Conformationally constrained analogues of diacylglycerol. 21. A solid-phase method of synthesis of diacylglycerol lactones as a prelude to a combinatorial approach for the synthesis of protein kinase C isozyme-specific ligands

Article abstract of DOI:10.1021/jm030610k

A solid-phase method for the synthesis of diacylglycerol lactones as protein kinase C ligands was developed, and a small array of nine compounds were selected with the idea of testing this methodology and forecasting the reliability of the biological data

Full text of DOI:10.1021/jm030610k

3
248
J . Med. Chem. 2004, 47, 3248-3254
Con for m a tion a lly Con str a in ed An a logu es of Dia cylglycer ol. 21. A Solid -P h a se
Meth od of Syn th esis of Dia cylglycer ol La cton es a s a P r elu d e to a Com bin a tor ia l
Ap p r oa ch for th e Syn th esis of P r otein Kin a se C Isozym e-Sp ecific Liga n d s
†
‡
†
‡
,†
Dehui Duan, Nancy E. Lewin, Dina M. Sigano, Peter M. Blumberg, and Victor E. Marquez*
Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute at Frederick,
National Institutes of Health, Frederick, Maryland 21702, and Laboratory of Cellular Carcinogenesis & Tumor Promotion,
Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892
Received December 13, 2003
A solid-phase method for the synthesis of diacylglycerol lactones as protein kinase C ligands
was developed, and a small array of nine compounds were selected with the idea of testing
this methodology and forecasting the reliability of the biological data as a preamble for the
construction of large chemical libraries to be synthesized under the same conditions. The process
started with the loading of 5-(hydroxymethyl)-5-[(4-methoxyphenoxy)methyl]-3,4,5-trihydro-
furan-2-one (1) to a 3,4-dihydro-2H-pyran resin packed inside IRORI MacroKan reactors. The
1
2
elements of diversity were introduced at the R-alkylidene (R ) and acyl (R ) positions using a
set of three different aldehydes and three different acid chlorides, respectively. An LDA-
1
mediated aldol condensation with R CHO in the presence of ZnCl
2
followed by a DBU-catalyzed
elimination of the triflate of the resulting aldol gave the R-alkylidene intermediates as mixtures
2
of geometric isomers. Removal of the aryl-protecting group followed by acylation with R COCl
introduced the second element of diversity. Acid-assisted cleavage of the compounds from the
resin afforded the final targets. The biological results obtained using the crude samples directly
obtained from the resin compared well with those from pure materials, as the K values between
i
the two sets varied only by a factor between 1.5 and 3.7.
In tr od u ction
(DAG-lactone, 7) where, depending on the nature of the
1
2
groups present at R or R , the compounds appear to
The central role of protein kinase C (PK-C) in cellular
signal transduction has established it as an important
therapeutic target for cancer and other diseases. PK-C
9
have some degree of isozyme specificity. Since among
the kinase and non-kinase C1 domains there are
important amino acid variations along the top rim of
the two â-loops that conform the area of the domain
thought to interact with the membrane, we surmised
that by employing a more extensive approach of explor-
1
isozymes, a family of related serine/threonine kinases,
contain in their regulatory domain one or two copies of
cysteine-rich motifs (C1 domains) about 50 amino acids
long, which bind to phorbol ester tumor promoters and
the second messenger diacylglycerol (DAG) exclusively
in the classic (R, â1, â2, and γ) and novel isoforms (δ, ꢀ,
η, and θ), resulting in their translocation and activa-
1
2
ing chemical diversity at R and R , we could improve
the chances of discovering specific C1-domain ligands
for each class of these multifaceted receptors.
To reach this goal, it was first necessary to develop a
robust solid-phase method of synthesis as a prelude to
the synthesis of a large number of compounds by a
combinatorial approach. The methodology that was
elaborated is presented in this manuscript, together
with the synthesis of a small, nine-member array of
compounds designed to test both the synthetic procedure
and the suitability for biological testing of the crude
materials directly obtained after cleavage from the
resin. For this purpose, each compound in the array was
purified and PK-C binding affinities for the R-isozyme
were compared with those obtained for the crude
products. Because phorbol esters and DAG-lactones
compete for the same site on the enzyme (the C1
2
tion. The atypical isoforms (ú and ι/λ) contain a single
nonfunctional C1 domain that fails to bind either
phorbol esters or DAG.2
It is now well established that there are other families
of proteins that contain C1 domains that are similarly
responsive to phorbol esters and DAG.3-8 The PKD/PKC
µ family represents kinases superficially similar to
PK-C; however, the kinase domains are not homologous
and show different selectivity. Other “non-kinase” pro-
teins that have a single copy of a functional C1 domain
and bind phorbol esters or DAG include the Rac GTPase
activating proteins (GAPs) R- and â-chimaerins, the Ras
exchange factor RasGRP, the Unc-13/Munc-13 family
of scaffolding proteins, and DAG kinases that abrogate
DAG signaling.
3
domain), displacement of PK-C-bound [20- H]phorbol-
1
2,13-dibutyrate (PDBU) by a DAG-lactone, expressed
During the past years, we have synthesized a number
of compounds built on a constrained glycerol scaffold
as Ki, provides a convenient assay for binding affinity.
The results described herein show that the method is
chemically sound and that the biological data obtained
from the crude samples provide valuable information,
as evidenced by the less than 4-fold variance in Ki
between purified and crude products.
*
To whom correspondence should be addressed. Phone: 301-846-
5
954. Fax: 301-846-6033. E-mail: marquezv@dc37a.nci.nih.gov.
†
Laboratory of Medicinal Chemistry.
‡
Laboratory of Cellular Carcinogenesis & Tumor Promotion.
1
0.1021/jm030610k This article not subject to U.S. Copyright. Published 2004 by the American Chemical Society
Published on Web 05/07/2004

Synthesis of Diacylglycerol Lactones
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12 3249
Sch em e 1a
a
Reagents and conditions: (a) lactone 1 (5 equiv), PPTS (4 equiv), 1,2-dichloroethane, 60 °C, 24 h (93%); (b) (i) LDA (10 equiv), THF,
1
-
78 °C, 6 h, (ii) ZnCl2 (12 equiv), -45 °C, 30 min, (iii) R CHO (12 equiv), -45 °C, 3 h, (iv) saturated NH4Cl solution, 90% (i f iv); (c) (i)
pyridine (20 equiv), (CF3SO2)2O (10 equiv), CH2Cl2, -20 °C, 20 h, (ii) DBU (20 equiv), toluene, -78 °C f room temp, 16 h, 75% (i f ii);
2
(
d) CAN (20 equiv), THF/H2O (4:1), 0 °C f room temp, 16 h, 100%; (e) R COCl (10 equiv), Et3N (20 equiv), 4-DMAP (1 equiv), CH2Cl2,
room temp, 20 h; (f) PPTS (10 equiv), n-butanol/1,2-dichloroethane (1:1), 60 °C, 16 h.
