1,4-Dihydroindeno[1,2-c]pyrazoles as potent checkpoint kinase 1 inhibitors: Extended exploration on phenyl ring substitutions and preliminary ADME/PK studies

Article abstract of DOI:10.1016/j.bmcl.2007.04.055

A study on substitutions at the four open positions on the phenyl ring of the 1,4-dihydroindeno[1,2-c]pyrazoles as potent CHK-1 inhibitors is described. Bis-substitution at both the 6- and 7-positions led to inhibitors with IC₅₀ values below 0.

Full text of DOI:10.1016/j.bmcl.2007.04.055

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3618–3623
1,4-Dihydroindeno[1,2-c]pyrazoles as potent checkpoint kinase 1
inhibitors: Extended exploration on phenyl ring substitutions
and preliminary ADME/PK studies
Yunsong Tong,* Akiyo Claiborne, Magdalena Pyzytulinska, Zhi-Fu Tao, Kent D. Stewart,
Peter Kovar, Zehan Chen, Robert B. Credo, Ran Guan, Philip J. Merta, Haiying Zhang,
Jennifer Bouska, Elizabeth A. Everitt, Bernard P. Murry, Dean Hickman, Tim J. Stratton,
Jian Wu, Saul H. Rosenberg, Hing L. Sham, Thomas J. Sowin and Nan-horng Lin
Cancer Research, Global Pharmaceutical R&D, R47S, AP10, Abbott Laboratories, 100 Abbott Park Road, Abbott Park,
IL 60064, USA
Received 12 March 2007; revised 13 April 2007; accepted 17 April 2007
Available online 25 April 2007
Abstract—A study on substitutions at the four open positions on the phenyl ring of the 1,4-dihydroindeno[1,2-c]pyrazoles as potent
CHK-1 inhibitors is described. Bis-substitution at both the 6- and 7-positions led to inhibitors with IC50 values below 0.3 nM. The
compound with the best overall activities (36) was able to potentiate the anti-proliferative effect of doxorubicin in HeLa cells by at
least 47-fold. Physicochemical, metabolic, and pharmacokinetic properties of selected inhibitors are also disclosed.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The concept of sensitizing cancer to the effects of DNA-
damaging agents through inhibition of checkpoint
kinase 1 (CHK-1) has been well established.1 CHK-1
is activated via phosphorylation by its upstream kinases
ATR and/or ATM upon DNA damage, leading to phos-
phorylation and degradation of CDC25A. The down-
stream event is the inhibition of cyclin E/Cdk2 or
cyclin B/Cdc2 kinases, which ultimately causes cell cycle
arrest at S or G2/M phase.2 The effectiveness of the
DNA-damaging agents faces deterioration since the
p53-deficient cancer cells have a mechanism of repairing
themselves at the S or G2/M phases. Therefore, inhibi-
tion of CHK-1 to abrogate S and/or G2/M checkpoints
can drive cancer cells into premature mitosis and apop-
tosis. On the other hand, the DNA-damaging agents
have little impact on normal cells since they have the
ability to arrest through p53-mediated G1 checkpoint.3
CHK-1 inhibitors (Chart 1).4 Our early study was
focused on investigating the structural biology of this
class of molecules. We learned that key extra hydrogen
bondings between the phenolic alcohol (or correspond-
ing carboxylic acid) of the inhibitors to the polar region
of the kinase in the active site were needed for high
potency, in addition to the bidentate hydrogen bonding
between the pyrazole part of the molecules and the hinge
region of the kinase. However, the substitution on the
phenyl ring of the tricyclic pyrazole core was limited
at the 6-position with narrow variations. In this paper,
we explore the expanded substitution types and posi-
tions around the fused phenyl ring while keeping the
bi-aryl phenol at the 3-position. In addition, we disclose
the metabolic, physicochemical, and pharmacokinetic
(PK) profiles of selected potent inhibitors and our initial
attempt to optimize the PK properties.
