Design, synthesis, and biological evaluation of new diaminoquinazolines as β-catenin/Tcf4 pathway inhibitors

Article abstract of DOI:10.1021/jm201494a

More than 50 new diaminoquinazoline derivatives have been synthesized and evaluated in a colon carcinoma cell growth inhibition assay using HCT116 and SW480 cells. Twenty compounds with good cell growth inhibitory activities (<4 μM) were tested as inhibitors of the β-catenin/T cell transcription factor 4 (Tcf4) signaling pathway using a HCT116 cell-based luciferase reporter assay. Results from the biological activities as well as the comparative molecular field analysis (CoMFA) of the properties of the molecules yielded a preliminary structure-activity relationship (SAR). Three potent compounds, 74, 78, and 86, showed IC₅₀ values <1 μM for growth inhibition of HCT116 cells and ～1 μM for SW480 cells, as well as IC₅₀ values of 1.5-2.5 μM for HCT116 cells with the luciferase reporter assay.

Full text of DOI:10.1021/jm201494a

Article
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of New
Diaminoquinazolines as β-Catenin/Tcf4 Pathway Inhibitors
Yongjun Mao,†,‡ Nan Lin,†,‡ Wang Tian,†,‡ Xiaofeng Han,†,‡ Xiaobing Han,†,‡ Ziwei Huang,†,‡
and Jing An*,†,‡
†Department of Pharmacology, State University of New York, Upstate Medical University, 750 East Adams Street, Syracuse,
New York 13210, United States
‡Upstate Cancer Research Institute, State University of New York, Upstate Medical University, 750 East Adams Street, Syracuse,
New York 13210, United States
ABSTRACT: More than 50 new diaminoquinazoline derivatives have been synthesized and evaluated in a colon carcinoma cell
growth inhibition assay using HCT116 and SW480 cells. Twenty compounds with good cell growth inhibitory activities (<4 μM)
were tested as inhibitors of the β-catenin/T cell transcription factor 4 (Tcf4) signaling pathway using a HCT116 cell-based
luciferase reporter assay. Results from the biological activities as well as the comparative molecular field analysis (CoMFA) of
the properties of the molecules yielded a preliminary structure−activity relationship (SAR). Three potent compounds, 74, 78,
and 86, showed IC50 values <1 μM for growth inhibition of HCT116 cells and ∼1 μM for SW480 cells, as well as IC50 values of
1.5−2.5 μM for HCT116 cells with the luciferase reporter assay.
signaling pathway presents a novel target for therapeutic inter-
vention in colorectal cancer.11,12
INTRODUCTION
■
The Wnt (wingless int-1) signaling pathway is involved in
multiple developmental events during embryogenesis and is
implicated in adult tissue homeostasis and tumorigenesis.1,2
Mutations or deregulated expression of components of the Wnt
pathway can induce diseases, most importantly cancers.3 The
Wnt signaling pathway is regulated by the Wnt ligands, the
adenomatous polyposis coli (APC)-Axin complex, and the
β-catenin.4 When canonical Wnt signaling is activated, cytosolic
β-catenin is stabilized and translocated into the nucleus, where
it functions together with Tcf proteins, such as LEF1
(lymphoid enhancer-binding factor 1), Tcf1, Tcf3, and Tcf4,
as a transcriptional activator for a large number of target genes
(e.g., c-Myc and cyclin D1).5 Transcriptional activation of
β-catenin/Tcf target genes is a hallmark of colorectal cancer
cells, and the constitutive activation of some of these genes is
essential for creating and maintaining the malignant phenotype.6
Colorectal cancer is the second leading cause of cancer
mortality in the United States. More than one million new cases
are diagnosed worldwide each year with nearly 16% of these in
the United States alone.7 Besides surgical resection, which is
rarely curative in advanced disease, current therapy for colon
cancer relies on traditional cytotoxic agents that show only limited
effects. Nearly 90% of all colorectal cancers have an activating
mutation of the canonical Wnt signaling pathway.8 Constant
activation of Wnt/β-catenin signaling is thought to be an initiating
event in colorectal carcinogenesis.9 Most colorectal tumors, and
certain other cancers, are characterized by an accumulation of
β-catenin in the nucleus and is accompanied by dysregulation of
the β-catenin/Tcf4 complex.10 Consequently, the β-catenin/Tcf4
Over the past decade, several small-molecule inhibitors of the
Wnt signaling pathway have been identified by high-throughput
screening (HTS) and visual screening.13−15 Most of these com-
pounds are naturally occurring substances in biological materials,
so that the structural modification is difficult. Recently, Dehnhardt
et al.16,17 reported the discovery of a novel class of small molecules
built on a quinazoline core that can inhibit the β-catenin/Tcf4
pathway. Although a couple of compounds showed inhibitory
activity on the growth of a β-catenin/RK3E tumor xenograft,
many of this series of compounds had no desired pharmacokinetic
(PK) properties.
