Design and synthesis of 8-octyl-benzolactam-V9, a selective activator for protein kinase Cε and η

Article abstract of DOI:10.1021/jm050857c

Conventional (α, βI, βII, γ) and novel (δ, ε, η, θ) protein kinase C (PKC) isozymes are main targets of tumor promoters, such as phorbol esters and indolactam-V (ILV). We have recently found that 1-hexyl derivatives of indolinelactam-V (2, 3), in which th

Full text of DOI:10.1021/jm050857c

J. Med. Chem. 2006, 49, 2681-2688
2681
Articles
Design and Synthesis of 8-Octyl-benzolactam-V9, a Selective Activator for Protein Kinase CE
and η
Yu Nakagawa,† Kazuhiro Irie,†,* Ryo C. Yanagita,† Hajime Ohigashi,† Ken-ichiro Tsuda,‡ Kaori Kashiwagi,§ and Naoaki Saito§
DiVision of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto UniVersity, Kyoto 606-8502, Japan, Bio-IT Business
Promotion Center, NEC Corporation, Tokyo 108-8001, Japan, and Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe
UniVersity, Kobe 657-8501, Japan
ReceiVed August 29, 2005
Conventional (R, âI, âII, γ) and novel (δ, ꢀ, η, θ) protein kinase C (PKC) isozymes are main targets of
tumor promoters, such as phorbol esters and indolactam-V (ILV). We have recently found that 1-hexyl
derivatives of indolinelactam-V (2, 3), in which the indole ring of ILV was replaced with the indoline ring,
showed a binding preference for novel PKCs over conventional PKCs. To develop a new ILV analogue
displaying increased synthetic accessibility and improved binding selectivity for novel PKCs, we have designed
8-octyl-benzolactam-V9 (4), a simple analogue without the pyrrolidine moiety of 2 and 3. Compound 4
showed significant binding selectivity for isolated C1B domains of novel PKCs. Moreover, 4 translocated
PKCꢀ and η from the cytoplasm to the plasma membrane of HeLa cells at 1 µM, whereas other PKC
isozymes did not respond even at 10 µM. These results indicate that 4 could be a selective activator for
PKCꢀ and η.
Introduction
Protein kinase C (PKC) is a family of serine/threonine kinases
involved in many cellular processes, such as cell cycle regula-
tion, gene expression, cell differentiation, and apoptosis.1,2 PKC
is also recognized as a main target of tumor promoters,3,4 such
as phorbol esters and indolactam-V (ILV).5,6 Eight PKC
isozymes have been identified to bind tumor promoters (Figure
1): calcium-dependent conventional PKCs R, âI, âII, and γ
and calcium-independent novel PKCs δ, ꢀ, η, and θ.7 Each
contains two binding sites of tumor promoters, designated as
C1A and C1B domains, with a cysteine-rich sequence of 50
amino acid residues.8 Although the mechanism of tumor
promotion is still under investigation, recent studies have
revealed that several novel PKCs (δ, ꢀ, η) might be involved
in tumor promotion9-11 and that they are activated by tumor
promoters that bind to the C1B domains.12-14 Selective activa-
tors for novel PKCs with binding preferences for their C1B
domains would facilitate the analysis of the mechanism of tumor
promotion.
Figure 1. PKC isozymes.
Figure 2. Indolactam-V (ILV) and its derivatives (1-3).
In this article, we describe the design and synthesis of a
simplified analogue of 2 and 3, 8-octyl-benzolactam-V9 (4)
without a pyrrolidine moiety. Compound 4 could be easily
synthesized from commercially available 2-bromo-6-nitrotoluene
and acted selectively upon PKCꢀ and η by binding to their C1B
domains. These results suggest that 4 might be a useful tool to
analyze the roles of these isozymes on tumor promotion.
We have recently synthesized isolated C1 domains of all PKC
isozymes (C1 peptides) in over 98% purity15,16 and found that
the 1-hexyl derivative of ILV (1, Figure 2) showed moderate
selectivity for the C1B domains of novel PKCs using the C1
peptide library.17,18 The binding selectivity for novel PKCs could
be improved by replacing the indole ring of 1 with an indoline
ring, as exemplified by 1-hexyl-indolinelactam-V (2, 3).17,18
However, their unique 1,3,4-trisubstituted indoline structures
hinder their synthetic accessibility and utility as lead compounds
for novel PKC-specific activators.
Results and Discussion
Both (3R)- and (3S)-1-hexyl-indolinelactam-V compounds (2,
3) showed significant binding selectivity for C1B peptides of
novel PKCs despite a large difference in their main conforma-
tions.17,18 The conformation of the 3R isomer (2) was character-
ized by a cis-amide geometry, whereas the 3S isomer (3) existed
in a trans-amide conformation in CDCl3 (Figure 3).17 However,
quite a small amount of the cis-amide conformer of 3 was
detected in CD3OD (data not shown). Because the ILV
* To whom correspondence should be addressed. Tel: +81-75-753-6282.
Fax: +81-75-753-6284. E-mail: irie@kais.kyoto-u.ac.jp.
† Kyoto University.
‡ NEC Corporation.
§ Kobe University.
10.1021/jm050857c CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/01/2006

2682 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Nakagawa et al.
Figure 4. Major conformer of 8-octyl-benzolactam-V9 (4) and its
structure. The conformation of 4 was determined by MM2 and PM3
calculations on the basis of the indicated NOE interactions followed
by the optimization using a Hartree-Fock calculation with 6-31G*.
The octyl group was displayed as a methyl group for convenience.
11 gave the aldehyde, which was subjected to an asymmetric
Strecker reaction23 to stereoselectively afford the (S)-amino
nitrile (12) (72%). The cyano group of 12 was then converted
to the methyl ester with HCl-saturated MeOH (59%). The
removal of the chiral auxiliary group and the deprotection of
the benzyl group was accomplished by hydrogenation. The
formation of the lactam ring using DPPA followed by the
reduction of the methyl ester gave 4 (25% in three steps).
The 1H NMR spectrum showed that 4 existed as two
conformers at room temperature in CDCl3 in the ratio 1:3.7.
