Discovery of 3-alkoxyamino-5-(pyridin-2-ylamino)pyrazine-2-carbonitriles as selective, orally bioavailable CHK1 inhibitors

Article abstract of DOI:10.1021/jm3012933

Inhibitors of checkpoint kinase 1 (CHK1) are of current interest as potential antitumor agents, but the most advanced inhibitor series reported to date are not orally bioavailable. A novel series of potent and orally bioavailable 3-alkoxyamino-5-(pyridin-

Full text of DOI:10.1021/jm3012933

Article
pubs.acs.org/jmc
Discovery of 3‑Alkoxyamino-5-(pyridin-2-ylamino)pyrazine-2-
carbonitriles as Selective, Orally Bioavailable CHK1 Inhibitors
Michael Lainchbury,§ Thomas P. Matthews,§ Tatiana McHardy,§ Kathy J. Boxall,§ Michael I. Walton,§
Paul D. Eve,§ Angela Hayes,§ Melanie R. Valenti,§ Alexis K. de Haven Brandon,§ Gary Box,§
G. Wynne Aherne,§ John C. Reader,# Florence I. Raynaud,§ Suzanne A. Eccles,§ Michelle D. Garrett,§
and Ian Collins*,§
§Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, 15 Cotswold Road, Sutton, SM2 5NG U. K.
#Sareum Ltd., Unit 2A Langford Arch, London Road, Pampisford, Cambridge CB22 3FX, U. K.
S
* Supporting Information
ABSTRACT: Inhibitors of checkpoint kinase 1 (CHK1) are of current interest as potential antitumor agents, but the most
advanced inhibitor series reported to date are not orally bioavailable. A novel series of potent and orally bioavailable 3-
alkoxyamino-5-(pyridin-2-ylamino)pyrazine-2-carbonitrile CHK1 inhibitors was generated by hybridization of two lead scaffolds
derived from fragment-based drug design and optimized for CHK1 potency and high selectivity using a cell-based assay cascade.
Efficient in vivo pharmacokinetic assessment was used to identify compounds with prolonged exposure following oral dosing.
The optimized compound (CCT244747) was a potent and highly selective CHK1 inhibitor, which modulated the DNA damage
response pathway in human tumor xenografts and showed antitumor activity in combination with genotoxic chemotherapies and
as a single agent.
forces proliferating cells to proceed to mitosis with unrepaired
DNA, resulting in aberrant cell division and death. Thus CHK1
inhibition can potentiate the cytotoxicity of genotoxic therapies,
as has been extensively demonstrated in preclinical studies with
CHK1 RNAi and small molecule CHK1 inhibitors.9,10 CHK1
inhibitors show high potentiation of the efficacy of anti-
metabolite DNA-damaging agents that act mainly in S-phase
(e.g., nucleotide analogues, folate synthesis inhibitors), and
selective inhibition of CHK1 over CHK2 has been shown to be
beneficial over simultaneous inhibition of CHK1 and CHK2.10
Recent studies have shown that some cancer cells carry a high
level of intrinsic DNA damage resulting from the particular
genetic defects underlying their transformation and are
dependent on CHK1-mediated DNA damage repair for
survival. CHK1 inhibition may confer synthetic lethality in
these tumors.11,12 For example, pediatric neuroblastomas driven
by amplification of the MYCN oncogenic transcription factor
have constitutive activation of the DNA damage response
pathway and are sensitive to single agent inhibition of CHK1.13
CHK1 inhibitors have been widely studied and a number of
compounds have reached early clinical trials.10 Notable among
these are the ATP-competitive inhibitors LY260361814 (1),
PF0047773615 (2), AZD776216 (3), SCH90077617 (4), and
LY260636818 (5) (Figure 1). However, of these agents, only 1
has so far progressed to phase II clinical trials,14 and the clinical
benefit of CHK1 inhibition remains to be tested. Most of these
compounds have low or no selectivity for inhibition of CHK1
over CHK2, and all are administered intravenously. Thus, there
is a need for CHK1 inhibitors with improved selectivity profiles,
INTRODUCTION
■
Checkpoint kinase 1 (CHK1) is an intracellular, serine/
threonine kinase that plays a central role in the DNA damage
response pathway.1,2 When single or double strand breaks are
formed in the DNA in proliferating cells, either by exogenous
DNA-damaging events (e.g., exposure to genotoxic chemicals
or ionizing radiation) or through faults in the DNA replication
process, a signaling cascade is triggered to halt the cell cycle and
initiate DNA repair. CHK1 is predominantly, but not
exclusively, activated by the upstream kinase, ataxia telangiec-
tasia and rad3 related (ATR), in response to single strand
breaks in DNA,3 and in turn CHK1 phosphorylates a number
of downstream proteins leading to cell cycle arrest in S-phase or
at the G2/M transition.4 As well as establishing S and G2/M
cell cycle checkpoints, CHK1 also promotes homologous
recombination repair of damaged DNA.5 Cell cycle arrest in
response to DNA damage may occur in G1, and the structurally
unrelated enzyme checkpoint kinase 2 (CHK2) plays a
significant part in the control of the G1 checkpoint.6 The
presence of alternative checkpoints and DNA repair mecha-
nisms reduces the sensitivity of normal cells to CHK1
inhibition. However, more than half of solid tumors are
deficient for the function of the tumor suppressor p537,8 or
contain other defects in cell cycle checkpoints and are more
reliant on the late phase cell cycle checkpoints and CHK1-
mediated DNA damage response pathways as a result.9
Inhibition of CHK1 is established as a potential therapy for
cancer in two distinct contexts: in combination with conven-
tional genotoxic chemotherapy or ionizing radiation, and as a
single agent in specific tumors with a genetic background that
leads to high levels of intrinsic DNA damage.10 CHK1
inhibition prevents effective repair of lesions in DNA and
Received: September 7, 2012
Published: October 19, 2012
© 2012 American Chemical Society
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potentiated genotoxic drug efficacy in cellular assays and in
human tumor xenografts. Although a potent and selective
CHK1 inhibitor, compound 6 lacked oral bioavailability. To
address this, we pursued a hybridization strategy, combining the
structural elements conferring CHK1 selectivity in 6 with an
alternative pyridine scaffold which had shown more promising
in vitro ADME properties. This approach generated a novel
series of 3-alkoxyamino-5-(pyridin-2-ylamino)pyrazine-2-car-
bonitriles, which we have optimized for potency and efficacy
in cells, and for ADME properties, leading to the highly
selective CHK1 inhibitor 26. Compound 26 has good oral
bioavailability and demonstrates biomarker modulation and
enhancement of genotoxic drug efficacy in multiple xenograft
models. Additionally, 26 shows strong single agent activity in a
MYCN-driven transgenic mouse model of pediatric neuro-
blastoma.
RESULTS AND DISCUSSION
■
The isoquinoline 6 was a potent CHK1 inhibitor with high
selectivity over inhibition of CHK2 and showed cellular effects
characteristic of selective CHK1 inhibition. However, the
compound suffered from high metabolism in mouse liver
microsomes (MLM) and had minimal oral bioavailability (F =
5%). We therefore sought to modify the chemical scaffold to
increase metabolic stability, with the aim of enhancing oral
bioavailability. During the evolution of the lead compound 6 by
scaffold morphing from a purine fragment hit,20 pyridines
represented by compound 7 had been prepared and
evaluated.19 Compound 7 lacked the potent cellular activity
or cellular selectivity of 6 but showed good microsomal stability
and equivalent permeability (Figure 2, Table 1). We therefore
investigated hybridization of the two scaffolds represented by 6
and 7 to give novel 3-alkoxyamino-5-(pyridin-2-ylamino)-
pyrazine-2-carbonitriles that could allow the maintenance of
the cellular potency and selectivity of 6 while conferring the
improved in vitro ADME properties observed for 7. Compound
6 had shown micromolar inhibition of the hERG ion channel,
whereas an apparent reduction in hERG inhibition was
observed for 7. An additional aim was therefore to minimize
hERG channel inhibition in the novel series of CHK1
inhibitors.