In this study, we reported a series of 2,4-diaminoquinazoline
analogues that were tested in a colon carcinoma cell growth
inhibition assay on two cell lines, HCT116 and SW480. The
compounds that showed good growth inhibitory activities were
also tested in a cell-based luciferase reporter assay in HCT116
cells. Therefore, we provided the first characterization of the
primary structure−activity relationship (SAR) based on the
biological activity and the Comparative Molecular Field Analysis
(CoMFA) results for these inhibitors of the β-catenin/Tcf4
signaling.
RESULTS AND DISCUSSION
Chemistry. The synthesis of the diaminoquinazoline ana-
logues 17−34 is shown in Scheme 1. The side chains 3−9 were
■
Received: November 4, 2011
Published: January 9, 2012
© 2012 American Chemical Society
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a
Scheme 1
a
Reagents and conditions: (a) (i) acyl chloride or sulfuryl chloride, TEA, CH2Cl2, rt, 2−8 h; (ii) TFA, rt, 3−12 h, 45−87%; (b) 2,4-
dichloroquinazoline (39), TEA, THF−CH2Cl2, rt, 3−6 h, 70−98%; (c) amine, THF or dioxane, sealed tube or reflux, 100−120 °C, 8−16 h, 20−
90%; (d) LiAlH4, THF, 68%; (e) Boc2O, TEA, CH2Cl2, 72%.
synthesized first through several steps from commercially
available materials.18,19 The quinazoline intermediates 10−15
were then obtained by the reaction of compounds 3−9 with 2,4-
dichloroquinazoline (39). Subsequent amination at the 2-position
of the quinazoline moiety was accomplished by heating with the
appropriate amines in THF or dioxane at 100−120 °C, mostly
carried out in sealed tubes, yield the final products 17−34.16,17
Another series of diaminoquinazoline and 6-methyl-
substituted analogues 62−91 were prepared from the benzoic
acid 4020 as depicted in Scheme 2. The side chains 41−48 and
the moiety 9821 were synthesized in a couple of steps from
commercially available materials using the traditional amidation
method.22 The intermediates 49−60 were synthesized by the
reaction of 41−48 with 39 or the 6-methylquinazoline 95,
which were prepared from o-aminobenzoic acid.23,24 The final
products 62−91 were obtained using the same methods used
for the preparation of compounds 17−34.
The 3-cyano-4-aminoquinoline intermediate 103 (Scheme 3)
was prepared by reaction of the side chain 3 with the 3-
cyanoquinoline moiety 102, which was synthesized from phenyl
isocyanate through a couple of steps.25,26 The final compounds
104 and 105 were obtained using the established method for
the preparation of 17−34. The 2,4-diaminopyrimidine
analogues 111−113 (Scheme 4) were prepared from the com-
mercially available dichloropyrimidines (107, 108) with the moiety
3, using the similar methods to those described above.
Biology and SAR Study. The chemical structures and the
bioactivities of the target compounds are depicted in Tables 1−8.
The IC50 values for cell growth inhibition and reporter assay
of compounds 17−29 are summarized in Table 1. The 4-fluo-
rophenyl-substituted compounds (17−22) had better activities
compared to the pyridine-4-yl analogues (23−25) and the
N-methylpiperazine derivatives (26, 27), which indicated that
the 4-fluorophenyl group was an optimal moiety. The 2-amino
substituents in the quinazoline core also had a relationship with
the activity. Secondary or tertiary amines, such as the
methylamino (17), dimethylamino (18), and n-propylamino
(21) groups, were both suitable for maintaining the activity
while the bulky dimethylaminopropylamino group (20)
resulted in a decrease of activity. Introducing a chloro
substituent into the molecule (28, 29) had no evident influence
on the bioactivity, which indicated that some modifications
could be made to the phenyl ring. We then tested three
promising compounds (17, 21, and 22) in a luciferase reporter
assay in HCT116 cells. The IC50 values were ∼2-fold higher
than in the related cell growth inhibition assay.
