Cyanopyridyl containing 1,4-dihydroindeno[1,2-c]pyrazoles as potent checkpoint kinase 1 inhibitors: Improving oral biovailability

Article abstract of DOI:10.1016/j.bmcl.2007.07.069

A series of 1,4-dihydroindeno[1,2-c]pyrazole compounds with a cyanopyridine moiety at the 3-position of the tricyclic pyrazole core was explored as potent CHK-1 inhibitors. The impact of substitutions at the 6 and/or 7-position of the core on pharmacokinetic properties was studied in detail. Compounds carrying a side chain with an ether linker at the 7-position and a terminal morpholino group, such as 29 and 30, exhibited much-improved oral biovailability in mice as compared to earlier generation inhibitors. These compounds also possessed desirable cellular activity in potentiating doxorubicin and will serve as valuable tool compounds for in vivo evaluation of CHK-1 inhibitors to sensitize DNA-damaging agents.

Full text of DOI:10.1016/j.bmcl.2007.07.069

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5665–5670
Cyanopyridyl containing 1,4-dihydroindeno[1,2-c]pyrazoles as
potent checkpoint kinase 1 inhibitors: Improving oral biovailability
Yunsong Tong,* Magdalena Przytulinska, Zhi-Fu Tao, Jennifer Bouska, Kent D. Stewart,
Chang Park, Gaoquan Li, Akiyo Claiborne, Peter Kovar, Zehan Chen, Philip J. Merta,
Mai-Ha Bui, Amanda Olson, Donald Osterling, Haiying Zhang, Hing L. Sham,
Saul H. Rosenberg, Thomas J. Sowin and Nan-horng Lin
Cancer Research, Global Pharmaceutical R&D, R47S, AP10, Abbott Laboratories, 100 Abbott Park, Abbott Park, IL 60064, USA
Received 12 June 2007; revised 17 July 2007; accepted 18 July 2007
Available online 21 August 2007
Abstract—A series of 1,4-dihydroindeno[1,2-c]pyrazole compounds with a cyanopyridine moiety at the 3-position of the tricyclic
pyrazole core was explored as potent CHK-1 inhibitors. The impact of substitutions at the 6 and/or 7-position of the core on phar-
macokinetic properties was studied in detail. Compounds carrying a side chain with an ether linker at the 7-position and a terminal
morpholino group, such as 29 and 30, exhibited much-improved oral biovailability in mice as compared to earlier generation inhib-
itors. These compounds also possessed desirable cellular activity in potentiating doxorubicin and will serve as valuable tool
compounds for in vivo evaluation of CHK-1 inhibitors to sensitize DNA-damaging agents.
Ó 2007 Elsevier Ltd. All rights reserved.
Cancer cells rely on various cell cycle checkpoints to re-
store their genomic integrity upon DNA-damaging
treatments including ionized radiation, UV radiation,
and chemotherapy. Studies have demonstrated that
checkpoint kinase 1 (CHK-1) plays an important role
in regulating G2/M and/or S phase checkpoints for com-
monly p53 deficient cancer cells in response to genotoxic
stresses.1 DNA damage triggers phosphorylation of
CHK-1, a serine/threonine kinase, by ataxia-telangiecta-
sia mutated (ATM) as well as ATM- and Rad3-related
(ATR) kinases.2 As a result, CHK-1 phosphorylates
and degrades CDC25A leading to cell cycle arrest at
G2/M or S phase.3 Inhibition of CHK-1 can thus cause
mitotic catastrophe to cancer cells since they are de-
prived of an opportunity to repair themselves at the
G2/M or S checkpoints.
bonding interactions with both the hinge and specificity
regions of the catalytic pocket of the kinase. Although
our earlier inhibitors possessed high inhibitory activity
(IC50 value as low as 0.2 nM) and desirable potencies
in both functional and mechanism-based cellular assays,
they were deficient in oral biovailability (F = 0–9% in
mice).5 In this study, we disclose our efforts leading to
a newer generation of potent CHK-1 inhibitors with
much improved oral biovailability.
