Discovery of 4′-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-benzonitriles and 4′-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-pyridine-2′-carbonitriles as potent checkpoint kinase 1 (Chk1) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.07.102

An extensive structure-activity relationship study of the 3-position of a series of tricyclic pyrazole-based Chk1 inhibitors is described. As a result, 4′-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-benzonitriles (4) and 4′-(1,4-dihydro-indeno[1,2-c]pyrazol-3

Full text of DOI:10.1016/j.bmcl.2007.07.102

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5944–5951
Discovery of 40-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-
benzonitriles and 40-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-
pyridine-20-carbonitriles as potent checkpoint
kinase 1 (Chk1) inhibitors
Zhi-Fu Tao,* Gaoquan Li, Yunsong Tong, Kent D. Stewart, Zehan Chen, Mai-Ha Bui,
Philip Merta, Chang Park, Peter Kovar, Haiying Zhang, Hing L. Sham,
Saul H. Rosenberg, Thomas J. Sowin and Nan-Horng Lin
Cancer Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064, USA
Received 19 June 2007; revised 20 July 2007; accepted 23 July 2007
Available online 25 August 2007
Abstract—An extensive structure–activity relationship study of the 3-position of a series of tricyclic pyrazole-based Chk1 inhibitors
is described. As a result, 40-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-benzonitriles (4) and 40-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-
pyridine-20-carbonitriles (29) emerged as new lead series. Compared with the original lead compound 2, these new leads fully retain
the biological activity in both enzymatic inhibition and cell-based assays. More importantly, the new leads 4 and 29 exhibit favorable
physicochemical properties such as lower molecular weight, lower ClogP, and the absence of a hydroxyl group. Furthermore, struc-
ture–activity relationship studies were performed at the 6- and 7-positions of 4, which led to the identification of ideal Chk1 inhib-
itors 49, 50, 51, and 55. These compounds not only potently inhibit Chk1 in an enzymatic assay but also significantly potentiate the
cytotoxicity of DNA-damaging agents in cell-based assays while they show little single agent activity. A cell cycle analysis by FACS
confirmed that these Chk1 inhibitors efficiently abrogate the G2/M and S checkpoints induced by DNA-damaging agent. The
current work paved the way to the identification of several potent Chk1 inhibitors with good pharmacokinetics that are suitable
for in vivo study with oral dosing.
Ó 2007 Elsevier Ltd. All rights reserved.
Cancer is a top killer of human beings. There is great ur-
gency to develop highly efficacious and minimally toxic
treatments for cancer. Although tremendous progress
has been achieved in the development of novel cancer
treatments, most of the current cancer drugs usually
exhibit high toxicity and are severely resisted by tumor
cells in the clinic. This dilemma is particularly true for
DNA-damaging agents, the mainstay of cancer treat-
ment.1 To improve the efficacy and lower the toxicity
of DNA-damaging anticancer drugs, the development
of adjuvant therapeutics has been aggressively pursued
in recent years.2,3 Such treatments may either sensitize
tumor tissue or protect normal tissue from DNA
damage.
Checkpoint kinase 1 (Chk1), a serine/threonine protein
kinase which plays a key role in DNA damage-induced
checkpoints,4,5 has emerged as an attractive chemosensi-
tization anticancer target.6,7 Chk1 inhibitors have been
demonstrated to abrogate the DNA damage-induced S
and G2 checkpoints and disrupt the DNA repair pro-
cess, resulting in premature chromosome condensation
and leading to cell death; this preferentially sensitizes
tumor cells, especially p53-null cells, to various DNA-
damaging agents.5–17 An optimal therapeutic window
may be achieved for Chk1 inhibitors in the clinic be-
cause normal cells can be arrested in the G1 phase and
are less affected by S and G2 checkpoint abrogation in
response to DNA damage.10
Several classes of Chk1 inhibitors have been recently
reported.10–27 UCN-01 is the most extensively studied
Chk1 inhibitor and is now in phase I/II clinical tri-
als.14,15 It abrogates both the S and G2 checkpoints
and sensitizes tumor cells to a wide spectrum of
Keywords: Antitumor agent; Chk1 inhibitors; Checkpoint kinase 1;
Pyrazole; Structure–activity relationship; DNA damage; Combination
therapy.
