Structure-activity relationships for 2-anilino-6-phenylpyrido[2,3-d] pyrimidin-7(8H)-ones as inhibitors of the cellular checkpoint kinase Wee1

Article abstract of DOI:10.1016/j.bmcl.2005.01.079

A series of 2-anilino-6-phenylpyrido[2,3-d]pyrimidin-7(8H)-ones were synthesized and evaluated for their inhibitory properties against the non-receptor kinase c-Src and the G2/M checkpoint kinase Wee1. Overall, the compounds were 10-100-fold more potent i

Full text of DOI:10.1016/j.bmcl.2005.01.079

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1931–1935
Structure–activity relationships for
2-anilino-6-phenylpyrido[2,3-d]pyrimidin-7(8H)-ones as
inhibitors of the cellular checkpoint kinase Wee1
Brian D. Palmer,a,* Jeff B. Smaill,a Gordon W. Rewcastle,a Ellen M. Dobrusin,b
Alan Kraker,b Charles W. Moore,b Randall W. Steinkampfb and William A. Dennya
aAuckland Cancer Society Research Centre, School of Medical Sciences, The University of Auckland, Private Bag 92019,
Auckland, New Zealand
bPfizer Global Research and Development, Ann Arbor Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48106-1047, USA
Received 30 August 2004; revised 27 January 2005; accepted 28 January 2005
Abstract—A series of 2-anilino-6-phenylpyrido[2,3-d]pyrimidin-7(8H)-ones were synthesized and evaluated for their inhibitory
properties against the non-receptor kinase c-Src and the G2/M checkpoint kinase Wee1. Overall, the compounds were 10–100-fold
more potent inhibitors of c-Src than Wee1, and variation of substituents on the 6-phenyl ring did not markedly alter this preference.
Solubilizing substituents off the 2-anilino ring in many cases increased Wee1 activity, thus lowering this preference to about 10-fold.
5-Alkyl substituted analogs were generally Wee1 selective, but at the expense of absolute potency.
Ó 2005 Elsevier Ltd. All rights reserved.
The general class of 2-anilino-6-phenylpyrido[2,3-d]pyr-
imidin-7(8H)-ones are broad-spectrum inhibitors of a
number of tyrosine kinase enzymes, including the recep-
tor kinases EGFr (erbB1), PDGFr, FGFr, and non-
receptor kinases such as c-Src. For example, compound
1 is reported1 to have IC50 values for inhibition of sub-
strate phosphorylation by isolated enzymes as follows;
PDGFr = 79 nM, bFGFr = 43 nM, EGFr = 44 nM,
and c-Src = 9 nM, with some other analogs being
equally potent.2 The breadth of this ꢀpan-kinaseꢁ activity
is likely due to their action as competitive inhibitors at
the ATP site,3 where they are proposed4 to form a key
bidentate H-bond between the 3-aza and 2-NH atoms
and a methionine residue (Met 341 in c-Src). Compound
1 also inhibited the formation of microcapillaries on
Matrigel-coated plastic at 10 nM concentrations, and
generated dose dependent inhibition of angiogenesis in
vivo in mice when given orally at doses between 1 and
25 mg/kg.5
We identified 1 in a mass screen as a potent inhibitor
(IC50 = 0.16 lM) of the Wee1 kinase.6 This enzyme is in-
volved in the maintenance of the G2/M checkpoint in
the cell cycle, through its inhibitory phosphorylation
of Cdc2 on Tyr-15. Normal cells arrest at both the
G1/S and G2/M checkpoints in response to DNA dam-
age caused by cytotoxic drugs or radiation, to allow time
to either repair the DNA or to activate cell death path-
ways if this is not possible.7,8 In contrast, many cancer
cells lacka functional p53 gene, which means that the
G1/S checkpoint is not functional.8 Inhibitors of
Wee1, via their abrogation of the G2/M checkpoint,
were hypothesized to preferentially enhance the cyto-
toxic effects of DNA damaging agents on p53-negative
cells. This was confirmed for 1, which inhibited radia-
tion-induced phosphorylation of Cdc2 at Tyr-15, and
enhanced the effect of radiation selectively in p53-nega-
tive cells.6,9
In this paper we report the synthesis of a number of ana-
logs of 1, and the first SAR study of Wee1 inhibitors. A
main aim of the study was to improve their selectivity
for inhibition of Wee1 over c-Src, since inhibition of
the latter has so many other cellular effects. Previous
studies with related compounds showed that the selecti-
vity for different enzymes can be significantly altered by
the substituent pattern on the pendant 6-phenyl ring.
