Pyridyl aminothiazoles as potent Chk1 inhibitors: Optimization of cellular activity

Article abstract of DOI:10.1016/j.bmcl.2012.01.120

Translation of significant biochemical activity of pyridyl aminothiazole class of Chk1 inhibitors into functional CEA potency required analysis and adjustment of both physical properties and kinase selectivity profile of the series. The steps toward optimization of cellular potency included elimination of CDK7 activity, reduction of molecular weight and polar surface area and increase in lipophilicity of the molecules in the series.

Full text of DOI:10.1016/j.bmcl.2012.01.120

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2613–2619
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyridyl aminothiazoles as potent Chk1 inhibitors: Optimization
of cellular activity
Vadim Y. Dudkin a, , Cheng Wang a, Kenneth L. Arrington a, Mark E. Fraley a, George D. Hartman a,
⇑
Steven M. Stirdivant b, Robert A. Drakas b, Keith Rickert b, Eileen S. Walsh b, Kelly Hamilton b,
Carolyn A. Buser b, James Hardwick b, Weikang Tao b, Stephen C. Beck b, Xianzhi Mao b, Robert B. Lobell b,
Laura Sepp-Lorenzino b
a Department of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
b Department of Cancer Research, Merck Research Laboratories, West Point, PA 19486, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 7 February 2012
Translation of significant biochemical activity of pyridyl aminothiazole class of Chk1 inhibitors into func-
tional CEA potency required analysis and adjustment of both physical properties and kinase selectivity
profile of the series. The steps toward optimization of cellular potency included elimination of CDK7
activity, reduction of molecular weight and polar surface area and increase in lipophilicity of the mole-
cules in the series.
Ó 2012 Elsevier Ltd. All rights reserved.
DNA-damaging agents have achieved widespread use as chemo-
therapy drugs and still constitute a basic tool set in the treatment of
cancer. Despite making a major impact on the survival of cancer
patients, this class of medicines has significant shortcomings,
including toxicity associated with lack of selectivity for tumors over
normal proliferating cells, as well as efficacy limitations. Therefore,
widening the therapeutic window of DNA-damaging chemothera-
pies would lead to significant improvement in cancer care.1 For
maximal efficacy to be achieved with such chemotherapy, a divid-
ing tumor cell should progress into mitosis after sustaining DNA
damage and subsequently undergo mitotic catastrophe and apop-
tosis. However, cells have the ability to halt cell cycling in G1, G2,
or S phases to allow for DNA repair.2,3 This survival mechanism
can normally be mediated through both p53 and Checkpoint Kinase
1 (Chk1) pathway activation.4 The p53 pathway is dysfunctional in
a large percentage of tumors.5 These p53- impaired tumors must
rely on Chk1-mediated G2/S arrest as a dominant defense mecha-
nism from DNA-damaging chemotherapy.2 Inhibition of Chk1 in
such p53-deficient cancer cells with damaged DNA would abrogate
the cell-cycle arrest and force the progression into mitosis resulting
in cell death, thus selectively sensitizing these tumor types to
chemotherapy. Accordingly, combination therapy comprising a
DNA-damaging agent with Chk1 inhibitor will potentially have sig-
nificantly higher therapeutic index than chemotherapy alone.6–8
The pyridyl aminothiazole class of Chk1 inhibitors9 originated from
lead structure 1 (Table 1), that emerged from a previous effort
directed at KDR kinase.10,11 Compounds belonging to pyridyl ami-
nothiazole series have demonstrated potential for high levels of
Chk1 enzyme inhibition in vitro.12 Here, we describe our efforts
aimed at translating the biochemical potency of aminothiazoles
into functional cellular activity in a Checkpoint Escape Assay
(CEA). CEA measures release of H1299 tumor cells from DNA-dam-
age induced cell-cycle arrest and progression into mitosis following
Chk1 inhibition.13
Investigation of CEA potency of the series initiated with lead 1
and its analogs bearing substitutions at C6 position of the aminopyr-
idine ring (2, 3, 4). To our surprise, none of the compounds 1–4 dem-
onstrated measurable cellular activity and only flat or bell-shaped
titration curves were observed in CEA experiment. However, 5 and
6, analogs with piperazine substitution at C4 of the pyridine ring,
were significantly active in checkpoint abrogation with intrinsic
potency levels equivalent to 1–4.
Especially puzzling was the comparison of isomers 4 and 5, dif-
fering only in the placement of piperazine moiety. While an
equally potent Chk1 inhibitor, 4 was inactive in CEA, 5 had an
EC50 of 1370 nM and a normal sigmoidal curve. Such close struc-
tural similarity suggested that differences in functional activity
could not be explained on the basis of cellular permeability, but
rather by some kind of off-target activity. A similar lack of cellular
potency was observed with several Chk1 inhibitors from the indo-
lyl indazole class.14 In this case, concomitant Cdk7 inhibition was
determined to be the culprit behind large shifts between functional
and biochemical potency levels.14 Cdk7 is a member of the cyclin-
dependent kinase family and plays a central role in regulating
progression of the cell cycle. Because Cdk7 activity is critical for
maintaining cell cycle transitions, it was proposed that Cdk7
⇑
Corresponding author.
E-mail address: vadim_dudkin@merck.com (V.Y. Dudkin).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.120

