Synthesis and binding selectivity of 7- and 15-decylbenzolactone-V8 for the C1 domains of protein kinase C isozymes

Article abstract of DOI:10.1016/S0960-894X(03)00637-1

Benzolactone-V8 (4) is a lactone analogue of the artificial tumor promoter benzolactam-V8 (1). To investigate the effect of hydrophobic substituents at positions 7 and 15 of 4 on binding selectivity for protein kinase C (PKC) isozymes, 7- and 15-decylbenz

Full text of DOI:10.1016/S0960-894X(03)00637-1

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3015–3019
Synthesis and Binding Selectivity of 7- and
15-Decylbenzolactone-V8 for the C1 Domains
of Protein Kinase C Isozymes
Yu Nakagawa,a Kazuhiro Irie,a,* Nobuhiro Yamanaka,a Hajime Ohigashia
and Ken-ichiro Tsudab
aDivision of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
bFundamental Research Laboratories NEC Corporation, 34 Miyukigaoka, Tsukuba 305-8501, Japan
Received 12 March 2002; accepted 10 May 2003
Abstract—Benzolactone-V8 (4) is a lactone analogue of the artificial tumor promoter benzolactam-V8 (1). To investigate the effect
of hydrophobic substituents at positions 7 and 15 of 4 on binding selectivity for protein kinase C (PKC) isozymes, 7- and 15-
decylbenzolactone-V8 (7, 8) were synthesized and their binding affinities for synthetic PKC isozyme C1 peptides were examined.
Compound 8 showed moderate selectivity for novel PKC isozymes similar to 9-decylbenzolactone-V8 (5), while 7 was less selective.
Compounds 7 and 8 showed no significant selectivity among novel PKC isozymes unlike 8-decylbenzolactone-V8 (6). These results
indicate that the introduction of a hydrophobic substituent at position 8 of 4 is most effective in the development of PKCe- and
PKCZ-selective binders.
# 2003 Elsevier Ltd. All rights reserved.
Protein kinase C (PKC) isozymes are serine/threonine-
specific protein kinases involved in cellular signal trans-
duction.1 PKC isozymes are subdivided into three
groups; conventional PKCs (a, bI, bII, g), novel PKCs
(d, e, Z, y), and atypical PKCs (z, ꢀ=l) (Fig. 1).2,3 Con-
ventional and novel PKCs are also major receptors of
tumor promoters and thus serve as therapeutic targets
in cancer. Tumor promoters such as 12-O-tetra-
decanoylphorbol 13-acetate (TPA),4 teleocidin B-4,5
and aplysiatoxin5 activate these PKC isozymes by
binding to two C1 domains (C1A, C1B) in the reg-
ulatory region, which are also the binding sites of the
endogenous second messenger 1,2-sn-diacylglycerol and
antineoplastic bryostatin 1.6 Recent studies revealed
that novel PKC isozymes (d, e, Z) are involved in tumor
promotion,7À10 and that the role of each C1 domain is
different among PKC isozymes in response to tumor
promoters.11À14 The design of agents with not only PKC
isozyme-selectivity but also C1 domain-selectivity is
thus indispensable to understand the precise mechanism
of tumor promotion and to develop anticancer drugs.
The artificial tumor promoter benzolactam-V8 (1)15 is a
promising lead compound for PKC isozyme- and C1
domain-selective agents since it has a simple structure,
and since its binding affinity for PKC isozymes can be
easily enhanced by the introduction of hydrophobic side
chains (Fig. 2). It is reported that the position of the
hydrophobic chain of benzolactam-V8s might affect the
selectivity of binding to PKC isozymes.16,17 The binding
affinities of 8-decylbenzolactam-V8 (2) were slightly
higher for conventional PKCs than novel PKCs, while
7-decylbenzolactam-V8 (3) showed moderate selectivity
for novel PKCs rather than conventional PKCs. Such
substituent effect was more significant in benzolactone-
V8 (4), the lactone analogue of 1.18 We have recently
synthesized 8- and 9-decylbenzolactone-V8s (5, 6) and
evaluated the selectivity of their binding to C1 domains
of PKC isozymes using synthetic PKC C1 peptides.19
The binding affinities of 9-decylbenzolactone-V8 (5)
were considerably higher for the C1B peptides of novel
PKCs than the other PKC C1 peptides. The selectivity
was especially improved in 6, which showed significant
selective binding of PKCe and Z. These results suggest
that benzolactone-V8s with hydrophobic substituents at
positions other than 8 and 9 might be candidates for
new agents with PKC isozyme- and C1 domain-selectivity.
