Synthesis, SAR studies, and pharmacological evaluation of 3-anilino-4-(3-indolyl) maleimides with conformationally restricted structure as orally bioavailable PKCβ-selective inhibitors

Article abstract of DOI:10.1016/j.bmc.2006.05.033

Conformationally restricted 3-anilino-4-(3-indolyl)maleimide derivatives were designed and synthesized aiming at discovery of novel protein kinase Cβ (PKCβ)-selective inhibitors possessing oral bioavailability. Among them, compounds having a fused five-me

Full text of DOI:10.1016/j.bmc.2006.05.033

Bioorganic & Medicinal Chemistry 14 (2006) 5781–5794
Synthesis, SAR studies, and pharmacological evaluation of
3-anilino-4-(3-indolyl) maleimides with conformationally
restricted structure as orally bioavailable PKCb-selective inhibitors
Masahiro Tanaka, Shoichi Sagawa, Jun-ichi Hoshi, Fumito Shimoma, Katsutaka Yasue,
Minoru Ubukata, Tomoyuki Ikemoto, Yasunori Hase, Mitsuru Takahashi,
Tomohiko Sasase, Nobuhisa Ueda, Mutsuyoshi Matsushita and Takashi Inaba*
Central Pharmaceutical Research Institute, Japan Tobacco Inc., 1-1, Murasaki-cho, Takatsuki Osaka 569-1125, Japan
Received 24 April 2006; revised 15 May 2006; accepted 16 May 2006
Available online 9 June 2006
Abstract—Conformationally restricted 3-anilino-4-(3-indolyl)maleimide derivatives were designed and synthesized aiming at discov-
ery of novel protein kinase Cb (PKCb)-selective inhibitors possessing oral bioavailability. Among them, compounds having a fused
five-membered ring at the indole 1,2-position inhibited PKCb2 with IC50 of nM-order and showed good oral bioavailability. One of
the most potent compounds was found to be PKCb-selective over other 6 isozymes and exhibited ameliorative effects in a rat dia-
betic retinopathy model via oral route.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
complications.1 A predominant factor of PKCb activa-
tion is enhanced de novo synthesis of diacylglycerol
(DAG) in hyperglycemia. Other factors associated with
the activation of PKCb include increased advanced gly-
cated end products (AGE) and elevated oxidative stress
in diabetic conditions.2 Thus, activated PKCb damages
microvascular systems and causes a variety of symptoms
of diabetic microvascular complications; hence PKCb-
selective inhibitors are expected to become therapeutic
agents for diabetic microvascular complications.3
Diabetes mellitus is one of the representative lifestyle-re-
lated diseases. The number of diabetic patients is over
150 million worldwide and continues to mount, especial-
ly in developed countries. Thanks to a variety of
medicines including sulfonylureas, biguanide, thiazolid-
inediones, and a-glycosidase inhibitors, it has become
possible to control patient’s blood glucose level. Even
with these medications, however, many patients are at
risk for developing diabetic microvascular complications
such as diabetic neuropathy, nephropathy, and retinop-
athy after suffering from long-term diabetes. Therefore,
there is a need for agents which directly block signal
transduction pathways leading to the onset of diabetic
microvascular complications independent of blood glu-
cose level control, thus improving the quality of life
for the patient with late-stage diabetes mellitus. Recent-
ly, the signal transduction pathway has become clearer
and protein kinase Cb (PKCb) activation was found
to be implicated in the onset of diabetic microvascular
The discovery of staurosporine, a non-selective PKC
inhibitor produced by Streptomyces sp., facilitated syn-
thesis of numbers of its structural analogues possessing
various inhibitory activities and selectivities.4 Among
them, ruboxistaurin3a,5 exhibited selective PKCb inhibi-
tory activity and ameliorative effects in diabetic compli-
cations in clinical studies, demonstrating significant
contribution of PKCb to the onset of these diseases.6
Ruboxistaurin selectively inhibits PKCb1 and b2 with
IC50 values of 4.7 and 5.9 nM, respectively, and IC50
for other PKC isoforms of 250 nM or greater.3a From
the point of view of structural features, the hitherto
synthesized staurosporine derivatives including ruboxis-
taurin were within a series having a pharmacophore
Keywords: PKCb selective inhibitor; 3-Anilino-4-(3-indolyl)maleimide;
Orally active compounds; Diabetic complications.
of bisindolylmaleimide (Ro 31-6233 (1))7 or
staurosporine-like carbazole substructure (Fig. 1). We
a
*
Corresponding author. Tel.: +81 72 681 9700; fax: +81 72 681
9725; e-mail: takashi.inaba@ims.jti.co.jp
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.05.033

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M. Tanaka et al. / Bioorg. Med. Chem. 14 (2006) 5781–5794
synthesized derivatives of 3 inhibited both isozymes to
H
N
H
N
approximately the same degree. The isozyme selectivity
was monitored using selectivity over PKCa, which
among the PKC family shares highest homology with
PKCb.10 Based on these in vitro data we selected com-
pound (R)-23g, and investigated a more detailed isozy-
me inhibitory profile as well as in vivo efficacy for this
compound.
O
O
O
N
N
N
N
O
H
Me
MeO
O
NMe
2
NHMe
Staurosporine
Ruboxistaurin
2. Design and synthesis
H
N
H
N
O
O
O
O
With the goal of obtaining more potent and orally avail-
able PKCb-selective inhibitors starting from 3, we de-
signed cyclic derivatives including 8-substituted-6,7,8,9-
tetrahydropyrido[1,2-a]indoles (series A), 7-substituted-
6,7,8,9-tetrahydropyrido[1,2-a]indoles (series B), and
2,3-dihydro-1H-pyrrolo[1,2-a]indoles (series C). In the
previous SAR study of acyclic derivatives of 3, we found
a dramatic rise in potency when hydrophilic groups (R)
were placed at the indole nitrogen of compound 2 via a
three- to four-carbon linker so that the hydrophilic
group could associate with the carboxylic acid residue
of Asp427 and/or the main chain amide of Asp470 of
PKCb.11 For instance, 3a and 3b were approximately
20 times and 10 times more potent than 2, respectively.
It was expected that, in these conformationally restricted
cyclic systems, substituting a hydrophilic group would
bring about elevated activity compared to the previous
acyclic series due to a smaller change in entropy upon
association with these amino acids in PKCb. We also
anticipated improvement in oral bioavailability in these
cyclic derivatives based on a report that demonstrated
that fewer rotatable bonds tended to have a positive ef-
fect on bioavailability in general.12 These previous stud-
ies led us to develop a rational synthesis for these
conformationally restricted cyclic compounds, with the
intention of improving potency as well as oral
bioavailability.
HN
N
H
N
H
N
H
Ro31-6233 (1)
2
H
N
H
N
O
O
O
O
HN
HN
N
N
R
R
3a: R=NMe
3b: R=OH
A
2
H
N
H
N
O
O
O
O
HN
HN
N
N
R
R
B
C
Figure 1. Structures of staurosporine and its derivatives.
reported a new structural class of PKCb-selective inhib-
itors, which possess anilino-monoindolylmaleimides (2)
as the pharmacophore.8 The representative compound
3a selectively inhibited PKCb1 and b2 with IC50 values
of 21 and 12 nM, respectively. Most recently, Zhang
et al. have also reported a series of indolylindazolylma-
leimides as an additional new structure class of PKCb-
selective inhibitors with inhibitory activity in the nM
range.9 We expected high in vivo efficacy for the previ-
ously reported derivatives of 3, however they were inef-
fective in all in vivo models because of a lack of oral
bioavailability. In this paper, we report the synthesis
of further derivatives of compound 3, which showed
good oral bioavailability as well as curative effects in
an animal model for diabetic complications. The most
potent compound (R)-23g not only exhibited strong
PKCb inhibition in vitro (IC50 of 1.4 nM at PKCb2),
but also improved retinal vascular blood flow rate (an
index of diabetic retinopathy) in the streptozotocin
(STZ) induced diabetic rat model via oral
administration.3a
8-Substituted-6,7,8,9-tetrahydropyrido[1,2-a]indoles
(series A, (S)-8b and (S)-9) were synthesized as shown in
Scheme 1. Both enantiomers, (S)- and (R)-4a, were
available in optically active forms using the method de-
scribed in the literature.13 After silyl protection of the
hydroxyl group of (S)-4a, coupling reaction with oxalyl
chloride followed by quenching with aqueous ammonia
gave (S)-5,14 which was then sequentially reduced with
NaBH4 and hydrosilane to provide (S)-6. Condensation
of (S)-6 with dimethyl oxalate in the presence of t-BuOK
gave (S)-7.15 The hydroxyl group of the maleimide of
(S)-7 was successfully replaced with aniline by heating
with excess aniline in acetic acid to afford (S)-8a. Then
the silyl protection was removed by TBAF to afford
(S)-8b. The regenerated hydroxyl group was replaced
with dimethylamine via the corresponding mesylate
(S)-8c to give (S)-9. The antipode was similarly prepared
from (R)-4a.
As shown in Scheme 2, the S and R enantiomers of
7-substituted-6,7,8,9-tetrahydropyrido[1,2-a]indoles
(series B, 14) were both synthesized from (R)-2-(2,2-
diethoxyethyl)-1,3-propanediol monoacetate (10)16
The SAR study was initially conducted based on
PKCb2 isozyme inhibitory activity since PKCb1 and
PKCb2 had high degrees of homology, and previously

