Design, synthesis and SAR of thienopyridines as potent CHK1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.10.105

A novel series of CHK1 inhibitors based on thienopyridine template has been designed and synthesized. These inhibitors maintain critical hydrogen bonding with the hinge and conserved water in the ATP binding site. Several compounds show single digit nanomolar CHK1 activities. Compound 70 shows excellent enzymatic activity of 1 nM.

Full text of DOI:10.1016/j.bmcl.2010.10.105

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7216–7221
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and SAR of thienopyridines as potent CHK1 inhibitors
Lianyun Zhao a, , Yingxin Zhang a, Chaoyang Dai a, Timothy Guzi a, Derek Wiswell b, Wolfgang Seghezzi b,
⇑
David Parry b, Thierry Fischmann c, M. Arshad Siddiqui a
a Department of Chemistry, Merck Research Laboratories, 320 Bent Street, Cambridge, MA 02141, USA
b Merck Research Laboratories, 901 California Avenue, Palo Alto, CA 94304, USA
c Drug Design Department, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of CHK1 inhibitors based on thienopyridine template has been designed and synthesized.
These inhibitors maintain critical hydrogen bonding with the hinge and conserved water in the ATP
binding site. Several compounds show single digit nanomolar CHK1 activities. Compound 70 shows
excellent enzymatic activity of 1 nM.
Received 2 September 2010
Revised 20 October 2010
Accepted 21 October 2010
Available online 26 October 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
CHK1 inhibitors
Thienopyridines
Antitutor agent
Kinase inhibitor
Development of DNA damage agents for cancer chemotherapy
is an area of great interest.1 However, mechanism based toxicity
to normal cells has limited their usage.2 Therefore, development
of antitumor agents with lower toxicity has been attracting more
attentions.3
Following DNA damage, normal cells arrest and attempt to re-
pair at the cell cycle checkpoints G1 and S phases through tumor
suppressor p53, and at G2 and S phases through checkpoint 1 ki-
nase (CHK1).4 However, the p53 gene is one of the most commonly
mutated gene in human cancer cells and most of the tumor cells
are short of p53 function and must rely on CHK1 to induce arrest
for repair and survival.5 Therefore, the inhibition of CHK1 should
selectively sensitize tumor cells to DNA damage while normal cells
still arrest at G1 and S phases.6 Thus, the identification of CHK1
inhibitors has attracted attention of researchers in the past few
years,7–13 and some of the CHK1 inhibitors are being evaluated in
clinical trials as an anticancer agent.14
inhibitor conformation in a putative six-membered ring. We rea-
soned that this conformation shown by X-ray crystal structure
could be further rigidified into the thienopyridine class of inhibi-
tors 2a (Fig. 1). It was expected that the constrained core would
likely maintain all key interactions with CHK1, including the H-
bond between the conserved water molecule and the nitrogen of
the thienopyridine ring as well as the hinge H-bonds with Glu85
and Cys87. The compound 2a was synthesized and the X-ray crys-
tal structure of 2a bound to CHK1 was determined (Fig. 2)18
confirming our design strategy. The chemistry and biological re-
sults of this series of compounds are presented in the following
sections.
Thienopyridine compounds
2 were prepared according to
Scheme 1. The Suzuki reaction of 3 with 4-chlorophenyl boronic
acid produced 4 in high yield. Compound 4 was reacted with
malonic acid in pyridine in the presence of catalytic amount of
piperidine using Knoevenagel reaction to provide acrylic acid 5.16
Compound 1 (Fig. 1) has been reported as a CHK1 inhibitor.15
The X-ray crystal structure of compound 1 (Fig. 2) indicated that
the carbonyl and NH2 of the urea group form hydrogen bonds
(H-bonds) with the hinge kinase backbone of Glu85 and Cys87.
Additionally, the carbonyl group of the amide forms H-bond inter-
actions with a conserved water molecule in the selectivity pocket
and the NH of the piperidine is involved in a network of H-bond
interactions with Asp148 and Glu134. An intramolecular H-bond
between the carbonyl of amide and the NH of urea freezes the
H
N
H
N
NH
NH
3
4
N
5
6
2
O
H
Cl
Cl
S
S
N
O
7
O
1
H2N
H2N
1
2a
⇑
Corresponding author. Tel.: +1 617 499 3569; fax: +1 617 499 3535.
E-mail address: lianyun.zhao@merck.com (L. Zhao).