Figure 1. Structures of the intravenous, clinical candidate checkpoint
kinase inhibitors LY2603618 (1), PF00477736 (2), AZD7762 (3),
SCH900776 (4), and LY2606368 (5).
while orally bioavailable compounds would provide flexibility
for dosing in combinations with conventional chemotherapies
and would also be advantageous in emerging single agent
contexts in oncology where more frequent administration may
be required. Oral CHK1 inhibitors have been recently reported
but not yet fully described.18
We have previously detailed the fragment-based discovery
and optimization of a series of 2-aminoisoquinoline CHK1
inhibitors, exemplified by SAR-02010619 (6, Figure 2), that
Compounds were assessed in an assay cascade designed to
identify inhibitors with potent and selective effects on CHK1
activity in cancer cells. As well as measuring inhibition of CHK1
in a biochemical assay, compounds were tested for their ability
Figure 2. Design of novel 3-alkoxyamino-5-(pyridin-2-ylamino)pyrazine-2-carbonitrile CHK1 inhibitors by hybridization of the 2-aminoisoquinoline
6 and the 2-aminopyridine 7.
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Table 1. CHK1 Inhibition and Cellular Activity of 3-Alkoxyamino-5-(5-carbomethoxypyridin-2-ylamino)pyrazine-2-
carbonitriles
potentiation of
gemcitabine
cytotoxicity
d
(fold)
a
b
c
e
no.
R1
R2
CHK1 IC50 (nM)
checkpoint abrogation IC50 (nM)
cellular selectivity (fold)
HT29
3.0
SW620
hERG IC50 (μM)
f
f
6
−
−
−
−
A
B
B
B
B
6.1 ( 1.2)
55 ( 19)
8.5
3.8
17
12
−
11
7.1
7.5
1.7
2.3
5
g
h
7
21 (15, 27)
7.0 (7.5, 6.4)
5.4 (5.3, 5.5)
8.8 (8.5, 9.0)
46.5 (47, 46)
18 (21, 14)
825 (950, 700)
40 (47, 33)
−
39% @10 μM
8
NHMe
NHMe
NMe2
OMe
6.9
3.6
4.1
1.2
1.6
5
9
28 (38, 18)
12
16
30
i
10
11
12
200
11
i
h
1700
8.0
44% @11 μM
i
h
SMe
390
6.8
25% @10 μM
a
b
Determined in Caliper microfluidic assay,40 mean of n = 2, individual values in parentheses. Abrogation of etoposide-induced G2 checkpoint arrest
in HT29 human colon cancer cells, mean of n = 2, individual values in parentheses. Ratio of cytotoxicity GI50 (measured by SRB assay41) to IC50 for
CHK1-mediated abrogation of etoposide-induced G2 checkpoint arrest in HT29 human colon cancer cells. Potentiation by CHK1 inhibitor of
gemcitabine cytotoxicity in HT29 or SW620 human colon cancer cells. Inhibition of hERG ion current in HEK cells overexpressing hERG ion
channel (PatchExpress, Millipore Inc.). Mean ( SD), n ≥ 3. Not determined. Percent inhibition at single concentration. Single determination.
c
d
e
f
g
h
i
Table 2. CHK1 Inhibition and Cellular Activity of (R)-3-((1-(Dimethylamino)propan-2-yl)oxy)-5-((5-substituted-4-
(methylamino)pyridin-2-yl)amino)pyrazine-2-carbonitriles
potentiation of
gemcitabine
cytotoxicity
d
(fold)
a
b
c
e
no.
R
CHK1 IC50 (nM)
checkpoint abrogation IC50 (nM)
126 (190, 62)
cellular selectivity (fold)
HT29 SW620 hERG IC50 (μM)
f
13
14
15
16
17
18
19
20
H
97 (93, 102)
20 (15, 25)
22
22
21
90
11
6.5
9.3
3.0
3.8
3.2
9
3.9
3.9
5.6
7.3
3.5
6.5
8.5
6.5
71% @10 μM
g
Cl
27
1
1
6
8
h
F3C
21 ( 4.5)
28 (21, 34)
10 (17, 3.7)
45 (32, 58)
65 (100, 29)
125 (140, 110)
cyc-C3H5
HCC
26 (23, 28)
11 (11, 11)
2.9 (3.1, 2.7)
36 (41, 31)
3.1 (3.5, 2.7)
3.2
2.8
3.3
6.9
f
HO(Me2)CCC
3-fluorophenyl
59%@10 μM
f
103% @11 μM
h
1-methyl-pyrazol-4-yl
27 ( 5)
10
12
a
b
Determined in Caliper microfluidic assay,40 mean of n = 2, individual values in parentheses. Abrogation of etoposide-induced G2 checkpoint arrest
in HT29 human colon cancer cells, mean of n = 2, individual values in parentheses. Ratio of cytotoxicity GI50 (measured by SRB assay41) to IC50 for
CHK1-mediated abrogation of etoposide-induced G2 checkpoint arrest in HT29 human colon cancer cells. Potentiation by CHK1 inhibitor of
gemcitabine cytotoxicity in HT29 or SW620 human colon cancer cells. Inhibition of hERG ion current in HEK cells overexpressing hERG ion
channel (PatchExpress, Millipore Inc.). Percent inhibition at single concentration. Single determination. Mean ( SD), n ≥ 3.
c
d
e
f
g
h
to abrogate an etoposide-induced G2 checkpoint arrest in
HT29 colon cancer cells, a specific CHK1-mediated effect.21 In
parallel, compounds were tested in an antiproliferative assay in
HT29 cells. Many cancer cell lines, including HT29, are not
anticipated to be sensitive to specific CHK1 inhibition. The
comparison of the checkpoint abrogation and antiproliferative
assays therefore informed on the degree of selectivity for CHK1
inhibition versus off-target effects in HT29 cells. Our target
profile was to achieve, as a minimum, CHK1 IC50 < 20 nM with
cellular checkpoint abrogation IC50 < 150 nM, and at least 5-
fold selectivity for checkpoint abrogation over antiproliferative
activity in the HT29 cells. Suitable compounds were further
tested for their ability to enhance the cytotoxicity of
gemcitabine in HT29 and SW620 colon cancer cells as a
measure of efficacy, where at least a 5-fold potentiation was
considered desirable.
Hybridization of the scaffolds represented by 6 and 7 led to
the pyridine ester 8 and analogues (Table 1). The importance
of the cyanopyrazine and aminoalkoxy groups in binding to
selectivity determinants in the CHK1 ATP site had previously
been shown by the X-ray structure of 6 complexed with
CHK1.19 The 4-aminomethyl substituent retained in the hybrid
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Table 3. CHK1 Inhibition and Cellular Activity of 3-Aminoalkoxy-5-((5-(1-methyl-1H-pyrazol-4-yl)-pyridin-2-
yl)amino)pyrazine-2-carbonitriles
potentiation of
gemcitabine
cytotoxicity
d
(fold)
a
b
c
e
no.
