Design and synthesis of novel diaminoquinazolines with in vivo efficacy for β-catenin/t-cell transcriptional factor 4 pathway inhibition

Article abstract of DOI:10.1021/jm901370m

We are introducing a novel series of 2,4-diaminoquinazolines as β-catenin/Tcf4 inhibitors which were identified by ligand-based design. Here we elucidate the SAR of this series and explain how we were able to improve key molecular properties such as solubility and cLogP leading to compound 9. Analogue 9 exhibited better biological activity and improved physical and pharmacological properties relative to the HTS hit 49. Furthermore, 9 demonstrated good cell growth inhibition against several human colorectal cancer lines such as LoVo and HT29. In addition, treatment with compound 9 led to gene expression changes that overlapped significantly with the transcriptional profile resulting from the pathway inhibition by siRNA knockdown of β-catenin or Tcf4. Subsequently, 9 was tested for efficacy in a β-catenin/RKE-mouse xenograft, where it led to more then 50% decrease in tumor volume. 2009 American Chemical Society.

Full text of DOI:10.1021/jm901370m

J. Med. Chem. 2010, 53, 897–910 897
DOI: 10.1021/jm901370m
Design and Synthesis of Novel Diaminoquinazolines with in Vivo Efficacy for β-Catenin/T-Cell
Transcriptional Factor 4 Pathway Inhibition
Christoph M. Dehnhardt,*,† Aranapakam M. Venkatesan,† Zecheng Chen,† Semiramis Ayral-Kaloustian,†
Osvaldo Dos Santos,† Efren Delos Santos,† Kevin Curran,† Max T. Follettie, Veronica Diesl, Judy Lucas,‡ Yi Geng,‡
Susan Quinn DeJoy,‡ Rosanne Petersen,‡ Inder Chaudhary,§ Natasja Brooijmans,† Tarek S. Mansour,† Kim Arndt,‡ and
Lei Chen‡
†Discovery Medicinal Chemistry, ‡Oncology Research, and §DSM, Wyeth Research, Pearl River, New York 10965, and Biological Technologies,
Wyeth Research, Cambridge, Massachusetts 02140
Received September 15, 2009
We are introducing a novel series of 2,4-diaminoquinazolines as β-catenin/Tcf4 inhibitors which were
identified by ligand-based design. Here we elucidate the SAR of this series and explain how we were able
to improve key molecular properties such as solubility and cLogP leading to compound 9. Analogue 9
exhibited better biological activity and improved physical and pharmacological properties relative to the
HTS hit 49. Furthermore, 9 demonstrated good cell growth inhibition against several human colorectal
cancer lines such as LoVo and HT29. In addition, treatment with compound 9 led to gene expression
changes that overlapped significantly with the transcriptional profile resulting from the pathway
inhibition by siRNA knockdown of β-catenin or Tcf4. Subsequently, 9 was tested for efficacy in a
β-catenin/RKE-mouse xenograft, where it led to more then 50% decrease in tumor volume.
Introduction
cytoplasmic β-catenin is captured by the APC/Axin complex
and is phosphorylated by kinases GSK3β (glycogen synthase
kinase 3β) and CK1R (casein kinase 1R) in the complex;
phosphorylated β-catenin is further ubiquitinated and tar-
geted for rapid proteasomal degradation. When cells are
stimulated by Wnt factors, the Wnt signaling cascade is
activated so that Axin is recruited to the membrane, resulting
in inhibition of the intrinsic kinase activity of the APC
complex. As a consequence, stable non-phosphorylated
β-catenin accumulates in the cell cytoplasm and subsequently
translocates into the nucleus, where it forms a complex with
Tcf4 (T-cell transcriptional factor 4) protein and constitu-
tively activates the transcription of a variety of Wnt target
genes.5 A number of genes have been proposed as critical
downstream effectors of Wnt signaling in cancer, including
c-myc,6 cyclin D1,7 MMP-7,8,9 axin2,10 and others.
Loss of function in APC has been found in 85% of color-
ectal cancers, and this marks the initiation step of a neoplastic
process toward carcinoma formation.1 In some cases of color-
ectal cancer in which APC is wild type, β-catenin is activated
by mutations in axin211,12 or by intragenic mutations that
abolish inhibitory phosphorylation sites at the N-terminus of
β-catenin;13 both mutations can prevent β-catenin from de-
gradation. All of these mutations result in accumulation of
non-phosphorylated β-catenin, thereby constitutively activat-
ing transcription of target genes and ultimately promoting
carcinogenesis. Either growthsuppressionor apoptosisoccurs
when wild-type APC was introduced into colon cancer cells
which have lost APC function, suggesting that these cells have
become dependent on elevated β-catenin/Tcf4 signaling.14
Taken together, the β-catenin/Tcf4 signaling pathway pre-
sents a novel target for therapeutic intervention in colorectal
cancer. The SW480 colorectal cancer cell line has a truncation
mutation in APC and thus has a constitutively activated
Colorectal cancer is a majorhealth burden. Each year, more
than one million new cases are diagnosed worldwide, and
155000 new cases develop in the United States.1 In Western
populations, 50% of people develop colorectal tumors by age
70, and about 10% of them have their tumors progress to
malignancy.2 Consequently, colorectal cancer is the second
leading cause of cancer mortality in the United States. Despite
the overall improvement in colorectal cancer therapy, the
survival outcome remains poor or unsatisfactory. Therefore,
there is a high unmet medical need for effective treatment of
colorectal cancer. Over the past decade, accumulating evi-
dence points to a role that activation of the canonical Wnta
(wingless int-1) signaling plays in a wide range of human
cancers, especially in colorectal cancers.2,3
First discovered in Drosophila,4 the canonicalWnt signaling
pathway is evolutionally conserved in mammalians, Xenopus,
Drosophila, and Caenorhabditis elegans.3 It not only controls
many events during embryonic development but also regu-
lates proliferation, morphology, motility, and cell fate at a
cellular level. As the key player of the pathway, the tumor
suppressor adenomatous polyposis coli (APC) forms a com-
plexwithAxin and regulates the stability ofβ-catenin, another
central component of the Wnt signaling pathway. The Wnt
pathway is activated by Wnt factors which form a large family
ofcell-signalingglycoproteins.3 Inthe absence of a Wnt signal,
*To whom correspondence should be addressed. Phone: 845-602-
2146. Fax: 845-602-5561. E-mail: dehnhac@wyeth.com.
a Abbreviations: Tcf4, T-cell transcriptional factor 4; APC, adeno-
matous polyposis coli; Wnt, wingless int-1; GSK3, glycogen synthase
kinase 3; CK1R, casein kinase 1R; MMP-7, matrix metalloprotease 7;
SV40, simian vacuolating virus 40; FF, firefly; R, renilla; siRNA, small
interfering RNA; DUSP, dual specificity phosphatase; MAP, mitogen-
activated protein; ip, intraperitoneal.
r
2009 American Chemical Society
Published on Web 12/21/2009
pubs.acs.org/jmc

898 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Scheme 1. Synthesis of Quinazoline Analogues 8-39a
Dehnhardt et al.
a For structures of 8-39, please see Tables 1-4. Reaction conditions: (a) NEt3, 4-aminobenzylamine, CHCl3; (b) ArCOCl; (c) 5-7, NEt3, CHCl3; (d)
HNR5R4, THF or dioxane, 70 °C, 24 h; (e) ArCOCl, NEt3; (f) TFA, CH2Cl2.
Scheme 2. Synthesis of 7-Substiuted Quinazoline Analogues 41-48a
a 41: R = -CN; ZnCN2, Pd(OAc)2, toluene, reflux, 70%. 42: R = -COCH3; (a) Bu3Sn-R-ethoxyvinyl, Pd(PPh3)4, toluene, reflux; (b) HCl, THF
reflux, 50% (2 steps). 43:R=(þ/-) -CH(OH)CH3; (a) Bu3Sn-R-ethoxyvinyl, Pd(PPh3)4, toluene, reflux; (b) HCl, THF reflux;(c) NaBH4, 40% (3 steps). 44:
R = -CHO; NaHCO2, Pd(OAc)2, CO (1 atm), DMF, 100 °C, 37%. 45: R = -CH2OH; (a) NaHCO2, Pd(OAc)2, DMF, 100 °C; (b) NaBH4, MeOH, 85% (2
steps). 46: R = -CH2NMe2; (a) NaHCO2, Pd(OAc)2, CO (1 atm), DMF, 100 °C; (b) HNMe2, NaBH3CN, AcOH, 23% (2 steps). 47: R = -CHdCH2;
Bu3Sn-vinyl, Pd(PPh3)4, toluene, reflux, 61%. 48: R = -C2H5; (a) Bu3Sn-vinyl, Pd(PPh3)4, toluene, reflux; (b) Pd/C, H2, MeOH, 59% (2 steps).
β-catenin/Tcf4 signaling pathway. We engineered a luciferase
reporter with Tcf4 binding sites in its promoter. The Tcf4-
luciferase reporter was subsequently integrated into the chro-
mosome of SW480, resulting in two reporter cell lines which
were used in a high-throughput screen. Recently, we reported
the discovery of a novel class of small molecules that inhibit the
β-catenin/Tcf4 pathway built on a quinazoline core.15 These
quinazolines exhibited good solubility, permeability, and oral
bioavailability. However, the compounds lacked in vivo effi-
cacy. Herein we report a novel and modified class of β-catenin/
Tcf4-pathway inhibitors that showed a similarly good in vitro
profile as the compounds described previously. However, in
this new series, we identified a compound that engaged the
β-catenin/Tcf4 pathway more effectively than the older analo-
gues and, more importantly, demonstrated in vivo efficacy.
iodo compound 39 with various organometallic reagents
and further functional group interconversion as shown in
Scheme 2. The iodo starting material 39 had been obtained
earlier as shown in Scheme 1.
Results and Discussion
All of the newly synthesized compounds were evaluated in
the Tcf4-luciferase reporter cell lines 33.13 and 22C11, and the
selectivity profile was established against the control cell line
5A8. Our Tcf4-luciferase reporter assay cell lines and the
control cell line were derived from the SW480 colorectal
cancer cell. The 33.13 line was generated by stepwise transfec-
tion of SW480 cells with the Tcf4-Firefly luciferase construct
(G418-resistant) and then with the Zeocin-resistant SV40-
Renilla luciferase construct, while the 22C11 lines was ob-
tained by cotransfection of the Tcf4-Firefly luciferase con-
struct and SV40-Renila luciferase construct under G418
selection. These two reporter lines represent two independent
stable clones of the desired integration events. Therefore,
assessment in both 33.13 and 22C11 cells provides a better
indication of a specific inhibition of the reporter rather than
some inhibition that was integration site-dependent. In gen-
eral, a selective inhibition of the Tcf4-luciferase expression in
both 33.13 and 22C11 lines but not in 5A8 was sought during
compound evaluation (for details see the Experimental
Section).
Chemistry
The synthesis of the novel 2,4-diamino analogues 8-39 is
shown in Scheme 1. These were synthesized20 by the reaction of
2,4-dichloroquinazolines 1-421 with 4-aminobenzylamine fol-
lowed by an in situ benzoylation (pathway A) or, alternatively,
by reaction with the fully assembled side chains 5-7 (pathway
B). The side chains 5-7 were synthesized in two steps from
commercially available N-BOC-4-aminobenzylamine 40, as
shown in Scheme 1. Subsequently, amination at the 2-position
of the quinazoline moiety was accomplished by heating with
the appropriate amines, in THF or dioxane, to yield 8-39.
A variety of 7-substituted 2-dimethylamino analogues 41-
48 were prepared from the 7-iodo derivative 39, as depicted in
Scheme 2. These 7-substituted quinazolines were synthesized
by the palladium-catalyzed cross-coupling reaction of the
Compound 49 was identified via HTS and showed potent
activity in the β-catenin/Tcf4 reporter assay (Table 5). In
addition, this compound showed acceptable TPSA but poor
solubilityand unfavorablephysicalproperties for oralabsorp-
tion such as high cLogP.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 899
Figure 1. Compound 49 was discovered from HTS. The aminomethylbiphenyl can be replaced with the aminomethylbenzamide substituent in
50 to improve potency and properties.
Furthermore, compound 49 exhibited poor metabolic stabi-
lity, which was caused by the hydrolysis of the methyl ester.
Ligand-based design led us to a new lead series where the
biphenyl substituent in 49 was replaced with a 4-benzamido
group (e.g., 50). Figure 1 shows the overlay of compounds 49
and 50. As one can see, the two structures have similar molecular
shapes, and the putative structure 50 offered an avenue for
novelty, better cLogP (6.0 for compound 49 vs 4.6 for compound
50), and, potentially, increased metabolic/chemical stability,
improved aqueous solubility, and a springboard from which to
develop more potent inhibitors of the β-catenin/Tcf4 pathway.
Table 1 summarizes the IC50 values of compounds 8-16 in
the Tcf4-luciferase reporter assay. Among the various 2-
methylamino-substituted quinazoline derivatives 8-16, the
4-halogen-substituted aryl derivatives 8 and 10 and the 6-
chloronicotinoyl compound 9 showed cell potencies below 1
μM in the reporter cell lines 33.13 and 22C11. The 4-chloro
regioisomer 10 was found to be 4-fold more potent than its
corresponding 3-chloro analogue 14 in the 33.13 cell line.
Similarly, the 6-chloronicotinoyl derivative 9 was marginally
more potent than the 2-chloropicolinyl compound 12. In
comparison with 49, compound 9 had improved water solu-
bility (Table 1). Similarly, the 4-fluorophenyl derivative 8 was
the most potent compound in this series and showed modest
selectivity over the 5A8 cell line. The other 4-substituted
analogues such as -CF3 (13), -CN (15), and -CH3 (16)
exhibited IC50 values of >1 μM in all of the above-mentioned
three cell lines. From the subset of analogues 8-10 with below
1 μM potency, we determined good microsomal stability for
10 and moderate stability for 8 and 9. In addition, 8 and 10
demonstrated good PAMPA permeability, accompanied by
low solubility, while 9 showed poor permeability and moder-
ate solubility.
Several 5-, 6-, 7-, and 8-methyl-substituted quinazoline
derivatives 17-28 were prepared according to Scheme 1,
and their in vitro activities are tabulated in Table 2. Methyl
groups were introduced at the 5-, 6-, 7-, and 8-position of the
quinazoline core, with varying “Ar” substituents. It is evident
from Table 2 that introduction of a methyl group to the
quinazoline core 5-, 6-, 7-, or 8-position can lead to improved
in vitro potency compared to the unsubstituted compounds
8-10. Among the various methyl-substituted derivatives, 5-
methyl derivatives 20 and 24 exhibited the lowest potency.
When the aryl moiety was 6-chloronicotinyl (derivatives
26-28), the 5-, 6-, and 7-methyl derivatives 26-28 were
equipotent with each other against 33.13 and 22C11. How-
ever, when the aryl moiety was 4-fluorophenyl, the 7-methyl
(18) and the 6-methyl (19) derivatives showed the best potency
against 33.13 and 22C11, which was followed by the 8-methyl
analogue (17), establishing the potency order as 7-Me > 6-Me
> 8-Me. For the 4-chlorophenyl analogues 21-24, the 7-
methyl compound 22 showed the best potency against 33.13.
Unfortunately, the introduction of a methyl group had, in
most cases, no impact on the selectivity against the 5A8 cell
line, which was around 3-9-fold for all of the analogues,
except 21, which showed 30-fold selectivity against 5A8.
However, all of the methyl analogues had low solubility,
and no improvement of microsomal stability or PAMPA
permeability was observed, with the exception of 18, when
compared to the demethyl derivatives 8-10. Compound 18
exhibited good microsomal stability but poor solubility and
permeability.
The data in Table 2 showed that methyl substitution in
the 7-position led to the best potency in the 33.13 reporter
line for compounds 18, 22, and 26. In this subset, only the 4-
fluoroaryl analogue 18 showed good microsomal stability.
Therefore, 18 was chosen for further optimization. This
SAR study also showed that the 7-position is best suited for
studying the introduction of other functional groups, in
order to improve properties, as shown in Table 4.
However, these initial results encouraged us to probe the
effect of the methyl substituent in the 5-, 6-, 7-, and 8-positions
of the quinazoline ring in compounds 8-10 in order to find
out which position is most suited for the introduction sub-
stitutents, to improve potency, stability, and solubility.

