Inhibition of cytopathic effect of human immunodeficiency virus type-1 by various phorbol derivatives

Article abstract of DOI:10.1248/cpb.50.523

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-O-acetylphorbol 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 13-decanoate (6a) (IC₁₀₀=15.6 μg/ml) vs. compound 6 (IC₁₀₀=0.0076 μg/ml), and 12-O-tetradecanoylphorbol 13,20-diacetate (8a) (IC₁₀₀=15.6 μg/ml) vs. 12-O- tetradecanoylphorbol 13-acetate (8) (IC₁₀₀=0.00048 μg/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 3β-hydroxyphorbol 12,13,20-triacetate (9f) (IC₁₀₀=500 μg/ml) vs. phorbol 12,13,20-triacetate (9d) (IC₁₀₀=62.5 μg/ml). Although 8 was equipotent in the inhibition of CPE, and activation of PKC, both activities were abruptly decreased by the acetylation of 20-OH 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.

Full text of DOI:10.1248/cpb.50.523

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
a
b
Norio NAKAMURA, Masao HATTORI, ,a Takuya KAWAHATA,b and Toru OTAKE
*
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 im-
munodeficiency virus (HIV)-1 induced cytopathic effects (CPE) on MT-4 cells, as well as their activation of pro-
tein 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-O-
acetylphorbol 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 13-
decanoate (6a) (IC100؍15.6 mg/ml) vs. compound 6 (IC100؍0.0076 mg/ml), and 12-O-tetradecanoylphorbol 13,20-
diacetate (8a) (IC100؍15.6 mg/ml) vs. 12-O-tetradecanoylphorbol 13-acetate (8) (IC100؍0.00048 mg/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-triac-
etate (9f) (IC100؍500 mg/ml) vs. phorbol 12,13,20-triacetate (9d) (IC100؍62.5 mg/ml). Although 8 was equipotent
in the inhibition of CPE, and activation of PKC, both activities were abruptly decreased by the acetylation of 20-
OH and methylation of 4-OH [as in 8a and 4-O-methyl-12-O-tetradecanoylphorbol 13,20-diacetate (8b), respec-
tively]. On the other hand, its positional isomer (12-O-acetylphorbol 13-tetradecanoate (8c) showed neither activ-
ities. The removal of a long acyl group in 8 led to a substantial loss of both activities, as shown in phorbol 13-ac-
etate (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-retro-
viral 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, signifi-
cant progress has been made towards the development of nat-
ural and synthetic agents that can directly inhibit HIV repli-
cation 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-(2-
methylbutyrate) (4) and 12-O-acetylphorbol 13-decanoate
(6) were found to potently inhibit an HIV-1-induced cyto-
pathic effect (CPE) on MT-4 cells without the activation of
protein kinase C (PKC) normally associated with tumor-pro-
moting action.7,8) This finding suggested that the develop-
ment 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 fo-
cused on the inhibition of CPE and activation of PKC.
R1
R2
R3
R4
1
H
Ac
Ac
Tig
Tig
C18H31O
C18H31O
C18H31O
H
H
1b Ac
H
2
3
4
H
H
Ac
H
C10H19O
2-Me butyryl
H
H
4a C10H19O
2-Me butyryl Ac
H
5
6
Tig
Ac
2-Me butyryl
C10H19O
H
H
H
H
6a Ac
6b Ac
7
8
C10H19O
Ac
H
C10H19O
Ac
Me
H
2-Me butyryl
C14H27O
C
12H23O
H
Ac
H
H
8a C14H27O
8b C14H27O
8c Ac
Ac
Ac
H
Ac
Ac
Me
H
C14H27O
C14H27O
H
H
8d Ac
9
9a Ac
9b
9c Ac
9d Ac
C14H27O
H
H
H
H
H
H
H
H
Ac
H
H
Ac
H
H
Ac
Ac
H
9e Ac
9g Ac
Ac
Ac
Me
Ac
H
Ac
Ac
10 Bz
11 Ac
11a Ac
12 Ac
12a Ac
13 Ac
13a Ac
Bz
Bz
Results
C6H11O
C6H11O
C9H17O
C9H17O
C12H23O
C12H23O
H
H
Synthesis of Phorbol and Isophorbol Derivatives Hy-
drolysis of a phorbol ester mixture from the seeds of Croton
tiglium with Ba(OH)2/MeOH9,10) yielded tetracyclic diter-
penes, phorbol (9), 4a-phorbol (14, isophorbol) and 4-
deoxy-4a-phorbol (23), which were identified by comparison
C6H11O
H
H
H
C9H17O
H
H
H
C12H23O
H
of their spectral data with those reported.11—13) Phorbol Chart 1. Chemical Structures of Phorbol and Its Derivatives
To whom correspondence should be addressed. e-mail: saibo421@ms.toyama-mpu.ac.jp
© 2002 Pharmaceutical Society of Japan

524
Vol. 50, No. 4
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 pre-
pared from 23, while 24a—d and 25 were obtained from 23b
(Chart 2).19)
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,
13-O-Acetylphorbol 20-octadecanoate (1), 4, 6 and 12-O-
tetradecanoylphorbol 13-acetate (8), showing appreciable in-
hibition 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-octa-
decanoate (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-O-
acetylphorbol 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 hy-
drolysis with 70% HClO4 in MeOH.11) The structures of
these compounds were established by various spectroscopic
means, including 2D-NMR.