Recently, we disclosed a new class of 1,4-dihydroinde-
no[1,2-c]pyrazole compounds as potent and selective
HN
N
3
Keywords: 1,4-Dihydroindeno[1,2-c]pyrazoles; Checkpoint kinase 1
inhibitors; Chk-1 inhibitors; Sensitizing DNA-damaging agents.
6
X
OH
*
Corresponding author. Tel.: +1 847 935 1252; fax: +1 847 935
5165; e-mail: yunsong.tong@abbott.com
Chart 1.
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.055

Y. Tong et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3618–3623
3619
SEM
H
O
H
N
N
N
N
i, ii
iii, iv
O
N
O
N
i, ii
iii, iv
O
N
H
Br
Cl
28
29
1
2
H
H
H
N
H
N
N
N
N
N
N
O
vi
N
v
O
v, vi
O
O
I
H
I
H
RHN
N
N
H
H
O
O
30
31
3
4
H
H
N
O
N
N
3
N
O
R
vii
H
N
RHN
N
N 6
O
O
vii, viii
O
H
I
HO
10-13
OH
33-37
32
OH
Scheme 1. Reagents and conditions: (i) NaH, SEMCl, DMF, 100%;
(ii) Pd(OAc)2, Xantphos, Cs2CO3, acetamide, 1,4-dioxane, 100 ꢁC,
79%; (iii) HCl, MeOH, 100%; (iv) NaHCO3, chloroacetyl chloride,
acetone, 78%; (v) NIS, DMF, 75 ꢁC, 43%; (vi) amines, EtOH, or
DMF, 50–100%; (vii) 4-biphenol pinacol borate, Pd(PPh3)2Cl2,
Na2CO3, EtOH/DME/H2O, microwave, 170 ꢁC, 500 s, ꢀ20–40%.
HN
N
O
ix, viii
31
R
OH
38-42
Scheme 2. Reagents and conditions: (i) NaH, ethyl formate, benzene,
91%; (ii) NH2NH2, AcOH, 90 ꢁC, 84%; (iii) Br2, AcOH, 95%; (iv)
PhLi, s-BuLi, THF, DMF, À78 ꢁC to rt, 99%; (v) NIS, DMF, 80 ꢁC,
68%; (vi) NaClO2, KH2PO4, NH2SO3H, H2O/1,4-dioxane, 65%; (vii)
1ꢁ amines, EDC, HOBt, Et3N, DMF, rt; or 2ꢁ amines, PyBOP, DIEA,
DMF, rt; (viii) 4-biphenol pinacol borate, Pd(PPh3)2Cl2, Na2CO3,
EtOH/DME/H2O, microwave, 160–180 ꢁC, 600–1000 s, ꢀ16–37%; (ix)
a—amines, TsOH, toluene, reflux; b—NaBH4, EtOH, THF.
Scheme 1 outlines how compounds 10–13, carrying an
acetylamino linker at the 6-position, were synthesized.
The pyrazole portion of the known compound 15 was
protected by SEM and the acetyl amino group was
introduced at the 6-position following a Buchwald pro-
tocol6 to give 2. Both the acetyl and the SEM groups
were removed with HCl. The aniline nitrogen was subse-
quently converted to the chloroacetamide. The resulting
compound 3 was iodonated at the 3-position using NIS
followed by nucleophilic substitution with various
amines to complete the side-chains at the 6-position.
The bi-aryl phenol was then installed via a Suzuki cou-
pling providing 10–13.
with good yield. The remaining transformations were
completed using well-developed chemistry resulting in
48. The yield for the final Suzuki coupling step was
much higher if the pyrazole nitrogen was protected.
The synthesis of compounds 49–52 (Scheme 4) was sim-
ilar to the procedures reported earlier.4
The synthesis of bis-substituted compounds 33–42 is
shown in Scheme 2. 6-Methoxy-1-indanone 28 was trea-
ted with NaH and ethyl formate followed by pyrazole
ring closure in the presence of hydrazine and acetic acid.