900 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Dehnhardt et al.
Table 1. Biological Data and Physical Properties for the Arylamido Analogues 8-16
IC50 (nM)a
sol, pH = 7.4
(μg/mL)b
micr stab
(rat), t1/2 (min)c
PAMPA
(pe ꢀ 10-6 cm/s)d
4.2
compd
Ar =
33.13
413
22C11
424
5A8
8
4-fluorophenyl
6-chloronicotinyl
4-chlorophenyl
4-biphenyl
>778
>2984
>2194
>5440
>5139
>1384
>5982
>12241
>25158
1
23
1
21
17
30
16
0
9
596
771
690
1188
0
10
11
12
13
14
15
16
1.4
1.2
0
941
972
>2720
931
1120
1
2-chloropicolinyl
4-CF3-phenyl
3-chlorophenyl
4-cyanophenyl
4-toluoyl
16
1
1038
2771
3647
>25158
30
15
10
16
5.3
3
2274
5642
16
6
0.2
5.8
>25158
2
a The values are an average of at least two separate determinations with a typical variation of less than (30%. b Aqueous solubility at pH = 7.4.16
c Rat microsomes t1/2 (min).17 d The effective permeability PAMPA was measured as described by Kerns et al.18
Table 2. Biological Data and Physical Properties for Alkyl-Substituted Quinazolines 17-28
IC50 (nM)a
sol, pH = 7.4
(μg/mL)b
micr stab
(rat), t1/2 (min)c
PAMPA
(pe ꢀ 10-6 cm/s)d
compd
R =
Ar =
33.13
22C11
5A8
17
18
19
20
21
22
23
24
25
26
27
28
8-methyl
7-methyl
6-methyl
5-methyl
8-methyl
7-methyl
6-methyl
5-methyl
8-methyl
7-methyl
6-methyl
5-methyl
4-fluorophenyl
4-fluorophenyl
4-fluorophenyl
4-fluorophenyl
4-chlorophenyl
4-chlorophenyl
4-chlorophenyl
4-chlorophenyl
6-chloronicotinyl
6-chloronicotinyl
6-chloronicotinyl
6-chloronicotinyl
942
299
415
1080
410
270
595
492
541
414
483
471
1113
329
469
1716
405
389
840
829
848
650
348
573
>3078
>752
1
0
10
>30
6
4.2
0.0
1.3
2.6
0
>3009
>3009
>11569
>832
10
2
15
11
18
6
1
1
0.0
2.4
0.9
0.2
0.3
0.0
1.4
>4019
>2894
>4366
>3850
>1444
>1203
11
0
12
2
2
7
8
3
13
22
1
a The values are an average of at least two separate determinations with a typical variation of less than (30%. b Aqueous solubility at pH = 7.4.16
c Rat microsomes t1/2 (min).17 d The effective permeability PAMPA was measured as described by Kerns et al.18
Next, in order to investigate the effect of different amino
substituents at the 2-position of the quinazoline core, a variety
of amines were introduced on the most potent methyl derivative
18, and the results are tabulated in Table 3. It is interesting
to note that compounds such as 29 and 31-33, bearing ter-
tiary amino groups in the 2-position, exhibited similar potency
against 33.13 and a comparable selectivity against 5A8
cell line. Analogues such as 29, 31, and 33 showed good
PAMPA permeability and poor solubility at physiological
pH. The piperazine containing analogue 35 had good selectiv-
ity, solubility, and modest PAMPA permeability. However,
compound 35 showed poor plasma levels when administered at
50 mpk, po, in female nude mice. This may be due to low
absorption or a high first-pass metabolism. Moreover, analo-
gues 29-38 showed poor rat microsomal stability, possibly due
to the formation of an oxidative metabolite which was
identified by LC/MS during in vitro microsomal incubation
studies.
However, most of the analogues 29-38 in this series
exhibited better selectivity (33.13 vs 5A8) compared to the
methylamino analogue 18. Therefore, it seemed prudent to
obtain further SAR for this series, carrying a tertiary amino
group in the 2-position.
In order to boost the potency and to improve the solubility,
several polar and nonpolar groups were introduced at the
7-position of the quinazoline moiety. The results are tabu-
lated in Table 4. The presence of iodo (39), cyano (41), acetyl
(42), and polar groups (analogues 43-46) is not tolerated.
However, the presence of nonpolar substituents such as vinyl
(47) or ethyl (48) groups is well tolerated. Especially com-
pound 47 exhibited good potency in both 33.13 and 22C11 cell
lines (239 and 337 nM, respectively) and very good selectivity

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 901
Table 3. Biological Data and Physical Properties for C-2-Substituted Analogues 29-38 in the 4-Fluorophenyl Series
IC50 (nM)a
sol, pH = 7.4
(μg/mL)b
micr stab
(rat), t1/2 (min)c
PAMPA
(pe ꢀ 10-6 cm/s)d
compd
NR1R2 =
33.13
22C11
5A8
29
30
31
32
33
34
35
36
37
38
dimethylamine
n-propylamine
azetidine
454
506
300
378
291
322
305
561
676
636
593
645
506
659
524
620
374
662
1008
1779
>2910
>2818
>2831
>2744
>2662
>2656
>4013
>3858
>9028
>10604
1
12
6
1.6
0.0
1.5
0.5
2.0
0.1
0.4
0.02
4.4
0
1
1
7
pyrrolidine
piperidine
piperazine
1
2
0
22
3
5
(R)-3-methylpiperazine
3,3-dimethylpiperazine
N-methylpiperazine
morpholine
27
>100
1
4
3
3
1
6
a The values are an average of at least two separate determinations with a typical variation of less than (30%. b Aqueous solubility at pH = 7.4.16
c Rat microsomes t1/2 (min).17 d The effective permeability PAMPA was measured as described by Kerns et al.18
Table 4. Biological Data and Physical Properties for 7-Substiuted Quinazoline Analogues 39 and 41-48
IC50 (nM)a
sol, pH = 7.4
(μg/mL)b
micr stab
(rat), t1/2 (min)c
PAMPA
(pe ꢀ 10-6 cm)d
compd
R =
33.13
22C11
5A8
39
41
42
43
44
45
46
47
48
I
4192
1479
2330
1900
884
6814
2138
2592
2012
895
>18472
>62900
>20244
>20162
>17940
>10374
>9166
>100
0.0
5.6
0.7
0.1
0
-CN
-COCH3
0
1
11
2
(þ/-) -CH(OH)CH3
18
1
6
-CHO
3
-CH2OH
855
811
4
22
3
0.0
0.4
1.1
0.3
-CH2NMe2
-CHdCH2
-C2H5
1222
239
1046
337
55
1
>11042
>2604
6
335
431
4
6
a The values are an average of at least two separate determinations with a typical variation of less than (30%. b Aqueous solubility at pH = 7.4.16
c Rat microsomes t1/2 (min).17 d The effective permeability PAMPA was measured as described by Kerns et al.18
against the 5A8 cell line. However, analogue 47 had poor
solubility, poor rat microsomal stability, and poor PAMPA
permeability.
Among the various compounds prepared in this series,
based on potency, selectivity (against 5A8), and solu-
bility, analogues 9 and 19 were chosen for further in vitro
evaluation.
Compounds 9, 19, and 49 were tested in a cell growth
inhibition assay with four different colon cancer cell lines
(33.13, HT29, DLD1, and LoVo) to investigate whether
these compounds could inhibit cell growth by inhibiting the
β-catenin/Tcf4 pathway. Listed in Table 5 are IC50 values
of compounds 9, 19, and 49. Notably, all of these three
compounds inhibited growth of the reporter line 33.13 at
concentrations similar to those at which they inhibited the
Tcf4-luciferase reporter activity. In addition, they all dis-
played potent inhibitory activity against three other colon
cancer cell lines.
To test if these lead compounds specifically inhibited
growth of cells that are dependent on activated β-catenin
signaling, we used the cell line RK3E, an adenovirus E1A-
immortalized epithelial cell line derived from neonatal rat
kidney that has been transformed by a mutant β-catenin
protein, β-cat/RK3E.19 This mutant β-catenin gene har-
bors a mutation at the GSK3β phosphorylation site at the
N-terminus of β-catenin, resulting in stabilization of the
β-catenin protein. The same RK3E line transformed by a
mutant K-ras called Kras/RK3E was used as a comparison.
All three compounds 9, 19, and 49 were about two to three
times more potent against the β-cat/RK3E than the Kras/
RK3E line, indicating a selective growth inhibition of the
cells that have a mutantβ-cateninproteinbythese compounds
(Table 5).
Oligonucleotide arrays were used to determine global
transcriptional responses, using Human Genome U133 Plus
2.0 Oligonucleotide Arrays, to siRNA gene knockdown

