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Abstract: New A-C8 and C-C2-linked 6-chloropurine-pyrrolo[2,1-c][1,4]benzodiazepine hybrids have been prepared and evaluated for their anticancer potential.
596

Letters in Drug Design & Discovery, 2007, 4, 596-604

Design, Synthesis and Biological Activity of A-C8/C-C2-Linked 6-Chloropurine-Pyrrolobenzodiazepine Hybrids as Anticancer Agents Ahmed Kamal*,1, N. Shankaraiah1, K. Laxma Reddy1, V. Devaiah1, Aarti Juvekar2 and Subrata Sen2 1

Biotransformation Laboratory, Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 007, India 2

Advanced Centre for Treatment, Research & Education in Cancer, Navy Mumbai, India Received September 20, 2007: Revised October 25, 2007: Accepted October 26, 2007

Abstract: New A-C8 and C-C2-linked 6-chloropurine-pyrrolo[2,1-c][1,4]benzodiazepine hybrids have been prepared and evaluated for their anticancer potential. The molecular modeling studies might be explained in terms of effect of the molecule on binding in the minor groove of DNA and also comparison to both A-C8/C-C2-position of the PBD. These PBD conjugates have shown DNA-binding ability and promising anticancer activity.

Keywords: Pyrrolo[2,1-c][1,4]benzodiazepines, 6-chloropurine nucleosides, DNA-binding affinity, Anticancer activity. INTRODUCTION The biologically important 6-chloropurine derivatives (1a-d) displayed more pronounced inhibition on the majority of cell lines. In particular, the A’-alkylidenebutenolide [1,2a,b], is a useful entity that is present in natural products such as fibrolides [3], dihydroxerulin [4], and protoanemonin [5]. Protoanemonin, its analogues, and its derivatives possess antiviral, antibiotic, and anticancer activities [5,6]. The chloro-substituted purine derivatives also selective adenosine receptor agonists for the A3AR are potentially useful for the treatment of stroke, neurodegenerative diseases, myocardial infarction, and cancer [7-11]. These also shows CDK inhibition property, like several 2,6,9-trisubstituted purine derivatives such as olomoucine (2a) [12], i-propyl-olomoucine, bohemine, R-roscovitine [13], H717 [14], and purvalanol B [15]. One of the derivative, i.e., R-roscovitine (2b, CYC202) has been approved for phase-II of clinical trials as an anticancer agent [16,17]. The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are naturally occurring antitumour antibiotics that have generated interest as potential anticancer and gene-targeting agents [18]. These compounds are a family of tricyclic low molecular weight DNA-interactive agents isolated from various Streptomyces species [19], well known examples include anthramycin, sibiromycin and DC-81 (3) (Fig. 1). These molecules are known to exert their cytotoxic effect by covalently binding to the C2-amino of guanine residues within the minor groove of DNA. Bonding occurs in sequence specific fashion with a preference for Pu-G-Pu motifs. Extensive studies have been carried out in both the solution [20] and solid phase [21] synthesis of PBDs, and a sound understanding of structure-activity relationships within the family has been developed [22]. Recently, Thurston and co-workers [23a] have reported A-C8/C-C2 amido-linked PBD dimers with marginal cross-linking ability. Recently, A-C8/C-C2*Address correspondence to this author at the Biotransformation Laboratory, Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 007, India; Tel: +91-40-27193157; Fax: +91-4027193189; E-mail: [email protected] 1570-1808/07 $50.00+.00

alkoxyamido-linked PBD dimers [23b,c] have been synthesized in this laboratory to explore their DNA-binding ability. This is in continuation to our efforts towards the structural modifications of PBD ring system apart from the development of new synthetic strategies [24] for the preparation of this ring system. In this context new A-C8 and C-C2linked 6-chloropurine-pyrrolobenzodiazepine hybrids have been designed, synthesized to explore their DNA-binding ability and anticancer activity. In view of that, our interest is to combine purine nucleoside moiety to A-C8 and C-C2position of pyrrolo[2,1-c][1,4]benzodiazepines (PBDs). These 6-chloropurine-PBD hybrids (4a,b and 5a,b) have shown enhanced DNA-binding ability and anticancer activity. RESULTS AND DISCUSSION Chemistry Synthesis of A-C8-linked 6-chloropurine-PBD hybrids have been carried out by employing 4-benzyloxy-5methoxy-2-nitrobenzoic acid (6) as the starting material, which has been obtained by the procedure described in the literature [25]. L-Proline methylester hydrochloride has been coupled to compound 6 to give the nitro ester 7. The ester group reduced with DIBAL-H followed by protection with EtSH/TMSCl, then debenzylation with BF3. OEt2/EtSH to afford the debenzylated diethylthioacetal 10. Etherification of Boc-protected various amino bromoalkanes followed by Boc-deprotection with TFA to afford the PBD precursors (12a,b). These compounds have been coupled [26] with 5amino-4,6-dichloropyrimidine in the presence of triethylamine followed by cyclization with triethylorthoformate to afford 14a,b. These 6-chloropurine-substituted nitrodithioacetal intermediates (14a,b) upon reduction with SnCl2 . 2H2O in methanol gives the aminothioacetal precursors 15a,b and these on deprotective cyclization employing HgCl2/CaCO3 affords the desired A-C8-linked 6chloropurine-PBD hybrids (4a,b) (Scheme 1).