The primary series of our earlier CHK-1 inhibitors fea-
turing a bi-aryl phenol unit at the 3-position of the tricy-
clic core (such as 1 in Table 1) have intrinsic
pharmacokinetic (PK) liability due to their high lipo-
philicity (ClogP at 5–6), potential metabolic instability,
and poor aqueous solubility. Since variation of the sub-
stitutions at both the 6 and 7-positions and small mod-
ifications to the bi-aryl phenol moiety failed to provide
compounds with acceptable oral biovailability,5 we
turned our attention to replacing the bi-aryl phenol in
order to optimize the overall physicochemical properties
of the inhibitors. To that end, we discovered that com-
pounds with a cyanopyridine group at the 3-position
(such as 2) were equally potent as those with the bi-aryl
phenol in the enzymatic biochemical assay.6 The cyano-
pyridine group compensates for the intricate hydrogen
We have discovered molecules with a 1,4-dihydroinde-
no[1,2-c]pyrazole core as selective and potent CHK-1
inhibitors.4,5 These compounds can engage in hydrogen
Keywords: 1,4-Dihydroindeno[1,2-c]pyrazole; Checkpoint kinase
inhibitors; CHK-1 inhibitors; Sensitizing DNA-damaging agents.
1
*
Corresponding author. Tel.: +1 847 935 1252; fax: +1 847 935
5165; e-mail: yunsong.tong@abbott.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.069

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Y. Tong et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5665–5670
Table 1. From bi-aryl phenol to cyanopyridine
O
O
iii, iv
i, ii
MeO
MeO
MeO
H
MeO
SEMO
7
N
MeO
MeO
N
H
3
4
N
MeO
6
SEM
N
H
N
3
N
N
N
v, vi
MeO
MeO
N
N
N
SEMO
SEMO
1
2
CN
Cl
CN
5
6
OH
H
N
N
vii, viii, ix
MeO
RO
Compound MW CHK-1
IC50 (nM) (lM) S/Ca (lM) S/Ca
MTS EC50 FACS EC50 ClogPb
N
CN
7-10
1
2
384 1.6 >59/1.0 >10/0.15
313 0.81 >59/1.1 >10/0.27
5.4
2.6
H
N
O
a S/C, single/combo.
b Calculated by Biobyte methods.
N
x
R'O
HO
RO
3
N
MeO
11, R=H
12, R=Me
CN
xi
13-15
bonding network lost by eliminating the interactions be-
tween the phenolic hydroxy group and Glu55 and Asn59
in the active site.7 The comparison between the two
compounds (1 and 2) shown in Table 1 reveals that with
regard to better drug-like properties, the cyanopyridine
group undoubtedly has superiority over the bi-aryl phe-
nol. Compound 2 is not only about two log units lower
in ClogP than 1 (2.6 vs 5.4), and possesses much lower
molecular weight, but also eliminates a potential site
(phenol) for glucuronidation. In addition, as compared
to 1, compound 2 shows slightly better enzymatic activ-
ity, similar cellular potency in sensitizing the anti-prolif-
erative potential of doxorubicin, a DNA-damaging
agent, by at least 53-fold in an MTS assay,8 and similar
cellular profile in a mechanism-based FACS assay (i.e.,
not disturbing normal cell cycle by themselves with sin-
gle EC50 > 10 lM but able to abrogate G2/M check-
point in combination with doxorubicin with combo
EC50 < 0.3 lM).9 Although the cyanopyridyl compound
2 shows poor PK properties itself (AUC = 0.18 lg h/mL
when dosed intraperitoneally in mice at 10 mg/kg), it
does provide a better platform for lead optimization to
discover potent CHK-1 inhibitors with desirable oral
biavailability.
O
O
MeO
xii
HO
18-31
(see Table 3 & 4)
17
16
Scheme 1. Reagents and conditions: (i) NaCN, DMSO, 145 °C, 53%;
(ii) SEMCl, DIEA, CH2Cl2, 72%; (iii) NaH, phenyl 6-chloronicotinate,
THF; (iv) AcOH, hydrazine, EtOH, 90 °C, 81% (two steps); (v) NaH,
SEMCl, DMF, 65%; (vi) Pd2dba3, dppf, Zn, Zn(CN)2, DMA, 120 °C,
80%; (vii) HCl, MeOH, CH2Cl2, rt, 100%; (viii) DBAD, Ph3P on
polymer support, alcohols, THF, ꢀ50–90%; (ix) HCl, EtOH, 75 °C,
ꢀ35–75%; (x) BBr3, CH2Cl2, 98%; (xi) MeI, Li2CO3, DMF, 55 °C,
72%; (xii) AlCl3, toluene, reflux, 85%.
at the 5-position using MeI and Li2CO3 transformed
11 into 12. Under similar conditions discussed above,
compounds 13–15 were synthesized. Compounds 18–
31, with mono-ether side chain at the 7-position, were
prepared from 6-methoxy-1-indanone 16. De-methyla-
tion with AlCl3 led to 17. The final compounds were pre-
pared following the well-established chemistry.