Ch em istr y
Rather, two individual steps consisting of triflation in
dichloromethane followed by a DBU-mediated elimina-
tion in toluene were necessary to obtain the polymer-
bound R-alkylidene lactone 4 in 75%. The quantitative
removal of the p-methoxyphenyl (PMP) group with
ammonium cerium(IV) nitrate (CAN) was successful in
THF/water (4:1), while the standard literature method
The parent DAG-lactone 1 [5-(hydroxymethyl)-5-[(4-
methoxyphenoxy)methyl]-3,4,5-trihydrofuran-2-one] was
dissolved in 1,2-dichloroethane and loaded onto a PL-
DHP (3,4-dihydro-2H-pyran) resin in the presence of
1
0
pyridinium p-toluenesulfonate (PPTS) (Scheme 1).
This operation was performed with the resin inside
IRORI MacroKan reactors in preparation for a future
large-scale combinatorial operation. The larger Macro-
Kan reactors were chosen with the purpose of obtaining
useful quantities of material suitable for purification by
column chromatography. A loading yield of 93% was
calculated from a single MacroKan by comparing the
1
3
using acetonitrile/water (4:1) failed, possibly because
of poor swelling of the cross-linked polystyrene resin in
this solvent system. Final removal of the aryl (PMP)
2
protection was followed by acylation with R COCl, thus
introducing the second element of diversity. PPTS-
assisted cleavage of the fully assembled DAG-lactone
from the resin afforded the final target compounds (7,
Table 1). During the entire process the MacroKan
reactors performed as filters. Hence, the solid-phase
reaction and its workup could be finished inside these
reactors, resulting in a simple operation without loss
of resin. This augured well for our future plans to use
this strategy in a radiofrequency (rf) tag-encoded com-
1
integration of H NMR signals of the cleaved product
(1) with those of dibromomethane used as an internal
standard. PPTS-assisted cleavage of 2 (Scheme 1) was
performed in a 1:1 n-butanol/1,2-dichloroethane solu-
1
0
tion. Yields for the rest of the intermediate steps were
similarly determined after cleaving the product from a
portion of the resin using the same PPTS-assisted
1
4
1
binatorial synthesis.
method. The first element of diversity (R ) was intro-
duced in two steps: (1) an LDA-mediated aldol conden-
sation with R CHO, which was facilitated by metal
The final compounds were obtained in sufficient
quantities to allow purification and full characterization
of each of the constituents of the nine-member set. As
far as the ratio of geometric isomers, the E-isomer was
major. Compounds 7a d , 7a e, 7bd , and 7be were puri-
fied as inseparable E/Z-mixtures with the E-isomer
predominating, whereas compounds 7a f, 7bf, 7cd , 7ce,
and 7cf were purified exclusively as E-isomers because
the amounts of the separable Z-isomers were negligible.
As we have previously observed, the vinyl proton for the
E-isomer appeared consistently ca. 0.5 ppm downfield
1
exchange with ZnCl2, and (2) a DBU-catalyzed elimina-
tion of the triflate of the aldol product (3) to give
mixtures of both E- and Z-R-alkylidene isomers (4). The
presence of ZnCl2 was critical to minimize the retro-
aldol reaction in the solid phase.1 Indeed, the aldol
reaction of the polymer-bound lactone 2 gave an increase
of 20% conversion when performed in the presence of
the Lewis acid. A one-pot mesylation of the polymer-
bound aldol product 3 followed by a DBU-catalyzed
1
1
2
12
elimination as reported in solution did not work well.
relative to the same signal for the Z-isomer. In cases

3
250 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12
Duan et al.
Ta ble 1. Chemical and Biological Parameters of Compounds
crude
yield (%)
E-isomer
Ki (nM),
pure
Ki (nM),
crude
Ki-based % purity
% HPLC purity
of crude at 254/210 nm
calcd
log P
a
b
compd
(%)
of crude
7
7
7
7
7
7
7
7
7
a d
a e
a f
bd
be
bf
cd
ce
cf
62
56
60
62
62
53
38
42
43
70
80
100
80
20.3 ( 4.8
19.1 ( 0.6
43.8 ( 1.2
25.7 ( 2.9
28.0 ( 2.0
43.3 ( 2.9
14.1 ( 1.8
22.7 ( 0.8
48.0 ( 2.1
37.0 ( 6.8
52.4 ( 2.1
92.5 ( 2.4
45.6 ( 2.7
41.8 ( 4.7
145.0 ( 7.3
41.0 ( 2.7
83.9 ( 5.2
152.3 ( 4.1
54.86
36.45
47.35
56.35
66.98
29.86
34.39
27.05
31.51
66.77/90.22
67.34/74.94
8.25/40.71
69.29/89.49
53.26/53.19
14.33/62.49
14.47/28.14
19.60/25.50
12.31/19.92
4.85
6.61
5.10
4.36
6.12
4.61
3.90
5.66
4.15
80
100
100
100
100
a
b
[
(Molar weight of crude product obtained from the resin)/(initial molar weight of DHP resin lactone 4 (0.25 mmol))] × 100. Calculated
1
5
log P values with the program KOWWIN 1.67.
where R1 was the bulky (4-phenylphenyl)methylene
chain, the normally preferred E-isomer became even
more dominant.
some compounds were purified as inseparable mixtures
of geometric E- and Z-isomers (vide supra). However,
since the same ratio of isomers also exists in the crude
samples, the biological comparisons between the two
sets are valid. The results show that the data obtained
from the crude samples provide valuable information
because the difference in Ki between the crude product
and purified compound was only a factor between 1.5
and 3.7 (Table 1). Finally, as a guide to interpreting the
biological data, the octanol/water partition coefficient
(log P) was also calculated for all target compounds
according to the fragment-based program KOWWIN
The purity of the crude samples obtained after cleav-
age from the resin was estimated by normal-phase
HPLC chromatography (Table 1). Because of the ex-
treme nonpolar nature of the compounds, reverse-phase
HPLC was not practical, and although normal-phase
HPLC is not ideal, purity could be effectively calculated
at two different wavelengths (210 and 254 nm) by
integration. Relative to the purity calculated on the
basis of Ki ratios for crude and pure samples (vide infra),
the HPLC purity determined at 254 nm approximated
the biological-based purity for compounds 7a d , 7bd ,
1
5
1.67.