M. Tanaka et al. / Bioorg. Med. Chem. 14 (2006) 5781–5794
5783
through 11c and 15c, respectively. 11c was prepared by
mesylation of the hydroxyl group of 10 followed by
replacement of the acetyl group with the silyl-protective
group, whereas 15c was obtained by silyl-protection of
the hydroxyl group of 10 prior to removal of the acetyl
group, and subsequent mesylation. Alkylation of indole-
3-acetamide with 11c yielded 12 which was then convert-
ed into (R)-13 via intramolecular Pictet-Spengler cycli-
zation17 and subsequent catalytic hydrogenation. The
target compound (S)-14 was achieved from (R)-13 as
in the preparation of (S)-9 from (S)-6 described in
Scheme 1. The antipode ((R)-14) was similarly obtained
from 15c.
O
CONH2
c,d
b
N
N
OR
OTBDPS
(S)-5
(S)-4a (R=H)
(S)-4b (R=TBDPS)
a
H
N
O
O
CONH2
e
f
OH
N
N
OTBDPS
As shown in Scheme 3, R and S enantiomers of 2,3-dihy-
dro-1H-pyrrolo[1,2-a]indoles (series C, 23) were both
prepared from optically active monoacetate (19a)
through (S)-21 and (R)-21, respectively. Commercially
available indole-2-carboxaldehyde (16) was transformed
into diester (17) by Knoevenagel condensation reaction
with diethylmalonate. Diol (18b) was obtained from 17
by 1,4-reduction with NaBH4, followed by reduction of
diethylesters with LiAlH4. Lipase-catalyzed asymmetric
acetylation16,18 of 18b was effective to obtain optically
active 19a, which was then cyclized into (S)-2119 through
conversion of the acetoxy group to a mesyl leaving group
by sequential procedures containing silyl-protection of
the hydroxyl group of 19a, saponification of the acetoxy
group, mesylation of the newly generated hydroxyl
group and cyclization with sodium hydride. Meanwhile,
(R)-21 was prepared through (R)-24, which was obtained
by cyclization of 19a after conversion of the hydroxyl
group into a mesyl leaving group. (S)-21 and (R)-21
were, respectively, transformed into (R)-23 and (S)-23
via (S)-22 and (R)-22 with the similar methods used in
Scheme 1. The enantioselectivity of this enzymatic ester-
ification was confirmed to be 90–95% by conversion of
(S)-21 into the corresponding MTPA ester.
OTBDPS
(S)-7
(S)-6
H
N
H
N
O
O
O
O
HN
HN
i
N
N
NMe2
OR
(S)-8a (R=TBDPS)
(S)-8b (R=H)
(S)-8c (R=Ms)
(S)-9
g
h
Scheme 1. Reagents: (a) TBDPSCl, imidazole; (b) i. (COCl)2, Et3N,
ii. aq NH3; (c) NaBH4; (d) TESH, TFA; (e) (CO2Me)2, t-BuOK; (f)
PhNH2, AcOH; (g) TBAF; (h) Ms2O, pyridine; (i) Me2NH.
HO
AcO
MsO
RO
OEt
OEt
OEt
OEt
a
d
10
11a (R=Ac)
11b (R=H)
b
c
11c (R=TBDPS)
CONH
CONH
2
2
The absolute chemistry of 23 resulted from the enzymat-
ic differentiation of the two symmetric primary hydroxyl
groups of 18b. To investigate the enzymatic preference
and determine the absolute configuration of 23, (S)-24
was synthesized using an alternative route as shown in
Scheme 4. The racemic mixture of cis-hydroxy ester 26
obtained by Pd-catalyzed reduction of 2520 was chro-
matographically separated into two diastereomers of
the Boc-L-alanine esters 27 and 28. The absolute struc-
ture of the crystalline isomer 28 was successfully deter-
OEt
OEt
N
e, f
N
OTBDPS
(R)-13
OTBDPS
12
H
N
O
O
HN
g - k
N
mined as depicted in Scheme
4
by X-ray
crystallographic analysis.21 Since (S)-24 obtained from
28 showed [a]D of +23.8°, the absolute stereochemistry
of (S)-23 derived from (R)-24 having [a]D of ꢀ20.4°
was determined to be S and that of antipode (R)-23
was assigned to R.
NMe
(S)-14
2
TBDPSO
RO
OEt
d - k
c
10
(R)-14
OEt
15a (R=Ac)
15b (R=H)
15c (R=Ms)
l
a
3. Results and discussion
3.1. In vitro SAR studies
Scheme 2. Reagents: (a) MsCl, Et3N; (b) aq NaOH; (c) TBDPSCl,
imidazole; (d) indole-3-acetamide, NaH; (e) TFA, H2O; (f) H2, 5% Pd–
C; (g) (CO2Me)2, t-BuOK; (h) PhNH2, AcOH; (i) TBAF; (j) Ms2O,
pyridine; (k) Me2NH; (l) K2CO3, EtOH.
It was expected that conformational restriction with cyc-
lic structures might provide more potent inhibitors than

5784
M. Tanaka et al. / Bioorg. Med. Chem. 14 (2006) 5781–5794
a
e
b
d
N
H
N
H
CHO
N
H
N
H
CO Et
R
2
R
EtO C
OAc
O
2
OR
16
17
18a (R=CO Et)
18b (R=CH OH)
19a (R=H)
2
c
2
H
N
H
N
O
O
O
o, p
HN
N
N
i - n
h
N
H
HN
1
N
OR
R
TBDPSO
TBDPSO
N
OH
(S)-22
2
R
20a (R=Ac)
20b (R=H)
20c (R=Ms)
(S)-21
(R)-23
f
g
1
2
(R)-23g (R =R =H)
q
1
2
(R)-23h (R =Ac, R =H)
g
o, p
f, e
h
i - n
N
19a (R=H)
19b (R=Ms)
(R)-22
(S)-23c
(R)-21
AcO
(R)-24
Scheme 3. Reagents: (a) CH2(CO2Et)2, AcOH, piperidine; (b) NaBH4; (c) LiAlH4; (d) Lipase PS, vinyl acetate; (e) TBDPSCl, imidazole; (f) K2CO3,
MeOH; (g) Ms2O, pyridine; (h) NaH, NaI; (i) i. (COCl)2, Et3N, ii. aq NH3; (j) NaBH4; (k) TESH, TFA; (l) (CO2Me)2, t-BuOK; (m) PhNH2, AcOH;
(n) TBAF; (o) Ms2O, pyridine or Tf2O, 2,4,6-collidine; (p) R1R2NH; (q) Ac2O.
ed PKCb2 five times more strongly than the
corresponding acyclic analogue (3b and 3a) showing
O
a
OH
b
N
N
IC50 values of 6 and 2 nM, respectively (Table 1). More-
over, these compounds were significantly more potent
than their enantiomers ((R)-8b and (R)-9). We initially
thought these facts implied that strict spatial arrange-
ments were required in the recognition of the hydrophil-
ic groups by Asp427 and/or Asp470 of the enzyme.11
This enantiomeric preference, however, was not extend-
ed to other series including 14 (series B), or 22 and 23
(series C). In series B, (S)-14 and its enantiomer (R)-14
equally inhibited the enzyme showing IC50 values of 4
and 6 nM, respectively. In the case of series C, (S)-22
and (R)-23c (enantiomers possessing hydrogen atom
for X) exhibited IC50 values of 4 and 5 nM, whereas
the IC50 of their enantiomers (R)-22 and (S)-23c (enan-
tiomers possessing hydrogen atom for Y) were 14 and
8 nM, respectively. The former set was scarcely more
potent than the latter one showing opposite enantiomer-
ic preference to that observed in series A. Although the
cyclic derivatives showed significantly higher potency
than their acyclic counterparts as expected (compare
dimethylamino derivatives (S)-9, (S)-14, and (R)-23a
with 3a, and hydroxyl derivatives (S)-8b and (S)-22 with
3b), the lack of obvious enantiomeric preference
throughout these three series complicated attempts to
gain insight into the Asp427 and Asp470 associable po-
sition. The increase in the inhibitory activity observed in
the cyclic inhibitors may be attributed to their smaller
number of rotatable bonds and the smaller change in
entropy factors in associating with the enzyme com-
pared to the acyclic inhibitors 3. The existing rotatable
bonds, including a C–C bond off the 5- and 6-membered
CO2Et
CO2Et
25
26
NHBoc
NHBoc
O
O
O
O
+
N
N
CO2Et
CO2Et
28
27
c
e
N
N
28
R
OAc
(S)-24
[α]20D +23.8 o
29a (R=CO2Et)
29b (R=CH2OH)
d
Scheme 4. Reagents: (a) H2, 5% Pd–C; (b) Boc-L-Ala, DCC, DMAP;
(c) H2 (2.7 atm), 10% Pd–C; (d) LiAlH4; (e) Ac2O, pyridine, DMAP.
acyclic inhibitor 3 by placing a hydrophilic group at a
more favorable position to associate with Asp427 and/
or Asp470 of PKCb.11 This idea was based upon our
previous report, in which we advocated the significant
effect of the linker length between the indole nitrogen
and a hydrophilic group upon the inhibitory activity
(in the case of 3, the best linkers are (CH2)3 and
(CH2)4).8 Supporting this idea, in 8-substituted-6,7,8,9-
tetrahydropyrido[1,2-a]indoles (series A), alcohol deriv-
ative (S)-8b and dimethylamino derivative (S)-9 inhibit-

M. Tanaka et al. / Bioorg. Med. Chem. 14 (2006) 5781–5794
5785
Table 1. PKCb2 and PKCa inhibitory activity of series A, B, C and the acyclic compounds
Series
Compound
X
Y
IC50 (nM)
b2
a
H
N
O
O
O
(S)-8b
(S)-9
CH2OH
CH2NMe2
H
6
2
394
96
HN
H
(R)-8b
(R)-9
H
H
CH2OH
CH2NMe2
17
25
2131
3057
N
X
Y
A
H
N
O
HN
(S)-14
(R)-14
H
CH2NMe2
CH2NMe2
H
4
6
252
445
N
X
B
Y
(S)-22
H
CH2OH
4
4
458
148
1955
481
542
152
212
95
(R)-23a
(R)-23b
(R)-23c
(R)-23d
(R)-23e
(R)-23f
(R)-23g
(R)-23h
(R)-22
H
CH2NMe2
CH2NEt2
CH2NMeEt
CH2-1-pyrrolidinyl
CH2NHMe
CH2NHEt
CH2NH2
CH2NHAc
H
H
N
H
32
5
O
O
H
H
8
2.2
HN
H
H
2.4
1.4
7.9
N
H
X
H
903
1021
613
Y
C
CH2OH
CH2NMeEt
14
8
(S)-23c
H
Acyclic
3a
3b
12
30
460
1620
rings, could account for the relatively large tolerance for
stereochemistry, ring size and substituent position
throughout these ring systems. Molecular modeling
studies implied that this flexibility allows the terminal
hydrophilic groups to associate with the Asp427 and/
or Asp470 in energetically acceptable conformations as
seen in a staurosporine-PKA co-crystal, in which the
lower NHMe group associates with Glu127 and
Glu170 of PKA22 and as also seen in the stauro-
sporine-PKCh co-crystal, in which the NHMe group
binds to the main chain carbonyl of Asp508 of PKCh.23
The PKCb2 selectivity of these cyclic compounds over
PKCa was around 50- to 100-fold, and this value did
not vary with the change in ring size, substituent posi-
tion or R/S stereochemistry inversion.
N(Me)Et ((R)-23c), and pyrrolidinyl ((R)-23d) resulted
in reduced inhibitory activity, and the amide derivatives
(R)-23h also showed lower activity than (R)-23a. In con-
trast, compounds having the smaller substituents
showed the stronger inhibitory activity (see (R)-23e,
(R)-23f, and (R)-23g). The size of the amino group did
not influence the PKCb2 selectivity over PKCa. The
most potent compound was (R)-23g, which exhibited
strong PKCb2 inhibition (IC50 of 1.4 nM) and good
selectivity over PKCa (67-fold). We selected this com-
pound, in its developmental salt form as the mesylate
(JTT-010), for more detailed investigation of the isozy-
me selectivity over other PKC isozymes (b1, c, d, e, f,
and l). As shown in Table 2, JTT-010 selectively inhib-
ited PKCb1 and PKCb2 with a selectivity profile similar
to that of ruboxistaurin.3a
To further increase the potency, derivatives having a
variety of hydrophilic groups (substituents X or Y in Ta-
ble 1) were investigated. We first synthesized and as-
sessed several derivatives of series A and B. However,
significant increase in activity was not observed in these
series, and more importantly, poor oral bioavailability
and unacceptable toxicity were found in series A and
B, respectively (data not shown). Therefore, we shifted
our focus to series C, and tested the effects of the hydro-
philic group on potency and selectivity using series C as
a platform. Replacement of the NMe2 group of (R)-23a
with larger substituents such as NEt2 ((R)-23b),
3.2. Ex vivo studies
Several potent compounds were selected for the rat ex vi-
vo studies to observe oral absorption as well as duration
of drug effect. After oral administration of the com-
pounds to rats at a dose of 2 mg/kg (1 mg/kg for (S)-
9), PKCb2 inhibitory activity in plasma was measured
at 2 and 6 h after dosing (Table 3). The inhibitory activ-
ity in animals receiving (S)-9 was marginal (30%) at 2 h
after administration and disappeared completely after
6 h, indicating poor oral bioavailability and short dura-