The structure and the bioactivity data of the benzsulfamide
analogues 30−34 are depicted in Table 2. However, these ana-
logues did not retain the bioactivity. The 2-N-methylpiperazine-
substituted compound 32 in particular lost almost all activities.
The other four derivatives had activities ∼4-fold lower than those
of the related benzamide compounds.
Another series of the benzamide derivatives 62−69 were
synthesized, and the bioactivities were evaluated to determine
which modification was the most suitable, as shown in Table 3.
Because these compounds can maintain the bioactivities, it
indicates that more linking patterns of the benzene ring are
available in the side chain. We investigated the effects of
different amino substituents at the 2-position of the quinazoline
core by introducing eight amines (e.g., cyclopropylamine,
n-butylamine, allylamine, 3-methylpiperazine) onto the most
potent 4-fluorophenylbenzamide quinazoline core. Most of
these analogues showed similar cell growth inhibition IC50
values for HCT116 and SW480 cells. The n-propylamino (65)-
and the n-butylamino (66)-substituted analogues were the most
potent, with IC50 values of 1−1.6 μM (cell survival) and 381−
684 nM (clonogenic growth). In contrast, the allylamino
derivative 67 was less active, with an IC50 value greater than
10 μM (cell survival). We also tested the effects of five potent
compounds on the luciferase reporter assay in HCT116 cells.
Compounds 65 and 66 showed IC50 values of 1.5−3 μM for
the luciferase reporter assay in HCT116 cells. However, the
3-methylpiperazine compound 69 had an IC50 value greater
than 5 μM.
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a
Scheme 2
a
Reagents and conditions: (a) (i) amine, HOBt, DIC, DIEA, CH2Cl2, rt, 6−14 h; (ii) TFA, rt, 3−12 h; (b) 39 or 95, TEA, THF−CH2Cl2, rt, 3−6 h,
70−98%; (c) amine, THF or dioxane, sealed tube or reflux, 100−120 °C, 8−16 h, 20−90%; (d) urea, 150−180 °C, 5 h; (e) POCl3, reflux, 3 h, 56%,
(f) (i) 4-fluorobenzoyl chloride or 4-fluorobenzyl chloride, TEA, CH2Cl2, rt, 2−8 h; (ii) TFA, rt, 12 h, 85−90%.
a
Scheme 3
a
Reagents and conditions: (a) ethyl cyanoacetate, TEA, DMF, rt, 1 h; (b) dichlorobenzene, reflux, 6 h; (c) POCl3, PCl5, reflux, 4 h, 35% for three
steps; (d) TEA, THF−CH2Cl2, rt, 3 h; (e) amine, THF, sealed tube, 100−120 °C, 14 h, 58−79%.
Next, we endeavored to improve the potency and diversity of
the chemical structures by preparing several 6-methylquinazo-
line derivatives, compounds 70−87, which had varying “Ar”
and 2-amino substituents. The structures and in vitro activities
are tabulated in Table 4. The 6-chloropyridin-3-yl substituent
analogues (72, 73) had activities equal or even higher than that
of the 4-fluorophenyl substituent (70), and the former had a
better water solubility. The benzyl, phenethyl, and phenyl-
cyclopropyl groups were introduced to the quinazoline core in
order to extend the side chain. This modification resulted in an
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a
Scheme 4
a
Reagents and conditions: (a) TEA, THF−CH2Cl2, rt, 3 h, 58−75%; (b) amine, THF, sealed tube, 100−120 °C, 14 h, 32−45%.
Table 1. Chemical Structure and Biological Activity of Compounds 17−29
a
IC50 (μM)
cell growth inhibition
compd
R1
Ar
NR2R3
methylamine
reporter assay: HCT116
3.5
HCT116
SW480
17
18
19
20
21
22
23
24
25
26
27
28
29
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
4-fluorophenyl
4-fluorophenyl
4-fluorophenyl
4-fluorophenyl
4-fluorophenyl
4-fluorophenyl
pyridin-4-yl
1.6
1.3
2.0
2.2
dimethylamine
4-methylpiperazine
dimethylaminopropylamine
n-propylamine
1.8
2.5
10.0
1.7
14.0
2.0
2.5
3.0
ethylamine
1.4
2.3
methylamine
>20
15.0
>20
10.0
>20
2.0
>20
11.0
>20
11.0
>20
2.3
pyridin-4-yl
dimethylamine
pyridin-4-yl
4-methylpiperazine
methylamine
4-[(4-methylpiperazin-1-yl)methyl]phenyl
4-[(4-methylpiperazin-1-yl)methyl]phenyl
4-fluorophenyl
dimethylaminopropylamine
methylamine
4-fluorophenyl
ethylamine
1.5
1.6
a
The IC50 values are the average of three separate experiments. Standard deviations were below 20%.