Although the intent of this work was to develop a
solid-phase synthesis method, the introduction of the
biphenyl moiety in the context of the DAG-lactone
template was also explored for the first time. The strong
preference for protein binding of the biphenyl moiety,
one of the most “privileged” substructures, has been
7
be, and 7bf ((12-16%). Better matches were obtained
at 210 nm for compounds 7a f, 7cd , 7ce, and 7cf ((2-
2%). For compound 7a e, the HPLC estimated purity
1
exceeded by more than 30% the biologically estimated
purity. Naturally, these numbers are to be used only
as a guide in determining problems with the synthesis
and, in the context of a larger library, to help in the
selection of potential targets to be resynthesized by
conventional solution techniques.
1
6
recognized in the literature. The most potent member
of this small matrix is compound 7cd (K ) 14.1 nM),
i
which does contain the biphenyl moiety as part of the
1
R-alkylidene group (R ). The most remarkable property
of 7cd is not only its potency, but also the fact that it is
the ligand with the lowest lipophilicity (log P ) 3.90).
Next in potency is 7a e (Ki ) 19.1 nM, log P ) 6.61),
but in this case the compound is nearly 3 orders of
magnitude more lipophilic! Although the biphenyl moi-
ety seems to work well both as acyl and R-alkylidene
groups, the latter seems to be the preferred location,
especially in combination with the phenyl moiety as R2
(7cd ). Combining both biphenyl moieties as acyl and
R-alkylidene groups did not improve binding affinity and
produced instead a weaker ligand (7ce, Ki ) 22.7 nM).
Another interesting observation is the difference a
Biologica l Resu lts
Binding affinity assays were done with intact recom-
binant PK-CR, an isozyme where both the C1a and C1b
domains are involved in ligand binding and membrane
translocation. The activity of the DAG-lactones was
assessed in terms of the ability of the ligand to displace
3
bound [20- H]phorbol-12,13-dibutyrate (PDBu) from a
recombinant single isozyme (PK-CR) in the presence of
phosphatidylserine.12 The inhibition curves obtained for
all ligands were of the type expected for competitive
inhibition, and the Ki values were calculated from the
ID50 values (see Experimental Section). For each com-
pound two sets of Ki values were obtained, one from the
pure fully characterized material and one from the same
target obtained in crude form directly after cleavage
from the resin (Table 1). It is important to note that
2
methylene group makes at the R position as one
compares the benzoyl and phenylacetyl moieties. In all
the cases where the phenylacetyl moiety is present (7a f,
7bf, and 7cf), the compounds are the weakest ligands,
whereas compounds with the benzoyl group (7a d , 7bd ,

Synthesis of Diacylglycerol Lactones
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12 3251
and 7cd ) are among the most potent. One important
point regarding the relationship between the E- and
Z-geometry of the R-alkylidene group is the generally
observed trend that the Z-isomers are ca. 2-fold more
potent than the E-isomers. This means that for those
cases where the Z-isomer is 20-30% of the mixtures,
the Ki values for the pure Z-isomer could be as low as
7ce, 7cf (2% f 50% 2-propanol/n-hexane, 1.7 mL/min, 40 min).
Samples of all nine compounds were further purified by column
chromatography on silica gel and fully characterized by FT-
1
13
IR, H NMR, C NMR, MS, and elemental analysis.
-(H yd r oxym et h yl)-5-[(4-m et h oxyp h en oxy)m et h yl]-
,4,5-tr ih yd r ofu r a n -2-on e (1). A solution of 5-[(4-methoxy-
5
3
17
phenoxy)methyl]-5-[(phenylmethoxy)methyl]-3,4,5-trihydrofuran-2-one
3.4 g, 10.0 mmol) in dichloromethane (200 mL) was cooled to
(
4
-6 nM.
-78 °C and treated with boron trichloride (1.0 M in dichlo-
romethane, 40 mL). After being stirred at -78 °C for 2 h, the
reaction mixture was quenched with a saturated solution of
sodium bicarbonate and immediately partitioned. The water
layer was extracted with diethyl ether (200 mL, 1×), and the
combined organic phase was washed with water (1×), dried
In comparison with the best DAG-lactones reported
9
to date, the compounds in Table 1 exhibit comparable
potencies in binding to PK-CR. However, a compound
such as 7cd is an attractive molecule for possible future
drug development because of its lower log P value
compared to most DAG-lactones. It is important to point
out that all the compounds made in this set are potential
2 4
(Na SO ), and concentrated in vacuo. The residue was purified
by flash column chromatography on silica gel with ethyl
acetate/hexane (1:1) as eluant to give 1 (2.3 g, 91%) as a white
solid: mp 60-62 °C (hexane/ethyl acetate); IR (neat) 3445
“hits” in their own right. With a DAG-lactone as an
-
1 1
(
OH), 1771 (CdO) cm ; H NMR (CDCl
OC OCH C), 4.04 (d, J ) 10.0 Hz, 1 H, CH
.99 (d, J ) 10.0 Hz, 1 H, CH OC OCHHC), 3.84 (dd, J )
2.1, 6.8 Hz, 1 H, HOCHHC), 3.68-3.78 (m, 4 H, HOCHHC
3
) δ 6.80 (s, 4 H, CH
3
-
invariant scaffold, it is very likely that the all com-
pounds will bind to PK-CR with varying degrees of
affinities. Our intent is that future tests with other
PK-C isozymes, as well as other families of proteins that
contain C1 domains, will reveal important individual
specificities.
6
H
4
2
3
OC OCHHC),
6 4
H
3
1
3
6 4
H
and CH OC H OCH C), 2.59-2.82 (m, 2 H, H-3 ), 2.15-2.34
3
6
4
2
a,b
1
3
(m, 2 H, H-4a,b), 1.93 (t, J ) 6.8 Hz, 1 H, HOCH
2
C); C NMR
) δ 177.13, 154.64, 152.58, 115.86, 114.92, 87.05, 71.13,
5.77, 55.92, 29.31, 25.88; FAB MS (m/z, relative intensity)
(CDCl
3
6
2
+
+•
53 (MH , 46), 252 (M , 100). Anal. (C13
16 5
H O ) C, H.
Con clu sion
P r oced u r e for Loa d in g 1 on th e P L-DHP Resin . Ten
MacroKan reactors, each containing 0.147 g (0.25 mmol) of
DHP resin, were placed in a 250 mL round-bottom flask
equipped with a stirring bar. Lactone 1 (3.2 g, 12.5 mmol, 5
equiv), pyridinium p-toluenesulfonate (PPTS, 2.5 g, 10 mmol,
We have developed a robust solid-phase method using
a PL-DHP resin for the synthesis of DAG-lactones using
1
2
an exploratory set of R and R groups at the R-alkyl-
idene and acyl positions, respectively. The use of Mac-
roKan reactors simplified the operation and provided
good reproducibility. The method was tested with the
synthesis of a small, nine-member array with the idea
of forecasting the reliability of the biological data that
could be obtained when using larger chemical libraries
synthesized under the same conditions. We conclude
that the biological results obtained using crude samples
directly obtained from the resin provide valuable infor-
mation because the difference in Ki between pure and
crude materials varied only by a factor between 1.5 and
4
equiv), and 1,2-dichloroethane (100 mL) were introduced into
the flask. The MacroKan reactors were degassed in the
solution by lowering the temperature to -30 °C in vacuo for 5
min. The temperature was then raised to 60 °C, and the
solution was stirred for 24 h. After reaching room temperature,
the solution was decanted into another flask to recover excess
unreacted lactone. The MacroKan reactors containing polymer-
bound lactone (2) were washed subsequently with dichlo-
romethane (1×), 1:1 DMF/water (4×), DMF (3×), dichlo-
romethane (3×) and finally dried in vacuo.
