Conformationally constrained analogues of diacylglycerol (DAG). 23. Hydrophobic ligand-protein interactions versus ligand-lipid interactions of DAG-lactones with protein kinase C (PK-C)

Article abstract of DOI:10.1021/jm049723+

The constrained glycerol backbone of DAG-lactones, when combined with highly branched alkyl chains, has engendered a series of DAG-lactone ligands capable of binding protein kinase C (PK-C) with affinities that approximate those of phorbol esters. These b

Full text of DOI:10.1021/jm049723+

4858
J . Med. Chem. 2004, 47, 4858-4864
Con for m a tion a lly Con str a in ed An a logu es of Dia cylglycer ol (DAG). 23†.
Hyd r op h obic Liga n d -P r otein In ter a ction s ver su s Liga n d -Lip id In ter a ction s of
DAG-La cton es w ith P r otein Kin a se C (P K-C)
Hirokazu Tamamura,‡,§ Dina M. Sigano,‡ Nancy E. Lewin,| Megan L. Peach,‡ Marc C. Nicklaus,‡
Peter M. Blumberg,| and Victor E. Marquez*,‡
Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer InstitutesFrederick, National Institutes of
Health, Building 376, Room 104, Frederick, Maryland 21702, and Laboratory of Cellular Carcinogenesis & Tumor Promotion,
Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 37, Room 3A01,
Bethesda, Maryland 20892
Received April 12, 2004
The constrained glycerol backbone of DAG-lactones, when combined with highly branched alkyl
chains, has engendered a series of DAG-lactone ligands capable of binding protein kinase C
(PK-C) with affinities that approximate those of phorbol esters. These branched chains not
only appear to be involved in making important hydrophobic contacts with the protein (specific
interactions) but also provide adequate lipophilicity to facilitate partitioning into the lipid-
rich membrane environment (nonspecific interactions). With the idea of minimizing the
nonspecific interactions without reducing lipophilicity, the present work explores the strategy
of relocating lipophilicity from the side chain to the lactone “core”. Such a transfer of lipophilicity,
exemplified by compounds 1 and 3, was conceived to allow the new hydrophobic groups on the
lactone to engage in specific hydrophobic contacts inside the binding pocket without any
expectation of interfering with the hydrogen-bonding network of the DAG-lactone pharma-
cophore. Surprisingly, both (E)-3 and (Z)-3 showed a significant decrease in binding affinity.
From the molecular docking studies performed with the new ligands, we conclude that the
binding pocket of the C1 domain of PK-C is sterically restricted and prevents the methyl groups
at the C-3 position of the lactone from engaging in productive hydrophobic contacts with the
receptor.
In tr od u ction a n d Ba ck gr ou n d
pholipids.3,4 This association is strongly facilitated by
the second messenger, sn-1,2-diacylglycerol (DAG), which
is generated as a result of a stimulus-initiated activation
of phospholipase C.5 The accepted mechanism is that
these isozymes are cytosolic in the inactive state and
translocate to the inner leaflet of the cellular membrane
as part of the activation process.6,7 The binding of PK-C
to the plasma membrane is transient and is regulated
by the association of its C1 domain with DAG in the
membrane.8
These C1 domains in the classical and novel PK-C
isozymes contain two small (∼50 residues) zinc-finger-
like structures (C1a and C1b), consisting of two â sheets
and a small R helix. The crystal structure of a single
C1b domain from PK-Cδ in complex with the PK-C
activator, phorbol-13-O-acetate,9 revealed that the ligand
is bound inside the pocket between the two hydrophobic
â sheets, thus capping the C1b domain and forming a
continuous hydrophobic surface that promotes the in-
sertion of the binary complex into the membrane. This
X-ray structure also confirmed the importance of hy-
drogen bonding of the main phorbol ester pharmacoph-
ores (C-3, C-4, and C-20) with amino acids inside the
binding pocket and furthermore revealed that the
lipophilic acyl chains at C-12 and C-13 are important
for activity because they are projected into the proper
position to interact with membrane lipids in the ternary
complex.10-13
The central role of protein kinase C (PK-C) in cell-
signal transduction has been well established over the
past two decades since its discovery.1,2 The PK-C family
is comprised of 10 isozymes grouped into 3 classes:
conventional (R, â1 and â2, γ), novel (δ, ꢀ, η, and θ), and
atypical (ú and ι/λ). In addition, PK-C µ and ν are
considered by some to constitute a fourth class and by
others to compose a distinct family called protein kinase
D.2 All isozymes contain a C-terminal kinase domain
with serine/threonine specific kinase activity and an
N-terminal regulatory domain. The regulatory domain
contains two key functionalities: an autoinhibitory
sequence and one or two membrane-targeting domains
(C1 and C2).
Both classical and novel PK-C isozymes are thought
to become activated as a result of association of the
cytosolic enzyme with membranes containing acid phos-
* Author to whom correspondence should be addressed. Tel: 301-
846-5954. Fax: 301-846-6033. E-mail: marquezv@mail.nih.gov.
† Because of a clerical error resulting in two publications designated
as number 19, there is no paper no. 22. See Nacro, K. et al.
Conformationally constrained analogues of diacylglycerol (DAG). Part
19: Asymmetric syntheses of (3R)- and (3S)-3-hydroxy-4,4- disubsti-
tuted heptono-1,4-lactones as protein kinase C (PK-C) ligands with
increased hydrophilicity. Tetrahedron 2002, 58, 5335-5345 and Choi,
Y. et al. Conformationally constrained analogues of diacylglycerol. 19.
Synthesis and protein kinase C binding affinity of diacylglycerol
lactones bearing an N-hydroxylamide side chain. J . Med. Chem. 2003,
46, 2790-2793. Paper nos. 20 and #21 have already been published.
‡ Laboratory of Medicinal Chemistry.
Docking of DAG into the same empty C1b domain
revealed a similar pattern of hydrogen bonding involv-
ing pharmacophores at C-1, C-2, and C-3. Modeling
§ Present address: Graduate School of Pharmaceutical Sciences,
Kyoto University, Sakyo-ku, Kyoto 606-8501, J apan.
| Laboratory of Cellular Carcinogenesis & Tumor Promotion.
10.1021/jm049723+ This article not subject to U.S. Copyright. Published 2004 by the American Chemical Society
Published on Web 08/27/2004

Conformationally Constrained Analogues of DAG
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20 4859
of DAG-lactones, 1 and 2, which are known as effective
PK-C ligands with affinities in the low nanomolar range
(Figure 2). The removal of two carbons from the side
chain of 1 (log P ) 5.03) to give 2 (log P ) 4.04) reduced
the log P by almost 1 unit and caused a ca. 2.5-fold
reduction in PK-CR binding affinity. If instead of
removing two carbon units from the side chain of 1 we
transfer them to the C-3 position on the lactone ring to
give 3, then one would maintain a nearly identical log
P (log P ) 4.91). In addition, if the receptor was flexible
enough to accommodate the ligand in the usual manner,
then perhaps this would allow the new hydrophobic
groups on the lactone to engage in specific hydrophobic
contacts inside the binding pocket, without any expecta-
tion of interfering with the hydrogen-bonding network.
