Discovery of 4-anilino-N-methylthieno[3,2-d]pyrimidines and 4-anilino-N-methylthieno[2,3-d]pyrimidines as potent apoptosis inducers

Article abstract of DOI:10.1016/j.bmcl.2009.04.145

We report the discovery of N-((benzo[d][1,3]dioxol-5-yl)methyl)-6-phenylthieno[3,2-d]pyrimidin-4-amine (2a) as an apoptosis inducer using our proprietary cell- and caspase-based ASAP HTS assay, and SAR study of HTS hit 2a which led to the discovery of 4-a

Full text of DOI:10.1016/j.bmcl.2009.04.145

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3536–3540
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 4-anilino-N-methylthieno[3,2-d]pyrimidines and
4-anilino-N-methylthieno[2,3-d]pyrimidines as potent apoptosis inducers
*
William Kemnitzer, Nilantha Sirisoma, Chris May, 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:
We report the discovery of N-((benzo[d][1,3]dioxol-5-yl)methyl)-6-phenylthieno[3,2-d]pyrimidin-4-
amine (2a) as an apoptosis inducer using our proprietary cell- and caspase-based ASAP HTS assay, and
SAR study of HTS hit 2a which led to the discovery of 4-anilino-N-methylthieno[3,2-d]pyrimidines and
4-anilino-N-methylthieno[2,3-d]pyrimidines as potent apoptosis inducers. Compounds 5d and 5e were
Received 24 March 2009
Revised 28 April 2009
Accepted 30 April 2009
Available online 5 May 2009
the most potent with EC50 values of 0.008 and 0.004 lM in T47D human breast cancer cells, respectively.
Compound 5d was found to be highly active in the MX-1 breast cancer model. Functionally, compounds
5d and 5e both induced apoptosis through inhibition of tubulin polymerization.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Apoptosis inducers
Anticancer agents
HTS
SAR
In vivo activity
Programmed cell death, or apoptosis, plays an important role in
the development of cancers.1 Excessive or improper inhibition of
apoptosis could lead to unchecked cell proliferation, and resistance
to cancer treatment.2 In addition, it is known that many anticancer
agents, including irinotecan3 and vinblastine4 kill tumors at least
partially through induction of apoptosis.5 Further exploration of
novel approaches to activate and promote apoptosis in cancer
cells,6 therefore, could lead to the discovery of new anticancer
agents.7
In our effort to discover and develop novel apoptosis inducers as
potential anti-cancer drugs, we have created a cell-based high-
throughput screening (HTS) assay for the identification of apopto-
sis inducers using our novel fluorescent caspase-3 substrate.8,9
Since caspase-3 is known to be the key effector caspase that
cleaves multiple protein substrates in cells leading to cell death,10
our HTS assay can discover apoptosis inducers that interact with
known or unknown targets through activation of any of the proa-
poptotic pathways upstream of caspase-3.
We have reported the discovery and structure–activity relation-
ship (SAR) studies of several series of apoptosis inducers interact-
ing with either known or novel molecular targets using our HTS
assay.11 These include gambogic acid (1a)12,13 4-aryl-4H-chrom-
enes (1b),14,15 and 3-aryl-5-aryl-1,2,4-oxadiazoles (1c)16,17 (Chart
1). More recently, we have reported the discovery of 2-chloro-4-
(4-methoxyanilino)-N-methylquinazoline (1d)18 and 4-(4-meth-
oxyanilino)-N,2-dimethylquinazoline (1e),19 a clinical candidate,
as potent apoptosis inducers with high in vivo efficacies in antican-
cer xenograft models.20 Herein we report the discovery of N-
((benzo[d][1,3]dioxol-5-yl)methyl)-6-phenylthieno[3,2-d]pyrimi-
din-4-amine (2a) as an apoptosis inducer using our HTS assay, and
SAR study around 2a which led to the discovery of a series of 4-ani-
lino-N-methylthieno[3,2-d]pyrimidines and 4-anilino-N-methyl-
thieno[2,3-d]pyrimidines as low nanomolar apoptosis inducers.
