Identification and characterisation of 2-aminopyridine inhibitors of checkpoint kinase 2

Article abstract of DOI:10.1016/j.bmc.2009.11.058

5-(Hetero)aryl-3-(4-carboxamidophenyl)-2-aminopyridine inhibitors of CHK2 were identified from high throughput screening of a kinase-focussed compound library. Rapid exploration of the hits through straightforward chemistry established structure-activity relationships and a proposed ATP-competitive binding mode which was verified by X-ray crystallography of several analogues bound to CHK2. Variation of the 5-(hetero)aryl substituent identified bicyclic dioxolane and dioxane groups which improved the affinity and the selectivity of the compounds for CHK2 versus CHK1. The 3-(4-carboxamidophenyl) substituent could be successfully replaced by acyclic ω-aminoalkylamides, which made additional polar interactions within the binding site and led to more potent inhibitors of CHK2. Compounds from this series showed activity in cell-based mechanistic assays for inhibition of CHK2.

Full text of DOI:10.1016/j.bmc.2009.11.058

Bioorganic & Medicinal Chemistry 18 (2010) 707–718
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification and characterisation of 2-aminopyridine inhibitors
of checkpoint kinase 2
Stephen Hilton a, , Sebastien Naud a, John J. Caldwell a, Kathy Boxall a, Samantha Burns a,
Victoria E. Anderson a, Laurent Antoni a, Charlotte E. Allen a, Laurence H. Pearl b, Antony W. Oliver b,
G. Wynne Aherne a, Michelle D. Garrett a, Ian Collins a,
*
a Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK
b Section of Structural Biology, Chester Beatty Laboratories, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
5-(Hetero)aryl-3-(4-carboxamidophenyl)-2-aminopyridine inhibitors of CHK2 were identified from high
throughput screening of a kinase-focussed compound library. Rapid exploration of the hits through
straightforward chemistry established structure–activity relationships and a proposed ATP-competitive
binding mode which was verified by X-ray crystallography of several analogues bound to CHK2. Variation
of the 5-(hetero)aryl substituent identified bicyclic dioxolane and dioxane groups which improved the
affinity and the selectivity of the compounds for CHK2 versus CHK1. The 3-(4-carboxamidophenyl) sub-
Received 14 September 2009
Revised 16 November 2009
Accepted 27 November 2009
Available online 6 December 2009
Keywords:
CHK2
Kinase inhibitor
High-throughput screening
Crystallography
stituent could be successfully replaced by acyclic
x-aminoalkylamides, which made additional polar
interactions within the binding site and led to more potent inhibitors of CHK2. Compounds from this ser-
ies showed activity in cell-based mechanistic assays for inhibition of CHK2.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
BRCA1 promotes double strand break repair,14 while phosphoryla-
tion of the transcription factor FOXM1 leads to increased expression
Checkpoint kinase 2 (CHK2) is a serine/threonine kinase which
plays an important part in the complex signalling networks respon-
sible for the maintenance of mammalian genomic integrity and re-
pair of damaged DNA.1–4 Signalling through CHK2 is activated in
cells in response to DNA damage caused by external agents such as
ionising radiation or genotoxic chemotherapy. Double strand breaks
in DNA lead to activation of the DNA-damage transducer ataxia and
telangiectasia mutated (ATM). ATM phosphorylates CHK2 on
Thr68,5,6 leading to homodimerisation of the protein and activation
through trans-autophosphorylation7,8 on Thr383 and Thr387 and
cis-autophosphorylation9,10 on Ser516. In turn, activated CHK2
phosphorylates multiple substrates that control cell cycle progres-
sion and DNA repair. In cells with a functional p53 tumour suppres-
sor pathway, phosphorylation of HDMX by CHK2 stabilises p53,
leading to a G1 cell cycle arrest and apoptotic cell death if the dam-
aged DNA is not repaired.11,12 Similarly, phosphorylation by CHK2
stabilizes the transcription factor E2F-1, promoting apoptosis.13
CHK2 also signals to the Cdc25 family of phosphatases that control
cell cycle progression in S, G2 and M phases through regulation of cy-
clin dependent kinases.3,4 At the same time, CHK2 activates several
DNA damage repair pathways. In particular, phosphorylation of
of proteins involved in the homologous recombination and base
excision repair mechanisms.15
There is current interest in the therapeutic potential of inhibi-
tors of CHK2 in several distinct contexts, but especially in cancer
where DNA-damaging agents remain a central component of treat-
ment regimes.1,2,16 The p53-mediated apoptotic response contrib-
utes to cell death in normal tissue in response to ionising
radiation and chemotherapies that cause double strand DNA
breaks. In contrast, many tumours lack a functional p53 pathway.17
There is therefore the potential for CHK2 inhibition to selectively
reduce p53-mediated cell death in normal tissue and suppress
the side-effects of these therapies. In support of this, chk2À/À trans-
genic mice show resistance to apoptosis after exposure to ionising
radiation.18,19 Importantly, no increased tumourigenesis is seen in
these CHK2-deficient animals, in contrast to p53-deficient mice. A
radioprotective effect on isolated mouse thymocytes and human T-
cells has been observed with selective small molecule inhibitors of
CHK2.20,21
It is also plausible that inhibition of CHK2 alone could exert an
antitumour effect. In some cancer cell lines, CHK2 is highly acti-
vated.3,22 There is the possibility that inhibition of CHK2 activity
would lead to cell death through inactivation of DNA repair path-
ways that are critical for the survival of the aberrant cells. Related
to the inhibition of DNA repair pathways is the recent finding that
replication of the human hepatitis C virus (HCV) is suppressed by
* Corresponding author. Tel.: +44 208 722 4317; fax: +44 208 722 4216.
E-mail address: ian.collins@icr.ac.uk (I. Collins).
Present address: The School of Pharmacy, University of London, UK.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.058

708
S. Hilton et al. / Bioorg. Med. Chem. 18 (2010) 707–718
siRNA knockdown of CHK2 or ATM.23 HCV infection is associated
with increased genomic instability and double strand breaks in
DNA.24
2. Kinase-focussed library screening
A commercial library of ca. 7000 small molecules designed as
potential ATP-competitive kinase inhibitors32 was screened against
purified recombinant full-length CHK2 in a high throughput Alpha-
Screen format.33 A total of 76 compounds were classified as hits
It has been suggested that selective inhibition of CHK2 in p53-
deficient tumour cells may radio- or chemosensitize the cells
through targeting of the G2 checkpoint.2 CHK2 is seen to be acti-
vated in a wide range of tumour cells in response to ionizing radi-
ation and DNA-damaging drugs. Experiments with one selective
small molecule inhibitor of CHK2, VRX0466617 (2, Fig. 1), did not
show an effect of CHK2 inhibition on the cell cycle distribution of
irradiated cells, nor potentiation of the cytotoxicity of the DNA-
damaging drugs cisplatin and doxorubicin.21 However, more re-
cently the selective CHK2 inhibitor PV1019, a cell-penetrant ana-
logue of NSC 109555, was shown to potentiate the cytotoxicity of
topotecan and camptothecin in ovarian cancer cell lines.56 More-
over, increased cytotoxicity of the CHK2 inhibitor alone was ob-
served in tumour cell lines with high endogenous levels of CHK2
compared to those expressing low levels of CHK2. The extent to
which CHK2 inhibition can synergize with DNA-damaging drugs
therefore remains unclear and is likely to depend on the interac-
tion of cellular genomic background, DNA-damaging modality
and inhibitor pharmacological profile.1
A limited number of small molecule inhibitors of CHK2 have
been reported to date, and thus the discovery of further inhibitor
chemotypes remains of interest. High-throughput screening has
identified 2-arylbenzimidazole-5-carboxamides (1),20,25 isothiaz-
ole carboxamidines (2)21,26 and bis-guanylhydrazones (3)27,56 as
selective, ATP-competitive inhibitors of CHK2 (Fig. 1). Compounds
that potently inhibit other kinases, particularly CHK1, in addition
to CHK2 have been described, including the staurosporine ana-
logue UCN-01,28 an indoloazepine derivative of the natural product
hymenialdisine29 and the dual inhibitor of CHK1 and CHK2,
AZD7762.30 Few crystal structures of CHK2 complexed with small
molecules are currently available in the public domain: CHK2-
ADP8 (PDB code: 2CN5), CHK2-debromohymenialdisine8 (PDB
code: 2CN8) and CHK2-NSC 10955531 (PDB code: 2WOJ). Given
the well established utility of multiple structures of protein–ligand
complexes to aid in selective kinase drug design, additional charac-
terisation of ligand binding modes to CHK2 would provide an
opportunity to enhance structure-based design of new inhibitors.
We have identified 3,5-diaryl-2-aminopyridine inhibitors of
CHK2 through high throughput screening of a kinase-focussed
compound library. We describe efficient synthetic routes to this
class of compounds and characterisation of their interaction with
CHK2. We have determined the structures of representative com-
pounds from the series bound to CHK2 to rationalise the observed
structure–activity relationships.
