3-(Indol-2-yl)indazoles as Chek1 kinase inhibitors: Optimization of potency and selectivity via substitution at C6

Article abstract of DOI:10.1016/j.bmcl.2006.08.118

The development of 3-(indol-2-yl)indazoles as inhibitors of Chek1 kinase is described. Introduction of amides and heteroaryl groups at the C6 position of the indazole ring system provided sufficient Chek1 potency and selectivity over Cdk7 to permit escape from DNA damage-induced arrest in a cellular assay. Enzyme potency against Chek1 was optimized by the incorporation of a hydroxymethyl triazole moiety in compound 21 (Chek1 IC₅₀ = 0.30 nM) that was shown by X-ray crystallography to displace one of three highly conserved water molecules in the HI region of the ATP-binding cleft.

Full text of DOI:10.1016/j.bmcl.2006.08.118

Bioorganic & Medicinal Chemistry Letters 16 (2006) 6049–6053
3-(Indol-2-yl)indazoles as Chek1 kinase inhibitors:
Optimization of potency and selectivity via substitution at C6
Mark E. Fraley,a,* Justin T. Steen,a Edward J. Brnardic,a Kenneth L. Arrington,a
Keith L. Spencer,a Barbara A. Hanney,a Yuntae Kim,a George D. Hartman,a
Steven M. Stirdivant,b Bob A. Drakas,b Keith Rickert,b Eileen S. Walsh,b
Kelly Hamilton,b Carolyn A. Buser,b James Hardwick,b Weikang Tao,b
Stephen C. Beck,b Xianzhi Mao,b Robert B. Lobell,b Laura Sepp-Lorenzino,b
Youwei Yan,c Mari Ikuta,c Sanjeev K. Munshi,c
Lawrence C. Kuoc and Constantine Kreatsoulasd
aDepartment of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
bDepartment of Cancer Research, Merck Research Laboratories, West Point, PA 19486, USA
cDepartment of Structural Biology, Merck Research Laboratories, West Point, PA 19486, USA
dDepartment of Molecular Systems, Merck Research Laboratories, West Point, PA 19486, USA
Received 13 July 2006; revised 29 August 2006; accepted 29 August 2006
Available online 15 September 2006
Abstract—The development of 3-(indol-2-yl)indazoles as inhibitors of Chek1 kinase is described. Introduction of amides and het-
eroaryl groups at the C6 position of the indazole ring system provided sufficient Chek1 potency and selectivity over Cdk7 to permit
escape from DNA damage-induced arrest in a cellular assay. Enzyme potency against Chek1 was optimized by the incorporation of
a hydroxymethyl triazole moiety in compound 21 (Chek1 IC50 = 0.30 nM) that was shown by X-ray crystallography to displace one
of three highly conserved water molecules in the HI region of the ATP-binding cleft.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Although effective, DNA damaging agents used in che-
motherapy show little selectivity for killing tumor cells
over normal proliferating cells. Therefore, strategies tar-
geting an increase in the therapeutic window of these
agents are warranted. In normal cells, DNA damage
causes cell cycle arrest through the tumor suppression
protein p53 and activation of checkpoint kinase Chek1,
allowing for repair.1–3 In tumor cells that have impaired
p53 function, a defect common to 50–70% of all cancers,
survival from DNA damage relies primarily on activa-
tion of Chek1 which ultimately leads to inactivation of
Cdc2 (Cdk1), a cyclin-dependent kinase whose activity
is critical to cell cycle progression. In these tumors,
Chek1 inhibition results in abrogation of arrest and pre-
mature cell cycle progression into mitosis where cell
death occurs through mitotic catastrophe and apoptotic
pathways. Accordingly, Chek1 inhibitors4 have the po-
tential to sensitize p53-impaired tumor cells to DNA
damaging agents during chemotherapy and thereby in-
crease the efficacy and alleviate the toxicity to normal
cells associated with such treatments. In this paper, we
report the optimization of Chek1 potency and selectivity
of lead 1 (Table 1) through modification of the indazole
substituent at C6.