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M. Tanaka et al. / Bioorg. Med. Chem. 14 (2006) 5781–5794
Table 3. PKCb2 inhibitory activity of rat plasma after oral dosing
tion. In contrast, strong inhibition was detected at 2 and
6 h after administration of compounds in series B and C.
Plasma from rats receiving (S)-14, (R)-14, and (S)-22 all
similarly inhibited the enzyme (64–79% inhibition at 2 h
and 45–70% inhibition at 6 h) suggesting that oral bio-
availability and duration of these three compounds were
similar given their nearly equal in vitro IC50 values
(ꢁ5 nM). Although (R)-23d, possessing a pyrrolidinyl
group at the terminal of the molecule, was also efficient-
ly absorbed, it appeared to be more rapidly cleared from
plasma than (S)-14, (R)-14, and (S)-22, considering the
lower inhibition (29%) after 6 h. Plasma from rats
receiving (R)-23a, which has a dimethylamino group,
strongly inhibited the enzyme and the effect lasted for
more than 6 h. This strong and long-lasting inhibitory
activity of (R)-23a in vivo was proven to be partially
attributable to its more potent des-methyl metabolites
(R)-23e and (R)-23g found in plasma of (R)-23a receiv-
ing animals. In response to this result, we conducted
the ex vivo test using the most potent (R)-23g, and
found that this compound almost completely inhibited
the enzyme even after 6 h. Subsequent pharmacokinetic
studies revealed that oral bioavailability and T1/2 of
(R)-23g in the rat were 57% and 5.9 h, respectively
(30 mg/kg, p.o. dosing). It could be speculated that
decreasing the number of rotatable bonds was effective
in increasing oral bioavailability in this series of PKCb
inhibitors.12
Series
Compound
Inhibitory activity
(%) of rat plasma
time after oral
dosinga
2 h
6 h
A
B
(S)-9
30b
ꢀ9b
(S)-14
(R)-14
65
79
58
70
C
(S)-22
64
84
82
92
97
45
82
29
91
93
(R)-23a
(R)-23d
(R)-23e
(R)-23g
a Dosing amount was 2 mg/kg otherwise noted.
b 1 mg/kg administration.
2.0
*
1.5
1.0
0.5
0.0
#
3.3. Ameliorative effect in STZ rat retinopathy model
PKCb activation is known to mediate retinal vascular
abnormalities including excessive vascular permeability
and neovascularization, which can cause macular ede-
mas and vascular occlusions, hallmarks of diabetic reti-
nopathy. Ruboxistaurin, a selective PKCb inhibitor,
was reported to suppress these retinal symptoms in dia-
betic animal models,24 demonstrating the involvement
of PKCb activity in diabetic retinopathy. Using the
streptozotocin (STZ) induced diabetic rat retinopathy
model, we investigated in vivo potency of our PKCb-se-
lective inhibitor (R)-23g, which was selected based on its
in vitro and ex vivo potency (vide ante). In this animal
model, mean circulation time (MCT) of fluorescein in
retinal vascular segments was measured as an index of
blood flow in retina.25 From the day subsequent to
STZ treatment, (R)-23g was orally given to the animals
twice a day for two weeks and MCT was measured. As
shown in Figure 2, STZ significantly prolonged MCT as
seen in the vehicle group. This blood flow deceleration
was, however, reversed in a dose-dependent manner by
oral administration of (R)-23g. A 1 mg/kg dose given
Non-treat
Vehicle
0.01 mg/kg
0.1 mg/kg
1mg/kg
STZ (65mg/kg i.p.) +(R)-23g
Figure 2. Effect of (R)-23g on retinal mean circulation time (MCT) in
STZ diabetic rats. MCT was measured after 2 weeks treatment of (R)-
23g. Values are expressed as means SE (N = 9–12). *significant
difference from non-treated group by LSD test (P < 0.05). #Significant
difference from STZ vehicle group by LSD test (P < 0.05).
to the STZ rats significantly shortened the MCT to the
levels comparable to the non-treated control group,
demonstrating the in vivo potency of (R)-23g.
4. Conclusion
Three new series of cyclic 3-anilino-4-(3-indolyl)malei-
mides, that is, series A, B, and C, were synthesized in
optically active form in order to find PKCb-selective
inhibitors with good oral bioavailability starting from
Table 2. Comparison of PKCb selectivity of (R)-23g mesylate, staurosporine, and ruboxistaurin
Compound
PKC isozyme IC50 (nM)
b1
4.0
11
4.7
b2
a
c
d
e
f
l
(R)-23g mesylate (JTT-010)
Staurosporine
Ruboxistaurina
2.3
4.0
5.9
86
8.7
360
110
11
54
4.3
250
490
7.4
600
1700
1700
>10000
24
NT
300
>100,000
a These data were cited from reference 3a.

M. Tanaka et al. / Bioorg. Med. Chem. 14 (2006) 5781–5794
5787
previously reported acyclic analogues 3 having no oral
bioavailability. Among these series, compounds in series
B and C exhibited good inhibitory activity as well as oral
bioavailability in ex vivo studies. In addition to this,
JTT-010 (mesylate of (R)-23g) in series C, which selec-
tively inhibited PKCb1 and b2 with IC50 values of 4.0
and 2.3 nM, respectively, remedied retinopathy in the
STZ induced diabetic rat model at a dose of 1 mg/kg,
oral administration. We have recently reported the ame-
liorative effects of JTT-010 also in diabetic neuropathy
and nephropathy animal models.26 It could be conclud-
ed that JTT-010 is a new structural class of orally bio-
available PKCb-selective inhibitors showing in vivo
curative effects in a variety of diabetic microvascular
complication models.
ethylamine (22.1 mL, 159 mmol) in CH2Cl2 (350 mL)
was added oxalyl chloride (12.8 mL, 147 mmol) drop-
wise over 15 min at 0 °C. After stirring for 30 min at
0 °C, the reaction mixture was poured into ice-cooled
28% aqueous NH3 (500 mL). The reaction mixture was
allowed to warm to room temperature over 1 h and
was partitioned between AcOEt and water. The organic
layer was separated, successively washed with water and
brine, dried over anhydrous Na2SO4, and concentrated
in vacuo to give crude ketoamide (S)-5 as a pale brown
amorphous solid, which was used directly in the next
step without any purification: 1H NMR (400 MHz,
CDCl3) d 1.08 (9H, s), 1.81–1.92 (1H, m), 2.08–2.19
(1H, m), 2.28–2.37 (1H, m), 2.94 (1H, dd, J = 10.5,
18.9 Hz), 3.53 (1H, ddd, J = 1.2, 4.9, 18.7 Hz), 3.66
(1H, dd, J = 7.2, 10.4 Hz), 3.77 (1H, dd, J = 5.6,
10.4 Hz), 3.95 (1H, dt, J = 4.8, 11.2 Hz), 4.29 (1H,
ddd, J = 2.8, 5.6, 12.4 Hz), 5.50 (1H, s), 6.68 (1H, s),
7.24–7.31 (3H, m), 7.35–7.46 (6H, m), 7.65–7.69 (4H,
m), 8.18 (1H, dd, J = 5.1, 8.5 Hz); MS (FAB) m/z 511
(M+H)+. To a stirring solution of all of resulting crude
(S)-5 in EtOH (1000 mL)–THF (250 mL) was added
NaBH4 (13.9 g, 367 mmol) portionwise at room temper-
ature. After stirring for 2 h at room temperature, the
reaction mixture was partitioned between AcOEt and
a 2 M aqueous KHSO4 solution. The organic layer
was separated, washed with brine, dried over anhydrous
Na2SO4, and concentrated in vacuo to give crude a-
hydroxyacetoamide as a pale brown oil. To a solution
of this compound in CH2Cl2 (800 mL) were successively
added triethylsilane (39 mL, 244 mmol) and trifluoro-
acetic acid (68 mL, 883 mmol) at room temperature.
After stirring for 2 h at room temperature, the reaction
mixture was quenched by addition of a NaHCO3 satu-
rated aqueous solution at 0 °C. The layers were separat-
ed and the aqueous layer was extracted with CHCl3. The
combined organic layers were washed with brine, dried
over anhydrous Na2SO4, and concentrated in vacuo.
The residue was chromatographed on silica gel eluting
with hexane–AcOEt (4:1–2:1) to give (S)-6 (28.5 g,
47% from (S)-4b) as a pale brown solid: 1H NMR
(300 MHz, CDCl3): d 1.08 (9H, s), 1.85 (1H, ddd,
J = 5.6, 11.3, 24.5 Hz), 2.01-2.19 (1H, m), 2.20-2.32
(1H, m), 2.53 (1H, dd, J = 10.8, 16.2 Hz), 3.00 (1H,
dd, J = 3.6, 16.2 Hz), 3.59 (1H, d, J = 23.1 Hz), 3.65
(1H, d, J = 23.1 Hz), 3.66-3.79 (2H, m), 3.88 (1H, dt,
J = 4.9, 11.7 Hz), 4.29 (1H, ddd, J = 3.0, 5.9, 11.7 Hz),
5.31 (1H, brs), 5.58 (1H, brs), 7.09-7.21 (2H, m), 7.28
(1H, d, J = 7.5 Hz), 7.35-7.47 (6H, m), 7.50 (1H, d,
J = 7.2 Hz), 7.66 (2H, d, J = 7.8 Hz), 7.67 (2H, d,
J = 7.2 Hz); MS (FAB) m/z 497 (M+H)+.
5. Experimental
5.1. Chemistry
Melting points were determined using a Yanagimoto mi-
¨
cro melting point apparatus or BUCHI B-545 melting
point instrument and were uncorrected. Proton nuclear
magnetic resonance spectra (1H NMR) were recorded
on a JEOL JNM-A300W, Bruker AMX-300 or JEOL
JNM-AL400 spectrometer in a solvent indicated. Chem-
ical shifts (d) are reported in parts per million relative to
internal standard tetramethylsilane. Elemental analysis
was performed with a Perkin-Elmer 2400 Series II
CHNS/O analyzer. Mass spectra (FAB+) were recorded
with a Finnigan TSQ 700 instrument and mass spectra
(ESI+) were recorded with a ThemoQuest LCQ mass
spectrometer. High-resolution mass spectra were ob-
tained with a JEOL SX 102A spectrometer. [a]D values
were obtained at 20 or 25 °C with a Perkin-Elmer 241
polarimeter or Rudolph Research Analytical AUTO-
POL V polarimeter.
5.1.1. (S)-8-(tert-Butyldiphenylsilyloxymethyl)-6,7,8,9-
tetrahydropyrido[1,2-a]indole ((S)-4b). To a solution of
(S)-4a13 (24.6 g, 122 mmol) and imidazole (20.0 g,
293 mmol) in DMF (300 mL), TBDPSCl (40.3 g,
146.6 mmol) was added at room temperature. The reac-
tion mixture was stirred overnight and was partitioned
between AcOEt and water. The organic layer was suc-
cessively washed with water and brine, dried over anhy-
drous Na2SO4, and concentrated in vacuo. The residue
was chromatographed on silica gel eluting with hex-
ane–AcOEt (100:1–50:1) to give (S)-4b (53.7 g, 100%)
1
as a pale yellow oil: H NMR (300 MHz, CDCl3): d
1.08 (9H, s), 1.84 (1H, ddd, J = 5.7, 13.0, 24.0 Hz),
2.03–2.18 (1H, m), 2.19–2.29 (1H, m), 2.68 (1H, dd,
J = 11.0, 16.0 Hz), 3.10 (1H, dd, J = 3.7, 16.0 Hz),
3.62–3.76 (2H, m), 3.85 (1H, dt, J = 4.9, 11.7 Hz), 4.27
(1H, ddd, J = 2.9, 5.7, 11.7 Hz), 6.19 (1H, s), 7.02–7.18
(2H, m), 7.26 (1H, d, J = 6.7 Hz), 7.32–7.47 (6H, m),
7.52 (1H, d, J = 7.2 Hz), 7.61–7.71 (4H, m); MS (FAB)
m/z 440 (M+H)+.
5.1.3. 3-[(S)-8-(tert-Butyldiphenylsilyloxymethyl)-6,7,8,9-
tetrahydropyrido[1,2-a]indol-10-yl]-4-hydroxy-1H-pyrrole-
2,5-dione ((S)-7). To a stirring solution of (S)-6 (24.5 g,
49.3 mmol) and dimethyl oxalate (6.40 g, 54.2 mmol)
in THF (250 mL) was added t-BuOK (12.18 g,
108.4 mmol) in two equal portions, 15 min part at
0 °C. The reaction mixture was allowed to warm to
room temperature over 1 h. To the resulting mixture
were successively added a 0.5 M aqueous KHSO4 solu-
tion and AcOEt, and the organic layer was separated.
The organic layer was successively washed with water
5.1.2. 2-[(S)-8-(tert-Butyldiphenylsilyloxymethyl)-6,7,8,9-
tetrahydropyrido[1,2-a]indol-10-yl]acetamide ((S)-6). To
a stirring solution of (S)-4b (53.7 g, 122 mmol) and tri-

