April 2002 Chem. Pharm. Bull. 50(4) 523—529 (2002) 523 Inhibition of Cytopathic Effect of Human Immunodeficiency Virus Type-1 by Various Phorbol Derivatives Sahar EL-MEKKAWY,a Meselhy Ragab MESELHY,a Atef Abdel-Monem ABDEL-HAFEZ,a Norio NAKAMURA,a Masao HATTORI,*,a Takuya KAWAHATA,b and Toru OTAKEb Institute of Natural Medicine, Toyama Medical and Pharmaceutical University,a 2630 Sugitani, Toyama 930–0194, Japan and Osaka Prefectural Institute of Public Health,b Osaka 537–0025, Japan. Received November 29, 2001; accepted January 7, 2002 Forty-eight derivatives of phorbol (9) and isophorbol (14) were evaluated for their inhibition of human immunodeficiency virus (HIV)-1 induced cytopathic effects (CPE) on MT-4 cells, as well as their activation of protein kinase C (PKC), as indices of anti-HIV-1 and tumor promoting activities, respectively. Of these compounds, the most potent inhibition of CPE was observed in 12-O-tetradecanoylphorbol 13-acetate (8) and 12-Oacetylphorbol 13-decanoate (6). The former also showed the strongest PKC activation activity, while the latter showed no activity at 10 ng/ml. Both activities were generally observed in those phorbol derivatives with an A/B trans configuration, but not in the isophorbol derivatives with an A/B cis configuration. Acetylation of 20-OH in the phorbol derivatives significantly reduced the inhibition of CPE, as shown in 12-O-, 20-O-diacetylphorbol 13decanoate (6a) (IC100515.6 m g/ml) vs. compound 6 (IC10050.0076 m g/ml), and 12-O-tetradecanoylphorbol 13,20diacetate (8a) (IC100515.6 m g/ml) vs. 12-O-tetradecanoylphorbol 13-acetate (8) (IC10050.00048 m g/ml), except in the case of 12-O-decanoylphorbol 13-(2-methylbutyrate) (4) and phorbol 12,13-diacetate (9c). The reduction of a carbonyl group at C-3 abruptly reduced the inhibition of CPE, as observed in 3b -hydroxyphorbol 12,13,20-triacetate (9f) (IC1005500 m g/ml) vs. phorbol 12,13,20-triacetate (9d) (IC100562.5 m g/ml). Although 8 was equipotent in the inhibition of CPE, and activation of PKC, both activities were abruptly decreased by the acetylation of 20OH and methylation of 4-OH [as in 8a and 4-O-methyl-12-O-tetradecanoylphorbol 13,20-diacetate (8b), respectively]. On the other hand, its positional isomer (12-O-acetylphorbol 13-tetradecanoate (8c) showed neither activities. The removal of a long acyl group in 8 led to a substantial loss of both activities, as shown in phorbol 13-acetate (9b). Of the 12-O-acetyl-13-O-acylphorbol derivatives, the highest inhibition of CPE was observed in 6, which has a dodecanoyl residue at C-13. Both an increase and decrease in the number of fatty acid carbon chains resulted in significant reduction of the inhibition of CPE. Key words human immunodeficiency virus; phorbol; protein kinase C; anti-HIV agent; 12-O-acetylphorbol 13-decanoate Current therapy for human immunodeficiency virus (HIV) infection relies primarily on the administration of anti-retroviral nucleoside analogues, either alone or in combination with HIV-protease inhibitors. Although these drugs have a clinical benefit, continuous therapy with these drugs causes to creates drug-resistant strains of the virus. Recently, significant progress has been made towards the development of natural and synthetic agents that can directly inhibit HIV replication or its essential enzymes.1—6) We previously reported the isolation of 8 phorbol diesters (1—8) from the seeds of Croton tiglium L., and 12-O-decanoylphorbol 13-O-(2methylbutyrate) (4) and 12-O-acetylphorbol 13-decanoate (6) were found to potently inhibit an HIV-1-induced cytopathic effect (CPE) on MT-4 cells without the activation of protein kinase C (PKC) normally associated with tumor-promoting action.7,8) This finding suggested that the development of 4 and 6 as lead compounds was a potential strategy for developing novel, therapeutically useful anti-HIV agents. In this paper, we report the chemical modification of phorbol (9) and isophorbol (14), and their biological activities focused on the inhibition of CPE and activation of PKC. Results Synthesis of Phorbol and Isophorbol Derivatives Hydrolysis of a phorbol ester mixture from the seeds of Croton tiglium with Ba(OH)2/MeOH9,10) yielded tetracyclic diterpenes, phorbol (9), 4a -phorbol (14, isophorbol) and 4deoxy-4a -phorbol (23), which were identified by comparison of their spectral data with those reported.11—13) Phorbol ∗ To whom correspondence should be addressed. 