P r oced u r e for Sta n d a r d P P TS-Assisted Clea va ge. One
MacroKan containing polymer-bound lactone (2, 0.25 mmol,
1
.0 equiv) was treated with PPTS (0.628 g, 2.5 mmol, 10.0
3
.7. Although the intent of this effort was not drug
equiv) in 1:1 n-butanol/1,2-dichloroethane (25 mL). After being
stirred at 60 °C for 16 h and cooling to room temperature, the
solution was transferred to a separatory funnel. The MacroKan
was washed with dichloromethane (3 × 20 mL), and the
combined organic phase was washed with a saturated aqueous
sodium bicarbonate solution (2×) and water (2×). The solution
discovery, a potentially interesting compound (7cd )
combining high affinity for PK-C with low lipophilicity
and bearing the “privileged” biphenyl moiety at the
R-alkylidene position was identified.
2 4
was then dried over Na SO and concentrated in vacuo to give
Exp er im en ta l Section
1
as colorless oil. A 93% loading yield was calculated from the
1
3
integration of the H NMR (CDCl ) signals of 1 compared to a
Gen er a l Tech n iqu es. All reagents and solvents purchased
were of the highest commercial quality and used without
further purification unless otherwise stated. MacroKan (IRORI)
reactors were purchased from Discovery Partners Interna-
tional, San Diego, CA. PL-DHP (3,4-dihydro-2H-pyran) resin
known amount of dibromomethane added as an internal
standard.
P r oced u r e for th e Ald ol Rea ction of P olym er -Bou n d
La cton e 2 w ith Decyl Ald eh yd e. MacroKan reactors con-
taining polymer-bound lactone 2 (9 MacroKan reactors, 2.25
mmol, 1 equiv) were suspended in THF (225 mL) in a round-
bottom flask equipped with a stirring bar. After the mixture
was degassed at -78 °C in vacuo for 5 min, LDA (2.0 M in
heptane/THF/ethylbenzene, 11.3 mL, 10 equiv) was added
while the temperature was maintained at -78 °C. After being
(
1.7 mmol/g, 150-300 µm) was purchased from Polymer
Laboratories, Ltd., Amherst, MA. All solid-phase reactions
were performed in MacroKan reactors filled with 147.1 mg
(0.25 mmol) of PL-DHP resin. The loading yield on the resin
was determined from an aliquot by comparing the integration
1
of H NMR signals of the cleaved product with those of an
internal standard (CH
2
Br
2
). Yields for intermediate steps of
stirred for 6 h at -78 °C, the solution was treated with ZnCl
2
the synthesis were similarly determined after cleavage of a
portion of the resin and calculation of the percent conversion
(0.5 M in THF, 54 mL, 12 equiv) and the reaction was allowed
to warm to -45 °C for 30 min. A solution of decyl aldehyde
(5.4 mL, 27 mmol, 12.0 equiv) in THF (18 mL) was then added,
and stirring continued at -45 °C for 3 h. The reaction was
quenched with a saturated solution of ammonium chloride, and
the mixture was allowed to reach room temperature. The
MacroKan reactors containing polymer-bound aldol product
3 were washed with 1:1 DMF/water (4×), DMF (3×), dichlo-
romethane (3×) and dried in vacuo. Standard PPTS-assisted
1
by integration of H NMR signals. Final crude products were
obtained after cleavage from the resin and evaporation of the
solvent. Purities of the crude products were determined by
normal-phase HPLC (see Table 1) under various conditions:
7
a d , 7a e, 7bd , 7be (1% 2-propanol/n-hexane, isocratic, 1.5 mL/
min); 7a f, (1% 2-propanol/n-hexane, isocratic, 1.7 mL/min); 7bf
2% f 10% 2-propanol/n-hexane, 1.7 mL/min, 45 min); 7cd ,
(

3
252 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12
Duan et al.
cleavage of a single MacroKan afforded the free aldol product
as a colorless solid (0.077 g, 87% yield). The crude product was
purified by flash column chromatography on silica gel with
ethyl acetate/hexane (2:3) as eluant to afford pure 3-(hydroxy-
decyl)-5-(hydroxylmethyl)-5-[(4-methoxyphenoxy)methyl]-3,4,5-
trihydrofuran-2-one as a white solid: mp 107-109 °C (hexane/
129.16, 129.03, 128.20, 127.77, 127.30, 126.99, 124.30, 115.88,
115.83, 113.91, 83.99, 70.52, 65.69, 55.91, 32.59; FAB MS (m/
+
+•
z, relative intensity) 417 (MH , 100%) 416 (M , 93). Anal.
) C, H.
(C26
24 5
H O
P r ocedu r e for Rem oval of th e p-Meth oxyph en yl (P MP )
Gr ou p fr om P olym er -Bou n d r-Alk ylid en e La ct on e 4.
