Stories from Staudinger: Synthesis of chiral beta-lactams

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:

1. 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;
2. 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