Extension the side chain of the 6-methyl quinazoline struc-
tures help to increase the bioactivity, another series of six
compounds 82−87 were synthesized. The 4-fluorobenzoylpi-
peridine and 4-fluorobenzylpiperidine substituents were in-
troduced into the molecule respectively. The benzoylpiperidine
analogues 82 and 83 had bioactivity lower than that of the
benzylpiperidine analogues 84−87, which showed IC50 values
of 2.5−3 μM for the luciferase reporter assay in HCT116 cells
and 1−2 μM for cell growth inhibition of the two cell lines. The
most potent 2-dimethylamino-substituted compound 86 had an
IC50 value of 0.8 μM in the HCT116 cells.
We explored the structure−activity relationships further
by preparing another four compounds 88−91. The chemical
structures and the biological data are depicted in Table 5.
The (4-fluorobenzyl)piperidinecarboxamide moiety was
introduced into the molecule to extend the side chain
further. However, these analogues had an IC50 value ∼5 μM
for the HCT116 and SW480 cell growth inhibition. One
possible reason is that the piperidine core in the molecule is
not a potential pharmacophore and the aromatic ring is more
suitable.
Table 2. Chemical Structure and Biological Activity of
Compounds 30−34
cell growth
inhibition, IC50
a
(μM)
compd
Ar
NR1R2
HCT116
SW480
30
31
32
33
34
4-methylphenyl
4-methylphenyl
4-methylphenyl
4-fluorophenyl
4-fluorophenyl
methylamine
5.8
11.0
>20
5.3
4.9
13.0
>20
4.8
dimethylamine
4-methylpiperazine
methylamine
cyclopropylamine
4.9
4.1
a
The IC50 values are the average of three separate experiments with
standard deviation below 20%.
improvement of the bioactivity. Most of these derivatives (74−
81) showed IC50 values of 1−3 μM for HCT116 and SW480
cell growth inhibition, as well as for suppression of the
luciferase reporter assay in HCT116 cells. The 2-dimethylami-
no-substituted compounds 74 and 78 had IC50 values of 0.9 μM
and 0.6 μM, respectively, in the HCT116 cell line.
Lastly, two other types of derivatives were synthesized and
evaluated with the cell growth inhibition assay as shown in
Table 6 and 7. The first type was the 3-cyano-2-aminoquinoline
structure 104 and 105. The purpose of this design was to find
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Table 3. Chemical Structure and Biological Activity of Compounds 62−69
a
IC50 (μM)
cell growth inhibition
compd
NR1R2
methylamine
reporter assay: HCT116
3.5
HCT116
SW480
62
63
64
65
66
67
68
69
2.9
1.9
4.1
2.5
ethylamine
cyclopropylamine
n-propylamine
n-butylamine
2.7
2.6
3.0
1.5
1.5
1.6
1.4
1.0
allylamine
14.0
1.9
11.0
2.6
dimethylamine
( )-3-methylpiperazine
3.0
>5.0
2.8
3.2
a
The IC50 values are the average of three separate experiments with standard deviation below 20%.
Table 4. Chemical Structure and Biological Activity of Compounds 70−87
a
IC50 (μM)
cell growth inhibition
compd
Ar
NR1R2
reporter assay: HCT116
HCT116
SW480
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
4-fluorophenyl
4-fluorophenyl
n-propylamine
allylamine
1.2
13.0
1.3
1.5
0.9
4.4
2.9
1.1
0.6
1.6
1.3
1.7
6.8
6.9
1.2
1.6
0.8
2.0
1.3
10.0
1.1
2.1
1.1
2.2
1.4
1.5
1.4
1.6
2.4
1.8
5.5
5.2
1.3
1.1
1.1
1.0
6-chloropyridin-3-yl
dimethylamine
n-propylamine
dimethylamine
n-propylamine
( )-3-methylpiperazine
methylamine
2.5
2.5
2.0
6-chloropyridin-3-yl
4-fluorobenzyl
4-fluorobenzyl
4-fluorobenzyl
4-fluorophenethyl
1.0
1.5
2.0
3.0
3.5
4-fluorophenethyl
dimethylamine
n-propylamine
dimethylamine
ethylamine
4-fluorophenethyl
trans-2-phenylcyclopropyl
trans-2-phenylcyclopropyl
1-(4-fluorobenzoyl)piperidine-4-yl
1-(4-fluorobenzoyl)piperidine-4-yl
1-(4-fluorobenzyl)piperidine-4-yl
1-(4-fluorobenzyl)piperidine-4-yl
1-(4-fluorobenzyl)piperidine-4-yl
1-(4-fluorobenzyl)piperidine-4-yl
n-propylamine
allylamine
methylamine
3.0
3.0
n-propylamine
dimethylamine
( )-3-methylpiperazine
2.5
>5.0
a
The IC50 values are the average of three separate experiments. Standard deviations were below 20%.