Lead 1 was designed originally as a KDR kinase inhib-
itor and was subsequently identified as an ATP-compet-
itive inhibitor of Chek1 from a directed screening
campaign. Our initial efforts to improve the potency of
1 involved the introduction of a basic sidechain to C50
of the indole ring system, a strategy successfully em-
ployed for this and closely related series of KDR kinase
inhibitors.5,6 Consistent with our previous findings,
addition of a benzylic amine to C50 provided a 10-fold
improvement in Chek1 potency with 2.7 Not surprising-
ly, 1 and 2 were devoid of cellular activity in both a
Keywords: Chek1 kinase; Checkpoint escape; DNA damage; CDK7
kinase; Oncology; Indazoles; Indoles.
*
Corresponding author. Tel.: +1 215 652 6937; fax: +1 215 652
7310; e-mail: mark_fraley@merck.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.118

6050
M. E. Fraley et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6049–6053
Table 1. Chek1 inhibitory and cellular activities of 1–5
Compound
R1
H
R2
Chek1 IC50 (nM)
27
Phos IC50 (nM)
>10,000
CEA IC50 (nM)
>10,000
1
2
3
4
5
2.3
17
>10,000
1000
—
>10,000
>10,000
—
CN
Cl
120
640
H
—
—
Chek1 autophosphorylation assay (Phos)8 and in a func-
tional checkpoint escape assay (CEA)9 that measures re-
lease of H1299 tumor cells from DNA damage-induced
cell cycle arrest and progression into mitosis following
Chek1 inhibition. The lack of activity of 1 and 2 in these
assays was attributed to a combination of low aqueous
solubility and high polarity engendered by the acidic tet-
razole group at C6. We postulated that cellular activity
would be gained by replacement of the tetrazole with a
neutral substituent, but potentially at the expense of en-
zyme potency, since an X-ray structure of 1 bound to
Chek1 showed the tetrazole engaged in an ionic interac-
tion with Lys38 located in hydrophobic region I (HI) of
the ATP-binding cleft.10 The Chek1 inhibitory activity
of an initial set of compounds confirmed the latter pre-
diction (Table 1), but encouragingly showed that the
cyano group in 3 provided significant retention in poten-
cy when exchanged with the tetrazole moiety.
this hypothesis was gained in a follow-up study where
we demonstrated that Cdk7 siRNA inhibited checkpoint
escape of a functionally active Chek1 inhibitor in this as-
say (data not shown). Consequently, to achieve check-
point escape, the desired cellular response to Chek1
inhibition, we set out to identify compounds in this series
that showed selectivity for Chek1 inhibition over Cdk7.
Our design strategy to gain selectivity for Chek1 cen-
tered on modification of the C6 substituent whose bond
vector is directed toward HI. The peptide sequences of
Chek1 and Cdk7 differ in this back region. Most nota-
bly, the residue equivalent to Leu84 of Chek1,15 the
so-called ‘gatekeeper’ residue which forms part of HI,
is a more sterically constraining Phe in Cdk7.16 In an at-
tempt to exploit this difference, we found that replace-
ment of the C6 cyano group with a methyl ester (7) or
small amides (8–10) retained Chek1 potency and affor-
ded a significant enhancement in selectivity over Cdk7
relative to 6 (Table 2). Of greater gratification was that
these compounds displayed cellular checkpoint escape
with EC50’s in the low micromolar range,17 a finding
consistent with our hypothesis concerning the impor-
tance of adequate Chek1 selectivity for functional activ-
ity in this assay. Heterocycles were also investigated as
replacements for the cyano group, although with mixed
results. Pyrazoles 11 and 13 and thiazole 14 were equi-
potent against Chek1 and Cdk7, and like 3 and 6 were
inactive in the CEA.18 Methylation of 11, providing