Many 4-anilinothieno[3,2-d]pyrimidines such as 1g,21 1h22 and
1i23 (Chart 1) have been reported as potent kinase inhibitors. Struc-
turally related compounds 3a, 3d and 3g–3h were prepared as
shown in Scheme 1 using previously reported procedures from
reaction of commercially available 4-chlorothieno[3,2-d]pyrimi-
dine (6a) with the corresponding substituted anilines in anhydrous
isopropanol (IPA) in the presence of concentrated HCl.18,19
N-((Benzo[d][1,3]dioxol-5-yl)methyl)thieno[3,2-d]pyrimidin-4-
amine (2b) and N-(benzo[d][1,3]dioxol-6-yl)-6-phenylthieno[3,2-
d]pyrimidin-4-amine (2c, Chart 2) were prepared similarly from
reaction of 6a with (benzo[d][1,3]dioxol-5-yl)methanamine, and
4-chloro-6-phenylthieno[3,2-d]pyrimidine (6b) with benzo[d]-
[1,3]dioxol-5-amine, respectively. Compound 2a was obtained from
Maybridge. Compounds 3b–3c and 3e–3f were prepared in two
steps via reaction of 6a with substituted anilines to produce com-
pounds 7a–7d, followed by methylation using reported procedures
(Scheme 2).18,19 Compounds 4a, 4c–4f were prepared from reaction
of the corresponding substituted 4-chlorothieno[3,2-d]pyrimidines
(6c–6g) with N-methyl-4-methoxyaniline (Scheme 3). The thie-
no[2,3-c]pyridine analog 4g (Table 2) was prepared under similar
conditions from reaction of 7-chlorothieno[2,3-c]pyridine with
N-methyl-4-methoxyaniline. Compound 4b was prepared from
* 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.2009.04.145

W. Kemnitzer et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3536–3540
3537
OMe
CO2H
O
OMe
MeO
Br
N
N
Cl
O
O
O
O
Cl
N
N
CN
S
N
R
O
N
OH
O
NH2
N
1d, R = Cl
1e, R = Me
1a
1b
1c
Cl
H
N
O
HN
N
Br
N
N
HN
N
S
HN
MeO2S
HN
N
OMe
N
S
N
S
N
F
Cl
O
N
1g
1f
1h
1i
Chart 1.
R2
R2
R1
Cl
N
R2
3a, R1 = Me, R2 = 4-OMe
3d, R1 = Me, R2 = 4-CO2Me
3g, R1 = Me, R2 = 2,5-OMe
3h, R1 = H, R2 = 4-OMe
N
N
Cl
N
HN
N
a
S
N
N
a
S
S
b
N
N
S
S
N
N
6a
N
6a
7a, R2 = 3-OMe
7b, R2 = 2-OMe
3b, R2 = 3-OMe
3c, R2 = 2-OMe
Scheme 1. Reagents: (a) R2-Ph-NHR1, i-PrOH, HCl.
7c, R2 = 3,4-OMe
7d, R2 = 3,5-OMe
3e, R2 = 3,4-OMe
3f, R2 = 3,5-OMe
coupling of 4a with pyridine-3-boronic acid using reported proce-
dure (Scheme 4).22 Compounds 5a–5h were prepared from reaction
of the commercial available substituted 4-chlorothieno[2,3-
d]pyrimidines (8a–8f) with N-methyl-4-methoxyaniline or 4-
methoxyaniline, respectively (Scheme 5).24
aConditions: a) R2-Ph-NH2, i-PrOH, HCl; b) MeI, NaH, DMF
Scheme 2. Reagents: (a) R2-Ph-NH2, i-PrOH, HCl; (b) MeI, NaH, DMF.
Theapoptosisinducingactivityof4-anilino-N-methylthieno[3,2-
d]pyrimidines, 4-anilino-N-methylthieno[2,3-d]pyrimidines and
related compounds was measured by our cell- and caspase-based
HTS assay25 in two cell lines, humanbreast cancer cells T47D and hu-
man non-small cell lung cancer cells H1299, and the results are sum-
OMe
4a, R1 = 6-I, R2 = H
Cl
N
4c, R1 = 7-Me, R2 = H
N
N
4d, R1 = H, R2 = Me
4e, R1 = H, R2 = Ph
4f, R1 = 7-Me, R2 = Me
a
marizedinTables1–3. Thescreeninghit2ahadanEC50 valueof 1 lM
S
S
N
N
in T47D cells. Compound 2a has a C log P value of 5.4, which is con-
sidered high for a drug candidate. We first explored the removal of
the bulky 6-phenyl group. Unfortunately, compound 2b was found
R1
R1
R2
R2
6c-6g
to be inactive in T47D cells up to 10 lM. To make the molecule more
Scheme 3. Reagents: (a) 4-OMe-Ph-NHMe, i-PrOH, HCl.
