Thalidomide (Thal: 1) and its two metabolites, 5-hydroxythalidomide (5-HT: 2) and N-hydroxythalidomide (N-HT: 3), showed an enhancing effect on all-trans retinoic acid (ATRA)-induced HL-60 cell differentiation. 5-HT and N-HT showed tubulin polymerization-inhibiting activity, but thalidomide did not.Key words thalidomide; hydroxythalidomide; retinoic acid; differentiation; tubulin Thalidomide (Thal: 1), a sedative drug which was once withdrawn from the market because of its teratogenicity, has been established to be effective for the treatment of various diseases, including leprosy, multiple myeloma (MM), AIDS, and various cancers.1-5) The anti-MM activity of Thal (1) is of particular interest, because Thal (1) overcomes the resistance of human MM cells to conventional drug therapy.6) Although various pharmacological effects elicited by Thal (1), including tumor necrosis factor (TNF)-a production-regulating activity, anti-angiogenic activity and cyclooxygenase (COX)-inhibiting activity, [1][2][3][4][5]7,8) have been reported, the mechanism of its anti-MM activity is unclear.During our structural development studies of Thal (1), we found that one of its major metabolites, 5-hydroxythalidomide (5-HT: 2), possesses tubulin polymerization-inhibiting activity, resulting in G1 arrest and apoptosis of myeloma cells. 9) We also previously reported that tubulin polymerization inhibitors, including colchicine, vinblastine, rhizoxin, maytansine, and ustiloxin, and tubulin depolymerization inhibitors, including taxol, enhance the chemically induced differentiation of human leukemia cell lines.10) Though the relationship between cell differentiation induction and tubulin polymerization/depolymerization inhibition is not clear, enhancement of chemically induced cell differentiation seems to be one of general features of tubulin disruptors.10) These findings led us to suspect that the enhancing effect of Thal (1) and/or its metabolites plays a role in the anti-MM activity of the drug, at least in part. Thal (1) is known to be metabolically unstable, and many metabolites have been identified, including 5-HT (2) and N-hydroxythalidomide (N-HT: 3). In this paper, we deal with the enhancing effect on all-trans retinoic acid (ATRA)-induced HL-60 cell differentiation and the tubulin polymerization-inhibiting activity of Thal (1), 5-HT (2) and N-HT (3).Thal (1) and 5-HT (2) were prepared as described previously.9,11) N-HT (3) was prepared by the method described by Robin et al. 12) Briefly, N-phthaloylglutamic acid anhydride was condensed with O-benzylhydroxylamine in the presence of dicyclohexylcarbodiimde (DCC) to give N-benzyloxythalidomide, which was deprotected by hydrogenation (H 2 gas, Pd/C) to afford N-HT (3). The structures of the synthesized compounds were confirmed by NMR and mass spectroscopy and elemental analysis. First, we investigated the effect of the compounds on tubulin polymerization using the method previously described.
9)Briefly, microtubulin was prepared from porcine brain and tubulin polymerization was followe...