Discovery of a novel class of 2-ureido thiophene carboxamide checkpoint kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.05.016

Checkpoint kinase-1 (Chk1, CHEK1) is a Ser/Thr protein kinase that mediates the cellular response to DNA-damage. A novel class of 2-ureido thiophene carboxamide urea (TCU) Chk1 inhibitors is described. Inhibitors in this chemotype were optimized for cellular potency and selectivity over Cdk1.

Full text of DOI:10.1016/j.bmcl.2008.05.016

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4242–4248
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Bioorganic & Medicinal Chemistry Letters
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Discovery of a novel class of 2-ureido thiophene carboxamide checkpoint
kinase inhibitors
a
a
a
a
a
James W. Janetka a, , Lynsie Almeida , Susan Ashwell , Patrick J. Brassil , Kevin Daly , Chun Deng ,
Thomas Gero a, Roberta E. Glynn a, Candice L. Horn a, , Stephanos Ioannidis a, Paul Lyne a,
Nicholas J. Newcombe b, Vibha B. Oza a, Martin Pass b, Stephanie K. Springer a,à, Mei Su a,
Dorin Toader a, Melissa M. Vasbinder a, Dingwei Yu a, Yan Yu a, Sonya D. Zabludoff a
*
a AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA
b AstraZeneca R&D Alderley Park, Cheshire, Macclesfield SK10 4TG, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Checkpoint kinase-1 (Chk1, CHEK1) is a Ser/Thr protein kinase that mediates the cellular response to
DNA-damage. A novel class of 2-ureido thiophene carboxamide urea (TCU) Chk1 inhibitors is described.
Inhibitors in this chemotype were optimized for cellular potency and selectivity over Cdk1.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 17 April 2008
Revised 1 May 2008
Accepted 2 May 2008
Available online 6 May 2008
Checkpoint kinase-1 (Chk1, CHEK1) is a Ser/Thr protein kinase
that mediates the cellular response to DNA-damage. In response
to DNA-damage, the ATM and ATR kinases activate Chk1 by phos-
phorylation on Ser-317 and/or Ser-345. Chk1 mediated signaling
then leads to S phase or G2/M cell-cycle arrest primarily driven
by Cdk inhibition. A Chk1 inhibitor would consequently permit a
cell with damaged DNA to progress through the cell-cycle ulti-
mately leading to mitotic catastrophe and/or apoptosis.1 Therefore,
Chk1 inhibitors have been highly sought after as chemo- or radio-
potentiation agents.2 We report here on the identification and SAR
found early on that a variety of substitutions on the 4-position
ether of the phenyl ring are tolerated (Table 1). Albeit not selective
for Chk1, the most potent analogs generally contain a basic second-
ary or tertiary amine sidechain as in compounds 3 and 4a–c. It is
interesting that the non-basic compounds 4d–h show only weak
inhibition of Chk1. Matched pair analysis of the compounds in Ta-
ble 1 shows that 3-position analogs are equipotent while those on
the 2-position lead to reduced potency.
To demonstrate the mechanism of action, Chk1 inhibitors were
profiled in a cellular assay where HT29 tumor cells were pretreated
with the DNA-damaging agent Camptothecin for 2 h causing cell-
cycle arrest at G2/M. The ability of a compound to abrogate this
cell-cycle arrest is then quantified by levels of the marker of mito-
sis (M phase), phospho histone-H3. Although compound 3 displays
potent enzyme inhibition, it does not abrogate the G2/M block
(EC50 > 12.5 lM), a marker of Chk1 inhibition at the cellular level.
Similarly, other closely related analogs fail to produce any substan-
tial cellular activity.
of
inhibitors.
Several diverse structural classes of Chk1 inhibitors shown in
a new class of thiophene carboxamide urea (TCU) Chk1
Figure 1 were identified via a high-throughput Chk1 kinase scintil-
lation proximity assay. The three major series are exemplified by
triazolones (1; IC50 0.8 lM), pyrazoles (2; IC50 0.6 lM), and thio-
phene carboxamides (3; IC50 0.15 lM). All three chemotypes
showed good potency inhibiting Chk1 activity and were pursued
as lead series. The discovery and initial optimization of the TCU
series exemplified by compound 3 will be discussed herein while
the optimization and SAR of other Chk1 inhibitor series will be re-
ported in the near future.