The conformer ratio of 4 is solvent- and concentration-
dependent. For example, the ratio of the major to minor
conformers was 4.3:1 in CDCl3 (0.031 M), whereas it was 3.4:1
in CD3OD (0.031 M) (Supporting Information). A set of
nonexchangeable protons almost coalesced at 55 °C in CDCl3
and at 70 °C in pyridine-d5 (Supporting Information). The signal
assignments in 4 were carried out in CDCl3 using 1H-1H COSY
and scalar heteronuclear experiments (HMBC and HMQC). The
highfield shifts of H-2 (δ 2.85) and H-4 (δ 4.58) signals of the
major conformer compared with those of the minor conformer
(δ 3.50 and 5.65, respectively) and a significant NOE enhance-
ment between them (δ 2.85 and 4.58) (Supporting Information),
which were characteristic of the trans-amide conformation in
3,17 were observed in the major conformer of 4, indicating that
the major conformation of 4 was similar to that of 3 with a
trans-amide geometry (Figure 4). Moreover, a distinctive NOE
enhancement between H-7â (δ 2.34) and H-16 (δ 2.71) was
detected. On the basis of these NOE data, we analyzed the major
conformation of 4 using molecular mechanics and quantum
mechanics calculations. The initial structure was calculated by
MM2 with the condition that the distances between H-2 and
H-4 and between H-7â and H-16 were fixed at 2 Å. The
resultant structure was optimized by PM3. Further optimization
was carried out by a Hartree-Fock calculation with a 6-31G*
basis set. The lactam conformation of the obtained structure of
4 was almost the same as that of 3 (Figures 3 and 4). The minor
conformer of 4 could not be fully assigned because of some
signal overlapping and a few characteristic NOE enhancements.
Because some signals of the minor conformer were a slightly
broadened, a variable-temperature NMR study of 4 at 300, 273,
253, and 243 K was conducted (Supporting Information).
Figure 3. Conformations of indolinelactam-V compounds. Top
panel: 3R isomer (2). Bottom panel: 3S isomer (3).
analogues could bind to PKC C1 peptides as a cis-amide
conformer,18-20 these results suggest that 3 would bind to PKC
isozymes in a cis-amide conformation as well as 2, and that
the deletion of π electrons in 1 at positions 2 and 3 might be
responsible for the selective binding of 2 and 3 to the C1B
domains of novel PKCs. Because the major conformation of 3
is the trans-amide, 3 might bind to the C1B peptides of novel
PKC isozymes as the trans-amide conformation. However, this
is unlikely because the trans-amide restricted analogue of
indolactam-V (5-chloro-1-hexyl-indolactam-V10) that we re-
cently synthesized was completely inactive,18 suggesting that 3
could bind to the C1B peptides as the cis-amide conformation.
On the basis of this consideration, we designed 8-octyl-
benzolactam-V9 (4) (Figure 4). In 4, the replacement of the
indole ring with a benzene ring could reduce the structural
complexity and, at the same time, remove the π electrons at
positions 2 and 3. The octyl group at position 8 of 4 would
compensate for the decreased hydrophobicity derived from the
removal of the pyrrole moiety of 1. Calculated log P values of
1-hexyl-ILV (1) and 4 were 5.83 and 7.20, respectively,
suggesting that 4 is more hydrophobic than 1.
8-Octyl-benzolactam-V9 (4) was synthesized from 2-bromo-
6-nitrotoluene (5) as shown in Scheme 1. After bromination at
the benzyl position of 5, a substitution reaction with sodium
diethyl malonate gave 6 (89% in two steps). The diester was
then hydrolyzed and decarboxylated under acidic conditions.
The resulting mono-carboxylic acid was converted to the ethyl
ester. A reduction of the ester group followed by the protection
of the resulting hydroxyl group with an acetyl group afforded
7 (99% in four steps). A modified Sonogashira reaction21 of 7
with 1-octyne gave the coupling product (56%), the triple bond,
and the nitro group, which were reduced by hydrogenation. The
resulting aniline derivative was formylated to give 9 (99% in
two steps). After the reduction of the formyl group and the
deprotection of the acetyl group of 9 (99% in two steps), the
valine unit was stereoselectively introduced by the SN2 reaction
of 10 with Kogan’s triflate22 (73%). Dess-Martin oxidation of

Design and Synthesis of 8-Octyl-benzolactam-V9
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2683
Scheme 1. Synthesis of 8-Octyl-benzolactam-V9 (4)
Table 1. Ki Values for the Inhibition of the Specific Binding of
[3H]PDBu by (3S)-1-Hexyl-indolinelactam-V (3) and
8-Octyl-benzolactam-V9 (4)
Although the chemical shifts of the signals for NH and OH
changed, the spectrum of 4 did not qualitatively change,
suggesting that the two sets of the signals could be assigned to
each conformer.
Ki (nM)
Kd (nM)
To estimate the minor conformer of 4, a conformation search
was carried out using the random search method implemented
in Sybyl 7.1 (Tripos, Inc.). The dielectric constant was set to
5.0, corresponding to the NMR solvent (CHCl3). The octyl group
of 4 was replaced with the ethyl group to simplify the
calculation. As a result, we obtained 11 conformers on the basis
of Itai’s nomenclature (Supporting Information).24 The global
minimum conformer (sofa) was identical with the major
conformer of 4 (Figure 4). In the minor conformer of 4, only
one significant NOESY cross-peak was observed between H-2
(δ 3.70) and H-11 (δ 7.13) (Supporting Information). Although
both the fold and trans-fold-like conformers satisfied this
constraint, the NOESY cross-peaks between H-4 and H-5, H-7â,
or H-16 were not observed in the minor conformer of 4,
suggesting that the minor conformer might be the fold form
(Supporting Information).
