Synthesis, conformation and PKC isozyme surrogate binding of indolinelactam-Vs, new conformationally restricted analogues of (-)-indolactam-V

Article abstract of DOI:10.1016/j.tet.2003.08.081

New conformationally restricted analogues of tumor promoter (-)-indolactam-V (1), indolinelactam-Vs (8, 11) and their hexyl derivatives at position 1 or 7 (9, 10, 12, 13), were synthesized from 1. (3R)-Indolinelactam-V (8) adopted a conformation similar to the twist form of 1 with a cis amide, while the conformation of (3S)-indolinelactam-V (11) was close to that of the sofa form of 1 with a trans amide. 7-Hexyl derivatives of 8 and 11 (10, 13) showed binding affinities for C1 domains of protein kinase C (PKC) isozymes compared to 1, but exhibited little selectivity among these PKC isozymes. However, introduction of the hexyl group at position 1 of 8 and 11 significantly enhanced their binding selectivity for novel PKC isozymes. The best selectivity for novel PKC isozymes was observed in (3S)-1-hexylindolinelactam-V (12) with a sofa-like conformation. These results suggest that a sofa-restricted analogue of 1 with a hydrophobic chain at an appropriate position would be a promising lead for designing agents with a high selectivity for novel PKC isozymes.

Full text of DOI:10.1016/j.tet.2003.08.081

Tetrahedron 60 (2004) 7077–7084
Synthesis, conformation and PKC isozyme surrogate binding of
indolinelactam-Vs, new conformationally restricted analogues of
(
2)-indolactam-V
a
Yu Nakagawa, Kazuhiro Irie, Yusuke Komiya, Hajime Ohigashi and Ken-ichiro Tsuda
a,*
a
a
b
a
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Fundamental Research Laboratories NEC Corporation, 34 Miyukigaoka, Tsukuba 305-8501, Japan
b
Received 29 May 2003; revised 19 August 2003; accepted 19 August 2003
Available online 24 June 2004
Abstract—New conformationally restricted analogues of tumor promoter (2)-indolactam-V (1), indolinelactam-Vs (8, 11) and their hexyl
derivatives at position 1 or 7 (9, 10, 12, 13), were synthesized from 1. (3R)-Indolinelactam-V (8) adopted a conformation similar to the twist
form of 1 with a cis amide, while the conformation of (3S)-indolinelactam-V (11) was close to that of the sofa form of 1 with a trans amide.
7
-Hexyl derivatives of 8 and 11 (10, 13) showed binding affinities for C1 domains of protein kinase C (PKC) isozymes compared to 1, but
exhibited little selectivity among these PKC isozymes. However, introduction of the hexyl group at position 1 of 8 and 11 significantly
enhanced their binding selectivity for novel PKC isozymes. The best selectivity for novel PKC isozymes was observed in (3S)-1-
hexylindolinelactam-V (12) with a sofa-like conformation. These results suggest that a sofa-restricted analogue of 1 with a hydrophobic
chain at an appropriate position would be a promising lead for designing agents with a high selectivity for novel PKC isozymes.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
it has a simple structure and shows a binding preference for
1
C1B domains over C1A domains of novel PKCs (Fig. 2).
4
Protein kinase C (PKC) isozymes are serine/threonine-
specific protein kinases involved in a variety of cellular
functions such as gene expression, growth, differentiation
(2)-Indolactam-V (1) exists as two stable conformers in
solution at room temperature; the twist form with a cis
amide geometry and the sofa form with a trans amide
1
15
and apoptosis. PKC isozymes are subdivided into three
groups; conventional PKCs (a, bI, bII, g), novel PKCs (d, e,
geometry. We have recently found that the sofa-restricted
6
analogue of 1 (5), not the twist-restricted analogue (4),
1
2
,3
14
h, u) and atypical PKCs (z, i/l) (Fig. 1). Conventional
and novel PKCs contain two cysteine-rich C1 domains
shows significant selectivity for novel PKCs. These results
suggest that the conformation of 1 plays a crucial role in its
binding selectivity for PKC isozymes.
(
C1A, C1B), both of which bind tumor promoters such as
4
1
ocidin B-4. Recent investigations have suggested that
2-O-tetradecanoylphorbol 13-acetate (TPA) and tele-
5
17,18
(3R)- and (3S)-2-Oxyindolactam-V (6, 7),
isolated from
novel PKCs (d, e, h) are involved in mouse skin tumor
6
the culture broth of Streptomyces blastmyceticum NA34-17
adopted slightly different conformations from both the twist
and the sofa forms of 1. However, these compounds were
inactive in several in vitro bioassays that correlate with in
vivo tumor promotion probably due to the steric hindrance
between the carbonyl group at position 2 and receptors such
–9
promotion
isozymes are the main targets of tumor promoters.
and that the C1B domains of these PKC
1
0,11
Design of new agents with high selectivity for C1B domains
of novel PKCs is thus indispensable in elucidating the
precise mechanism of skin-tumor promotion.
1
9,20
as PKC isozymes.
