In vitro platelet-activating factor receptor binding inhibitory activity of pinusolide derivatives: A structure-activity study

Article abstract of DOI:10.1021/jm970569j

Pinusolide, a labdane-type diterpene lactone isolated from Biota orientalis, was found to be a potent platelet-activating factor (PAF) receptor binding antagonist. To investigate the structure-activity relationship and find derivatives with improved pharmacological profiles, 17 pinusolide derivatives were prepared and tested for their ability to inhibit the PAF receptor binding. The results demonstrated that the carboxymethyl ester group at C-19, the integrity of the α,β-unsaturated butenolide ring, and the exocyclic olefinic function of pinusolide are all necessary for its maximum PAF receptor binding inhibitory activity. Among the derivatives, the 17-nor-8-oxo derivative 8 was found to be as potent as pinusolide. The results also suggested that several derivatives warrant further pharmaceutical and pharmacological studies due to their improved water solubility (8 and 11) and apparent lack of susceptibility to Michael-type nucleophilic addition (13 and 18).

Full text of DOI:10.1021/jm970569j

2
626
J . Med. Chem. 1998, 41, 2626-2630
Notes
In Vitr o P la telet-Activa tin g F a ctor Recep tor Bin d in g In h ibitor y Activity of
P in u solid e Der iva tives: A Str u ctu r e-Activity Stu d y
Byung Hoon Han,* Hyun Ok Yang, Young-Hwa Kang, Dae-Yeon Suh, Hyun J ung Go, Wan-J in Song,
Yong Chul Kim, and Man Ki Park†
Natural Products Research Institute, Seoul National University, 28, Yeongun-dong, J ongro-ku, Seoul 110-460, Korea, and
College of Pharmacy, Seoul National University, Seoul 151-742, Korea
Received August 25, 1997
Pinusolide, a labdane-type diterpene lactone isolated from Biota orientalis, was found to be a
potent platelet-activating factor (PAF) receptor binding antagonist. To investigate the
structure-activity relationship and find derivatives with improved pharmacological profiles,
1
7 pinusolide derivatives were prepared and tested for their ability to inhibit the PAF receptor
binding. The results demonstrated that the carboxymethyl ester group at C-19, the integrity
of the R,â-unsaturated butenolide ring, and the exocyclic olefinic function of pinusolide are all
necessary for its maximum PAF receptor binding inhibitory activity. Among the derivatives,
the 17-nor-8-oxo derivative 8 was found to be as potent as pinusolide. The results also suggested
that several derivatives warrant further pharmaceutical and pharmacological studies due to
their improved water solubility (8 and 11) and apparent lack of susceptibility to Michael-type
nucleophilic addition (13 and 18).
Platelet-activating factor (PAF, 1-O-alkyl-2(R)-acetyl-
glyceryl-3-phosphorylcholine) is a potent phospholipid
autacoid released by platelets, leukocytes, and other
stimulated cells.1 This factor exerts a wide range of
biological activities such as platelet aggregation, bron-
choconstriction, hypotension, and an increase in vascu-
lar permeability. Further, PAF has been reported to
be involved in many pathological conditions including
inflammation, allergy, anaphylaxis, endotoxin shock,
Ch em istr y
A total of 18 pinusolide (1) derivatives were prepared
for evaluation of their PAF receptor binding inhibitory
activities through reduction (3a ,b, 9, 13, 14, 15, 16),
oxidation (3, 6, 7, 8, 10, 11, 12, 17), hydration (5), and
methylation (4, 18) of 1 or its derivatives. Pinusolidic
acid (2) was isolated from the leaf extract of Biota
7
orientalis.
2
Reduction of 1 with [(C2H5)2AlH2]Na afforded the diol
and transplanted organ rejection. It acts by specifically
8
3
a and triol 3b. The major product, 3b, was further
binding to a high-affinity G protein-coupled receptor,
9
which was recently cloned.3 Therefore, PAF antagonists
oxidized by silver carbonate on Celite. In accordance
9
a
with the literature, two different lactones, 3 and 17,
were obtained with 17 being the major product (69%)
resulting from the oxidation of the less hindered -C(15)-
H2OH group of 3b. The -C(19)H2OH group of 17 was
further treated with J ones reagent and CH2N2 to yield
which block the specific binding to the receptor have
been extensively sought, and a number of synthetic and
natural antagonists with various chemical structures
1
b,4
were found.