*
Corresponding author. Tel: +1 847 938 6772; fax: +1 847 935 5165;
e-mail: Zhi-Fu.Tao@abbott.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.102

Z.-F. Tao et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5944–5951
5945
DNA-damaging agents.16,17 Unfortunately, UCN-01 also
potently inhibits a number of other kinases and exhibits
strong single agent activity, and it has a high binding
affinity for human plasma protein, which could limit
its use in the clinic.18 It remains a great challenge to
identify ideal Chk1 inhibitors that show no single agent
activity, but significantly potentiate DNA-damaging
antitumor agents. We have discovered 40-(1,4-dihydro-
indeno[1,2-c]pyrazol-3-yl)-biphenyl-4-ols, exemplified
by 1 and 2, as a novel class of Chk1 inhibitors.25–27
SAR studies on the 5-, 6-, and 7-positions disclosed that
analogues with substituents on both the 6- and 7-posi-
tions stood out as the most potent subseries of tricyclic
pyrazole-based Chk1 inhibitors.26 For example, 2 is not
only highly potent in the inhibition of Chk1 in an enzy-
matic assay, but also significantly potentiates the cyto-
toxicity of DNA-damaging agents to tumor cells
without showing single agent activity.26 However, these
compounds show very poor pharmacokinetics (PK).27
Preliminary ADME studies indicated that they exhibit
low solubility due to the high lipophilicity of the biphe-
nyl moiety.27 In addition, the hydroxyl group is poten-
tially labile to phase II biotransformations such as
O-glucuronidation and O-sulfation. We have therefore
performed extensive SAR studies on the 3-position in
order to replace the biphenyl moiety of 2. As a result,
40-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-benzonitriles
and 40-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-pyridine-
20-carbonitriles were identified as new series of potent
Chk1 inhibitors.
Table 1. Structure–activity relationship of phenyl analogues at the C-3
position
N
NH
O
O
7
3
6
R
Compound
R=
Chk1 inhibition
(IC50, nM)a
2
2
OH
3
4
5
OH
CN
25
7
CO2H
2
6
25
OH
NH2
7
779
8
355
NH2
NH2
9
10
11
>10,000
73
Br
>10,000
N
O
H
1
N
HO
N
1: R1=
N
12
13
14
1288
6223
4579
H
NH
3
2'
N
R2
R1
7
3'
R2=H. IC50 = 6 nM
N
N
6
2: R1=R2=OMe
OH
5
O
IC50 = 2 nM
Table 1 shows the SAR results of analogues in which the
terminal phenol group of 2 was replaced. Compounds
3–6, which have polar substituents, showed potent
Chk1 inhibition activity, suggesting possible hydrogen
bonds in the polar region. However, the replacement
of the hydroxyl group with an amino group (7, 8) re-
sulted in a substantial decrease in potency. Moving the
amino group to the 30-position of the phenyl ring pro-
duced a completely inactive compound (9). Other sub-
stituents such as bromide (10), pyridyl (11, 12),
morpholinyl (13), 40-hydroxyl piperidinyl (14) caused
considerable reduction of potency. Compounds with a
variety of five-member ring heterocyclic substituents
(15–21) were also evaluated. Only compound 15, which
contains the tetrazole, exhibits comparable potency as 2.
The analogues containing pyrazole (16) and pyrrole (17)
are slightly less potent than 15. Analogues containing
imidazole (18–20) and furan (21) are much weaker
Chk1 inhibitors.
OH
N
15
16
NH
11
47
N
N
NH
N
H
N
17
77
NH
18
19
1260
1374
N
N
N
N
O
20
21
3709
3003
N
COOH
Analogues with an alkyne spacer between the tricyclic
core and the phenol are depicted in Table 2. These com-
pounds (22–27) retain rigidity, are less lipophilic, and
have lower molecular weight compared with the biphe-
a IC50 was measured by using 2 lM of substrate peptide in the presence
of c-[33P]ATP (5 lM). For detailed enzymatic procedures, see
Ref. 10.

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Z.-F. Tao et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5944–5951
Table 2. Structure–activity relationship at the C-3 position with an
alkynyl spacer
inhibitor. The nitrogens at the 1- and 2-positions of 1
provide the hinge hydrogen-bonding replacement for
the urea carbonyl and a NH of the macrocyclic inhibi-
tor, respectively. The 40-N of the urea pyrazinyl ring is
situated in approximately the same position as C-30 of
1. We reasoned that a pyridyl analogue such as 28 would
possess better activity by forming water-mediated
hydrogen bonds in the water pocket (Table 3). Indeed,
the potency of 2 was improved 4-fold by incorporation
of a nitrogen atom at the 30-position (28). The replace-
ment of the terminal hydroxylphenyl group with a cyano
group (29) fully retained the potency. Other substitu-
tions on the pyridyl ring resulted in reduction of potency
(30–36). Moving the N atom to the other position in the
ring considerably decreased the activity (29 vs 37). The
introduction of an additional N atom into the ring
was also detrimental to potency (38 vs 30, 39 vs 30).