(3R)- and (3S)-Indolinelactam-Vs (8,
The naturally occurring tumor promoter (2)-indolactam-V
1
11), which lack a carbonyl group at position 2, might be new
conformationally restricted analogues of 1 and may exhibit
different binding selectivity for PKC isozymes from that of
2,13
(
1)
is a promising lead compound for such agents since
1
, 4 and 5. We report here the synthesis, conformation and
Keywords: (2)-Indolactam-V; Indolinelactam-V; Phorbol ester; Protein
kinase C; Tumor promoter.
PKC isozyme surrogate binding of indolinelactam-Vs (8,
11) and their hexyl derivatives at positions 1 and 7 (9, 10,
12, 13) (Fig. 3).
*
Corresponding author. Tel.: þ81-75-753-6282; fax: þ81-75-753-6284;
e-mail address: irie@kais.kyoto-u.ac.jp
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.08.081

7078
Y. Nakagawa et al. / Tetrahedron 60 (2004) 7077–7084
Figure 1. Structures of PKC isozymes.
Figure 2. Structures of indolactam forms.
Figure 3. Structures of 2-oxyindolactam-Vs (6,7) and indolinelactam-Vs (8–13).
2
. Results and discussion
a significant NOE interaction between H-3 (d 3.17) and H-9
1
5
1
(
d 4.25) protons. As observed in the sofa form of 1, the H
(
3R)- and (3S)-Indolinelactam-Vs (8, 11) were synthesized
from (2)-indolactam-V (1). Reduction of the indole ring of
proceeded easily with sodium cyanoborohydride in acetic
NMR signals for H-10 (d 4.92) and H-12 (d 3.08) in 11
shifted upfield and an NOE interaction was observed
between them. These results indicate that the conformation
of 11 is close to the sofa form of 1 with a trans amide.
1
2
1
acid to give two diastereomers 8 and 11 (31 and 20%,
1
respectively). The H NMR spectra of 8 and 11 in CDCl
3
showed that each compound existed as a single conformer at
room temperature and at 240 8C (Table 1). A significant
NOE interaction between H-3 (d 3.84) and H-12 (d 4.37)
was observed in 8, suggesting that 8 is the (3R) isomer. The
To determine the conformations of 8 and 11 precisely, we
performed conformational studies using molecular
mechanics and quantum mechanics calculations. The initial
structure of 8 was calculated by MM2 with the distances
between H-3 and H-12, and between H-9 and H-18 fixed at
1
downfield shift of the H NMR signals H-10 (d 7.31) and
H-12 (d 4.37), and the lack of an NOE interaction between
˚
2 A in accordance with the NOE data. The resultant
1
5
these protons, are characteristic of the twist form of 1 and
indicate that 8 adopts the twist-like conformation with a cis
amide. On the other hand, 11 was the (3S) isomer because of
structure was optimized by PM3. The initial structure of
11 was similarly determined with the three NOE inter-
actions between H-3 and H-9, H-3 and H-18, and H-10 and

Y. Nakagawa et al. / Tetrahedron 60 (2004) 7077–7084
Table 1. H NMR data of (2)-indolactam-V (1) and (3R)- and (3S)-indolinelactam-Vs (8, 11) in CDCl (500 MHz, 300 K)
7079
1
3
No.
d (multiplicity, J in Hz)
(2)-Indolactam-V (1)
(3R)-Indolinelactam-V (8)
(3S)-Indolinelactam-V (11)
a
a
b
c
Twist
Sofa
Twist-like
Sofa-like
1
2
7.99 (br. s)
6.89 (s)
8.27 (br. s)
7.06 (s)
3.75 (br. s)
3.18 (dd, J¼8.9, 0.9)
3.73 (br. s)
3.21 (d, J¼8.4)
3.52 (dd, J¼8.4, 6.6)
3.17 (m)
6.52 (d, J¼7.8)
7.02 (t, J¼7.8)
6.48 (d, J¼7.8)
1.87 (dt, J¼12.2, 1.8)
2.06 (q, J¼12.2)
4.25 (m)
3
.77 (t, J¼8.9)
3
5
6
7
8
3.84 (m)
6.51 (d, J¼7.5)
7.06 (t, J¼7.5)
7.06 (d, J¼8.2)
7.17 (t, J¼8.2)
7.28 (d, J¼8.2)
6.23 (d, J¼7.9)
6.98 (t, J¼7.9)
6.21 (d, J¼7.9)
1.70 (dt, J¼12.6, 2.0)
1.92 (dt, J¼12.6, 4.6)
4.01 (m)
6.91 (d, J¼7.5)
3.00 (dd, J¼17.4, 3.8)
2.83 (d, J¼14.0)
3.11 (dd, J¼14.0, 4.8)
4.46 (m)
3
.20 (d, J¼17.4)
9
4.30 (m)
6.59 (br. s)
10
12
14
4.72 (d, J¼10.8)
2.99 (d, J¼10.8)
3.44 (m)
3.44 (m)
2.40 (m)
1.25 (d, J¼7.1)
0.94 (d, J¼7.1)
2.75 (s)
7.31 (d, J¼6.2)
4.37 (d, J¼8.6)
3.43 (m)
3.64 (m)
2.43 (m)
1.10 (d, J¼6.4)
0.90 (d, J¼6.8)
2.84 (s)
4.92 (br.s)
3.08 (d, J¼10.7)
4.39 (d, J¼10.2)
3.54 (m)
3.74 (m)
3.37 (m)
3.56 (m)
2.26 (m)
15
16
17
18
2.62 (m)
0.93 (d, J¼6.4)
0.63 (d, J¼6.8)
2.92 (s)
1.14 (d, J¼6.7)
0.94 (d, J¼6.5)
2.62 (s)
a
b
c
Twist: Sofa¼1.0: 0.4 (0.004 M).
