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有英文文献好多求找有关1,4-苯并二氮杂卓类衍生物的合成方法并翻译
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Synthesis and DNA binding affinity of novel A-C8/C-C2-exo unsaturated alkoxyamido-linked pyrrolo[2,1-c][1,4]benzodiazepine dimers Ahmed Kamal,* O. Srinivas, P. Ramulu, G. Ramesh, P. Praveen Kumar and M. Shiva Kumar Biotransformation Laboratory, Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500007, India Received 17 May 2004; revised 10 June 2004; accepted 11 June 2004 Available online 3 July 2004 Abstract—The synthesis of novel A-C8/C-C2-exo unsaturated alkoxyamido-linked pyrrolo[2,1-c][1,4]benzodiazepine dimers is reported and these dimers show significant DNA binding affinity and they also exhibit moderate anticancer activity. 2004 Elsevier Ltd. All rights reserved. 1. Introduction In the last decade there has been increasing interesttowards the synthesis of DNA sequence selective agents, particularly low molecular weight antitumour antibiotics. Pyrrolo[2,1-c][1,4]benzodiazepines are naturally occurring antitumour antibiotics that interact within the minor groove of DNA forming monocovalent adducts.1 Recently, there has been tremendous interest in the design and synthesis of DNA interstrand cross-linking agents that are likely to enhance the sequence selectivity and in turn could increase selectivity for tumour cells.2 In this context a large number of PBD dimers have been designed and synthesized with a view to explore their DNA cross-linking ability and their sequence selectivity. 3 These PBD dimers have been joined through different positions such as A-C7/A-C70, A-C8/A-C80 (3) and C-C2/C-C20 (2), amongst these A-C8/A-C80 linked PBD dimers have shown promising cytotoxicity and efficient cross-linking property.4 Further it is observed from the literature that C2-exo unsaturation as in case of tomaymycin significantly enhances the anticancer potential and DNA binding ability5 in comparison to non-C2-exo unsaturation (DC-81) and this feature has been extensively investigated for many synthetic analogues.6 Thurston and co-workers7 have developed new pyrrolobenzodiazepine dimers with remarkable DNA crosslinking ability. Moreover, the same group has reported the first example of A-C8/C-C2 amido-linked PBD dimer8 (4) and recently we have reported A-C8/C-C2 alkoxyamido-linked tail to head type PBD dimer (5) with significant DNA binding ability and low anticancer activity.9 Therefore, introduction of C2-exo unsaturation is an important component that probably is causing the flattening of the C-ring to produce an improved isohelical fit within the DNA minor groove.10 In view of these findings and in conjunction with our pursuit towards the design and synthesis of PBD hybrids,11 we have considered the synthesis of A-C8/C-C2-exo unsaturated alkoxyamido linked PBD dimers (6a–b and 7a–b) as a significant and important aspect in the development of novel PBD dimers with potential anticancer activity and DNA binding ability. Further, we have also prepared such PBD dimers (8a–b) with amide functionality at N10–C11position of A-C8 component to investigate the effect of the absence of one of the imine functionality on the DNA binding profile. 2. Results and discussion 2.1. Synthesis Synthesis of A-C8/C-C2-exo unsaturated alkoxyamidolinked PBD dimers 6a–b has been carried out by Keywords: Pyrrolobenzodiazepine dimers; DNA binding affinity; Cytotoxicity. * Corresponding author. Tel.: +91-40-27193157; fax: +91-40-271931- 89; e-mail: ahmedkamal@iict.res.in 0968-0896/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2004.06.013 Bioorganic & Medicinal Chemistry 12 (2004) 4337–4350 amidation of the A-C8 alkoxyamine components (14a–b) and the C-C2-exo unsaturated acid component (22a). The C8 alkoxyamine has been synthesized in the following manner; the precursor methyl 4-benzyloxy-5- methoxy-2-nitrobenzoate 9 has been prepared by employing the literature method,12 which upon debenzylation with BF3ÆOEt2–EtSH gives compound 10 and etherification with Boc-protected bromoalkylamines provide 11a–b. These compounds have been hydrolyzed and coupled with (2-S)-pyrrolidinecarboxaldehyde diethyl thioacetal to give 13a–b, which upon deprotection provide the desired intermediate amines 14a–b (Scheme 1). The other precursor C2-exo unsaturated acid (22a) has been prepared by employing 4-benzyloxy-5-methoxy- 2-nitrobenzoic acid 15a as the starting material, which has been obtained by the procedure described in the literature.12 trans-4-Hydroxy-L-proline methyl ester hydrochloride has been coupled to compound 15a to give the nitro ester 16a. The hydroxy group is protected with TBDMS-Cl followed by reduction with DIBAL-H to produce the corresponding aldehyde, which is protected with EtSH/TMS-Cl. Interestingly, in this reaction protection of aldehyde to diethyl thioacetal and deprotection of TBDMS takes place in the same step to afford compound 