Synthesis of new homochiral bispyrrolidines as potential DNA cross-linking antitumour agents.

We are seeking to develop more effective bifunctional alkylating agents as antitumour agents. We previously synthesized conformationally restricted nitrogen mustards containing one piperidine ring, then bispiperidine derivatives were designed and prepared with varying lengths of carbon chain between the two rings and structure-activity relationships in these systems were studied. A bispiperidine with the shortest bridge of two carbon atoms was the most reactive bifunctional alkylating agent. In order to extend this work and investigate the effects of a change in the size of the heterocyclic systems, new bispyrrolidine salts 17-23 with chloromethyl groups at the 2-positions and a bridge between the two nitrogen atoms of 2-8 carbon atoms were synthesized from L-proline so that only the LL-enantiomers were produced. The free bases were designed to be bifunctional alkylating agents via aziridinium ion formation with different distances between the two alkylating sites. All of the bispyrrolidines were efficient cross-linkers of naked DNA apart from those with three-carbon (18) and four-carbon (19) bridges, in contrast to the results with the bispiperidines. A piperazine derivative 24 with two potential alkylating sites was also shown to be an efficient cross-linker, as was an alicyclic compound 25 with six carbon atoms between the two alkylating sites. Compounds 26 and 30 with an extra carbon atom between the nitrogen and the leaving group were not cross-linkers, as expected if aziridinium ion formation is crucial for cross-linking ability. The preformed aziridine 27 with a further alkylating site was an efficient cross-linker. Compounds 28-29 with only one potential alkylating centre were not cross-linkers of DNA. None of the compounds, however, produced significant cytotoxicity in human tumour cells in vitro.

Cross-linking and sequence-specific alkylation of DNA by aziridinylquinones. 3. Effects of alkyl substituents.

The cytotoxicities and DNA cross-linking abilities of several alkyl-substituted diaziridinylquinones have been investigated. The cytotoxicities were determined in DT-diaphorase-rich (H460 and HT29) and -deficient (H596 and BE) cell lines. It was shown that the cytotoxicities in these cell lines correlated with the relative rates of reduction by the purified human enzyme and with the cross-linking efficiencies. The rates of reduction by DT-diaphorase were more dependent on the structures of the compounds than the reduction potentials, as determined by cyclic voltammetry. A computer model was also used to explain high efficiency of cross-linking and the GNC sequence selectivity of the reduced methyl-substituted diaziridinylquinones.

DNA alkylation and interstrand cross-linking by treosulfan.

The anti-tumour drug treosulfan (L-threitol 1,4-bismethanesulphonate, Ovastat) is a prodrug for epoxy compounds by converting non-enzymatically to L-diepoxybutane via the corresponding monoepoxide under physiological conditions. The present study supports the hypothesis that this conversion of treosulfan is required for cytotoxicity in vitro. DNA alkylation and interstrand cross-linking of plasmid DNA is observed after treosulfan treatment, but this is again produced via the epoxide species. Alkylation occurs at guanine bases with a sequence selectivity similar to other alkylating agents such as the nitrogen mustards. In treosulfan-treated K562 cells, cross-links form slowly, reaching a peak at approximately 24 h. Incubation of K562 cells with preformed epoxides shows faster and more efficient DNA cross-linking.

Synthesis of new bifunctional compounds which selectively alkylate guanines in DNA.

The aim of this work was to develop new bifunctional alkylating agents which damage DNA in a selective manner. In order to extend our previously published work on conformationally restricted nitrogen mustards containing one piperidine ring, new bispiperidine derivatives were designed with varying lengths of carbon chain between the two rings and structure-activity relationships in these systems were studied. Thus samples of new bispiperidine salts 22-26 with chloromethyl groups at the 2-positions and a bridge between the two nitrogen atoms of 2-6 carbon atoms were synthesized. The analogous new bis(p-nitrophenylcarbamates) 17-21 were also prepared. The free bases were designed to be bifunctional alkylating agents via aziridinium ion formation with different distances between the two alkylating sites. The bispiperidines 22-24 were shown to alkylate guanines at the 7-position in the major groove of DNA more selectively than melphalan. The bispiperidine 22 with the shortest two carbon bridge was the most reactive but it was less cytotoxic than melphalan in a human colon carcinoma cell line (IC50 value approximately 30 microM) and in a human chronic myeloid leukaemia cell line (IC50 value approximately 12 microM). The most cytotoxic compound in the latter cell line was the carbamate 17, with an IC50 value of approximately 0.3 microM, and carbamates 17, 19 and 20 were most potent in a panel of human ovarian carcinoma cell lines. These compounds also showed circumvention of acquired cisplatin resistance in three paired cell lines. The carbamates 17-21, however, were less efficient at alkylating and cross-linking naked DNA than the bispiperidines 22-26.

A monofunctional derivative of melphalan: preparation, DNA alkylation products, and determination of the specificity of monoclonal antibodies that recognize melphalan-DNA adducts.

Bifunctional alkylating agents, such as those based on nitrogen mustard, form important parts of many anti-cancer chemotherapy protocols and are responsible for increased incidences of secondary tumors in successfully treated patients. These drugs generally form a majority of monofunctional DNA adducts, although the bifunctional adducts appear to be necessary for their powerful cytotoxic and antitumor effects. The relative importance of bifunctional as opposed to monofunctional adducts in the varied biological consequences of drug exposure has not been studied in detail, particularly in relation to the role and specificity of biochemical responses to therapy-related DNA damage. A simple method is described for the preparation of useful quantities of a pure monofunctional derivative of the nitrogen mustard-based drug melphalan. Monohydroxymelphalan was prepared by partial hydrolysis, purified by reversed phase chromatography, and characterized by MS, NMR, and HPLC. Contamination with melphalan was

Cross-linking and sequence specific alkylation of DNA by aziridinyl quinones. 2. Structure requirements for sequence selectivity.

The cytotoxicities and DNA sequence selectivity for guanine-N7 alkylation of 22 mono- and disubstituted 2,5-diaziridinyl-1,4-benzoquinones have been investigated. Several quinones produced patterns of alkylation following reduction with a selectivity for 5'-TGC-3' sequences. This sequence selectivity appeared to be dependent only on the presence of a hydrogen in position-6 of the quinone. A computer model, based on published crystallographic data, was used to explain this selectivity. The sequence selective quinones were generally more cytotoxic that the quinones which reacted randomly.

Cross-linking and sequence specific alkylation of DNA BY aziridinylquinones. 1. Quinone methides.

The cytotoxicities and DNA cross-linking abilities of 16 1,4-benzoquinones have been investigated. All of the alkylmonoaziridinyl-1,4-benzoquinones were able to interstrand crosslink DNA after reduction and were cytotoxic in vitro. Compounds lacking an aziridine group were unable to cross-link DNA and were less cytotoxic. The methyl analogues were shown to preferentially react at TGC sequences. From comparing the structural requirements for crosslinking and the cytotoxicities, a mechanism has been proposed wherein some hydroquinones can associate and react at TGC sequences in DNA. These hydroquinones can subsequently autoxidize to form a reactive quinone methide which reacts at the opposite strand to form a cross-link.