OMeOMeMeO

CHO

6 7

9a: Z = CH2, R1 = OTBDMS, R2 = OMe, R3 = H9b: Z = CH, R1 = OMe, R2 = OTBDMS, R3 = OMe

9ab

10a: Z = CH2, R1 = OH, R2 = OMe, R3 = H10b: Z = CH, R1 = OMe, R2 = OH, R3 = OMe

OMeOMeMeO

NOH

R2

R1

R3

Z

MeO

MeOOMe

ON

ZR1

R2

R3

8a,b

(i)

(ii)

(iii)

5a: Z = CH2, R1 = OMe, R2 = OH, R3 = OMe

11: Z = CH2, R1 = OMe, R2 = O(CH2)5Br, R3 = OMe

+

(iv)

8a: Z = CH2, R1 = OTBDMS, R2 = OMe, R3 = H8b: Z = CH, R1 = OMe, R2 = OTBDMS, R3 = OMe

Scheme 1. Reagents and conditions: (i) NH2OH.HCl, NaHCO3, CH3OH:H2O (3:1), 0 oC, then rt, 6 h;(ii) 13% aq NaOCl, Et3N, CH2Cl2, 0 oC, then rt, 24 h; (iii) TBAF, THF, rt, 2 h; (iv) 1,5-dibromo pentane,K2CO3, DMF, rt, 24 h.

27

The synthesis of dihydroquinazolinones is outlined in Scheme 2. Compounds

12a,b have been treated with benzamide in presence of N,N-dimethylacetamide

(DMAC) to give compounds 13a,b respectively.14 These upon etherification with

dibromoalkanes using K2CO3 in DMF provide the 2,3-dihydroquinazolinone

precursors (14a–d). Compound 14d has been coupled with N-boc piperazine to

obtain compound 15, which upon deprotection gives compound 16.

The synthesis of hybrid compounds 17a–d and 18a–d has been carried out from

the compounds 14a–d and 3,5-diaryl isoxazoline/isoxazole precursors (5a,b and

10a,b) using K2CO3 in DMF. The other hybrid (19) has been prepared from

compounds 11 and 16 as shown in Scheme 3.

R1

R2

O

HNH

NH

O

HR1

R2

(i) (ii)

NH

NH

O

HR1

R212a,b 13a,b 14ad12a: R1 = OMe, R2 = OH12b: R1 = OH, R2 = OMe 14a: R1 = OMe, R2 =

14b: R1 = OMe, R2 =14c: R1 = OMe, R2 =14d: R2 = OMe, R1 =

O (CH2)3BrO (CH2)4BrO (CH2)5BrO (CH2)4Br

NH

NH

O

O N N-Boc

NH

NH

O

O N NH

(iii)

(iv)

14d

15

16

OMe

OMe

( )

( )

n

n

n = 2

Scheme 2. Reagents and conditions: (i) Benzamide, p-toulene sulfonicacid monohydrate,N, N-dimethylacetamide, rt, 2 h; (ii) Br(CH2)nBr, K2CO3, DMF, rt, 24 h; (iii) N-boc piperazine,K2CO3, DMF, rt, 24 h; (iv) TFA, CH2Cl2, rt, 12 h.

28

19: m = 3; n = 2

NO

MeOOMe OMe

O N N ONH

HN

O

( )m

( )n

MeO

MeO OMe

14ac

NO

MeOOMe

MeO OMe

OOMe

17a: n = 117b: n = 217c: n = 3

O( )n

5aNH

HN

O

MeO

NO

MeOOMe

MeO O

OMe

17d: n = 2

NH

HN

O

MeO

O

(i)

ON

OMe

O ( )n10a

NH

HN

O

ON

MeOOMe

ONH

HN

O

MeO

O

OMe

OMe( )

n

O

MeO

MeO

MeO

MeO

OMe

(i)

14d

5b

(i)

10b

(i)

( )n

11 + 16(i)

18a: n = 118b: n = 218c: n = 3

18d: n = 2

Scheme 3. Reagents and conditions: (i) K2CO3, DMF, rt, 24 h.

