Conformationally constrained analogues of diacylglycerol (DAG). Effect on protein kinase C (PK-C) binding by the isosteric replacement of sn-1 and sn-2 esters in DAG-lactones

Article abstract of DOI:10.1016/S0968-0896(03)00156-1

In order to determine the importance of the two ester pharmacophores in high affinity, conformationally constrained DAG-lactones (Lac-1-5) as PK-C ligands, we have independently replaced the sn-1 and sn-2 carbonyl esters in these compounds by ketone (2, 10, 11), amide (3, 25-28), and hydroxyl (12, 13) isosteres. Although the ketone analogue of the sn-1 ester (2) exhibited comparable activity to the parent Lac-1 when taking into account the difference in lipophilicities, the other isosteres were significantly poorer PK-Cα ligands compared to the parent DAG-lactones. This study demonstrates that the ester functionality in DAG-lactone plays an important role in the ligand's capacity to form a strong hydrogen bond with Gly253 at the active site. The discrete K_i analysis from the sn-1 and sn-2 isosteres further confirms that the DAG-lactones bind preferentially to the C1-domain in the sn-2 binding mode, as previously suggested.

Full text of DOI:10.1016/S0968-0896(03)00156-1

Bioorganic & Medicinal Chemistry 11 (2003) 2529–2539
Conformationally Constrained Analogues of Diacylglycerol
(DAG). Effect on Protein Kinase C (PK-C) Binding by the
Isosteric Replacement of Sn-1 and Sn-2 Esters in DAG-lactones
Ji-Hye Kang,a Hye-Eun Chung,a Su Yeon Kim,a Yerim Kim,a Jeewoo Lee,a,*
Nancy E. Lewin,b Larry V. Pearce,b Peter M. Blumbergb and Victor E. Marquezc
aLaboratory of Medicinal Chemistry, College of Pharmacy, Seoul National University, Seoul 151-742, South Korea
bLaboratory of Cellular Carcinogenesis and Tumor Promotion, NCI, NIH, Bethesda, MD 20892, USA
cLaboratory of Medicinal Chemistry, NCI, NIH, Frederick, MD 21701, USA
Received 3 February 2003; revised 7 March 2003; accepted 10 March 2003
Abstract—In order to determine the importance of the two ester pharmacophores in high affinity, conformationally constrained
DAG-lactones (Lac-1–5) as PK-C ligands, we have independently replaced the sn-1 and sn-2 carbonyl esters in these compounds by
ketone (2, 10, 11), amide (3, 25–28), and hydroxyl (12, 13) isosteres. Although the ketone analogue of the sn-1 ester (2) exhibited
comparable activity to the parent Lac-1 when taking into account the difference in lipophilicities, the other isosteres were sig-
nificantly poorer PK-Ca ligands compared to the parent DAG-lactones. This study demonstrates that the ester functionality in
DAG-lactone plays an important role in the ligand’s capacity to form a strong hydrogen bond with Gly253 at the active site. The
discrete Ki analysis from the sn-1 and sn-2 isosteres further confirms that the DAG-lactones bind preferentially to the C1-domain in
the sn-2 binding mode, as previously suggested.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
scaffold, unlike the flexible glycerol backbone of DAG,
is able to specifically direct the hydrophilic pharmaco-
phores (Fig 1).
The Protein Kinase C (PK-C) family of serine/threonine
kinases is implicated in growth factor- and G-protein-
coupled receptor signaling and plays a key regulatory
role in a variety of cellular functions, including cell
growth, differentiation, apoptosis, and cell-cycle con-
trol.1ꢀ3 As an endogenous ligand, diacylglycerol (DAG)
activates both the calcium-dependent classical PKC
isoforms a, b, and g and the novel or calcium-indepen-
dent PKC isoforms d, e, Z and y by binding to the C1
domains of the enzymes and promoting association with
the membrane phospholipids.4 Phorbol esters, which are
used as powerful pharmacological tools for studying
PK-C function, bind to the same DAG-binding site of
PK-Cs in a competitive manner and display binding
affinities several orders of magnitude higher than that of
DAG.5,6 Phorbol esters function as potent structural
mimetics of DAG because their conformationally rigid
Over the past several years, we have explored con-
formationally constrained DAG analogues to identify
structurally simple PK-C ligands with affinities similar
to those of the phorbol esters. Among these, we have
reported that 5-acyloxymethyl-5-hydroxymethyl-tetra-
hydro-2-furanones, a class of DAG-lactones, display
low nanomolar binding affinities for PK-Ca.7 Extensive
molecular modeling studies, extending our insights
derived from the crystal structure of the PK-Cd–phor-
bol-13-acetate complex,8 revealed that when these
DAG-lactones are docked into the empty receptor, only
one of the two non-equivalent carbonyl esters, sn-1 or
sn-2 (nomenclature based on the corresponding DAG
backbone as shown in Fig. 2), forms hydrogen bonds
with Gly253. The two energetically equivalent orienta-
tions are defined, respectively, as sn-1 and sn-2 binding
modes depending on which carbonyl group is engaged
in binding to the protein.9 Although in either binding
mode one of the carbonyls of the DAG lactones
*Corresponding author. Tel.: +82-2-880-7846; fax: +82-2-888-0649;
e-mail: jeewoo@snu.ac.kr
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00156-1

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J.-H. Kang et al. / Bioorg. Med. Chem. 11 (2003) 2529–2539
for PK-C may contain either a different oxygen type,
namely a ketone (phorbol’s C3-C¼O and ingenol’s C9-
C¼O), or an amide (teleocidin’s C11-CONH) (Fig. 1). In
the present study, we therefore sought to replace both
sn-1 and sn-2 carbonyls with ketone, amide and
hydroxyl groups to gain additional insight into the
importance of these structural changes on a DAG-lac-
tone template for PK-C binding affinity in either the sn-1
or sn-2 binding modalities.
Chemistry
The DAG-lactones (Lac-1–5), selected as the parent
compounds, were previously reported11ꢀ13 or synthe-
sized by a similar approach. Starting with optically
active (E)-5-benzyloxymethyl-5-formyl-3-[(Z)-9-octadec-
enyldene]tetrahydro-2-furanone (1) reported pre-
viously,12 a,b-unsaturated ketone (2) and acetamide (3)
analogues were synthesized as sn-1 isosteres of DAG-
lactone Lac-1 as outlined in Scheme 1.
Syntheses of cyclopentanone and cyclopentanol ana-
logues as sn-2 isosteres are represented in Scheme 2.
Alkylation of diethylmalonate with cis-1,4-dichloro-2-
butene afforded the cyclopentene 4, whose diester func-
tion was reduced and then protected as the acetonide to
afford compound 5. Dihydroxylation of the olefin in 5
and selective monoprotection of a single hydroxyl as a
p-methoxybenzyl (PMB) ether group was followed by
oxidation of the remaining hydroxyl to ketone 7. Wittig
olefination of 7 with tetradecyl triphenylphosphonium
bromide and deprotection of PMBfollowed by oxida-
tion of the corresponding hydroxyl produced an E/Z-
mixture of the a-alkylidene cyclopentanones 8 and 9 in
71% yield with a ratio of 1:1.6. The geometric isomers
were readily separated by column chromatography at
this stage, and their structural assignments were deter-
mined by 1H NMR spectroscopy where the b-vinyl
proton of the E-isomer (8) appears ca. 0.6 ppm down-
field from that of the Z-isomer (9). Each isomer was
individually hydrolyzed to remove the isopropylidene
group and selectively acylated with pivaloyl chloride to
give the racemic cyclopentanone products 10 (E-isomer)
Figure 1.
remained uninvolved in the binary complex with the
protein, the removal of either the sn-1 or sn-2 carbonyl
in a DAG-lactone caused a dramatic drop in binding
affinity, emphasizing the importance of the additional
carbonyl group.10 We hypothesize that the additional
carbonyl function, like the C-9(OH)/C-13(C¼O) motif
in phorbol esters which also appears free of contact with
the protein in the phorbol ester/C1b complex, interacts
with the phospholipid head groups required for high
affinity binding under the conditions of the biological
assays.
Although two of the three pharmacophores in DAG-
lactones are ester carbonyls, their counterpart
pharmacophores in other natural products with affinity
Figure 2.

J.-H. Kang et al. / Bioorg. Med. Chem. 11 (2003) 2529–2539
2531
Scheme 1. Reagents and conditions: (a) CH3COCHPPh3, CH2Cl2, rt; (b) BCl3, CH2Cl2, ꢀ78 ꢁC; (c) NaBH4, MeOH, 0 ꢁC; (d) MsCl, NEt3, CH2Cl2;
(e) NaN3, DMF, 100 ꢁC; (f) H2, Lindler’s cat, Ac2O, EtOAc.