R1
R2
R3
R4
14
H
H
H
Ac
H
H
H
OH
OH
OH
OH
OH
OAc
OH
OH
OH
OH
OH
OH
OH
H
14a
14b
14c
14d
14e
15
16
17
18
19
20
21
23
23a
23b
23c
24
Ac
Ac
Ac
C4H7O
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
H
Ac
Ac
C4H7O
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
H
C4H7O
Ac
C8H15O
C10H19O
10-Undecenoyl
C12H23O
C14H27O
C17H33O
1-Adamantanoyl
H
H
H
Ac
Ac
Ac
Ac
Ac
Ac
H
H
H
H
H
Ac
Ac
Ac
Ac
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
C10H19O
C14H27O
25
H
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 (mg/ml)
% Activation
of PKCa)
No.
R1
R2
R3
R4
IC100
CC0
14
H
H
H
Ac
C4H7O
Ac
C8H15O
C10H19O
10-Undecenoyl
C12H23O
C14H27O
C17H33O
1-Adamantanoyl
H
H
H
OH
OH
OH
OH
OH
OAc
OH
OH
OH
OH
OH
OH
OH
NE
500
Ͼ1000
Ͼ1000
500
0
14a
14b
14c
14d
14e
15
16
17
18
19
20
21
22
23
23a
23b
23c
24
Ac
Ac
Ac
C4H7O
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
NE
NE
250
NE
NE
NT
NT
0
0
0
NT
NT
NT
NT
NT
NT
NT
0
Ac
Ac
C4H7O
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
62.5
500
500
500
500
31.25
125
125
NE
NE
NE
250
NE
NE
62.5
62.5
7.81
31.25
62.5
1000
1000
Ͼ1000
500
500
500
1000
500
250
H
H
H
Ac
C10H19O
C14H27O
H
H
Ac
Ac
Ac
Ac
H
H
H
H
H
H
H
0
Ac
Ac
Ac
Ac
Ac
NT
NT
NT
NT
NT
1000
1000
25
a) At 10 ng/ml, relative to that shown by TPA (100% inhibition) (8); NE, not effective; NT, not tested.

April 2002
525
Table 2. Inhibition of HIV-1 Induced CPE and Activation of PKC by Phorbol and Its Derivatives
Inhibition of CPE (mg/ml)
Activation of PKC
R1
R2
R3
R4
a)
IC100
CC0
%
MAC (mg/ml)
1
1b
2
3
4
4a
5
6
6a
6b
7
H
Ac
H
Ac
C10H19O
C10H19O
Tig
Ac
Ac
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
C18H31O
C18H31O
C18H31O
H
H
Ac
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
Me
H
H
H
H
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
62.5
62.5
62.5
0
0
14
16
0
10
10
0
11
0
16
100
0
0
0
0
8
13
0
57
0
0
0
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
Ͼ50
Ͼ50
H
Ͼ100
Ac
Ac
H
Ac
2-Me butyryl
8
C14H27O
C14H27O
C14H27O
Ac
Ac
H
Ac
H
Ac
Ac
H
0.01
8a
8b
8c
8d
9
9a
9b
9c
9d
9e
9g
Ac
Ac
H
C14H27O
H
H
H
H
Ac
Ac
Ac
Bz
NE
125
H
H
H
Me
Ac
H
Ͼ1000
Ͼ1000
125
NE
62.5
31.3
125
NE
Ac
Ac
Bz
Ac
Ac
Bz
125
250
31.3
10
100
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 Ͼ1000, respectively.
(Table 1), while phorbol esters 9a—g showed variable activi-
ties (Table 2). Of the isophorbol derivatives, 12-O-octa-
noylphorbol 13,20-diacetate (15), as well as mono- and di-
acetyl derivatives of 23 (23a, b), were moderately active
against CPE (IC100 value of 62.5 mg/ml), and the activity of
23b was remarkably enhanced by further acetylation (as in
23c, IC100 value of 7.81 mg/ml). Decanoyl and tetradecanoyl
derivatives of 23 (24, 25) were moderately active (IC100 val-
ues of 31.25 and 62.5 mg/ml, respectively). As for the phor-
bol derivatives, 12-O-acetyl derivative (9a) was inactive,
while the 13-O-acetyl counter part (9b) showed weak inhibi-
tion 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 mg/ml). Methylation of
the triacetate 9d enhanced the inhibition of CPE (as in 9e,
IC100 value of 31.3 mg/ml), while reduction of a carbonyl
group at C-3 sharply reduced the activity, as in 9f (IC100
value of 500 mg/ml, CC0 value of 1000 mg/ml, without PKC
activation). A two-fold increase in the inhibitory activity
against CPE was observed for 1a (IC100 value of 7.81 mg/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).