(>60% inhibition of CHK2 activity at 30 lM test concentration)
and assessed for potential non-specific inhibition by re-screening
in triplicate in the presence of 0.01% Triton X-100 detergent.34
From this, 36 compounds showed limited reduction of activity
(>50% inhibition) when the detergent was added. These com-
pounds were counterscreened for their ability to interfere with
the AlphaScreen format by conducting the assay in the presence
of the inhibitors and a control phosphopeptide.35 A reduction in
the AlphaScreen signal was seen with 25 compounds, indicative
of interference in the assay readout. The activity of the remaining
11 compounds was confirmed in a DELFIA assay and IC50 values
were determined (Fig. 2, Table 1). The identities and purities of
the HTS samples were confirmed by mass spectrometry and HPLC.
Interestingly, although two weakly active and chemically unre-
lated singleton hits were identified (not shown), the majority of
the compounds (4–12) were from one well-defined class of 3,5-
diaryl-2-aminopyridines. Only 2-aminopyridines bearing a 3-(4-
carboxamido)phenyl (4–8) or a 3-(4-hydroxyphenyl) (9–12) sub-
stituent gave substantial CHK2 inhibition. The specificity for these
Figure 2. Structures of 2-aminopyridine hits from HTS against CHK2.
Table 1
CHK2 inhibition and calculated ligand efficiencies of 2-aminopyridine screening hits
and selected analogues in the library
L.E.b
a
Compound
CHK2 IC50
(
lM)
4
5
6
7
8
0.61 ( 0.12)c
2.7
2.1
2.4
1.8
2.3
3.0
4.4
6.3
0.40
0.34
0.34
0.31
0.27
0.29
0.27
0.28
0.31
—
9
10
11
12
13
14
15
>100
>100
>100
—
—
a
Single determination in DELFIA assay format. Standard inhibitor staurosporine
gave mean ( SEM) IC50 = 27 ( 2.6) nM, n = 10 determinations.
b
L.E. = ligand efficiency.43
Mean ( SEM), n = 5 determinations.
c
Figure 1. Structures of reported CHK2 inhibitors.

S. Hilton et al. / Bioorg. Med. Chem. 18 (2010) 707–718
709
Simple nitrile (17) and ester (18) replacements for the amide func-
tionality of 4 were introduced by incorporation of different 4-
substituted arylboronates. The ester 18 was hydrolysed to yield
the acid (19). Starting with 3,5-dibromopyridine, sequential cou-
pling as described above gave the analogue lacking the 2-amino
substituent (20, Table 2).
To introduce substituted amides, the benzoate 18 was directly
aminated with methylamine in the presence of 1,5,7-triazabicy-
clo[4.4.0]dec-5-ene44 to give 21 in excellent yield (Scheme 2). An
alternative synthesis of the scaffold was developed beginning by
coupling 4-(methoxycarbonyl)-benzeneboronic acid to 2-amino-
3-bromopyridine 22. Subsequent bromination of the pyridine at
C-5 was achieved in high yield using 1,3-dibromo-5,5-dimethylhy-
dantoin.45 Reaction of the intermediate ester 23 with dimethyl-
amine yielded 24, which was coupled with thiophene-3-boronic
acid to give 25.
To introduce alkyl substitution on the 2-amino group of 4, the
2-alkylaminopyridines 26 and 27 were subject to bis-bromination
with 1,3-dibromo-5,5-dimethylhydantoin45 at low temperature
(Scheme 3). In the case of 26, the C-5 monobrominated product
was also isolated (23%). Suzuki coupling of 4-carbamoylphenylbo-
ronic acid to the dibromopyridine 28 occurred preferentially at the
C-3 halogen, analogous to the reaction of 16, leading to the 3-bro-
mo derivative 30 as the major product isolated after chromatogra-
phy. Introduction of the 3-thienyl substituent was realised by a
final Suzuki coupling to yield the 2-(methylamino)pyridine 32.
Applying the same sequence to 29 gave the 2-(dimethylamino)-
pyridine 33.
Figure 3. Structures of selected inactive analogues in the screening set.
groups was confirmed by re-screening structurally related 2-
aminopyridines in the focussed library that had not emerged as
hits in the primary screen (Fig. 3, Table 1). For example, the 3-(3-
carboxamidophenyl) analogue 13, secondary amide 14, and benzyl
alcohol 15 were all inactive in the DELFIA assay. Remining of the
HTS data identified a number of 2-aminopyridines that had not
reached the cut-off for selection as primary hits but which pos-
sessed the 4-carboxamide and 4-phenol structural motifs and
had activities weaker than the confirmed hits.
We chose to investigate the 3-(4-carboxamidophenyl)-2-
aminopyridines in more detail. 3,5-Diaryl(or heteroaryl)-2-amino-
pyridines have been described as kinase inhibitor scaffolds in the
recent patent literature,36–42 although to our knowledge detailed
descriptions of structure–activity relationships and crystallo-
graphic characterisation of the binding modes have not been
reported.
The preliminary structure–activity from the HTS data indicated
a specific and well-defined interaction with CHK2 for this scaffold,
with a variety of substituents tolerated at C-5 of the pyridine. The
calculated ligand efficiencies of these hits, ranging from 0.27 to
0.40 kcal molÀ1 heavy atomÀ1, were suitable for further progres-
sion.43 In contrast, the activity of the 3-(4-hydroxyphenyl)-2-
aminopyridines appeared reliant on the presence of the 3,4,5-tri-
methoxyphenyl group or similar substituents. The ligand efficien-
cies of this group of hits were generally lower than that of the
benzamido-substituted analogues.
The regiochemistries of these sequential couplings were con-
firmed by NOE determinations for the products 32 and 33. In 32,
NOEs were observed between the pyridyl H-6 proton (d
8.35 ppm) and protons on the thiophene (H-2 d 7.53 ppm; H-4 d
7.42 ppm). No NOEs were seen between the pyridyl H-6 and pro-
tons in the 4-carbamoylphenyl group. An analogous pattern of
NOEs was observed in 33. Furthermore, reduction of the interme-
diate bromides 30 and 31 (H2, Pd–C) generated the expected 2-
alkylamino-3-(4-carbamoylphenyl)pyridines. Although the first
Suzuki coupling is selective for the apparently more hindered 3-
We used two approaches to explore the 3-(4-carboxamidophe-
nyl)-2-aminopyridine series, initially exploiting straightforward
synthetic routes to prepare a library of analogues designed to sys-
tematically define structure–activity relationships. In parallel we
investigated co-crystallisation of the inhibitors with CHK2 to
determine the bound conformation of the ligands.
3. Synthetic chemistry
The 2-aminopyridine 4 and close analogues were initially pre-
pared by selective, sequential palladium-mediated cross coupling
of aryl boronates to 3-iodo-5-bromo-2-pyridine 16 (Scheme 1).
Scheme 2. Reagents and conditions: (i) MeNH2, 1,5,7-triazabicyclo[4.4.0]dec-5-
ene, THF, 100 °C, microwave, 89%; (ii) 4-(methoxycarbonyl)-phenylboronic acid,
Pd(PPh3)4, Na2CO3 aq, DME, 135 °C, microwave, 68%; (iii) 1,3-dibromo-5,5-dimeth-
ylhydantoin, DMF, rt, 75%; (iv) Me2NH, 1,5,7-triazabicyclo[4.4.0]dec-5-ene, THF,
100 °C, microwave, 79%; (v) (3-thienyl)B(OH)2, Pd(PPh3)4, Na2CO3 aq, DME, 135 °C,
microwave, 47%.
Scheme 1. Reagents and conditions: (i) ArB(OH)2, Pd(PPh3)4, Na2CO3 aq, DME,
135 °C, microwave, 36–68%; (ii) (3-thienyl)B(OH)2, Pd(PPh3)4, Na2CO3 aq, DME,
135 °C, microwave, 31–64%; (iii) NaOH aq, THF, rt 69%.

710
S. Hilton et al. / Bioorg. Med. Chem. 18 (2010) 707–718
OEt
O
Br
CO2Et
NH2
Ar
i
N
N
NH2
57
58
59
: Ar = 3-thienyl
: Ar =
S
O
O
ii, iii
Ar
R
HN
O
N
NH2
60
: Ar = 3-thienyl, R = CONH2
61: Ar = 3-thienyl, R = NMe2
62
Scheme 3. Reagents and conditions: (i) 1,3-dibromo-5,5-dimethylhydantoin,
CH2Cl2, À78 °C, 51–54%; (ii) (4-carbamoyl-phenyl)B(OH)2, Pd(PPh3)4, Na2CO3 aq,
DME, 135 °C, microwave, 33–39%; (iii) (3-thienyl)B(OH)2, Pd(PPh3)4, Na2CO3 aq,
DME, 135 °C, microwave, 37–38%.
