Imidazo[1,2-a]pyridines. Part 2: SAR and optimisation of a potent and selective class of cyclin-dependent kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.02.015

Exploration of SAR and optimisation of the imidazo[1,2-a]pyridine CDK inhibitors has lead to the discovery of novel, potent and selective inhibitors of the cyclin-dependent kinase CDK2. Understanding of SAR has identified positions of substitution, which allow modification of physical properties and offer the potential for in vivo optimisation.

Full text of DOI:10.1016/j.bmcl.2004.02.015

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2245–2248
Imidazo[1,2-a]pyridines. Part 2: SAR and optimisation of a potent
and selective class of cyclin-dependent kinase inhibitors
Kate F. Byth, Janet D. Culshaw, Stephen Green, Sandra E. Oakes and Andrew P. Thomas*
AstraZeneca, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK
Received 11 November 2003; revised 27 January 2004; accepted 3 February 2004
Abstract—Exploration of SAR and optimisation of the imidazo[1,2-a]pyridine CDK inhibitors has lead to the discovery of novel,
potent and selective inhibitors of the cyclin-dependent kinase CDK2. Understanding of SAR has identified positions of substitution,
which allow modification of physical properties and offer the potential for in vivo optimisation.
Ó 2004 Elsevier Ltd. All rights reserved.
The normal progression through the cell cycle leading to
cell division is a remarkably ordered process regulated
by the sequential activation and deactivation of several
members of the cyclin-dependent kinase (CDK) family.1
For example, CDK2 plays an important role in several
stages of the cell cycle. The CDK2/cyclin E complex
contributes to pRb phosphorylation to enable the G1/S-
phase transition and activate the transcription factor
E2F.2 CDK2 then associates with cyclin A promoting
uninterrupted passage through the S-phase and appro-
priately timed deactivation of E2F.3 Recent studies
using a dominant negative CDK2 have shown that
CDK2 also plays an important role in the entry and
possibly progression in the G2/M-phase.4
number of groups have identified CDK inhibitors
from a variety of structure classes.6–17 In an earlier
communication we described the discovery of prototype
2-anilino-4-(3-imidazo[1,2-a]pyridyl)pyrimidine CDK
inhibitors.17 In this paper we describe the optimisation
and structure–activity relationships (SAR) of these im-
idazo[1,2-a]pyridines, as a potent and selective class of
CDK inhibitors.
The initial lead structures of this series17 are represented
by structure 1, where R2 ¼ Me.
N
H
N
R1
N
It is a feature of most cancer cells that they exhibit a
deregulation of CDK function and cell cycle control.
Virtually all cancers exhibit at least one alteration in
CDK function, through upregulation of cyclin effectors
such as cyclins D and E, the loss of negative regulators
such as p16 and p27 or genetic mutations to CDK
substrates.3;5 Evidence from in vitro studies suggests
that inhibition of CDK2 selectively kills tumour cells
with deregulated E2F-1 activity.6 CDK inhibitors are
therefore particularly attractive as potential therapeutic
agents7 and may offer new opportunities for selective
and tolerable therapy for human cancer.
N
R2
N
1
The important contribution of the aniline and pyrim-
idine groups to the binding and activity of these com-
pounds has been shown in our earlier work.17 Our initial
goals in this work were to develop chemistry that would
allow exploration of the role of the aniline substituent
(R1 on compound 1) and the substitution on imidazo-
pyridine ring; and to further improve potency. The ini-
tial synthetic route to compounds 1 is shown in Scheme
1: the 3-acetyl imidazo[1,2-a]pyridines A were prepared
by reaction of 2-amino pyridine with 3-chloro-2,4-pen-
tanedione for the case R2 is methyl or by acetylation of
the parent imidazo[1,2-a]pyridine for the case where R2
is hydrogen.18 The latter reaction gave varying degrees
of conversion and although it was possible to force this
As a result of these findings, there is much interest in the
development of small molecule CDK inhibitors and a
Keywords: CDK2 inhibitor.
*Corresponding author. Tel.: +44-(0)1625-515170; fax: +44-(0)1625-
513910; e-mail: andrew.p.thomas@astrazeneca.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.015

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K.F.Byth et al./ Bioorg.Med.Chem.Lett.14 (2004) 2245–2248
position of the imidazo[1,2-a]pyridine leads to a signif-
N
icant increase in potency (compare compounds 1a,c,e
with 1f,g,h). Summarising a broad range of aniline
substituents that were investigated, substitution at the 2-
position of the aniline group was found to be detri-
mental to CDK2 activity (compare 1a with 1b) while
potency was relatively insensitive to substitution of rel-
atively small and nonpolar substituents at the 3- and
4-positions (compare 1a with 1c and 1d). However, the
4-sulfamoyl group was found to significantly improve
activity, compounds 1e and 1h. Similar SAR has been
previously reported.21 The importance of the imi-
dazo[1,2-a]pyridine group to binding was also investi-
gated. Introduction of alternative heterocycles that did
not have an equivalent to the N-1 hydrogen bond
acceptor of the imidazo[1,2-a]pyridine were very much
less active (data not shown), while even the apparently
modest isomeric change to a benzimidazole group
(compound 2) led to a significant decrease in activity
(compare 2 with 1g). The straightforward, though low
yielding, synthesis of compound 2 is shown in Scheme 2.