Table 3. Effects of Acyl Chain Length at C-13 of 12-O-Acetyl-13-O-
acylphorbol Derivatives on Inhibition of HIV-1 Induced CPE and Activation
of PKC
Inhibition of CPE (mg/ml)
% Activation
No.
n
of PKCa)
IC100
CC0
9c
11
12
6
0
4
7
NE
NE
31.3
0.0076
Ͼ1000
57
27
10
0
62.5
31.3
62.5
8
13
8c
10
12
250
NE
500
125
35
0
a) At 10 ng/ml, relative to that shown by TPA (100% inhibition) (8); NE, not effec-
tive.
group at C-20 (as in 4a, IC100 value of 3.9 mg/ml).
Although 8 was found to be equipotent in terms of the in-
hibition 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-
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

526
Vol. 50, No. 4
isolated from C. tiglium,30) and ostodin and 12-O-undeca-
dienoylphorbol 13-acetate isolated from Ostodes panicu-
lata,31) were potent antitumor agents. 12-Deoxyphorbol 13-
decadienoate, isolated from Excoecaria agallocha as an anti-
HIV principle, was also a potent displacer of [3H]-phorbol
dibutyrate from rat brain membrane.32) However, other deriv-
atives (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 discrepan-
cies have been postulated to reflect a difference in the spe-
cific 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 re-
ceptor(s) other than/or in addition to PKC.
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 mg/ml) (Table 2).
Table 3 shows the comparison of inhibitory activity of
CPE and activation of PKC among various 12-O-acetyl-13-
O-acylphorbols having different chain lengths of fatty acid
residue (C6 : 0, C9 : 0, C12 : 0 and C14 : 0). The maximum in-
hibition 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 inhi-
bition and PKC-activation activities are influenced by the
configuration of the diterpene ester, in which all active phor-
bol derivatives are of the A/B trans configuration. The A/B
cis analogs (isophorbol type) showed no remarkable in-
hibitory effects on CPE. The finding that an anti-CPE activ-
ity of 1 was enhanced 2-times after homoallylic rearrange-
ment of an a-(acetoxycyclopropyl)carbinol group (as in 1a)
Experimental
Instruments Optical rotations were obtained on a DIP-360 automatic
polarimeter (Jasco, Tokyo, Japan). IR spectra were recorded on an FT/IR-
suggested that this group is not a critical requirement for the 230 spectrophotometer (Jasco). UV spectra were measured with a UV-2200
UV-VIS recording spectrophotometer (Shimadzu, Kyoto, Japan). NMR
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 activa-
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
tion, 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 HIV-
1-induced CPE, but showed no activation of PKC, suggested
that the difference in chain lengths of acyl groups and its rel-
ative 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.
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)ϫ30 m] (J & W Scientific,
U.S.A.), coupled to an automass system II benchtop quadrupole mass spec-
trometer (JEOL) under the following conditions: column temperature, 50 °C
for 10 min and gradient to 250 °C (10 °C/min) for 20 min; injection tempera-
ture, 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
Chowdhury et al.21) found that the cocultivation of MOLT-
4 and MOLT-4/HIV-1HTLV-IIIB cells at concentrations of more
than 0.01 ng/ml of TPA (8) for 20 h strikingly inhibited HIV-
induced 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 down- followed by heating.