© 2007 Bentham Science Publishers Ltd.

Design, Synthesis and Biological Activity

Letters in Drug Design & Discovery, 2007, Vol. 4, No. 8

Ph

Cl 1' N

7' 5' N

6'

597

Ph NH

NH

8'

2'

N 4' N 9' 3' 6

N

N O

4

1 O

5 3 R2O

HN

2 OR1

1a: R1 = R2 = Me 1b: R1 = Me, R2 = 4-(NO2)Bzl 1c: R1 = 4-(NO2)Bzl, R2 = Me 1d: R1 = R2 = 4-(NO2)Bzl

H3CO 7 Cl

10 N

9 8

A 5 O

3

N

N

O

11 H 1

N C 4 3

2 N

R1

H

n

N

H3CO

H N

R2

N

4a: n = 2 4b: n = 3

N

N

2b

B 11a

6

N N

N H

2a

OH HO

HO

N

N

N

N

N N

O R1,R2

O

5a: = OCH3 5b: R1 = OBn, R2 = OCH3

Cl N

N

Fig. (1). Chemical structures of purine nucleoside derivatives (2b, CYC202), DC-81 (3) 6-chloropurine-PBD hybrids (4a,b and 5a,b). 6

i

7 ii 8 iii 9 BnO

iv

NO2

NO2

HO

10

O 10

NO2 CH(SEt)2

O n

N

H3CO

N

H3CO 6

H N

Boc

v

OH

H3CO

CH(SEt)2

O

11a: n = 2 11b: n = 2

O

11a,b

vi

12a,b vii Cl

N

NH2 NO2

O

N

N

14a,b

N

N

N

N

ix

O

CH(SEt)2

N

13a,b

NH2

CH(SEt)2 x

n

15a,b

NO2

O

O N N

N N

N

H3CO

O

H3CO

N

Cl N

N

H N n

viii

H3CO Cl

CH(SEt)2

n

N

Cl

13a,b

O

N

H

n N

H3CO

O 4a: n = 2 4b: n = 3

O

Scheme 1. Reagents and conditions: (i) (a) SOCl2, C6H6, 1-3 drops DMF, rt, 2 h; (b) Et3N, L-proline methylester hydrochloride, THF-H2O, 0 o C-rt, 2 h, 85%; (ii) DIBAL-H, CH2Cl2, -78 oC, 1 h, 71%; (iii) EtSH/TMSCl, CH2Cl2, rt, 8 h, 80%; (iv) BF3. OEt2, EtSH, CH2Cl2, rt, 12 h, 75%; (v) Boc-protected aminobromoalkanes, K2CO3, DMF, 48 h, rt; (vi) TFA, CH2Cl2, rt, 6 h; (vii) 5-amino-4,6-dichloropyrimidine, Et3N, nBuOH, reflux, 48 h, 58%; (viii) CH(OEt)3, HCl (10%), rt, 16 h, 62%; (ix) SnCl2. 2H2O, MeOH, reflux, 6 h, 85-87%; (x) HgCl2-CaCO3, CH3CN-H2O (4:1), rt, 12 h, 68-71%.

598 Letters in Drug Design & Discovery, 2007, Vol. 4, No. 8

Kamal et al.