Figure 1. Structures of compound 1 and designed compound 2a.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.105

L. Zhao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7216–7221
7217
Acyl azide formation of 5 yielded precursors 6. Curtius rearrange-
ment followed by intramolecular cyclization in phenyl ether with
the presence of NBu3 at 230 °C gave the thienopyridone 7 in 86%
yield. Regioselective bromination with NBS provided compound 8
in 78% yield. Compound 8 was treated with CuCN followed by FeCl3
to yield thienopyridone nitrile 9. Chlorination of 9 with P(O)Cl3 fol-
lowed by displacement with desired amines and hydrolysis of
nitrile with concentrated H2SO4 gave the desired compounds
2a–l, listed in Table 1.
In order to explore SAR at 2-position of the thienopyridine ring,
analogs listed in Table 2 were prepared following Scheme 2. Selec-
tive bromination of compound 12 with NBS followed by the treat-
ment with CuCN gave thienopyridone nitrile 14. Regioselective
bromination of compound 14 with NBS at 60 °C afforded
compound 15. Chlorination of 15 followed by amination with
(S)-1-Boc-3-aminopiperidine provided the key intermediate 17.
Suzuki coupling of 17 with different boronic acids followed by
hydrolysis of nitrile group yielded compounds 19–57 shown in
Table 2. Starting from 14, compound 59 was obtained with H at
2-position. Whereas compound 58 was derived from 15 through
P(O)Cl3 mediated chlorination, amination with 1-Boc-3-aminopi-
peridine and hydrolysis.
To explore the effect of the sulfur atom positions in thienopyri-
dine ring 60, an efficient synthetic route was devised. Preparation
of compound 60 in Scheme 3 began with 3-(3-thienyl)acrylic acid
61 with the similar method described in Scheme 2. Acyl azide for-
mation followed by Curtius rearrangement and intramolecular
cyclization at high temperature gave compound 62. Bromination
of 62 with NBS followed by treatment with CuCN and subsequent
bromination gave compound 65. Chlorination of 65 with P(O)Cl3
followed by amination with (S)-1-Boc-3-aminopiperidine and
Suzuki coupling with 4-chlorophenyl boronic acid provided
intermediate 68. Compound 60 was obtained through the treat-
ment of 68 with concentrated H2SO4.
To further understand the role of nuclear nitrogen in thiophene
ring, the thiazolopyridine analog 69 was prepared according to
Scheme 4 by following methods and conditions used for compound
2 in Scheme 1 starting from 2-bromo-5-formylthiazole.
Figure 2. X-ray structures of compounds 1 and 2a in CHK1 enzyme. Pictures
generated with PyMol.17,18
H
a
b
c
d
S
6
H
N3
S
HO
S
4
Cl
Br
S
Cl
O
O
Cl
O
O
3
5
Cl
O
O
O
g
e
f
N
HN
HN
Cl
Cl
Cl
HN
Cl
S
8
S
9
S
S
Br
CN
CN
10
7
R1 R2
N
R2 R1
N
h
i
N
N
Cl
Cl
S
S
CN
H2N
O
11
2a-o
Scheme 1. Reagents and conditions: (a) 4-chlorophenyl boronic acid, Pd(dppf)Cl2, K3PO4, dioxane, 80 °C, overnight, 83%; (b) malonic acid, piperidine, Py, reflux, overnight,
83%; (c) Et3N, ethyl chloroformate, acetone, 0 °C to rt, 20 min then NaN3, 0 °C to rt, 30 min; (d) NBu3, phenyl ether, 230 °C, 30 min, 65% in two steps; (e) NBS, DMF, rt,
overnight, 90%; (f) CuCN, DMF, reflux, overnight, then FeCl3, HCl, 70 °C, 15 min, 75%; (g) P(O)Cl3, reflux, overnight; 83% (h) amines, K2CO3, NMP, 80 °C, 1.5 h, 88%; (i) concd
H2SO4, rt, 30 min, 66%.