1 1b 2 3 4 4a 5 6 6a 6b 7 8 8a 8b 8c 8d 9 9a 9b 9c 9d 9e 9g 10 11 11a 12 12a 13 13a Chart 1. R1 R2 H Ac H Ac C10H19O C10H19O Tig Ac Ac Ac 2-Me butyryl C14H27O C14H27O C14H27O Ac Ac H Ac H Ac Ac Ac Ac Bz Ac Ac Ac Ac Ac Ac Ac Ac Tig Tig 2-Me butyryl 2-Me butyryl 2-Me butyryl C10H19O C10H19O C10H19O C12H23O Ac Ac Ac C14H27O C14H27O H H Ac Ac Ac Ac Ac Bz C6H11O C6H11O C9H17O C9H17O C12H23O C12H23O R3 C18H31O C18H31O C18H31O H H Ac H H Ac Ac H H Ac Ac H C14H27O H H H H Ac Ac Ac Bz H C6H11O H C9H17O H C12H23O R4 H H H H H H H H H Me H H H Me H H H H H H H Me Ac H H H H H H H Chemical Structures of Phorbol and Its Derivatives e-mail: saibo421@ms.toyama-mpu.ac.jp © 2002 Pharmaceutical Society of Japan 524 Vol. 50, No. 4 12,13,20-triacetate (9d) and phorbol 12,13,20-tribenzoate (10) were synthesized from 9, while phorbol 12-acetate (9a), phorbol 12,13-diacetate (9c) and its 4-methyl ether (9e), 3b hydroxyphorbol 12,13,20-triacetate (9f), and phorbol 4,12, R1 H H H Ac C4H7O Ac C8H15O C10H19O 10-Undecenoyl C12H23O C14H27O C17H33O 1-Adamantanoyl H H H Ac C10H19O C14H27O 14 14a 14b 14c 14d 14e 15 16 17 18 19 20 21 23 23a 23b 23c 24 25 R2 R3 R4 H Ac Ac Ac C4H7O Ac Ac Ac Ac Ac Ac Ac Ac H Ac Ac Ac Ac Ac H H Ac Ac C4H7O Ac Ac Ac Ac Ac Ac Ac Ac H H Ac Ac Ac Ac OH OH OH OH OH OAc OH OH OH OH OH OH OH H H H H H H 13,20-tetraacetate (9g) were from 9d (Chart 1),14—18) and isophorbol derivatives 14a—e and 15—21 were from 14 and isophorbol 13-acetate (14b), respectively,19) while a photo product 22 was obtained from isophorbol 12,13,20-triacetate (14c) after irradiation with UV light at 254 nm. On the other hand, 4-deoxy-4a -phorbol derivatives (23a—c) were prepared from 23, while 24a—d and 25 were obtained from 23b (Chart 2).19) 13-O-Acetylphorbol 20-octadecanoate (1), 4, 6 and 12-Otetradecanoylphorbol 13-acetate (8), showing appreciable inhibition of CPE, were selected for further modification. Compound 1 was treated with mesyl chloride in pyridine at room temperature to afford a ring fission product (1a),20) and this was converted to 12-O-,13-O-diacetylphorbol 20-octadecanoate (1b) on acetylation. Furthermore, the acetylation of 4, 6, and 8 gave the respective 20-O-acetyl derivatives 4a, 6a and 8a, and the methylation of 6a and 8a with MeI and Ag2O afforded 4-O-methyl derivatives 6b and 8b. Various 13-O-acyl derivatives of 12-O-acetylphorbol, such as 12-Oacetylphorbol 13-tetradecanoate (8c), 12-O-acetylphorbol 13-hexanoate (11), 12-O-acetylphorbol 13-octanoate (12) and 12-O-acetylphorbol 13-dodecanate (13) were prepared from 9a with various acyl chlorides followed by partial hydrolysis with 70% HClO4 in MeOH.11) The structures of these compounds were established by various spectroscopic means, including 2D-NMR. Inhibition of HIV-1 Induced CPE and Activation of PKC Un-acylated phorbols (9, 14, 23) did not show any significant inhibition of HIV-induced CPE or activation of PKC (Tables 1 and 2). Most of the derivatives of isophorbol and 4-deoxy-4a -isophorbol (14a, b, 15—25) were inactive Chart 2. Chemical Structures of Isophorbol and Its Derivatives Table 1. Inhibition of HIV-1 Induced CPE and Activation of PKC by Isophorbol and Its Derivatives Inhibition of CPE (m g/ml) No. 14 14a 14b 14c 14d 14e 15 16 17 18 19 20 21 22 23 23a 23b 23c 24 25 R1 R2 R3 R4 H H H Ac C4H7O Ac C8H15O C10H19O 10-Undecenoyl C12H23O C14H27O C17H33O 1-Adamantanoyl H Ac Ac Ac C4H7O Ac Ac Ac Ac Ac Ac Ac Ac H H Ac Ac C4H7O Ac Ac Ac Ac Ac Ac Ac Ac OH OH OH OH OH OAc OH OH OH OH OH OH OH H H H Ac C10H19O C14H27O H H Ac Ac Ac Ac H Ac Ac Ac Ac Ac H H H H H H a) At 10 ng/ml, relative to that shown by TPA (100% inhibition) (8); NE, not effective; NT, not tested. IC100 CC0 NE NE NE 250 NE NE 31.25 125 125 NE NE NE 250 NE NE 62.5 62.5 7.81 31.25 62.5 500 .1000 .1000 500 62.5 500 500 500 500 1000 1000 .1000 500 500 500 1000 500 250 1000 1000 % Activation of PKCa) 0 NT NT 0 0 0 NT NT NT NT NT NT NT 0 0 NT NT NT NT NT April 2002 Table 2. 525 Inhibition of HIV-1 Induced CPE and Activation of PKC by Phorbol and Its Derivatives Inhibition of CPE (m g/ml) R1 R2 R3 R4 IC100 1 1b 2 3 4 4a 5 6 6a 6b 7 8 8a 8b 8c 8d 9 9a 9b 9c 9d 9e 9g 10 H Ac H Ac C10H19O C10H19O Tig Ac Ac Ac 2-Me butyryl C14H27O C14H27O C14H27O Ac Ac H Ac H Ac Ac Ac Ac Bz Ac Ac Tig Tig 2-Me butyryl 2-Me butyryl 2-Me butyryl C10H19O C10H19O C10H19O C10H19O Ac Ac Ac C14H27O C14H27O H H Ac Ac Ac Ac Ac Bz C18H31O C18H31O C18H31O H H Ac H H Ac Ac H H Ac Ac H C14H27O H H H H Ac Ac Ac Bz H H H H H H H H H Me H H H Me H H H H H H H Me Ac H CC0 15.6 7.81 7.81 125 7.81 3.90 31.3 0.0076 15.6 NE 15.6 0.00048 15.6 NE NE 62.5 NE NE 125 NE 62.5 31.3 125 NE 62.5 62.5 62.5 500 31.3 15.6 62.5 62.5 31.3 1.95 62.5 31.3 62.5 15.6 125.0 125.0 1000 500 .1000 .1000 125 125 250 31.3 Activation of PKC %a) 0 0 14 16 0 10 10 0 11 0 16 100 0 0 0 0 8 13 0 57 0 0 0 100 MAC (m g/ml) .50 .50 .100 0.01 a) At 10 ng/ml, relative to that shown by TPA (100% inhibition) (8); MAC, minimum concn. for maximum activation of PKC; NE, not effective. Under the same conditions, dextrine sulfate DS 8000 (positive control) showed IC100 and CC0 values of 3.90 and .1000, respectively. (Table 1), while phorbol esters 9a—g showed variable activities (Table 2). Of the isophorbol derivatives, 12-O-octanoylphorbol 13,20-diacetate (15), as well as mono- and diacetyl derivatives of 23 (23a, b), were moderately active against CPE (IC100 value of 62.5 m g/ml), and the activity of 23b was remarkably enhanced by further acetylation (as in 23c, IC100 value of 7.81 m g/ml). Decanoyl and tetradecanoyl derivatives of 23 (24, 25) were moderately active (IC100 values of 31.25 and 62.5 m g/ml, respectively). As for the phorbol derivatives, 12-O-acetyl derivative (9a) was inactive, while the 13-O-acetyl counter part (9b) showed weak inhibition of CPE (Table 2). Although phorbol 12,13-diacetate (9c) and the