Compound 29 was lithiated and treated with DMF to
afford the aldehyde 30. Iodonation using NIS gave 31.
The formyl group at the 6-position was oxidized to the
carboxylic acid, which was in turn converted into an
amide using either HOBT/EDC or PyBOP coupling
conditions. The bi-aryl phenol unit was introduced in
the final step to complete the synthesis of 33–37. Mean-
while, reductive amination on the formyl group of 31
followed by Suzuki coupling afforded compounds
38–42.
Our earlier work on 1,4-dihydroindeno[1,2-c]pyrazoles4
had limited substitution patterns on the phenyl ring of
the tricyclic core since all of the inhibitors carried
extended side-chains with either an aminomethyl or an
aminocarbonyl linker at the 6-position, exemplified by
6 or 7 (Table 1). As indicated in the same table, a smaller
substitution such as a hydroxyl or a hydroxymethyl
showed similar potency. Both 8 and 9 possessed IC50
values below 10 nM. However, the unsubstituted com-
pound (5) suffered a dramatic potency loss (IC50
=
1373 nM). Compounds having side-chains linked by an
acetylamino group (10–13) uniformly performed well
in the enzymatic assay. With an IC50 value at 0.74 nM,
inhibitor 13 was the most potent compound containing
a 6-position substitution.
The synthesis of 48 (Scheme 3) with a gem-dimethyl
group at the 4-position of the tricyclic core started with
43 prepared via a known protocol.7 The phenolic alco-
hol of 43 was protected with a SEM group followed
by pyrazole formation to provide 44. Protection of the
pyrazole nitrogen and selective removal of the SEM
on the phenol gave 45. The resulting hydroxyl group
was converted into an amide via a triflate intermediate
We also investigated substitutions on the remaining
three open positions (5, 7, and 8) of the phenyl ring with
results displayed in Table 2. Within the CHK-1 binding
pocket,10 the substitution at the 5-position is sand-
wiched by protein walls with a narrow opening. The
small groups were well tolerated at the 5-position as

3620
Y. Tong et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3618–3623
Table 1. Substitutions at 6-position: extended study
HN
O
O
OH
O
i
ii, iii, iv
N
O
OH
+
HO
43
6
X
OH
H
SEM
N
N
N
vii, viii
N
v, vi
O
O
a
CHK-1 IC50 (nM)
Compound
X
5
H
1373
SEMO
HO
44
45
H
SEM
N
N
N
N
ix, x
6
7
24
O
O
N
N
O
H
H
I
N
N
H
N
N
9.3
4.4
7.7
6.4
1.2
2.1
0.74
O
HO
HO
O
46
47
H
N
88
98
10
11
12
13
OH
N
O
xi
H
N
4
CH2OH
O
HO
OH
48
NH
N
Scheme 3. Reagents and conditions: (i) polyphosphoric acid, 40–
110 ꢁC, 39%; (ii) SEMCl, DIEA, CH2Cl2, 93%; (iii) NaH, ethyl
formate, THF, 50 ꢁC, 74%; (iv) NH2NH2 monohydrate, AcOH,
EtOH, 85 ꢁC, 89%; (v) SEMCl, NaH, THF, 95%; (vi) HCl, MeOH,
50%; (vii) PhN(OTf)2, NaH, THF, 40 ꢁC, 96%; (viii) CO,
H
N
O
NH
HO
N
H
O
PdCl2(dppf)ÆCH2Cl2,
trans-4-aminocyclohexanol,
THF,
TEA,
NH
245 psi, 120 ꢁC, 77%; (ix) HCl, EtOH, 60 ꢁC, 50%; (x) NIS, 1,4-
dioxnae, 90 ꢁC, 34%; (xi) 4-biphenol pinacol borate, Pd(PPh3)2Cl2,
Na2CO3, EtOH/DME/H2O, microwave, 180 ꢁC, 1000 s, 7%.