Molecular modeling indicates that three hydrogen
bonds (between the CN and Lys38, between the pyridyl
N and Ser147, and water-mediated hydrogen bonds be-
tween the pyridyl N and Asn59) may greatly contribute
to the high potency of 29.
N
NH
O
O
R
Compound R=
Chk1 inhibition (IC50, nM)
5140
22
H
23
19
OH
O
24
25
26
27
5
OH
>10,000
1743
>10,000
N
O
N
NH2
NH2
H
O
Cell-based assays reveal that 4 and 29 significantly sen-
sitize tumor cells to DNA-damaging agents while
showing little cytotoxicity alone (vide infra). Thus,
extensive SAR studies were performed on the 5-, 6-,
and 7-positions to further optimize these two series.
Table 4 shows the SAR results at the 6-position of
compound 4. Analogues with polar groups (40–43) at
the 6-position exhibit low single digital nanomolar
IC50 values against Chk1. Analogues with heteroaro-
matic groups (44–49) at the 6-position also potently in-
hibit Chk1 and their potencies were moderately
affected by the type of pendant ring as well as the lin-
ker length between the tricyclic core and the pendant
aromatic ring. Analogues with cyclic substituents (50–
52) are the most potent compounds in this series.
The potency difference between 53 and 54 suggests an
important role of the carbonyl group of 53. The
above-discussed SAR results indicate that elaboration
of the 6-position not only significantly improves the
enzymatic activity (52, 50, 51, 49 vs 4) but also the
nol 2. The hydroxylphenyl analogues (23, 24) exhibit
comparable potency to 2 in an enzymatic assay, but
had inferior cellular activity (vide infra). The replace-
ment of the hydroxyl group with other polar groups
such as cyano (25), urea (26), and amide (27) lost the
Chk1 inhibition activity.
Examination of the X-ray co-crystal structure of 1-Chk1
complex revealed that the hydroxyl group forms a
hydrogen bond with Asn59 in the water pocket, an area
of interest in gaining both potency and selectivity.7,25
Similar interaction in the water pocket was reported
for macrocyclic urea Chk1 inhibitors.13 The 40-N of
the pyrazinyl ring of the macrocyclic inhibitors points
to the water pocket and forms water-mediated hydrogen
bonds with Asn59.13 Figure 1a shows the overlay of
co-crystal structures of 1 and a macrocyclic urea Chk1
a
b
Asn59
Glu85
A
Cys87
Cl
H
Glu55
B
C
O
N
H
N
N
N
H
Lys38
N
O
N
N
O
Figure 1. (a) Overlay of crystal structures of 1 (magenta carbons) and macrocyclic inhibitor (orange carbons) bound to Chk1 kinase. The connolly
surface outlining the active site is shown in gray. Hinge hydrogen bonds for 1 are shown with dotted lines. Glu85 and Cys87 comprising the hinge and
Lys38, Glu55, and Asn59 are shown with thick bonds. Ser147 is located directly behind the pyrazine of the macrocyclic inhibitor and is obscured by
the active site surface. Three water molecules as observed in the crystal structure of the macrocyclic inhibitor, denoted A–C, are shown with one
possible assignment of hydrogen positions. This network of intermolecularly hydrogen-bonded waters within the pocket is characteristic of Chk1
kinase subfamily within the kinase enzyme family. (b) The chemical structure of the macrocyclic urea compound.

Z.-F. Tao et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5944–5951
5947
Table 3. Structure–activity relationship at the C-3 position with a
heteroaromatic ring spacer
Table 4. Structure–activity relationship at the 6-position of
compound 4
N
N
NH
NH
O
R
7
O
O
7
R
3
6
6
CN
Compound
R=
Chk1 inhibition
(IC50, nM)
Compound
R=
Chk1 inhibition
(IC50, nM)
N
N
40
4
O
O
28
29
30
31
32
33
34
35
0.5
0.8
15
4
OH
N
41
42
43
44
45
46
47
3
O
N
HO
HO
CN
3
O
N
HO
HO
6
O
N
O
14
20
10
77
N
Cl
N
2
O
N
F
O
N
N
N
288
288
16
Br
O
N
N
O
CF3
48
49
9
2
S
N
N
Cl
N
Cl
Cl
50
51
52
53
3
2
1
3
36
>10,000
N
O
O
Cl
O
N
O
37
38
39
44
89
44
CN
O
O
H
N
N
O
O
N
N
O
54
55
17
2
N
H
N
OH
potentiation ratio in the cellular assay (Table 5). SAR
studies were also performed on the 7-position. In gen-
eral, analogues with modifications at the 7-position are
less potent than those with the same modifications at
the 6-position (data not shown).