On the basis of these widely varying
structural patterns of the PAF antagonists, a receptor
model for PAF antagonist binding was postulated to be
a multipolarized cylinder with a set of ‘cache-oreilles’
1
8, which has a â-substituted lactone as compared to
the R-substituted lactone in 1.
Anti-Markovnikov hydration of the exocyclic double
bond of 1 was effected by hydroboration with
1
0-12 Å apart and a hydrophobic pocket.5
In the course of the screening of novel PAF antago-
nists from medicinal plants, we have previously reported
that pinusolide ((4S,5R,9S,10R)-8(17),13-labdadien-
(
5
CH3)2S‚BH3 followed by alkaline H2O2 oxidation to give
1
0
.
The axial C-20 methyl group and bulky alkyl chain
attached to C-9 impose a large degree of steric hindrance
on the â-face of the C(8)-C(17) exocyclic double bond
of 1. The facts that hydroboration occurs with attack
taking place from the less hindered face (R-face in the
1
6,15-olid-19-oic acid methyl ester, 1) possesses a potent
6
PAF antagonistic activity. It is a labdane-type diter-
penoid with an R,â-unsaturated butenolide ring. In this
study, various derivatives of 1 were synthesized and
3
case of 1) and that there are no other peaks around
tested for their ability to displace [ H]PAF-specific
1
2
.5-4 ppm in H NMR except a multiplet at 3.71 ppm
binding from rabbit washed platelets in order to study
strongly suggested that 5 contains an axial -CH2OH,
thus S configuration at C-8. Similarly, the configuration
at C-8 of 6 and 7 was assumed to be 8R, where oxidative
attack by m-CPBA and OsO4 occurred from the less
hindered R-face.
the structure-activity relationship.
*
Author for correspondence. Tel: 82-2-743-8349. Fax: 82-2-744-
4
243. E-mail: natural@plaza.snu.ac.kr.
†
College of Pharmacy.
S0022-2623(97)00569-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/05/1998

Notes
Extensive model studies by others indicated that
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2627
Ta ble 1. Structure and in Vitro PAF Receptor Binding
Inhibitory Activity of Pinusolide Derivatives
equitorial substituents R to the flattened cyclic ketones
such as 8 promote equitorial attack of hydride (“flat-
tening rule”) to give predominant formation of less
stable axial alcohol.11 On the basis of this and the
chemical shift (δ 4.03) and coupling constant (bd, J ) 2
Hz) of the H-8 proton, the configuration at C-8 of 9 was
assigned to be S, where C-8 contains an equitorial
proton and an axial hydroxyl group. On the other hand,
since the Baeyer-Villiger reaction occurs with retention
1
2
of stereochemistry at the migrating carbon center, the
configuration at C-9 of 10 was assigned to be R.
On treatment with HIO3 in aqueous acetone, 1 was
converted to 11. On the basis of results obtained with
model compounds, this reaction was proposed to proceed
via the 8-hydroxy-17-iodo intermediate that would be
+
formed by electrophilic attack of I ion, presumably
donated by HOI-like species generated in situ by reduc-
tion of acetone by HIO3. Antielimination of a water
molecule from the axial hydroxyl group on C-8 and axial
H-9 and subsequent hydrolysis of the allylic iodine
would yield 11. Detailed accounts of this novel reaction
will be reported elsewhere.13 Subsequent oxidation with
PDC furnished the aldehyde derivative 12.14
When 1 was reduced using Co(II) and NaBH4, the
trisubstituted double bond in the lactone ring was
selectively reduced to give 13, while the disubstituted
exocyclic double bond remained unreacted.15 The ob-
served selectivity is most likely due to the electronic
effect of the electron-withdrawing carbonyl function.