Keywords: Pyridopyrimidine; Checkpoint inhibitor; Wee1 kinase
inhibitor.
*
Corresponding author. Tel.: +64 9 373 7599; fax: +64 9 373 7502;
e-mail: b.palmer@auckland.ac.nz
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.01.079

1932
B. D. Palmer et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1931–1935
For example, in the related urea series, the 20,60-dichloro
analog (2) is a potent inhibitor of both PDGFr (IC50
1.1 lM) and FGFr (IC50 0.13 lM), whereas the 30,50-
diOMe analog (3) is selective for FGFr (IC50s > 50
and 0.06 lM, respectively).10 In the analogous napthyr-
idine series,11 unsubstituted 3-phenyl analogs were non-
specific inhibitors of c-Src, FGFr and PDGFr tyrosine
kinases, whereas 20,60-dichloro analogs were most effec-
tive against c-Src and FGFr, and 30,50-dimethoxy deriv-
atives showed high selectivity for FGFr alone (e.g.,
IC50s 0.03 and >50 lM for 4 against FGFr and c-Src,
respectively).
20,60-disubstituents against the 20,60-dichloro analog 7,
which is a moderate Wee1 inhibitor (IC50 2.6 lM) but
a much more potent c-Src inhibitor (IC50 0.024 lM).
A crystal structure of the related 20,60-dichloro urea ana-
log 2, which is substantially more potent than the unsub-
stituted compound, shows the phenyl ring essentially
orthogonal to the chromophore.10 Compounds 7–13 ex-
plore a range of symmetrically substituted compounds
using substituents of varied electronic and steric proper-
ties. There were no consistent correlations between
Wee1 or c-Src potencies, or Wee1/c-Src selectivity, and
various measures of substituent electronic (r), lipophilic
2'
Cl
Cl
4'
5
3
O
Et2N
N
N
6'
Cl
6
Cl
N 2
H
N
N
O
H2N
N
N
NH
Me
1
2
O
NHtBu
OMe
OMe
N
OMe
N
OMe
H2N
N
N
NH
NHtBu
N
H
N
NH
NHtBu
N
3
O
O
4
O
Most of the compounds were prepared from the
known12 2-(methylthio)pyrido[2,3-d]pyrimidin-7(1H)-
one (5) (Method 1, Scheme 1), or from 4-(methyl-
amino)-2-(methylsulfanyl)-5-pyrimidinecarbaldehyde (6)
by reaction with various phenylacetonitriles, followed
by oxidation of the methylsulfanyl group and subse-
quent displacement with anilines13 (Method 2). In Meth-
od 1, different bases were used in the Suzuki coupling,
depending on the reactivity of the arylboronic acid (i.
K2CO3; ii. Ba(OH)2; iii. CsF).
(p) or steric (MR) properties. Extending this analysis to
include the unsymmetrical 20,60-disubstituted com-
pounds (14–20) did not change the result.
