An optimized protein kinase C activating diacylglycerol combining high binding affinity (Ki) with reduced lipophilicity (log P)

Article abstract of DOI:10.1021/jm010052e

A small, focused combinatorial library encompassing all possible permutations of acyl branched alkyl chains - small and large, saturated and unsaturated - was generated from the active diacylglycerol enantiomer (S-DAG) to help identify the analogue with t

Full text of DOI:10.1021/jm010052e

1892
J . Med. Chem. 2001, 44, 1892-1904
An Op tim ized P r otein Kin a se C Activa tin g Dia cylglycer ol Com bin in g High
Bin d in g Affin ity (Ki) w ith Red u ced Lip op h ilicity (log P )
Kassoum Nacro,†,‡ Dina M. Sigano,‡ Shunqi Yan,‡ Marc C. Nicklaus,‡ Larry L. Pearce,§ Nancy E. Lewin,§
Susan H. Garfield,# Peter M. Blumberg,§ and Victor E. Marquez*,‡
Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute at Frederick,
Frederick, Maryland 21702, and Laboratory of Cellular Carcinogenesis and Tumor Promotion and Laboratory of
Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health,
Bethesda, Maryland 20892
Received J anuary 31, 2001
A small, focused combinatorial library encompassing all possible permutations of acyl branched
alkyl chainsssmall and large, saturated and unsaturatedswas generated from the active
diacylglycerol enantiomer (S-DAG) to help identify the analogue with the highest binding
affinity (lowest Ki) for protein kinase C (PK-C) combined with the minimum lipophilicity (log
P). The selected ligand (3B) activated PK-C more effectively than sn-1,2-dioctanoylglycerol
(diC8) despite being 1.4 log units more hydrophilic. Compound 3B indeed represents the most
potent, hydrophilic DAG ligand to date. With the help of a green fluorescent protein (GFP)-
tagged PK-CR, 3B was able to translocate the full length protein to the membrane with an
optimal dose of 100 µM in CHO-K1 cells, while diC8 failed to achieve translocation even at
doses 3-fold higher. Molecular modeling of 3B into an empty C1b domain of PK-Cδ clearly
showed the existence of a preferred binding orientation. In addition, molecular dynamic
simulations suggest that binding discrimination could result from a favorable van der Waals
(VDW) interaction between the large, branched sn-1 acyl group of 3B and the aromatic rings
of Trp252 (PK-Cδ) or Tyr252 (PK-CR). The DAG analogue of 3B in which the acyl groups are
reversed (2C) showed a decrease in binding affinity reflecting the capacity of PK-C to effectively
discriminate between alternative orientations of the acyl chains.
In tr od u ction a n d Ba ck gr ou n d
Protein kinase C (PK-C) is a family of serine/threo-
nine-specific isozymes that are regulated by phospho-
rylation, by calcium, and by association with phospho-
lipids and 1,2-diacylglycerol (DAG).1-4 Typically, these
isozymes are cytosolic in the inactive state and, as part
of the activation process, translocate to the inner leaflet
of the cellular membrane.5,6 Classical (R, â, and γ) as
F igu r e 1. Hydrogen bonding interaction of DAG at the C1
domain of PK-Cδ in the alternative binding modes of sn-1 and
sn-2. Numbering of the DAG carbon backbone is shown.
well as novel (δ, ꢀ, η, and θ) PK-C isozymes become
activated as a result of the association of the cytosolic
enzyme with membranes containing acid phospho-
lipids.7,8 This association is strongly facilitated by the
lipophilic second messenger, DAG, which is generated
as a result of a stimulus-initiated activation of phos-
pholipase C.9 The binding of PK-C to the plasma
membrane is transient and regulated by its association
with DAG in the membrane.10
sible binding orientations when DAG was docked into
the empty C1b domain. These two apparently compa-
rable binding modes, sn-1 and sn-2, form an identical
network of hydrogen bonds with amino acids Thr242,
Leu251, and Gly253, as was observed with phorbol-13-
O-acetate in the crystal structure of the complex, and
are shown schematically in Figure 1. Based on the two
nonequivalent carbonyl moieties of DAG, the sn-1
binding mode is defined as that in which the sn-1
carbonyl and the hydroxyl group form hydrogen bonds
to the protein. In the alternative sn-2 binding mode, the
sn-2 carbonyl and the hydroxyl group are engaged in
hydrogen bonding to the protein. A prediction is that
these two binding orientations can direct the hydropho-
bic groups (R1 and R2) of the ligand in rather different
directions and that differences in binding affinities
should reflect preferences between these two binding
modes (Figure 1). Consistent with this prediction, we
sought to discriminate between these two binding
alternatives by using an array of different alkyl chains
DAG binds to the C1 domain of both the classical and
novel members of the PK-C family to activate their
downstream pathways.11 Modeling experiments using
the crystal coordinates of the C1b domain of PK-Cδ in
complex with phorbol-13-O-acetate12 revealed two pos-
* Corresponding author. Phone: 301-846-5954. Fax: 301-846-6033.
E-mail: marquezv@mail.nih.gov.
† Present address: Research Bldg., Suite W224A, Lombardi Cancer
Center, Georgetown University, 3970 Reservoir Rd., NW, Washington,
DC 20007.
‡ National Cancer Institute at Frederick.
§ Laboratory of Cellular Carcinogenesis and Tumor Promotion,
National Institutes of Health.
#
Laboratory of Experimental Carcinogenesis, National Institutes
of Health.
10.1021/jm010052e This article not subject to U.S. Copyright. Published 2001 by the American Chemical Society
Published on Web 05/12/2001

Protein Kinase C Activating Diacylglycerol
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1893
Sch em e 1
F igu r e 2. Common commercially available DAGs.
on DAG. An additional objective was also to select from
this array a DAG ligand with adequate solubility (low
lipophilicity) suitable for crystallographic and NMR
studies with the C1b domain of PK-Cδ. Finally, the
biological activity of the selected ligand was to be
compared to that of a prototypic DAG in terms of PK-C
activation and ability to translocate the enzyme from
the cytosol to the membrane.
It is well-known that the phorbol esters function as
potent and metabolically stable DAG surrogates. In fact,
the phorbol esters bind to PK-C with affinities that are
several orders of magnitude greater than those of the
commonly studied DAGs.13 Among the most commonly
used DAGs in PK-C studies are the commercially
available 1,2-dioctanoyl-sn-glycerol (diC8), 1-oleoyl-2-
acetyl-sn-glycerol (OAG), and 1,2-dioleoyl-sn-glycerol
(diolein) (Figure 2).
Since the calculated lipophilicities for PDBU (phorbol-
12,13-O-dibutyrate) and prostratin (12-deoxyphorbol-13-
O-acetate) expressed as log P values (see Table 2) are
lower than that of the least lipophilic, commercially
available DAG (diC8), we undertook the task of iden-
tifying a DAG candidate where a decrease in the log P
value would not compromise binding affinity. Hence, by
reducing lipophilicity, nonspecific binding interactions
between DAG and the membrane would be minimized,
and differences in Ki values would be expected to reflect
changes in specific interactions with the protein.
Resu lts a n d Discu ssion
Over the past few years, we have attempted to bridge
the affinity gap between phorbol esters and DAGs by
two independent approaches. One approach includes
reducing the entropic penalty associated with DAG
binding by constraining the glycerol backbone into DAG-
lactones.14 A second approach includes increasing the
bulk of the acyl groups by incorporating highly branched
alkyl chains capable of interacting more efficiently with
a group of conserved amino acids in the space between
the two â sheets of the C1b domain.14-16 Since branched
alkyl chains are less lipophilic than their linear coun-
terparts, we have decided to reduce the lipophilicity (log
P) of DAG further to levels that would allow the
detection of differences between the sn-1 and sn-2
binding modes by combining small and large, branched
alkyl chains.
While the role of the alkyl chain in DAGs has been
principally correlated with providing adequate lipophi-
licity to facilitate partitioning into a lipid-rich environ-
ment, the docking of DAG into the C1b domain of PK-
Cδsas in the case of the DAG-lactonessrevealed
potentially important hydrophobic contacts with the
protein which could extend into the protein-membrane
interface (vide infra). Thus, any increase in binding
affinity due to the branched alkyl chains in DAG would
probably result from a combination of two factors:
adequate membrane partitioning and hydrophobic con-
tacts with the protein. We anticipated that by reducing
the lipid dependency required for membrane distribu-
tion to an absolute minimum, the compounds would be
more effectively discriminated by the protein in terms
of the two possible binding modes (sn-1 versus sn-2).
For that reason, we designed a small combinatorial
library based on a uniform “core” S-DAG enantiomer
with variable R1 and R2 groups containing branched
alkyl chains, small and large, saturated and unsatur-
ated (Scheme 1, Table 1).
Syn th esis. A small combinatorial library of 16 DAGs
was prepared to help identify the optimal DAG for PK-C
combining the highest binding affinity with the lowest
log P value. In selecting the R1 and R2 alkyl groups,
consideration was also given to the possibility of reduc-
ing the entropy of binding by restricting the rotation
about the R,â-bond of the alkyl chain. Thus, analogous
alkyl chains were designed in which the R,â σ-bond was
replaced with a π-bond to analyze its effect on binding
affinity (see Scheme 1).
The target compounds were synthesized in three
simple steps starting from commercially available (R)-
(+)-3-benzyloxy-1,2-propanediol (Scheme 1). Monoacyl-
ation at the sn-1 position was followed by a second
acylation step at the sn-2 position, and debenzylation
gave the asymmetrically substituted target compounds.
In cases where the acyl groups at both the sn-1 and sn-2
positions were the same, the target compounds were
easily synthesized in just two steps. Hence, by using 2
equiv of the activated acid, both the sn-1 and sn-2
positions could be acylated in the same reaction. De-
benzylation then gave the symmetrically substituted
target compounds.
Biologica l Activity. The interaction of all of DAGs
with PK-C was assessed in terms of the ability of the
ligand to displace bound [20-3H]phorbol-12,13-dibu-
tyrate (PDBU) from a recombinant single isozyme (PKC-
R) in the presence of phosphatidylserine. The inhibition
curves obtained for all ligands were of the type expected
for competitive inhibition, and the ID50 values were
determined by fit of the data points to the theoretical
noncooperative competition curve. The Ki’s for inhibition
of binding (Table 1) were calculated from the ID50
values. The octanol/water partition coefficients (log P)
were calculated according to the fragment-based pro-
gram KOWWIN 1.63.17

1894 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Nacro et al.
Ta ble 1. Small Combinatorial DAG Library with Log P and Ki Value (in nM)
Ta ble 2. Commonly Used DAGs and Phorbol Esters with Log
P and Ki Value.
The compounds in quadrant I (1A, 1B, 2A, and 2B)
are all substituted with the smaller isopropyl and
isopropenyl chains. These compounds contain similar
size branched alkyl groups, and all combinations of
saturated and unsaturated chains are shown. The Ki
values of these compounds are in the micromolar range
and log P values are around 2. The poor binding affinity
shown by the compounds in quadrant I can be attributed
to a lack of adequate lipophilicity due to the small
isopropyl/isopropenyl groups. These groups may be too
small to meet a minimum required lipophilic threshold
and hence cannot properly partition the DAG into the
membrane.
compound
log Pa Ki ( SEM (nM)b
diolein (1,2-dioleoyl-sn-glycerol)
OAG (1-oleoyl-2-acetate-sn-glycerol)
diC8 (sn-1,2-dioctanoylglycerol)
PDBu (phorbol-12,13-dibutyrate)
prostratin
14.6
7.0
5.3
3.4
1.9
87.2 ( 1.6
50.4 ( 4.2
33.0 ( 1.46
0.2 ( 0.03
4.8 ( 0.425
a
b
Octanol/water partition coefficients (log P).17 Values repre-
sent the mean ( standard error (triplicate).
All of the possible combinations using large and small,
saturated and unsaturated, branched alkyl chains are
represented in Table 1 and can be visualized in four
quadrants. The sn-1 substituent remains constant across
the rows, while the sn-2 substituent remains constant
down the columns.
On the other extreme, the compounds in quadrant IV
(3C, 3D, 4C, and 4D) are all substituted with the larger
and equally bulky diisopropylethyl and diisopropyl-
ethenyl, branched alkyl chains. All combinations of