ii

17a,b 18a,b

R1

i

OH

R2

NO2

R1

NO2

iii

COOCH3

N

R2

O 16a: R1, R2 = OCH3 16b: R1 = OBn, R2 = OCH3

COOCH3

N

R2

OH

O

NO2

R1

19a,b

N3

O

17a,b

19a,b 19a,b

iv

20a,b v 21a,b vi

22a,b

vii NO2

R1

CH(SEt)2

NO2

R1 viii

N

R2

N

O 24a,b

NH

Cl N

CH(SEt)2

N

R2

N

23a,b

NH2

O

Cl N

23a,b

N

N

ix

R1

NH2

O 25a,b

H

x

N

R2

N

R1

CH(SEt)2

N

R2

N N

O

Cl N

N

R1,

R2

5a: = OCH3 5b: R1 = OBn, R2 = OCH3

N N

Cl N

N

Scheme 2. Reagents and conditions: (i) SOCl2, C6H6, trans- 4-hydroxy L-proline methylester hydrochloride, THF-H2O, O oC-rt, 2 h, 85%; (ii) mesyl chloride, Et3N, CH2Cl2, rt, 6 h, 85%; (iii) NaN3, DMF, 50-60 oC, 6 h, 80%; (iv) DIBAL-H, CH2Cl2, -78 oC, 1 h, 71%; (v) EtSH/TMSCl, CH2Cl2, rt, 8 h, 72%; (vi) TPP, THF-H2O, rt, 24 h, 75%; (vii) 5-amino-4,6-dichloropyrimidine, Et3N, n-BuOH, reflux, 48 h, 58%; (viii) CH(OEt)3, HCl (10%), rt,16 h, 62%; (ix) SnCl2. 2H2O, MeOH, reflux, 6 h, 85-87%; (x) HgCl2-CaCO3, CH3CN-H2O (4:1), rt, 12 h, 68-72%.

Synthesis of C-C2-linked 6-chloropurine-PBD hybrid (5a) has been carried out by employing the commercially available 4,5-dimethoxy-2-nitrobenzoic acid (16a). This has been further coupled to trans-4-hydroxy L-proline methylester hydrochloride to afford the compound 17a. Mesylation of C2-hydroxy group followed by azidation (bimolecular nucleophilic substitution reaction SN2) with NaN3 affords 19a. This upon reduction with DIBAL-H, protection with EtSH/TMSCl gives 21a. The compound 21a reduction with TPP followed by coupling with 5-amino-4,6-dichloropyrimidine in the presence of Et3N, cyclization with CH(OEt)3 affords 24a. Then subsequent reduction followed by deprotection of diethylthioacetal group affords the desired compound 5a. Compound 5b has also been obtained in the similar procedure by employing 4-benzyloxy-5-methoxy-2nitrobenzoic acid (16b) (Scheme 2).

by thermal denaturation studies using calf thymus (CT) DNA at pH 7.0, incubated at 37 oC. It is interestingly to observe that C8-linked 6-chloropurine-PBD hybrids (4a,b) elevate the helix melting temperature of CT-DNA by 2.1-2.2 oC after incubation for 18 h. In the same experiment the naturally occurring DC-81 exhibit a Tm of 0.7 oC. Further, the C-C2linked 6-chloropurine-PBD hybrids (5a,b) do not exhibit any significant variation in Tm values. These results are as shown in Table 1. Molecular Modeling Studies

DNA-Interactions: Thermal Denaturation Studies

The program GOLD (V 2.1) was used to perform molecular docking studies to understand in detail the interactions between protein and synthesized ligands. Gold is a well-known automated ligand-docking program that uses a genetic algorithm to explore the full range of ligand conformational flexibility with partial flexibility of the protein side chains [27,28].

The DNA-binding ability of these novel A-C8 and C-C2linked 6-chloropurine-PBD hybrids have been investigated

In continuation the molecular modeling of the complexes of 6-chloropurine-PBDs 4a and 5a with B-DNA duplex

Design, Synthesis and Biological Activity

Table 1.

Letters in Drug Design & Discovery, 2007, Vol. 4, No. 8

599

Thermal Denaturation Data for A-C8/C-C2-Linked 6-Chloropurine-PBD Hybrids Employing Calf Thymus (CT) DNA

PBD

 Tm (˚C)a after incubation at 37 ˚C for

[PBD]/[DNA] molar ratiob

0h

18 h

4a

1:5

0.9

2.1

4b

1:5

1.1

2.2

5a

1:5

0.0

0.5

5b

1:5

0.3

0.6

DC-81

1:5

0.7

0.7

For CT-DNA alone at pH 7.00 ± 0.01, Tm = 69.6 °C ± 0.01 (mean value from 10 separate determinations), all Tm values are ± 0.1 - 0.2 °C. For a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concentration = 100 μM and ligand concentration = 20 μM in aqueous sodium phosphate buffer [10 mM sodium phosphate + 1 mM EDTA, pH 7.00 ± 0.01]. a

b

Fig. (2). Projection diagram showing the docking of DNA with PBD hybrids 4a and 5a.

