2-(6-Phenyl-1H-indazol-3-yl)-1H-benzo[d]imidazoles: Design and synthesis of a potent and isoform selective PKC-ζ inhibitor

Article abstract of DOI:10.1016/j.bmcl.2008.11.105

The inhibition of PKC-ζ has been proposed to be a potential drug target for immune and inflammatory diseases. A series of 2-(6-phenyl-1H indazol-3-yl)-1H-benzo[d]imidazoles with initial high crossover to CDK-2 has been optimized to afford potent and selec

Full text of DOI:10.1016/j.bmcl.2008.11.105

Bioorganic & Medicinal Chemistry Letters 19 (2009) 908–911
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-(6-Phenyl-1H-indazol-3-yl)-1H-benzo[d]imidazoles: Design and synthesis
of a potent and isoform selective PKC-f inhibitor
a,
a
a
a
a
John I. Trujillo a, , James R. Kiefer , Wei Huang , Atli Thorarensen , Li Xing , Nicole L. Caspers ,
Jacqueline E. Day a, Karl J. Mathis b, Kuniko K. Kretzmer b, Beverley A. Reitz b, Robin A. Weinberg b,
Roderick A. Stegeman a, Ann Wrightstone c, Lori Christine c, Robert Compton c, Xiong Li b
*
*
a Department of Medicinal Chemistry, Pfizer Global Research and Development, 700 Chesterfield Pkwy, AA236, Chesterfield, MO 63017, USA
b Department of Biochemical Pharmacology, Pfizer Global Research and Development, Chesterfield, MO 63017, USA
c Department of Biochemistry/Enzymology, Pfizer Global Research and Development, Chesterfield, MO 63017, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The inhibition of PKC-f has been proposed to be a potential drug target for immune and inflammatory
diseases. A series of 2-(6-phenyl-1H indazol-3-yl)-1H-benzo[d]imidazoles with initial high crossover to
CDK-2 has been optimized to afford potent and selective inhibitors of protein kinase c-zeta (PKC-f).
The determination of the crystal structures of key inhibitor:CDK-2 complexes informed the design and
analysis of the series. The most selective and potent analog was identified by variation of the aryl substi-
tuent at the 6-position of the indazole template to give a 4-NH2 derivative. The analog displays good
Received 8 October 2008
Revised 24 November 2008
Accepted 26 November 2008
Available online 6 December 2008
Keywords:
PKC-f
PKC isoforms
CDK-2
selectivity over other PKC isoforms (
a
, bII,
c, d,
e
,
l
, h,
g
and
i
/k) and CDK-2, however it displays marginal
selectivity against a panel of other kinases (37 profiled).
Ó 2008 Elsevier Ltd. All rights reserved.
NF-jb
Indazole
Surrogate kinases
X-ray crystallography
The PKC family of kinases is comprised of 11 members that are
grouped into three categories based upon their molecular structure
authors described their lead compound as possessing significant
crossover onto CDK-2 despite possessing good GSK-3 activity.
The strategy employed to enhance the selectivity of their lead
was to explore variation of the 6-aryl ring by replacement with
alternate heterocycles and substituted phenyl derivatives. Indeed,
the Glaxo group was successful in identifying analogs which
spared CDK-2, as well as other derivatives to provide a compound
with excellent selectivity against a panel of 23 other kinases. Given
and mode of activation, conventional PKCs ( , bI, bII,
a
c), novel PKCs
(d, , h and ), and atypical PKCs (f and i
e
,
l
g
/k).1 Recent literature
has implicated the atypical PKCs in important cellular signaling
events, PKC-f in particular has been proposed as an intermediary
in the activation of the NF-
The NF- b pathway is important in immune and inflammatory dis-
j
b pathway and IL-4/Stat6 pathway.2
j
eases, therefore an inhibitor of PKC-f may serve to reduce the
severity of these types of diseases.3 Recently we identified com-
pound 1, a 6-phenyl-3-benzimidazole through a screening of our
corporate compound collection. The compound was profiled
against a variety of kinases and found to be especially active
against PKC-f (IC50 = 22 nM) and CDK-2 (IC50 = 102 nM).
Thus, a key objective of our chemistry effort was to enhance the
PKC-f selectivity of our lead. Recent publications by GlaxoSmithK-
line disclosed the identification of GSK-3 kinase inhibitors belong-
ing to the 6-aryl-pyrazolo[3,4-b]-pyridine structural class, 2,
analogous to our 6-aryl indazoles.4 In these publications the
* Corresponding authors. Tel.: +1 636 244 1937 (J.I.T.); tel.: +1 636 247 6173
(J.R.K.).