Using the 20,60-dichloro motif to anchor the phenyl ring
orthogonal to the chromophore, compounds 21–41 ex-
plored SAR for a variety of additional substituents in
the 30- and 40-positions. The 20,60-dichloro motif was
chosen for ease of synthesis, and also because it was
one of the most active. A few examples using the 20,60-
diMe motif were also prepared, but these compounds
were generally less active; compare 39, 41 (Table 1)
and 1, 46 (Table 2). There were cases of improved com-
parative activity (10/7 and 40/38), but these pairs
showed much lower absolute potencies. Calculations
with the 30- and 40-substituted 20,60-dichloro analogs
again showed no correlations between kinase potency
or selectivity and measures of substituent lipophilic,
electronic, or steric properties. In the 30-substituted ser-
ies (21–30), the 3-OH compound 27 stood out as being
35-fold more potent than the next most active com-
pound against Wee1 (although it was also 10-fold more
potent as a c-Src inhibitor as well). The remarkable con-
trast with the 3-CH2OH and 30-OMe analogs 23 and 28
suggests the likely existence of a geometrically con-
strained H-bond acceptor in this region of the protein.
A similar but less striking pattern is shown by the
40-OH and 40-OMe analogs 32 and 34. This suggests
interaction with the same binding site, which is better
positioned for the 30- than the 40-OH group.
Further derivatization of some substituted 6-phenyl
compounds, via standard synthetic methods, gave addi-
tional analogs (see footnotes to Table 1). Esters 48–50
were prepared from the acid 51 via activation with
SOCl2 and coupling with the appropriate alcohols
(Method 3). The 5-methyl analogs (Table 3) were pre-
pared as shown in Scheme 2 (Method 4). All compounds
had satisfactory analyses, or MS/HPLC purity >95%.
IC50 values for inhibition of Wee16 and c-Src1 were
determined by the published methods.
Table 1 gives data on the Wee1 and c-Src inhibitory
properties of a wide range of phenyl-substituted com-
pounds. The initial study was a comparison of various
R
Br
N
i
N
ii
iv
compounds
of Table 1
MeS
N
N
H
O
R
N
N
Me
O
5: R=H
R=MeS
iii
R=Br
R=PhNH
Despite these exceptions, varying the substituents on the
6-phenyl ring did not generally significantly alter the
selectivity of the compounds for Wee1 over c-Src. This
Scheme 1. Reagents: (i) NBS/DMF; (ii) Me2SO4/Cs2CO3; (iii) m-
CPBA, then excess PhNH2; (iv) ArB(OH)2/Pd(PPh3)4/AsPh3.

B. D. Palmer et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1931–1935
1933
Table 1. 2-Anilino-6-phenylpyrido[2,3-d]pyrimidin-7(8H)-ones: variation of 6-phenyl substituents
3'
Z
4'
5'
2'
N
6'
N
N
O
N
H
Me
No.
Z
Mp (°C)
Ma
IC50 (lM)b
Wee1
c-Src
0.024
7
20,60-DiCl
Ref. 1
2.6
9.7
8
20,60-DiF
267–269
230–232
207–209
246
1ii
2
0.066
0.047
0.014
8.1
9
20,60-DiBr
0.41
0.99
41
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
20,60-DiMe
1i
20,60-DiCF3
2
20,60-DiOH
269–270
264–266
278–280
218–220
234–236
235–237
268–270
224–228
251
On 13c
1iii
27
0.72
0.98
0.043
0.012
0.21
0.12
0.022
0.058
0.05