Early on it was determined that benzylic amine substitution in
the 5-position of the thiophene led to increased potency against
Chk1 (Table 2), which is consistent with the previous result indi-
cating that ether-linked amines were preferred substituents. Once
again, a variety of substituents are tolerated. Unfortunately, this set
of compounds also showed only limited cellular activity (5f; EC50
0.27 lM).
TCU 3, while showing good potency toward Chk1, also showed
activity against other kinases. This was not a surprise since the
TCU’s have been previously reported as kinase inhibitors.3 We
It was hypothesized that limited or lack of cellular activity could
indicate poor cellular permeability, an efflux liability, or additional
off-target activities (e.g., Cdk1) which would mask the cellular ef-
fects of Chk1 inhibition. At this juncture, it was concluded that
the combination of Chk1 selectivity and cellular potency could
not be achieved simply through modulation of substitution on
the 5-position aryl ring.
* Corresponding author. Tel.: +1 781 839 4434; fax: +1 781 839 4230.
E-mail address: james.janetka@astrazeneca.com (J.W. Janetka).
Present address: Lilly, Indianapolis, IN, USA.
à
Present address: Amgen, Cambridge, MA, USA.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.016

J. W. Janetka et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4242–4248
4243
O
O
H2N
S
O
NH
N
N
NH
N
O
N
N
NH2
N
H
O
O
H
N
1
3
2
Figure 1. Chk1 inhibitors identified from high-throughput screening.
Table 1
Table 2
Aryl ether substituted thiophene carboxamide ureas
Benzylamine thiophene carboxamide ureas
H2N
O
H2N
O
NH
S
NH
S
R1
NH2
NH2
O
R2
R2
O
Compound
R1
Chk1 IC50 (lM)
Compound
Chk1 IC50 (lM)
N
5a
NMe
>0.3
3
0.15
O
5b
5c
5d
0.072
0.031
0.039
O
O
NH
NMe
4a
4b
4c
0.36
0.23
0.23
N
H
HO
O
O
N
NH
N
MeO
NH
5e
5f
0.029
0.037
4d
4e
4f
23
O
O
O
N
N
4.1
6.7
O
O
O
MeN
5g
5h
0.041
0.017
O
N
4g
4h
OMe
OCF3
5
2.6
Prior work on this series3 had revealed that other kinase activity
could be diminished by amide substitution and it was postulated
that these inhibitors could possess a different binding mode to
Chk14 that would accommodate substitution at this position.
Therefore, a small set of exploratory amides was prepared holding
the para-substituted basic aryl sidechain or ‘solvent tail’ constant
as seen in Table 3. Indeed, a variety of amide substituents were
not only allowed but also afforded improved potency and selectiv-
ity against Cdk1. The lack of activity against Cdk1 is important for
an effective Chk1 inhibitor since Cdk1 inhibition leads to cell-cycle
arrest at G2/M phase of the cell-cycle and is counterproductive to
the desired G2/M checkpoint abrogation. The piperidinyl amides
6d and 6e both displayed excellent potency but only 6e showed
modest cellular activity. The large drop-off from enzyme to cell po-
tency was partly attributed to the dibasic nature of these inhibitors
likely leading to poor cellular permeability. Having shown that that
Chk1 selectivity and potency was possible through amide modifi-
cation and that basic moieties were preferred, the basic solvent tail
was eliminated with the goal of improving cellular potency by
eliminating the dibasic attribute of these inhibitors. Therefore, a di-
verse library slightly biased toward amides with basic amines and
holding the 5-position phenyl constant was synthesized as out-
lined in Scheme 1. The original route utilized a Gewald5 synthesis
consisting of a Knovenagel condensation of benzaldehyde 8 with
methylcyanoacetate followed by cyclization with elemental sulfur
under basic conditions to construct the 2-amino-3-carboxy-5-phe-
nyl thiophene 9. Hydrolysis of ester 9 required forcing conditions
