Discovery of substituted 4-anilino-2-arylpyrimidines as a new series of apoptosis inducers using a cell- and caspase-based high throughput screening assay. 2. Structure-activity relationships of the 2-aryl group

Article abstract of DOI:10.1016/j.bmcl.2009.02.074

As a continuation of our efforts to discover and develop the apoptosis inducing 4-anilino-2-(2-pyridyl)pyrimidines as potential anticancer agents, we explored replacing the 2-pyridyl group by other aryl groups. SAR studies showed that the 2-pyridyl group

Full text of DOI:10.1016/j.bmcl.2009.02.074

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2305–2309
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of substituted 4-anilino-2-arylpyrimidines as a new series
of apoptosis inducers using a cell- and caspase-based high throughput
screening assay. 2. Structure–activity relationships of the 2-aryl group
Nilantha Sirisoma, Azra Pervin, Bao Nguyen, Candace Crogan-Grundy, Shailaja Kasibhatla,
*
Ben Tseng, John Drewe, Sui Xiong Cai
EpiCept Corporation, 6650 Nancy Ridge Drive, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
As a continuation of our efforts to discover and develop the apoptosis inducing 4-anilino-2-(2-pyri-
dyl)pyrimidines as potential anticancer agents, we explored replacing the 2-pyridyl group by other aryl
groups. SAR studies showed that the 2-pyridyl group can be replaced by a 3-pyridyl, 4-pyridyl and 2-pyr-
azinyl group, and that the SAR for the anilino group was similar to that of the 2-pyridyl series. However,
replacement of the 2-pyridyl group by a phenyl group, a 3,5-dichloro-4-pyridyl group, or a saturated ring
led to inactive compounds. Several potent compounds, including 2f, 3d, 3j and 4a, with EC50 values of
Received 30 January 2009
Revised 17 February 2009
Accepted 19 February 2009
Available online 23 February 2009
Keywords:
Apoptosis inducers
Anticancer agents
HTS
0.048–0.024
studies. In a tubulin polymerization assay, compound 2f, which was active against all the three cell lines
tested (T47D, HTC116 and SNU398), inhibited tubulin polymerization with an IC50 value of 0.5 M, while
compound 2a, which was active against T47D cells but not active against HTC116 and SNU398 cells, was
not active in the tubulin assay at up to 50 M.
lM in the apoptosis induction assay against T47D cells, were identified through the SAR
l
SAR
l
Ó 2009 Elsevier Ltd. All rights reserved.
Apoptosis, also called programed cell death, is a highly regu-
lated process of cellular suicide and is known to play a vital role
for the elimination of damaged or excess cells. Inadequate apopto-
sis is one of the hallmarks of cancers that result in excessive tumor
cell growth and tumor resistance to chemotherapeutic treatment.1
The pathways of apoptosis involve the activation of a family of cys-
teine proteases called caspases that is essential for the initiation as
well as execution of apoptosis.2 Caspase-3 is the main executioner
of apoptosis and cleaves many protein substrates leading to cell
death.3 Since it is known that many chemotherapeutics induce
apoptosis in cancer cells,4 targeting apoptosis regulators to pro-
mote apoptosis is a promising strategy for anticancer drug discov-
studies of the anilino group showed that a small and electron-donat-
ing group, such as an OMe and OH group, at the meta-position is
important for apoptosis inducing activity. An anilino substituted
with small groups at the 2,5-positions, such as 2,5-dimethoxy (1g),
was found to increase the potency >10-fold versus the mono-
meta-substituted analogs.13 Herein we report the SAR of the 2-aryl
group of 4-anilino-2-arylpyrimidines as apoptosis inducers.
The 4-anilino-2-(3-pyridyl)-6-(trifluoromethyl)pyrimidines
(2a–n) (Table 1) were synthesized as shown in Scheme 1, from
reaction of 4-chloro-2-(3-pyridyl)-6-(trifluoromethyl)pyrimidine
(7) with a substituted aniline.13 Compound 7 was prepared follow-
ing the literature procedure14 from reaction of nicotinamidine (8)
with ethyl 4,4,4-trifluorobut-2-ynoate (9) in the presence of KOH
in EtOH to produce the intermediate 2-(3-pyridyl)-6-(trifluoro-
methyl)pyrimidin-4(3H)-one (10), followed by reaction with POCl3
to convert the ketone to the chloride. The 4-anilino-2-(4-pyridyl)-
6-(trifluoromethyl)pyrimidines (3a–j) (Table 2), 4-anilino-2-(2-
pyrazinyl)-6-(trifluoromethyl)pyrimidines (4a–d) (Table 3) were
prepared similar via reaction of 4-chloro-2-(4-pyridyl)-6-(trifluo-
romethyl)pyrimidine (11) and 4-chloro-2-(4-pyrazinyl)-6-(trifluo-
romethyl)pyrimidine (12) with a substituted aniline (Scheme 2).
