2,4-Diamino-quinazolines as inhibitors of β-catenin/Tcf-4 pathway: Potential treatment for colorectal cancer

Article abstract of DOI:10.1016/j.bmcl.2009.07.070

The synthesis and SAR of a series of 2,4-diamino-quinazoline derivatives as β-catenin/Tcf-4 inhibitors are described. This series was developed by modifying the initial lead 1, which was identified by screening of our compound library and found to inhibit

Full text of DOI:10.1016/j.bmcl.2009.07.070

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4980–4983
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2,4-Diamino-quinazolines as inhibitors of b-catenin/Tcf-4 pathway: Potential
treatment for colorectal cancer
a
a
a
Zecheng Chen a, , Aranapakam M. Venkatesan , Christoph M. Dehnhardt , Osvaldo Dos Santos ,
Efren Delos Santos a, Semiramis Ayral-Kaloustian a, Lei Chen b, Yi Geng b, Kim T. Arndt b, Judy Lucas b,
Inder Chaudhary c, Tarek S. Mansour a
*
a Wyeth Research, Chemical Sciences, 401 N. Middletown Road, Pearl River, NY 10965, USA
b Wyeth Research, Oncology, 401 N. Middletown Road, Pearl River, NY 10965, USA
c Wyeth Research, Drug Metabolism, 401 N. Middletown Road, Pearl River, NY 10965, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and SAR of a series of 2,4-diamino-quinazoline derivatives as b-catenin/Tcf-4 inhibitors are
described. This series was developed by modifying the initial lead 1, which was identified by screening of
our compound library and found to inhibit the b-catenin/Tcf-4 pathway. Replacement of the biphenyl
moiety in compound 1 with the N-phenylpiperidine-4-carboxamide chain as in 2, resulted in a number
of new analogues, which are potent inhibitors of the b-catenin/Tcf-4 pathway. Compound such as 16k
exhibited good cellular potency, solubility, metabolic stability and oral bioavailability.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 9 June 2009
Revised 8 July 2009
Accepted 14 July 2009
Available online 17 July 2009
Keywords:
b-Catenin/Tcf-4 pathway
Inhibitors
2,4-Diamino-quinazoline
Colorectal cancer is the second leading cause of cancer deaths in
the United States, and nearly 150,000 patients are diagnosed with
colorectal cancer and over 55,000 Americans die of this disease
each year.1 Besides surgical resection, current therapy for colon
cancer relies on traditional cytotoxic agents with limited success.
However, there is still a high unmet medical need for effective
treatment of colorectal cancer. Over the past decades, accumulat-
ing evidence has suggested that activation of the canonical Wnt
signaling is implicated in a wide range of human cancers, espe-
cially in colorectal cancers.2,3
The canonical Wnt signaling pathway was first discovered in
Drosophila.4 This signaling pathway is evolutionally conserved in
mammalians, Xenopus, Drosophila and Caneorhabditis elegans.3 It
controls many events during embryonic development and regulates
proliferation, morphology, motility and cell fate at a cellular level.
Within the pathway, the tumor suppressor Adenomatous Polyposis
Coli (APC) in complex with Axin is required to regulate the stability
of b-catenin, a central component of the Wnt signaling pathway.5 In
the absence of a Wnt signal, cytoplasmic b-catenin is captured by
APC and the resulting Axin complex is phosphorylated by kinases
the APC complex is inhibited. As a consequence, stable non-phos-
phorylated b-catenin accumulates in the cytoplasm and subse-
quently translocates into the nucleus, where it binds to Tcf-4 (T-
cell transcriptional factor-4) protein and activates the transcription
of a variety of Wnt target genes.6 Numerous candidate genes have
been proposed as critical downstream effectors of Wnt signaling
in cancer, including c-myc,7 cyclin D1,8 and axin2,9 etc.
Notably, most of colorectal cancers have loss of function in APC
which initiates a neoplastic process towards carcinoma formation.1
In some cases of colorectal cancer in which APC is wild type, b-cate-
nincanbe activatedbymutationsinaxin210,11 orby intragenic muta-
tionsthat abolish inhibitoryphosphorylation sites at the N-terminus
of b-catenin.12 Under both situations b-catenin is no longer de-
graded. Allthesemutationsresultinaccumulationof non-phosphor-
ylated b-catenin, thereby constitutively activating transcription of
genes, and ultimately promoting carcinogenesis. Introduction of
wild type APC into cells which have lost APC function has been
shown to result in either growth suppression or apoptosis, suggest-
ing that these cells have become dependent on elevated b-catenin/
Tcf-4 signaling.5a,13
GSK3b and CK1
a
. The phosphorylated b-catenin is then ubiquitinat-
Taken together, the b-catenin/Tcf-4 signaling pathway presents
a novel target for potential therapeutic intervention of colorectal
cancer. We herein report our medicinal chemistry efforts, which
led to identification of 2,4-diamino-quinazoline derivatives that in-
hibit the b-catenin/Tcf-4 pathway.
ed and targeted for rapid proteosomal degradation. In the presence
of Wnt factors, the Wnt signaling cascade is activated so that Axin is
recruited to the membrane, thereby the intrinsic kinase activity of
Compound 1 was identified14 as a potent inhibitor of b-catenin/
Tcf-4 through screening of our compound library. However, the use
* Corresponding author.
E-mail addresses: chenzecheng@hotmail.com, chenz1@wyeth.com (Z. Chen).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.070