0
0
.079 M.
.046 M.
H-12 used to restrict the distance between these proton pairs
˚
to 2 A. Further optimization of these initial structures was
carried out by a Hartree–Fock calculation with the 6-31G*
basis set to give the optimized structures of 8 and 11 as
shown in Figure 4. The conformations of 8 and 11 resembled
the twist and the sofa forms of 1, respectively. However, the
spatial arrangement of the methylene group at position 8 in
8 and 11 was quite different from that of the twist and the
Figure 4. Stable conformations of twist (upper left) and sofa (upper right) form of (-)-indolactam-V (1), (3R)-indolinelactam-V (8, lower left) and (3S)-
indolinelactam-V (11, lower right). The initial structures were determined by MM2 and PM3 calculations based on the indicated NOE interactions.
Optimization was carried out by a Hartree-Fock calculation with the 6-31G basis set.
p

7080
Y. Nakagawa et al. / Tetrahedron 60 (2004) 7077–7084
3
Values for inhibition of the specific binding of [ H]PDBu by (2)-indolactam-V (1), the twist- and sofa-restricted analogues of 1 (4, 5), and (3R)-
Table 2. K
i
and (3S)-indolinelactam-Vs (8, 11)
PKC C1 peptide
i
K (nM)
a
1
a
4
a
5
8
11
Conventional PKC
b
c
d
a-C1A (72-mer)
a-C1B
21 (1.0)
4000 (870)
19 (4.5)
39 (3.9)
4500 (1200)
25 (2.6)
330 (21)
110 (8.0)
270 (26)
6000 (730)
11,000 (900)
8200 (830)
321 (25)
12,000 (2400)
790 (140)
13,000 (2500)
.10,000
8900 (1800)
16,000 (2500)
.10,000
ND
ND
ND
ND
ND
ND
b-C1A (72-mer)
b-C1B
g-C1A
140 (4.4)
140 (14)
210 (5.0)
g-C1B
17,000 (3800)
Novel PKC
d-C1A
d-C1B
e-C1A
e-C1B
h-C1A
h-C1B
u-C1A
u-C1B
1900 (190)
8.3 (1.1)
4100 (50)
7.7 (1.2)
2900 (570)
31 (6.6)
2500 (850)
29 (4.1)
1300 (2.0)
14 (3.2)
NT
23,000 (4300)
21 (4.4)
15,000 (3100)
12 (2.4)
8600 (1400)
6.2 (0.4)
NT
.10,000
660 (45)
8200 (2100)
920 (190)
11,000 (730)
310 (42)
NT
ND
5000 (960)
ND
7700 (2200)
ND
2200 (400)
NT
7700 (600)
3800 (480)
5.5 (0.6)
e
NT
8.7 (1.2)
43 (6.8)
26 (2.2)
400 (52)
a
These data are cited from Ref. 14.
Ten 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
b
low.
Standard deviation of at least two separate experiments.
c
d
e
Not detected.
Not tested. The K
3
value of [ H]PDBu to u-C1A could not be measured because of its very weak binding affinity.
d
2
3
sofa forms of 1, respectively. These results indicate that 8
and 11 are new conformationally restricted analogues of 1.
Sharkey and Blumberg. Table 2 summarizes the K values of
i
8 and 11 along with those of (2)-indolactam-V (1) and its
conformationally restricted analogues (4, 5).
Binding affinities of 8 and 11 for PKC isozyme C1 domains
were evaluated by inhibition of the specific binding of
(3R)- and (3S)-Indolinelactam-Vs (8, 11) showed quite low
binding affinities for all PKC C1 peptides compared with
those of 1, 4 and 5. The binding affinities of 8 for almost all
PKC C1 peptides were more than 50-fold lower than those
of 1, and 11 exhibited little binding affinity for all PKC C1
peptides. Since introduction of a substituent at position 2 in
1 abolished the tumor-promoting activity due to steric
hindrance, the quite low PKC binding affinities of 8 and 11
might be due to the steric hindrance between the two
hydrogen atoms at position 2 and the C1 peptides of PKC
isozymes. Interestingly, the selectivity of 8 for the C1B
peptides of novel PKCs (d, e, h, u), relative to both C1
3
[
peptides (about 50–70 amino acids) of all PKC isozymes as
H]phorbol 12,13-dibutyrate (PDBu) to the synthetic C1
1
1,22,23
reported previously.