rigid, we eliminated the methylene linker in 2a, resulting in 2c that
also was inactive in T47D cells. We observed some structural simi-
larity between 2b–2c and 1d–1e and decided to introduce a methyl
group into the nitrogen of the anilino group (Chart 2), which was
known to be important for the apoptotic activity of 1d and 1e.18,19
Since compound 1f, with an extra methylene linker, has been found
to be >30-fold less active than compound 1d,18 we decided to con-
centrate our SAR studies on compounds with an anilino and not with
a benzylamino group.
R1
OMe
O
N
O
O
O
O
R1
N
HN
HN
N
O
HN
S
and
N
S
S
and
N
N
R2
N
S
N
R2
Ph
R4
Ph
N
N
R3
S
R3
3a-3g, 4a-4f
N
N
2c
2a
2b
5a-5f
Chart 2.

3538
W. Kemnitzer et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3536–3540
Table 2
OMe
Activity of 4-(4-methoxyanilino)-N-methylthieno[3,2-d]pyrimidines and related
B(OH)2
compound in the caspase activation assay
a
N
N
4a
+
S
OMe
N
N
N
N
4b
4
S
N
2
Scheme 4. Reagents: (a) DMF, K2CO3, DPPF, (PhCN)2PdCl2.
R1
6
A
R3
7
R2
OMe
R1
R2
R3
A
EC50 (lM)
a
Compd #
R4
R1
Cl
N
R1
N
N
T47D
H1299
a
4a
4b
4c
4d
4e
4f
I
H
H
Me
H
H
H
H
H
Me
Ph
Me
H
N
N
N
N
N
N
C
>10
>10
>10
>10
N
R2
N
3-Pyb
H
R2
R3
S
0.56 0.04
0.014 0.001
0.34 0.01
0.24 0.01
>10
0.56 0.04
0.032 0.008
0.63 0.02
0.31 0.03
>10
R3
S
H
H
H
H
8a-8f
5a-5h
Me
H
4g
Scheme 5. Reagents: (a) 4-OMe-Ph-NHMe or 4-OMe-Ph-NH2, i-PrOH, HCl.
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
3-Pyridyl.
Table 1
Activity of 4-anilinothieno[3,2-d]pyrimidines and related compounds in the caspase
activation assay
Table 3
R4
Activity of 4-(4-methoxyanilino)thieno[2,3-d]pyrimidines in the caspase activation
assay
R3
R2
R5
OMe
N
R4
4
R1
S
7
R1
N
N
N
6
2
N
R2
N
R3
S
Compd #
R1
R2
R3
R4
R5
EC50 (lM)
a
R1
R2
R3
R4
EC50 (lM)
a
Compd #
T47D
H1299
T47D
H1299
2a
2b
2c
3a
3b
3c
3d
3e
3f
NAb
NA
NA
H
H
OMe
H
H
H
OMe
H
NA
NA
NA
H
OMe
H
NA
NA
NA
OMe
H
NA
NA
NA
H
H
H
H
H
OMe
OMe
H
NA
NA
NA
Me
Me
Me
Me
Me
Me
Me
H
1.0 0.1
>10
>10
0.052 0.004
>10
>10
0.19 0.01
1.7 0.1
>10
2.6 0.1
>10
0.70 0.06
>10
>10
0.24 0.10
>10
>10
0.34 0.01
2.8 0.2
>10
5.0 0.1
>10
5a
5b
5c
5d
5e
5f
5g
5h
H
H
H
Me
H
H
Me
H
H
NA
NA
H
H
H
Me
Me
Me
H
Me
NA
NA
Me
Me
Me
Me
Me
Me
H
H
NA
NA
0.022 0.003
0.015 0.003
0.024 0.004
0.008 0.0002
0.004 0.0001
0.021 0.005
>10
>10
0.002 0.0001
0.009 0.001
0.044 0.004
0.025 0.007
0.053 0.002
0.016 0.001
0.015 0.003
0.091 0.032
>10
>10
0.006 0.001
0.05 0.01
Me
Me
H
Me
Me
Me
Me
NAb
NA
H
H
CO2Me
OMe
H
H
OMe
OMe
OMe
H
3g
3h
1e
H
Colchicine
a
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).