: Ar = 3-thienyl, R = NH2
63: Ar =
R = NMe2
S
O
O
S
64
: Ar =
R = NH2
O
O
bromo substituent in 28 and 29, this position is presumably ren-
dered more reactive to palladium (0) insertion by steric repulsion
from the adjacent substituent weakening the C3–Br bond.
A wide range of 3-(4-benzamido)-2-aminopyridines (36–55)
were prepared by Suzuki cross coupling of 4-carbo-
xamidophenylboronic acid to 3-bromo-2-aminopyridine 22
(Scheme 4). Bromination at C-5 of the pyridine 34 using 1,3-dibro-
mo-5,5-dimethylhydantoin at room temperature was followed by
the introduction of the second aryl substituent through palla-
dium-catalysed cross couplings. These reactions were conveniently
carried out in parallel using a microwave reactor and automated
MPLC purification. In general, higher yields were obtained for the
coupling of boronic acids compared to arylstannanes. The analogue
bearing the amine and carboxamidophenyl groups in a 1,4 rela-
tionship (56, Table 2) could be prepared by applying the same reac-
tion sequence starting from 5-bromo-2-aminopyridine, or by the
route shown in Scheme 1 for compound 4 but reversing the order
of coupling of the boronic acid reagents to 16.
Ester and amide replacements for the 3-(4-carboxamidophenyl)
substituent were prepared by Suzuki couplings to ethyl 5-bromo-
2-aminopyridine-3-carboxylate 57 (Scheme 5). Conversion of the
ethyl esters 58 and 62 to the amides was again achieved using di-
rect amination in the presence of 1,5,7-triazabicyclo[4.4.0]dec-5-
ene. The primary amines 62 and 64 were introduced using Boc-
protected ethylenediamine, and the protecting group was removed
by treatment with HCl in methanol.
Scheme 5. Reagents and conditions: (i) ArB(OH)2, Pd(PPh3)4, Na2CO3 aq, DME,
135 °C, microwave, 40–59%; (ii) RCH2CH2NH2, 1,5,7-triazabicyclo[4.4.0]dec-5-ene,
THF, 100 °C, microwave, 20–71%; (iii) 62 and 64 only, 4 M HCl, MeOH, rt, 52%.
4. Results and discussion
Attempted co-crystallisation of the initial HTS hits with CHK2 to
elucidate the interactions with the protein was not successful. This
was attributed to the poor aqueous solubility of the compounds
relative to their weak affinity for the enzyme. However based on
literature precedent,20,25,46–49 two plausible binding modes for
the 3,5-diaryl-2-aminopyridines were considered (Fig. 4).
The role of the 2-aminopyridine group as a bidentate hinge-
binding substituent has been shown by crystallography for sev-
eral kinase inhibitors, although the orientation of the bifunc-
tional 2-aminopyridine relative to the hinge region of the
kinase varies, particularly when the 2-aminopyridine bears only
one other substituent.46–49
A general preference for placing
unsubstituted amino groups facing into the interior of the ATP-
binding site (towards the gatekeeper) has been noted across a
wide selection of hinge-binding scaffolds.50 Thus a likely binding
mode for the 3,5-diaryl-2-aminopyridines was envisaged in
Scheme 4. Reagents and conditions: (i) (4-carbamoylphenyl)B(OH)2, Pd(PPh3)4,
Na2CO3 aq, DME, 135 °C, microwave, 31%; (ii) 1,3-dibromo-5,5-dimethylhydantoin,
DMF, rt, 61%; (iii) ArB(OH)2, Pd(PPh3)4, Na2CO3 aq, DME, 135 °C, microwave, 4–62%
or ArSnBu3, Pd(PPh3)4, DMF, 130 °C, microwave, 4–16%.
Figure 4. Possible binding modes considered for the 2-aminopyridines.

S. Hilton et al. / Bioorg. Med. Chem. 18 (2010) 707–718
711
Table 3
which the 2-aminopyridine bound to the hinge with the amino
group oriented towards the gatekeeper residue. As a result, the
4-benzamido substituent would be directed towards the con-
served lysine and DFG aspartate in CHK2 (Fig. 4A). However,
an alternative binding mode has been proposed for the potent
2-arylbenzimidazole-5-carboxamides 1, in which the carboxam-
ide could bind to the hinge region of CHK2.20,25 It was consid-
ered that if the 3,5-diaryl-2-aminopyridines 4–8 adopted an
equivalent orientation, the 4-carboxamidophenyl functionality
could serve as a hinge-binding motif while the 2-aminopyridine
group would be directed into the ribose pocket (Fig. 4B). Simple
overlays of the 2-aminopyridines with the structure of ADP
bound to CHK28 indicated that the ligands might be accommo-
dated in either of these orientations.
To investigate the possible roles of the carboxamide and 2-
aminopyridine groups, analogues of the potent hit 6 were pre-
pared which varied the hydrogen bonding potential of these
functionalities (Table 2). Mono- or dimethylation of the 2-amino
group to give 32 and 33, respectively, led to only weakly active
compounds, while removal of the amine (20) gave an eightfold
reduction in potency. Translocation of the amine to the 6-posi-
tion of the pyridine also led to a significant reduction in activity
(56). In contrast, variation of the 4-carboxamide was better tol-
erated. Although translocation of the primary amide to the 3-po-
sition of the aryl ring (13, Fig. 3) abolished activity, the
secondary and tertiary amides 21 and 25 displayed some CHK2
inhibition. Replacement of the amides by the weaker hydrogen
bond accepting ester 18 or the geometrically distinct nitrile 17
was not possible, although the acid 19 retained some activity.
Taken together, these results suggested a specific requirement
for bidentate hydrogen bonding compatible with the 2-amino-
pyridine interacting with the hinge region of the kinase, with
an additional requirement for an appropriate hydrogen bond
acceptor group at the 4-position of the aryl ring (Fig. 4A).
With apparently limited scope for variation of the 2-amino and
3-(4-carboxamidophenyl) substituents of the pyridine, we pre-
pared a larger number of analogues which explored changes to
the 5-substituent (Table 3). Based on the model in Figure 4A, we
expected that more polar heteroaromatic rings or substituents
might be tolerated at this position, since this would be anticipated
to be directed towards the solvent exposed surface at the opening
to the ATP-binding pocket. Replacement of the thiophen-3-yl
group by thiophen-2-yl (36) and various thiazolyl (37–39) or pyr-
idyl (40–42) groups was possible, with the five-membered hetero-
aromatics generally favoured. Substitution of the prototype phenyl
analogue 5 (Table 1) identified the 3-methoxyphenyl derivative 43
as more active, and a longer methoxyethylether solubilising chain
In vitro activity of 5-aryl and 5-heteroaryl substituted 2-amino-3-(4-
carboxamidophenyl)pyridines
a
b
No.
CHK2 IC50
(lM)
CHK1 IC50 (lM)
4
36
37
38
39
40
41
42
43
44
45
46
47
48
3-Thienyl
2-Thienyl
2-Thiazolyl
4-Thiazolyl
5-Thiazolyl
2-Pyridyl
3-Pyridyl
4-Pyridyl
3-MeO-Ph
4-MeO-Ph
3-(MeOCH2CH2O)-Ph
4-(MeOCH2CH2O)-Ph
3,4-(MeO)2-Ph
3-(MeO)-4-(HO)-Ph
0.61 ( 0.27)c
0.35
0.99
1.1
3.0
4.0
d
—
—
2.0
—
—
—
—
—
3.5
0.68
2.2
2.2
1.6
0.60
1.4
0.42
1.1
0.27
0.31
2.3
1.1
49
50
51
52
1.35
0.83
—
8.9
0.21 (0.20, 0.22)e
8.5 (5.9, 11.0)e
2.2
—
53
0.092 ( 0.018)f
5.2 (4.5, 5.9)e
54
55
>100
>10
—
—
a
See footnote to Table 1.
Single determination in DELFIA assay format. Standard inhibitor staurosporine
b
gave mean ( SEM) IC50 = 2.1 ( 0.41) nM, n = 26 determinations.
c
Mean ( SEM), n = 5 determinations.
Not determined.
Mean of n = 2, individual determinations in parentheses.
Mean ( SEM), n = 3 determinations.
d
e
f
Table 2
(45) was also tolerated at this position. The 3,4-dimethoxyphenyl
analogue 47 and the related 3-methoxy-4-phenol 48 gave more ac-
tive compounds.
Effect of variation of the hydrogen-bonding capacity of 6 on CHK2 inhibition
Elaboration of the dialkoxy substituent patterns to fused dioxa-
cycles (49–52) identified the 1,3-benzodioxole 51 as a potent
inhibitor, while a 10-fold drop in activity was observed for the
ring-expanded 1,2-dihydro-1,4-benzodioxin analogue 52. How-
ever, when the phenyl ring of 52 was replaced by a 2-thiophene,
the most potent compound in this compound set was identified
(53, CHK2 IC50 = 92 nM, L.E. = 0.38 kcal molÀ1 heavy atomÀ1),
although with only marginally improved activity to the hit 4. Fu-
sion of an aromatic ring to the active thiophenes 4 or 36 to give
the benzothiophenes 54 and 55 led to a complete loss of CHK2
inhibitory activity.
a
No.