*Corresponding author. Tel.: +81-75-753-6282; fax: +81-75-753-
6284; e-mail: irie@kais.kyoto-u.ac.jp
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00637-1

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Y. Nakagawa et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3015–3019
Figure 1. Structures of PKC isozymes.
Figure 2. Structures of benzolactam-V8s and benzolactone-V8s.
We report here the synthesis of 7- and 15-decylbenzo-
lactone-V8 (7 and 8) and their binding affinities for the
C1 peptides of all PKC isozymes.
tion of 15 with d-valine-derived triflate21 to give two
diastereomeric esters 16 (84%). The primary hydroxyl
group of 16 was selectively protected with a TBDMS
group at 0 ꢀC (83%). Hydrogenolysis of the benzyl
group of 17, followed by intramolecular esterification with
1,3-dicyclohexylcarbodiimide (DCC) and 4-(dimethyl-
amino)pyridine (DMAP), gave diastereomeric lactones
18 (37%). To avoid intramolecular transesterification
observed in the synthesis of benzolactone-V8 (4),18
deprotection of the TBDMS group of 18 was carried
out with tetrabutylammonium fluoride (TBAF) at
À20 ꢀC. The two diastereomers were easily separated by
column chromatography to give 7 (41%)22 and its C-5
epimer 19 (23%),23 which were identified according to
the NOESY spectra; significant NOE enhancement
between the H-2( d 3.22) and H-5 (d 4.51) protons was
observed in 19 but not in 7.
7-Decylbenzolactone-V8 (7) was synthesized from 2-
bromo-6-nitrotoluene (9) as shown in Scheme 1. SN2
substitution of 9 with diethyl oxalate gave the ethyl
pyruvate derivative 10 (93%). After reduction of both
the ketone and the ester groups of 10 (91%), the resulting
two hydroxyl groups were protected with acetyl groups
(94%). Modified Sonogashira coupling20 of 12 with 1-
decyne gave 13 in a slightly lower yield (40%) because
of steric hindrance with the ortho substituent. Hydro-
genation of 13 followed by N-formylation gave 14
(62%), the formyl group of which was reduced with
borane, followed by alkaline hydrolysis to yield 15
(81%). The valine subunit was introduced by substitu-
Scheme 1. Synthesis of 7-decylbenzolactone-B8 (7).

Y. Nakagawa et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3015–3019
3017
Scheme 2. Synthesis of 15-decylbenzolactone-V8 (8).
Synthesis of 8 is shown in Scheme 2. The starting mate-
rial 20 was prepared as reported previously.18 After the
diacetylation of 20, the nitro group was reduced by
hydrogenation to give 21 (91%). Undecanoylation of 21
(99%) and reduction of the amide group with borane
gave the undecyl aniline derivative, two acetyl groups of
which were deprotected by alkaline hydrolysis (81%).
SN2substitution of 23 with d-valine-derived triflate
(76%), followed by selective TBDMS protection of the
primary hydroxyl group, gave 25 (91%). After removal
of the benzyl group of 25, intramolecular esterification
easily proceeded (60%) since the carboxyl group could
access the diol moiety due to steric repulsion between
the undecyl group and the TBDMS group. Deprotec-
tion of the TBDMS group of 26 with TBAF at À20 ꢀC
gave three products 27,24 8,25 and 2826 (26% for 27,
41% for 8 and 28). 1H NMR and NOESY spectra
showed that 27 was epi-15-decylbenzolactone-V8 with
the R configuration at position 5 because significant
NOE enhancement between the H-2( d 3.26) and H-5 (d
4.72) protons was observed. Unexpectedly, 8 and 28
interconverted easily at room temperature. The ratio of 8
to 28 was 1:3 in deuterioacetonitrile. The free hydroxyl-
methylene signal (d 3.59) was observed in 8, indicating
that 8 was 15-decylbenzolactone-V8. On the other hand,
the hydroxymethylene signals of 28 were shifted down-
field (d 3.85, 4.40). These results indicated that 28 was a
nine-membered lactone deduced to be formed by intra-
molecular transesterification. Since the nine-membered
lactone adopted a quite different conformation from
Figure 3. Stable conformations of benzolactone-V8 (4, left), 7-decylbenzolactone-V8 (7, center) and 15-decylbenzolactone-V8 (8, right). The decyl
groups of 7 and 8 were displayed as butyl groups for convenience. The initial structures were determined by MM2calculation on the condition that
the distance between H-2and H6 a was fixed to 2A followed by PM3 optimization. Further optimization was carried out by Hartree-Fock calcu-
lation with 6-31G* basis set.