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
b
NA, not applied, please see Chart 2 for structure.
NA, not applied.
Table 1 shows that compound 3a, with an N-methyl group and a
4-methoxy group at the 4-position of the anilino ring, was highly
Keeping the preferred N-methyl-4-(4-methoxyanilino) group at
the 4-position, we explored substitutions at the 2-, 6- and 7-posi-
tions of the thieno[3,2-d]pyrimidine ring (Table 2). Compounds 4a
and 4b, with an iodo or 3-pyridyl group in the 6-position, were
active with an EC50 value of 0.052
compound 3h, without the N-methyl group, was inactive up to
10 M, at least 200-fold less active than that of 3a, indicting that
lM in T47D cells. In comparison,
l
the N-methyl group is critical for the apoptotic activity of 4-anili-
no-N-methylthieno[3,2-d]pyrimidines. Exploration of substitution
at the anilino ring (3b–3g) showed that a small substitution at
the 4-position was important for apoptotic activity, while substitu-
tions at the 2- and 3-positions were not preferred. The SAR for sub-
stitutions at the anilino ring is similar to that of 4-anilino-N-
methylquinazolines,18,19 indicating that a thieno[3,2-d]pyrimidine
ring system can be used to replace the quinazoline ring system
in 1d and 1e.
found to be inactive up to 10 lM in T47D cells, suggesting that sub-
stitution at the 6-position might not be tolerated. Compound 4c,
with a methyl group at the 7-position, had an EC50 value of
0.56 lM, about 10-fold less active than 3a, indicating that substitu-
tion at the 7-position is better tolerated than the 6-position. The 2-
methyl analog 4d was found to be about fourfold more potent than
3a, while the 2-phenyl analog 4e was >20-fold less active than 4d,
indicating that a small group such as methyl at the 2-position
might be preferred, which is similar to the reported 2-position

W. Kemnitzer et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3536–3540
3539
SAR of 4-anilino-N-methylquinazolines.18,19 The 2,7-dimethyl ana-
log 4f was >2-fold more active than 4c, confirming that the 2-
methyl group contributed positively to the apoptotic activity. The
thieno[2,3-c]pyridine analog 4g was inactive up to 10 lM, indicat-
2000
1500
1000
500
0
Vehicle
7 mg/kg
35 mg/kg
50 mg/kg
75 mg/kg
ing that the nitrogen at the 1-position is important for activity.
We then explored the replacement of the thieno[3,2-d]pyrimi-
dine system in 3a by the isosteric thieno[2,3-d]pyrimidine system
(Table 3). Compound 5a was >2-fold more potent than 3a, suggest-
ing that the thieno[2,3-d]pyrimidine system might be preferred
over the thieno[3,2-d]pyrimidine system for apoptotic activity.
By maintaining the N-methyl-4-(4-methoxyanilino) group at the
4-position, substitutions at the 2-, 5- and 6-positions of the thie-
no[2,3-d]pyrimidine ring were explored. The 5-methyl analog 5b
was slightly more active than 5a, indicating that a small group is
preferred at the 5-position. The 5,6-dimethyl analog 5c was about
twofold less active than 5b, indicating that a small group at the 6-
position is well tolerated, and suggesting that the SAR of the 6-po-
sition of 4-anilino-N-methylthieno[2,3-d]pyrimidines is not the
same as that of 4-anilino-N-methylthieno[3,2-d]pyrimidines. The
2-methyl analog 5d was about threefold more potent than 5a,
which is similar to the observed 2-position SAR of 4-anilino-N-
methylthieno[3,2-d]pyrimidines as well as that of 4-anilino-N-
methylquinazolines.18,19 Combining the preferred methyl group
at the 2- and 5-positions led to compound 5e, which was highly ac-
0
2
4
6
8
10
12
14
16
Day After Initial Dose
Figure 1. Compound 5d inhibited the growth of established (ꢀ100 mm3) MX-1
tumor xenografts in Crl:Nu/Nu-nuBR mice. Compound 5d was dosed iv at 7 mg/kg
intravenous days 1–5, or 35, 50 and 75 mg/kg single intravenous administrations at
day 1. P value as calculated by Student’s t-test is <0.001 for 75 mg/kg.
