S. Huang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4818–4821
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imide to generate bromide 2, which was converted to bor-
onate 3 using bis(pinacolato) diboron in the presence of
Pd(II)Cl2(dppf).6 It is noteworthy that NH protection
with the benzenesulfonyl group was critical because the
reactions with unprotected 7-azaindole or BOC protected
7-azaindole failed to generate the desired product. Cou-
pling of compound 3 with 2,4-dichloropyrimidine using
Pd(0)(PPh3)4 produced biaryl intermediate 4. This reac-
tion proceeded with superior regioselectivity and high
yield. Next, the chloride of compound 4 was displaced
with various amines to provide the desired analogues
5a–o. Two equiv of amine was used in this step because
cleavage of the benzenesulfonyl group also consumed
one equivalent of amine reagent.
activity. Compound 5i had an IC50 of 3 nM for
CDK1. However, shifting methyl group to C-3 or C-4
position of the phenyl ring reduced kinase binding by
more than 10-fold. Compounds 5j and 5k had double-
digit nM IC50s for CDK1. Adding aminoalkyl or
hydroxyalkyl side chain to C-4 position of the 2-methyl-
phenyl ring generated positive effect on both CDK1 and
HeLa cell potency. The enhanced cellular activities for
analogues 7a–c could be resulted from their improved
solubility and cellular permeability. Attaching cyclohex-
yl (5l) or aminocyclohexyl (5n) to the pyrimidine ring
also generated potent compounds, though an extra
methylene linkage between cyclohexyl and the NH
group seemed to be harmful to potency.
Solubilizing amino side chains were installed using the
chemistry shown in Scheme 2. The hydroxyl compound
5o was reacted with methanesulfonyl chloride to gener-
ate mesylate 6, which was then treated with various
amines to give the desired derivatives 7a–c.
The NH of the 7-azaindolyl ring was derivatized with sev-
eral groups in order to assess its role in CDK1 binding.
Using the chemistry shown in Scheme 3, compound 5l
was treated with KOt-Bu, then various electrophiles were
added to produce the corresponding products 8a–d.
Alkylation of the NH with methyl or dimethylaminoethyl
group resulted in a near complete loss of potency. As
shown in Table 2, compounds 8a and 8d had an IC50 of
>10 lM for CDK1. Acetylation or methanesulfonylation
also reduced potency, though to a lesser extent.
Therefore, an unsubstituted NH is crucial for both
CDK1 and cellular antiproliferation activity.
All analogues were tested for kinase and cellular anti-
proliferative activity.7 CDK1 activity was measured
using CDK1 in complex with cyclin B to phosphorylate
a histone-H1 biotinylated peptide substrate. Inhibition
of CDK1 activity was measured by observing a reduced
amount of 33P-c-ATP incorporated into the immobi-
lized substrate in a Flashplate assay format. In the cell
proliferation assay, the HeLa (cervical carcinoma) cell
line was used. The IC50 for inhibition of cell prolifera-
tion was determined by quantifying the incorporation
of 14C-thymidine into cellular DNA. The data are
shown in Table 1. Compound 5a with an unsubstituted
phenyl group showed moderate activity for CDK1 with
an IC50 of 54 nM. Addition of an extra group at C-2
position of the phenyl ring generated mixed results.
For example, a hydroxyl group (5b) was detrimental
to kinase inhibition, while methoxy (5c), fluoro (5d), tri-
fluoromethyl (5e) or ethyl group (5f) had little effect on
potency. On the other hand, chloro (5g), bromo (5h) or
methyl (5i) group at C-2 position was beneficial to
A methyl group was added to C-2 position of the 7-aza-
indolyl ring using the chemistry shown in Scheme 4.
Compound 4 was treated with LDA at À78 °C, followed
by addition of methyl iodide to afford compound 9.
Next the chloride was displaced with trans-1,4-cyclohex-
anediamine to generate diamine 10. In contrast to the
facile removal of the N-benzenesulfonyl group observed
in the synthesis of compounds 5a–o, the adjacent C-2
methyl group caused the N-benzenesulfonyl group to
be stable under the hot aminolysis conditions. There-
fore, an extra deprotecting step with potassium carbon-
ate was used to produce final product 11. Based on the
SAR shown by two closely related series imidazo
[1,2-a]pyridine and imidazo[1,2-a]pyridazine,8 it was
expected that introduction of a methyl group to the
C-2 position would be tolerated. However, the results
for compound 11 showed a 140-fold reduction for
CDK1 inhibition compared to compound 5n, which
indicates that there is a significant difference in SAR be-
tween our series and the two series reported previously
from AstraZeneca group.8
To evaluate the kinase selectivity of this series, selected
compounds 5i and 5l were screened against a panel of
100 kinases with 2 lM of ATP used. At the concentra-
tion of 1 lM, both compounds displayed >50% inhibi-
tion of 61 kinases in the panel, and >80% inhibition of
33 kinases. Strong inhibition for other CDK family
members (including CDK2/cyclinA, CDK2/cyclinE,
CDK 3/cyclinE, CDK5/p35 and CDK6/cyclinD3) was
observed. In addition, many other kinases implicated
in cancer and other diseases were strongly inhibited,
indicating these compounds are promiscuous kinase
inhibitors. The selectivity data highlight a commom
issue: generating selective CDK inhibitors in order to
Scheme 2. Synthesis of analogues 7a–c with amino side chains.
Reagents: (a) methanesulfonyl chloride, Et3N, CH2Cl2, 95%; (b)
RNH2, DMF, 80–90%.
Huang, Shenlin
