The amide hydrogen of (-)-indolactam-V and benzolactam-V8's plays a critical role in protein kinase C binding and tumor-promoting activities

Article abstract of DOI:10.1016/S0960-894X(01)00047-6

To investigate the role of the amide hydrogen of (-)-indolactam-V (1) and benzolactam-V8's on protein kinase C (PKC) binding and tumor promotion. 8-decylbenzolactone-V8 (6), a new lactone analogue of 8-decylbenzolactam-V8 (4), was synthesized from 2-nitrophenylpyruvic acid (7) in 11 steps. The PKC binding ability and tumor-promoting activities in vitro of 6 were much lower than those of 1 and 4, suggesting that the amide hydrogen of 1 and benzolactam-V8's plays a critical role in tumor promotion, However, it is noteworthy that 6 showed significant selectivity in the PKC isozyme surrogate binding.

Full text of DOI:10.1016/S0960-894X(01)00047-6

Bioorganic & Medicinal Chemistry Letters 11 (2001) 723±728
The Amide Hydrogen of (À)-Indolactam-V and Benzolactam-V8's
Plays a Critical Role in Protein Kinase C Binding
and Tumor-Promoting Activities
Yu Nakagawa,a Kazuhiro Irie,a,* Yoshimasa Nakamurab and Hajime Ohigashia
aDivision of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
bLaboratory of Food and Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
Received 30 November 2000;accepted 13 January 2001
AbstractÐTo investigate the role of the amide hydrogen of (À)-indolactam-V (1) and benzolactam-V8's on protein kinase C (PKC)
binding and tumor promotion, 8-decylbenzolactone-V8 (6), a new lactone analogue of 8-decylbenzolactam-V8 (4), was synthesized
from 2-nitrophenylpyruvic acid (7) in 11 steps. The PKC binding ability and tumor-promoting activities in vitro of 6 were much
lower than those of 1 and 4, suggesting that the amide hydrogen of 1 and benzolactam-V8's plays a critical role in tumor promo-
tion. However, it is noteworthy that 6 showed signi®cant selectivity in the PKC isozyme surrogate binding. # 2001 Published by
Elsevier Science Ltd.
Tumor-promoting (À)-indolactam-V (1)1,2 is the mini-
mal basic structure exhibiting tumor promotion and
activation of protein kinase C (PKC),3 a crucial enzyme
involved in cellular signal transduction (Fig. 1). Inten-
sive structure±activity studies on 1 revealed almost all
structural factors required for tumor promotion and
PKC binding except for the role of the amide subunit.4
Conformation studies led to the ®nding that 1 exists as
two stable conformers in solution at room temperature;5
the active twist conformer with a cis amide geometry
and the inactive sofa conformer with a trans amide
geometry.6,7 The tumor promoter binding site of PKC
was also identi®ed;phorbol ester type tumor promoters
bind to the cysteine-rich C1 domains designated as C1A
and C1B.8,9 Based on these ®ndings coupled with X-ray
crystal structure analysis of PKCd-C1B in complex with
phorbol 13-acetate as a ligand,10 computational docking
studies indicated that both the amide hydrogen and the
carbonyl oxygen of 1 interact with the binding site of
PKCd-C1B.11,12 However, there are no experimental
results on the role of the amide group in the PKC isozyme
binding and tumor promotion. Although we previously
synthesized indolactone-V (2), a lactone analogue of 1,
we could not determine whether the amide hydrogen of 1
is necessary for tumor promotion or not since 2 existed as
only the inactive sofa conformer.13
Endo et al. and Kozikowski et al. independently repor-
ted that benzolactam-V8 (3) with an eight-membered
lactam containing a benzene ring instead of the nine-
membered lactam of 1 reproduced the active conforma-
tion of 1. 8-Decylbenzolactam-V8 (4) showed signi®cant
tumor-promoting activities in vitro comparable to
1.6,14,15 They also independently found that the binding
mode of 3 to PKCd-C1B is quite similar to that of 1 by
computational docking studies.11,15 These ®ndings
prompted us to synthesize 8-decylbenzolactone-V8 (6), a
new lactone analogue of 4, and to examine its con-
formation, PKC binding ability, and tumor-promoting
activities in vitro.
