Nineteen-step total synthesis of (+)-phorbol.

Phorbol, the flagship member of the tigliane diterpene family, has been known for over 80 years and has attracted attention from many chemists and biologists owing to its intriguing chemical structure and the medicinal potential of phorbol esters. Access to useful quantities of phorbol and related analogues has relied on isolation from natural sources and semisynthesis. Despite efforts spanning 40 years, chemical synthesis has been unable to compete with these strategies, owing to its complexity and unusual placement of oxygen atoms. Purely synthetic enantiopure phorbol has remained elusive, and biological synthesis has not led to even the simplest members of this terpene family. Recently, the chemical syntheses of eudesmanes, germacrenes, taxanes and ingenanes have all benefited from a strategy inspired by the logic of two-phase terpene biosynthesis in which powerful C-C bond constructions and C-H bond oxidations go hand in hand. Here we implement a two-phase terpene synthesis strategy to achieve enantiospecific total synthesis of (+)-phorbol in only 19 steps from the abundant monoterpene (+)-3-carene. The purpose of this synthesis route is not to displace isolation or semisynthesis as a means of generating the natural product per se, but rather to enable access to analogues containing unique placements of oxygen atoms that are otherwise inaccessible.

Highly potent, synthetically accessible prostratin analogs induce latent HIV expression in vitro and ex vivo.

Highly active antiretroviral therapy (HAART) decreases plasma viremia below the limits of detection in the majority of HIV-infected individuals, thus serving to slow disease progression. However, HAART targets only actively replicating virus and is unable to eliminate latently infected, resting CD4(+) T cells. Such infected cells are potentially capable of reinitiating virus replication upon cessation of HAART, thus leading to viral rebound. Agents that would eliminate these reservoirs, when used in combination with HAART, could thus provide a strategy for the eradication of HIV. Prostratin is a preclinical candidate that induces HIV expression from latently infected CD4(+) T cells, potentially leading to their elimination through a virus-induced cytopathic effect or host anti-HIV immunity. Here, we report the synthesis of a series of designed prostratin analogs and report in vitro and ex vivo studies of their activity relevant to induction of HIV expression. Members of this series are up to 100-fold more potent than the preclinical lead (prostratin) in binding to cell-free PKC, and in inducing HIV expression in a latently infected cell line and prostratin-like modulation of cell surface receptor expression in primary cells from HIV-negative donors. Significantly, selected members were also tested for HIV induction in resting CD4(+) T cells isolated from infected individuals receiving HAART and were found to exhibit potent induction activity. These more potent agents and by extension related tunable analogs now accessible through the studies described herein should facilitate research and preclinical advancement of this strategy for HIV/AIDS eradication.

Artificial analogs of naturally occurring tumor promoters as biochemical tools and therapeutic leads.

Tumor promoters are non-carcinogenic chemicals that enhance tumor formation when administered repeatedly after a low dose of a carcinogen. Phorbol esters, teleocidins, and aplysiatoxins are typical examples of naturally occurring tumor promoters. All of them share the ability to bind and activate protein kinase C (PKC) despite the differences in their chemical structures. A variety of analogs with unique chemical and biological properties have been developed to analyze the molecular mechanism of tumor promotion through PKC activation. Moreover, coupled with the emerging significance of PKC in the pathological processes of Alzheimer's disease (AD) and acquired immune deficiency syndrome (AIDS) as well as cancer, several efforts have been made recently to generate analogs of tumor promoters with therapeutic potential. This review focuses on artificial analogs of phorbol esters, teleocidins, and aplysiatoxins, and discusses their potential as biochemical tools and therapeutic leads.

Design and physicochemical properties of new fluorescent ligands of protein kinase C isozymes focused on CH/pi interaction.