R1
R2
R3
CHK2 IC50
(
lM)
17
18
19
20
21
25
32
33
56
H
H
H
H
H
H
H
H
NH2
NH2
NH2
H
NH2
NH2
NHMe
NMe2
H
CN
CO2Me
CO2H
CONH2
CONHMe
CONMe2
CONH2
CONH2
CONH2
30%@100b
27%@100
9.8
5.2
6.7
6.1
85
16%@100
22
NH2
The addition of the potential solubilising groups led to the suc-
cessful co-crystallisation of a number of the inhibitors with CHK2
(Fig. 5). The structure of 48 (PDB code 2WCT) bound to CHK2
a
See footnote to Table 1.
% Inhibition at maximum concentration tested.
b

712
S. Hilton et al. / Bioorg. Med. Chem. 18 (2010) 707–718
Figure 5. Crystal structures of 48 (A; PDB code 2WCT), 51 (B; PDB code 2WTD), 53 (C; PDB code 2WTI) and 63 (D; PDB code 2WTJ) bound to CHK2. Key interacting residues
are shown for each structure, with CHK2 carbon atoms coloured green (48), orange (51), cyan (53) or blue (63), nitrogen in blue, oxygen in red, and sulphur in yellow. Water
molecules involved in hydrogen-bonding networks with the compound are shown as dark blue spheres. Residues comprising the DFG motif are highlighted in magenta. Omit
map (brown mesh) contoured at 1.5
r (48), 2.8 r (51), 4 r (53), 2.5 r (63).
was solved at 3.0 Å resolution and clearly showed a binding mode
equivalent to that proposed in Figure 4A and consistent with the
structure–activity relationships discussed above. The inhibitor
occupied the ATP-binding site of CHK2 and was sandwiched be-
tween the hydrophobic side chains of Leu309 and Leu354
(Fig. 5A). The 2-aminopyridine was hydrogen bonded to the back-
bone carbonyl of Leu303 and the backbone amide NH of Met304.
The ligand carboxamide substituent accepted a hydrogen bond
from Lys249, which was also involved in hydrogen bonding to
Glu273 in the
aC-helix, indicative of an ‘active’ conformation for
the kinase domain. The crystal structures of 51 (2.75 Å resolution;
PDB code 2WTD) and 53 (2.5 Å resolution; PDB code 2WTI) showed

S. Hilton et al. / Bioorg. Med. Chem. 18 (2010) 707–718
713
Table 4
Replacement of the 3-(4-carboxamidophenyl) group by 3-carboxy and 3-amido substituents
R1
R2
CHK2 IC50
(
lM)
CHK1 IC50 (lM)
a
a
No.
58
60
61
62
3-Thienyl
3-Thienyl
3-Thienyl
3-Thienyl
CO2Et
17
0.41
3.6
—
3.4
—
CONH(CH2)2CONH2
CONH(CH2)2NMe2
CONH(CH2)2NH2
0.039b (0.051, 0.027)
0.7
0.78
63
64
CONH(CH2)2NMe2
CONH(CH2)2NH2
2.5
2.5
0.028b (0.028, 0.0284)
a
See footnote to Table 1.
Mean of n = 2, individual determinations in parentheses.
b
identical binding modes and key interactions to the protein (Fig. 5B
and C). An additional hydrogen bond was observed between the
carboxamide NH2 group and the side chain of Asp368 in the DFG
motif for the most potent compound 53.
pared bearing terminal hydrogen bonding functionality in an acy-
clic 3-substituent to replace the 3-(4-carboxamidophenyl) group
(Table 4). Intramolecular hydrogen bonding between the 2-amino
and 3-amido substituents was anticipated to hold the 3-substitu-
ent in a favourable position to interact with Lys249 or Asp368.
Some 3-carboxamido-2-aminopyridines have been described in
patents as kinase inhibitor scaffolds.51,52
While the intermediate ester 58 had only weak activity, the
addition of a terminal amide 60 gave CHK2 inhibition comparable
to the original 4-carboxamidophenyl HTS hit 4. Substitution of the
amide by the dimethylamine 61 reduced activity, but replacement
of the amide by a primary amine gave a potent CHK2 inhibitor (62;
L.E. = 0.55 kcal molÀ1 heavy atomÀ1). When the 3-thienyl group
was replaced by the 2,3-dihydrothieno[3,4-b]-1,4-dioxin-5-yl sub-
stituent the dimethylamine 63 showed better CHK2 inhibition in
this case. Affinity for the kinase was improved further for the pri-
mary amine 64 to give the most potent inhibitor identified in this
study (64; L.E. = 0.46 kcal molÀ1 heavy atomÀ1).
The dioxole and dioxin rings of 51 and 53 approached the sur-
face defined by Leu303 and Met304 and, although partially ex-
posed to solvent, provide a better fit to this region of the protein
than an unsubstituted five- or six-membered ring. The better inhi-
bition by the 1,3-benzodioxole 51 and the 2,3-dihydrothieno[3,4-
b]-1,4-dioxin 53 compared to the 1,2-dihydro-1,4-benzodioxin
analogue 52 can be rationalised by a preference for a coplanar ori-
entation of the 5-substituent and the central pyridine ring. This is
possibly energetically less favourable for the [6,6] bicyclic group of
52 due to a closer approach of atoms in the dioxacylic ring to the
H-6 proton of the pyridine. The failure of the benzothiophenes
54 and 55 to show significant inhibition is perhaps surprising,
but may reflect a mismatch between these large hydrophobic
groups and their solvent exposed position.
With the exception of the C-5 substituent, the 2-aminopyridines
are close mimics of ADP bound to CHK2. The compounds can also be
compared to the recently determined structure of the symmetrical
bis-guanylhydrazone 3 (NSC 109555; PDB code 2WOJ) bound to
CHK2.31 Here, interaction to the hinge region of the kinase was
through contacts from the urea carbonyl via a single water molecule
to the carbonyl of Glu302 and amide of Met304. In the present com-
pounds thisnetwork of hydrogenbondsis replacedby direct interac-
tionsof thebifunctional 2-aminopyridines. Interestingly, thesolvent
exposed methyl substituent of 3 occupies a similar space to the car-
bon atoms of the dioxole or dioxin rings of 51 and 53, suggesting a
productive hydrophobic association with the protein in this region.
The backbone of CHK2 around the ATP-binding site is similar when
the bis-guanylhydrazone or 2-aminopyridines are bound, and the
greatest differences in protein conformation are seen in the area
around the carboxamide group of the 2-aminopyridines. For exam-
ple, the carboxamide 53 interacts with both Lys249 and Asp368,
whereas the larger guanylhydrazone functionality of 3 displaces
and fills the space occupied by Lys249 in CHK2-ADP and interacts
The crystal structure of 63 (PDB code 2WTJ) confirmed that the
2-aminopyridine remained anchored to the hinge region (Fig. 5D).
The orientation of the 3-amido group was consistent with an intra-
molecular hydrogen bond from the 2-amino NH to the amide C@O.
In addition, the carbonyl of the amide participated in a water med-
iated hydrogen bond to Asp368, which was rotated from its posi-
tion in the previously determined structures. The Asp368
carboxylate functionality also interacted with the terminal
dimethylamine of the ligand, expected to be protonated at physio-
logical pH (calcd pKa = 8.96). The methyl groups did not appear to
interact productively with the protein, providing a rationale for the
improved affinity of the unsubstituted amines 62 and 64. A water
molecule on the opposite side of the 3-amide group mediated an
interaction to Glu308 not possible with the original 3-(4-carbox-
amidophenyl) substituted scaffold. This network of new interac-
tions within the ATP pocket appeared to compensate for the
reduction in rigidity of the 3-substituent, such that good affinity
was achieved with a more polar and more flexible scaffold (63,
L.E. = 0.34 kcal molÀ1 heavy atomÀ1).
with Glu273 from the
a
C-helix. The glycine-rich loop, which coordi-
Inhibition of the checkpoint kinase inhibitor CHK1 was investi-
gated for selected 3,5-diaryl-2-aminopyridines with submicromo-
lar potency for CHK2 inhibition (Tables 3 and 4). Where dual
inhibitors of CHK1 and CHK2 have been examined in cellular as-
says, the effects of CHK1 inhibition have often been observed to
dominate.1 Selectivity for CHK2 was generally between 3 and 11-
fold for the hit 4 and direct analogues, with the 3-carboxamido-
2-aminopyridines 60, 62 and 63 showing a similar pattern of inhi-
nates the phosphate groups of ATP, is disordered in each of our struc-
tures, as in those reported for 3 and debromohymenialdisine. The
conformational flexibility of this region, and the lack of direct inter-
action by each of the bound inhibitor molecules, is the most likely
explanation for this phenomenon.