Scheme 2. Reagents and conditions: (a) NaOEt, cis-ClCH2CH¼CHCH2Cl, EtOH; (b) LiAlH4, Et2O; (c) CH2¼C(OCH3)CH3, p-TsOH, DMF;
(d) 4-NMO, OsO4, acetone; (f) PMBCl, NaH, THF; (g) PCC, 4 A mol. sieve, CH2Cl2; (h) C14H29PPh3Br, t-BuOK, THF, benzene; (i) DDQ,
CH2Cl2–H2O; (j) p-TsOH, MeOH; (k) (CH3)3CCOCl, pyridine, CH2Cl2; (l) NaBH4, CeCl3–7H2O, CH2Cl2–MeOH.
and 11 (Z-isomer), respectively. The ensuing selective
reduction of (E)-cyclopentanone 10 gave a diastereo-
meric mixture of cyclopentanols 12 and 13. Assignment
of their structures was confirmed by COSY and
NOESY experiments in which, for instance, the NOE
enhancement between the H-1 and methylene protons
of CH2OH in 12 was examined.
E/Z mixture of 21 and 22, which were readily separated
by column chromatography and were assigned as
described for the previous lactone series. Following the
same procedure used for the synthesis of cyclopenta-
none analogues, 21 and 22 were converted to the final
target compounds 25–28, respectively.
The synthesis of lactam analogues as an additional sn-2
isostere is outlined in Scheme 3. Commercially available
trishydroxymethylaminomethane (14) was protected
with the t-BOC group followed by acetonide protection
of the diol to give 16. The remaining primary alcohol of
16 was oxidized to the aldehyde 17, which was then
extended by two carbons to give 18 after Wittig olefi-
nation and subsequent hydrogenation of the double
bond. Base-catalyzed cyclization of 18 with sodium
methoxide formed the lactam ring which occurred con-
comitantly with removal of the t-BOC to give 19. This
compound was reprotected with t-BOC to give 20, and
the incorporation of an a-alkylidene chain into lactam
20 proceeded via the aldol product to give the expected
Results and Discussion
The interaction of the sn-1 and sn-2 isosteres of DAG-
lactone 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 the recombinant single isozyme,
PK-Ca, in the presence of phosphatidylserine as pre-
viously described.9 The inhibition curves obtained for all
ligands were of the type expected for competitive inhibi-
tion, and the IC50 values were determined by fitting the
data points to the theoretical noncooperative competi-
tive curve. The Ki values for inhibition of binding were
calculated from the corresponding IC50 values (Table 1).
The parent compounds, Lac-1–5, and their isosteres

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J.-H. Kang et al. / Bioorg. Med. Chem. 11 (2003) 2529–2539
Scheme 3. Reagents and conditions: (a) Boc2O, NEt3, MeOH; (b) p-TsOH, acetone; (c) PCC, 4 A mol. sieve, CH2Cl2; (d) PPh3CHCO2CH3, CH2Cl2;
(e) H2, Pd/C, EtOAc; (f) NaOCH3, MeOH; (g) Boc2O, NEt3, DMAP, THF; (h) LiHMDS, THF; C17H33CHO; (i) MsCl, NEt3, CH2Cl2; DBU (j)
CF3CO2H, THF–H2O; (k) CH3COCl (or C7H15COCl), pyridine, CH2Cl2.
Table 1.
Chirality/Geometry
X
Y
Z
R1
R2
cLogPa
Ki (nM)
Sn1
Lac-1
2
E (5S)
E (5S)
E (5S)
CH¼CH
CH¼CH
CH2NH
O
O
O
C¼O
C¼O
C¼O
OCH3
CH3
CH3
C17H33
C17H33
C17H33
7.38
7.06
6.04
20 (ꢂ2.9)b
56.6 (ꢂ7.2)
1740 (ꢂ180)
3
Sn-2
Lac-2
10
12
13
Lac-3
11
Lac-4
25
27
E
CH2O
CH2O
CH2O
CH2O
CH2O
CH2O
CH2O
CH2O
CH2O
CH2O
CH2O
CH2O
O
C¼O
C¼O
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
CH3
C13H27
C13H27
C13H27
C13H27
C13H27
C13H27
C17H33
C17H33
C17H33
C17H33
C17H33
C17H33
6.72
7.95
8.58
8.58
6.72
7.95
7.11
7.10
10.04
7.11
7.10
10.04
7.83 (ꢂ0.55)c
4926 (ꢂ85)
3850 (ꢂ160)
6480 (ꢂ620)
7.15 (ꢂ0.51)c
605 (ꢂ98)
E
E (1R*,4S*)
E (1S*,4R*)
CH2
CH2
CH2
O
CH2
O
NH
NH
O
NH
NH
CH–OH
CH–OH
C¼O
Z
Z
E
E
E
Z
Z
Z
C¼O
C¼O
28 (ꢂ2.2)d
C¼O
CH3
5990 (ꢂ660)
3789 (ꢂ75)
24 (ꢂ2.9)d
C¼O
C7H15
CH3
CH3
Lac-5
26
28
C¼O
C¼O
3640 (ꢂ140)
1970 (ꢂ100)
C¼O
C7H15
acalculated according to the fragment-based program KOWWIN (http://esc.syrres.com).
bref 13.
cunpublished result.
dref 11.
contain a combination of acetyl or pivaloyl as R1 on the
sn-1 ester and tetradecyl or oleyl as R2 on the sn-2 ester.
The binding affinities of the isosteric ligands were com-
pared with those of the parent compounds with identical
R1 and R2 groups. As expected, all the parent com-
pounds were identified as high affinity ligands for PK-
Ca, with a range of Ki values of 7.15–28 nM.
Substitution of the sn-1 ester in Lac-1 (Ki=20 nM) with
a ketone caused a 3-fold reduction in binding affinity for
compound 2 (Ki=56.6 nM). Considering the relative
calculated log P values for Lac-1 (log P=7.38) and 2
(log P=7.06), the intrinsic binding affinities of these
compounds are probably very comparable since there is
a positive correlation with lipophilicity for this range of

J.-H. Kang et al. / Bioorg. Med. Chem. 11 (2003) 2529–2539
2533
potencies.14 Substitution of the sn-1 ester in Lac-1 for
an amide resulted in an almost two orders of magnitude
loss in the binding affinity of compound 3 (Ki=1740
nM). Although both compounds 2 and 3 contain a car-
bonyl group capable of effectively hydrogen bonding
the NH group of Gly253 based on our previous model,7
the replacement of the ether oxygen in the sn-1 ester of
DAG-lactones with either a carbon or nitrogen atom
hampered both derivatives from favorably binding to
the enzyme. The result might be explained in two ways.
One hypothesis is that the ether oxygen of the ester
might play an important role as a hydrogen bonding
acceptor for binding with PK-C, because the amide
analogue 3, bearing N–H as the hydrogen bonding
donor, was a poorer ligand than the ketone analogue 2.
The alternative is that the electron effect of the ether
oxygen on the carbonyl, acting as a hydrogen bonding
acceptor in enzyme binding, would be more favorable
than those of the carbon and amine groups. However,
since both the oxygen and amine are electron-donating
atoms and have similiar electronic properties, the latter
scenario appears to be less convincing.
moderate binding affinity to PK-Ca (Ki=>300 nM
depending on Log P).15 In addition, their molecular
modeling study revealed that the N–H of the pyrroli-
done skeleton formed an additional hydrogen bond
with the C¼O of Leu251 in PK-Cd, which participated
in hydrogen bonding with the C20-OH of phorbol in the
X-ray structure of PK-Cd C1b in complex with phorbol
13-acetate. Although they demonstrated that the pyrro-
lidone template might be another surrogate of teleo-
cidin, a natural PK-C activator, the binding affinities
(Ki) of their g-lactam analogues were much poorer than
those of our g-lactone analogues, which have affinities
in the low nM range. Therefore, it might be concluded
that the oxygen of the ester has an advantage over the
nitrogen of the amide in interacting with the C1-domain
of PK-C. Even though the sn-1 and sn-2 isosteric ana-
logues were not profitable as PK-C ligand candidates,
the degree of the reduction in affinity provides informa-
tion regarding the two binding modes of DAG-lactone
to the enzyme proposed previously.9 The reductions in
the sn-1 ester isosteres were calculated as ca. 3-fold in 2
(ketone) and 87-fold in 3 (amide) as compared with the
parent compound (ester), Lac-1. However, those in the
sn-2 ester were severely reduced, as represented by the
630-fold loss in 10 (ketone) and the 216-fold loss in 25
(amide) as compared with Lac-2 and Lac-4, respec-
tively. The comparison indicates that the parent DAG-
lactones probably bind to the enzyme in the sn-2 mode,
since its modification caused more severe losses in
binding affinity than that of the sn-1 mode. This result
also confirmed our previous suggestion that DAG-lac-
tones usually favor the sn-2 binding mode to the crys-
tallographic position of phorbol 13-acetate in the C1b
domain.9
In the study of the sn-2 ester isosteres in DAG-lactone,
the lactones of Lac-2–5 as the parent compounds were
replaced by cyclopentanone 10, 11, cyclopentanol 12,
13, and lactams 25–28, respectively. However, they dis-
played even lower binding affinities than those of the
sn-1 type. For the geometric E-isomer, the affinity of the
DAG-lactone with tetradecylidene in R2 (Lac-2,
Ki=7.83 nM) dropped 630-fold in cyclopentanone 10
(Ki=4926 nM), and 490- and 830-fold, respectively, in
cyclopentanols 12 (Ki=3850 nM) and 13 (Ki=6480
nM). Likewise, the affinity of the DAG-lactone with
oleylidene (Lac-4, Ki=28 nM) was also reduced 214-
fold in the corresponding lactam 25 (Ki=5990 nM) and
135-fold in the lipophilic lactam 27 (Ki=3789 nM). The
results suggest that the sn-2 ester of the DAG-lactone is
more sensitive to isosteric modification than the sn-1
ester, in terms of binding affinity, and would be regar-
ded as the more crucial pharmacophore as a hydrogen-
bonding acceptor.