(a)
NH2
N
O
(c)
R2 = Me
O
N
R2
N
(b)
R2
N
N
B
A
N
R2 = H
N
(d)
N
SMe
N
H
N
N
N
(e)
N
R2
R1
N
N
C
R2
N
1
Scheme 1. Synthesis of imidazo[1,2-a]pyridines 1. Reagents and con-
ditions: (a) 3-chloro-2,4-pentanedione, Et2O/THF, reflux, 12 h, 44%;
(b) acetic anhydride, AlCl3, CH2Cl2, 5 °C, reflux, 2 h, 40–80%; (c)
DMF, DMA, reflux, 3 days, 50–70%; (d) (i) thiourea, NaOMe,
n-BuOH, 85 °C, 2–20 h, (ii) cool to 30 °C, MeI, 3 h, 80–93%; (e)
R1-substituted aniline, NaN(SiMe3)2, NMP, 150 °C, 3 h, 3–12%.
Given the interesting levels of activity shown by sul-
fonamide 1h and the particularly poor reaction in the
conversion of intermediate C to D in Scheme 1,
improvements in synthetic route were required to facil-
itate preparation of required compounds. Alternative
routes from the aminopropenone intermediate B to final
targets were developed (Schemes 3 and 4).
reaction close to completion, it was practically easier to
carry forward crude product to the following reaction.
Condensation of methyl ketone A with N,N-dimethyl-
formamide dimethyl acetal gave aminopropenone B,
which could be cyclised with thiourea and methylated in
the same pot to give the key intermediate C.19 The thio-
methyl group could be displaced, albeit in low yield, by
reaction with aniline anions at high temperature to give
the compounds 1.
N
H
N
N
Cl
Cl
N
H
N
(b)
(a)
N
N
N
Compounds were assayed using a scintillation proximity
assay (SPA) to detect the inhibition of CDK2-cyclin E
catalysed [c-33-P]-phosphorylation of a GST–Rb sub-
strate.20
N
N
N
2
Scheme 2. Synthesis of benzimidazole 2. Reagents and conditions: (a)
NaH, 2,4-dichloropyrimidine, DMF, 0 °C 3 h, 8%; (b) 3-chloroaniline,
n-BuOH, reflux, 5 h, 13%.
Initial results demonstrated the sensitivity of substitu-
ents R1 and R2 in structure 1 (Table 1). These results
show that removal of the methyl group from the 2-
N
N
NH2
O
N
Table 1. Structures and enzyme activity for imidazo[1,2-a]pyridines
(b)
(c)
N
N
N
H
N
N
H
N
N
B
N
D
Cl
N
R1
N
(a)
(d)
N
N
N
N
R2
Cl
N
H
N
N
2
N
1
(e)
N
CDK2 IC50 (lM)a
R1
Compd
R1
H
R2
N
N
1a
1b
1c
1d
1e
1f
1g
1h
2
Me
Me
Me
Me
Me
H
0.21
8.8
N
1
N
E
2-Cl
3-Cl
4-Cl
0.15
0.62
Scheme 3. Improved synthesis of imidazo[1,2-a]pyridines 1 (R2 ¼ H).
Reagents and conditions: (a) Phenylguanidine, H2CO3, DMA, 130 °C,
24 h, 49–74%; (b) guanidine, HCl, NaOMe, n-BuOH/MeOH, reflux,
48 h, 67%; (c) substituted bromo (or iodo)benzene, Pd2(dba)3, BINAP,
NaO-t-Bu, Toluene, 100 °C, 24 h, 30–42%; (d) (i) NaNO2, AcOH,
60 °C, 3 h, 83%; (ii) POCl3, PCl5, reflux, 24 h, 69–80%; (e) R1-substi-
tuted aniline, Pd2(dba)3, BINAP, NaO-t-Bu, Toluene, 100 °C, 24 h, 40–
53%.