Benzolactone-V8 (5), the core structure of 8-decylbenzo-
lactone-V8 (6), was synthesized from 2-nitrophenyl-
pyruvic acid (7) as shown in Scheme 1. Reduction of
both the ketone and the carboxyl group of 7 with
*Corresponding author. Tel.: +81-75-753-6282;fax: +81-75-753-
6284;e-mail: irie@kais.kyoto-u.ac.jp
Figure 1. Structures of (À)-indolactam-V, benzolactam-V8's, and their
lactone derivatives.
0960-894X/01/$ - see front matter # 2001 Published by Elsevier Science Ltd.
PII: S0960-894X(01)00047-6

724
Y. Nakagawa et al. / Bioorg. Med. Chem. Lett. 11 (2001) 723±728
Scheme 1. Synthesis of benzolactone-V8 (5).
borane gave a diol (8, 73.5%), whose primary hydroxyl
group was protected selectively with a tert-butyl-
dimethylsilyl (TBDMS) group (80.5%). The nitro group
of 9 was reduced by catalytic hydrogenation using 10%
palladium on carbon to give the aniline derivative 10
(95.3%). The valine subunit was introduced by sub-
stitution of 10 with d-valine-derived tri¯ate16 to give
two diastereomeric esters (11, 71.2%). Since separation
of these diastereomers was quite dicult at this point,
we proceeded to the next cyclization step. After depro-
tection of the benzyl group of 11 by hydrogenation in
acetonitrile, intramolecular esteri®cation was accom-
plished with DCC, HOBt, and triethylamine in di-
chloromethane to give 12 (28.7%). This slightly lower
cyclization yield was expected since the synthesis of an
eight- or a nine-membered lactone is generally quite
dicult.17 The presence of bulky substituents like iso-
propyl and TBDMS groups might be another reason for
this low yield. Methylation of the diastereomeric lac-
tones (12) by the method of Kozikowski et al.15 gave
two diastereomers (13, 14), which were easily separated
by silica gel column chromatography.
protons in the NOESY spectrum suggested that 15 was
epi-benzolactone-V818 with the R con®guration at posi-
1
tion 5. On the other hand, the H±1H COSY spectrum
revealed that 16 was not the expected benzolactone-V8
but the nine-membered lactone19 because of the lack of
the hydroxymethyl proton signals. This nine-membered
lactone (16) was deduced to be formed by the intramo-
lecular transesteri®cation under the acidic condition of
the TBDMS deprotection step (1 N HCl±dioxane). We
attempted to deprotect the TBDMS group of 14 with
TBAF in THF at room temperature to get a major
product identi®ed as 16 (63.8%) along with a minor
product 5 (6.9%), which was thought to be a desired
eight-membered lactone. To suppress the transester-
i®cation, we conducted this reaction at À20 ꢀC and suc-
1
ceeded in obtaining mainly 5 (76.0%). The H NMR
spectrum showed that 5 existed as a single conformer in
deuteriochloroform at room temperature. The cross
peak between the hydroxyl group and the methylene
protons at position 11 was observed in the 1H±1H
COSY spectrum, suggesting that 5 is an eight-mem-
bered lactone. No NOE enhancement between H-2 and
H-5 protons in the NOESY spectrum strongly indicated
that 5 is benzolactone-V820 with the S con®guration at
position 5. In addition, signi®cant NOE enhancements
between H-2 and H-6 protons, and between H-5 and H-
15 protons, which are characteristic of benzolactam-V8
(3),6 were observed, indicating that the conformation of
5 is very close to that of 3. Benzolactone-V8 (5) did not
convert to 16 even at room temperature in Tris or
phosphate bu€er (pH 7.4) which is used in the bioassays
shown below.