In view of the importance of the sn-2 (or lactone) ether
oxygen and the favorable sn-2 binding mode in DAG-
lactones indicated by the above SAR, we examined the
possibility that the ether oxygen of the lactone may
participate in hydrogen bonding with the enzyme. A
molecular modeling study of Lac-3 indicated that the
two oxygens in the lactone may interact with the amide
proton of Gly253 in a bifurcated hydrogen bonding
mode, the so-called three-centered interaction,16,17 as
shown in Figure 3.18 Even though the distance between
the ring oxygen and the proton was 2.89 A, which is a
little longer than the optimal hydrogen bonding range
(1.5–2.5 A), it would be sufficient for them to participate
in the three-centered interaction, once the enzyme con-
formation is induced.
We reported previously that the replacement of the sn-1
and sn-2 carbonyl esters in DAG-lactones with sulfo-
nate esters as an isostere indicated that their isosteric
properties were structurally dependent.10 While the
C¼O and SO2 groups appeared to be true isosteres
when forced to reside near the lipid interface by the
adjacent hydrophobic alkyl chain, they did not show
equivalent isosteric properties when they existed away
from the lipid bilayer, at a site where they are expected
to bind to the C1 domain of PK-C. However, in con-
trast to the aspects of the sulfonate ester discussed pre-
viously, most of the ester isosteres utilized in these
study, including the ketone, hydroxyl, and amide,
except for the ketone analogue of the sn-1 ester, did not
serve as adequate isosteres regardless of their proximity
to the lipid interface. Regarding the lactam template as
a PK-C ligand, Kozikowski’s group recently reported
that 5(S)-hydroxymethyl-2,3-disubstituted-pyrrolidone
analogues designed as PK-C ligands, which have a
similar template as our g-lactams 25–28, displayed a
In summary, we have replaced the sn-1 and sn-2 car-
bonyl esters of DAG-lactone, identified as a potent lead
PK-C ligand, by isosteres including ketone, amide, and
alcohol, respectively. None of the compounds surpassed
the strong potency of the parent DAG-lactones in terms
of their binding affinities to PK-Ca, although the ketone
analogue of the sn-1 ester showed comparable activity
to the parent DAG-lactone when considering the
reduction in lipophilicity. This finding indicated that
both esters in DAG-lactones were crucial to bind tightly
with the enzyme through favorable hydrogen bonding.
The comparative analysis of the sn-1 and sn-2 isosteric

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J.-H. Kang et al. / Bioorg. Med. Chem. 11 (2003) 2529–2539
>C¼CH–CH2), 1.2–1.5 (m, 22H), 1.19 (s, 9H,
C(CH3)3), 0.88 (distorted t, 3H).; IR (neat) 3426 (OH),
1726 (C¼O), 1681 cmꢀ1; MS (EI) m/z 424 (M+).
1
Lac-2. A colorless oil, H NMR (CDCl3) d 6.25 (m,
1H, >C¼CH), 4.28 (d of AB, 1H, J=11.3 Hz,
CH2OCO), 4.13 (d of AB, 1H, J=11.3 Hz, CH2OCO),
3.66 (ddd of AB, 2H, CH2OH), 2.90 (m, 1H, H-4a),
2.65–2.85 (m, 3H, H-3b and >C¼CH–CH2), 2.09 (t,
1H, OH), 1.2–1.5 (m, 22H), 1.19 (s, 9H, C(CH3)3), 0.88
(distorted t, 3H); IR (neat) 3438 (OH), 1737 (C¼O),
1681 cmꢀ1; MS (EI) m/z 424 (M+).
(S)-5-(Hydroxymethyl)-5-[2-(methylcarbonyl)-(E)-ethenyl]-
3-{(E)-[(Z)-9-octadecenyldene]}tetrahydro-2-furanone (2).
A solution of 1 (80 mg, 0.166 mmol) prepared by the
method reported in previous literature12 in CH2Cl2 (5
mL) was treated with 1-triphenylphosphoranylidene-2-
propanone (106 mg, 0.332 mmol) and stirred for 16 h at
room temperature. The mixture was concentrated in
vacuo and the residue was purified by flash column
chromatography on silica gel with EtOAc/hexanes (1:3)
as eluant to give the ketone as an oil (68 mg, 78%). The
compound (68 mg, 0.13 mmol) was dissolved in CH2Cl2
(4 mL) and cooled to ꢀ78 ꢁC. The solution was treated
with boron trichloride (1.0 M in CH2Cl2, 0.65 mL, 0.65
mmol) slowly and stirred for 90 min at ꢀ78 ꢁC. The
reaction mixture was quenched with saturated NaHCO3
solution and immediately partitioned between ether and
pH 7 buffer solution. The organic layer was washed with
H2O, dried over MgSO4 and concentrated in vacuo. The
residue was purified by flash column chromatography on
silica gel with EtOAc/hexanes (2:3 to 1:1) as eluant to
give 2 as an oil (44 mg, 77%): [a]D +17.5 (c 0.32, CHCl3);
1H NMR (CDCl3) d 6.76 (m, 1H, >C¼CH), 6.73 (d, 1H,
J=15.9 Hz, CH¼CHCOMe), 6.39 (d, 1H, J=15.9 Hz,
CH¼CHCOMe), 5.33 (m, 2H, CH2CH¼CHCH2), 3.77
(d of AB, 1H, J=12.1 Hz, CH2OH), 3.64 (d of AB, 1H,
J=12.1 Hz, CH2OH), 3.05 (m, 1H, H-4a), 2.68 (m, 1H,
H-4b), 2.26 (s, 3H, COCH3), 2.14 (m, 2H, >C¼CH-
CH2), 1.9–2.05 (m, 4H, CH2CH¼CHCH2), 1.1–1.6 (m,
22H), 0.86 (distorted t, 3H); 13C NMR (CDCl3) d 197.18,
169.66, 143.04, 142.87, 130.04, 129.65, 129.51, 124.93,
83.76, 66.81, 38.53, 38.45, 32.35, 31.87, 30.34, 29.73,
29.68, 29.49, 29.29, 29.17, 29.12, 29.00, 28.65, 28.00,
27.20, 27.12, 26.49, 26.42, 22.66, 14.09; IR (neat) 3383
(OH), 1731 and 1681 (C¼O), 1673 (C¼C) cmꢀ1. Anal.
calcd for C27H44O4: C, 74.95; H, 10.25. Found: C, 73.86;
H, 10.29.
Figure 3. sn-2 binding mode of DAG-lactone (Lac-3) with bifurcated
hydrogen bonding.
analogues support the conclusion that the parent DAG-
lactones bind to the C1-domain of PK-C more favor-
ably in the sn-2 binding mode, as previously speculated.
Experimental
All chemical reagents were commercially available.
Melting points were determined on a Melting Point
Buchi B-540 apparatus and are uncorrected. Silica gel
column chromatography was performed on silica gel 60,
230–400 mesh, Merck. Proton NMR spectra were
recorded on a JEOL JNM-LA 300 at 300 MHz. Chem-
ical shifts are reported in ppm units with Me4Si as a
reference standard. Infrared spectra were recorded on a
Perkin-Elmer 1710 Series FTIR. Mass spectra were
recorded on a VG Trio-2 GC-MS. Elemental analyses
were performed with an EA 1110 Automatic Elemental
Analyzer, CE Instruments. The syntheses of (5S)-5-
(hydroxymethyl)-5-[2-(methoxycarbonyl)-(E)-ethenyl]-3-
{(E)-[(Z)-9-octadecenyldene]} tetrahydro-2-furanone
(Lac - 1), 5 - [(acetyloxy)methyl] - 5 - hydroxymethyl - 3 -
[(E,9Z)-9-octadecenylidene]tetrahydro-2-furanone (Lac-
4) and 5-[(acetyloxy)methyl]-5-hydroxymethyl-3-[(Z,9Z)-
9-octadecenylidene]tetrahydro-2-furanone (Lac-5) were
described in our previous report.11,13
5-Hydroxymethyl-5-pivaloyloxymethyl-3-[(E)-tetradecyli-
dene]tetrahydro-2-furanone (Lac-2) and 5-hydroxymethyl
-5-pivaloyloxymethyl-3-[(Z)-tetradecylidene]tetrahydro-
2-furanone (Lac-3). These compounds were prepared by
following our procedure described in ref 11.
(S)-5-(Hydroxymethyl)-5-[(acetylamino)methyl]-3-{(E)-
[(Z)-9-octadecenyldene]}tetrahydro-2-furanone (3).