d]pyrimidines as potent apoptosis inducers. 4-(4-Methoxyanili-
no)-N,2,5-trimethylthieno[2,3-d]pyrimidine 5e was found to be
tive with an EC50 value of 0.004 lM in T47D cells, a potency
the most potent compound having an EC50 value of 0.004 lM in
approaching that of 1e. The 2,5,6-trimethyl analog 5f was sev-
eral-fold less active than 5e. Compounds 5g and 5h, without the
N-methyl group, were both inactive up to 10 lM, >500-fold less ac-
T47D cells, and to inhibit tubulin polymerization, which most
probably is its main mechanism of action for apoptosis induction.
Compound 5d, one of the potent apoptosis inducers identified in
the series, was highly efficacious in a MX-1 human xenograft mod-
el and could be a potential clinical candidate.
tive than the corresponding N-methyl analogs 5b and 5e, confirm-
ing that similar to the SAR of 4-anilino-N-methylquinazolines,18,19
the N-methyl group is critical for apoptotic activity of 4-anilino-N-
methylthieno[3,2-d]pyrimidines as well as that of 4-anilino-N-
methylthieno[2,3-d]pyrimidines.
The activities of these compounds towards the human non-
small cell lung cancer cell line H1299 roughly parallel the activ-
ity in T47D cells. The compounds that were inactive in T47D
cells also were inactive in H1299 cells. Compounds 5d and 5e,
the most active ones in this series against T47D cells, were also
the most active ones against H1299 cells with EC50 values of
References and notes
1. Fischer, U.; Schulze-Osthoff, K. Pharmacol. Rev. 2005, 57, 187.
2. O’Driscoll, L.; Linehan, R.; Clynes, M. Curr. Cancer Drug Targets 2003, 3, 131.
3. Grivicich, I.; Regner, A.; da Rocha, A. B.; Grass, L. B.; Alves, P. A.; Kayser, G. B.;
Schwartsmann, G.; Henriques, J. A. Oncol. Res. 2005, 15, 385.
4. Kolomeichuk, S. N.; Bene, A.; Upreti, M.; Dennis, R. A.; Lyle, C. S.; Rajasekaran,
M.; Chambers, T. C. Mol. Pharmacol. 2008, 73, 128.
5. Kolenko, V. M.; Uzzo, R. G.; Bukowski, R.; Finke, J. H. Apoptosis 2000, 5, 17.
6. Fesik, S. W. Nat. Rev. Cancer 2005, 5, 876.
7. (a) Zobel, K.; Wang, L.; Varfolomeev, E.; Franklin, M. C.; Elliott, L. O.; Wallweber,
H. J.; Okawa, D. C.; Flygare, J. A.; Vucic, D.; Fairbrother, W. J.; Deshayes, K. ACS
Chem. Biol. 2006, 1, 525; (b) Shangary, S.; Qin, D.; McEachern, D.; Liu, M.; Miller,
R. S.; Qiu, S.; Nikolovska-Coleska, Z.; Ding, K.; Wang, G.; Chen, J.; Bernard, D.;
Zhang, J.; Lu, Y.; Gu, Q.; Shah, B. R.; Pienta, K. J.; Ling, X.; Kang, S.; Guo, M.; Sun,
Y.; Yang, D.; Wang, S. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 3933.
8. Cai, S. X.; Zhang, H.-Z.; Guastella, J.; Drewe, J.; Yang, W.; Weber, E. Bioorg. Med.
Chem. Lett. 2001, 11, 39.
9. Zhang, H.-Z.; Kasibhatla, S.; Guastella, J.; Drewe, J.; Tseng, B.; Cai, S. X.
Bioconjugate Chem. 2003, 14, 458.
10. Thornberry, N. A. Chem. Biol. 1998, 5, R97.
11. Cai, S. X.; Drewe, J.; Kasibhatla, S. Curr. Med. Chem. 2006, 13, 2627.
12. Zhang, H.-Z.; Kasibhatla, S.; Wang, Y.; Herich, J.; Guastella, J.; Tseng, B.; Drewe,
J.; Cai, S. X. Bioorg. Med. Chem. 2004, 12, 309.