A small number of 5-(3-thienyl)- and 5-(2,3-dihydrothieno[3,4-
b]-1,4-dioxin-5-yl)-3-carboxamido-2-aminopyridines were pre-

714
S. Hilton et al. / Bioorg. Med. Chem. 18 (2010) 707–718
Figure 6. Selectivity of 51, 53, 62 and 64 for inhibition of a panel of 24 kinases at 1
l
M concentration of test compound. Red = >70% inhibition, Orange = 70–40% inhibition,
Green = <40% inhibition.
bition. More robust selectivity was seen for the benzodioxole 51
(42-fold) and 2,3-dihydrothieno[3,4-b]-1,4-dioxin 53 (56-fold).
The combination of the 2,3-dihydrothieno[3,4-b]-1,4-dioxin 5-sub-
stituent with the (2-aminoethyl)amide 3-substituent in 64 gave
the highest selectivity (ca. 89-fold).
The inhibition of a panel of 24 kinases was determined for com-
pounds 51, 53, 62 and 64 using a microfluidic substrate phosphor-
ylation assay53 (Fig. 6). All compounds showed generally good
selectivity for CHK2 compared to other kinases in the set, however
the 3-carboxamido-2-aminopyridines 62 and 64 were notably
more selective than the 3-(4-carboxamidophenyl) analogues 51
and 53. This was reflected in the higher Gini coefficients for 62
and 64 (0.75 and 0.77, respectively) compared to 51 and 53 (0.42
and 0.46, respectively). The Gini coefficient assesses the cumula-
tive inhibition measured for the compounds across all the kinases
in the panel, with values close to 1 indicating specific inhibition of
a single kinase.54
5. Conclusion
5-(Hetero)aryl-3-(4-carboxamidophenyl)-2-aminopyridine
inhibitors of CHK2 were identified from high throughput screening
of a kinase-focussed compound library. Structure–activity relation-
ships were established by rapid exploration of analogues of the hits
through straightforward chemistry. X-ray crystallography of sev-
eral analogues bound to CHK2 confirmed a predicted ATP-compet-
itive binding mode for the compounds, consistent with the
observed SAR. Variation of the 3-(hetero)aryl substituent identified
Compounds 51, 53 and 64 were investigated in cellular assays.
HT29 cells were treated with the topoisomerase II inhibitor etopo-
side to activate the DNA damage repair signalling pathway. Activa-
tion of CHK2 was shown by the appearance of Thr68
phosphorylation and a bandshift of the CHK2 protein on the elec-
trophoresis gel. Inhibition of CHK2 was measured by assessing
the ability of the compounds to inhibit the DNA-damage-induced
bandshift and the specific CHK2 autophosphorylation on S516
(Fig. 7). Both 51 and 53 inhibited CHK2 activity in cells, with half
maximal effects between 10–20 lM and 5–10 lM, respectively.
The difference between the potency of the compounds as mea-
sured in the in vitro biochemical assay and the potency for inhibi-
tion of CHK2-specific biomarkers in cells is consistent with the
increase in [ATP] between the biochemical assay and cells, taking
into account the Km,ATP of the CHK2 enzyme.20,55 Compound 64
also showed good cellular inhibition of CHK2 activity despite the
increased polarity and hydrogen bond donor functionality in the
scaffold, with half maximal effects at approximately 5
pounds 51, 53 and 64 also showed weak antiproliferative activity
in HT29 cells (GI50 = 27 M, 26 M and 28 M, respectively), how-
lM. Com-
l
l
l
ever the single agent cytotoxicity of the compounds may include
non-CHK2 related effects. Notwithstanding this caveat, these com-
pounds may prove useful for further investigating the potential for
CHK2 inhibition to synergize with the antiproliferative effects of
DNA-damaging agents in cancer cells.
Figure 7. Western blot showing inhibition of CHK2 in HT29 cells by compounds 51
(Panel A), 53 (Panel B) and 64 (Panel C) following activation of the DNA damage
response pathway by treatment with etoposide (50 lM).

S. Hilton et al. / Bioorg. Med. Chem. 18 (2010) 707–718
715
bicyclic dioxins and dioxanes which gave gains in affinity for CHK2,
and improved the selectivity for CHK2 versus CHK1. The 3-(4-car-
boxamidophenyl) substituent could be successfully replaced by an
(6.96 mg, 6.02 lmol) in DME (3 mL) was heated at 135 °C in a
microwave reactor for 30 min. The reaction mixture was parti-
tioned between water (30 mL) and EtOAc (3 Â 30 mL). The organic
extracts were dried, filtered and concentrated. MPLC, eluting with
hexane/EtOAc 3:1, gave 18 (0.022 g, 0.071 mmol, 59%) as a light
yellow powder; mp 152–154 °C; 1H NMR (500 MHz, CDCl3) d
8.38 (d, J = 2.3, 1H), 8.17 (d, J = 8.5, 2H), 7.61–7.59 (m, 3H), 7.42
(dd, J = 3.0, 5.0, 1H), 7.38 (dd, J = 1.4, 3.0, 1H), 7.34 (dd, J = 1.4,
5.0, 1H), 4.65 (s, 2H), 3.97 (s, 3H); 13C NMR (126 MHz, CDCl3) d
166.6, 154.4, 144.8, 142.3, 138.7, 136.3, 130.5, 129.8, 128.8,
126.7, 125.7, 123.4, 120.9, 119.2, 52.3; LC–MS (6+ min) m/z 311
[M+H]+, tR = 3.38 min, purity >95%; HRMS found [M+H]+
311.0848, C17H15N2O2S requires 311.0849.
acyclic
x-aminoalkylamide which made additional polar interac-
tions within the binding site. The 3-(carboxamido) derivatives
were selective inhibitors of CHK2 over other kinases. Compounds
from this series showed activity in cell-based mechanistic assays
for inhibition of CHK2.
6. General experimental
Starting materials and solvents were purchased from commer-
cial suppliers and were used without further purification. Micro-
wave reactions were carried out in
a Biotage Initiator 60
7.3. 4-(2-Amino-5-(thiophen-3-yl)pyridin-3-yl)benzoic acid
(19)
microwave reactor. Organic solutions were dried over MgSO4 or
Na2SO4. Flash silica chromatography was performed using Merck
Silica Gel 60 (0.025–0.04 mm). Ion exchange chromatography
was performed using Isolute Flash SCX-II (acidic) or Flash NH2 (ba-
sic) resin cartridges. Automated MPLC was performed on a Biotage
SP1 Instrument using prepacked silica cartridges and UV-triggered
fraction collection (254 nm). 1H NMR spectra were recorded on a
Bruker AMX500 instrument using an internal deuterium lock. 13C
NMR spectra were recorded on a Bruker AMX500 instrument at
126 MHz using an internal deuterium lock. Chemical shifts (d)
are reported relative to TMS (d = 0) and/or referenced to the sol-
vent 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 electrospray ioni-
zation (+ve or Àve ion mode as indicated), and with HPLC per-
formed using Supelco DISCOVERY C18, 50 mm  4.6 mm or
30 mm  4.6 mm i.d. columns, at a temperature of 22 °C with gra-
dient elution of 10–90% MeOH/0.1% aqueous formic acid at a flow
rate of 1 mL/min (run time of 4 min or 6 min as indicated). Com-
pounds were detected at 254 nm using a Waters 2487 dual k absor-
bance detector. High-resolution mass spectra were measured on an
1 M NaOH aq (1.77 mL, 1.77 mmol) was added to a solution of
18 (0.055 g, 0.177 mmol) in THF (5 mL) at rt and the mixture was
stirred for 24 h. THF was removed by evaporation and water
(30 mL) was added. The solution was washed with ethyl acetate
(2 Â 30 mL). The aqueous phase was acidified with citric acid aq
(10%) and extracted with EtOAc (3 Â 20 mL). The organic extracts
were dried, filtered and concentrated. Ion exchange chromatogra-
phy on acidic resin gave 19 (0.036 g, 0.121 mmol, 69%) as a light
brown solid; mp 286–288 °C; 1H NMR (500 MHz, DMSO) d 8.37
(d, J = 2.3, 1H), 8.00 (d, J = 8.1, 2H), 7.76 (dd, J = 1.1, 2.9, 1H), 7.70
(d, J = 2.3, 1H), 7.59 (dd, J = 2.9, 5.0, 1H), 7.56–7.54 (m, 3H), 5.72
(s, 2H); 13C NMR (126 MHz, DMSO) d 168.0, 155.4, 144.9, 140.5,
138.9, 135.2, 129.7, 128.2, 126.8, 125.6, 121.2, 119.9, 118.5
(1 Â quaternary C not observed); LC–MS (4 min) m/z 297 [M+H]+,
tR = 1.82 min, purity >95%; found [M+H]+ 297.0688, C16H13N2O2S
requires 297.0692.