902 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Table 5. Biological Activity of Compounds 9, 19, and 49
Dehnhardt et al.
IC50 (nM)a
reporter assay
colon cell growth inhibition
cell differential assay
compd
22C11
33.13
5A8
33.13
HT29
DLD1
LoVo
β-cat
Kras
Kras/β-cat
49
9
561
690
469
455
596
415
>6273
>2984
>3009
385
648
447
594
1375
767
1086
1739
620
850
1400
750
1037
749
3036
2096
2021
2.9
2.8
2.1
19
912
a The values are an average of at least two separate determinations with a typical variation of less than (30%.
to determine pathway-specific biomarkers and their corre-
lation with compound activity in HT29 colon cancer cells.
Pathway-responsive genes were identified by transcrip-
tional profiling of triplicate cell samples 48 h following
siRNA-mediated knockdown of either β-catenin or Tcf4
mRNA in comparison to a combination of mock transfec-
tion or Non-targeting siRNA controls. Specific knock-
down of siRNA target gene mRNA was confir-
med for both β-catenin and Tcf4 (57% and 53% de-
crease, respectively) as shown in Supporting Information
Figure 1. Following targeted siRNA knockdown of
β-catenin or Tcf4 in HT29 cells, modulated 310 or 193 tiled
gene probe sets were modulated from 1.8- to 13-fold (P <
0.05), respectively. Of the 435 unique tiled probe sets
modulated by either β-catenin or Tcf4 siRNA knockdown,
66% were modulated more than 1.5-fold by both pathway-
specific knockdowns, demonstrating the anticipated over-
lap in pathway activity. These genes thereby represent
pathway-specific transcriptional biomarkers to compare
with compound activity in HT29 cells.
Compound-responsive transcription was similarly iden-
tified by comparison of mRNA levels in HT29 cells treated
for 24 h with either 9 or 19 relative to vehicle controls.
Compound treatment generated a robust transcriptional
response with 1229 or 1028 unique genes modulated by
either 9 or 19 by minimally 3-fold (P < 0.05; Table 2).
Approximately 75% of the genes were modulated in com-
mon by the two compounds and are candidate biomarkers
of target activity. Cluster analysis of normalized mRNA
expression levels20 identified sets of genes either modulated
in common or preferentially by the individual compounds
(Figure 2 and Supporting Information Figure 2). Both
compounds led to a generally common decreased gene
expression that reflected the strong antiproliferative activ-
ity of these compounds including multiple cyclins
(CCNB1, CCNA2), phosphatases (CDC25B, CDC25C),
and transcription factors (FoxM1) involved in G2/M cell
cycle progression. The compound induced-gene expression
had a component in common including induction of anti-
proliferative stress activated genes (ATF3, GADD34,
HSP40) and induction of multiple DUSP phosphatases
(DUSP1, DUSP5) that both inactivate proliferation-
related MAP kinases.
transcription was normalized to mean expression, and
matrix cluster analysis is shown in Figure 3. All control
samples including mock transfection, nontargeting oligo-
nucleotides, and vehicle treated cells clustered on a unique
node clearly distinct from gene expression in the com-
pound-treated or pathway siRNA knockdown samples.
Analogue 9 clustered closer to the samples following
pathway knockdown and more closely recapitulated the
β-catenin knockdown profile relative to 19. While the
mechanism of action, the kinetics, and the degree of
resultant protein inhibition differ between the siRNA-
mediated and small molecule antagonists, the overlap in
transcriptional signatures provides additional evidence of
β-catenin pathway-specific targeting.
Compared to compound 19, 9 exhibited gene expression
profile changes more similar to those mediated by the
biological pathway knockdown. Therefore, we wanted to
set the stage for in vivo studies by evaluating routes of
administration. However, in subsequent exposure studies
compounds 9 and 19 were each dosed orally at 50 mg/kg in
nude mice; only poor plasma and tumor levels of drug
substance were observed for both. These findings could be
rationalized by the poor PAMPA permeability of 9, lead-
ing to poor absorption, and by the poor solubility and poor
metabolic stability of 19, which could lead to either poor
absorption or high absorption and high clearance. Hence,
in order to conduct a proof of concept study, we settled on
the ip (intraperitoneal) route of administration for an in
vivo efficacy study with compound 9 in a β-cat/RK3E
tumor xenograft model (Figure 3).
When compound 9 was administered once a week at
increasing doses by ip injections, tumor growth inhibition
was observed in a dose-dependent manner. Up to 66%
suppression of tumor growth was achieved when 150 mg/kg
body weight was dosed to mice, compared to the vehicle
control tumors. In addition, a daily low dose of 18.75 mg/kg
by ip for 7 days also led to more than 50% decrease of
tumor volume. At the maximal tolerated dose (150 mg/kg
once a week), several animals died with no other visible signs
of mortality. However, at all other efficacious doses we
observed neither deaths nor weight loss or other signs of
toxicity.
This in vivo study demonstrated that colon cancer cells,
whose growth is dependent on activated β-catenin/
Tcf4 activity, could be inhibited by intervention with a
small molecule. However, the series requires further
optimization to obtain an orally bioavailable drug candi-
date that is efficacious against other colon cancer xeno-
grafts, such as HT29 and LoVo. Clearly, future compounds
will need to address crucial PK issues in order to
bring forward a successful β-catenin/Tcf4 inhibitor. We
will report studies with further optimized compounds
that exhibit better PK properties and potency in the near
future.
Compound-mediated alterations of the expression of the
435 probe sets modulated by either β-catenin and/or Tcf4
siRNA targeted knockdown were interrogated to deter-
mine correlation in pathway-specific gene expression and
candidate antagonist activity. Compound treatment with 9
or 19 resulted in a common minimal 50% induction or
repression for 125 pathway-induced and 99 pathway-re-
pressed probe sets, respectively. Overall, 52% of the path-
way-specific transcription signature responded in a similar
direction following treatment with the β-catenin antago-
nists. The pathway and compound coordinately modulated

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 903
Figure 3. Compound 9 inhibits in vivo growth of a β-catenin/RK3E
tumor xenograft. Nude mice bearing subcutaneous β-catenin/
RK3E tumors were dosed ip with vehicle and compound 9 at
indicated doses once a week and at 18.75 mg/kg once daily for
7 days.
instability and cLogP, which resulted in acceptable metabolic
stability and solubilty. Compounds such as 9 demonstrated
in vivo efficacy in a β-catenin/RK3E mouse xenograft model.
This is the first example which offers a proof of concept that
inhibiting the β-catenin/Tcf4 signaling pathway might be a
viable therapy for treating colon cancer. Further gene profil-
ing analysis demonstrated that compound treatment caused
changes in the pathway-specific gene expression profile simi-
lar to those by siRNA knockdown of β-catenin or Tcf4,
suggesting that these compounds exerted their antiproliferative
effects by inhibiting the β-catenin/Tcf4 pathway specifically.
Experimental Section
General Methods. Melting points were determined in an open
capillary tube on a Meltemp melting point apparatus and are
1
uncorrected. H NMR spectra were determined with a Bruker
DPX-400 spectrometer at 400 MHz. Chemical shifts δ are
reported in parts per million (δ) relative to residual chloroform
(7.26 ppm), TMS (0 ppm), or dimethyl sulfoxide (2.49 ppm) as
an internal reference with coupling constants (J) reported in
hertz (Hz). The peak shapes are denoted as follows: s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; br, broad. Electro-
spray (ES) mass spectra were recorded in positive or negative
mode on a Micromass platform spectrometer. Electron impact
(EI) and high-resolution mass spectra were obtained on a
Finnigen MAT-90 spectrometer. Combustion analyses were
obtained using a Perkin-Elmer Series II 2400 CHNS/O analyzer.
The purity of compounds 8-30, 35, 36, 39, and 41-48 was
determined by combustion analysis. The purity of compounds
31-34, 37, and 38 was determined by analytical LC/MS using
an Agilent 1100 LC, equipped with an Agilent MSD mass spec-
trometer, and an Aquasil C18 column, using MeCN/H2O eluent
at 0.8 mL/min flow (containing 0.1% formic acid), 5.5 min
gradient 0% MeCN to 100% MeCN, monitoring UV absorp-
tion at 254 nm. The purity of compounds 8-39 and 41-48 was
found to be >95%. Reverse-phase HPLC purifications were
performed on a Gilson preparative HPLC system controlled by
Unipoint software using a Phenomenex Gemini 100 ꢀ 30.0 mm.
Thin-layer chromatography (TLC) was performed on Merck
PLC prescored plates 60F254. The terms “concentrated” and
“evaporated” refer to removal of solvents using a rotary eva-
porator at water aspirator pressure with a bath temperature
equal to or less than 40 °C. Unless otherwise noted, reagents
were obtained from commercial sources and were used without
further purification.
Figure 2. Overlap in pathway and compound-mediated gene
expression. Genes modulated in common direction following
pathway knockdown (β-catenin or Tcf4 siRNA) or compound
treatment 9 or 19 were selected and mRNA expression levels
Z normalized to mean expression and clustered by the method
of Eisen.20 Red and green represent mRNA expression levels higher
or lower, respectively, to mean expression. All mock, Non-targeting
oligo and vehicle-treated controls clustered on a distinct node,
while both compound-treated and siRNA knockdown samples
coclustered.
Conclusions
A series of novel diaminoquinazoline derivatives have been
prepared and evaluated as inhibitors of the β-catenin/Tcf4
signaling pathway. Structural modifications were targeted
primarily at improving the pharmaceutical and pharmacoki-
netic properties of the lead compound 49. Replacement of the
biphenyl methyl ester group solved the issue of hydrolytic

904 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Dehnhardt et al.
General Procedure for the Preparation of 2,4-Diaminoquinazo-
line Analogues. Procedure A (Step1). To a stirred solution of 4-
aminobenzylamine (122 mg, 1 mmol) and triethylamine (202
mg, 2 mmol) in CHCl3 (5 mL) at 20 °C was added (1 mmol) of
the appropriately substituted 2,4-dichloroquinazoline, and the
mixture was stirred for a minimum of 3 h. After TLC showed the
complete disappearance of the 2,4-dichloroquinazoline, aroyl
chloride (1 mmol) in CHCl3 (1 mL) was added slowly. The
reaction mixture was allowed to stir overnight. At the end, the
reaction mixture was quenched with CHCl3 (50 mL) and water
(15 mL). The reaction mixture was washed with water, and the
chloroform layer was dried over MgSO4. After removal of
MgSO4 by filtration and evaporation of solvents the crude
product was purified by column chromatography with hex-
ane/CH2Cl2/EA to give the 2-chloroquinazolines in yields be-
tween 50% and 95%.
Procedure A (Step 2). The appropriately substituted 2-chlor-
oquinazoline derivatives (1 mmol) obtained by procedure A
(step 1) was taken up either in a sealed tube or in a round-bottom
flask and was suspended in THF (5 mL) or dioxane. (If the
reactant amine is monomethylamine or dimethylamine, a sealed
tube was used, and for other amines, a round-bottom flask can
be used.) The appropriate amine was added, and the mixture
was heated over 16 h to 100 °C or alternatively heated for 40 min
to 120 °C using a microwave. After reaction was completed, the
solvents were removed in vacuo, and the crude compound was
purified by silica gel column chromatography by using CH2Cl2/
MeOH/NH3 mixtures as eluent to give the diaminoquinazolines
in yields between 65% and 95%.
Procedure B (Step 1). To a stirred suspension of N-[4-(amino-
methyl)phenyl]-4-fluorobenzamide (1.40 g, 5.73 mmol) and
triethylamine (2 mL) in THF (20 mL) at room tempera-
ture was added 7-methyl-2,4-dichloroquinazoline (1.22 g,
5.73 mmol) dissolved in CHCl3 (10 mL), and the mixture
was kept stirring for a minimum of 3 h. After TLC showed
the complete disappearance of the 7-methyl-2,4-dichloroquina-
zoline, CHCl3 (250 mL) and water (25 mL) were added. The
layers were separated, the aqueous layer was extracted twice
with CHCl3 (25 mL), and the combined organic layers were
dried over MgSO4. After removal of MgSO4 by filtration
and evaporation of solvents the crude product was purified
by column chromatography with hexane/CH2Cl2/EA to give
N-(4-{[(2-chloro-7-methylquinazolin-4-yl)amino]methyl}phe-
nyl)-4-fluorobenzamide (1.80 g, 73% yield). MS (ESI) m/z
421.2.
Procedure B (Step 2). N-(4-{[(2-Chloro-7-methylquinazolin-
4-yl)amino]methyl}phenyl)-4-fluorobenzamide (1 mmol) ob-
tained by procedure B (step 1) was taken up either in a sealed
tube or in a round-bottom flask and was suspended in an
appropriate solvent (THF, 5 mL, or dioxane, DMF, 2-propa-
nol, etc., 5 mL). (If the reactant amine is monomethylamine or
dimethylamine, a sealed tube was used, and for other amines, a
round-bottom flask can be used.) The appropriate amine or
amine hydrochloride was added, and the mixture was heated
under stirring over 2-16 h to 100-120 °C. After the reaction
was completed, the solvents were removed in vacuum, and the
crude compound was purified by preparative HPLC (high-
pressure liquid chromatography) using ACN/water/NH3 or
TFA gradients as eluent or column chromatography with
CH2Cl2/MeOH/NH3 to give the diaminoquinazolines in yields
between 65% and 95%.
1H NMR (DMSO) δ 2.81 (d, J = 4.4 Hz, 3H), 4.69 (d, J = 5.0
Hz, 2H), 6.45 (m, 1H), 7.03 (t, J = 7.2 Hz), 7.28 (m, 1H), 7.36 (m,
4H), 7.48 (t, J = 7.2 Hz, 1H), 7.69 (d, J = 8.1 Hz, 2H), 8.03 (m,
3H), 8.38 (s, 1H), 10.25 (s, 1H) ppm; MS (ESI) m/z 402.1. Anal.
Calcd for C23H20FN5O (%): C, 68.81; H, 5.02; F, 4.73; N, 17.45.
Found: C, 69.10; H, 4.83; N, 17.16.
Preparation of 6-Chloro-N-[4-({[2-(methylamino)quinazolin-
4-yl]amino}methyl)phenyl]nicotinamide (9). 6-Chloro-N-[4-({[2-
(methylamino)quinazolin-4-yl]amino}methyl)phenyl]nicotin-
amide was prepared starting from 2,4-dichloroquinazoline, 4-
aminobenzylamine, and 6-chloronicotinoyl chloride following
procedure A (step 1). The intermediate product from step 1 was
aminated using monomethylamine following procedure A (step 2)
to yield the final product. Starting from 2,4-dichloroquinazoline
(0.15 g, 0.75 mmol), 140 mg (95% yield) of the final product was
isolated. 1H NMR (DMSO) δ 2.81 (d, J = 5.0 Hz, 3H), 4.70 (d,
J = 5.0 Hz, 2H), 6.47 (br s, 1H), 7.29-7.24 (m, 1H), 7.39-
7.35 (m, 2H), 7.50-7.47 (m, 1H), 7.71-7.67 (m, 4H), 8.04-8.0 (m,
2H), 8.47-8.30 (m, 2H), 8.93 (d, J = 3.0 Hz, 1H), 10.47 (s, 1H)
ppm; MS (ESI) m/z 419.1. Anal. Calcd for C22H19ClN6O (%): C,
63.08; H, 4.57; N, 20.06. Found: C, 63.31; H, 4.84; N, 20.25.
Preparation of 4-Chloro-N-[4-({[2-(methylamino)quinazolin-
4-yl]amino}methyl)phenyl]benzamide (10). 4-Chloro-N-[4-({[2-
(methylamino)quinazolin-4-yl]amino}methyl)phenyl]benza-
mide was prepared starting from 2,4-dichloroquinazoline, 4-
aminobenzylamine, and 4-chlorobenzoyl chloride following
procedure A (step 1). The intermediate product from step 1
was aminated using monomethylamine following procedure A
(step 2) to yield the final product. Starting from 2,4-dichlor-
oquinazoline (100 mg, 0.5 mmol), 20 mg (41% yield) of the final
product was isolated. 1H NMR (DMSO) δ 2.49 (d, J = 5.0 Hz,
3H), 4.71-4.66 (m, 2H), 6.51-6.41 (m, 1H), 7.04 (t, J = 7.8 Hz,
1H), 7.29-7.22 (m, 1H), 7.39 (d, J = 8.8 Hz, 2H), 7.49 (t, J = 7.8
Hz, 1H), 7.61 (d, J = 9.1 Hz, 2H), 7.69 (d, J = 8.8 Hz, 2H), 7.97
(d, J = 9.1 Hz, 2H), 8.01 (d, J = 7.8 Hz, 1H), 8.40 (s, 1H), 10.29
(s, 1H) ppm; MS (ESI) m/z 418.1. Anal. Calcd for C23H20ClN5O
(%): C, 66.10; H, 4.82; N, 16.76. Found: C, 65.97; H, 5.06; N,
16.80.
Preparation of N-[4-({[2-(Methylamino)quinazolin-4-yl]ami-
no}methyl)phenyl]biphenyl-4-carboxamide (11). N-[4-({[2-(Methyl-
amino)quinazolin-4-yl]amino}methyl)phenyl]biphenyl-4-carbox-
amide was prepared starting from 2,4-dichloroquinazoline,
4-aminobenzylamine, and biphenyl-4-carbonyl chloride following
procedure A (step 1). The intermediate product from step 1 was
aminated using monomethylamine following procedure A (step 2)
to yield the final product. Starting from 2,4-dichloroquinazoline
(0.17 g, 0.85 mmol), 50 mg (49% yield) of the final product was
isolated. 1H NMR (DMSO) δ 2.81 (d, J = 5.0 Hz, 3H), 4.72-4.67
(m, 2H), 6.53-6.43 (m, 1H), 7.07-7.01 (m, 1H), 7.52-7.22 (m,
7H), 7.86-7.73 (m, 6H), 8.08-8.00 (m, 3H), 8.45-8.33 (s, 1H),
10.28 (s, 1H) ppm; MS (ESI) m/z 460.2. Anal. Calcd for C29H25-
N5O (%): C, 75.80; H, 5.48; N, 15.24. Found: C, 76.03; H, 5.73; N,
15.13.
Preparation of 2-Chloro-N-[4-({[2-(methylamino)quinazolin-
4-yl]amino}methyl)phenyl]isonicotinamide (12). 2-Chloro-N-[4-
({[2-(methylamino)quinazolin-4-yl]amino}methyl)phenyl]iso-
nicotinamide was prepared starting from 2,4-dichloroquinazoline,
4-aminobenzylamine, and 2-chloroisonicotinoyl chloride fol-
lowing procedure A (step 1). The intermediate product from
step 1 was aminated using monomethylamine following proce-
dure A (step 2) to yield the final product. Starting from 2,
4-dichloroquinazoline (0.18 g, 0.9 mmol), 40 mg (24% yield) of
the final product was isolated. 1H NMR (DMSO) δ 9.36 (br s,
1H), 9.31 (t, J = 3.0 Hz, 1H), 8.34 (d, J = 9.0 Hz, 1H), 8.27 (d,
J = 9.0 Hz, 1H), 7.92 (d, J = 9.0 Hz, 2H), 7.85-7.79 (m, 1H),
7.65 (d, J = 12.0 Hz, 1H), 7.58-7.53 (m, 2H), 7.38-7.31 (m,
3H), 7.14-7.10 (m, 1H), 6.72-6.66 (m, 1H), 4.76 (d, J = 5.0 Hz,
2H), 2.83 (d, J = 5.0 Hz, 3H) ppm; MS (ESI) m/z 419.8. Anal.
Calcd for C22H19ClN6O (%): C, 63.08; H, 4.57; N, 20.06.
Found: C, 63.21; H, 4.78; N, 20.16.
Preparation of 4-Fluoro-N-[4-({[2-(methylamino)quinazolin-4-
yl]amino}methyl)phenyl]benzamide (8). 4-Fluoro-N-[4-({[2-(me-
thylamino)quinazolin-4-yl]amino}methyl)phenyl]benzamide
was prepared starting from 2,4-dichloroquinazoline, 4-amino-
benzylamine, and 4-fluorobenzoyl choride following procedure
A (step 1). The intermediate product from step 1 was aminated
using monomethylamine following procedure A (step 2) to yield
the final product. Starting from 2,4-dichloroquinazoline (0.38 g,
1.9 mmol), 100 mg (55% yield) of the final product was isolated.