R1
R2
CHK1 IC50 (nM)
3.1 (3.5, 2.7)
checkpoint abrogation IC50 (nM)
cellular selectivity (fold)
HT29
6.9
SW620
6.5
hERG IC50 (μM)
f
20
21
22
23
24
25
26
27
28
29
NHMe
NHMe
NHMe
NHMe
NHMe
NHMe
OMe
A
C
B
D
E
F
27 ( 5)
10
6.4
6.7
7.1
11
5.9
21
15
20
12
f
g
h
169 ( 30)
2200
3.5
7.4
4.1
5.6
8.4
8.3
4.8
7.3
9.5
1.5
8.4
13
−
g
i
<1
210
22% @ 10 μM
i
<1
210 (240, 180)
110 (130, 90)
135 (150, 120)
37% @ 10 μM
4.4 (4.1, 4.7)
3.4 (3.9, 3.0)
8.6
10
24
19
5
f
f
A
B
E
A
7.7 ( 3.1)
29 ( 8)
11
h
OMe
<1
29 (38, 19)
56 (54, 57)
5.5
7.5
4.3
−
OMe
6.3 (6.1, 6.5)
27 (28, 26)
16
g
SMe
110
19
8
a
b
Determined in Caliper microfluidic assay,40 mean of n = 2, individual values in parentheses. Abrogation of etoposide-induced G2 checkpoint arrest
in HT29 human colon cancer cells, mean of n = 2, individual values in parentheses. Ratio of cytotoxicity GI50 (measured by SRB assay41) to IC50 for
CHK1-mediated abrogation of etoposide-induced G2 checkpoint arrest in HT29 human colon cancer cells. Potentiation by CHK1 inhibitor of
gemcitabine cytotoxicity in HT29 or SW620 human colon cancer cells. Inhibition of hERG ion current in HEK cells overexpressing HERG ion
channel (PatchExpress, Millipore Inc.) Mean ( SD), n ≥ 3. Single determination. Not determined. Percent inhibition at single concentration.
c
d
e
f
g
h
i
molecule was anticipated to generate a pseudobicycle to replace
the isoquinoline of 6 through forming an intramolecular
hydrogen bond to the adjacent ester. Compound 8 showed
equivalent potency and cellular activities to 6. The microsomal
stability of 8 was somewhat improved over 6 (MLM: 49%
metabolized @30 min) as anticipated, although not to the level
of 7. Previous studies with 6 had shown that the (S)-1-
(dimethylamino)propan-2-oxy side chain could be productively
replaced by pyrrolidin-3-ols, and CHK1 inhibition and cellular
activity were retained in the pyrrolidine 9. Variation of the
pyridine 4-substituent was examined on this scaffold (9−12,
Table 1), showing that the 4-aminomethyl substituent was
more favorable for cellular activity than the dimethylamino,
methoxy, or thiomethyl groups. Selectivity for CHK1 inhibition
over CHK2 and CDK1 was high for both 6 and 7 (Figure 2),
and greater than 100-fold selectivity was maintained in the
hybrid compounds 9 and 10 over both CHK2 and CDK1 (data
not shown). Encouragingly, hERG inhibition was generally
lower for the hybrid pyridine esters than for 6 (Table 1).
We investigated variation of the pyridine 5-substituent in the
new series. Although ester 7 had shown microsomal stability,
we wished to replace this functionality to avoid possible
hydrolysis by plasma or intracellular esterases.22 The vector of
the pyridine 5-substituent in these compounds is aligned to a
solvent-accessible surface that is part of the canonical ATP
binding site pharmacophore for protein kinases.23 In CHK1,
this region is tolerant of a range of functional groups. Analysis
of the known or predicted binding modes of published CHK1
inhibitors18 suggested that the small halo or methyl substituents
of 1, 4, and 6, the larger fluorophenyl group of 3, or the 1-
methylpyrazole of 2 could all occupy the relevant space, and
these analogues were therefore prepared. In addition to these
extremes of substituent size and shape, groups of intermediate
size, such as cyclopropyl and substituted alkynyl were also
studied (13−20, Table 2).
The complete removal of the 5-substituent, as in 13,
compromised potency and cellular efficacy, while larger groups
were better tolerated. Potency increased generally with size,
except that the large, lipophilic 3-fluorophenyl analogue 19
showed a drop in biochemical and cellular activity. The potent
CHK1 biochemical inhibition by the 2-methylpent-3-yn-2-ol 18
failed to translate to increased checkpoint abrogation in cells.
This may reflect the incorporation of an additional hydrogen
bond donor in 18, leading to compromised membrane
permeability compared to other molecules in this set. Of the
compounds studied, the 1-methylpyrazole 20 was the most
favorable replacement for the ester, giving a balance of high
potency and cellular efficacy with reduced hERG activity
relative to 6. The 3-fluorophenyl group 19 and the small
lipophilic substituents (14, 15) were associated with increased
hERG activity. Compound 20 maintained selectivity for CHK1
over CHK2 (IC50 = 3.2 μM) and CDK1 (IC50 ∼ 10 μM).
Importantly, 20 also showed low microsomal turnover and
moderate permeability (Table 4), achieving the original aim of
combining the in vitro ADME performance of the pyridines
(e.g., 7) with the in vitro activity profile of the isoquinolines
(e.g., 6).
To generate a broader range of compounds suitable for in
vivo testing, the 5-(1-methylpyrazolyl) substituent of 20 was
kept constant and conservative variations of the pyrazine
aminoalkoxy group and the 4-substituent of the pyridine were
explored (21−29, Table 3). The enantiomer of 20, compound
21, was a significantly less potent CHK1 inhibitor. This
mirrored the difference in activity we had observed between 6
and its enantiomer.19 Subnanomolar CHK1 inhibition was seen
when the aminoalkoxy group of 20 was replaced with either
enantiomer of pyrrolidin-3-ol to give 22 and 23, but neither
compound retained potent cell cycle checkpoint abrogation
activity. Removal of a hydrogen bond donor functionality by N-
methylation of the pyrrolidines gave compounds 24 and 25,
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Table 4. In Vitro ADME and Mouse in Vivo Plasma Concentrations for Selected Potent CHK1 Inhibitors
d
in vitro stability and permeability
mouse plasma levels following 10 mg/kg iv or po
mouse liver microsome metabolism
CaCo-2 permeability (10−6
efflux
c
a
b
no.
(%)
cm/s)
ratio
iv, 1 h (nM) iv, 6 h (nM) po, 1 h (nM) po, 6 h (nM)
e
f
f
9
17 ( 9)
43 ( 4)
42 ( 16)
40 ( 5)
30 ( 13)
12 ( 23)
17 ( 4)
19 ( 6)
38 ( 13)
18 ( 13)
−
−
>6
877 ( 215)
74 ( 19)
33 ( 11)
29 ( 2)
7 ( 1)
90 ( 64)
24 ( 15)
26 ( 2)
8 ( 2)
15
16
17
20
23
24
25
26
28
<4.6
1044 ( 106)
417 ( 81)
833 ( 58)
809 ( 114)
80 ( 30)
−
−
−
−
4.8
439 ( 112)
6 ( 2)
g
h
h
h
h
8.8 ( 0.4)
910 ( 23)
45 ( 10)
53 ( 59)
ij
<20 ,
555 ( 82)
57 ( 7)
−
−
−
−
254 ( 61)
3 ( 1)
2 ( 1)
i
320 ( 30)
193 ( 72)
18 ( 8)
12 ( 9)
4 ( 1)
147 ( 5)
8 ( 1)
27 ( 6)
7 ( 3)
29
2.4
1850 ( 217)
1184 ( 61)
1125 ( 264)
264 ( 168)
52 ( 15)
−
−
6 ( 2)
a
b
c
Percent metabolized after 30 min incubation, mean ( SD), n ≥ 3. Permeability A→B across CaCo-2 cell monolayer, single determination. Ratio
of permeability A→B/B→A across CaCo-2 cell monolayer. Plasma levels at 1 and 6 h following 10 mg/kg iv or po of test compound. Not
determined. Normalized from 5 mg/kg dose. Mean ( SD), n ≥ 3. Data abstracted from full PK determination. Normalized from 1 mg/kg dose.
d
e
f
g
h
i
j
Below level of detection.
which had cellular potency better than that of 22 and 23,
despite somewhat reduced CHK1 inhibition in the biochemical
assay. It was notable that the increased polarity of 22 and 23
was associated with minimal hERG inhibition.
clearance (greater than mouse liver blood flow), and had a high
volume of distribution. To carry out a comparative assessment
of the in vivo pharmacokinetics of other compounds efficiently,
a limited sampling strategy was validated on a series of test
compounds, whereby plasma levels from various paired time
points were evaluated for their potential to predict plasma
clearance. We established that analysis of plasma levels at 1 and
6 h after intravenous and oral dosing of the compounds was
useful and applied this to the CHK1 inhibitors (Table 4).
Although this approach was not expected to accurately predict
the full in vivo pharmacokinetic profiles, certain features could
be used for comparison between compounds within the series.