Protein Kinase C Activating Diacylglycerol
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1895
saturated and unsaturated chains are represented. The
more efficient binding reflected by the smaller Ki values
(compared to the compounds in quadrant I) can be
attributed to the increased lipophilicity (log P ) ca. 5.7
versus ca. 2) associated with the larger alkyl groups.
Thus, these compounds appear to have significant
hydrophobicity to properly concentrate at the membrane
and are able to bind to the protein with comparable high
binding affinities. The log P and Ki values closely match
the corresponding values for diC8 (see Table 2). These
results appear to indicate that when the branched alkyl
chains are of similar size, as they are within quadrants
I and IV, there is little or no discrimination in binding
mode preference. Perhaps this can be ascribed to the
inability of the PK-C:membrane complex to distinguish
between sn-1 and sn-2 alkyl groups of similar size. Thus,
there would not be a strong preference for one binding
mode over the other if DAG chooses a preferred binding
mode based on the nature of its alkyl groups.
F igu r e 3. Stimulation of PK-CR kinase activity as a function
of ligand concentration. Kinase activity of PK-CR was assayed
in the presence of the indicated concentrations of 3B (open
circles) or diC8 (filled squares). Results are from a single
experiment, with each point determined in triplicate. Two
additional experiments gave similar results.
When the sn-1 and sn-2 positions are unsymmetri-
cally substituted and therefore not of the same size, as
shown within quadrants II and III, and the compounds
contain fully saturated, or completely unsaturated alkyl
chains (1C, 2D, 3A, and 4B), the absolute position (sn-1
versus sn-2) of the larger and smaller branched alkyl
groups does not seem to have a significant effect either.
However, when one of the unsymmetrical alkyl chains
is unsaturated at either the sn-1 or sn-2 positions, the
level of discrimination is quite significant as demon-
strated along the diagonal (1D, 2C, 3B, and 4A). This
can be attributed to a reduction in the degrees of
freedom at either the sn-1 or sn-2 position induced by
the double bond compared to its saturated counterpart.
Such an effect, combined with the reduced lipophilicity
of the compounds, enhances the level of discrimination,
allowing the detection of preferential binding interac-
tions between the ligand and the protein.
(δ, ꢀ, η, and θ) PK-C isozymes is strictly conserved and
that DAG has similar effects on all these isoforms. The
only difference between the C1b domains of the R and
δ isozymes is that Trp252 in PK-Cδ is replaced by
Tyr252 in PK-CR. The Ki value for 3B against PK-Cδ
was in fact lower (20.8 nM ( 1.8), reflecting perhaps a
more efficient VDW interaction between the branched
sn-1 acyl group and the wider aromatic ring of Trp252.
Further evaluation of compound 3B showed that it
stimulated PK-CR enzymatic activity to a similar level
as did diC8 (Figure 3). In addition, its half-maximally
effective concentration for stimulation under these assay
conditions was slightly less, reflecting better potency
(3B, 9.5 ( 0.8 µM, n ) 3; diC8, 16.4 ( 2.9 µM, n ) 3).
The values in parentheses represent mean ( SEM for
triplicate experiments. These results indicate that 3B
functions as an effective activator of PK-C.
In intact cells, phorbol esters and other activators of
PK-C induce translocation from the cytosol to the
membrane and other cellular compartments. This trans-
location can be visualized in the intact cells with fusion
constructs between PK-C isoforms and a fluorescent tag,
green fluorescent protein.18 Using green fluorescent
protein (GFP)-tagged PK-C, the dynamics of PK-C
translocation can be monitored in living cells and in real
time. In Chinese hamster ovary (CHO-K1) cells trans-
fected with PK-CR-GFP, 3B induced translocation with
an optimal dose of 100 µM (Figure 4); less extensive
translocation was observed at 30 µM; and variable,
limited translocation was seen at 10 µM (data not
shown). For comparison, diC8 at 300 µM induced no
response, and translocation by 300 nM PDBu resembled
that by 100 µM 3B. These relative potencies for trans-
location generally agree with those obtained in the
ligand binding assay. The difference between diC8 and
3B is worth remarking since Oancea and Meyer have
reported that while diC8 effectively translocated the
GFP-tagged C1 domain of PK-Cγ to the plasma mem-
brane, it had no measurable effect on the full-length PK-
Cγ-GFP, even at doses 10-fold higher than those used
The increased efficiency of PK-C in discriminating
between DAG analogues along the 1D, 2C, 3B, 4A
diagonal could be attributed to either the ligand being
optimized for one binding mode over the other (Figure
1) or to the ability of PK-C to discriminate between
different alkyl chain orientations in a single, preferred
binding mode. Compound 3B represents the DAG with
the most efficient binding affinity; indeed, its log P value
was reduced 1.5 log units relative to diC8 (log P3B
)
3.8 versus log PdiC8 ) 5.3) without compromising binding
affinity (Ki(3B) ) 39.9 nM ( 1.9 versus Ki(diC8) ) 33.0 nM
( 1.46). In 3B the sn-1 alkyl group is large, while the
sn-2 alkyl group is small and conformationally re-
stricted. The reversal of these groups, as in 2C, results
in a ca. 4-fold decrease in binding affinity reflecting a
remarkable discriminating ability by PK-C. Computa-
tional docking and molecular dynamic simulations (vide
infra) suggest that binding discrimination could result
from a favorable van der Waals (VDW) interaction
between the large, branched sn-1 acyl group of 3B
and the aromatic ring of Trp252 (PKC-δ) or Tyr252
(PKC-R).
Since the C1b domain of PK-Cδ is used as a “general
receptor” for modeling, while actual binding affinities
are measured against the complete R isozyme, it is
important to remember that the C1b domain (amino
acids 242-253) among classical (R, â, and γ) and novel

1896 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Nacro et al.
Ta ble 3. Percent of Docking Simulations in sn-1 and sn-2
Binding Modes53
ligand
sn-1
sn-2
other
1A
1B
1C
1D
2A
2B
2C
2D
3A
3B
3C
3D
4A
4B
4C
4D
42
90
45
73
82
57
49
54
89
93
63
61
83
91
56
42
6
8
52
2
10
16
6
19
23
9
10
7
17
22
9
8
12
17
45a
11a
12a
34a
38a
39a
1
0
20a
17a
8a
1
32a
41a
a
Very weak sn-2 binding with
a distance between atoms
involved in hydrogen bonding of 2.70-3.40 Å.
F igu r e 4. Translocation of PK-CR-GFP in response to 3B and
other ligands. PK-CR-GFP was expressed in CHO-K1 cells.
Cells were treated as indicated with 3B, with PDBU, or with
diC8, and the localization of PK-CR-GFP was determined by
confocal microscopy as a function of time. Images shown are
of cells before treatment or after treatment for the times
indicated. At least three experiments were performed and
images shown are representative.
in this work.10 They explained their observation on the
basis of the lower affinity of PK-C for DAG. However,
despite the different PK-C isozyme involved, our results
would suggest that this observation is only valid for
diC8 since 3B is a DAG analogue capable of trans-
locating the full length PK-CR-GFP.
F igu r e 5. Docking results showing hydrogen-bonding interac-
tions of 3B with the C1b domain of PK-Cδ in the sn-1 binding
mode.
binding modes for each ligand are shown in Table 3.
The results suggest that the sn-1 mode is preferred. In
terms of the hydrogen-bonding network, the sn-2 bind-
ing mode appears weaker since most distances involved
in hydrogen bonding were found to be greater than 2.70
Å. Figure 5 depicts the docking results obtained with
3B displaying optimal hydrogen bond distances.
F lexible Dock in g. The sn-1 binding mode for the
DAGs was also found to be energetically favorable by
flexible docking with the Merck Molecular Force Field
(MMFF) available in Macromodel.20 The low-mode
(LMOD) algorithm implemented in Macromodel was
applied for the conformational search. Each ligand,
together with six residues of the C1b domain (Ser240,
Thr242, Leu251, Trp252, Gly253, and Leu254), was
allowed to move freely during the simulation, while the
rest of the residues were kept fixed. Each ligand was
subjected to explicit translation/rotation in the binding
site via the MOLS command in BatchMin. The simul-
Com p u ta tion a l Mod elin g
Au tod ock Stu d y. Docking studies were performed
using version 2.4 of AutoDock.19 The structure of the
empty C1b domain of PK-Cδ, grid setup, and param-
eters necessary for AutoDock and cluster analysis were
the same as previously reported.15 Each ligand was
subjected to 1000 steps of a Monte Carlo (MC) confor-
mational search using the program Macromodel 7.0.20
The minimum conformation reached by MC was then
fully optimized at the semiempirical level using the AM1
Hamiltonian within the program InsightII,21 and the
charges necessary for AutoDock were derived at this
level. The docking simulation was repeated 100 times
for each ligand starting with a randomly different
conformation each time. The number of sn-1 and sn-2

Protein Kinase C Activating Diacylglycerol
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1897
Ta ble 4. Electrostatic (elec), van der Waals (VDW), and Total
Energies (kcal/mol) Calculated from MD Simulations for
Ligands 2C, 3B, and 3C in the Alternative sn-1 and sn-2
Binding Modesa
2C
3B
3C
total
elec
sn-1
sn-2
sn-1
sn-2
sn-1
sn-2
-38.12
-36.48
-13.14
-12.57
-24.98
-23.92
-41.54
-34.83
-15.23
-12.72
-26.25
-22.11
-41.63
-36.73
-15.62
-11.73
-25.90
-25.00
VDW
a
The simulations were performed at 298 K for 37-40 ns.
taneous translation and rotation of the ligand randomly
varied between 0.1 and 0.5 Å and 0-90°, respectively.
A 5000-step search was performed for the flexible
docking of each of the 16 DAGs listed in Table 1. For
DAGs 3B and 2C, a lengthy 25000-step search led to
the same global minimum as the 5000-step search. For
each ligand, more than 100 structures were found
within a 30 kJ energy range, and in every case, more
than 98% of the structures appeared with the ligand
bound in the sn-1 binding mode at much lower energies
than in the alternative sn-2 binding mode.
Molecu la r Dyn a m ic Sim u la tion s. Molecular dy-
namics (MD) simulations of approximately 40 ns using
the program CHARMM28A122 were performed for 3B,
2C, and the symmetrically branched 3C for both sn-1
and sn-2 binding modes inside the C1b domain of PK-
Cδ. The CHARMM force field, parametrized with the
all-hydrogen PARAM2723 parameter set, provided the
potential energies. A dielectric constant of 2.0 was used
to mimic the lipid environment of a transmembrane
protein. No explicit water (solvent) molecules were
added. The cutoff distance for nonbonded interactions
was set to 14.0 Å. The cutoff distance at which the
switching function eliminates all contributions from an
interactive pair in calculating energies was set to 12.0
Å. A SHAKE algorithm was applied to constrain cova-
lent bonds to hydrogens to 10-8 Å. Molecular dynamic
simulations were carried out in parallel with 8 CPUs
of an SGI Origin computer by heating for 80 ps and then
equilibrating for 80 ps more at 298 K. A time step of 1
fs was used and the simulations were performed at 298
K for ca. 37-40 ns.
Zinc finger geometries ((Cys)3-Zn-His) of PK-C were
calculated at the Density Functional Theory (DFT) level
using the B3LYP/6-311+G** basis set in Gaussian.24
Ligands 3B and 2C were fully optimized with the level
of B3LYP/6-31G* basis set in the gas phase, and the
electrostatic potential-derived (ESP) charges were cal-
culated from these geometries with either J aguar 3.5
or 4.0 versions.20 The corresponding parameters for zinc
fingers and the ligands for MD simulations were derived
from these optimized structures. The interaction ener-
gies between the protein and the ligand were calculated
using the CHARMM command INTERACT. They are
shown in Table 4 in kcal/mol.
F igu r e 6. Close approach between Trp252 and the branched
sn-1 acyl chain of 3B observed during MD simulation.
the order 3C < 3B < 2C, which is in good agreement
with the measured Ki values. The small energy differ-
ence between 3C and 3B correlates well with the similar
experimentally measured Ki values (28.6 nM and 39.9nM,
respectively, Table 1). The lower Ki for 3C relative to
3B is probably the result of a more effective membrane
partitioning due to the higher lipophilicity of the former;
however, the nearly 100-fold difference in calculated
lipophilicity (∆log P ca. 2) would not seem to be
commensurate with the relative small difference in
potency. In terms of an ideal relationship of Ki versus
log P, 3B remains the most potent DAG analogue.
Ligand 3B is also the most discriminating in terms of
the binding energies for sn-1 versus sn-2 modes (com-
pare 2C versus 3B, Table 4). Among other factors that
could account for this phenomenon is a favorable VDW
interaction between the large, branched sn-1 acyl chain
and the flexible aromatic ring of Trp252 that was
observed during the MD simulation with the C1b
domain of PK-Cδ (Figure 6). Relative to its position in
the crystal structure (see Figure 5), Trp252 moves closer
to the branched sn-1 acyl chain of 3B to achieve a
favorable hydrophobic contact during the MD simula-
tion. As mentioned before, a similar favorable contact
in the C1b domain of PK-CR could also occur between
the branched sn-1 acyl chain of 3B and Tyr252.
It is remarkable how all the structural factors must
play a concerted role in determining affinity. Indeed,
all the compounds in the same quadrant as 3B have
either a similar or equivalent branched acyl chain as
3B, and yet they show weaker binding affinities. It is
interesting to remark that according to the docking
results (Table 3), compound 3B has the highest percent
of occupancy in the sn-1 binding mode. This means that
a combination of factors, including hydrogen bonding,
VDW interactions, and proper membrane partitioning,
appears to have been optimized in 3B.
For each representative ligand studied (3B, 2C, and
3C), the sn-1 binding mode appears to be energetically
more favorable than the sn-2 binding mode. Even for
the weakest ligand of this triad (2C), both electrostatic
and VDW interactions favor the sn-1 binding mode. The
same preference for the sn-1 mode was also observed
for the better ligands, 3B and 3C, and the calculated
energies in the sn-1 mode for the three ligands follow
Obser va tion s a n d Con clu sion s
By employing a combination of nonequivalent, large
and small, branched alkyl chains as acyl groups of DAG,
we have been able to decrease lipophilicity up to 1.5 log
units lower than that of diC8 without sacrificing po-
tency. However, a log P value lower than ca. 2 appears
to be below the minimum lipophilic threshold required

