The enzyme protein kinase C (PKC) is thought to play a major role in cellular signal transduction and has been shown to exist as several isoforms. PKC is activated in cells by diacylglycerol and other diverse tumor promotors such as phorbol 12-myristate-13-acetate (1; PMA). Computer modeling studies on PMA have suggested that carbonyl group at C-3, hydroxyl groups at C-4, C-9, and C-20, and a long-chain hydrophobic portion at C-12 would be important to bind with PKC. However these structural hypotheses have not been confirmed by determining the actual PKC binding affinity or activity relationships of such substituted phorbols. We report here the entire stereospecific syntheses of phorbol analogs with the proposed necessary structural features in PKC activation. In particular, these include the syntheses of the analogs 7 and 10 having long-chain hydrophobic moieties at C-12 of their phorbol C-rings. The key intermediate 18 for the synthesis of these analogs is prepared from (+)-3-carene in a stereocontrolled manner. For the synthesis of 10, treatment of 18 with the phosphonate carbanion generated from 19 resulted in stereospecific formation of the E-stereoisomer of 20. DIBAH reduction of 20 and protection of the resulting primary alcohol, as its tert-butyldiphenylsilyl ether, yielded 21. A three-step series of reactions involving tosylation, iodide substitution, and nitration gave 22. The next crucial step was intramolecular cyclization of 22 to the corresponding isoxazolin 23, which was subsequently hydrogenated to the hydroxy ketone 24 in 85% yield (2 steps). Internal cerium alkoxide assisted propynylation of 24 was then followed by oxidation and conversion of the resulting intermediate into the ethyl-ketone 26. Intramolecular aldol cyclization of 26 and dehydration of the resulting aldol mixture in one-pot, followed by deprotection of the silyl ethers, gave the analog 9. Its allylic alcohol functionality was protected as its trityl ether, and the long-chain myristate group was then introduced at C-12. Selective deprotection of the trityl group of the resulting intermediate afforded the phorbol analog 10. The remaining hydrophobic analog 7 was obtained from 18 using similar methods to those used in the conversion of 18 to 10. The in vitro binding affinity of the phorbol analogs 7 and 10, relative to that of PMA (1), was then evaluated. The analog 7 proved to be very weak with respect to its affinity to bind to PKC, and an approximate 100 fold concentration of 10 was required. These results suggested that either the additional methyl and/or the acetoxyl groups at C-11 and C-13 respectively of 1 are also responsible for its high binding affinity to PKC. They may also be attributed to the presence of too much hydrophobic character due to the long chain at C-12 of the deacetoxy analog 10 resulting in a different hydrophobic interaction with phosphatidyl serine or PKC. In fact, an analog which has octanoyl group at C-12 shows higher binding affinity than that of 10.
