Acyclic N-(azacycloalkyl)bisindolylmaleimides: Isozyme selective inhibitors of PKCβ

Article abstract of DOI:10.1016/S0960-894X(03)00286-5

The synthesis and structure-activity relationship (SAR) trends of a new class of N-(azacycloalkyl)bisindolylmaleimides 1, acyclic derivatives of staurosporine, is described. The representative compound for this series (1e) exhibits an IC₅₀ of 40-50 nM against the human PKCβ₁ and PKCβ₂ isozymes and selectively inhibits the PKCβ isozymes in comparison to other PKC isozymes (α, γ, δ, ε, λ, and η). The series is also kinase selective for PKC in comparison to other ATP-dependent kinases. A comparison of the PKC isozyme and kinase activity of the series is made to the kinase inhibitor staurosporine.

Full text of DOI:10.1016/S0960-894X(03)00286-5

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Bioorganic & Medicinal Chemistry Letters 13 (2003) 1857–1859
Acyclic N-(azacycloalkyl)bisindolylmaleimides: Isozyme Selective
Inhibitors of PKCꢀ
Margaret M. Faul,a,* James R. Gillig,b Michael R. Jirousek,c Lawrence M. Ballas,d
Theo Schotten,e Astrid Kahle and Michael Mohre
aGlobal Chemical Process Research and Development Division, Lilly Research Laboratories, A Division of Eli Lilly and Company,
Indianapolis, IN 46285, USA
bDiscovery Chemistry Research and Technologies, Lilly Research Laboratories, A Division of Eli Lilly and Company,
Indianapolis, IN 46285, USA
cLa Jolla Laboratories, Pfizer Global Research and Development, San Diego, CA, USA
dSphinx Laboratories, Research Triangle Park, NC, USA
eLilly Forschung, A Division of Eli Lilly and Company, Hamburg, Germany
Received 3 January 2003; revised 17 March 2003; accepted 19 March 2003
Abstract—The synthesis and structure–activity relationship (SAR) trends of a new class of N-(azacycloalkyl)bisindolylmaleimides 1,
acyclic derivatives of staurosporine, is described. The representative compound for this series (1e) exhibits an IC50 of 40–50 nM
against the human PKCb1 and PKCb2 isozymes and selectively inhibits the PKCb isozymes in comparison to other PKC isozymes
(a, g, d, e, l, and Z). The series is also kinase selective for PKC in comparison to other ATP-dependent kinases. A comparison of
the PKC isozyme and kinase activity of the series is made to the kinase inhibitor staurosporine.
# 2003 Elsevier Science Ltd. All rights reserved.
Protein kinase C (PKC), a family of serine/threonine
specific kinases,1 composed of at least 12 isozymes, are
involved in signal transduction pathways that govern a
wide range of physiological processes including differ-
entiation, proliferation, gene expression, brain function,
membrane transport and the organization of cytoskele-
tal and extracellular matrix proteins which regulate
vascular function.2À4 Therapeutically, antagonists that
possess kinase selectivity for PKC in addition to PKC
isoenzyme selectivity, are potentially useful pharmaco-
logical agents for treatment of a variety of diseases
including diabetes and cancer.5,6
addition, ruboxistaurin was found to be several orders
of magnitude more selective for inhibition of PKCb
relative to the other kinases, and is currently being
evaluated in the clinic for treatment of diabetic compli-
cations.
In our continued evaluation of the biological activity of
compounds in this area, we identified a series of acyclic
(N-azacycloalkyl)bisindolylmaleimides 1a–k, that are
The natural product staurosporine, although a potent
inhibitor of PKC, has limited selectivity in vitro for
both ATP-dependent kinases and individual PKC iso-
zymes (Fig. 1).7 Recently, our group identified a novel
class of macrocyclic bisindolylmaleimides, represented
by ruboxistaurin mesylate (LY333531), that were com-
petitive reversible inhibitors of PKC b1 and b2.3,8À10 In
*Corresponding author. Tel.:+1-317-277-1150; fax:+1-317-276-3014;
e-mail: faul_margaret_m@lilly.com
Figure 1. Chemical structures for (+)-staurosporine and ruboxis-
taurin mesylate.