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Y. Nakagawa et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3015–3019
Table 1. Ki values for inhibition of the specific binding of [3H]PDBu by 7-decylbenzolactone-V8 (7), 15-decylbenzolactam-V8 (8), 9-decylbenzo-
lactone-V8 (5) and 8-decylbenzolactone-V8 (6)
PKC C1 peptide
Ki (nM)
Kd (nM)
7-Decylbenzolactone-V8 15-Decylbenzolactone-V8 9-Decylbenzolactone-V8 8-Decylbenzolactone-V8
(5)a
PDBu
(7)
(8)
(6)a
Conventional PKC
a-C1A (72-mer)b
a-C1B
b-C1A (72-mer)
b-C1B
g-C1A
g-C1B
870 (340)c
>10,000
539 (57)
2690 (330)
970 (45)
1720 (320)
4580 (982)
>10,000
4690 (390)
2070 (157)
5750 (198)
2820 (146)
412 (6)
4050 (153)
1140 (216)
610 (24)
2360 (187)
675 (23)
>10,000
>10,000
>10,000
1.12(0.04)
7.44 (0.27)
1.31 (0.26)
1.34 (0.41)
1.50 (0.57)
1.19 (0.29)
22,000 (1900)
6320 (536)
17,600 (1650)
Novel PKC
d-C1A
d-C1B
e-C1A
e-C1B
Z-C1A
Z-C1B
y-C1A
y-C1B
>10,000
92(8)
30,300 (1480)
415 (49)
5360 (626)
411 (31)
6330 (2580)
189 (5)
6150 (304)
149 (1)
4830 (112)
83 (4)
1590 (95)
37 (1)
NT
70,600 (3320)
1160 (46)
>10,000
262 (4)
51.9 (15.6)
0.53 (0.14)
5.60 (0.62)
0.81 (0.03)
4.30 (0.18)
0.45 (0.12)
>200
7880 (1330)
540 (70)
4250 (780)
111 (5)
>10,000
120 (7)
NT
NTd
167 (14)
NT
358 (33)
62(5)
1100 (53)
0.72(0.14)
aThese data are cited from ref 19.
bTen residues from both N and C-termini of the previous a-C1A and b-C1A were elongated as 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 for y-C1A could not be measured because of a very weak binding affinity.
those of eight-membered lactones, and since the 10-
decyl derivative of the nine-membered lactone did not
bind to C1 peptides of novel PKC isozymes at all
(unpublished data), 28 would lack the ability to bind to
PKC isozymes. Therefore, the binding selectivity of 8
for the C1 peptides of PKC isozymes would be esti-
mated without separation of 8 from 28 though the pre-
cise binding constants of 8 for each PKC C1 peptides
cannot be determined.
PKC isozymes.28 Using the PKC C1 peptides, the con-
centration required to cause 50% inhibition of the
[3H]PDBu binding (IC50) was measured. The binding affi-
nities of 7 and 8 for each PKC C1 peptide were expressed
as Ki values calculated from the IC50 and the Kd value of
[3H]PDBu as reported by Sharkey and Blumberg.29 Table
1 summarizes the Ki values of 7 and 8 along with those of
9- and 8-decylbenzolactone-V8 (5, 6).19
The binding affinities of 15-decylbenzolactone-V8 (8)
were more than 10-fold higher for the C1B peptides of
novel PKCs (d, e, Z, y) than for the other PKC C1
peptides. This selectivity of 8 was similar to that of 9-
decylbenzolactone-V8 (5) though the binding affinities
of 8 were 10-fold weaker than those of 5. In contrast,
the selectivity of 7-decylbenzolactone-V8 (7) for the
C1B peptides of novel PKCs was relatively low because
of strong binding to the C1A peptides of conventional
PKCs. This result indicates that the decyl group at
position 7 could effectively interact with the C1A pep-
tides of conventional PKCs. On the other hand, the
decyl group at position 8 might evoke steric hindrance
in binding with the C1 peptides of conventional PKCs
since 8-decylbenzolactone-V8 (6) exhibited poor binding
affinity for these C1 peptides. Moreover, significant
binding selectivity for the C1B peptides of PKCe and Z
was also observed in 6 although 7 and 8 along with 5
showed little selectivity among novel PKCs. Since the
ring conformation of four decylbenzolactone-V8s (5, 6,
7, 8) was quite similar to each other, these results indi-
cate that the position of the hydrophobic substituent in
the benzolactone-V8 skeleton plays a critical role in the
binding selectivity for PKC isozymes.