out whether the 3-cyanoquinoline core could mimic the
quinazoline structure. The second class was the 2,4-diaminopyr-
imidine structure 111−113. This design was intended to confirm
whether the benzene ring of the quinazoline core dominates the
inhibitory action. Neither of these structures showed any cell
growth inhibition when supplied to HCT116 and SW480 cells.
These biological activity data tell us that the quinazoline core is
the key pharmacophore of the inhibitors.
The top five compounds with the growth inhibition activities,
65, 66, 74, 78, and 86 were selected to test the clonogenic
assays on HCT116 cell line, which are shown in Table 8. The
five compounds have the IC50 values of 264−684 nM for the
assays.
CoMFA Analysis. The SAR of the synthesized compounds
was further studied using 19 analogues with diverse struc-
tures and bioactivities by applying CoMFA. The calculation
method is described in the Experimental Section. These
selected compounds include 18, 20, 23, 24, 30, 65, 67, 70,
71, 74, 77, 78, 86, 90, 104, 105, and 111−113, which are
shown in Tables 1−7.
Analysis of the 3D-QSAR Model. The CoMFA contour map
of this series of compounds is shown in Figure 1. The CoMFA
model gave a good cross-validated correlation with a q2 value of
0.612 with five optimum components. The conventional
correlation coefficient r2 was 0.960, F5,13 was 62.629, and the
estimated standard error was 0.194. The calculated steric and
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Table 5. Chemical Structure and Biological Activity of
Compounds 88−91
cell growth inhibition,
a
IC50 (μM)
compd
R1
NR2R3
HCT116
SW480
88
89
90
91
H
dimethylamine
n-propylamine
dimethylamine
n-propylamine
5.7
5.5
5.6
4.6
5.5
5.7
5.6
5.2
H
Me
Me
Figure 1. The CoMFA contour map of the selected 19 compounds.
a
The IC50 values are the average of three separate experiments with
be enhanced by increasing the steric bulk on the benzene ring
of the quinazoline core. The bioactivities of the parent nucleus
6-methylquinazoline and quinazoline were much higher than
those of the 3-cyano-2,4-diaminoquinoline and 2,4-diaminopyr-
imidine core. (2) The large yellow area indicated that the
activity could be improved by decreasing the size of the 2-amino
group of the quinazoline core. Generally, methylamine, dimethyl-
amine, ethylamine, and n-propylamine groups had bioactivities
better than those of the bulky substituents, such as 1-piperazine
and 3-dimethylaminopropylamine. (3) The red area showed the
difference between the two kinds of benzamides. As usual, better
bioactivities were obtained for the N-phenyl-4-[(quinazolin-
4-ylamino)methyl]benzamides (compounds 70−87) than for
their reversed amide bond analogues (compounds 17−29). The
benzamide linker also had activities higher than those of the
benzsulfamide structure. (4) The substituents at the 4-position of
quinazoline core also played an important role in the contribution
of bioactivities, whereby properly extending the 4-substituents
will enhance the activities. So far, the 4-fluorobenzyl, 4-
fluorophenethyl, 4-fluorobenzylpiperidine, phenylcyclopropyl,
and 6-chloropyridin-3-yl substituents have activities better than
those of the other groups such as 4-pyridine and 4-methylphenyl.
standard deviation below 20%.
Table 6. Chemical Structure and Biological Activity of
Compounds 104 and 105
a
cell growth inhibition, IC50 (μM)
compd
R1
HCT116
SW480
104
105
Me
>20
>20
>20
>20
n-Pr
a
The IC50 values are the average of three separate experiments.
Standard deviations were below 20%.