The aim of the present work was to synthesize target
compound 3 and test the basis of this hypothesis.
F igu r e 1. Structures of phorbol esters, DAG, and DAG-
lactone.
alone, however, does not explain the substantial differ-
ence in binding affinity (g3 orders of magnitude) that
exists between phorbol esters and DAG (Figure 1).14,15
Over the past few years, we have attempted to bridge
this 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 (Figure 1).16,17 A second approach involves
increasing the bulk of the acyl and R-alkylidene groups
in these DAG-lactones by incorporating highly branched
alkyl chains that are capable of making important
hydrophobic contacts with a group of conserved hydro-
phobic amino acids in the space between the two â
sheets of the C1b domain.16-19 An additional role for
these alkyl chains is to provide adequate lipophilicity,
expressed as a log P value, to facilitate partitioning into
the lipid-rich membrane environment of the cell. The
log P (octanol/water partition coefficient) is a measure
of the hydrophobic/hydrophilic balance of a molecule,
and in the present work, it is calculated by the frag-
ment-based program KOWWIN 1.67.20,21 Because of the
dual role of the alkyl chains, a higher binding affinity
due to increased lipophilicity is probably the result of
these two important factors: adequate membrane par-
titioning (nonspecific interactions) and hydrophobic
contacts with the protein (specific interactions). To
understand this complex relationship between affinity
(expressed as Ki) and hydrophobicity (log P), we have
examined plots of 1/Ki versus log P and determined that
the optimal log P for DAG-lactones lies somewhere
between 5 and 6.18 With the idea of reducing the
nonspecific interactions of the side chains without
dropping below the minimum log P threshold required
for adequate membrane distribution, we decided to
explore a strategy of transferring lipophilic methyl
groups from the side chain to the lactone “core”. Thus,
compound 3 was designed on the basis of the properties
Resu lts a n d Discu ssion
Ch em istr y. The essential methyl groups on the
lactone ring in 3 were introduced early in the synthesis
via a radical exo-trig cyclization from intermediate 4
(Scheme 1). This approach was based on the work of
Ueno and co-workers, who developed a particularly
useful synthesis of γ-butyrolactones via the stereose-
lective radical cyclization of bromoacetals in the pres-
ence of AIBN and tin hydride.22 Ueno’s methodology has
been considered to be particularly advantageous in cases
such as ours, where a sterically hindered carbon is
involved because it is known that this type of carbon-
carbon bond-forming reaction generally gives poor re-
sults. The exclusive exo-trig cyclization anticipated from
the Baldwin rules for ring closure ensured an efficient
route to our functionalized γ-butyrolactones. Racemic
intermediate 5, obtained from the Grignard reaction of
1-(benzyloxy)-3-(4-methoxyphenoxy)acetone23 and iso-
propenyl bromide, provided the requisite bromoacetal
(6) via alkoxybromination with ethyl vinyl ether in the
presence of NBS. Immediate radical cyclization led to
the dimethylated ethyl glycoside (7) in excellent yield.
Hydrolysis to the lactol (8) followed by oxidation gave
the essential dimethyl lactone (9). Aldol condensation
of 9 with 4-methyl-3-(methylethyl)pentan-1-one using
LDA gave the expected aldol product, but elimination
to the olefin using our standard protocol proved difficult.
Indeed, mesylation followed by attempted DBU elimi-
nation gave a mixture of unreacted alcohol, the corre-
sponding mesylate ester, and the lactone product of the
reverse aldol reaction with no elimination products
observed. However, when this reaction mixture was
further treated with NaH in DMF, the desired R-alky-
F igu r e 2. Structures of DAG-lactones 1-3 showing their corresponding log P values and PK-CR binding affinities (Ki) for
compounds 1 and 2.

4860 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20
Tamamura et al.
Sch em e 1a
Ta ble 1. Binding Affinities (Ki) of (Z)-3 and (E)-3 for the Intact
R-Isozyme and the Isolated C1b Domain of the δ-Isozymea
(Z)-3
(E)-3
(Z)-1
(E)-1
PK-CR
680 ( 47.3 1668 ( 17.2
2.90 ( 0.4
3.25 ( 0.15
(C1a + C1b)
PK-Cδ
1457 ( 95.4 3473 ( 102.8 1.16 ( 0.07 0.90 ( 0.07
(C1b)
a
Ki values are in nM.
In addition, such transfer of lipophilicity from the side
chain to the lactone core was also expected to increase
specific protein-ligand interactions with the receptor,
provided that the newly introduced methyl groups
would not interfere with the hydrogen-bonding network
involving the DAG-lactone pharmacophores. Surpris-
ingly, the corresponding (Z)-3 isomer showed a ca. 230-
fold decrease in binding affinity to PK-CR and an even
higher ca. 1250-fold drop in binding affinity to the
isolated C1b domain of PK-Cδ, relative to that of
compound 1 (Table 1). The (E)-3 isomer experienced
binding reductions of ca. 500-fold and ca. 3800-fold to
PK-CR and PK-Cδ(C1b), respectively (Table 1).
Docking of either (Z)-3 or (E)-3 into the empty C1b
domain of PK-Cδ, whose coordinates were obtained from
the crystal structure of the binary complex of this
domain with phorbol-13-O-acetate,9 showed that the
methylated lactone does not fit as well as the unsub-
stituted lactone into the C1 domain. The lactone ring
is forced to tilt up slightly and is unable to penetrate
deeply into the binding pocket because of steric clashes
between the methyl groups and Pro 241 (Figure 3), and
as a result, optimal distances of important hydrogen
bonds in the complex are affected. Although such an
unexpected steric barrier has been observed only for the
two isozymes (R and δ) reported here, the highly
conserved nature of this proline throughout the entire
set of isozymes, except for the atypical PK-Cú and PK-
Cι/λ, which do not bind DAG, suggests this would hold
true for the rest of the PK-C family.
a
(a) isopropenylmagnesium bromide, THF, -78 f -25 °C
(50%); (b) ethyl vinyl ether, 4-Å molecular sieves, NBS, CH2Cl2, 0
°C (22%); (c) n-Bu3SnH, AIBN, C6H6, ∆ (91%); (d) HCl, THF, H2O
0 °C (77%); (e) TPAP, NMO, 4Å ms, CH2Cl2/CH3CN, 0 °C f rt
(66%); (f) LDA, i-Pr2CHCH2CHO, THF, -78 °C; (g) MsCl, Et3N,
CH2Cl2, 0 °C f rt; (h) DBU, 0 °C f rt; (i) NaH, DMF (26% (Z)-10
and 13% (E)-10 for four steps); (j) CAN, CH3CN/H2O, 0 °C (72%
(Z)-11 and 57% (E)-11); (k) (CH3)3C(O)Cl, DMAP, pyridine (88%
(Z)-12 and 92% (E)-12); (l) BCl3, CH2Cl2, -78 °C (92% (Z)-3 and
93% (E)-3).
