Organic Chemical Creation of Substances with Novel Function and Its Application

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The heading numbers are same as those described in 2000〜2001 research projects.(1) Studies on solitary wasp venoms have been postponed due tithe urgency in the studies on A-ring modified vitamin D analogues.2-1) All eight possible A-ring diastereomers of 2-methyl-1, 25-dihydroxyvitamin D_3 and their 20-epimers were convergently synthesized by Trost's Pd coupling reaction of A-ring precursors enynes with CD ring portions. The synthesized analogues were biologically evaluated both in vitro and in vivo. The potency was highly dependent on the stereochemistry of each isomer. In particular, 2α-Me-1α,25-(OH)_2D_3 exhibited 4-fold higher potency than 1α,25(OH)_2D_3 (natural hormone) (1) both in VDR (Vitamin D Receptor) binding and in elevation of rat serum calcium concentration and was twice as potent as 1 in HL-60 cell differentiation. Furthermore, 20-epi-2α-Me-1α,25-(OH)_2D_3, exhibited exceptionally high activities : 12-fold higher in VDR binding affinity, 7-fold higher in calcium mobiliza … More tion, and 590-fold higher in HL-60 cell differentiation, as compared to 1. Conformational analysis of the A-ring of eight diastereomers by ^1H-NMR and molecular mechanics calculation (MM2^*) were carried out : α-form (60:40) was predominant over β-form in 2α-Me analogue, β-form was predominant over α-form(75:25) in 2β-Me analogue. On the other hand, α-ring in 2α-Me-1 α,25-(OH)_2D_3 existed exclusively in the β-form by an X-ray crystallographic analysis. These findings did not permit any decisive conclusion to the well-known hypothesis "the A-ring of natural hormone equilibrates between two chair conformers, the α-form and β-form (1:1) and the β-form , in which the 1 α-OH occupies the equatorial position, may be responsible for biological activity". In further evaluation of biological activities, we found that the structural requirement for inducing apoptosis of HL-60 ells was clearly different from that for inducing differentiation of these cells. Therefore, these findings provided useful information not only for structure-function studies of 1α,25(OH)2_D_3 analogues but also the development of therapeutic agents for the treatment of leukemia and other cancers.2-2) In connection with 19-nor-lα 2b(OH)_2D_3 analogue as a candidate for cancer therapy all possible A-ring stereoisomers of 5, 6-trans-2-methyl-1, 25-(OH)_2D_3 and their 20-epimers were synthesized and biologically evaluated. Introduction of the pseudo 2α-Me to 5, 6-trans-1, 25- (OH)_2D_3 enhanced the potency. Based on Moras's X-ray structure of the complex of 1 with VDR, computational docking studies of 2α-Me-analogues to VDR by molecular mechanics calculation revealed the activity-strengthening effect of 2α-Me group.2-3) In order to investigate the activity-strengthening effect of 2α-Me group, 2α-alkyl, 2α-(ω-hydroxyalkyl), and 2α-(co-hydroxyalkoxy)- 1α,25(OH)_2D_3 analogues were convergently synthesized by Pd-coupling reactions of CD ring portion with A-ring precursors enynes, which were stereoselectively synthesized starting from sugars, and then biologically evaluated. 2α-hydroxypropyl analogue exhibited 3-fold higher potency than in VDR binding , 2.4 fold higher in HL-60 cell differentiation, and surprisingly 508-fold higher in Ca mobilization, as compared to 1. 2α hydroxybutyl- and 2α-hydroxypropoxy-analogues also showed higher VDR binding affinity than 1. Computational docking studies of three analogues to VDR revealed the activity-strengthening effect of these group.3) New synthetic route to A-ring precursor in convergent synthesis of 1α,25-(OH)_2D_3analogues was developed starting from furansulfolene. Less