The binding affinities of 4 for the PKC C1 peptides were
evaluated by the inhibition of the specific binding of [3H]phorbol
12,13-dibutyrate (PDBu) to these peptides by the method
reported previously.14,15,25 Table 1 shows the inhibition constants
(Ki) of 4 as well as 3 for the PKC C1 peptides. The binding
affinities of 4 for the C1B peptides of novel PKCs were about
10-fold lower than those of 3, probably because of the high
flexibility of its lactam ring. However, the binding selectivity
for these C1 peptides was quite high; 4 showed little or no
binding to both the C1 peptides of conventional PKCs and the
C1A peptides of novel PKCs. Because 4 as well as 3 seems to
bind to the C1B domains as the cis-amide conformation,18 these
results indicate that the deletion of the π electrons at positions
2 and 3 of ILV analogues could be effective for increasing the
binding selectivity for the C1B peptides of novel PKCs.
PKC activation is tightly coupled with its translocation from
the cytoplasm to the plasma membrane.26 The binding of a PKC
(3S)-1-hexyl-
8-octyl-
PKC C1 peptide indolinelactam-V (3)a benzolactam-V9 (4) PDBu
R-C1A (72-mer)b
R-C1B
550 (44)c
>10000
1200 (27)
250
1800 (290)
1600
>10000
16 (1.5)
>10000
14 (1.0)
>10000
12 (2.0)
NTd
>10000
>10000
>10000
>10000
>10000
5200 (810)
>10000
216 (9.0)
>10000
510 (39)
>10000
144 (13)
NT
1.1
5.3
1.3
1.3
1.5
1.2
52
0.53
5.6
â-C1A (72-mer)
â-C1B
γ-C1A
γ-C1B
δ-C1A
δ-C1B
ꢀ-C1A
ꢀ-C1B
0.81
4.3
0.45
>200
0.72
η-C1A
η-C1B
θ-C1A
θ-C1B
12 (1.3)
290 (15)
a These data are cited from ref 17. b Ten residues from both N- and
C-termini of the previous R-C1A and â-C1B were elongated because the
solubility of the orignal 52-mer peptides was extremely low.14 c Standard
deviation of at least two separate experiments. d Not tested.
activator, such as a tumor promoter, to an inactive PKC in the
cytoplasm increases its membrane affinity. The membrane
association causes a conformational change in PKC and results
in the release of the autoinhibitory sequence from its kinase
domain to activate the enzyme. Recently, Wada et al.27 reported
that a phorbol ester analogue with a glycol moiety in the ester
chain at position 12 strongly bound to PKCδ but did not
translocate the enzyme, indicating that the binding and activation
abilities of PKC ligands do not fully correspond to each other.
It is thus important to examine the translocation-inducing
abilities as well as the binding abilities of PKC ligands.
The translocation assay using a fusion protein of a PKC
isozyme and a green fluorescent protein (GFP) in living cells
is one of the promising methods to evaluate the translocation-

2684 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Nakagawa et al.
Figure 5. Translocation of GFP-tagged PKCꢀ by 8-octyl-benzolactam-V9 (4). The fluorescence of GFP-tagged PKCꢀ in HeLa cells: (a) before
and (b) after stimulation by 4 (10 µM). The fluorescence of tandem DsRed2-tagged MARCKS in HeLa cells: (c) before and (d) after stimulation
by 4 (10 µM).
Table 2. Translocation of GFP-Tagged PKC Isozymes Induced by
as well as one of conventional PKCs (PKCâI). However,
Various Concentrations of (3S)-1-Hexyl-indolinelactam-V (3) Expressed
compound 4 selectively translocated PKCꢀ and η at 1 µM. The
by the R Valuesa
minimum concentration of 4 to induce PKCδ translocation was
20 µM, and conventional PKCs (PKCR, âI, γ) did not respond
at the same concentration (Table 3).
PKC isozymes
10 µM
1 µM
0.5 µM
NTc
0.95 (0.03)
NT
NT
2.42 (0.36)
1.01 (0.26)
PKCR
PKCâI
PKCγ
PKCδ
PKCꢀ
PKCη
1.79 (0.33)b
2.38 (0.31)
1.53 (0.29)
2.41 (0.41)
4.29 (1.25)
1.92 (0.23)
0.97 (0.01)
2.39 (0.29)
0.96 (0.05)
0.90 (0.06)
2.73 (0.18)
1.84 (0.06)
The activation of PKCꢀ and η was confirmed by monitoring
the translocation of the myristoylated alanine-rich C-kinase
substrate (MARCKS), one of the most popular PKC substrates
that changes its distribution from the plasma membrane to the
cytoplasm in a PKC phosphorylation-dependent manner.31 We
examined the translocation of tandem DsRed2-tagged MARCKS
using HeLa cells expressing GFP-tagged PKCꢀ stimulated by
4 at 1 µM. PKCꢀ in the cytoplasm translocated to the plasma
membrane after the stimulation of 4, and the membrane-
distributed MARCKS translocated to the cytoplasm as the
membrane target of PKCꢀ (Figure 5). PKCη also translocated
to the plasma membrane by 4 at 1 µM and changed the
distribution of MARCKS (data not shown). These results suggest
that only 4 can selectively activate PKCꢀ and η in living cells.
a The values (R ) Ipm/Icyt) represent the ratio of the fluorescent intensities
between the cytosol (Icyt) and the plasma membrane (Ipm). The details are
described in the Experimental Section. b Standard deviation of at least three
measurements. c Not tested.