13. Kasibhatla, S.; Jessen, K.; Maliartchouk, S.; Wang, J.; English, N.; Drewe, J.; Qui,
L.; Archer, S.; Ponce, A.; Sirisoma, N.; Jiang, S.; Zhang, H.-Z.; Gehlsen, K.; Cai, S.
X.; Green, D. R.; Tseng, B. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 12095.
14. (a) Kemnitzer, W.; Kasibhatla, S.; Jiang, S.; Zhang, H.; Wang, Y.; Zhao, J.; Jia, S.;
Herich, J.; Labreque, D.; Storer, R.; Meerovitch, K.; Bouffard, D.; Rej, R.; Denis,
R.; Blais, C.; Lamothe, S.; Attardo, G.; Gourdeau, H.; Tseng, B.; Drewe, J.; Cai, S.
X. J. Med. Chem. 2004, 47, 6299; (b) Kemnitzer, W.; Drewe, J.; Jiang, S.; Zhang,
H.; Zhao, J.; Crogan-Grundy, C.; Xu, L.; Lamothe, S.; Gourdeau, H.; Denis, R.;
Tseng, B.; Kasibhatla, S.; Cai, S. X. J. Med. Chem. 2007, 50, 2858; (c) Kemnitzer,
W.; Drewe, J.; Jiang, S.; Zhang, H.; Crogan-Grundy, C.; Labreque, D.; Bubenick,
M.; Attardo, G.; Denis, R.; Lamothe, S.; Gourdeau, H.; Tseng, B.; Kasibhatla, S.;
Cai, S. X. J. Med. Chem. 2008, 51, 417.
15. (a) Kasibhatla, S.; Gourdeau, H.; Meerovitch, K.; Drewe, J.; Reddy, S.; Qiu, L.;
Zhang, H.; Bergeron, F.; Bouffard, D.; Yang, Q.; Herich, J.; Lamothe, S.; Cai, S. X.;
Tseng, B. Mol. Cancer Ther. 2004, 3, 1365; (b) Gourdeau, H.; Leblond, L.;
Hamelin, B.; Desputeau, C.; Dong, K.; Kianicka, I.; Custeau, D.; Bourdeau, C.;
Geerts, L.; Cai, S. X.; Drewe, J.; Labrecque, D.; Kasibhatla, S.; Tseng, B. Mol.
Cancer Ther. 2004, 3, 1375.
0.016 and 0.015 lM, respectively. In general, H1299 cells were
slightly less sensitive (about two to fourfold less as indicated
by the higher EC50 values) to the compounds than T47D cells
in this assay.
We have found that 1d, 1e and related compounds are tubulin
inhibitors that bind at or close to the colchicine site of b-tubu-
lin.18,19 Compounds 3a, 4d and 5a–5f, which were highly active
in the caspase activation assay, were tested in the tubulin polymer-
ization assay.26 All the compounds were found to inhibit tubulin
polymerization with IC50 values of less than 1 lM, which is similar
to that of compounds 1d and 1e. In comparison, colchicine, a well
known tubulin inhibitor was found to be highly active in our cas-
pase activation assay with an EC50 value of 9 nM against T47D cells
(Table 3), and had a similar potency in the tubulin assay (0.5
lM).
Therefore these 4-(4-methoxyanilino)-N-methylthieno[3,2-
d]pyrimidines and 4-(4-methoxyanilino)-N-methylthieno[2,3-
d]pyrimidines most probably induce apoptosis through inhibition
of tubulin polymerization.
Compound 5d was tested in a MX-1 xenograft breast cancer
model. The MX-1 in vivo experiment was performed as described
previously.18 Compound 5d inhibited tumor growth dose depen-
dently and produced 90% tumor growth inhibition when dosed
intravenous with a single administration at a dose of 75 mg/kg
(Fig. 1), and is well tolerated with maximum body weight decrease
of <10%.
16. Zhang, H.-Z.; Kasibhatla, S.; Kuemmerle, J.; Kemnitzer, W.; Oliis-Mason, K.; Qui,
L.; Crogran-Grundy, C.; Tseng, B.; Drewe, J.; Cai, S. X. J. Med. Chem. 2005, 48,
5215.
In conclusion, we have discovered a series of 4-anilino-N-meth-
ylthieno[3,2-d]pyrimidines and 4-anilino-N-methylthieno[2,3-

3540
W. Kemnitzer et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3536–3540
17. Jessen, K.; English, N.; Wang, J.; Qui, L.; Brand, R.; Maliartchouk, S.; Drewe, J.;
Kuemmerle, J.; Zhang, H.-Z.; Gehlsen, K.; Tseng, B.; Cai, S. X.; Kasibhatla, S. Mol.
