Organic Chemical Investigation of Novel Biologically Active Compounds, Syntheses and Reactions, Evaluation of Biological Activities.

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(1) The specimens of female solitary wasps were collected in Sagamiko, Ibaraki, and Kyoto. The lyophilized venom sacs were extracted and subjected repeatedly to reverse-phase HPLC, accompanied by testing neuro activity using neuromuscular preparations of walking leg of lobster, to give purified venoms. The structures of the isolated venoms were estimated mainly by the leading tequniques of mass spectrometry, MALDI-TOF-CID method andlor Ladder sequence method, and finally determined by solid phase synthesis based on the Fmoc- L-amino acid strategy. Eumenine mastoparan-AF was isolated from the venom of the Eumenine wasp. Anterhynchium flavomarginatum micado. Both the structure and theneuroactivity were similar to those ofmastoparan of a hornet wasp. Novel and uniqueneurotoxins, alpha- and beta-pompilidotoxins were isolated from the venoms of the Pompilidaewasps, Anoplius samariensis and Batozonellus maculifrons, These venoms potentiatessynaptic transmission of lobster leg muscle by the p … More resynaptic mechanisms.(2) All eight possible A-ring diastereomers of both 2-methyl- and 4-methyl- 1,25-dihydroxyvitamin D_3 were synthesized to investigate the relationship of the A-ring conformation [alpha-form<double arrow>beta-form (active form ; lalpha-OH is equatorial)] to the biological activities C/DR affinity as an index). Also, all eight possible A- ring diastereomers of 2-methyl-20-epi-l, 25-dihydroxyvitamin D3 were synthesized. Introduction of 2-methyl- and 4-methyl-group would result in equibrium shift to either one way in certain extent. Starting from either (S)- or (R)-isomer of methyl 3-hydroxy-2-methyl-propionate, the A-ring portions(enynes) were obtained in good overall yields, which were subsequently coupled with the CD-ring-side chain portion by the Trost' Procedure to give the 2-methyl analogues. Similarly, starting from 1,3-propanediol, the A-ring portions of 4-methyl analogues were prepared as racemic forms, which, after separation, were subjected to the same procedure to give 4-methyl steroisomers. In VDR affinity using bovine thymus, the potency of 2-methyl isomers was highly dependent on the stereo-chemistry of the A-ring substituents. Thus, 2alpha-Me-1alpha-OH,3betaisomer showed 4-fold higher affinity than natural lalpha, 25-(OH)_2-D_3 (normalized, and so forth). This isomer also exhibited 4-fold higher potency in Ca mobilization, and 2-fold higher potency in HL-60 cell differentiation. Furthermore, double modified steroisomer of A-ring and side chain, 2alpha-Me-20-epi-1alpha, 25-(OH) _2-D_3 showed 12 fold higher in VDR affinity, 7 fold higher in Ca mobilization and 590 times higher potency in HL-60 cell differentiation, which is the most potent analogue reported to date. In contrast to 2-methyl series, VDR affinities of all diastereomers of 4-methyl-1,25-(OH) _2-D_3 were less than 10^21-10^3. From the conformational analysis by NMR spectroscopy, chair-wform is predominant in A-ring coconformation of 2alpha-Me-1alpha, 25-(OH) _2-D_3, while 2beta-Me isomer prefer half-boat-beta-form in A-ring. Further synthetic (various substituents in 2-position, conformational restricted) and biological (apotosis, transcriptional potency) studies are in progress. Less