Z. Chen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4980–4983
4981
of this compound was limited due to a variety of issues including
poor aqueous solubility, poor oral exposure, and metabolic insta-
bility. During the course of SAR exploration of this diamino-qui-
nazoline series it was discovered that replacement of the
biphenyl moiety with a N-phenylpiperidine-4-carboxamide was
feasible (e.g., 1 vs 2, Fig. 1) with minimal loss of potency (IC50 for
depicted in Scheme 1). Replacement of 2-Cl with different amino
substitutions under microwave irradiation conditions resulted in
compounds 7a–k (Table 1) in good to moderate yields.
All the compounds were tested in a cell-based reporter assay:
two cell lines (Tcf22C11 and Tcf33.13) where luciferase expression
is driven by multimerized Tcf-4 binding sites and one control cell
line (SV5A8) where luciferase expression is driven by the SV40 pro-
moter.17 The desired profile of a compound is to selectively inhibit
the reporter activity in Tcf22C11 and Tcf33.13 but not inhibit the
reporter activity in SV5A8.
The effect of different amino substitutions is summarized in Ta-
ble 1. Among the different amino substitutions screened, the
dimethylamino and pyrrolidinyl substitutions turned out to be
the optimal. The potency against 22C11 cell line for compound 2
(dimethylamino substitution) and 7c (pyrrolidinyl substitution)
22C11 cell line: 0.60 lM vs 0.74 lM). The resulting amides offered
several advantages including: (1) increased metabolic/chemical
stability; (2) improved aqueous solubility; (3) a springboard from
which to develop more potent inhibitors of the b-catenin/Tcf-4
pathway.
In an effort to improve potency, initial modifications were made
at the 2-position of the quinazoline by preparing different amino
substituted derivatives. Analogues 7a–k were synthesized by the
method outlined in Scheme 1. 2-Amino-4-methylbenzoic acid
was heated with urea to gave the 7-methylquinazoline-2,4-dione
3.15,16 Chlorination of 3 with POCl3 resulted in the dichloride 4.
Assembly of the side chain was achieved by selective substitution
of 4-Cl with 5 (readily prepared from the commercially available
ethyl 1-benzylpiperidine-4-carboxylate by a three step process as
were found to be 0.74
M. Piperidinyl substitution (7k) was also tolerated. Compound
7a (ethylamino substitution) exhibited moderate potency against
22C11 and 33.13 with IC50 1.38 and 1.13 M, respectively, but
lM and 0.66 lM, respectively, both below
1
l
l
showed very limited selectivity against the control cell line 5A8
NH
N
N
N
N
N
N
O
O
N
N
H
H
N
O
H
2
1
Initial Lead:
IC50 (22C11): 0.60 µM
IC50(22C11): 0.74 µM
Figure 1.
Cl
O
b
HO2C
H2N
a
N
HN
+
5
Cl
N
O
N
H
4
3
Ph
R2
N
R1
N
Ph
O
Cl
N
N
N
N
N
c
d
N
N
H
H
N
O
N
H
H
7
6
HO
O
HN Boc
O
e
N
H2N
+
N
Ph
Ph
O
9
8
NH2
f, g
HN
N
Ph
O
5
Scheme 1. Reagents and conditions: (a) urea, 180 °C, 2 h, 85%; (b) POCl3, reflux, 16 h, 45%; (c) 5 (1.2 equiv), Et3N (3 equiv), CH2Cl2, rt, 12 h, 74%; (d) R1R2NH (10 equiv), THF,
100 °C, 30 min, microwave irradiation (300 W), 45–90%; (e) KOH (3 equiv), MeOH, 60 °C, 3 h, 99%; (f) (1) (COCl)2 (1.5 equiv), DMF (cat.), CH2Cl2, 1 h; (2) Et3N (3 equiv), 9
(1.0 equiv), CH2Cl2, rt, 12 h, 93%; (g) TFA, CH2Cl2, 0 °C to rt, 2 h, 99%.