N
H
O
HO
NH
N
H
O
H
H
N
a Compound concentration needed to cause 50% inhibition of Cdc25C
phosphorylation in the presence of recombinant CHK-1 protein
(ATP concentration is 5 lM).9
N
N
N
ref. 4
I
H
R1
R2
O
OH
Interestingly, substitutions at both the 6- and 7-posi-
tions appeared to have some additive effect. As shown
in Table 3, a methoxy group at the 7-position coupled
with a solubilizing group at the 6-position led to com-
pounds with IC50 values mostly less than 1 nM (33–
42). For the most potent compounds, 33, 36, and 40,
the IC50 values were in the range of 0.2–0.3 nM, which
represents at least a 10-fold improvement over most of
the mono-substituted inhibitors. While both 6- and 7-
positions are solvent exposed, the 7-position is closer
to the protein. The 7-OMe group can bind to the enzyme
by making van der Waals contact within a small hydro-
phobic pocket formed by the side-chain of Tyr86 and
the methylene of Gly90. We speculate that the presence
of the 6-substitution may limit the 7-OMe group to be
conformationally organized into the requisite binding
orientation and resulted in inhibitors with superior
potency.
H
N
H
49: R1=
R2=F 50: R1=
R2=F 52: R1=
R2=CN
N
,
,
,
,
N
N
N
O
O
51: R1=
R2=CN
N
N
N
Scheme 4.
14 and 15 possessed IC50 values between 5 and 8 nM.
However, the potency deteriorated upon increasing the
size of the substitution. As such, compound 16 suffered
a greater than 20-fold drop in its activity, while 17 and
18 with long and bulky side-chains completely lost
potency. Substitutions at the 7-position are outside of
the binding cavity and extended into the solvent similar
to those at the 6-position. Consequently, groups with
variable size and length all resulted in potent inhibitors
(19–26) with IC50 values ranging from 2 to 34 nM. The
8-position is very close to the protein backbone in the
active site and was not considered to be an ideal site
for the SAR study. Compound 27 with a hydroxy group
in that position was a much weaker inhibitor with an
IC50 value of 671 nM as anticipated.
Potent inhibitors in the enzymatic assay were further
evaluated in a functional and a mechanism-based cellu-
lar assay.11 The functional assay was a cell proliferation
assay (MTS assay) in HeLa cells. In this assay, the anti-
proliferation effect was measured in the form of EC50
values for a CHK-1 inhibitor as a single agent and also

Y. Tong et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3618–3623
Table 2. Substitutions at 5-, 7-, or 8-positiona
3621
HN
N
8
7
X
5
OH
Compound
X position
X
CHK-1 IC50 (nM)
14
15
16
17
18
19
20
21
22
23
24
25
26
27
5
5
5
5
5
7
7
7
7
7
7
7
7
8
Acetylamino
CH2OH
5.5
7.8
2,2-Dimethyl-propylamino-methyl
(trans-4-Hydroxy-cyclohexylamino)-carbonyl
(Phenyl-methylamino)-carbonyl
CH2OH
209
>10,000
>10,000
4.4
2,2-Dimethyl-propylamino-methyl
2-Hydroxy-ethylamino-methyl
(2-N,N-Dimethylamino-ethyl)amino-methyl
(2-Pyrrolidin-1-yl-ethylamino)-methyl
(2-Hydroxy-ethylamino)-carbonyl
Methylamino-carbonyl
34
12
5.2
7.1
5.0
2.3
Morpholin-4-yl-carbonyl
OH
91
671
a The compounds were synthesized in the same fashion as those with substitutions at the 6-position.