EC50 values and potentiation ratios of representative
compounds. These compounds could be classified into
three categories: (1) compounds that do not show cellu-
lar activity at all (5, 15, 23) or only show little or no
potentiation of the cytotoxicity of Dox (24, 54, 32); (2)
compounds that show significant potentiation but also
have single agent activity (28, 43); (3) compounds that
Potent Chk1 inhibitors were further evaluated in a MTS
assay using p53-deficient HeLa cells in the absence or
presence of doxorubicin (Dox).28 Table 5 shows the

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Z.-F. Tao et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5944–5951
Table 5. Cellular activities in the presence and absence of Dox
abrogated camptothecin (CPT)-induced S arrest (data
not shown).
Compound
MTS EC50
Potentiation
ratio
FACS EC50
(lM) Cmpd +
Dox/(ꢀDox)
1.3/>50
>10/>10
>10/>10
4.8/>10
6.1/>10
0.3/3.5
(lM) Cmpd +
Dox/(ꢀDox)
1.3/33.6
The syntheses of 3–15 and 17–21 are shown in Scheme 1.
Compound 10, prepared by a literature procedure,26 was
smoothly coupled with various boronic acids under Su-
zuki conditions in a microwave synthesizer to provide 8,
9, 11, and 17–21. A similar Suzuki coupling of 57, pre-
pared from 5,6-dimethoxy-indan-1-one in three steps,
gave 3 and 5–7.
4
5
26
>5.9/>59.3
>5.9/>59.3
>5.9/>59.3
4.4/2.5
N/A
N/A
N/A
0.5
13
15
23
24
28
29
30
31
32
40
41
42
43
49
50
51
52
54
0.8/10.6
1.1/>59.3
4.8/>59.3
3.9/>59.3
2.1/14.2
>54
>12
>15
7
0.3/>10
4.2/>10
1.4/>10
0.68/8.2
1.3/>10
2.7/>10
2.7/>10
2.1/>10
0.7/>10
2.6/>10
1.9/>10
1.3/>10
1.3/>10
Compounds 12–14 were obtained by amination of 10
under Buckwald conditions in a microwave synthesizer.
The displacement of bromide from 10 by cyanide was ef-
fected in Pd(PPh3)4–Zn(CN)2–DMF to produce 4. Com-
pound 15 was obtained through cyanylation of 10
followed by in situ cyclization with sodium azide in
one pot under microwave conditions.
1.8/14.2
1.5/13.9
8
9
1.4/10.8
1.0/18.3
8
18
1.0/42.0
1.9/>59.3
0.8/46.5
42
>31
57
Scheme 2 depicts the synthesis of 16. Protected tricyclic
pyrazole bromide 60 (the protecting group could be on
either of the N atoms, only one isomer is shown), pre-
pared from 10, was transformed into boronic ester 61
in excellent yield. Compound 61 was coupled with io-
dide 59 to provide 62. Deprotection of the two pyraz-
oles of 62 produced the final product 16 in quantitative
yield.
1.6/>59.3
4.0/16.3
>37
4
show little or no antiproliferative activity alone but sig-
nificantly potentiate Dox (4, 29, 49, 50, 51, and 52),
which have been defined as ideal Chk1 inhibitors. The
lack of a good correlation between Chk1 enzymatic
inhibition potency and cellular antiproliferative activity
may be due to variation in physicochemical properties
such as cellular permeability and potential off-target
activity.
The syntheses of tricyclic pyrazoles bearing an alkyne
spacer are shown in Scheme 3. The syntheses featured
two Sonogashira reactions. Coupling of compound 57
with TMS-protected acetylene followed by deprotection
provided 22. A second Sonogashira reaction between 22
and various arylbromides in a microwave synthesizer
smoothly produced 23–27.