From this result, we conclude that the binding pocket
of the C1 domain of PK-C is probably narrower, steri-
cally restricted, and more rigid than previously antici-
pated. Such an apparent rigidity and narrowness of the
binding pocket must, therefore, be considered in the
future design of new DAG-lactones. In previous work,24
when a single hydroxyl group was introduced at C-3 of
the lactone, in both stereochemical dispositions, the
resulting compounds were also weaker ligands, and
binding affinities dropped 10- to 200-fold. This drop in
binding affinity reflected the absence of additional
hydrophilic contacts between the hydroxyl groups with
the receptor and the corresponding desolvation penalty
incurred during binding in a lipid milieu. From this
work, we have now learned that adding lipophilic
methyl groups to the same position also reduces binding
affinity suggesting that the C-3 position of the DAG-
lactones must remain intact without substitution. This
finding also highlights the structural rigidity of the first
loop in the binding site (residues 238-243) perhaps due
in part to the presence of the conserved proline 241
residue discussed above.
lidene DAG-lactone 10 was obtained as a mixture of E
and Z isomers, which were easily separated by silica
gel column chromatography. Consistent with previously
synthesized DAG-lactones, the vinyl proton of the Z
isomer displayed a characteristic triplet at δ 6.0 in its
1H NMR spectra, whereas the corresponding signal of
the E isomer appeared more downfield at δ ) 6.7.18
Removal of the protecting p-methoxyphenyl group from
each isomer with CAN was followed by acylation with
pivaloyl chloride to give penultimate intermediates (E)-
12 and (Z)-12, which after a final deprotection with BCl3
to remove the benzyl groups afforded the desired target
dimethyl DAG-lactones (E)-3 and (Z)-3 in excellent
yield.
Biologica l Resu lts a n d Discu ssion
The synthetic strategy described above essentially
resulted in the transferring of two hydrophobic methyl
groups of ca. 0.5 log P units each from the R-alkylidene
side chain of high-affinity PK-C ligand 1 to the lactone
core of slightly weaker DAG-lactone 2. The intent of this
exercise, leading to DAG-lactone 3, was to minimize the
nonspecific interactions involving the branched side
chain with the membrane while maintaining the favor-
able log P of DAG-lactone 1, which was indeed cor-
roborated by the 0.12 log P difference between 1 and 3.
Con clu sion s
It is clear from this study that geminal methyl groups
at the C-3 position of the lactone hinder penetration of

Conformationally Constrained Analogues of DAG
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20 4861
F igu r e 3. Overlapping of (Z)-2 (green) and (Z)-3 (yellow) showing the poorer penetration of (Z)-3 due to its slightly twisted
position (left) and top view showing the steric interaction between the methyl groups of (Z)-3 (yellow) and Pro 241 (right).
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. 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, Georgia.
1-(4-Meth oxyp h en oxy)-3-m eth yl-2-[(p h en ylm eth oxy)-
m eth yl]bu t-3-en -2-ol (5). A -78 °C stirring solution of
1-(benzyloxy)-3-(4-methoxyphenoxy)acetone23 (1.18 g, 4.1 mmol)
in THF (5 mL) was treated dropwise with isopropenylmagne-
sium bromide. The temperature was raised to -25 °C, and the
solution was stirred at this temperature for 2 h. The reaction
was quenched with saturated aqueous NH4Cl and concentrated
in vacuo. The residue was extracted with EtOAc, washed with
water, dried over MgSO4, and concentrated. Purification by
flash column chromatography gave 5 (675 mg, 50%) as an oil.
IR (neat) 3571 (OH), 3019 (CH), 2934 (CH), 2868 (CH), 1643
the ligand into the active site, fail to engage in positive
hydrophobic contacts with the receptor, and possibly
weaken the hydrogen-bonding network of the DAG-
lactone pharmacophores. It can also be concluded that
the binding pocket must be quite narrow and not very
flexible because, despite having an adequate lipophilic-
ity, compounds (Z)-3 and (E)-3 cannot bind effectively
inside the pocket. We can surmise that the C-3 position
of the DAG-lactones must remain intact without sub-
stitution, and such constraints must, therefore, be
considered in the future design of new DAG-lactones.
Exp er im en ta l Section
Molecu la r Mod elin g. The structure for the methylated
lactone (compound (Z)-3) was built in Sybyl25 and minimized
with the MMFF94 force field and partial charges.26 Docking
was then performed, using FlexX27 through its SYBYL module,
into the crystal structure of the C1b domain of PK-Cδ.9 The
binding site was defined as residues 238-243, 250-254, and
257. The ring structure of the ligand was treated flexibly, and
all other options were set to their default values. Figures were
generated using MolScript28 and Raster3D.29
1
(CdC) cm-1; H NMR (CDCl3) δ 1.95 (s, 3 H, CC(CH3)dCH2),
2.92 (br s, 1 H, COH), 3.72-3.84 (app q, 2 H, PhCH2OCH2C),
3.86 (s, 3 H, CH3OC6H4OCH2C), 4.12 (s, 2 H, CH3OC6H4-
OCH2C), 4.69 (s, 2 H, PhCH2OCH2C), 5.16 (br s, 1 H,
CC(CH3)dCHH), 5.33 (br s, 1 H, CC(CH3)dCHH), 5.33-6.98
(m, 4 H, CH3OC6H4OCH2C), 7.34-7.45 (m, 5 H, PhCH2-
OCH2C);13C NMR (CDCl3) δ 19.65, 55.64, 71.74, 72.84, 73.55,
76.15, 112.87, 114.48, 115.66, 127.56, 127.61, 128.29, 137.79,
145.39, 152.76, 153.94; FAB-MS (m/z, relative intensity): 328
(MH+•, 39), 91 (100). Anal. (C20H24O4) C, H.
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 a n d log P . Enzyme-ligand interac-
tions were analyzed by competition with [3H]PDBU binding
for the single isozyme PK-CR essentially as described previ-
ously.18 The ID50 values were determined by least-squares
fitting of the theoretical sigmoidal competition curve to the
binding data. The Ki was calculated from the ID50 values
according to the relationship
2-Br om o-1-m eth oxy-1-{1-[(4-m eth oxyph en oxy)m eth yl]-
2-m et h yl-1-[(p h en ylm et h oxy)m et h yl]p r op -2-en yloxy}-
eth a n e (6). Under an argon atmosphere, compound 5 (679
mg, 2.07 mmol) was added to a room-temperature solution of
ethylvinyl ether (1.5 mL, 15.6 mmol) in CH2Cl2 (3 mL) in a
flask containing dried and activated 4-Å molecular sieves (2
g). The reaction was cooled to 0 °C, NBS (552 mg, 3.10 mmol)
was added, and the reaction was stirred for 2 h. After reaching
ambient temperature, the solution was filtered, and the filtrate
was reduced to dryness in vacuo. Following purification by
silica gel flash column chromatography, compound 6 (221 mg,
22%) was obtained as an inseparable mixture of diastereomers.