(15-mer sequence 5’-GGGGAGAGAGAGGGG-3) has been carried out. The building fitness score between DNA and PBDs of 4a and 5a is 59.64 and 54.42 k.cal/mol. It can be seen the compound 4a reduces more stability to the complex compared to the compound 5a. This property correlates with experimentally determined values of the DNA melting temperature in these complexes (Fig. 2). Molecules 4a has a binding fitness score of 5.22 k.cal/mol more than that of molecule 5a, even though mole-

cule 5a forming two hydrogen bonds. This might be explained in terms effect of the structure of the molecule on binding in the minor groove of the DNA. Molecule 5a has a rigid structure at the C-C2-position that might be creating hindrance in the formation of more stable bonding with DNA; ultimately the bonding energy is low in contrast to this. The molecule possesses a flexible bond at C8-position enabling a stronger bond with DNA. Hence it may be concluded that presence of a flexible structure at A-C8-position favors the binding ability of molecule with DNA.

600 Letters in Drug Design & Discovery, 2007, Vol. 4, No. 8

Table 2.

Kamal et al.

Percentage Inhibitiona as Compared to Control. Molar Concentrations of the Test Compounds is Taken

Cell Line and tissue of origin

Hop62 (lung)

KB, (oral)

MCF7 (breast)

Colo205 (colon)

PC3 (prostate)

Compd. (Molar conc.)

4a

4b

ADR

10-6

0.0

0.0

16.7

10

-5

11.2

2.8

26.1

10

-4

12.8

14.3

39.6

10

-6

3.5

5.0

33.0

10

-5

52.3*

50.7*

90.2*

10

-4

58.8*

61.0*

94.1*

10

-6

16.2

12.0

17.5

10-5

38.8

28.5

52.2*

10-4

50.7*

55.9*

78.6*

10

-6

4.4

3.2

37.3

10

-5

13.7

6.7

42.6

10

-4

50.9*

46.8

57.2*

10

-6

0.0

2.0

29.4

10

-5

12.8

18.2

59.9*

51.3*

55.2*

70.4*

10

-6

0.0

0.0

43.3

10

-5

3.3

2.3

68.2*

10

-4

59.7*

51.2*

78.8*

10

-6

0.5

0.6

59.2*

10

-5

12.1

18.4

89.4*

10

-4

63.9*

39.9

95.6*

10

-6

0.0

0.0

29.2

10

-5

10-4

A549 (lung)

Gurav (oral)

Zr-75-1 (breast)

A2780 (ovary)

0.4

2.4

46.4

10-4

61.4*

38.0

75.3*

10-6

0.0

0.0

20.8

10

-5

0.0

0.0

54.8*

10

-4

55.1*

18.1

83.7*

0.0

0.0

11.1

10-6 DWD (oral)

SiHa (cervix)

10

-5

1.5

2.4

35.3

10-4

77.5*

66.0*

86.3*

10-6

59.8*

37.3

83.2*

42.1

44.3

79.4*

55.2*

59.1*

81.9*

10

-5

10-4 %Inhibition values are mean of three experiments with SD  5%. % Inhibition values 50 indicate activity. ADR, adriamycine. a

*

Cytotoxicity The A-C8-linked 6-chloropurine-PBD hybrids (4a,b) have also been evaluated against eleven human tumour cell lines derived from seven cancer types. The compound exhibiting at least 50% growth inhibition at 10-5 M concentration are considered as active against the corresponding the cell lines. These compounds exhibited growth inhibition in selected human cancer cell lines and results are illustrated in

Table 2. However the compounds 5a,b does not possess growth inhibition on these cell lines. The antiproliferative activity has been assayed by using the SRB protocol as described [29,30]. CONCLUSION In conclusion, the design and synthesis of 6chloropurine-PBD hybrids linked at A-C8-position of PBD

Design, Synthesis and Biological Activity

by various alkyl amino spacers has been described. These AC8-linked 6-chloropurine-PBD hybrids have shown DNAbinding affinity and also exhibit significant anticancer activity in all the three oral cancer cell lines used in the studies. The observation strongly suggests activity of the compounds on some unidentified target selectively expressed in the oral cancer cell lines. However, the C-C2-linked 6-chloropurinePBD hybrids do not exhibit DNA-binding as well as anticancer activity. The molecular modeling studies also explain the DNA binding ability of these molecules. The detailed mechanistic studies are in progress. EXPERIMENTAL SECTION Reaction progress was monitored by thin-layer chromatography (TLC) using GF254 silica gel with fluorescent indicator on glass plates. Visualization was achieved with UV light and iodine vapor unless otherwise stated. Chromatography was performed using Acme silica gel (100-200 and 60120mesh). The majority of reaction solvents were purified by distillation under nitrogen from the indicated drying agent and used fresh: dichloromethane (calcium hydride), tetrahydrofuran (sodium benzophenone ketyl), methanol (magnesium methoxide), and acetonitrile (calcium hydride).