Article
Preparation of N-[4-({[2-(Methylamino)quinazolin-4-yl]amino}-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 905
Hz, 1H), 8.02 (m, 2H), 8.28 (s, 1H), 10.22 (s, 1H) ppm; MS (ESI)
m/z 416.1. Anal. Calcd for C24H22FN5O (%): C, 69.38; H, 5.34;
N, 16.86. Found: C, 69.53; H, 5.58; N, 16.57.
methyl)phenyl]-4-(trifluoromethyl)benzamide (13).N-[4-({[2-(Methyl-
amino)quinazolin-4-yl]amino}methyl)phenyl]-4-(trifluoromethyl)-
benzamide was prepared starting from 2,4-dichloroquinazoline,
4-aminobenzylamine, and 4-trifluoromethylbenzoyl chloride follow-
ing procedure A (step 1). The intermediate product from step 1 was
aminated using monomethylamine following procedure A (step 2) to
yield the final product. Starting from 2,4-dichloroquinazoline (0.19 g,
1.0 mmol), 50 mg (43% yield) of the final product was isolated. 1H
NMR (DMSO) δ 3.07 (d, J = 5.0 Hz, 3H), 4.65-4.63 (m, 2H),
6.62-6.53 (m, 1H), 7.41-7.32 (m, 4H), 7.62-7.51 (m, 4H),
7.79-7.64 (m, 4H), 7.94-7.89 (m, 2H) ppm; MS (ESI) m/z 452.2.
Anal. Calcd for C24H20F3N5O (%): C, 63.85; H, 4.47; N, 15.51.
Found: C, 63.56; H, 4.65; N, 15.43.
Preparation of 4-Fluoro-N-[4-({[7-methyl-2-(methylamino)-
quinazolin-4-yl]amino}methyl)phenyl]benzamide (18). 4-Fluoro-
N-[4-({[7-methyl-2-(methylamino)quinazolin-4-yl]amino}me-
thyl)phenyl]benzamide was prepared by amination of N-(4-{[(2-
chloro-7-methylquinazolin-4-yl)amino]methyl}phenyl)-4-fluo-
robenzamide (250 mg, 0.6 mmol) and 2 M methylamine in THF
following procedure B (step 2). After purification by HPLC and
solvent removal the final product (150 mg, 76% yield) was
1
isolated. H NMR (DMSO) δ 2.34 (s, 3H), 2.79 (d, J = 4.5
Hz, 3H), 4.67 (d, J = 5.6 Hz, 2H), 6.47 (s, 1H), 6.88 (m, 1H), 7.08
(s, 1H), 7.36 (m, 4H), 7.69 (d, J = 8.3 Hz, 2H), 7.91 (d, J = 8.3
Hz, 1H), 8.02 (m, 2H), 8.32 (s, 1H), 10.23 (s, 1H) ppm; MS (ESI)
m/z 416.2. Anal. Calcd for C24H22FN5O (%): C, 69.38; H, 5.34;
N, 16.86. Found: C, 69.24; H, 5.52; N, 16.93.
Preparation of 3-Chloro-N-[4-({[2-(methylamino)quinazolin-
4-yl]amino}methyl)phenyl]benzamide (14). 3-Chloro-N-[4-({[2-
(methylamino)quinazolin-4-yl]amino}methyl)phenyl]benza-
mide was prepared starting from 2,4-dichloroquinazoline,
4-aminobenzylamine, and 3-chlorobenzoyl chloride following
procedure A (step 1). The intermediate product from step 1 was
aminated using monomethylamine following procedure A (step
2) to yield the final product. Starting from 2,4-dichloroquinazo-
line (100 mg, 0.5 mmol), 30 mg (30% yield) of the final product
Preparation of 4-Fluoro-N-[4-({[6-methyl-2-(methylamino)-
quinazolin-4-yl]amino}methyl)phenyl]benzamide (19). 4-Fluoro-
N-[4-({[6-methyl-2-(methylamino)quinazolin-4-yl]amino}methyl)-
phenyl]benzamide was prepared starting from 6-methyl-2,
4-dichloroquinazoline, 4-aminobenzylamine, and 4-fluoroben-
zoyl chloride following procedure A (step 1). The intermediate
product from step 1 was aminated using monomethylamine
following procedure A (step 2) to yield the final product.
Starting from 410 mg (2.0 mmol) of 6-methyl-2,4-dichloroqui-
nazoline, 200 mg (58% yield) of the final product was isolated.
1H NMR (DMSO) δ 2.34 (s, 3H), 2.81 (d, J = 5.0 Hz, 3H), 4.68
(d, J = 5.0 Hz, 3H), 6.49 (br s, 1H), 7.23-7.19 (m, 1H),
7.38-7.33 (m, 3H), 7.71-7.68 (m, 2H), 7.86 (s, 1H), 8.06-
8.00 (m, 2H), 8.49-8.30 (br s, 1H), 10.25 (s, 1H) ppm; MS (ESI)
m/z 416.2. Anal. Calcd for C24H22FN5O (%): C, 69.38; H, 5.34;
N, 16.86. Found: C, 69.21; H, 5.63; N, 16.75.
Preparation of 4-Fluoro-N-[4-({[5-methyl-2-(methylamino)-
quinazolin-4-yl]amino}methyl)phenyl]benzamide (20). 4-Fluoro-
N-[4-({[5-methyl-2-(methylamino)quinazolin-4-yl]amino}methyl)-
phenyl]benzamide was prepared starting from 5-methyl-2,
4-dichloroquinazoline (426 mg, 2 mmol), 4-aminobenzylamine,
and 4-fluorobenzoyl chloride following procedure A (step 1)
to give the 4-fluoro-N-[4-({[2-chloro-5-methylquinazolin-4-yl]-
amino}methyl)phenyl]benzamide (490 mg, 58% yield). The
intermediate product (250 mg, 0.6 mmol) from step 1 was ami-
nated with monomethylamine following procedure A (step 1) to
obtain the final product (98 mg, 40% yield). mp 228-230 °C; 1H
NMR (DMSO) δ 2.50 (s, 3H), 2.78 (s, 6H), 4.69 (d, J = 5.8 Hz,
2H), 6.43 (s, 1H), 6.82 (d, J = 6.8 Hz, 1H), 7.11 (m, 1H),
7.30-7.43 (m, 5H), 7.69 (d, J = 7.8 Hz, 2H), 8.02 (m, 2H), 10.23
(s, 1H) ppm; MS (ESI) m/z 416.3. Anal. Calcd for C24H22FN5O
(%): C, 69.38; H, 5.34; N, 16.86. Found: C, 69.42; H, 5.43; N,
16.90.
Preparation of 4-Chloro-N-[4-({[8-methyl-2-(methylamino)-
quinazolin-4-yl]amino}methyl)phenyl]benzamide (21). 4-Chloro-
N-[4-({[8-methyl-2-(methylamino)quinazolin-4-yl]amino}methyl)-
phenyl]benzamide was prepared starting from 8-methyl-2,4-
dichloroquinazoline, 4-aminobenzylamine, and 4-chlorobenzoyl
chloride following procedure A (step 1). The intermediate
product from step 1 was aminated using monomethylamine
following procedure A (step 1) to yield the final product.
Starting from 500 mg (2.35 mmol) of 8-methyl-2,4-dichloroqui-
nazoline, 320 mg (32% yield) of the final product was isolated.
1H NMR (DMSO) δ 2.40 (s, 3H), 2.83 (d, J = 4.6 Hz, 3H), 4.68
(d, J = 6.0 Hz, 2H), 6.40 (m, 1H), 6.92 (t, J = 7.8 Hz, 1H), 7.35
(m, 4H), 7.60 (d, J = 7.8 Hz, 2H), 7.68 (d, J = 8.8 Hz, 2H), 7.85
(d, J = 7.8 Hz, 1H), 7.97 (d, J = 8.8 Hz, 2H), 8.29 (s, 1H), 10.27 (s,
1H) ppm; MS (ESI) m/z 432.1. Anal. Calcd for C24H22ClN5O (%):
C, 66.74; H, 5.13; N, 16.21. Found: C, 66.53; H, 5.35; N, 16.11.
Preparation of 4-Chloro-N-[4-({[7-methyl-2-(methylamino)-
quinazolin-4-yl]amino}methyl)phenyl]benzamide (22). 4-Chloro-
N-[4-({[7-methyl-2-(methylamino)quinazolin-4-yl]amino}methyl)-
phenyl]benzamide was prepared starting from 7-methyl-2,
1
was isolated. H NMR (DMSO) δ 3.05 (d, J = 5. 0 Hz, 3H),
4.81-4.76 (m, 2H), 5.89 (br s, 1H), 7.11-7.04 (m, 1H), 7.44-
7.38 (m, 6H), 7.56-7.49 (m, 2H), 7.61-7.58 (m, 2H), 7.76-7.72
(m, 1H), 7.91-7.82 (m, 2H) ppm; MS (ESI) m/z 418.1. Anal.
Calcd for C23H20ClN5O (%): C, 66.10; H, 4.82; N, 16.76.
Found: C, 65.82; H, 5.05; N, 16.53.
Preparation of 4-Cyano-N-[4-({[2-(methylamino) quinazolin-
4-yl]amino}methyl)phenyl]benzamide (15). 4-Cyano-N-[4-({[2-
(methylamino)quinazolin-4-yl]amino}methyl)phenyl]benza-
mide was prepared starting from 2,4-dichloroquinazoline,
4-aminobenzylamine, and 4-cyanobenzoyl chloride following
procedure A (step 1). The intermediate product from step 1 was
aminated using monomethylamine following procedure A (step
2) to yield the final product. Starting from 2,4-dichloroquinazo-
line (190 mg, 1.0 mmol), 30 mg (16%yield) of the final product
1
was isolated. H NMR (DMSO) δ 3.08-3.06 (m, 3H), 4.67-
4.65 (m, 2H), 6.47-6.41 (m, 1H), 7.22-7.16 (m, 1H), 7.40-7.36
(m, 2H), 7.69-7.53 (m, 5H), 7.79-7.73 (m, 4H), 7.92-7.89 (m,
2H) ppm; MS (ESI) m/z 409. Anal. Calcd for C24H20N6O (%):
C, 70.57; H, 4.94; N, 20.58. Found: C, 70.29; H, 5.20; N, 20.45.
Preparation of 4-Methyl-N-[4-({[2-(methylamino)quinazolin-
4-yl]amino}methyl)phenyl]benzamide (16). 4-Methyl-N-[4-({[2-
(methylamino)quinazolin-4-yl]amino}methyl)phenyl]benza-
mide was prepared starting from 2,4-dichloroquinazoline,
4-aminobenzylamine, and 4-methylbenzoyl choride following
procedure A (step 1). The intermediate product from step 1 was
aminated using monomethylamine following procedure A (step
2) to yield the final product. Starting from of 2,4-dichloroqui-
nazoline (500 mg, 2.5 mmol), 200 mg (40% yield) of the final
product was isolated. 1H NMR (DMSO) δ 2.50 (s, 3H), 3.35 (d,
J = 3.5 Hz, 3H), 4.44 (d, J = 5.0 Hz, 2H), 7.36-7.24 (m, 6H),
7.90-7.68 (m, 6H), 8.97-8.92 (m, 1H), 10.14 (s, 1H) ppm; MS
(ESI) m/z 398.2. Anal. Calcd for C24H23N5O (%): C, 72.52; H,
5.83; N, 17.62. Found: C, 72.41; H, 6.09; N, 17.90.
Preparation of 4-Fluoro-N-[4-({[8-methyl-2-(methylamino)-
quinazolin-4-yl]amino}methyl)phenyl]benzamide (17). 4-Flu-
oro-N-[4-({[8-methyl-2-(methylamino)quinazolin-4-yl]ami-
no}methyl)phenyl]benzamide was prepared starting from
8-methyl-2,4-dichloroquinazoline, 4-aminobenzylamine, and
4-fluorobenzoyl chloride following procedure A (step 1). The
intermediate product from step 1 was aminated using mono-
methylamine following procedure A (step 2) to yield the final
product. Starting from 500 mg (2.35 mmol) of 8-methyl-2,4-
dichloroquinazoline, 274 mg (28% yield) of the final product
was isolated. 1H NMR (DMSO) δ 2.40 (s, 3H), 2.83 (d, J = 4.8
Hz, 3H), 4.68 (d, J = 5.8 Hz, 2H), 6.39 (m, 1H), 6.92 (t, J = 7.8
Hz, 1H), 7.35 (m, 4H), 7.68 (d, J = 8.6 Hz, 2H), 7.85 (d, J = 7.8