ID50
Ki )
L
1 +
Kd
where L is the concentration of free [3H]PDBU at the ID50 and
Kd is the dissociation constant for [3H]PDBU under the assay
conditions.30 Values represent the mean ( standard error
(three determinations). The octanol/water partition coefficients
(log P) were calculated according to the fragment-based
program KOWWIN 1.67 (http://www.epa.gov/oppt/exposure/
docs/episuitedl.htm).21
Gen er a l P r oced u r es. All chemical reagents were com-
mercially available. AIBN ) 2,2′-azobisisobutyronitrile, CAN
) cerium(IV) ammonium nitrate, DMF ) N,N-dimethylform-
amide, DMAP ) dimethylamino pyridine, LDA ) lithium
diisopropylamide, MPM ) methoxyphenylmethyl, MsCl )
methanesulfonyl chloride, NBS ) N-bromosuccinimide, NMO
) 4-methylmorpholine N-oxide, and TPAP ) tetrapropyl-
ammonium perruthenate. Column chromatography was per-
formed on silica gel 60, 230-400 mesh (E. Merck). 1H and 13C
IR (neat) 3020 (CH), 2979 (CH), 2935 (CH), 1642 (CdC) cm-1
;
FAB-MS (m/z, relative intensity): 478 (MH+•, 30), 91 (100).
Anal. (C24H31BrO5) C, H.
1-({5-Eth oxy-3,3-d im eth yl-2-[(p h en ylm eth oxy)m eth yl]-
oxola n -2-yl}m eth oxy)-4-m eth oxyben zen e (7). According to
a literature procedure,22 n-Bu3SnH (165 µL, 0.61 mmol) was
added dropwise over 30 min, under a blanket of argon, to a
solution of 6 (196 mg, 0.41 mmol) and AIBN (1.34 mg, 0.008
mmol) in benzene (2 mL) at room temperature. After 4 h, the
reaction mixture was heated to 45 °C for 4 h and then stirred
at room temperature for 1 day. Additional n-Bu3SnH (200 µL,

4862 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20
Tamamura et al.
0.75 mmol) was then added, and the reaction was again heated
to 50 °C for 4 h and then stirred at room temperature for 1
day. The reaction mixture was applied directly to a silica gel
flash chromatography column to give compound 7 (150 mg,
91%) as an inseparable mixture of diastereomers. IR (neat)
3019 (CH), 2963 (CH), 2934 (CH) cm-1; FAB-MS (m/z, relative
intensity): 400 (MH+•, 46), 91 (100). Anal. (C24H32O5‚0.8H2O)
C, H.
3 H, CdCHCH2CH(CH(CH3)2)2), 0.97 and 1.00 (d, J ) 1.7 Hz,
3H,CdCHCH2CH(CH(CH3)2)2),1.15-1.26(m,1H,CdCHCH2CH-
(CH(CH3)2)2), 1.31 and 1.34 (s, 3 H, lactone-CH3), 1.73-1.97
(m, 2 H, CdCHCH2CH(CH(CH3)2)2), 2.78 (d, J ) 5.9 Hz, 1 H,
CdCHCHHCH(CH(CH3)2)2), 2.81 (d, J ) 6.1 Hz, 1 H, CdCH-
CHHCH(CH(CH3)2)2), 3.79-3.89 (m, 2 H, PhCH2OCH2C), 3.85
(s, 3 H, CH3OC6H4OCH2C), 4.21 (AB quartet, J ) 9.8 Hz, 2 H,
CH3OC6H4OCH2C), 4.64 (AB quartet, J ) 12.2 Hz, 2 H, PhCH2-
OCH2C), 6.02 (t, J ) 7.3 Hz, 1 H, CdCHCH2CH(CH(CH3)2)2),
6.89 (s, 4 H, CH3OC6H4OCH2C), 7.33-7.40 (m, 5 H, PhCH2-
OCH2C);13C NMR (CDCl3) δ 19.45, 21.70, 21.72, 23.88, 24.21,
25.81, 29.34, 29.38, 44.16, 51.35, 55.63, 68.85, 70.25, 73.60,
85.22, 114.47, 115.53, 127.35, 127.51, 128.21,134.89, 137.57,
141.77, 152.29, 154.07, 168.88; FAB-MS (m/z, relative inten-
sity): 494 (M+•, 51), 91 (100). Anal. (C31H42O5) C, H. (E)-10,
5-[(4-Meth oxyp h en oxy)m eth yl]-4,4-d im eth yl-5-[(p h en -
ylm et h oxy)m et h yl]oxola n -2-ol (8). HCl (2 N, 7 mL) was
added at 0 °C to a solution of 7 (222 mg, 0.55 mmol) in THF
(10.5 mL) and H2O (4 mL). After 5 h at 0 °C, the reaction was
allowed to reach room temperature and was stirred overnight.
The reaction was quenched with solid NaHCO3 followed by
H2O. The aqueous solution was extracted with CH2Cl2, dried,
and concentrated. Purification by flash column chromatogra-
phy gave 8 (140 mg, 77%) as an inseparable mixture of
diastereomers. IR (neat) 3484 (OH), 3020 (CH), 2956 (CH),
IR (neat) 3020 (CH), 2958 (CH), 2891 (CH), 1750 (CdO) cm-1
;
1H NMR (CDCl3) δ 0.92 and 0.95 (d, J ) 2.0 Hz, 3 H,
CdCHCH2CH(CH(CH3)2)2), 0.97 and 0.99 (br s, 3 H, CdCH-
CH2CH(CH(CH3)2)2), 1.25-1.37 (m, 1 H, CdCHCH2CH(CH-
(CH3)2)2), 1.49 and 1.51 (s, 3 H, lactone-CH3), 1.82-1.92 (m, 2
H, CdCHCH2CH(CH(CH3)2)2), 2.38 (t, J ) 6.6 Hz, 2 H, CdCH-
CH2CH(CH(CH3)2)2), 3.82-3.94 (m, 2 H, PhCH2OCH2C), 3.85
(s, 3 H, CH3OC6H4OCH2C), 4.24 (AB quartet, J ) 9.8 Hz, 2 H,
CH3OC6H4OCH2C), 4.63 (s, 2 H, PhCH2OCH2C), 6.72 (t, J )
7.1 Hz, 1 H, CdCHCH2CH(CH(CH3)2)2), 6.84-6.92 (m, 4 H,
CH3OC6H4OCH2C), 7.35-7.42 (m, 5 H, PhCH2OCH2C);13C
NMR (CDCl3) δ 19.50, 19.60, 21.43, 22.64, 22.76, 25.30, 28.91,
28.96, 43.72, 50.87, 55.64, 69.21, 70.58, 73.66, 85.13, 114.48,
115.38, 127.28, 127.52, 128.22, 134.83, 137.51, 140.39, 152.21,
154.06, 170.38; FAB-MS (m/z, relative intensity): 495 (MH+,
49), 91 (100). Anal. (C31H42O5‚0.2H2O) C, H.