Letters in Drug Design & Discovery, 2007, Vol. 4, No. 8

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4.18 Hz); 4.53-4.62 (m, 1H); 4,05-4.11 (t, 2H, J = 5.86 Hz); 3.87 (s, 3H); 3.50-3.74 (m, 2H); 3.08-3.20 (m, 2H); 2.562.77 (m, 4H); 1.71-2.23 (m, 6H); 1.14-1.31 (m, 6H); FABMS: m/z 584 [M++H]. (2S)-N-{[4-(6-Chloropurinyl)ethoxy]-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehy dediethylthioacetal (14a) A mixture of 13a (340 mg, 0.59 mmol), HC(OEt)3 (7.74 mL, 52.49 mmol) and aq. HCl (12 M: 0.15 mL) were stirred at room temperature for 16 h. The solvent was removed then the residue was dissolved in THF (20 mL) and treated with aq. HCl (0.5 M, 10 mL) at room temperature for 2 h. The reaction mixture was neutralized with aq. NaOH (0.5 M) and the solvents were evaporated in vacuo. The crude product was purified by column chromatography using ethyl acetatehexane (8:2) to give 14a (308 mg, 62%); 1H NMR (300 MHz, CDCl3):  8.72 (s, 1H); 8.40 (s, 1H); 7.61 (s, 1H); 6.79 (s, 1H); 4.75-4.83 (m, 2H); 4,57-4.61 (m, 1H); 4.41-4.45 (m, 2H); 3.92 (s, 3H); 3.12-3.26 (m, 2H); 2.65-2.82 (m, 4H); 2.21-2.33 (m, 2H); 1.91-2.00 (m, 2H); 1.78 (m, 1H); 1.261.46 (m, 6H); FABMS: m/z 581 [M++H]. (2S)-N-{[4-(6-Chloropurinyl)propoxy]-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal (14b)

H NMR spectra were recorded on Varian Gemini 200 MHz and Avance 300 MHz spectrometer using tetramethyl silane (TMS) as an internal standard. Chemical shifts are reported in parts per million (ppm) down field from tetramethyl silane. Spin multiplicities are described as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet). Coupling constants are reported in Hertz (Hz). Low-resolution mass spectra were recorded on VG-7070H Micromass mass spectrometer at 200 oC, 70 eV with trap current of 200 A and 4 KV acceleration voltage.

This compound was prepared according to the procedure described for the earlier preparation of 14a employing a mixture of 13b (330 mg, 0.42 mmol) HC(OEt)3 (6.00 mL, 37.04 mmol) and aq. HCl (12 M: 0.12 mL) was stirred at room temperature for 16 h to give 14b (300 mg, 69%); 1H NMR (300 MHz, CDCl3):  8.64 (s, 1H); 8.17 (s, 1H); 7.58 (s, 1H); 6.78 (s, 1H); 4.80-4.83 (d, 1H, J = 4.08 Hz); 4.61-4.65 (m, 1H); 4,57-4.61 (t, 2H, J = 5.35, 8.24 Hz); 4.02-4.21 (m, 2H); 3.92 (s, 3H); 3.19-3.25 (m, 2H); 2.64-2.82 (m, 4H); 2.492.59 (m, 2H); 1.98-2.31 (m, 3H); 1.68-1.89 (m, 1H); 1.251.40 (m, 6H); FABMS: m/z 595 [M++H].

(2S)-N-{[4-(5-Amino-6-chloro-4-pyrimidinyl)aminoethoxy]5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal (13a)

(2S)-N-{[4-(6-Chloropurinyl)ethoxy]-5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal (15a)

1

To a stirred solution of 12a (440 mg, 1.0 mmol) in anhydrous n-BuOH (15 mL), 5-amino-4,6-dichloropyrimidine (246 mg, 1.5 mmol) and anhydrous Et3N (0.70 mL, 5.0 mmol) were added. The reaction mixture was refluxed under nitrogen atmosphere for 48 h. The solvent was evaporated in vacuo and the crude product was purified by column chromatography using ethyl acetate-hexane (7:3) to give 13a (348 mg, 58%); 1H NMR (300 MHz, CDCl3):  7.78 (s, 1H); 7.31 (s, 1H); 6.74 (s, 1H); 5.8 (s, 1H); 5.25 (s, 2H); 4.83 (d, 1H, J = 3.71 Hz); 4.61-4.70 (m, 1H); 4,09-4.23 (m, 2H); 3.91 (s, 3H); 3.18-3.28 (m, 2H); 2.64-2.84 (m, 4H); 1.78-2.35 (m, 4H); 1.21-1.39 (m, 6H); FABMS: m/z 571 [M++H]. (2S)-N-{[4-(5-Amino-6-chloro-4-pyrimidinyl)aminopropoxy]5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal (13b) This compound was prepared according to the procedure described for the earlier preparation of 13a employing 12b (450 mg, 1.0 mmol) in anhydrous BuOH (15 mL), 5-amino4,6-dichloropyrimidine (246 mg, 1.5 mmol) and anhydrous Et3N (0.70 mL, 5.0 mmol) to give 13b (339 mg, 58%); 1H NMR (300 MHz, CDCl3):  7.84 (s, 1H); 7.47 (s, 1H); 6.66 (s, 1H); 5.89-5.92 (t, 1H, J = 5.02 Hz); 4.71-4.73 (d, 1H, J =