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00286-5

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
1858
M. M. Faul et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1857–1859
also more selective kinase inhibitors than staurosporine
and have selectivity for PKCb comparable to ruboxis-
taurin. This manuscript will describe the results of this
SAR that has resulted in identification of 1e as a selec-
tive inhibitor of PKCb. This compound is currently
being evaluated in the clinic as an antiangiogenic agent
for the treatment of cancer, either as stand-alone therapy
or in combination with traditional oncolytic therapies.
cium calmodulin, casein kinase and src-tryosine kinase)
was used in tandem with the PKC isozyme panel to
demonstrate the kinase selectivity of the acyclic (N-aza-
cycloalkyl)bisindolylmaleimides 1a–k.
Staurosporine is a potent, non selective PKC inhibitor
(Table 1). However, as reported previously,8 ruboxis-
taurin shows remarkable selectivity for the b isozymes,
especially over the other calcium dependent isozymes
(PKCa and PKCg), which have a high degree of shared
homology in the ATP binding region.
Acyclic (N-azacycloalkyl) bisindolylmaleimides 1b–k
were prepared in 6–35% yield (unoptimized) by a two-
step sequence involving (i) conversion of the N-(aza-
cycloalkyl)indole 2b–k into the corresponding glyoxyl
chloride 3b–k; (ii) reaction of 3b–k with the appro-
priately substituted indolyl-3-acetimidate 4b–k in the
presence of a suitable base to generate an intermediate
hydroxypyrroline that upon dehydration with p-TsOH
or TFAA/pyridine afforded 1b–k (Scheme 1).11,12 The
unsubstituted piperidine derivative 1a, was prepared
from 1g by removal of the Boc-protecting group under
acidic conditions.13 An improved procedure for the
synthesis of these compounds has been developed and
will be reported in the future.14
The acyclic (N-azacycloalkyl)bisindolylmaleimides 1a–k
produced selective inhibition of PKCb when assayed in
the isozyme panel, but were about 10-fold less active
than ruboxistaurin for the PKCb isozymes. These com-
pounds showed good PKCb selectivity with regard to
PKCb inhibition and PKC isozyme selectivity. The
SAR comparing the 5-membered pyrrolidine derivative
1d with the 6-membered piperidine 1c, showed that
comparable results were obtained for PKCb inhibition
and PKC selectivity, thus the SAR was completed with
the achiral piperidine derivatives. The SAR around the
R1 groups attached to the piperidine ring shows that the
larger groups (benzyl 1c, pyridyl 1e and 1h, Boc 1g) are
well tolerated compared to the smaller groups (H 1a,
Me 1b and 1i) and these large substituents do not
change the PKCb activity but decrease PKCa and
PKCg inhibition. The benzyl group also decreases
activity across the panel of calcium independent PKCs
(d, e and Z) (e.g., 1c).
In analogy to our work on the macrocyclic series, a
panel of eight cloned human PKC isozymes (a, b1, b2, g,
d, e, z, and Z) were employed to optimize this series of
PKCb selective antagonists.8 The panel is representative
of the calcium dependent PKC isoforms (a, b and g),
the calcium independent isoforms (d, e and Z) and the
atypical isoforms (z). Using assay conditions that simu-
late the endogenous activation of PKC with DAG and
phosphatidylserine, compounds 1a–k were found to be
PKCb selective in comparison to most of the other iso-
zymes in the panel. Acyclic bisindolylmalimides have
previously been reported to be competitive inhibitors of
ATP-binding, presumably by interacting at the ATP-
binding site.15 An additional criterion for selectivity is
thus the preferential inhibition of the PKCb isozymes
versus other ATP-dependent kinases. An ATP depen-
dent kinase panel consisting of four kinases (PKA, cal-