Chemicals and Enzymes Compounds 1—8 were obtained from the
modulating 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 HIV-
1induced 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 phor-
bol derivatives showed different spectra of biological re-
sponses, 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
seeds of Croton tiglium as reported previously.8) Rat brain protein kinase C
(PKC, specific activity, 100 units/ml), staurosporine and L-a-phosphatidyl-L-
serine were purchased from Sigma (St. Louis, U.S.A.). A protein kinase en-
zyme assay system (code RPN 77 kit) was purchased from Amersham Inter-
national (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,NЈ,NЈ-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), sup-
plemented with 10% fetal calf serum (FCS, Flow Laboratories, North Ryde,
Australia), 100 mg/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
inhibit CPE. The diesters 12-O-tigloylphorbol 13-decanoate CPE was measured by the method of Harada et al.34) MT-4 cells were in-

April 2002
527
had the following physicochemical and spectroscopic properties: oil. [a]D
ϩ3° (cϭ0.05, CHCl3). IR nmax cmϪ1: 3450 (OH), 2960 and 2930 (CϭC),
2850, 1730 (ester CϭO), 1600 (a,b-unsaturated ketone). UV lmax (log e)
nm: 243 (3.55). EI-MS m/z 650 [M]ϩ, 632 [MϪH2O]ϩ, 590 [MϪAcOH]ϩ,
572 [MϪAcOHϪH2O]ϩ, 370 [MϪlinoleic acid]ϩ, 352 [MϪlinoleic
acidϪH2O]ϩ, 310 [MϪlinoleic acidϪAcOH]ϩ. 1H-NMR (CDCl3) d: 0.90
(3H, m, CH3, linoloyl), 1.10 (3H, d, Jϭ7.4 Hz, H3-18), 1.32 (14H, br d,
7ϫCH2, 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–CHϭCH–CH2–CHϭCH–CH2, linoloyl), 2.35 (2H, m, CO–CH2,
linoloyl), 2.38 (1H, d, Jϭ19.0 Hz, Hb-5), 2.57 (1H, d, Jϭ19.0 Hz, Ha-5), 2.76
(2H, dd, Jϭ13 and 3.8 Hz, ϭCH–CH2–CHϭ, linoloyl), 3.1 (1H, t,
Jϭ2.2 Hz, H-10), 3.25 (1H, m, H-11), 3.40 (1H, d, Jϭ8.8 Hz, H-14), 3.50
(1H, dd, Jϭ8.8 and 5.5 Hz, H-8), 4.44 (2H, ABq, Jϭ15.0 Hz, H2-20), 4.90
(1H, s, Ha-16), 5.00 (1H, s, Hb-16), 5.10 (1H, t, Jϭ2.1 Hz, H-12), 5.34 (4H,
m, –(CHϭCH)2, linoloyl), 5.50 (1H, d, Jϭ5.5 Hz, H-7), and 7.60 (1H, t,
Jϭ2.2 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 (CHϭCH–CH2–CHϭCH–, linoloyl),
28.3 (CH2–CHϭCH–CH2–CHϭCH–CH2, linoloyl), 30.3—30.8 (4ϫCH2,
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
(CHϭCH, linoloyl), 130.0 (C-7), 131.1 and 131.2 (CHϭCH, linoloyl),
134.6 (C-2), 136.5 (C-6), 144.1 (C-13), 148.0 (C-15), 160.1 (C-1), 172.1
(CϭO, linoloyl), 175.5 (CϭO, COCH3), and 215.5 (C-3).
fected for 1h with the LAI strain of HIV-1 at TCID50 of 0.001/cell (deter-
mined 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 1.5ϫ105
cells/ml in RPMI-1640 medium. Then, 200 ml/well of the cell suspension
was cultured for 5 d in a 96-well culture plate containing various concentra-
tions of a test compound. Control assays were performed without the test
compounds in HIV-1-infected and -uninfected cultures. On day 5, the con-
centration of the test compound that completely prevented CPE (IC100) and
the concentration that reduced the viability of MT-4 cells (CC0) were deter-
mined 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 pep-
tide, 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 com-
pounds (10 ng/ml) dissolved in DMSO (a final concentration of DMSO did
not exceed 0.02%). The reaction mixture (55 ml) 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 mg/ml phenyl methyl sulphonyl fluoride, 4.2 mM benza-
midine, 1.3 mM calcium acetate, 75 mM peptide, 34 mg/ml L-a-phosphatidyl-
L-serine, 3.4 mM DTT, 0.68 mM sodium azide, and 6.5 nM MgCl2. After the
addition of 0.55 nM [g-32P]ATP (50ϫ103 cpm/nmol), the reaction mixture
was mixed and maintained at 37 °C for 30 min. Reactions were terminated
by the addition of 10 ml of an ice-cold stop reagent. Aliquots of 35 ml 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 du-
plicate 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 mg/ml L-a-phosphatidyl-L-serine, 1.3 mM Ca ac-
etate and 2.7 mg/ml TPA, PKC activity was completely inhibited by stau-
rosporine at a concentration of 180 nM.
Preparation of Phorbol (9), Isophorbol (14) and 4-Deoxy-4a-isophor-
bol (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 chromatog-
raphy 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 4-
deoxy-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-Triac-
etate (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 so-
lution of 9d (50 mg) in 0.05 M KOH/MeOH (10 ml) was stirred at room tem-
perature for 1 h to give phorbol 12-acetate (9a) (40 mg, 80%). Selective hy-
drolysis 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 [MϪCH3COOH]ϩ, 328
[MϪ2ϫCH3COOH]ϩ. 1H-NMR (CDCl3) d: 2.0 and 2.12 (each 3H, s,
2ϫCH3CO–), 5.4 (1H, d, Jϭ10.2, H-12) and 4.0 (2H, ABq, Jϭ12.5, H2-
20)].