tetraacetate (9g) were inactive, the triacetate (9d) was moderately active (IC100 value of 62.5 m g/ml). Methylation of the triacetate 9d enhanced the inhibition of CPE (as in 9e, IC100 value of 31.3 m g/ml), while reduction of a carbonyl group at C-3 sharply reduced the activity, as in 9f (IC100 value of 500 m g/ml, CC0 value of 1000 m g/ml, without PKC activation). A two-fold increase in the inhibitory activity against CPE was observed for 1a (IC100 value of 7.81 m g/ml) by treatment of 1 with mesyl chloride. Similar enhancement of the inhibitory activity was also observed after introducing an acetyl group at C-12, as in 12-O-,13-O-diacetylphorbol 20-decanoate (1b). Acetylation of 6 (as in 6a) and 4-O-methylation (as in 6b) significantly reduced the inhibitory activity against CEP, but enhanced the inhibitory activity of 4 by introducing an acetyl Table 3. Effects of Acyl Chain Length at C-13 of 12-O-Acetyl-13-Oacylphorbol Derivatives on Inhibition of HIV-1 Induced CPE and Activation of PKC Inhibition of CPE (m g/ml) No. n IC100 9c 11 12 6 13 8c 0 4 7 8 10 12 NE NE 31.3 0.0076 250 NE CC0 .1000 62.5 31.3 62.5 500 125 % Activation of PKCa) 57 27 10 0 35 0 a) At 10 ng/ml, relative to that shown by TPA (100% inhibition) (8); NE, not effective. group at C-20 (as in 4a, IC100 value of 3.9 m g/ml). Although 8 was found to be equipotent in terms of the inhibition of CPE and the activation of PKC, both activities were dramatically decreased by introducing an acetyl group at C-20 (as in 8a) and by the methylation of a free hydroxyl group at C-4 (as in 8b) (Table 2). On the other hand, its posi- 526 tional isomer (8c) was almost inactive. Removal of the long chain acyl group from 8 resulted in a substantial loss of both activities, as in 9b (IC100 value of 125 m g/ml) (Table 2). Table 3 shows the comparison of inhibitory activity of CPE and activation of PKC among various 12-O-acetyl-13O-acylphorbols having different chain lengths of fatty acid residue (C6 : 0, C9 : 0, C12 : 0 and C14 : 0). The maximum inhibition of CPE was observed for compound 6 (C10 : 0), and both an increase and decrease in the chain length resulted in an abrupt decrease in the inhibitory activity against CPE. Similarly, the activation of PKC was appreciably influenced by an acyl group attached at C-13. Discussion The present study suggests that HIV-1 induced CPE inhibition and PKC-activation activities are influenced by the configuration of the diterpene ester, in which all active phorbol derivatives are of the A/B trans configuration. The A/B cis analogs (isophorbol type) showed no remarkable inhibitory effects on CPE. The finding that an anti-CPE activity of 1 was enhanced 2-times after homoallylic rearrangement of an a -(acetoxycyclopropyl)carbinol group (as in 1a) suggested that this group is not a critical requirement for the anti-CPE activity of these compounds. The observation that TPA (8, with C14 and C2 at C-12 and C-13, respectively) was equipotent in terms of both CPE inhibition and PKC activation, while 6 (with C2 and C10 at C-12 and C-13) and 4 (with C10 and C5 at C-12 and C-13) were potent inhibitors of HIV1-induced CPE, but showed no activation of PKC, suggested that the difference in chain lengths of acyl groups and its relative positions significantly influenced both activities. Both activities were dramatically decreased by introducing an acetyl group at C-20 (as in 8a), while removal of a long chain acyl group from 8 resulted in a substantial loss of both activities, as in 9b. Chowdhury et al.21) found that the cocultivation of MOLT4 and MOLT-4/HIV-1HTLV-IIIB cells at concentrations of more than 0.01 ng/ml of TPA (8) for 20 h strikingly inhibited HIVinduced syncytia formation through down-modulation of CD4 molecules, and that these effects were abrogated by staurosporine, a PKC inhibitor. They reached the conclusion that TPA (8) acted through the activation of PKC in downmodulating CD4 molecules and syncytia formation. Their findings are, in most respects, consistent with our findings that TPA (8) at a concentration of 0.4 ng/ml inhibited HIV1induced CPE on MT-4 cells, and demonstrated the maximal activation of PKC. Phorbol and its esters (9, 9d, g), which were previously reported as non-tumor promoters, were also very weak activators of PKC in the present experiment, though their CPE activity was variable. Although there is strong biochemical evidence that PKC is the major receptor for phorbol esters,22—29) the behavior of these compounds in biological systems clearly indicates that both activities cannot be explained by a single, homogenous class of well behaved receptors. For example, different phorbol derivatives showed different spectra of biological responses, and for a single compound, different concentrations may be required to induce different activities. Although 8 was an equipotent inhibitor of CPE and activator of PKC, 10, which demonstrated the maximal activation of PKC, failed to inhibit CPE. The diesters 12-O-tigloylphorbol 13-decanoate Vol. 50, No. 4 isolated from C. tiglium,30) and ostodin and 12-O-undecadienoylphorbol 13-acetate isolated from Ostodes paniculata,31) were potent antitumor agents. 