Synthesis of anilino-monoindolylmaleimides as potent and selective PKCβ inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.07.061

The synthesis and enzyme inhibitory activity of PKCIβ-selective inhibitors possessing the novel pharmacophore of anilino-monoindolylmaleimide are described.

Full text of DOI:10.1016/j.bmcl.2004.07.061

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5171–5174
Synthesis of anilino-monoindolylmaleimides as potent
and selective PKCb inhibitors
Masahiro Tanaka, Shoichi Sagawa, Jun-ichi Hoshi, Fumito Shimoma, Isamu Matsuda,
Kenji Sakoda, Tomohiko Sasase, Masanori Shindo and Takashi Inaba*
Central Pharmaceutical Research Institute, Japan Tobacco Inc., 1-1, Murasaki-cho, Takatsuki, Osaka 569-1125, Japan
Received 30 June 2004; revised 26 July 2004; accepted 26 July 2004
Available online 14 August 2004
Abstract—We report herein synthesis of PKCb-selective inhibitors possessing the novel pharmacophore of anilino-monoindolyl-
maleimide. Several compounds of this series exhibited IC50Õs as low as 50nM against human PKCb2. One of the most potent com-
pounds, 6l, inhibited PKCb1 and PKCb2 with IC50 of 21 and 5nM, respectively, and exhibited selectivity of more than 60-fold in
favor of PKCb2 relative to other PKC isozymes (PKCa, PKCc, and PKCe).
Ó 2004 Elsevier Ltd. All rights reserved.
There have been reported 12 isozymes in a protein kin-
ase C (PKC) family, and they play indispensable roles in
organisms regulating cellular signal transduction path-
ways although specific physiological roles of each iso-
zyme remain unclear.1 Since the discovery of
staurosporine, a nonselective PKC inhibitor produced
by Streptomyces sp., a number of its analogues possess-
ing various inhibitory activity have been synthesized.2
Among these analogues, ruboxistaurin (PKCb1; IC50
of 4.7nM, PKCb2; IC50 of 5.9nM), a macrocyclic bis-
indolylmaleimide, is widely known because of its
marked PKCb selectivity.3 More importantly, it was re-
ported that this compound showed ameliorative effects
on diabetic vascular dysfunction including retinopathy
in the clinical studies through inhibition of activated
PKCb2 in diabetic hyperglycemic conditions.4 More re-
cently enzastaurin, another bisindolylmaleimide, was re-
ported as a PKCb-selective inhibitor, which is now
evaluated for the treatment of cancer.5 Thus, PKCb
has been now proven to be a novel target for the treat-
ment of diabetic complications and cancer,6 but no
highly potent PKCb-selective inhibitors have heretofore
been reported other than ones with the Ro 31-6233 (1)7
pharmacophore (Fig. 1). In this paper, we report a
new class of staurosporine-related PKCb-selective inhib-
itors, anilino-monoindolylmaleimides represented by the
structure of 4 and 6.8 One of our most potent com-
pounds, 6l, showed an IC50 of 21 and 5nM for human
PKCb1 and PKCb2, respectively, with selectivity for
PKCb2 of more than 60-fold compared to the a and e
isozymes.
Anilino-monoindolylmaleimides 4 were prepared via
two steps as shown in Scheme 1. 3-Hydroxymaleimide
3, which was obtained by treatment of 3-indolylacet-
amide 2 with (CO2Me)2 in the presence of t-BuOK,9
was converted into 4 by heating with an excess amount
of amine (R1R2NH) in AcOH.10 The yield of represent-
ative compound 4c (R1 = Ph, R2 = H) was 37% from
2.11 One of the most potent compounds, 6l, was pre-
pared as shown in Scheme 2. Alkylation of 2 with
Br(CH2)3OTBDMS gave 5, which was then converted
into 6e via the anilino-maleimide formation step with
the similar conditions used in the preparation of 4 and
deprotection step. The terminal hydroxyl group was re-
placed with imidazole via bromination step to afford
compound 6l.12 Compounds 6a–d,f–k were similarly ob-
tained in 10–38% from 2.10
Although no crystal structure of the catalytic domain of
PKC is available to date, the crystal structure of PKA
bound to staurosporine13 allowed us to speculate how
staurosporine inhibits PKC since PKA and PKC are
both inhibited by staurosporine in an ATP-competitive
manner and the ATP binding catalytic domains of these
kinases are well conserved. This crystal structure sug-
gests that the minimal structural elements required for
Keywords: PKCb inhibitor.