MacroKan reactors containing polymer-bound R-alkylidene
lactone 4 (8 MacroKan reactors, 2 mmol, 1.0 equiv) were
-1 1
ethyl acetate); IR (neat) 3432 (OH), 1715 (CdO) cm ; H NMR
CDCl ) δ 6.79 (s, 4 H, CH OC OCH C), 3.95-4.05 (m, 2 H,
CH OC OCH C), 3.78-3.85 (m, 1 H, HOCHHC), 3.65-3.75
m, 5 H, HOCHHC, CH OC OCH C and CH(OH)C ),
.83-3.01 (m, 1 H, H-3), 2.34-2.42 (m, 1 H, H-4 ), 1.95-2.06
), 0.84
); C NMR (CDCl ) δ
(
3
3
6
H
4
2
3
6
H
4
2
2
suspended in a 4:1 THF/H O mixture (200 mL) inside a round-
(
3
6
H
4
2
8
H
16CH
3
bottom flask equipped with a stirring bar. After the mixture
was cooled to 0 °C, ammonium cerium(IV) nitrate (CAN, 21.9
g, 40 mmol, 20 equiv) was introduced into the flask and stirring
continued at room temperature for 20 h. Finally, the MacroKan
reactors containing polymer-bound diol-R-alkylidene lactone
5 were washed with a 5% solution of sodium thiosulfate (3×),
THF (1×), 1:1 DMF/water (4×), DMF (3×), dichloromethane
(3×) and dried in vacuo. Standard PPTS-assisted cleavage of
one MacroKan afforded the free diol-R-alkylidene lactone as a
yellow oil (0.039 g, 100% yield). The crude product was purified
by flash column chromatography on silica gel with ethyl
acetate/methanol (100:1) as eluant to afford pure 5,5-bis-
(hydroxymethyl)-3-decylidene-4,5-dihydrofuran-2-one as a white
solid: mp 57-58 °C (hexane); IR (neat) 3393 (OH), 1746
2
a
(
(
1
6
m, 1 H, H-4
b
8 3
), 1.15-1.55 (m, 16 H, CH(OH)C H16CH
1
3
t, J ) 7.2 Hz, 3 H, CH(OH)C
79.23, 154.67, 152.48, 115.86, 114.93, 85.51, 72.45, 70.96,
5.62, 55.91, 45.83, 34.97, 32.08, 29.78, 25.30, 22.87, 14.31;
8
H
16CH
3
3
+
+•
FAB MS (m/z, relative intensity) 409 (MH , 77) 408 (M , 100).
Anal. (C23 ) C, H.
36 6
H O
P r oced u r e for Tr ifyla tion of P olym er -Bou n d Ald ol
P r od u ct 3 F ollow ed by DBU-Assisted Elim in a tion . Mac-
roKan reactors containing polymer-bound aldol product 3 (3
MacroKan reactors, 0.75 mmol, 1.0 equiv) were placed in a
round-bottom flask equipped with a stirring bar and suspended
in dichloromethane (75 mL). After the mixture was degassed
at -78 °C in vacuo for 5 min, pyridine (1.2 mL, 15 mmol, 20
equiv) and trifluoromethylsulfonic anhydride (1.3 mL, 7.5
mmol, 10 equiv) were subsequently introduced into the flask
at the same temperature. The resulting mixture was stirred
at -20 °C for 20 h. The reaction was then quenched with a
saturated solution of sodium bicarbonate, and the mixture was
allowed to reach room temperature. The MacroKan reactors
were washed with dichloromethane (1×), 1:1 DMF/water (4×),
DMF (3×), dichloromethane (3×) and dried in vacuo. The
MacroKan reactors were then immediately suspended in
toluene (75 mL), and after degassing at -78 °C for 5 min, DBU
-
1
1
(CdO) cm
CdCH), 6.17 (t, J ) 7.6 Hz, 0.1 H, (Z)-CdCH), 3.72 (d, J )
12.1 Hz, 2 H, CH OH), 3.62 (d, J ) 12.1 Hz, 2 H, CH OH),
2.70 (s, 0.2 H, (Z)-H-4a,b), 2.65 (s, 1.8 H, (E)-H-4a,b), 2.57 (br s,
3
; H NMR (CDCl ) δ 6.64-6.73 (m, 0.9 H, (E)-
2
2
2
1
CH
8
H, HOCH
.46 (m, 14 H, CH C H14CH
2
C), 2.10 (irregular q, 2 H, CH
), 0.81 (t, J ) 6.8 Hz, 3 H,
); C NMR (CDCl ) δ 170.99, 142.45, 126.43,
2 7 3
C H14CH ), 1.12-
2 7 3
1
3
2
C
7
H
14CH
3
3
5.52, 65.29, 32.04, 30.50, 29.58, 28.25, 22.85, 14.29; FAB MS
+
(
0
28 4
m/z, relative intensity) 285 (MH , 100%). Anal. (C16H O ‚
.2H
2
O) C, H.
P r oced u r e for Acyla tion of P olym er -Bou n d Diol La c-
(2.25 mL, 15 mmol, 20 equiv) was added at the same temper-
ton e 5 w ith Ben zoyl Ch lor id e. A MacroKan containing
polymer-bound diol-R-alkylidene lactone 5 (1 MacroKan, 0.25
mmol, 1.0 equiv) was suspended in CH Cl (25 mL) in a round-
2 2
ature. The MacroKan reactors were stirred at room temper-
ature for 16 h, after which time the reaction was quenched
with a saturated solution of ammonium chloride. The Macro-
Kan reactors containing polymer-bound R-alkylidene lactone
bottom flask equipped with a stirring bar. After the mixture
was degassed at -78 °C for 5 min, triethylamine (0.7 mL, 5
mmol, 20 equiv), 4-DMAP (30.5 mg, 0.25 mmol, 1 equiv), and
benzoyl chloride (0.29 mL, 2.5 mmol, 10 equiv) were subse-
quently introduced into the flask. The mixture was stirred at
room temperature for 20 h, after which time the MacroKan
was washed with dichloromethane (1×), 1:1 DMF/water (4×),
DMF (3×), dichloromethane (3×) and dried in vacuo. The
polymer-bound target DAG-lactone 6 was treated with the
standard PPTS-assisted cleavage protocol to provide the final
crude DAG-lactone as a yellow oil (60 mg, 90%). The crude
product was purified by flash column chromatography on silica
gel with ethyl acetate/hexane (2:3) as eluant to afford pure
[4-decylidene-2-(hydroxymethyl)-5-oxo-2-2,3-dihydrofuryl]-
methyl benzoate (7a d ) as a colorless solid: mp 77-81 °C
4
were washed with dichloromethane (1×), 1:1 DMF/water
(
4×), DMF (3×), dichloromethane (3×) and dried in vacuo.