1898 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Nacro et al.
A1 represents basal activity, A2 represents maximal stimulated
activity, X represents the concentration of activator, and X0
represents the half-maximally effective dose.
for proper partitioning of DAG into the membrane.
When both branched alkyl substituents are of the same
size, there appears to be little discrimination in binding.
On the other hand, when the alkyl chains are not of the
same size and the compound is neither completely
saturated nor unsaturated, the best combination is for
the sn-1 substituent to be the larger, saturated branched
alkyl group, and for the sn-2 substituent to be the
smaller, unsaturated branched alkyl group.
Cell Cu ltu r e. CHO-K1 cells (CCL 61) were obtained from
the American Type Culture Collection (Manassas, VA). The
cells were cultured at 37 °C in Dulbecco’s modified Eagle’s
medium supplemented with 4500 mg/L glucose, 4 mM L-
glutamine, and 10% fetal bovine serum (Life Technologies, Inc.
Rockville, MD) in a humidified atmosphere containing 5% CO2.
The cells were subcultured twice a week and reseeded at 106
cells/T-75 tissue culture flask.
From these studies we found compound 3B to be the
most potent DAG analogue in Table 1 with the lowest
log P value. The combination of asymmetric alkyl
branches and the specific location of the double bond in
the small alkyl branch allow this molecule to discrimi-
nate effectively between two alternative binding modes
while interacting with PK-C. Based on the overwhelm-
ing preference for the sn-1 binding mode demonstrated
by three different modeling approaches, we conclude
that DAGs prefers to bind in this modality and that the
contrast between 3B and its transposed analogue (2C)
reflects the ability of PK-C to discriminate between
different orientations of the two alkyl branches. Com-
pound 2C could also achieve the same disposition for
the two alkyl branches as in 3B but at the higher price
of binding in the less favorable sn-2 binding mode.
Therefore, when mixed binding modes are possible they
are expected to produce lower binding affinities. We
conclude that compound 3B appears to have the struc-
tural attributes to overwhelmingly prefer the sn-1
binding mode (see Table 3).
Compound 3B represents the most potent, hydrophilic
DAG ligand to date. It activates PK-CR ca. 2-fold better
than diC8, and it is capable of translocating the full
length protein efficiently to the membrane. This com-
pound represents an ideal candidate for structural
analysis in a complex with a C1 domain. The realization
of this objective and a continuing comprehensive study
of the biology of 3B are issues of high priority in our
laboratory.
Exp r ession of P KCR-GF P F u sion P r otein . The cDNA
encoding bovine PK-CR fused to a variant of green fluorescent
protein (GFP) was constructed as reported for PK-Cδ GFP.26
CHO-K1 cells were grown on 40 mm round coverslips
(Bioptechs, Inc., Butler, PA) at a seeding density of 2.5 × 105
cells per dish. Transient transfection was conducted using
LipofectAMINE PLUS (Life Technologies, Inc. Rockville, MD)
according to the manufacturer’s protocol. The fluorescence
became detectable 24 h after transfection, and all experiments
were performed 48 h after transfection. Before imaging of the
cells, the medium was aspirated from the cells and replaced
with DMEM plus 10% fetal bovine serum without phenol red
(Life Technologies, Rockville, MD). All PK-C activators were
diluted in the same medium with the final concentration of
solvent (DMSO) always less than 0.1%. The cells were perfused
up to 1 h with specified concentrations of the activators phorbol
12,13-dibutyrate (LC Services, Woburn, MO), 1,2-dioctanoyl-
glycerol (Avanti Polar Lipids, Alabaster, AL), and 3B.
Con foca l Im a gin g. Confocal fluorescent images were col-
lected with a Bio-Rad MRC 1024 confocal scan head (Bio-Rad,
Hercules, CA) mounted on a Nikon Optiphot microscope with
a
×60 planapochromat lens. Excitation at 488 nm was
provided by a krypton-argon gas laser with a 522/32 emission
filter for green fluorescence. For live cell imaging, a Biotechs
Focht Chamber System (Bioptechs, Butler, PA) was inverted
and attached to the microscope stage with a custom stage
adapter. The cells plated on the 40-mm round coverslip were
enclosed in the chamber and connected to a temperature
controller set at 37 °C, and medium was perfused through the
chamber with a Lambda microperfusion pump. Sequential
images of the same cells were collected at one minute time
points using LaserSharp software.
Gen er a l P r oced u r es. All chemical reagents were com-
mercially available. Melting points were determined on a
MelTemp II apparatus, Laboratory Devices, USA, and are
uncorrected. Silica gel chromatography was performed on silica
gel 60, 230-400 mesh (E. Merck). 1H and 13C NMR spectra
were recorded on a Bruker AC-250 instrument at 250 and 62.9
MHz, respectively. Spectra are referenced to the solvent in
which they were run (7.24 ppm for CDCl3). Infrared spectra
were recorded on a Perkin-Elmer 1600 series FT-IR. Optical
rotations were recorded on a Perkin-Elmer model 241 pola-
rimeter at room temperature with a path length ) 1 dm.
Positive ion fast-atom bombardment mass spectra (FAB-MS)
were obtained on a VG 7070E mass spectrometer at an
accelerating voltage of 6 kV and a resolution of 2000. Glycerol
was used as the sample matrix and ionization was effected by
a beam of xenon atoms. Elemental analyses were performed
by Atlantic Microlab, Inc., Atlanta, GA.
Exp er im en ta l Section
An a lysis of In h ibition of [3H]P DBU Bin d in g by Non -
r a d ioa ctive Liga n d s. Enzyme-ligand interactions were
analyzed by competition with [3H]PDBU binding essentially
as described previously with the single isozyme PK-CR.15
Kin a se Assa y. Protein kinase C activity was assayed by
measuring the incorporation of 33P from [γ-33P 3000 Ci/mmol]
ATP (ICN, Costa Mesa, CA) into PK-CR pseudosubstrate
peptide [ser25] PKC (Life Technologies, Rockville, MD) in the
presence of PK-CR, which was partially purified as described.25
The assay buffer contained 20 mM Tris-C1 pH 7.5, 7.5 mM
magnesium acetate, 0.1 mM CaC12, 0.25 mg/mL bovine serum
albumin (fraction V) (Sigma, St. Louis, MO), 10 µg/mL phos-
phatidylserine, and 90 µg/mL phosphatidylcholine (Avanti
Polar Lipids, Alabaster, AL) and activators phorbol 12-
myristate,13-acetate (LC Laboratories, Woburn, MA), 1,2-
dioctanoylglycerol (Avanti Polar Lipids, Alabaster, AL), or 3B.
The reaction was started with the addition of ATP (50 µM final
concentration, 1 µCi) and 300 nM pseudosubstrate peptide and
incubated at 30 °C for 10 min. The reaction was stopped by
chilling on ice. A 25 µL aliquot was spotted onto DE81 ion
exchange chromatography paper (Whatman Ltd., Maidstone,
England). The paper was washed three times in 0.5% phos-
phoric acid. The bound radioactivity was measured in a
scintillation counter. In each experiment, each ligand concen-
tration was assayed in triplicate. The dose response curve was
fitted to the relationship y ) [(A1-A2)/(1+(X/X0)P)] + A2, where
4-Met h yl-3-(isop r op yl)p en t -2-en oyl Ch lor id e. Under
argon, oxalyl chloride (1.8 mL) and pyridine (52 µL) were
added dropwise to a 0 °C solution of 4-methyl-3-(isopropyl)-
pent-2-enoic acid (2.0 g) in Et2O (26 mL). The solution was
allowed to reach room temperature and stirred 44 h. The
reaction mixture was then filtered through Celite, and the
filtrate was concentrated to give the acid chloride as a thin
pale yellow oil (2.03 g, 91%). This compound was used without
further purification. IR (neat) 2963 (CH), 1774 (CdO), 1600
1
(CdC), 996 (CCl) cm-1; H NMR (250 MHz, C6D6) δ 0.66 and
0.73 (d, J ) 6.8 Hz, 12 H, ClC(O)CHdC(CHMe2)2), 2.06 and
3.65 (heptet, J ) 6.8 Hz, 2 H, ClC(O)CHdC(CHMe2)2), 5.92
(s, 1 H, ClC(O)CHdC(CHMe2)2); 13C NMR (62.5 MHz, C6D6) δ
19.39, 23.02, 29.55, 30.99, 119.66, 163.23, 182.36.