Similarly, compounds 5a–b and 6a–d (Table 4) were prepared via
reaction of the corresponding substituted 4-chloropyrimidine with
the corresponding substituted aniline. Compound 5c was obtained
from hydrogenation of 1f under acidic conditions (Scheme 3).
ery.5 We have therefore developed
a cell-based Anti-cancer
Screening Apoptosis Program (ASAP) for the discovery of apoptosis
inducers,6 utilizing HTS assays with our proprietary fluorescent
caspase-3 substrate.7
Applying this assay, we have discovered several series of novel
apoptosis inducers including N-phenyl nicotinamide (1a),8 4-aryl-
4H-chromenes (1b),9 gambogic acid (1c),10 3-aryl-5-aryl-1,2,4-oxa-
diazoles (1d)11 and 4-anilinoquinazolines (1e)12 (Chart 1). More re-
cently, we have reported the discovery and SAR study of 4-anilino-2-
(2-pyridyl)pyrimidines (1f) as potent apoptosis inducers.13 SAR
* Corresponding author. Tel.: +1 858 202 4006; fax: +1 858 202 4000.
E-mail address: scai@epicept.com (S.X. Cai).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.074

2306
N. Sirisoma et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2305–2309
O
OMe
OH
MeO
Br
NO2
O
H
N
O
O
O
N
O
CN
O
OEt
N
O
NH2
OH
1c
1a
1b
CF3
OMe
R
O
Cl
N
N
N
CF3
N
N
N
N
N
S
H
O
N
Cl
1f, R = H
1g, R = OMe
1d
1e
Chart 1.
The apoptosis inducing activity of these 4-anilino-2-arylpyrim-
idines was measured by our cell- and caspase-based HTS assay8 in
human breast cancer cells T47D, human colon cancer cells HCT116
and hepatocellular carcinoma cancer cells SNU398 and the results
are summarized in Tables 1–4. Since it has been found that a small
and electron-donating group at the 3-position of the anilino ring
was important for the apoptosis inducing activity of 4-anilino-2-
(2-pyridyl)pyrimidines,13 we first explored the SAR of substituents
at the 3-position of the anilino ring of 4-anilino-2-(3-pyridyl)-6-
(trifluoromethyl)pyrimidines (Table 1). Table 1 shows that the 3-
in T47D, and is inactive up to 10
l
M against HCT116 and SNU398
cells, showing that 2a is selective against certain cancer cell lines.
The 3-OH analog 2b is about threefold less active than 2a. With an
electron-withdrawing group or a large group at the 3-position,
analogs 2c (3-Cl) and 2d (3-Ph) were inactive up to 10 lM. Simi-
larly, the 4-methoxy analog 2e also was inactive. These data indi-
cate that the SAR of the anilino group for the 3-pyridyl compounds
is similar to the previously reported SAR of 2-pyridyl compounds.13
We then explored 2,5-disubstituted-anilino analogs of 2a. Sim-
ilar to what was observed previously for 4-anilino-2-(2-pyri-
dyl)pyrimidines,13 2,5-disubstituted analogs of 2a in general
OMe analog 2a had an EC50 value of 0.34 lM for caspase activation
Table 1
SAR of 4-anilino-2-(3-pyridyl)-6-(trifluoromethyl)pyrimidines in the caspase activation assay
CF3
R4
H
R3
R2
N
N
N
N
H
R1
Entry
R1
R2
R3
R4
H
OMe
H
H
H
H
H
OMe
Me
Cl
OMe
OPh
EC50 (l
M)a
T47D
HCT116
SNU398
1fb
1gb
2a
2b
2c
2d
2e
2f
H
OMe
H
H
H
H
H
OMe
OMe
OMe
Cl
OMe
H
OMe
OH
Cl
Ph
H
H
H
H
H
H
H
H
H
H
H
H
OMe
H
H
0.36 0.17
0.018 0.001
0.34 0.01
0.97 0.02
>10
6.0 0.2
0.19 0.03
>10
>10
>10
3.7 0.7
0.11 0.02
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.024 0.005
0.096 0.009
0.36 0.02
0.14 0.01
1.5 0.1
0.17 0.03
0.13 0.01
0.61 0.05
0.93 0.23
3.3 0.3
0.13 0.01
0.13 0.03
0.44 0.04
0.82 0.22
5.0 0.5
2g
2h
2i
H
H
H
2j
OMe
N
2k
H
H
OMe
>10
>10
>10
2l
2m
2n
Me
Cl
H
H
H
OMe
H
H
H
OH
OH
OMe
0.087 0.010
0.23 0.04
0.082 0.003
>10
>10
2.9 0.6
5.5 0.1
>10
1.4 0.1
a
Cells were treated with the test compounds for 48 h, data are the mean of three or more experiments and are reported as mean standard error of the mean (SEM).