*
Corresponding author. Tel.: +81 72 681 9700; fax: +81 72 681
9725; e-mail: takashi.inaba@ims.jti.co.jp
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.061

5172
M. Tanaka et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5171–5174
PKA numbering), and (ii) widely spread two aromatic
H
H
N
N
O
O
N
O
H
rings bound to the 3- and 4-positions of the maleimide
(or the five-membered lactam) filling hydrophobic re-
gions of the enzyme. Based on these requirements, we
initially investigated the inhibitory activity of bis-
indolylmaleimide 1 against PKC b1, b2, a, and e because
1 was the smallest inhibitor that meets the pharmaco-
phoric requirements, and is a substructure of staurospo-
rine-related PKC inhibitors including ruboxistaurin and
enzastaurin.7 In our assay conditions, 1 exhibited mod-
erate PKCb inhibitory activity (PKCb1; IC50 of 304nM,
PKCb2; IC50 of 212nM). Taking this activity as a crite-
rion, one of the indoles of 1 was replaced with aliphatic
amines or aniline in order to find novel pharmacophores
with the guide of PKCb2 inhibitory activity as shown in
Scheme 1 and Table 1.14
N
N
N
O
Me
MeO
O
NMe2
NHMe
Staurosporine
Ruboxistaurin
H
N
O
O
H
N
O
O
N
N
Me
Compound 4a, obtained using benzylamine, did not in-
hibit the enzyme up to a concentration of 10lM. The
inhibitory activity of cyclohexyl analogue 4b was also
10-fold less potent than 1.15 In marked contrast to these
aliphatic amines, anilino-monoindolylmaleimide 4c
showed an IC50 of 223nM and this was found to be as
potent as 1. More importantly, 4c was significantly more
selective than 1 over PKC a and e (Table 2).16 It is spec-
ulated that the inhibitory activity and selectivity of 4c
are imparted by the sp2-like character of the nitrogen-
linker, which allows the plane of the aromatic system
to take an appropriate angle, moderately deviated from
the plane of the maleimide. The cocrystal structure of
PKA-staurosporine suggests that the two indoles in sta-
urosporine, which are strictly placed on the same plane
of the five-membered lactam, are important to exert its
PKA inhibitory activity. As for PKCb inhibition, how-
ever, this planarity requirement is not likely to be very
strict since nonplanar ruboxistaurin and enzastaurin
also show good affinity to the enzyme. The nonplanarity
should be rather speculated to be preferable from the
point of view of PKCb selectivity. Thus, a nonplanar
conformation of 4c, in which the phenyl ring adopts
N
H
N
N
H
N
Ro 31-6233 (1)
Enzastaurin
Figure 1. Structure of staurosporine, ruboxistaurin, enzastaurin, and
Ro 31-6233.
H
H
H2NOC
N
N
O
O
O
O
a
b
R1
N
OH
R2
N
H
N
N
H
H
2
3
4
Scheme 1. Reagents and conditions: (a) (CO2Me)2, t-BuOK, DMF,
0°C to rt; (b) R1R2NH, AcOH, D.
H
H2NOC
N
N
O
O
a
b, c, d
Table 1. PKCb2 inhibitory activity of compounds 4
HN
2
Compds R1
R2
PKCb2 IC50 (nM)
N
4a
4b
4c
4d
4e
4f
–CH2Ph
Cyclohexyl
H
>10,000
2448
223
3
TBDMSO
3
H
HO
5
6e
Phenyl
Phenyl
H
H
–CH3
H
939
N
O
O
2-Methylphenyl
3-Methylphenyl
4-Methylphenyl
3-Fluorophenyl
3-Chlorophenyl
3-Methoxyphenyl
3-Trifluoromethylphenyl
3-(i-Propyl)phenyl
2146
151
e, f
H
4g
4h
4i
H
480
249
HN
H
N
H
198
747
4j
H
N
3 6l
N
4k
4l
H
1450
2863
H
Scheme 2. Reagents and conditions: (a) Br(CH2)3OTBDMS, NaH,
DMF, 0°C to rt, 92%; (b) (CO2Me)2, t-BuOK, THF, 0°C to rt; (c)
PhNH2, AcOH, D; (d) TBAF, THF; (e) NBS, PPh3, THF; (f)
imidazole, NaH, DMF, 31% overall from 5.
Table 2. Comparison of PKCb selectivity of 1 and 4c
Compds
PKC IC50 (nM)
b1
b2
a
e
PKC inhibition are (i) maleimide or five-membered lac-
tam part, which forms rigid hydrogen bonds to the pep-
tide backbone of the enzyme (Glu121 and Val123in
1
4c
304
395
212
223
1599
7233
7516
>10,000

M. Tanaka et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5171–5174
5173
an appropriately deviation from the plane of the male-
imide, is considered to be crucial not only for the inhib-
itory activity but also for the selectivity. Methylation of
the nitrogen atom of 4c might oust the phenyl group too
far from the maleimide plane resulting in lower inhibi-
tory activity (see compound 4d, Table 1).