Protein Kinase C Activating Diacylglycerol
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1899
Gen er a l P r oced u r e for th e Syn th esis of 1-Su bstitu ted -
3-ben zyl Glycer ols. Under argon, dibutyltinoxide (2.0 equiv)
was added to a stirring solution of (R)-(+)-3-benzyloxy-1,2-
propanediol (1.0 equiv) in toluene (15 mL/mmol) containing 4
Å molecular sieves. The mixture was then heated to reflux for
3 h, after which it was cooled to 0 °C, and the acyl chloride
(1.1 equiv) was added dropwise. The ice bath was removed,
and the reaction was stirred for 90 min. The reaction was
quenched with pH 7 buffer solution and diluted with CHCl3.
The mixture was washed with brine, extracted twice with
CHCl3, dried over MgSO4, and concentrated. Purification by
silica gel column chromatography (2:1 hexane:ether) gives the
named product as a colorless oil.
1-acyl-3-benzyl-sn-glycerol (1.0 equiv) in CH2Cl2 (10 mL/mmol).
The mixture was then cooled to 0 °C, and acyl chloride (4.0
equiv) was added dropwise. The ice bath was removed, and
the reaction was warmed to room temperature and was
monitored by TLC. The crude reaction was then filtered
through a plug of silica gel and concentrated. Further purifica-
tion by silica gel column chromatography (5% ether/hexane)
gives the named product as a colorless oil.
1-(3-Meth ylbu tan oyl)-2-(3-m eth ylbu t-2-en oyl)-3-ben zyl-
sn -glycer ol. (R)-(+)-(1-(3-methylbutanoyl)-3-benzyl-sn-glyc-
erol and 3-methylbut-2-enoyl chloride were reacted to give the
title compound in 32% yield. [R]20 ) +11.34 (c 0.67, CHCl3);
D
IR (neat) 2959 (CH), 1735 (CdO), 1721 (CdO), 1654 (CdC)
1
cm-1; H NMR (250 MHz, CDCl3) δ 1.01 (d, J ) 6.4 Hz, 6 H,
1-(3-Met h ylb u t a n oyl)-3-b en zyl-sn -glycer ol. (R)-(+)-3-
benzyloxy-1,2-propanediol and 3-methylbutanoyl chloride were
reacted to give the title compound in 83% yield. [R]20D ) +2.55
(c 2.9, CHCl3); IR (neat) 3454 (OH), 2953 (CH), 1730 (CdO)
C(O)CH2CHMe2), 1.98 (d, J ) 1.2 Hz, 3 H, C(O)CHdCMe2),
2.05-2.35 (m, 3 H, C(O)CH2CHMe2 containing δ 2.25 (d, J )
1.2 Hz, 3 H, C(O)CHdCMe2)), 3.70 (d, J ) 5.4 Hz, 2 H, OCH2-
CH(O-)CH2O), 4.25-4.50 (m, 2 H, OCH2CH(O-)CH2O), 4.63
(AB quartet, J ) 9.3 Hz, 2 H, PhCH2O), 5.30-5.38 (m, 1 H,
OCH2CH(O-)CH2O), 5.78 (t, J ) 1.2 Hz, 1H, C(O)CHdCMe2),
7.39 (s, 5 H, PhCH2O); 13C NMR (62.5 MHz, CDCl3) δ 20.26,
22.31, 25.57, 27.41, 43.13, 62.60, 68.23, 69.12, 73.21, 115.53,
127.50, 127.58, 128.25, 137.64, 157.66, 165.46, 172.53. FAB-
MS (m/z, relative intensity): 349 (MH+, 36), 91 (71), 83 (100).
HRMS (FAB) calcd for C20H29O5 (MH+): 349.2015, found
349.2021.
1
cm-1; H NMR (250 MHz, CDCl3) δ 1.03 (d, J ) 6.6 Hz, 6 H,
C(O)CH2CHMe2), 2.12-2.25 (m, 1 H, C(O)CH2CHMe2), 2.30
(d, J ) 6.6 Hz, 2 H, C(O)CH2CHMe2), 2.61 (d, J ) 4.9 Hz, 1 H,
OCH2CH(OH)CH2O), 3.61 (2 overlapping AB quartets, 2 H,
OCH2CH(OH)CH2O), 4.01-4.18 (m, 1 H, OCH2CH(OH)CH2O),
4.23-4.27 (m, 2 H, OCH2CH(OH)CH2O), 4.64 (s, 2 H, PhCH2O),
7.42 (s, 5 H, PhCH2O); 13C NMR (62.5, CDCl3) δ 22.32, 25.59,
43.12, 65.16, 68.73, 70.87, 73.36, 127.61, 127.69, 128.30,
137.56, 173.00,; FAB-MS (m/z, rel intensity) 267 (MH+, 50),
91 (100). Anal. (C15H22O4) C, H.
1-(3-Me t h ylb u t a n oyl)-2-(3-isop r op yl-4-m e t h ylp e n -
ta n oyl)-3-ben zyl-sn -glycer ol. According to the general pro-
cedure, 1-(3-methylbutanoyl)-3-benzyl-sn-glycerol and 3-iso-
propyl-4-methylpentanoyl chloride were reacted to give the
1-(3-Meth ylbu t-2-en oyl)-3-ben zyl-sn -glycer ol. (R)-(+)-3-
benzyloxy-1,2-propanediol and 3-methylbut-2-enoyl chloride
were reacted to give the title compound in 85% yield. [R]20
)
D
title compound in 56% yield. [R]20 ) +11.9 (c 2.7, CHCl3); IR
+5.21 (c 1.84, CHCl3); IR (neat) 3459 (OH), 3031 and 2913
D
(CH), 1715 (CdO), 1652 (CdC) cm-1 1H NMR (250 MHz,
;
(neat) 2963 (CH), 2871 (CH), 1739 (CdO) cm-1; 1H NMR (250
MHz, CDCl3) δ 0.89 (dd, J ) 6.6, 1.5 Hz, 6 H, C(O)CH2CH-
(CHMe2)2), 0.99 (d, J ) 6.6 Hz, 6 H, C(O)CH2CH(CHMe2)2),
1.02 (d, J ) 6.3 Hz, 6 H, C(O)CH2CHMe2), 1.66-1.91(m, 3 H,
C(O)CH2CH(CHMe2)2), 2.09-2.33 (m, 1 H, C(O)CH2CHMe2
containing δ 2.25 (d, J ) 6.1 Hz, 2 H, C(O)CH2CH(CHMe2)2)
and δ 2.28 (d, J ) 5.6 Hz, 2 H, C(O)CH2CHMe2)), 3.68 (d, J )
4.9 Hz, 2 H, OCH2CH(O-)CH2O), 4.28 (dd, J ) 11.8, 6.5 Hz,
1 H, OCH2CH(O-)CH2O), 4.44 (dd, J ) 12.0, 3.7 Hz, 1 H,
OCH2CH(O-)CH2O), 4.62 (AB quartet, J ) 12.1 Hz, 2 H,
PhCH2O), 5.28-5.36 (m, 1 H, OCH2CH(O-)CH2O), 7.40 (s, 5
H, PhCH2O); 13C NMR (62.5 MHz, CDCl3) δ 18.67, 18.70, 21.28,
21.30, 22.31, 25.53, 29.29, 29.35, 32.94, 43.07, 46.61, 62.54,
68.15, 69.96, 73.17, 127.46, 127.58, 128.23, 137.56, 172.38,
173.86; FAB-MS (m/z, relative intensity): 407 (MH+, 9), 91
(100). Anal. (C24H38O5) C, H.
CDCl3) δ 1.98 and 2.25 (s, 6 H, C(O)CHdCMe2), 2.60 (br s, 1
H, OCH2CH(OH)CH2O), 3.62 (2 overlapping AB quartets, 2
H, OCH2CH(OH)CH2O), 4.10-4.20 (m, 1 H, OCH2CH(OH)-
CH2O), 4.20-4.33 (m, 2 H, CH2CH(OH)CH2O), 4.65 (s, 2 H,
PhCH2O), 5.79 (s, 1 H, C(O)CHdCMe2), 7.42 (s, 5 H, PhCH2O);
13C NMR (62.5 MHz, CDCl3) δ 20.23, 27.36, 64.66, 68.88, 70.99,
73.35, 115.44, 127.59, 127.64, 128.29, 137.64, 157.46, 166.41.
FAB-MS (m/z, rel intensity): 265 (MH+, 53), 91 (78), 83 (100).
Anal. (C15H20O4‚0.16H2O) C, H.
1-(3-Isop r op yl-4-m et h ylp en t a n oyl)-3-b en zyl-sn -glyc-
er ol. (R)-(+)-3-benzyloxy-1,2-propanediol and 4-methyl-3-(iso-
propyl)pentanoyl chloride were reacted to give the title com-
pound in 87% yield. [R]20 ) +1.48 (c 2.16, CHCl3); IR (neat)
D
3452 (OH), 2956 (CH), 1733 (CdO) cm-1; 1H NMR (250 MHz,
CDCl3) δ 0.89 and 0.98 (d, J ) 6.8 Hz, 12 H, C(O)CH2CH-
(CHMe)2), 1.63-1.93 (m, 3 H, C(O)CH2CH(CHMe)2), 2.29 (d,
J ) 5.9 Hz, 2 H, C(O)CH2CH(CHMe)2), 3.61 (2 overlapping
AB quartets, 2 H, OCH2CH(OH)CH2O), 4.05-4.31 (m, 3 H,
OCH2CH(OH)CH2O), 4.64 (s, 2 H, PhCH2O), 7.42 (s, 5 H,
PhCH2O); 13C NMR (62.5 MHz, CDCl3) δ 18.70, 21.31, 29.33,
32.79, 46.69, 65.35, 68.77, 70.90, 73.38, 127.61, 127.70, 128.31,
137.52, 174.80. FAB-MS (m/z, rel intensity): 323 (MH+, 29),
91 (100). Anal. (C19H30O4) C, H.
1-(3-Met h ylb u t a n oyl)-2-(3-isop r op yl-4-m et h ylp en t -2-
en oyl)-3-ben zyl-sn -glycer ol. According to the general pro-
cedure, 1-(3-methylbutanoyl)-3-benzyl-sn-glycerol and 3-iso-
propyl-4-methylpentanoyl chloride were reacted to give the
title compound in 77% yield. [R]20 ) +7.23 (c 4.73, CHCl3);
D
IR (neat) 2963 (CH), 2871 (CH), 1740 (CdO), 1719 (CdO), 1637
(CdC) cm-1; 1H NMR (250 MHz, CDCl3) δ 1.01 (d, J ) 6.6 Hz,
6 H, C(O)CH2CHMe2), 1.13 (d, J ) 6.9 Hz, 6 H, C(O)CHd
C(CHMe2)2), 1.15 (d, J ) 6.3 Hz, 6 H, C(O)CHdC(CHMe2)2),
2.08-2.33 (m, 3 H, C(O)CH2CHMe2), 2.63 (heptet, J ) 6.7 Hz,
1 H, C(O)CHdC(CHMe2)2), 3.71 (d, J ) 5.1, 2 H, OCH2CH-
(O-)CH2O), 4.12 (heptet, J ) 6.8 Hz, 1 H, C(O)CHd
C(CHMe2)2), 4.25-4.50 (m, 2 H, OCH2CH(O-)CH2O), 4.62 (AB
quartet, J ) 9.7 Hz, 2 H, PhCH2O), 5.29-5.40 (m, 1 H,
OCH2CH(O-)CH2O), 5.74 (s, 1 H, C(O)CHdC(CHMe2)2), 7.38
(s, 5 H, PhCH2O-); 13C NMR (62.5 MHz, CDCl3) δ 20.18, 20.22,
22.30, 24.08, 25.57, 29.00, 29.74, 43.14, 62.57, 68.21, 69.06,
73.16, 112.25, 127.45, 127.48, 127.56, 128.23, 137.63, 165.84,
172.44, 176.98; FAB-MS (m/z, relative intensity): 405 (MH+,
22), 139 (100), 91 (91). Anal. (C24H36O5‚0.2 H2O) C, H.
1-[4-Meth yl-3-(isopr opyl)pen t-2-en oyl]-3-ben zyl-sn -glyc-
er ol. (R)-(+)-3-benzyloxy-1,2-propanediol and 4-methyl-3-(iso-
propyl)pent-2-enoyl chloride were reacted to give the title
compound in 58% yield. [R]20D ) +3.88 (c 3.07 CHCl3); IR (neat)
3415 (OH), 2964 (CH), 2871 (CH), 1714 (CdO), 1637 (CdC)
1
cm-1; H NMR (250 MHz, CDCl3) δ 1.14 (d, J ) 6.8 Hz, 6 H,
C(O)CHdC(CHMe2)2), 1.16 (d, J ) 6.6 Hz, 6 H, C(O)CHd
C(CHMe2)2), 2.59 (br s, 1 H, OCH2CH(OH)CH2O), 2.64 (heptet,
J ) 6.8 Hz, 2 H, C(O)CHdC(CHMe2)2), 3.63 (2 overlapping
AB quartets, 2 H, OCH2CH(OH)CH2O), 4.07-4.33 (m, 3 H,
OCH2CH(OH)CH2O), 4.66 (s, 2 H, PhCH2O), 5.76 (s, 1 H, C(O)-
CHdC(CHMe2)2), 7.42 (s, 5 H, PhCH2O); 13C NMR (62.5 MHz,
CDCl3) δ 20.22, 24.11, 28.98, 29.70, 64.76, 68.89, 71.01, 73.33,
112.15, 127.57, 127.64, 128.28, 137.65, 166.79, 176.88. FAB-
MS (m/z, rel intensity) 321 (MH+, 54), 139 (100), 91 (57). Anal.
(C19H28O4) C, H.
1-(3-Meth ylbu t-2-en oyl)-2-(3-m eth ylbu tan oyl)-3-ben zyl-
sn -glycer ol. According to the general procedure, 1-(3-meth-
ylbut-2-enoyl)-3-benzyl-sn-glycerol and isovaleryl chloride were
reacted to give the title compound in 80% yield. [R]20D ) +9.29
(c 5.31, CHCl3); IR (neat) 2959 (CH), 2871 (CH), 1736(CdO),
Gen er a l P r oced u r e for th e Syn th esis of 1,2-Disu bsti-
tu ted -3-ben zyl Glycer ols. Under argon, pyridine (4.0 equiv)
and DMAP (0.1 equiv) were added to a stirring solution of
1
1723 (CdO), 1654 (CdC) cm-1; H NMR (250 MHz, CDCl3) δ
1.03 (d, J ) 6.4 Hz, 6 H, C(O)CH2CHMe2), 1.96 (s, 3 H, C(O)-