906 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Dehnhardt et al.
4-dichloroquinazoline (500 mg, 3.0 mmol), 4-aminobenzyla-
mine, and 4-chlorobenzoyl chloride following procedure A
(step 1) to give 4-chloro-N-[4-({[2-chloro-7-methylquinazolin-
4-yl]amino}methyl)phenyl]benzamide (845 mg, 82% yield). The
product (200 mg, 0.46 mmol) from step 1 was aminated with
monomethylamine hydrochloride to obtain the final product
(150 mg, 35% yield). mp 264-266 °C; 1H NMR (DMSO) δ 2.34
(s, 3H), 2.80 (d, J = 4.3 Hz, 3H), 4.67 (d, J = 5.8 Hz, 2H), 6.44
(s, 1H), 6.88 (dd, J = 1.8, 8.3 Hz, 1H), 7.08 (s, 1H), 7.35 (d, J =
8.3 Hz, 2H), 7.60 (d, J = 8.3 Hz, 2H), 7.69 (d, J = 8.6 Hz, 2H),
7.91 (d, J = 8.6 Hz, 1H), 7.07 (d, J = 8.6 Hz, 2H), 8.32 (s, 1H),
10.28 (s, 1H) ppm; MS (ESI) m/z 432.2. Anal. Calcd for
C24H22ClN5O (%): C, 66.74; H, 5.13; N, 16.21. Found: C,
66.96; H, 5.34; N, 16.11.
Preparation of 4-Chloro-N-[4-({[6-methyl-2-(methylamino)-
quinazolin-4-yl]amino}methyl)phenyl]benzamide (23). 4-Chloro-
N-[4-({[6-methyl-2-(methylamino)quinazolin-4-yl]amino}methyl)-
phenyl]benzamide was prepared starting from 6-methyl-2,
4-dichloroquinazoline, 4-aminobenzylamine, and 4-chloroben-
zoyl chloride following procedure A (step 1). The intermediate
product from (step 1 was aminated using monomethylamine
following procedure A (step 2) to yield the final product.
Starting from 6-methyl-2,4-dichloroquinazoline (700 mg, 3.5
mmol), 400 mg (61% yield) of the final product was isolated. 1H
NMR (DMSO) δ 2.49 (s, 3H), 3.08-3.03 (d, J = 5.0 Hz, 3H),
5.91-5.85 (m, 1H), 6.82-6.76 (m, 1H), 7.30-7.24 (m, 4H),
7.50-7.43 (m, 4H), 7.84-7.78 (m, 2H), 7.99-7.97 (m, 1H),
12.10 (s, 1H) ppm; MS (ESI) m/z 432.3. Anal. Calcd for
C24H22ClN5O (%): C, 66.74; H, 5.13; N, 16.21. Found: C,
66.53; H, 5.36; N, 16.46.
Preparation of 4-Chloro-N-[4-({[5-methyl-2-(methylamino)-
quinazolin-4-yl]amino}methyl)phenyl]benzamide (24). 4-Chloro-
N-[4-({[5-methyl-2-(methylamino)quinazolin-4-yl]amino}methyl)-
phenyl]benzamide was prepared starting from 5-methyl-2,
4-dichloroquinazoline (426 mg, 2 mmol), 4-aminobenzylamine,
and 4-chlorobenzoyl chloride following procedure A (step 1) to
give the 4-chloro-N-[4-({[2-chloro-5-methylquinazolin-4-yl]-
amino}methyl)phenyl]benzamide (155 mg, 18% yield). The
product (120 mg, 0.28 mmol) from step 1 was aminated with
monomethylamine to obtain the final product (71 mg, 60%
yield). 1H NMR (DMSO) δ 2.80 (m, 6H), 4.72 (m, 2H), 6.90 (d,
J = 7.6 Hz, 1H), 7.17 (d, J = 7.6 Hz, 1H), 7.38 (m, 1H), 7.59 (d,
J = 8.3 Hz, 2H), 7.60 (d, J = 8.3 Hz, 2H), 7.70 (d, J = 9.0 Hz,
2H), 7.97 (d, J = 8.3 Hz, 2H), 10.29 (s, 1H) ppm; MS (ESI) m/z
432.3. Anal. Calcd for C24H22ClN5O (%): C, 66.74; H, 5.13; N,
16.21. Found: C, 66.53; H, 5.45; N, 16.34.
mL) was added, and the mixture was filtered over Celite. The
solvent were removed in vacuum to obtain (4-aminobenzyl)(2-
methylamino-7-methyl-quinazolin-4-yl)amine, which was sus-
pended in THF (10 mL) and NEt3 (0.4 mL) and cooled to 0 °C.
6-Chloronicotinoyl chloride (280 mg, 1.59 mmol) was added.
After completion 1 N NaOH (0.5 mL) was added, and the layers
were separated. The organic layer was extracted twice with
THF/ethyl acetate (10 mL), and the combined organic layers
were dried over MgSO4. The crude material was purified by
column chromatography using CH2Cl2/MeOH/NH3 to give
product (230 mg, 31% yield). 1H NMR (DMSO) δ 2.33 (s,
3H), 2.79 (d, J = 4.5 Hz, 3H), 4.67 (d, J = 5.8 Hz, 2H), 6.38 (m,
1H), 6.87 (d, J = 7.8 Hz, 1H), 7.07 (m, 1H), 7.36 (d, J = 7.8 Hz,
2H), 7.69 (m, 3H), 7.90 (d, J = 7.8 Hz, 1H), 8.29 (s, 1H), 8.34 (d,
J = 7.8 Hz, 1H), 8.93 (s, 1H) ppm; MS (ESI) m/z 433.2. Anal.
Calcd for C23H21ClN6O (%): C, 63.81; H, 4.89; N, 19.41.
Found: C, 64.10; H, 5.05; N, 19.32.
Preparation of 6-Chloro-N-[4-({[6-methyl-2-(methylamino)-
quinazolin-4-yl]amino}methyl)phenyl]nicotinamide (27). 6-Chloro-
N-[4-({[6-methyl-2-(methylamino)quinazolin-4-yl]amino}methyl)-
phenyl]nicotinamide was prepared starting from 6-methyl-2,
4-dichloroquinazoline (639 mg, 3.0 mmol), 4-aminobenzylamine,
and 6-chloronicotinoyl chloride following procedure A (step 1)
to give the 6-chloro-N-[4-({[2-chloro-8-methylquinazolin-4-yl]-
amino}methyl)phenyl]nicotinamide (490 mg, 37% yield). The
product (200 mg, 0.46 mmol) from step 1 was aminated with
monomethylamine hydrochloride following procedure B (step 2)
to obtain the final product (90 mg, 43% yield). mp 233-236 °C; 1H
NMR (DMSO) δ 2.51 (s, 3H), 2.95 (s, 3H), 4.76 (br s, 2H),
7.45-7.40 (m, 2H) 7.65-7.61 (m, 1H) 7.78-7.68 (m, 3H),
8.22-8.16 (m, 1H), 8.39-8.35 (m, 1H), 8.95 (m, 1H), 10.18 (br
s, 1H), 9.83-9.70 (m, 1H) 10.58 (s, 1H), 12.30 (br s, 1H) ppm; MS
(ESI) m/z 432.3. Anal. Calcd for C23H21ClN6O (%): C, 63.81; H,
4.89; N, 19.41. Found: C, 63.52; H, 5.07; N, 19.47.
Preparation of 6-Chloro-N-[4-({[8-methyl-2-(methylamino)-
quinazolin-4-yl]amino}methyl)phenyl]nicotinamide (28). 6-Chloro-
N-[4-({[8-methyl-2-(methylamino)quinazolin-4-yl]amino}methyl)-
phenyl]nicotinamide was prepared starting from 8-methyl-2,
4-dichloroquinazoline, 4-aminobenzylamine, and 6-chloronicoti-
noyl chloride following procedure A (step 1). The intermediate
product from step 1 was aminated using monomethylamine
following procedure A (step 2) to yield the final product. Starting
from 8-methyl-2,4-dichloroquinazoline (213 mg, 0.1 mmol), 250
mg (23% yield) of the final product was isolated. 1H NMR
(DMSO) δ 2.40 (s, 3H), 2.83 (d, J = 4.8 Hz, 3H), 4.70 (d, J =
5.5 Hz, 2H), 6.42 (s, 1H), 6.93 (m, 1H), 7.37 (m, 3H), 7.69 (m, 3H),
7.86 (d, J = 7.8 Hz, 1H), 8.31 (s, 1H), 8.34 (dd, J = 2,5 ; 8.3 Hz,
1H), 8.93 (d, J = 2.5 Hz, 1H), 10.46 (s, 1H) ppm; MS (ESI) m/z
433.2. Anal. Calcd for C23H21ClN6O (%): C, 63.81; H, 4.89; Cl,
8.19; N, 19.41; O, 3.70. Found: C, 64.03; H, 5.04; N, 19.62.
Preparation of N-[4-({[2-(Dimethylamino)-7-methylquinazo-
lin-4-yl]amino}methyl)phenyl]-4-fluorobenzamide (29). N-[4-({[2-
(Dimethylamino)-7-methylquinazolin-4-yl]amino}methyl)phenyl]-
4-fluorobenzamide was prepared by amination of N-(4-{[(2-
chloro-7-methylquinazolin-4-yl)amino]methyl}phenyl)-4-fluoro-
benzamide (150 mg, 0.36 mmol) with 2 M dimethylamine
hydrochloride in 2-propanol following procedure B (step 2). After
purification by column chromatography and solvent removal the
final product (110 mg, 71% yield) was obtained as a white solid.
1H NMR (DMSO) δ 2.36 (s, 3H), 2.63 (s, 6H), 4.68 (d, J = 5.5 Hz,
2H), 6.93 (m, 1H), 7.15 (m, 1H), 7.36 (m, 4H), 7.69 (d, J = 8.5 Hz,
2H), 7.95 (d, J = 8.1 Hz, 1H), 8.02 (m, 2H), 10.23 (s, 1H) ppm; MS
(ESI) m/z 430.3. Anal. Calcd for C25H24FN5O (%): C, 69.91; H,
5.63; F, 4.42; N, 16.31. Found: C, 69.73; H, 5.87; N, 16.23.
Preparation of 4-Fluoro-N-[4-({[7-methyl-2-(propylamino)-
quinazolin-4-yl]amino}methyl)phenyl]benzamide (30). 4-Fluoro-
N-[4-({[7-methyl-2-(propylamino)quinazolin-4-yl]amino}methyl)-
phenyl]benzamide was prepared by amination of N-(4-
{[(2-chloro-7-methylquinazolin-4-yl)amino]methyl}phenyl)-4-
fluorobenzamide (150 mg, 0.36 mmol) and propylamine in THF
Preparation of 6-Chloro-N-[4-({[5-methyl-2-(methylamino)-
quinazolin-4-yl]amino}methyl)phenyl]nicotinamide (25). 6-Chloro-
N-[4-({[5-methyl-2-(methylamino)quinazolin-4-yl]amino}methyl)-
phenyl]nicotinamide was prepared starting from 8-methyl-2,
4-dichloroquinazoline (639 mg, 3.0 mmol), 4-aminobenzylamine,
and 6-chloronicotinoyl chloride following procedure A (step 1) to
give 6-chloro-N-[4-({[2-chloro-8-methylquinazolin-4-yl]amino}-
methyl)phenyl]nicotinamide (490 mg, 37% yield). The product
(200 mg, 0.46 mmol) from step 1 was aminated with monomethy-
lamine hydrochloride following procedure B (step 2) to obtain the
1
final product (66 mg, 33% yield). mp 233-236 °C; H NMR
(DMSO) δ 2.79 (m, 6H), 4.70 (d, J = 5.3 Hz, 2H), 6.42 (s, 1H),
6.82 (d, J = 7.0 Hz, 1H), 7.11 (d, J = 8.6 Hz, 1H), 7.32 (t, J = 7.7
Hz, 1H), 7.42 (d, J= 8.3 Hz, 2H), 7.69 (d, J= 8.6 Hz, 1H), 7.70 (d,
J = 8.3 Hz, 2H), 8.34 (dd, J = 3.0, 8.8 Hz, 1H), 8.93 (d, J = 2 Hz,
1H), 10.45 (s, 1H) ppm; MS (ESI) m/z 432.3. Anal. Calcd for
C23H21ClN6O (%): C, 63.81; H, 4.89; N, 19.41. Found: C, 63.62;
H, 5.03; N, 19.23.
Preparation of 6-Chloro-N-[4-({[7-methyl-2-(methylamino)-
quinazolin-4-yl]amino}methyl)phenyl]nicotinamide (26). (2-N-
Dimethylamino-7-methylquinazolin-4-yl)(4-nitrobenzyl)amine
(540 mg, 1.67 mmol) was dissolved in THF (30 mL), Pd-C (10%
wet wt) (100 mg) was added, and the mixture was hydrogenated
under 1 atm of pressure for 24 h. When completed MeOH (500