12-Deoxyphorbol 13decadienoate, isolated from Excoecaria agallocha as an antiHIV principle, was also a potent displacer of [3H]-phorbol dibutyrate from rat brain membrane.32) However, other derivatives (4, 6) were found to potently inhibit the HIV-1-induced CPE without activating PKC. Kubinski et al.33) reported that although chemically related compounds tend to change the properties of the microsomal membrane in a similar way, TPA (8) decreased the amount of bound DNA 5-fold, while phorbol 12,13-didecanoate, the second strongest promoter, increased this amount by about one-third. These discrepancies have been postulated to reflect a difference in the specific site of action, and led us to suggest that except for TPA (8), the inhibition of CPE and activation of PKC by these compounds are not parallel in potency, and that the anti-CPE activity shown by 4 and 6 is mediated through different receptor(s) other than/or in addition to PKC. Experimental Instruments Optical rotations were obtained on a DIP-360 automatic polarimeter (Jasco, Tokyo, Japan). IR spectra were recorded on an FT/IR230 spectrophotometer (Jasco). UV spectra were measured with a UV-2200 UV-VIS recording spectrophotometer (Shimadzu, Kyoto, Japan). NMR spectra were obtained on a Varian Unity plus 500 (1H-, 500 MHz; 13C-, 125 MHz) spectrometer, and chemical shifts are given in d ppm relative to tetramethylsilane (TMS). Electron impact (EI) mass spectra were obtained with a JMS-AX 505 HAD spectrometer (JEOL) at an ionization voltage of 70 eV. Electrospray ionization (ESI) mass spectra were measured with a PE SCIEX API III biomolecular mass analyzer. Chromatography Column chromatography: Silica gel 60 (70—230 mesh, Merck), and ODS Cosmosil 140 C18-OPN (Nacalai Tesque, Kyoto, Japan). Medium pressure liquid chromatography (MPLC) was performed on a LiChroprep Si 60 column or LiChroprep RP-18 column (both size A, Merck, Darmstadt, Germany). Gas chromatography-mass spectra (GC-MS) were obtained using a GC-17A gas chromatograph (Shimadzu, Kyoto, Japan) fitted with a DB-1 column [0.25 mm (i.d)330 m] (J & W Scientific, U.S.A.), coupled to an automass system II benchtop quadrupole mass spectrometer (JEOL) under the following conditions: column temperature, 50 °C for 10 min and gradient to 250 °C (10 °C/min) for 20 min; injection temperature, 250 °C or isothermal at 30 °C for 30 min, and 170 °C for methyl esters of short chain fatty acids; carrier gas, He (flow rate, 15 ml/min). Thin-layer chromatography (TLC) was performed with Silica gel 60 F254 and RP-18 F254 S plates, both 0.25 mm thickness (Merck, Darmstadt), and spots were detected under UV light or after spraying with anisaldehyde-H2SO4 reagent followed by heating. Chemicals and Enzymes Compounds 1—8 were obtained from the seeds of Croton tiglium as reported previously.8) Rat brain protein kinase C (PKC, specific activity, 100 units/ml), staurosporine and L-a -phosphatidyl-Lserine were purchased from Sigma (St. Louis, U.S.A.). A protein kinase enzyme assay system (code RPN 77 kit) was purchased from Amersham International (Buckinghamshire, England). [g -32P]ATP was supplied at specific activity of 370 MBq/ml (10 mCi/ml) from Amersham. Benzamidine HCl was obtained from Tokyo Kasei Org. Chemicals (Tokyo, Japan). Ethylene diamine tetraacetic acid (EDTA), ethylene glycol bis(b -aminoethylether)N,N,N9,N9-tetra-acetic acid (EGTA), phenyl methylsulphonyl fluoride, b mercaptoethanol, Tris/HCl and orthophosphoric acid were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Fatty acid methyl esters were from Nacalai Tesque (Kyoto, Japan). Cells The HTLV-I-carrying cell line MT-4 cells and the human leukemia T-cell line MOLT-4 cells were used. They were maintained at 37 °C under 5% CO2 in RPMI-1640 medium (Flow Laboratories, Irvine, Scotland), supplemented with 10% fetal calf serum (FCS, Flow Laboratories, North Ryde, Australia), 100 m g/ml of streptomycin (Meiji Seika, Tokyo) and 100 U/ml of penicillin G (Banyu Pharmaceutical, Tokyo). Virus The LAI strain of HIV-1 was obtained from a culture supernatant of MOLT-4 cells that had been persistently infected with the LAI strain. Inhibition of CPE on MT-4 Cells The inhibition of HIV-1-induced CPE was measured by the method of Harada et al.34) MT-4 cells were in- April 2002 fected for 1h with the LAI strain of HIV-1 at TCID50 of 0.001/cell (determined by MT-4 cells on day 5 after infection), and the non-adsorbed virus was removed by washing. Then, the cells were re-suspended at 1.53105 cells/ml in RPMI-1640 medium. Then, 200 m l/well of the cell suspension was cultured for 5 d in a 96-well culture plate containing various concentrations of a test compound. Control assays were performed without the test compounds in HIV-1-infected and -uninfected cultures. On day 5, the concentration of the test compound that completely prevented CPE (IC100) and the concentration that reduced the viability of MT-4 cells (CC0) were determined through an optical microscope. Activation of Protein Kinase C The PKC activation was assayed by measuring the incorporation of 32P radioactivity from [g -32P]ATP into a peptide, Arg-Lys-Arg-Thr-Leu-Arg-Arg-Leu-OH, using a Biotrak PKC enzyme assay system, except that TPA in the kit was replaced by the tested compounds (10 ng/ml) dissolved in DMSO (a final concentration of DMSO did not exceed 0.02%). The reaction mixture (55 m l) contained 2 milli units of PKC, 50 mM Tris/HCl (pH 7.5), 0.13% w/v mercaptoethanol, 2.1 mM EDTA, 4.2 mM EGTA, 20.9 m g/ml phenyl methyl sulphonyl fluoride, 4.2 mM benzamidine, 1.3 m M calcium acetate, 75 m M peptide, 34 m g/ml L-a -phosphatidylL-serine, 3.4 mM DTT, 0.68 m M sodium azide, and 6.5 nM MgCl2. After the addition of 0.55 nM [g -32P]ATP (503103 cpm/nmol), the reaction mixture was mixed and maintained at 37 °C for 30 min. Reactions were terminated by the addition of 10 m l of an ice-cold stop reagent. Aliquots of 35 m l were transferred to the center of peptide binding discs. After 10 min, the discs were washed two times with 75 mM orthophosphoric acid. The radioactivity of 32P-labeled samples was counted in 10 ml of a scintillation fluid for 1 min. In the presence of TPA, lipid and Ca acetate, PKC activation represents 100% and corresponds to 8400 nmol/mg/min (positive control). Values for the activation of PKC by the tested compounds are given as the mean of duplicate determinations, and calculated relative to that of the positive control (TPA). Blank (in the absence of PKC) and control (in the absence of TPA or tested samples) tests were also carried out. One unit of PKC was defined as that amount of enzyme which incorporated 1 nmol of phosphate from ATP into its substrate, peptide, per min under the assay conditions described above. In the presence of 34 m g/ml L-a -phosphatidyl-L-serine, 1.3 m M Ca acetate and 2.7 m g/ml TPA, PKC activity was completely inhibited by staurosporine at a concentration of 180 nM. Preparation of Phorbol (9), Isophorbol (14) and 4-Deoxy-4a -isophorbol (23) A hexane-soluble fraction (50 g) of the methanol extract of the seeds of C. tiglium, as reported previously,8) was mixed with a solution of Ba(OH)2 · 8H2O (2.2% in MeOH, 500 ml) and stirred under an atmosphere of argon for 20 h at room temperature. The procedure was followed as reported previously9,10) to obtain a crude phorbol fraction (10 g). Column chromatography of the fraction on SiO2 using CHCl3–MeOH (9.5 : 0.5—7 : 3) as an eluent and subsequent purification with MPLC (RP-18, MeOH–H2O, 4 : 6) yielded phorbol (9, 537 mg),11—13) isophorbol (14, 185 mg)11—13) and 4deoxy-4a -phorbol (23, 10 mg).35,36) Preparation of Phorbol 12-Acetate (9a), Phorbol 12,13-Diacetate (9c), Phorbol 12,13,20-Triacetate (9d), 4-O-Methylphorbol 12,13,20-Triacetate (9e), 3b -Hydroxyphorbol 12,13,20-Triacetate (9f ), and Phorbol 12,13,20-Tribenzoate (10) Acetylation of 9 with Ac2O/pyridine at 90 °C for 1 h gave phorbol 12,13,20-triacetate (9d).16,17) Methylation of 9d with CH3I/Ag2O in DMF at room temperature for 20 h gave 4-O-methylphorbol 12,13,20-triacetate (9e),14) while the reduction of 9d with NaBH4 gave 3b hydroxyphorbol 12,13,20-triacetate (9f).13) When Ac2O/p-TsOH was used, 9d gave phorbol 4,12,13,20-tetraacetate (9g),16) while the reaction of 9 with benzoyl chloride/pyridine gave phorbol 12,13,20-tribenzoate (10).15,16) A solution of 9d (50 mg) in 0.05 M KOH/MeOH (10 ml) was stirred at room temperature for 1 h to give phorbol 12-acetate (9a) (40 mg, 80%). Selective hydrolysis of 9d (30 mg) with HClO4 afforded phorbol 12,13-diacetate (9c, 24 mg, in a yield of 80%) [oil. EI-MS m/z 338 [M2CH3COOH]1, 328 [M223CH3COOH]1. 1H-NMR (CDCl3) d : 2.0 and 2.12 (each 3H, s, 23CH3CO–), 5.4 (1H, d, J510.2, H-12) and 4.0 (2H, ABq, J512.5, H220)]. Preparation of Phorbol 13-Acetate (9b) Selective hydrolysis of 1 with HClO4 afforded 9b.18) Preparation of Isophorbol 12,13,20-Triacetate (14c), Isophorbol 12,13,20-Tributylate (14d), Isophorbol 4,12,13,20-Tetraacetate (14e), and a Photo Product (22) Acylation of 14 (36 mg) with butyryl chloride in pyridine afforded 14d (18 mg, in a yield of 50%). Acetylation of 14 gave 14c35) and 14e.19) Irradiation of 14c with UV light (254 nm/5 h) afforded 22.19) Preparation of 1a Compound 1a was obtained from 1 by treatment with mesyl chloride/pyridine at room temperature for 21 h.20) The compound 527 had the following physicochemical and spectroscopic properties: oil. [a ]D 13° (c50.05, CHCl3). IR n max cm21: 3450 (OH), 2960 and 2930 (C5C), 2850, 1730 (ester C5O), 1600 (a ,b -unsaturated ketone). UV l max (log e ) nm: 243 (3.55). EI-MS m/z 650 [M]1, 632 [M2H2O]1, 590 [M2AcOH]1, 572 [M2AcOH2H2O]1, 370 [M2linoleic acid]1, 352 [M2linoleic acid2H2O]1, 310 [M2linoleic acid2AcOH]1. 1H-NMR (CDCl3) d : 0.90 (3H, m, CH3, linoloyl), 1.10 (3H, d, J57.4 Hz, H3-18), 1.32 (14H, br d, 73CH2, linoloyl), 1.57 (3H, s, H3-17), 1.60 (2H, m, CO–CH2–CH2, linoloyl), 1.80 (3H, m, H3-19), 2.08 (3H, s, COCH3), 2.10 (4H, m, CH2–CH5CH–CH2–CH5CH–CH2, linoloyl), 2.35 (2H, m, CO–CH2, linoloyl), 2.38 (1H, d, J519.0 Hz, Hb-5), 2.57 (1H, d, J519.0 Hz, Ha-5), 2.76 (2H, dd, J513 and 3.8 Hz, 5CH–CH2–CH5, linoloyl), 3.1 (1H, t, J52.2 Hz, H-10), 3.25 (1H, m, H-11), 3.40 (1H, d, J58.8 Hz, H-14), 3.50 (1H, dd, J58.8 and 5.5 Hz, H-8), 4.44 (2H, ABq, J515.0 Hz, H2-20), 4.90 (1H, s, Ha-16), 5.00 (1H, s, Hb-16), 5.10 (1H, t, J52.1 Hz, H-12), 5.34 (4H, m, –(CH5CH)2, linoloyl), 5.50 (1H, d, J55.5 Hz, H-7), and 7.60 (1H, t, J52.2 Hz, H-1). 