4982
Z. Chen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4980–4983
Table 1
Activity of amino substituents at the 2-position of the quinazoline ringa
b
Compds
R1R2N
22C11 IC50
(
lM)
33.13 IC50
(
lM)
5A8 IC50 (lM)
2
Me2N
EtNH
Azetidinyl
Pyrrolidinyl
0.74
1.38
1.00
0.66
1.04
2.26
1.41
1.58
1.26
1.92
2.27
0.96
0.76
1.13
0.80
0.79
0.96
1.44
1.31
1.32
1.12
1.46
2.12
0.85
>7.05
>2.14
>8.50
>7.79
>6.88
>8.83
>8.62
>17.7
>13.0
>8.86
>8.42
>8.09
7a
7b
7c
7d
7e
7f
7g
7h
7i
Azepanyl
Me2N(CH2)2NMe
Me2N(CH2)3NMe
4-Methylpiperazinyl
4-Ethylpiperazinyl
(s)-3-Methyl-piperzinyl
3-(2-Hydroxyethyl)-piperazinyl
4-Pyrrolidinyl-piperidinyl
7j
7k
a
Values are means of at least two experiments.
The number reported behind the ‘>’ sign indicates the minimal dose at which cells started to be killed due to toxicity, as indicated by the Renilla luciferase signal.
b
(IC50 >2.14
l
M), which indicated some off-target toxicity. Replace-
2. Introduction of a fluorine atom in the phenyl ring resulted in a
significant increase in potency in both the 22C11 and 33.13 cell
lines when compared to compound 2. Among the three F substi-
tuted analogues (16k, 16l and 16m), the 4-F substituted compound
ment of the pyrrolidinyl group by the azetidinyl (7b) or azepanyl
(7d) led to a slight decrease in potency. A variety of piperazine sub-
stitutions (7g–j) resulted in a decrease in potency, so did the linear
diamino groups (7e–f).
16k is the most potent with an IC50 value of 0.22 lM against the
In comparison with 1, compound 2 had an improved water sol-
ubility (43 lg/mL at pH 7.4, and >100 lg/mL at pH 3.0) and was
found to have an oral bioavailability of 41% in female nude mice
(25 mg/kg, po).
The in vivo metabolite identification studies in nude mouse of
compound 2 indicated that the N-debenzylation was the major
metabolic pathway. To address this metabolic liability, replacing
the benzyl group by a variety of substitutions was explored. The
new analogues 16a–p were prepared by a modified route as out-
lined in Scheme 2, and their in vitro potencies are shown in Table
22C11 cell line, which is about threefold more potent than com-
pound 2. The 2-F substituted compound (16l) is less potent than
the 3-F analogue (16m) against the 22C11 cell line, but in the
33.13 cell line 16l and 16m are equally potent. As in the case of
the fluoro substitution, introduction of 4-Cl in the phenyl ring also
led to an increase in potency (16n, IC50 0.39 lM). The disubstituted
analogue 16p, with a 2-Cl-4-F phenyl ring, exhibited comparable
activity to 16k against the 33.13 cell line, however, 16p is less po-
tent than 16k against the 22C11 cell line. Introduction of chlorine
on the pyridine ring resulted in an increase in potency in both cell
O
HO
Cl
O
O
N
O
O
O
N
a,b
10
NH
Cl
Cl
H2N
12
O
NH2
NH
O
O
O
O
11
Cl
N
Cl
N
N
d
c
N
+
12
N
H
Cl
N
N
H
4
13
O
N
O
N
N
H
N
N
N
N
N
e
N
H
N
H
N
O
N
H
O
15
H
14
N
R
N
N
N
f
N
H
N
H
O
16
Scheme 2. Reagents and conditions: (a) 11 (1 equiv), Et3N (3 equiv), HOBt (1.5 equiv), EDCI (1.5 equiv), THF, rt, 88%; (b) Zn powder, HOAc, rt, 1 h, 95%; (c) 12 (1.2 equiv), Et3N
(3 equiv), CH2Cl2, rt, 12 h, 84%; (d) Me2NH (10 equiv), THF, 100 °C, 30 min, microwave irradiation (300 W), 90%; (e) TFA, CH2Cl2, 0 °C to rt, 2 h, 96%: (f) R3R4CO or ArCHO
(2 equiv), ZnCl2 (1.5 equiv), NaBH3CN (1.5 equiv), THF, rt, 2 h, 56–78%.