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 907
following procedure B (step 2). After purification by HPLC and
solvent removal, the final product (150 mg, 76% yield) was
obtained as an off-white solid. mp 170-174 °C; 1H NMR
(DMSO) δ 0.89 (m, 3H), 1.52 (m, 2H), 2.44 (s, 3H), 3.36 (m,
3H), 4.76 (d, J = 5.5 Hz, 2H), 7.25 (m, 2H), 7.37 (m, 4H), 7.73 (d,
J = 8.3 Hz, 2H), 7.97 (m, 1H), 8.02 (m, 2H), 8.20 (m, 1H), 10.28 (s,
1H) ppm; MS (ESI) m/z 444.1. Anal. Calcd for C26H26FN5O (%):
C, 70.41; H, 5.91; N, 15.79. Found: C, 70.24; H, 6.03; N, 15.85.
Preparation of N-(4-{[(2-Azetidin-1-yl-7-methylquinazolin-4-
yl)amino]methyl}phenyl)-4-fluorobenzamide (31). N-(4-{[(2-Aze-
tidin-1-yl-7-methylquinazolin-4-yl)amino]methyl}phenyl)-4-
fluorobenzamide was prepared by amination of N-(4-{[(2-
chloro-7-methylquinazolin-4-yl)amino]methyl}phenyl)-4-fluo-
robenzamide (42 mg, 0.1 mmol) with azetidine hydrochloride
and NEt3 in dioxane following procedure B (step 2). After
purification by HPLC and solvent removal, the final product
(20 mg, 74% yield) was obtained as an off-white solid. Analy-
tical LC/MS using an Aquasil C18 column, ACN/H2O eluent at
0.8 mL/min flow (containing 0.1% formic acid), 5.5 min gra-
dient 0% ACN to 100% ACN, monitored by UV absorption at
254 nm showed >99.5% purity. MS (ESI) m/z 442.2.
Preparation of 4-Fluoro-N-(4-{[(7-methyl-2-pyrrolidin-1-yl-
quinazolin-4-yl)amino]methyl}phenyl)benzamide (32). 4-Fluoro-
N-(4-{[(7-methyl-2-pyrrolidin-1-ylquinazolin-4-yl)amino]methyl}-
phenyl)benzamide was prepared by amination of N-(4-{[(2-
chloro-7-methylquinazolin-4-yl)amino]methyl}phenyl)-4-fluo-
robenzamide (42 mg, 0.1 mmol) and pyrrolidine in dioxane
following procedure B (step 2). After purification by HPLC and
solvent removal, the final product (16 mg, 56% yield) was
obtained as an off-white solid. Analytical LC/MS using an
Aquasil C18 column, ACN/H2O eluent at 0.8 mL/min flow
(containing 0.1% formic acid), 5.5 min gradient 0% ACN to
100% ACN, monitored by UV absorption at 254 nm showed
>99.5% purity. MS (ESI) m/z 456.5.
(m, 1H), 2.69 (m, 1H), 2.86 (d, J = 11.9 Hz, 1H), 4.56 (d, J = 11.9
Hz, 2H), 4.63 (t, J = 5.3 Hz, 2H), 6.89 (d, J = 8.3 Hz, 1H), 7.07 (s,
1H), 7.36 (m, 4H), 7.69 (d, J = 8.0 Hz, 2H), 7.91 (d, J = 8.6 Hz,
1H), 8.02 (m, 2H), 8.43 (t, J = 5.3 Hz, 1H), 10.22 (s, 1H) ppm; MS
(ESI) m/z 485.6. Anal. Calcd for C28H29FN6O (%): C, 69.40; H,
6.03; F, 3.92; N, 17.34; O, 3.30. Found: C, 69.15; H, 6.14; N, 17.17.
Preparation of N-[4-({[2-(3,3-Dimethylpiperazin-1-yl)-7-
methylquinazolin-4-yl]amino}methyl)phenyl]-4-fluorobenzamide
(36). 4-N-[4-({[2-(3,3-Dimethylpiperazin-1-yl)-7-methylquina-
zolin-4-yl]amino}methyl)phenyl]-4-fluorobenzamide was pre-
pared by amination of N-(4-{[(2-chloro-7-methylquinazolin-4-yl)-
amino]methyl}phenyl)-4-fluorobenzamide (150 mg, 0.36 mmol)
and 2,2-dimethylpiperazine in THF following procedure B
(step 2). After purification by HPLC and solvent removal, the
final product (105 mg, 59% yield) was obtained as an off-white
1
solid. H NMR (DMSO) δ 0.98 (s, 6H), 2.34 (s, 3H), 2.79 (m,
2H), 3.52 (s, 2H), 3.70 (m, 2H), 4.62 (d, J = 5.5 Hz, 2H), 6.89
(dd, J = 1.5, 8.3 Hz, 1H), 7.06 (s, 1H), 7.35 (m, 4H), 7.60 (d, J =
8.6 Hz, 2H), 7.91 (d, J = 8.3 Hz, 1H), 8.02 (m, 2H), 8.45 (t, J =
5.5 Hz, 1H), 10.22 (s, 1H) ppm; MS (ESI) m/z 499.3. Anal. Calcd
for C29H31FN6O (%): C, 69.86; H, 6.27; N, 16.86. Found: 69.63;
H, 6.52; N, 17.01.
Preparation of 4-Fluoro-N-[4-({[7-methyl-2-(4-methylpipe-
razin-1-yl)quinazolin-4-yl]amino}methyl)phenyl]benzamide (37).
4-Fluoro-N-[4-({[7-methyl-2-(4-methylpiperazin-1-yl)quinazolin-
4-yl]amino}methyl)phenyl]benzamide was prepared by amina-
tion of N-(4-{[(2-chloro-7-methylquinazolin-4-yl)amino]methyl}-
phenyl)-4-fluorobenzamide (42 mg, 0.1 mmol) and 1-methylpi-
perazine in dioxane following procedure B (step 2). After
purification by HPLC and solvent removal, the final product
(20 mg, 67% yield) was obtained as an off-white solid. Analy-
tical LC/MS using an Aquasil C18 column, ACN/H2O eluent at
0.8 mL/min flow (containing 0.1% formic acid), 5.5 min gra-
dient 0% ACN to 100% ACN, monitored by UV absorption at
254 nm showed >99.5% purity. MS (ESI) m/z 485.6.
Preparation of 4-Fluoro-N-(4-{[(7-methyl-2-piperidin-1-ylqui-
nazolin-4-yl)amino]methyl}phenyl)benzamide (33). 4-Fluoro-
N-(4-{[(7-methyl-2-piperidin-1-ylquinazolin-4-yl)amino]methyl}-
phenyl)benzamide was prepared by amination of N-(4-{[(2-
chloro-7-methylquinazolin-4-yl)amino]methyl}phenyl)-4-fluo-
robenzamide (42 mg, 0.1 mmol) and piperidine in dioxane
following procedure B (step 2). After purification by HPLC
and solvent removal, the final product (11 mg, 38% yield) was
obtained as an off-white solid. Analytical LC/MS using an
Aquasil C18 column, ACN/H2O eluent at 0.8 mL/min flow
(containing 0.1% formic acid), 5.5 min gradient 0% ACN to
100% ACN, monitored by UV absorption at 254 nm showed
>99.5% purity. MS (ESI) m/z 470.6.
Preparation of 4-Fluoro-N-(4-{[(7-methyl-2-morpholin-4-yl-
quinazolin-4-yl)amino]methyl}phenyl)benzamide (38). 4-Fluoro-
N-(4-{[(7-methyl-2-morpholin-4-ylquinazolin-4-yl)amino]methyl}-
phenyl)benzamide was prepared by amination of N-(4-{[(2-
chloro-7-methylquinazolin-4-yl)amino]methyl}phenyl)-4-fluo-
robenzamide (42 mg, 0.1 mmol) and morpholine in dioxane
following procedure B (step 2). After purification by HPLC and
solvent removal, the final product (17 mg, 60% yield) was
obtained as an off-white solid. Analytical LC/MS using an
Aquasil C18 column, ACN/H2O eluent at 0.8 mL/min flow
(containing 0.1% formic acid), 5.5 min gradient 0% ACN to
100% ACN, monitored by UV absorption at 254 nm showed
>99.5% purity. MS (ESI) m/z 472.5.
Preparation of 4-Fluoro-N-(4-{[(7-methyl-2-piperazin-1-ylqui-
nazolin-4-yl)amino]methyl}phenyl)benzamide (34). 4-Fluoro-
N-(4-{[(7-methyl-2-piperazin-1-ylquinazolin-4-yl)amino]methyl}-
phenyl)benzamide was prepared by amination of N-(4-{[(2-
chloro-7-methylquinazolin-4-yl)amino]methyl}phenyl)-4-fluo-
robenzamide (42 mg, 0.1 mmol) and piperazine in dioxane
following procedure B (step 2). After purification by HPLC
and solvent removal, the final product (21 mg, 72% yield) was
obtained as an off-white solid. Analytical LC/MS using an
Aquasil C18 column, ACN/H2O eluent at 0.8 mL/min flow
(containing 0.1% formic acid), 5.5 min gradient 0% ACN to
100% ACN, monitored by UV absorption at 254 nm showed
96.0% purity. MS (ESI) m/z 471.6.
Preparation of 4-Fluoro-N-{4-[({7-methyl-2-[(3R)-3-methylpi-
perazin-1-yl]quinazolin-4-yl}amino)methyl]phenyl}benzamide (35).
4-Fluoro-N-{4-[({7-methyl-2-[(3R)-3-methylpiperazin-1-yl]qui-
nazolin-4-yl}amino)methyl]phenyl}benzamide was prepared by
amination of N-(4-{[(2-chloro-7-methylquinazolin-4-yl)amino]-
methyl}phenyl)-4-fluorobenzamide (25 mg, 0.06 mmol) and
2-(R)-methylpiperazine in dioxane following procedure B (step
2). After purification by HPLC and solvent removal, the final
product (19 mg, 64% yield) was obtained as an off-white solid. 1H
NMR (DMSO) δ 0.98 (d, J = 6.5 Hz, 3H), 2.34 (m, 5H), 2.54
Preparation of N-[4-({[2-(Dimethylamino)-7-iodoquinazolin-
4-yl]amino}methyl)phenyl]-4-fluorobenzamide (39). Step 1: Syn-
thesis of Ethyl {[(3-Iodophenyl)amino]carbonothioyl}carbamate.
To a solution of 3-iodoaniline (11.4 g, 52 mmol) in CH2Cl2
(150 mL) was added a solution of ethyl isothiocyanato-
formate (6.14 mL, 52 mmol) in CH2Cl2 (20 mL). The mixture
was stirred at 20 °C for 2 h and concentrated under reduced
pressure to give an off-white solid (18.2 g, 100% yield). MS (ESI)
m/z 351.1.
Step 2: Synthesis of 2-(Ethylthio)-7-iodoquinazolin-4(3H)-one.
To a solution of ethyl {[(3-iodophenyl)amino]carbonothi-
oyl}carbamate (18.2 g, 52 mmol) in acetone (200 mL) was
added dropwise EtI (4.16 mL, 52 mmol) and K2CO3 (21.5 g,
156 mmol). The resulting mixture was stirred at room tempera-
ture overnight and filtered through a pad of Celite. The filtration
was concentrated in vacuum, and the residue was dissolved
in CH2Cl2 (200 mL). The solution was washed with water
and brine and dried over (MgSO4). Evaporation of solvent
gave a yellow oil (19.0 g), which was dissolved in phenyl ether
(200 mL). The mixture was heated at 200 °C overnight and
cooled to 20 °C, during which time a lot of white precipitate was
formed. The mixture was diluted with hexanes and filtered to