Standard PPTS-assisted cleavage of one MacroKan afforded
the free R-alkylidene lactone as a yellow oil (0.071 g, 80%
yield). The crude product was purified by flash column
chromatography on silica gel with ethyl acetate/hexane (2:3)
as eluant to afford pure 3-decylidene-5-(hydroxylmethyl)-5-[(4-
methoxyphenoxyl)methyl]-4,5-dihydrofuran-2-one as a white
solid: mp 67-68 °C (hexane); IR (neat) 3441 (OH), 1755
-
1
1
(
CdO) cm
;
H NMR (CDCl
C), 6.71-6.78 (m, 0.9 H, (E)-CdCH), 6.18 (t, J ) 7.6 Hz,
OC
OCHHC), 3.84
d, J ) 12.2 Hz, 1 H, HOCHHC), 3.75 (d, J ) 12.2 Hz, 1 H,
HOCHHC), 3.73 (s, 3 H, CH OC OCH C), 2.89 (d, J ) 17.2
Hz, 1 H, H-4 ), 2.76 (d, J ) 17.2 Hz, 1 H, H-4 ), 2.16 (irregular
q, 2 H, CH ), 1.16-1.55 (m, 14 H, CH ),
) δ
3
) δ 6.79 (s, 4 H, CH
3 6 4
OC H -
OCH
0
2
.1 H, (Z)-CdCH), 4.04 (d, J ) 9.6 Hz, 1 H, CH
3
6 4
H -
OCHHC), 3.95 (d, J ) 9.6 Hz, 1 H, CH OC
(
3
6 4
H
-1
(hexane); IR (neat) 3426 (OH), 1759 (CdO), 1724 (CdO) cm ;
1
H
4
2
H NMR (CDCl
7.54 (t, J ) 7.5 Hz, 1 H, PhCO
H, PhCO CH C), 6.72-6.81 (m, 0.7 H, (E)-CdCHCH
(t, J ) 7.6 Hz, 0.3 H, (Z)-CdCHCH ), 4.45 (AB q, J ) 11.9 Hz,
2 H, PhCO CH C), 3.75 (AB q, J ) 12.1 Hz, 2 H, HOCH C),
2.71-2.99 (m, 2 H, H-4a,b), 2.60-2.69 (m, 0.6 H, (Z)-
3
) δ 7.94 (d, J ) 7.5 Hz, 2 H, PhCO
CH C), 7.40 (t, J ) 7.5 Hz, 2
), 6.21
2 2
CH C),
3
6
a
b
2
2
2
C
7
H
14CH
3
2
C
7
H
14CH
3
2
2
2
1
3
0
1
7
.85 (t, J ) 6.8 Hz, 3 H, CH
70.40, 154.59, 152.62, 142.23, 126.29, 115.81, 114.89, 83.85,
0.47, 65.64, 55.91, 32.05, 30.48, 29.67, 28.28, 22.84, 14.29;
2
C
7
H
14CH
3
); C NMR (CDCl
3
2
2
2
2
+
CdCHCH ), 2.34 (br s, 1 H, HOCH C), 2.07-2.19 (m, 1.4 H,
(E)-CdCHCH
6
1
6
FAB MS (m/z, relative intensity) 390 (MH , 100%). Anal.
) C, H.
A similar procedure was used to obtain and purify 5-(hy-
2
2
(C
23
H
36
O
6
2
), 1.12-1.46 (m, 14 H, C
7 3 3
7
H
14CH
3
), 0.84 (t, J )
1
3
.9 Hz, 3 H, C H14CH ); C NMR (CDCl ) δ 170.19, 166.50,
45.88, 142.40, 133.72, 129.99, 129.30, 128.72, 126.00, 83.50,
droxymethyl)-5-[(4-methoxyphenoxy)methyl]-3-[(4-phenylphe-
nyl)methylene]-4,5-dihydrofuran-2-one, which was obtained as
a white solid: mp 151-153 °C (hexane/ethyl acetate); IR (neat)
6.31, 65.01, 32.03, 30.50, 30.12, 29.50, 28.22, 22.84, 14.30;
+
FAB MS (m/z, relative intensity) 389 (MH , 89%), 105 (100%).
-
1
1
32 5
Anal. (C23H O ) C, H.
3
8
(
(
4
371 (OH), 1729 (CdO) cm ; H NMR (CDCl
3
) δ 7.91 (d, J )
F in a l P P TS-Assisted Clea va ge. The rest of the target
DAG-lactones were obtained after following the same standard
PPTS-assisted cleavage method described above.
.2 Hz, 0.4 H, Ar), 7.64 (d, J ) 8.4 Hz, 1.6 H, Ar), 7.58-7.62
m, 4.8 H, Ar and (E)-CdCH), 7.36-7.46 (m, 3 H, Ar), 7.05
m, 0.2 H, (Z)-CdCH), 6.77-6.82 (m, 4 H, CH OC OCH C),
.10 (d, J ) 9.6 Hz, 1 H, CH OC OCHHC), 4.02 (d, J ) 9.6
Hz, 1 H, CH OC OCHHC), 3.80-3.97 (m, 2 H, HOCH C),
.72 (s, 3 H, CH OC OCH C), 3.15-3.35 (m, 2 H, H-4a,b),
.02 (t, J ) 6.8 Hz, 1 H, HOCH
54.67, 152.54, 142.971, 140.16, 137.17, 133.63, 131.51, 130.87,
3
6
H
4
2
3
6
H
4
[4-Decylid en e-2-(h yd r oxym eth yl)-5-oxo-2-2,3-d ih yd r o-
fu r yl]m eth yl 4-P h en ylben zoa te (7a e). Colorless solid, mp
116-120 °C (hexane/ethyl acetate); IR (neat) 3409 (OH), 1729
3
6
H
4
2
3
2
1
3
6
H
4
2
1
3
-1 1
2
C); C NMR (CDCl
3
) δ 171.49,
(CdO), 1714 (CdO) cm ; H NMR (CDCl
3
) δ 7.97 (d, J ) 8.2
Hz, 2 H, ArH), 7.48-7.65 (m, 4 H, ArH), 7.26-7.45 (m, 3 H,

Synthesis of Diacylglycerol Lactones
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12 3253
ArH), 6.70-6.78 (m, 0.8 H, (E)-CdCHCH
2
), 6.17 (t, J ) 7.6
(E )-{2-(H yd r oxym e t h yl)-5-oxo-4-[(4-p h e n ylp h e n yl)-
m et h ylen e]-2-2,3-d ih yd r ofu r yl}m et h yl Ben zoa t e (7cd ).
Colorless solid, mp 140-143 °C (hexane/ethyl acetate); IR
Hz, 0.2 H, (Z)-CdCHCH
2
), 4.43 (AB q, J ) 11.8 Hz, 2 H,
C), 2.68-
.96 (m, 2 H, H-4a,b), 2.48-2.65 (m, 0.4H, (Z)-CdCHCH ), 2.27
br s, 1 H, HOCH C), 2.02-2.18 (m, 1.6 H, (E)-CdCHCH ),
.05-1.44 (m, 14 H, C ), 0.79 (t, J ) 6.9 Hz, 3 H,
) δ 170.19, 166.41, 146.50, 145.89,
ArCO
2
(
1
2
CH
2
C), 3.71 (AB q, J ) 12.1 Hz, 2 H, HOCH
2
-
1 1
2
(neat) 3382 (OH), 1714 (CdO) cm ; H NMR (CDCl
(d, J ) 8.4 Hz, 2 H, Ar), 7.46-7.65 (m, 8 H, Ar and CdCH),
7.28-7.44 (m, 5 H, Ar), 4.54 (d, J ) 11.9 Hz, 1 H, PhCO CHHC),
4.41 (d, J ) 11.9 Hz, 1 H, PhCO CHHC), 3.83 (dd, J ) 12.3,
6.9 Hz, 1 H, HOCHHC), 3.76 (dd, J ) 12.3, 6.9 Hz, 1H,
HOCHHC), 3.28 (dd, J ) 17.6, 3.1 Hz, 1 H, H-4 ), 3.11 (dd, J
) 17.6, 3.1 Hz, 1H, H-4 ), 2.21 (t, J ) 6.9 Hz, 1 H, HOCH C);
3
) δ 7.89
2
2
7
H
14CH
3
3
2
1
3
C
7
H
14CH
3
); C NMR (CDCl
2
1
6
42.41, 139.95, 130.53, 129.18, 128.51, 127.47, 126.03, 83.53,
6.33, 65.03, 32.01, 30.51, 30.16, 29.47, 28.25, 22.84, 14.29;
a
+
FAB MS (m/z, relative intensity) 465 (MH , 32%), 181 (100%).