Cancer Ther. 2005, 4, 761.
18. Sirisoma, N.; Kasibhatla, S.; Pervin, A.; Zhang, H.; Jiang, S.; Willardsen, J. A.;
Anderson, M.; Baichwal, V.; Mather, G. G.; Jessing, K.; Hussain, R.; Hoang, K.;
Pleiman, C. M.; Tseng, B.; Drewe, J.; Cai, S. X. J. Med. Chem. 2008, 51, 4771.
19. Sirisoma, N.; Pervin, A.; Zhang, H.; Jiang, S.; Willardsen, A. J.; Anderson, M.;
Mather, G. G.; Pleiman, C. M.; Kasibhatla, S.; Tseng, B.; Drewe, J.; Cai, S. X. J.
Med. Chem. 2009, 52, 2341.
20. Kasibhatla, S.; Baichwal, V.; Cai, S. X.; Roth, B.; Skvortsova, I.; Skvortsov, S.;
Lukas, P.; English, N. M.; Sirisoma, N.; Drewe, J.; Pervin, A.; Tseng, B.; Carlson, R.
O.; Pleiman, C. M. Cancer Res. 2007, 67, 5865.
21. Showalter, H. D.; Bridges, A. J.; Zhou, H.; Sercel, A. D.; McMichael, A.; Fry, D. W.
J. Med. Chem. 1999, 42, 5464.
Shewchuk, L. M.; Spehar, G. M.; Truesdale, A. T.; Vanderwall, D. E.; Wood, E. R.;
Uehling, D. E. Bioorg. Med. Chem. Lett. 2009, 19, 817.
24. Synthesis procedure for N-(4-methoxyphenyl)-N,2-dimethylthieno[2,3-
d]pyrimidin-4-amine (5d). To an oven-dried one-neck reaction flask charged
with a magnetic stir bar at room temperature under argon was added 4-chloro-
2-methylthieno[2,3-d]pyrimidine (0.100 g, 0.542 mmol), isopropanol (2.7 mL),
N-methyl-p-anisidine (0.082 g, 0.60 mmol) and 2.0 M HCl in ether (0.260 mL).
The brown solution was heated at 80 °C for 4 h, cooled to rt and diluted with
EtOAc (70 mL). The organic layer was washed with saturated NaHCO3
(2 Â 25 mL), brine (2 Â 20 mL), dried over Na2SO4, filtered and concentrated
to yield a brown oil. Purification by flash column chromatography (4 g pre-
packed silica gel column, elution with EtOAc:hexanes, 1:9) gave 0.047 g (30%)
of 5d as a white solid: mp 87–88 °C; 1H NMR (CDCl3): 7.20–7.17 (m, 2H), 6.97–
6.94 (m, 2H), 6.73 (dd, J = 6.0 and 0.5 Hz, 1H), 5.55 (dd, J = 6.0 and 0.8 Hz, 1H),
3.86 (s, 3H), 3.55 (s, 3H), 2.65 (s, 3H).
22. Munchhof, M. J.; Beebe, J. S.; Casavant, J. M.; Cooper, B. A.; Doty, J. L.; Higdon, R.
C.; Hillerman, S. M.; Soderstrom, C. I.; Knauth, E. A.; Marx, M. A.; Rossi, A. M.;
Sobolov, S. B.; Sun, J. Bioorg. Med. Chem. Lett. 2004, 14, 21.
25. Cai, S. X.; Nguyen, B.; Jia, S.; Guastella, J.; Reddy, S.; Tseng, B.; Drewe, J.;
Kasibhatla, S. J. Med. Chem. 2003, 46, 2474.
23. Rheault, T. R.; Caferro, T. R.; Dickerson, S. H.; Donaldson, K. H.; Gaul, M. D.;
Goetz, A. S.; Mullin, R. J.; McDonald, O. B.; Petrov, K. G.; Rusnak, D. W.;
26. Barron, D. M.; Chatterjee, S. K.; Ravindra, R.; Roof, R.; Baloglu, E.; Kingston, D. G.
I.; Bane, S. Anal. Biochem. 2003, 315, 49.