Phorbol ester-type tumor promoters such as indolactam-V (IL-V, 1) bind to the C1 domains of protein kinase C (PKC) isozymes. A more convenient method to investigate the interaction between each tumor promoter and PKC C1 domain is needed. Focusing on our recent finding that the indole ring of IL-V is involved in the CH/pi interaction with Pro-11 of the PKCdelta-C1B domain, we developed new fluorescent probes (2-4) from IL-V by forming a pyrroloindazole ring. Compound 2 without a substituent at the pyrroloindazole ring bound most strongly to PKC C1 domains with a potency similar to IL-V, but its fluorescent intensity was the weakest of any of the probes. Although the binding affinity of 3 with a methyl group was significantly weaker than that of IL-V, 4 with a trifluoromethyl group showed moderate affinity and the most potent fluorescence intensity. The fluorescence intensity and emission maxima of 4 changed significantly when bound to the PKCdelta-C1B peptide in both the presence and absence of phosphatidylserine. These results suggest that 4 could be a useful probe for analyzing the interaction of tumor promoters with PKC C1 domains.

Protein kinase C ligands based on tetrahydrofuran templates containing a new set of phorbol ester pharmacophores.

A series of substituted tetrahydrofurans with an embedded glycerol backbone carrying additional tetrahydrofuranylideneacetate or tetrahydrofuranylacetate motifs were grouped into four distinct templates (I-IV) according to stereochemistry. The compounds were designed to mimic three essential pharmacophores (C(3)-C=O, C(20)-OH and C(13)-C=O) of the phorbol esters according to a new, revised model. The tetrahydrofuran ring was constructed from glycidyl 4-methoxyphenyl ether, and the structures of the isomeric templates were assigned by NMR spectroscopy, including NOE. The binding affinity for protein kinase C (PKC) was assessed in terms of the ability of the ligands to displace bound [(3)H-20]phorbol 12, 13-dibutyrate (PDBU) from a recombinant alpha isozyme of PKC. Geometric Z- and E-isomers (1 and 3, respectively) containing a tetrahydrofuranylideneacetate motif were the most potent ligands with identical K(i) values of 0.35 microM. Molecular modeling studies of the four templates showed that the rms values when fitted to a prototypical phorbol 12,13-diacetate ester correlated inversely with affinities in the following order: I approximately II > III > IV. These compounds represent the first generation of rigid glycerol templates seeking to mimic the binding of the C(13)-C=O of the phorbol esters. The binding affinities of the most potent compounds are in the same range of the diacylglycerols (DAGs) despite the lack of a phorbol ester C(9)-OH pharmacophore surrogate. This finding confirms that mimicking the binding of the C(13)-C=O pharmacophore of phorbol is a useful strategy. However, since the C(9)-OH and C(13)-C=O in the phorbol esters appear to form an intramolecular hydrogen bond that functions as a combined pharmacophore, it is possible the lack of this combined motif in the target templates restricts the compounds from reaching higher binding affinities.

Cyclohexane diester analogues of phorbol ester as potential activators of protein kinase C.

Phospholipid-dependent, Ca2(+)-sensitive protein kinase (protein kinase C) is activated by the plant product phorbol ester at nanomolar concentrations and also in vivo at micromolar concentrations by diacylglycerols. We designed and synthesized cyclohexane diester analogues of the phorbol ester C ring as potential high-affinity activators of protein kinase C. We proposed that the necessary pharmacophore of phorbol ester could be mimicked by diesters of appropriately substituted cyclohexanediols. A series of 1,2-cyclohexanediol diesters with different substituents at position 4 was synthesized. These substituents were designed to mimic the 6,7-double bond and C-20 hydroxy of phorbol ester. Competitive binding vs [3H]phorbol dibutyrate determined that these compounds have an affinity for protein kinase C of 1 mM or more, and thus they do not bind to nor are they activators of this enzyme.

Synthesis and evaluation of phorboid 20-homovanillates: discovery of a class of ligands binding to the vanilloid (capsaicin) receptor with different degrees of cooperativity.