Preparation of 12-O-,13-O-Diacetylphorbol 20-(9Z,12Z-Tetradeca-
dienoate) (1b), 12-O-Decanoyl-13-O-(2-methylbutyryl)phorbol 20-Ac-
etate (4a), 12-O-,20-O-Diacetylphorbol 13-Decanoate (6a) and 12-O-
Tetradecanoylphorbol 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%), re-
spectively. These compounds had the following properties: 1b: oil. EI-MS
m/z, 710 [M]ϩ, 650 [MϪCH3COOH]ϩ. 1H-NMR (CDCl3) d: 2.0—212 (6H,
s, 2ϫCH3CO–) and 5.4 (1H, d, Jϭ10.2 Hz, H-12); 4a: oil. EI-MS m/z 644
1
[M]ϩ, 584 [MϪCH3COOH]ϩ. H-NMR (CDCl3) d: 2.1 (3H, s, CH3CO–)
and 4.42 (2H, ABq, Jϭ13.5 Hz, H2-20); 6a: oil. EI-MS m/z 602 [M]ϩ, 542
[MϪCH3COOH]ϩ, 482 [MϪ2ϫCH3COOH]ϩ. 1H-NMR (CDCl3) d: 2.04
and 2.1 (each 3H, s, CH3CO–) and 4.43 (2H, ABq, Jϭ15.0 Hz, H2-20);
8a: oil. EI-MS m/z 658 [M]ϩ, 598 [MϪCH3COOH]ϩ, 538 [MϪ2ϫ
1
CH3COOH]ϩ. H-NMR (CDCl3) d: 2.06—2.10 (each 3H, s, CH3CO–) and
4.42 (2H, ABq, Jϭ12.5 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]ϩ. 1H-NMR (CDCl3) d: 2.08 and 2.13 (each 3H,
1
s, CH3CO–), and 3.27 (3H, s, CH3O–); 8b: oil. EI-MS m/z 672 [M]ϩ. H-
NMR (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), 12-
O-Acetylphorbol 13,20-Dihexanoate (11a), 12-O-Acetylphorbol 13,20-
Dinonanoate (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 re-
spective 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]ϩ, 766 [MϪCH3COOH]ϩ,
598 [MϪCH3(CH2)12COOH]ϩ, 370 [MϪ2ϫCH3–(CH2)12ϪCOOH]ϩ. 1H-
NMR (CDCl3) d: 2.06 (3H, s, CH3CO–), 5.45 (1H, d, Jϭ10.5 Hz, H-12),
4.45 (2H, ABq, Jϭ12.0 Hz, H2-20), and signals for two tetradecanoyl moi-
eties at d 2.3 (4H, 2ϫCH2), 1.60 (4H, 2ϫCH2), 1.2 (40H, 2ϫ10CH2) and
0.09 (6H, 2ϫCH3); 11a: oil (32.5 mg, 65% from 9a). EI-MS m/z 602
[M]ϩ, 542 [MϪCH3COOH]ϩ, 486 [MϪCH3(CH2)4COOH]ϩ, 370 [MϪ2ϫ
CH3(CH2)4COOH]ϩ. 1H-NMR (CDCl3) d: 2.06 (3H, s, CH3CO–), 5.45 (1H,
d, Jϭ10.0 Hz, H-12), 4.43 (2H, ABq, Jϭ12.0 Hz, H2-20), and signals for
two hexanoyl moieties at 2.3 (4H, 2ϫCH2), 1.60 (4H, 2ϫCH2), 1.2 (12H,
6ϫCH2) and 0.09 (6H, 2ϫCH3); 12a: oil (40 mg, 80% from 9a). EI-MS m/z
626 [M]ϩ, 528 [MϪCH3(CH2)7COOH]ϩ, 370 [MϪ2ϫCH3(CH2)7COOH]ϩ.
1H-NMR (CDCl3) d: 2.06 (3H, s), 5.45 (1H, d, Jϭ10.0 Hz, H-12), 4.41 (2H,
ABq, Jϭ12.0 Hz, H2-20), and signals for two octanoyl moieties at 2.30 (4H,
2ϫCH2), 1.62 (4H, 2ϫCH2), 1.2 (20H, 10ϫCH2) and 0.09 (6H, 2ϫCH3);
13a: oil (37.5 mg, 75% from 9a). EI-MS m/z 770 [M]ϩ, 570 [MϪ
CH3(CH2)10COOH]ϩ, 510 [MϪCH3COOHϪCH3(CH2)10COOH]ϩ, 370
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
1
[MϪ2ϫCH3(CH2)10COOH]ϩ. H-NMR (CDCl3) d: 2.06 (3H, s), 5.45 (1H,
with mesyl chloride/pyridine at room temperature for 21 h.20) The compound

528
Vol. 50, No. 4
d, Jϭ10.0 Hz, H-12), 4.45 (2H, ABq, Jϭ14.0 Hz, H2-20), and signals of two Jϭ10.3 Hz, H-12), and signals for dodecanoyl moiety at d: 0.87 (3H, t,
dodecanoyl moieties at 2.30 (4H, 2ϫCH2), 1.60 (2H, CH2), 1.20 (32H,
16ϫCH2) and 0.09 (6H, 2ϫCH3).