7218
L. Zhao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7216–7221
Table 2
Table 1
SAR at 2-position of thienopyridine
SAR at 4-position of thienopyridine
H
N
R2 R1
N
NH
O
N
Cl
N
Ar
S
S
H2N
O
H2N
IC50
Compound
R1
—
R2
CHK1 IC50 (nM)
2
Compound Ar
Compound Ar
IC50
(nM)
1
—
(nM)
H
N
F
Cl
2a
H
3
2a
3
39
40
9
5
Cl
NH
O
HN
HN
CF3
2b
2c
H
H
14
19
20
24
5
NH
F
F
O
707
NH
S
41
2
H2N
O
F
2d
2e
746
3
N
CN
H
21
22
23
4
2
2
42
43
44
7
5
9
N
Cl
H
F
NH
NH
NH
O
HN
H
N
2f
H
H
H
7
292
197
O
CF3
H
N
NH2
N
2g
2h
N
N
24
25
3
2
45
46
60
4
H
N
NH
NH
F
F
Cl
H
N
F
2i
2j
H
937
148
S
26
27
5
4
47
48
2
N
O
NH2
N
67
N
Cl
H
N
28
29
21
5
49
50
34
1
N
2k
2l
H
211
NH
O
NH
O
NH2
NH
NH2
H
27,143
O
30
31
19
3
51
52
5
8
N
N
In order to explore the effect of additional nitrogen in the
pyridine part of the thienopyridine scaffold, the thienopyridazine
70 was prepared via a synthetic route devised as shown in
Scheme 5. Compound 71 was treated with LDA at À78 °C followed
by diethyl oxalate, then cyclization with hydrazine in ethanol at
60 °C gave product 72. Chlorination of 72 with P(O)Cl3 followed
by amination with (S)-1-Boc-3-aminopiperidine provided key
intermediate 74. The desired compound 70 was obtained by treat-
ing 74 with concentrated ammonia in sealed tube at 80 °C followed
by deprotection with 4 N HCl in dioxane.
Table 1, containing compounds 2a–l, summarizes the SAR at
4-position of the thienopyridine. Compound 2a, with (S)-3-amin-
opiperidine-3-yl group at 4-position of thienopyridine ring, showed
potent enzymatic activity (IC50 = 3 nM).19 However, its enantiomer
2k with (R)-3-aminopiperidine-3-yl group showed approximately
100-fold loss in enzyme potency suggesting that the NH of the
piperidine ring needs to be correctly aligned to form key H-bond
interactions with Asp148 and Glu134, as shown by the X-ray crystal
structure (Fig. 2). Also, compounds 2b and 2e, with five- and seven-
membered ring amines with S stereochemistry maintained most of
O
N
32
3
53
3
OH
O
33
34
3
4
54
55
1
3
N
N
S
O
O O
S N
O
Ph
35
36
37
38
4
56
57
20
16
O
N
S
NH 60
O
H
N
16
58
59
Br
H
56
F
9
717
F

L. Zhao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7216–7221
7219
Boc
N
O
Cl
O
O
O
e
NH
a
b
c
d
HN
N
HN
HN
Br
Br
HN
N
S
S
S
S
Br
S
CN
15
CN
16
S
CN
14
Br
13
CN
17
12
H
N
Boc
N
NH
NH
g
f
N
N
Ar
Ar
S
S
H2N
O
CN
18
19-57
H
N
H
N
O
O
NH
NH
d, e, g
d, e, g
HN
HN
Br
N
N
Br
S
S
S
S
CN
14
CN
15
H2N
58
O
H2N
59
O
Scheme 2. Reagents and conditions: (a) NBS, DMF, rt, overnight, 95%; (b) CuCN, DMF, reflux, overnight, then FeCl3, HCl, 70 °C, 15 min, 73%; (c) NBS, DMF, 60 °C, 30 min, 89%;
(d) P(O)Cl3, reflux, overnight, 87%; (e) (S)-1-Boc-3-aminopiperidine, K2CO3, NMP, 80 °C, 1.5 h, 85%; (f) boronic acids, Pd(dppf)Cl2, K3PO4, dioxane, 80 °C, overnight, yield
65–97%; (g) concd H2SO4, rt, 30 min, yield 33–83%.
O
O
O
O
Cl
O
OH
a, b
S
S
S
S
d
f
c
HN
HN
HN
N
e
S
Br
Br
HN
Br
CN
CN
CN
S
61
62
63
64
65
66
H
N
Boc
N
Boc
N
NH
g
h
i
NH
NH
S
N
S
S
N
N
Cl
Cl
Br
CN
67
CN
68
H2N
O
60
Scheme 3. Reagents and conditions: (a) Et3N, ethyl chloroformate, acetone, 0 °C to rt, 20 min then NaN3, 0 °C to rt, 30 min; (b) NBu3, phenyl ether, 230 °C, 30 min, 66% in two
steps; (c) NBS, DMF, rt, overnight, 88%; (d) CuCN, DMF, reflux, overnight, then FeCl3, HCl, 70 °C, 15 min, 77%; (e) NBS, DMF, 60 °C, 30 min, 89%; (f) P(O)Cl3, reflux, overnight,
83%; (g) (S)-1-Boc-3-aminopiperidine, K2CO3, NMP, 80 °C, 1.5 h, 88%; (h) 4-chlorophenyl boronic acid, Pd(dppf)Cl2, K3PO4, dioxane, 80 °C, overnight, 79%; (i) concd H2SO4, rt,
30 min, 63%.