0.27
2.8
20,60-DiOMe
>50
2.4
20-Cl, 60-F
20-Cl, 60-Me
2
2
1.9
20-Cl, 60-CF3
2
1ii
>50
3.4
20-Cl, 60-OMe
20-Cl, 60-OH
On 17c
1.5
4.5
20-Me, 60-Br
On 9d
20-OMe, 60-OH
20,60-DiCl, 30-Cl
20,60-DiCl, 30-Me
2,60-DiCl, 30-CH2OH
20,60-DiCl, 30-CH2NH2
2,60-DiCl, 30-CO2H
20,60-DiCl, 30-CONH2
20,60-DiCl, 30-OH
20,60-DiCl, 30-OMe
20,60-DiCl, 30-NH2
20,60-DiCl, 30-NHAc
20,60-DiCl, 40-Cl
20,60-DiCl, 40-OH
20,60-DiMe, 40-OH
20,60-DiCl, 40-OMe
20,60-DiMe, 40-OMe
20,60-DiCl, 40-NH2
20,60-DiCl, 4-NHAc
20,60-DiCl, 30,50-diOMe
20,60-DiCl, 30,50-diOH
20,60-DiMe, 30,50-diOMe
20,60-DiMe, 30,50-diOH
On 13e
11
232
2
>50
50
243–244
250–252
205–215
326–330
180–190
300–304
268–270
284
2
2
3.5
31
0.039
0.062
0.28
0.13
0.009
0.54
0.09
0.78
0.33
0.073
0.017
6.5
On 23f
On 23g
On 25h
On 28i
2
3.2
8.6
0.074
>50
2.6
On 30j
2
160–163
239–241
318–321
286–288
244–246
234
>50
8.6
2
On 34c
On 35c
2
0.22
0.58
>50
>50
3.7
1i
0.25
0.27
1.5
281–283
313–315
323–325
318–321
255–258
288–291
On 37j
2
36
2
>50
0.14
33
>50
0.011
39
0.11
On 38k
2
On 40k
1.0
a Method of synthesis; see text.
b IC50 values were determined for both Wee16 and c-Src1 inhibition by the published methods cited. Values are the average of two or more
independent determinations, with a variance of 20%.
c 6 equiv BBr3/DCM/reflux/16 h.
d (CH3)4Sn/Pd(PPh3)4/DMF/100 °C/3 h.
e 3 equiv BBr3/DCM/rt/2 h.
f (i) PPh3Cl2/Et3N/DCM/2 h, (ii) NH3(g)/i-PrOH/110 °C/18 h.
g Jones reagent/acetone/2 h.
h (i) SOCl2/cat. DMF/reflux/3 h, (ii) c. NH3/THF.
i PyridineÆHCl/200 °C/1 h.
j Concd HCl/reflux/1 h.
k PyridineÆHCl/200 °C/10 min.
is evident from the overall relationship between these
two activities for the compounds of Table 1 for which
both IC50 values were available
Workin related series of compounds 10,11 has shown that
solubilizing substituents off the 2-anilino ring in many
cases provide substantial increases in potency, and this
was explored in the 6-phenylpyrido[2,3-d]pyrimidin-
7(8H)-ones with the compounds of Table 2 (ortho- and
meta- X-substituted compounds were also evaluated,
but were less active than the para-substituted com-
pounds reported; data not shown). Compounds 42–45
log IC50 ðWee1Þ ¼ 0:75ðÆ0:14Þ log IC50 ðSrcÞ
þ 1:27ðÆ0:14Þ
½n ¼ 26; r ¼ 0:74; F ¼ 30ꢀ:

1934
B. D. Palmer et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1931–1935
c-Src. This is less clear in the 20,60-diMe series, but even
Me
Me
R
the cationic analog 62 has a Wee1/c-Src IC50 ratio of
1.5-fold, compared with 43-fold for the corresponding
5-H analog 46.
R
O
Ar
O
iii
vi
N
N
N
Ar'
MeS
N
NHMe
MeS
N
N
Me
N
H
N
N
Me
6: R=CHO
i
ii
iv
v
R=H
compounds of Table 3
The primary finding from this study is that variation of
the substituents on the 6-phenyl ring does not signifi-
cantly improve the selectivity of the compounds for
Wee1 over c-Src, which was a primary goal. Solubilizing
substituents off the 2-anilino ring in many cases did pro-
vide substantial increases in potency for Wee1, with an
overall improvement of the Wee1/c-Src IC50 ratio to
about 10-fold, but few compounds were better than this.