A large increase in kinase selectivity is observed in going
from staurosporine to the (N-azacycloalkyl)bisindolyl-
maleimides 1a–k. Larger R1 groups (1c, 1e, 1g, and 1h)
increase the selectivity versus pKA and src-tryosine
kinase, while the smaller R1 groups (1a, 1b, and 1f)
showed decreased or similar selectivity for these iso-
zymes. In terms of kinase selectivity we were most con-
Table 2. ATP dependent kinase IC50 values and IC50 values for
inhibition of rat brain PKCa
Kinase IC50 (mM)
Table 1. PKC isozyme IC50 valuesa
PKC isozyme IC50 (mM)
b
Compd
pKA Ca calmodc Casein Kd src-Tke RB-PKCf
1a
1b
1c
1d
1e
1f
1i
1j
1k
1
7
2
2
>100
>100
>40
>100
>100
>100
91
>100
>100
14
nt
>100
>100
nt
>100
68
83
0.4
0.2
4
Compd
a
bI
bII
g
d
e
z
Z
>100 >40
1a
1b
1c
1d
1e
1f
1g
1i
1j
1k
0.3 0.03 0.03
2
0.4
1
4
4
0.2
0.05
>100
>100
>100
4
8
>100
59
10
25
3
7
7.5
2.8
0.7
0.3
0.2
0.2
0.25
0.19
0.32
0.4 0.02 0.01 0.5 0.4 0.4
0.05 0.03
3.8 0.022 0.024 3.5 4.2 9.4
0.8 0.03 0.03 0.3
0.3 0.03 0.01 0.4 0.4 0.9
0.2 0.08
0.2 0.02 0.01 0.5 0.4 0.05
0.4 0.02 0.005 0.3 0.3 0.4
0.34 0.019 0.026 1.3 0.97 0.39
6
7
6
4
>100 0.5
>100 0.042
2
1
8
0.4
0.05
8
0.04
0.04
11
nt
9
>100
6
5
7
6
Staurosporine 0.10
Ruboxistaurin >100
0.004
6.2
0.001
>100
5
7
>100
9.3 0.13
>1.5 0.005
>100 0.052
aThe values are the average of at least three independent determina-
tions from eight-point titration curves.
Staurosporine 0.045 0.023 0.019 0.11 0.028 0.018
Ruboxistaurin 0.36 0.0047 0.0059 0.3 0.25 0.6
bBovine heart cAMP dependent protein kinase catalytic subunit kinase
assay.
aMeasurements are the average of at least three independent determi-
nations from eight-point titration curves. The typical standard
deviation was 30% of the IC50 value. Cloned human isozymes were
used in the assay that were activated by DAG using phosphatidyl-
serine vesicles incubated with [32P]ATP and histone or myelin basic
protein as a substrate.
cPurified mammalian brain calcium calmodulin dependent protein
kinase assay.
dPurified rat brain casein protein kinase II assay.
esrc protein tryrosine kinase assay.
fPurified rat brain protein kinase C assay.

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
M. M. Faul et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1857–1859
1859
Scheme 1. Method employed for the synthesis of acyclic (N-azacycloalkyl)bisindolylmaleimide.
5. Goekjian, P. G.; Jirousek, M. R. Exp. Opin. Invest. Drugs
2001, 10, 2117.
6. Van Gijn, R.; Zuidema, X.; Bult, A.; Beijnen, J. H. J.
Oncol. Pharm. Pract. 1999, 5, 166.
7. Gribble, G. W., Berthel, S. J. Stud. Nat. Prod. Chem. 1993,
(Stereoselective Synthesis (Pt. H)), 365.
cerned with the inhibition of calcium calmodulin
because of the possibility of toxicological problems and
wanted to advance a compound that was at least as
selective for calcium calmodulin as ruboxistaurin.
The second criterion employed in evaluating our com-
pounds was rat brain PKC inhibition. Although we
were most interested in inhibition of the human iso-
zymes, a compound that approximated the activity of
ruboxistaurin versus rat brain PKC would help us to
decide which compound to carry forward.16 Compar-
ison of rat brain PKC inhibition data and kinase selec-
tivity data (Table 2), indicated that only compound 1e
produced the required kinase selectivity, PKC selectiv-
ity, and rat brain PKC inhibition and was selected as
the clinical candidate.
8. Jirousek, M. R.; Gillig, J. R.; Gonzalez, C. M.; Heath,
W. F.; McDonald, J. H. R.; Neel, D. A.; Rito, C. J.; Singh, U.;
Stramm, L. E.; Melikian-Badalian, A.; Baevsky, M.; Ballas,
L. M.; Hall, S. E.; Winneroski, L. L.; Faul, M. M. J. Med.
Chem. 1996, 39, 2664.
9. Danis, R. P.; Bingaman, D. P.; Jirousek, M. R.; Yang, Y.
Invest. Ophthal. Vis. Sci. 1998, 39, 171.