2614
V. Y. Dudkin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2613–2619
Table 1
Inhibitory profile and CEA activities of 1–7
3
H
R1
N
N
4
N2
S
5
1
6
R3
R2
Compd
R1
R2
R3
Chk1 IC50
Cdk IC50
1760
CEA EC50
>50,000
O
N
N
N
N
O
1
H
47
N
H
NH2
N
2
3
H
H
27
33
870
26
>16,670
>16,670
H
N
N
H
N
N
N
N
4
5
6
7
H
38
1000
>16,670
1370
1530
—
N
H
N
H
H
37
>20,000
11,000
410
N
H
N
29
N
N
N
H
H
N
N
H
160
N
inhibition resulted in cellular arrest that precluded checkpoint
abrogation event mediated by Chk1 inhibition. This hypothesis
was further supported by an experiment where Cdk7 siRNA inhib-
ited CEA effect of functionally active Chk1 inhibitor.14 Therefore,
counterscreening of 1–6 for Cdk7 inhibition was warranted. In
the event, 1 and all C6 substituted analogs 2, 3, 4, 7 demonstrated
similar levels of Cdk7 and Chk1 inhibition (Table 1). As expected,
functionally active C4 substituted compounds were virtually de-
void of Cdk7 activity.
This discovery has prompted us to discontinue development of
compounds modified at C6 of the western pyridine ring and imple-
ment Cdk7 assay as a routine counterscreen for the series. Having
fixed the substitution on western pyridine at C4, we then turned
our attention to investigation of cellular activity of analogs bearing
eastern pyridine ring modified at C30 (Table 2). A series of com-
pounds with a biaromatic motif attached to the thiazole ring was
prepared by Suzuki couplings with corresponding bromides, for
example, 8.
While bromide 8 itself is a Cdk7 inhibitor with no CEA activ-
ity, the majority of compounds carrying an aromatic ring at this
position did not show detectable potency against Cdk7. However,
even in the in the absence of Cdk7 inhibition shifts between po-
tency in CEA and Chk1 enzymatic assay remained very large, for
example, 9, 10, and 11 showed little to no functional activity.
Some improvement was observed with triazole 12 and pyrazole
13 modified with piperazine as R1, however only micromolar
CEA levels were achieved even with Chk1 potency now in the
single nM range. The best cellular potency in this series was
demonstrated by bipyridine 14 with CEA EC50 = 570 nM and a
5 nM IC50 in the Chk1 enzyme assay. A major issue for this sub-
series emerged when Chk1 biochemical activity in the presence
of 30% human serum was tested. Many triaryl compounds dem-
onstrated significant drop in potency in this experiment (data
not shown), probably due to high amount of nonspecific protein
binding. The findings helped explain some of the lackluster CEA
results, since CEA assay is also performed in the presence of ser-
um (10%). Thus, while some gains in functional activity with het-
eroaryl derivatives at C30 were achieved; a superior replacement
needed to be identified.
After some experimentation, amides on the C30 of the eastern
pyridyl ring were found to provide significant gain in biochemical
potency for this class (Table 3). Both the precursor carboxylic acid
15 and primary amide 16, despite being relatively potent Chk1
inhibitors at 16 and 10 nM, respectively, showed no CEA activity.
We were therefore pleased to find that simple methylation of the
amide afforded a compound (18) with noticeable improvement
in CEA potency (EC50 = 296 nM). Even higher levels of cellular
activity were expected from replacement of the methyl amide with
ethylenediamine amide, since it afforded compounds with pM
activity levels in Chk1 assay. For example, with R1 = morpholine
(19) 50 pM IC50 was achieved, and both sulfonamide 20 and N-
acetylpiperazine 21 showed 30 pM IC50s.