7.4. 4-(2-Amino-5-(thiophen-3-yl)pyridin-3-yl)-N-
methylbenzamide (21)
Agilent 6210 ToF HPLC-MS with a Phenomenex Gemini 3 lm C18
(3 cm  4.6 mm i.d) column. Melting points were measured on a
A
mixture of 18 (0.070 g, 0.226 mmol), 1,5,7-triazabicy-
Leica Gallen III apparatus and are uncorrected.
clo[4.4.0]dec-5-ene (0.010 g, 0.072 mmol), 2 M MeNH2 in THF
(2 mL, 4.00 mmol) and THF (1 mL) was heated in a microwave
reactor at 100 °C for 60 min. Silica gel was added and solvent
was evaporated. MPLC, eluting with CH2Cl2/EtOAc 1:1, gave 21
(0.062 g, 0.20 mmol, 89%) as a colourless solid; mp 142–144 °C;
1H NMR (500 MHz, CDCl3) d 8.38 (d, J = 2.3, 1H), 7.89 (d, J = 8.3,
2H), 7.60–7.59 (m, 3H), 7.41 (dd, J = 3.0, 5.0, 1H), 7.37 (dd, J = 1.4,
3.0, 1H), 7.34 (dd, J = 1.4, 5.0, 1H), 6.20 (br s, 1H), 4.61 (s, 2H),
3.07 (d, J = 4.8, 3H); 13C NMR (126 MHz, CDCl3) d 167.6, 154.6,
145.5, 141.1, 139.0, 136.0, 134.2, 129.0, 127.7, 126.6, 125.7 123.4,
120.7, 119.0, 26.9; LC–MS (6 min) m/z 310 [M+H]+, tR = 2.63 min,
purity >95%; HRMS found [M+H]+ 310.1006, C17H16N3OS requires
310.1009.
7. Representative methods for the preparation of 2-amino-3,5-
disubstituted-pyridines
7.1. 4-(2-Amino-5-(thiophen-3-yl)pyridin-3-yl)benzamide (4)
A mixture of 35 (0.042 g, 0.144 mmol), thiophen-3-ylboronic
acid (0.020 g, 0.156 mmol), Na2CO3 (0.032 g, 0.30 mmol) and
Pd(PPh3)4 (0.017 g, 0.015 mmol) in 1,4-dioxane (1.5 mL) and water
(0.4 mL) was heated at 120 °C in a microwave reactor for 1 h. The
reaction mixture was partitioned between water (20 mL) and
EtOAc (20 mL). The organic layer was dried, filtered and concen-
trated. Preparative TLC, eluting with CH2Cl2/MeOH 9:1, gave 4
(0.013 g, 0.044 mmol, 31%) as a beige solid. mp 112–115 °C; 1H
NMR (500 MHz, DMSO) d 8.38 (s, 1H), 8.00–7.98 (m, 3H), 7.76 (d,
J = 2.0, 1H), 7.72 (d, J = 2.0, 1H), 7.61–7.59 (m, 3H), 7.55 (d, J = 5.0,
1H), 7.36 (s, 1H), 5.81 (br s, 2H); 13C NMR (126 MHz, DMSO) d
167.4, 155.2, 144.4, 140.7, 138.7, 135.6, 133.1, 128.5, 128.1,
126.8, 125.6, 121.2, 119.8, 118.7; LC–MS (6 min) m/z 296 [M+H]+,
tR = 2.43 min, purity >95%; HRMS found [M+H]+ 296.0860,
C16H14N3OS requires 296.0852.
7.5. Methyl 4-(2-amino-5-bromopyridin-3-yl)benzoate (23)
Method A: A mixture of 5-bromo-3-iodopyridin-2-amine 16
(0.113 g, 0.378 mmol), and 4-(methoxycarbonyl)phenylboronic
acid (0.077 g, 0.428 mmol), 2 M Na2CO3 aq (0.68 mL, 1.36 mL)
and Pd(PPh3)4 (0.022 g, 0.019 mmol) in EtOH (2 mL) and toluene
(4 mL) was heated at 135 °C in a microwave reactor for 25 min.
The reaction mixture was partitioned between water (30 mL) and
EtOAc (3 Â 30 mL). The organic extracts were dried, filtered and
concentrated. MPLC, eluting with hexane/EtOAc 3:1, gave 23
(0.058 g, 0.189 mmol, 50%) as a colourless powder. mp 157–
159 °C; 1H NMR (500 MHz, CDCl3) d 8.19–8.13 (m, 3H), 7.54 (d,
J = 8.3, 2H), 7.51 (d, J = 2.3, 1H), 4.63 (s, 2H), 3.97 (s, 3H); 13C
NMR (126 MHz, CDCl3) d 166.5, 154.3, 148.4, 141.4, 139.8, 130.5,
7.2. Methyl 4-(2-amino-5-(thiophen-3-yl)pyridin-3-yl)benzoate
(18)
A mixture of 23 (0.037 g, 0.120 mmol), thiophen-3-ylboronic
acid (0.017 g, 0.133 mmol), 2 M Na2CO3 aq (0.13 mL) and Pd(PPh3)4

716
S. Hilton et al. / Bioorg. Med. Chem. 18 (2010) 707–718
130.1, 128.6, 122.3, 108.6, 52.3; LC–MS (6 min) m/z 308, 309
[M+H]+, tR = 4.62 min, purity = 94%; HRMS found [M+H]+
307.0078, C13H12BrN2O2 requires 307.0077.
concentrated. MPLC, eluting with an EtOAc/hexane gradient, gave
28 (2.63 g, 9.89 mmol, 51%) as a colourless solid; mp 47–49 °C;
1H NMR (500 MHz, CDCl3) d 8.12 (d, J = 2.1, 1H), 7.70 (d, J = 2.1,
1H), 5.04 (br s, 1H), 3.01 (d, J = 4.9, 3H); 13C NMR (126 MHz, CDCl3)
d 154.0, 147.3, 140.9, 105.6, 105.9, 28.8; LC–MS (4 min) m/z 265,
267, 269 [M+H]+, tR = 2.54 min, purity >95%; HRMS found [M+H]+
264.8975, C6H7Br2N2 requires 264.8971.
Also isolated, 5-bromo-N-methylpyridin-2-amine: colourless
solid (1.0 g, 23%), mp 72–74 °C; 1H NMR (500 MHz, CDCl3) d 8.12
(d, J = 2.5, 1H), 7.49 (dd, J = 2.5, 8.8, 1H), 6.30 (d, J = 8.8, 1H), 4.66
(br s, 1H), 2.90 (d, J = 5.1, 3H); 13C NMR (126 MHz, CDCl3) d
158.1, 148.7, 139.7, 107.6, 106.8, 29.1; LC–MS (4 min) m/z 187,
189 [M+H]+, tR = 0.78 min, purity >95%; HRMS found [M+H]+
186.9864, C6H8BrN2 requires 186.9865.
Method B: A mixture of 3-bromopyridin-2-amine 22 (1.61 g,
9.31 mmol), 4-(methoxycarbonyl)phenylboronic acid (1.84 g,
10.2 mmol), 2 M Na2CO3 aq (7.0 mL, 14 mmol) and Pd(PPh3)4
(0.538 g, 0.465 mmol) in DME (8 mL) was heated at 135 °C in a
microwave reactor for 40 min. The reaction mixture was partitioned
between water (50 mL) and EtOAc (3 Â 50 mL). The organic extracts
were dried, filtered and concentrated. MPLC, eluting with an EtOAc/
hexane gradient, gave methyl 4-(2-aminopyridin-3-yl)benzoate
(1.45 g, 6.35 mmol, 68%) as a colourless solid; mp 127–129 °C; 1H
NMR (500 MHz, CDCl3) d 8.11 (d, J = 8.3, 2H), 8.08 (dd, J = 1.7, 5.0,
1H), 7.54 (d, J = 8.3, 2H), 7.37 (dd, J = 1.7, 7.4, 1H), 6.75 (dd, J = 5.0,
7.4, 1H), 4.67 (s, 2H), 3.94 (s, 3H); 13C NMR (126 MHz, CDCl3) d
166.6, 155.6, 148.0, 142.9, 137.8, 130.3, 129.5, 128.7, 120.7, 114.5,
52.2; LC–MS (4 min) m/z 229 [M+H]+, tR = 1.34 min, purity >95%;
HRMS found [M+H]+ 229.0977, C13H13N2O2 requires 229.0972. 1,3-
Dibromo-5,5-dimethylhydantoin (0.197 g, 0.689 mmol) was added
to a solution of methyl 4-(2-aminopyridin-3-yl)benzoate (0.262 g,
1.15 mmol) in CH2Cl2 (10 mL) at À78 °C and the resulting mixture
was stirred for 2 h. The mixture was concentrated and purified by
MPLC, eluting with hexane/EtOAc 3:1, to give 23 (0.264 g,
0.860 mmol, 75%) as a pale yellow solid.