2-Pyridyl compounds, please see Chart 1 for structure.
b

N. Sirisoma et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2305–2309
2307
CF3
CF3
N
CF3
NH
NH2
CF3
N
N
N
R
KOH, EtOH
O
R-Ph-NH2
H2O/HCl
+
O
R
N
N
Cl
Ar
N
Ar
H
H
N
O
N
11, Ar = 4-pyridyl
12, Ar = 2-pyrazinyl
3a-3j, Ar = 4-pyridyl
4a-4d, Ar = 2-pyrazinyl
8
9
10
CF3
CF3
N
Scheme 2.
N
N
R-Ph-NH2
H2O/HCl
POCl3
N
N
Cl
N
N
H
were found to be more potent than 3-substituted analogs. The 2,5-
dimethoxy compound 2f was the most potent analog, with an EC50
7
2a-2n
value of 0.024
than 2a. Compounds 2g, 2h and 2i also were highly active with
EC50 values of 0.096–0.36 M at T47D cells. Interestingly, these
lM at T47D cells, and was >10-fold more potent
Scheme 1.
l
2,5-disubstituted analogs were active in all the three cell lines
tested, suggesting that the 2,5-disubstituted analogs and the 3-
substituted analogs may not have the same mechanism of action.
2,5-Disubstituted analogs with a large group at the 5-position
(2j, OPh) or 2-position (2k, piperidino) were >50-fold less active
than 2f. Interestingly, compounds 2l and 2m, with a 5-OH group
and a 2-Me or 2-Cl group, respectively, were highly active in
T47D cells but had very low activity in HCT116 and SNU398 cells,
indicating that they were selective against certain cancer cells sim-
ilar to that of 2a. The 3,5-dimethoxy compound 2n also was highly
Table 2
SAR of 4-anilino-2-(4-pyridyl)-6-(trifluoromethyl)pyrimidines in the caspase activa-
tion assay
CF3
R4
H
R3
R2
N
active in the T47D cells with an EC50 value of 0.082 lM. Interest-
N
N
H
ingly, it was >15-fold less active in the other two cell lines tested,
indicating that it was selective against certain cancer cells.
We then explored the SAR of 4-anilino-2-(4-pyridyl)-6-(trifluo-
romethyl)pyrimidines (Table 2). Similar to what is observed for the
2-pyridyl and 3-pyridyl analogs, the 3-OMe analog 3a and 3-OH
analog 3b were active in the T47D cells while the 4-methoxy ana-
log 3c was inactive. 2,5-Disubstituted analogs (3d–g) were found
to be highly potent, with 3d the most potent one with an EC50 va-
N
R1
Entry
R1
R2
R3
R4
EC50 (l
M)a
T47D
HCT116
SNU398
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
H
H
Me
Cl
OMe
Me
Me
Cl
OMe
OH
H
H
H
OMe
H
H
H
H
H
H
H
H
H
OMe
OMe
Me
Me
OH
OH
OMe
1.3 0.8
0.68 0.01
>10
0.025 0.004
0.10 0.01
0.076 0.01
0.12 0.01
0.048 0.008
0.077 0.007
0.034 0.016
>10
3.1 0.7
>10
0.12 0.01
0.96 0.26
0.16 0.03
0.61 0.39
2.0 0.2
1.1 0.3
0.24 0.09
>10
NDb
>10
ND
ND
ND
ND
ND
ND
ND
H
H
H
H
H
H
lue of 0.025 lM. Similar to what was observed for compounds 2l
and 2m, analogs 3 h and 3i, with an OH group at the 5-position,
were highly active in T47 D cells but had low activity in HCT116
cells. The 3,5-dimethoxy compound 2j also was highly active in
the T47D cells with an EC50 value of 0.034 lM.