Jϭ6.6 Hz, CH3), 1.27 (19H, m, H-16, 8ϫCH2), 1.67 (2H, m, CH2), and 2.36
(2H, m, CH2); 19: oil (42 mg, 57%). EI-MS m/z 658 [M]ϩ, 640 [MϪH2O]ϩ,
Preparation of 12-O-Acetylphorbol 13-Tetradecanoate (8c), 12-O- 597 [MϪH2OϪCH3CO–]ϩ, 538 [MϪH2OϪCH3COOϪCH3CO–]ϩ, 429
Acetylphorbol 13-Hexanoate (11), 12-O-Acetylphorbol 13-Octanoate [MϪH2OϪC14H27O]ϩ. H-NMR (CDCl3) d: 2.09, 2.07 (6H, 2s, COCH3),
(12) and 12-O-Acetylphorbol 13-Dodecanoate (13) by Partial Hydrolysis 4.33 (2H, ABq, Jϭ12.4 Hz, H-20), 5.47 (1H, d, Jϭ10.3 Hz, H-12), and sig-
1
The 12-O-acetyl-13-O-,20-O-diacylphorbol derivatives obtained (8d, 11a,
12a and 13a) were separately hydrolyzed (each 30 mg) with 70%
nals for tetradecanoyl moiety at d: 0.88 (3H, t, Jϭ6.8 Hz, CH3), 1.26 (23H,
m, H-16, 9ϫCH2), 1.64 (2H, m, CH2), and 2.32 (2H, m, CH2); 20: Oil
HClO4/MeOH to give the respective 12-O-acetyl-13-O-acylphorbol deriva- (50 mg, 64%). EI-MS m/z 700 [M]ϩ, 682 [MϪH2O]ϩ, 587 [MϪH2OϪ
tives (8c, 11, 12 and 13) in yields of 60—70%. All products were purified by CH3COO–]ϩ, 569 [MϪ2H2OϪCH3COO–]ϩ, 429 [MϪH2OϪC17H33O]ϩ.
column chromatography on SiO2, followed by MPLC (RP-18). The struc- 1H-NMR (CDCl3) d: 2.09, 2.07 (6H, 2s, COCH3), 4.33 (2H, ABq, Jϭ
tures of these compounds were established by spectroscopic methods and 12.4 Hz, H-20), 5.47 (1H, d, Jϭ10.3 Hz, H-12), and signals for heptade-
their characteristics are described below. 8c: oil (21 mg, 70% from 8d). EI- canoyl moiety at d: 0.88 (3H, t, Jϭ6.8 Hz, CH3), 1.25 (29H, m, H-16,
MS m/z 616 [M]ϩ, 556 [MϪCH3COOH]ϩ, 388 [MϪCH3(CH2)12COOH]ϩ, 13ϫCH2), 1.63 (2H, m, CH2), and 2.31 (2H, m, CH2); 21: oil (45 mg, 66%).
370 [MϪH2OϪCH3(CH2)12COOH]ϩ. 1H-NMR (CDCl3) d: 2.06 (3H, s, EI-MS m/z 610 [M]ϩ, 592 [MϪH2O]ϩ, 532 [MϪH2OϪCH3COOH]ϩ, 429
CH3CO–), 5.45 (1H, d, Jϭ10.0 Hz, H-12), 4.00 (2H, ABq, Jϭ12.0 Hz, H2- [MϪH2O-1-(1-adamantnoyl)]ϩ. 1H-NMR (CDCl3) d: 2.09, 2.07 (6H, 2s,
20), and signals for tetradecanoyl moiety at d: 2.3 (2H, CH2), 1.62 (2H,
COCH3), 4.33 (2H, ABq, Jϭ12.0 Hz, H-20), 5.44 (1H, d, Jϭ10.5 Hz, H-12),
CH2), 1.2 (20H, 10ϫCH2) and 0.09 (3H, CH3); 11: oil (18 mg, 60% and signals for 1-adamantanoyl moiety at d: 1.73 (7H, m), 1.91 (6H, m, H-
from 11a). EI-MS m/z 504 [M]ϩ, 444 [MϪCH3COOH]ϩ, 388 [MϪ 11), and 2.06 (3H, m).