2876 (CH) cm-1; FAB-MS (m/z, relative intensity): 372 (MH+•
7), 91 (100). Anal. (C22H28O5‚0.1H2O) C, H.
,
5-[(4-Meth oxyp h en oxy)m eth yl]-4,4-d im eth yl-5-[(p h en -
ylm eth oxy)m eth yl]-3,4,5-tr ih yd r ofu r a n -2-on e (9). Accord-
ing to a literature procedure,31 solid TPAP (5 mol %, 506 mg,
0.16 mmol) was added in one portion to a stirred mixture of 8
(119 mg, 0.32 mmol), NMO (60 mg, 0.48 mmol), and powdered
4-Å molecular sieves (1650 mg) in CH2Cl2/CH3CN (9/1, 0.65
mL) at 0 °C under argon. After 4.5 h, the reaction mixture
was warmed to room temperature and stirred for 1 day.
Additional NMO (35 mg) was added, and the reaction was
stirred for an extra day. The crude mixture was then filtered
through Celite, and the filtrate was concentrated. Purification
by silica gel flash column chromatography gave 9 (78 mg, 66%)
as an oil. IR (neat) 3021 (CH), 2938 (CH), 2877 (CH), 1774
(CdO) cm-1; 1H NMR (CDCl3) δ 1.32 and 1.36 (s, 3 H, lactone-
CH3), 4.63 (AB quartet, J ) 17.2 Hz, 2 H, lactone-CH2), 3.90
(AB quartet, J ) 10.2 Hz, 2 H, PhCH2OCH2C), 3.85 (s, 3 H,
CH3OC6H4OCH2C), 4.23 (AB quartet, J ) 10.0 Hz, 2 H, CH3-
OC6H4OCH2C), 4.64 (s, 2 H, PhCH2OCH2C), 6.92 (s, 4 H, CH3-
OC6H4OCH2C), 7.34-7.47 (m, 5 H, PhCH2OCH2C);13C NMR
(CDCl3) δ 24.00, 24.11, 40.55, 45.28, 55.64, 69.82, 71.32, 73.75,
87.54, 114.58, 115.45, 127.45, 127.70, 128.34, 137.37, 152.07,
(Z)-5-(Hyd r oxym eth yl)-4,4-dim eth yl-3-[4-m eth yl-3-(m e-
th yleth yl)p en tylid en e]-5-[(p h en ylm eth oxy)m eth yl]-4,5-
d ih yd r ofu r a n -2-on e ((Z)-11). According to a literature pro-
cedure,23 ammonium cerium(IV) nitrate was added to a 0 °C
solution of (Z)-10 (104 mg, 0.21 mmol) in CH3CN/H2O (4/1,
3.5 mL). After 30 min, the reaction mixture was diluted with
CH2Cl2, and the layers were separated. The organic layer was
washed with brine, dried, and concentrated. Purification by
silica gel flash column chromatography gave (Z)-11 (59 mg,
72%) as an oil. IR (neat) 3591 (OH), 3022 (CH), 2961 (CH),
2873 (CH), 1750 (CdO), 1664 (CdC) cm-1; 1H NMR (CDCl3) δ
0.91, 0.94, 0.96 and 0.99 (s, 3 H, CdCHCH2CH(CH(CH3)2)2),
1.12-1.22 (m, 1 H, CdCHCH2CH(CH(CH3)2)2), 1.25 and 1.27
(s, 3 H, lactone-CH3), 1.78-1.91 (m, 2 H, CdCHCH2CH-
(CH(CH3)2)2), 2.42 (br s, 1 H, HOCH2C), 2.68-2.79 (m, 2 H,
CdCHCH2CH(CH(CH3)2)2), 3.71-3.93 (overlapping AB quar-
tets, J ) 12.0 and 9.7 Hz, 4 H, C6H5CH2OCH2C and HOCH2C),
4.62 (AB quartet, J ) 12.2 Hz, 2 H, C6H5CH2OCH2C), 6.03 (t,
J ) 7.3 Hz, 1 H, CdCHCH2CH(CH(CH3)2)2), 7.34-7.42 (m, 5
H, PhCH2OCH2C);13C NMR (CDCl3) δ 19.37, 19.40, 21.69,
21.72, 22.82, 24.41, 25.85, 29.36, 29.39, 44.01, 51.32, 63.41,
69.42, 73.69, 127.45, 127.65, 128.28, 134.47, 137.42, 142.78,
169.04; FAB-MS (m/z, relative intensity): 389 (MH+, 40), 91
(100). Anal. (C24H36O4‚0.5H2O) C, H.
(E)-5-(Hyd r oxym eth yl)-4,4-dim eth yl-3-[4-m eth yl-3-(m e-
th yleth yl)p en tylid en e]-5-[(p h en ylm eth oxy)m eth yl]-4,5-
d ih yd r ofu r a n -2-on e ((E)-11). According to the same litera-
ture procedure as used for (Z)-11,23 ammonium cerium(IV)
nitrate was added to a 0 °C solution of (E)-10 (52 mg, 0.11
mmol) in CH3CN/H2O (4/1, 1.8 mL) to give (E)-11 (23 mg, 57%)
as an oil. IR (neat) 3692 (OH), 3020 (CH), 2959 (CH), 2870
(CH), 1734 (CdO), 1664 (CdC) cm-1; 1H NMR (CDCl3) δ 0.92
and 0.95 (d, J ) 1.2 Hz, 3 H, CdCHCH2CH(CH(CH3)2)2), 0.97
and 0.99 (d, J ) 3.4 Hz, 3 H, CdCHCH2CH(CH(CH3)2)2), 1.24-
1.34 (m, 1 H, CdCHCH2CH(CH(CH3)2)2), 1.42 and 1.48 (s, 3
H, lactone-CH3), 1.80-1.92 (m, 2 H, CdCHCH2CH(CH(CH3)2)2),
2.13 (br s, 1 H, HOCH2C), 2.34-2.39 (irregular t, 2 H, CdCH-
CH2CH(CH(CH3)2)2), 3.82 (AB quartet, J ) 10.0 Hz, 4 H, C6H5-
CH2OCH2C), 3.92 (s, 2 H, HOCH2C), 4.62 (AB quartet, J )
12.0 Hz, 2 H, C6H5CH2OCH2C), 6.72 (t, 1 H, J ) 6.9 Hz, CdC-
154.22, 175.74; FAB-MS (m/z, relative intensity): 370 (M+•
,
98), 91 (100). Anal. (C22H26O5) C, H.