basic amines (2g–i) where aminomethyl group were connected to
the 4-position of thienopyridine core showed modest activity
against CHK1 enzyme, again presumably due to the misalignment
of the basic NH group of inhibitors with Asp148 and Glu134. In
summary, specific orientation of basic groups is required for more
favorable interactions with CHK1 protein and the most optimal
group at the 4-position of the thienopyrididne was found to be
(S)-3-aminopiperidine-3-yl group (compound 2a).
See the method
and conditions of
the synthesis of
compound 2 in
scheme 1
H
N
NH
N
S
N
S
N
Br
Cl
O
H2N
O
69
Table 2 lists compounds prepared to explore SAR at the 2-posi-
tion of the thienopyridine while keeping (S)-3-aminopiperidine-3yl
group at the 4-position of the core. In general, diverse aromatic
hydrophobic groups are preferred at this position providing
compounds with activity against CHK1 enzyme in low nM range.
The predominant interaction is the hydrophobic packing between
these aromatic groups and Leu15, located at the ‘roof’ of the binding
pocket (not shown in Fig. 2). The aromatic groups project into the
solvent-exposed opening of the active site of CHK1. The absence
of the hydrophobic groups at 2-position of thienopyridine core re-
sulted in significant loss of potency (compound 59). It is interesting
Scheme 4. Reagents and conditions are the same as described in Scheme 1.
the potency at 3 and 14 nM, respectively, presumably preserving H-
bond interactions with Asp148 and Glu134. Compound 2c with
four-membered ring showed significant loss of activity with IC50
of 707 nM, perhaps due to the inaccessibility of the azetidine NH
to form key H-bond interactions with Asp148 and Glu134. Intro-
duction of a flexible 2-methylaminoethylamine group (compound
2f) maintained most of the activity at 7 nM. Inhibitors containing

7220
L. Zhao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7216–7221
Boc
N
Cl
O
O
NH
O
N
N
a, b
Cl
c
HN
N
e
d
Cl
Cl
N
N
S
Cl
S
S
S
O
O
O
f
O
O
O
72
71
73
74
H
N
Boc
N
NH
O
NH
N
N
N
N
Cl
Cl
S
S
H2N
H2N
O
75
70
Scheme 5. Reagents and conditions: (a) LDA, diethyl oxalate, THF, À78 °C, 30 min; (b) hydrazine, ethanol, 60 °C, 10 min, followed by rt, 30 min, 57% in two steps; (c) P(O)Cl3,
90 °C, 3 h, 87%; (d) (S)-1-Boc-3-aminopiperidine, K2CO3, NMP, 80 °C, 1.0 h, 91%; (e) amonnia, 80 °C, overnight, 83%; (f) 4 N HCl in dioxane, rt, 10 min, 97%.
to note that CHK1 activity does not alter significantly by 2-position
hydrophobic groups with electron-donating groups, like compound
32 with methoxy group at 4-postion of the phenyl ring, or electron-
withdrawing groups, like compound 43 with fluoro substituted
group. Compounds 45 and 48 with 2-pyrimidine and 3-pyrido-al-
kyne groups, respectively, slightly deviate from above mentioned
SAR trends where there was slight loss of CHK1 activity. In sum-
mary, SAR at the 2-position of thienopyridine core reflects its adja-
cency to the solvent-exposed opening of CHK1 and it is relatively
insensitive to changes in aromatic hydrophobic groups.
Table 3 summarized the SAR of the core modification. Switching
position of sulfur atom in thienopyridine core from the 1-position
to the 3-position (compound 60) resulted in dramatic loss of po-
tency, presumably due to misalignment of key interactions of
Table 3
SAR of core modification of thienopyridine
Figure 3. X-ray structures of compound 70 in CHK1 enzyme.17,18
Compound
Structure
CHK1 IC50 (nM)
H
N
inhibitor with the CHK1 protein and the different direction angle
of aromatic ring at 2-position led to the hydrophobic packing be-
tween these aromatic groups and Leu15 was not favorable to the
potency. Introduction of an additional nitrogen atom at 3-position
in thienopyridine ring (compound 69) had very little impact on the
potency. Whereas insertion of an additional nitrogen at the 6-posi-
tion of the thienopyridine ring (compound 70) showed improve-
ment in potency, due to interaction of the additional nitrogen
with the conserved water molecule as determined by X-ray crys-
tallography (Fig. 3).18 Since the conserved water molecule which
has interaction with the additional water molecule also has the
interaction with the primary amide, it may further locked the con-
firmation of the amide to benefit the potency. Compare to com-
pound 1, the compound 70 showed improved enzymatic activity
(1 nM) while compound 1 showed 2 nM activity at biochemical
assay.