Anal. (C29 ) C, H.
E)-[4-Decylid en e-2-(h yd r oxym eth yl)-5-oxo-2-2,3-d ih y-
d r ofu r yl]m eth yl 2-P h en yla ceta te (7a f). Colorless solid, mp
b
2
1
3
H
36
O
5
3
C NMR (CDCl ) δ 171.31, 166.52, 143.08, 140.09, 137.32,
33.74, 133.43, 130.81, 130.00, 129.25, 129.17, 128.73, 128.24,
27.79, 127.29, 123.98, 83.64, 66.27, 64.98, 32.68; FAB MS (m/
z, relative intensity) 415 (MH , 100%). Anal. (C26
1
1
(
+
-
1
H
22
O
5
2
‚0.2H O)
6
8-70 °C (hexane); IR (neat) 3383 (OH), 1730 cm (CdO);
1
C, H.
H NMR (CDCl
.75 (m, 1 H, CdCHCH
CH CO CHHC), 4.16 (d, J ) 11.9 Hz, 1 H, PhCH
.62 (d, J ) 12.1 Hz, 1 H, HOCHHC), 3.61 (s, 2 H, PhCH
CO CH C), 3.54 (d, J ) 12.1 Hz, 1 H, HOCHHC), 2.70 (d, J )
7.0 Hz, 1 H, H-4 ), 2.52 (d, J ) 17.0 Hz, 1 H, H-4 ), 2.08
irregular q, 2 H, CdCHCH ), 1.92 (br s, 1 H, HOCH C), 1.12-
.46 (m, 14 H, C ), 0.85 (t, J ) 6.8 Hz, 3 H, C );
) δ 171.52, 169.99, 146.10, 142.51, 133.56,
3
) δ 7.18-7.31 (m, 5 H, PhCH
), 4.27 (d, J ) 11.9 Hz, 1 H, C
CO CHHC),
2 2 2
CO CH C), 6.65-
(
E )-{2-(H yd r oxym e t h yl)-5-oxo-4-[(4-p h e n ylp h e n yl)-
m eth ylen e]-2-2,3-d ih ydr ofu r yl}m eth yl 4-P h en ylben zoa te
7ce). Colorless solid, mp 234-236 °C (hexane/ethyl acetate);
6
2
6 5
H -
2
2
2
2
(
3
2
-
-
1 1
IR (neat) 3390 (OH), 1765 (CdO), 1716 (CdO) cm ; H NMR
CDCl ) δ 7.96 (d, J ) 8.4 Hz, 2 H, Ar), 7.46-7.65 (m, 11 H,
Ar and CdCH), 7.28-7.44 (m, 6 H, Ar), 4.57 (d, J ) 11.9 Hz,
H, ArCO CHH), 4.44 (d, J ) 11.9 Hz, 1 H, ArCO CHH), 3.84
dd, J ) 12.3, 6.9 Hz, 1 H, HOCHHC), 3.77 (dd, J ) 12.3, 6.9
Hz, 1 H, HOCHHC), 3.29 (dd, J ) 17.8, 2.9 Hz, 1 H, H-4 ),
.13 (dd, J ) 17.8, 2.9 Hz, 1H, H-4 ), 2.22 (t, J ) 6.9 Hz, 1 H,
HOCH ) δ 171.33, 166.42, 146.51, 143.08,
C); 13C NMR (CDCl
40.09, 137.33, 133.44, 130.82, 130.54, 129.16, 128.51, 128.24,
27.90, 127.81, 127.48, 127.39, 127.29, 123.98, 83.67, 66.28,
2
2
(
3
1
(
a
b
2
2
1
2
2
1
7
H
14CH
3
7 3
H14CH
(
1
3
C NMR (CDCl
3
a
1
3
29.44, 128.91, 127.55, 125.74, 83.06, 65.89, 65.02, 41.56,
3
b
2.06, 30.48, 29.59, 28.26, 22.86, 14.30; FAB MS (m/z, relative
+
2
3
intensity) 403 (MH , 100%). Anal. (C24
34 5
H O ) C, H.
1
1
6
1
[
4-Non ylid en e-2-(h yd r oxym eth yl)-5-oxo-2-2,3-d ih yd r o-
fu r yl]m eth yl Ben zoa te (7bd ). Colorless solid, mp 80-81 °C
+
4.98, 32.70; FAB MS (m/z, relative intensity) 491 (MH , 49),
81 (100). Anal. (C32 ‚0.3H O) C, H.