In particular, the differences between plasma levels at 1 and 6 h
allowed for the identification of compounds most likely to have
persistent oral levels, considered desirable because sustained
inhibition of CHK1 may be required for optimal potentiation of
genotoxic efficacy.18 A comparison of the plasma levels at the 1
h time point following iv and po dosing was used to rank
compounds for the likely degree of oral absorption.
A range of oral pharmacokinetic behaviors was observed for
the compounds tested, despite generally similar in vitro MLM
stabilities, emphasizing the value of an efficient in vivo
screening approach to complement in vitro ADME assays.
The ester 9 showed limited oral exposure compared to the 5-
pyrazolyl analogue 20. Substitutions at C-5 by cyclopropyl or
alkynyl groups 16 and 17, respectively, were less favorable for
sustained oral exposure. The 5-trifluoromethyl analogue 15
maintained the oral bioavailability of 20, although there was a
suggestion from the CaCo-2 cell permeability data that this
compound could have an increased potential for active efflux.
The expected importance of high membrane permeability was
demonstrated by the unsubstituted pyrrolidine 23 which, in line
with its poor cellular activity, showed negligible oral exposure.
N-Methylation to give the pyrrolidine 24, a change which had
restored cellular activity, also improved the plasma levels
achieved on oral dosing but not to the extent of the parent (S)-
1-(dimethylamino)propan-2-oxy analogue 20. Replacement of
the 4-methylamino substituent of 20 by 4-methoxy in 26,
removing one hydrogen bond donor, gave a 2-fold enhance-
ment in plasma levels at the 1 h time point. A similar change
was seen when comparing the N-methylpyrrolidine 28 with the
parent unsubstituted pyrrolidine 24. Determination of the full
pharmacokinetic profile of 26 (Table 5) confirmed the
Given the apparent dependence of the translation of
biochemical activity to cellular potency on total hydrogen
bond donor count, we examined the 4-thiomethyl and 4-
methoxy substituents on the pyridine as replacements for the 4-
methylamino group. With the optimized 5-(1-methylpyrazolyl)
substituent in place of the ester, the change from 4-
methylamino to 4-methylthio in 29 was better tolerated than
in other examples but still resulted in reduced biochemical and
cellular activity. Incorporation of the 4-methoxy group gave
excellent potency in the (S)-1-(dimethylamino)propan-2-oxy
analogue 26 along with high potentiation of the efficacy of
gemcitabine in two cell lines. However, the replacement of the
4-methylamino group by 4-methoxy did not confer any
reduction in hERG inhibition, and 26 had in vitro hERG
activity similar to the isoquinoline 6. The 4-methoxy-
substituted pyrrolidine 27 also showed improved cellular
potency compared to the 4-methylamino derivative 22 but
with a higher differential between biochemical and cellular
activities than that of 26 and reduced potentiation of genotoxic
efficacy.
Selected compounds from this series that met the target
potencies and showed good cellular effects were assessed for
MLM stability (Table 4). The in vitro metabolic stability of
these pyridine-based inhibitors was improved over the
isoquinolines represented by 6, as had been anticipated.
Compounds were screened in vivo for oral exposure to
determine if the increased metabolic stability would consis-
tently lead to oral bioavailability. A full pharmacokinetic profile
was obtained for 20 (Table 5). Gratifyingly, the compound
showed substantial oral bioavailability, despite high in vivo
Table 5. Mouse in Vivo Pharmacokinetic Data for
Compounds 20 and 26
a
a
PK parameter
20
26
Cl (L/h)
Vss (L)
F (%)
0.132
0.209
0.125
0.113
48
61
a
Determined following 10 mg/kg iv and po dosing.
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moderately enhanced oral bioavailability of this compound
relative to the analogue 20.
On the basis of their oral pharmacokinetic profiles, in
particular the ability to sustain plasma levels at 6 h following a
10 mg/kg dose, and the potency and cellular efficacy of the
compounds, 20 and 26 were identified as the most promising
compounds and were progressed to more detailed studies.
Human plasma protein binding (PPB) was determined as
moderate for both compounds (20, PPB = 70%; 26, PPB =
82%), indicating that a substantial fraction of the plasma levels
would be available as unbound drug. The thermodynamic
solubilities of 20 and 26 in water were 0.1 mg/mL and 0.07
mg/mL, respectively. In line with the presence of a basic amine
in the molecules, in pH 6.5 aqueous phosphate buffer their
solubilities increased to >8 mg/mL and 6.2 mg/mL. To allow
further in vivo studies, the maximum tolerated doses (MTDs)
in mice were determined; when administered as single doses in
suspension, the individual MTDs were 160 mg/kg po and >300
mg/kg po for 20 and 26, respectively.
The ability of 20 and 26 to inhibit DNA damaging agent-
induced CHK1 signaling in human tumors xenografts in
athymic mice after oral dosing was assessed (Figure 3). The
compounds were given as suspensions at their respective
MTDs to athymic mice bearing SW620 human colon cancer
xenografts, followed by dosing of gemcitabine (60 mg/kg iv)
after 1 h. Plasma and tumor samples were collected at 6 and 12
h after dosing the genotoxic agent. Tumor lysates were
analyzed by Western blot for total CHK1 protein, phospho-
S317 CHK1 as a marker of activation of CHK1 by the upstream
kinase ATR, and phospho-S296 CHK1 autophosphorylation to
demonstrate inhibition of CHK1 kinase function.21 Activation
of the DNA damage response resulting from gemcitabine
treatment was shown by the increase in phospho-S317 CHK1
and was sustained over 12 h. CHK1 autophosphorylation on
S296 was also seen at both time points in response to
gemcitabine treatment. Both 20 and 26 at their MTDs strongly
inhibited CHK1 S296 autophosphorylation at 6 h following
gemcitabine treatment. Compound 26 continued to show
robust inhibition of CHK1 at the 12 h time point, while a less
powerful effect was seen for 20 (Figure 3A). Analysis of the
drug levels in plasma and tumors showed micromolar plasma
concentrations and high distribution to tumor for both
compounds (Figure 3B). The 2-fold higher dose of 26 was
associated with an increased exposure compared to 20, as
expected. However, a significantly greater fall in drug
concentration between 6 and 12 h was seen for 20 (ca. 20-
fold decrease) than with 26 (ca. 2−3-fold decrease). The more
sustained levels of 26 in plasma and tumor correlated with
stronger inhibition of CHK1 autophosphorylation at 12 h.
While 26 had shown a slightly lower clearance than 20 at low
dose (Table 5), it is possible that the difference in behavior of
26 and 20 in the pharmacodynamic experiment could indicate
saturation of metabolism at the high dose of 26. Further
pharmacodynamic studies showed that single doses of 100−300
mg/kg po 26 could maintain inhibition of gemcitabine-induced
pS296 CHK1 for up to 24 h in HT29 colon tumor xenografts.24
The kinase selectivity of 26 was studied in detail. In common
with most of the compounds tested in this novel series (data
not shown), 26 exhibited excellent selectivity for inhibition of
CHK1 versus CHK2 (IC50 >10 μM) and CDK1 (IC50 >10
μM). To expand on this, the compound was profiled against a
panel of 140 representative kinases using a radiometric assay
format.25 At a test concentration of 1 μM, greater than 50%
Figure 3. (A) Effect of single doses of 20 (160 mg/mg po) and 26
(300 mg/kg po) on gemcitabine (60 mg/kg, iv)-induced CHK1
phosphorylation in SW620 human cancer colon xenografts in nude
mice, at 6 and 12 h after dosing the genotoxic. (B) Plasma and tumor
levels of 20 and 26 at 6 and 12 h after genotoxic drug administration.
inhibition was seen for only 13 enzymes, including CHK1, of
which just 7 were strongly inhibited (>80%; CHK1, RSK1,
RSK2, AMPK, BRSK1, IRAK1, TrkA). The high selectivity of
26 was confirmed by a similar screen against 121 kinases at an
increased test concentration of 10 μM. This indicated that the
selectivity of 26 for CHK1 was >1000-fold against 89 out of the
121 enzymes.