Deprotection of each TBDMS group of 13 and 14 with
1 N HCl in dioxane gave a single product, 15 and 16,
1
respectively, whose H NMR spectra in deuteriochloro-
form showed that each compound existed as a single
1
conformer at room temperature (Table 1). The H±1H
COSY spectrum indicated that 15 was an eight-mem-
bered lactone since the cross peak between the hydroxyl
group and methylene protons at position 11 was
observed. The signi®cant NOE between H-2 and H-5

Y. Nakagawa et al. / Bioorg. Med. Chem. Lett. 11 (2001) 723±728
725
Table 1. 1H NMR spectra of benzolactam-V8 (3), benzolactone-V8 (5), epi-benzolactam-V8, and epi-benzolactone-V8 (15) in deuteriochloroform
(500 MHz, 300 K)
d (Multiplicity, J in Hz)
Benzolactam-V8
(3)a
Benzolactone-V8
(5)b
epi-Benzolactam-V8c
epi-Benzolactone-V8
(15)d
No
2
5
3.46 (1H, d, J=8.6)
4.05 (1H, m)
3.34 (1H, d, J=10.2)
4.82 (1H, m)
3.18 (1H, d, J=10.6)
3.83 (1H, m)
3.28 (1H, d, J=10.8)
4.65 (1H, m)
6a
6b
7
8
9
10
11a
11b
12
13
14
15
2.81 (1H, dd, J=16.9, 2.2)
3.08 (1H, dd, J=16.9, 8.0)
7.02 (1H, d, J=7.6)
7.18 (1H, t, J=7.6)
6.88 (1H, t, J=7.6)
7.04 (1H, d, J=7.6)
3.52 (1H, m)
3.70 (1H, m)
2.43 (1H, m)
1.06 (3H, d, J=6.5)
0.89 (3H, d, J=6.8)
2.79 (3H, s)
2.98 (1H, dd, J=16.3, 5.1)
3.05 (1H, dd, J=16.3, 3.7)
7.08 (1H, d, J=7.6)
7.22 (1H, t, J=7.6)
7.04 (1H, t, J=7.6)
7.09 (1H, d, J=7.6)
3.69 (1H, m)
3.69 (1H, m)
2.23 (1H, m)
1.02 (3H, d, J=6.6)
0.99 (3H, d, J=6.5)
2.80 (3H, s)
2.86 (1H, d, J=15.2)
2.92 (1H, dd, J=15.2, 6.5)
7.10 (1H, d, J=7.4)
7.19 (1H, t, J=7.4)
6.95 (1H, t, J=7.4)
7.12 (1H, d, J=7.4)
3.76 (1H, m)
3.76 (1H, m)
2.41 (1H, m)
0.97 (3H, d, J=6.6)
0.87 (3H, d, J=6.5)
2.93 (3H, s)
2.82 (1H, dd, J=15.5, 2.4)
2.94 (1H, dd, J=15.5, 5.8)
7.11 (1H, d, J=7.3)
7.22 (1H, t, J=7.3)
7.06 (1H, t, J=7.3)
7.19 (1H, d, J=7.3)
3.77 (1H, dd, J=11.9, 4.2)
3.82 (1H, dd, J=11.9, 7.2)
2.32 (1H, m)
1.06 (3H, d, J=6.6)
0.88 (3H, d, J=6.5)
2.91 (3H, s)
a0.082 M.
b0.102 M.
c0.067 M.
d0.061 M.
Scheme 2. Synthesis of 8-decylbenzolactone-V8 (6).
A decyl group was introduced at position 8 of 14 as
shown in Scheme 2. Unexpectedly, iodination of 14
using iodine in pyridine did not proceed at all though
11-O-acetylbenzolactam-V8 was iodinated by the same
reaction conditions.15 Since transesteri®cation might
occur in the conventional iodination and bromination
reactions under strong acidic conditions, we used a
strong bromination reagent, benzyltrimethylammonium
tribromide (BTMABr3),21 under neutral conditions.
Bromination of 14 using BTMABr3 mildly proceeded at
room temperature to give 11-O-TBDMS-8-bromo-
benzolactone-V8 (17, 69.4%). Coupling reaction of 17
with 1-decene was accomplished by the method of Endo
et al.6 to give two coupling products. Hydrogenation of
these alkenes using 5% palladium on carbon followed
by deprotection of the TBDMS group with TBAF at
À20 ꢀC gave 8-decylbenzolactone-V8 (6, 34.4%)22 and
its regio isomers (18, 12.8%). The 1H NMR and
NOESY spectra showed that 6 existed as a single con-
former in deuteriochloroform at room temperature, and
that its ring conformation is quite similar to that of
benzolactam-V8 (3).