Z. Chen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4980–4983
4983
Table 2
20–35% tumor growth inhibition was observed for the compound
2 at the end, after a 200 mg/kg oral dose. No significant tumor
growth inhibition was found by using compound 16k at a 50 mg/
kg po dose even though 16k is threefolds more potent than 2,
against the 22C11 and 33.13 cell lines. These results were surpris-
ing given the excellent exposures of compounds 2 and 16k in tu-
mor bearing nude mice (Table 3). Another related experiment
showed that there were no major pathway-related gene changes
in 2 and 16k treated tumors, while these changes were observed
in in vitro gene chip analyses. The reason for this disconnection
is not clear.
In summary, initial SAR exploration of the lead diamino-quinaz-
oline has led to a number of potent in vitro inhibitors of the b-cate-
nin/Tcf-4 pathway with improved in vivo pharmacokinetic
properties. Compound 16k exhibited improved potency (IC50
0.22 lM against the 22C11 cell line and 0.19 lM against the
33.13 cell line), good solubility, permeability and oral bioavailabil-
Activity of different substitutions on the piperidine ringa
b
Compds
R
22C11 IC50
M)
33.13 IC50
M)
5A8 IC50
M)
(
l
(
l
(l
16a
16b
16c
16d
16e
16f
16g
16h
16i
16j
16k
16l
16m
16n
16o
Me
iPr-CH2
n-Butyl
Cyclohexyl
1.92
0.65
1.47
0.75
0.76
1.83
>20
1.18
0.47
0.52
0.22
0.53
0.33
0.39
0.57
1.67
0.96
1.17
0.81
0.85
1.93
>20
0.90
0.50
0.45
0.19
0.32
0.35
0.32
0.41
>17.3
>10.5
>10.5
>8.32
>4.82
>20
Boc
2-Furanylmethyl
2-Imidazolylmethyl
4-Pyridinylmethyl
4-Methylbenzyl
4-Methoxybenzyl
4-Fluorobenzyl
2-Fluorobenzyl
3-Fluorobenzyl
4-Chlorobenzyl
(5-Chloropyridin-2-
yl)methyl
>20
>8.07
>4.15
>6.18
>4.82
>6.67
>4.17
>3.18
>5.40
ity (46%), and showed excellent blood and tumor exposure, follow-
16p
2-Chloro-4-fluorobenzyl 0.52
0.21
>5.91
ing oral dosage. However, neither compound
2 nor 16k
a
Values are means of at least two experiments.
The number reported behind the ‘>’ sign indicates the minimal toxic dose at
demonstrated significant in vivo efficacy in HT29 and HCT116
xenograft models. Further optimization in potency and PK proper-
ties of this series is ongoing, and the results will be reported in due
course.
b
which cells started to be killed due to toxicity, as indicated by the Renilla luciferase
signal.
Table 3
Plasma and tumor concentrations of compounds 2 and 16k
Acknowledgment
Compds Dose (po,
mg/kg)
Plasma conc. 8 h
(ng/mL)
Tumor conc. 8 h
(ng/mL)
Tumor/
plasma ratio
We thank Wyeth Chemical Technologies Department for com-
pound identification and the pharmaceutical profiling results.
2
200
50
2973
440
90,736
70,306
40
220
16k
References and notes
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lines (16o vs 16h). Imidazole substitution was not tolerated, and
led to a dramatic loss in potency (IC50 >20 M for 16g). Replacing
phenyl with furanyl resulted in a loss in potency in both cell lines
(16f vs 2). In addition to aromatic substitutions, some alkyl substi-
tutions were also explored. Interestingly, the linear alkyl substitu-
l
tions such as methyl (16a, IC50 1.92
1.47 M) led to a loss in potency, but the branched or cyclic alkyl
groups such as iso-butyl (16b, IC50 0.57 M) and cyclohexyl (16d,
IC50 0.75 M) are both better tolerated. Carbamate analogue 16e
is also tolerated with an IC50 value of 0.76 M.
lM) and n-butyl (16c, IC50
l
l
l
l
As shown in Table 3, the exposure studies showed that the plas-
ma concentration of compound 2 in nude mouse (n = 4) is 2973 ng/
mL at 8 h following a single oral dose (200 mg/kg), while the tumor
concentration is 90,736 ng/mL, with a favorable tumor/plasma ra-
tio of 40. Compound 16k also exhibited good exposure in blood
and in tumor at 8 h after a single po dose (50 mg/kg). Compound
16k was preferentially distributed to the tumor with a tumor/plas-
ma ratio of 220. The pharmacokinetic studies in female nude mice
showed that both compounds exhibited reasonable bioavailability
(41% for 2, and 46% for 16k, respectively).
The in vivo efficacy of compounds 2 and 16k was then assessed
in nude mice bearing HT29 or HCT116 tumors grown subcutane-
ously. In this experiment, tumor volume was measured after po
dose of 25, 50, 100, or 200 mg/kg once daily for 17 days. Only