It was notable that for the new compounds prepared in this
study, many of those with reduced in vitro hERG activity
relative to the original isoquinoline 6 did not fulfill the target
criteria for CHK1-mediated potentiation of genotoxic cellular
efficacy or had suboptimal exposure on oral dosing. Thus, 26
showed micromolar inhibition of the hERG ion channel (Table
3). A wider assessment of the compound’s effect on seven other
human cardiac ion channels was carried out.26 Pleasingly, 26
showed minimal inhibition of all the other ion channels tested
(hNav1.5, hKv4.3/hKChIP2, hCav1.2, hKv1.5, hKCNQ1/
hminK, hHCN4; hKir2.1 <25% inhibition @ 10 μM). The
potential significance of the in vitro hERG inhibition by 26 for
further development would require more detailed assessment in
advanced models of cardiac arrhythmic effects.27
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Table 6. In Vivo Potentiation of Cytotoxic Drug Efficacy by 26 in Human Tumor Xenografts
c
tumor growth delay (days)
a
b
tumor
schedule
control tumor growth (days)
gemcitabine, 100 mg/kg iv
irinotecan, 12.5 mg/kg i.p.
26, 75 mg/kg po
combination
d
e
HT29
q7d × 3
q4d × 3
q7d × 3
q7d × 3
8.8 ( 4.8)
5.3 ( 1.3)
6.9 ( 1.6)
6.9 ( 1.5)
2.3 ( 3.3)
1.6 ( 2.2)
7.2 ( 6.2)
−
−
−
−1.8 ( 2.1)
2.8 ( 2.2)
11.4 ( 3.3)***
6.0 ( 2.4)*
15.9 ( 2.5)**
f
e
SW620
g
e
Calu6
0.10 ( 1.2)*
0.30 ( 1.4)***
e
HT29
−
6.4 ( 4.6)
13.5 ( 2.4)***
a
b
Each cycle of treatment consisted of the genotoxic agent (dosed at T = 0 h) followed by two doses of 26 (at T = 24 h and T = 48 h). Time to
c
reach 300% of starting tumor volume, mean ( SD), for four to six mice per treatment group. Delay in time to reach 300% of starting tumor volume
relative to vehicle-treated controls, mean ( SD), for four to six mice per treatment group. Statistical significance of growth delay was determined
using a one-way ANOVA with Tukey’s multiple comparison test: *, P < 0.05; **, P < 0.01; ***, P < 0.001, significantly different from cytotoxic
treatment alone. HT29 human colon cancer xenograft. Not applicable. SW620 human colon cancer xenograft. Calu6 human non-small cell lung
cancer xenograft.
d
e
f
g
Compound 26 showed good oral bioavailability and robust
inhibition of CHK1 signaling over 12−24 h in human tumor
xenografts following a single dose and was therefore studied for
oral antitumor efficacy in combination with genotoxic chemo-
therapies in vivo.24 It has been suggested that prolonged
inhibition of the DNA-damage response following acute
treatment with a genotoxic agent is required to maximize the
additional cell death from the combination treatment.18,28
Some previously reported CHK1 inhibitors have been studied
preclinically using schedules involving delayed administration
of the CHK1 inhibitor until up to 24 h after the genotoxic drug
dose.15,28,29 The phase I clinical trial of 1 involved sequential
treatment with pemetrexed and 1 separated by a 24 h interval.14
We investigated the scheduling of 6 and 26 in combination
with gemcitabine in SW620 cells in vitro.24 These studies
showed that continuous exposure of the cells to the CHK1
inhibitors during the period 24 − 48 h after gemcitabine
addition was required to obtain the maximum in vitro
potentiation of gemcitabine cytotoxicity. Provided this late
period of exposure was maintained, the degree of potentiation
was similar following either simultaneous exposure of the
genotoxic and 26 or with a 24 h delay before addition of 26.
On the basis of the above data, we used a treatment schedule
in vivo that involved giving 26 for two daily doses, beginning 24
h after each genotoxic treatment. Three cycles of treatment
were administered with each cycle beginning at seven- or four-
day intervals. For multiple doses over an extended period, the
MTD of 26 in combination with gemcitabine was determined
as 75 mg/kg po. Pleasingly, and in agreement with the in vitro
data, strong potentiation of the efficacy of gemcitabine by 26
(75 mg/kg po) was observed in HT29 and SW620 human
colon cancers, and in Calu6 human non-small cell lung cancers,
grown as xenografts in athymic mice (Table 6).24 Additionally,
potentiation of the efficacy of the topoisomerase I inhibitor
irinotecan was also observed in the HT29 xenograft model.
Importantly, there were minimal effects on tumor growth of the
CHK1 inhibitor alone, reflecting the weak single-agent
cytotoxicity observed in the cellular studies. The treatments
were well tolerated and neither the single agent nor
combination-treated cohorts showed any body weight loss
over the period of the experiment. Because single agent activity
of CHK1 inhibitors has been proposed in tumors over-
expressing the MYC oncogene,11−13 we also investigated the
oral efficacy of 26 as a single agent in a hemizygotic mouse
transgenic model of neuroblastoma (TH-MYC30) which
overexpresses MYCN. Pleasingly, tumors arising in these
animals underwent significant regression in response to
treatment with 26 (100 mg/kg po; qd7d) with the 26-treated
cohort showing a statistically significant reduction in tumor
burden compared with solvent (vehicle)-treated controls (T/C
= 13%; p < 0.001).24
CONCLUSIONS
■
A novel series of orally active 3-alkoxyamino-5-(pyridin-2-
ylamino)pyrazine-2-carbonitrile CHK1 inhibitors was designed
through the hybridization of potent and selective 2-amino-
isoquinolines with a more metabolically stable 2-aminopyridine
scaffold. Such a lead generation strategy is most effective when
the desirable properties of the parent moieties translate
independently into the merged structure. In this case, the
high potency and selectivity of the 2-aminoisoquinolines such
as 6 were known to depend to a high degree on the binding of
the 5-amino-3-(alkoxyamino)pyrazine-2-carbonitrile group to
specific residues in the ATP site of CHK1,19,31 and the
potential for these interactions was retained in the merged
structures. The in vitro microsomal stability and good
membrane permeability of the prototype 2-aminopyridine 7
was observed to be retained in the merged structures, leading to
an orally bioavailable hybrid series.
The in vitro optimization of the 3-alkoxyamino-5-(pyridin-2-
ylamino)pyrazine-2-carbonitriles demonstrates the usefulness
of a cell-based mechanistic assay cascade to guide the
achievement of high selectivity in a series of ATP-competitive
kinase inhibitors, a task often considered challenging.32 Seeking
well-defined functional selectivity in cells may provide a
potential alternative to iterative large-scale kinome profiling
during lead optimization.17 Ultimately, the optimized molecule
in this study, 26, indeed showed high biochemical kinase
selectivity as well as selectivity for CHK1-dependent mecha-
nism-of-action in human cancer cells. For CHK1, specificity of
binding in the ATP site is aided by the presence of structural
features, such as protein-bound water molecules and polar
residues deep in the ATP pocket, that are unique to CHK1 and
are effectively targeted by the 5-amino-3-(alkoxyamino)-
pyrazine-2-carbonitrile group.19,20,31,33,42
Some inhibition of the hERG ion channel was observed with
several of the inhibitors in this series. Increased polarity
generally led to decreased hERG inhibition34 but was often
accompanied by reduced cellular potency or efficacy,
particularly where the polarity arose from an increase in the
number of hydrogen bond donors. The micromolar hERG
inhibition observed for 26 requires further studies to determine
the therapeutic index relevant to potential clinical development,
but it is notable that other cardiac ion channels were not
inhibited by 26.
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Significantly improved in vitro microsomal stability,
compared to that of the parent isoquinoline 6, was observed
in the hybrid compounds. This led to oral bioavailability
provided that high membrane permeability was maintained.