Resu lts a n d Discu ssion
We have previously reported pinusolide (1) and pi-
nusolidic acid (2) as PAF antagonists.6 In this study,
various derivatives of 1 were prepared, and their PAF
receptor binding inhibitory activities were measured
,7
Ta ble 2. Structure and in Vitro PAF Receptor Binding
Inhibitory Activity of Pinusolide Derivatives Not Having the
R-Substituted, 5-Membered Lactone Ring
3
using rabbit platelets as the receptor source and [ H]-
PAF as a ligand. CV 3988 (Takeda) was used as the
reference compound throughout the study.17
IC50 values of derivatives were obtained, and the
mean values of at least two independent measurements
are shown in Table 1. The presence of a free acidic
group (2) or a hydroxyl group (3) at C-19 significantly
increased the IC50 values. This detrimental effect of a
hydroxyl group at C-19 on the binding to the receptor
was also observed with 17 (IC50 ) 110 µM) as compared
to 18 (IC50 ) 6.0 µM). However, the binding affinity
was much regained when the hydroxyl group was
methylated as in 4 (IC50 ) 1.7 µM).
Replacement of the R,â-unsaturated butenolide with
a furan ring (16) (Table 2) resulted in a complete loss
of the binding affinity, whereas â-substituted R,â-
unsaturated butenolide derivative 18 was about 20
times less active than 1, which has an R-substituted
butenolide ring. The increased IC50 values of deriva-
tives 3a , 13, and 15 indicated that the integrity of the
butenolide structure is beneficial to the binding. How-
ever, since 13 and 15 are epimeric mixtures of unknown
proportions at C-13, the exact potency of each epimer
could not be determined.
Other modifications resulted in 4.0-32 times increases
in the IC50 values, indicating the exocyclic olefinic
function is preferable in binding to the PAF receptor.
Interestingly, the ketonic derivative 8 showed a com-
parable inhibitory activity to the lead compound 1.
A purpose of this study was to find derivatives of 1
Exocyclic olefinic bond, C(8-17), in 1 was subjected
to various reactions including hydration and oxidation
(
(
5-12). When the olefinic bond was oxidized to a glycol
7), it resulted in a complete loss in the binding affinity.

2
628 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Notes
that might exhibit improved pharmaceutical/pharma-
cological profiles, that is, longer biological half-life and
improved oral activity. In line with this, derivatives 13,
19-Meth oxy-8(17),13-la bd a d ien -16,15-olid e (4). To a
solution of 3 (50 mg, 0.16 mmol) in ether (1 mL) were added
(
C
2
H
5
)
2
O‚BH
After 1 h of stirring, aliquots of NH
reaction mixture, and the mixture was partitioned to H
3
(0.1 mL) and CH
2
N
2
in ether (excess) at 0 °C.
OH were added to the
O (30
4
1
5, and 18 received our attention, since these deriva-
2
tives should not be susceptible to Michael-type nucleo-
philic addition at C-14, whereas 1 might form the
Michael-type adduct with nucleophilic biomolecules
mL) and ether (20 mL). The ether layer was washed with
brine, dried over Na SO , and chromatographed over a silica
2
4
gel column (hexane-CHCl
3
-EtOAc, 4:10:0.1) to give 4 (R )
f
6
b
0.3, 10 mg): colorless crystal, mp 62-4 °C; EIMS (m/z) 332
resulting in short duration of in vivo activity.
detailed study in this regard is being investigated.
A
+
+
1
(
M ), 287 (M - CH
2
OCH
H, d, J ) 9 Hz, -C(19)H
.57, 4.81 (each 1H, s, dC(17)H
3
); H NMR (CDCl
OCH ), 3.27 (3H, s, -C(19)H
), 4.76-4.78 (2H, m, dCHC-
32 3
O-), 7.09 (1H, t, J ) 2.0 Hz, dC(14)H-). Anal. (C21H O )
3
) δ 3.09, 3.41 (each
1
4
2
3
2
OCH ),
3
Another approach to this end was to find derivatives
that are less hydrophobic and have comparable inhibi-
tory activity. It turned out that derivatives 8 and 11
whose log P(octanol/water) values are 0.86 and 0.26,
respectively, were less hydrophobic than 1 (log P(oc-
tanol/water) ) 3.12).
In conclusion, the carboxymethyl ester group at C-19,
the integrity of the R,â-unsaturated butenolide ring, and
the exocyclic olefinic function of 1 are all necessary for
its maximum PAF receptor binding inhibitory activity.
On the other hand, the results revealed that derivatives
2
(15)H
2
C, H.