13C-NMR (CDCl3) d : 11.0 (C-19), 14.9 (CH3, linoloyl), 17.3 (C-18), 18.6 (C-17), 21.4 (COCH3), 23.6 (CH2–CH3, linoloyl), 26.1 (CO–CH2–CH2, linoloyl), 26.7 (CH5CH–CH2–CH5CH–, linoloyl), 28.3 (CH2–CH5CH–CH2–CH5CH–CH2, linoloyl), 30.3—30.8 (43CH2, linoloyl), 33.1 (CH2, linoloyl), 32.7 (CH2, linoloyl), 35.4 (CO–CH2, linoloyl), 37.8 (C-11), 44.1 (C-8), 50.0 (C-14), 57.2 (C-10), 70.7 (C-20), 78.6 (C-4), 86.5 (C-9), 117.4 (C-16), 121.3 (C-12), 129.1 and 129.2 (CH5CH, linoloyl), 130.0 (C-7), 131.1 and 131.2 (CH5CH, linoloyl), 134.6 (C-2), 136.5 (C-6), 144.1 (C-13), 148.0 (C-15), 160.1 (C-1), 172.1 (C5O, linoloyl), 175.5 (C5O, COCH3), and 215.5 (C-3). Preparation of 12-O-,13-O-Diacetylphorbol 20-(9Z,12Z-Tetradecadienoate) (1b), 12-O-Decanoyl-13-O-(2-methylbutyryl)phorbol 20-Acetate (4a), 12-O-,20-O-Diacetylphorbol 13-Decanoate (6a) and 12-OTetradecanoylphorbol 13,20-Diacetate (8a) The respective acetylation of 1, 4, 6, and 8 gave 1b (85% in yield), 4a (90%), 6a (85%), and 8a (70%), respectively. These compounds had the following properties: 1b: oil. EI-MS m/z, 710 [M]1, 650 [M2CH3COOH]1. 1H-NMR (CDCl3) d : 2.0—212 (6H, s, 23CH3CO–) and 5.4 (1H, d, J510.2 Hz, H-12); 4a: oil. EI-MS m/z 644 [M]1, 584 [M2CH3COOH]1. 1H-NMR (CDCl3) d : 2.1 (3H, s, CH3CO–) and 4.42 (2H, ABq, J513.5 Hz, H2-20); 6a: oil. EI-MS m/z 602 [M]1, 542 [M2CH3COOH]1, 482 [M223CH3COOH]1. 1H-NMR (CDCl3) d : 2.04 and 2.1 (each 3H, s, CH3CO–) and 4.43 (2H, ABq, J515.0 Hz, H2-20); 8a: oil. EI-MS m/z 658 [M]1, 598 [M2CH3COOH]1, 538 [M223 CH3COOH]1. 1H-NMR (CDCl3) d : 2.06—2.10 (each 3H, s, CH3CO–) and 4.42 (2H, ABq, J512.5 Hz, H2-20). Preparation of 12-O-,20-O-Diacetyl-4-O-methylphorbol 13-Decanoate (6b) and 4-O-Methyl-12-O-tetradecanoylphorbol 13,20-Diacetate (8b) Methylation of 6 and 8 with MeI and Ag2O gave 6b (6 mg, 30%) and 8b (5 mg, 25%), respectively. These compounds had the following properties. 6b: oil. EI-MS m/z 616 [M]1. 1H-NMR (CDCl3) d : 2.08 and 2.13 (each 3H, s, CH3CO–), and 3.27 (3H, s, CH3O–); 8b: oil. EI-MS m/z 672 [M]1. 1HNMR (CDCl3) d : 2.06 and 2.1 (each 3H, s, CH3CO–) and 3.27 (3H, s, CH3O–). Preparation of 12-O-Acetylphorbol 13,20-Ditetradecanoate (8d), 12O-Acetylphorbol 13,20-Dihexanoate (11a), 12-O-Acetylphorbol 13,20Dinonanoate (12a) and 12-O-Acetylphorbol 13,20-Didodecanoate (13a) A pyridine solution (1.5 ml) containing 2 mM acyl chloride and 0.12 mM of 9a (50 mg) was stirred at 0 °C under an atmosphere of argon, and then at room temperature for 5 d, with products monitored by TLC, to give the respective 12-O-acetyl-13-O-,20-O-diacyl derivatives in yields of 60—80%. 8d: oil (30 mg, 60% from 9a). EI-MS m/z 826 [M]1, 766 [M2CH3COOH]1, 598 [M2CH3(CH2)12COOH]1, 370 [M223CH3–(CH2)122COOH]1. 1HNMR (CDCl3) d : 2.06 (3H, s, CH3CO–), 5.45 (1H, d, J510.5 Hz, H-12), 4.45 (2H, ABq, J512.0 Hz, H2-20), and signals for two tetradecanoyl moieties at d 2.3 (4H, 23CH2), 1.60 (4H, 23CH2), 1.2 (40H, 2310CH2) and 0.09 (6H, 23CH3); 11a: oil (32.5 mg, 65% from 9a). EI-MS m/z 602 [M]1, 542 [M2CH3COOH]1, 486 [M2CH3(CH2)4COOH]1, 370 [M223 CH3(CH2)4COOH]1. 1H-NMR (CDCl3) d : 2.06 (3H, s, CH3CO–), 5.45 (1H, d, J510.0 Hz, H-12), 4.43 (2H, ABq, J512.0 Hz, H2-20), and signals for two hexanoyl moieties at 2.3 (4H, 23CH2), 1.60 (4H, 23CH2), 1.2 (12H, 63CH2) and 0.09 (6H, 23CH3); 12a: oil (40 mg, 80% from 9a). EI-MS m/z 626 [M]1, 528 [M2CH3(CH2)7COOH]1, 370 [M223CH3(CH2)7COOH]1. 1 H-NMR (CDCl3) d : 2.06 (3H, s), 5.45 (1H, d, J510.0 Hz, H-12), 4.41 (2H, ABq, J512.0 Hz, H2-20), and signals for two octanoyl moieties at 2.30 (4H, 23CH2), 1.62 (4H, 23CH2), 1.2 (20H, 103CH2) and 0.09 (6H, 23CH3); 13a: oil (37.5 mg, 75% from 9a). EI-MS m/z 770 [M]1, 570 [M2 CH3(CH2)10COOH]1, 510 [M2CH3COOH2CH3(CH2)10COOH]1, 370 [M223CH3(CH2)10COOH]1. 1H-NMR (CDCl3) d : 2.06 (3H, s), 5.45 (1H, 528 d, J510.0 Hz, H-12), 4.45 (2H, ABq, J514.0 Hz, H2-20), and signals of two dodecanoyl moieties at 2.30 (4H, 23CH2), 1.60 (2H, CH2), 1.20 (32H, 163CH2) and 0.09 (6H, 23CH3). Preparation of 12-O-Acetylphorbol 13-Tetradecanoate (8c), 12-OAcetylphorbol 13-Hexanoate (11), 12-O-Acetylphorbol 13-Octanoate (12) and 12-O-Acetylphorbol 13-Dodecanoate (13) by Partial Hydrolysis The 12-O-acetyl-13-O-,20-O-diacylphorbol derivatives obtained (8d, 11a, 12a and 13a) were separately hydrolyzed (each 30 mg) with 70% HClO4/MeOH to give the respective 12-O-acetyl-13-O-acylphorbol derivatives (8c, 11, 12 and 13) in yields of 60—70%. All products were purified by column chromatography on SiO2, followed by MPLC (RP-18). The structures of these compounds were established by spectroscopic methods and their characteristics are described below. 8c: oil (21 mg, 70% from 8d). EIMS m/z 616 [M]1, 556 [M2CH3COOH]1, 388 [M2CH3(CH2)12COOH]1, 370 [M2H2O2CH3(CH2)12COOH]1. 1H-NMR (CDCl3) d : 2.06 (3H, s, CH3CO–), 5.45 (1H, d, J510.0 Hz, H-12), 4.00 (2H, ABq, J512.0 Hz, H220), and signals for tetradecanoyl moiety at d : 2.3 (2H, CH2), 1.62 (2H, CH2), 1.2 (20H, 103CH2) and 0.09 (3H, CH3); 11: oil (18 mg, 60% from 11a). EI-MS m/z 504 [M]1, 444 [M2CH3COOH]1, 388 [M2 CH3(CH2)4COOH]1, 370 [M2H2O2CH3(CH2)4COOH]1. 