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Gossypol 1, gossypolone 2, and a series of bis 3 and half Schiff's bases 4 of gossypol were synthesised and tested for anti-proliferative and anti-oxidant activity. (-)-Gossypol (-)-1 was the most potent inhibitor of the proliferation of the HPV-16 keratinocyte cell line (using an MTT viability assay) with a GI50 of 4.8 microM. The bis Schiff's base of (-)-gossypol with L-tyrosine ethyl ester (-)-3b was the most potent inhibitor of iron/ascorbate dependent lipid peroxidation (using the thiobarbituric acid test), with an IC50 of 11.7 microM, with (-)-gossypol being the next most potent of the series, with an IC50 of 13.1 microM. The results from these initial assays suggest that gossypol, as either a racemic mixture rac-1, or the individual atropisomers (-)-1 or (+)-1, has potential for the treatment of psoriasis.

Gossypol [(2,2'-binaphthalene)-8,8'-dicarboxaldehyde-1,1',6,6',7,7'-hexahydroxy-5,5'-diisopropyl-3,3'-dimethyl] 1a is a naturally occurring compound extracted from the cotton plant and has been extensively studied as an oral male contraceptive. Its favorable toxicity profile, and the more recent demonstration of anti-tumor activity in animals and humans, prompted us to investigate the role of the aldehyde groups in a structure-activity study in cultured tumor cells. Four racemic compounds were evaluated: gossypol 1a, gossypolone 2, the bis Schiff's base of L-phenylalanine methyl ester with gossypol (bis Schiff's base) 1c and apogossypol 1b. The former two compounds both retain the aldehyde functional groups at positions 8 and 8' of the molecule whilst in the latter two compounds the aldehydes are blocked or absent, respectively. In addition, the l- and d-isomers of gossypol 1a, the bis Schiff's base 1c and the half Schiff's base 1d (one aldehyde blocked) were tested. The cell lines studied included melanoma (SK-mel-19), cervix (Sihas), small cell lung (H69) and myelogenous leukemia (K562). Cytotoxicity was measured using the MTT and flow cytometric viability assays. Racemic gossypol 1a and gossypolone 2 induced similar dose-dependent decreases in cell viability in all the cell lines with IC50 values of 23-46 and 28-50 microM, respectively. In contrast, the racemic bis Schiff's base derivative of gossypol 1c and apogossypol 1b showed minimal activity in any cell line up to 50 microM. The l-enantiomer of gossypol 1a was significantly more active than the d-enantiomer (IC50 of 20 versus > 50 microM, respectively). When one aldehyde of either enantiomer was blocked 1d cytoxicity was comparable to the l-enantiomer of gossypol. The data suggest that only one aldehyde group is required for the cytotoxicity of gossypol 1a, irrespective of the stereoconfiguration.

The naturally occurring compound, gossypol, has been previously used as a male oral contraceptive, for the treatment of benign gynaecological conditions and cancer patients. Long-term daily dosing with gossypol is associated with minimal side effects and no myelosuppression. Since gossypol exhibits atropisomerism due to the restricted rotation about the 2,2' carbon bond, we have isolated the l- and d-isomers by Schiff's base formation using a chiral amine and regenerated the enantiomers by acid hydrolysis. The enantiomers and the proposed oxidative metabolite, gossypolone, were characterized by HPLC, 1H-NMR and optical rotation. The cytotoxicity was assessed in cell cultures derived from melanoma, lung, breast, cervix, and leukaemia using the MTT viability assay. The cytotoxicity of gossypolone was similar to racemic gossypol in five out of the six cell lines studied. The l-enantiomer of gossypol induced a dose-dependent cell kill in all cell lines with a mean IC50 of 20 microM and was significantly more potent than racemic gossypol, the d-enantiomer of gossypol and gossypolone. In addition, when the leukaemia line was exposed to l-gossypol (0.5-10 microM) over a 4-day period, a schedule-dependent decrease in cell viability was observed. l-Gossypol was also compared with respective drugs used to treat patients with melanoma, lung cancer and leukaemia. The data indicate that l-gossypol was significantly more active than cisplatin, melphalan and dacarbazine in the two melanoma lines, cisplatin and daunorubicin in the lung line and hydroxyurea and busulphan in the leukaemia line. Preliminary studies using one melanoma line showed that the l-isomer induced cell shrinkage, membrane blebbing and DNA fragmentation, characteristics suggestive of apoptotic cell death.

The cytotoxicity of the (-)- and (+)-isomers and the quinone metabolite gossypolone prepared from the naturally occurring polyphenolic dialdehyde gossypol were compared using two human melanoma cell lines (SK-mel-19 and SK-mel-28) with a similar growth rate, one melanotic (melanin content of 69 pg/cell) and one amelanotic (melanin content of 10 pg/cell). Results from two viability assays (MTT and flow cytometry) showed that the cytotoxicity of racemic gossypol was identical for both cell lines (50% inhibition of cell growth IC50 = 22 microM). Gossypolone at equimolar concentrations was inactive in the amelanotic cell line and as potent as racemic gossypol in the melanotic cell line. (-)-Gossypol was significantly more active in both cell lines compared with the (+)-isomer. The cytotoxic effect of (-)-gossypol was both concentration and time dependent. Under serum-free conditions, the cytotoxicity of both enantiomers was increased, suggesting that serum protein binding may play a role in the differential toxicity of these isomers in vitro. Morphological changes after exposure to (-)-gossypol included shrinkage and loss of adherence. Cell sensitivity to the (-)-isomer was five-fold greater (IC50 = 4 microM) using a clonogenic assay. At equimolar concentrations, (-)-gossypol was more cytotoxic to both cell lines than the clinically used drugs cisplatin, dacarbazine and melphalan. The results of this study suggest that (-)-gossypol may be of potential therapeutic benefit in melanoma patients.