However, the in vivo behavior of the compounds was not
uniform, with varying levels of oral exposure and in vivo
clearance observed even where similar metabolic stability was
observed in vitro. The limited sampling strategy for in vivo
pharmacokinetics presented here was useful in optimizing
compound oral pharmacokinetics and distinguishing between
compounds with similar in vitro profiles, without the need for
determining multiple full PK profiles.
Scheme 1. Synthesis of 5-Amino-3-alkoxypyrazine-2-
carbonitrile Intermediates
a
We focused on achieving sustained high concentrations of
the compounds in plasma and xenograft tumors over at least a 6
h period, because sustained inhibition of CHK1 signaling is
postulated to be necessary for efficacy.10,18,28 This approach
successfully translated into sustained inhibition of CHK1
signaling in vivo, as shown by the inhibition of genotoxic
agent-induced biomarkers of the DNA damage response by
compounds 20 and 26. Importantly, the pharmacodynamic
effects of 26 in xenograft tumors translated to efficacy in a
range of tumor xenograft models. As anticipated from the
cellular activities, the selective oral CHK1 inhibitor 26
potentiated the antiproliferative effect of the DNA-damaging
agents gemcitabine and irinotecan in p53-defective solid
tumors. Additionally, oral compound 26 was effective as a
single agent in a genetically engineered model of MYCN-driven
neuroblastoma, confirming with a distinct chemical class of
CHK1 inhibitor the results reported for the intravenous
inhibitor 2 in a tumor xenograft neuroblastoma model.13
In summary, we have identified a novel series of potent and
highly selective, orally bioavailable CHK1 inhibitors with oral
antitumor activity in vivo. This represents one of the first series
of orally bioavailable and efficacious CHK1 inhibitors to be
described.18 The profile of the highlighted compound 26
(CCT244747) shows this molecule to be an excellent CHK1-
selective research tool for in vitro and in vivo pharmacological
studies35 and to possess many of the properties desirable in a
clinical development compound.
a
Reagents and conditions: (i) R−OH, NaH, dioxane, 90 °C (16−
69%).
a
Scheme 2. Synthesis of Compounds 8−12
a
Reagents and conditions: (i) MeNH2 or Me2NH, MeCN, 0 °C to rt,
(71−80%); (ii) MeONa, THF, rt (56%); (iii) MeSNa, THF−H2O, rt
(56%); (iv) 30, Pd2dba3, xantphos, Cs2CO3, toluene, 130 °C,
microwave (92%); (v) (a) 32, Pd2dba3, xantphos, Cs2CO3, toluene
or dioxane, 130 °C, microwave, and then (b) CF3CO2H, CH2Cl2, rt
(16−31%).
EXPERIMENTAL SECTION
■
Synthetic Chemistry. Modular syntheses using sequential chemo-
or regioselective SNAr reactions and palladium-catalyzed couplings
were developed to prepare the novel 3-alkoxyamino-5-(pyridin-2-
ylamino)pyrazine-2-carbonitriles. 5-Amino-3-(alkoxy)pyrazine-2-car-
bonitriles 30−35 were synthesized from 5-amino-3-chloropyrazine-2-
carbonitrile19 by SNAr displacement with the appropriate alcohol
under basic conditions (Scheme 1). The enantiomerically pure 1-
(dimethylamino)propan-2-ols were prepared from the appropriate
enantiomer of glycidol as previously described.19
prepared starting with the selective SNAr displacement of the 4-iodo
substituent of 2-chloro-4-iodo-5-trifluoromethylpyridine 43 with
methylamine to give 2-chloro-N-methyl-5-(trifluoromethyl)pyridin-4-
amine 44, followed by Buchwald coupling to 30.
To vary the 5-substituent on the pyridine ring more widely, 2-
chloro-5-iodo-N-methylpyridin-4-amine 46 was prepared as a key
intermediate (Scheme 3). Iodination of 2-chloropyridin-4-amine with
iodine monochloride on a multigram scale gave a mixture of 3-iodo-, 5-
iodo-, and 3,5-diiodopyridine products, from which 45 was isolated in
moderate yield (43%) by column chromatography.36 Monomethyla-
tion of 45 was achieved by reductive amination using paraformalde-
hyde and sodium triacetoxyborohydride. Suzuki and Sonogashira
coupling reactions of 46 were found to occur selectively at the 5-iodo
substituent, and subsequent Buchwald coupling to the remaining 2-
chloro group gave compounds 16−19. For compound 20 and
analogues 21−25, the 5-(1-methylpyrazol-3-yl) substituent was
introduced first by Suzuki coupling, followed by Buchwald coupling
to a variety of 5-amino-3-(alkoxy)pyrazine-2-carbonitriles.
To prepare the 5-(1-methylpyrazol-3-yl)-4-methoxy-substituted
pyridines 26−28, commercially available 2-chloro-4-methoxypyridine
48 was first iodinated with N-iodosuccinimide in sulfuric acid to give
2-chloro-5-iodo-4-methoxypyridine, which was then subject to a
chemoselective Suzuki coupling at the iodo substituent to give the
5-substituted intermediate 49 (Scheme 4). For small-scale reactions,
The 5-carboxymethyl-substituted compounds 8−12 were made
from methyl 4,6-dichloronicotinate 36 by displacement of the more
reactive 4-chloro substituent with methylamine, dimethylamine,
methanol, or methanethiol, followed by Buchwald coupling to the
appropriate 5-amino-3-(alkoxy)pyrazine-2-carbonitrile 30 or 32 and
N-deprotection where required (Scheme 2).
To prepare the 5-unsubstituted pyridine analogue 13, commercially
available 2-chloro-N-methylpyridin-4-amine 41 was coupled with the
aminopyrazine 30 (Scheme 3). The 5-chloropyridine 14 was also
prepared starting from 41, which was chlorinated with N-
chlorosuccinimide to give a mixture of 2,3-dichloro-, 2,5-dichloro-,
and 2,3,5-trichloropyridine products, from which 2,5-dichloro-N-
methyl-pyridin-4-amine 42 was isolated by column chromatography.
Subsequent Buchwald reaction of 42 proceeded selectively at the more
reactive 2-chloro substituent. The 5-trifluoromethyl analogue 15 was
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a
Scheme 3. Synthesis of Compounds 15−25
a
Reagents and conditions: (i) 30, 31, 34, or 35, Pd2dba3, xantphos, Cs2CO3, toluene or dioxane, 130 °C, microwave (6−66%); (ii) NCS, KOAc,
AcOH, 80 °C (13%); (iii) MeNH2, MeOH, 130 °C, microwave (31%); (iv) ICl, KOAc, AcOH, 80 °C (43%); (v) (H2CO)n, NaB(OAc)3H, AcOH,
40 °C (78%); (vi) cyc-(C3H5)B(OH)2, Pd(PPh3)4, Na2CO3, MeCN, 100 °C, microwave (49%); (vii) R-CC-H, PdCl2(PPh3)2, CuI, Et3N, DMF, 120
°C, microwave (50−75%); (viii) 3-F-(C6H4)B(OH)2, Pd(PPh3)4, Na2CO3, MeCN, 100 °C, microwave (79%); (ix) 1-methyl-4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)-1H-pyrazole, Pd(PPh3)4, Na2CO3, MeCN−H2O, 100 °C, microwave (96%); (x) (a) 32 or 33, Pd2dba3, xantphos, Cs2CO3,
toluene or dioxane, 130 °C, microwave, and then (b) CF3CO2H, CH2Cl2, rt (31−47%).
microwave heating and homogeneous tetrakis(triphenylphosphine)-
palladium(0) catalyst provided suitable conditions for these couplings.
For larger scale preparations of 26, the use of a microencapsulated
palladium source (Pd EnCat TPP30; palladium(II) acetate and
triphenylphosphine microencapsulated in polyurea matrix)37,38 as the
catalyst and a standard heat source simplified the preparation and
purification. Buchwald couplings to the remaining chloro group of 49
using microwave heating gave 26 and related target compounds on a
small scale. For large scale preparations of 26, the Buchwald coupling
could be conveniently carried out using several microwave reactions in
parallel, which were combined for purification, or alternatively by
nonmicrowave conditions. To introduce a 4-thiomethyl substituent to
the pyridine, 2-chloro-5-iodopyridine 50 was selectively lithiated at the
4-position39 and quenched with dimethyl disulfide to give 2-chloro-5-
iodo-4-(methylthio)pyridine 51, which was coupled with 1-methyl-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole to yield 52.