1900 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Nacro et al.
CHdCMe2), 2.12-2.30 (m, 3 H, C(O)CH2CHMe2 containing δ
2.23 (s, 3 H, C(O)CHdCMe2)), 3.69 (d, J ) 5.1 Hz, 2 H, OCH2-
CH(O-)CH2O), 4.36 (2 overlapping AB quartets, 2 H, OCH2-
CH(O-)CH2O), 4.61 (AB quartet, J ) 7.3 Hz, 2 H, PhCH2O),
5.32-5.40 (m, 1 H, OCH2CH(O-)CH2O), 5.72 (s, 1 H, C(O)-
CHdCMe2), 7.40 (s, 5 H, PhCH2O); 13C NMR (62.5 MHz,
CDCl3) δ 20.18, 22.26, 25.67, 27.33, 43.34, 61.92, 68.33, 70.01,
73.15, 115.38, 127.46, 127.56, 128.23, 137.62, 157.26, 165.84,
172.13; FAB-MS (m/z, relative intensity): 349 (MH+, 27), 91
(94), 83 (100). Anal. (C20H28O5) C, H.
CH(O-)CH2O), 4.26 (dd, J ) 12.0, 6.6 Hz, 1 H, OCH2CH(O-)-
CH2O), 4.43 (dd, J ) 11.8, 3.8 Hz, 1 H, OCH2CH(O-)CH2O),
4.62 (AB quartet, J ) 7.1 Hz, 2 H, PhCH2O), 5.30-5.38 (m, 1
H, OCH2CH(O-)CH2O), 7.34-7.43 (m, 5 H, PhCH2O); 13C
NMR (62.5 MHz, CDCl3) δ 18.68, 21.29, 22.29, 25.67, 29.30,
32.75, 43.32, 46.66, 62.75, 68.25, 69.87, 73.21, 127.49, 127.62,
128.27, 137.53, 172.16, 174.26; FABMS (m/z, relative inten-
sity): 407 (MH+, 11), 91 (99), 57 (100). Anal. (C24H38O5) C, H.
1-(3-Isop r op yl-4-m et h ylp en t a n oyl)-2-(3-m et h ylb u t -2-
en oyl)-3-ben zyl-sn -glycer ol. According to the general pro-
cedure, 1-(3-isopropyl-4-methylpentanoyl)-3-benzyl-sn-glycerol
and 3,3-dimethylacryloyl chloride were reacted to give the title
compound contaminated by (CH3)2CdCHCOOH (ca. 1:1) in
74% yield. This product was used directly in the next step
without further purification.
1,2-Di-(3-m eth ylbu t-2-en oyl)-3-ben zyl-sn -glycer ol. Ac-
cording to the general procedure, (R)-(+)-3-benzyloxy-1,2-
propanediol and 3,3-dimethylacryloyl chloride (3 equiv) were
reacted to give the title compound in quantitative yield. [R]20
D
) +3.59 (c 1.95, CHCl3); IR (neat) 2914 (CH), 1721 (CdO),
1
1652 (CdC) cm-1; H NMR (250 MHz, CDCl3) δ 1.97 (d, J )
1,2-Di-(3-isop r op yl-4-m et h ylp en t a n oyl)-3-b en zyl-sn -
glycer ol. Under argon, 3-isopropyl-4-methylpent-2-enoyl chlo-
ride was added to a 0 °C solution of 3-isopropyl-4-methylpen-
tanoyl chloride in CH2Cl2 and stirred overnight warming to
room temperature. The crude reaction mixture was then
concentrated in vacuo, and the products were separated by
column chromatography to give the desired title compound in
64% yield, along with the monoacylated product in 33% yield.
This compound was used directly in the next step without
1.2 Hz, 3 H, C(O)CHdCMe2), 1.98 (d, J ) 1.3 Hz, 3 H, C(O)-
CHdCMe2), 2.23 and 2.25 (d, J ) 1.2 Hz, 6 H, C(O)CHdCMe2),
3.72 (d, J ) 5.2 Hz, 2 H, OCH2O(O-)CH2O), 4.34 (dd, J )
11.8, 6.1 Hz, 1 H, OCH2CH(O-)CH2O), 4.44 (dd, J ) 11.8, 4.2
Hz, 1 H, OCH2CH(O-)CH2O), 4.63 (AB quartet, J ) 9.3 Hz, 2
H, PhCH2O), 5.32-5.40 (m, 1 H, OCH2CH(O-)CH2O), 5.73-
5.75 (m, J ) 1.3 Hz, 1 H, C(O)CHdCMe2), 5.78-5.80 (m, J )
1.3 Hz, 1 H, C(O)CHdCMe2), 7.40 (s, 5 H, PhCH2O); 13C NMR
(62.5 MHz, CDCl3) δ 20.17, 20.22, 27.30, 27.34, 62.04, 68.35,
69.33, 73.16, 115.52, 115.67, 127.45, 127.49, 128.20, 137.73,
157.02, 157.39, 165.46, 165.90. FABMS (m/z, relative inten-
sity): 347 (MH+, 42), 247 (100), 91 (97), 83 (100). Anal.
(C20H26O5) C, H.
1
further purification. H NMR (250 MHz, CDCl3) δ 0.87-1.03
(m, 24 H, C(O)CH2CH(CHMe2)2), 1.52-1.90 (m, 6 H, C(O)-
CH2CH(CHMe2)2), 2.24 (d, J ) 5.9 Hz, 2 H, C(O)CH2CH-
(CHMe2)2), 2.28 (d, J ) 5.6 Hz, 2 H, C(O)CH2CH(CHMe2)2),
3.69 (d, J ) 5.1 Hz, 2 H, OCH2CH(O-)CH2O), 4.27 (dd, J )
12.0, 6.3, 1 H, OCH2CHCH2O), 4.43 (dd, J ) 12.0, 3.8, 1 H,
OCH2CH(O-)CH2O), 4.62 (AB quartet, J ) 7.3 Hz, PhCH2O),
5.30-5.38 (m, 1 H, OCH2CH(O-)CH2O), 7.40 (s, 5 H, PhCH2O);
13C NMR (62.5 MHz, CDCl3) δ 18.68, 21.29, 29.31, 32.70, 32.94,
46.62, 62.72, 68.19, 69.96, 73.18, 127.44, 127.56, 128.23,
137.56, 173.84, 174.16.
1-(3-Met h ylb u t -2-en oyl)-2-(3-isop r op yl-4-m et h ylp en -
ta n oyl)-3-ben zyl-sn -glycer ol. According to the general pro-
cedure, 1-(3-methylbut-2-enoyl)-3-benzyl-sn-glycerol and 3-iso-
propyl-4-methylpentanoyl chloride were reacted to give the
title compound in 40% yield. [R]20 ) +9.80 (c 1.53, CHCl3);
D
IR (neat) 2960 (CH), 2874 (CH), 1727 (CdO), 1653 (CdC) cm-1
;
1H NMR (250 MHz, CDCl3) δ 0.89 and 1.01 (d, J ) 6.6 Hz, 12
H, C(O)CH2CH(CHMe2)2), 1.71 (pentet, J ) 5.7 Hz, 1 H, C(O)-
CH2CH(CHMe2)2), 1.81 (heptet, J ) 6.6 Hz, 2 H, C(O)CH2CH-
(CHMe2)2), 1.97 and 2.23 (d, J ) 0.7 Hz, 6 H, C(O)CHdCMe2),
2.29 (d, 2 H, J ) 5.6 Hz, C(O)CH2CH(CHMe2)2), 3.70 (d, J )
5.1 Hz, 2 H, OCH2CH(O-)CH2O), 4.32 (dd, J ) 12.0, 6.3 Hz,
1 H, OCH2CH(O-)CH2O), 4.42 (dd, J ) 12.0, 3.9 Hz, 1 H,
OCH2CH(O-)CH2O), 4.60 (AB quartet, J ) 8.1 Hz, 2 H,
PhCH2O), 5.31-5.39 (m, 1 H, OCH2CH(O-)CH2O), 5.72 (s, 1
H, C(O)CHdCMe2), 7.40 (s, 5 H, PhCH2O); 13C NMR (62.5
MHz, CDCl3) δ 18.69, 18.71, 20.20, 21.27, 27.34, 29.29, 29.35,
33.00, 46.63, 61.94, 68.31, 70.09, 73.18, 115.41, 127.45, 127.54,
128.23, 137.64, 157.21, 165.91, 173.96. FABMS (m/z, relative
intensity): 405 (MH+, 14), 91 (72), 83 (100). Anal. (C24H36O5)
C, H.
1-(3-Met h ylb u t -2-en oyl)-2-(3-isop r op yl-4-m et h ylp en t -
2-en oyl)-3-ben zyl-sn -glycer ol. Under argon, DCC (0.35 g,
1.70 mmol), DMAP (0.14 g, 1.14 mmol), and a solution of 1-(3-
methylbut-2-enoyl)-3-benzyl-sn-glycerol (0.33 g, 1.13 mmol) in
toluene (4 mL) were added to a solution of 3-isopropyl-4-
methylpent-2-enoic acid (0.27 g, 1.7 mmol) in toluene (8 mL)
and heated to 80 °C for 3 h. After cooling to room temperature,
the crude reaction mixture was concentrated to dryness in
vacuo, dissolved in Et2O, and recrystallized. Purification by
silica gel chromatography (gradient elution from 5% EtOAc
in hexane to 25% EtOAc in hexane) gave the title compound
contaminated by DCC/DCU which was used directly as in the
next step without further purification.
1-(3-Isop r op yl-4-m et h ylp en t a n oyl)-2-(3-isop r op yl-4-
m eth ylp en t-2-en oyl)-3-ben zyl-sn -glycer ol. According to the
general procedure, 1-(3-isopropyl-4-methylpentanoyl)-3-benzyl-
sn-glycerol and 3-isopropyl-4-methylpent-2-enoyl chloride were
reacted to give the title compound in 36% yield. [R]20D ) +7.89
(c ) 0.71, CHCl3); IR (neat) 2961 (CH), 2360 (CH), 1728 (Cd
O), 1637 (CdC) cm-1; 1H NMR (250 MHz, CDCl3) δ 0.88 (d, J
) 6.8 Hz, 6 H, C(O)CH2CH(CHMe2)2), 0.97 (d, J ) 6.6 Hz, 6
H, C(O)CH2CH(CHMe2)2), 1.15 (irreg t, 12 H, C(O)CHd
C(CHMe2)2), 1.60-1.75 (m, 1 H, C(O)CH2CH(CHMe2)2), 1.75-
1.90 (m, 2 H, C(O)CH2CH(CHMe2)2), 2.25 (d, J ) 5.6 Hz, 2 H,
C(O)CH2CH(CHMe2)2), 2.64 (heptet, J ) 6.8 Hz, 1 H, C(O)-
CHdC(CHMe2)2), 3.72 (d, J ) 5.1 Hz, 2 H, OCH2CH(O-)-
CH2O), 4.13 (heptet, J ) 6.8 Hz, 1 H, C(O)CHdC(CHMe2)2),
4.32 (dd, J ) 11.8, 6.2 Hz, 1 H, OCH2CH(O-)CH2O), 4.43 (dd,
J ) 11.8, 3.9 Hz, 1 H, OCH2CH(O-)CH2O), 4.64 (AB quartet,
J ) 10.8 Hz, 2 H, PhCH2O), 5.30-5.40 (m, 1 H, OCH2CH(O-)-
CH2O), 5.75 (s, 1 H, -C(O)CHdC(CHMe2)2), 7.40 (s, 5 H,
PhCH2O); 13C NMR (62.5 MHz, CDCl3) δ 18.66, 18.71, 20.22,
21.30, 21.31, 24.09, 29.03, 29.30, 29.35, 29.76, 32.82, 46.73,
62.80, 68.31, 69.09, 73.19, 112.27, 127.47, 127.56, 128.24,
137.66, 165.86, 174.27, 177.00; FAB-MS (m/z, relative inten-
sity): 461 (MH+, 7), 139 (100), 91 (46). Anal. (C28H44O5) C, H.
1-(3-Isop r op yl-4-m et h ylp en t -2-en oyl)-2-(3-m et h ylb u -
ta n oyl)-3-ben zyl-sn -glycer ol. According to the general pro-
cedure, 1-(3-isopropyl-4-methylpent-2-enoyl)-3-benzyl-sn-glyc-
erol and isovaleryl chloride were reacted to give the title
compound in 85% yield. [R]20D +8.08 (c 2.53, CHCl3); IR (neat)
2963 (CH), 2871 (CH), 1740 (CdO), 1719 (CdO), 1637 (CdC)
1-(3-Isop r op yl-4-m e t h ylp e n t a n oyl)-2-(3-m e t h ylb u -
ta n oyl)-3-ben zyl-sn -glycer ol. According to the general pro-
cedure, 1-(3-isopropyl-4-methylpentanoyl)-3-benzyl-sn-glycerol
and isovaleryl chloride were reacted to give the title compound
1
cm-1; H NMR (250 MHz, CDCl3) δ 1.03 (d, J ) 6.4 Hz, 6 H,
C(O)CH2CHMe2), 1.13 and 1.14 (d, J ) 6.8 Hz, 12 H, C(O)-
CHdC(CHMe2)2), 2.21 (heptet, J ) 6.6 Hz, 1 H, C(O)-
CH2CHMe2), 2.30 (d, J ) 6.4 Hz, 2 H, C(O)CH2CHMe2), 2.62
(heptet, J ) 6.8 Hz, 1 H, C(O)CHdC(CHMe2)2), 3.69 (d, J )
4.9 Hz, 2 H, OCH2CH(O-)CH2O), 4.11 (heptet, J ) 6.8 Hz, 1
H, C(O)CHdC(CHMe2)2), 4.31 (dd, J ) 12.0, 6.6 Hz, 1 H, OCH2-
CH(O-)CH2O), 4.41 (dd, J ) 12.0, 4.0 Hz, 1 H, OCH2CH(O-)-
CH2O), 4.62 (AB quartet, J ) 7.7 Hz, 2 H, PhCH2O), 5.33-
5.41 (m, 1 H, OCH2CH(O-)CH2O), 5.69 (s, 1 H, C(O)CHd
in 88% yield. [R]20 ) +8.57 (c 1.54, CHCl3); IR (neat) 2960
D
(CH), 2873 (CH), 1740 (CdO) cm-1; 1H NMR (250 MHz, CDCl3)
δ 0.88 and 0.97 (d, J ) 6.8 Hz, 12 H, C(O)CH2CH(CHMe2)2),
1.04 (d, J ) 6.4 Hz, 6 H, C(O)CH2CHMe2), 1.63-1.72 (m, J )
5.7 Hz, 1H, C(O)CH2CH(CHMe2)2), 1.80 (heptet, J ) 6.6 Hz, 2
H, C(O)CH2CH(CHMe2)2), 2.13-2.33 (m, 5 H, C(O)CH2CH-
(CHMe2)2 and C(O)CH2CHMe2), 3.68 (d, J ) 5.1 Hz, 2 H, OCH2-