by refluxing in 3 N methanolic sodium hydroxide. EDC coupling
of the resultant carboxylic acid with various amines produced
the desired amides 10a. It is important to note, however, that in
many cases the use of HOBt in these couplings resulted in the
generation of an unreactive benzotriazole intermediate6 and only
a small amount of amide product. Finally, installation of the
urea was accomplished via a two-step reaction of aminothiophene
10a with trichloroacetyl isocyanate to give 11 followed by
deprotection with ammonia yielding the desired TCU’s 12. An
improved, more divergent synthesis using a Weinreb amidation
reaction7 as the key step was also utilized to synthesize the target
compounds 12. Reaction of the trichloroacetyl protected urea 13

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J. W. Janetka et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4242–4248
Table 3
0.17 lM. Furthermore, piperidine 12n was shown to potently
Ethylamino phenyl ether substituted amides
abrogate the G2/M checkpoint in the abrogation assay with an
EC50 of 20 nM versus only weak abrogation with 12o with an
EC50 of 1.9 lM. Substitution with tertiary amines like methylpi-
peridine 12p and quinuclidine 12w diminishes both enzyme
and cellular Chk1 activity. In addition, tertiary amides exempli-
fied by morpholine 12g, piperazine 12h, and 12l all show reduced
or weak Chk1 activity.
R1
HN
O
NH
N
S
NH2
O
This exciting result with compound 12n prompted a com-
prehensive evaluation of SAR around the 5-position substitu-
tion of the 2-ureido thiophene while keeping the amide
constant as 3S-piperidinyl. In order to improve the synthetic
efficiency, a more divergent synthesis employing a Suzuki cou-
pling as the key pivotal step was designed (shown in Scheme
2). Reaction of 2-amino thiophene ester 14 with trichloroacetyl
isocyanate followed by bromination afforded bromide 15 in
85% yield over two steps. Subsequent urea deprotection gave
urea 16 which was then subjected to Weinreb amidation using
2–3 equiv of chiral N-1-Boc-3S-aminopiperidine to give the
amide and key intermediate 17. An excess of amine was
required to obtain good yields. Suzuki coupling of bromide
17 under standard conditions was accomplished at 80 °C. In
some cases especially for more difficult couplings a microwave
reaction was employed. Finally, deprotection of the tert-butox-
ycarbonyl carbamate produced the target compound 18 in
excellent yields.
O
Compound
R1
Chk1
Cdk1
IC50 (lM)
Abrogation
EC50 (lM)
IC50 (lM)
OH
6a
6b
0.05
>100
>12.5
>12.5
0.1
44
N
NH
6c
0.1
>100
>69
>12.5
>12.5
H
N
6d
0.035
6e
0.01
4.8
0.71
NH
A summary of the compounds with results is compiled in
Table 5. A number of diverse functional groups on the 5-posi-
tion phenyl are tolerated and in many cases substitution in-
creases potency in the Chk1 enzyme assay. For example, a
variety of other simple aryl substituents such as F (18d–f), Cl,
(18g–h) Me (18i), CN (18j), and CF3 (18k) are all essentially
equipotent. Anilines and ethers are also tolerated as seen with
18a–c and 18l–o. With the exception of dimethylaniline 18m,
all these analogs show a drop-off in cellular potency. It is inter-
esting that 18c and 18l–o all contain a weakly acidic residue. It
was later elucidated that these analogs are efflux substrates
which could rationalize this result. A similar result was found
with the amides 18v–z0. Compounds 18a and 18b both have a
basic amine linker and as a result are both dibasic compounds.
It is presumable that the lack of cellular potency seen in this
with trimethyl-aluminate complex of primary or secondary amine
results in dual amide formation and deprotection to afford com-
pounds 12 in good yield.