(
8S)-17-Hydr oxy-13-labden -16,15-olid-19-oic Acid Meth -
yl Ester (5). To a stirred solution of 1 (30 mg, 0.087 mmol)
in anhydrous THF (1 mL) was added (CH
3
)
2
S‚BH
3
2
(2.6 mL of
(10 mL)
2
.0 M in THF) at 0 °C under N . After 2 h 30% H
2
O
2
and 3 N NaOH (30 mL) were added to the reaction mixture,
and the mixture was stirred for another 1 h at 50 °C.
Following ether extraction, the organic layer was concentrated,
and the residue was subjected to silica gel column chroma-
tography (hexanes-EtOAc, 1:1) to give 5 (R ) 0.2, 23 mg):
f
8
, 11, 13, and 18 warrant further pharmaceutical and
needles, mp 88-90 °C; HREIMS m/z 346.2142 [∆ -0.2 mmu
+
1
(
(
7
M
- H
2H, m, -C(17)H
.13 (1H, s-like, dC(14)H-). Anal. (C21
8R)-8,17-Ep oxy-13-la bd en -16,15-olid -19-oic Acid Meth -
yl Ester (6). A solution of 1 (0.15 g, 0.46 mmol) and m-CPBA
0.12 g, 0.92 mmol) in dry CH Cl (3 mL) was stirred for 4 h
2
O)]; H NMR (CDCl
OH), 4.79-4.80 (2H, m, dCHC(15)H
) C, H.
3
) δ 3.63 (3H, s, -OCH
3
), 3.71
pharmacological studies due to their improved water
solubility and/or apparent lack of susceptibility to
Michael-type addition. Furthermore, the results ob-
tained in this study will provide useful information for
the interaction between the PAF receptor and its
ligands.
2
2
O-),
32 5
H O
(
(
2
2
under N . The reaction mixture was diluted with CH Cl (20
2
2
2
mL) and saturated NaHCO
layer was washed with brine, dried over Na
3
solution (10 mL). The organic
SO , and chro-
matographed over a silica gel column (hexanes-EtOAc, 2:1)
to give 6 (R ) 0.2, 0.1 g): needles, mp 131-3 °C; HREIMS
m/z 362.2092 [∆ -0.1 mmu (M )]; H NMR (CDCl
d, J ) 4.2 Hz, -C(17)HH-O-), 2.72 (1H, dd, J ) 1.8, 4.2 Hz,
C(17)HH-O-), 3.64 (3H, s, -OCH ), 4.74-4.76 (2H, m,
dCHC(15)H O-), 7.18 (1H, t, J ) 1.6 Hz, dC(14)H-). Anal.
) C, H.
8R)-8,17-Dih yd r oxy-13-la bd en -16,15-olid -19-oic Acid
Meth yl Ester (7). A solution of 1 (150 mg, 0.43 mmol) and
OsO (1.1 g, 0.43 mmol) in dioxane (11 mL) was stirred for 5
h under N . The reaction mixture was treated with saturated
NaHSO , and the EtOAc extract was chromatographed over
a silica gel column (CHCl -MeOH, 25:1) to give 7 (R ) 0.3,
0 mg): needles, mp 121-2 °C; HREIMS m/z 362.2095 [∆ +0.2
Exp er im en ta l Section
2
4
1
5,16-Dih yd r oxy-8(17),13-la bd a d ien -19-oic Acid Meth -
f
yl Ester (3a ) a n d 8(17),13-La bd a d ien e-15,16,19-tr iol (3b).
To a solution of 1 (1 g, 2.9 mmol) in ether (150 mL) was added
dropwise sodium diethyldihydroaluminate (4.3 mL, 8.7 mmol)
+
1
3
) δ 2.54 (1H,
-
3
2
at room temperature under N , and the resulting gel-like
2
precipitate was stirred for 2 h. The reaction mixture was
digested by addition of 1 N HCl (30 mL) on ice. Following
ether extraction, the organic layer was washed with brine,
21 30 5
(C H O
(
dried over Na
column (CHCl
2
SO
4
, and chromatographed over a silica gel
) 0.2, 150 mg)
4
3
-MeOH, 30:1) to give 3a (R
f
2
and 3b (R
(
f
) 0.05, 300 mg). 3a : colorless oil; EIMS (m/z) 350
M ); H NMR (CDCl ) δ 0.51 (3H, s, -C(20)H ), 1.19 (3H, s,
), 3.62 (3H, s, -OCH ), 4.16 (2H, d-like, J ) 2.1 Hz,
OH), 4.21 (2H, d, J ) 6.8 Hz, dCHC(15)H OH), 4.55,
), 5.61 (1H, t-like, J ) 6.8 Hz,
OH). Anal. (C21 ) C, H. 3b: mp 93-7 °C;
EIMS (m/z) 322 (M ); H NMR (CDCl ) δ 0.65 (3H, s, -C(20)-
), 0.98 (3H, s, -C(18)H ), 3.39, 3.75 (each 1H, d, J ) 7.5
Hz, -C(19)H OH), 4.18 (2H, d, J ) 2.1 Hz, -C(16)H OH), 4.21
2H, d, J ) 6.8 Hz, -C(15)H OH). Anal. (C20 ) C, H.
