Synthesis and biological evaluation of 1-(2,4,5-trisubstituted phenyl)-3-(5-cyanopyrazin-2-yl)ureas as potent Chk1 kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.01.028

Based on the X-ray crystallography of our lead compound 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-cyanopyrazin-2-yl)urea in the checkpoint kinase 1 (Chk1) enzyme, we modified R⁴, and to a lesser extent, R², and R⁵ of the phenyl ring, and made a variety of N-aryl-N′-pyrazinylurea Chk1 inhibitors. Enzymatic activity less than 20 nM was observed in 15 of 41 compounds. Compound 8i provided the best overall results in the cellular assays as it abrogated doxorubicin-induced cell cycle arrest (IC₅₀ = 1.7 μM) and enhanced doxorubicin cytotoxicity (IC₅₀ = 0.44 μM) while displaying no single agent activity.

Full text of DOI:10.1016/j.bmcl.2006.01.028

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2293–2298
Synthesis and biological evaluation of
1-(2,4,5-trisubstituted phenyl)-3-(5-cyanopyrazin-2-yl)ureas
as potent Chk1 kinase inhibitors
Gaoquan Li,* Lisa A. Hasvold, Zhi-Fu Tao, Gary T. Wang, Stephen L. Gwaltney, II,
Jyoti Patel, Peter Kovar, Robert B. Credo, Zehan Chen, Haiying Zhang, Chang Park,
Hing L. Sham, Thomas Sowin, Saul H. Rosenberg and Nan-Horng Lin
Cancer Research, Global Pharmaceutical Research & Development, Abbott Laboratories, Abbott Park, IL 60064, USA
Received 11 November 2005; revised 3 January 2006; accepted 6 January 2006
Available online 30 January 2006
Abstract—Based on the X-ray crystallography of our lead compound 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-cyanopyrazin-2-yl)
urea in the checkpoint kinase 1 (Chk1) enzyme, we modified R4, and to a lesser extent, R2, and R5 of the phenyl ring, and made
a variety of N-aryl-N0-pyrazinylurea Chk1 inhibitors. Enzymatic activity less than 20 nM was observed in 15 of 41 compounds.
Compound 8i provided the best overall results in the cellular assays as it abrogated doxorubicin-induced cell cycle arrest
(IC50 = 1.7 lM) and enhanced doxorubicin cytotoxicity (IC50 = 0.44 lM) while displaying no single agent activity.
Ó 2006 Elsevier Ltd. All rights reserved.
When DNA is damaged by radiation or chemical
reagents, normal cells arrest in the G1 phase, via the
tumor suppressor protein p53, and attempt repair.1,2 Tu-
mor cells, however, often have mutated p53 and thus,
must rely on the S and G2 checkpoints to repair their
DNA.1–3 Checkpoint kinase 1 (Chk1) is a human nucle-
ar serine/threonine protein kinase. Upon DNA damage,
Chk1 is activated and can phosphorylate and destabilize
Cdc25A. This phosphorylation is necessary for S and
G2 arrest.4,5 Inhibition of Chk1 results in abrogation
of arrest in the S and G2 phases, thereby allowing the
DNA-damaged cells to progress prematurely into mito-
sis resulting in mitotic catastrophe or apoptosis.6–8
Therefore, abrogation of the S and G2 checkpoints
should lead to an increased and selective sensitivity of
cancer cells to DNA damaging reagents in p53-deficient
cells.1–3,9 Thus, selective inhibitors of Chk1 may be of
great therapeutic value in cancer treatment.
of Chk1 inhibitors. Our previous work focused on the
modification of R2 of the phenyl ring12,13 (1 wherein
R4 = H, Fig. 1). We also synthesized 1-(5-chloro-
2,4-dimethoxyphenyl)-3-(5-cyanopyrazin-2-yl)urea (2,
Fig. 1) as a potent Chk1 kinase inhibitor (Chk1
IC50 ꢀ 7 nM).12 Tolerance of the methoxy substituent
at R4 of the phenyl ring indicates that some amount of
space exists in this region of the Chk1 protein and that
other groups may be tolerated, as well. X-ray crystallog-
raphy of compound 2 in the Chk1 enzyme16 (Fig. 2)
showed that R2 points toward the ribose pocket of the
Chk1 enzyme, while R4 points toward the solvent front.