12, was attempted to improve Chek1 selectivity but
was found detrimental to Chek1 potency. On the other
hand, thiazole 15 maintained potent Chek1 inhibitory
activity, displayed 80-fold selectivity for Chek1 over
Cdk7, and accordingly demonstrated checkpoint escape
in the functional assay. Incorporation of an N1-linked
1,2,3-triazole in 16 enhanced Chek1 potency and appar-
ently provided sufficient selectivity to render this com-
While 3 was active in the Phos assay, indicating that its
intrinsic potency and cell membrane permeability11 were
sufficient to inhibit Chek1 activity in cells, it was found to
be functionally inactive in the CEA. A potential explana-
tion for the unexpected lack of cellular response in the
CEA was revealed through the profiling of 3 against a
broad panel of kinases where strong inhibitory activity
was measured against Cdk7 (IC50 = 39 nM) in particu-
lar.12 Cdk7 is a member of the cyclin-dependent kinase
family that plays a central role in the regulation of cell
cycle transitions.13,14 Specifically, Cdk7 activates other
Cdks critical to cell cycling, including Cdc2, through
phosphorylation of key threonine residues. Because
Cdk7 activity is essential to cell cycle progression, we
proposed that the inactivity of 3 in the CEA was due
to counterproductive Cdk7 inhibition resulting in cellu-
lar arrest that precluded the expected cell cycle progres-
sion mediated by Chek1 inhibition. Further support for

M. E. Fraley et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6049–6053
6051
Table 2. Inhibitory profiles of 6–21
Compound
X
R
Chek1 IC50 (nM)
Cdk7 IC50 (nM) (ratio)
CEA EC50 (nM)
6
7
O
O
O
O
O
CN
30
62
12
13
80
11 (0.37)
6500 (100)
>50,000
1930
980
CO2Me
CONH2
CONHMe
CONMe2
8
1300 (110)
4900 (380)
>100,000 (>1200)
9
10
650
4000
11
12
13
14
15
16
O
10
530
59
25 (2.5)
—
>50,000
—
O
O
34 (0.58)
18 (1.5)
4000 (83)
57 (22)
>50,000
>10,000
2000
CH2
CH2
CH2
12
48
2.6
350
17
18
19
CH2
CH2
CH2
30
110
34
420 (14)
2300 (22)
480 (14)
1400
>10,000
4500
20
21
CH2
CH2
45
7000 (160)
56 (190)
5000
690
0.30
pound active in the CEA. Interestingly, methylation of
the triazole to give 17 maintained an adequate selectivity
ratio for functional activity, but reduced enzyme poten-
cy by an order of magnitude. N-Linked pyrazole 18
highlighted the importance of the 3-nitrogen atom of tri-
azole 16 with regard to Chek1 and Cdk7 potency, while
19 showed that some of the loss could be recovered with
an N2-linked 1,2,3-triazole. While the phenyl group of
20 provided adequate selectivity to induce checkpoint
escape, potency was found to be only modest. Surpris-
ingly, subnanomolar enzymatic potency for Chek1 was
realized with the functionally active derivative 21, a
compound bearing a unique hydroxymethyl triazole
group at C6.19 To summarize, our data showed that
while compounds equipotent against Chek1 and Cdk7
(6, 11, 13, and 14) were inactive in the CEA, inhibitors
with Cdk7/Chek1 IC50 ratios as low as 14 (17 and 19)
were functionally active.
three conserved water molecules that make up a key
hydrogen bonding network in HI. While desolvation
of the hydroxyl group may be an unfavorable thermody-
namic event, the entropic gain of resolvation of the
Insight into the superior binding affinity of 21 was
gained through analysis of an inhibitor-bound X-ray
structure (Fig. 1).20 The structure revealed that the
hydroxyl group of the triazole had displaced one of
Figure 1. X-ray crystallographic structure of 21 (green) bound to
Chek1 (light brown) with water molecules shown (red spheres and
surface). Water molecule displaced by 21, but present in X-ray
structure of 1 shown as a mesh sphere (magenta).