A
cooled solution of 1 (145 mg, 0.3 mmol) in MeOH (6
mL) in an ice-bath was treated portionwise with sodium
borohydride (46 mg, 1.2 mmol) and stirred for 1 h at
0 ꢁC. The reaction mixture was quenched with excess
acetone and concentrated in vacuo. The residue was
purified by flash column chromatography on silica gel
with EtOAc/hexanes (1:2) as eluant to give the alcohol
(125 mg, 86%). A solution of the above alcohol (96 mg,
0.2 mmol) in CH2Cl2 (5 mL) was treated with triethyl-
amine (0.084 mL, 0.6 mmol) and methanesulfonyl
1
Lac-2. A colorless oil, H NMR (CDCl3) d 6.77 (m,
1H, >C¼CH), 4.29 (d of AB, 1H, J=11.7 Hz,
CH2OCO), 4.14 (d of AB, 1H, J=11.7 Hz, CH2OCO),
3.68 (ddd of AB, 2H, CH2OH), 2.83 (m, 1H, H-4a), 2.66
(m, 1H, H-3b), 2.26 (t, 1H, OH), 2.17 (dd of AB, 2H,

J.-H. Kang et al. / Bioorg. Med. Chem. 11 (2003) 2529–2539
2535
chloride (0.03 mL, 0.4 mmol) and stirred for 2 h. The
mixture was concentrated in vacuo and the residue was
purified by flash column chromatography on silica gel
with EtOAc/hexanes (1:2) as eluant to give the mesylate.
The mesylate was dissolved in DMF (3 mL) and treated
with sodium azide (65 mg, 1 mmol). After being stirred
for 16 h at 100 ꢁC, the mixture was diluted with H2O
and extracted with EtOAc several times. The combined
organic layers were washed with H2O and brine, dried
over MgSO4 and concentrated in vacuo. The residue
was purified by flash column chromatography on silica
gel with EtOAc/hexanes (1:2) as eluant to give the azide
(86 mg, 84%).
8,8-dimethyl-7,9-dioxaspiro[4.5]dec-2-ene (5). A cooled
solution of lithium aluminum hydride (2.07 g, 54.8
mmol) at 0 ꢁC in diethyl ether (50 mL) was dropwise
treated with a solution of 4 (2.9 g, 13.7 mmol) in diethyl
ether (20 mL). After being stirred for 2 h at room tem-
perature, the reaction mixture was cooled in an ice-bath
and quenched with H2O (2.07 mL), 15% NaOH (2.07
mL) and H2O (6.21 mL), successively. The suspension
was stirred for 1 h, filtrated with ether and the filtrate
was concentrated in vacuo. The residue was purified by
flash column chromatography on silica gel with EtOAc/
hexanes (1:1) as eluant to give the diol as a solid (1.4 g,
1
84%); H NMR (CDCl3) d 5.56 (s, 2H, CH¼CH), 3.81
(bs, 2H, 2ꢃOH), 3.58 (s, 4H, 2ꢃCH2OH), 2.12 (s, 4H,
2ꢃCH2).
1H NMR (CDCl3) d 6.75 (m, 1H, >C¼CH), 5.32 (m,
2H, CH2CH¼CHCH2), 4.55 (s, 2H, PhCH2O), 3.60 (q
of AB, 2H, CH2N3), 3.55 (s, 2H, BnOCH2), 3.05 (m,
1H, H-4a), 2.65 (m, 1H, H-4b), 2.15 (m, 2H, >C¼CH-
CH2), 1.9–2.05 (m, 4H, CH2CH¼CHCH2), 1.1–1.6 (m,
22H), 0.86 (distorted t, 3H).
The diol (1.474 g, 11.5 mmol) was dissolved in cooled
DMF (5 mL) at 0 ꢁC and treated with 2-methoxy-
propene (2.2 mL, 23 mmol) and a catalytic amount of p-
TsOH. After being stirred for 4 h at room temperature,
the reaction mixture was treated with solid NaHCO3
and extracted with EtOAc several times. The combined
organic layers were washed with H2O and brine, dried
over MgSO4 and concentrated in vacuo. The residue
was purified by flash column chromatography on silica
gel with EtOAc/hexanes (1:4) as eluant to give 5 as an
oil (1.88 g, 98%); 1H NMR (CDCl3) d 5.58 (s, 2H,
CH¼CH), 3.63 (s, 4H, 2ꢃOCH2), 2.23 (s, 4H, 2ꢃCH2),
1.40 (s, 6H, 2ꢃCH3).
A mixture of the azide (82 mg, 0.16 mmol), acetic
anhydride (0.06 mL, 0.64 mmol) and Lindler’s catalyst
(16 mg) in EtOAc (5 mL) was hydrogenated under a
balloon of hydrogen for 2 h, filtered and concentrated in
vacuo. The residue was purified by flash column chro-
matography on silica gel with EtOAc/hexanes (2:1) as
eluant to give the acetamide (66 mg, 78%) as an oil,
which was debenzylated by following the debenzylation
method described for the synthesis of 2 and purified by
flash column chromatography on silica gel with EtOAc/
hexanes (5:1) to give 3 as an oil in 92% yield:
3-[(4-Methoxybenzyl)oxy]-8,8-dimethyl-7,9-dioxaspiro[4.5]-
decan-2-ol (6). A solution of 5 (1.89 g, 10 mmol) in ace-
tone (20 mL) was treated with 4-methylmorpholine N-
oxide (2.34 g, 20 mmol) and a catalytic amount of
osmium tetroxide and stirred for 16 h at room tem-
perature. The reaction mixture was quenched by aqu-
eous Na2S2O3 solution and extracted with EtOAc
several times. The combined organic layers were washed
with H2O and brine, dried over MgSO4 and con-
centrated in vacuo. The residue was purified by flash
column chromatography on silica gel with EtOAc/hex-
anes (1:1) as eluant to give the corresponding diol as a
1
[a]D=+12.5 (c 0.5, CHCl3); H NMR (CDCl3) d 6.77
(m, 1H, >C¼CH), 6.17 (bt, 1H, NH), 5.33 (m, 2H,
CH2CH¼CHCH2), 3.99 (d of AB, 1H, J=8.1 Hz,
NHCH2), 3.93 (d of AB, 1H, J=8.1 Hz, NHCH2), 3.57
(d of AB, 1H, J=12.0 Hz, CH2OH), 3.29 (d of AB, 1H,
J=12.0 Hz, CH2OH), 2.9–3.1 (m, 1H, H-4a and OH),
2.48 (m, 1H, H-4b), 2.05 (s, 3H, NHCOCH3), 1.9–2.2
(m, 6H, >C¼CH–CH2 and CH2CH¼CHCH2), 1.1–1.6
(m, 22H), 0.86 (distorted t, 3H); 13C NMR (CDCl3) d
172.43, 170.00, 143.23, 130.03, 129.66, 125.29, 84.12,
63.38, 44.26, 34.38, 31.87, 31.80, 31.68, 30.30, 29.73,
29.67, 29.49, 29.29, 29.17, 29.12, 29.00, 28.85, 28.05,
27.20, 27.13, 26.69, 26.63, 22.89, 22.65, 14.09; IR (neat)
1
solid (2.87 g, 71%); H NMR (CDCl3) d 4.07 (m, 2H,
2ꢃCHOH), 3.70 (s, 2H, OCH2), 3.52 (s, 2H, OCH2),
3.09 (bs, 2H, 2ꢃOH), 1.78 (dd, 2H, J=6.1, 14 Hz,
>CH2CH), 1.66 (dd, 2H, J=5.1, 14 Hz, >CH2CH),
1.40 (s, 6H, 2ꢃCH3).
3384 (OH), 1730 and 1680 (C¼O), 1665 (C¼C) cmꢀ1
.
Anal. calcd for C26H45O4N: C, 71.68; H, 10.41; N, 3.22.
Found: C, 71.94; H, 10.44; N. 3.20.
A solution of the above diol (1.21 g, 6.0 mmol) in THF
(4 mL) was treated with NaH (0.24 g, 6.0 mmol) and
stirred for 30 min at room temperature. The mixture
was treated with p-methoxybenzyl chloride (0.8 mL, 6.0
mmol) followed by tetrabutylammonium iodide (1.1 g,
3.0 mmol) and stirred for 20 h at room temperature.
The reaction mixture was concentrated in vacuo and
diluted with CH2Cl2. The organic layers were washed
with H2O and brine, dried over MgSO4 and con-
centrated in vacuo. The residue was purified by flash
column chromatography on silica gel with EtOAc/hex-
Diethyl 3-cyclopentene-1,1-dicarboxylate (4). To
a
sodium ethoxide solution prepared from sodium (2.3 g,
100 mmol) and ethanol (40 mL) was added diethylmal-
onate (6 g, 37 mmol) followed by 1,4-dichloro-2-butene
(3.94 mL, 37 mmol). After being refluxed for 16 h, the
reaction mixture was cooled and concentrated in vacuo.
The residue was extracted with ether several times. The
combined organic layers were washed with H2O and
brine, dried over MgSO4, and concentrated in vacuo.