The synthesis of compounds 37 or 38 carrying a side
chain with an amino or acetylamino linker at the 7-posi-
tion and a terminal morpholine group is outlined in
Scheme 2. Iron reduction converted 6-nitro-1-indanone
32 into the amino indanone 33, which was in turn trans-
formed into the intermediate 35 using similar procedures
shown in Scheme 1. Compound 35 was treated with
chloroacetaldehyde under reductive amination condi-
tions to furnish 36. The terminal chlorine was substi-
tuted by morpholine and subsequent SEM de-
protection led to 37. Compound 38 was derived from
a HATU-mediated amide coupling reaction involving
35 and 3-morpholinopropanoic acid.
The chemistry leading to compounds with side chains at
the 6 and/or 7-position of the tricyclic pyrazole core via
an ether linker is shown in Scheme 1. 5,6-Dimethoxy-1-
indanone 3 was selectively de-methylated at the 5-posi-
tion in the presence of NaCN at 145 °C and the resulting
hydroxy group was protected by a SEM group. Com-
pound 4 was treated with NaH and phenyl 6-chloronic-
otinate followed by hydrazine and acetic acid in one pot
to provide 5. The pyrazole moiety of 5 was SEM-pro-
tected. The chloropyridine fragment was subsequently
converted into a cyanopyridine under Pd-catalyzed con-
ditions. The SEM on the hydroxy group of the interme-
diate 6 was removed selectively with HCl at room
temperature. Next, the ether side chain at the 6-position
was installed using a Mistunobu protocol. The acidic
cleavage of SEM on the pyrazole led to compounds 7–
10. In a separate route, 3 was first de-methylated at both
the 5 and 6-positions using BBr3. Selective methylation
Intermediate 39 could be readily made from 6-hydroxy-
1-indanone 17 in a similar fashion as described earlier
(Scheme 3). The hydroxy group of 39 was converted into
a triflate in the presence of NaH and PhN(OTf)2. A
Sonogashira reaction yielded compound 40. The hydroxy

Y. Tong et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5665–5670
5667
O
O
As we have demonstrated earlier, substitutions at both
the 6 and 7-positions extend into the solvent exposed
area in the kinase active site and can tolerate substantial
variations.5 In addition, the attachment of bis-alkoxy
groups to an aromatic ring is known to exist in marketed
orally active kinase inhibitors such as IressaÒ and Tarc-
evaÒ. Therefore, our initial strategy to improve the PK
profile of 2 was to replace one of its methoxy groups
with a different alkoxy moiety.10 During our study,
intraperitoneal (ip) dosing was the primary choice for
the initial PK screening in mice. As shown in Table 2,
although all the inhibitors (7–15) have potent inhibitory
activity, the plasma exposure of representative com-
pounds is low (AUC 6 0.93 lg h/mL). The location of
the methoxy (6 or 7-position) and the nature of the ter-
minal groups on the longer alkoxy side chain have min-
imal impact on the AUC values. These findings led us to
investigate compounds with only one alkoxy substitu-
tion at the 7-position with elimination of the methoxy
group, a potential metabolic liability. A series of com-
pounds (18–28, Table 3) were prepared mostly with a
solubilizing group capping the alkoxy side chain. Inter-
estingly, the basicity of the side chain has a profound
impact on the cellular anti-proliferative activity (data
from the MTS assay) of the inhibitors. Non-basic side
chain moieties such as thiophene and thiazole (18 and
19) led to compounds with no cellular potency (combo
EC50 > 5.9 lM). On the other hand, basic functional
ii, iii
i
O2N
H2N
iv, v
32
33
SEM
H
N
N
N
N
H2N
H2N
N
N
Cl
CN
34
35
H
N
H
N
vi
vii, viii
N
Cl
N
36
CN
H
O
N
H
N
N
N
N
X
37: X=H, H
38: X=O
35
CN
ix, viii
Scheme 2. Reagents and conditions: (i) Fe, NH4Cl, EtOH, water,
90 °C, 68%; (ii) NaH, phenyl 6-chloronicotinate, THF; (iii) AcOH,
hydrazine, EtOH, 90 °C, 66% (two steps); (iv) NaH, SEMCl, DMF,
73%; (vi) Pd2dba3, dppf, Zn, Zn(CN)2, DMA, 180 °C, microwave,
20 min, 54%; (vi) chloroacetaldehyde in water, H2SO4, NaBH4, THF,
0 °C, 28%; (vii) morpholine, Et3N, CH3CH2CN, KI, 85 °C, 32%; (viii)
HCl, CH2Cl2, EtOH, 75 °C, 68%; (ix) 3-morpholinopropanoic acid,
HATU, Et3N, THF, 71%.