V. Y. Dudkin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2613–2619
2615
Table 2
lenediamine amide even in the absence of solvent front engaging
polar groups at the western pyridine ring, as exemplified by com-
pound 22 (Table 4). Thus, notable decrease in PSA and number of
basic nitrogens was achieved leading to a more reasonable CEA/
Chk1 shift of 12ꢀ. Analysis of X-ray structures of aminothiaz-
oles12,17 and pyrazoloquinolinones19 suggested an improvement
in potency could be obtained by relocating the Cl atom in 22 one
position closer to the aminogroup. Gratifyingly, the o-substituted
23 was 20 times more potent Chk1 inhibitor than m-substituted
22 with identical PSA and Cdk7 inhibitory values.
However, only a modest 2.8-fold improvement in cellular
activity (942–329 nM) was observed as CEA/Chk1 shift has per-
plexingly increased 10 fold, driven solely by the change in the
Cl placement. An analog with Me substitution at the same posi-
tion (24) was shown to have virtually identical profile to 23 in
terms of Chk1, Cdk7 inhibition and PSA, and a similar CEA
potency of 431 nM.
Chk1 and CEA activity of triaromatic compounds 8–14
H
R1
N
N
N
S
5'
4'
6'
R2
3'
1' N
Chk1 IC50
2'
Compd
R1
R2
Br
Cdk7 IC50
CEA EC50
>50,000
H
N
8
21
480
260
N
H
N
9
8
5530
Our second strategy was to study the effect of removal of nitro-
gen atoms from the pyridyl aminothiazol system on Chk1 binding,
PSA and CEA shift parameters. Representative compounds stem-
ming from this work are shown in Table 5.
HN
N
N
N
O
10
11
12
19
37
9
>10,000
>10,000
>10,000
>10,000
>10,000
1390
A significant synthetic effort was expended in preparation of
these analogs resulting in a decrease in PSA to ca. 100 Å range for
most compounds. However, Chk1 biochemical potency was also
dramatically affected by all such manipulations. For example, sub-
stitution of eastern (25) and western (27) side pyridine nitrogens
with CH caused 150 and 110 fold drop in potency, respectively,
and displacement of carboxamide moiety afforded CH2-linked com-
pound 26 that was 240 times less active than the corresponding
amide 19. Accordingly, most similarly ‘denitrogenated’ molecules
had little measurable CEA activity. Replacement of terminal amino
group with hydroxyl (e.g., 28) was also not successful and led to
300 fold potency loss. These derivatives showed no cellular potency
either.
The third approach commenced with screening of possible fluo-
rinated replacements for the ethylenediamine group while the
western pyridine C4 substitution was fixed as morpholine (Table
6). Of these, trifluoroethylamine amides proved to be the best
derivatives in terms of cellular activity as we were encouraged to
find that some decrease in Chk1 inhibitory activity for 29 was com-
pensated by low Chk1/CEA shift, resulting in CEA EC50 = 200 nM.
With trifluoroethylamine in place, the screening of western C4
derivatives was next carried out and provided sulfonamide 30 with