inducing ability of PKC ligands.28-30 We expressed a GFP-
fusion protein of each PKC isozyme in HeLa cells and examined
its translocation by the stimulation of 3 and 4 at various
concentrations. Membrane translocation of the GFP-fusion
protein of PKCꢀ induced by 10 µM 4 is shown in Figure 5a
and b as a typical example. To quantify the translocation, the
relative fluorescence intensity in the plasma membrane (R) was
defined as R ) Ipm/Icyt, where Ipm and Icyt are the plasma
membrane fluorescence intensity and the average cytosolic
fluorescence intensity, respectively. After each compound (3,
4, and ILV) was added at various concentrations, the R values
were plotted against time (Supporting Information). The maxi-
mum R values of 3 and 4 at various concentrations are
summarized in Tables 2 and 3. Unexpectedly, significant
selectivity for novel PKCs was not observed in 3; Compound
3 (1 µM) translocated two isozymes of novel PKCs (PKCꢀ, η)
Conclusion
On the basis of the hypothesis that the deletion of π electrons
at positions 2 and 3 of 1-hexyl-indolactam-V (1) could increase
the binding selectivity for the C1B domains of novel PKCs as
exemplified for 1-hexyl-indolinelactam-V compounds (2, 3),17
we designed 8-octyl-benzolactam-V9 (4) without the pyrrole
moiety of 1. Compound 4 showed significant binding selectivity
for the C1B peptides of novel PKCs comparable to those of 2
and 3. We have recently found that the indole ring of ILV could
Table 3. Translocation of GFP-Tagged PKC Isozymes Induced by Various Concentrations of 8-Octyl-benzolactam-V9 (4) Expressed by the R Valuesa
PKC isozymes
30 µM
20 µM
10 µM
5 µM
NTc
NT
NT
NT
2.67 (0.59)
1.78 (0.30)
1 µM
NT
NT
NT
NT
1.66 (0.20)
1.46 (0.12)
0.5 µM
NT
NT
NT
NT
1.65 (0.11)
0.97 (0.02)
PKCR
PKCâI
PKCγ
PKCδ
PKCꢀ
PKCη
0.98 (0.01)b
2.40 (0.52)
0.97 (0.01)
NT
NT
NT
0.97 (0.01)
0.97 (0.01)
0.97 (0.01)
2.22 (0.16)
NT
0.98 (0.01)
0.97 (0.01)
0.97 (0.01)
0.95 (0.03)
1.96 (0.36)
1.72 (0.11)
NT
a The values (R ) Ipm/Icyt) represent the ratio of the fluorescent intensities between the cytosol (Icyt) and the plasma membrane (Ipm). The details are
described in the Experimental Section. b Standard deviation of at least three measurements. c Not tested.

Design and Synthesis of 8-Octyl-benzolactam-V9
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2685
120 °C for 15 h. The reaction mixture was poured into H2O and
extracted with EtOAc. The collected EtOAc layer was washed with
brine, dried over Na2SO4, and concentrated to give the crude
monocarboxylic acid. The crude monocarboxylic acid in EtOH (10
mL) was added to a solution of SOCl2 (10 mL) in dry ethanol (20
mL) at 0 °C. The mixture was refluxed for 1.5 h at 100 °C and
then concentrated. The residue was poured into saturated aqueous
NaHCO3 and extracted with EtOAc. The collected EtOAc layer
was washed with brine, dried over Na2SO4, and concentrated. The
residue was purified by column chromatography on Wakogel C-200
using hexane and increasing amounts of EtOAc to give the ethyl
ester (5.96 g, 19.7 mmol). To a solution of the ethyl ester (2.85 g,
9.44 mmol) in THF (20 mL) was added LiBH4 (519 mg, 23.6 mmol)
in three portions at 0 °C. The mixture was then stirred at room
temperature for 10 h. The reaction was quenched by the addition
of 0.2 N HCl, and the mixture was extracted with EtOAc. The
collected EtOAc layer was washed with brine, dried over Na2SO4,
and concentrated to give the crude alcohol. To a solution of this
alcohol in pyridine (7.0 mL) was added Ac2O (5.0 mL) at 0 °C,
and the mixture was stirred for 30 min at 0 °C. The reaction was
quenched by the addition of H2O, and the mixture was concentrated.
The residue was purified by column chromatography on Wakogel
C-200 using hexane and increasing amounts of EtOAc to give 7
(3.11 g, 10.3 mmol, 99% in four steps). Compound 7: 1H NMR δ
(500 MHz, 300 K, CDCl3, 0.062 M): 2.05 (2H, m, ArCH2CH2-),
2.08 (3H, s, Ac), 2.99 (2H, m, ArCH2-), 4.18 (2H, t, J ) 6.2 Hz,
-CH2Ac), 7.23 (1H, t, J ) 8.1 Hz, Ar), 7.73 (1H, dd, J ) 8.1, 1.0
Hz, Ar), 7.80 (1H, dd, J ) 8.0, 0.9 Hz, Ar); HRMS-FAB m/z:
302.0081 (MH+, calcd for C11H13NO4Br, 302.0027).
To a mixture of 7 (3.10 g, 10.3 mmol), PdCl2(PPh3)2 (145 mg,
0.207 mmol), 1-octyne (1.80 mL, 12.2 mmol) and triethylamine
(10 mL) was added CuI (78.0 mg, 0.411 mmol) at room temperature
under an Ar atmosphere. The reaction mixture was stirred for 18 h
at 50 °C and then filtered. The filtrate was concentrated and purified
by column chromatography on Wakogel C-200 using hexane and
increasing amounts of EtOAc to give 8 (1.92 g, 5.8 mmol, 56%).
Compound 8: 1H NMR δ (500 MHz, 300 K, CDCl3, 0.077 M):
0.91 (3H, t, J ) 7.0 Hz, octynyl), 1.31-1.34 (4H, m, octynyl), 1.46
(2H, m, octynyl), 1.62 (2H, m, octynyl), 2.05 (2H, m, ArCH2CH2-
), 2.06 (3H, s, Ac), 2.46 (2H, t, J ) 7.1 Hz, octynyl), 3.04 (2H, m,
ArCH2-), 4.15 (2H, t, J ) 6.4 Hz, -CH2OAc), 7.26 (1H, t, J )
8.0 Hz, Ar), 7.60 (1H, dd, J ) 8.0, 0.9 Hz, Ar), 7.71 (1H, dd, J )
8.0, 0.9 Hz, Ar); HRMS-FAB m/z: 332.1863 (MH+, calcd for
C19H26NO4, 332.1862).
be involved in the CH/π interaction with the hydrogen atom at
position 4 of Pro-11 of the PKCδ C1B domain.32 Although Pro-
11 is preserved in all C1 domains of conventional and novel
PKCs, the spatial position of Pro-11 in PKC isozymes might
be different from each other. The selective binding of 2, 3, and
4 for the C1B domains of novel PKCs, thus, might reflect, in
part, the difference of the CH/π interaction with Pro-11 between
conventional and novel PKCs in addition to the steric hindrance
at position 1.18
The PKC translocation assay using GFP-tagged PKC isozymes
revealed that 4 could selectively activate PKCꢀ and η, whereas
3 did not show any selective activation for novel PKCs. Garcia-
Bermejo et al. reported that new diacylglycerol-lactones did not
show any binding selectivity between PKCR and δ but
selectively activated PKCR in LNCaP prostate cancer cells.33
These results indicate that it is important to evaluate the
translocation-inducing ability of PKC ligands as well as their
PKC-binding ability. Compound 4 is the first PKC ligand that
could selectively activate PKCꢀ and η by binding to their C1B
domains. Because PKCꢀ and η play important roles in tumor
promotion,10,11 4 would be useful for analyzing the mechanism
of tumor promotion.