5788
M. Tanaka et al. / Bioorg. Med. Chem. 14 (2006) 5781–5794
and brine, dried over anhydrous Na2SO4 and concen-
trated in vacuo. Thus obtained residue was chromato-
graphed on silica gel eluting with hexane–AcOEt (4:1–
2:1) to give (S)-7 (24.4 g, 90%) as a brown amorphous
dd, J = 11.2, 16.1 Hz), 2.33–2.43 (0.5H, m), 2.60–2.70
(0.5H, m), 3.19 (3H, s), 3.31–3.54 (1H, m), 3.69–3.80
(0.5H, m), 3.94–4.27 (3H, m), 6.57–6.64 (2H, m), 6.72
(3H, s), 6.98 (1H, t, J = 7.5 Hz), 7.06 (1H, t,
J = 7.5 Hz), 7.30 (2H, t, J = 8.3 Hz), 9.28 (0.5H, s),
9.33 (0.5H, s), 10.64 (1H, s).
1
solid: H NMR (400 MHz, DMSO-d6): d 1.02 (9H, s),
1.75–1.88 (1H, m), 2.03–2.15 (1H, m), 2.17–2.26 (1H,
m), 2.63 (1H, dd, J = 11.1, 17.2 Hz), 3.00 (1H, dd,
J = 3.3, 16.9 Hz), 3.71 (2H, d, J = 6.0 Hz), 3.88 (1H,
dt, J = 4.9, 12.0 Hz), 4.29–4.38 (1H, m), 7.02 (1H, dt,
J = 1.2, 8.4 Hz), 7.10 (1H, dt, J = 1.2, 7.2 Hz), 7.34–
7.39 (2H, m), 7.40–7.49 (6H, m), 7.61–7.67 (4H, m),
10.49 (1H, s), 11.70 (1H, br s); MS (ESI) m/z 551
(M+H)+.
5.1.6. 3-Anilino-4-[(S)-8-[(dimethylamino)methyl]-6,7,8,9-
tetrahydropyrido[1,2-a]indol-10-yl]-1H-pyrrole-2,5-dione
((S)-9). To a solution of (S)-8c (80 mg, 0.172 mmol) in
THF (1.5 mL) was added 50% aqueous dimethylamine
(0.774 mL, 8.60 mmol) at room temperature. The mix-
ture was sealed in a stainless steel pressure tube and
heated at 65 °C for 16 h. The reaction mixture was then
allowed to cool to room temperature and was parti-
tioned between AcOEt and water. The organic layer
was separated, washed with brine, dried over anhydrous
Na2SO4, and concentrated in vacuo. Thus obtained res-
idue was purified by preparative thin-layer chromatog-
raphy on silica gel (hexane–AcOEt (1:4) and then
CHCl3–MeOH (6:1)) to give (S)-9 (51 mg, 72%) as an
orange solid: mp 240–242 °C; 1H NMR (400 MHz,
DMSO-d6): d 0.88–1.01 (0.5H, m), 1.02–1.14 (0.5H,
m), 1.36–1.54 (1H, m), 1.82–2.02 (4H, m), 2.07 (3H, s),
2.09 (3H, s), 2.21–2.31 (0.5H, m), 2.57–2.68 (0.5H, m),
3.41–3.52 (0.5H, m), 3.69–3.80 (0.5H, m), 4.02–4.11
(0.5H, m), 4.12–4.21 (0.5H, m), 6.54–6.63 (2H, m),
6.72 (3H, br s), 6.92–7.10 (2H, m), 7.25–7.36 (2H, m),
9.23 (0.5H, s), 9.29 (0.5H, s), 10.63 (1H, s); MS (FAB)
m/z 415 (M+H)+; Anal. Calcd for C25H26N4O2 Æ0.25-
H2O: C, 71.66; H, 6.37; N, 13.37. Found: C, 71.69; H,
5.1.4. 3-Anilino-4-[(S)-8-(hydroxymethyl)-6,7,8,9-tetra-
hydropyrido[1,2-a]indol-10-yl]-1H-pyrrole-2,5-dione ((S)-
8b). To a solution of (S)-7 (24.4 g, 44.3 mmol) in AcOH
(100 mL), aniline (20.2 mL) was added. The reaction
mixture was stirred at 100 °C for 1 h, and concentrated
in vacuo. The residue was chromatographed on silica gel
eluting with hexane–AcOEt (20:1–4:1) to give (S)-8a
(23.0 g, 83%) as a red-orange amorphous solid. To a
solution of all of resulting (S)-8a in THF (250 mL)
was added a 1 M solution of tetrabutylammonium fluo-
ride in THF (128.8 mL, 128.8 mmol) at room tempera-
ture. After stirring at room temperature for 2 h, a
0.5 M aqueous KHSO4 solution was added to the result-
ing mixture, which was then extracted with AcOEt. The
separated organic layer was washed with brine, dried
over anhydrous Na2SO4, and then concentrated in vac-
uo. Thus obtained residue was chromatographed on sil-
ica gel eluting with hexane–AcOEt (1:1–1:3) to give (S)-
8b (13.8 g, 97%) as a red-orange solid: mp 243–246 °C;
1H NMR (400 MHz, DMSO-d6): d 0.92–1.12 (1H, m),
1.42–1.56 (1H, m), 1.67–1.80 (0.5H, m), 1.86–2.04 (1H,
m), 2.06–2.19 (0.5H, m), 2.26–2.36 (0.5H, m), 2.53–
2.64 (0.5H, m), 3.09–3.31 (2H, m), 3.43–3.54 (0.5H,
m), 3.63–3.75 (0.5H, m), 4.07–4.23 (1H, m), 4.59 (1H,
d, J = 6.0 Hz), 6.60 (2H, d, J = 8.6 Hz), 6.71 (3H, br
s), 6.96 (1H, t, J = 7.2 Hz), 7.03 (1H, t, J = 7.5 Hz),
7.28 (2H, t, J = 6.8 Hz), 9.23 (0.5H, s), 9.26 (0.5H, s),
10.58 (1H, br s); MS (FAB) m/z 388 (M+H)+; Anal.
Calcd for C23H21N3O3: C, 71.30; H, 5.46; N, 10.85.
6.28; N, 13.39; HRMS (FAB–) calcd for C25H25N4O2
413.1978. Found: 413.2019; ½aꢂ
25
D
ꢀ36.4° (c 0.23,
CHCl3–MeOH 1:4).
(R)-8b and (R)-9 were also obtained from (R)-4a13 with
the similar procedures used for the preparation of (S)-8b
and (S)-9. All spectral data other than optical rotation
25
D
25
D
((R)-8b: ½aꢂ + 25.1° (c 0.21, MeOH) and (R)-9: ½aꢂ
+
33.7° (c 0.20, CHCl3–MeOH 1:4)) were consistent with
those of (S)-8b and (S)-9.
5.1.7. (S)-2-(tert-Butyldiphenylsilyloxymethyl)-4,4-dieth-
oxybutyl methanesulfonate (11c). To a stirring solution
of 1016 (1.09 g, 4.67 mmol) and Et3N (1.30 mL,
9.34 mmol) in THF (20 mL), methanesulfonyl chloride
(0.398 mL, 5.14 mmol) was added at 0 °C. The reaction
mixture was stirred for 25 min at 0 °C, and then was
partitioned between AcOEt and NaHCO3 saturated
water. The organic layer was separated, washed with
brine, dried over anhydrous MgSO4, and concentrated
in vacuo to give crude 11a (1.53 g) as a pale yellow oil.
To a solution of thus obtained crude 11a (1.53 g) in
1,4-dioxane (22 mL)–H2O (11 mL) was added 4 M
NaOH (1.40 mL, 5.60 mmol) at 0 °C. The reaction mix-
ture was stirred for 1 h at 0 °C and then was partitioned
between AcOEt and brine. The organic layer was sepa-
rated, successively washed with NaHCO3 saturated
water and brine, dried over anhydrous MgSO4, and con-
centrated in vacuo to give crude 11b (1.31 g) as a pale
yellow oil. To a stirring solution of all of the crude
11b and imidazole (636 mg, 9.34 mmol) in DMF
(5 mL), TBDPSCl (1.42 g, 5.14 mmol) was added at
Found: C, 70.88; H, 5.44; N, 10.73; HRMS (FAB–)
25
calcd for C23H20N3O3 386.1505. Found 386.1523; ½aꢂ
D
ꢀ27.3° (c 0.26, MeOH).
5.1.5.
3-Anilino-4-[(S)-8-(methanesulfonyloxymethyl)-
6,7,8,9-tetrahydropyrido[1,2-a]indol-10-yl]-1H-pyrrole-
2,5-dione ((S)-8c). To a solution of (S)-8b (13.8 g,
35.6 mmol) in THF (270 mL) were added pyridine
(8.64 mL, 129 mmol) and methanesulfonic anhydride
(12.4 g, 71.2 mmol) sequentially at room temperature.
The reaction mixture was heated to reflux for 2 h. The
reaction mixture was cooled to room temperature and
quenched with a 1 M aqueous KHSO4 solution. The
organic layer was separated, sequentially washed with
water and brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. Thus obtained residue was chro-
matographed on silica gel eluting with hexane–AcOEt
(1:1–1:2) to give (S)-8c (12.4 g, 75%) as a red-orange sol-
1
id: H NMR (400 MHz, DMSO-d6): d 1.09–1.26 (1H,
m), 1.54–1.72 (1H, m), 1.88–2.15 (1H, m), 2.22 (0.5H,