E-mail addresses: john.i.trujillo@pfizer.com (J.I. Trujillo), james.r.kiefer@pfizer.-
Figure 1. Structure and biological activity of PKC-f lead molecule and GSK
com (J.R. Kiefer).
compound.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.105

J. I. Trujillo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 908–911
909
Scheme 1. Preparation of 6-aryl/heteroaryl indazole-benzimidazoles. Reagents and
conditions: (a) NaNO2, 2 N HCl, acetone:H2O (1:1), 55%; (b) S, DMF, 80 °C, 71% or
Na2S2O5, EtOH:H2O (10:1), 85%; (c) Boc2O, CH3CN, DMAP, 95%; (d) RB(OH)2,
PdCl2(dppf), CH3CN:H2O (10:1), Na2CO3, 50–70%; (e) 30% TFA, CH2Cl2, rt, 50–80%
yield.
a range of aryl/heteroaryl boronic acids afforded the corresponding
coupled products, which were then treated with 30% TFA in CH2Cl2,
followed by purification via RP-HPLC to give the final compounds 1
and 3–25.
Figure 2. Schematic of binding interactions of compound 1 with PKC-f predicted by
homology modeling and molecular docking.8
Initially, we investigated the role of the 2-OMe and thus pre-
pared compounds 3 and 4; not surprisingly neither derivative dis-
played enhanced selectivity for PKC-f over CDK-2; rather, both
compounds were more potent against both enzymes relative to
compound 1 (Table 1). Subsequently, to further understand the
role of the 4-OH substituent, an analog with the 4-OH converted
to a 4-OMe 6, and two analogs with the 4-OH removed entirely,
were prepared (7 and 8). The analogs lost significant activity
against both kinases, thus pointing to the need for a substituent
capable of donating an H-bond. In order to probe the area further,
analogs capable of making H-bonds but differing in size and donor/
acceptor status were prepared 5( , 9, 13, 15, 17, 20, 23, and 25, Table
1). Notably, compound 9 displayed very good PKC-f activity com-
bined with a reduction in CDK-2 activity, thus resulting in a selec-
tivity ratio of ꢀ200-fold. Analogs of compound 9 were prepared to
further investigate the area surrounding the 4-NH2 (11, 12, 15, and
16).
The data from these analogs suggests a high degree of sensitiv-
ity of the PKC-f kinase to steric and electronic variations. For in-
stance, addition of the 2-OMe moiety to either the 4-OH (1 and
4) or the 4-NH2 (9 and 17) both result in approximately a 4-fold
loss of potency against PKC-f but also show a net 2-fold improve-
ment in selectivity versus CDK-2. By contrast, substitution of 4-OH
for 4-NH2 (1 and 17, 4 and 9) does not affect potency for PKC-f, but
the change causes 70- to 100-fold weaker inhibition of CDK-2. To
investigate the basis for the observed selectivity, we determined
the crystal structures of key compounds either bound by CDK-2
alone (9 and 17) or the CDK-2:CyclinA complex (9; Fig. 3 and
Supplementary Materials).
Glaxo’s success in identifying a selective inhibitor of GSK-3, we
sought to apply a similar strategy in the development of a selective
PKC-f inhibitor.
