2-Aminoquinazoline inhibitors of cyclin-dependent kinases

Article abstract of DOI:10.1016/j.bmcl.2005.05.131

The inhibition of cyclin-dependent kinase 4 (Cdk4) causes cell cycle arrest and restores a checkpoint that is absent in the majority of tumor cells. Compounds that inhibit Cdk4 selectively are targeted for treating cancer. Appropriate substitution of 2-aminoquinazolines is demonstrated to produce high levels of selectivity for Cdk4 versus closely related serine-threonine kinases.

Full text of DOI:10.1016/j.bmcl.2005.05.131

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3881–3885
2-Aminoquinazoline inhibitors of cyclin-dependent kinases
Yadagiri Bathini,a Inderjit Singh,a Patricia J. Harvey,b Paul R. Keller,b Rajeshwar Singh,a
Ronald G. Micetich,a David W. Fry,b Ellen M. Dobrusinc and Peter L. Toogoodc,*
aNAEJA Pharmaceutical Inc., #2, 4290-91A Street, Edmonton, Alberta, Canada T65VE
bCancer Pharmacology, Pfizer Global Research and Development, Michigan Laboratories, 2800 Plymouth Road,
Ann Arbor, MI 48105, USA
cDepartment of Medicinal Chemistry, Pfizer Global Research and Development, Michigan Laboratories, 2800 Plymouth Road,
Ann Arbor, MI 48105, USA
Received 25 March 2005; revised 25 May 2005; accepted 26 May 2005
Available online 29 June 2005
Abstract—The inhibition of cyclin-dependent kinase 4 (Cdk4) causes cell cycle arrest and restores a checkpoint that is absent in the
majority of tumor cells. Compounds that inhibit Cdk4 selectively are targeted for treating cancer. Appropriate substitution of 2-
aminoquinazolines is demonstrated to produce high levels of selectivity for Cdk4 versus closely related serine-threonine kinases.
Ó 2005 Elsevier Ltd. All rights reserved.
Cyclin-dependent kinases (cdks) are key regulatory com-
ponents of the cell division cycle.1–5 As such they repre-
sent potential molecular targets for new antiproliferative
agents that may be used to treat cancer or other prolif-
erative diseases. Of the cell cycle associated Cdks, Cdk4
especially has been targeted due to its fundamental role
in the response to growth signals and the initiation of
cell division.6,7 Expression of the regulatory protein, cy-
clin D, is increased in response to growth factor signal-
ing, resulting in the activation of Cdk4 kinase activity
and sequestration of the Cdk inhibitor protein p27.
The major substrate for phosphorylation by Cdk4 is
the retinoblastoma protein (pRb). pRb is phosphory-
lated first by Cdk4/cyclin D and subsequently by
Cdk2/cyclin E. Hyperphosphorylated pRb dissociates
from the E2F/DP transcription factor complex, releas-
ing it to transactivate the expression of genes essential
for DNA synthesis including dihydrofolate reductase,
thymidylate synthase, and RNA polymerase.8,9 This
pathway, the so-called Rb pathway, represents a key
regulatory pathway controlling cell proliferation. The
importance of this pathway is underscored by the obser-
vation that almost 100% of human tumors possess some
defect in this regulatory function.10 These defects mani-
fest as mutation or deletion of pRb, overexpression of
cyclin D, activating mutations in Cdk4, or loss of the
Cdk4 inhibitor protein, p16. Biochemical experiments
with dominant negative cyclin D, or cyclin D antisense,
and experiments to replace p16 through adenoviral
expression, all indicate that restoration of integrity to
the Rb pathway can inhibit tumor cell proliferation.6
On the basis of these results, inhibitors of Cdk4 are
expected to be good candidates for antitumor thera-
py.11–15
Our interest in finding inhibitors of Cdk4/cyclin D led
us to investigate a series of 2, 7, 8-substituted quinazo-
lines. These compounds were modeled directly on our
successful inhibitors from a series of pyrido[2,3-d]pyr-
imidin-7-ones that have been reported earlier.16–21
Our anticipation was that the quinazolines would dis-
play a similar structure–activity relationship (SAR)
profile to the pyrido[2,3-d]pyrimidin-7-ones, but might
offer a different profile of physicochemical and phar-
macokinetic properties.
A survey of published procedures for the preparation of
appropriately substituted quinazolines rapidly led us to
the conclusion that it would be necessary to develop a
new route to the compounds that we were targeting.
This route has been outlined in a previous communica-
tion.22 The conversion of intermediate quinazolines 1,
wherein R1 and R2 may be hydrogen or methyl, and
R3 is a cyclopentyl or an isopropyl group, to final com-
pounds containing appropriate side chains at the C-2
Keywords: Quinazoline; Cdk.
*
Corresponding author. Tel.: +1 734 761 6279; fax: +1 734 622
5165; e-mail: peter.toogood2@pfizer.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.05.131