5-[(4-Meth oxyph en oxy)m eth yl]-4,4-dim eth yl-3-[4-m eth -
yl-3-(m et h ylet h yl)p en t ylid en e]-5-[(p h en ylm et h oxy)m e-
th yl]-4,5-d ih yd r ofu r a n -2-on e (10). A stirred solution of 9
(514 mg, 1.39 mmol) in THF (4 mL) was treated dropwise,
under a blanket of argon at -78 °C, with LDA (1.11 mL, 2 M,
in heptane/THF/ethylbenzene). After stirring at -78 °C for 2
h, a solution of 3,3-diisopropylpropionaldehyde18 in THF (1 mL)
was added dropwise at the same temperature. After stirring
at -78 °C for 1 day, the reaction was quenched with saturated
aqueous NH4Cl, and then allowed to reach ambient temper-
ature. The aqueous layer was extracted with diethyl ether, and
the extract was washed with H2O, dried, and concentrated to
give an oil, which was then immediately taken up in CH2Cl2
(15 mL). Et3N (774 µL, 5.5 mmol) was added, and the resulting
solution was cooled to 0 °C. MsCl was then added dropwise,
and the solution was stirred at the same temperature for 30
min and then at room temperature for 2 h. The reaction
mixture was cooled again to 0 °C, DBU (1.04 mL, 6.94 mmol)
was added, and the resulting solution was stirred at room
temperature for 3 h. Concentration and purification by silica
gel flash column chromatography gave a mixture of products
that were immediately dissolved in DMF (0.4 mL) and treated
with NaH (6 mg, 60% immersion in mineral oil) at 0 °C for 1
h. The reaction was then cooled to -78 °C, quenched with
saturated aqueous NH4Cl, and warmed to room temperature.
The resulting mixture was extracted with diethyl ether, and
the extract washed with brine, dried, and concentrated.
Purification by silica gel flash column chromatography gave
(Z)-10 (52 mg, 26%) and (E)-10 (104 mg, 13%) after four steps.
(Z)-10, IR (neat) 3020 (CH), 2959 (CH), 2872 (CH), 1751
(CdO) cm-1; 1H NMR (CDCl3) δ 0.93 and 0.95 (d, J ) 2.2 Hz,
HCH2CH(CH(CH3)2)2), 7.36-7.42 (m,
5
H, PhCH2O-
CH2C);13C NMR (CDCl3) δ 19.48, 19.54, 21.36, 21.50, 22.03,
22.79, 25.40, 28.93, 28.99, 43.62, 50.88, 63.55, 69.49, 73.74,

Conformationally Constrained Analogues of DAG
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20 4863
86.09, 127.37, 127.64, 128.27, 134.26, 137.42, 141.99, 170.41;
FAB-MS (m/z, relative intensity): 389 (MH+, 91), 91 (100).
Anal. (C24H36O4‚0.2H2O) C, H.
relative intensity): 383 (MH+, 73), 57 (100). Anal. (C22H38O5‚
0.1H2O) C, H.
(E )-{2-(h yd r oxym e t h yl)-3,3-d im e t h yl-4-[4-m e t h yl-3-
(m et h ylet h yl)p en t ylid en e]-5-oxo-2-2,3-d ih yd r ofu r yl}-
m eth yl 2,2-d im eth ylp r op a n oa te ((E)-3). A solution of BCl3
(0.08 mL, 1 M in CH2Cl2) was added slowly to a -78 °C
solution of (E)-12 (20.4 mg, 0.04 mmol) and was stirred for 2
h at the same temperature. Following a similar work up as
that for (Z)-3, compound (E)-3 (15.3 mg, 93%) was obtained
as an oil. IR (neat) 3589 (OH), 3020 (CH), 2962 (CH), 2875
(CH), 1751 (CdO), 1664 (CdC) cm-1; 1H NMR (CDCl3) δ 0.93
and 0.96 (d, J ) 3.2 Hz, 3 H, CdCHCH2CH(CH(CH3)2)2), 0.98
and 1.01 (s, 3 H, CdCHCH2CH(CH(CH3)2)2), 1.28 (s, 9 H,
(CH3)3CO2CH2C), 1.30-1.36 (m, 1 H, CdCHCH2CH(CH-
(CH3)2)2), 1.46 and 1.48 (s, 3 H, lactone-CH3), 1.85-2.10 (m, 2
H, CdCHCH2CH(CH(CH3)2)2), 2.10 (br s, 1 H, HOCH2C),
2.35-2.41 (m, 2 H, CdCHCH2CH(CH(CH3)2)2), 3.89 (s, 2 H,
HOCH2C), 4.41 (AB quartet, J ) 12.1 Hz, 2 H, (CH3)3-
CO2CH2C), 6.76 (irregular t, J ) 7.0 Hz, 1 H, CdCHCH2CH-
(CH(CH3)2)2);13C NMR (CDCl3) δ 19.41, 19.60, 21.38, 21.41,
22.45, 22.72, 25.44, 27.00, 28.89, 28.97, 38.73, 43.43, 50.84,
62.60, 63.60, 85.29, 134.08, 142.30, 169.96, 177.92; FAB-MS
(m/z, relative intensity): 383 (MH+, 77), 57 (100). Anal.
(C22H38O5) C, H.
(Z)-{3,3-Dim et h yl-4-[4-m et h yl-3-(m et h ylet h yl)p en t yl-
id en e]-5-oxo-2-[(p h en ylm eth oxy)m eth yl]-2-2,3-d ih yd r o-
fu r yl}m et h yl-2,2-d im et h ylp r op a n oa t e ((Z)-12). Under a
blanket of argon, trimethylacetyl chloride (59 µL, 0.0.48 mmol)
was added to a 0 °C solution of (Z)-11 (460 mg, 0.12 mmol)
and DMAP (5 mg, 0.041 mmol) in pyridine (539 µL). The
resulting solution was allowed to warm to room temperature
over 7 h and then was concentrated. Purification by silica gel
flash column chromatography gave (Z)-12 (50 mg, 88%) as an
oil. IR (neat) 3020 (CH), 2962 (CH), 2872 (CH), 1752 (CdO),
1
1665 (CdC) cm-1; H NMR (CDCl3) δ 0.92 and 0.94 (s, 3 H,
CdCHCH2CH(CH(CH3)2)2), 0.97 (d, J ) 4.4 Hz, 3 H, CdCH-
CH2CH(CH(CH3)2)2), 0.99 (d, J ) 4.6 Hz, 3 H, CdCHCH2CH-
(CH(CH3)2)2), 1.24 (s, 9 H, (CH3)3CO2CH2C), 1.27 (br s, 6 H,
lactone-CH3), 1.78-1.94 (m, 2 H, CdCHCH2CH(CH(CH3)2)2),
2.51-2.62 (m, 1 H, CdCHCHHCH(CH(CH3)2)2), 2.91-3.03 (m,
1 H, CdCHCHHCH(CH(CH3)2)2), 3.72 (AB quartet, J ) 9.5
Hz, 2 H, PhCH2OCH2C), 4.35 (AB quartet, J ) 12.2 Hz, 2 H,
(CH3)3CO2CH2C), 4.59 (s, 2 H, PhCH2OCH2C), 6.02 (dd, 1 H,
J ) 8.3, 6.4 Hz, CdCHCH2CH(CH(CH3)2)2), 7.34-7.41 (m, 5
H, PhCH2OCH2C);13C NMR (CDCl3) δ 19.22, 19.58, 21.57,
21.79, 23.45, 24.54, 25.77, 26.99, 29.24, 29.45, 38.69, 44.03,
51.31, 64.49, 69.72, 73.65, 84.52, 127.42, 127.65, 128.26,
134.54, 137.27, 142.33, 168.64, 177.60; FAB-MS (m/z, relative
intensity): 473 (MH+, 28), 91 (100). Anal. (C29H44O5) C, H.