The compound 14a (300 mg, 0.54 mmol) dissolved in MeOH (20 mL), SnCl2. 2H2O (613 mg, 2.71 mmol) was added and refluxed for 6 h or until the TLC indicated that reaction was complete. The methanol was evaporated by vacuum and the aqueous layer was then carefully adjusted to pH 8 with 10% NaHCO3 solution and separated the tin salts through celite. This upon extracted with ethyl acetate (3x30 mL). The combined organic phase was dried over anhydrous Na2SO4 and evaporated under vacuum to afford the amino diethyl thioacetal 15a (241 mg, 85%). (2S)-N-{[4-(6-Chloropurinyl)propoxy]-5-methoxy-2-aminobenzoyl}pyrrolidine- 2- carboxaldehyde diethylthioacetal (15b) The compound 15b was prepared according to the method described for the compound 15a employing the compound 14b (295 mg, 0.52 mmol) and SnCl2. 2H2 O (0.586 g, 2.60 mmol) to afford the amino diethyl thioacetal 15b (228 mg, 87%). (11aS)-N-{[4-(6-Chloropurinyl)ethoxy]-5-methoxy-1,2,3,11atetrahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5-one (4a) A solution of 15a (241 mg, 0.43 mmol), HgCl2 (295 mg, 1.09 mmol) and CaCO3 (109 mg, 1.09 mmol) in CH3CN/H2O (4:1, 15 mL) was stirred at room temperature

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for 12 h until TLC (ethyl acetate), indicates complete loss of starting material. The reaction mixture was diluted with EtOAc (20 mL) and filtered through a celite bed. The clear yellow organic supernatant was extracted with saturated 5% NaHCO3 (20 mL) and the combined organic phase was dried in Na2SO4. The organic layer was evaporated in vacuum and purified by column chromatography (95% EtOAc-MeOH) to afford the compound 4a (110 mg, 68%); 1H NMR (200 MHz, CDCl3)  8.69 (s, 1H); 8.45 (s, 1H); 7.61 (d, 1H, J = 4.34 Hz); 7.46 (s, 1H); 6.66 (s, 1H); 4.76-4.80 (m, 2H); 4.244.50 (m, 2H); 3.91 (s, 3H); 3.42-3.82 (m, 3H); 2.20-2.36 (m, 2H); 1.89-2.18 (m, 2H); FABMS: m/z 427 [M++H].

at room temperature for 16 h. The solvent was removed then the residue was dissolved in THF (20 mL) and treated with aq. HCl (0.5 M: 10 mL) at room temperature for 2 h. The mixture was neutralized with aq. NaOH (0.5 M) and the solvents were evaporated in vacuo. The crude product thus obtained was purified by column chromatography using ethyl acetate-hexane (8:2) to give 24a (311 mg, 62%); 1H NMR (200 MHz, CDCl3)  8.70 (s, 1H); 8.20 (s, 1H); 7.64 (s, 1H); 6.90 (s, 1H); 5.09-5.19 (m, 2H); 4.92-4.99 (m, 1H); 3.99 (s, 3H); 3.96 (s, 3H); 3.80-3.88 (m, 1H); 3.01-3.06 (m, 1H); 2.70-2.81 (m, 6H) 1.38-1.44 (m, 6H); FABMS: m/z 567 [M++H].

(11aS)-N-{[4-(6-Chloropurinyl)propoxy]-5-methoxy-1,2,3, 11a-tetrahydro-5H-pyrrolo[2,1-c]-[1,4]benzodiazepin-5-one (4b)

(2S,4S)-N-4[(6-Chloropurinyl]-5-benzyloxy-4-methoxy-(2nitrobenzoyl)pyrrolidine-2-carboxaldehyde diethylthioacetal (24b)