908 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Dehnhardt et al.
give the title compound as an off-white solid (10.8 g, 63% yield).
MS (ESI) m/z 333.1.
DMF (5 mL) was added PdCl2(PPh3)2 (26 mg, 5 mol %) as
catalyst, followed by addition of tributyl(1-ethoxyvinyl)tin
(0.27 mL, 0.81 mmol). The resulting mixture was heated at
90 °C under nitrogen for 4 h. Upon completion, the reaction
mixture was cooled to 20 °C and then poured into cold water.
The resulting solid was collected by filtration. The solid was
dissolved in MeOH (3 mL) and 6 N HCl (0.5 mL) and heated at
70 °C for 3h. The mixture was cooled to 20 °C and concentrated
in vacuum. The resulting residue was purified by flash chroma-
tography (CH2Cl2:CH3OH = 90:10) to give an off-white solid
(168 mg, 50% yield). mp 195 °C; 1H NMR (DMSO) δ 2.67 (s,
3H), 3.28 (s, 6H), 4.78 (d, J = 5.8 Hz, 2H), 7.36 (t, J = 9.0 Hz,
2H), 7.42 (d, J = 8.3 Hz, 2H), 7.74 (d, J = 8.3 Hz, 2H), 7.96 (d,
J = 8.3 Hz, 1H), 8.03 (m, 2H), 8.38 (s, 1H), 8.51 (d, J = 8.3 Hz,
1H), 10.30 (s, 1H), 10.34 (s, 1H), 11.91 (s, 1H) ppm; MS (ESI) m/
z 458.2. Anal. Calcd for C26H24FN5O2 (%): C, 68.26; H, 5.29; N,
15.31. Found: C, 68.14; H, 5.45; N, 15.23.
Step 3: Synthesis of 7-iodoquinazoline-2,4(1H,3H)-dione. To a
solution of 2-(ethylthio)-7-iodoquinazolin-4(3H)-one (10.8 g,
32.5 mmol) in ethanol (40 mL) was added 6 N HCl (40 mL). The
mixture was heated at 80 °C overnight and then cooled to 20 °C,
during which time a lot of white precipitate was formed. The
resulting solid was collected by filtration to give an off-white
solid (8.97 g, 96% yield). MS (ESI) m/z 289.0.
Step 4: Synthesis of 2,4-Dichloro-7-iodoquinazoline. POCl3
(10 mL) was added to 7-iodoquinazoline-2,4(1H,3H)-dione
(3.78 g, 13.1 mmol), followed by addition of N,N-dimethylani-
line (1 mL). The resulting mixture was heated at 115 °C for 6 h.
After cooling to 20 °C, most of POCl3 was removed by distilla-
tion under reduced pressure. The residue was poured into
ice-water; ammonium hydroxide was added to adjust the pH
to 5-7. The mixture was extracted several times with CH2Cl2,
and the combined extracts were washed with brine and dried
over MgSO4. The solvent was removed under reduced pressure,
and the residue was purified by flash chromatography (EtOAc/
CH2Cl2 = 5:95) to give 2,4-dichloro-7-iodoquinazoline as an
off-white solid (3.56 g, 84% yield). mp 163 °C; MS (ESI) m/z
324.9.
Step 5: Synthesis of N-(4-{[(2-Chloro-7-iodoquinazolin-4-
yl)amino]methyl}phenyl)-4-fluorobenzamide. To a solution of
N-[4-(aminomethyl)phenyl]-4-fluorobenzamide (537 mg, 2.2
mmol) in CH2Cl2 (10 mL) was added Et3N (0.84 mL, 6.0 mmol),
followed by addition of 2,4-dichloro-7-iodoquinazoline (648
mg, 2.0 mmol). The resulting mixture was stirred at room
temperature for 20 h, during which time a lot of precipitate
was formed. The resulting solid was collected by filtration and
washed with a small amount of EtOAc and water to give the
expected product as an off-white solid (745 mg, 70% yield). MS
(ESI) m/z 533.0.
Step 6: Synthesis of N-[4-({[2-(Dimethylamino)-7-iodoquina-
zolin-4-yl]amino}methyl)phenyl]-4-fluorobenzamide (39). To a
solution of N-(4-{[(2-chloro-7-iodoquinazolin-4-yl)amino]-
methyl}phenyl)-4-fluorobenzamide (532 mg, 1 mmol) in THF
(2 mL) was added dimethylamine solution (40% in water, 0.6
mL, 5 mmol). The resulting mixture was heated at 100 °C in a
sealed tube for 26 h and then cooled to 20 °C. The solvent was
evaporated under reduced pressure, and the residue was purified
by flash chromatography (CH2Cl2:CH3OH = 90:10) to give an
off-white solid (487 mg, 90% yield). 1H NMR (DMSO) δ 3.23 (s,
6H), 4.75 (d, J = 6.0 Hz, 2H), 7.37 (m, 4H), 7.72 (d, J = 8.5 Hz,
1H), 7.81 (d, J = 8.3 Hz, 1H), 8.02 (m, 3H), 8.10 (s, 1H), 10.0 (s,
1H), 10.26 (s, 1H), 11.48 (s, 1H) ppm; MS (ESI) m/z 542.1. Anal.
Calcd for C24H21FIN5O (%): C, 53.25; H, 3.91; N, 12.94.
Found: C, 53.07; H, 4.17; N, 12.76.
Preparation of N-[4-({[2-(Dimethylamino)-7-(1-hydroxyethyl)-
quinazolin-4-yl]amino}methyl)phenyl]-4-fluorobenzamide (43).
To a solution of N-[4-({[7-acetyl-2-(dimethylamino)quina-
zolin-4-yl]amino}methyl)phenyl]-4-fluorobenzamide (84 mg,
0.18 mmol) in MeOH (5 mL) was added NaBH4 (14 mg, 0.36
mmol). The resulting mixture was stirred at 20 °C for 6 h. The
reaction mixture was concentrated under reduced pressure, and
the residue was treated with saturated aqueous NaHCO3 solu-
tion and extracted with EtOAc. The organic phases were washed
with brine and dried over MgSO4. The solvent was removed, and
the residue was purified by HPLC and converted to the HCl salt
1
to give an off-white solid (71 mg, 80% yield). mp 232 °C; H
NMR (DMSO) δ 1.37 (d, J = 6.3 Hz, 3H), 3.24 (s, 6H), 4.77 (d,
J = 5.8 Hz, 2H), 4.86 (m, 1H), 5.56 (d, J = 3.8 Hz, 1H), 7.37
(m, 5H), 7.73 (d, J = 9.1 Hz, 2H), 7.82 (s, 1H), 8.03 (m, 2H), 8.28
(d, J = 8.8 Hz, 1H), 10.04 (s, 1H), 10.29 (s, 1H), 11.62
(s, 1H) ppm; MS (ESI) m/z 460.2. Anal. Calcd for C26H26FN5O2
(%): C, 67.96; H, 5.70; N, 15.24. Found: C, 68.12; H, 5.84; N,
15.15.
Preparation of N-[4-({[2-(Dimethylamino)-7-formylquinazo-
lin-4-yl]amino}methyl)phenyl]-4-fluorobenzamide (44). To a so-
lution of N-[4-({[2-(dimethylamino)-7-iodoquinazolin-4-
yl]amino}methyl)phenyl]-4-fluorobenzamide (417 mg, 0.75
mmol) in DMF 3 (mL) were added HCO2Na (102 mg, 1.5
mmol) and PdCl2(PPh3)2 (26 mg, 5 mol %). The resulting
mixture was heated at 100 °C under CO (gas, 1 atm) for 5 h.
Upon completion, the reaction mixture was cooled to 20 °C and
poured into cold water. The resulting solid was collected by
filtration and purified by HPLC to give an off-white solid (123
mg, 37% yield). 1H NMR (DMSO) δ 3.26 (s, 6H), 4.80 (d, J =
5.8 Hz, 2H), 7.34-7.42 (m, 4H), 7.73 (d, J = 8.6 Hz, 2H), 7.92 (s,
1H), 8.02 (m, 2H), 8.18 (s, 1H), 8.48 (d, J = 7.5 Hz, 1H), 10.14 (s,
1H), 10.28 (s, 1H), 10.79 9s, 1H) ppm; MS (ESI) m/z 444.1. Anal.
Calcd for C25H22FN5O2 (%): C, 67.71; H, 5.00; N, 15.79.
Found: C 67.53; H, 5.18; N, 15.55.
Preparation of N-[4-({[7-Cyano-2-(dimethylamino)quinazolin-
4-yl]amino}methyl)phenyl]-4-fluorobenzamide (41). To a solu-
tion of N-[4-({[2-(dimethylamino)-7-iodoquinazolin-4-yl]amino}-
methyl)phenyl]-4-fluorobenzamide (252 mg, 0.46 mmol) in
DMF 9 (mL) were added Zn(CN)2 (66 mg, 0.56 mmol) and
Pd(PPh3)4 (26 mg, 5 mol %). The resulting mixture was heated at
80 °C under N2 for 5 h. Upon completion, the reaction mixture
was cooled to 20 °C and poured into cold water. The resulting
solid was collected by filtration, purified by HPLC, and then
converted to the HCl salt to give an off-white solid (156 mg, 70%
yield). mp 150 °C; 1H NMR (DMSO) δ 3.28 (s, 6H), 4.78 (d, J =
5.6 Hz, 2H), 7.36 (t, J = 8.5 Hz, 2H), 7.41 (d, J = 8.5 Hz, 2H),
7.73 (d, J = 9.3 Hz, 2H), 7.88 (d, J = 8.8 Hz, 1H), 8.02 (d, J =
9.1 Hz, 1H), 8.03 (d, J = 8.5 Hz, 1H), 8.24 (s, 1H), 8.53 (d, J =
8.1 Hz, 1H), 10.29 (s, 1H), 10.36 (s, 1H), 12.08 (s, 1H) ppm; MS
(ESI) m/z 441.1. Anal. Calcd for C25H21FN6O (%): C, 68.17; H,
4.81; N, 19.08. Found: C, 67.95; H, 5.03; N, 19.03.
Preparation of N-[4-({[2-(Dimethylamino)-7-(hydroxymethyl)-
quinazolin-4-yl]amino}methyl)phenyl]-4-fluorobenzamide (45).
To a solution of N-[4-({[2-(dimethylamino)-7-formylquinazo-
lin-4-yl]amino}methyl)phenyl]-4-fluorobenzamide (100 mg,
0.23 mmol) in MeOH (5 mL) was added NaBH4 (17 mg, 0.46
mmol). The resulting mixture was stirred at 20 °C for 2 h. The
reaction mixture was concentrated under reduced pressure, and
the residue was treated with saturated aqueous NaHCO3 solu-
tion and extracted with EtOAc. The organic phases were washed
with brine and dried over MgSO4. The solvent was removed, and
the residue was purified by HPLC and converted to the HCl salt
1
to give an off-white solid (94 mg, 85% yield). mp 158 °C; H
NMR (DMSO) δ 3.25 (s, 6H), 4.66 (s, 2H), 4.76 (d, J = 5.8 Hz,
2H), 5.61 (s, 1H), 7.37 (m, 5H), 7.73 (d, J = 8.3 Hz, 2H), 7.81 (s,
1H), 8.03 (m, 2H), 8.29 (d, J = 8.3 Hz, 1H), 10.06 (t, J = 5.8 Hz,
1H), 10.29 (s, 1H), 11.65 (s, 1H) ppm; MS(ESI) m/z 446.2. Anal.
Calcd for C25H24FN5O2 (%): C, 67.40; H, 5.43; N, 15.72.
Found: C, 67.52; H, 5.68; N, 15.96.
Preparation of N-[4-({[7-Acetyl-2-(dimethylamino)quinazolin-
4-yl]amino}methyl)phenyl]-4-fluorobenzamide (42). To a solu-
tion of N-[4-({[2-(dimethylamino)-7-iodoquinazolin-4-yl]amino}-
methyl)phenyl]-4-fluorobenzamide (400 mg, 0.74 mmol) in