Experimental Section
General Remarks. The following spectroscopic and analytical
instruments were used: UV, Shimadzu UV-2200A; [R]D, Jasco DIP-
1000; 1H NMR, Bruker ARX500, AVANCE400 (reference to
TMS); HPLC, Waters Model 600E with Model 2487 UV detector;
(HR) EIMS and HRMS-FAB JEOL JMS-600H. The NOESY
spectrum was measured using the pulse sequence noesytp in
BRUKER ARX500 with a mixing time of 800 ms (Supporting
Information). Essentially similar spectra were obtained by varying
the D8 parameter (500 and 300 ms) (Supporting Information).
HPLC was carried out on a YMC-packed SH-342-5 (ODS, 20
mm i.d. × 150 mm) column (Yamamura Chemical Laboratory).
Wakogel C-200 (silica gel, Wako Pure Chemical Industries) and
YMC A60-350/250 gel (ODS, Yamamura Chemical Laboratory)
were used for column chromatography. [3H]PDBu (19.0 Ci/mmol)
was purchased from Perkin-Elmer Life Science. All the other
chemicals and reagents were purchased from chemical companies
and used without further purification.
Synthesis of 8-Octyl-benzolactam-V9 (4). To a suspension of
2-bromo-6-nitrotoluene (5.0 g, 23.1 mmol) and NBS (4.5 g, 25.4
mmol) in CCl4 (25 mL) was added AIBN (760 mg, 4.62 mmol).
The reaction mixture was refluxed at 100 °C for 15 h and then
filtered. The filtrate was concentrated under reduced pressure and
purified by column chromatography on Wakogel C-200 using
hexane and increasing amounts of EtOAc to give a benzyl bromine
derivative (6.33 g, 21.1 mmol). NaH in oil (1.11 g, 27.7 mmol)
was washed with hexane and suspended in dry DMF (10 mL) under
an Ar atmosphere. Diethyl malonate (3.90 mL, 25.7 mmol) was
added in three portions at 0 °C, and the resulting suspension was
stirred at 0 °C for 10 min. A solution of the benzyl bromine
derivative (6.33 g, 21.1 mmol) in DMF (20 mL) was added
dropwise. The reaction mixture was stirred for 2 h at 0 °C and
then poured into EtOAc and H2O. The EtOAc layer was collected,
and the aqueous layer was extracted with EtOAc. The combined
EtOAc layer was washed with brine, dried over Na2SO4, and
concentrated. The residue was purified by column chromatography
on Wakogel C-200 using hexane and increasing amounts of EtOAc
to give 6 (7.08 g, 18.9 mmol, 89% in two steps). Compound 6: 1H
NMR δ (500 MHz, 300 K, CDCl3, 0.027 M): 1.23 (6H, t, J ) 7.1
Hz, -CO2CH2CH3 × 2), 3.69 (2H, d, J ) 7.6 Hz, ArCH2-), 3.82
(1H, t, J ) 7.6 Hz, -CH2CH(CO2Et)2), 4.18 (4H, dd, J ) 14.2,
7.1 Hz, -CO2CH2CH3 × 2), 7.28 (1H, t, J ) 8.1 Hz, Ar), 7.78
(1H, d, J ) 8.1 Hz, Ar), 7.80 (1H, d, J ) 8.1 Hz, Ar); HRMS-
FAB m/z: 374.0260 (MH+, calcd for C14H17NO6Br, 374.0239).
To a solution of 6 (7.01 g, 18.9 mmol) in AcOH (20 mL) was
added concentrated HCl (20 mL), and the mixture was refluxed at
A mixture of 8 (1.92 g, 5.80 mmol), 10% Pd-C (192 mg) in
EtOH (13 mL) was stirred vigorously under 1 atm of H2 at room
temperature for 2 h. The reaction mixture was filtered and then
concentrated to give a crude amine. To a solution of the amine in
THF (6.8 mL) was added acetic formic anhydride (2.6 mL) at 0
°C. The mixture was stirred at 0 °C for 1 h and concentrated. The
residue was poured into saturated aqueous K2CO3 and extracted
with EtOAc. The collected EtOAc layer was washed with brine,
dried over Na2SO4, and concentrated. The residue was purified by
column chromatography on Wakogel C-200 using hexane and
increasing amounts of EtOAc to give 9 (1.91 g, 5.74 mmol, 99%
in two steps), in which two conformers existed in a ratio of 1:0.4.
Compound 9: 1H NMR δ (500 MHz, 300 K, CDCl3, 0.077 M) for
the major conformer: 0.88 (3H, t, J ) 6.9 Hz, octyl), 1.27-1.31
(10H, m, octyl), 1.56 (2H, m, octyl), 1.85 (2H, m, ArCH2CH2-),
2.14 (3H, s, Ac), 2.61 (2H, t, J ) 7.5 Hz, octyl), 2.74 (2H, t, J )
7.9 Hz, ArCH2-), 4.11 (2H, t, J ) 6.0 Hz, -CH2OAc), 7.00 (1H,
d, J ) 7.7 Hz, Ar), 7.09 (1H, d, J ) 7.0 Hz, Ar), 7.17 (1H, t, J )
7.8 Hz, Ar), 7.91 (1H, br.d, J ) 11.0 Hz, -NHCHO), 8.49 (1H, d,
J ) 11.0 Hz, -NHCHO); HRMS-FAB m/z: 333.2308 (M+, calcd
for C20H31NO3, 333.2304).