Based upon the binding mode observed for similar compounds
in other kinases, we surmised that compound 1 would form three
hydrogen bonds to the hinge region of PKC-f and potentially an
additional hydrogen bond between its phenol and Glu300 of helix
C of the kinase (Fig. 2). A comparison of the protein sequences in
the binding sites of CDK-2 with the PKC isoforms identified multi-
ple residues that differ between CDK-2 and PKC-f as well as several
amino acid differences between the PKC isoforms. Since helix C of
kinases can be in at least two distinct conformations, depending on
the state of the enzyme (active or inactive),5 and since the struc-
tures of PKC family kinases were not available at the time of our
work (since then, three different isoforms now have known struc-
tures),6 we could not be certain of that hydrogen bond forming. In
order to validate the assumptions of the binding mode and to test
the hypothesis that variation of the C-6-substituent would lead to
a CDK-2 sparing compound, a series of analogs were prepared
(compounds 3–25). Crystal structures of select analogs were then
determined in complex with CDK-2.7
The 6-ary/heteroaryl indazole-benzimidazoles were prepared
according to a modification of procedures described previously,9
and as described in Scheme 1. Thus the 6-bromo-1H-indazole-3-
carbaldehyde 27 was prepared by oxidation of 6-bromo-1H-indole
26 with NaNO2/HCl in 55% yield.9b Formation of the benzimidazole
ring system was accomplished via one of two routes: (i) elemental
sulfur in DMF at 80 oC9; or (ii) sodium thiosulfate in EtOH:H2O10
using 3-(4-methylpiperazin-1-yl)benzene-1,2-diamine5 as the dia-
mine partner 28. Following formation of the benzimidazole ring
system the free NH of the indazole and benzimidazole were pro-
tected as their Boc-derivative in quantitative yield to give the
key intermediate 29. Suzuki cross-coupling of the bromide 29 with
In both the active (CDK-2:CyclinA complex) and inactive (CDK-2
alone) forms of the crystal structures, the inhibitor binds in nearly
the identical position, the largest difference being a ꢀ10° rotation
of the plane of the compound in the binding site when comparing 9
complexed with CDK-2:CyclinA and 17 bound by CDK-2 alone. The
center of the rotation was approximately N1 of the indazole ring.

910
J. I. Trujillo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 908–911
Table 1
Inhibition of hPKC-f and hCDK-2 by selected indazole analogs-phenyl derivativesa
1, 3-21
22-25
Compound
R
PKC-f
CDK-2
Ratio
1
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
2-OMe, 4-OH
3-OMe, 4-OH
4-OH
22
2.26
5.6
290
12,400
5520
19,000
5.18
1510
55.5
102
4.1
13.3
4.6
1.8
2.4
74
0.8
1.8
0.5
201
4
31
4.8
1.1
1.1
186
—
4-CH2OH
21,500
10,000
10,000
10,000
1040
6100
1730
247
10,000
10,000
109,000
—
2-OMe, 4-OMe
2-OMe, 4-H
2-OMe, 4-F
4-NH2
3-NH2
3-F, 4-NH2
3-OMe, 4-NH2,
4-CH2NH2
4-NHCOMe
4-NHMe
4-NMe2
2-OMe, 4-NH2
4-CO2H
51.3
31,100
8800
585
1310
23.3
27,300
13,600
11,800
8930
4080
6840
1070
3020
10,000
10,000
10,000
—
429
0.4
0.7
—
4-COMe
4-CONH2
4-SO2NH2
5-Indolyl
6-Quinolyl
6-Indazole
6-Benzofuran
—
—
10,000
10,000
—
2.5
1.5
—
—
—
a
Values are in nM.
While the compound positions were conserved, the conformation
of the kinases in their DFG loop, flap, and C helix regions increas-
ingly differed. In the structure with CDK-2 alone, helix C was ob-
served in its inactive conformation with hydrophobic side chains
Ile52 and Leu55 projecting inward towards the aniline substituent
of the compound. Only the three predicted hinge hydrogen bonds
are formed between 9 and the protein in its complex with CDK-2
alone. By contrast, in the CDK-2:CyclinA complex with 9, six hydro-
gen bonds are formed in total, three between backbone atoms of
the hinge region and the three adjacent nitrogens of the indazole
and benzimidazole rings, one between the terminal piperazine
nitrogen and Asp86, and the final bonds between the aniline nitro-
gen of 9 and the side chain of Glu51 (Glu300 in PKC-f) of helix C
and the backbone nitrogen of Phe146 of the DFG loop. This binding
mode agrees with both the SAR of the series as well as the model
for the binding of 1 to PKC-f (Fig. 1).
As has been observed in previous reports of the transition from
inactive to active CDK-2, helix C both pivots and rotates to present
a different, hydrophilic face (Glu51) to the active site. While the
structures of the CDK-2:CyclinA complex most closely matched
the SAR for the series, those crystals typically diffracted X-rays to
only around 3 Å resolution. By contrast, the crystals of CDK-2 alone
routinely produce data to better than 2 Å resolution. For that rea-
son, we primarily pursued structures of CDK-2 alone to probe the
structural aspects of this series.
Figure 3. Crystal structure of compound 9 in complex with CDK-2:CyclinA. (a)
Comparison of the structure of compound 9 bound to CDK-2 alone (tan) and in
complex with CyclinA (blue). (b) CDK-2:CyclinA complex structure showing sites of
amino acid variation between CDK-2 (tan) and PKC-f (green) are shown as
elaborated side chains.