The residue was purified by flash column chromato-
graphy on silica gel with EtOAc/hexanes (1:4) as eluant
1
anes (1:1) as eluant to give 6 as an oil (1.01 g, 52%); H
1
to give 4 as an oil (5.8 g, 75%); H NMR (CDCl3) d
NMR (CDCl3) d 7.27 (d, J=8.4 Hz, 2H, Ar), 6.90 (d,
J=8.4 Hz, 2H, Ar), 4.57 (d, J=11.3 Hz, 1H, OCH2Ar),
4.45 (d, J=11.3 Hz, 1H, OCH2Ar), 4.12 (m, 1H,
5.60 (s, 2H, CH¼CH), 4.20 (q, 4H, 2ꢃCO2CH2CH3),
3.01 (s, 4H, 2ꢃCH2), 1.25 (m, 6H, 2ꢃCO2CH2CH3).

2536
J.-H. Kang et al. / Bioorg. Med. Chem. 11 (2003) 2529–2539
CHOPMB), 3.8–3.9 (m, 4H, CHOH and OCH3), 3.74
(dd of AB, 2H, CH2O), 3.54 (dd of AB, 2H, CH2O),
2.44 (d, 1H, OH), 1.90 (ddd of AB, 2H,
CH2CHOPMB), 1.63 (ddd of AB, 2H, CH2CHOH),
1.43 (s, 6H, 2ꢃCH3).
2H, H-1), 2.29 (s, 2H, H-4), 2.16 (dd of AB, 2H,
>C¼CH-CH2), 1.47 (s, 3H, CH3), 1.45 (s, 3H, CH3),
1.25–1.3 (m, 22H), 0.90 (distorted t, 3H).
1
9. Z-Isomer, H NMR(CDCl3) d 6.03 (t, 1H, J=7.3
3-[(4-Methoxybenzyl)oxy]-8,8-dimethyl-7,9-dioxaspiro
[4.5]decan-2-one (7). A suspension of 6 (0.818 g, 2.54
mmol) and 4 A molecular sieve (1.095 g) in CH2Cl2 (8
mL) was treated with pyridinium chlorochromate (1.095
g, 5.08 mmol) and stirred for 12 h at room temperature.
The mixture was quenched with ether and Celite and
stirred for 30 min. It was filtrated through a silica gel
pad with an aid of ether and the filtrate was con-
centrated in vacuo. The residue was purified by flash
column chromatography on silica gel with EtOAc/hex-
anes (1:2) as eluant to give 7 as a solid (0.789 g, 97%);
1H NMR (CDCl3) d 7.27 (d, J=8.4 Hz, 2H, Ar), 6.87
(d, J=8.4 Hz, 2H, Ar), 4.74 (d, J=11.5 Hz, 1H,
OCH2Ar), 4.56 (d, J=11.5 Hz, 1H, OCH2Ar), 3.86 (t,
1H, J=7.8 Hz, CHOPMB), 3.80 (s, 3H, OCH3), 3.72
(dd of AB, 2H, CH2O), 3.57 (dd of AB, 2H, CH2O),
2.35 (s, 2H, CH2CO), 2.18 (dd of AB, 1H,
CH2CHOPMB), 1.68 (dd of AB, 1H, CH2CHOPMB),
1.44 (s, 3H, CH3), 1.40 (s, 3H, CH3).
Hz, >C¼CH), 3.68 (ddd of AB, 4H, 2ꢃOCH2), 2.6–2.7
(m, 4H, H-1 and >C¼CH–CH2), 2.27 (s, 2H, H-4), 1.47
(s, 3H, CH3), 1.45 (s, 3H, CH3), 1.25–1.3 (m, 22H), 0.90
(distorted t, 3H).
4-Hydroxymethyl-4-pivaloyloxymethyl-2-[(E)-tetradecyli-
dene]cyclopentanone (10) and 4-hydroxymethyl-4-piva-
loyloxymethyl-2-[(Z)-tetradecylidene]cyclopentanone (11).
A solution of 8 (188 mg, 0.5 mmol) and p-TsOH (10 mg,
0.05 mmol) in MeOH (4 mL) was stirred for 2 h at room
temperature. The mixture was neutralized with solid
NaHCO3, filtered and the filtrate was concentrated in
vacuo. The residue was purified by flash column chro-
matography on silica gel with EtOAc/hexanes (3:1) as
eluant to give the diol as an oil (122 mg, 70%). The diol
(122 mg, 0.36 mmol) was dissolved in CH2Cl2 (6 mL),
treated with pyridine (0.06 mL, 0.72 mmol) and stirred
for 2 h at room temperature. The mixture was treated
with pivaloyl chloride (0.44 mL, 0.36 mmol), stirred for
2 h at room temperature and concentrated in vacuo.
The residue was purified by flash column chromato-
graphy on silica gel with EtOAc/hexanes (1:4) as eluant
to give 10 as an oil (114 mg, 75%).
3-[(E or Z)-Tetradecylidene]-8,8-dimethyl-7,9-dioxaspiro[4.5]-
decan-2-one (8, 9). A mixture of 7 (1.24 g, 3.88 mmol)
and tetradecyl triphenylphosphonium bromide (4.18 g,
7.76 mmol) was dried overnight under vacuum and dis-
solved in benzene (30 mL). The suspension was refluxed
and treated with potassium tert-butoxide in THF until
starting material was consumed as determined by TLC.
The mixture was cooled and concentrated in vacuo. The
residue was purified by flash column chromatography
on silica gel with EtOAc/hexanes (1:10) as eluant to give
the corresponding alkene as an oil (1.39 g, 72%). A
solution of the above alkene (1.188 g, 2.37 mmol) in
CH2Cl2-H2O (19 mL, 18:1) was treated with 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone (0.808 g, 3.56
mmol) and stirred for 2 h at room temperature. The
reaction mixture was quenched by aqueous Na2S2O3
and extracted with CH2Cl2 several times. The combined
organic layers were washed with H2O and brine, dried
over MgSO4 and concentrated in vacuo. The residue
was purified by flash column chromatography on silica
gel with EtOAc/hexanes (1:10) as eluant to give the
corresponding alcohol as an oil (0.603 g, 67%). A solu-
tion of the above alcohol (0.393 g, 1.033 mmol) in
CH2Cl2 (15 mL) was treated with 4 A molecular sieve
(0.668 g) and pyridinium chlorochromate (0.668 g, 3.1
mmol) and stirred for 6 h at room temperature. The
mixture was quenched with ether and Celite, stirred for
an addition 30 min, filtered through a short pad of silica
gel and the filtrate was concentrated in vacuo. The resi-
due was purified by flash column chromatography on
silica gel with EtOAc/hexanes (1:10) as eluant to give 8
(E-isomer, 0.278 g, 27%) and 9 (Z-isomer, 0.455 g,
44%) as an oil, respectively.
10. E-Isomer; 1H NMR(CDCl3) d 6.63 (m, 1H,
>C¼CH), 4.18 (d of AB, 1H, J=11.5 Hz, CH2OCO),
4.02 (d of AB, 1H, J=11.5 Hz, CH2OCO), 3.44 (s, 2H,
CH2OH), 2.58 (m, 1H, H-3a), 2.44 (m, 1H, H-3b), 2.32
(s, 2H, H-5), 2.13 (dd of AB, 2H, >C¼CH–CH2), 1.25–
1.5 (m, 22H), 1.21 (s, 9H, C(CH3)3), 0.88 (distorted t,
3H); IR (neat) 3443 (OH), 1731, 1715 (C¼O) cmꢀ1; MS
(EI) m/z 423 (M+). Anal. calcd for C26H46O4: C, 73.89;
H, 10.97; Found: C, 74.02; H, 10.99.
11. Z-Isomer, prepared from 9 by following the pro-
cedure described for the synthesis of 10 in 53% yield; 1H
NMR(CDCl3) d 6.60 (m, 1H, >C¼CH), 4.21 (d of AB,
1H, J=11.2 Hz, CH2OCO), 4.00 (d of AB, 1H, J=11.2
Hz, CH2OCO), 3.41 (dd of AB, 2H, CH2OH), 2.63 (t,
2H, H-5), 2.4–2.55 (m, 4H, H-3 and >C¼CH–CH2),
1.25–1.6 (m, 22H), 1.21 (s, 9H, C(CH3)3), 0.88 (distorted
t, 3H); IR (neat) 3443 (OH), 1730, 1715 (C¼O) cmꢀ1
;
MS (EI) m/z 423 (M+). Anal. calcd for C26H46O4: C,
73.89; H, 10.97; Found: C, 74.04; H, 11.00.
(rel-1R,4S)-4-Hydroxymethyl-4-pivaloyloxymethyl-2-
[(E)-tetradecylidene]cyclopentanol (12) and (rel-1S,4R)-
4-hydroxymethyl-4-pivaloyloxymethyl-2-[(Z)-tetradecyli-
dene]cyclopentanol (13). A cooled solution of 10 (105
mg, 0.24 mmol) in CH2Cl2–MeOH (8 mL, 2:1) at 0 ꢁC
was treated with cerium(III) chloride heptahydrate (96
mg, 0.264 mmol) followed by sodium borohydride (10
mg, 0.264 mmol) and stirred for 30 min at 0 ꢁC. The
reaction mixture was quenched by 1 N HCl and
extracted with CH2Cl2 several times. The combined
1
8. E-Isomer, H NMR(CDCl3) d 6.63 (t, 1H, J=7.5
Hz, >C=CH), 3.66 (dd of AB, 4H, 2ꢃOCH2), 2.61 (s,

J.-H. Kang et al. / Bioorg. Med. Chem. 11 (2003) 2529–2539
2537
organic layers were washed with H2O and brine, dried
over MgSO4 and concentrated in vacuo. The residue
was purified by flash column chromatography on silica
gel with EtOAc/hexanes (1:2) as eluant to give 12 (36
mg, 35%) and 13 (30 mg, 30%) as white solids,
respectively.