Conformational analyses of 7 and 8 were performed
using molecular mechanics and quantum mechanics
calculations. Since significant NOE enhancement
between the H-2( d 3.40 for 7, d 3.32for 8) and H-6a (d
2.97 for 7, d 2.96 for 8) protons was observed in both
compounds, the initial structures were determined by
MM2calculation on the condition that the distance
between H-2and H-6 a was fixed to 2A followed by
PM3 optimization. Further optimization was carried
out by Hartree–Fock calculation with 6-31G* basis set.
The calculated structures of 7 and 8 along with 418 are
shown in Figure 3. The ring conformation of both
compounds was quite similar to that of 4. These results
suggest that introduction of the decyl group at position
7 or 15 does not influence the lactone ring conformation
though the decyl group at position 15 of 8 lowered the
relative thermochemical stability of the eight-membered
lactone ring.
Binding affinities of 7 and 8 for PKC isozymes were
evaluated by inhibition of the specific binding of
[3H]phorbol 12,13-dibutyrate (PDBu) to the synthetic
C1 peptides of all PKC isozymes as reported pre-
viously.27À29 PKC C1 peptides consisting of about 50
amino acids are PKC C1 domain surrogates, which
exhibit PDBu binding affinities comparable to the whole
In summary, we have synthesized 7- and 15-decylbenzo-
lactone-V8 (7, 8) to investigate the effect of the hydro-

Y. Nakagawa et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3015–3019
3019
phobic substituent at positions 7 and 15 of benzolactone-
V8 (4) on the binding selectivity for PKC isozymes.
Although 7 and 8 along with 9-decylbezolactone-V8 (5)16
bound more strongly to the C1B domains of novel
PKCs (d, e, Z, y) than the other PKC C1 domains, sig-
nificant selectivity among novel PKCs was not observed
unlike 8-decylbenzolactone-V8 (6),18,19 which shows
high selectivity for PKCe and PKCZ. The present
results indicate that the introduction of a hydrophobic
substituent at position 8 of 4 is most effective in
increasing the PKC isozyme- and C1 domain-selectivity
and to develop PKCe- and PKCZ-selective binders,
which might be useful for analyzing the mechanism of
tumor promotion.
Glazer, R. I.; Bogi, K.; Acs, P.; Modarres, S.; Lewin, N. E.;
Blumberg, P. M. J. Med. Chem. 1997, 40, 1316.
17. Ma, D.; Tang, G.; Kozikowski, A. P. Org. Lett. 2002, 4,
2377.
18. Nakagawa, Y.; Irie, K.; Nakamura, Y.; Ohigashi, H.
Bioorg. Med. Chem. Lett. 2001, 11, 723.
19. Nakagawa, Y.; Irie, K.; Masuda, A.; Ohigashi, H. Tetra-
hedron 2002, 58, 2101.
20. Thorand, S.; Krause, N. J. Org. Chem. 1998, 63, 8551.
21. Kogan, T. P.; Somers, T. C.; Venuti, M. C. Tetrahedron
1990, 46, 6623.
22. 7-Decylbenzolactone-V8 (7): [a]D À88.0ꢀ (c 0.45, MeOH,
29.7 ꢀC); UV lmax (MeOH) nm (e) 255 (5100), 212 (15,600); 1H
NMR (500 MHz, CDCl3, 0.079 M, 27 ꢀC) d 0.88 (3H, t,
J=7.0 Hz), 0.96 (3H, d, J=6.6 Hz), 1.02(3H, d, J=6.5 Hz),
1.28–1.36 (14H, m), 1.53 (2H, m), 2.27 (1H, m), 2.34 (1H, br.s),
2.57 (2H, m), 2.83 (3H, s), 2.97 (1H, dd, J=17.2, 6.5 Hz), 3.12
(1H, dd, J=17.1, 2.7 Hz), 3.40 (1H, d, J=9.6 Hz), 3.72(2H, m),
4.80 (1H, m), 6.90 (1H, d, J=7.5 Hz), 6.95 (1H, d, J=7.7 Hz),
7.12(1H, t, J=7.7 Hz); HR-FABMS m/z 404.3159 (MH+
calcd for C25H42NO3, 404.3165).