Based on this, we decided to modify R4 for possible
improvement of physical properties such as polarity
and solubility while keeping similar potency (1,
Fig. 1). Initially, R4 was altered, while R2 and R5 were
held constant as the methoxy and chloro moieties,
Several compounds such as UCN-01 have been reported
as Chk1 inhibitors.10,11 We12,13 and others14,15 recently
discovered N-aryl-N0-pyrazinylureas to be a new class
R5
R2
Cl
O
R4
N
N
CN
O
N
N
CN
O
O
N
N
N
N
H
H
H
H
Keywords: Urea; Chk1 inhibitor.
*
1
2
Corresponding author. Tel.: +1 847 938 8114; fax: +1 847 935
7551; e-mail: Gaoquan.Li@abbott.com
Figure 1. Structure of compounds 1 and 2.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.028

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G. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2293–2298
Table 1. IC50 of compounds 6, 7, 8a–v, 14, and 15
Cl
R4
N
N
CN
O
N
N
H
H
O
Compound
R4–
Chk1 IC50 (nM)
6
7
–NO2
–NH2
42
5
O
N
8a
8b
8c
8d
8e
8f
3
22
N
H
O
O
O
N
H
O
O
O
18
O
N
H
9
N
H
O
O
O
Figure 2. X-ray crystal structure of compound 2 in the Chk1 enzyme.
15
N
H
O
O
O
O
23
N
respectively (Table 1). Later, R2 and R4 were concomi-
tantly altered, while R5 was held constant as the chloro
moiety or was removed completely (Table 2). The
synthesis and biological results are presented.
H
O
8g
8h
8i
>10,000
11
N HN
O
N
HN
The synthesis of the urea compounds with various
amide substituents at R4 of the phenyl ring is shown
in Scheme 1. Methylation of compound 4 provided com-
pound 5, which was refluxed with one equivalent of
compound 3 in toluene12,13 to give urea compound 6.
Reduction of the nitro group gave intermediate 7, which
was subsequently reacted with a variety of acid chlorides
to provide compounds 8a–v.
O
N
8
HN
O
N
8j
9
N
H
O
8k
8l
111
42
O
HN
O
S
HN
The preparation of compound 15 is shown in Scheme 2.
Methylation of compound 9 was followed by nitro
group reduction and protection of the resulting amino
group with TFAA. The methyl group was then trans-
formed through bromination to benzyl bromide 11, sub-
sequent hydrolysis and oxidation to the corresponding
aldehyde 12. Following removal of the trifluoroacetyl-
protecting group, urea compound 14 was synthesized.
Finally, reductive amination achieved compound 15.
O
8m
8n
8o
8p
8q
8r
8s
8t
58
N
H
O
S
91
N
H
S
O
68
N
H
O
243
328
72
N
H
H
N
The preparation of compounds of structure 19 in
Scheme 3 began with replacement of the 2-chloro substi-
tuent of 2,4,5-trichloronitrobenzene with one equivalent
of pyrrolidine.17 Diversity was introduced into the mol-
ecule in the next step by replacement of the 4-chloro
moiety with a selection of alkyl alcohols. Subsequent
reduction followed by urea formation provided com-
pounds 19a–e.
O
O
N
O
O
H
O
51
N
H
O
O
O
1365
7
N
H
O
S
O
8u
8v
14
15
N
H
O
Scheme 4 illustrates the synthesis of compounds of
structure 22. Compound 20 was obtained via reflux of
compound 16 and 10 equiv of NaOH in water for 3
days.18 Aromatic nucleophilic substitution with 2°
amines was followed by Mitsunobu reaction with a
variety of alcohols and intermediate 21. Nitro group
reduction and urea formation completed the
synthesis of compounds 22a–d.