9-Hyd r oxy-8(17),13-la bd a d ien -16,15-olid e (3) a n d 19-
Hyd r oxy-8(17),13-la bd a d ien -15,16-olid e (17). Compound
b (0.1 g, 0.31 mmol) was oxidized by refluxing for 1 h with
Ag CO (0.85 g, 3.1 mmol) and Celite (0.4 g) in benzene (7 mL)
under dark and N atmosphere. The reaction mixture was
+
1
3
3
3
3
f
-
C(18)H
C(16)H
3
2
3
7
-
2
+
1
mmu (M - H
1 Hz, -C(17)HHOH), 3.63 (3H, s, -OCH
11 Hz, -C(17)HHOH), 4.76-4.78 (2H, m, dCHC(15)H
.16 (1H, t, J ) 1.8 Hz, dC(14)H-). Anal. (C21 ) C, H.
17-Nor -8-oxo-13-la bd en -16,15-olid -19-oic Acid Meth yl
Ester (8). To a stirred solution of 7 (0.14 g, 0.37 mmol) in
MeOH (2.5 mL) was added NaIO (0.2 g, 0.93 mmol) in 1 N
SO (2 mL). After 2 h the reaction mixture was diluted with
2
O)]; H NMR (CDCl
3
) δ 3.54 (1H, dd, J ) 1.5,
), 3.67 (1H, d, J )
O-),
4
.85 (each 1H, s, dC(17)H
2
1
3
dC(14)HCH
2
34 4
H O
+
1
2
3
7
32 6
H O
H
3
3
2
2
(
2
34 3
H O
1
4
H
2
4
ether (15 mL), and the organic layer was washed with brine,
dehydrated, and chromatographed over a silica gel column
3
2
3
(
1
(
H
CHCl
31-2 °C; HREIMS m/z 348.1942 [∆ +0.5 mmu (M )]; H NMR
CDCl ) δ 3.62 (3H, s, -OCH ), 4.76-4.77 (2H, m, dCHC(15)-
O-), 7.14 (1H, t, J ) 2.1 Hz, dC(14)H-); C NMR (CDCl
) C, H.
(8S)-17-Nor -8-h yd r oxy-13-la bd en -16,15-olid -19-oic Acid
Meth yl Ester (9). To a stirred solution of 8 (30 mg, 0.086
mmol) in MeOH (2 mL) was added NaBH (0.96 g, 25 mmol).
3
-MeOH, 25:1) to give 8 (R
f
) 0.3, 90 mg): needles, mp
2
+
1
filtered to remove black silver and silver carbonate. Filter cake
was washed with ether, and the organic layer was washed with
3
3
1
3
) δ
brine, dried over Na
gel column (CHCl -benzene-MeOH, 1:1:0.5) to give 3 (R
.2, 20 mg) and 17 (R ) 0.1, 45 mg). 3: needles, mp 99-102
C; HREIMS m/z 318.2193 [∆ -0.2 mmu (M )]; H NMR
CDCl ) δ 0.66 (3H, s, -C(20)H ), 0.98 (3H, s, -C(18)H ), 3.40,
.75 (each 1H, d, J ) 11 Hz, -CH OH), 4.58, 4.86 (each 1H, s,
), 4.78 (2H, m, dCHC(15)H O-), 7.10 (1H, s-like, dC-
14)H-). Anal. (C20 ) C, H. 17: white crystals, mp 162-4
2 4
SO , and chromatographed over a silica
2
3
6
28 5
2.8 (C-9), 211.7 (C-8). Anal. (C20H O
3
f
)
0
f
+
1
°
(
3
3
3
4
3
2
After 15 h the reaction mixture was digested by addition of 1
N HCl (2 mL) and extracted with EtOAc. The EtOAc layer
was washed with brine, dehydrated, and chromatographed
dC(17)H
2
2
(
°
30 3
H O
+
+
+
C; EIMS (m/z) 318 (M ), 300 (M - H
2
O), 287 (M - CH
); H NMR (CDCl ) δ 3.39 (1H, dd, J
10.8, 4.5 Hz, -C(19)HHOH), 3.74 (1H, dd, J ) 10.8, 5.8 Hz,
C(19)HHOH), 4.45, 4.87 (each 1H, s, dC(17)H ), 4.71-4.72
O-), 5.85 (1H, s-like, dC(14)H-). Anal.