The compound 4b was prepared according to the method described for the compound 4a employing 15b (247 mg, 0.43 mmol), HgCl2 (295 mg, 1.09 mmol) and CaCO3 (109 mg, 1.09 mmol) to afford 4b (100 mg, 70%); 1H NMR (200 MHz, CDCl3)  8.65 (s, 1H); 8.11 (s, 1H); 7.60 (d, 1H, J = 4.32 Hz); 7.45 (s, 1H); 6.67 (s, 1H); 4.48-4.56 (t, 2H, J = 5.08 Hz); 4.01-4.11 (m, 1H); 3.88 (s, 3H); 3.45-3.78 (m, 4H); 2.40 (m, 2H); 2.25 (m, 2H); 1.95 (m, 2H); FABMS: m/z 441 [M++H]. (2S,4S)-N-4[(5-Amino-6-chloro-4-pyrimidinyl)amino]-4,5dimethoxy-(2-nitrobenzoyl)pyrrolidine-2-carboxaldehyde diethylthioacetal (23a) To a stirred solution of 22a (500 mg, 1.16 mmol) in anhydrous n-BuOH (15 mL), 5-amino-4,6-dichloropyrimidine (286 mg, 1.74 mmol) and anhydrous Et3N (0.80 mL, 5.80 mmol) were added. The reaction mixture was refluxed under nitrogen atmosphere for 48 h. The solvent was evaporated in vacuo and the crude product was purified by column chromatography using ethyl acetate-hexane (7:3) to give 23a (376 mg, 58%); 1H NMR (200 MHz, CDCl3)  7.95 (s, 1H); 7.68 (s, 1H); 6.72 (s, 1H); 5.48-5.52 (t, 1H, J = 9.67 Hz); 4.94 (d, 1H, J = 2.54 Hz); 4.75-4.86 (m, 2H); 4.56-4.68 (m, 1H); 3.98 (s, 3H); 3.96 (s, 3H); 3.68-3.78 (m, 1H); 3.45 (brs, 2H); 3.08-3.16 (m, 1H); 2.70-2.80 (m, 4H) 2.01-2.10 (m, 1H); 1.25-1.43 (m, 6H); FABMS: m/z 557 [M++H]. (2S,4S)-N-4[(5-Amino-6-chloro-4-pyrimidinyl)amino]-5benzyloxy-4-methoxy-(2-nitrobenzoyl)pyrrolidine-2-carboxaldehyde diethylthioacetal (23b) This compound was prepared according to the procedure described for the earlier preparation of 23a employing 22b (500 mg, 0.99 mmol) in anhydrous n-BuOH (15 mL), 5amino-4,6-dichloropyrimidine (243 mg, 1.48 mmol) and anhydrous Et3N (0.68 mL, 4.95 mmol) to give 23b (376 mg, 60%); 1H NMR (200 MHz, CDCl3)  7.91 (s, 1H); 7.72 (s, 1H); 7.33-7.42 (m, 5H); 6.78 (s, 1H); 5.43-5.53 (t, 1H, J = 8.65 Hz); 5.14 (s, 2H) 4.91 (d, 1H, J = 2.67 Hz); 4.72-4.82 (m, 1H); 4.52-4.62 (m, 1H); 3.97 (s, 3H); 3.62-3.78 (m, 1H); 3.45 (brs, 2H); 3.05-3.14 (m, 1H); 2.62-2.90 (m, 4H); 2.012.08 (m, 2H); 1.37-1.41 (m, 6H); FABMS: m/z 633 [M++H]. (2S,4S)-N-4[(6-Chloropurinyl]-4,5-dimethoxy-(2-nitrobenzoyl)pyrrolidine-2-carboxaldehyde diethylthioacetal (24a) A mixture of 23a (360 mg, 0.64 mmol), HC(OEt)3 (8.4 mL, 56.87 mmol) and aq. HCl (12 M: 0.15 mL) were stirred