CH2Cl2 (100 mL) was treated with 4 A molecular sieve
(12.6 g) and stirred for 30 min at room temperature. To
the mixture was added pyridinium chloroformate (10.4
g, 48 mmol) portionwise. After stirring for 12 h at room
temperature, the reaction mixture was treated with
Celite and ether, stirred for 30 min and filtered through
a short pad of silica gel. The filtrate was concentrated in
vacuo and the residue was purified by flash column
chromatography on silica gel with EtOAc/hexanes (1:4)
as eluant to give 17 as a solid (4.90 g, 78%): mp 62 ꢁC;
1H NMR (CDCl3) d 9.63 (s, 1H, CHO), 5.57 (bs, 1H,
NH), 4.07 (d, 2H, J=11.7 Hz, CH2O), 3.95 (d, 2H,
J=11.7 Hz, CH2O), 1.46–1.52 (m, 15H, C(CH3)3 and
C(CH3)2); 13C NMR (CDCl3) d 199.25, 155.40, 98.67,
80.82, 62.55, 59.77, 28.15, 27.11, 19.58.
12. Mp 40 ꢁC; 1H NMR (CDCl3) d 5.55 (m, 1H,
>C¼CH), 4.51 (m, 1H, CHOH), 4.20 (dd of AB, 2H,
J=9.7 Hz, CH2OCO), 3.29 (d, 2H, J=5.6 Hz, CH2OH),
2.54 (t, 1H, OH), 2.24 (dd of AB, 2H, H-3), 1.99 (dd of
AB, 2H, >C¼CH–CH2), 1.90 (dd of AB, 2H, J=6.8,
13.9 Hz, H-5), 1.25–1.6 (m, 22H), 1.21 (s, 9H, C(CH3)3),
0.88 (distorted t, 3H); IR (KBr) 3440, 1730, 1711 cmꢀ1
;
MS (EI) m/z 424 (M+). Anal. calcd for C26H48O4: C,
73.54; H, 11.39; Found: C, 73.79; H, 11.41.
Methyl 3-{5-[(tert-butoxycarbonyl)amino]-2,2-dimethyl-
1,3-dioxan-5-yl}propanoate (18). A solution of 17 (4.45
g,
17
mmol)
and
methyl
(triphenylphos-
13. Mp 40 ꢁC; 1H NMR (CDCl3) d 5.56 (m, 1H,
>C¼CH), 4.49 (m, 1H, CHOH), 3.96 (dd of AB, 2H,
J=11.2 Hz, CH2OCO), 3.54 (dd of AB, 2H, J=11 Hz,
CH2OH), 2.44 (d of AB, 1H, H-3a), 2.14 (d of AB, 1H,
H-3b), 1.9–2.0 (m, 4H, >C¼CH–CH2 and H-5), 1.25–
1.65 (m, 22H), 1.21 (s, 9H, C(CH3)3), 0.88 (distorted t,
3H); IR (KBr) 3439, 1731, 1710 cmꢀ1; MS (EI) m/z 424
(M+). Anal. calcd for C26H48O4: C, 73.54; H, 11.39;
Found: C, 73.82 ; H, 11.43.
phoranylidene)acetate (8.53 g, 25.5 mmol) in CH2Cl2
(150 mL) was stirred for 20 h at room temperature. The
mixture was concentrated in vacuo and the residue was
purified by flash column chromatography on silica gel
with EtOAc/hexanes (1:4) as eluant to give the a,b-
unsatuꢁrated methyl ester as a white solid (4.27 g, 80%):
mp 85 C; 1H NMR (CDCl3) d 6.93 (d, 1H, J=16.1 Hz,
CH¼CHCO2CH3), 5.98 (d, J=16.1 Hz, 1H,
CH¼CHCO2CH3), 5.20 (bs, 1H, NH), 3.88 (t, 4H,
J=14.2 Hz, 2ꢃCH2O), 3.74 (s, 3H, CO2CH3), 1.46–1.52
(m, 15H, C(CH3)3 and C(CH3)2).
tert-Butyl
carbamate (15).
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-
solution of trishydroxy-
A
methylaminomethane (14) (6.0 g, 50 mmol) in a mixture
of triethylamine and methanol (1:9, 150 mL) was trea-
ted with di-tert-butyl dicarbonate (13.1 g, 60 mmol) and
stirred at room temperature for 3 h. The reaction mix-
ture was concentrated in vacuo, diluted with H2O and
extracted with EtOAc three times. The combined
organic layer was washed with H2O and brine, dried
over MgSO4, and concentrated in vacuo. The residue
was purified by flash column chromatography on silica
gel with CH2Cl2/MeOH (9:1)ꢁas eluant to give 15 as a
The above compound (3.96 g, 12.6 mmol) was dis-
solved in EtOAc (40 mL), treated with 10% Pd/C (0.4
g) and hydrogenated under a balloon of hydrogen for 5
h. The reaction mixture was filtered through Celite and
the filtrate was concentrated in vacuo. The residue was
purified by flash column chromatography on silica gel
with EtOAc/hexanes (1:3) as eluant to give 18 as a
ꢁ
1
white solid (3.6 g, 90%); mp 65 C; H NMR (CDCl3)
d 4.95 (bs, 1H, NH), 3.78 (d, J=14.4 Hz, 2H, CH2O),
3.57 (d, J=14.4 Hz, 2H, CH2O), 3.56 (s, 3H,
CO2CH3), 2.23 (t, J=7.6 Hz, 2H, CH2CO2CH3), 1.93
(t, J=8.3 Hz, 2H, CH2CH2CO2CH3), 1.34–1.40 (m,
15H, C(CH3)3 and C(CH3)2); 13C NMR (CDCl3) d
173.45, 154.56, 98.03, 78.94, 65.50, 51.28, 50.81, 28.02,
27.42, 26.47, 26.21, 20.21; IR (KBr) 1735 and 1715
1
solid (8.06 g, 72%): mp 147 C; H NMR (CDCl3) d
5.50 (bs, 1H, NH), 3.68 (m, 6H, 3ꢃCH2), 3.33 (bs, 3H,
3ꢃOH), 1.45 (s, 9H, C(CH3)3).
tert-Butyl
N-[5-(hydroxymethyl)-2,2-dimethyl-1,3-di-
oxan-5-yl]carbamate (16). A solution of 15 (8.06 g, 36
mmol) in acetone (150 mL) was treated with a catalytic
amount of p-toluene sulfonate and stirred for 18 h at
room temperature. The reaction mixture was alkalinized
with solid NaHCO3, filtered and concentrated in vacuo.
The residue was purified by flash column chromato-
graphy on silica gel with EtOAc/hexanes (1:1) as eluant
to give 16 as a solid (7.18 g, 76%): mp=99 ꢁC; 1H NMR
(CDCl3) d 5.34 (s, 1H, NH), 3.85 (d, 2H, J=11.5 Hz,
CH2O), 3.80 (d, 2H, J=11.5 Hz, CH2O), 3.72 (s, 2H,
CH2OH), 1.41–1.50 (m, 15H, C(CH3)3 and C(CH3)2);
13C NMR (CDCl3) d 156.43, 98.75, 80.45, 64.71, 64.38,
53.32, 28.33, 26.81, 20.31; IR (KBr) 3421 (OH), 1715
(C¼O) cmꢀ1
.
8,8-Dimethyl-7,9-dioxa-1-azaspiro[4.5]decan-2-one (19).
A solution of 18 (3.0 g, 9.45 mmol) in methanol (100
mL) was treated with 28% sodium methoxide (1.62 mL,
28.35 mmol) and refluxed for 12 h. After cooling, the
reaction mixture was concentrated in vacuo. The residue
was dissolved in ethyl acetate, filtrated with Celite and
concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel with EtOAc/
MeOH (9:1) as eluant to give 19 as a white solid (1.34 g,
77%); mp 174 ꢁC; H NMR (CDCl3) d 6.41 (bs, 1H,
1
NH), 3.84 (d, 2H J=11.5 Hz, CH2O), 3.63 (d, 2H,
J=11.5 Hz, CH2O), 2.41 (t, 2H, J=7.8 Hz,
CH2CONH), 1.77 (t, 2H, J=8.8 Hz, CH2CH2CONH),
1.46 (s, 3H, CH3), 1.42 (s, 3H, CH3); IR (KBr) 3187
(C¼O) cmꢀ1
.
tert-Butyl N-(5-formyl-2,2-dimethyl-1,3-dioxan-5-yl)car-
bamate (17). A solution of 16 (6.3 g, 24 mmol) in
(NH), 1715, 1650 (C¼O) cmꢀ1
.