10. Faul, M. M.; Winneroski, L. L.; Krumrich, C. A.; Sulli-
van, K. A.; Gillig, J. R.; Neel, D. A.; Rito, C. J.; Jirousek,
M. R. J. Org. Chem. 1998, 63, 1961.
11. Bit, R. A.; Crackett, P. H.; Harris, W.; Hill, C. H. Tet.
Lett. 1993, 34, 5623.
12. Preparation of 1b–k: These compounds were prepared by
a slight modification of the process described in ref 11. To an
ice-cooled solution of the indole derivative 2b,c,d,e,f,i, j and k
(2.5 mmol) in 6 mL of absolute Et2O under argon was added
oxalyl chloride (1.1 equiv) dropwise. Stirring was continued
for 30 min at 0 ꢀC. The crystalline precipitate of the corre-
sponding indol-3-yl glyoxyl chloride thus obtained was filtered
off under an argon atmosphere with exclusion of moisture and
rinsed with a small amount of Et2O. The precipitate then was
redissolved or suspended in 15 mL of dry DCM and 2.5 mmol
(1.0 equiv) of the imidate ester hydrochloride 4 added. The
mixture was cooled to 0 ꢀC and a solution of 12.6 mmol (5
equiv) of Et3N in 4 mL of DCM added under stirring, main-
taining the temperature at 0 ꢀC. The mixture was warmed to rt
and stirring was continued for 4 h. Excess of TsOH mono-
hydrate (7 equiv) was added under cooling (slightly exother-
mic) and the reaction stirred for 3.5 h. The organic solution
was washed successively with saturated aqueous Na2CO3,
brine and water and dried over Na2SO4. After evaporation the
residue was further purified via column chromatography or by
recrystallization from dioxane, ethyl acetate or Et2O.
13. Preparation of 1a: Boc-protecting group was cleaved as
follows: 0.13 mmol of the maleimide was added to a mixture of
0.5 mL of TFA and 0.05 mL ethane thiol and stirred for 7 min
while monitored by TLC. A saturated aqueous solution of
Na2CO3 was added carefully, followed by DCM. The precipitate
obtained was filtered, thoroughly rinsed with water and dried.
14. Faul, M. M.; Grutsch, J. L.; Kobierski, M. E.; Kopach,
M. E.; Krumrich, C. A.; Staszak, M. A.; Sullivan, K. A.;
Udodong, U.; Vicenzi, J. T. Tetrahedron 2003, submitted for
publication.
In conclusion, a new series of acyclic (N-azacycloalkyl)
bisindolylmaleimides 1a–k have been synthesized. The
piperidinyl derivative 1e exhibits nanomolar activity
against PKCb and at least >26-fold selectivity for
PKCb versus PKCa. Kinase selectivity is also main-
tained in this series and 1e is >300-fold more selective
for PKCb relative to calcium calmodulin and >3000-fold
more selective for src-tyrosine kinase. Therefore, 1e was
identified as a clinical candidate and is currently under
evaluation in the clinic for the treatment of cancer.
Acknowledgements
The authors would like to acknowledge Dr. Kirk Ways
for helpful discussions during the course of this work.
References and Notes
1. Lester, D. S.; Epand, R. M. Protein Kinase C; Current
Concepts and Future Perspectives; Ellis Harwood: New York,
1992.
2. Carter, C. A. Curr. Drug Targets 2000, 1, 163.
3. Ishii, H.; Jirousek, M. R.; Koya, D.; Takagi, C.; Xia, P.;
Clermont, A.; Bursell, S. E.; Kern, T. S.; Ballas, L. M.; Heath,
W. F.; Stramm, L. E.; Feener, E. P.; King, G. L. Science 1996,
272, 728.
15. Kennedy, M. B.; McGuinness, T.; Greengard, P. J. Neu-
rosci. 1983, 3, 818.
16. PKCs are ubiquitously expressed, although the levels of
individual isoaymes vary considerable among different cells
and tissues. PKCa is found in most cells and tissues. PKCb is
mainly expressed in immune function cells as well as some
epithelial cells.
4. Hisayama, T.; Nakayama, K.; Saito, N.; Kihara, Y.; Nish-
izawa, S.; Obara, K.; Ishizuka, T. Nippon Yakurigaku Zasshi
2002, 119, 65.