The biological activities of 6 along with 1 and 4, which
was synthesized by the method of Endo et al.14 with a
slight modi®cation, were examined by three in vitro
bioassays related to in vivo tumor promotion: binding
to the PKC C1 domains,23,24 Epstein-Barr virus early
antigen (EBV-EA)-inducing ability,25,26 and superoxide
À
(O2 ) generation-inducing ability in di€erentiated HL-
60 cells.27,28 The binding anity to the PKC C1
domains was evaluated by inhibition of the speci®c
binding of [3H]phorbol-12,13-dibutyrate (PDBu) to
these C1 domains as reported by Sharkey and Blum-

726
Y. Nakagawa et al. / Bioorg. Med. Chem. Lett. 11 (2001) 723±728
berg.24 We have recently synthesized individual C1A
and C1B domains of all PKC isozymes consisting of
about 50 amino acids by the solid-phase synthesis and
measured the dissociation constants (Kd) of
[3H]PDBu.23,29,30 Using these PKC C1 peptides, the
concentration required to cause 50% inhibition, IC50, of
the [3H]PDBu binding was measured. The binding a-
nity of 4 and 6 to each PKC C1 peptide was expressed
as the Ki values calculated from the IC50 and the Kd
values of [3H]PDBu as reported previously.24,29,30
observed with typical tumor promoters.7 Superoxide
(O2 ) generation is triggered by 12-O-tetra-
À
decanoylphorbol-13-acetate (TPA) in epithelial cells and
leukocytes through the xanthine oxidase31 and NADPH
oxidase systems,32 respectively. The ability is expressed
À
as the level of O2 production. Under our experimental
conditions, TPA produced 1.32 nmol/mL/min of O2 at
À
10À7 M. Tables 2±4 summarize the results of these
assays.
8-Decylbenzolactam-V8 (4) showed slightly weaker
binding anities than 1 for almost all PKC C1 peptides.
On the other hand, the binding anities of 8-decylben-
zolactone-V8 (6) for all PKC C1 peptides were far lower
than those of 1 and 4. These results indicate that the
amide hydrogen of 1 and benzolactam-V8's is necessary
to amplify the binding anities for all PKC isozymes.
EBVs are under the strict control of the host human
lymphoblastoid Raji cells. They are activated by tumor
promoters to produce the early antigen (EA).25,26 The
EBV-EA-inducing activity is expressed as the percen-
tage of EA-positive cells. Under our experimental con-
ditions, about 30% of maximum EA-induction was
Table 2. Ki Values for inhibition of the speci®c binding of [3H]PDBu by 8-decylbenzolactam-V8 (4) and 8-decylbenzolactone-V8 (6)
PKC C1 peptide
Ki (nM)
8-Decylbenzolactam-V8
(4)
8-Decylbenzolactone-V8
(6)
(À)-Indolactam-V
(1)a
a-C1A(72-mer)b
a-C1B
322.5 (36.1)c
4690 (201)
442.4 (34.7)
260.8 (16.7)
1664 (99.7)
150.6 (6.8)
2771 (240)
14.6 (1.6)
8361 (385)
13.1 (0.2)
2489 (74)
6.4 (1.2)
>10,000
>10,000
>10,000
22,010 (1896)
6321 (536)
17,574 (1648)
70,641 (3322)
1155 (46)
>10,000
262.3 (3.5)
>10,000
119.9 (6.7)
NT
1097 (53)
126.9
4000
173.5
135.6
137.9
212.6
1900
8.3
4110
7.7
3770
5.5
NT
b-C1A(72-mer)b
b-C1B
g-C1A
g-C1B
d-C1A
d-C1B
e-C1A
e-C1B
Z-C1A
Z-C1B
y-C1A
y-C1B
NTd
13.2 (2.6)
8.7
aData taken from ref 30.
bTen residues from both N- and C-termini of the previous a-C1A and b-C1A23 were elongated since the solubility of the original 52-mer peptides
was extremely low.
cStandard deviation of at least two separate experiments.
dNot tested. The Kd value of [3H]PDBu to y-C1A could not be measured because of its very weak binding anity.