2538
J.-H. Kang et al. / Bioorg. Med. Chem. 11 (2003) 2529–2539
tert-Butyl 8,8-dimethyl 2-oxo-7,9-dioxa-1-azaspiro[4.5]
decane-1-carboxylate (20). A solution of 19 (0.648 g,
3.5 mmol), di-tert-butyl dicarbonate (2.3 g, 10.5 mmol),
triethylamine (2.5 mL, 17.5 mmol) and 4-dimethyl ami-
nopyridine (0.085 g, 0.7 mmol) in THF (10 mL) was
refluxed for 24 h. The reaction mixture was cooled and
concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel with EtOAc/hex-
anes (1:2) as eluant to give 20 as a white solid (0.572 g,
57%): mp 106 ꢁC; 1H NMR (CDCl3) d 4.62 (d, 2H,
J=11 Hz, OCH2), 3.46 (d, 2H, J=11 Hz, OCH2), 2.48
(t, 2H, J=8.3 Hz, CH2CONH), 2.28 (t, 2H, J=8 Hz,
CH2CH2CONH), 1.55 (m, 12H, C(CH3)3 and (CH3)2C),
1.41 (s, 3H, (CH3)2C); 13C NMR (CDCl3) d 174.43,
150.27, 98.60, 83.80, 64.28, 60.41, 29.68, 28.16, 28.05,
Hz, OCH2C), 3.40 (d, 2H, J=11.2 Hz, OCH2C), 2.91
(m, 2H, H-4), 2.72 (m, 2H, >C¼CHCH2), 2.0 (m, 4H,
CH2CH¼CHCH2), 1.56 (bs, 12H, NCO2C(CH3)3 and
C(CH3)2), 1.41 (s, 3H, C(CH3)2), 1.25–1.5 (m, 22H),
0.88 (distorted t, 3H).
13C NMR (CDCl3) d 166.96, 151.05, 144.55, 129.92,
129.81, 126.24, 98.44, 83.56, 64.56, 57.45, 37.08, 31.89,
29.75, 29.51, 29.38, 29.30, 29.22, 28.49, 28.14, 27.65,
27.19, 22.67, 19.23, 14.10; IR (neat) 1739 and 1714
(C¼O), 1456 cmꢀ1
.
5,5-Bis(hydroxymethyl)-3-[(E,9Z)-9-octadecenylidene]-2-
pyrrolidinone (23). A solution of 21 (0.107 g, 0.2 mmol)
in THF (2 mL) and H2O (0.5 mL) was treated with tri-
fluoroacetic acid (0.25 mL) at 0 ꢁC, and stirred for 12 h
at room temperature. The reaction mixture was cooled
and alkalinized with solid NaHCO3. The mixture was
filtrated and the filtrate was concentrated in vacuo. The
residue was purified by flash column chromatography
on silica gel with EtOAc as eluant to give 23 (0.057 g,
27.68, 19.42; IR (KBr) 1754 and 1709 (C¼O) cmꢀ1
.
tert-Butyl-3-[(E,9Z)-9-octadecenylidene]-8,8-dimethyl-2-
oxo-7,9-dioxa-1-azaspiro[4.5]decane-1-carboxylate (21)
and
tert-butyl-3-[(Z,9Z)-9-octadecenylidene]-8,8-dime-
thyl-2-oxo-7,9-dioxa-1-azaspiro[4.5]decane-1-carboxylate
(22). A solution of 20 (0.574 g, 2.0 mmol) in THF (8 mL)
was cooled to ꢀ78 ꢁC and treated slowly with lithium
bis(trimethylsilyl)amide (1 M in THF, 2.4 mL, 2.4
mmol). After stirring for 30 min, a solution of oleyl
aldehyde (0.7 g, 2.6 mmol) in THF (2 mL) was added and
stirring was continued for 4 h at ꢀ78 ꢁC. The reaction
mixture was quenched with a solution of ammonium
chloride and diluted with ether. The organic layer was
washed with H2O, dried over MgSO4, and concentrated
in vacuo. The residue was purified by flash column
chromatography on silica gel with EtOAc/hexanes (1:4)
as eluant to give the intermediate b-hydroxy lactam
(0.785 g, 71%) as an oil. The compound was dissolved in
CH2Cl2 (8 mL), cooled to 0 ꢁC, and treated with triethyl-
amine (0.82 mL, 5.68 mmol) and methanesulfonyl chlo-
ride (0.22 mL, 2.84 mmol). After stirring for 3 h at room
temperature, the reaction mixture was cooled to 0 ꢁC
and 1,8-diazabicyclo[5,4,0]undec-7-ene (0.637 mL, 4.26
mmol) was added. After refluxing for 12 h, the reaction
mixture was concentrated in vacuo. The residue was
diluted with ether, washed with 1 N HCl and H2O, dried
over MgSO4, and concentrated in vacuo. The residue
was purified by flash column chromatography on silica
gel with EtOAc/hexanes (1:10) as eluant to give 21 (0.425
g, 56%) and 22 (0.228 g, 30%), respectively, as oils.
72%) as a white solid: mp 37 ꢁC; H NMR (acetone-d6)
1
d 7.48 (bs, 1H, NH), 6.32 (m, 1H, >C¼CH), 5.34 (m,
2H, CH¼CH), 3.60 (d, 2H, J=11 Hz, CH2OH), 3.53 (d,
2H, J=11 Hz, CH2OH), 2.53 (m, 2H, H-4), 1.98–2.15
(m, 6H, >C¼CHCH2 and CH2CH¼CHCH2), 1.25–1.5
(m, 22H), 0.87 (distorted t, 3H).
5,5-Bis(hydroxymethyl)-3-[(Z,9Z)-9-octadecenylidene]-di-
hydro-1H-pyrrol-2-one (24). This compound was pre-
pared from 22 by following the above procedure in 79%
yield as a white solid: mp 39 ꢁC; 1H NMR (acetone-d6) d
6.70 (bs, 1H, NH), 5.77 (m, 1H, >C¼CH), 5.30 (m, 2H,
CH¼CH), 3.48 (m, 4H, CH2OH), 2.53 (m, 2H, H-4),
1.98–2.15 (m, 6H, >C¼CHCH2 and CH2CH¼CHCH2),
1.25–1.5 (m, 22H), 0.87 (distorted t, 3H).
{2-(Hydroxymethyl)-4-[(E,9Z)-9-octadecenylidene]-5-oxo-
2-pyrrolidinyl}methyl acetate (25). A solution of 23
(0.126 g, 0.32 mmol) and pyridine (0.05 mL, 0.64 mmol) in
CH2Cl2 (6 mL) was stirred for 2 h at room temperature,
cooled to 0 ꢁC, and treated with acetyl chloride (0.023 mL,
0.32 mmol). After stirring for 16 h at room temperature,
the mixture was concentrated in vacuo and the residue
was purified by flash column chromatography on silica gel
with EtOAc/MeOH (9:1) as eluant to give 25 (0.072 g,
52%) as an oil: 1H NMR (CDCl3) d 6.53 (m, 1H,
>C¼CH), 6.07 (bs, 1H, NH), 5.35 (m, 2H, CH¼CH),
4.22 (d, 1H, J=11.2 Hz, AcOCH2), 4.07 (d, 1H, J=11.2
Hz, AcOCH2), 3.56 (d, 1H, J=11 Hz, CH2OH), 3.48 (d,
1H, J=11 Hz, CH2OH), 2.65 (m, 2H, >C¼CHCH2),
2.51 (m, 2H, H-4), 1.9–2.05 (m, 4H, CH2CH¼CHCH2),
1.2–1.4 (m, 22H), 0.83 (distorted t, 3H); 13C NMR
(CDCl3) d 171.27, 170.62, 135.98, 130.03, 129.75, 125.03,
66.28, 65.58, 59.52, 32.21, 30.45, 29.77, 29.75, 29.53, 29.46,
29.34, 29.21, 27.24, 27.19, 23.43, 22.69, 20.75, 14.12.; IR
(neat) 3438 (OH), 1745 and 1666 (C¼O), 1456 cmꢀ1; MS
(EI) m/z 435 (M+). Anal. calcd for C26H45NO4: C, 71.68;
H, 10.41; N, 3.22. Found: C, 72.12; H, 10.45; N, 3.19.
21. E-Isomer, 1H NMR (CDCl3) d 6.74 (m, 1H,
>C¼CH), 5.35 (m, 2H, CH¼CH), 4.74 (d, 2H, J=11
Hz, OCH2C), 3.37 (d, 2H, J=11 Hz, OCH2C), 2.91 (bs,
2H, H-4), 2.18 (m, 2H, >C¼CHCH2), 2.0 (m, 4H,
CH2CH¼CHCH2), 1.58 (s, 3H, C(CH3)2), 1.56 (s, 9H,
NCO2C(CH3)3), 1.44 (s, 3H, C(CH3)2), 1.25–1.5 (m,
22H), 0.88 (distorted t, 3H); 13C NMR (CDCl3) d 167.37,
151.07, 140.13, 130.01, 129.77, 128.34, 98.46, 83.65,
64.91, 57.59, 33.53, 31.92, 29.78, 29.74, 29.53, 29.48,
29.32, 29.20, 28.61, 28.33, 28.12, 27.24, 27.19, 22.69,
19.16, 14.12; IR (neat) 1746 and 1714 (C¼O), 1457 cmꢀ1
.