Su p p or tin g In for m a tion Ava ila ble: Elemental analysis
data for 5-10, (E)- and (Z)-3, 11, 12, and (E)-10. This material
is available free of charge via the Internet at http://
pubs.acs.org.
(E)-{3,3-Dim et h yl-4-[4-m et h yl-3-(m et h ylet h yl)p en t yl-
id en e]-5-oxo-2-[(p h en ylm eth oxy)m eth yl]-2-2,3-d ih yd r o-
fu r yl}m eth yl-2,2-d im eth ylp r op a n oa te ((E)-12). Following
the same procedure as that used for (Z)-12, (E)-11 (20 mg, 0.05
mmol) was converted to give (E)-12 (23 mg, 92%) as an oil. IR
(neat) 3020 (CH), 2962 (CH), 2874 (CH), 1731 (CdO), 1665
Refer en ces
(1) Castagna, M.; Takai, Y.; Kaibuchi, K.; Sano, K.; Kikkawa, U.;
Nishizuka, Y. Direct activation of calcium-activated, phospho-
lipid-dependent protein kinase by tumor-promoting phorbol
esters. J . Biol. Chem. 1982, 257, 7847-7851.
(CdC) cm-1 1H NMR (CDCl3) δ 0.92 and 0.95 (s, 3 H,
;
CdCHCH2CH(CH(CH3)2)2), 0.96 and 0.99 (d, J ) 4.4 Hz, 3 H,
CdCHCH2CH(CH(CH3)2)2), 1.24 (s, 9 H, (CH3)3CO2CH2C),
1.26-133 (m, 1 H, CdCHCH2CH(CH(CH3)2)2), 1.43 (s, 3 H,
lactone-CH3), 1.49 (s, 3 H, lactone-CH3), 1.83-1.93 (m, 2 H,
CdCHCH2CH(CH(CH3)2)2), 2.34-2.39 (m, 2 H, CdCHCH2CH-
(CH(CH3)2)2), 3.77 (s, 2 H, PhCH2OCH2C), 4.35 (AB quartet,
J ) 12.2 Hz, 2 H, (CH3)3CO2CH2C), 4.60 (s, 2 H, PhCH2-
OCH2C), 6.72 (irregular t, 1 H, J ) 6.9 Hz, CdCHCH2CH-
(CH(CH3)2)2), 7.34-7.45 (m, 5 H, PhCH2OCH2C); 13C NMR
(CDCl3) δ 19.32, 19.68, 21.27, 21.45, 22.45, 22.82, 25.31, 26.94,
28.79, 28.97, 38.68, 43.55, 50.77, 65.15, 70.18, 73.73, 84.49,
12.737, 127.66, 128.27, 134.60, 137.20, 140.98, 170.04, 177.61;
FAB-MS (m/z, relative intensity): 473 (MH+, 45), 91 (100).
Anal. (C29H44O5) C, H.
(2) Newton, A. C. Protein kinase C: Structural and spatial regula-
tion by phosphorylation, cofactors, and macromolecular interac-
tions. Chem. Rev. 2001, 101, 2353-2364.
(3) Newton, A. C.; Keranen, L. M. Phosphatidyl-L-serine is necessary
for protein-kinase Cs high-affinity interaction with diacylglyc-
erol-containing membranes. Biochemistry 1994, 33, 6651-6658.
(4) Newton, A. C. Regulation of protein kinase C. Curr. Opin. Cell
Biol. 1997, 9, 161-167.
(5) Nishizuka, Y. Intracellular signaling by hydrolysis of phospho-
lipids and activation of protein-kinase-C. Science 1992, 258,
607-614.
(6) Oancea, E.; Teruel, M. N.; Quest, A. F. G.; Meyer, T. Green
fluorescent protein (GFP)-tagged cysteine-rich domains from
protein kinase C as fluorescent indicators for diacylglycerol
signaling in living cells. J . Cell Biol. 1998, 140, 485-498.
(7) Sakai, N.; Sasaki, K.; Ikegaki, N.; Shirai, Y.; Ono, Y.; Saito, N.
Direct visualization of the translocation of the gamma-subspecies
of protein kinase C in living cells using fusion proteins with
green fluorescent protein. J . Cell Biol. 1997, 139, 1465-1476.
(8) Oancea, E.; Meyer, T. Protein kinase C as a molecular machine
for decoding calcium and diacylglycerol signals. Cell 1998, 95,
307-318.
(9) Zhang, G. G.; Kazanietz, M. G.; Blumberg, P. M.; Hurley, J . H.
Crystal-structure of the Cys2 activator-binding domain of protein-
kinase C-delta in complex with phorbol ester. Cell 1995, 81,
917-924.
(10) Van Duuren, B. L.; Tseng, S.-S.; Segal, A.; Smith, A. C.;
Melchionne, S.; Seidman, I. Effects of structural changes on the
tumor-promoting activity of phorbol myristate acetate on mouse
skin. Cancer Res. 1979, 39, 2644-2646.
(11) Baird, W. M.; Boutwell, R. K. Tumor-promoting activity of
phorbol and four diesters of phorbol in mouse skin. Cancer Res.
1971, 31, 1074-1079.
(12) El-Mekkawy, S.; Meselhy, M. R.; Nakamura, N.; Hattori, M.;
Kawahata, T.; Otake, T. 12-O-acetylphorbol-13-decanoate po-
tently inhibits cytopathic effects of human immunodeficiency
virus type 1 (HIV-1), without activation of protein kinase C.
Chem. Pharm. Bull. 1999, 47, 1346-1347.
(13) Krauter, G.; VonderLieth, C. W.; Schmidt, R.; Hecker, E.
Structure/activity relationships of polyfunctional diterpenes of
the tigliane typesA pharmacophore model for protein-kinase-C
activators based on structure/activity studies and molecular
modeling of the tumor promoters 12-O- tetradecanoylphorbol
13-acetate and 3-O-tetradecanoylingenol. Eur. J . Biochem. 1996,
242, 417-427.