PKC C1 peptides exhibit PDBu
binding affinities comparable to the whole PKC isozymes
enabling evaluation of PKC isozyme selectivity, as well as
C1 domain selectivity of PKC C1 domain-binding com-
pounds. Using the PKC C1 peptides, the concentration
2
4
3
required to cause 50% inhibition of the [ H]PDBu binding
(
each PKC C1 peptide were expressed as K values calculated
IC ) was measured. The binding affinities of 8 and 11 for
50
i
3
from the IC50 and the K value of [ H]PDBu as reported by
d
Scheme 1. Synthesis of 1-hexylindolinelactam-Vs (9,12).

Y. Nakagawa et al. / Tetrahedron 60 (2004) 7077–7084
7081
Scheme 2. Synthesis of 7-hexylindolinelactam-Vs (10,13).
peptides of conventional PKCs (a, b, g), was considerably
higher than that of 1 and 4. This result was unexpected since
the conformation of 8 has many points of similarity to that
of 4, which showed low selectivity for the C1B peptides of
novel PKCs relative to the C1A peptides of conventional
PKCs. It is not clear whether the conformational difference
between 4 and 8, such as the spatial arrangement of the
methylene group at position 8, or the possible steric
hindrance of hydrogen atoms at position 2, produces the
binding selectivity of 8. However, 8 could be a lead
compound for a new agent that selectively binds to the C1B
domains of novel PKCs.
TBDMS group with tetrabutylammonium fluoride (TBAF)
to give (3R)-1-hexylindolinelactam-V (9, 57%) and its (3S)-
isomer (12, 11%). Synthesis of the 7-hexyl derivatives (10,
2
5
13) is shown in Scheme 2. 7-Hexylindolactam-V (3) was
synthesized in three steps (14% from 1) according to our
2
4
previously reported method. Hydride reduction of 3 was
then carried out to give the desired two diastereomers (15%
for 10, 23% for 13). Proton NMR spectroscopy showed that
each of the 1- and 7-hexyl derivatives of 8 and 11 existed as
a single conformer in CDCl at room temperature and at
3
240 8C. Conformer analysis similar to that mentioned
above indicated that the conformations of 9 and 10 were
almost the same as the twist-like form of 8. On the other
hand, the calculated conformation of 12 and 13 was the
sofa-like form similar to that of 11. These results indicate
that introduction of the hexyl group at position 1 or 7 does
not influence the conformations of 8 and 11.
Since introduction of a hydrophobic alkyl chain at position 1
or 7 in 1 dramatically increases its binding affinity for
2
4
phorbol ester receptors such as PKC isozymes, we
attempted to enhance the PKC binding ability of 8 and 11
by introducing a hexyl group at position 1 or 7. The 1-hexyl
derivatives of 8 and 11 (9, 12) were synthesized from (2)-
indolactam-V (1) in four steps as shown in Scheme 1. After
protection of the hydroxyl group of 1 with a tert-
butyldimethylsilyl (TBDMS) group (83%), a hexyl group
Recent investigations suggest that the C1A domain of
conventional PKCs (a, b, g) is mainly involved in phorbol
ester binding and translocation from the cytosol to plasma
2
6,27
membrane,
e, h, u) plays a predominant role in translocation in
whereas the C1B domain of novel PKCs (d,
was introduced at position 1 by S 2 reaction of 14 with
N
1-iodohexane (79%). The indole ring of 15 was reduced
with sodium cyanoborohydride followed by removal of the
2
8
response to TPA. Our binding study using PKC C1
peptides also supports these results; the major binding site
3
Values for inhibition of the specific binding of [ H]PDBu by 1-hexyl derivatives
Table 3. K
i
PKC C1 peptide
i
K (nM)
(
2)-1-Hexylindolactam-V (2)
(3R)-1-Hexylindolinelactam-V (9)
(3S)-1-Hexylindolinelactam-V (12)
Conventional PKC
a-C1A (72-mer)
a-C1A (72-mer)
g-C1A
a
5.8 (1.1)
9.8 (1.6)
18 (2.4)
170 (25)
240 (14)
570 (90)
550 (44)
1200 (27)
1800 (290)
Novel PKC
d-C1B
e-C1B
h-C1B
u-C1B
0.22 (0.04)
0.47 (0.12)
0.34 (0.11)
1.41 (0.03)
4.7 (0.5)
7.0 (0.8)
4.9 (0.8)
7.0 (0.6)
16 (1.5)
14 (0.9)
12 (2.0)
12 (1.3)
a
Standard deviation of at least two separate experiments.

7082
Y. Nakagawa et al. / Tetrahedron 60 (2004) 7077–7084
3
Values for inhibition of the specific binding of [ H]PDBu by 7-hexyl derivatives
Table 4. K
i
PKC C1 peptide
i
K (nM)
(
2)-7-Hexylindolactam-V (3)
(3R)-7-Hexylindolinelactam-V (10)
(3S)-7-Hexylindolinelactam-V (13)
Conventional PKC
a-C1A (72-mer)
b-C1A (72-mer)
g-C1A
a
1.2 (0.1)
2.5 (0.1)
2.6 (0.2)
15 (1.2)
35 (5.4)
89 (12)
65 (14)
91 (17)
136 (29)
Novel PKC
d-C1B
e-C1B
h-C1B
u-C1B
0.21 (0.02)
0.36 (0.07)
0.18 (0.05)
0.59 (0.05)
4.7 (0.2)
7.0 (0.8)
2.9 (0.3)
6.1 (0.9)
12 (0.5)
13 (1.3)
13 (3.1)
13 (1.4)
a
Standard deviation of at least two separate experiments.