even higher CEA activity of 106 nM. Introduction of fluorine in this
part of the molecule was also explored. We were happy to discover
that exposure of fluorinated piperidines to the solvated area of the
binding cleft did not result in the loss of intrinsic activity as com-
pared to morpholine and piperazine. For example, monofluoro-
and difluoropiperidine derivatives 31 and 32 were highly potent
Chk1 inhibitors with IC50 of 70 and 50 pM, respectively, and dis-
played over 6000 fold selectivity over Cdk7. Once again, fluorina-
tion helped significantly improve CEA/Chk1 shift, with difluoro
derivative 32 being significantly more potent than monofluoro 31
with EC50 = 135 nM. Overall, the fluorination strategy proved most
successful in translating biochemical potency of pyridyl aminothi-
azole Chk1 inhibitors into functional cellular activity.
N
O
N
O
N
N
N
N
N
N
H
N
13
14
6
5
>2000
1330
570
N
N
N
H
N
>10,000
N
N
Unfortunately, this remarkable boost in intrinsic potency did
not translate into comparable levels of cellular activity, even
though high selectivity against Cdk7 was maintained with C30
amides. We postulated that one reason for the disappointing in-
crease in the CEA/Chk1 shift15 could be that gains in intrinsic po-
tency resulting from ethylenediamine introduction came at the
expense of dramatically increased polar surface area (PSA).16 In-
deed, PSA values for 19–21 were now in the range of 130–160 Å,
compared to ca. 100 Å for methyl amide 18. Elevated PSA and mul-
tiple basic functionalities in Chk1 inhibitors have been previously
correlated with decreased cellular permeability and high CEA/
Chk1 shift ratios, and PSA of less than 100 Å was suggested as a
prerequisite for achieving cellular potency.17,18 Accordingly, both
19 and 20 only achieved ca. 500 nM CEA IC50s, while 21 showed
worse than 1000 nM activity in this assay.
Further improvements in amide series could therefore only be
realized through enhancements in physical properties of the com-
pounds necessary for better cell membrane permeability. To this
end, we designed a strategy to address the issue in three ways:
decreasing molecular weight, removing one or more nitrogen
atoms from the core, and introducing fluorination to increase gen-
eral lipophilicity of the compounds.
The synthesis of C30 amides subset of aminothiazole series is
shown on Scheme 1.20 A bromonicotinamide 33 can be coupled
with protected aminothiazole 34 in a Heck-type reaction cata-
lyzed by Pd(PPh3)4 to give biaromatic compound 35 in modest
to good yields and complete regioselectivity. Removal of pivaloyl
group was then performed in acidic conditions and was accom-
panied by concomitant hydrolysis of the amide affording amino-
acid 36 as a hydrochloride. The amine in 36 was then converted
into chloride through the intermediate diazonium salt formed in
the presence of copper(II) chloride.
The first approach was driven by realization that significant
Chk1 activity could be maintained through incorporation of ethy-