Table 3. Bis-substitutions at 6- and 7-positions
HN
N
MeO
OH
X
Compound
X
CHK-1 IC50 (nM)
33
34
35
36
37
38
39
40
41
42
(4-Hydroxy-piperidin-1-yl)-carbonyl
(4-Methyl-piperazin-1-yl)-carbonyl
(2-Pyrrolidin-1-yl-ethylamino)-carbonyl
0.24
2.3
0.83
0.24
0.55
0.43
0.51
0.21
0.56
1.1
(trans-4-Hydroxy-cyclohexylamino)-carbonyl
Morpholin-4-yl-carbonyl
2-Hydroxy-ethylamino-methyl
(4-Methyl-piperazin-1-yl)-methyl
(2-Pyrrolidin-1-yl-ethylamino)-methyl
(trans-4-Hydroxy-cyclohexylamino)-methyl
(4-Hydroxy-piperidin-1-yl)-methyl
for an inhibitor in the presence of doxorubicin
(150 nM), a DNA-damaging agent. For the combina-
tion study, the base line of the regression for EC50 calcu-
lation was adjusted to the inhibition level determined for
150 nM doxrubicin alone, a concentration known to
cause G2/M arrest in HeLa cells. The ratio of the two
corresponding EC50s (combo/single) represents a rela-
tive potentiation scale of a CHK-1 inhibitor to sensitize
doxorubicin. The mechanism for the anti-proliferative
function of the inhibitors was examined by a cell cycle
analysis (FACS assay) in H1299 cells. The EC50 values
were measured for an inhibitor alone and also for an
inhibitor in combination with doxorubicin. The latter
value indicates how effectively an inhibitor can reduce
the doxorubicin-induced G2/M cell population. The cel-
lular activity profiles for a group of potent CHK-1
inhibitors are summarized in Table 4 where compounds
such as 7 and 36 represent the desirable profiles. These
examples had no single agent activity (i.e., not cytotoxic)
in either assay (EC50 > 22 lM in MTS, EC50 > 10 lM in
FACS). In combination with doxorubicin, however,
they were able to potentiate the anti-proliferative effect
of doxorubicin in the MTS assay by a large margin
(92-fold for 7 and at least 47-fold for 36). Meantime,
the results from the FACS assay confirmed that the ob-
served potentiation effect was through abrogating G2/M
cell arrest with combo-EC50 values at 1.3 and 0.61 lM
for 7 and 36, respectively. Some compounds (9, 15,
and 19) showed very weak anti-proliferative potentials,
while others (6, 12, 23, 33, 40, and 41) displayed desir-
able doxorubicin sensitization but appeared to exhibit
single agent cyto-toxicity (EC50 value between 0.62
and 2.8 lM in the MTS assay), an indication of certain
off-target activities. The clinical implications of single
agent activity remain unknown at this stage. It is
noteworthy to mention that a lack of good correlation
between the intrinsic enzymatic potency and the func-
tional cellular potency was observed. In the literature,12
some CHK-1 inhibitors have shown diminished cellular
activities due to their poor kinase selectivity profile. This

3622
Y. Tong et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3618–3623
Table 4. Cellular activity of selected CHK-1 inhibitors
Table 6. Metabolic stability and permeability profile of 7
Rat liver microsomal stabilitya Caco-2b
Compound
MTS assay EC50
(lM)
FACS assay EC50
(lM)
T1/2 (min) Kelimination
(1/min)
% Remaining Papp (1 · 10À6 cm/s)
Single
With Doxa
Single
With Dox
(30 min)
(A to B)
6
7
1.7
23
1.2
>10
>10
NTb
>10
>10
>10
NT
>10
>10
NT
5.2
>10
1.3
25
8
0.0294 0.0100 40 11
13.26 1.36
0.25
28
a 1 lM compound concentration, 0.1 mg/mL protein concentration.
b 1 lM compound concentration.