2
4
of (2)-indolactam-V (1) is the C1A domain for conven-
4
sponding to the structure–activity studies of 1, 7-hexyl
derivatives (10, 13) bound to conventional PKCs (a, b, g)
more strongly than 1-hexyl derivatives (9, 12) probably due
to steric hindrance between the hexyl group at position 1
and conventional PKCs. However, a similar effect was not
observed in novel PKCs (d, e, h, u); the binding affinities of
1- and 7-hexyl derivatives of 8 and 11 for novel PKC C1B
peptides were similar to each other. (3S)-1-Hexylindoline-
lactam-V (12), with a sofa-like conformation, exhibited
about 50–300 fold greater selectivity for C1B domains of
novel PKCs than for C1A domains of conventional PKCs,
indicating that sofa-restricted analogues of 1 with a
hydrophobic chain at an appropriate position, such as 5
and 12, would be likely lead compounds for the rational
design of novel PKC selective modulators. The present
results provide a basis for the synthesis and exploitation of
such compounds as medicinal agents.
1
tional PKCs and the C1B domain for novel PKCs. Thus,
binding affinities of 9, 10, 12 and 13, along with (2)-1- and
(
2)-7-hexylindolactam-Vs (2, 3), were evaluated for C1A
peptides of conventional PKCs and C1B peptides of novel
PKCs (Tables 3 and 4). The binding affinities of 7-hexyl
derivatives (3, 10, 13) for all PKC C1 peptides dramatically
increased compared with those of the corresponding core
compounds (1, 8, 11). Unexpectedly, the selectivity of 10
for novel PKCs was lower than that of 8, and 13 did not
show significant selectivity, regardless of its sofa-like
conformation. This conformation is similar to 5, which
showed increased selectivity for novel PKCs. In contrast to
the 7-hexyl derivatives, the selectivity for novel PKCs was
improved in the 1-hexyl derivatives. These derivatives (2, 9,
1
2) showed lower binding affinities for conventional PKCs
compared with the corresponding 7-hexyl derivatives (3, 10,
3), but similar binding affinities for novel PKCs. These
1
results suggest that for indolactam derivatives the position
of the hydrophobic chain is more important than the
conformation for exhibiting novel PKC selectivity. How-
ever, 12, which has a sofa-like conformation, showed the
highest selectivity for novel PKC C1B peptides; the binding
affinities of 12 for PKCh- and u-C1B peptides were
approximately 50-fold and more than 100-fold higher than
those for C1A peptides of PKCa, b and g, respectively.
Since the sofa-restricted form of 1 (5) bound selectively to
novel PKCs, sofa analogues of 1 with a hydrophobic chain
at an appropriate position may be promising agents that
exhibit high selectivity not only for novel PKCs but also
among novel PKCs.
4
. Experimental
4
.1. General remarks
The following spectroscopic and analytical instruments
were used: UV, Shimadzu UV-2200A; Digital Polarimeter,
Jasco DIP-1000; H NMR, Bruker ARX500 (ref. TMS);
HPLC, Waters Model 600E with Model 2487 UV detector;
1
(
HR) EI-MS, JOEL JMS-600H. HPLC was carried out on a
YMC packed SH-342-5 (ODS, 20 mm i.d. £250 mm)
column (Yamamura Chemical Laboratory). Wako C-200
gel (silica gel, Wako Pure Chemical Industries) and YMC
A60-350/250 gel (ODS, Yamamura Chemical Laboratory)
3
were used for column chromatography. [ H]PDBu
3
. Conclusions
(17.0 Ci/mol) was purchased from PerkinElmer Life
Sciences Research Products. All other chemicals and
reagents were purchased from chemical companies and
used without further purification.
We have synthesized (3R)- and (3S)-indolinelactam-Vs (8,
1) along with their hexyl derivatives (9, 10, 12, 13) as new
conformationally restricted analogues of (2)-indolactam-V
1) in order to find new lead compounds with a high
selectivity for novel PKC isozymes. (3R)-Indolinelactam-
Vs (8–10) adopted a conformation similar to the twist form
of 1 with a cis amide, and the conformation of the (3S)-
isomers (11–13) was close to that of the sofa form with a
trans amide. Although the binding affinities of 8 and 11 to
PKC C1 peptides were far less than those of 1, the
introduction of a hexyl group at position 1 or 7 resulted in
PKC binding affinities comparable to those of 1. Corre-
1
(
4.1.1. Synthesis of (3R)- and (3S)-indolinelactam-Vs (8,
11). To a solution of (2)-indolactam-V (1, 48.8 mg,
162 mmol) in acetic acid (1 ml) was added NaCNBH3
(25 mg, 397 mmol) at room temperature. After stirring for
1.5 h, another NaCNBH (25 mg, 397 mmol) was added and
3
the reaction mixture was stirred for another 2.5 h. The
reaction was quenched by the addition of H O (1 ml) and the
2
mixture was poured into saturated NaHCO aq (20 ml),
3
followed by extraction with EtOAc. The EtOAc layer was

Y. Nakagawa et al. / Tetrahedron 60 (2004) 7077–7084
7083
washed with brine, dried over Na SO and concentrated.