M. Tanaka et al. / Bioorg. Med. Chem. 14 (2006) 5781–5794
5789
0 °C. The reaction mixture was allowed to warm to
room temperature, stirred overnight, and then diluted
with AcOEt. The mixture was washed with NaHCO3
saturated water and brine successively, and concentrated
in vacuo. The residue mixture was chromatographed on
silica gel eluting with hexane–AcOEt (4:1) to give 11c
(2.11 g, 89% from 10) as a colorless oil: 1H NMR
(400 MHz, CDCl3): d 1.06 (9H, s), 1.16 (3Hx2, t,
J = 7.2 Hz), 1.68 (2H, t, J = 6.0 Hz), 2.07–2.17 (1H,
m), 2.93 (3H, s), 3.37–3.48 (2H, m), 3.53–3.65 (3H, m),
3.72 (1H, dd, J = 4.8, 10.4 Hz), 4.33 (1H, dd, J = 6.0,
(1H, dt, J = 1.4, 7.1 Hz), 7.27 (1H, d, J = 8.4 Hz), 7.32–
7.48 (6H, m), 7.50 (1H, d, J = 7.8 Hz), 7.63–7.69 (4H,
m); MS (ESI) m/z 497 (M+H)+.
5.1.10.
3-Anilino-4-{(S)-7-[(dimethylamino)methyl]}-
6,7,8,9-tetrahydropyrido[1,2-a]indol-10-yl}-1H-pyrrole-
2,5-dione ((S)-14). (S)-14 was obtained from (R)-13 in
43% yield as an orange-red solid with similar procedures
used for the preparation of (S)-9 from (S)-6: mp 200–
1
202 °C; H NMR (300 MHz, DMSO-d6): d 0.75 (1H,
m), 1.24 (1H, m), 1.50–2.60 (5H, m), 2.12 (6H, s), 3.30
(1H, m), 4.06 (1H, m), 6.60 (br d, 2H, J = 6.6 Hz),
6.72 (3H, br s), 6.98 (1H, t, J = 7.3 Hz), 7.05 (1H, t,
J = 7.3 Hz), 7.28 (1H, d, J = 7.3 Hz), 7.33 (1H, d,
J = 7.3 Hz), 9.24 (1H, s), 10.61 (1H, br s); MS (FAB)
m/z 415 (M+H)+; Anal Calcd for C25H26N4O2Æ0.6H2O:
C, 70.60; H, 6.45; N, 13.17. Found: C, 70.32; H, 6.37;
N, 13.01; HRMS (FAB–) calcd for C25H26N4O2
9.6 Hz), 4.39 (1H, dd, J = 5.2, 9.6 Hz), 4.50 (1H, t,
20
J = 6.0 Hz), 7.35–7.46 (6H, m), 7.62–7.67 (4H, m); ½aꢂ
D
+1.25° (c 0.72, CHCl3).
5.1.8. 2-{1-[(R)-2-(tert-Butyldiphenylsilyloxymethyl)-4,4-
diethoxybutyl]-1H-indol-3-yl}acetamide (12). To a solu-
tion of indole-3-acetamide (1.08 g, 6.23 mmol) in DMF
(10 mL) was added NaH (249 mg, 60% in mineral oil,
6.23 mmol) at 0 °C, and the reaction mixture was stirred
for 15 min at 0 °C. To the resulting mixture, 11c (2.11 g,
4.15 mmol) and NaI (62 mg, 0.415 mmol) were added.
After stirring overnight at room temperature, the reac-
tion mixture was poured into water and extracted with
AcOEt. The organic layer was separated, washed with
brine, dried over anhydrous MgSO4, and concentrated
in vacuo. The residue was chromatographed on silica
gel eluting with hexane–AcOEt (1:1–1:3) to give 12
413.1978. Found: 413.2020; ½aꢂ25 +35.4° (c 0.28, MeOH).
D
5.1.11. (S)-2-(tert-Butyldiphenylsilyloxymethyl)-4,4-di-
ethoxybutyl acetate (15a). To a stirring solution of 1016
(1.00 g, 4.27 mmol) and imidazole (581 mg, 8.54 mmol)
in DMF (5 mL) was added TBDPSCl (1.29 g,
4.70 mmol) at 0 °C. The reaction mixture was stirred
overnight and then poured into NaHCO3 saturated
water. The mixture was extracted with AcOEt and the
organic layer was separated, washed with brine, and
concentrated in vacuo. The residue was chromato-
graphed on silica gel eluting with hexane–AcOEt (9:1)
1
(1.42 g, 57%) as a colorless oil: H NMR (400 MHz,
CDCl3): d 1.10 (3H, t, J = 7.2 Hz), 1.11 (9H, s), 1.14
(3H, t, J = 7.2 Hz), 1.60–1.69 (1H, m), 1.72–1.82 (1H,
m), 2.21–2.30 (1H, m), 3.27–3.38 (2H, m), 3.44–3.57
(4H, m), 3.64 (2H, s), 4.16 (1H, dd, J = 6.4, 14.4 Hz),
4.25 (1H, dd, J = 8.0, 14.4 Hz), 4.35 (1H, t,
J = 5.6 Hz), 5.21 (1H, br s), 5.52 (1H, br s), 6.90 (1H,
s), 7.13 (1H, t, J = 7.2 Hz), 7.21 (1H, t, J = 7.8 Hz),
7.30–7.37 (4H, m), 7.39–7.45 (3H, m), 7.53 (1H, d,
J = 8.0 Hz), 7.57–7.63 (4H, m).
1
to give 15a (2.00 g, 99%) as a colorless oil: H NMR
(400 MHz, CDCl3): d 1.05 (9H, s), 1.16 (6H, t,
J = 7.2 Hz), 1.63 (1H, dt, J = 6.0, 14.4 Hz), 1.74 (1H,
dt, J = 6.2, 14.4 Hz), 1.98 (3H,s), 1.99–2.07 (1H, m),
3.38–3.49 (2H, m), 3.55–3.65 (3H, m), 3.69 (1H, dd,
J = 4.8, 10.4 Hz), 4.16 (1H, d, J = 6.0 Hz), 4.54 (1H, t,
J = 5.8 Hz), 7.34–7.45 (6H, m), 7.62–7.66 (4H, m).
5.1.12. (S)-2-(tert-Butyldiphenylsilyloxymethyl)-4,4-dieth-
oxybutan-1-ol (15b). To a solution of 15a (2.00 g,
4.23 mmol) in EtOH (20 mL) was added potassium car-
bonate (643 mg, 4.65 mmol) at room temperature. The
reaction mixture was stirred overnight at room temper-
ature and then poured into brine. The mixture was
extracted with toluene, and the organic layer was sepa-
rated, washed with brine, and concentrated in vacuo.
The residue was chromatographed on silica gel eluting
with hexane–AcOEt (4:1) to give 15b (1.79 g, 98%) as
a colorless oil: 1H NMR (400 MHz, CDCl3): d 1.06
(9H, s), 1.16 (3H, t, J = 7.2 Hz), 1.18 (3H, t,
J = 7.2 Hz), 1.65 (2H, t, J = 5.8 Hz), 1.90–2.00 (1H,
m), 2.77 (1H, t, J = 6.0 Hz), 3.39–3.49 (2H, m), 3.54–
3.69 (3H, m), 3.70–3.77 (3H, m), 4.52 (1H, t,
J = 5.6 Hz), 7.35–7.46 (6H, m), 7.63–7.69 (4H, m).
5.1.9. 2-[(R)-7-(tert-Butyldiphenylsilyloxymethyl)-6,7,8,9-
tetrahydropyrido[1,2-a]indol-10-yl]acetamide ((R)-13). To
a solution of 12 (1.42 g, 2.36 mmol) in CHCl3 (108 mL)
was added a 50% aqueous trifluoroacetic acid solution
(7.1 mL, 92.2 mmol) at room temperature. The reaction
mixture was stirred vigorously for 30 min at room
temperature. The reaction mixture was poured into
NaHCO3 saturated water and extracted with CHCl3.
The organic layer was separated, dried over anhydrous
MgSO4, and concentrated in vacuo. Thus obtained resi-
due was dissolved in EtOH (20 mL) and the solution
was stirred for 3 h at room temperature under H2 atmo-
sphere in the presence of 50%-wet 5% palladium-carbon
(100 mg). Then the insoluble catalyst was removed by
filtration and the filtrate was concentrated in vacuo.
The residue was chromatographed on silica gel eluting
with hexane–AcOEt (1:2–1:3) to give (R)-13 (1.12 g,
95%) as an off-white solid: 1H NMR (400 MHz, CDCl3):
d 1.09 (9H, s), 1.58-1.69 (1H, m), 2.00–2.08 (1H, m), 2.28–
2.40 (1H, m), 2.78 (1H, ddd, J = 6.0, 11.2, 16.8 Hz), 3.00
(1H, dt, J = 4.6, 17.2 Hz), 3.63 (2H, dd, J = 18.5,
24.5 Hz), 3.70–3.75 (2H, m), 3.81 (1H, dd, J = 5.4,
10.2 Hz), 4.30 (1H, dd, J = 5.6, 12.3 Hz), 5.20 (1H, br
s), 5.57 (1H, br s), 7.15 (1H, dt, J = 1.3, 7.5 Hz), 7.20
5.1.13.
3-Anilino-4-{(R)-7-[(dimethylamino)methyl]}-
6,7,8,9-tetrahydropyridol[1,2-a]indol-10-yl]-1H-pyrrole-
2,5-dione ((R)-14). To a solution of 15b (1.78 g,
4.13 mmol) in THF (20 mL) were successively added
Et3N (1.15 mL, 8.26 mmol) and methanesulfonyl chlo-
ride (0.352 mL, 4.54 mmol) at 0 °C. The reaction mix-
ture was stirred for 2 h at 0 °C and was partitioned
between NaHCO3 saturated water and AcOEt. The