5-Alkyl substitution did improve the ratio, but at the ex-
R=C(Me)OH
R=COMe
R=Br
R=Ar
Scheme 2. Reagents: (i) MeMgBr/THF; (ii) MnO2; (iii) (EtO)2-
P(O)CH2CO2Et/NaH; (iv) NBS/(PhCO2)2; (v) ArB(OH)2/Pd(PPh3)4/
AsPh3/Ba(OH)2; (vi) m-CPBA, then Ar0NH2.
use neutral amide side chains, and were about fivefold
more potent than 7 against Wee1 with little change in
c-Src potency, improving the ratio from about 100-fold
to only about 10-fold in favor of c-Src. Compounds 1,
46–50 explored cationic side chains of varying pKa,
but while in general these showed increased Wee1 po-
tency, c-Src activity also increased. The anionic analogs
51–53 showed better ratios, with the butyric acid 51
being equally potent toward both Wee1 and c-Src in
the enzyme inhibition assay. Compounds 54–57 incor-
porate a cationic or anionic side chain with the most po-
tent of the 6-phenyl ring substitution patterns, but
comparison of 54, 55 with 27, and 56, 57 with 33, sug-
gests the motifs are not additive.
Table 3. 2-Anilino-5-methyl-6-phenylpyrido[2,3-d]pyrimidin-7(8H)-ones
3'
Z
4'
5'
2'
Me
5
X
N
6'
N
N
O
N
H
Me
No.
X
Z
Mp (°C) Ma IC50 (lM)b
Wee1 c-Src
Finally, in Table 3 we explore a limited series of 5-
methyl substituted compounds. Difficulties of synthesis
precluded the preparation of all but a few analogs
(20,60-dichloro compounds could not be obtained), but
for the 20-Cl series that could be prepared the 5-methyl
group clearly distinguishes in favor of Wee1 over
58
59
60
61
62
H
H
H
250–253
258–262
182–184
4
4
4
4
4
>50
0.41
0.55
1.2
>50
>50
>50
15
20-Cl
O(CH2)2NEt2 20-Cl
H
2,60-DiMe 215–217
O(CH2)2NEt2 20,60-DiMe 155–159
0.54
0.36
a,b As for Table 1.
Table 2. 2-Anilino-6-phenylpyrido[2,3-d]pyrimidin-7(8H)-ones: solubilizing substituents
3'
Z
Cl
4'
5'
X
N
Cl
N
N
O
N
H
Me
No.
X
Z
Ma
Mp °C
IC50 (lM)b
Wee1
c-Src
42
43
44
45
1
CH2CONH2
H
H
H
H
H
Hc
H
H
H
H
H
H
H
2
2
2
2
268–270
141–144
188–193
199–201
Ref. 1
0.12
0.19
0.013
0.030
0.028
0.014
0.007
0.023
0.004
0.004
0.007
0.011
0.032
0.009
0.025
0.006
0.099
0.008
0.006
(CH2)2CONH2
(CH2)4CONH2
OCH2CONH2
0.26
0.25
O(CH2)2NEt2
O(CH2)2NEt2
O(CH2)3CO2H
0.165
0.99
46c
47
48
49
50
51
52
53
54
55
56
57
2
2
3
3
3
2
2
2
2
2
2
2
122–125
146–149
152
0.086
0.095
0.124
0.142
0.032
0.09
(CH2)3CO2(CH2)2Nmorph
(CH2)3CO2(CH2)2NMe2
(CH2)3CO2(CH2)2Npip
(CH2)3CO2H
143–146
147–149
231–237
242
CH2CH(NH2)CO2H
(CH2)3tetrazole
O(CH2)2NEt2
178
0.069
0.15
0.04
30-OH
30-OH
40-OH
40-OH
180–186
185–192
220–228
210–212
O(CH2)3CO2H
O(CH2)2NEt2
O(CH2)3CO2H
0.08
0.04
a,b As for Table 1.
c 20,60-Dimethyl derivative.

B. D. Palmer et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1931–1935
1935
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analogs also showed potent inhibition of other kinases,
including Chk1, PhosK, and PKC (data not shown).
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