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Y. Bathini et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3881–3885
position was achieved as shown in Scheme 1. Diazotiza-
tion and hydrolysis of the amine function at C-2 in
compounds 1, provided hydroxyquinazolines 2. Chlori-
nation with POCl3 produced coupling partners 3 that al-
lowed the introduction of a variety of amine groups at
the C-2 position, albeit under relatively forcing condi-
tions (heating to 110 °C in a sealed tube). Where neces-
sary, the side chain protecting group in product 4
(typically a Boc group) was removed using standard
procedures (e.g., TFA). Demethylation of the C-7 meth-
yl ether was effected using sodium ethanethiolate to pro-
vide the phenols 5 (see Scheme 1).
As seen in Tables 1 and 2, the unsubstituted anilines
themselves (compounds 6 and 15) were quite potent
inhibitors of Cdk4 with IC50 values of 0.125 and
0.166 lM, respectively.24 Introduction of a pyrrolidine
ring at the para-position of the aniline was detrimental
to inhibition of Cdk4 with the potency of compound
16 dropping by an order of magnitude compared to
compound 15. In contrast, incorporation of a piperidine
at the same position (9, 18) led to an increase in potency
against Cdk4 with the cyclopentyl derivative (18) dis-
playing an IC50 = 0.011 lM. The difference between
the potencies for the pyrrolidine and piperidine analogs
may reflect better Van der Waals contacts between the
six-membered ring and the protein at the mouth of the
ATP binding site, and a steric clash between the protein
and the pyrrolidine as a consequence of the lower con-
formational flexibility of this ring. The morpholine ana-
logs (10, 19), were comparable to the piperidines,
however, a substantial further increase in potency
against Cdk4 was observed in switching to the pipera-
zine derivatives (12, 20). This increase in potency does
not appear to be associated with an ionic interaction be-
tween the protein and a positively charged piperazine
nitrogen because acylation of the piperazine secondary
amine with either acetate (13 and 22) or glycine (14
and 23) had no significant effect on the potency of
Cdk4 inhibition (less than 2-fold difference in IC50 val-
ues). Similarly, fluorination of the aromatic ring had
no detrimental effect on Cdk4 inhibition. The 30-amino-
pyrrolidine analog that bears an isopropyl substituent at
C-8 (compound 8: IC50 = 0.011 lM) is equipotent with
the isomeric piperazine (compound 11: IC50 = 0.016
Our initial expectation was that the quinazoline series of
compounds would bind to the ATP site of Cdks in a
similar manner to the pyrido[2,3-d]pyrimidin-7-ones we
reported previously.16 Therefore, our SAR studies be-
gan with an exploration of substituted aryl amine
groups appended to the C-2 position (Table 1). These
compounds were prepared with either a cyclopentyl
group or an isopropyl group attached to C-8 since these
two groups were anticipated to provide a good balance
of potency and drug-like physical properties for this
chemical series. Similarly, by extension from the related
pyrido[2,3-d]pyrimidin-7-one series, electron-rich para-
substituted aniline side chains at the C-2 position were
anticipated to provide the most potent Cdk4 inhibitors.
Thus, the initial choice of target compounds was very
much grounded in experience obtained previously in a
related chemical class bearing a similar substitution pat-
tern, and was not influenced by SAR from previously
disclosed 4-anilinoquinazoline Cdk inhibitors.23
Scheme 1. Synthetic route used to produce quinazoline Cdk inhibitors. R1, R2, and R3 are H or methyl. R3 is isopropyl or cyclopentyl. R4
substituents are illustrated in Figure 1. Reagents: (a) tetrafluoroboric acid, NaNO2, (b) POCl3, (c) R4-NH2, CH3CN, sealed tube 110 °C, (d) EtSNa.
Figure 1. Side chains used at position R4.