Subsequent Buchwald coupling to 52 gave the 4-thiomethyl analogue
29.
carried out using a Biotage Initiator microwave reactor. Flash silica
column chromatography was performed using Merck silica gel 60
(0.025−0.04 mm). Gradient silica column chromatography was
performed with a Biotage SP1 medium pressure chromatography
system, using prepacked silica gel cartridges. Ion exchange
chromatography was performed using Isolute Flash SCX-II (acidic)
or Flash NH2 (basic) resin cartridges. 1H NMR spectra were recorded
on a Bruker AMX500 instrument at 500 MHz or on a Bruker Avance
instrument at 400 MHz using internal deuterium locks. Chemical
shifts (δ) are reported relative to TMS (δ = 0) and/or referenced to
the solvent in which they were measured. Combined HPLC-MS
analyses were recorded using a Waters Alliance 2795 separations
module and Waters/Micromass LCT mass detector with HPLC
performed using Supelco DISCOVERY C18, 50 mm × 4.6 mm or 30
mm × 4.6 mm i.d. columns, or using an Agilent 6210 TOF HPLC-MS
with a Phenomenex Gemini 3 μm C18 (3 cm × 4.6 mm i.d.) column.
Both HPLC systems were run at a temperature of 22 °C with gradient
elution of 10−90% MeOH/0.1% aqueous formic acid at a flow rate of
1 mL/min and a run time of 3.5, 4, or 6 min as indicated. UV
detection was at 254 nm, and ionization was by positive or negative
ion electrospray as indicated. The molecular weight scan range was
50−1000 amu. All tested compounds gave >95% purity as determined
General Synthetic Chemistry. Reactions were carried out under
N2. Organic solutions were dried over MgSO4 or Na2SO4. Starting
materials and solvents were purchased from commercial suppliers and
were used without further purification. Microwave reactions were
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a
half by evaporation. Water was added to the mixture, followed by
basification with NaHCO3. The mixture was extracted with EtOAc (3
× 70 mL), the combined organic layers were dried and absorbed onto
silica gel, and solvent was removed by evaporation. Gradient silica
column chromatography, eluting with 5−10% EtOAc in cyclohexane,
Scheme 4. Synthesis of Compounds 26−29
1
gave 46 (1.65 g, 6.14 mmol, 78%) as a white powder. H NMR (500
MHz, CDCl3) δ 8.27 (s, 1H), 6.41 (s, 1H), 4.85 (brs, 1H), 2.93 (d, J =
5.0, 3H); LC-MS (3.5 min) tR = 1.90 min; m/z (ESI) 268 (M + H);
HRMS m/z calcd for C6H7ClIN2 (M + H) 268.9337, found 268.9339.
2-Chloro-N-methyl-5-(1-methyl-1H-pyrazol-4-yl)pyridin-4-amine
(47). A mixture of 46 (0.083 g, 0.309 mmol), 1-methyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.068 g, 0.309
mmol), Pd(PPh3)4 (0.018 g, 0.015 mmol), and aq Na2CO3 (0.5 M;
0.927 mL) in MeCN (2.1 mL) was heated at 100 °C in a microwave
reactor for 30 min. The cooled reaction mixture was absorbed onto
silica gel, and solvent was removed by evaporation. Gradient silica
column chromatography, eluting with 1−10% MeOH in CH2Cl2, gave
1
47 (0.066 g, 0.296 mmol, 96%) as a colorless solid. H NMR (500
MHz, CDCl3) δ 7.83 (s, 1H), 7.56 (s, 1H), 7.45 (s, 1H), 6.48 (s, 1H),
4.68 (brs, 1H), 3.96 (s, 3H), 2.84 (d, J = 5.1 Hz, 3H). LC-MS (3.5
min) tR = 1.09 min; m/z (ESI) 223 (M + H). HRMS m/z calcd for
C10H12ClN4 (M + H) 223.0745, found 223.0744.
(R)-3-((1-(Dimethylamino)propan-2-yl)oxy)-5-((5-(1-methyl-1H-
pyrazol-4-yl)-4-(methylamino)pyridin-2-yl)amino)pyrazine-2-car-
bonitrile (20). A mixture of 47 (0.066 g, 0.296 mmol), 3019 (0.066 g,
0.296 mmol), xantphos (0.035 g, 0.059 mmol), Pd2dba3 (0.027 g,
0.030 mmol), and Cs2CO3 (0.193 g, 0.593 mmol) in toluene (2.12
mL) was heated at 130 °C in a microwave reactor for 45 min. The
cooled reaction mixture was diluted with MeOH (10 mL) and
absorbed onto an SCX-2 acidic ion exchange column (2 g). The
column was eluted with MeOH (4 × 20 mL), then NH3 in MeOH (2
M; 4 × 20 mL). The basic eluent fractions were combined and
absorbed onto silica gel. Solvent was removed by evaporation.
Gradient silica column chromatography, eluting with 1−15% MeOH
1
in CH2Cl2, gave 20 (0.040 g, 0.076 mmol, 26%) as a yellow solid. H
a
Reagents and conditions: (i) NIS, H2SO4, 55 °C (22%); (ii) 1-
methyl-4-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)-1H-pyrazole,
PdEnCat TPP30, Na2CO3, MeCN−H2O, 105 °C (59%); (iii) 30,
Pd2dba3, xantphos, Cs2CO3, dioxane, 100 °C (71%); (iv) 32, Pd2dba3,
xantphos, Cs2CO3, toluene, 130 °C, microwave, and then (b)
CF3CO2H, CH2Cl2, rt (27%); (v) LDA, MeSSMe, THF, −78 °C −
rt (11%); (vi) 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)-1H-pyrazole, Pd(PPh3)4, Na2CO3, MeCN, 100 °C, microwave
(72%); (vii)) 30, Pd2dba3, xantphos, Cs2CO3, dioxane, 130 °C,
microwave (5%).
NMR (500 MHz, CDCl3) δ 8.23 (brs, 1H), 7.85 (s, 1H), 7.58 (s, 1H),
7.47 (s, 1H), 7.03 (s, 1H), 5.48−5.37 (m, 1H), 4.72 (q, J = 5 Hz, 1H),
3.98 (s, 3H), 2.90 (d, J = 5 Hz, 3H), 2.74 (dd, J = 13.4, 7.2 Hz, 1H),
2.51 (dd, J = 13.4, 4.4 Hz, 1H), 2.31 (s, 6H), 1.41 (d, J = 6.3 Hz, 3H).
LC-MS (3.5 min) tR = 1.09 min; m/z (ESI) 408 (M + H). HRMS m/z
calcd for C20H26N9O (M + H) 408.2255, found 408.2255.
Synthesis of Compound 26. 2-Chloro-4-methoxy-5-(1-methyl-
1H-pyrazol-4-yl)pyridine (49). NIS (25.3 g, 107 mmol) was added
portion wise at rt to a solution of 2-chloro-4-methoxypyridine 48
(15.35 g, 107 mmol) in H2SO4 (76 mL). The mixture was heated and
stirred at 55 °C for 2 h. The reaction mixture was poured onto ice−
water (400 mL), and aq NaOH (8 M; 500 mL) was added slowly to
basify the mixture (pH ∼ 14), after which the dark brown solution
became pale yellow. The mixture was extracted with CH2Cl2 (2 × 600
mL). The combined organic layers were washed with brine (300 mL)
and absorbed onto silica gel. Solvent was removed by evaporation. Dry
flash silica column chromatography, eluting with 50% EtOAc in
cyclohexane and collecting the first major product (Rf = 0.66 in 50%
EtOAc/cyclohexane), followed by flash silica column chromatography
of mixed fractions, gave 2-chloro-5-iodo-4-methoxypyridine as a white
by these methods. All purified synthetic intermediates gave >95%
purity as determined by these methods except where indicated in the
text.