Protein Kinase C Activating Diacylglycerol
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1901
C(CHMe2)2), 7.39 (s, 5 H, PhCH2O); 13C NMR (62.5 MHz,
CDCl3) δ 20.21, 22.26, 24.09, 25.69, 28.94, 29.71, 43.36, 61.97,
68.36, 70.01, 73.15, 112.09, 127.44, 127.56, 128.23, 137.63,
166.27, 172.10, 176.64; FAB-MS (m/z, relative intensity): 405
(MH+, 9), 139 (100), 91 (55). Anal. (C24H36O5) C, H.
68.34, 69.35, 73.15, 112.24, 112.38, 127.46, 127.52, 128.23,
137.75, 165.96, 166.44, 176.39, 176.78. FAB-MS (m/z, relative
intensity): 459 (MH+, 11), 139 (100), 91 (97). Anal. (C28H42O5)
C, H.
Gen er a l P r oced u r e for th e Deben zyla tion of Un sa tu r -
a ted 1,2-Disu bstitu ted -3-ben zyl Glycer ols. Under argon,
BCl3 (4.0 equiv) was added dropwise to a -78 °C stirring
solution of 1,2-disubstituted-3-benzyl-sn-glycerol (1.0 equiv) in
CH2Cl2 (40 mL/mmol). The reaction was monitored by TLC
and complete within 1-2 h. The reaction was then diluted with
Et2O (40 mL/mmol) and poured into a pH ) 7 buffer solution.
After washing with a pH ) 7 buffer solution (3×), the organic
layer was dried over MgSO4 and concentrated. Purification by
silica gel column chromatography (3 hexane:1 Et2O) gives the
named product as a colorless oil.
1-(3-Isop r op yl-4-m eth ylp en t-2-en oyl)-2-(3-m eth ylbu t-
2-en oyl)-3-ben zyl-sn -glycer ol. According to the general
procedure, 1-(3-isopropyl-4-methylpent-2-enoyl)-3-benzyl-sn-
glycerol and 3,3-dimethylacryloyl chloride were reacted to give
the title compound in 70% yield. [R]20 +4.22 (c 2.02, CHCl3);
D
IR (neat) 2965 (CH), 2870 (CH), 1719 (CdO), 1654 (CdC), 1637
(CdC) cm-1; 1H NMR (250 MHz, CDCl3) δ 1.14 (t, J ) 6.6 Hz,
12 H, C(O)CHdC(CHMe2)2, 1.98 (d, J ) 0.7 Hz, 3 H, C(O)-
CHdCMe2), 2.25 (d, J ) 1.0 Hz, 3 H, C(O)CHdCMe2), 2.63
(heptet, J ) 6.8 Hz, 1 H, C(O)CHdC(CHMe2)2), 3.73 (d, J )
5.1 Hz, 2 H, OCH2CH(O-)CH2O), 4.10 (heptet, J ) 6.8 Hz, 1
H, C(O)CHdC(CHMe2)2), 4.34 (dd, J ) 11.8, 6.0 Hz, 1 H, OCH2-
CH(O-)CH2O), 4.43 (dd, J ) 11.8, 4.2 Hz, 1 H, OCH2CH(O-
)CH2O), 4.64 (AB quartet, J ) 9.7 Hz, 2 H, PhCH2O), 5.33-
5.41 (m, 1 H, OCH2CH(O-)CH2O), 5.71 (s, 1 H, C(O)CHd
C(CHMe2)2), 5.81 (irreg t, 1H, C(O)CHdCMe2), 7.40 (s, 5 H,
PhCH2O); 13C NMR (62.5 MHz, CDCl3) δ 20.22, 20.25, 24.12,
27.38, 28.92, 29.75, 62.10, 68.35, 69.37, 73.17, 112.21, 115.67,
127.46, 127.52, 128.23, 137.73, 157.44, 165.51, 166.44, 176.45;
FAB-MS (m/z, relative intensity): 403 (MH+, 7), 139 (100), 91
(42). Anal. (C24H34O5) C, H.
1,2-Di-(3-m eth ylbu ta n oyl)-sn -glycer ol (1A). Palladium
on carbon (50 mg, 10%) was added to a solution of 1,2-di-(3-
methylbut-2-enoyl)-3-benzyl-sn-glycerol (0.5 g, 1.44 mmol) in
methanol (20 mL), and the mixture was agitated under H2
pressure (44 psi) for 2 h. The palladium was filtered off, and
the filtrate was concentrated under reduced pressure. Purifi-
cation by silica gel chromatography (gradient elution from 20%
EtOAc/hexane to 30% EtOAc/hexane) gave 1A (375 mg, 1.44
mmol, 100%). [R]20 ) -3.92 (c 3.34, CHCl3); IR (neat) 3467
D
(OH), 2961 (CH), 2874 (CH), 1740 (CdO) cm-1; 1H NMR (250
MHz, CDCl3) δ 1.03 (d, J ) 2.7 Hz, 6 H, C(O)CH2CHMe2), 1.05
(d, J ) 2.4 Hz, 6 H, C(O)CH2CHMe2), 2.05-2.35 (m, 6 H, C(O)-
CH2CHMe2), 3.81 (d, J ) 5.1 Hz, 2 H, HOCH2CH(O-)CH2O),
4.30 (dd, J ) 12.0, 5.6 Hz, 1 H, HOCH2CH(O-)CH2O), 4.41
(dd, J ) 12.0, 4.4 Hz, 1 H, HOCH2CH(O-)CH2O), 5.17 (m, 1
H, HOCH2CH(O-)CH2O); 13C NMR (62.5 MHz, CDCl3) δ
22.19, 22.24, 25.51, 25.61, 43.03, 43.22, 61.20, 62.08, 71.91,
172.52, 172.82. FAB-MS (m/z, relative intensity): 261 (MH+,
36), 159 (100), 85 (100). Anal. (C13H24O5) C, H.
1-(3-Isop r op yl-4-m eth ylp en t-2-en oyl)-2-(3-isop r op yl-4-
m eth ylp en ta n oyl)-3-ben zyl-sn -glycer ol. According to the
general procedure, 1-(3-isopropyl-4-methylpent-2-enoyl)-3-ben-
zyl-sn-glycerol and 3-isopropyl-4-methylpentanoyl chloride
were reacted to give the title compound in 40% yield. [R]20
D
+8.18 (c 1.04, CHCl3); IR (neat) 2963 (CH), 2873 (CH), 1735
(CdO), 1719(CdO), 1637 (CdC) cm-1 1H NMR (250 MHz,
;
CDCl3) δ 0.89 (dd, J ) 6.7, 0.6 Hz, 6 H, C(O)CH2CH(CHMe2)2),
0.99 (d, J ) 6.8 Hz, 6 H, C(O)CH2CH(CHMe2)2), 1.13 and 1.15
(d, J ) 6.8 Hz, 12 H, C(O)CHdC(CHMe2)2), 1.72 (pentet, J )
5.7 Hz, 1 H C(O)CH2CH(CHMe2)2), 1.82 (heptet, 2 H, C(O)-
CH2CH(CHMe2)2), 2.29 (d, J ) 5.6 Hz, 2 H, C(O)CH2CH-
(CHMe2)2), 2.63 (heptet, J ) 6.8 Hz, 1 H, C(O)CHdC(CHMe2)2),
3.71 (d, J ) 5.1 Hz, 2 H, OCH2CH(O-)CH2O), 4.12 (heptet, J
) 6.8 Hz, 1 H, C(O)CHdC(CHMe2)2), 4.31 (dd, J ) 12.0, 6.6
Hz, 1 H, OCH2CH(O-)CH2O), 4.42 (dd, J ) 12.0, 3.9 Hz, 1 H,
OCH2CH(O-)CH2O), 4.63 (AB quartet, J ) 9.0 Hz, 2 H,
PhCH2O), 5.31-5.40 (m, 1 H, OCH2CH(O-)CH2O), 5.69 (s, 1
H, C(O)CHdC(CHMe2)2), 7.40 (s, 5 H, PhCH2O); 13C NMR
(62.5 MHz, CDCl3) δ 18.68, 18.73, 20.22, 21.32, 24.11, 28.93,
29.31, 29.38, 29.71, 33.01, 46.67, 62.00, 68.32, 70.13, 73.15,
112.07, 127.45, 127.56, 128.24, 137.65, 166.34, 173.98, 176.74;
FAB-MS (m/z, relative intensity): 461 (MH+, 9), 139 (100), 91
(100). Anal. (C28H44O5) C, H.
1-(3-Meth ylbu ta n oyl)-2-(3-m eth ylbu t-2-en oyl)-sn -glyc-
er ol (1B). According to the general procedure, 1-(3-methylbu-
tanoyl)-2-(3-methylbut-2-enoyl)-3-benzyl-sn-glycerol was depro-
tected to give 1B in 50% yield. [R]20 ) -14.8 (c 0.18, CHCl3);
D
IR (neat) 3449 (OH), 2960 (CH), 1719 (CdO), 1654 (CdC),
cm-1; 1H NMR (250 MHz, CDCl3) δ 1.03 (2 d, J ) 6.6 Hz, 6 H,
C(O)CH2CHMe2), 1.99 (d, J ) 1.2 Hz, 3 H, C(O)CHdCMe2),
2.10-2.35 (m, 3 H, C(O)CH2CHMe2 containing 2.25 (d, J )
1.2 Hz, 3 H, C(O)CHdCMe2), 3.81 (br s, 2 H, HOCH2CH(O-)-
CH2O), 4.37 (2 overlapping AB quartets, 2 H, HOCH2CH(O-)-
CH2O), 5.14-5.22 (m, 1 H, HOCH2CH(O-)CH2O), 5.75-5.79
(m, 1 H, -C(O)CHdCMe2); 13C NMR (62.5 MHz, CDCl3) δ
20.29, 22.29, 25.58, 27.41, 43.10, 61.64, 61.99, 71.35, 115.25,
158.33, 165.77, 172.87; FAB-MS (m/z, relative intensity): 259
(MH+, 27), 83 (100). Anal. (C13H22O5) C, H.
1-(3-Me t h ylb u t a n oyl)-2-(3-isop r op yl-4-m e t h ylp e n -
ta n oyl)-sn -glycer ol (1C). Palladium on carbon (100 mg, 10%)
was added to a suspension of the intermediate 1-(3-methylbu-
tanoyl)-2-(3-isopropyl-4-methylpentanoyl)-3-benzyl-sn-glyc-
erol (525 mg) in methanol (20 mL), and the mixture was stirred
under H2 (45 psi) overnight. The crude reaction mixture was
filtered, and the filtrate was concentrated under reduced
pressure. Purification by silica gel chromatography (gradient
elution from 5% EtOAc/hexane to 50% EtOAc/hexane) gave
1,2-Di-(3-isop r op yl-4-m eth ylp en t-2-en oyl)-3-ben zyl-sn -
glycer ol. Under argon, a solution of (R)-(+)-3-benzyloxy-1,2-
propanediol (1.0 g, 5.49 mmol) in toluene (4 mL) was added to
a solution of 4-methyl-3-(isopropyl)pent-2-enoic acid (2.17 g,
13.71 mmol, 2.5 equiv) in toluene (38.4 mL) containing DCC
(2.83 g, 13.72 mmol, 2.5 equiv) and DMAP (1.34 g, 10.96 mmol,
2 equiv). The mixture was heated at 80 °C for 3 h and then
concentrated under reduced pressure. The residue was recrys-
tallized from Et2O several times to partially eliminate DCC/
DCU. Further purification by silica gel chromatography
(gradient elution from 2% EtOAc in hexane to 25% EtOAc in
hexane) gave the title compound (0.36 g, 0.79 mmol, 58%)
along with two other monoacylated products (0.964 and 0.23
1C (0.15 g, 0.46 mmol, 45%). [R]20 ) -2.44 (c 1.27, CHCl3);
D
IR (neat) 3463 (OH), 2960 (CH), 2875 (CH), 1740 (CdO) cm-1
;
1H NMR (250 MHz, CDCl3) δ 0.90 and 0.99 (d, J ) 6.6 Hz, 12
H, C(O)CH2CH(CHMe2)2), 1.04 (d, J ) 6.3 Hz, 6 H, C(O)CH2-
CHMe2), 1.70 (pentet, J ) 5.8 Hz, 1 H, C(O)CH2CH(CHMe2)2),
1.83 (heptet, J ) 6.5 Hz, 2 H, C(O)CH2CH(CHMe2)2), 2.08-
2.33 (m, 5 H, C(O)CH2CHMe2 and C(O)CH2CH(CHMe2)2), 3.81
(d, J ) 4.9 Hz, 2 H, HOCH2CH(O-)CH2O), 4.32 (dd, J ) 12.0,
5.7 Hz, 1 H, HOCH2CH(O-)CH2O), 4.41 (dd, J ) 12.0, 4.4 Hz,
1 H, HOCH2CH(O-)CH2O), 5.05-5.20 (m, 1 H, HOCH2CH(O-)-
CH2O); 13C NMR (62.5 MHz, CDCl3) δ 18.63, 18.68, 21.26,
21.28, 22.28, 25.54, 29.28, 29.35, 32.92, 43.05, 46.71, 61.30,
61.99, 72.08, 172.84, 174.34; FAB-MS (m/z, relative inten-
sity): 317 (MH+, 16), 57 (100). Anal. (C17H32O5) C, H.
g). [R]20 ) +6.19 (c 1.68, CHCl3); IR (neat) 2964 (CH), 2874
D
(CH), 1718 (CdO), 1637 (CdC) cm-1
;
1H NMR (250 MHz,
CDCl3) δ 1.10-1.20 (m, 24 H, C(O)CHdC(CHMe2)2), 2.63 and
2.64 (heptet, J ) 6.8 Hz, 2 H, C(O)CHdC(CHMe2)2), 3.74 (d,
J ) 5.1 Hz, 2 H, OCH2CH(O-)CH2O), 4.00-4.21 (m, 2 H, C(O)-
CHdC(CHMe2)2), 4.35 (dd, J ) 12.0, 5.9 Hz, 1 H, OCH2CH-
(O-)CH2O), 4.43 (dd, J ) 12.0, 4.3 Hz, 1 H, OCH2CH(O-)-
CH2O), 4.65 (AB quartet, J ) 9.4 Hz, 2 H, PhCH2O), 5.32-
5.40 (m, 1 H, OCH2CH(O-)CH2O), 5.71 and 5.77 (s, 1 H,
-C(O)CHdC(CHMe2)2), 7.40 (s, 5H, PhCH2O-); 13C NMR (62.5
MHz, CDCl3) δ 20.22, 20.25, 24.10, 28.95, 29.05, 29.78, 62.10,
1-(3-Met h ylb u t a n oyl)-2-(3-isop r op yl-4-m et h ylp en t -2-
en oyl)-sn -glycer ol (1D). According to the general procedure,