Table 3. EBV-EA-inducing activities of 8-decylbenzolactam-V8 (4) and 8-decylbenzolactone-V8 (6)a
Compound
% of EA-positive cells
10À5
10À7
M
10À6
M
M
10À4.5 Mb
(À)-Indolactam-V (1)
16.8 (2.1)c
33.5 (4.1)
6.7 (0.1)
0.8 (0.2)
28.9 (0.5)
11.3 (0.5)
1.9 (0.9)
8-Decylbenzolactam-V8 (4)
8-Decylbenzolactone-V8 (6)
17.6 (1.3)d
5.0 (1.6)
aThis assay was done by the method reported previously.25,26 The cell viability exceeded 80% in all experiments except for 4 at 10À4.5 M.
bData at 10À4 M could not be obtained because the cell viability was 0%.
cStandard deviation.
dCell viability: 65.7%.
Table 4. Superoxide generation-inducing activities of 8-decylbenzolactam-V8 (4) and 8-decylbenzolactone-V8 (6)a
À
Compound
O2 generation (nmol/mL min)
10À7
M
10À6
M
10À5
M
10À4
M
(À)-Indolactam-V (1)
0.05 (0.07)b
0.33 (0.29)
3.19 (0.10)
0.31 (0.18)
0.11 (0.02)
3.18 (0.08)
3.04 (0.14)
0.20 (0.08)
8-Decylbenzolactam-V8 (4)
8-Decylbenzolactone-V8 (6)
0.19 (0.03)
aThis assay was done by the method reported previously28 with slight modi®cation.
bStandard deviation.

Y. Nakagawa et al. / Bioorg. Med. Chem. Lett. 11 (2001) 723±728
5. Endo, Y.;Shudo, K.;Itai, A.;Hasegawa, M.;Sakai, S.
727
However, it is noteworthy that 6 bound to Z-C1B more
Tetrahedron 1986, 42, 5905.
6. Endo, Y.;Ohno, M.;Hirano, M.;Itai, A.;Shudo, K.
Am. Chem. Soc. 1996, 118, 1841.
selectively than 1 and 4;the binding anity of 6 for Z-
C1B was about 2-fold, 10-fold, and more than 50-fold
higher than those for e-C1B, d- and y-C1B, and the
other PKC C1 peptides, respectively. These results sug-
gest that relative contribution of the amide hydrogen of
1 and benzolactam-V8's to the Z-C1B binding is smaller
than that to the other PKC C1 peptide binding.
J.
7. Irie, K.;Isaka, T.;Iwata, Y.;Yanai, Y.;Nakamura, Y.;
Koizumi, F.;Ohigashi, H.;Wender, P. A.;Satomi, Y.;Nish-
ino, H. J. Am. Chem. Soc. 1996, 118, 10733.
8. Ono, Y.;Fujii, T.;Igarashi, K.;Kuno, T.;Tanaka, C.;
Kikkawa, U.;Nishizuka, Y. Proc. Natl. Acad. Sci. U.S.A.
1989, 86, 4868.
Compound 4 showed about 10-fold lower activities than
1 in both EBV-EA induction test and superoxide gen-
eration test. This indicates that 4 might be 10-fold
weaker as a tumor promoter than 1. However, 6 was
inactive even at 10À4.5 M in the EBV-EA induction test
and at 10À4 M in the superoxide generation test. These
results also support the theory that the amide hydrogen
of 1 and benzolactam-V8's plays a critical role in tumor
promotion. It is recently reported that PKCa and bII
are expressed in Raji B cells and that PKCb is essential
for the superoxide generation in di€erentiated HL-60
cells.33,34 These data are consistent with quite weak
binding abilities of 6 to the C1 peptides of PKCa and b.
9. Hurley, J. H.;Newton, A. C.;Perker, P. J.;Blumberg,
P. M.;Nishizuka, Y. Protein Sci. 1997, 6, 477.
10. Zhang, G.;Kazanietz, G. M.;Blumberg, M. P.;Hurley,
H. J. Cell 1995, 81, 917.
11. Endo, Y.;Takehara, S.;Ohno, M.;Driedger, P. E.;Sta-
bel, S.;Mizutani, M. Y.;Tomioka, N.;Itai, A.;Shudo, K.