9
>59
>59
1.7
NT
5.4
11
12
13
15
19
22
23
33
36
40
41
48d
3.6
0.56
3.3
1.6
2.0
>59
>59
>59
>59
2.8
>5.9
8.0
NT
5.5
gated the solubility, permeability, and metabolism
properties of selected compounds. The solubilizing
side-chains of 6 and 7 had only marginal impact on
aqueous solubility at neutral pH. Both compounds pos-
sessed solubility less than 0.03 lg/mL. Although the
inadequate water solubility may not be the only reason
for the poor oral bioavailability, we made an effort to
improve solubility via disrupting the planarity of the tri-
cyclic pyrazole core by preparing the C4 gem-dimethy-
lated compound 48 (Scheme 3). It was observed that
this change of geometry did bring a 10-fold boost in
aqueous solubility after a direct comparison between
the two compounds having similar side-chains as 36/48
(data not shown). Unfortunately, the addition of the
gem-dimethyl moiety resulted in less enzymatic activity
of the inhibitor (IC50 = 0.24 nM for 36, 18 nM for 48),
but more importantly, also diminished the cellular
anti-proliferative activity of the compound (combo-
EC50 in MTS assay more than 5.9 lM as compared to
0.89 lM for 36, Table 4).
2.5
1.4
>10
NT
0.89
0.61
1.8
1.4
0.73
0.89
0.19
0.27
>5.9
>59c
0.91
0.62
8.6
>10
5.1
3.1
>10
1.0
2.6
a Doxorubicin.
b Not tested.
c Second test result is 38.
d IC50 = 18 nM.
class of CHK-1 inhibitors, however, is generally selec-
tive against other Ser/Thr kinases as reported earlier.4
Low cellular activities of compounds with high inhibi-
tory potency may reflect their improper physicochemical
properties.
Based on the above assay profiles, compounds 6, 7, and
36 were selected for PK evaluation in mice. As shown in
Table 5, both mono-substituted compounds, 6 and 7,
had high plasma clearance (CL), moderate-to-large vol-
umes of distribution (Vd), and short half-lives. Both
compounds exhibited moderate plasma exposure when
dosed intraperitoneally with AUC values being
2.7 lg h/mL for 6 and 4.4 lg h/mL for 7. However, the
oral bioavailability was poor for both. The bis-substi-
tuted compound 36 showed even higher clearance and
volume of distribution while its oral bioavailability
was reduced to an undetectable level. Noticeably, all
three compounds are highly lipophilic with clogP
(ACD) values ranging from 4.8 to 6.0.
The in vitro whole-cell permeability of 7 was assessed
via a Caco-2 assay. The apparent permeability (apical
to basal) is 13.3 · 10À6 cm/s (Table 6), suggesting that
the molecule should have moderate absorption.13 In
the presence of rat liver microsomes, compound 7
showed a half-life of 25 min (Table 6). Major routes of
hepatic metabolism included oxidation of the tricyclic/
biphenyl rings (data not shown). As reported earlier,14
the metabolic stability of compounds containing phenol
can be enhanced by installing electron-withdrawing
groups next to the phenolic hydroxyl group to reduce
glucuronidation (phase II metabolism). In addition, this
strategy also has the potential to reduce the oxidative
metabolism (phase I metabolism) on the aromatic rings
due to reduced electron density. To this end, compounds
In an attempt to further evaluate other factors poten-
tially associated with PK of these molecules, we investi-
Table 5. Pharmacokinetic profiles
Compound
CHK-1
IC50 (nM)
Mouse iva
T1/2
(h)
Mouse ipb
AUC
Mouse poc
CL
(L/h kg)
Vd
(L/kg)
AUC
(lg h/mL)
Cmax
(lM)
Cmax
(lM)
AUC
(lg h/mL)
F
(%)
(lg h/mL)
7
49
50
6
9.3
27
5.4
3.6
—
4.7
2.4
—
0.60
0.46
—
1.2
1.7
—
2.0
2.8
—
4.4
3.8
—
0.25
<0.05
—
0.16
0
4
0
9091
24
—
—
9
4.7
11
3.4
9.2
—
0.50
0.60
—
1.5
0.69
—
1.1
1.3
—
2.7
1.6
—
0.09
0.08
—
0.45
0.13
—
51
52
36
28
>10,000
0.24
6
—
11
—
0
6.0
0.39
0.57
1.5
4.2
<0.1
0
a Intravenous dosing, 3.0 mg/kg.
b Intraperitoneal dosing, 10 mg/kg.
c Oral dosing, 10 mg/kg.

