Synthesis of seco-B-ring bryostatin analogue WN-1 via C-C bond-forming hydrogenation: critical contribution of the B-ring in determining bryostatin-like and phorbol 12-myristate 13-acetate-like properties.

The seco-B-ring bryostatin analogue, macrodiolide WN-1, was prepared in 17 steps (longest linear sequence) and 30 total steps with three bonds formed via hydrogen-mediated C-C coupling. This synthetic route features a palladium-catalyzed alkoxycarbonylation of a C2-symmetric diol to form the C9-deoxygenated bryostatin A-ring. WN-1 binds to PKCα (Ki = 16.1 nM) and inhibits the growth of multiple leukemia cell lines. Although structural features of the WN-1 A-ring and C-ring are shared by analogues that display bryostatin-like behavior, WN-1 displays PMA-like behavior in U937 cell attachment and proliferation assays, as well as in K562 and MV-4-11 proliferation assays. Molecular modeling studies suggest the pattern of internal hydrogen bonds evident in bryostatin 1 is preserved in WN-1, and that upon docking WN-1 into the crystal structure of the C1b domain of PKCδ, the binding mode of bryostatin 1 is reproduced. The collective data emphasize the critical contribution of the B-ring to the function of the upper portion of the molecule in conferring a bryostatin-like pattern of biological activity.

[Phorbols: chemical synthesis and chemical biology].

The phorbol esters, such as phorbol 12- myristate 13-acetate (PMA), are known to be powerful tumor promoters and activators of protein kinase C (PKC). First discovered by Nishizuka et al., PKC is a phospholipid- and calcium-dependent serine/threonine kinase, phisiologically activated by 1,2-diacyl-sn-glycerol (DAG). PKC is also known to be an important target for other structurally diverse tumor promoters such as ingenols, teleocidins, and aplysiatoxins. Structure-activity analyses of a variety of analogs of DAG and these tumor promoters have been carried out. Although many pharmacophore models have been proposed from molecular modeling, no information about specific amino acid residues that interact with these ligands is available. Moreover it has been shown that the biological activity of 11-demethyl-13-deoxyphorbol esters 1, which were synthesized by our group, was not fully consistent with the pharmacophore models so far. Thus, we are now interested in determining the importance of the 13-acetoxy group in phorbol ester-PKC complexes. This has led us to design new photoaffinity probes 66 and 67 and to carry out previously unprecedented photoaffinity labeling of PKC. Photoaffinity labeling of protein kinase C isozymes by both the probes resulted in specific cross-linking. Although the cross-linking yield is not very high, we suppose that determination of the cross-linking site can be realized by taking advantage of subpicomole order analysis by mass spectrometry and other methodologies to clarify the role of individual cysteine rich domein (CRD) in native PKC. We have also designed a new phorbol ester-phosphatidylserine hybrid molecule 69. Because phosphatidylserines in phospholipid membranes are known to have specific interactions with phorbol ester-PKC complexes, such a hybrid molecule can be expected to act as a specific inhibitor of PKC by preventing PKC from interacting with phospholipid membranes. The hybrid molecule was synthesized and preliminary biological activities were examined to inhibit PKC. A catalytic asymmetric synthesis of phorbol PMA is also currently under investigation. Progress is discussed.