7.8. 4-(5-Bromo-2-(methylamino)pyridin-3-yl)benzamide (30)
A mixture of 28 (0.309 g, 1.16 mmol), 4-carbamoylphenylbo-
ronic acid (0.211 g, 1.28 mmol), Na2CO3 aq (2 M, 1.16 mL) and
Pd(PPh3)4 (0.067 g, 0.058 mmol) in DME (3 mL) was heated at
135 °C in a microwave reactor for 30 min. The reaction mixture
was partitioned between water (30 mL) and EtOAc (3 Â 30 mL).
The organic extracts were dried, filtered and concentrated. MPLC,
eluting with hexane/EtOAc 5:1, gave 4-(5-bromo-2-(methyl-
amino)pyridin-3-yl)benzamide (0.140 g, 0.457 mmol, 39%) as a
colourless solid; mp 85–87 °C; 1H NMR (500 MHz, CDCl3) d 8.20
(d, J = 2.4, 1H), 7.93 (d, J = 8.3, 2H), 7.47 (d, J = 8.3, 3H), 7.36 (d,
J = 2.4, 1H), 6.15 (s, 2H), 4.55 (s, 1H), 2.92 (d, J = 4.9, 3H); 13C
NMR (126 MHz, CDCl3) d 168.7, 154.7, 148.1, 140.4, 138.8, 133.2,
129.1, 128.4, 122.8, 106.8, 28.7; LC–MS (6 min) m/z 306 [M+H]+,
tR = 3.25 min, purity >95%; HRMS found [M+H]+ 306.0240,
C13H13BrN3O requires 306.0237.
7.6. 4-(2-Amino-5-(thiophen-3-yl)pyridin-3-yl)-N,N-
dimethylbenzamide (25)
A
mixture of 23 (0.199 g, 0.648 mmol), 1,5,7-triazabicy-
clo[4.4.0]dec-5-ene (0.042 g, 0.302 mmol), 2 M Me2NH in THF
(2 mL, 37.7 mmol) and THF (1 mL) was heated in a microwave
reactor at 100 °C for 1 h. Silica was added and solvent was evapo-
rated. MPLC, eluting with an EtOAc/hexane gradient, gave 4-(2-
amino-5-bromopyridin-3-yl)-N,N-dimethylbenzamide
24
7.9. 4-(2-(Methylamino)-5-(thiophen-3-yl)pyridin-3-
yl)benzamide (32)
(0.163 g, 0.509 mmol, 79%) as a colourless solid; mp 219–221 °C;
1H NMR (500 MHz, CDCl3) d 8.13 (d, J = 2.4, 1H), 7.55–7.48 (m,
5H), 4.62 (br s, 2H), 3.15 (br s, 3H), 3.04 (br s, 3H); 13C NMR
(126 MHz, CDCl3) d 170.9, 154.5, 148.1, 139.8, 138.0, 136.4,
128.6, 128.0, 122.6, 108.5, 39.6, 35.4; LC–MS (6 min) m/z 319
[M+H]+, tR = 3.41 min, purity = 90%; HRMS found [M+H]+
A mixture of 30 (0.172 g, 0.562 mmol), 3-thiopheneboronic acid
(0.072 g, 0.562 mmol), Na2CO3 aq (2 M, (0.562) and Pd(PPh3)4)
(0.032 g, 0.028 mmol) in DME (3 mL) was heated in a microwave
reactor at 135 °C for 35 min. The mixture was partitioned between
water (30 mL) and EtOAc (3 Â 30 mL). The organic extracts were
dried, filtered and concentrated. MPLC, eluting with hexane/EtOAc
1:1, gave 32 (0.065 g, 0.210 mmol, 37%) as a colourless solid; mp
128–130 °C; 1H NMR (500 MHz, CD3OD) d 8.35 (d, J = 2.3, 1H),
8.01 (d, J = 8.2, 2H), 7.65 (d, J = 2.3, 1H), 7.60 (d, J = 8.2, 2H), 7.53
(dd, J = 2.9, 1.2, 1H), 7.49 (dd, J = 2.9, 5, 1H), 7.42 (dd, J = 1.2, 5,
1H), 2.92 (s, 3H); 13C NMR (126 MHz, CDCl3) d 169.0, 155.1,
145.3, 141.6, 139.3, 135.1, 133.0, 132.0, 129.2, 128.4, 126.4,
125.6, 121.2, 118.4, 28.8; LC–MS (6 min) m/z 310 [M+H]+,
tR = 2.39 min, purity >95%; HRMS found [M+H]+ 310.1003,
C17H16N3OS requires 310.1009.
320.0397, C14H15BrN3O requires 320.0393.
A mixture of 24
(0.093 g, 0.290 mmol), thiophen-3-ylboronic acid (0.041 g,
0.320 mmol), 2 M Na2CO3 aq (0.68 mL, 1.36 mmol) and Pd(PPh3)4
(0.017 g, 0.015 mmol) in DME (3.0 mL) was heated at 135 °C in a
microwave reactor for 30 min. The reaction mixture was parti-
tioned between water (30 mL) and EtOAc (3 Â 30 mL). The com-
bined organic extracts were dried, filtered and concentrated.
MPLC, eluting with CH2Cl2/EtOAc 1:1, gave 25 (0.044 g,
0.136 mmol, 47%) as a colourless solid. mp 126–128 °C; 1H NMR
(500 MHz, CDCl3) d 8.34 (d, J = 2.0, 1H), 7.63–7.46 (m, 5H), 7.44–
7.30 (m, 3H), 4.73 (s, 2H), 3.14 (s, 3H), 3.05 (s, 3H); 13C NMR
(126 MHz, CDCl3) d 171.0, 154.8, 145.2, 139.2, 139.0, 136.0,
128.8, 128.0, 126.5, 125.7, 123.3, 120.9, 118.9, 39.6, 35.4 (1 Â qua-
7.10. 4-(2-Amino-5-bromopyridin-3-yl)benzamide (35)
ternary
C
not observed); LC–MS (6 min) m/z 324 [M+H]+,
tR = 2.70 min, purity >95%; HRMS found [M+H]+ 324.1165,
Method A: A mixture of 5-bromo-3-iodopyridin-2-amine 16
(0.16 g, 0.535 mmol), 4-carbamoylphenyl-boronic acid (0.097 g,
0.589 mmol), Na2CO3 aq (2 M, 0.68 mL) and Pd(PPh3)4 (0.031 g,
0.027 mmol) in DME (6.0 mL) was heated at 135 °C in a microwave
reactor for 20 min. The reaction mixture was partitioned between
water (30 mL) and EtOAc (3 Â 30 mL). The organic extracts were
dried, filtered and concentrated. MPLC, eluting with CH2Cl2/EtOAc
1:1, gave 35 (0.107 g, 0.366 mmol, 68%) as a light yellow powder.
mp 233–235 °C; 1H NMR (500 MHz, DMSO) d 8.04 (d, J = 2.4, 1H),
8.02 (br s, 1H), 7.97 (d, J = 8.0, 2H), 7.5–7.54 (m, 3H), 7.37 (br s,
1H), 5.90 (s, 2H); 13C (126 MHz, DMSO) d 168.2, 156.4, 148.4,
140.4, 140.0, 134.3, 129.2, 129.0, 122.6, 106.9; LC–MS (6 min) m/
C18H18N3OS requires 324.1165.
7.7. 3,5-Dibromo-N-methylpyridin-2-amine (28)
1,3-Dibromo-5,5-dimethylhydantoin (5.56 g, 19.5 mmol) was
added to a solution of 2-(methylamino)pyridine 26 (2.00 mL,
19.5 mmol) in CH2Cl2 (10 mL) at À78 °C and the mixture was stir-
red for 2 h. The mixture was filtered and concentrated. The residue
was mixed with CH2Cl2 (50 mL) and satd NaHCO3 aq (50 mL). Solid
sodium thiosulfate and water (50 mL) were added and the organic
phase was separated. The organic extract was dried, filtered and

S. Hilton et al. / Bioorg. Med. Chem. 18 (2010) 707–718
717
z 292 [M+H]+, tR = 3.00 min, purity >95%; HRMS found [M+H]+
292.0086, C12H11BrN3O requires 292.0080.
organic extracts were dried, filtered and concentrated. MPLC, elut-
ing with CH2Cl2/MeOH 9:1, gave 53 (0.077 g, 0.218 mmol, 58%) as a
light yellow solid; mp 125–127 °C; 1H NMR (500 MHz, DMSO) d
8.24 (d, J = 2.3, 1H), 7.98 (d, J = 8.3, 2H), 7.53–7.55 (m, 3H), 6.51
(s, 1H), 4.27–4.20 (m, 4H); 13C NMR (126 MHz, DMSO) d 167.3,
155.1, 144.4, 142.1, 140.6, 137.2, 134.6, 133.1, 128.3, 128.1,
119.4, 118.6, 113.4, 96.2, 64.6, 64.1; LC–MS (4 min) m/z 354
[M+H]+, tR = 2.07 min, purity >95%; found [M+H]+ 354.0900,
C18H16N3O3S requires 354.0907.