Table 2. Potency and PK evaluation of 6,7-substituted inhibitors
H
N
N
SEM
N
R1
O
N
HO
N
i, ii
HO
RO
R2
6
N
CN
CN
17
39
SEM
N
N
v, vi
Compound R1
R2
CHK-1
IC50 (nM) (lg h/mL)
AUCa
N
N
CN
7
8
MeO
3.7
0.47
40: R=H
O
iii, iv
41: R=4-morpholino
O
N
MeO
3.8
—
O
H
N
O
N
N
S
O
9
MeO
MeO
2.5
0b
N
N
CN
42
O
10
13
3.1
3.0
0.30
HO
Scheme 3. Reagents and conditions: (i) NaH, PhN(OTf)2, THF, 87%;
(ii) 3-butyn-1-ol, CuI, Pd(dppf)Cl2, Et3N, DMF, 85 °C, 59%; (iii)
MsCl, Et3N, THF, 85%; (iv) morpholine, DMF, 90 °C, 52%; (v) Pd/C,
H2, THF, 81%; (vi) HCl, EtOH, 75 °C, 59%.
O
N
MeO
0.93
O
S
N
O
14
15
MeO
MeO
1.1
2.6
—
group on 40 was mesylated and displaced by morphol-
ine. The acetylene moiety of 41 was reduced via catalytic
hydrogenation followed by removal of the SEM to give
42, a compound having a side chain with a carbon–car-
bon bond linker at the 7-position and a terminal mor-
pholine group.
O
—
HO
a Intraperitoneal dosing in mouse (10 mg/kg) except for 9. Vehicle (for
all mouse PK study unless indicated otherwise): 2.5% EtOH, 5%
Tween 80, 25% PEG400, PBS.
b Oral dosing in rat (5 mg/kg).

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Y. Tong et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5665–5670
groups, such as piperidyl and homomorpholino, with
pKa values over 9 resulted in compounds (25–28) with
weak to fair potency in combination with doxorubicin.
However, these compounds exhibited some cytotoxicity
by themselves (single EC50 value between 2.7 and
6.2 lM). Only alkoxy side chains with pKa around 6–7
gave inhibitors (20–24) with desirable results in the
MTS assay, that is, weak or no single agent activity
but high potentiation effect (at least 24-fold) on doxoru-
bicin. More importantly, among this group of inhibi-
tors, compound 20 possesses an AUC value (via ip
dosing) in mice more than 6 times higher (5.7 vs
0.93 lg h/mL) than that of 13, which has an extra meth-
oxy at the 6-position. Therefore, compound 20 emerged
as a new lead for further PK optimizations.
Table 4 summarizes the modification efforts around 20.
The carbon bridge between the morpholino group and
the oxygen on the side chain of 20 was extended from
two carbons up to five. Compounds 29 (with a three-car-
bon bridge) and 30 (with a four-carbon bridge) dis-
played plasma exposure comparable to 20 when dosed
intraperitoneally in mice. Both compounds also pos-
sessed desirable cellular potency. Compound 29 was
not cytotoxic as a single agent and was able to potenti-
ate doxorubicin with an EC50 value at 1.6 lM. Mean-
Table 3. Evaluation of inhibitors with an ether linker at the 7-position
H
N
N
O
R
N
CN
a
Compound
R
pKa
1.6
3.3
5.7
6.1
6.1
6.9
7.2
9.1
9.1
9.1
11
CHK-1
IC50 (nM)
MTS EC50
(lM) S/Cb
AUCc
(lg h/mL)
18
19
20
21
22
23
24
25
26
27
28
52
>59/ > 5.9
7.6/ > 5.9
>59/1.5
29/1.2
—
S
S
6.4
5.9
1.8
1.0
1.5
1.1
1.5
0.92
2.6
1.0
—
N
N
5.7
1.7
3.0
2.2
—
O
O
O
N
N
>52/1.6
33/0.51
>59/1.4
6.2/>5.9
5.4/1.1
O
O
N
N
N
N
N
—
N
0.031d
—
N
O
N
4.0/1.3
2.7/2.9
—
HN
a Used PharmaAlgorithms protocol in calculation.