Med. Chem. 1998, 41, 1476.
12. Wang, S.;Liu, M.;Lewin, N. E.;Lorenzo, P. S.;Bhatta-
charrya, D.;Qiao, L.;Kozikowski, A. P.;Blumberg, P. M. J.
Med. Chem. 1999, 42, 3436.
13. Nakagawa, Y.;Irie, K.;Nakamura, Y.;Ohigashi, H.;
Hayashi, H. Biosci. Biotech. Biochem. 1997, 61, 1415.
14. Endo, Y.;Ohno, M.;Takehara, S.;Driedger, P. A.;Sta-
bel, S.;Shudo, K. Chem. Pharm. Bull. 1997, 45, 424.
15. Kozikowski, A. P.;Wang, S.;Ma, D.;Yao, J.;Ahmad, S.;
Glazer, R. I.;Bogi, K.;Acs, P.;Modarres, S.;Lewin, N. E.;
Blumberg, P. M. J. Med. Chem. 1997, 40, 1316.
16. Kogan, T. P.;Somers, T. C.;Venuti, M. C. Tetrahedron
1990, 46, 6623.
J.
In summary, we have synthesized 8-decylbenzolactone-
V8 (6), a lactone analogue of 8-decylbenzolactam-V8
(4), to investigate the role of the amide hydrogen of
(À)-indolactam-V (1) and benzolactam-V8's on PKC
binding and tumor promotion. Compound 6 was far
less active than either 1 or 4 in the three in vitro bioas-
says related to in vivo tumor promotion, indicating that
the amide hydrogen of 1 and benzolactam-V8's plays a
critical role in the PKC binding and tumor promotion.
The PKC surrogate binding assay also revealed that the
role of the amide hydrogen of 1 and benzolactam-V8's
is signi®cantly di€erent among the PKC isozymes. The
present results provide the basis for the rational design
of new medicinal agents with PKC isozyme selectivity.
Compound 6 might be a lead compound for a PKCZ
selective modulator.
17. Rousseau, G. Tetrahedron 1995, 51, 2777.
18. epi-Benzolactone-V8 (15): [a]D À174.0ꢀ (c=0.57, MeOH,
27.8 ꢀC);UV lmax (MeOH) nm (e) 256 (5000), 207 (13,800);
HR-EIMS m/z: 263.1500 (M+, calcd for C15H21NO3,
263.1521).
19. Nine-membered benzolactone (16): [a]D À75.0ꢀ (c=0.40,
MeOH, 27.8 ꢀC);UV lmax (MeOH) nm (e) 270 (2900), 235
(2500), 206 (11,500); 1H NMR (500 MHz, CDCl3, 0.061 M,
27 ꢀC) d ppm: 1.00 (3H, d, J=6.6 Hz), 1.13 (3H, d, J=6.7 Hz),
2.01 (1H, d, J=7.3 Hz), 2.23 (1H, m), 2.71 (3H, s), 2.79 (1H,
dd, J=12.7, 2.7 Hz), 3.04 (1H, dd, J=12.7, 10.4 Hz), 3.14
(1H, d, J=9.7 Hz), 4.00 (1H, br.s), 4.17 (1H, m), 4.57 (1H,
br.s), 7.15±7.24 (4H, m);HR-EIMS m/z: 263.1497 (M+ calcd
for C15H21NO3, 263.1521).
20. Benzolactone-V8 (5) : [a]D À124.0ꢀ (c=0.68, MeOH,
27.8 ꢀC);UV lmax (MeOH) nm (e) 254 (4300), 207 (14,000);
HR-EIMS m/z: 263.1496 (M+, calcd for C15H21NO3,
263.1521).
21. Kajigaeshi, S.;Kakinami, T.;Inoue, K.;Kondo, M.;
Nakamura, H.;Fujikawa, M.;Okamoto, T. Bull. Chem. Soc.
Jpn. 1988, 61, 597.
Acknowledgements
This work was supported by a Grant-in-Aid for Scien-
ti®c Research (C) (2) (No. 11660109) and on Priority
Areas (A) (2) (No. 12045241) from the Ministry of
Education, Science, Culture, and Sports of Japan (for
K.I.), and from the Japan Society for the Promotion of
Science for Young Scientists (Y.N.).