In summary, we have rationally designed and synthesized po-
tent CHK1 inhibitors based on the thienopyridine scaffold. SAR
studies at the 4-position of the thienopyridine scaffold identified
(S)-3-aminopiperidine-3-yl group as one of the optimal groups
whereas at the 2-position most aromatic groups were tolerated
including 4-chlorophenyl. Scaffold changes from thienopyridine
to thiazolopyridine (compound 69) and to thienopyridazine (com-
pound 70) were tolerated. Thienopyridazine compound 70 showed
the highest CHK1 potency.
NH
N
2a
3
Cl
Cl
Cl
Cl
S
S
H2N
O
NH
O
H
N
60
69
70
4582
N
H2N
H
N
H
O
N
9
1
N
S
N
H2N
H
N
NH
N
N
S
H2N
O

L. Zhao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7216–7221
7221
15. Ashwell, S.; Thomas, G.; Ioannidis, S.; Janetka, J.; Lyne, P.; Su, M.; Toader, D.; Yu,
Y. WO2005066163, 2005.
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crystallized by hanging drop using 2.0 ll protein mixed with 2.0 ll
precipitant. Optimized precipitant consists of 0.1 M Tris–Cl, pH 7.5; 1%
1-butanol; 2.5% DMSO; 25% glycerol; 2.5 mM TCEP-Cl; 10 mM di-sodium
dithionite and 6–12% PEG 3.25 K. Co-structures with inhibitor obtained by
soaking for 5–7 days a crystal in a solution containing the ligand of interest at a
concentration of 1 mM. The structures have been determined at a resolution of
1.4 Å (Rwork = 18.6% Rfree = 20.8%), 1.9 Å (Rwork = 15.0% Rfree = 25.5%) and 1.55 Å
(Rwork = 19.3% Rfree = 21.3%) for compounds 1, 2a and 70, respectively. The
respective PDB access codes are 3PA3, 3PA4 and 3PA5.
19. CHK1 SPA assay. An in vitro assay utilizing recombinant His-CHK1 expressed in
the baculovirus expression system as an enzyme source and biotinylated
peptide based upon CDC25C as substrate. His-CHK1 was diluted to 32 nM in
kinase buffer containing 50 mM Tris pH 8.0, 10 mM MgCl2, and 1 mM DTT.
CDC25C (CDC25 Ser216 C-term biotinylated peptide, Research Genetics)
peptide was diluted to 1.93
20 of 32 nM CHK1 enzyme solution and 20
solution were mixed and combined with 10 L of compound diluted in 10%
lM in kinase buffer. For each kinase reaction,
lL
l
L
of 1.926 substrate
lM
l
DMSO, making final reaction concentrations of 6.2 nM CHK1, 385 nM CDC25C
and 1% DMSO after addition of start solution. The reaction was started by
addition of 50
ATP (Amersham, UK), making a final reaction concentration of 1
0.2 Ci of 33P-ATP per reaction. Kinase reactions ran for 2 h at room
temperature and were stopped by the addition of 100 L of stop solution
l
L of start solution consisting of 2
lM ATP and 0.2
lCi of 33P-
M ATP, with
l
l
l
consisting of 2 M NaCl, 1% H3PO4, and 5 mg/mL Streptavidin-coated SPA beads
(Amersham, UK). SPA beads were captured using a 96-well GF/B filter plate
(Packard/Perkin Elmer Life Sciences) and
(Packard/Perkin Elmer Life Sciences). Beads were washed twice with 2 M NaCl
and twice with 2 M NaCl with 1% phosphoric acid. Signal was then assayed
using a TopCount 96 well liquid scintillation counter (Packard/Perkin Elmer
Life Sciences). Dose–response curves were generated from duplicate 8 point
serial dilutions of inhibitory compounds. IC50 values were derived by nonlinear
regression analysis.
a Filtermate universal harvester