Natalie Brooks

SJG-136 (NSC 694501), a Novel Rationally Designed DNA Minor Groove Interstrand Cross-Linking Agent with Potent and Broad Spectrum Antitumor Activity Part 1: Cellular Pharmacology, In vitro and Initial In vivo Antitumor Activity

SJG-136 (NSC 694501) is a rationally designed pyrrolobenzodiazepine dimer that binds in the minor groove of DNA. It spans 6 bp with a preference for binding to purine-GATC-pyrimidine sequences. The agent has potent activity in the National Cancer Institute (NCI) anticancer drug screen with 50% net growth inhibition conferred by 0.14 to 320 nmol/L (7.4 nmol/L mean). Sensitive cell lines exhibit total growth inhibition and 50% lethality after treatment with as little as 0.83 and 7.1 nmol/L SJG-136, respectively. COMPARE and molecular target analysis of SJG-136 data versus that of >60,000 compounds tested in the NCI 60 cell line screen shows that, although the agent has similarity to other DNA binding agents, the pattern of activity for SJG-136 does not fit within the clusters of any known agents, suggesting that SJG-136 possesses a distinct mechanism of action. Testing in the NCI standard hollow fiber assay produced prominent growth inhibition in 20 of 24 i.p. and 7 of 24 s.c. test combinations with 5 of 12 cell lines exhibiting cell kill. In addition, SJG-136 produced antitumor activity in mice bearing CH1 and CH1cisR xenografts, a cisplatin-resistant human ovarian tumor model, and also in mice bearing LS174T xenografts, a human colon tumor model. SJG-136 produces DNA interstrand cross-links between two N-2 guanine positions on opposite strands and separated by 2 bp. In human tumor cell lines, the cross-links form rapidly and persist compared with those produced by conventional cross-linking agents such as nitrogen mustards. In mice bearing the LS174T human colon xenograft, DNA interstrand cross-links can be detected in tumor cells using a modification of the single cell gel electrophoresis (comet) assay after administration of a therapeutic dose. Cross-links in the tumor increase with dose and are clearly detectable at 1 hour after i.v. administration. The level of cross-linking persists over a 24-hour period in this tumor in contrast to cross-links produced by conventional cross-linking agents observed over the same time period.

The role of the N-(hydroxymethyl)melamines as antitumour agents: Mechanism of action studies

The hexamethylmelamine analogue trimelamol (tris-hydroxymethyl[trimethyl]melamine) and its equicytotoxic stable analogues CB 7547, CB 7639 and CB 7669 have been used to clarify the mechanism of action for the N-(hydroxymethyl)melamines as antitumour agents. Two main mechanisms have been proposed and explored: (i) formation of a reactive iminium species forming covalent adducts with DNA; and (ii) local formaldehyde release leading to cytotoxic damage. 32P-postlabelling and thermal denaturation experiments showed these compounds to be interactive with cytosine and guanine. Trimelamol gave rise to DNA-interstrand crosslinks in naked plasmid DNA and in cultured cell lines, whereas the analogues failed to do so under a variety of experimental conditions. Along with our observations that cell lines with acquired resistance to the N-(hydroxymethyl)melamines showed no significant cross-resistance to classical bifunctional alkylating agents, DNA crosslinking may play only a minor role in their mechanism of action. In cultured cell lines treatment with formaldehyde, trimelamol and CB 7639 gave rise to high levels of DNA-protein crosslinks with a gradual disappearance over a 24 hr period. Along with our earlier observation that resistance to trimelamol coincides with cross-resistance to formaldehyde, we conclude that formaldehyde-release may be an important factor in their cytotoxicity. Further, the cytotoxicity of trimelamol or formaldehyde towards human ovarian cancer cells was not influenced by glutathione depletion. As the precise mechanism of action for the N-(hydroxymethyl)melamines is apparently not shared by many commonly used anticancer agents, this may confer their broad-spectrum activity versus heavily pretreated tumours.