CH3(CH2)4COOH]ϩ, 370 [MϪH2OϪCH3(CH2)4COOH]ϩ. 1H-NMR (CDCl3)
d: 2.06 (3H, CH3CO–), 5.45 (1H, d, Jϭ10.0 Hz, H-12), 4.00 (2H, ABq,
Preparation of 13-O-Acetyl(4-deoxy-4a-phorbol) (23a), 13-O-,20-O-
Diacetyl(4-deoxy-4a-phorbol) (23b) and 12-O-,13-O-,20-O-Triacetyl(4-
Jϭ12.0 Hz, H2-20), and signals for hexanoyl moiety at 2.3 (2H, CH2), 1.62 deoxy-4a-phorbol) (23c) Acetylation of 23 with Ac2O/pyridine at 60 °C
(2H, CH2), 1.2 (4H, 2ϫCH2) and 0.09 (3H, CH3); 12: oil (21 mg, 70% for 1 h afforded 23a (21%), 23b (35%) and 23c (11%). 23a: oil. EI-MS m/z
from 12a). EI-MS m/z 546 [M]ϩ, 486 [MϪCH3COOH]ϩ, 388 [MϪ 390 [M]ϩ, 372 [MϪH2O]ϩ, 354 [MϪ2H2O]ϩ, 329 [MϪH2OϪCH3CO–]ϩ.
CH3(CH2)7COOH]ϩ, 370 [MϪH2OϪCH3(CH2)7COOH]ϩ. 1H-NMR (CDCl3)
1H-NMR (CDCl3) d: 2.09 (3H, s, COCH3); 23b: oil. EI-MS m/z 432 [M]ϩ,
1
d: 2.06 (3H, s, CH3–CO–), 5.45 (1H, d, Jϭ10.0 Hz, H-12), 4.00 (2H, ABq, 414 [MϪH2O]ϩ, 354 [MϪH2OϪCH3COOH]ϩ. H-NMR (CDCl3) d: 2.12,
Jϭ12.0 Hz, H2-20), and signals for octanoyl moiety at 2.30 (2H, CH2), 1.61
2.11 (6H, 2s, COCH3), and 4.47 (2H, ABq, Jϭ11.8 Hz, H-20); 23c36): oil.
(2H, CH2), 1.2 (10H, 5ϫCH2) and 0.09 (3H, CH3); 13: oil (19.5 mg, EI-MS m/z 474 [M]ϩ, 431 [MϪCH3CO–]ϩ, 413 [MϪH2OϪCH3CO–]ϩ, 353
65% from 13a). EI-MS m/z 570 [M]ϩ, 510 [MϪCH3COOH]ϩ, 388
[MϪH2OϪCH3COOHϪCH3CO–]ϩ. H-NMR (CDCl3) d: 2.12, 2.11, 2.07
1
[MϪCH3(CH2)10COOH]ϩ, 370 [MϪH2OϪCH3(CH2)10COOH]ϩ. 1H-NMR (9H, 3s, COCH3), 4.40 (2H, ABq, Jϭ12.7 Hz, H-20), and 5.44 (1H, d,
(CDCl3) d: 2.06 (3H, s), 5.45 (1H, d, Jϭ10.0 Hz, H-12), 4.00 (2H, ABq, Jϭ10.5 Hz, H-12).
Jϭ12.0 Hz, H2-20), and signals for dodecanoyl moiety at 2.3 (2H, CH2),
1.62 (2H, CH2), 1.20 (16 H, 8ϫCH2) and 0.09 (3H, CH3).
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)
Preparation of 13-O-Acetylisophorbol (14a) and 13-O-,20-O-Di- Acyl chloride (400 ml, 0.48 mmol) was added to an ice cooled solution of
acetylisophorbol (14b) Acetic anhydride (50 ml) was added to an ice 23b (50 mg, 0.12 mmol) in pyridine (1 ml), and the mixture was stirred at
cooled solution of isophorbol (14, 50 mg, 0.14 mmol) in pyridine (1 ml), and
room temperature for 24 h. The reaction mixture was worked up as usual
the mixture was stirred at 60 °C for 1 h. The reaction mixture was worked up to give 24 (71%) and 25 (68%). 24: oil. EI-MS m/z 586 [M]ϩ, 543
as usual to afford 14a (8 mg, 14%) and 14b (22 mg 36%). 14a: oil. EI-MS [MϪCH3CO–]ϩ, 526 [MϪCH3COOH]ϩ, 413 [MϪH2OϪC10H19O]ϩ. 1H-
m/z 406 [M]ϩ, 388 [MϪH2O]ϩ, 370 [MϪ2H2O]ϩ, 352 [MϪ3H2O]ϩ, 309
NMR (CDCl3) d: 2.12, 2.06 (6H, 2s, COCH3), 4.40 (2H, ABq, Jϭ12.5 Hz,
1
[MϪ3H2OϪCH3CO–]ϩ. H-NMR (CDCl3) d: 2.10 (3H, s, COCH3); 14b: H-20), 5.47 (1H, d, Jϭ10.5 Hz, H-12), and signals for decanoyl moiety at d:
oil. EI-MS m/z 448 [M]ϩ, 430 [MϪH2O]ϩ, 412 [MϪ2H2O]ϩ, 369 0.88 (3H, t, Jϭ7.1 Hz, CH3), 1.28 (12H, m, 6ϫCH2), 2.36 (2H, m, CH2),
1
[MϪ2H2OϪCH3CO–]ϩ. H-NMR (CDCl3) d: 2.11, 2.09 (6H, 2s, COCH3), and 2.59 (2H, t, Jϭ7.8 Hz, CH2); 25: oil. EI-MS m/z 642 [M]ϩ, 599
4.33 (2H, ABq, Jϭ12.4 Hz, H-20).