To a solution of 9 (1.91 g, 5.74 mmol) in THF (30 mL) was
added dropwise 1.0 M BH3 in THF solution (17 mL) at 0 °C, and
the reaction mixture was stirred for 2 h at 0 °C. The reaction was
quenched by the addition of 10% aqueous citric acid (20 mL), and
the mixture was extracted with EtOAc. The collected EtOAc layer
was washed with brine, dried over Na2SO4, and then concentrated

2686 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Nakagawa et al.
to give the crude N-methylaniline. To a solution of the N-
methylaniline in MeOH (20 mL) was added 1 N NaOH (5.0 mL).
The reaction mixture was stirred at room temperature for 1 h and
then concentrated. The residue was poured into H2O, and the
mixture was extracted with EtOAc. The collected EtOAc layer was
washed with brine, dried over Na2SO4, and then concentrated. The
residue was purified by column chromatography on Wakogel C-200
using hexane and increasing amounts of EtOAc to give 10 (1.59 g,
5.74 mmol, 99% in two steps). Compound 10: 1H NMR δ (500
MHz, 300 K, CDCl3, 0.077 M): 0.88 (3H, t, J ) 6.9 Hz, octyl),
1.22-1.34 (10H, m, octyl), 1.55 (2H, m, octyl), 1.77 (2H, m,
ArCH2CH2-), 2.57 (2H, m, octyl), 2.66 (2H, t, J ) 7.6 Hz, ArCH2-
), 2.84 (3H, s, N-CH3), 3.64 (2H, t, J ) 5.8 Hz, -CH2OH), 6.53
(1H, dd, J ) 8.0, 0.8 Hz, Ar), 6.62 (1H, dd, J ) 7.9, 0.9 Hz, Ar),
7.09 (1H, t, J ) 7.9 Hz, Ar); HR-EIMS m/z: 277.2403 (M+, calcd
for C18H31NO, 277.2406).
7.26-7.35 (8H, m, Ar); HRMS-FAB m/z: 612.4204 (MH+, calcd
for C39H54N3O3, 612.4165).
A solution of 12 (1.40 g, 2.29 mmol) in HCl-saturated MeOH
(25 mL) was stirred at room temperature for 36 h. The reaction
was quenched by neutralization with cool 1N NaOH. The mixture
was poured into brine (30 mL) and extracted with EtOAc. The
collected EtOAc layer was washed with brine, dried over Na2SO4,
and then concentrated. The residue was purified by column
chromatography on Wakogel C-200 using hexane and increasing
amounts of EtOAc to give 13 (860 mg, 1.34 mmol, 59%).
Compound 13: [R]D -44.2° (c ) 0.31, MeOH, 30.9 °C); 1H NMR
δ (500 MHz, 300 K, CDCl3, 0.036 M): 0.87 (3H, d, J ) 6.8 Hz,
-CH(CH3)2), 0.88 (3H, t, J ) 6.8 Hz, octyl), 1.08 (3H, d, J ) 6.7
Hz, -CH(CH3)2), 1.26-1.34 (10H, m, octyl), 1.52 (2H, m, octyl),
1.61 (1H, br.s, OH), 1.69 (1H, m, ArCH2CH2-), 1.82 (1H, m,
ArCH2CH2-), 2.11 (1H, br.s, NH), 2.20 (1H, m, -CH(CH3)2), 2.49
(1H, dt, J ) 12.5, 3.5 Hz, ArCH2-), 2.50 (2H, m, octyl), 2.75
(3H, s, N-CH3), 2.92 (1H, dt, J ) 12.5, 5.1 Hz, ArCH2-), 3.16
(1H, d, J ) 9.9 Hz, N-CH(i-Pro)CO2Bn), 3.18 (1H, t, J ) 6.7 Hz,
N-CH(Ph)CH2OH), 3.54 (1H, m, N-CH(CO2CH3)CH2-), 3.69 (3H,
s, -CO2CH3), 3.73 (2H, m, -CH2OH), 4.97 (2H, ABq, J ) 12.5
Hz, -CO2CH2Ph), 6.86 (1H, dd, J ) 7.6, 1.5 Hz, Ar), 6.92 (1H,
dd, J ) 7.5, 1.5 Hz, Ar), 6.95 (1H, t, J ) 7.5 Hz, Ar), 7.08 (2H, m,
Ar), 7.23-7.36 (8H, m, Ar); HRMS-FAB m/z: 645.4256 (MH+,
calcd for C40H57N2O5, 645.4267).
A mixture of 13 (202 mg, 0.313 mmol) and 10% Pd-C (20 mg)
in MeOH (5.0 mL) was stirred vigorously under 1 atm of H2 at
room temperature for 18 h. The reaction mixture was filtered and
then concentrated to give the crude amino acid. To a solution of
the amino acid in DMF (9.0 mL) was added diphenylphosphoryl
azide (135 µL, 0.627 mmol) and triethylamine (131 µL, 0.942
mmol) at 0 °C. The solution was stirred at room temperature for
27 h. The reaction mixture was poured into H2O and extracted with
EtOAc. The collected EtOAc layer was washed with brine, dried
over Na2SO4, and then concentrated. The residue was purified by
column chromatography on Wakogel C-200 using hexane and
increasing amounts of EtOAc to give a lactam. To a solution of
the lactam in THF (1.0 mL) was added LiBH4 (5.0 mg, 0.228 mmol)
at 0 °C, and the mixture was stirred at room temperature for 1 h.
The reaction mixture was poured into H2O, and the mixture was
extracted with EtOAc. The EtOAc layer was washed with brine,
dried over Na2SO4, and then concentrated. The residue was purified
by HPLC on YMC-SH-342-5 using 85% MeOH to give 4 (24.7
mg, 64.0 µmol, 84%) in which two conformers existed in a ratio
of 3.7:1.0. The purity of 4 was more than 95%, which was
confirmed by two diverse HPLC systems on SH-342-5 using 85%
MeOH (flow rate of 8.0 mL/min; retention time of 36.0 min) and
70% MeCN (flow rate of 8.0 mL/min; retention time of 40.7 min).