b S/C, Single/Combo.
c Intraperitoneal dosing in mouse (10 mg/kg) except for 26.
d Oral dosing in mouse (10 mg/kg).

Y. Tong et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5665–5670
5669
Table 4. Optimizing 20
Compounds 29 and 30 were chosen to be further evalu-
ated for their PK properties dosed via IV and PO in
mice (Table 5). They both showed reasonable oral
AUC values (3.0 lg h/mL for 29 and >3.311 for 30)
and oral biovailability (F = 44% and > 50% for 29
and 30, respectively). These numbers represented a sig-
nificant improvement from the earlier generation inhib-
itors with a bi-aryl phenol moiety at the 3-position5 (oral
AUC = 0–0.45 lg h/mL, F = 0–9%). The oral exposure
of compound 30 was also studied in the Nude/Nude
mice (Chart 1), a more appropriate model relevant to fu-
ture in vivo efficacy studies. Dosed in two different vehi-
cles (NMP and Phosal), the compound exhibited
significant plasma exposures with Cmax around 5 or 10
times the cellular EC50 values at 25 or 50 mkd dosage,
respectively.
H
N
N
7
6
N
R
CN
Compound R
CHK-1
MTS EC50 AUCb
IC50 (nM) (lM) S/Ca (lg h/mL)
O
O
O
O
N
20
5.9
3.2
0.75
1.4
7.0
>59/1.5
>59/1.6
21/0.54
8.1/1.9
5.7
2.6
O
7
N
29
n=2
O
7
7
7
In summary, we explored a newer series of 1,4-dihydr-
oindeno[1,2-c]pyrazole compounds with a cyanopyri-
dine moiety at the 3-position as potent CHK-1
inhibitors. Replacing the bi-aryl phenol in the earlier
series with the cyanopyridine not only retained the
good enzymatic potency12 for the inhibitors but also
provided a better foundation for the PK improvement.
The compounds with bis-alkoxy side chains failed to
enhance PK properties. However, installing mono-
30
10.4c
—
N
n=3
n=4
O
N
31
O
NH
O
N
37
>59/>5.9
33/2.3
—
—
O
7
7
38
42
43
44
16
N
N
NH
7
O
O
Table 5. Further pharmacokinetic studies on 29 and 30
Compound Mouse IVa
Mouse POb
1.2
6.5
6.4
9.7/1.6
—
—
—
n=2
CLc Vd T1/2 (h) AUCe Cmax AUCe F (%)
d
f
O
O
29
30
3.8
3.4
7.6 1.4
6.9 1.4
2.0
2.1
0.66
0.77
3.0
>3.3
44
>50
N
N
>59/>5.9
>59/>5.9
O
O
6
a 3.0 mg/kg.
b 10 mg/kg.
c L/h kg.
d L/kg.
e lg h/mL.
f lM.
n=2
6
a S/C, Single/Combo.
b Intraperitoneal dosing in mouse (10 mg/kg).
c Vehicle, 10% NMP, 20% Solutol, 70% PBS to achieve better
solubility.
5
4
3
2
while, compound 30 had a slightly different profile with
weak single agent activity (EC50 = 21 lM) but stronger
anti-proliferative potency in the presence of doxorubicin
(combo EC50 = 0.54 lM). Like its close analogs, 20 and
29, compound 30 showed desired properties in the
mechanism-based cellular assay (FACS analysis) with
single-agent EC50 value more than 10 lM and combo
EC50 value at 0.58 lM. When the carbon bridge was ex-
tended to five carbons, the inhibitor (31) started to show
a deteriorated potentiation ratio (EC50 S/C) in the MTS
assay. The linker between the side chain and the tricyclic
core was also investigated. To this end, compounds with
an amino (37), an acetylamino (38), and a carbon (42)
linker were synthesized. Unfortunately, they showed
no or only marginal potentiation of doxorubicin. The
morpholino-capped alkoxy side chain was also moved
from the 7 to the 6-position. The resulting compounds
(43 and 44) showed no cellular activity at all, a dramatic
contrast to their corresponding analogs at the 7-position
(20 vs 43, and 29 vs 44).