{5-(Hydroxymethyl)-3-[(Z,9Z)-9-octadecenylidene]-2-ox-
otetrahydro-3H-pyrrol-5yl}methyl acetate (26). This
compound was prepared from 24 by following the above
22. Z-Isomer, 1H NMR (CDCl3) d 6.11 (m, 1H,
>C¼CH), 5.35 (m, 2H, CH¼CH), 4.67 (d, 2H, J=11.2

J.-H. Kang et al. / Bioorg. Med. Chem. 11 (2003) 2529–2539
2539
1
procedure in 50% yield as an oil; H NMR (CDCl3) d
Acknowledgements
6.49 (bs, 1H, NH), 5.93 (m, 1H, >C¼CH), 5.34 (m, 2H,
CH¼CH), 4.19 (d, 1H, J=11.2 Hz, AcOCH2), 4.05 (d,
1H, J=11.2 Hz, AcOCH2), 3.56 (d, 1H, J=11.5 Hz,
CH2OH), 3.47 (d, 1H, J=11.5 Hz, CH2OH), 2.70 (m,
2H, >C¼CHCH2), 2.58 (m, 2H, H-4), 2.10 (s, 3H,
CH3CO2), 1.9–2.1 (m, 4H, CH2CH¼CHCH2), 1.2–1.5
(m, 22H), 0.88 (distorted t, 3H); 13C NMR (CDCl3) d
171.30, 171.01, 140.39, 129.93, 129.86, 125.03, 66.11,
65.42, 59.13, 34.02, 32.21, 31.91, 29.77, 29.70, 29.53,
29.45, 29.32, 29.28, 27.23, 27.07, 22.69, 20.75, 14.12; IR
(neat) 3438 (OH), 1745 and 1694 (C¼O), 1455 cmꢀ1; MS
(EI) m/z 435 (M+). Anal. calcd for C26H45NO4: C, 71.68;
H, 10.41; N, 3.22. Found: C, 72.13; H, 10.44; N, 3.19.
This research was supported by grant KOSEF R01-
2000-00176 from the Korea Science and Engineering
Foundation. We thank Justin Kung and Kathy Hughes
for technical assistance.
References and Notes
1. Protein Kinase C. Current and Future Perspectives; Lester,
D. S., Espand, R. M., Eds. Ellis Horwood: New York, 1992.
2. Protein Kinase C; Kuo, J. F. Ed. Oxford University Press:
New York, 1994.
3. Livneh, E.; Fishman, D. D. Eur. J. Biochem. 1997, 248, 1.
4. Newton, A. C. Regulation of Protein Kinase. Curr. Opin.
Cell. Biol. 1997, 9, 161.
5. Blumberg, P. M. Mol. Carcinog. 1991, 4, 339.
6. Kazanietz, M. G.; Caloca, M. J.; Eroles, P.; Fujii, T.; Gar-
cia-Bermejo, M. L.; Reilly, M.; Wang, H. B. Biochem. Phar-
macol. 2000, 60, 1417.
7. Marquez, V. E.; Nacro, K.; Benzaria, S.; Lee, J.; Sharma,
R.; Teng, K.; Milne, G. W. A.; Bienfait, B.; Wang, S.; Lewin,
N. E.; Blumberg, P. M. Pharmacol. Ther. 1999, 82, 251.
8. Zhang, G.; Kazanietz, M. G.; Blumberg, P. M.; Hurley,
J. H. Cell 1995, 81, 917.
9. 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. J. Med. Chem. 2000, 43, 921.
10. Benzaria, S.; Bienfait, B.; Nacro, K.; Wang, S.; Lewin, N.;
Beheshti, M.; Blumberg, P. M.; Marquez, V. E. Bioorg. Med.
Chem. Lett. 1998, 8, 3403.
11. Sharma, R.; Lee, J.; Wang, S.; Milne, G. W. A.; Lewin,
N. E.; Blumberg, P. M.; Marquez, V. E. J. Med. Chem. 1996,
39, 19.
{2-(Hydroxymethyl)-4-[(E,9Z)-9-octadecenylidene]-5-oxo-
2-pyrrolidinyl}methyl octanoate (27). This compound
was prepared from 23 by following the procedure
1
described for synthesis of 25 in 57% yield as an oil: H
NMR (CDCl3) d 6.56 (bs, 1H, NH), 6.50 (m, 1H,
>C¼CH), 5.35 (m, 2H, CH¼CH), 4.22 (d, 1H, J=11.2
Hz, CO2CH2), 4.06 (d, 1H, J=11.2 Hz, CO2CH2), 3.57
(d, 1H, J=11.5 Hz, CH2OH), 3.47 (d, 1H, J=11.5 Hz,
CH2OH), 2.54 (m, 2H, H-4), 2.34 (t, 2H, J=7.3 Hz,
CH2CO2), 1.9–2.2 (m, 6H, >C¼CHCH2 and
CH2CH¼CHCH2), 1.2–1.5 (m, 32H), 0.88 (distorted t,
6H); 13C NMR (CDCl3) d 174.20, 170.51, 136.00, 130.04,
129.76, 125.05, 66.06, 65.58, 59.56, 34.14, 32.62, 31.92,
31.65, 29.78, 29.76, 29.54, 29.47, 29.34, 29.23, 29.11,
28.90, 27.25, 27.20, 24.91, 22.70, 22.61, 14.13, 14.06; IR
(neat) 3438 (OH), 1746 and 1665 (C¼O), 1456 cmꢀ1; MS
(EI) m/z 519 (M+). Anal. calcd for C32H57NO4: C, 73.94;
H, 11.05; N, 2.69. Found: C, 74.21; H, 11.10; N, 2.65.
12. Lee, J.; Wang, S.; Milne, G. W. A.; Sharma, R.; Lewin,
N. E.; Blumberg, P. M.; Marquez, V. E. J. Med. Chem. 1996,
39, 29.
13. Lee, J.; Sharma, R.; Wang, S.; Milne, G. W. A.; Lewin,
N. E.; Szallazi, Z.; Blumberg, P. M.; George, C.; Marquez,
V. E. J. Med. Chem. 1996, 36.
14. Marquez, V. E.; Lee, J.; Sharma, R.; Teng, K.; Wang, S.;
Lewin, N.; Blumberg, P. M. Bioorg. Med. Chem. Lett. 1994, 4,
355.
15. Qiao, L.; Zhao, L.-Y.; Rong, S.-B.; Wu, X.-W.; Wang, S.;
Fujii, T.; Kazanietz, M. G.; Rauser, L.; Savage, J.; Roth, B. L.;
Flippen-Anderson, J.; Kozikowski, A. P. Bioorg. Med. Chem.
Lett. 2001, 11, 955.
16. Rozas, I.; Alkorta, I.; Elguero, J. J. Phys. Chem. A 1998,
102, 9925.
17. Goutev, N.; Matsuura, H. J. Phys. Chem. A 2001, 1005,
4741.
18. Lac-3 as DAG-lactone was docked into the empty PK-Cd
C1b domain using the program DOCK in SYBYL in sn-2
mode and then minimized with the constraints reported pre-
viously.9
{5-(Hydroxymethyl)-3-[(Z,9Z)-9-octadecenylidene]-2-oxo-
tetrahydro-3H-pyrrol-5-yl}methyl octanoate (28). This
compound was prepared from 24 by following the pro-
cedure described for synthesis of 26 in 58% yield as an
oil: 1H NMR (CDCl3) d 6.15 (bs, 1H, NH), 5.95 (m, 1H,
>C¼CH), 5.34 (m, 2H, CH¼CH), 4.19 (d, 1H, J=11.5
Hz, CO2CH2), 4.05 (d, 1H, J=11.5 Hz, CO2CH2), 3.51
(d, 1H, J=11.2 Hz, CH2OH), 3.46 (d, 1H, J=11.2 Hz,
CH2OH), 2.70 (m, 2H, >C¼CHCH2), 2.58 (m, 2H, H-
4), 2.34 (t, 2H, J=7.3 Hz, CH2CO2), 1.9–2.1 (m, 4H,
CH2CH¼CHCH2), 1.2–1.5 (m, 32H), 0.88 (distorted t,
6H); 13C NMR (CDCl3) d 174.23, 170.68, 140.37, 129.94,
129.87, 127.02, 65.88, 65.42, 59.05, 34.15, 34.09, 31.92,
31.65, 29.80, 29.72, 29.54, 29.46, 29.34, 29.30, 29.11,
28.90, 27.24, 27.08, 24.91, 22.70, 22.61, 14.13, 14.07; IR
(neat) 3437 (OH), 1745 and 1664 (C¼O), 1455 cmꢀ1; MS
(EI) m/z 519 (M+). Anal. calcd for C32H57NO4: C, 73.94;
H, 11.05; N, 2.69. Found: C, 74.23; H, 11.11; N, 2.66.