2
increasing amounts of EtOAc to give 16 (28.5 mg,
56.9 mmol, 73%) as a mixture of two diastereomers.
4
The residue was purified by HPLC on YMC SH-342-5 using
2
5% CH CN to give 8 (15.1 mg, 50 mmol, 31%) and
3
1
1 (9.6 mg, 32 mmol, 20%). Compound 8: [a]D 2478.08
TBAF·5H O (108 mg, 338 mmol) was added to 16
2
(
(
5
4
1
c¼0.700, MeOH, 302.5 K); UV l
(MeOH) nm (e): 303
2000), 238 (24,000); C NMR d (CDCl , 0.079 M,
(28.5 mg, 56.9 mmol) in THF (1.4 ml) at 0 8C. After stirring
for 40 min at 0 8C, the reaction mixture was poured into 5%
KHSO aq and extracted with EtOAc. The EtOAc layer was
max
1
3
3
00 MHz, 300 K) ppm: 19.65, 21.85, 30.50, 34.87, 39.30,
0.12, 52.87, 53.95, 66.25, 68.16, 102.02, 107.87, 117.97,
29.23, 150.85, 152.83, 175.70; HR-EI-MS m/z: 303.1937
4
washed with brine, dried over Na SO and concentrated.
2
4
The residue was purified by HPLC on YMC SH-342-5 using
80% MeOH to give 9 (11.7 mg, 30.2 mmol, 53%) and 12
(2.5 mg, 6.5 mmol, 11%). Compound 9: [a]D 2435.08
þ
(
M , calcd for C H N O , 303.1947). Compound 11: [a]
1
7
25
3
2
D
þ146.08 (c¼0.477, MeOH, 300.2 K); UV l
(MeOH) nm
e): 298 (2600), 244 (9500); C NMR d (CDCl , 0.046 M,
max
1
3
(
(c¼0.435, MeOH, 301.2 K); UV l
(MeOH) nm (e): 310
max
(2500), 246 (30,000); H NMR d (CDCl , 0.060 M,
3
3
1
5
4
1
00 MHz, 300 K) ppm: 19.06, 19.87, 24.11, 34.77, 40.29,
0.58, 54.43, 57.22, 66.11, 76.10, 106.68, 119.35, 129.04,
30.39, 150.87, 152.46, 170.88; HR-EI-MS m/z: 303.1918
500 MHz, 300 K) ppm: 0.90 (3H, t, J¼6.8 Hz), 0.91 (3H,
d, J¼6.8 Hz), 1.10 (3H, d, J¼6.4 Hz), 1.34–1.37 (6H, m),
1.55 (2H, m), 1.63 (1H, t, J¼12.2 Hz), 1.92 (1H, dt, J¼12.2,
þ
(
M , calcd for C H N O , 303.1947).
17 25 3 2
4.6 Hz), 2.43 (1H, m), 2.83 (3H, s), 2.88 (1H, m), 3.13 (1H,
4
.1.2. Synthesis of (3R)- and (3S)-1-hexylindolinelactam-
d, J¼8.6 Hz), 3.23 (1H, m), 3.43 (1H, t, J¼8.6 Hz), 3.44
(1H, m), 3.50 (1H, t, J¼6.0 Hz), 3.65 (1H, m), 3.73 (1H, m),
4.02 (1H, m), 4.36 (1H, d, J¼8.4 Hz), 6.05 (1H, d,
J¼8.0 Hz), 6.17 (1H, d, J¼8.0 Hz), 7.02 (1H, t,
Vs (9, 12). To a mixture of (2)-indolactam-V (1, 44.0 mg,
46 mmol) and imidazole (30.0 mg, 441 mmol) in dry DMF
0.5 ml) was added TBDMS-Cl (24.1 mg, 160 mmol) at
8C. After stirring for 1 h at 0 8C, the reaction mixture was
1
(
0
1
3
J¼8.0 Hz), 7.41 (1H, d, J¼6.4 Hz); C NMR d (CDCl ,
3
poured into H O and extracted with EtOAc. The EtOAc
2
layer was washed with brine, dried over Na SO and
2
concentrated. The residue was purified by column chroma-
tography on Wako gel C-200 using hexane and increasing
amounts of EtOAc to give 14 (50.3 mg, 121 mmol, 83%).