More importantly the selectivity in favor of PKCb2 ver-
sus PKC a and e was as high as that of 4c. Introduction
of a hydroxyl group was also effective in raising the po-
tency, although 6e has half the potency of 6b. Modifica-
tion of 3-position of the aniline part of 6b was well
tolerated, and 6f and 6g were as potent as 6b. These re-
sults were consistent with the SAR found in 4. Replace-
ment of the dimethylamine part of 6b with cyclic amines
was also conducted. However, 6h, 6i, and 6j had IC50
around 20nM, which have half the potency of 6b. Aim-
ing at finding more potent compounds, the isothiourea
salt 6k was prepared. This functional group had been
used as a hydrophilic part of bisindolylmaleimide type
PKC inhibitors by the Roche group and reported to
be effective in increasing the PKC inhibitory activity
remarkably.17 As anticipated, 6k showed IC50 of 2nM
for PKCb2, but the selectivity over PKC a and e was sig-
nificantly decreased. The most potent compound with
remarkable isozyme selectivity was the imidazole deriv-
ative 6l, which showed IC50 of 5nM for PKCb2
(21nM for PKCb1) and selectivity over PKC a and e
was 66-fold and 560-fold, respectively. IC50Õs of 6b and
6l against PKCc, which is one of the conventional
PKC isozymes, were found to be greater than 1lM.
Next, the effect of the substituents on the benzene ring
was investigated (Table 1). Although no significant in-
crease in potency was found in this approach, com-
pounds possessing a small substituent at 3-position (4f,
4h, 4i) were as potent as nonsubstituted 4c. The larger
substituents at this position, however, were not well tol-
erated (4j, 4k, 4l). Introduction of a methyl group at the
2-position (4e) also resulted in loss of activity by 10-fold
reflecting the importance of the torsional angle of the
phenyl plane relative to the maleimide plane as seen in
4d.
In order to increase the activity of 4c, hydrophilic
groups were attached to the molecule using the indole
NH as a handhold (Scheme 2 and Table 3). It could
be predicted from earlier SAR studies on staurospo-
rine-related compounds3b,17 that attaching hydrophilic
substituent(s) on the appropriate position of the modest
PKCb2 inhibitor 4c would generate much more potent
inhibitors. Initially dimethylamine was introduced with
linkers of varying lengths to afford 6a–d. As expected,
the potency was increased dramatically by this ap-
proach. Compounds 6b and 6c showed IC50 values of
12 and 15nM, respectively, indicating linkers of C3
and C4 length were appropriate for PKCb2 inhibition.
In conclusion, a new class of anilino-monoindolylmale-
imides was synthesized and evaluated for their PKCb2
inhibitory activity and selectivity over other isozymes.
Representative compounds 6b and 6l exhibited IC50 of
12 and 5nM for PKCb2, respectively, with remarkable
selectivity over the other calcium-dependent isozymes
(PKCa and PKCc) and the calcium-independent iso-
zyme (PKCe). The pharmacokinetic studies of these
compounds remain to be conducted for further evalua-
tion of their ameliorative effects in diabetic animals.
Table 3. Enzyme inhibitory activity of compounds 6
H
N
O
O
Acknowledgements
HN
R1
The authors wish to thank our Analytical Research and
Development Laboratories for collecting analytical data
and Mr. Yasunori Hase for fruitful discussion. Dr.
Jun-ichi Haruta, Dr. Hidetsura Cho, and Dr. Itsuo
Uchida are also acknowledged for their continuous
encouragement.
N
R2
Compds R1
R2
PKC IC50 (nM)
b2
a
e
6a
6b
6c
6d
6e
6f
H
H
H
H
H
–(CH2)2NMe2
–(CH2)3NMe2
–(CH2)4NMe2
–(CH2)5NMe2
–(CH2)3OH
28 2157 >10,000
12
15
460
730
3456
5866
3905
6322
3455
2605
References and notes
41 2232
30 1620
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was reported as dual inhibitors of PKCb and GSK-3b: (c)
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3-Me –(CH2)3NMe2
3-Cl
15
10
727
558
6g
–(CH2)3NMe2
6h
6i
6j
H
18
17
662 >10,000
-(CH2)3
-(CH2)3
-(CH2)3
N
N
N
H
H
726
3425
23968 >10,000
NMe
HBr
NH
6k
6l
H
H
2
5
27
58
-(CH2)3S
NH2
N
331
2807
-(CH2)3
N

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M. Tanaka et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5171–5174
Derian, S. A.; Addo, M. F.; Andrade-Gordon, P.;
(2H, t, J = 4.0Hz), 6.71–6.80 (4H, m), 6.85–7.03(4H, m),
7.13–7.18 (4H, m), 7.42 (1H, s), 7.54–7.61 (2H, m). Anal.