This compound was prepared according to the procedure described for the earlier preparation of 24a employing a mixture of 23b (360 mg, 0.56 mmol), HC(OEt)3 (7.4 mL, 50.04 mmol) and aq. HCl (12 M: 0.15 mL) to give 32b (330 mg, 60%); 1H NMR (200 MHz, CDCl3)  8.71 (s, 1H); 8.18 (s, 1H); 7.70 (s, 1H); 7.32-7.41 (m, 5H); 6.78 (s, 1H); 5.18 (s, 2H); 4.90-5.00 (m, 2H); 3.98 (s, 3H); 3.80-3.89 (m, 2H); 2.62-3.08 (m, 5H); 2.01-2.08 (m, 2H); 1.36-1.42 (m, 6H); FABMS: m/z 643 [M++H]. (2S,4S)-N-4(6-Chloropurinyl)-4,5-dimethoxy-(2-aminobenzoyl)pyrrolidine-2-carboxaldehyde diethylthioacetal (25a) The compound 24a (300 mg, 0.47 mmol) dissolved in MeOH (20 mL) and added SnCl2. 2H2O (533 mg, 2.36 mmol) was added and refluxed for 6 h or until the TLC indicated that reaction was complete. The methanol was evaporated by vacuum and the aqueous layer was then carefully adjusted to pH 8 with 10% NaHCO3 solution then filtered through celite. This upon extracted with ethyl acetate (3x30 mL), the combined organic phase was dried over anhydrous Na2SO4 and evaporated under vacuum to afford the amino diethyl thioacetal 25a (221 mg, 85%). (2S,4S)-N-4(6-Chloropurinyl)-5-benzyloxy-4-methoxy-(2aminobenzoyl)pyrrolidine-2-carboxaldehyde diethylthioacetal (25b) The compound 25b was prepared according to the method described for the compound 25a employing the compound 24b (320 mg, 0.49 mmol) and SnCl2. 2H2O (561 mg, 2.48 mmol) to afford the amino diethyl thioacetal 25b (216 mg, 86%). (4S,11aS)-N-4-(6-Chloropurinyl)-4,5-dimethoxy-1,2,3,11atetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (5a) A solution of 25a (221 mg, 0.41 mmol), HgCl2 (278 mg, 1.02 mmol) and CaCO3 (102 mg, 1.02 mmol) in CH3CN/H2O (4:1, 25 mL) was stirred at room temperature for 12 h until TLC (ethyl acetate), indicates complete loss of starting material and purified by column chromatography (90% EtOAc-MeOH) to afford the compound 5a (102 mg, 68%); 1H NMR (200 MHz, CDCl3)  8.58 (s, 1H), 8.01 (s, 1H), 7.60 (d, 1H, J = 4.16), 7.52 (s, 1H), 6.82 (s, 1H), 5.085.32 (m, 1H), 4.22-4.54 (m, 1H), 3.75-3.89 (s, 6H), 3.013.18 (m, 2H); 1.45 (m, 2H); FABMS: m/z 413 [M++H].

Design, Synthesis and Biological Activity

Letters in Drug Design & Discovery, 2007, Vol. 4, No. 8

(4S,11aS)-N-4-(6-Chloropurinyl)-4-benzyloxy-5-methoxy1,2,3,11a-tetrahydro-5H-pyrrolo[2,-1-c][1,4]benzodiazepin5-one (5b) The compound 5b was prepared according to the method described for the compound 5a employing 25b (216 mg, 0.35 mmol), HgCl2 (238 mg, 0.88 mmol) and CaCO3 (88 mg, 0.88 mmol) to afford 5b (108 mg, 70%); 1H NMR (200 MHz, CDCl3)  8.59 (s, 1H); 8.00 (s, 1H); 7.62 (d, 1H, J = 4.18); 7.52 (s, 1H); 7.33-49 (m, 5H); 6.88 (s, 1H); 5.12 (s, 2H); 5.14-5.33 (m, 1H); 4.21-4.53 (m, 3H); 3.96 (s, 3H); 3.023.19 (m, 2H); FABMS: m/z 489 [M++H]. Anticancer (SRB) Assay The cell lines have grown in RPMI 1640 medium containing 10% fetal bovine serum and 2 mM L-glutamine and have inoculated into 96 well micro titer plates in 90 L at plating densities depending on the doubling time of individual cell lines. The micro titer plates have incubated at 37 °C, 5% CO2, 95% air and 100% relative humidity for 24 h prior to addition of experimental drugs. Aliquots of 10 L of the drug dilutions have added to the appropriate micro titer wells already containing 90 L of cells, resulting in the required final drug concentrations. Plates have incubated for further 48 h and assay has terminated by the addition of 50 L of cold TCA. (final concentration, 10% TCA) and has incubated for 60 minutes at 4 °C. The plates have washed five times with tap water and air-dried. Sulforhodamine B (SRB) solution (50 L) at 0.4% (w/v) in 1% acetic acid was added to each of the wells, and plates has incubated for 20 minutes at room temperature. The residual dye has removed by five times washing with 1% acetic acid. The plates have air-dried. Bound stains subsequently eluted with 10 mM trizma base, and the absorbance has read on an ELISA plate reader at a wavelength of 540 nm with 690 nm reference wavelengths. Percent growth has calculated on a plate-by-plate basis for test wells relative to control wells. Percent Growth as expressed as the (ratio of average absorbance of the test well to the average absorbance of the control wells) x100. ACKNOWLEDGEMENTS

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We are thankful to the Department of Biotechnology (BT/PR/7037/Med/14/933/2006), New Delhi for financial assistance. Three of the authors N. S., K. L. R. and V. D. are thankful to CSIR, New Delhi for the award of research fellowships. REFERENCES [1] [2]

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