O
228
14
N
H
–CHO
N
12

G. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2293–2298
2295
Table 2. IC50 of compounds 19a–e, 22a–c, 25, 28, and 29a–e
Cl
Cl
O
H
Cl
O
Cl
Br
O
O
O
O
a,b
c,d
e,f
X5
NH2
H
N
CF3
N
CF3
NO2
R4
N
N
CN
H
H
O
O
OH
N
N
H
9
10
11
12
R2
H
Cl
O
Cl
Compound R2–
R4–
X5– Chk1
IC50 (nM)
H
Cl
O
N
O
O
N
NH
N
NH
g
h
i
H
H
O
NH2
N
N
19a
–Cl
–Cl
–Cl
–Cl
–Cl
–Cl
–Cl
–Cl
–Cl
–H
–H
–H
–H
–H
10
N
N
O
N
N
O
O
CN
CN
15
13
14
19b
19c
19d
19e
22a
22b
22c
25
25
64
38
15
66
38
43
32
45
10
96
73
13
N
O
N
N
N
N
N
Scheme 2. Reagents and conditions: (a) MeI, K2CO3, DMF, rt, 72 h;
(b) SnCl2, MeOH, 50 °C, 24 h; (c) TFAA, pyridine, CH2Cl2, 0 °C, 3 h;
(d) NBS, AIBN, CCl4, reflux, 5 h; (e) CeliteÒ, dioxane, H2O, reflux,
4 h; (f) Dess–Martin periodinane, CH2Cl2, rt, 5 min; (g) K2CO3,
MeOH, H2O, rt, 2 h; (h) 3, toluene, 100 °C, 12 h; (i) piperidine,
NaBH3CN, CH2Cl2, MeOH, AcOH, 50 °C, 24 h.
N
N
O
O
O
O
O
Cl
Cl
Cl
O
Cl
R
O
N
O
Cl
O
N
R
Cl
a
b
c,d
N
NH
O
N
N
N
H
NO2
NO2
N
NO2
N
N
Cl
N
N
N
O
CN
16
17
18
19
Scheme 3. Reagents and conditions: (a) pyrrolidine, CH3CN, reflux,
3 hrs; (b) alcohols, NaOH, 70 °C, 12 h; (c) Raney Ni, H2NNH2, EtOH,
rt, 8 h; (d) 3, toluene, reflux, 3 h.
N
O
N
N
O
O
O
2-dimethylaminoethanol. Subsequent nitro reduction
and urea formation provided compound 25.
28
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–CHO
N
29a
29b
29c
29d
29e
Preparation of compound 29, as shown in Scheme 6, be-
gan by amidation of compound 26 with MeNHOMe.
LiAlH4 reduction resulted in intermediate 27. Urea
formation provided core compound 28. Reductive
amination using a variety of secondary amines afforded
compounds 29a–e.
N
O
N
N
Results of the enzymatic assay12,13 of compounds 8a–v
are listed in Table 1. Compounds 8a–f, which contain
flexible chains, provided excellent enzymatic activity
ranging from 3 to 23 nM. Compound 8a carries a tertia-
ry aliphatic amine, which is very hydrophilic, and
showed the best enzymatic activity (IC50 = 3 nM).
Compounds 8b, 8c, and 8f vary in chain length from
5 to 9 atoms, yet they showed little variance in
enzymatic activity with IC50’s of 22, 18, and 23 nM,
respectively. Another subset of compounds we
looked at were pyridine-containing compounds 8g–j.
Compounds 8h–j showed excellent enzymatic activity
N
N
–H 112
N
The synthesis of compound 25 is shown in Scheme 5.
Replacement of the 2-chloro moiety was effected by
reacting 2,4,5-trichloronitrobenzene with excess (3-
methyl-oxetan-3-yl)-methanol and one equivalent of
NaOH.18 The 4-chloro moiety was then replaced using
Cl
Cl
O
Cl
O
H
O
N
O2N
H2N
R
N
Cl
O
N
O
N
O
N
O
NH
O
O2N
b
c
d
N
NH
N
NH
N
NH
+
H
H
H
O
NH2
N
OR
4 R=H
N
N
N
CN
CN
CN
CN
3
a
6
7
8
5 R=Me
Scheme 1. Reagents and conditions: (a) MeI, K2CO3, DMF, rt, 72 h; (b) 3, toluene, reflux, 2 h; (c) SnCl2, MeOH, 50 °C, 12 h; (d) acid chlorides,
pyridine, CH2Cl2, rt, 2 h.

2296
G. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2293–2298
R1 Cl
N
Cl
Cl
R1 Cl
N
R2
O
N
Cl
Cl
R2
N
NH
a
b
c,d,e
H
O
NO2
NO2
NO2
R3
Cl
OH
OH
N
21
22
20
16
CN
Scheme 4. Reagents and conditions: (a) NaOH, H2O, reflux, 72 h; (b) 2° amines, CH3CN, reflux, 3 h; (c) alcohols, PPh3-polymer supported, di-tert-
butyl azodicarboxylate, THF, rt, 12 h; (d) Raney Ni, H2NNH2, EtOH, rt, 8 h; (e) 3, toluene, reflux, 2 h.