2
-
over a silica gel column (CHCl
3
-MeOH, 15:1) to give 9 (R )
f
+
1
OH), 221 (M - C
5
H
5
O
2
3
0.3, 27 mg): rodlike crystals, mp 120-2 °C; HREIMS m/z
+
1
)
-
(
350.2092 [∆ -0.1 mmu (M )]; H NMR (CDCl
-C(20)H ), 3.69 (3H, s, -OCH ), 4.03 (1H, bd, J ) 2.0 Hz,
-C(8)HeqOH), 4.76-4.79 (2H, m, dCHC(15)H O-), 7.14 (1H,
t, J ) 1.8 Hz, dC(14)H-). Anal. (C20 ) C, H.
3
) δ 0.81 (3H, s,
2
3
3
2H, m, dC(16)H
) C, H.
2
2
(C
20
H
30
O
3
30 5
H O

Notes
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2629
(
9R)-8,9-E p oxy-17-n or -8-oxo-13-la b d en -16,15-olid -19-
m, -C(13)HCOO-), 3.63 (3H, s, -OCH
m, -C(15)H ) δ 30.5 (C-14), 63.2 (C-9),
O-); 13C NMR (CDCl
66.6 (C-15), 177.3 (C-19), 179.8 (C-16), 211.6 (C-8). Anal.
) C, H.
15,16-Ep oxy-8(17),14,16-la bd a tr ien -19-oic Acid Meth yl
Ester (16). To a solution of 1 (17 mg, 0.049 mmol) in dry
THF (2 mL) was added DIBALH (0.05 mmol) in hexane at -30
°C. After 5 h the reaction mixture was digested by addition
3
), 4.19, 4.34 (each 1H,
oic Acid Meth yl Ester (10). To a stirred solution of 8 (52
2
3
mg, 0.15 mmol) and catalytic amounts of p-TsOH in CHCl
mL) was added m-CPBA (37 mg, 0.30 mmol) in CHCl (2 mL)
under N After 24 h at room temperature the reaction
mixture was diluted with CHCl (20 mL) and washed with
saturated NaHCO . The organic layer was dehydrated and
chromatographed over a silica gel column (hexanes-EtOAc,
:1) to give 10 (R ) 0.3, 50 mg): needles, mp 110 °C; HREIMS
m/z 364.1890 [∆ +0.4 mmu (M )]; H NMR (CDCl
s, -C(20)H ), 3.66 (3H, s, -OCH ), 4.01 (1H, dd, J ) 2.7, 11.4
Hz, -C(9)HO-), 4.79-4.80 (2H, m, dCHC(15)H O-), 7.19 (1H,
) δ 86.7 (C-9), 175.7
3
(1
3
30 5
(C20H O
2
.
3
3
1
f
of 1 mL of 10% H
2 4
SO , and the EtOAc extract was chromato-
+
1
3
) δ 0.77 (3H,
graphed over a silica gel column (hexanes-EtOAc, 3:1) to give
+
+
3
3
16 (R
COOCH
-OCH ), 4.57, 4.89 (each 1H, s-like, dC(17)H
) 1.6 Hz, dC(14)H-), 7.19 (1H, s, dC(16)HO-), 7.35 (1H, d, J
1.6 Hz, dC(15)HO-). Anal. (C21 ) C, H.
8(17),13-La bd a d ien -15,16-olid -19-oic Acid Meth yl Ester
f
) 0.2, 10 mg): solids; EIMS (m/z) 330 (M ), 271 (M -
1
+
2
3
), 81 (C
5
H
5
O ); H NMR (CDCl
3
) δ 3.61 (3H, s,
), 6.25 (1H, d, J
1
3
t, J ) 1.8 Hz, dC(14)H-); C NMR (CDCl
C-8). Anal. (C20 ) C, H.