1H-NMR (CDCl3) d : 2.06 (3H, CH3CO–), 5.45 (1H, d, J510.0 Hz, H-12), 4.00 (2H, ABq, J512.0 Hz, H2-20), and signals for hexanoyl moiety at 2.3 (2H, CH2), 1.62 (2H, CH2), 1.2 (4H, 23CH2) and 0.09 (3H, CH3); 12: oil (21 mg, 70% from 12a). EI-MS m/z 546 [M]1, 486 [M2CH3COOH]1, 388 [M2 CH3(CH2)7COOH]1, 370 [M2H2O2CH3(CH2)7COOH]1. 1H-NMR (CDCl3) d : 2.06 (3H, s, CH3–CO–), 5.45 (1H, d, J510.0 Hz, H-12), 4.00 (2H, ABq, J512.0 Hz, H2-20), and signals for octanoyl moiety at 2.30 (2H, CH2), 1.61 (2H, CH2), 1.2 (10H, 53CH2) and 0.09 (3H, CH3); 13: oil (19.5 mg, 65% from 13a). EI-MS m/z 570 [M]1, 510 [M2CH3COOH]1, 388 [M2CH3(CH2)10COOH]1, 370 [M2H2O2CH3(CH2)10COOH]1. 1H-NMR (CDCl3) d : 2.06 (3H, s), 5.45 (1H, d, J510.0 Hz, H-12), 4.00 (2H, ABq, J512.0 Hz, H2-20), and signals for dodecanoyl moiety at 2.3 (2H, CH2), 1.62 (2H, CH2), 1.20 (16 H, 83CH2) and 0.09 (3H, CH3). Preparation of 13-O-Acetylisophorbol (14a) and 13-O-,20-O-Diacetylisophorbol (14b) Acetic anhydride (50 m l) was added to an ice cooled solution of isophorbol (14, 50 mg, 0.14 mmol) in pyridine (1 ml), and the mixture was stirred at 60 °C for 1 h. The reaction mixture was worked up as usual to afford 14a (8 mg, 14%) and 14b (22 mg 36%). 14a: oil. EI-MS m/z 406 [M]1, 388 [M2H2O]1, 370 [M22H2O]1, 352 [M23H2O]1, 309 [M23H2O2CH3CO–]1. 1H-NMR (CDCl3) d : 2.10 (3H, s, COCH3); 14b: oil. EI-MS m/z 448 [M]1, 430 [M2H2O]1, 412 [M22H2O]1, 369 [M22H2O2CH3CO–]1. 1H-NMR (CDCl3) d : 2.11, 2.09 (6H, 2s, COCH3), 4.33 (2H, ABq, J512.4 Hz, H-20). Preparation of 12-O-Octanoylisophorbol 13,20-Diacetate (15), 12-ODecanoylisophorbol 13,20-Diacetate (16), 12-O-(10-Undecenoyl)isophorbol 13,20-Diacetate (17), 12-O-Dodecanoylisophorbol 13,20-Diacetate (18), 12-O-Tetradecanoylisophorbol 13,20-Diacetate (19), 12-O-Heptadecanoylisophorbol 13,20-Diacetate (20) and 12-O-(1-Adamantanoyl)isophorbol 13,20-Diacetate (21) Acyl chloride (400 m l, 0.44 mmol) was added to an ice cooled solution of 14a (50 mg, 0.11 mmol) in pyridine (1 ml), and the mixture was stirred at room temperature for 1—3 d. The reaction mixture was worked up as usual to give the respective products in yields of 43—74%. 15: oil (45 mg, 70%). EI-MS m/z 574 [M]1, 556 [M2H2O]1, 514 [M2H2O2CH3CO–]1, 496 [M22H2O2CH3CO–]1, 429 [M2H2O2C8H15O]1. 1H-NMR (CDCl3) d : 2.10, 2.07 (6H, 2s, COCH3), 2.33 (2H, m, CH2), 4.33 (2H, ABq, J512.2 Hz, H-20), 5.47 (1H, d, J510.2 Hz, H-12), and signals for octanoyl moiety at d : 0.87 (3H, t, J56.6 Hz, CH3), 1.31 (8H, m, 43CH2), 1.68 (2H, m, CH2); 16: oil (43 mg, 64%). EI-MS m/z 602 [M]1, 583 [M2H2O]1, 524 [M2H2O2CH3COOH]1, 481 [M2H2O2CH3COOH2CH3COO–]1, 429 [M2H2O2C10H19O]1. 1HNMR (CDCl3) d : 2.09, 2.07 (6H, 2s, COCH3), 2.35 (2H, m, CH2), 4.33 (2H, ABq, J512.5 Hz, H-20), 5.47 (1H, d, J510.3 Hz, H-12), and signals for decanoyl moiety at d : 0.88 (3H, t, J56.6 Hz, CH3), 1.27 (15H, m, H-16, 63CH2), and 1.67 (2H, m, CH2); 17: oil (51 mg, 74%). EI-MS m/z 614 [M]1, 596 [M2H2O]1, 536 [M2H2O2CH3COOH]1, 429 [M2H2O2(10undecenoyl)]1. 1H-NMR (CDCl3) d : 2.09, 2.06 (6H, 2s, COCH3), 4.34 (2H, ABq, J512.4 Hz, H-20), 5.47 (1H, d, J510.3 Hz, H-12), and signals for 10undecenoyl moiety at d : 1.3—1.6 [(2H, m, CH25CH–(CH2)7–CH2–), 2.03 [(2H, m, CH25CH–(CH2)7–CH2–), 2.6 [(2H, t, J58.04 Hz, CH25CH– (CH2)7–CH2–), 4.94 [(2H, m, CH25CH–(CH2)8–), and 5.81 [(1H, m, CH25CH–(CH2)8–); 18: oil (47 mg, 67%). EI-MS m/z 630 [M]1, 612 [M2H2O]1, 534 [M22H2O2CH3COOH]1, 475 [M22H2O2 CH3COOH2CH3COO–]1, 429 [M2H2O2C12H23O]1. 1H-NMR (CDCl3) d : 2.10, 2.08 (6H, 2s, COCH3), 4.32 (2H, ABq, J512.5 Hz, H-20), 5.46 (1H, d, Vol. 50, No. 4 J510.3 Hz, H-12), and signals for dodecanoyl moiety at d : 0.87 (3H, t, J56.6 Hz, CH3), 1.27 (19H, m, H-16, 83CH2), 1.67 (2H, m, CH2), and 2.36 (2H, m, CH2); 19: oil (42 mg, 57%). EI-MS m/z 658 [M]1, 640 [M2H2O]1, 597 [M2H2O2CH3CO–]1, 538 [M2H2O2CH3COO2CH3CO–]1, 429 [M2H2O2C14H27O]1. 1H-NMR (CDCl3) d : 2.09, 2.07 (6H, 2s, COCH3), 4.33 (2H, ABq, J512.4 Hz, H-20), 5.47 (1H, d, J510.3 Hz, H-12), and signals for tetradecanoyl moiety at d : 0.88 (3H, t, J56.8 Hz, CH3), 1.26 (23H, m, H-16, 93CH2), 1.64 (2H, m, CH2), and 2.32 (2H, m, CH2); 20: Oil (50 mg, 64%). EI-MS m/z 700 [M]1, 682 [M2H2O]1, 587 [M2H2O2 CH3COO–]1, 569 [M22H2O2CH3COO–]1, 429 [M2H2O2C17H33O]1. 1 H-NMR (CDCl3) d : 2.09, 2.07 (6H, 2s, COCH3), 4.33 (2H, ABq, J5 12.4 Hz, H-20), 5.47 (1H, d, J510.3 Hz, H-12), and signals for heptadecanoyl moiety at d : 0.88 (3H, t, J56.8 Hz, CH3), 1.25 (29H, m, H-16, 133CH2), 1.63 (2H, m, CH2), and 2.31 (2H, m, CH2); 21: oil (45 mg, 66%). EI-MS m/z 610 [M]1, 592 [M2H2O]1, 532 [M2H2O2CH3COOH]1, 429 [M2H2O-1-(1-adamantnoyl)]1. 1H-NMR (CDCl3) d : 2.09, 2.07 (6H, 2s, COCH3), 4.33 (2H, ABq, J512.0 Hz, H-20), 5.44 (1H, d, J510.5 Hz, H-12), and signals for 1-adamantanoyl moiety at d : 1.73 (7H, m), 1.91 (6H, m, H11), and 2.06 (3H, m). Preparation of 13-O-Acetyl(4-deoxy-4a -phorbol) (23a), 13-O-,20-ODiacetyl(4-deoxy-4a -phorbol) (23b) and 12-O-,13-O-,20-O-Triacetyl(4deoxy-4a -phorbol) (23c) Acetylation of 23 with Ac2O/pyridine at 60 °C for 1 h afforded 23a (21%), 23b (35%) and 23c (11%). 