EFFICIENT SYNTHESIS OF (±)-seco-CYCLOPROPANEINDOLINE ANALOGS OF CC-1065

An efficient method for the preparation of racemic seco-cyclopropaneindoline, or seco-CI, analogs of the anticancer agent CC1065 is described. The syntheses of seco-CI compounds containing either 5,6,7-trimethoxyindole-2-carbonyl, 4, or 5-(benzofuran-2carboxamido)indole-2-carbonyl, 10, or 2-(4-N,N-diethyl)aminophenyl)benzimidazole-6carbonyl, 11, or 4-(4-butanamido-l-methylpyrrole-2-carboxamido)-l-methylpyrrole-2-carbonyl, 12, subunit are detailed. At μΜ concentrations, compounds 4,10-12 inhibited the growth of human leukemic K562 cells in culture.

Stable analogues of the antitumour agent trimelamol retain in vitro cytotoxicity in drug-sensitive and resistant rodent and human cell lines

In spite of clinical activity in heavily-pretreated ovarian cancer, the antitumour s-triazine trimelamol [TM; tris(hydroxymethyl)-tris(methyl)melamine] had to be withdrawn from further clinical studies due to formulation difficulties related to instability. A synthetic programme has produced tris(hydroxymethyl) analogues containing electron-withdrawing groups in place of methyl-triscyanomethyl CB 7669, tristrifluoroethyl CB 7639, CB 7529 and trispropargyl CB 7547, all showing markedly superior stability to TM. Chemosensitivity testing of analogues (MTT assay, continuous exposure) using a panel of rodent and human cell lines showed activity close to that of TM, e.g. for the CH1 human ovarian cancer cell line. IC50 values were TM 23.4 microM, CB 7639 30.5 microM, CB 7529 29.5 microM, CB 7547 28.5 microM and CB 7669 27.3 microM. CB 7669 and CB 7639 required prolonged exposure (> 12 h) in order to exhibit equivalent cytotoxicity to a 2-h exposure to TM. Thus, rather than administration as a single daily dose, the stable analogues may be more suited to prolonged infusion, which was suggested as being a more beneficial regimen in clinical trials with TM. In line with clinical observations indicating the efficacy of TM in platinum-refractory ovarian cancer, we saw no significant cross-resistance to TM or CB 7529 in a range of platinum-sensitive and acquired-resistant cell line pairs or in an alkylating-agent resistant cell line, despite TMs ability to crosslink DNA. Data obtained using cell lines with acquired resistance to TM, CB 7669 and formaldehyde (released in the breakdown of TM) suggest a pivotal role for formaldehyde and a more minor role for alkylating activity in the mechanism of action of the N-(hydroxymethyl)melamines in vitro. Further clinical trials of these compounds are eagerly awaited, and their usefulness as second-line chemotherapy for heavily pretreated ovarian cancer deserves further investigation.

Pre-clinical development of the anti-tumour agent CB 7646, bis N-(hydroxymethyl) trimethylmelamine, a stable analogue of trimelamol

Formulation difficulties prevented the otherwise promising clinical development of the anti‐tumour agent trimelamol (TM; tris‐[hydroxymethyl]trimethylmelamine). A synthetic analogue programme resulted in the identification of CB 7646 (bis N‐[hydroxymethyl]trimethylmelamine) as a compound with improved stability and favourable formulation characteristics. The in vitro and in vivo activity of CB 7646 was shown to be very similar to that of TM. In addition, curative activities were shown in the PXN/65 human ovarian cancer xenograft and the MX‐1 and T‐61 human breast cancer xenograft models. The effectiveness of the N‐(hydroxymethyl)melamines against platinum‐refractory disease was noted in the phase I clinical trial of TM. In line with this finding, the present study confirmed the effective activity of both TM and CB 7646 against various forms of platinum resistance in in vitro models. Curative activity for TM and CB 7646 was seen in the HX110P human ovarian cancer xenograft with acquired carboplatin resistance. Animal studies indicated less neurotoxicity for CB 7646 than for TM. The pharmacokinetic profile of CB 7646 indicated a decreased plasma elimination, indicative of slower in vivo degradation than for TM. CB 7646, therefore, represents a promising candidate for clinical development, designed to supersede TM.