(Table 2), with only the PKC-
i
isoform exhibiting less than 10-fold
is most closely related to PKC-f,
selectivity. Interestingly, PKC-
i
only differing by a single amino acid in the binding site. However,
the other PKC isoforms and CDK-2 differ from PKC-f at several
other positions, many of which flank the 6-substituent of the inda-
Table 2
PKC isoform selectivity for compounds 1 and 9a
Compound
f
a
bII
c
d
e
g
i
h
With the selectivity for CDK-2 (>200-fold) achieved, the next
step was to profile the compounds against a panel of PKC isoforms,
as well as against other relevant kinases. Indeed, when 9 was pro-
filed against a panel of PKC isoforms good selectivity was achieved
1
9
1
1
109
8000
126
20,671
1
711
6
918
88
1895
1695
12,424
7
10
4
1218
a
Values are fold selectivity (IC50-PKC isoform/IC50-PKC-f).

J. I. Trujillo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 908–911
911
Figure 4. Selectivity profiling12 of compounds 1 and 9 against 37 different kinases (upstate panel). Values are % inhibition at 10
lM using ATP = Km.
WO 2004/104217A2.; (e) Redig, A. J.; Platanias, L. C. J. Interferon Cytokine Res.
2007, 27, 622.
zole, poised to read out the changes discussed above (Fig. 3). In
spite of this progress on selectivity against both closely and dis-
tantly related kinases, when 9 was profiled against a broader set
of other kinases (Upstate panel), significant crossover was ob-
served (Fig. 4).
4. (a) Witherington, J.; Bordas, V.; Garland, S. L.; Hickey, D. M. B.; Ife, R. J.; Liddle,
J.; Saunders, M.; Smith, D. G.; Ward, R. Bioorg. Med. Chem. Lett. 2003, 13, 1577;
(b) Witherington, J.; Bordas, V.; Haigh, D.; Hickey, D. M. B.; Ife, R. H.; Rawlings,
A. D.; Sligsby, B. P.; Smith, D. G.; Ward, R. W. Bioorg. Med. Chem. Lett. 2003, 13,
1581; (c) Witherington, J.; Bordas, V.; Gaiba, A.; Garton, N. S.; Naylor, A.;
Rawlings, A. D.; Slingsby, B. P.; Smith, D. G.; Takle, A. K.; Ward, R. W. Bioorg.
Med. Chem. Lett. 2003, 13, 3055; (d) Witherington, J.; Bordas, V.; Gaiba, A.;
Naylor, A.; Rawlings, A. D.; Slingsby, B. P.; Smith, D. G.; Takle, A. K.; Ward, R.
Bioorg. Med. Chem. Lett. 2003, 13, 3059.
5. (a) Jeffrey, P. D.; Russo, A. A.; Polyak, K.; Gibbs, E.; Hurwitz, J.; Massague, J.;
Pavletich, N. P. Nature 1995, 376, 313; (b) Lawrie, A. M.; Noble, M. E.; Tunnah,
P.; Brown, N. R.; Johnson, L. N.; Endicott, J. A. Nat. Struct. Biol. 1997, 4, 796.
6. (a) Xu, Z. B.; Chaudhary, D.; Olland, S.; Wolfrom, S.; Czerwinski, R.; Malakian,
K.; Lin, L.; Stahl, M. L.; Joseph-McCarthy, D.; Benander, C.; Fitz, L.; Greco, R.;
Somers, W. S.; Mosyak, L. J. Biol. Chem. 2004, 279, 50401; (b) Messerschmidt, A.;
Macieira, S.; Velarde, M.; Baedeker, M.; Benda, C.; Jestel, A.; Brandstetter, H.;
Neuefeind, T.; Blaesse, M. J. Mol. Biol. 2005, 352, 918; (c) Grodsky, N.; Li, Y.;
Bouzida, D.; Love, R.; Jensen, J.; Nodes, B.; Nonomiya, J.; Grant, S. Biochemistry
2006, 45, 13970.
7. The crystal structures of compounds 9 and 17 bound at the active sites of CDK-
2 or CDK-2 in complex with CyclinA have been deposited in the Protein
Databank (Accession codes: 3EZV, 3EZR, 3FX5). Data for those experiments
were in part collected at IMCA-CAT (17-ID) beamline at the Advanced Photon
Source and was supported by the companies of the Industrial Macromolecular
Crystallography Association through a contract with the Center for Advanced
Radiation Sources at the University of Chicago.