(Z)-{2-(H yd r oxym et h yl)-3,3-d im et h yl-4-[4-m et h yl-3-
(m et h ylet h yl)p en t ylid en e]-5-oxo-2-2,3-d ih yd r ofu r yl}-
m eth yl 2,2-d im eth ylp r op a n oa te ((Z)-3). A solution of BCl3
(0.17 mL, 1 M in CH2Cl2) was added slowly to a -78 °C
solution of (Z)-12 (39.6 mg, 0.09 mmol) in CH2Cl2 (1.5 mL) and
was stirred for 2 h at the same temperature. The buffer
solution (pH 7.2, 1 mL) was slowly added at -78 °C, and then
the mixture was diluted with diethyl ether. The layers were
separated, and the aqueous layer was extracted with CHCl3
(5 mL), dried, and concentrated. Purification by silica gel
(neutralized with 2% Et3N/hexane) flash column chromatog-
raphy gave (Z)-3 (29.5 mg, 92%) as an oil. IR (neat) 3587 (OH),
3020 (CH), 2962 (CH), 2874 (CH), 1752 (CdO), 1664 (CdC)
cm-1 1H NMR (CDCl3) δ 0.92 and 0.95 (s, 3 H, CdCHCH2-
;
CH(CH(CH3)2)2), 0.97 and 1.00 (d, J ) 3.2 Hz, 3 H, CdCHCH2-
CH(CH(CH3)2)2), 1.12-1.25 (m, 1 H, CdCHCH2CH(CH-
(CH3)2)2), 1.27 (s, 3 H, lactone-CH3), 1.28 (s, 9 H, (CH3)3CO2-
CH2C), 1.31 (s, 3 H, lactone-CH3), 1.78-1.90 (m, 2 H,
CdCHCH2CH(CH(CH3)2)2), 2.23 (br s, 1 H, HOCH2C), 2.57-
2.68 (m, 1 H, CdCHCHHCH(CH(CH3)2)2), 2.86-2.98 (m, 1 H,
CdCHCHHCH(CH(CH3)2)2), 3.82 (s, 2 H, HOCH2C), 4.38 (s,
2 H, (CH3)3CO2CH2C), 6.05-6.11 (m, 1 H, CdCHCH2CH(CH-
(CH3)2)2);13C NMR (CDCl3) δ 19.26, 19.51, 21.60, 21.76, 23.50,
24.19, 25.89, 27.04, 29.29, 29.44, 38.76, 43.86, 51.33, 62.42,
62.99, 85.31, 134.09, 143.42, 168.50, 177.98; FAB-MS (m/z,

4864 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20
Tamamura et al.
(14) Kazanietz, M. G.; Krausz, K. W.; Blumberg, P. M. Differential
irreversible insertion of protein kinase C into phospholipid
vesicles by phorbol esters and related activators. J . Biol. Chem.
1992, 267, 20878-20886.
(23) Lee, J .; Kang, J . H.; Lee, S. Y.; Han, K. C.; Torres, C. M.;
Bhattacharyya, D. K.; Blumberg, P. M.; Marquez, V. E. Protein
kinase C ligands based on tetrahydrofuran templates containing
a new set of phorbol ester pharmacophores. J . Med. Chem. 1999,
42, 4129-4139.
(15) Sharkey, N. A.; Blumberg, P. M. Comparison of the activity of
phorbol 12-myristate 13-acetate and the diglyceride glycerol
1-myristate 2-acetate. Carcinogenesis 1986, 7, 677-679.
(16) Marquez, V. E.; Nacro, K.; Benzaria, S.; Lee, J .; Sharma, R.;
Teng, K.; Milne, G. W.; Bienfait, B.; Wang, S.; Lewin, N. E.;
Blumberg, P. M. The transition from a pharmacophore-guided
approach to a receptor-guided approach in the design of potent
protein kinase C ligands. Pharmacol. Ther. 1999, 82, 251-261.
(17) Marquez, V. E.; Blumberg, P. M. Synthetic diacylglycerols (DAG)
and DAG-lactones as activators of protein kinase C (PK-C). Acc.
Chem. Res. 2003, 36, 434-443.
(18) Nacro, K.; Bienfait, B.; Lee, J .; Han, K. C.; Kang, J . H.; Benzaria,
S.; Lewin, N. E.; Bhattacharyya, D. K.; Blumberg, P. M.;
Marquez, V. E. Conformationally constrained analogues of
diacylglycerol (DAG). 16. How much structural complexity is
necessary for recognition and high binding affinity to protein
kinase C? J . Med. Chem. 2000, 43, 921-944.
(19) Nacro, K.; Bienfait, B.; Lewin, N. E.; Blumberg, P. M.; Marquez,
V. E. Diacylglycerols with lipophilically equivalent branched acyl
chains display high affinity for protein kinase C (PK-C). A direct
measure of the effect of constraining the glycerol backbone in
DAG lactones. Bioorg. Med. Chem. Lett. 2000, 10, 653-655.
(20) Meylan, W.; Howard, P. Fragment Contribution Method for
Estimating Octanol-Water Partition Coefficients. J . Pharm. Sci.
1995, 84, 83-92.
(24) Nacro, K.; Lee, J .; Barchi, J . J .; Lewin, N. E.; Blumberg, P. M.;
Marquez, V. E. Conformationally constrained analogues of
diacylglycerol (DAG). Part 19: Asymmetric syntheses of (3R)-
and (3S)-3-hydroxy-4,4-disubstituted heptono-1,4-lactones as
protein kinase C (PK-C) ligands with increased hydrophilicity.
Tetrahedron 2002, 58, 5335-5345.
(25) Tripos, Inc. (www.tripos.com).
(26) Halgren, T. A. Merck molecular force field. 1. Basis, form, scope,
parametrization, and performance of MMFF94. J . Comput.
Chem. 1996, 17, 490-519.
(27) Rarey, M.; Kramer, B.; Lengauer, T.; Klebe, G. A fast flexible
docking method using an incremental construction algorithm.
J . Mol. Biol. 1996, 261, 470-489.
(28) Kraulis, P. J . Molscriptsa program to produce both detailed and
schematic plots of protein structures. J . Appl. Crystallogr. 1991,
24, 946-950.
(29) Merritt, E. A.; Bacon, D. J . Raster3D: Photorealistic molecular
graphics. Methods Enzymol. 1997, 277, 502-524.
(30) Lewin, N. E.; Blumberg, P. M. [3H]-Phorbol 12,13-dibutyrate
binding assay for protein kinace C and related proteins. Methods
Mol. Biol. 2003, 233, 129-156.
(31) Ley, S. V.; Norman, J .; Griffith, W. P.; Marsden, S. P. Tetra-
propylammonium perruthenate, Pr4n+Ruo4-, Tpapsa catalytic
oxidant for organic-synthesis. Synthesis 1994, 639-666.
(21) Meylan, W.; Howard, P. KOWWIN 1.63; Syracuse Research
Corp, 1995.
(22) Ueno, Y.; Moriya, O.; Chino, K.; Watanabe, M.; Okawara, M.
General synthetic route to gamma-butyrolactones via stereo-
selective radical cyclization by organotin species. J . Chem. Soc.,
Perkin Trans. 1 1986, 1351-1356.
J M049723+