7-Hydr oxy-8,13-labdadien -16,15-olid-19-oic Acid Meth -
yl Ester (11). To a stirred solution of 1 (3 g, 8.6 mmol) in
acetone (80 mL) was slowly added HIO (1.52 g, 8.6 mmol) in
O (20 mL) at room temperature. After 3 h the reaction
mixture was diluted with H O (150 mL). The ether extract
was washed with brine, dried over Na SO , and chromato-
graphed over a silica gel column (CHCl -MeOH, 100:1) to give
1 (R ) 0.2, 2.5 g): white crystals, mp 134-6 °C; HREIMS
m/z 362.2090 [∆ -0.3 mmu (M) ]; H NMR (CDCl
s, -C(20)H ), 3.67 (3H, s, -OCH ), 4.11, 4.24 (each 1H, d, J )
1 Hz, -C(17)H OH), 4.83 (2H, m, dCHC(15)H O-), 7.19 (1H,
s-like, dC(14)H-); CMR (CDCl ) δ 60.1 (C-17), 128.7 (C-9),
39.1 (C-8). Anal. (C21 ) C, H.
7-Oxo-8,13-la bd a d ien -16,15-olid -19-oic Acid Meth yl
Ester (12). To a stirred solution of 11 (30 mg, 0.082 mmol)
in CH Cl (2 mL) was added PDC (31 mg, 0.082 mmol) at room
temperature. After 1 h the reaction mixture was diluted with
saturated Na SO (2 mL). The CH Cl extract was washed
with brine, dried over Na SO , and chromatographed over a
silica gel column (hexanes-EtOAc, 2:1) to give 12 (R ) 0.3,
4 mg): white crystals, mp 141-3 °C; HREIMS m/z 360.1937
3
3
2
(
28 6
H O
)
30 3
H O
1
(18). To a solution of 17 (0.10 g, 0.31 mmol) in acetone (4 mL)
was added J ones reagent (1.25 mmol) at room temperature.
After 6 h the reaction was quenched by addition of saturated
3
H
2
2
Na
trated. The crude reaction product thus obtained was further
treated with CH in ether for 1 h at 0 °C. Following addition
of glacial acetic acid (0.1 mL) to stop the reaction, the ether
extract was washed with brine, dried over Na SO , and
chromatographed over a silica gel column (hexanes-EtOAc,
5:1) to give 18 (R ) 0.3, 40 mg): colorless oil; HREIMS m/z
2 3
SO solution (5 mL), and the ether extract was concen-
2
4
3
1
f
2 2
N
+
1
3
) δ 0.84 (3H,
3
3
2
4
1
2
2
1
3
3
f
+
1
1
H
30
O
5
346.2143 [∆ -0.1 mmu (M )]; H NMR (CDCl
OCH ), 4.46, 4.91 (each 1H, s, dC(17)H ), 4.71-4.72 (2H, m,
dC(16)H ) δ
O-), 5.85 (1H, s-like, dC(14)H-); 13C NMR (CDCl
3.2 (C-16), 106.8 (C-17), 115.4 (C-14), 147.6 (C-8), 171.2 (C-
3), 174.4 (C-15), 177.9 (C-19). Anal. (C21 ) C, H.
In Vitr o P AF Recep tor Bin d in g Assa y. The PAF recep-
3
) δ 3.62 (3H, s,
-
3
2
1
2
3
7
1
2
2
30 4
H O
2
3
2
2
3
tor binding assay using washed rabbit platelets and [ H]PAF
was carried out according to the method of Valone with some
modification.16 The reaction mixture consisted of 200 µL of
2
4
f
2
[
H
7
8
+
1
washed rabbit platelet suspension (2 × 10 cells/mL), 25 µL of
∆ +0.0 mmu (M) ]; H NMR (CDCl
), 3.65 (3H, s, -OCH ), 4.80-4.81 (2H, m, dCHC(15)H
.19 (1H, s-like, dC(14)H-), 10.15 (1H, s, -C(17)HO); CMR
CDCl ) δ 130.1, 130.2 (C-8, C-13), 161.4 (C-9), 189.9 (C-17).
Anal. (C21 ) C, H.