4-SO2NH2
H
3-Cl
0.038
<0.003
0.005
<0.003
0.68
H
4-SO2NH2
H
a Average of at least two measurements; enzyme protocol.20

K.F.Byth et al./ Bioorg.Med.Chem.Lett.14 (2004) 2245–2248
2247
N
The relatively similar activity of compounds 3e and 3f
demonstrates that the NH of the sulfonamide is not
essential for CDK2 activity. This result confirms the
SAR shown by compounds 1f and 1h (Table 1) where
the sulfonamide group does not appear to add greatly to
potency in the more optimised unsubstituted (R2 ¼ H)
imidazo[1,2-a]pyridines, this is despite the sulfonamide
group showing three hydrogen bonding interactions
with the CDK2 protein.17 From this we conclude that
the sulfonamide group acts primarily to increase kinase
selectivity rather than increase potency in more opti-
mised compounds. Increased selectivity being due to the
fact that only those kinases, which are able to accom-
modate the hydrogen bonding needs of the sulfonamide
group can bind these inhibitors. This proposition is
confirmed by selectivity measurements in kinase panels
and in cellular assays. For example, the sulfonamides
such as 3c and 3d show greater differential effects on the
viability of proliferating cells relative to nonproliferating
cells than imidazopyridines that do not carry a sulfon-
amide substituent (data not shown). Proliferating cells
being sensitive to CDK inhibition while nonproliferat-
ing cells should not be.
H
N
N
H
N
N
N
R2
N
N
N
S
R1
N
N
O
O
1f
3
Scheme 4. Synthesis of sulfonamides 3. Reagents and conditions:
(i) ClSO3H, SOCl2, reflux, 1 h; (ii) evaporate; (iii) add R1(R2)NH,
MeOH, 0–20 °C, 1 h, 43–87%.
Reaction of the aminopropenone B with simple phenyl
guanidines (R1 ¼ H or 3-Cl) proceeded smoothly.19
Alternatively the aminopyrimidine D could be similarly
be prepared from guanidine. Intermediate D could be
converted in to target 1 (where R1 ¼ N-alkyl-sulfamoyl)
by two routes. Direct coupling with 4-bromo (or iodo)
phenyl-sulfonamides under Buchwald conditions22 pro-
ceeded in moderate yield. Alternatively D could be con-
verted to the 2-chloropyrimidine E by hydrolysis23 and
chlorination. Chloropyrimidine E was surprisingly inert
to conventional acid or base catalysed displacement
reactions, however, target compounds 1 could be pre-
pared by using Buchwald conditions.22 Finally, simple
variation of the sulfonamide substituent could be
achieved from the parent aniline 1f by chlorosulfonation
and in situ reaction with the required amine24 (Scheme 4).
From understanding of the binding interactions with the
CDK2 enzyme17, it was of interest to explore the effect
of substitution at the 5-position of the pyrimidine and
5-position of the imidazo[1,2-a]pyridine; R1 and R2,
respectively, in structure 4. Compounds 4a and 4b were
prepared from reaction of D with the appropriate
N-halosuccinimide and conversion to final product by
steps (d) and (e) in Scheme 3. Compound 4c was pre-
pared from 4b by displacement of the bromo group with
sodium phenylthiolate. 2-Amino-5-bromopyridine was
condensed with 2-bromoacetaldehyde diethylacetal to
give 5-bromoimidazo[1,2-a]pyridine, which was simi-
larly taken through to Compound 4d. Compounds 4e–g
were prepared from 4d either by palladium catalysed
cyanide displacement25 or by sodium thiolate displace-
ment.
Representative results from sulfonamides of structure 3
(Table 2) show that CDK2 enzyme activity and effects
on cellular proliferation are relatively insensitive to
sulfonamide substitution. This is consistent with the
binding mode shown by compound 1h in the crystal
structure of its complex with CDK2,17 where the sul-
fonamide substituent is directed out towards solvent.
However, this does allow such substituents to provide
the means to modify physical properties by incorpora-
tion of basic residues such as in 3d and 3e. The reduced
serum protein binding of these two compounds con-
tributes to the increased cellular potency shown by these
compounds.
The activity of these compounds (Table 3) shows that
relatively small substituents in the 5-position of the
Table 2. Structures and biological activity for sulfonamides 3
N
H
N
Table 3. Structures and enzyme activity for imidazo[1,2-a]pyridines 4
N
R2
N
N
H
N
N
S
R1
R1
N
N
O
O
R2
H
N
3
N
S
O
Compd
R1
R2
CDK2 IC50
(lM)a
MCF-7 prolif.
IC50 (lM)b
N
O
O
4
1h
3a
3b
3c
3d
3e
3f
H
Me
H
<0.003
<0.003
0.012
0.26
0.26
0.39
0.6
Compd
R1
R2
CDK2 IC50 (lM)a
H
(CH2)2OMe
(CH2)3OMe
(CH2)2NMe2
(CH2)3NMe2
(CH2)3NMe2
H
3b
4a
4b
4c
4d
4e
4f
H
H
0.005
<0.003
<0.003
>10
H
0.004
0.005
Cl
Br
SPh
H
H
H
0.07
0.07
0.44
H
H
Me
<0.003
0.005
H
Br
CN
SEt
SPh
<0.003
<0.003
<0.003
0.016
H
a Average of at least two measurements; enzyme protocol.20
b IC50 for inhibition of BrdU incorporation to MCF-7 cells following
3 day exposure to test compound; average of at least two measure-
ments.