6052
M. E. Fraley et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6049–6053
Table 3. Inhibitory activities of 22–24
Compound
R1
R2
Chek1 IC50 (nM)
0.30
Cdk7 IC50 (nM)
180
CEA EC50 (nM)
340
22
23
24
0.11
0.25
300
310
610
92
ordered water molecule released from HI compensates
for it.21 Moreover, the X-ray structure of 21 showed that
the hydroxyl group made an additional hydrogen bond
to the protein backbone at Asp148. Taken together, we
concluded that the gain in binding affinity with 21 was
largely due to the ‘anchoring’ of the hydroxyl group
within the depicted hydrogen bonding array.
yl triazole. Interestingly, an X-ray structure of 24
showed displacement of all three water molecules from
HI by the phenolic group, consistent with the reported
finding.
The synthesis of 21 is shown in Figure 2.24 The route be-
gan with the preparation of 6-bromoindazole by the
method of Bartsch and Yang.25 Iodination followed by
a regioselective Suzuki cross-coupling reaction with the
depicted indole boronic acid proceeded smoothly to
form the 3-(indol-2-yl)indazole core structure. Palladi-
um-catalyzed coupling of the product bromide with
propargyl alcohol in pyrrolidine26 and subsequent oxi-
dation to the aldehyde set the stage for the construction
of the triazole by way of a 3 + 2 cyclization with sodium
azide.27 Sodium borohydride reduction of the resulting
aldehyde then provided 21.
In exploring the scope of this finding, we found that the
aminomethyl triazole of 23 provided a small gain in
Chek1 potency in the enzyme assay, but offered no
improvement in cellular activity in the CEA relative to
22 (Table 3).22 In 24, we introduced the phenolic group
that was recently reported as a potency-enhancing fea-
ture for a related series of Chek1 inhibitors.23 For this
series, the phenol conferred similar enzyme potency
and greater cellular activity relative to the hydroxymeth-
Figure 2. Synthesis of 21.

M. E. Fraley et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6049–6053
6053
antibody-coated, bead-based assay. For assay details, see
Fraley, M. E. et al. Bioorg. Med. Chem. Lett. 2006, 16,
1775, Ref. 16. The values of EC50 were measured with 10-
point, half-log dilution series and are reported as an
average of tetraplicate determinations.
In summary, we have described the effects of C6 sub-
stitution on Chek1 potency and selectivity for a series
of 3-(indol-2-yl)indazoles. We showed that selectivity
for Chek1 over Cdk7 was required for functional
activity in a cell-based checkpoint escape assay for this
series of compounds. The hydroxymethyl triazole
group provided enhanced binding affinity, apparently
through participation in a hydrogen bonding network
in HI following displacement of a conserved water
molecule.
10. Traxler, P.; Furet, P. Pharmacol. Ther. 1999, 82, 195.
11. Compound
3
displayed high passive permeability
(Papp = 30 · 10ꢀ6 cm/s) across LLC-PK1 cell monolayers.
For assay description, see Hochman, J. H.; Yamazaki, M.;
Ohe, T.; Lin, J. H. Curr. Drug Metab. 2002, 3, 257.
12. Of note, compound 3 showed relatively weak activity
against Cdc2 (IC50 = 4200 nM). Cdk7 and Cdc2 were
assayed using IMAP-FP kits from Molecular Devices at
0.05 lM and 0.03 lM ATP, respectively. Enzymes were
purchased from Upstate Biotechnology.
References and notes
13. Fisher, R. P.; Morgan, D. O. Cell 1994, 78, 713.
14. Harper, J. H.; Elledge, S. J. Genes Dev. 1998, 12, 285.
15. Chen, P.; Luo, C.; Deng, Y.; Ryan, K.; Register, J.;
Margosiak, S.; Tempczyk-Russell, A.; Nguyen, B.; Myers,
P.; Lundgren, K.; Kan, C.-C.; O’Connor, P. M. Cell 2000,
100, 681.
1. Niida, H.; Nakanishi, M. Mutagenesis 2006, 21, 3.
2. Zhou, B.-B. S.; Bartek, J. Nat. Rev. Cancer 2004, 4, 1.
3. Bartek, J.; Lukas, J. Cancer Cell 2003, 3, 421.
4. For recent reviews on published Chek1 inhibitors, see: (a)
Prudhomme, M. Recent Patents Anti-Cancer Drug Discov.