3j
H
OMe
H
The 4-anilino-2-(2-pyrazinyl)-6-(trifluoromethyl)pyrimidines
(Table 3) were found to have a similar SAR as the pyridyl-pyrimi-
dines (Table 1 and 2). 2,5-Di-methoxy analog 4a was highly potent,
a
Cells were treated with the test compounds for 48 h, data are the mean of three
or more experiments and are reported as mean standard error of the mean (SEM).
b
ND, not determined.
with an EC50 value of 0.048 lM. Compounds 4b and 4c, with a OH
group at the 5-position, were highly active in T47 D cells but had
low activity in HCT116 and SNU398 cells. The 3,5-dimethoxy com-
pound 4d also was highly active in the T47D cells but inactive in
HCT116 and SNU398 cells up to 10 lM.
We then explored the replacement of the 2-pyridyl group in 1f
by other ring systems (Table 4). Compound 5a, with the 2-pyridyl
Table 3
SAR of 4-anilino-2-(2-pyrazinyl)-6-(trifluoromethyl)pyrimidines in the caspase acti-
vation assay
group in 1f replaced by a phenyl group, was inactive up to 10 lM,
R4
indicating that the nitrogen in 1f, as well as in 2a and 3a, is impor-
tant for the apoptosis inducing activity. Replacing the 4-pyridyl
group in 3e by a 3,5-dichloro-4-pyridyl group also led to an inac-
tive compound (5b), suggesting that substitutions in this ring are
not preferred. Compound 5c, with a saturated 2-piperidyl group
CF3
H
R3
R2
N
N
N
N
N
H
R1
Entry
R1
R2
R3
R4
EC50 (l
M)a
OMe
OMe
CF3
CF3
T47D
HCT116
SNU398
H2/HCl/Pd
MeOH
N
N
H
4a
4b
4c
4d
OMe
Me
Cl
H
H
H
OMe
H
H
H
H
OMe
OH
OH
0.048 0.006
0.19 0.01
0.56 0.22
0.13 0.01
0.41 0.19
>10
>10
0.28 0.03
>10
>10
N
N
N
N
N
N
H
H
H
OMe
>10
>10
1f
5c
a
Cells were treated with the test compounds for 48 h, data are the mean of three
or more experiments and are reported as mean standard error of the mean (SEM).
Scheme 3.

2308
N. Sirisoma et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2305–2309
Table 4
SAR of substituted 4-anilinopyrimidines in the caspase activation assay
R2
N
OMe
R3
N
R1
N
H
R4
Entry
R1
R2
R3
H
R4
EC50 (l
M)a
T47D
>10
HCT116
SNU398
>10
5a
CF3
H
>10
Cl
5b
5c
6a
6b
6c
CF3
H
H
H
Cl
>10
>10
>10
N
Cl
H
N
CF3
H
>10
>10
>10
N
N
N
N
OMe
0.42 0.12
0.33 0.21
0.16 0.04
H
OMe
OMe
H
H
>10
>10
>10
H
OMe
>10
>10
>10
N
6d
Me
Cl
>10
>10
>10
O
a
Cells were treated with the test compounds for 48 h, data are the mean of three or more experiments and are reported as mean standard error of the mean (SEM).
replacing the 2-pyridyl group in 1f, was inactive up to 10
cating that a plainer aromatic ring is needed for activity.
l
M, indi-
assays in T47D, HCT116 and SNU398 cells were run in a 96-well
microtiter plate as described previously6 and the data are summa-
rized in Table 5. Compound 2a was found to be highly active with a
We also explored other substitutions in the pyrimidine ring. Pre-
viously, we found that the trifluoromethyl group in 1f and 1g can be
replaced by a methyl group.13 Compound 6a, with the trifluoro-
methyl group in 1g replaced by a 2-pyridyl group, was >10-fold less
active than 1g in T47D cells, but had similar activity in the HCT116
and SNU398 cells, indicating that the relatively large 2-pyridyl
group is tolerated at the 6-position of the pyrimidine ring. Com-
pounds 6b and 6c, with the 6-trifluoromethyl group in 1f and 1g re-
GI50 value of 0.094 lM in T47D cells, and was >50-fold less active
against HCT116 and SNU398 cells, confirming that 2a was selective
against certain cancer cells. Compounds 2n and 3j, found to be
selective in the caspase assay, were also selective in the growth
inhibition assay. In comparison, compounds 2f and 6a were equi-
potent in all three cell lines, confirming that they are broadly
active.
placed by a 5-methoxy group, were not active up to 10
suggesting that substitution in the 5-position is not preferred. Com-
l
M,
The apoptosis-inducing activity of the potent analog 2f was also
characterized by cell cycle analysis. T47D cells were treated with
pound 6d, with a morpholino group at the 2-position of pyrimidine,
0.05 lM of compound 2f for 24 h or 48 h at 37 °C, then stained
also was inactive up to 10
l
M, confirming the above observation
with propidium iodide and analyzed by flow cytometry (Fig. 1).