22. 8-Decylbenzolactone-V8 (6)
:
[a]D À84.0ꢀ (c=0.42,
CHCl3, 24.3 ꢀC);UV lmax (MeOH) nm (e) 260 (2500); 1H
NMR (500 MHz, CDCl3, 0.088 M, 27 ꢀC) d ppm: 0.88 (3H, t,
J=6.9 Hz), 0.97 (3H, d, J=6.6 Hz), 1.05 (3H, d, J=6.6 Hz),
1.23±1.31 (14H, m), 1.58 (2H, m), 2.17 (2H, m), 2.53 (2H, t,
J=7.8 Hz), 2.76 (3H, s), 2.87 (1H, dd, J=15.8, 5.1 Hz), 3.01
(1H, dd, J=15.8, 4.0 Hz), 3.28 (1H, d, J=10.2 Hz), 3.69 (2H,
m), 4.87 (1H, m), 6.86 (1H, s), 7.02 (2H, s); 13C NMR (125
MHz, CDCl3, 0.088 M, 27 ꢀC) d ppm: 14.12, 19.28, 19.87,
22.70, 26.68, 29.34, 29.41, 29.52, 29.62, 29.64, 31.52, 31.93,
35.27, 35.31, 36.39, 64.49, 74.66, 77.54, 126.74, 127.93, 132.17,
133.41, 139.68, 148.30, 170.68;HR-EIMS m/z: 403.3064 (M+,
calcd for C25H41NO3, 403.3086).
References and Notes
1. Endo, Y.;Shudo, K.;Okamoto, T.
1982, 30, 3457.
2. Irie, K.;Hirota, M.;Hagiwara, N.;Koshimizu, K.;Haya-
shi, H.;Marao, S.;Tokuda, H.;Ito, Y.
1984, 48, 1269.
3. Nishizuka, Y. Nature 1984, 308, 693.
Chem. Pharm. Bull.
23. Irie, K.;Oie, K.;Nakahara, A.;Yanai, Y.;Ohigashi, H.;
Wender, P. A.;Fukuda, H.;Konishi, H.;Kikkawa, U. J. Am.
Chem. Soc. 1998, 120, 9159.
24. Sharkey, N. A.;Blumberg, P. M. Cancer Res. 1985, 45, 19.
25. Ito, Y.;Yanase, S.;Fujita, J.;Harayama, T.;Takashima,
M.;Imanaka, H. Cancer Lett. 1981, 13, 29.
Agric. Biol. Chem.
4. Irie, K.;Koshimizu, K. Comments Agric. Food Chem. 1993,
3, 1.

728
Y. Nakagawa et al. / Bioorg. Med. Chem. Lett. 11 (2001) 723±728
26. zur Hausen, H.;Bornkamm, G. W.;Schmidt, R.;Hecker,
E. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 782.
30. Nakagawa, Y.;Irie, K.;Ohigashi, H.;Hayashi, H.;Wen-
der, P. A. Bioorg. Med. Chem. Lett. 2000, 10, 2087.
31. Reiners, J. J., Jr.;Pence, B. C.;Barcus, M. C. S.;Cantu,
A. R. Cancer Res. 1987, 47, 1775.
27. Market, M.;Andrews, P. C.;Babior, B. M.
Enzymol. 1984, 105, 358.
Methods
28. Nakamura, Y.;Murakami, A.;Ohto, Y.;Torikai, K.;
Tanaka, T.;Ohigashi, H. Cancer Res. 1998, 58, 4832.
29. Irie, K.;Nakahara, A.;Ikawa, Y.;Tanaka, M.;Naka-
gawa, Y.;Nakamura, Y.;Ohigashi, H.;Wender, P. A. Biosci.
Biotechnol. Biochem. 2000, 64, 2429.
32. Cross, A. R.;Jones, O. T. G. Biochim. Biophys. Acta 1991,
1057, 281.
33. Setterblad, N.;Onyango, I.;Pihlgren, U.;Rask, L.;
Andersson, G. J. Immunol. 1998, 161, 4819.
34. Korchak, H. M.;Rossi, M. W.;Kilpatrick, L. E. J. Biol.
Chem. 1998, 273, 27292.