19a. Then C2-hydroxy group is oxidized with TPAP/NMO to give compound 20a, which upon Horner–Emmons olefination with methyl diethylphosphonoacetate affords compound 21a. In this reaction, (E) ester3d (21a) has been obtained exclusively, which upon hydrolysis affords the corresponding acid 22a. The key intermediates 23a–b have been prepared by amidation of compound 22a with 14a–b. The compounds 23a–b have been reduced with SnCl2Æ2H2O to afford the corresponding amino diethyl thioacetal (25a– b). Deprotection of amino diethyl thioacetal with HgCl2/ CaCO3 provides the target molecules 6a–b (Scheme 2). The compounds 7a–b have been prepared in the same manner by employing with commercially available 4,5-dimethoxy-2-nitrobenzoic acid (Scheme 2). The compounds 30a–b have been prepared by amidation of compounds 22a and 29a–b, then subsequent reduction followed by deprotection of diethyl thioacetal group affords the desired compounds 8a–b (Scheme 3). 2.2. DNA interactions: thermal denaturation studies The DNA binding ability for these A-C8/C-C2-exo unsaturated alkoxyamido-linked PBD dimers has been determined by thermal denaturation studies using calf thymus (CT) DNA. Interestingly, in this assay four of these PBD dimers that is, 6a,b, 7a and 7b elevate the helix melting temperature of CT-DNA by a 5.7, 8.3, 5.6 and 6.4 C, respectively, after incubation for 18 h at 37 C. On the other hand, the A-C8/A-C80 PBD dimer, DSB-120 exhibits a DTm of 15.3 C after 18 h of incubation. Further, the values of Tm enhances with the increase of incubation time. This demonstrates that these PBD dimers show significant DNA binding affinity but not as high as DSB-120. On the contrary, the imine– amide dimers (8a and 8b) do not exhibit significant DNA binding ability (Table 1). 2.3. Cytotoxicity Compounds 6a and 8b have been evaluated for their in vitro cytotoxicity against 60 human tumour cell lines derived from nine cancer types (leukaemia, nonsmall cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer). According to the in vitro screening data from the National Cancer Institute (NCI) initial primary test, the compounds 6a and 8b have cytotoxic potency against many cell lines. Compound 6a exhibits a wide spectrum activity against 32 cell lines in nine cell panels, with GI50 values of <10 lM, whereas compound 8b exhibits activity against 24 cell lines in seven cancer cell panels, with GI50 values of <50 lM. A reference compound of the same family DRG-16 ranges from 0.001 to 7.94nM (mean 0.12 nM).7 The GI50 values of compounds 6a and 8b for different cell lines have been illustrated in Table 2. O MeO NO2 N CH(SEt)2 O HN BOC BnO MeO NO2 OMe O HO MeO NO2 OMe O O MeO NO2 OMe O HN BOC n O MeO NO2 OH O HN BOC n n O MeO NO2 N CH(SEt)2 O H2N n i ii iii iv v 9 10 11a-b 12 a-b 13 a-b 14 a-b n = 2, 3 Scheme 1. Reagents and conditions: (i) EtSH–BF3OEt2, CH2 Cl2, 12 h, rt, 75%; (ii) Boc-protected bromo alkylamine, K2CO3, DMF, rt, 24 h, 85– 88%; (iii) 1N LiOH, THF–H2O–MeOH, 2 h, rt, 80–83%; (iv) 2(S)-pyrrolidinecarboxaldehyde diethyl thioacetal, EDCl–HOBt, CH2Cl2–H2O, 24 h, rt, 60–65%; (v) TFA, CH2Cl2, 8 h, rt. 4338 A. Kamal et al. / Bioorg. Med. Chem. 12 (2004) 4337–4350 2.4. RED100-restriction endonuclease digestion assay Many studies have employed restriction endonuclease inhibition to confirm the relative binding affinity of DNA-interactive small molecule ligands.13–16 A quantitative restriction enzyme digest (RED100) assay was developed in which the inhibition of DNA cleavage by BamH1 was used to probe the DNA binding capability of PBD monomers.17 We have earlier investigated this assay for preferences of base pair selectivity of the imine–amide PBD dimers.18 Recently this technique has also been used to study the covalent DNA interaction of PBD dimers and it is capable to distinguish between the monomeric and dimeric families.7 The BamH1 cleavage sequence G#GATCC overlaps with several favoured PBD binding sites suggesting ligand binding has the potential to inhibit the BamH1 cleavage activity. The study has been carried out to determine the ability of A-C8/C-C2-exo unsaturated alkoxyamido-linked PBD dimers, which inhibit the DNA linearization by BamH1. The results of this experiment for compounds 6a,b, 7a and 7b shown in Figure 2 suggest that the PBD dimers inhibit BamH1. There are differences in the inhibitory activity exhibited by PBDs evaluated in this assay. It is observed that the ranking order is 6b>7b>6a>7a for inhibition of BamH1 cleavage is in agreement with the DNA binding affinity as determined by thermal denaturation. These results clearly demonstrate that as the linker chain increases from two to three carbon spacer as in case of 6b there is an enhancement in the inhibitory activity (Fig. 1). 2.5. Conclusions The increase of alkane spacer from two to three carbons in the newly designed A-C8/C-C2-exo unsaturated |
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