Organic Chemical Creation of Substances with Novel Function and Its Application

(1)由于A环修饰维生素D类似物研究的紧迫性，对单黄蜂毒液的研究已被推迟。2-1)所有8个可能的2-甲基-1，25-二羟基维生素D_3及其20-异构体的A环非对映异构体都是通过A环前体苯炔与CD环部分的Trost PD偶联反应而聚合合成的。对合成的类似物进行了体外和体内生物学评价。其效力高度依赖于每个异构体的立体化学。其中，2-α-Me-1α，25-(OH)_2D_3(天然激素)比1α，25(OH)_2D_3(天然激素)(1)对维生素D受体(VdR)结合和升高大鼠血清钙浓度均表现出4倍以上的活性，在HL-60细胞分化中的活性是1的两倍。此外，20-epi-2α-me-1α，25-(OH)_2D_3显示出极高的活性：VdR结合亲和力提高12倍，钙动员…提高7倍对8个非对映异构体的A环进行了构象分析：在2个α-Me类似物中以β形式为主(60：40)，在2个β-Me类似物中以β形式为主(75：25)。X-射线单晶分析表明，α环在2α-Me-1α，25-(OH)_2D_3中仅以β形式存在。这些发现不允许对著名的假设得出任何决定性的结论，即自然激素的A环在两个椅子构象α-形式和β-形式(1：1)和β-形式之间平衡，其中1个α-OH占据赤道位置，可能与生物活性有关。在进一步的生物活性评估中，我们发现诱导HL-60细胞凋亡的结构要求与诱导这些细胞分化的结构要求明显不同。因此，这些发现不仅为1-α，25(OH)2_D_3类似物的结构和功能研究提供了有用的信息，也为开发治疗白血病和其他癌症的治疗药物提供了有用的信息。2)结合19-Nor-Lα2b(OH)_2D_3类似物作为抗癌候选化合物，合成了所有可能的A环立体异构体5，6-反式-2-甲基-1，25-(OH)_2D_3及其20-同分异构体，并对其进行了生物活性评价。在5，6-α-1，25-(OH)_2D_3中引入伪2-TRAN-Me增强了活性。2)为了考察2个α-Me基团、2个α-Me基团(2个α-Me基，2个α-烷基，2个α-(ω-羟基烷基)和2个α-(co-羟基烷氧基)-1α，25(OH)_2D_3类似物的活性增强作用，以糖为起始原料，通过CD环部分的Pd偶联反应，聚合合成了25(OH)_2D_3类似物。2.与1.2相比，α-羟丙基类似物的结合活性提高了3倍，在HL-60细胞中的分化能力提高了2.4倍，钙动员能力提高了508倍。2.α羟丁基和2-α-羟丙氧基类似物也显示了比1更高的VdR结合亲和力。3)以呋喃舒叶烯为起始原料，发展了1-α，25-(OH)_2D_3类似物A环前体的聚合合成路线。较少

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