Compound 4: [R]D +118.9° (c ) 1.09, MeOH, 31.3 °C); UV λmax
(MeOH) nm (ꢀ): 274 (2,600), 238 (3,000), 204 (21,900); 13C NMR
δ (125 MHz, 300 K, CDCl3, 0.112 M): 14.12, 19.00, 19.69, 22.68,
23.80, 24.46, 29.27, 29.49, 29.91, 31.17, 31.88, 32.97, 35.26, 36.02,
54.49, 65.02, 76.14, 127.31, 128.82, 142.99, 143.43, 151.34, 170.42;
1H NMR δ (500 MHz, 300 K, CDCl3, 0.112 M) for the major
conformer: 0.88 (3H, t, J ) 6.7 Hz, octyl), 0.90 (3H, d, J ) 6.3
Hz, -CH(CH3)2), 1.16 (3H, d, J ) 6.6 Hz, -CH(CH3)2), 1.21-
1.37 (10H, m, octyl), 1.50 (3H, m, octyl, ArCH2CH2-), 2.05 (1H,
br.s, OH), 2.27 (2H, m, ArCH2CH2- CH(CH3)2), 2.34 (1H, dd, J
) 14.8, 9.1 Hz, ArCH2-), 2.47 (1H, m, octyl), 2.56 (1H, m, octyl),
2.71 (3H, s, N-CH3), 2.76 (1H, dd, J ) 14.8, 8.9 Hz, Ar-CH2),
2.85 (1H, d, J ) 10.9 Hz, NCH(i-Pr)CONH-), 3.40 (1H, m, -CH2-
OH), 3.57 (1H, m, -CH2OH), 4.36 (1H, m, NCH(CH2OH)CH2-
), 4.58 (1H, br.d, J ) 10.6 Hz, NH), 6.96 (1H, d, J ) 7.6 Hz, Ar),
7.02 (1H, d, J ) 6.7 Hz, Ar), 7.09 (1H, t, J ) 7.6 Hz, Ar); for the
minor conformer: 1.12 (3H, d, J ) 6.9 Hz, -CH(CH3)2), 1.19 (3H,
d, J ) 6.7 Hz, -CH(CH3)2), 1.88 (1H, dd, J ) 13.5, 3.8 Hz,
ArCH2CH2-), 2.61 (3H, s, N-CH3), 2.76 (1H, m, ArCH2-), 3.10
(1H, br.s, OH), 3.20 (1H, t, J ) 12.8 Hz, ArCH2-), 3.51 (2H, m,
-CH2OH), 3.70 (1H, d, J ) 5.9 Hz, NCH(i-Pr)CONH-), 4.67 (1H,
m, NCH(CH2OH)CH2-), 5.65 (1H, br.d, J ) 10.2 Hz, NH), 6.96
(1H, m, Ar), 7.07 (1H, m, Ar), 7.13 (1H, d, J ) 7.8 Hz, Ar). Other
A mixture of 10 (1.56 g, 5.63 mmol), 2,6-lutidine (1.3 mL, 11.2
mmol), and Val-Tf 22 (2.26 g, 6.65 mmol) in 1,2-dichloroethane
(20 mL) was refluxed at 80 °C for 16 h and then concentrated.
The residue was purified by column chromatography on Wakogel
C-200 using hexane and increasing amounts of EtOAc to stereospe-
cifically give 11 (1.93 g, 4.13 mmol, 73%). Compound 11: [R]D
1
-16.5° (c ) 0.73, MeOH, 30.9 °C); H NMR δ (500 MHz, 300
K, CDCl3, 0.073 M): 0.89 (3H, t, J ) 7.3 Hz, octyl), 0.91 (3H, d,
J ) 6.6 Hz, -CH(CH3)2), 1.18 (3H, d, J ) 6.7 Hz, -CH(CH3)2),
1.27-1.39 (10H, m, octyl), 1.55 (2H, m, octyl), 1.70 (1H, m,
ArCH2CH2), 1.82 (1H, m, ArCH2CH2), 2.25 (1H, m, -CH(CH3)2),
2.54 (1H, m, ArCH2), 2.67 (2H, m, ArCH2), 2.74 (1H, dd, J ) 7.9,
4.7 Hz, OH), 2.84 (3H, s, N-CH3), 2.89 (1H, m, ArCH2), 3.21 (1H,
d, J ) 10.4 Hz, N-CH(i-Pro)CO2Bn), 3.39 (1H, m, CH2OH), 3.57
(1H, m, CH2OH), 4.92 (2H, s, -CO2CH2Ph), 6.91 (1H, dd, J )
7.1, 2.2 Hz, Ar), 6.97 (2H, m, Ar), 7.02 (2H, m, Ar), 7.26 (3H, m,
Ar); HR-EIMS m/z: 467.3403 (M+, calcd for C30H45NO3, 467.3399).
