4048
T. P. Matthews et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4045–4049
Table 4
Kinase profiling for selected compounds by mobility shift assay and IC50 determinations
a % Inhibition of kinases in a mobility shift assay21at 10
lM concentration of test compound and [ATP] = Km,ATP for each individual kinase. Single point determination. Orange
>80% inhibition, yellow = 50–80% inhibition.
b
IC50 determined in DELFIA assay.7 Mean of two independent determinations unless otherwise stated.
IC50 determined in Z’-Lyte assay.23
c
d
e
Single determination.
Mean ( SEM)% inhibition for internal standard H89, a pan kinase inhibitor,24 n = 15 determinations.
reaction temperature to 150 °C allowed the 3-chloro substituent to
be subsequently derivatised. By carrying out the two reactions in
tandem and using automated HPLC purification for the final com-
pounds, a larger number of compounds were prepared (Table 3).
Compound 31g was shown by crystallography to bind identically
to the previous examples (Fig. 2C).
in changes in orientation of the core between kinase targets. It
may however be possible to modulate the selectivity profile of
the imidazo[1,2-a]pyrazines by specific choice of the 3- and
6-substituents.
In summary, scaffold hopping from a very weak benzisoxazole
hit generated novel 3,6-di(hetero)aryl imidazo[1,2-a]pyrazine
ATP-competitive inhibitors of CHK1. By developing synthetic
routes that allowed late stage derivatisation of the scaffold, we
quickly synthesised analogues with improved potency and ligand
efficiency, leading to micromolar inhibitors of CHK1. Kinase profil-
ing showed that several of the imidazo[1,2-a]pyrazines inhibited
other kinases with equivalent or better potency. The selectivity
profile of the compounds varied with the nature of the pendant
substituents. The 3,6-di(hetero)aryl imidazo[1,2-a]pyrazine scaf-
fold may therefore be generally useful for the development of
new kinase inhibitors.
Despite a variety of functionality introduced onto the 3- and
6-substituents, the activity for these compounds at CHK1 was not
improved over that of 24. In addition, counterscreening against the
structurally distinct enzyme CHK2, also a potential oncology tar-
get,18 indicated little selectivity. For example, compounds 24, 31g
and 32g gave CHK2 IC50 values7 of 0.48
lM, 1.3 lM and 0.12 lM,
respectively (Table 4). Further selectivity profiling revealed other
significant kinase inhibitory activities associated with the scaffold.
Aselection of11analoguesweretestedat10 lM inhibitor concen-
tration against a panel of 24 kinases using a mobility shift assay20 (Ta-
ble 4, [ATP] = Km,ATP for each kinase,21,22 see Supplementary data). At
this concentration none of the selected imidazo[1,2-a]pyrazines
showed greater than 50% inhibition against MAPKAPK2, PKC, PRAK,
MET, ERK2, PKA, AKT2, INSR, p38a, AKT1 and MSK1. However activity
was observed against the remaining 13 kinases by one or more of the
compounds. A group of structurally similar analogues (32g, 32h, 32i)
Acknowledgements
We thank Dr. A. Mirza, M. Richards and Dr. M. Liu for assistance
in the spectroscopic characterisation of test compounds and Dr M.
Lamers for the preparation of CHK1 protein. This work was sup-
ported by Cancer Research UK [CUK] Grant Numbers C309/
A2187, C309/A8274 and C309/A8365 and by The Institute of Can-
cer Research. We acknowledge NHS funding to the NIHR Biomedi-
cal Research Centre.
stronglyinhibited(>94%@10 lM)thetyrosinekinasesABL,FYN, LYN,
LCK and SRC. Compounds 22, 24, 31g and again 32i were inhibitors of
AurA. To validate the screening results, IC50 determinations were
made for the most potent inhibitors against CHK2, ABL1 and GSK3b,23
and good agreement was seen between these data and the single-
point inhibitions. Notably, sub-micromolar potency was observed
for inhibition of ABL1 and CHK2 with these compounds.
Supplementary data
The elaboration of other template screen hits, which were eval-
uated in parallel with 1, led to potent and selective CHK1 inhibitors
which were able to bind in the interior pocket of the enzyme.19 In
view of this, and given the low CHK1 potency and selectivity seen
with the compounds described here, the imidazo[1,2-a]pyrazines
were not progressed further against CHK1. Our experience shows
how scaffold morphing from fragment-like hits can generate new
inhibitor scaffolds, but also highlights the benefit of monitoring ki-
nase selectivity during the development of the new scaffolds. We
speculate that the single explicit hydrogen bond from the core imi-
dazo[1,2-a]pyrazine to the hinge region may result in an increased
importance of the interactions formed by the two pendant aryl
groups to determine affinity and selectivity, and may also result
Supplementary data associated with this article can be found, in
References and notes
1. Cohen, P. Nat. Rev. Drug Disc. 2002, 1, 309.
2. Pitt, W. R.; Parry, D. M.; Perry, B. G.; Groom, C. R. J. Med. Chem. 2009, 52, 2952.
3. Blasina, A.; Hallin, J.; Chen, E.; Arango, M. E.; Kraynov, E.; Register, J.; Grant, S.;
Ninkovic, S.; Chen, P.; Nichols, T.; O’Connor, P.; Anderes, K. Mol. Cancer Ther.
2008, 7, 2394.
4. Ashwell, S.; Zabludoff, S. Clin. Cancer Res. 2008, 14, 4032.
5. Bucher, N.; Britten, C. D. Br. J. Cancer 2008, 98, 523.
6. Janetka, J. W.; Ashwell, S.; Zabludoff, S.; Lyne, P. Curr. Opin. Drug Discov. Devel.
2007, 10, 473.