Abstract
Combretastatin A-4 (CA-4) is the most potent antimitotic agent of the Combretastatin A series; a group of diaryl stilbenes isolated from the wood of the South African tree Combretum caffrum. [i] It has significant anticancer activity through inhibition of tubulin polymerization and microtubule assembly. [ii] While cis-stilbene structures demonstrate superior biological activity[iii], the corresponding trans derivatives are inherently more stable. Isomerization of cis CA-4 to the trans form is observed both during storage and in vivo during metabolism which dramatically reduces antitumour activity. [iv] Our group has previously employed the Staudinger reaction to synthesize novel 3-hydroxy-1,4-diaryl-2-azetidinones. The problem of CA-4’s Cis- Trans isomerization has been overcome via chemical manipulation of CA-4’s alkene bridge; utilizing a beta-lactam ring to induce cis restriction. A number of analogues have shown potent nanomolar antiproliferative activity in MCF-7 and HL-60 cells with enhanced activity relative to CA-4. [v] Typical Staudinger reactions form mixtures of cis and trans isomers depending on reaction conditions employed, and additionally; at the 3-hydroxy position’s chiral center; racemic mixtures of R&S enantiomers. Trans isomers of 3-substituted-2-azetidinones have been shown to be up to 50 times more potent than the corresponding cis derivatives[vi] emphasizing the requirement to optimize the Staudinger approach to minimize yields of the undesirable cis isomer. Levo- and dextro-rotatory enantiomers hold potential to display lesser or greater biological activity relative to one another. Our current work aims to: Improve the available yield for chiral resolution by determining the necessary conditions to achieve stereoselective synthesis of trans 3-hydroxy-1,4-diaryl-2-azetidinones in the Staudinger reaction; Purification of racemic mixtures using N-(tert-butoxycarbonyl)-L-Proline as a chiral resolving agent to afford optically pure enantiomers for further biological evaluation. Trans 3-hydroxy b-lactams have been separated from cis derivatives using chromatographic purification. We have since optimized the Staudinger reaction to return relative yields of 95:% relative ratio of trans: cis isomers; as indicated by integration of protons at position 3 and 4 of the beta-lactam on 1 H-NMR. Diastereomeric resolution using flash column chromatography followed by hydrolysis of chiral resolving agents has successfully yielded enantiomers of EMCL001&2. Preliminary biochemical data for the enantiomers in breast cancer cells will be reported. References [i] Watt, J. M.; Gerdina, M., The Medicinal and Poisonous Plants of Southern and Eastern Africa. E. & S. Livingstone Ltd: Edinburgh and London, U.K., 1962. [ii] Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.; Garcia-Kendall, D., Isolation and structure of the strong cell growth and tubulin inhibitor combretastatin Experientia 1988, (45). [iii] Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.; Garcia-Kendall, D., Isolation and structure of the strong cell growth and tubulin inhibitor combretastatin A-4 ~. Experientia 1988, (45). [iv] Ohsumi, K.; Hatanaka, T.; Fujita, K.; Nakagawa, R.; Fukuda, Y.; Nihei, Y.; Suga, Y.; Morinaga, Y.; Akiyama, Y.; Tsuji, T., Synthesis and antitumor activity of cis-restricted combrestatins: 5-membered heterocyclic analogues. YNTHESES AND ANTITUMOR ACTIVITY OF CISRESTRICTED COMBRETASTATINS: 5-MEMBERED HETEROCYCLIC ANALOGUES Bioorg. Med. Chem. Lett. 1998, 8, 3153-3158 [v]N.M. O’Boyle, M. Carr, L.M. Greene, O. Bergin, S.M. Nathwani, T.McCabe, D.G. Lloyd, D.M. Zisterer, M.J. Meegan, Synthesis and evaluation of azetidinone analogues of combretastatin A-4 as tubulin targeting agents, J. Med. Chem., 53 (2010), p. 8569 [vi] Azizah M. Malebari Lisa M. Greene, S. M. N., Darren Fayne, Niamh M. O'Boyle , Shu Wang , Brendan Twamley , Daniela M. Zisterer , Mary J. Meegan beta-Lactam analogues of combretastatin A-4 prevent metabolic inactivation by glucuronidation in chemoresistant HT-29 colon cancer cells. Eur. J. Med. Chem. 2017, 130, 261-285
Highlights
Combretastatin A-4 (CA-4) is a potent anticancer drug isolated from the wood of the South African tree Combretum caffrum acting by inhibition of tubulin polymerisation
Determine the necessary conditions to optimise the yield of the trans isomer of 3hydroxy-1,4-diaryl-2-azetidinones in the Staudinger reaction
Relative stereochemistry is determined by the rate of ring closure; controlled by two competing factors: 1. Competition for direct ring closure 2
Summary
Combretastatin A-4 (CA-4) is a potent anticancer drug isolated from the wood of the South African tree Combretum caffrum acting by inhibition of tubulin polymerisation. Isomerization of cis CA-4 to the trans form is observed both during storage and in vivo during metabolism. Our group has previously synthesized novel 3-hydroxy-1,4-diaryl-2-azetidinones by Staudinger reaction, inducing cis-restriction and overcoming the problem of isomerisation of CA-4. A number of trans beta-lactams have shown potent nanomolar antiproliferative activity in MCF-7 breast cancer cells with enhanced activity relative to CA-4
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