10 mkd NMP AUC = 6
10 mkd Phosal AUC = 5
25 mkd NMP AUC = 12
25 mkd Phosal AUC = 15
50 mkd NMP AUC = 25
50 mkd Phosal AUC = 32
1
EC50 (MTS)
0
0.5
1
3
6
9
Time (hr)
Chart 1. Plasma levels of 30 in Nu/Nu mice via oral dosing (vehicle:
NMP or Phosal).

5670
Y. Tong et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5665–5670
7. See Ref. 4 for detailed structural biology information.
8. The MTS assay in HeLa cells was used as the functional
assay. In this assay, the anti-proliferative effect was
measured in the form of EC50 values for a CHK-1
inhibitor as a single agent and also for an inhibitor in the
presence of doxorubicin (150 nM), a DNA-damaging
agent. For the combination study, the baseline of the
regression for EC50 calculation was adjusted to the
inhibition level determined for 150 nM doxorubicin alone,
a concentration known to cause G2/M arrest in HeLa
cells. The ratio of the two corresponding EC50s (combo/
single) represents a relative scale of a CHK-1 inhibitor’s
function to potentiate doxorubicin. See Ref. 4 for assay
conditions.
9. The cell cycle analysis (FACS assay) in H1299 cells
examines the mechanism for the anti-proliferative function
of the inhibitors. An EC50 was either the concentration of
a CHK-1 inhibitor that reduces doxorubicin (500 nM)-
induced G2/M cell population by half (combo), or it was
measured in the absence of doxorubicin (single). See Ref. 4
for assay conditions and representative raw data.
alkoxy with a morpholino terminal at the 7-position
led to compounds, such as, 20, 29, and 30, with much
improved plasma exposures upon ip dosing in mice as
well as desirable cellular anti-proliferative potentiation
of doxorubicin. The window for achieving balanced
properties appeared to be small since heterocyclic
groups other than morpholine at the end of the side
chains, extra alkoxy groups at the 6-position, linkers
other than the ether off the tricyclic core, and placing
the optimized side chains at the 6 instead of the 7-po-
sition all resulted in compounds with inferior overall
profiles. With compounds 29 and 30 having oral
biovailability at 44% or higher in mice, we have
achieved tricyclic pyrazole compounds with much im-
proved oral biovailability while maintaining good
potencies. These two molecules will be used as tool
compounds for in vivo evaluation of CHK-1 inhibi-
tors to sensitize DNA-damaging agents.
10. For compounds with bis-alkoxy groups at the 6 and 7-
positions and a bi-aryl phenol moiety at the 3-position of
this class of CHK-1 inhibitors, see Tao, Z.-F.; Li, G.; Tong,
Y.; Chen, Z.; Merta, P.; Kovar, P.; Zhang, H.; Rosenberg,
S. H.; Sham, H. L.; Sowin, T. J.; Lin, N.-H. Bioorg. Med.
Chem. Lett. 2007. doi:10.1016/j.bmcl.2007.05.027.
References and notes
1. For reviews, see (a) Zhou, B. B.; Bartek, J. Nat. Rev.
Cancer 2004, 4, 1; (b) Tao, Z-F.; Lin, N-H. Anti-Cancer
Agents Med. Chem. 2006, 6, 377.
11. The plasma concentration was sustained during the last
several sampling time points.
2. For reviews, see (a) Abraham, R. T. Genes Dev. 2001, 15,
2177; (b) Shiloh, Y. Nat. Rev. Cancer 2003, 3, 155.
3. (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.;
12. 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
biotinylated 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
EDTA (pH 8.0). A 40 lL aliquot of the reaction 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 mag-
nesium chloride and incubated at room temperature for
10 min. The plate was then washed 3 times in PBS with
0.05% of Tween 20 and counted in a Packard TopCount
counter (Packard BioScience Company, Meriden, CT).
˚
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6. The medicinal chemistry effort leading to discover of the
cyanopyridine moiety will be published in a separate
account.