As seen in Table 4, a large array of substitution is allowed
including alkyl, aryl, benzyl, and various heterocycles. However,
as determined previously alkyl linked and cyclic amines were
preferred. In this set of compounds, a clear preference for the
3S-piperidinyl amide was seen with compound 12n. Incredibly,
amide 12n has an IC50 of 7 nM for Chk1 while its enantiomer
12o shows a 24-fold drop in activity with an IC50 of only
O
OMe
a
b
c, d
O
CO2Me
CHO
NH2
S
7
8
9
R1
R1
O
R1
O
N
N
N
f
R2
NH2
R2
NH
e
R2
O
NH
S
S
S
CCl3
NH2
N
H
O
O
10a
12
11
O
g
OMe
e
O
NH
9
S
CCl3
N
O
H
13
Scheme 1. Synthesis of 5-phenyl thiophene substituted amides. Reagents and conditions: (a) DIBAL, toluene, ꢀ78 °C; (b) NCCH2CO2Me, S, DIEA, DMF; (c) 3 N NaOH, MeOH,
reflux; (d) R1R2NH, EDCI, DMF; (e) trichloroacetyl isocyanate, THF; (f) NH3, MeOH; (g) AlMe3, R1R2NH, THF.

J. W. Janetka et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4242–4248
4245
Table 4
Table 4 (continued)
Library of 5-phenyl thiophene substituted amides
Compound
R1
Chk1 IC50 (lM)
Abrogation EC50 (lM)
R1
N(at)
H
N
12s
0.3
NDa
O
O
NH
S
H
N
NH2
O
12t
0.003
23
0.11
>13
NDa
NDa
NDa
Compound
R1
Chk1 IC50 (lM)
Abrogation EC50 (lM)
12a
Me
0.89
NDa
12u
12v
12w
O
O
N
12b
12c
1
NDa
NDa
H
H
N
>10
0.11
0.13
0.98
N
H
12d
2.1
NDa
O
N
12e
12f
3.9
5.1
NDa
NDa
N
OH
12x
a
ND, not determined.
O
12g
12h
12i
>30
7.1
NDa
NDa
0.96
0.58
4.1
N(at)
case could be rationalized by a lack of cellular permeability.
Some of the most potent analogs identified were heterocyclic
replacements for the phenyl ring including thiophenes 18r–s
and pyridyls 18t–u. As seen before with a number of other ana-
logs, compounds with pyridyl substitution such as 18t and 18u
are also prone to cellular efflux. This is in contrast with the
high cellular potency observed with the thiophenes 18r and
18s. Furthermore, all the Chk1 inhibitors in Table 5 exhibit
excellent selectivity over the cell-cycle kinase Cdk1. In sum-
mary, a large number of very potent Chk inhibitors were iden-
tified from this 5-position library. In addition, this class of 3S-
piperidinyl amide TCU Chk inhibitors, as exemplified by lead
12n, displays excellent physical and drug-like properties, includ-
ing high solubility, low protein binding, and good in vivo PK
(Fig. 2).
In conclusion, a novel class of potent thiophene carboxamide
urea or TCU Chk1 inhibitors was discovered and optimized for
Chk1 selectivity and cellular potency through SAR exploration
of the 5-position aryl ring and 3-position amide substitution.
A preference for the chiral 3S-aminopiperidinyl amide was dis-
covered resulting in large improvements in both Chk1 enzyme
and cellular potency. This series of TCU Chk1 inhibitors possess
drug-like properties and further optimization of lead compound
12n resulted in the identification of the candidate drug check-
point kinase inhibitor AZD77628 that is currently undergoing
phase I clinical evaluation in combination with various DNA-
damaging agents. Further details on the optimization of the
TCU series of Chk1 inhibitors and discovery of AZD7762 are
forthcoming.