Synthesis of Compound 20. 2-Chloro-5-iodopyridin-4-amine
(45). ICl (10.0 g, 61.6 mmol) was added to a solution of 2-
chloropyridin-4-amine (6.60 g, 51.3 mmol) and KOAc (10.1 g, 103
mmol) in AcOH (325 mL). The reaction was heated to 80 °C and
stirred for 3 h. AcOH was removed by evaporation. Toluene was
added, and the mixture was evaporated to remove residual AcOH
(×2). The crude products were dissolved in CH2Cl2/MeOH and
absorbed on to silica gel. The silica gel was dried under high vacuum
for 18 h. Gradient silica column chromatography, eluting with 5−20%
EtOAc in cyclohexane and collecting the second major component (Rf
= 0.5 in 1:1 EtOAc/cyclohexane), gave 45 (5.66 g, 22.2 mmol, 43%) as
1
solid (6.20 g, 23.0 mmol, 22%). H NMR (500 MHz, DMSO) δ 8.52
(s, 1H), 7.19 (s, 1H), 3.96 (s, 3H); LC-MS (4 min) tR = 2.56 min; m/z
(ESI) 270 (M + H). HRMS m/z calcd for C6H5ClIN2O2 (M + H)
269.9177, found 299.9181. Aqueous Na2CO3 (0.5M; 24.5 mL, 12.25
mmol) was added to a mixture of 2-chloro-5-iodo-4-methoxypyridine
(2.20 g, 8.16 mmol), 1-methyl-4-(4,4,5,5-tetramethyl1,3,2-dioxabor-
olan-2-yl)-1H-pyrazole (1.70 g, 8.16 mmol), and Pd EnCat TPP30
(Sigma-Aldrich Co.; 2.04 g, 0.816 mmol) in MeCN (48.6 mL) and
EtOH (12.4 mL). N2 was bubbled through the mixture for 15 min.
The reaction mixture was heated at reflux (105 °C) for 3 h. The
mixture was cooled and filtered to recover the microencapsulated
palladium catalyst. The filtrate was absorbed onto silica gel, and
solvent was removed by evaporation. Gradient silica column
chromatography, eluting with 1−10% EtOH in CH2Cl2, gave the
partially purified product, which was subjected to ion exchange
1
an off white powder. H NMR (500 MHz, CD3OD) δ 8.18 (s, 1H),
6.68 (s, 1H), 4.81 (s, 2H); LC-MS (3.5 min) tR = 1.66 min; m/z (ESI)
255 (M + H); HRMS m/z calcd for C5H5ClIN2 (M + H) 254.9180,
found 254.9189.
2-Chloro-5-iodo-N-methylpyridin-4-amine (46). 45 (2.00 g, 7.86
mmol) and paraformaldehyde (0.472 g, 15.7 mmol) were dissolved in
AcOH (56.1 mL) and stirred for 2.5 h at 40 °C. NaB(OAc)3H (3.66 g,
17.3 mmol) was added, and the mixture was stirred at 40 °C for 1.5 h.
Further NaB(OAc)3H (3.66 g, 17.3 mmol) was added, and the mixture
was stirred for 19 h. The reaction mixture was reduced in volume by
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Journal of Medicinal Chemistry
Article
chromatography on acidic SCX-II resin, eluting with MeOH and then
with 2 M NH3 in MeOH. The basic fractions were combined and
concentrated to give 49 (1.08 g, 4.84 mmol, 59%) as an off white solid.
1H NMR (500 MHz, CDCl3) δ 8.39 (s, 1H), 7.84 (s, 1H), 7.78 (s,
1H), 6.87 (s, 1H), 3.97 (s, 3H), 3.95 (s, 3H). LC-MS (3.5 min) tR =
2.38 min; m/z (ESI) 224 (M + H). HRMS m/z calcd for
C10H11ClN3O (M + H) 224.0585, found 224.0583.
transgenic models of MYCN-neuroblastoma. This work was
supported by Cancer Research UK [CUK] grant numbers
C309/A8274 and C309/A11566, and by The Institute of
Cancer Research. We acknowledge NHS funding to the NIHR
Biomedical Research Centre.
ABBREVIATIONS USED
(R)-3-((1-(Dimethylamino)propan-2-yl)oxy)-5-((4-methoxy-5-(1-
methyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)pyrazine-2-carbonitrile
(26). N2 was passed through a mixture of 49 (0.532 g, 2.38 mmol),
3019 (0.526 g, 2.38 mmol), xantphos (0.275 g, 0.476 mmol), Pd2dba3
(0.218 g, 0.238 mmol), and Cs2CO3 (1.55 g, 0.593 mmol) in dioxane
(24 mL) for 10 min. The mixture was heated at 100 °C for 17 h. The
cooled mixture was absorbed onto SCX-II acidic ion-exchange resin
(20 g) and eluted with MeOH/CH2Cl2 and then with 2 M NH3 in
MeOH and finally with 7 M NH3 in MeOH. The basic eluent fractions
containing the crude product were absorbed onto silica gel, and
solvents were removed by evaporation. Gradient silica column
chromatography, eluting with 1−14% MeOH in CH2Cl2, gave 26
■
ATP, adenosine triphosphate; ATR, ataxia telangiectasia and
rad3 related; CDK1, cyclin dependent kinase 1; CHK1,
checkpoint kinase 1; CHK2, checkpoint kinase 2; DELFIA,
dissociation-enhanced lanthanide fluorescent immunoassay;
ELISA, enzyme-linked immunosorbent assay; hERG, human
ether-a-go-go related gene product; MLM, mouse liver
microsomes; MPM2, M-phase phosphoprotein 2; MYCN, V-
myc myelocytomatosis viral related oncogene, neuroblastoma
derived; RNAi, RNA interference; SRB, sulforhodamine B
1
(0.693 g, 1.68 mmol, 71%) as a yellow solid. H NMR (500 MHz,
REFERENCES
■
CDCl3/CD3OD) δ 9.14 (s, 1H), 8.35 (s, 1H), 7.83 (s, 1H), 7.76 (s,
1H), 7.06 (s, 1H), 5.87−5.75 (m, 1H), 4.00 (s, 3H), 3.94 (s, 3H), 3.35
(dd, J = 13.7, 6.7 Hz, 1H), 3.21 (d, J = 13.7 Hz, 1H), 2.87 (s, 6H), 1.53
(d, J = 6.4 Hz, 3H). LC-MS (3.5 min) tR = 2.11 min; m/z (ESI) 409
(M + H). HRMS m/z calcd for C20H25N8O2 (M + H) 409.2095,
found 409.2103.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental methods for the synthesis and characterization of
compounds 8−19, 21−25, 27−35, 37−40, 42, 44, 51, and 52;
experimental methods for the determination of CHK1 and
CHK2 inhibition; experimental methods for the determination
of checkpoint abrogation, antiproliferative activity, and
potentiation of genotoxic drug efficacy in cancer cell lines;
experimental method for the determination of compound
concentrations in vivo at selected time points following iv and
oral dosing; kinase inhibition profile for 26 at 1 μM and 10 μM
test concentrations. This material is available free of charge via
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AUTHOR INFORMATION
Corresponding Author
*Fax: +44 208 722 4216. E-mail: ian.collins@icr.ac.uk.
■
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Notes
The authors declare the following competing financial
interest(s): M. Lainchbury, T. P. Matthews, T. McHardy, K.
J. Boxall, M. I. Walton, P. D. Eve, A. Hayes, M. R. Valenti, A. K.
de Haven Brandon, G. Box, G. W. Aherne, F. I. Raynaud, S. A.
Eccles, M. D. Garrett, I. Collins are or have been employees of
The Institute of Cancer Research which has a commercial
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Research are subject to a “Rewards to Inventors Scheme” which
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ACKNOWLEDGMENTS
■
We thank Dr. A. Mirza, M. Richards, and Dr. M. Liu for
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