A number of phorboid 20-homovanillates were prepared by condensation of phorbol 12,13-diesters and 12-dehydrophorbol 13-esters with Mem-homovanillic acid followed by removal of the protecting group with SnCl4 in THF. These compounds were evaluated for their ability to inhibit [3H]resiniferatoxin (RTX) binding to rat spinal cord membranes. Compounds bearing a lipophilic ester group on ring C were considerably active, but a surprising tolerance of the vanilloid receptor toward the location and the orientation of this ester group was disclosed. Unexpectedly, these ligands could also diminish, to a variable degree, the positive cooperativity which characterizes RTX binding to the vanilloid receptor. Phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV, 6a), a compound which abolished binding cooperativity, was further tested in a variety of in vivo assay used to characterize vanilloid-like activity. PPAHV showed only a marginal pungency and failed to induce a measurable hypothermia response at doses (up to 200 mg/kg) at which it effectively desensitized against neurogenic inflammation. These data suggest that the peculiar binding behavior of these ligands might be associated with a distinct spectrum of biological activity.

Inter- and intramolecular reductive coupling reactions: an approach to the phorbol skeleton.

[reaction: see text] An expeditious convergent route to the ABC-tricyclic core of the phorbol esters is described. The chemistry capitalizes upon both inter- and intramolecular reductive coupling processes promoted electrochemically and via the use of samarium diiodide.

A new class of simplified phorbol ester analogues: synthesis and binding to PKC and eta PKC-C1B (eta PKC-CRD2).

[formula: see text] A unique class of simplified phorbol ester analogues is described for the first time. A highly efficient retro-annelation sequence was developed in order to remove the five-membered ring from the phorbol diterpene core, allowing access to BCD ring analogues of the phorbol esters. The binding of these analogues to protein kinase C (PKC) and the truncated peptide eta PKC-C1B (eta PKC-CRD2) is also reported.

[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.

Practical synthesis of prostratin, DPP, and their analogs, adjuvant leads against latent HIV.

Although antiretroviral therapies have been effective in decreasing active viral loads in AIDS patients, the persistence of latent viral reservoirs prevents eradication of the virus. Prostratin and DPP (12-deoxyphorbol-13-phenylacetate) activate the latent virus and thus represent promising adjuvants for antiviral therapy. Their limited supply and the challenges of accessing related structures have, however, impeded therapeutic development and the search for clinically superior analogs. Here we report a practical synthesis of prostratin and DPP starting from phorbol or crotophorbolone, agents readily available from renewable sources, including a biodiesel candidate. This synthesis reliably supplies gram quantities of the therapeutically promising natural products, hitherto available only in low and variable amounts from natural sources, and opens access to a variety of new analogs.

Novel phorbol analogs which bind to protein kinase C (PKC) without activation.

13-Deacetoxy-11-demethyl-phorbol derivatives with acyl groups of various lengths (from hexanoyl to tetradecanoyl) at the C-12 position were synthesized in an optically active form. Although considerable binding affinities to PKC were observed by analogs 3-7, no activation of PKC was seen even at 10 microM.

Protein kinase C translocation by modified phorbol esters with functionalized lipophilic regions.

Several novel phorbol esters were prepared with polar functional groups terminating their C12 and/or C13 acyl chains. Designed to be inhibitory protein kinase C (PKC) ligands, these phorbol analogues contain various polar functional groups (amide, ester, carboxylic acid, or quaternary ammonium salt) to prevent membrane insertion of the PKC-phorbol ester complex. All phorbol derivatives were synthesized with use of diterpene starting materials obtained from croton oil, the seed oil of Croton tiglium. The ability of these derivatives to recruit PKC to the lipid bilayer-a usual requirement for enzyme activation-was determined by using a sucrose-loaded vesicle assay. Phorbol 12-octanoate-13-acetate derivatives translocate PKC-betaII to increasing degrees as the functionality on the C12 ester becomes more hydrophobic. Likewise, PKC translocation by carboxylic acid-containing phorbol esters was dependent upon length and saturation of the hydrocarbon tether. The most promising PKC inhibitors had short carboxylic acids capping their C12 and C13 acyl chains, since these compounds did not recruit PKC to any appreciable extent.