MeN
N(at)
NH2
0.03
0.10
0.11
Me
12j
N
H
Me
N
12k
Me
NH2
(N(at) =
NMe)
12l
5.5
NDa
NDa
12m
0.11
NH
12n
0.007
0.02
N
H
12o
12p
0.17
0.16
1.9
2.3
N
H
Me
N
12q
12r
0.1
0.1
2.1
6.2
N
H
Acknowledgments
We thank Anne White and Graham Walker for their respec-
tive works on the development and implementation of the
high-throughput Chk1 kinase screening assay. We also thank
Elizabeth Mouchet for the development of the cellular abroga-
tion assay.
N
H

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J. W. Janetka et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4242–4248
O
O
O
O
O
O
d
c
a, b
O
Br
NH
70%
Br
NH
S
95%
85%
NH2
S
Cl
Cl
S
NH2
N
O
O
Cl
H
16
14
15
boc
H
N
N
O
O
e, f
N
H
N
H
ClH
60-80%
Br
NH
S
Ar
NH
S
NH2
NH2
O
17
O
18
Scheme 2. Synthesis of 5-arylthiophene (3S)-piperidinyl amides. Reagents and conditions: (a) trichloroacetyl isocyanate, THF; (b) Br2, AcOH; (c) NH3, MeOH; (d) AlMe3, N-1
Boc-3S-aminopiperidine, THF; (e) ArB(OH)2, Pd(Ph3P)4, Cs2CO3, dioxane, H20, 80 °C; (f) 4 N HCl, dioxane.
Table 5
Library of 5-aryl thiophene 3S-piperidinyl amides
H
N
O
N
H
R1
NH
S
NH2
O
Compound
R1
Chk1 IC50 (lM)
Cdk1 IC50 (lM)
Cdk1/Chk1 IC50 ratio
Abrogation EC50 (lM)
18a
0.01
27
2700
2.60
N
N
O
(racemic)
O
18b
18c
12n
<0.01
0.007
0.007
3.6
10.2
5.1
>360
1457
729
1.10
1.20
0.02
OH
F
18d
18e
18f
18g
0.009
0.011
0.009
0.014
NDa
5.6
3.3
8.8
—
0.08
0.02
0.03
0.04
F
509
367
629
F
Cl
18h
0.010
3.7
370
0.04
Cl

J. W. Janetka et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4242–4248
4247
Table 5 (continued)
Compound
R1
Chk1 IC50 (lM)
Cdk1 IC50 (lM)
Cdk1/Chk1 IC50 ratio
550
Abrogation EC50 (lM)
18i
18j
18k
18l
0.010
5.5
0.07
Me
CN
0.006
0.019
0.013
3.6
600
284
—
0.08
0.17
0.92
5.4
CF3
NH2
NDa
18m
18n
0.041
0.008
8.4
205
0.09
1.80
NMe2
Me
NDa
—
N
O
H
H
N
O
18o
18p
18q
18r
18s
18t
18u
0.041
0.006
0.007
0.003
0.006
0.006
0.004
NDa
2.2
NDa
5.6
5.3
3.3
1.8
—
2.20
0.20
S
O
Me
O
S
367
O
Me
Me
—
0.06
O
1867
883
550
450
<0.008
<0.009
0.30
S
S
N
0.34
N
NDa
NDa
—
—
8.10
O
18v
0.005
0.005
NH2
O
18w
3.20
NH2
(continued on next page)

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J. W. Janetka et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4242–4248
Table 5 (continued)
Compound
R1
Chk1 IC50 (lM)
Cdk1 IC50 (lM)
Cdk1/Chk1 IC50 ratio
Abrogation EC50 (lM)
O
18x
18y
0.009
NDa
—
4.30
N
H
Me
Me
O
0.009
3.7
411
0.32
N
Me
O
O
18z
N
0.010
0.011
NDa
10.8
—
0.61
0.13
O
18z0
N
982
a
ND= not determined.