Y. Bathini et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3881–3885
Table 1. SAR at the C-2 position when the N-8 substituent is isopropyl
3883
R4
IC50 (lM)
CDK1/B
CDK2/A
CDK2/E
CDK4/D
6
7
a
b
c
d
e
f
1.04
>5
0.253
1.60
1.08
>5
0.125
0.560
0.011
0.052
0.052
0.016
0.007
0.025
0.010
8
1.30
>5
0.296
0.410
0.665
0.185
0.141
NA
2.55
3.10
4.4
9
10
11
12
13
14
1.97
0.506
0.743
NA
1.20
0.655
0.900
1.10
g
h
i
0.800
0.191
For an explanation of the R4 substituents see Figure 1.
NA, not available.
Table 2. SAR at the C-2 position when the N-8 substituent is cyclopentyl
R4
IC50 (lM)
CDK1/B
CDK2/A
CDK2/E
CDK4/D
15
16
17
18
19
20
21
22
23
a
b
c
d
e
f
0.801
>5
0.398
>5
0.202
>5
0.650
>5
0.166
1.75
0.203
0.384
0.384
0.028
0.062
NA
0.775
>5
0.115
0.011
0.018
0.001
0.004
0.002
0.002
1.59
0.132
0.214
0.650
0.250
0.300
0.265
0.450
g
h
i
>5
0.270
0.095
For an explanation of the R4 substituents see Figure 1.
NA, not available.
lM). However, in the C-8 cyclopentyl series, the 30-
aminopyrrolidine 17 is significantly less potent than
the isomeric piperazine 20. In both the series the, amino-
pyrrolidine is more potent than the pyrrolidine. Com-
pounds 16 and 17 are the only two C-8 cyclopentyl
quinazolines that are less potent inhibitors of Cdk4 than
the corresponding C-8 isopropylquinazoline analogs.
The reason for this change in potency order is unclear.
Overall, as in the pyrido[2,3-d]pyrimidin-7-one series, a
C-2 side chain containing a para-piperazinyl aniline is
preferred for potent inhibition of Cdk4. Consistent with
these SAR trends, a crystal structure of compound 11
bound to Cdk2 showed the ligand located in the ATP-
binding site in a similar orientation to the corresponding
pyrido[2,3-d]pyrimidin-7-one.16
7-one series. Consequently, several analogs were
prepared in which the methyl ether was cleaved to the
parent phenol. A comparison of these phenols with their
methyl ether counterparts revealed that the phenols were
generally at least as potent as the corresponding ethers
(Table 3). For example, compounds 25, 26, and 27,
within experimental error, were all as potent as the most
potent methyl ether (Compound 20, Table 2). In fact,
these compounds may actually be more potent than they
appear since their IC50 values approach the concentra-
tion of enzyme used in the assay. Thus, the compounds
are essentially titrating the enzyme, which creates a
minimum IC50 despite any further increase in affinity.
Besides potent inhibition of Cdk4, another goal of this
program was to obtain compounds that are selective
for Cdk4 versus other kinases, including other Cdks.
Compounds 26 and 27 are both around 100-fold selec-
tive for Cdk4 versus Cdk2A, an enzyme against which
The methyl ether at C-7 in the compounds described
here was considered a poor replacement for the amide
carbonyl in the corresponding pyrido[2,3-d]pyrimidin-

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Y. Bathini et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3881–3885
Table 3. SAR for C-7 hydroxyquinazolines
R3
R4
IC50 (lM)
CDK1/B
CDK2/A
CDK2/E
CDK4/D
24
25
26
27
2-Propyl
a
e
f
0.813
1.94
0.250
0.18
0.550
0.635
0.320
0.270
0.325
0.003
0.001
0.001
Cyclopentyl
Cyclopentyl
Cyclopentyl
0.030
0.761
0.082
0.112
g
For an explanation of the R4 substituents see Figure 1.
it is especially difficult to achieve selectivity with these
ATP competitive inhibitors. Notably, the phenols ap-
pear to be slightly more selective for Cdk4 than the cor-
responding ethers by 3- to 8-fold.
28, compound 30, was about 2-fold more potent an
inhibitor of Cdk4 and similarly selective versus other cy-
clin-dependent kinases. Compound 30 displays similar
potency and enzyme selectivity to the corresponding
pyrido[2,3-d]pyrimidin-7-one analog, compound 31. In
addition, compound 30 displays antiproliferative activi-
ty in HCT-116 human carcinoma cells with
IC50 = 100 nM. Notably, however, the quinazolines are
generally more lipophilic and less soluble (in pH 6.5
phosphate buffer) than the corresponding pyrido[2,3-
d]pyrimidin-7-one analogs as illustrated by the represen-
tative data provided in Table 4. Thus, while differing
from the pyrido[2,3-d]pyrimidin-7-ones in physical
properties, the quinazoline series does not offer any
obvious advantages.
To further improve the selectivity for Cdk4 versus other
kinases, substitution at C-5 was examined. Once again
the impetus for these changes was the increase in selec-
tivity observed with C-5 methyl pyrido[2,3-d]pyrimi-
din-7-ones versus their unsubstituted homologs. C-5
substituted quinazolines were obtained following the
chemistry outlined previously and in Scheme 1 above
starting with dimethoxy toluene. The C-5 methyl deriv-
atives 28 and 29 were both less potent than their C-5 H
counterparts, however, compound 28 was still an effec-
tive inhibitor of Cdk4 and was quite selective for Cdk4
versus Cdk2A, Cdk2E, and Cdk1B. In contrast, com-
pound 29 was a significantly less active inhibitor of
Cdk4, possibly highlighting a size limitation in the
ATP-binding site that restricts access to a compound
with substitution both on the quinazoline ring and on
the side chain piperazine. The C-7 phenol analog of
In conclusion, a series of 2, 7, 8-substituted quinazolines
has been shown to inhibit cyclin-dependent kinases with
a similar SAR to that displayed by a related series of
pyrido[2,3-d]pyrimidin-7-ones. This observation enabled
the rapid identification of potent and selective Cdk4
inhibitors with activity in cell-based assays.
Table 4. C-5 methylquinazolines
IC50 (lM)
c Log P
Log D(pH 7.4)
Solubility (lg/mL)
CDK1/B
CDK2/A
CDK2/E
CDK4/D
28
29
30
31
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
0.055
4.13
5.86
5.46
5.96
3.52
2.32
3.77
1.39
0.63
2
NA
1
0.020
0.014
13
NA, not available.

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3885
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