Preparation of retinoic acid esters of phorbol derivatives.

The synthesis of 12-O-retinoylphorbol-13-acetate (RPA), an incomplete tumor promoter (second stage promoter) is described. The preparation starts with phorbol-13-acetate-20-tritylether which is acylated by a carbodiimide method to yield its 12-retinoate. The latter is detritylated by acidic methanol to give RPA. Following an analogous procedure, the 4-methyl-ether of RPA is prepared from 4-O-methylphorbol-13-acetate-20-tritylether.

Skin irritant effects of esters of phorbol and related polyols.

The skin irritant potencies of several naturally occurring and semi-synthetic polyfunctional esters based on the tigliane hydrocarbon skeleton were studied using a mouse ear irritancy assay. The compounds used were esters of 12-deoxyphorbol, 12-deoxy-16-hydroxyphorbol, and phorbol. Observations were also made on the time interval between application of these compounds in acetone solution to the mouse ear, and the attainment of maximum response. Structure/activity relationships are discussed and compared with previous observations on the skin irritant potencies of esters based on the closely related daphnane hydrocarbon skeleton. The possible nature of the site of action in the target membrane is also discussed.

Fluorescent tetradecanoylphorbol acetate: a novel probe of phorbol ester binding domains.

Protein kinase C (PKC) has a prominent role in signal transduction of many bioactive substances. We synthesized the fluorescent derivative, phorbol-13-acetate-12-N-methyl-N-4-(N,N'-di(2-hydroxyethyl)amino)-7-n itr obenz-2-oxa-1,3-diazole-aminododecanoate (N-C12-Ac(13)) of 12-O-tetradecanoylphorbol-13-acetate (TPA) to monitor the location of phorbol ester binding sites and evaluate its potential use as a probe of PKC in viable cells. The excitation maximum wavelength of N-C12-Ac(13) is close to 488 nm, facilitating its use in argon-ion laser flow and imaging cytometry. When incubated with 100 nM N-C12-Ac(13) at 25 degrees C, P3HR-1 Burkitt lymphoma cells accumulated the dye rapidly, reaching maximum fluorescence within 25 min, 20-fold above autofluorescence. Addition of unlabeled TPA significantly decreased the fluorescence of N-C12-Ac(13) stained cells in a dose-dependent manner indicating specific displacement of the bound fluoroprobe. Competitive displacement of [3H]-phorbol-12,13-dibutyrate ([3H]-PBu2) from rat brain cytosol with N-C12-Ac(13) gave an apparent dissociation constant (Kd) of 11 nM. N-C12-Ac(13) possessed biological activity similar to TPA. Like TPA (final concentration 65 nM) N-C12-Ac(13), at a lower concentration (51 nM), induced expression of Epstein-Barr viral glycoprotein in P3HR-1 cells, differentiation of promyelocytic HL60 cells, and caused predicted changes in the mitotic cycle of histiocytic DD cells. Microspectrofluorometric images of single cells labeled with N-C12-Ac(13) showed bright fluorescence localized intracellularly and dim fluorescence in the nuclear region, consistent with dye binding mainly to cytoplasmic structures and/or organelles and being mostly excluded from the nucleus. Because of the high level of non-specific binding of N-C12-Ac(13), this probe is not ideal for visualizing PKC in intact cells, but would be a valuable fluoroprobe to investigate the kinetic properties of purified PKC. Also, knowledge gained from these studies allows us to predict structures of fluorescent phorbols likely to have less non-specific binding and, consequently, be potentially useful for monitoring PKC in viable cells.