2616
V. Y. Dudkin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2613–2619
Table 3
Cellular and biochemical potency of C30 amides 15–21
H
N
N
N
S
R1
1
R
N
O
Compd
15
R1
R2
Chk1 IC50 (nM)
Cdk7 IC50 (nM)
>10,000
PSA
117
CEA EC50 (nM)
>10,000
H
N
OH
16
N
O
16
17
NH2
10
12
>3700
119
114
>10,000
920
N
O
NH2
>10,000
N
O
S
18
19
NHMe
7
>10,000
340
104
130
300
460
N
O
HN
NH2
NH2
0.05
N
SO2Me
N
HN
HN
20
21
0.03
0.03
170
210
162
140
500
N
Ac
N
NH2
1210
N
Table 4
Potency and selectivity profile of 22–24
N
R
H
O
N
NH2
S
N
H
N
Compd
R
Chk1 IC50 (nM)
Cdk7 IC50 (nM)
940
PSA
111
CEA EC50 (nM)
940
Cl
N
N
N
22
16
Cl
23
24
0.3
0.4
960
910
110
110
330
430
Me

V. Y. Dudkin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2613–2619
2617
Table 5
Profiles of aminothiazoles with N replacements 25–28
Compd
Structure
Chk1 IC50 (nM)
Cdk7 IC50 (nM)
PSA
103
CEA EC50 (nM)
F
F
H
N
N
N
N
S
25
8
160
>10,000
H
N
H2N
O
O
N
H
N
N
N
S
26
27
28
12
33
15
>10,000
>10,000
>10,000
102
—
N
H2N
Cl
H
N
N
S
97
>10,000
N
H
N
H2N
O
O
N
H
N
N
N
S
125
>10,000
N
N
HO
O
Table 6
Examples of fluorinated aminothiazoles 29–32
H
N
N
N
S
R1
N
R2
O
Compd
29
R1
R2
Chk1 IC50 (nM)
1.6
Cdk7 IC50 (nM)
705
PSA
101
CEA EC50 (nM)
198
O
N
N
CF3
CF3
H
SO2Me
N
N
H
30
1.7
2100
131
106
N
(continued on next page)

2618
V. Y. Dudkin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2613–2619
Table 6 (continued)
Compd
R1
R2
Chk1 IC50 (nM)
0.07
Cdk7 IC50 (nM)
PSA
116
CEA EC50 (nM)
F
N
H
NH2
NH2
31
32
350
484
N
F
F
N
H
0.05
317
116
134
N
Cl-
N
S
Br
N
PivHN
Pd(PPh3)4
KOAc
H3N+
aq. HCl
N
S
N
N
O
DMA
33
NH2
60-70%
O
NH2
O
OH
N
S
35
36
PivHN
O
N
CuCl2
66%
tBuONO
DMSO
34
NaH
DMSO
30-80%
N
S
N
S
Cl
N
N
O
N
H
N
N
40
i. H2NCH2CH2NHBoc
PyBop
Hunig's base
DMF
O
O
OH
O
OH
37
N
38
NH2
N
N
ii. TFA
80%
N
N
H
S
N
Cl
19
NH2
O
N
H
N
COOH
39
Scheme 1. Synthesis of ethylenediamine amide 19.
The resulting chlorothiazole 37 was then coupled in the pres-
ence of sodium hydride with substituted aminopyridine 38, itself
prepared from pyridinecarboxylic acid 39. Ethylenediamine moiety
was finally introduced through amide bond formation followed by
Boc deprotection. Alternatively, when screening of pyridine sub-
stituents is desired, 37 can be efficiently coupled with a 4- or 6-
chloro-2-aminopyridine, followed by SNAr displacement of the
chloride with an appropriate amine.
In summary, translation of the intrinsic potency of the pyridyl
aminothiazole series of Chk1 inhibitors into functional cellular
activity required investigation and tuning of parameters governing
both selectivity and cell membrane permeability. In particular,
Cdk7 inhibition was again identified as a factor impeding check-
point abrogation activity. Elevated PSA values can be generally
considered as a potential indicator of low cellular permeability
and therefore a predictor of high shifts between cellular and bio-
chemical potency. In case of pyridyl aminothiazoles, fluorination
proved to be the most effective strategy to combat this problem.
Through this approach, several fluorinated derivatives were dis-
covered with optimized cellular activity profile.
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