5790
M. Tanaka et al. / Bioorg. Med. Chem. 14 (2006) 5781–5794
organic layer was separated, washed with NaHCO3 sat-
urated water and brine, dried over anhydrous MgSO4,
and concentrated in vacuo to give crude 15c (2.14 g) as
at room temperature, the reaction mixture was passed
through a Celite-filter. The filtrate was concentrated in
vacuo to give crude 19a (9.72 g) as a brown oil. No puri-
fication was attempted on this compound, which was
used directly in the next step. An analytical sample
was purified by column chromatography on silica gel
eluting with hexane–AcOEt (5:1–1:1) to afford pure 19a
as a pale yellow solid: mp 66–69 °C; 1H NMR
(300 MHz, CDCl3): d 2.10 (3H, s), 2.15–2.27 (1H, m),
2.77–2.91 (2H, m), 3.55–3.69 (2H, m), 4.18 (2H, d,
J = 5.9 Hz), 6.27 (1H, s), 7.01–7.16 (2H, m), 7.31 (1H,
a pale yellow oil, which was converted to (R)-14 showing
25
D
½aꢂ of ꢀ33.3° (c 0.23, MeOH) in 35% yield (nine steps
from 15b) with the same procedures used for the trans-
formation of 11c into (S)-14. All spectral data other
than optical rotation were consistent with those of (S)-
14.
5.1.14. Diethyl (1H-indol-2-ylmethylene)malonate (17). A
mixture of 16 (10.0 g, 68.9 mmol), diethylmalonate
d, J = 8.0 Hz), 7.53 (1H, d, J = 7.5 Hz), 8.43 (1H, br s);
25
MS (ESI) m/z 248 (M+H)+; ½aꢂ +8.33° (c 0.48, CHCl3).
(12.5 mL,
82.7 mmol),
piperidine
(0.684 mL,
D
˚
6.89 mmol), and powdered 4 A molecular sieves
(10.0 g) in toluene (150 mL) was refluxed for 3 h. The
reaction mixture was allowed to cool to room tempera-
ture, insoluble molecular sieves were removed by filtra-
tion, and then the filtrate was concentrated in vacuo.
The residue was purified by column chromatography
on silica gel eluting with hexane–AcOEt (4:1) followed
by precipitation from hexane–iPrOH to give 17 (10.0 g,
5.1.17. (S)-2-(tert-Butyldiphenylsilyloxymethyl)-3-(1H-
indol-2-yl)-propan-1-ol (20b). To a solution of crude
19a and imidazole (2.61 g, 38.3 mmol) in DMF
(35 mL), TBDPSCl (8.98 mL, 38.3 mmol) was added at
room temperature. The reaction mixture was stirred
for 1.5 h and was partitioned between AcOEt and brine.
The organic layer was separated, successively washed
with water and brine, dried over anhydrous Na2SO4,
and concentrated in vacuo to give crude 20a (19.1 g)
as a brown oil. Thus obtained 20a was dissolved in
MeOH (70 mL), and K2CO3 (4.81 g, 34.8 mmol) was
added to the solution. After stirring for 40 min at room
temperature, brine and a 1 M aqueous KHSO4 solution
were added to the reaction mixture at 0 °C, and the
resulting mixture was extracted with AcOEt. The organ-
ic layer was separated, washed with brine and concen-
trated in vacuo. The residue was chromatographed on
silica gel eluting with hexane–AcOEt (4:1) to give 20b
(11.6 g, 75% from 17) as a colorless oil: 1H NMR
(400 MHz, CDCl3): d 1.10 (9H, s), 2.03–2.11 (1H, m),
2.15 (1H, br s), 2.82 (1H, dd, J = 6.8, 14.8 Hz), 2.93
(1H, dd, J = 7.6, 14.8 Hz), 3.68 (1H, dd, J = 6.0,
11.2 Hz), 3.72–3.85 (3H, m), 6.18 (1H, s), 7.05 (1H, dt,
J = 1.2, 7.6 Hz), 7.10 (1H, dt, J = 1.2, 8.0 Hz), 7.22
(1H, d, J = 8.0 Hz), 7.34–7.48 (6H, m), 7.50 (1H, d,
1
51%) as a yellow solid: H NMR (300 MHz, CDCl3): d
1.36 (3H, t, J = 7.1 Hz), 1.38 (3H, t, J = 7.1 Hz), 4.32
(2H, q, J = 7.2 Hz), 4.41 (2H, q, J = 7.2 Hz), 6.97 (1H,
d, J = 1.2 Hz), 7.11 (1H, dt, J = 1.2, 8.1 Hz), 7.30 (1H,
dt, J = 1.2, 8.4 Hz), 7.41 (1H, dd, J = 0.9, 8.4 Hz), 7.64
(1H, dd, J = 0.9, 8.1 Hz), 7.76 (1H, s), 10.49 (1H, br s);
MS (ESI) m/z 288 (M+H)+.
5.1.15. 2-(1H-Indol-2-ylmethyl)propane-1,3-diol (18b).
To a suspension of NaBH4 (1.32 g, 34.8 mmol) in THF
(50 mL), a solution of 17 (10.0 g, 34.8 mol) in THF
(50 mL) was added at 0 °C. After stirring for 3.5 h at
0 °C, brine, and a 1 M aqueous KHSO4 solution were
added to the reaction mixture. The organic layer was sep-
arated and the aqueous layer was extracted with AcOEt.
The combined organic layers were washed with brine,
and concentrated in vacuo to give crude 18a (10.0 g) as
a yellow-orange solid. A solution of resulting crude 18a
in THF (70 mL) was added to a suspension of LiAlH4
(2.64 g, 69.6 mmol) in THF (80 mL) at 0 °C. After stir-
ring for 2 h at 0 °C, crushed ice, brine and a 1 M aqueous
KHSO4 solution were successively added to the reaction
mixture. The organic layer was separated and the aque-
ous layer was extracted with AcOEt. The combined
organic layers were washed with brine and concentrated
in vacuo to give crude 18b. Thus obtained crude 18b was
azeotroped with vinyl acetate to afford AcOEt-free 18b
as a brown oil. No further purification was attempted
on this compound, which was used directly in the next
step. An analytical sample was purified by column chro-
matography on silica gel eluting with hexane–AcOEt
(1:2–1:3) to afford pure 18b as an off-white solid: mp
77–80 °C; 1H NMR (300 MHz, CDCl3); d 2.06 (1H,
m), 2.45 (2H, br s), 2.84 (2H, d, J = 7.3 Hz), 3.62–3.73
(2H, m), 3.76–3.86 (2H, m), 6.25 (1H, s), 7.01–7.17
(2H, m), 7.29 (1H, d, J = 7.9 Hz), 7.52 (1H, d,
J = 7.5 Hz), 8.51 (1H, br s); MS (ESI) m/z 206 (M+H)+.
J = 7.6 Hz), 7.62–7.70 (4H, m), 8.27 (1H, br s); MS
20
(ESI) m/z 444 (M+H)+; ½aꢂ +5.10° (c 1.11, CHCl3).
D
5.1.18. (S)-2-(tert-Butyldiphenylsilyloxymethyl)-2,3-dihy-
dro-1H-pyrrolo[1,2-a]indole ((S)-21). To a stirring solu-
tion of 20b (11.6 g, 26.1 mmol) in THF (200 mL) were
successively added pyridine (6.30 mL, 78.3 mmol) and
methanesulfonic anhydride (9.09 g, 52.2 mmol) at 0 °C.
The reaction mixture was allowed to warm to room tem-
perature over 2 h and then was partitioned between
brine and AcOEt. The organic layer was washed with
a 1 M aqueous KHSO4 solution and brine, dried over
anhydrous Na2SO4, and concentrated in vacuo to give
crude 20c as a yellow-orange amorphous solid. Thus ob-
tained crude 20c was dissolved in DMF (200 mL), and
NaH (60% in mineral oil, 1.15 g, 6.23 mmol) and NaI
(391 mg, 2.61 mmol) were added to the stirring solution
at 0 °C. The reaction mixture was stirred for 40 min at
0 °C and for 12 h at room temperature. To the reaction
mixture, a 1 M aqueous KHSO4 solution was added and
the product was extracted with AcOEt. The organic
layer was separated, successively washed with water
and brine, and concentrated in vacuo. The residue was
chromatographed on silica gel eluting with hexane–
5.1.16.
(R)-3-Hydroxy-2-(1H-indol-2-ylmethyl)propyl
acetate (19a). To a solution of thus obtained AcOEt-free
18b in vinyl acetate (100 mL), Lipase PS16,18 (348 mg)
was added at room temperature. After stirring for 13 h