All the new 3,5-diaryl isoxazoline/isoxazole linked 2,3-dihydroquinazolinone

hybrids (17a–d, 18a–d and 19) through different alkane spacers as well as through

piperazine side-armed alkane spacers. Among the hybrid compounds prepared,

compound 17c has exhibited significant anticancer activity against nineteen cancer

29

cell lines with GI50 values less than 1 M. The other compounds have also exhibited

potent anticancer activity against MCF7 and PC3 cancer cell lines. The most

promising compound among this series has been evaluated for cell cycle effects on

MCF7 cell lines. (Patent filed 2602/DEL/08 (India), PCT/IN2009/000490, Manuscript

under preparation)

SECTION B

SYNTHESIS OF 3,5-DIARYL ISOXAZOLINE/ISOXAZOLE LINKED STYRYLQUINAZOLINONE

HYBRIDS

The synthesis of styrylquinazolinones is outlined in Scheme 1. The first synthetic

step involved the condensation of anthranilic acid (1) with acetic anhydride to afford

the desired benzoxazinone (2) in quantitative yield. After evaporation of the excess

of anhydride under reduced pressure, the crude product was used without any

further purification. The compound 2 on heating with 4-aminophenol at 160–180 °C

for 2 h in oil bath gives 3, which upon etherification by employing dibromoalkanes

yields compounds 4ac. These analogues on reflux with benzaldehyde in acetic acid

provide the compounds 5ac.

30

OH

NH2

O

N

O

O

N

N

OOH

N

N

OO Br

N

N

OO Br( ) ( )

nn

(i) (ii)

(iii)

(iv)

1 2 3

4ac5ac

n = 1, 2, 3

Scheme 1. Reagents and conditions: (i) (CH3CO)2O, reflux, 1 h; (ii) p-aminophenol, 180 oC, 2 h;(iii) dibromo alkanes, K2CO3, acetone, reflux, 12 h; (iv) benzaldehyde, CH3COOH, reflux, 12 h.

The synthesis of precursors 3,5-diaryl-isoxazoline and isoxazole precursors 6a,b

and 7ac has been carried out as described in the chapter IV section A. The synthesis

of styrylquinazolinone linked 3,5-diaryl-isoxazoline/isoxazole hybrids (8ad, and

9ad) has been carried out from compounds 5ac and isoxazoles/ isoxazolines (6ab

and 7ac) using K2CO3 in acetone as shown in Scheme 2. The other hybrids (13a and

13b) have been prepared from the reaction between 10b,c which has been prepared

by etherification of hydroxyl isoxazoles (7a,c) with 1,5 dibromo pentane and

compound 12, which in turn has been obtained by deprotection of compound 11,

prepared from 5c by reaction with N-boc piperazine in DMF and K2CO3 at room

temperature as shown in Scheme 3 .

31

5ac

NO

MeOOMe

MeO OMe

OOMe

8a: n = 18b: n = 28c: n = 3

O( )n

6a

NO

MeOOMe

MeO O

OMe

8d: n = 3

O

(i)

ON

OMe

O ( )n

ON

MeOOMe

O O

OMe

OMe( )

n

O

MeO

MeO

MeO

OMe

6b

( )n

9a: n = 19b: n = 29c: n = 3

9d: n = 3

Scheme 2. Reagents and conditions: (i) K2CO3, DMF, rt, 24 h.

N

N

O

N

N

O

N

N

O

N

N

O

NO

MeOOMe

MeO OMe

OHOMe

NO

MeOOMe

MeO OH

OMe 5c(i)

ON

OMe

OH

MeO

MeO

OMe

(i)

5ac

7a

ON

MeOOMe

OH

OMe

OMe

MeO

7c

5c(i)

The compounds prepared have exhibited significant anticancer activity against

MCF7 and PC3 cancer cell lines with GI50 values ranging from <0.1 to >100 M

concentration. Amongst all the hybrids, the two promising compounds 8a and 9a

have been evaluated for cell cycle effects on MCF7 cell lines. The compounds 8a and

9a have shown more cells in sub G1 phase indicating cell death. Further biological

studies related to mechanism aspects are in progress (Manuscript under

preparation)

32

Scheme 3. Reagents and conditions: (i) 1,5-dibromo pentane, K2CO3, DMF, rt, 24 h; (ii) N-boc piperazineK2CO3, DMF, rt, 24 h; (iii) TFA, CH2Cl2, rt, 12 h; (iv) 10b,c, K2CO3, DMF, rt, 24 h.

ON

Z

OMe

OMe

MeO

MeO

OH

(i)

7b,cOMe

7b: Z = CH27c: Z = CH

ON

Z

OMe

OMe

MeO

MeO

O

10b,c

OMeBr( )

m

13a,b

(ii)5c

( )n

11 12

(iii)

For 13a: Z = CH2For 13b: Z = CH

n

m = 3 and n = 3

N

N

OO N NBoc ( )

n

N

N

OO N NH

(iv)ON

Z

OMe

OMe

MeO

MeO

OOMe

( ) N N ( )m

m = 3

O

N

N

O

In this study, by employing hybridization concept, we have identified potent

anticancer compounds that are more efficient than their individual parent moieties.

Hence, this concept might through more promising for the development of lead

molecules.