To a solution of 11 (1.93 g, 4.13 mmol) in CH2Cl2 (20 mL) was
added the Dess-Martin reagent (2.07 g, 4.88 mmol) at room
temperature under an Ar atmosphere. The reaction mixture was
stirred for 30 min and then concentrated. The residue was poured
into saturated aqueous Na2SO4 and extracted with EtOAc. The
collected EtOAc layer was washed with brine, dried over Na2SO4,
and then concentrated. The residue was purified by column
chromatography on Wakogel C-200 using hexane and increasing
amounts of EtOAc to give an aldehyde. A mixture of the aldehyde
and (R)-phenylglycinol (518 mg, 3.78 mmol) in MeOH (6.3 mL)
was stirred at room temperature for 30 min before it was heated to
40 °C. At this temperature, trimethylsilyl cyanide (0.687 mL, 5.16
mmol) was added dropwise, and the mixture was stirred for a further
1 h at the same temperature. The reaction mixture was poured into
brine, and the mixture was extracted with EtOAc. The collected
EtOAc layer was washed with brine, dried over Na2SO4, and then
concentrated. The residue was purified by column chromatography
on Wakogel C-200 using hexane and increasing amounts of EtOAc
to give a diasteriomeric mixture of amino nitriles. A solution of
the diastereomeric mixture in MeOH (20 mL) was refluxed at 80
°C for 3 h, and then concentrated. The residue was purified by
column chromatography on Wakogel C-200 using hexane and
increasing amounts of EtOAc to give (S,S) isomer 12 as a single
diastereomer (1.79 g, 2.93 mmol, 85% in two steps). Compound
1
12: [R]D +51.5° (c ) 0.20, MeOH, 30.9 °C); H NMR δ (500
MHz, 300 K, CDCl3, 0.041 M): 0.87 (3H, d, J ) 6.6 Hz, -CH-
(CH3)2), 0.88 (3H, t, J ) 7.1 Hz, octyl), 1.03 (3H, d, J ) 6.7 Hz,
-CH(CH3)2), 1.26-1.38 (10H, m, octyl), 1.55 (2H, m, octyl), 1.75
(2H, m, ArCH2CH2-), 2.05 (1H, m, -CH(CH3)2), 2.21 (2H, m,
NH, OH), 2.57 (2H, m, octyl), 2.67 (1H, dt, J ) 12.3, 4.3 Hz,
ArCH2-), 2.72 (3H, s, N-CH3), 2.89 (1H, dt, J ) 12.3, 5.1 Hz,
ArCH2-), 3.14 (1H, d, J ) 9.7 Hz, N-CH(i-Pro)CO2Bn), 3.30 (1H,
t, J ) 6.8 Hz, N-CH(Ph)CH2OH), 3.58 (1H, t, J ) 10.0 Hz, -CH2-
OH), 3.79 (1H, dd, J ) 10.0, 4.0, -CH2OH), 4.13 (1H, dd, J )
9.1, 4.0 Hz, N-CH(CN)CH2-), 4.96 (2H, ABq, J ) 12.3 Hz,
CO2CH2Ph), 6.87 (1H, dd, J ) 7.6, 1.4 Hz, Ar), 6.94 (1H, dd, J )
7.6, 1.4 Hz, Ar), 6.98 (1H, t, J ) 7.6 Hz, Ar), 7.08 (2H, m, Ar),

Design and Synthesis of 8-Octyl-benzolactam-V9
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2687
peaks had weak intensities and overlapped those of the major
conformer. HR-EIMS m/z: 388.3089 (M+, calcd for C24H40N2O2,
388.3090).
compound was added at various concentrations, the R values were
plotted against time. The resulting curves represent the time course
of plasma membrane translocation of GFP-tagged PKC subtypes
of each compound (Supporting Information). The maximum R value
represents the maximum membrane translocation of each PKC in
response to the compound at various concentrations and are
summarized in Table 2.
Conformational Analysis of 8-Octyl-benzolactam-V9 (4). The
main conformation of 8-octyl-benzolactam-V9 (4) was estimated
by the Chem 3D (Cambridge Soft) and AMOSS-H11 (NEC
quantum chemistry group) programs. The initial structures were
calculated by molecular mechanics calculations using the MM2
theory with the distance between two protons (H-2 and H-4, and
H-7â and H-16) fixed at 2 Å, congruent with NOE data. The
resultant structures were optimized by a semiempirical quantum
mechanics calculation using the PM3 theory. Further optimization
of these calculated structures was carried out by ab initio molecular
orbital schemes using a Hartree-Fock theory with the 6-31G* basis
set to give the most stable conformation.
Acknowledgment. This research was partly supported by
Grants-in-Aid for Scientific Research (A) 15208012 (to H.O.
and K.I.), for Scientific Research on Priority Areas 17035046
( to K.I.), and for the Promotion of Science for Young Scientists
(to Y.N.) from the Ministry of Education, Science, Culture,
Sports and Technology of the Japanese Government.
A conformation search was carried out using the random search
method implemented in Sybyl 7.1 (Tripos, Inc.). The octyl group
of 4 was replaced with the ethyl group to simplify the calculation.
The following conditions and parameters were used: all rotatable
bonds were perturbed; the energy cut off was set to 15 kcal/mol;
the maximum number of search iterations and the maximum number
of hits were both set to 100 000; and the RMS threshold was set to
0.2 Å. The conformations produced by the random conformational
search were fully optimized using the MMFF94s force field. The
dielectric constant (ꢀ) was set to 5.0 (CHCl3). Powell’s method
was used for energy minimization until the gradient value was
smaller than 0.001 kcal/mol‚Å. As a result, we obtained 244
conformations with energy values within 10 kcal/mol from the
global minimum, and they were classified into 11 groups (sofa,
r-trans-fold-like, fold, r-trans-fold, cis-sofa, trans-fold-like, r-sofa,
r-cis-sofa, cis-sofa-like, r-fold, and twist) on the basis of Itai’s
nomenclature (Supporting Information).24 The global minimum
(sofa) was identical to that of the major conformer of 4 (Figure 4).
Inhibition of Specific [3H]PDBu Binding to PKC Isozyme C1
Peptides. The [3H]PDBu binding to the PKC isozyme C1 peptides
was evaluated using the procedure of Sharkey and Blumberg24 with
modifications, as reported previously,14 under the following condi-
tions: 50 mM Tris-maleate buffer (pH 7.4 at 4 °C), 5-20 nM a
PKC isozyme C1 peptide, 20-40 nM [3H]PDBu (19.0 Ci/mmol),
50 µg/mL 1,2-di(cis-9-octadecenoyl)-sn-glycero-3-phospho-L-serine,
3 mg/mL bovine γ-globulin, and various concentrations of 8-octyl-
benzolactam-V9 (4). The binding affinity was evaluated by the
concentration required to cause 50% inhibition of the specific [3H]-
PDBu binding, IC50, which was calculated by a computer program
(SAS) with a probit procedure. The binding constant, Ki, was
calculated using the method of Sharkey and Blumberg.24
Supporting Information Available: Translocation of all GFP-
tagged PKC isozymes induced by 1 µM of ILV and 3 and 10 µM
of 4 in HeLa cells along with NMR and conformational analyses
of 4. This material is available free of charge via the Internet at
http://pubs.acs.org.
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