1902 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Nacro et al.
1-(3-methylbutanoyl)-2-(3-isopropyl-4-methylpent-2-enoyl)-3-
benzyl-sn-glycerol was deprotected to give 1D in 44% yield.
[R]20D ) -4.6 (c 1.36, CHCl3); IR (neat) 3478 (OH), 2963 (CH),
2873 (CH), 1722 (CdO), 1634 (CdC) cm-1; 1H NMR (250 MHz,
CDCl3) δ 1.02 (d, J ) 6.6 Hz, 6 H, C(O)CH2CHMe2), 1.13 and
1.14 (d, J ) 6.8 Hz, 12 H, C(O)CHdC(CHMe2)2), 2.16 (heptet,
J ) 6.6 Hz, 1 H, C(O)CH2CHMe2), 2.28 (d, J ) 6.6 Hz, 2 H,
C(O)CH2CHMe2), 2.44 (br t, J ) 7.3 Hz, 1 H, HOCH2CH(O-
)CH2O), 2.64 (heptet, J ) 6.8 Hz, 1 H, C(O)CHdC(CHMe2)2),
3.83 (t, J ) 5.4 Hz, 2 H, HOCH2CH(O-)CH2O), 4.10 (heptet,
J ) 6.8 Hz, 1 H, C(O)CHdC(CHMe2)2), 4.37 (2 overlapping
doublets, J ) 5.3, 4.9 Hz, 2 H, HOCH2CH(O-)CH2O), 5.10-
5.25 (m, 1 H, HOCH2CH(O-)CH2O), 5.73 (s, 1 H, C(O)CHd
C(CHMe2)2); 13C NMR (62.5 MHz, CDCl3) δ 20.13, 20.20, 22.29,
24.07, 25.59, 29.05, 29.79, 43.11, 61.61, 62.01, 71.31, 111.93,
166.18, 172.87, 177.82; FAB-MS (m/z, relative intensity): 315
(MH+, 31), 139 (100). Anal. (C17H30O5) C, H.
3.79-3.81 (m, 2 H, HOCH2CH(O-)CH2O), 4.07 (heptet, J )
6.8 Hz, 1 H, C(O)CHdC(CHMe2)2), 4.36 (d, J ) 5.1 Hz, 2 H,
HOCH2CH(O-)CH2O), 5.13-5.20 (m, 1 H, HOCH2CH(O-)-
CH2O), 5.71 (s, 1H, C(O)CHdC(CHMe2)2), 5.79 (t, J ) 1.2 Hz,
1 H, C(O)CHdCMe2); 13C NMR (62.5 MHz, CDCl3) δ 20.17,
20.27, 24.08, 27.40, 28.97, 29.77, 61.40, 61.47, 71.53, 111.89,
115.38, 158.13, 165.82, 166.76, 177.36; FAB-MS (m/z, relative
intensity): 313 (MH+,40), 139 (97), 83 (100). Anal. (C17H28O5)
C, H.
1-(3-Isop r op yl-4-m e t h ylp e n t a n oyl)-2-(3-m e t h ylb u -
ta n oyl)-sn -glycer ol (3A). According to the general procedure,
1-(3-isopropyl-4-methylpentanoyl)-2-(3-methylbutanoyl)-3-ben-
zyl-sn-glycerol was deprotected to give 3A in 77% yield. [R]20
D
) -3.80 (c 2.12, CDCl3); IR (neat) 3468 (OH), 2960 (CH), 2874
(CH), 1741 (CdO) cm-1; 1H NMR (250 MHz, CDCl3) δ 0.86 (d,
J ) 6.6 Hz, 6 H, C(O)CH2CH(CHMe2)2), 0.95 (d, J ) 6.8 Hz, 6
H, C(O)CH2CHMe2), 1.02 (d, J ) 6.6 Hz, 6 H, C(O)CH2CH-
(CHMe2)2), 1.65 (pentet, J ) 5.8 Hz, 1H, C(O)CH2CH(CHMe2)2),
1.80 (heptet, J ) 6.5 Hz, 2 H, C(O)CH2CH(CHMe2)2), 2.10-
2.29 (m, 5 H, C(O)CH2CH(CHMe2)2 and -C(O)CH2CHMe2),
2.54 (br s, 1 H, HOCH2CH(O-)CH2O), 3.78 (d, J ) 5.1 Hz, 2
H, HOCH2CH(O-)CH2O), 4.25 (dd, J ) 12.0, 5.9 Hz, 1 H,
HOCH2CH(O-)CH2O), 4.38 (dd, J ) 12.0, 4.2 Hz, 1H, HOCH2-
CH(O-)CH2O), 5.11-5.19 (m, 1 H, HOCH2CH(O-)CH2O-);
13C NMR (62.5 MHz, CDCl3) δ 18.63, 21.24, 22.24, 25.64, 29.27,
32.72, 43.24, 46.68, 61.35, 62.23, 71.99, 172.54, 174.64. FAB-
MS (m/z, relative intensity): 317 (MH+, 32), 159 (100), 57
(100). Anal. (C17H32O5) C, H.
1-(3-Meth ylbu t-2-en oyl)-2-(3-m eth ylbu ta n oyl)-sn -glyc-
er ol (2A). According to the general procedure, 1-(3-methylbut-
2-enoyl)-2-(3-methylbutanoyl)-3-benzyl-sn-glycerol was depro-
tected to give 2A in 67% yield. [R]20 ) -11.2 (c 0.92, CHCl3);
D
IR (neat) 3478 (OH), 2960 (CH), 2873 (CH), 1722 (CdO), 1651
1
(CdC) cm-1; H NMR (250 MHz, CDCl3) δ 1.02 (2 d, J ) 6.6
Hz, 6 H, C(O)CH2CHMe2), 1.97 (s, 3 H, C(O)CHdCMe2), 2.11-
2.31 (m, 3 H, C(O)CH2CHMe2 containing 2.23 (d, J ) 1.0 Hz,
3 H, C(O)CHdCMe2)), 2.55 (br s, 1 H, HOCH2CH(O-)CH2O),
3.79 (d, J ) 4.9 Hz, 2 H, HOCH2CH(O-)CH2O), 4.36 (2
overlapping AB quartets, 2 H, HOCH2CH(O-)CH2O), 5.13-
5.21 (m, 1 H, HOCH2CH(O-)CH2O), 5.75 (m, 1 H, C(O)CHd
CMe2); 13C NMR (62.5 MHz, CDCl3) δ 20.27, 22.23, 25.67,
27.39, 43.08, 61.22, 61.33, 72.10, 115.10, 158.08, 166.24,
172.54; FAB-MS (m/z, relative intensity): 259 (MH+, 29), 83
(100). Anal. (C13H22O5) C, H.
1-(3-Isop r op yl-4-m et h ylp en t a n oyl)-2-(3-m et h ylb u t -2-
en oyl)-sn -glycer ol (3B). According to the general procedure,
1-(3-isopropyl-4-methylpentanoyl)-2-(3-methyl-but-2-enoyl)-3-
benzyl-sn-glycerol was deprotected to give 3B in 94% yield.
[R]20 ) -10.24 (c 1.69, CDCl3); IR (neat) 3467 (OH), 2960
D
1,2-Di-(3-m eth ylbu t-2-en oyl)-sn -glycer ol (2B). According
(CH), 1723 (CdO), 1653 (CdC) cm-1 1H NMR (250 MHz,
;
to the general procedure, 1,2-di-(3-methylbut-2-enoyl)-3-ben-
CDCl3) δ 0. 89 (d, J ) 6.8 Hz, 6 H, C(O)CH2CH(CHMe2)2), 0.98
(d, J ) 6.6 Hz, 6 H, C(O)CH2CH(CHMe2)2), 1.69 (pentet, J )
5.7 Hz, 1H, C(O)CH2CH(CHMe2)2), 1.75-1.95 (m, 2 H, C(O)-
CH2CH(CHMe2)2), 1.99 and 2.26 (d, J ) 0.74 Hz, 6 H, C(O)-
CHdCMe2), 2.28 (d, J ) 5.6 Hz, 2 H, C(O)CH2CH(CHMe2)2),
3.84 (d, J ) 4.9 Hz, 2 H, HOCH2CH(O-)CH2O), 4.36 (2
overlapping AB quartets, 2 H, HOCH2CH(O-)CH2O), 5.15-
5.23 (m, 1 H, HOCH2CH(O-)CH2O), 5.79 (s, 1 H, C(O)CHd
CMe2);13C NMR (62.5 MHz, CDCl3) δ 18.66, 20.29, 21.25, 27.41,
29.29, 32.77, 46.70, 61.66, 62.23, 71.39, 115.29, 158.27, 165.80,
174.66. FAB-MS (m/z, relative intensity): 315 (MH+, 18), 83
(100). Anal. (C17H30O5‚0.2H2O) C, H.
zyl-sn-glycerol was deprotected to give 2B in 92% yield. [R]20
D
) -27.07 (c 3.65, CHCl3); IR (neat) 3481 (OH), 2941 (CH), 1716
1
(CdO), 1652 (CdC) cm-1; H NMR (250 MHz, CDCl3) δ 1.98
and 2.24 (s, 12 H, C(O)CHdCMe2), 2.48 (br s, 1 H, HOCH2-
CH(O-)CH2O), 3.81 (d, J ) 4.9 Hz, 2 H, HOCH2CH(O-)-
CH2O), 4.38 (d, J ) 5.1 Hz, 2 H, HOCH2CH(O-)CH2O), 5.13-
5.21 (m, 1 H, HOCH2CH(O-)CH2O), 5.77 and 5.79 (br s, 2 H,
C(O)CHdCMe2); 13C NMR (62.5 MHz, CDCl3) δ 20.24, 27.36,
61.37, 61.40, 71.46, 115.22, 115.39, 157.83, 158.02, 165.80,
166.28. FAB-MS (m/z, relative intensity): 257 (MH+, 31), 83
(100). Anal. (C13H20O5) C, H.
1-(3-Met h ylb u t -2-en oyl)-2-(3-isop r op yl-4-m et h ylp en -
ta n oyl)-sn -glycer ol (2C). According to the general procedure,
1-(3-methylbut-2-enoyl)-2-(3-isopropyl-4-methylpentanoyl)-3-
benzyl-sn-glycerol was deprotected to give 2C in quantitative
1,2-Di-(3-isopr opyl-4-m eth ylpen tan oyl)-sn -glycer ol (3C).
This compound was prepared as previously reported in ref 16.
1-(3-Isop r op yl-4-m et h ylp en t a n oyl)-2-(3-isop r op yl-4-
m eth ylp en t-2-en oyl)-sn -glycer ol (3D). According to the
general procedure, 1-(3-isopropyl-4-methylpentanoyl)-2-(3-iso-
propyl-4-methylpent-2-enoyl)-3-benzyl-sn-glycerol was depro-
tected to give 3D in 90% yield. [R]20D ) -4.97 (c ) 0.93, CDCl3);
IR (neat) 3482 (OH), 2960 (CH), 2879 (CH), 1718 (CdO), 1637
(CdC) cm-1; 1H NMR (250 MHz, CDCl3) δ 0.88 (d, J ) 6.8 Hz,
6 H, C(O)CH2CH(CHMe2)2), 0.96 (d, J ) 6.6 Hz, 6 H, C(O)-
CH2CH(CHMe2)2), 1.13 and 1.14 (d, J ) 6.8 Hz, 12 H, CHd
C(CHMe2)2), 1.20-1.80 (m, 3 H, C(O)CH2CH(CHMe2)2), 2.27
(d, J ) 5.6 Hz, 2 H, C(O)CH2CH(CHMe2)2), 2.42 (br s, 1 H,
HOCH2CH(O-)CH2O), 2.63 (heptet, J ) 7.1 Hz, 1 H, C(O)-
CHdC(CHMe2)2), 3.83 (d, J ) 4.9 Hz, 2 H, HOCH2CH(O-)-
CH2O), 4.11 (heptet, J ) 7.1 Hz, 1 H, C(O)CHdC(CHMe2)2),
4.36 (2 overlapping AB quartets, 2 H, HOCH2CH(O-)CH2O),
5.12-5.21 (m, 1 H, HOCH2CH(O-)CH2O), 5.73 (s, 1 H, C(O)-
CHdC(CHMe2)2); 13C NMR (62.5 MHz, CDCl3) δ 18.64, 18.68,
20.15, 20.20, 21.29, 24.07, 29.05, 29.29, 29.33, 29.78, 32.78,
46.73, 61.68, 62.22, 71.37, 111.95, 166.18, 174.65, 177.80; FAB-
MS (m/z, relative intensity): 371 (MH+, 17), 139 (100). Anal.
(C21H38O5) C, H.
yield. [R]20 ) -11.16 (c 0.65, CHCl3); IR (neat) 3487 (OH),
D
2960 (CH), 1724 (CdO), 1654 (CdC) cm-1; 1H NMR (250 MHz,
CDCl3) δ 0.90 and 0.99 (d, J ) 6.6 Hz, 12 H, C(O)CH2CH-
(CHMe2)2), 1.71 (pentet, J ) 5.7 Hz, 1 H, C(O)CH2CH(CHMe2)2),
1.82 (heptet, J ) 6.3 Hz, 2 H, C(O)CH2CH(CHMe2)2), 1.99 (d,
J ) 0.74 Hz, 3 H, C(O)CHdCMe2), 2.25 (d, J ) 1.0 Hz, 3 H,
C(O)CHdCMe2), 2.31 (d, J ) 5.6 Hz, 2 H, C(O)CH2CH-
(CHMe2)2), 3.81 (d, J ) 4.9 Hz, 2 H, HOCH2CH(O-)CH2O),
4.38 (d, J ) 5.1 Hz, 2 H, HOCH2CH(O-)CH2O), 5.13-5.21
(m, 1 H, HOCH2CH(O-)CH2O), 5.76 (t, J ) 1.2 Hz, 1 H,
C(O)CHdCMe2); 13C NMR (62.5 MHz, CDCl3) δ 18.67,
18.70, 20.29, 21.29, 27.41, 29.31, 29.36, 32.96, 46.74, 61.12,
61.37, 72.24, 115.11, 158.13, 166.27, 174.35 FAB-MS (m/z,
relative intensity): 315 (MH+, 19), 83 (100). Anal. (C17H30O5)
C, H.
1-(3-Met h ylb u t -2-en oyl)-2-(3-isop r op yl-4-m et h ylp en t -
2-en oyl)-sn -glycer ol (2D). According to the general proce-
dure, 1-(3-methylbut-2-enoyl)-2-(3-isopropyl-4-methylpent-2-
enoyl)-3-benzyl-sn-glycerol was deprotected to give 2D in 95%
yield. [R]20 ) -26.53 (c 0.48, CHCl3); IR (neat) 3482 (CO),
D
2965 (CH), 2872 (CH), 1719 (CdO), 1638 (CdC), cm-1; 1H NMR
(250 MHz, CDCl3) δ 1.09-1.14 (m, 12 H, C(O)CHdC(CHMe2)2),
1.96 and 2.23 (d, J ) 1.0 Hz, 6 H, C(O)CHdCMe2), 2.50-2.55
(m, 2 H, C(O)CHdC(CHMe2)2 and HOCH2CH(O-)CH2O),
1-(3-Isop r op yl-4-m et h ylp en t -2-en oyl)-2-(3-m et h ylb u -
ta n oyl)-sn -glycer ol (4A). According to the general procedure,
1-(3-isopropyl-4-methylpent-2-enoyl)-2-(3-methylbutanoyl)-3-
benzyl-sn-glycerol was deprotected to give 4A in 89% yield.