(17)-La bd en -16,15-olid -19-oic Acid Meth yl Ester (13).
To an ice-cold solution of 1 (20 mg, 0.057 mmol) and CoCl ‚-
O (14 mg, 0.059 mmol) in dry EtOH (2 mL) was added
NaBH (4.3 mg, 0.11 mmol). After 3 h the reaction mixture
3
) δ 0.91 (3H, s, -C(20)-
3
[
H]PAF (0.6 nM, 60 000 dpm) with or without unlabeled PAF
3
3
2
O-),
3
1
3
(500-fold excess over [ H]PAF), and 25 µL of sample in 2%
DMSO. After 1-h incubation at room temperature, the free
and bound ligands were separated by filtration using What-
man GF/C glass fiber filters. The difference between total
(
3
28 5
H O
8
3
radioactivities of bound [ H]PAF in the absence and presence
2
of excess unlabeled PAF is defined as specific binding of the
6
H
2
3
radiolabeled ligand. In a set of experiments, [ H]PAF was
4
incubated with different concentrations of sample, and the
inhibitory effect of the sample on the specific binding is
expressed as percent inhibition of the control. The IC50 value
of a sample was defined as the final concentration of the
was treated with 3 N HCl (3 mL). The ether extract was
concentrated, and the residue was chromatographed over a
silica gel column (hexanes-EtOAc, 7:1) to give 13 (R
f
) 0.2,
1
+
5 mg): C(13)-epimeric mixture; HREIMS m/z 348.2302 [∆
3
sample required to block 50% of the specific [ H]PAF binding
+
1
0.2 mmu (M) ]; H NMR (CDCl
3
) δ 2.45 (1H, m, -C(13)-
C(15)H O-),
); CMR (CDCl ) δ 29.4
29.6) (pair, C-14), 39.4 (C-13), 66.40 (66.45) (pair, C-15), 177.9
C-19), 179.7 (C-16). Anal. (C21 ) C, H.
La bd a n -16,15-olid -19-oic Acid Meth yl Ester (14).
0.35 g, 1 mmol) was catalytically hydrogenated by stirring
0 min in 3 mL of dry THF with 0.32 g of 10% Pd/C under H
to rabbit platelet receptors, and the mean values obtained from
at least duplicate determinations (n ) 2-4) are given together
with standard deviations.
HCOO-), 3.62 (3H, s, -OCH
3
), 4.32 (2H, m, -CH
2
2
1
3
4
(
(
.53, 4.88 (each 1H, s-like, dC(17)H
2
3
32 4
H O
Ack n ow led gm en t. This work was supported by the
Korean Science Engineering Foundation through the
Center for Biofunctional Molecules, POSTECH, and by
the Ministry of Health and Welfare.
1
(
3
2
gas. The reaction mixture was filtered and worked up to
obtain 14 (0.35 g): needles, C(13)-epimeric mixture, mp 65-7
Su p p or tin g In for m a tion Ava ila ble: IR, EIMS, and 13C
NMR spectral data (3 pages). Ordering information is given
on any current masthead page.
+
1
°
C; HREIMS m/z 350.2457 [∆ +0.0 mmu (M) ]; H NMR
(
CDCl
3
) δ 0.64 (3H, s, -C(17)H
.60 (3H, s, -OCH ), 4.17, 4.33 (each 1H, m, -C(15)H
) δ 14.1, 14.7 (14.8) (C-20, C-17), 29.17 (29.21)
pair, C-14), 39.2 (C-13), 52.0, 53.0 (C-8, C-9), 66.4 (66.5) (pair,
3
), 2.42 (1H, m, -C(13)HCOO-),
3
3
2
O-);
1
3
CMR (CDCl
3
Refer en ces
(
C-15), 178.2 (C-19), 179.6, (179.8) (pair, C-16). Anal. (C21
C, H.
H
34
O
4
)
(1) (a) Benveniste, J .; Henson, P. M.; Cochrane, C. G. Leucocyte
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7-Nor -8-oxo-la bd a n -16,15-olid -19-oic Acid Meth yl Es-
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1
36, 1356-1377. (b) Braquet, P.; Tougui, L.; Shen, T. Y.;
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+
1
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3
3
) δ 0.53 (3H, s, -CH ), 2.42 (1H,

2
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2 4
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(
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J M970569J