Method B: A mixture of 3-bromo-2-aminopyridine 22 (1.14 g,
6.59 mmol),
4-carbamoylphenyl-boronic
acid
(1.196 g,
7.25 mmol), Na2CO3 aq (7.25 mL, 14.5 mmol) and Pd(PPh3)4
(0.381 g, 0.329 mmol) in DME (8 mL) was heated at 135 °C in a
microwave reactor for 40 min. The reaction mixture was parti-
tioned between water (50 mL) and EtOAc (3 Â 50 mL). The organic
extracts were dried, filtered and concentrated. The residue was
washed with hot EtOAc to give 4-(2-aminopyridin-3-yl)benzamide
34 (0.44 g, 2.06 mmol, 31%) as a pale yellow solid; mp 219–221 °C;
1H NMR (500 MHz, DMSO) d 8.04–7.91 (m, 4H), 7.52 (d, J = 8.3, 2H),
7.35–7.37 (m, 2H), 6.67 (dd, J = 4.9, 7.3, 1H), 5.60 (s, 2H); 13C NMR
(126 MHz, DMSO) d 167.5, 156.3, 147.4, 141.1, 137.6, 133.0, 128.2,
128.1, 119.6, 113.1; LC–MS (4 min) m/z 214 [M+H]+, tR = 0.82 min,
purity >95%;; HRMS found [M+H]+ 214.0970, C12H12N3O requires
7.14. Ethyl 2-amino-5-(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-
yl)nicotinate (59)
A mixture of ethyl 2-amino-5-bromonicotinate 57 (0.276 g,
1.13 mmol), 5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-2,3-
dihydrothieno-[3,4-b][1,4]dioxine (0.362 g, 1.35 mmol), Pd(PPh3)4
(0.065 g, 0.056 mmol) and 2 M Na2CO3 aq (0.845 mL, 1.69 mmol)
in DME (4 mL) was heated at 135 °C in a microwave reactor for
40 min. Solvent was removed by evaporation and the residue
was purified by MPLC, eluting with hexane/EtOAc 3:1, to give 59
(0.202 g, 0.659 mmol, 59%) as a pale yellow solid; mp 165–
167 °C; 1H NMR (500 MHz, CDCl3) d 8.59 (d, J = 2.4, 1H), 8.39 (d,
J = 2.4, 1H), 6.57 (br s, 2H), 6.28 (s, 1H), 4.39 (q, J = 7.1, 2H), 4.33–
4.29 (m, 2H), 4.28–4.23 (m, 2H), 1.42 (t, J = 7.1, 3H); 13C NMR
(126 MHz, CDCl3) d 166.9, 157.8, 151.2, 142.2, 137.6, 137.3,
119.1, 113.8, 106.2, 96.7, 64.8, 64.5, 61.0, 14.3; LC–MS (4 min) m/
z 307 [M+H]+, tR = 2.59 min, purity >95%; HRMS found [M+H]+
307.0744, C14H15N2O4S requires 307.0747.
214.0975.
1,3-Dibromo-5,5-dimethylhydantoin
(0.100 g,
0.349 mmol) was added to a solution of 34 (0.149 g, 0.699 mmol)
in DMF (3 ml) at 20 °C and the resulting mixture was stirred for
24 h. The mixture was concentrated and purified by MPLC, eluting
with CH2Cl2/EtOAc 1:1, to give 35 (0.125 g, 0.428 mmol, 61%).
7.11. 4-(2-Amino-5-(thiophen-2-yl)pyridin-3-yl)benzamide
(36)
A mixture of 35 (0.081 g, 0.277 mmol), thiophen-2-ylboronic
acid (0.039 g, 0.305 mmol), Na2CO3 aq (2 M, 0.31 mL) and
Pd(PPh3)4 (0.016 g, 0.014 mmol) in DME (4 mL) was heated at
135 °C in a microwave reactor for 20 min. The reaction mixture
was partitioned between water (30 mL) and EtOAc (3 Â 30 mL).
The organic extracts were dried, filtered and concentrated. MPLC,
eluting with hexane/EtOAc 4:1, gave 36 (0.040 g, 0.135 mmol,
7.15. 2-Amino-5-(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)-N-
(2-(dimethylamino)ethyl)nicotinamide (63)
49%) as
a
light yellow powder; mp 222–224 °C; 1H NMR
A mixture of 59 (0.067 g, 0.22 mmol), 1,5,7-triazabicy-
(500 MHz, DMSO) d 8.29 (d, J = 2.2, 1H), 8.02 (s, 1H), 7.99 (d,
J = 8.3, 2H), 7.58–7.60 (m, 3H), 7.43 (d, J = 5.0, 1H), 7.39–7.37 (m,
2H), 7.09 (dd, J = 3.5, 5.0, 1H), 5.89 (s, 2H); 13C NMR (126 MHz,
DMSO) d 167.4, 155.9, 144.3, 140.8, 140.4, 134.7, 133.2, 128.4,
128.2, 128.1, 123.9, 122.2, 119.9, 119.7; LC–MS (4 min) m/z 296
[M+H]+, tR = 2.67 min, purity >95%; HRMS found [M+H]+
296.0857, C16H14N3OS requires 296.0852.
clo[4.4.0]dec-5-ene (0.012 g, 0.087 mmol) and N,N-dimethylethyl-
enediamine (0.096 mL, 0.875 mmol) in THF (4 mL) was heated at
100 °C in a microwave reactor for 60 min. Solvent was evaporated
and the residue was purified by MPLC, eluting with hexane/EtOAc
1:1, to give 63 (0.044 g, 0.126 mmol, 58% yield) as a colourless oil;
1H NMR (500 MHz, CDCl3) d 8.44 (d, J = 2.3, 1H), 7.94 (d, J = 2.3,
1H), 7.02 (br s, 1H), 6.44 (br s, 2H), 6.24 (s, 1H), 4.28–4.23 (m,
4H), 3.52–3.49 (m, 2H), 2.56 (t, J = 6.0, 2H), 2.30 (s, 6H); 13C NMR
(126 MHz, CDCl3) d 167.8, 157.1, 149.1, 142.2, 137.5, 133.3,
118.8, 113.9, 110.5, 96.6, 64.8, 64.5, 57.7, 45.1, 37.0; LC–MS
(4 min) m/z 349 [M+H]+, tR = 1.18 min, purity >95%; HRMS found
[M+H]+ 349.1321, C16H21N4O3S requires 349.1329.
7.12. 4-(2-Amino-5-(thiazol-2-yl)pyridin-3-yl)benzamide (37)
Pd(PPh3)4 (7.91 mg, 0.0685 mmol) was added to a solution of 2-
(tributylstannyl)thiazole (24 ll, 0.075 mmol) and 35 (20 mg,
0.068 mmol) in DMF (0.200 mL). The reaction mixture was heated
at 130 °C in a microwave reactor for 45 min. The crude mixture
was concentrated. MPLC, eluting with CH2Cl2/MeOH 95/5–90/10,
followed by preparative HPLC gave 37 (3.0 mg, 0.010 mmol, 15%)
as a white solid; mp 226–227 °C; 1H NMR (500 MHz, CD3OD) d
8.58 (d, J = 2.3, 1H), 8.04 (d, J = 8.4, 2H), 7.96 (d, J = 2.3, 1H), 7.83
(d, J = 3.3, 1H), 7.65 (d, J = 8.4, 2H), 7.54 (d, J = 3.3, 1H); 13C NMR
(126 MHz, CD3OD) d 171.8, 167.6, 159.0, 146.8, 144.1, 142.1,
137.1, 134.8, 130.0, 129.6, 122.4, 121.5, 119.5; LC–MS (4 min) m/
z 297 [M+H]+, tR = 1.47 min, purity >95%; HRMS found [M+H]+
297.0806, C15H13N4OS requires 297.0805.
Acknowledgments
We thank Dr. A. Mirza and Mr. M. Richards for assistance in the
characterisation of test compounds. This work was supported by
Cancer Research UK [CUK] Grant Numbers C309/A2187, C309/
A2874, C309/A8365 and C302/A8265 and Infrastructure Support
Grant C302/A7803, and by the Institute of Cancer Research (stu-
dentship to CEA). We acknowledge NHS funding to the NIHR Bio-
medical Research Centre.
Supplementary data
7.13. 4-(2-Amino-5-(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-
yl)pyridin-3-yl)benzamide (53)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.11.058.
A mixture of 5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-
2,3-dihydrothieno-[3,4-b][1,4]dioxine (0.151 g, 0.565 mmol), 35
(0.11 g, 0.77 mmol), Na2CO3 aq (0.377 mL, 0.753 mmol) and
Pd(PPh3)4 (0.022 g, 0.019 mmol) in DME (3 mL) was heated in a
microwave reactor at 135 °C for 40 min. The residue was parti-
tioned between water (30 mL) and ethyl acetate (3 Â 30 mL). The
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