H
4g
H
a Average of at least two measurements; enzyme protocol.20

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K.F.Byth et al./ Bioorg.Med.Chem.Lett.14 (2004) 2245–2248
Table 4. Biological characterisation of compounds 3c and 3d
Compd
CDK2 IC50 (lM)
CDK4 IC50 (lM)
CDK1 IC50 (lM)
MCF-7 prolif. IC50 (lM)a
S249-T252 Phos. IC50 (lM)b
3c
3d
0.004
0.005
3.1
0.26
0.006
0.015
0.6
0.07
0.4
0.05
a IC50 for inhibition of BrdU incorporation to MCF-7 cells following 3 day exposure to test compound; average of at least two measurements.
b IC50 for inhibition of phosphorylation of S249-T252 site on Rb protein in MCF-7 cells following 2 h exposure to test compound.
Chen, H.; Chang, C.-H.; Seitz, S. P.; Trainor, G. L.
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10. Honma, T.; Hayashi, K.; Aoyama, T.; Hashimoto, N.;
Machida, T.; Fukasawa, K.; Iwama, T.; Ikeura, C.;
pyrimidine (compounds 4a and 4b) are tolerated or
beneficial for activity while larger groups such as in 4c
are very much less active. This is consistent with this
substituent approaching Phe80 at the closed end of the
binding pocket.17 Substitution at the 5-position of the
imidazo[1,2-a]pyridine, compounds 4d–g, is less sensitive
to the introduction of larger groups, which is consistent
with this group being directed towards solvent at the
open end of the binding pocket.17
Suzuki-Takahashi, I. J.Med.Chem.
4627.
2001, 44, 4615–
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Shan, W.; Mitt, T.; Cai, Z.-W.; Poss, M. A.; Zhu, H.;
Sack, J. S.; Tokarski, J. S.; Chang, C. Y.; Pavletich, N.;
Kamath, A.; Humphreys, W. G.; Marathe, P.; Bursuker,
I.; Keller, K. A.; Roongta, U.; Batorsky, R.; Mulheron,
J. G.; Bol, D.; Fairchild, C. R.; Lee, F. Y.; Webster, K. R.
J.Med.Chem. 2002, 45, 3905–3927.
13. Furet, P.; Meyer, T.; Strauss, A.; Raccuglia, S.; Rondeau,
J.-M. Bioorg.Med.Chem.Lett. 2002, 12, 221–224.
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Boyle, F. T.; Curtin, N. J.; Davies, T. G.; Endicott, J. A.;
Gibson, A. E.; Golding, B. T.; Griffin, R. J.; Jewsbury, P.;
Johnson, L. N.; Newell, D. R.; Noble, M. E. M.; Wang,
Finally, the biological profiles of compounds 3c and 3d
were more fully characterised (Table 4). These results
(Table 4) show that compounds 3c and 3d inhibit CDK1
with similar potency to CDK2 though show selectivity
with respect to CDK4. Both compounds block the cell
cycle at G1, S and G2/M-phases (data not shown), and
at concentrations that inhibit proliferation show inhi-
bition of CDK-dependent phosphorylation of the Rb
protein within 2 h of drug exposure. These observations
are consistent with these compounds acting as direct
CDK inhibitors in cells.
L.; Hardcastle, I. R. Bioorg.Med.Chem.Lett.
217–222.
15. Li, X.; Huang, P.; Cui, J. J.; Zhang, J.; Tang, C. Bioorg.
Med.Chem.Lett. 2003, 13, 1939–1942.
2003, 13,
In conclusion, imidazo[1,2-a]pyridines have been opti-
mised and characterised as potent inhibitors of CDK
enzymes and provide useful leads for the discovery of
orally active CDK inhibitors.
16. Engler, A. A.; Furness, K.; Mulhotra, S.; Sanchez-
Martinez, C.; Shih, C.; Xie, W.; Zhu, G.; Zhou, X.;
Conner, S.; Faul, M. M.; Sullivan, K. A.; Kolis, S. P.;
Brooks, H. B.; Patel, B.; Schultz, R. M.; DeHahn, T. B.;
Kirmani, K.; Spencer, C. D.; Watkins, S. A.; Considine,
E. L.; Dempsey, J. A.; Ogg, C. A.; Stamm, N. B.;
Anderson, B. D.; Campbell, R. M.; Vasudevan, V.; Lytle,
M. L. Bioorg.Med.Chem.Lett. 2003, 13, 2261–2267.
17. Anderson, M.; Beattie, J. F.; Breault, G. A.; Breed, J.;
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