Protein Kinase C Activating Diacylglycerol
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1903
[R]20D ) -46.3 (c 0.58, CHCl3); IR (neat) 3478 (OH), 2964 (CH),
2873 (CH), 1722 (CdO), 1634 (CdC) cm-1; 1H NMR (250 MHz,
CDCl3) δ 1.02 (d, J ) 6.6 Hz, 6 H, C(O)CH2CHMe2), 1.11 (d, J
) 6.8 Hz, 6 H, C(O)CHdC(CHMe2)2), 1.13 (d, J ) 6.6 Hz, 6 H,
C(O)CHdC(CHMe2)2), 2.17 (heptet, J ) 6.6 Hz, 1 H, C(O)-
CH2CHMe2), 2.30 (d, J ) 6.6 Hz, 2 H, C(O)CH2CHMe2), 2.62
(heptet, J ) 6.8 Hz, 1 H, C(O)CHdC(CHMe2)2), 3.79 (d, J )
4.2 Hz, 2 H, HOCH2CH(O-)CH2O), 4.08 (heptet, J ) 6.9 Hz,
1 H, C(O)CHdC(CHMe2)2), 4.34 (2 overlapping AB quartets,
2 H, HOCH2CH(O-)CH2O), 5.14-5.23 (m, 1 H, HOCH2CH(O-
)CH2O), 5.70 (s, 1 H, C(O)CHdC(CHMe2)2); 13C NMR (62.5
MHz, CDCl3) δ 20.17, 22.24, 24.06, 25.68, 28.98, 29.76, 43.28,
61.27, 61.36, 72.12, 111.79, 166.64, 172.56, 177.53. FAB-MS
(m/z, relative intensity): 315 (MH+, 33), 139 (100). Anal.
(C17H30O5‚0.4H2O) C, H.
1-(3-Isop r op yl-4-m eth ylp en t-2-en oyl)-2-(3-m eth ylbu t-
2-en oyl)-sn -glycer ol (4B). According to the general proce-
dure, 1-(3-isopropyl-4-methylpent-2-enoyl)-2-(3-methyl but-2-
enoyl)-3-benzyl-sn-glycerol was deprotected to give 4B in 81%
yield. [R]20D ) -46.26 (c 5.8, CHCl3); IR (neat) 3492 (OH), 2966
(CH), 2873 (CH), 1722 (CdO), 1651 (CdC) cm-1; 1H NMR (250
MHz, CDCl3) δ 1.13 (t, J ) 6.3 Hz, 12 H, C(O)CHdC(CHMe2)2),
1.97 and 2.24 (d, J ) 0.7 Hz, 6 H, C(O)CHdCMe2), 2.52 (br s,
1 H, HOCH2CH(O-)CH2O), 2.63 (heptet, J ) 6.8 Hz, 1 H,
C(O)CHdC(CHMe2)2), 3.82 (d, J ) 4.9 Hz, 2 H, HOCH2CH-
(O-)CH2O), 4.08 (heptet, J ) 6.8 Hz, 1 H, C(O)CHd
C(CHMe2)2), 4.37 (d, J ) 5.1 Hz, 2 H, HOCH2CH(O-)CH2O),
5.13-5.21 (m, 1 H, HOCH2CH(O-)CH2O), 5.72 (s, 1 H, C(O)-
CHdC(CHMe2)2), 5.80, (br s, 1H, -C(O)CHdCMe2); 13C NMR
(62.5 MHz, CDCl3) δ 20.17, 20.28, 24.08, 27.40, 28.99, 29.79,
61.37, 61.52, 71.55, 111.89, 115.37, 158.13, 165.82, 166.76,
177.38. FAB-MS (m/z, relative intensity): 313 (MH+, 23), 139
(100). Anal. (C17H28O5) C, H.
Advanced Biomedical Computing Center, National Can-
cer Institute at Frederick, National Institutes of Health,
Frederick, MD. Some of the computational studies were
performed on the SGI Origin 2000 system at the Center
for Information Technology, National Institutes of Health,
Bethesda, MD.
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(3) Mellor, H.; Parker, P. J . The extended protein kinase C super-
family. Biochem. J . 1998, 332, 281-292.
(4) Ron, D.; Kazanietz, M. G. New insights into the regulation of
protein kinase C. and novel phorbol ester receptors. FASEB J .
2000, 13, 1658-1676.
(5) Sakai, N.; Sadaki, K.; Ikegaki, N.; Shirai, Y.; Ono, Y.; Saito, N.
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(6) Oancea, E.; Teruel, M. N.; Quest, A. F. G.; Meyer, T.
Green fluorescent protein (GFP)-tagged cysteine-rich domains
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gylcerol signaling in living cells. J . Cell Biol. 1998, 140, 485-
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(9) Nishizuka, Y. Intracelluar signaling by hydrolysis of phospho-
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S.; Lewin, N. E.; Bhattacharyya, D. K.; Blumberg, P. M.,
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1-(3-Isop r op yl-4-m eth ylp en t-2-en oyl)-2-(3-isop r op yl-4-
m eth ylp en ta n oyl)-sn -glycer ol (4C). According to the gen-
eral procedure, 1-(3-isopropyl-4-methylpent-2-enoyl)-2-(3-
isopropyl-4-methylpentanoyl)-3-benzyl-sn-glycerol was depro-
tected to give 4C in 86% yield. [R]20D ) -10.04 (c 0.44 CHCl3);
IR (neat) 3475 (OH), 2962 (CH), 2873 (CH), 1720 (CdO), 1637
1
(CdC) cm-1; H NMR (250 MHz, CDCl3) δ 0.90 and 0.99 (d,
J ) 6.6 Hz, 12 H, C(O)CH2CH(CHMe2)2), 1.13 (d, J ) 6.8 Hz,
6 H, C(O)CHdC(CHMe2)2), 1.15 (d, J ) 6.6 Hz, 6 H, C(O)CHd
C(CHMe2)2), 1.65-1.91 (m, 3 H, C(O)CH2CH(CHMe2)2), 2.27
(d, J ) 5.6 Hz, 2 H, C(O)CH2CH(CHMe2)2), 2.64 (heptet, J )
6.8 Hz, 1 H, C(O)CHdC(CHMe2)2), 3.82 (d, J ) 4.9 Hz, 2 H,
HOCH2CH(O-)CH2O), 4.11 (heptet, J ) 6.8 Hz, 1 H, C(O)-
CHdC(CHMe2)2), 4.33-4.41 (m, 2 H, HOCH2CH(O-)CH2O),
5.13-5.21 (m, 1 H, HOCH2CH(O-)CH2O), 5.72 (s, 1H, C(O)-
CHdC(CHMe2)2); 13C NMR (62.5 MHz, CDCl3) δ 18.66, 18.72,
20.20, 21.31, 24.09, 29.02, 29.32, 29.38, 29.79, 32.97, 46.76,
61.17, 61.44, 72.29, 111.78, 166.68, 174.39, 177.64; FAB-MS
(m/z, relative intensity): 371 (MH+, 10), 139 (100). Anal.
(C21H38O5) C, H.
1,2-Di-(3-isop r op yl-4-m e t h ylp e n t -2-e n oyl)-sn -glyc-
er ol (4D). According to the general procedure, 1,2-di-(3-
isopropyl-4-methylpent-2-enoyl)-3-benzyl-sn-glycerol was depro-
tected to give 4D in 99% yield. [R]20D ) -15.58 (c 1.54, CHCl3);
IR (neat) 3474 (OH), 2965 (CH), 2875 (CH), 1717 (CdO), 1637
1
(CdC) cm-1; H NMR (250 MHz, CDCl3) δ 1.12-1.18 (m, 24
H, C(O)CHdC(CHMe2)2), 2.31 (br s, 1 H, HOCH2CHCH2O),
2.64 (heptet, J ) 6.8 Hz, 1 H, C(O)CHdC(CHMe2)2), 2.65
(heptet, J ) 6.8 Hz, 1 H, C(O)CHdC(CHMe2)2), 3.85 (d, J )
4.9 Hz, 2 H, HOCH2CH(O-)CH2O), 4.10 (heptet, J ) 6.8 Hz,
1 H, C(O)CHdC(CHMe2)2), 4.12 (heptet, J ) 6.8 Hz, 1 H, C(O)-
CHdC(CHMe2)2), 4.39 (d, J ) 5.1 Hz, 2 H, HOCH2CH(O-)-
CH2O), 5.13-5.21 (m, 1 H, HOCH2CH(O-)CH2O), 5.75 and
5.77 (s, 1 H, C(O)CHdC(CHMe2)2); 13C NMR (62.5 MHz,
CDCl3) δ 20.16, 20.20, 24.07, 29.01, 29.07, 29.80, 61.35, 61.54,
71.57, 111.91, 112.07, 166.22, 166.76, 177.35, 177.56; FAB-
MS (m/z, relative intensity): 369 (MH+, 14), 139 (100). Anal.
(C21H36O5) C, H.
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