8. Molecular graphics images prepared with PyMol v0.98 (DeLano, W. L.;
www.pymol.org) and Pov-Ray v3.6 (www.povray.org).
9. (a) Kania, R. S.; Bender, S. L.; Borchardt, A. J.; Braganza, J. F.; Cripps, S. J.; Hua, Y.;
Johnson, D. M.; Johnson, T. O.; Luu, H. T.; Palmer, C. L.; Reich, S. H.; Tempczyk-
Russell, A. M. WO 2001/02369A2.; (b) Buchi, G.; Lee, G. C. M.; Yang, D.;
Tannenbaum, S. R. J. Am. Chem. Soc. 1986, 108, 4115.
While the molecular determinants of kinase selectivity can
occasionally be linked to a single amino acid difference, in most
cases multiple amino acid changes and subtle shifts of conforma-
tion of the protein govern the answer. Comparing the PKC-f dock-
ing model for 1 and the CDK-2:CyclinA complex with 9 reveals a
tilt in the position of the compounds in the binding site (see Sup-
plementary Materials). This shift allows the phenyl ring of 1 to ro-
tate ꢀ70° and its 4-OH group to come within 3.9 Å of the side chain
of Asp394. Conversion of the hydroxyl moiety of 1 to an amine as
in 9 and 17 would provide a second hydrogen bond donor that
could, with slight shifts to the protein or ligand position, allow it
to form an interaction with that Asp residue. Interestingly, CDK-2
and the PKC isoforms with decreased affinity for 9 and 17 com-
pared to 1 all differ from PKC-f and PKC-i at Phe304, Ile330,
Thr393, and Tyr395: amino acid positions flanking the phenyl ring
of the inhibitors (Fig. 1). However, comparison of amino acid iden-
tity across those positions in the broader panel of kinases assayed
does not reveal a clear trend to predict selectivity in this series.
In conclusion, by varying the nature of the 6-substituent of the
indazole-benzimidazole11 1, a potent and highly PKC isoform
selective compound 9 was identified.13 The compound unfortu-
nately does not possess the desired selectivity across a broader
10. (a) Ozden, S.; Atabey, D.; Yildiz, S.; Goker, H. Bioorg. Med. Chem. 2005, 13, 1587;
(b) Thompson, L. K.; Ramaswamy, B. S.; Seymour, E. A. Can. J. Chem. 1977, 55,
878.
range of other kinases (<50% inhibition at 10 lM). Structure based
design of selectivity against these other kinases is currently
underway.
11. (a) For other reports of kinase inhibitors belonging to the indazole-
benzimidazole structural class, see: Georges, G.; Goller, B.; Limberg, A.;
Rueger, P.; Rueth, M.; Schuell, C.; Stahl, M. WO 2007/107346A1.; (b) Teng,
M.; Zhu, J.; Johnson, M. D.; Chen, P.; Kornmann, J.; Chen, E.; Blasina, A.;
Register, J.; Anderes, K.; Rogers, C.; Deng, Y.; Ninkovic, S.; Grant, S.; Hu, Q.;
Lundgren, K.; Peng, Zhengwei; Kania, R. S. J. Med. Chem. 2007, 50, 5253; (c)
Georges, G.; Goller, B.; Kuenkele, K.; Limberg, A.; Reiff, U.; Rueger, P.; Rueth, M.;
Schuell, C. WO 2006/063841A2.; (d) McBride, C. M.; Renhowe, P. A.; Gesner, T.
G.; Jansen, J. M.; Lin, J.; Ma, S.; Zhou, Y.; Shafer, C. M. Bioorg. Med. Chem. Lett.
2006, 16, 3789; (e) McBride, C. M.; Renhowe, P. A.; Heise, C.; Jansen, J. M.;
Lapointe, G.; Ma, S.; Pineda, R.; Vora, J.; Wiesmann, M.; Shafer, C. M. Bioorg.
Med. Chem. Lett. 2006, 16, 3595; (f) Foloppe, N.; Fisher, L. M.; Francis, G.;
Howes, R.; Kierstan, P.; Potter, A. Bioorg. Med. Chem. 2006, 14, 1792.
12. Heat map prepared using Spotfire DecisionSite 8.1.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.11.105.
References and notes
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2005/079300A2. For a recent report of a family of natural products that inhibit
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