Cl
compound 6 to the amino group of compound 7 impart-
O
Cl
Cl
O
Cl
N
O
N
ed an 8-fold increase in activity. Alternatively, the in-
creased bulk of compound 15 from the reductive
amination of compound 14 does not impart a significant
change in activity. Compounds 15 and 14 showed very
similar potency, 12 and 14 nM, respectively.
Cl
O
Cl
N
N
NH
a
b
c,d
O
H
O
NO2
NO2
NO2
O
O
O
Cl
N
CN
16
23
24
25
Scheme 5. Reagents and conditions: (a) (3-methyl-oxetan-3-yl)-meth-
anol, NaOH, 70 °C, 12 h; (b) 2-dimethylamino-ethanol, NaOH, 70 °C,
12 h; (c) Raney Ni, H2NNH2, EtOH, 0 °C, 0.5 h; (d) 3, toluene, reflux,
2 h.
The SAR of compounds 19a–e, 22a–c, and 25 are listed
in Table 2. Overall, these compounds showed very good
to excellent enzymatic activity with IC50’s ranging from
10 to 66 nM. The heteroaliphatic-substituted com-
pounds 19a–c showed decreased activity with increased
chain length. Their IC50’s are 10, 25, and 64 nM, respec-
tively. In compounds 22a–c, R4 was held constant as the
methylpiperidinyl moiety, while R2 was modified.
Although the R2 modifications ranged in size, polarity,
and flexibility, compounds 22a–c do not differ signifi-
cantly in activity (IC50 = 38–66 nM).
(IC50’s = 8–11 nM). However, compound 8g is inactive
(IC50 > 10 lM). Compound 8g differs from compounds
8h and 8i only by placement of the pyridine nitrogen
atom. It may be, in this case, that the nitrogen atom
of the pyridine ring is unable to make the critical inter-
action needed to impart activity. In compounds 8k–v, a
variety of furan, thiophene, substituted, and unsubsti-
tuted phenyl rings were introduced into the molecule.
In general, the activities of these compounds are worse
than those seen for the compounds that contain hetero-
aliphatic chains (8a–f) or pyridine rings (8h–j, 8g is an
exception). However, compound 8u, which possesses
the highly polar methylsulfonyl group, is an exception
with an IC50 of 7 nM. Compound 8j is 6.4-fold more ac-
tive than compound 8m. This indicates pyridine rings
may provide better interaction with the enzyme than thi-
ophene rings. Compounds 8o, 8r, and 8s, all of which
have a phenethyl group, showed similar potencies de-
spite addition or placement of a methoxy substituent
with IC50’s of 68, 72, and 51 nM, respectively. Com-
pounds 8t and 8u form an interesting comparison. Com-
pound 8u is 195-fold more active than compound 8t.
This difference in activity stems from a single structural
change, compound 8u has a methylsulfonyl group at the
para position of the benzyl ring, while compound 8t has
a methoxy group.
The SAR of compounds 28 and 29a–e are also shown in
Table 2. Removal of the chloro group from R5 was not
detrimental to the ability of these compounds to inhibit
Chk1. Direct comparison of compounds 28 and 29a
with their chloro-containing counterparts, compounds
14 and 15, respectively, showed a 3-fold decrease for
one compound and a slight increase in activity for the
other compound. Among compounds 29a–e, 29a, and
29d gave the best enzymatic activity, 10 and 13 nM,
respectively. Compounds 29a–c all have six-membered
heteroaromatic rings, however, compound 29b has a
morpholine ring and is 9.6-fold less active than com-
pound 29a. Compound 29c has a methylpiperidine ring
and is 7.3-fold less active than compound 29a.
A selection of compounds possessing the best IC50 val-
ues was also tested in one or more cellular assays,
including a MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carb-
oxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt) cell proliferation assay and FACS (fluores-
cence-activated cell sorting).12,13 The MTS assay mea-
sures the amount of surviving cells as an assessment
The enzymatic activity of compounds 6, 7, 14, and 15 is
also shown in Table 1. Reduction of the nitro moiety of
H
R42
N
O
N
OH
H
O
O
N
R41
N
NH
O
O
c
N
NH
d
a,b
H
O
H
O
NH2
NH2
N
O
O
N
CN
26
27
CN
28
29
Scheme 6. Reagents and conditions: (a) MeNHOMeÆHCl, EDC, DMF/Et3N, rt, 12 h; (b) LiAlH4, THF, 0 °C, 1 h; (c) 3, toluene, reflux, 2 h; (d) 2°
amines, NaBH3CN, CH2Cl2, MeOH, AcOH, 50 °C, 24 h.

G. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2293–2298
Table 3. Results of the MTS and FACS cellular assays (lM)
2297
Compound
IC50 (nM)
MTSa (lM)
FACSb (lM)
Cpd alone
Cpd/Doxc
Ratio
EC50
Cpd/Doxd
Ratio
7
5
>58.9
8.65
15.0
0.89
24.0
>59.3
0.44
12.2
0.41
0.76
0.22
4.66
6.55
>3.9
9.7
8a
8b
8f
3
22
23
8
>59.3
>59.3
>59.3
>58.9
7.86
>2.5
>10
>10
>10
1.71
8i
>136
>4.8
19.1
2.0
>5.9
8j
9
8u
15
19a
29a
29d
7
12
10
10
13
1.54
0.83
>10
3.66
>10
0.14
0.58
1.45
>71
6.3
3.8
3.1
14.5
16.4
>6.9
2.5
a Tested using HeLa cells.
b Tested using H1299 cells.
c The Dox concentration was 100 nM.
d The Dox concentration was 500 nM.
for cytotoxicity. FACS analysis measures abrogation of
the G2 checkpoint as an indicator of Chk1-based cellu-
lar mechanism for the compounds. These data, along
with the enzymatic assay data (IC50’s), are presented
in Table 3 as IC50 values at which the compound reduces
cell growth or decreases G2 cells by half. We have
defined an ideal result as a compound possessing little
or no antiproliferative activity when dosed alone
(>59.3 lM in the MTS assay and >10 lM in the FACS
assay), but possessing high antiproliferative activity in
the presence of doxorubicin (Dox) (ideally 61 lM), thus
providing the highest ratio.
the compounds showed activity in both assays. Of the
compounds tested, 8i showed the best overall results
among all three assays possessing an IC50 of 8 nM,
MTS single agent activity for compound alone of
>59.3 lM and combination activity with doxorubicin
of 0.44 lM, and a ratio of single agent to combination
activity of >10.0 lM/1.7 lM in the FACS assay. In
addition, compounds 8a and 8u also showed moderate
to good activity having 9.7- and 19.1-fold MTS single/
combination ratios, respectively.
In summary, we have modified R4 of the urea phenyl
ring with a variety of chemical structures, and R2 and
R5 to some degree. Several compounds showed very
promising results as potent and selective Chk1 inhibi-
tors. In particular, compound 8i showed very good re-
sults throughout the enzymatic and cellular assays.
Although we have progressed toward our goal of finding
a potent and selective Chk1 inhibitor, additional work is
needed to obtain greater insight into the SAR of these
compounds.
From the data in Table 3, it is seen that good activity in
the enzymatic assay does not always translate into good
cellular activity. Factors such as cell permeability can
contribute to these differences. Compounds 7, 8b, 8f,
and 8j all have potent IC50’s ranging from 5 to 23 nM,
however, compounds 7, 8b, and 8j have only very weak
combination cellular activity: 15, 24.0, and 12.2 lM,
respectively. Compound 8f did not show any combina-
tion or single agent cellular activity in either the MTS
or FACS assay. Overall, the two cellular assays corrob-
orate well with each other. Compound 8i had no single
compound activity and showed strong combination
activity in the MTS assay. FACS analysis also showed
little single compound activity and had some combina-
tion activity. An apparent discrepancy between the
MTS and FACS assays was observed for compounds
15, 19a, and 29a. However, the different cell lines and
compound concentrations used in the two assays could
explain the limited variations. The MTS assay uses
HeLa cells that are more sensitive than the H1299 cells
used in the FACS assay. For example, a single com-
pound EC50 value of 0.83 lM for compound 19a in
the MTS assay became 3.66 lM in the FACS assay.
Moreover, the MTS assay measures proliferation, while
FACS analysis evaluates Chk1-based cellular mecha-
nism. The two parameters may vary in the cell lines.
The single compound activities for compounds 15 and
29a in the MTS assay were not detected in the FACS as-
say because the maximum concentration for FACS was
only 10 lM. For combination treatments, all three of
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