Y. Tong et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3618–3623
3623
Science 2000, 288, 1425; (c) Zhao, H.; Watkins, J. L.;
Piwnica-Worms, H. Proc. Natl. Acad. Sci. U.S.A. 2002,
99, 14795.
with fluoro and cyano groups ortho to the phenolic
hydroxyl group were prepared (49–52, Scheme 4). The
data in Table 5 revealed that, compared to the parent
molecules (7 and 6), compounds with the fluoro substi-
tution (49 and 51) maintained the enzymatic inhibition
level but the overall PK parameters were generally the
same, if not worse. The cyano group, however, was
not tolerated in the binding pocket since the resulting
compounds (50 and 52) lost their inhibitory activity. It
is speculated that the combination of poor aqueous sol-
ubility and high lipophilicity of the molecules has led to
the poor PK profiles. The high lipophilicity may have
additionally contributed to the high clearance rate.
3. (a) Powell, S. N.; DeFrank, J. S.; Connell, P.; Eogan, M.;
Preffer, F.; Dombkowski, D.; Tang, W.; Friend, S. Cancer
Res. 1995, 55, 1643; (b) Kuerbitz, S. J.; Plunkett, B. S.;
Walsh, W. V.; Kastan, M. B. Proc. Natl. Acad. Sci. U.S.A.
1992, 89, 7491.
4. Tong, Y.; Claiborne, A.; Stewart, K. D.; Park, P.; Kovar,
P.; Chen, Z.; Credo, R. B.; Gu, W.-Z.; Gwaltney, S. L., II;
Judge, R. A.; Zhang, H.; Rosenberg, S. H.; Sham, H. L.;
Sowin, T. J.; Lin, N. H. Bioorg. Med. Chem. 2007, 15,
2759.
5. Doyle, K.; Rafferty, P.; Steele, R.; Turner, A.; Wilkins, D.;
Arnold, L. Therapeutic agents. US 6297238.
6. Yin, J.; Buchwald, S. L. Org. Lett. 2000, 2, 1101.
7. Anastasis, P.; Brown, P. E. J. Chem. Soc. Perkin Trans. 1
1982, 2013.
8. Compounds 8 and 9 were synthesized from known starting
materials, 5-hydroxy-2,3-dihydro-1H-inden-1-one and
3-iodo-1,4-dihydroindeno[1,2-c]pyrazole-6-carbaldehyde,
respectively, following similar procedures shown in this
article.
9. CHK-1 enzymatic inhibition assay. The assay was carried
out using a recombinant CHK-1 kinase domain protein
with amino acids from residue 1–289. A human biotin-
ylated Cdc25C peptide was used as the substrate
(Synpep Catalog# 02-1-22-1-ABB). The reaction mixture
contained 25 mM of HEPES at pH 7.4, 10 mM MgCl2,
0.08 mM Triton X-100, 0.5 mM DTT, 5 lM ATP, 4 nM
33P ATP, 5 lM Cdc25C peptide substrate, and 5 nM of
the recombinant CHK-1 protein. For potent compound
with Ki below 1 nM, 0.5 nM of the recombinant CHK-1
protein and 8 nM of 33P were used. The concentration
of the vehicle, DMSO, in the final reaction is 2%. After
30 min at room temperature, the reaction was stopped
by the addition of equal volume of 4 M NaCl and 0.1 M
In summary, we have systematically studied the substi-
tution patterns on the phenyl ring of the tricyclic core.
The substitutions at the 6- or 7-position were more tol-
erated than positions 5 and 8. The 5-position could only
accommodate smaller groups, while even minor substi-
tution at the 8-position led to significant potency loss.