Calcd for C24H21N5O2: C, 70.06; H, 5.14; N, 17.02.
Found: C, 69.89; H, 5.38; N, 17.07.
Eckardt, A. J.; Conway, B. R.; Westover, L.; Xu, J. Z.;
Look, R.; Demarest, K. T.; Emanuel, S.; Maryanoff, B. E.
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Med. Chem. 1992, 35, 177.
8. 3-Anilino-4-arylmaleimides were reported as GSK-3 selec-
tive inhibitors, but there was no mention of PKCb
inhibition or of anilino-monoindolylmaleimides: Smith,
D. G.; Buffet, M.; Fenwick, A. E.; Haigh, D.; Ife, R. J.;
Saunders, M.; Slingsby, B. P.; Stacey, R.; Ward, R. W.
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13. Prade, L.; Engh, R. A.; Girod, A.; Kinzel, V.; Huber, R.;
Bossemeyer, D. Structure 1997, 5, 1627.
14. PKC enzyme assay system (Amersham Biosciences) and
[c-32P]-ATP were used for enzyme assay. All PKC
isozymes (recombinant human PKC a, b1, b2, c, and e)
were purchased from EMD Biosciences. The assay mix-
ture (final volume 21lL) contained 1.2mM calcium
acetate for PKC a, b1, b2, and c or 0.1mM EGTA for
PKCe, 0.6lg L-a-phosphatidyl-L-serine, 0.05lg phorbol
12-myristate 13-acetate (PMA), 3mM dithiothreitol,
0.1mM ATP, 7mM MgCl2, 2.5kBq [c-32P]-ATP, 90mM
substrate peptide (RKRTLRRL-OH), enzyme (PKC a:
0.9mU, b1: 0.4mU, b2: 9.5mU, c: 28.7mU, e: 5.1mU),
and compound dissolved in DMSO (final concentration
4.8%). Assay mixture was incubated for 15min at 37°C,
and reaction was stopped by addition of 300mM H3PO4.
Assay mixture was blotted onto phosphocellulose paper
(Whatman), washed with 75mM H3PO4 and evaluated by
IP-autoradiography (Fujifilm). The IC50 value was calcu-
lated in a semi-logarithmic proportional manner from the
two points enclosing 50% inhibition.
15. Aliphatic amine derivatives were reported as micromolar
PKC inhibitors: He, M.; Buisine, E.; Tartar, A.; Sergh-
eraert, C. Bioorg. Med. Chem. Lett. 1994, 4, 2845.
16. Neither compound 1 nor 4c inhibited PKA up to the
concentration of 10lM. MESACUP protein kinase assay
kit (Medical & Biological Laboratories) was used for PKA
enzyme assay. PKCe was chosen as a representative of
calcium-independent PKCs to discuss the selectivity since
this isozyme possesses the highest degree of overall
identity with PKCb among this type of PKCs.
9. (a) Rooney, C. S.; Randall, W. C.; Streeter, K. B.; Ziegler,
C.; Cragoe, E. J.; Schwam, H.; Michelson, S. R.; William,
H. W.; Eichler, E.; Duggan, D. E.; Ulm, E. H.; Noll, R.
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M. R.; McDonald, J. H., III Bioorg. Med. Chem. Lett.
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10. All compounds gave appropriate physical spectra data (1H
NMR, IR, and elemental analysis and/or FAB-MS).
11. 3-Anilino-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione (4c): H
1
NMR (300MHz, DMSO-d6) d 6.64–6.95 (6H, m), 7.22
(2H, d, J = 8.1Hz), 7.32 (2H, d, J = 8.0Hz), 9.04 (1H, s),
10.60 (1H, s), 11.16 (1H, br s). Anal. Calcd for
C18H13N3O2: C, 71.28; H, 4.32; N, 13.85. Found: C,
71.10; H, 4.70; N, 13.86.
12. 3-[1-(3-Imidazol-1-ylpropyl)-1H-indol-3-yl]-4-anilino-1H-
pyrrole-2,5-dione (6l): 1H NMR (300MHz, CDCl3) d 2.20
(2H, quintet, J = 4.0Hz), 3.77 (2H, t, J = 4.0Hz), 3.97
17. Davis, P. D.; Elliott, L. H.; Harris, W.; Hill, C. H.; Hurst,
S. A.; Keech, E.; Kumar, M. K. H.; Lawton, G.; Nixon,
J. S.; Wilkinson, S. E. J. Med. Chem. 1992, 35, 994.