Fluorescence-activated cell sorting (FACS) analysis of
cell cycle profiles was used to study the mechanism of
action of Chk1 inhibitors.29 Table 5 shows the EC50 val-
ues for abrogation of G2/M caused by Dox in the pres-
ence of various concentrations of Chk1 inhibitors. In
accord with the antiproliferative activity cited above,
ideal Chk1 inhibitors such as 4, 29, 49, 50, 51, and 52
do not affect the regular cell cycle profile at high concen-
tration (up to 50 lM), but efficiently abrogated the Dox-
induced-G2/M checkpoint with nanomolar to low single
digit micromolar EC50 values. Furthermore, we con-
firmed that representative Chk1 inhibitors efficiently
The synthesis of heteroaromatic analogues is shown in
Scheme 4. 6-Chloronicotinic acid 64 was activated by
DCI and then coupled with 5,6-dimethoxy-indanone in
the presence of NaH to provide the diketone 66. This
diketone was treated with hydrazine in ethanol to pro-
duce 31. Compound 28 was obtained by the Suzuki cou-
pling of 31 with 4-hydroxyphenyl boronic acid. The
cyanylation of 31 provided 29. Compounds 32–38 were
prepared by using synthetic procedures analogous to
H
H
H
N
N
N
N
N
N
MeO
c
MeO
MeO
MeO
MeO
d
NH
R
MeO
10
8, 9, 11
17-21
R
Br
12-14
a
b
4
15
H
H
N
N
O
N
N
O
O
O
O
O
e
c
f
3
5-7
I
89%
64%
O
56
57
Scheme 1. Reagents: (a) Pd(PPh3)4, Zn(CN)2, DMF; (b) Pd(PPh3)4, Zn(CN)2, DMF; then, NaN3, NH4Cl, microwave; (c) boronic acids, Na2CO3,
Pd(PPh3)2Cl2, DME–EtOH–H2O; (d) corresponding amines, biphenyl-2-yldi-tert-butylphosphine, NaOt-Bu, Pd(OAc)2, toluene; (e) formic acid ethyl
ester, NaH, benzene; then, NH2NH2, HOAc, EtOH; (f) NIS, DMF.

Z.-F. Tao et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5944–5951
5949
R
H
O
N
O
N
a
N
I
N
I
72%
R=
58
59
R
N
R
N
N
N
MeO
MeO
MeO
a
b
O
10
B
O
83%
88%
61
MeO
60
Br
R
N
HN
N
N
MeO
MeO
d
MeO
MeO
c
N
N
95%
65%
NH
16
62
N
R
Scheme 2. Reagents and condition: (a) 4,40-(chloromethylene)bis(methoxybenzene), TEA, THF; (b) bis(pinacolato)dibrone, PdCl2(dppf)CH2Cl2;
(c) 59, Na2CO3, Pd(PPh3)2Cl2, DME–EtOH–H2O, microwave, 160 °C; (d) HCl, dioxane.
H
H
N
N
N
N
O
O
O
O
c
a
b
23 - 27
57
TMS
63
22
Scheme 3. Reagents and condition: (a) (trimethylsilyl)acetylene, PdCl2(PPh3)2, CuI, PPh3, TEA, DMF, microwave, 120 °C; (b) TBAF, THF;
(c) arylbromides, PdCl2(PPh3)2, CuI, PPh3, TEA, DMF, microwave, 120 °C.
O
O
O
O
MeO
MeO
b
N
a
N
N
N
HO
N
Cl
65
66
Cl
Cl
64
c
H
N
H
N
N
N
MeO
MeO
MeO
MeO
e
d
28
N
N
29
31
Cl
CN
Scheme 4. Reagents and condition: (a) DCI, DMF; (b) 5,6-dimethoxy-indanone, NaH, THF; (c) NH2NH2, HOAc, ethanol; (d) 4-hydroxyphenyl
boronic acid, Na2CO3, Pd(PPh3)2Cl2, DME–EtOH–H2O, microwave, 160 °C; (e) Zn(CN)2, Pd(PPh3)4, DMF.
those described for 31. Compounds 30 and 39 were syn-
thesized by Suzuki coupling of 57 with the correspond-
ing boronic acids.
Scheme 6 depicts the synthesis of 49, 53, and 54. 5-Hy-
droxy-6-methoxy-indanone (72)26 was converted into
trifilate 73 in excellent yield. Carbonylation of 73 was
smoothly achieved under palladium-catalyzed condi-
tions to provide 74. Intermediate 75 was obtained in
two steps by following a sequence similar to that de-
scribed for 31. The saponification of 75 gave acid 76.