6. No reaction seen even after heating with excess amine. Proposed benzotriazole
H
N
LogD 1.28
ester intermediate 10b or rearranged N-oxide amide 10c:
Solubility >2000 μM
PPB (% free) 7.7%
O
N
N+
N
N
O
H
Mouse/Dog Cl 35.7/12.8 mL/min/kg
Mouse/Dog t1/2 1.5/5.2h
Dog F 78.8%
N
O
N
N
NH
S
O
O
NH2
O
hERG IC50 >31.6 μM
CYP IC50 all isoforms >10 μM
NH2
NH2
S
S
12n
Figure 2. Lead TCU Chk1 inhibitor properties.
10b
10c
References and notes
Mahmoud, K. A.; Long, Y-T. Y.-T.; Schatte, G.; Kraatz, H-B. H.-B. Eur. J. Inorg.
Chem. 2005, 1, 173.
1. (a) Bucher, N.; Britten, C. D.Br. J. Cancer 2008, 98, 523; (b) Luo, Y.; Leverson, J. D.
Expert Rev. Anticancer Ther. 2005, 5, 333. (and references cited within).
2. (a) Janetka, J. W.; Ashwell, S.; Zabludoff, S.; Lyne, P. Curr. Opin. Drug Discov. Dev.
2007, 10, 473; (b) Arrington, K. L.; Dudkin, V. Y. ChemMedChem 2007, 2, 1571; (c)
Tao, Z.-F.; Lin, N.-H. Anti-Cancer AgentsMed. Chem. 2006, 6, 377; (d) Prudhomme,
M. Recent Patents Anti-Cancer Drug Discov. 2006, 11, 55.
3. Baxter, A.; Brough, S.; Cooper, A.; Floettmann, E.; Foster, S.; Harding, C.; Kettle, J.;
McInally, T.; Martin, C.; Mobbs, M.; Needham, M.; Newham, P.; Paine, S.; St.-
Gallay, S.; Salter, S.; Unitt, J.; Xue, Y. Bioorg. Med. Chem. Lett. 2004, 14, 2817.
4. Lyne, P. D.; Kenny, P. W.; Cosgrove, D. A.; Deng, C.; Zabludoff, S.; Wendoloski, J.
J.; Ashwell, S. J. Med. Chem. 2004, 47, 1962.
7. (a) Levin, J. I.; Turos, E.; Weinreb, S. M. Synth. Commun. 1982, 12, 989; (b) Lipton,
M. F.; Basha, Anwer; Weinreb, S. M. Org. Synth. 1980, 59, 49.
8. (a) Ashwell, S. Abstracts of Papers. 2007 AACR Annual Meeting, Los Angeles,
CA, April 14–18, 2007.; (b) Almeida, L.; Ashwell, S.; Ayres, D. W.; Brassil, P. J.;
Daly, K.; Deng, C.; Gero, T.; Glynn, R. E.; Horn, C. L.; Ioannidis, S.; Janetka, J.
W.; Lyne, P.; Oza, V. B.; Springer, S. K.; Su, M.; Toader, D.; Vasbinder, M. M.;
Yu, D.; Yu, Y.; Zabludoff, S. D. Abstracts of Papers. 2007 EORTC-NCI-AACR
International Meeting on Molecular Targets and Therapeutics, San Francisco,
CA, October 22–26, 2007, p A233.; (c) Ashwell, S.; Caleb, B. L.; Green, S.;
Grondine, M. R.; Haye, H. R.; Horn, C. L.; Janetka, J. W.; Liu, D.; Mouchet, E.;
Ready, S.; Rosenthal, J. L.; Queva, C.; Taylor, K. J.; Sheehy, A. M.; Walker, G. E.;
White, A. M.; Zabludoff, S. D. Abstracts of Papers. 2007 EORTC-NCI-AACR
International Meeting on Molecular Targets and Therapeutics, San Francisco,
CA, October 22–26, 2007, p A232.
5. (a) Buchstaller, H-P. H.-P.; Siebert, C. D.; Lyssy, R. H.; Frank, I.; Duran, A.;
Gottschlich, R.; Noe, C. R. Monatshefte fuer Chemie 2001, 132, 279; (b) Sabnis, R.
W.; Rangnekar, D. W.; Sonawane, N. D. J. Heterocyclic Chem. 1999, 36, 333.