Acknowledgements
This work was partly supported by a Grant-in-Aid for
Scientific Research on Priority Areas (2) (No. 13024245)
(for K.I.) and Scientific Research (A) (2) (No.
15208012) (for H.O.) from the Ministry of Education,
Culture, Sports, Science and Technology, the Japanese
Government, and from the Japan Society for the Pro-
motion of Science for Young Scientists (Y.N.).
23. epi-7-Decylbenzolactone-V8 (19): [a]D À121.0ꢀ (c 0.79,
MeOH, 29.7 ꢀC); UV lmax (MeOH) nm (e) 256 (4900), 213
(14,800); H NMR (500 MHz, CDCl3, 0.079 M, 27 ꢀC) d 0.87
1
(3H, t, J=6.5 Hz), 0.89 (3H, d, J=6.9 Hz), 1.03 (3H, d,
J=6.6 Hz), 1.27–1.39 (14H, m), 1.50 (2H, m), 2.43 (2H, m),
2.57 (2H, m), 2.77 (1H, dd, J=15.9, 5.5 Hz), 2.89 (3H, s), 3.00
(1H, dd, J=15.9, 1.8 Hz), 3.22 (1H, d, J=10.5 Hz), 3.83 (1H,
dd, J=11.7, 9.0 Hz), 3.89 (1H, dd, J=11.7, 7.3 Hz), 4.51 (1H,
m), 6.94 (1H, d, J=7.3 Hz), 7.05 (1H, d, J=7.7 Hz), 7.12(1H,
t, J=7.7 Hz); HR-FABMS m/z 404.3161 (MH+ calcd for
C25H42NO3, 404.3165).
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(11,700); H NMR (500 MHz, CDCl3, 0.049 M, 27 ꢀC) d 0.86
1
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(3H, d, J=6.5 Hz), 0.87 (3H, t, J=7.1 Hz), 1.10 (3H, d,
J=6.5 Hz), 1.19–1.32 (18H, m), 2.07 (1H, br.s), 2.21 (1H, m),
2.87 (2H, m), 3.22 (2H, m), 3.26 (1H, d, J=10.6 Hz), 3.73 (2H,
m), 4.72 (1H, m), 7.10 (2H, m), 7.22 (2H, m); HR-FABMS m/z
404.3164 (MH+ calcd for C25H42NO3, 404.3165).
25. 15-Decylbenzolactone-V8 (8): [a]D À58.0ꢀ (c 0.79, EtOH,
22.1 ꢀC); UV lmax (EtOH) nm (e) 260 (2500), 205 (11,100); 1H
NMR (500 MHz, CD3CN, 0.071 M, 27 ꢀC) d 0.87 (3H, t,
J=6.9 Hz), 0.92(3H, d, J=6.5 Hz), 1.08 (3H, d, J=6.7 Hz),
1.15–1.29 (18H, m), 2.07 (1H, m), 2.96 (4H, m), 3.04 (1H, br.t,
J=6.2Hz), 3.32 (1H, d, J=10.0 Hz), 3.59 (2H, m), 4.85 (1H,
m), 7.09–7.25 (4H, m); HR-FABMS m/z 404.3161 (MH+
calcd for C25H42NO3, 404.3165).
26. Nine-membered benzolactone (28): 1H NMR (500 MHz,
CD3CN, 0.071 M, 27 ꢀC) d 0.87 (3H, t, J=7.0 Hz), 0.98 (3H,
d, J=6.6 Hz), 1.08 (3H, d, J=6.7 Hz), 1.15–1.30 (18H, m),
2.28 (1H, m), 2.65 (1H, dd, J=13.1, 2.7 Hz), 2.88 (1H, m),
2.96 (2H, m), 3.12 (1H, d, J=9.3 Hz), 3.40 (1H, br.d,
J=5.2Hz), 3.85 (1H, dd, J=11.1, 5.4 Hz), 4.06 (1H, m), 4.40
(1H, br.s), 7.12–7.24 (4H, m).
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