M. Tanaka et al. / Bioorg. Med. Chem. 14 (2006) 5781–5794
5791
AcOEt (19:1) to give (S)-21 (8.94 g, 80%) as a colorless
oil: H NMR (400 MHz, CDCl3): d 1.06 (9H, s), 2.81
10.62 (1H, s); MS (ESI) m/z 429 (M+H)+; Anal. Calcd
for C26H28N4O2Æ0.4H2O: C, 71.67; H, 6.67; N, 12.86.
Found: C, 71.72; H, 6.54; N, 12.93; HRMS (FAB–) calcd
1
(1H, ddd, J = 0.8, 6.0, 16.8 Hz), 3.08 (1H, ddd,
J = 0.8, 8.4, 16.8 Hz), 3.17–3.27 (1H, m), 3.73 (1H, dd,
J = 7.6, 10.4 Hz), 3.79 (1H, dd, J = 6.4, 10.4 Hz), 3.95
(1H, dd, J = 5.6, 10.0 Hz), 4.17 (1H, dd, J = 8.0,
10.4 Hz), 6.12 (1H, s), 7.05 (1H, dt, J = 1.2, 7.6 Hz),
7.11 (1H, dt, J = 1.2, 7.2 Hz), 7.22 (1H, d, J = 8.0 Hz),
25
for C26H27N4O2 427.2134. Found: 427.2139; ½aꢂ ꢀ50.4°
D
(c 0.24, MeOH).
5.1.22. 3-Anilino-4-((R)-2-{[ethyl(methyl)amino]methyl}-
2,3-dihydro-1H-pyrrolo[1,2-a]indol-9-yl)-1H-pyrrole-2,5-
dione ((R)-23c). (R)-23c was similarly obtained as an or-
7.33–7.46 (6H, m), 7.53 (1H, d, J = 7.6 Hz), 7.62–7.66
20
D
(4H, m); MS (ESI) m/z 426 (M+H)+; ½aꢂ +48.5° (c
ange-red solid in 49% yield: mp 184–185 °C; H NMR
1
1
0.84, CHCl3). (S)-MTPA ester derived from (S)-21: H
NMR (300 MHz, CDCl3): d 2.77 (1H, ddd, J = 1.1,
5.9, 16.1 Hz), 3.15 (1H, ddd, J = 1.1, 8.4, 16.1 Hz),
3.30–3.44 (1H, m), 3.53 (3H, s), 3.78 (1H, dd, J = 5.5,
10.3 Hz), 4.14 (1H, dd, J = 7.7, 10.6 Hz), 4.43 (2H, d,
J = 6.6 Hz), 6.14 (1H, s), 7.01–7.19 (3H, m), 7.46–7.55
(3H, m), 7.66–7.77 (3H, m).
(300 MHz, DMSO-d6): d 0.93 (3H, t, J = 7.0 Hz), 2.01
(3H, m), 2.09 (3H, s), 2.22 (2H, m), 2.58 (1H, dd,
J = 8.4, 16.5 Hz), 2.73 (1H, m), 3.63 (1H, dd, J = 5.8,
10.3 Hz), 3.99 (1H, dd, J = 7.3, 10.3 Hz), 6.60–6.82
(5H, m), 6.91 (1H, t, J = 7.0 Hz), 7.00 (1H, t,
J = 7.0 Hz), 7.22 (1H, d, J = 7.7 Hz), 7.35 (1H, d,
J = 7.7 Hz), 9.17 (1H, s), 10.61 (1H, s); MS (FAB) m/z
415 (M+H)+; Anal. Calcd for C25H26N4O2: C, 72.44;
H, 6.32; N, 13.52. Found: C, 72.17; H, 6.07; N, 13.49;
5.1.19. 3-Anilino-4-[(S)-2-(hydroxymethyl)-2,3-dihydro-
1H-pyrrolo[1,2-a]indol-9-yl]-1H-pyrrole-2,5-dione ((S)-
22). (S)-22 was obtained as an orange-red solid in 42%
yield from (S)-21 with the same procedures used for
the preparation of (S)-8b from (S)-4b: mp 248–251 °C;
1H NMR (300 MHz, DMSO-d6): d 2.21 (1H, dd,
J = 5.7, 16.5 Hz), 2.57 (1H, dd, J = 9.9, 16.5 Hz), 2.73
(1H, m), 3.09 (1H, m), 3.20–3.32 (1H, m), 3.73 (1H,
dd, J = 5.5, 10.2 Hz), 3.99 (1H, dd, J = 7.7, 10.2 Hz),
4.75 (1H, t, J = 5.1 Hz), 6.62–6.73 (3H, m), 6.75–6.83
(2H, m), 6.88 (1H, t, J = 7.3 Hz), 6.98 (1H, t,
J = 7.5 Hz), 7.20 (1H, d, J = 8.0 Hz), 7.30 (1H, d,
J = 7.7 Hz), 9.15 (1H, s), 10.60 (1H, br s); MS (FAB)
m/z 374 (M+H)+; Anal Calcd for C22H19N3O3: C,
70.76; H, 5.13; N, 11.25. Found: C, 70.34; H, 5.07; N,
HRMS (FAB–) calcd for C25H25N4O2 413.1978. Found:
25
D
413.1991; ½aꢂ ꢀ39.4° (c 0.31, MeOH).
5.1.23.
3-Anilino-4-[(R)-2-(pyrrolidin-1-ylmethyl)-2,3-
dihydro-1H-pyrrolo[1,2-a]indol-9-yl]-1H-pyrrole-2,5-dione
((R)-23d). (R)-23d was similarly obtained as an orange-
red solid in 44% yield: mp 159–164 °C; 1H NMR
(300 MHz, DMSO-d6): d 1.67 (4H, br s), 2.10 (3H, m),
2.37 (4H, m), 2.60 (1H, dd, J = 8.4, 16.1 Hz), 2.72
(1H, m), 3.67 (1H, dd, J = 5.5, 10.2 Hz), 4.01 (1H, dd,
J = 7.3, 10.2 Hz), 6.64–6.82 (5H, m), 6.90 (1H, dd,
J = 7.0, 8.1 Hz), 7.00 (1H, dd, J = 7.0, 8.1 Hz), 7.23
(1H, d, J = 8.1 Hz), 7.35 (1H, d, J = 8.1 Hz), 9.17 (1H,
s), 10.61 (1H, s); MS (ESI) m/z 427 (M+H)+; Anal.
Calcd for C26H26N4O2Æ0.5H2O: C, 71.70; H, 6.25; N,
12.86. Found: C, 71.90; H, 6.53; N, 12.53; HRMS
11.20; HRMS (FAB–) calcd for C22H18N3O3 372.1348.
25
D
Found: 372.1384; ½aꢂ ꢀ52.8° (c 0.22, MeOH).
(FAB–) calcd for C26H25N4O2 425.1978. Found:
25
D
5.1.20.
3-Anilino-4-{(R)-2-[(dimethylamino)methyl]-2,3-
425.2012; ½aꢂ ꢀ31.2° (c 0.23, MeOH).
dihydro-1H-pyrrolo[1,2-a]indol-9-yl}-1H-pyrrole-2,5-dione
((R)-23a). (R)-23a was obtained as an orange-red solid
in 57% yield from (S)-22 with the similar procedures
used for the preparation of (S)-9 from (S)-8b: mp
203–206 °C; 1H NMR (300 MHz, DMSO-d6): d 1.93
(2H, d, J = 7.0 Hz), 2.08 (1H, m), 2.08 (6H, s), 2.58
(1H, dd, J = 8.4, 16.2 Hz), 2.73 (1H, m), 3.65 (1H, dd,
J = 5.5, 10.3 Hz), 3.99 (1H, dd, J = 7.3, 10.3 Hz),
6.64–6.81 (5H, m), 6.91 (1H, dd, J = 7.0, 7.7 Hz), 7.00
(1H, dd, J = 7.0, 7.7 Hz), 7.22 (1H, d, J = 7.7 Hz), 7.35
(1H, d, J = 7.7 Hz), 9.17 (1H, s), 10.61 (1H, s); MS
(ESI) m/z 401 (M+H)+; Anal. Calcd for C24H24N4O2: C,
71.98; H, 6.04; N, 13.99. Found: C, 71.55; H, 6.01; N,
5.1.24.
3-[(R)-2-(Aminomethyl)-2,3-dihydro-1H-pyr-
rolo[1,2-a]indol-9-yl]-4-anilino-1H-pyrrole-2,5-dione ((R)-
23g). (R)-23g was similarly obtained as an orange-red
solid in 34%: mp 225–229 °C; 1H NMR (300 MHz,
DMSO-d6): d 2.19 (1H, dd, J = 8.7, 19.5 Hz), 2.25–2.42
(2H, m), 2.51–2.65 (2H, m), 3.69 (1H, dd, J = 5.1,
10.5 Hz), 3.99 (1H, dd, J = 7.2, 10.2 Hz), 6.64–6.72 (3H,
m), 6.74–6.81 (2H, m), 6.88 (1H, dt, J = 1.2, 7.5
Hz), 6.99 (1H, dt, J = 0.9, 7.5 Hz), 7.19 (1H, d,
J = 8.1 Hz), 7.30 (1H, d, J = 7.5 Hz), 9.13 (1H, br s);
MS (ESI) m/z 373 (M+H)+; Anal. Calcd for
C22H20N4O2Æ0.5EtOH: C, 69.85; H, 5.86; N, 14.17.
13.95; HRMS (FAB–) calcd for C24H23N4O2 399.1821.
Found: 399.1838; ½aꢂ ꢀ33.8° (c 0.20, MeOH).
Found: C, 69.49; H, 5.47; N, 14.42; HRMS (FAB–) calcd
25
D
25
D
for C22H19N4O2 371.1508. Found: 371.1477; ½aꢂ ꢀ10.9°
(c 0.26, MeOH).
5.1.21.
3-Anilino-4-{(R)-2-[(diethylamino)methyl]-2,3-
dihydro-1H-pyrrolo[1,2-a]indol-9-yl}-1H-pyrrole-2,5-dione
((R)-23b). (R)-23b was similarly obtained as an orange-
red solid in 49% yield: mp 167–169 °C; 1H NMR
(300 MHz, DMSO-d6): d 0.90 (6H, t, J = 7.0 Hz),
1.90–2.15 (3H, m), 2.25–2.61 (5H, m), 2.70 (1H, m),
3.65 (1H, dd, J = 5.4, 10.3 Hz), 3.97 (1H, dd, J = 7.4,
10.3 Hz), 6.64–6.81 (5H, m), 6.91 (1H, dd, J = 7.0,
7.9 Hz), 7.00 (1H, dd, J = 7.0, 7.9 Hz), 7.22 (1H, d,
J = 7.9 Hz), 7.35 (1H, d, J = 7.9 Hz), 9.20 (1H, s),
5.1.25.
3-Anilino-4-{(R)-2-[(methylamino)methyl]-2,3-
dihydro-1H-pyrrolo[1,2-a]indol-9-yl}-1H-pyrrole-2,5-dione
((R)-23e). To a stirring solution of (S)-22 (100 mg,
0.268 mmol) and 2,4,6-collidine (0.106 mL, 0.802 mmol)
in THF (1.0 mL) was added dropwise trifluoromethane-
sulfonic anhydride (0.135 mL, 0.802 mmol) at ꢀ78 °C.
After stirring for 1.5 h at ꢀ78 °C, 40% methylamine in
MeOH (0.820 mL, 8.04 mmol) was added to the reac-
tion mixture at the same temperature. The reaction mix-