0.060 M, 125 MHz, 300 K) ppm: 14.07, 19.68, 21.84, 22.66,
26.85, 27.31, 30.60, 31.70, 35.03, 38.34, 39.52, 48.47,
54.00, 58.63, 66.25, 68.27, 99.45, 107.04, 118.06, 129.18,
4
þ
150.64, 153.71, 175.92; HR-EI-MS m/z: 387.2884 (M ,
calcd for C H N O , 387.2886). Compound 12: [a]
2
3
37
3
2
D
þ215.08 (c¼0.052, MeOH, 300.8 K); UV l
(MeOH) nm
(e): 307 (2800), 259 (13,900); H NMR d (CDCl , 0.013 M,
max
1
NaH in oil (11.6 mg, 290 mmol) was washed with hexane
and suspended in dry DMF (0.5 ml) under an Ar atmosphere
To this suspension was added 14 (50.3 mg, 121 mmol) in
dry DMF (0.5 ml) at 0 8C. After stirring for 10 min,
1-iodohexane (21.4 ml, 145 mmol) was added dropwise
and the reaction mixture was stirred for 1 h at 0 8C. The
3
500 MHz, 300 K) ppm: 0.90 (3H, t, J¼6.6 Hz), 0.95 (3H, d,
J¼6.5 Hz), 1.14 (3H, d, J¼6.7 Hz), 1.32–1.37 (6H, m),
1.56 (2H, m), 1.88 (1H, dd, J¼14.1, 2.5 Hz), 2.03 (1H, q,
J¼13.2 Hz), 2.26 (1H, m), 2.62 (3H, s), 2.76 (1H, m), 3.11
(1H, d, J¼10.7 Hz), 3.14 (3H, m), 3.22 (1H, m), 3.41 (1H,
m), 3.58 (1H, m), 4.24 (1H, m), 4.85 (1H, br.s), 6.29 (1H, d,
J¼7.8 Hz), 6.44 (1H, d, J¼7.8 Hz), 7.06 (1H, t, J¼7.8 Hz);
mixture was poured into H O (20 ml) and extracted with
2
EtOAc. The EtOAc layer was washed with brine, dried over
Na SO and concentrated. The residue was purified by
column chromatography on Wako gel C-200 using hexane
1
3
C NMR d (CDCl , 0.013 M, 125 MHz, 300 K) ppm:
2
4
3
14.06, 19.07, 19.85, 22.63, 24.15, 26.91, 27.16, 31.67,
34.88, 39.07, 41.05, 49.15, 54.83, 62.44, 66.30, 76.05,
104.25, 117.95, 129.13, 130.46, 150.51, 153.52, 171.33;
and increasing amounts of EtOAc to give 15 (47.4 mg,
9
MeOH, 298.3 K); UV l (MeOH) nm (e): 307 (8200),
5 mmol, 79%). Compound 15: [a]D 2107.08 (c¼0.188,
þ
HR-EI-MS m/z: 387.2868 (M , calcd for C H N O ,
23 37 3 2
max
1
2
30 (25,000); H NMR d (CDCl , 0.068 M, 500 MHz, 300
3
387.2886).
K, twist:sofa¼3.3:1) ppm for twist conformer: 0.03 (3H, s),
0
.05 (3H, s), 0.63 (3H, d, J¼6.8 Hz), 0.87 (9H, s), 0.88 (3H,
4.1.3. Synthesis of (3R)- and (3S)-7-hexylindolinelactam-
2
5
t, J¼7.0 Hz), 0.92 (3H, d, J¼6.4 Hz), 1.30–1.36 (6H, m),
Vs (10, 13). (2)-7-Hexylindolactam-V (3) was treated in a
manner similar to that described for the synthesis of 9 and
12 to give 10 (2.5 mg, 6.5 mmol, 15%) and 13 (3.8 mg,
9.8 mmol, 23%). Compound 10: [a]D 2397.08 (c¼0.165,
(MeOH) nm (e): 305 (3000),
max
1
2
9
.81 (2H, m), 2.61 (1H, m), 2.87 (1H, dd, J¼17.4, 3.5 Hz),
.91 (3H, s), 3.14 (1H, d, J¼17.4 Hz), 3.45 (1H, dd, J¼10.1,
.8 Hz), 3.63 (1H, dd, J¼10.1, 4.3 Hz), 3.98 (2H, t,
J¼7.4 Hz), 4.21 (1H, m), 4.38 (1H, d, J¼10.2 Hz), 6.16
MeOH, 299.4 K); UV l
1
(
1H, br.s), 6.49 (1H, d, J¼7.9 Hz), 6.76 (1H, s), 6.84 (1H,
237 (26,000); H NMR d (CDCl , 0.013 M, 500 MHz,
3
J¼7.9 Hz), 7.07 (1H, t, J¼7.1 Hz); HR-EI-MS m/z:
300 K) ppm: 0.89 (3H, t, J¼6.8 Hz), 0.92 (3H, d,
þ
4
99.3574 (M , calcd for C H N O Si, 499.3594).
29 49 3 2
J¼6.8 Hz), 1.10 (3H, d, J¼6.4 Hz), 1.32 (4H, m), 1.37
(
2H, m), 1.57 (2H, m), 1.70 (1H, t, J¼12.2 Hz), 1.89 (1H, dt,
To a solution of 15 (39.1 mg, 78 mmol) in acetic acid
0.5 ml) was added NaCNBH (9.0 mg, 140 mmol) at room
J¼12.2, 4.5 Hz), 2.36 (2H, t, J¼7.8 Hz), 2.45 (1H, m), 2.84
(3H, s), 2.97 (1H, br.s), 3.22 (1H, d, J¼8.9 Hz), 3.44 (1H,
m), 3.58 (1H, br.s), 3.64 (1H, m), 3.78 (1H, t, J¼8.9 Hz),
3.83 (1H, m), 4.04 (1H, m), 4.33 (1H, d, J¼8.4 Hz), 6.22
(1H, d, J¼8.2 Hz), 6.84 (1H, d, J¼8.2 Hz), 6.97 (1H, d,
(
3
temperature. After stirring for 1.5 h additional NaCNBH3
9.0 mg, 140 mmol) was added and the reaction mixture was
stirred for another 2.5 h. The reaction was quenched by the
(
1
3
addition of H O (1 ml) and the mixture was poured into
2
saturated NaHCO aq (20 ml) and extracted with EtOAc.