An increase in G2/M population (from 13% to 53%) was observed
after 24 h treatment with 2f, together with an increased apoptotic
sub-G1 population (from 1% to 7%). Sub-G1 population was in-
creased to 19% after 48 h treatment with 2f, indicating that many
with 5c that a saturated ring is not tolerated at that position.
Selected compounds were also tested by the traditional inhibi-
tion of cell growth (GI50) assay to confirm that the active com-
pounds can inhibit tumor cell growth. The growth inhibition

N. Sirisoma et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2305–2309
2309
Table 5
Since compound 2f arrested cells at G2/M followed by induction
of apoptosis, which is similar to known tubulin inhibitors such as
colchicine, we suspected that 2f might be a tubulin inhibitor. In a
tubulin polymerization assay,9b compound 2f was found to inhibit
Inhibition of cell growth of 4-anilino-2-(3-pyridyl)-6-(trifluoromethyl)pyrimidines
Entry
GI50 (l
M)a
T47D
HCT116
SNU398
6.4 0.1
0.068 0.010
1.0 0.1
0.27 0.01
0.41 0.09
polymerization of tubulin with an IC50 value of 0.5 lM, indicating
2a
2f
2n
3j
0.094 0.007
0.095 0.010
0.061 0.031
0.036 0.006
0.24 0.01
>10
that 2f most probably induces apoptosis through inhibiting tubulin
polymerization, which is in agreement with its broad activity
against the three cancer cell lines tested. In comparison, compound
2a, which was active against T47D cells but was inactive against
colon cancer HCT116 and liver cancer SNU398 cells, was found
0.18 0.04
1.3 0.2
0.27 0.02
0.13 0.02
6a
a
Cells were treated with the test compounds for 48 h, data are the mean of three
or more experiments and are reported as mean standard error of the mean (SEM).
not to inhibit tubulin polymerization at up to 50 lM, suggesting
that 2a should have a different mechanism of action from that of
2f. Similarly, compounds 2l and 2n, both of which showed activity
against T47D cells but were much less active against colon cancer
HCT116 and liver cancer SNU398 cells, were found not to inhibit
tubulin polymerization at up to 50 lM.
In conclusion, we have explored the SAR of the 2-aryl group of
4-anilino-2-arylpyrimidines as apoptosis inducers. Our studies
showed that the 2-pyridyl group in our previously reported apop-
tosis inducing 4-anilino-2-(2-pyridyl)pyrimidines can be replaced
by a 3-pyridyl, 4-pyridyl and 2-pyrazinyl group, and these com-
pounds maintain similar SAR for the anilino group. However,
replacement of the 2-pyridyl group by a phenyl group, or a 3,5-di-
chloro-4-pyridyl group, or a saturated 2-piperidyl or morpholino
group, resulted in inactive compounds. In addition, replacement
of the 6-trifluoromethyl group in the pyrimidine ring by a 6-(2-
pyridyl) group was tolerated while its replacement by a 5-methoxy
group led to inactive compounds. Through these studies, several
low nanomolar compounds, both selective and nonselective were
identified in the apoptosis induction assay against T47D cells.
Compounds that were active against all the three cell lines
(T47D, HCT116 and SNU398), such as 2f, were found to be inhibi-
tors of tubulin polymerization, which most probably is the mech-
anism of action in inducing apoptosis. Compounds that were
selective against T47D cells, such as 2a, 2l and 2n, were found to
be inactive in the tubulin assay and the primary cellular target
remains to be defined.
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(%)
Entry
1A
Sub(%)
G1
S(%) G2/M(%)
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1
7
78
7
6
13
53
1B
32
31
1C
19
7
43
Figure 1. Drug-induced apoptosis in T47D cells as measured by flow cytometric
analysis. The x-axis is the fluorescence intensity and the y-axis is the number of
cells with that fluorescence intensity. (A) Control cells showing most of the cells in
10. Zhang, H.-Z.; Kasibhatla, S.; Wang, Y.; Herich, J.; Guastella, J.; Tseng, B.; Drewe,
J.; Cai, S. X. Bioorg. Med. Chem. 2004, 12, 309.
G1 phase of the cell cycle. (B) Cells treated with 0.05
showing most of the cells arrested in G2/M phase. (C) Cells treated with 0.05
compound 2f for 48 h showing a progression from G2/M to cells with sub-diploid
l
M of compound 2f for 24 h
M of
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DNA content, which are apoptotic cells with fragmented nuclei.
cells were apoptotic. These data showed that compound 2f ar-
rested cancer cells in G2/M, indicative of an antimitotic effect, fol-
lowed by induction of apoptosis.