The coupling of 76 and 4-aminocyclohexanol in the
presence of Bop reagent provided the final product 53.
Compound 78, prepared by the reductive amination of
77, was coupled with the cyanophenyl boronic acid un-
der Suzuki conditions to provide 54. Compound 79, ob-
tained by the SEM protection of 75 (the SEM group
could be on either of the nitrogen atoms; for clarity,
only one isomer is shown), was reduced to alcohol 80.
The coupling of 80 and DCI in acetonitrile gave 81.
The removal of the SEM group from 80 under acidic
conditions provided the final product 49.
Scheme 5 outlines the synthesis of compounds 40–48,
50–52, and 55. Starting from the O-protected indanone
67,26 tricyclic pyrazole 68 was synthesized in two steps
by a synthetic procedure analogous to that described
for 31. The treatment of 68 with SEMCl produced the
protected pyrazole 69 as a mixture of two regioisomers
with the SEM group attached to either of the nitrogen
atoms (for clarity, only one isomer is shown). Selective
removal of the SEM group at the 6-position of 69 in a
diluted HCl–ethanol system produced the key interme-
diate 70. Compound 71, prepared by coupling of 70 with
an alcohol under Mitsunobu conditions, was deprotec-
ted to provide Chk1 inhibitors 48, 50–52. Removal of
both SEM groups from 69 gave 55.

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Z.-F. Tao et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5944–5951
SEM
H
N
N
O
N
N
O
O
O
O
O
b
a
O
69
SEM
CN
68
SEM
CN
67
SEM
c
SEM
SEM
N
N
N
N
O
O
40 - 48
50 - 52
e
d
RO
HO
71
CN
70
CN
Scheme 5. Reagents: (a) 5-(imidazole-1-carbonyl)-pyridine-2-carbonitrile, NaH, THF; then, NH2NH2, HOAc, ethanol; (b) SEMCl, NaH, DMF;
(c) HCl (diluted), dioxane; (d) ROH, DBAD, polymer-supported PPh3, THF; (e) HCl, EtOH.
O
O
O
O
O
O
O
a
b
O
O
77%
94%
HO
74
73
Tf
72
c
81%
H
N
H
N
N
N
O
O
d
e
O
53
O
85%
68%
HO
O
CN
75
76
CN
H
N
H
N
O
N
g
N
f
O
I
54
I
78
OHC
NH
77
HO
SEM
SEM
N
N
N
N
O
i
O
h
75
O
80
CN
HO
79
CN
H3CO
SEM
N
N
O
j
k
49
81
CN
N
N
Scheme 6. Reagents and conditions: (a) triflic anhydride, 2,6-lutidine, 4-DMAP; (b) CO, PdCl2(dppf)-CH2Cl2, TEA, CH3OH; (c) 5-(imidazole-1-
carbonyl)-pyridine-2-carbonitrile, NaH, THF; then, NH2NH2, HOAc, ethanol; (d) NaOH, H2O, EtOH; (e) 4-aminocyclohexanol, TEA, Bop
reagent, DMF; (f) trans-4-aminocyclohexanol, K2CO3, EtOH; then NaBH4; (g) 4-cyanophenyl boronic acid, Na2CO3, Pd(PPh3)2Cl2, DME–EtOH–
H2O, microwave, 160 °C; (h) SEMCl, NaH, DMF; (i) NaBH4, THF–MeOH; (j) DCI, CH3CN, 80 °C, 24 h; (k) HCl, EtOH.
In summary, an extensive structure–activity relationship
at the 3-position of tricyclic pyrazole-based Chk1 inhib-
itors was described. In this endeavor, 40-(1,4-dihydro-in-
deno[1,2-c]pyrazol-3-yl)-benzonitriles (4) and 40-(1,4-
dihydro-indeno[1,2-c]pyrazol-3-yl)-pyridine-20-carbo-
nitriles (29) emerged as new lead series. Compared with
the original lead compound 2, these new leads fully re-
tain the biological activity in both enzymatic inhibition
and cell-based assays. More importantly, the new leads
4 and 29 exhibit favorable physicochemical properties
such as lower molecular weight, lower ClogP, and the
absence of the hydroxyl group. SAR on the 6-position
of 5 suggested that both the enzymatic activity (49, 50,
51, 55 vs 4) and the potentiation ratio in the cellular as-
say could be improved significantly by modifying the 6-
position. The discovery of lead compound 29 has paved

Z.-F. Tao et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5944–5951
5951
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