E )-{2-(H yd r oxym e t h yl)-5-oxo-4-[(4-p h e n ylp h e n yl)-
m eth ylen e]-2-2,3-d ih yd r ofu r yl}m eth yl 2-P h en yla ceta te
7cf). Colorless solid, mp 110-114 °C (hexane/ethyl acetate);
1
(
hexane); IR (neat) 3420 (OH), 1765 (CdO), 1730 (CdO); H
NMR (CDCl ) δ 7.94 (d, J ) 7.5 Hz, 2 H, PhCO CH C), 7.54
t, J ) 7.5 Hz, 1 H, PhCO CH C), 7.39 (t, J ) 7.5 Hz, 2 H,
PhCO CH C), 6.71-6.79 (m, 0.8 H, (E)-CdCHCH ), 6.21 (t, J
7.6 Hz, 0.2 H, (Z)-CdCHCH ), 4.45 (AB q, J ) 11.9 Hz, 2 H,
PhCO CH C), 3.75 (AB q, J ) 12.1 Hz, 2 H, HOCH C), 2.66-
.99 (m, 2 H, H-4a,b), 2.60-2.64 (m, 0.4 H, (Z)-CdCHCH ), 2.40
br s, 1 H, HOCH C), 2.04-2.19 (m, 1.6 H, (E)-CdCHCH ),
.14-1.44 (m, 12 H, C ), 0.84 (t, J ) 6.9 Hz, 3 H,
) δ 170.20, 166.50, 142.39, 133.72,
H
26
O
5
2
3
2
2
(
(
2
2
2
2
2
(
)
2
-1
1
IR (neat) 3364 (OH), 1735 (CdO) cm ; H NMR (CDCl ) δ
7
3
2
2
2
.65 (d, J ) 8.2 Hz, 2 H, Ar), 7.60 (d, J ) 8.2 Hz, 2 H, Ar),
2
2
7.54 (t, J ) 2.9 Hz, 1 H, CdCH), 7.34-7.52 (m, 5 H, Ar), 7.13-
(
2
2
7.19 (m, 5 H, Ar), 4.32 (d, J ) 11.9 Hz, 1 H, PhCH CO CHHC),
2
2
1
C
6
H
12CH
3
3
4.24 (d, J ) 11.9 Hz, 1H, PhCH CO CHHC), 3.66 (dd, J ) 12.1,
2
2
1
3
6
H
12CH
3
); C NMR (CDCl
7.0 Hz, 1 H, HOCHHC), 3.58 (dd, J ) 12.1, 7.0 Hz, 1H,
HOCHHC), 3.56 (s, 2 H, PhCH CO CH C), 3.06 (dd, J ) 17.8,
1
3
29.98, 129.30, 128.72, 126.01, 83.51, 66.31, 65.01, 32.00,
2
2
2
0.49, 30.11, 29.49, 29.31, 28.22, 22.81, 14.27; FAB MS (m/z,
2.9 Hz, 1 H, H-4 ), 2.88 (dd, J ) 17.8, 2.9 Hz, 1 H, H-4 ), 2.01
13
a
b
+
relative intensity) 375 (MH , 100). Anal. (C22
H
30
O
5
) C, H.
(t, J ) 6.7 Hz, 1 H, HOCH C); C NMR (CDCl ) δ 171.45,
2
3
1
1
6
71.17, 143.08, 140.11, 137.20, 133.50, 130.89, 129.35, 129.20,
28.87, 128.27, 127.77, 127.49, 127.31, 123.78, 83.35, 65.89,
5.10, 41.42, 32.30; FAB MS (m/z, relative intensity) 429 (MH ,
[
4-Non ylid en e-2-(h yd r oxym eth yl)-5-oxo-2-2,3-d ih yd r o-
fu r yl]m eth yl 4-P h en ylben zoa te (7be). Colorless solid, mp
21-123 °C (hexane/ethyl acetate); IR (neat) 3409 (OH), 1729
+
1
-
1
1
100). Anal. (C H O ‚0.7H O) C, H.
(CdO), 1714 (CdO) cm ; H NMR (CDCl
3
) δ 8.01 (d, J ) 8.2
27 24
5
2
3
An a lysis of In h ibition of [ H]P DBU Bin d in g by Non -
Hz, 2 H, ArH), 7.30-7.68 (m, 7 H, ArH), 6.72-6.81 (m, 0.8 H,
r a d ioa ctive Liga n d s. Enzyme-ligand interactions were
(
(
E)-CdCHCH
AB q, J ) 11.9 Hz, 2 H, ArCO
2
, 6.22 (t, J ) 7.6 Hz, 0.2 H, (Z)-CdCHCH
2
), 4.48
3
analyzed by competition with [ H]PDBU binding for the single
2
CH C), 3.80 (dd, J ) 12.1, 6.5
2
1
2
isozyme PK-CR essentially as described previously. The ID50
values were determined by least-squares fitting of the theo-
retical sigmoidal competition curve to the binding data. The
Hz, 1H, HOCHHC), 3.75 (dd, J ) 12.1, 6.5 Hz, 1 H, HOCHHC),
.66-2.99 (m, 2 H, H-4a,b), 2.48-2.65 (m, 0.4 H, (Z)-
CdCHCH ), 2.37 (t, J ) 6.5 Hz, 1 H, HOCH C), 2.05-2.21
m, 1.6 H, (E)-CdCHCH ), 1.05-1.45 (m, 12 H, C ),
) δ 170.21,
2
2
2
K
i
was calculated from the ID50 values according to the
(
2
6 3
H12CH
1
3
relationship
0
1
1
2
4
6 3 3
.82 (t, J ) 6.9 Hz, 3 H, C H12CH ); C NMR (CDCl
66.41, 146.49, 142.39, 139.95, 130.53, 129.16, 128.51, 127.97,
27.47, 126.05, 83.55, 66.34, 65.03, 32.00, 30.51, 30.15, 29.54,
9.52, 28.25, 22.80, 14.26; FAB MS (m/z, relative intensity)
51 (MH , 54%), 181 (100%). Anal. (C28
ID50
K )
i
1
+ L/Kd
+
34 5
H O ) C, H.
(
E)-[4-Non ylid en e-2-(h yd r oxym eth yl)-5-oxo-2-2,3-d ih y-
d r ofu r yl]m eth yl 2-P h en yla ceta te (7bf). Colorless solid, mp
4-57 °C (hexane); IR (neat) 3384 (OH), 1757 (CdO), 1730
3
where L is the concentration of free [ H]PDBU at the ID50 and
3
d
K is the dissociation constant for [ H]PDBU under the assay
conditions. Values represent the mean ( standard error
5
1
8
-
1
1
cm (CdO); H NMR (CDCl
CO CH C), 6.65-6.75 (m, 1 H, CdCHCH
Hz, 1 H, PhCH CO CHHC), 4.16 (d, J ) 11.9 Hz, 1 H, PhCH
CO CHHC), 3.61 (s, 2 H, PhCH CO CH
.5 Hz, 1 H, HOCHHC), 3.54 (dd, J ) 12.1, 6.5 Hz, 1 H,
HOCHHC), 2.69 (dd, J ) 17.0, 2.8 Hz, 1 H, H-4 ), 2.51 (dd, J
17.0, 2.8 Hz, 1H, H-4 ), 1.97-2.14 (m, 3 H, HOCH C and
), 1.08-1.49 (m, 12 H, C ), 0.85 (t, J ) 6.8
) δ 171.49, 169.92, 142.49,
3
) δ 7.08-7.38 (m, 5 H, PhCH
2
-
(three determinations).
2
2
2
), 4.26 (d, J ) 11.9
2
2
2
-
Su p p or tin g In for m a tion Ava ila ble: Elemental analysis
data. This material is available free of charge via the Internet
at http://pubs.acs.org.
2
2
2
2
C), 3.59 (dd, J ) 12.1,
6
a
)
b
2
Refer en ces
CdCHCH
Hz, 3 H, C
2
6 3
H12CH
3
1
3
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H
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4
(
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1.35, 32.01, 30.47, 29.88, 29.53, 28.25, 22.83, 14.28; FAB MS
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+
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(
23 32 5
C H O

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