23a: oil. EI-MS m/z 390 [M]1, 372 [M2H2O]1, 354 [M22H2O]1, 329 [M2H2O2CH3CO–]1. 1 H-NMR (CDCl3) d : 2.09 (3H, s, COCH3); 23b: oil. EI-MS m/z 432 [M]1, 414 [M2H2O]1, 354 [M2H2O2CH3COOH]1. 1H-NMR (CDCl3) d : 2.12, 2.11 (6H, 2s, COCH3), and 4.47 (2H, ABq, J511.8 Hz, H-20); 23c36): oil. EI-MS m/z 474 [M]1, 431 [M2CH3CO–]1, 413 [M2H2O2CH3CO–]1, 353 [M2H2O2CH3COOH2CH3CO–]1. 1H-NMR (CDCl3) d : 2.12, 2.11, 2.07 (9H, 3s, COCH3), 4.40 (2H, ABq, J512.7 Hz, H-20), and 5.44 (1H, d, J510.5 Hz, H-12). Preparation of 12-O-Decanoyl(4-deoxy-4a -phorbol) 13,20-Diacetate (24) and 12-O-Tetradecanoyl(4-deoxy-4a -phorbol) 13,20-Diacetate (25) Acyl chloride (400 m l, 0.48 mmol) was added to an ice cooled solution of 23b (50 mg, 0.12 mmol) in pyridine (1 ml), and the mixture was stirred at room temperature for 24 h. The reaction mixture was worked up as usual to give 24 (71%) and 25 (68%). 24: oil. EI-MS m/z 586 [M]1, 543 [M2CH3CO–]1, 526 [M2CH3COOH]1, 413 [M2H2O2C10H19O]1. 1HNMR (CDCl3) d : 2.12, 2.06 (6H, 2s, COCH3), 4.40 (2H, ABq, J512.5 Hz, H-20), 5.47 (1H, d, J510.5 Hz, H-12), and signals for decanoyl moiety at d : 0.88 (3H, t, J57.1 Hz, CH3), 1.28 (12H, m, 63CH2), 2.36 (2H, m, CH2), and 2.59 (2H, t, J57.8 Hz, CH2); 25: oil. EI-MS m/z 642 [M]1, 599 [M2CH3CO–]1, 582 [M2CH3COOH]1, 522 [M22CH3COOH]1, 413 [M2H2O2C14H27O]. 1H-NMR (CDCl3) d : 2.12, 2.06 (6H, 2s, COCH3), 4.14 (2H, ABq, J512.5 Hz, H-20), 5.47 (1H, d, J510.8 Hz, H-12), and signals for tetradecanoyl moiety at d : 0.88 (3H, t, J57.1 Hz, CH3), 1.27 (16H, m, 63CH2), 2.36 (2H, m, CH2), and 2.59 (2H, t, J57.8 Hz, CH2). References 1) Che C.-T., “Economic and Medicinal Plant Research,” Vol. 5, ed. by Wagner H., Farnsworth N. R., Academic Press, London, 1991, pp. 167—251. 2) Schinazi R. F., “Natural Products as Antiviral Agents,” ed. by Chu C.-K., Cutler H. G., Plenum, New York, 1992, pp. 1—29. 3) Nasr M., Cradock J., Johnson M. “Natural Products as Antiviral Agents,” ed. by Chu C.-K., Cutler H. G., Plenum, New York, 1992, pp. 31—56. 4) El-Mekkawy S., Meselhy M. R., Kusumoto I. T., Kadota S., Hattori M., Namba T., Chem. Pharm. Bull., 43, 641—648 (1995). 5) El-Mekkawy S., Meselhy M. R., Nakamura N., Tezuka Y., Hattori M., Kakiuchi N., Shimotohno K., Kawahata T., Otake T., Phytochemistry, 49, 1651—1657 (1998). 6) Ng T. B., Huang B., Fong W. P., Yeung H. W., Life Sci., 61, 933—949 (1997). 7) El-Mekkawy S., Meselhy M. R., Nakamura N., Hattori M., Kawahata T., Otake T., Chem. Pharm. Bull., 47, 1346—1347 (1999). 8) El-Mekkawy S., Meselhy M. R., Nakamura N., Hattori M., Kawahata T., Otake T., Phytochemistry, 54, 457—464 (2000). 9) Cairnes D. A., Mirvish S. S., Wallcave L., Nagel D. L., Smith J. W., Cancer Lett., 14, 85—91 (1981). 10) Mishra N. C., Estensen R. D., Abdel-Monem M. M., J. Chromatogr., 369, 435—439 (1986). 11) Tseng S.-S., Van Duuren B. L., Solomon J. J., J. Org. Chem., 42, 3645—3649 (1977). April 2002 12) Evans F. J., Kinghorn A. D., J. Chromatogr., 87, 443—448 (1973). 13) Hecker E., Bartsch H., Gschwendt M., Harle E., Kreibich G., Kubinyi H., Schairer H. U., Szczepanski Ch.-V., Thielmann H. W., Tetrahedron Lett., 1967, 3165—3170. 14) Bartsch H., Snatzke G., Hecker E., Z. Naturforschg., 23b, 1453—1460 (1968). 15) Crombie L., Games M. L., Pointer D. J., J. Chem. Soc. C, 1968, 1347—1362. 16) Hecker E., Szczepanski C. V., Kubinyi H., Bresch H., Harle E., Schairer H. U., Bartsch H., Z. Naturforschg., 21b, 1204—1214 (1966). 17) Hecker E., Kubinyi H., Szczepanski Ch.-V., Harle E., Bresch H., Tetrahedron Lett., 1965, 1837—1842. 18) Zayed S., Sorg B., Hecker E., Planta Med., 50, 65—69 (1984). 19) Hecker E., Harle E., Schairer H. U., Jacobi P., Hoppe W., Gassmann J., Rohrl M., Abel H., Angew. Chem., 80, 913—914 (1968). 20) Bartsch H., Hecker E., Z. Naturforschg., 24b, 91—98 (1969). 21) Chowdhury M. I., Koyanagi Y., Kobayashi S., Hamamoto Y., Yoshiyama H., Yoshida T., Yamamoto N., Virology, 176, 126—132 (1990). 22) Nishizuka Y., Nature (London), 308, 693—698 (1984). 23) Nishizuka Y., Science, 233, 305—312 (1986). 529 24) 25) 26) 27) 28) 29) 30) 31) 32) 33) 34) 35) 36) Nishizuka Y., Nature (London), 334, 661—668 (1988). Bell R. M., Cell, 45, 631—632 (1986). Weinstein I. B., Cancer Res., 48, 4135—4143 (1988). Rahmsdorf H. J., Herrlich P., Pharmacol. Ther., 48, 157—188 (1990). Raineri R., Simsiman R. C., Boutwell R. K., Cancer Res., 33, 134— 139 (1973). Castagna M., Takai Y., Kaibuchi K., Sano K., Kikkawa U., Nishizuka Y., J. Biol. Chem., 257, 7847—7851 (1982). Kupchan S. M, Uchida I., Branfman A. R., Daily R. G., Yu-Fei B., Jr., Science, 191, 571—572 (1976). Handa S. S., Kinghorn A. D., Cordell G. A., Farnsworth N. R., J. Nat. Prod., 46, 123—126 (1983). Erickson K. L., Beutler J. A., Cardellina II J. H., McMahon J. B., Newman D. J., Boyd M. R., J. Nat. Prod., 58, 769—772 (1995). Kubinski H., Strangstalein M. A., Baird W. M., Boutwell R. K., Cancer Res., 33, 3103—3107 (1973). Harada S., Koyanagi Y., Yamamoto N., Science, 229, 563—566 (1985). Jacobi P., Härle E., Schairer H. U., Hecker E., Liebigs Ann. Chem., 741, 13—32 (1970). Fürsteinberger G., Hecker E., Tetrahedron Lett., 1977, 925—928.