Article
Preparation of N-{4-[({2-(Dimethylamino)-7-[(dimethylamino)-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 909
cell lines were established by selection to contain the above
luciferase reporters integrated into their chromosomes as fol-
lows: 33.13 cell line (Tcf4-Luc and SV40-R-Luc), 22C11 cell line
(Tcf4-Luc and SV40-R-Luc), and 5A8 cell line (SV40-Luc and
SV40-R-Luc). The 22C11 and 5A8 cell lines were grown in the
presence of 500 μg/mL G418, and 33.13 was grown in the
presence of 500 μg/mL G418 þ 125 μg/mL Zeocin to maintain
the integrated reporters. The cells were plated at 1 ꢀ 104 cells/
well in solid white 96-well plates. After overnight incubation at
37 °C, 5% CO2, compounds were added at titrated concentra-
tions between 10 ng/mL and 10 μg/mL. The luciferase signal was
detected using Promega’s Dual-Glo luciferase assay system.
After 24 h of incubation with compound, culture media were
removed by aspiration and 75 μL of fresh medium was added
into each well. Dual-Glo luciferase reagent (75 μL) was subse-
quently added per well, mixing with shaking for 10 min on a
plate shaker. The firefly luminescence was measured on a CCD
camera imaging device or a luminometer. After quantifying the
firefly luminescence, 75 μL of Dual-Glo Stop & Glo reagent
(Promega) was added to each well, and the plate was mixed by
shaking for 10 min. R luciferase was then quantified using a
CCD camera or a luminometer. The luminescence signal from
each well was expressed as percent of control for both firefly and
Renilla luciferase activity. The ratio of FF/R was then calcu-
lated and averaged between duplicate plates. Data were plotted
using the LSW Data Analysis program (model 33) to determine
the IC50 of each compound. A compound was considered
positive only if it selectively inhibited the reporter activity in
33.13 and 22C11 but did not inhibit reporter activity in 5A8.
Colon Carcinoma Cell Growth Inhibition Assay. The colon cell
growth inhibition assay was an in vitro assay that helped to
determine if the lead compound inhibited the β-catenin/Tcf4
signaling pathway as indicated by growth inhibition of colon cell
lines at concentrations similar to its IC50 in the reporter assay or
if its activity was through another mechanism of action. The
colon cell growth inhibition assay used four different colon
cancer cell lines: HT29, DLD1, LoVo, and the SW480-derived
33.13 line which was one of the reporter cell lines carrying the
integrated Tcf4-luciferase reporter. A compound of interest
would be inhibitory at the concentrations similar to its IC50 in
the reporter assay. The assay readout was based on Promega’s
Cell Titer-Glo luminescent cell viability assay. This is a homo-
geneous method to determine the number of viable cells in
culture based on the quantification of ATP present. ATP signals
the presence of metabolically active cells and can be measured
with this system. Since monooxygenation of luciferin is cata-
lyzed by luciferase in the presence of Mg2þ, ATP, and molecular
oxygen, the resulting luminescence can be quantified. All four
lines are grown in DMEM þ 10% FBS þ penicillin/streptomy-
cin. Cells were trypsinized and plated at 2 ꢀ 103 cells/well in
opaque 96-well plates. Compounds were added 24 h later so that
concentration curves could be generated starting at 10 μg/mL
with nine subsequent 2-fold dilutions. The plates were incubated
with compound for 3 days, at which time the cells were lysed
with Promega’s Cell Titer-Glo reagent. The resulting lumines-
cence was read on a CCD camera imaging device or a lumin-
ometer.
methyl]quinazolin-4-yl}amino)methyl]phenyl}-4-fluorobenzamide
(46). To a solution of N-[4-({[7-(aminomethyl)-2-(dimethyl-
amino)quinazolin-4-yl]amino}methyl)phenyl]-4-fluorobenza-
mide HCl salt (110 mg, 0.21 mmol) in MeOH (2 mL) were added
formaldehyde (37%, 68 μL, 0.84 mmol), NaBH3CN (13 mg,
0.21 mmol), and ZnCl2 (14 mg, 0.10 mmol). The resulting
mixture was stirred at 20 °C overnight. The mixture was filtered
and washed with methanol. The resulting filtrate was concen-
trated in vacuum and subjected to HPLC separation. The
product was converted to the HCl salt, which was an off-white
solid (68 mg, 60% yield). mp 94 °C; 1H NMR (DMSO) δ 2.73 (s,
6H), 3.26 (s, 6H), 4.40 (s, 2H), 4.77 (d, J = 5.6 Hz, 2H), 7.37 (m,
4H), 7.73 (d, J = 9.1 Hz, 2H), 7.87 (s, 1H), 8.02 (d, J = 6.0 Hz,
1H), 8.04 (d, J = 6.0 Hz, 1H), 8.44 (s, 1H), 10.29 (s, 1H), 10.89 (s,
1H), 11.98 (s, 1H) ppm; MS (ESI) m/z 473.3. Anal. Calcd for
C27H29FN6O (%): C, 68.62; H, 6.19; N, 17.78. Found: C, 68.46;
H, 6.49; N, 17.83.
Synthesis of N-[4-({[2-(Dimethylamino)-7-vinylquinazolin-4-
yl]amino}methyl)phenyl]-4-fluorobenzamide (47). To a solution
of N-[4-({[2-(dimethylamino)-7-iodoquinazolin-4-yl]amino}-
methyl)phenyl]-4-fluorobenzamide (541 mg, 1 mmol) in DMF
(5 mL) was added PdCl2(PPh3)2 (35 mg, 5 mol %) as catalyst,
followed by addition of tributyl(vinyl)tin (0.35 mL, 1.2 mmol).
The resulting mixture was heated at 90 °C under nitrogen for 3 h.
Upon completion, the reaction mixture was cooled to room
temperature and then poured into cold water. The resulting solid
was collected by filtration and then purified by flash chroma-
tography (CH2Cl2:CH3OH = 90:10) to give an off-white solid,
which was treated with HCl in methanol to form the HCl salt as
1
an off-white solid (292 mg, 61% yield). mp 145 °C; H NMR
(DMSO) δ 3.33 (s, 6H), 4.74 (d, J = 5.8 Hz, 2H), 5.50 (d, J =
11.6 Hz, 1H), 6.04 (d, J = 18.1 Hz, 1H), 6.85 (dd, J = 18.1, 11.6
Hz, 1H), 7.37 (m, 4H), 7.51 (s, 1H), 7.61 (s, 1H), 7.72 (d, J = 8.6
Hz, 2H), 8.02 (m, 2H), 8.21 (d, J = 7.5 Hz, 1H), 10.27 (s, 1H),
11.61 (s, 1H) ppm; MS (ESI) m/z 442.1. Anal. Calcd for
C26H24FN5O (%): C, 70.73; H, 5.48; N, 15.86. Found: C,
70.47; H, 5.64; N, 15.95.
Preparation of N-[4-({[2-(Dimethylamino)-7-ethylquinazolin-
4-yl]amino}methyl)phenyl]-4-fluorobenzamide (48). To a solu-
tion of N-[4-({[2-(dimethylamino)-7-vinylquinazolin-4-yl]amino}-
methyl)phenyl]-4-fluorobenzamide (239 mg, 0.5 mmol) in
MeOH (10 mL) was added Pd/C (10%) catalyst (48 mg). The
resulting mixture was hydrogenated at room temperature for
2 h. The reaction mixture was filtered through a pad of Celite
and washed with methanol, and the resulting filtrate was con-
centrated under reduced pressure. The residue was subjected to
HPLC separation to give an off-white solid, which was con-
verted to the HCl salt as an off-white solid (229 mg, 96% yield).
mp 270 °C; 1H NMR (DMSO) δ 1.24 (t, J = 7.3 Hz, 3H), 2.77 (q,
J = 7.3 Hz, 2H), 3.25 (s, 6H), 4.77 (d, J = 6.6 Hz, 2H), 7.36 (m,
5H), 7.62 (s, 1H), 7.73 (d, J = 8.6 Hz, 2H), 8.03 (dd, J = 9.4, 5.8
Hz, 2H), 8.25 (d, J = 8.3 Hz, 2H), 10.0 (t, J = 6.6 Hz, 1H), 10.28
(s, 1H), 11.58 (s, 1H) ppm; MS (ESI) m/z 444.2. Anal. Calcd for
C26H26FN5O (%): C, 70.41; H, 5.91; N, 15.79. Found: C, 70.23;
H, 6.12; N, 15.53.
Reporter Assay. The SW480 colorectal cancer cell line has a
truncation mutation in APC and thus has a constitutively
activated β-catenin/Tcf4 signaling pathway, which can be mon-
itored by a luciferase reporter with Tcf4 binding sites in its
promoter. Our Tcf4-luciferase reporter assay utilizes three
different cell lines that were derived from SW480 by transient
transfection with the following luciferase reporter constructs:
(1) Tcf4-Luc, a promoter containing Tcf4 binding sites that
drive firefly luciferase in the pGL3 vector, to measure the
activity of β-catenin/Tcf4; (2) SV40-Luc, SV40 promoter driv-
ing firefly luciferase in the pGL3 vector, serving as a control for
nonspecific inhibitors; (3) SV40-R-Luc, SV40 promoter driving
Renilla luciferase in the pGL3 vector, serving as an internal
control for cell number and toxicity. After transfection, stable
Differential Cell Assay. The RK3E differential adherent assay
is an in vitro assay which helped to determine if the lead
compound selectively inhibited the β-catenin/Tcf4 signaling
pathway. This assay used rat kidney epithelial cells transformed
with either mutant β-catenin or K-ras to look for a differential
effect of compound between the two lines. A compound of
interest would be inhibitory against the β-catenin transformed
line but not against the Kras transformed line. This assay used
two cell lines which were kindly provided by Dr. B. Vogelstein’s
laboratory: a transformed RK3E subclone expressing an acti-
vated K-rasG12 V (KRAS) and a transformed RK3E subclone
expressing a mutant β-catenin harboring the S33Y mutation,
which stabilizes β-catenin protein (β-cat). The assay readout is

910 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Dehnhardt et al.
based on Promega’s Cell Titer-Glo luminescent cell viability
assay as described above. Both lines were grown in DMEM þ
10% FBS þ penicillin/streptomycin. Cells were plated at 2 ꢀ 104
cells/well in opaque 96-well plates. Twenty-four hours later,
compounds were added so that a concentration curve could
be generated starting at 10 μg/mL with nine subsequent 2-fold
dilutions. Cells were incubated with compound for 3 days,
at which time the cells were lysed and luminescence was
recorded.
Xenograft Tumor Efficacy Studies. Female nude mice
(Charles River Laboratories) bearing β-cat/RK3E cells inocu-
lated subcutaneously on the flank at 5 million cells per mouse
were staged and randomized into treatment groups (n = 15 for
vehicle; n = 12 for each compound treatment group). Mice were
dosed on day 0 by a single ip injection with vehicle or compound
9 at 18.75, 37.5, 75, and 150 mg/kg formulated in 0.5%
methhocel/0.4% Tween. In addition, compound 9 was adminis-
tered at 18.75 mg/kg once daily for 7 days by ip. Tumor growth
was monitored for 1 week.
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siRNA Knockdown. HT29 cells were seeded at 5 ꢀ 106 in a T25
flask in DMEM/10% FBS and grown overnight at 37 °C with
5% CO2. Media were removed and cells treated with 100 nmol of
targeting siRNA (Dharmacon) or fluorescently labeled nontar-
geting BLOCK-IT oligo (Invitrogen). siRNA oligos and Lipo-
fectamine 2000 (Invitrogen) lipid were suspended in Opti-MEM
(Invitrogen) and mixed following the manufacturer’s instruc-
tions. Cells were treated with 1.3 mL of the lipid/oligonucleotide
mixtures for 4 h prior to addition of 1.3 mL of DMEM/20%
FBS and grown overnight. Cells were provided fresh DMEM/
10% FBS media and incubated for an additional 48 h at 37 °C
with 5% CO2 prior to harvesting and RNA purification.
Oligonucleotide-Array mRNA Expression Profiling. Purifica-
tion of RNA and amplification of biotin-labeled cRNA were as
described previously.21 For each sample, 10 μg of fragmented
biotin-labeled cRNA was hybridized to Human Genome U133
Plus 2.0 GeneChip Oligonucleotide Arrays (Affymetrix) and
scanned with an GeneChip scanner 3000 (Affymetrix) using
buffers and conditions recommended by the manufacturer.
Fluorescent data were collected and converted to gene-specific
signal intensities using MicroArray Suite 4.0 software and
converted to mRNA frequency by comparison to the spiked
standard curve and normalized to the mean total frequency
across all samples.22 For analysis, mean mRNA frequency
values of replicate samples were determined for each of the
experimental groups. Genes were initially filtered to remove
those probes not called present by the MAS4 software in all
samples of at least one cohort. Mean mRNA frequency values
were subsequently compared between experimental groups to
identify genes with an average fold change typically greater than
2-fold (P < 0.05).
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L. A.; Qian, C.; Christensen, E.; Schmidt, S. S.; Roche, P. C.;
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Acknowledgment. The authors thank Dr. Jerry Sun for the
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