2006, 1, 55; (b) Kawabe, T. Mol. Cancer Ther. 2004, 3,
513.
16. Lolli, G.; Lowe, E. D.; Brown, N. R.; Johnson, L. N.
Structure 2004, 12, 2067.
17. Note that Chek1 inhibitory activity in the enzyme assay is
measured at Km for ATP (0.1 mM). In the cellular assays,
the ATP concentration is 2.0 mM resulting in an inherent
10-fold shift in potency between the enzyme and cellular
assays.
18. Functionally inactive compounds 6, 11, 13, and 14 were
cell permeable as indicated by their activity in the Phos
assay (IC50s 200, 2800, 700, and 2300 nM, respectively).
19. The NH of the triazole is arbitrarily assigned.
20. PDB coordinates for 21: 2HOG.
5. Hanney, B. A.; Kim, Y.; Hartman, G. D. Presented at the
229th National Meeting of the American Chemical Soci-
ety, San Diego, CA, March 2005; Abstract MEDI-121.
See also WO 2003024969.
6. Fraley, M. E.; Arrington, K. L.; Buser, C. A.; Ciecko, P.
A.; Coll, K. E.; Fernandes, C.; Hartman, G. D.; Hoffman,
W. F.; Lynch, J. J.; McFall, R. C.; Rickert, K.; Singh, R.;
Smith, S.; Thomas, K. A.; Wong, B. K. Bioorg. Med.
Chem. Lett. 2004, 14, 351.
7. Chek1 inhibitory activity was measured using a homoge-
neous time-resolved fluorescence assay that detects phos-
phorylation of a biotinylated GSK-3 peptide as described
by Barnett, S. F. et al. Biochem. J. 2005, 385, 399. The
assay was run at 0.5 nM enzyme and 0.1 mM ATP. IC50
values are reported as averages of at least two independent
determinations; standard deviations are within 25–50%
of IC50 values.
8. NCI-H1299 lung carcinoma cells were incubated with
Chek1 inhibitors for 2 h and then treated with campto-
thecin for an additional 4 h. Inhibition of Chek1 auto-
phosphorylation was measured by cyto-blot analysis using
an antibody against phospho-S296 Chek1. For assay
details, see WO2006086255. The values of IC50 were
measured with 10-point, 3-fold dilution series and are
reported as an average of triplicate determinations.
9. NCI-H1299 lung carcinoma cells were arrested with 16 h
treatment of camptothecin and then treated with Chek1
inhibitors for additional 8 h. Escape from arrest and
progression into mitosis were measured by quantifying the
mitosis-specific phosphorylation of nucleolin using an
21. Dunitz, J. D. Science 1994, 264, 670.
22. The substantial shift in cellular activity relative to enzyme
potency for 21–23 is believed to be due to suboptimal cell
permeability as a result of high polar surface area
2
˚
(PSA > 120 A , calculated by the method of Clark, D. E.
J. Pharm. Sci. 1999, 88, 807). For discussions on the
inverse relationship between PSA and cell permeability,
see Papageorgiou, C.; Camenisch, G.; Borer, X. Bioorg.
Med. Chem. Lett. 2001, 11, 1549, and references therein.
23. Foloppe, N.; Fisher, L. M.; Francis, G.; Howes, R.;
Kierstan, P.; Potter, A. Bioorg. Med. Chem. 2006, 14,
1792.
24. For full experimental detail on compounds described in
this paper, see WO2006086255.
25. Bartsch, R. A.; Yang, I.-W. J. Heterocycl. Chem. 1984, 21,
1063.
26. Alami, M.; Ferri, F.; Linstrunelle, G. Tetrahedron Lett.
1993, 34, 6403.
27. Journet, M.; Cai, D.; Kowal, J. J.; Larsen, R. D.
Tetrahedron Lett. 2001, 42, 9117.