3
J¼6.5 Hz); C NMR d (CDCl , 0.013 M, 125 MHz, 300 K)
3
ppm: 14.12, 19.72, 21.83, 22.67, 29.07, 29.46, 30.67, 30.81,
31.77, 35.07, 39.47, 40.14, 52.88, 53.81, 66.31, 68.26,
108.08, 116.07, 118.11, 128.58, 148.80, 150.55, 175.71;
The EtOAc layer was washed with brine, dried over Na SO
2
4
and concentrated. The residue was purified by column
chromatography on Wako gel C-200 using hexane and
þ
HR-EI-MS m/z: 387.2889 (M , calcd for C H N O ,
23 37 3 2

7084
Y. Nakagawa et al. / Tetrahedron 60 (2004) 7077–7084
3
87.2886). Compound 13: [a] þ114.08 (c¼0.151, MeOH,
References and notes
D
3 (MeOH) nm (e): 298 (3300), 243
9700); H NMR d (CDCl , 0.020 M, 500 MHz, 300 K)
00.4 K); UV l
max
1
(
1. Nishizuka, Y. FASEB J. 1995, 9, 484–496.
3
ppm: 0.89 (3H, t, J¼6.8 Hz), 0.94 (3H, d, J¼6.5 Hz), 1.14
3H, d, J¼6.7 Hz), 1.32 (4H, m), 1.36 (2H, m), 1.56 (2H,
m), 1.89 (1H, dd, J¼13.2, 2.2 Hz), 2.06 (1H, q, J¼13.2 Hz),
.24 (1H, m), 2.40 (2H, t, J¼7.8 Hz), 2.62 (3H, s), 3.06 (1H,
d, J¼10.6 Hz), 3.18 (1H, m), 3.25 (1H, d, J¼8.3 Hz), 3.42
1H, m), 3.52 (1H, t, J¼7.4 Hz), 3.58 (1H, m), 3.60 (1H,
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(
2
(
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6
br.s), 4.25 (1H, m), 4.62 (1H, br.s), 6.49 (1H, d, J¼8.0 Hz),
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1
3
6
1
2
.87 (1H, d, J¼8.0 Hz); C NMR d (CDCl , 0.020 M,
3
25 MHz, 300 K) ppm: 14.11, 19.08, 19.85, 22.65, 24.16,
9.01, 29.36, 30.91, 31.74, 34.82, 40.59 (two carbon signals
7
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overlapped), 54.55, 57.17, 66.24, 76.25, 119.18, 120.89,
1
3
28.89, 129.81, 148.43, 150.04, 171.10; HR-EI-MS m/z:
þ
8
. Reddig, P. J.; Dreckschmidt, N. E.; Bourguignon, S. E.;
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87.2884 (M , calcd for C H N O , 387.2886).
3 37 3 2
2
9
4
.2. Conformer analysis
2
003, 63, 2404–2408.
The most stable conformations of indolinelactam-Vs
8–13) were estimated by the Chem 3D (Cambridge Soft)
and AMOSS-H11 (NEC quantum chemistry group) pro-
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2
001, 9, 2073–2081.
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1
3
8
–10, H-3 and H-9 protons, H-3 and H-18 protons and H-10
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˚
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3
4.3. Inhibition of specific [ H]PDBu binding to PKC
isozyme C1 peptides
1
1
1
3
The [ H]PDBu binding to the PKC isozyme C1 peptides
was evaluated using the procedure of Sharkey and
2
3
11
Blumberg with modifications as reported previously
under the following conditions: 50 mM Tris–maleate buffer
pH 7.4 at 4 8C), 5–20 nM a PKC isozyme C1 peptide, 20–
(
3
4
0 nM [ H]PDBu (17.0 Ci/mmol), 50 mg/ml 1,2-di(cis-9-
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tration required to cause 50% inhibition of the specific
3
[
computer program (SAS) with a probit procedure. The
H]PDBu binding, IC , which was calculated by a
50
binding constant, K , was calculated using the method of
i
Sharkey and Blumberg.
23. Sharkey, N. A.; Blumberg, P. M. Cancer Res. 1985, 45,
19–24.
2
3
2
2
2
2
2
4. Irie, K.; Koshimizu, K. Memb. Colloid Agric. Kyoto Univ.
1
988, 132, 1–59.
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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. and K.I.) from the Ministry of Education, Culture,
Sports, Science and Technology, the Japanese Government,
and from the Japan Society for the Promotion of Science for
Young Scientists (Y.N.).
9
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(
(
2
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