Bis-substitution at both the 6- and 7-positions generally
led to compounds with higher enzymatic potency (IC50
value mostly below 1 nM), while the most potent com-
pounds (33, 36, and 40) exhibited IC50 values between
0.2 and 0.3 nM. The best compound, 36, was able to
potentiate the anti-proliferative effect of doxorubicin in
the MTS assay by at least 47-fold. Its mechanism of ac-
tion was through the abrogation of the cell cycle arrest
at the G2/M phase based on the FACS analysis. PK
studies in mice revealed that this class of CHK-1 inhib-
itors had high clearance, moderate bioavailability when
dosed intraperitoneally, but poor oral bioavailability.
Attempts have been made to analyze the causes for the
inadequate PK profiles and specific compounds were
synthesized accordingly. While the strategy to improve
solubility via disruption of planarity was successful,
weaker cellular potency prevented advancement of these
analogs. Substituting electron-withdrawing groups such
as fluoro next to the phenolic hydroxyl group to poten-
tially alleviate metabolism did not help to improve the
PK. It is likely that the combination of low aqueous sol-
ubility and high lipophilicity was the primary culprit for
the deficient oral bioavailability. In order to address
these issues, our future work will call for more dramatic
changes to the structure of the molecules including iden-
tifying new moieties to replace the lipophilic bi-aryl phe-
nol, while maintaining hydrogen bond interactions with
the polar region of the kinase active site necessary for
the high potency.
EDTA (pH 8.0).
A 40 lL aliquot of the reaction
mixture was added to a well in a Flash Plate (NEN
Life Science Products, Boston, MA) containing 160 lL
of phosphate-buffered saline (PBS) without calcium
chloride and magnesium chloride and incubated at
room temperature for 10 min. The plate was then
washed three times in PBS with 0.05% of Tween 20
and counted in a Packard TopCount counter (Packard
BioScience Company, Meriden, CT).
10. See Ref. 4 for detailed X-ray co-crystal structures with
coordinated deposited in the PDB.
11. See Ref. 4 for assay conditions and representative raw
data.
12. Fraley, M. E.; Steen, J. T.; Brnardic, E. J.; Arrington,
K. L.; Spencer, K. L.; Hanney, B. A.; Kim, Y.;
Hartman, G. D.; Stirdivant, S. M.; Drakas, B. A.;
Rickert, K.; Walsh, E. S.; Hamilton, K.; Buser, C. A.;
Hardwick, J.; Tao, W.; Beck, S. C.; Mao, X.; Lobell, R.
B.; Sepp-Lorenzino, L.; Yan, Y.; Ikuta, M.; Munshi, S.
K.; Kuo, L. C.; Kreatsoulas, C. Bioorg. Med. Chem.
Lett. 2006, 16, 6049.
References and notes
13. Yee, S. Pharm. Res. 1997, 14, 763.
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Cancer 2004, 4, 1; (b) Tao, Z.-F.; Lin, N.-H. Anti-Cancer
Agents Med. Chem. 2006, 6, 377.
2. (a) Xiao, Z.; Chen, Z.; Gunasekera, A. H.; Sowin, T. J.;
Rosenberg, S. H.; Fesik, S.; Zhang, H. J. Biol. Chem.
2003, 278, 21767; (b) Mailand, N.; Falck, J.; Lukas, C.;
14. Madsen, P.; Ling, A.; Plewe, M.; Sams, C. K.; Knudsen,
L. B.; Sidelmann, U. G.; Ynddal, L.; Brand, C. L.;
Anderson, B.; Murphy, D.; Teng, M.; Truesdale, L.; Kiel,
D.; May, J.; Kuki, A.; Shi, S.; Johnson, M. D.; Teston, K.
A.; Feng, J.; Lakis, J.; Anderes, K.; Gregor, V.; Lau, J.
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Syljuasen, R. G.; Welcker, M.; Bartek, J.; Lukas, J.