5792
M. Tanaka et al. / Bioorg. Med. Chem. 14 (2006) 5781–5794
ture was allowed to warm to room temperature over 2 h.
To the reaction mixture, NaHCO3 saturated water was
added and the product was extracted with AcOEt. The
organic layer was separated, dried over anhydrous
Na2SO4, and concentrated in vacuo. Thus obtained res-
idue was purified by preparative thin-layer chromatog-
raphy on silica gel (CHCl3–MeOH–28% aqueous NH3
150:20:2) to give (R)-23e (72 mg, 70%) as an orange sol-
added pyridine (0.098 mL, 1.21 mmol) and methanesulf-
onic anhydride (211 mg, 1.82 mmol) at 0 °C. The reac-
tion mixture was allowed to warm to room
temperature and stirred for 12 h. The reaction mixture
was partitioned between brine and AcOEt, and the
organic layer was separated, successively washed with
2 M HCl and brine, and concentrated in vacuo. The res-
idue was chromatographed on silica gel eluting with
hexane–AcOEt (2:1) to give 19b (174 mg, 88%) as a col-
1
id: mp 218–222 °C; H NMR (300 MHz, DMSO-d6): d
1
2.08–2.34 (3H, br m), 2.23 (3H, s), 2.54–2.78 (2H, m),
3.67 (1H, dd, J = 5.3, 10.3 Hz), 3.99 (1H, dd, J = 7.5,
10.3 Hz), 6.62–6.83 (5H, m), 6.89 (1H, t, J = 7.0 Hz),
6.99 (1H, t, J = 7.0 Hz), 7.20 (1H, d, J = 8.1 Hz), 7.32
(1H, d, J = 8.0 Hz), 9.16 (1H, s); MS (FAB) m/z 387
(M+H)+; Anal. Calcd for C23H22N4O2: C, 71.48; H,
5.74; N, 14.50. Found: C, 71.12; H, 5.74; N, 14.40;
orless oil: H NMR (300 MHz, CDCl3): d 2.10 (3H, s),
2.42–2.55 (1H, m), 2.90 (2H, d, J = 7.2 Hz), 3.02 (3H,
s), 4.08–4.30 (4H, m), 6.30 (1H, s), 7.08 (1H, dt,
J = 1.2, 8.4 Hz), 7.15 (1H, dt, J = 1.5, 7.5 Hz), 7.33
(1H, d, J = 8.1 Hz), 7.53 (1H, d, J = 7.5 Hz), 8.35 (1H,
br s).
HRMS (FAB–) calcd for C23H21N4O2 385.1665. Found:
385.1693; ½aꢂ ꢀ30.8° (c 0.28).
5.1.29. (R)-2,3-Dihydro-1H-pyrrolo[1,2-a]indol-2-ylmeth-
yl acetate ((R)-24). To a stirring solution of 19b (164 mg,
0.504 mmol) in DMF (5 mL) were successively added
NaH (60% in mineral oil, 22.2 mg, 0.554 mmol) and
NaI (8.3 mg, 0.0554 mmol) at 0 °C and the reaction mix-
ture was stirred for 1 h at 0 °C. To the reaction mixture,
a 1 M aqueous KHSO4 solution was added and the mix-
ture was extracted with AcOEt. The organic layer was
separated, successively washed with water and brine
and concentrated in vacuo. The residue was chromato-
graphed on silica gel eluting with hexane–AcOEt
(20:1–10:1) to give (R)-24 (98 mg, 85%) as a colorless
25
D
5.1.26. 3-Anilino-4-{(R)-2-[(ethylamino)methyl]-2,3-dihy-
dro-1H-pyrrolo[1,2-a]indol-9-yl}-1H-pyrrole-2,5-dione
((R)-23f). (R)-23f was similarly obtained as an orange-
red solid in 38% yield: mp 227–230 °C; 1H NMR
(300 MHz, DMSO-d6): d 0.98 (3H, t, J = 7.2 Hz),
2.08–2.28 (2H, m), 2.30–2.39 (2H, m), 2.40–2.50 (2H,
m), 2.52–2.76 (2H, m), 3.68 (1H, dd, J = 5.4,
10.2 Hz), 4.00 (1H, dd, J = 7.2, 10.2 Hz), 6.61–6.82
(5H, m), 6.89 (1H, t, J = 7.5 Hz), 6.99 (1H, t,
J = 7.4 Hz), 7.20 (1H, d, J = 7.5 Hz), 7.32 (1H, d,
J = 7.8 Hz), 9.15 (1H, br s); MS (FAB) m/z 401
(M+H)+; Anal. Calcd for C24H24N4O2: C, 71.98; H,
6.04; N, 13.99. Found: C, 71.55; H, 6.10; N, 13.73;
1
oil: H NMR (400 MHz, CDCl3): d 2.09 (3H, s), 2.83
(1H, dd, J = 6.4, 16.4 Hz), 3.19 (1H, ddd, J = 0.8, 8.4,
16.0 Hz), 3.28–3.40 (1H, m), 3.88 (1H, dd, J = 6.0,
10.4 Hz), 4.17 (1H, dd, J = 7.6, 11.6 Hz), 4.21 (1H, dd,
J = 8.0, 10.4 Hz), 4.27 (1H, dd, J = 6.0, 11.2 Hz), 6.17
(1H, s), 7.07 (1H, t, J = 7.6 Hz), 7.13 (1H, t,
HRMS (FAB–) calcd for C24H23N4O2 399.1821.
25
D
Found: 399.1807; ½aꢂ ꢀ21.5° (c 0.26, MeOH) for
(R)-23fÆHCl.
J = 7.6 Hz), 7.22 (1H, d, J = 8.0 Hz), 7.55 (1H, d,
20
J = 8.0 Hz); MS (ESI) m/z 230 (M+H)+; ½aꢂ ꢀ20.4° (c
D
5.1.27. N-{[(R)-9-(4-Anilino-2,5-dioxo-2,5-dihydro-1H-
pyrrol-3-yl)-2,3-dihydro-1H-pyrrolo[1,2-a]indol-2-yl]methyl}-
acetamide ((R)-23h). To a suspension of (R)-23g
(100 mg, 0.269 mmol) in THF (1.0 mL) was added acetic
anhydride (30.4 lL, 0.323 mmol) at room temperature.
After stirring for 3 h at room temperature, the reaction
mixture was concentrated in vacuo. To the residue,
EtOH (2 mL) was added and the resulting suspension
was stirred at room temperature for 0.5 h. Deposited
solid was collected by filtration to give (R)-23h (57 mg,
51%) as an orange-red solid: mp 153–155 °C; 1H
NMR (300 MHz, DMSO-d6): d 1.80 (3H, s), 2.23 (1H,
dd, J = 5.7, 16.5 Hz), 2.55–2.79 (2H, m), 2.81–3.00
(2H, m), 3.64 (1H, dd, J = 5.4, 10.5 Hz), 4.00 (1H, dd,
J = 7.2, 10.2 Hz), 6.65–6.72 (3H, m), 6.74–6.82 (2H,
m), 6.87 (1H, dt, J = 1.2, 7.5 Hz), 6.98 (1H, dt, J = 1.2,
7.7 Hz), 7.18 (1H, d, J = 7.8 Hz), 7.28 (1H, d,
J = 8.1 Hz), 7.97 (1H, t, J = 5.6 Hz), 9.19 (1H, s),
10.62 (1H, s); MS (ESI) m/z 415 (M+H)+; Anal. Calcd
for C24H22N4O3Æ0.75 H2O: C, 67.35; H, 5.53; N, 13.09.
0.51, CHCl3).
(R)-22 and (S)-23c were obtained from (R)-24 with the
similar procedures used for the preparation of (S)-22
and (R)-23c. All spectral data other than optical rota-
25
tion ((R)-22: ½aꢂ +57.9 (c 0.15, MeOH) and (S)-23c:
D
25
D
of (S)-22 and (R)-23c.
½aꢂ +34.2 (c 0.12, MeOH)) were consistent with those
5.1.30. Ethyl (1R*,2R*)-1-hydroxy-2,3-dihydro-1H-pyr-
rolo[1,2-a]indole-2-carboxylate (26). To a solution of
2520 (30 g, 123 mmol) in THF (200 mL)-EtOH
(100 mL) was added 50%-wet 5% palladium-carbon
(6.0 g). The reaction mixture was stirred for 22.5 h
at room temperature under hydrogen atmosphere.
After removal of the catalyst by filtration, the filtrate
was concentrated in vacuo. The residue was chromato-
graphed on silica gel eluting with hexane–AcOEt
(4:1–2:1) to give 26 (22.7 g, 75%) as an off-white
solid: 1H NMR (400 MHz, CDCl3): d 1.35 (3H, t,
J = 7.2 Hz), 2.61 (1H, br s), 3.87 (1H, dt, J = 6.0,
8.8 Hz), 4.30 (2H, q, J = 7.2 Hz), 4.34 (1H, dd, J =
8.5, 10.3 Hz), 4.49 (1H, dd, J = 8.9, 10.5 Hz), 5.48 (1H,
d, J = 6.0 Hz), 6.47 (1H, s), 7.10 (1H, dt, J = 0.9,
8.0 Hz), 7.20 (1H, dt, J = 0.9, 8.2 Hz), 7.29 (1H, d,
J = 8.2 Hz), 7.62 (1H, d, J = 8.0 Hz); MS (ESI) m/z
246 (M+H)+.
Found: C, 67.59; H, 5.34; N, 13.21; HRMS (FAB–)
25
D
calcd for C24H21N4O3 413.1614. Found: 413.1641; ½aꢂ
ꢀ29.1 (c 0.11, MeOH).
5.1.28. (S)-3-(1H-Indol-2-yl)-2-{[(methylsulfonyl)oxy]meth-
yl}propyl acetate (19b). To a stirring solution of 19a
(150 mg, 0.607 mmol) in THF (6 mL) were successively

M. Tanaka et al. / Bioorg. Med. Chem. 14 (2006) 5781–5794
5793
5.1.31. Ethyl (1R,2R)-1-{[(S)-N-(tert-butoxyoxycarbon-
yl)alanyl]oxy}-2,3-dihydro-1H-pyrrolo[1,2-a]indole-2-car-
boxylate (28). To a stirring solution of 26 (10.0 g,
40.8 mmol), Boc-L-alanine (8.50 g, 44.9 mmol), and
DMAP (498 mg, 4.49 mmol) in CH2Cl2 (200 mL),
DCC (9.26 g, 44.9 mmol) dissolved in CH2Cl2 (50 mL)
was added at 0 °C. After stirring for 9 h at room temper-
ature, the reaction mixture was filtered through a Celite-
pad. The filtrate was concentrated in vacuo, and result-
ing residue was chromatographed on silica gel eluting
with hexane–AcOEt (10:1–6:1) to give 28 (3.70 g, 22%)
as a colorless solid. Thus obtained 28 was recrystallized
from a mixed solvent of hexane and AcOEt to give fine
crystals that were used for X-ray crystallographic analy-
sis:21 1H NMR (300 MHz, CDCl3): d 1.31 (3H, d,
J = 7.3 Hz), 1.33 (3H, t, J = 7.3 Hz), 1.41 (9H, s), 4.05
(1H, dd, J = 8.4, 15.0 Hz), 4.13–4.35 (3H, m), 4.38
(1H, dd, J = 8.1, 10.2 Hz), 4.57 (1H, t, J = 9.8 Hz),
4.95–5.10 (1H, m), 6.42 (1H, d, J = 6.3 Hz), 6.51 (1H,
s), 7.11 (1H, t, J = 7.4 Hz), 7.23 (1H, t, J = 8.1 Hz),
7.31 (1H, d, J = 7.8 Hz), 7.61 (1H, d, J = 8.1 Hz).
OH) or 2 lg PKC-e substrate peptide (EMD Biosci-
ences) or Syntide 2 (PLARTLSVAGLPGKK, Sigma–
Aldrich), recombinant human PKC-a, -b1, -b2, -c, -d,
-e, -f or -l (EMD Biosciences), and compound dissolved
in DMSO (final concentration 4.8%), was incubated for
15 min at 37 °C. Reaction was stopped by addition of
300 mM H3PO4. The assay mixture was blotted onto
phosphocellulose paper (Whatman), washed with
75 mM H3PO4, and evaluated by IP-autoradiography
(Fujifilm). The IC50 value was calculated in a semi-loga-
rithmic proportional manner from the two points
enclosing 50% inhibition. All the assays were carried
out in duplicate or triplicate and the average results
are presented.
5.3. Ex vivo studies
Compounds were suspended in 0.5% methylcellulose
and orally administered to 7-week-old male Sprague–
Dawley rats (Charles River Japan) at a dose of 2 mg/
kg. PKCb2 inhibitory activity of plasma at 2 and 6 h
after administration was evaluated as described above.
5.1.32. (S)-2,3-Dihydro-1H-pyrrolo[1,2-a]indol-2-ylmeth-
yl acetate ((S)-24). To a solution of 28 (3.65 g,
8.76 mmol) in THF (50 mL) was added 50%-wet 10%
palladium-carbon (1.2 g). The reaction mixture was stir-
red for 6 h at room temperature under hydrogen atmo-
sphere (2.7 kgf/cm2). After removal of the catalyst by
filtration, the filtrate was concentrated in vacuo, and
the residue was chromatographed on silica gel eluting
with hexane–AcOEt (10:1) to give 29a (1.61 g, 80%) as
a colorless solid. To a stirring suspension of lithium alu-
minum hydride (4.0 mg, 0.105 mmol) in THF (0.3 mL),
a solution of 29a (30 mg, 0.131 mmol) in THF (0.3 mL)
was added at 0 °C. After stirring for 2 h at 0 °C, brine
and a 1 M aqueous KHSO4 solution were added to
the reaction mixture. The resulting mixture was extract-
ed with AcOEt and the organic layer was separated,
washed with brine, and concentrated in vacuo to give
crude 29b. Thus obtained crude 29b was dissolved in
THF (0.5 mL), and to the solution were added pyridine
(32 lL, 0.393 mmol), acetic anhydride (25 lL,
0.262 mmol), and DMAP (1.6 mg, 0.0131 mmol) at
room temperature. After stirring for 6 h at room tem-
perature, the reaction mixture was poured into brine
and extracted with AcOEt. The organic layer was sepa-
rated, successively washed with a 1 M aqueous KHSO4
solution and brine, and concentrated in vacuo. The res-
idue was purified by preparative thin-layer chromatog-
5.4. Retinal mean circulation time (MCT) in diabetic rats
Diabetes was induced with a single intraperitoneal injec-
tion of 65 mg/kg streptozotocin (STZ, Sigma–Aldrich)
dissolved in 0.05 M citric acid buffer (pH 4.5) to 8-
week-old male Sprague–Dawley rats. Animal protocols
used in the study complied with our Laboratory Guide-
lines for Animal Experimentation. Distilled water or
compound (R)-23g (0.01, 0.1, and 1 mg/kg) was admin-
istered twice per day for 14 days from the day following
STZ injection. The day after the last administration, vid-
eo fluorescein angiography was performed to evaluate
retinal mean circulation time (MCT). Animals were
anesthetized with sodium pentobarbital (20 mg/kg, i.p.)
and 30 lL of 10% fluorescein was injected via catheter
inserted in the jugular vein. Fluorescence circulation in
retinal artery and vein was recorded by video angiogram
and MCT was calculated.25
Acknowledgments
The authors thank our Analytical Research and Devel-
opment Laboratories for collecting analytical data. Dr.
Jun-ichi Haruta, Dr. Hidetsura Cho, and Dr. Itsuo Uch-
ida are also acknowledged for their continuous
encouragement.
raphy (hexane–AcOEt 4:1) to give (S)-24 (22 mg, 73%
20
D
from 29a) as an off-white solid showing ½aꢂ of +23.8°
(c 0.52, CHCl3). All of spectral data other than optical
rotation were consistent with those of (R)-24.
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