[MϪCH3CO–]ϩ, 582 [MϪCH3COOH]ϩ, 522 [MϪ2CH3COOH]ϩ, 413
Preparation of 12-O-Octanoylisophorbol 13,20-Diacetate (15), 12-O- [MϪH2OϪC14H27O]. 1H-NMR (CDCl3) d: 2.12, 2.06 (6H, 2s, COCH3),
Decanoylisophorbol 13,20-Diacetate (16), 12-O-(10-Undecenoyl)isophor- 4.14 (2H, ABq, Jϭ12.5 Hz, H-20), 5.47 (1H, d, Jϭ10.8 Hz, H-12), and sig-
bol 13,20-Diacetate (17), 12-O-Dodecanoylisophorbol 13,20-Diacetate nals for tetradecanoyl moiety at d: 0.88 (3H, t, Jϭ7.1 Hz, CH3), 1.27 (16H,
(18), 12-O-Tetradecanoylisophorbol 13,20-Diacetate (19), 12-O-Heptade- m, 6ϫCH2), 2.36 (2H, m, CH2), and 2.59 (2H, t, Jϭ7.8 Hz, CH2).
canoylisophorbol 13,20-Diacetate (20) and 12-O-(1-Adamantanoyl)-
isophorbol 13,20-Diacetate (21) Acyl chloride (400 ml, 0.44 mmol) was References
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 re-
action 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]ϩ, 556
[MϪH2O]ϩ, 514 [MϪH2OϪCH3CO–]ϩ, 496 [MϪ2H2OϪCH3CO–]ϩ, 429
[MϪH2OϪC8H15O]ϩ. 1H-NMR (CDCl3) d: 2.10, 2.07 (6H, 2s, COCH3),
2.33 (2H, m, CH2), 4.33 (2H, ABq, Jϭ12.2 Hz, H-20), 5.47 (1H, d,
Jϭ10.2 Hz, H-12), and signals for octanoyl moiety at d: 0.87 (3H, t,
Jϭ6.6 Hz, CH3), 1.31 (8H, m, 4ϫCH2), 1.68 (2H, m, CH2); 16: oil (43 mg,
64%). EI-MS m/z 602 [M]ϩ, 583 [MϪH2O]ϩ, 524 [MϪH2OϪCH3COOH]ϩ,
481 [MϪH2OϪCH3COOHϪCH3COO–]ϩ, 429 [MϪH2OϪC10H19O]ϩ. 1H-
NMR (CDCl3) d: 2.09, 2.07 (6H, 2s, COCH3), 2.35 (2H, m, CH2), 4.33 (2H,
ABq, Jϭ12.5 Hz, H-20), 5.47 (1H, d, Jϭ10.3 Hz, H-12), and signals for de-
canoyl moiety at d: 0.88 (3H, t, Jϭ6.6 Hz, CH3), 1.27 (15H, m, H-16,
6ϫCH2), and 1.67 (2H, m, CH2); 17: oil (51 mg, 74%). EI-MS m/z 614
[M]ϩ, 596 [MϪH2O]ϩ, 536 [MϪH2OϪCH3COOH]ϩ, 429 [MϪH2OϪ(10-
undecenoyl)]ϩ. 1H-NMR (CDCl3) d: 2.09, 2.06 (6H, 2s, COCH3), 4.34 (2H,
ABq, Jϭ12.4 Hz, H-20), 5.47 (1H, d, Jϭ10.3 Hz, H-12), and signals for 10-
undecenoyl moiety at d: 1.3—1.6 [(2H, m, CH2ϭCH–(CH2)7–CH2–), 2.03
[(2H, m, CH2ϭCH–(CH2)7–CH2–), 2.6 [(2H, t, Jϭ8.04 Hz, CH2ϭCH–
(CH2)7–CH2–), 4.94 [(2H, m, CH2ϭCH–(CH2)8–), and 5.81 [(1H,
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