Table 7. Chemical Structure and Biological Activity of
Compounds 111−113
CONCLUSIONS
■
More than 50 new diaminoquinazoline derivatives have been
prepared and evaluated in HCT116 and SW480 cells using the
colon carcinoma cell growth inhibition assay. Among these
derivatives, 20 compounds with good growth inhibitory acti-
vities (<4 μM) were also tested as inhibitors of the β-catenin/
Tcf4 signaling pathway in a cell-based luciferase reporter assay
in the HCT116 cell line. The biological activity results, as well
as the CoMFA analysis of the properties of the molecules,
identified a preliminary SAR. (1) Compounds substituted with
secondary or tertiary amines at the 2-position of the
quinazoline core maintain the bioactivity, whereas substitution
with bulky substituents reduce the activity. (2) The benzsul-
famide analogues generally have activities ∼4-fold lower than
those of the related benzamide compounds. (3) The 3-cyano-
2-aminoquinoline and 2,4-diaminopyrimidine structures showed
no cell growth inhibition activities, indicating that the quina-
zoline core is important for retaining bioactivity. (4) Generally
the 4-[(quinazolin-4-ylamino)methyl]benzamides (70−87) had
bioactivity better than that of their reversed amide bond
analogues (17− 29). (5) Introducing suitably long side chains
to the 4-position of the quinazoline core can boost the bio-
activity. Three potent compounds, 74, 78, and 86, had IC50
values of 264−313 nM for the clonogenic assay and 1.5−2.5 μM
cell growth inhibition, IC50
a
(μM)
compd
R2
R3
HCT116
SW480
111
112
113
Me
Me
Br
Me
Et
>20
>20
>20
>20
>20
>20
Me
a
The IC50 values are the average of three separate experiments.
Standard deviations were below 20%.
Table 8. Clonogenic Assays of the Five Compounds on
HCT116 Cells
compd
65
66
74
78
86
a
IC50 (nM)
684
381
298
264
313
a
The IC50 values are the average of three separate experiments.
Standard deviations were below 20%.
electrostatic fields contributed 48% and 52%, respectively, to
the variations in the percent inhibition.
The CoMFA analysis and the biological activity data
produced a preliminary SAR result, as shown in Figure 1 and
Table 9. (1) The large green area showed that the activity could
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Table 9. Preliminary SAR Results of the Synthesized Compounds
The product was separated by column chromatography with hexane/
ethyl acetate and then suspended in CH2Cl2 (20 mL). TFA (10 mL)
was added, and the reaction solution was stirred at room temperature
for 6 h. The solvent was removed, and the residue was taken into
CH2Cl2 (100 mL) and ice (50 g) and the pH adjusted to >10 with
20% NaOH aqueous solution. The layers were allowed to separate, and
the aqueous layer was extracted once with CH2Cl2 (50 mL). The
combined organic layers were washed once with brine and dried over
anhydrous Na2SO4. Removal of Na2SO4 by filtration and evaporation of
solvents produced 4-aminomethyl-N-(4-fluorophenyl)benzamide 41 (1.83 g,
75%). 1H NMR (DMSO-d6) δ 3.78 (s, 2H), 7.17 (m, 2H), 7.46 (d, J =
9.0 Hz, 2H), 7.78 (m, 2H), 7.89 (d, J = 9.0 Hz, 2H), 10.21 (s, 1H).
Procedure C. A stirred suspension of the benzamide 41 (1.22 g,
5 mmol) obtained from procedure B and TEA (1.4 mL, 10 mmol) in
THF (20 mL) at room temperature was combined with 2,4-
dichloroquinazoline 39 (1.0 g, 5 mmol) dissolved in CH2Cl2 (10 mL),
and the mixture was stirred for 3−6 h to complete the reaction. The
reaction solution was diluted with CH2Cl2 (200 mL) and washed with
water and brine in turn. The organic layer was then separated and dried
over anhydrous Na2SO4. After removal of Na2SO4 by filtration and
evaporation of the solvents, the crude product was purified by column
chromatography with hexane/CH2Cl2 to give 4-[[(2-chloroquinazolin-
4-yl)amino]methyl]-N-(4-fluorophenyl)benzamide 49 (1.50 g, 71% yield).
MS (ESI) m/z 407.2.
Procedure D. The 2-chloroquinazoline intermediate 49 (0.42 g,
1 mmol) obtained from procedure C was taken up in 2.0 M
methylamine solution in THF (8 mL) in a sealed tube. (A sealed tube
was used if the reactant amine was methylamine, dimethylamine,
ethylamine, n-propylamine, cyclopropylamine, or allylamine; THF or
dioxane was the solvent. For other amines, the reactions were carried
out in dioxane in round-bottom flask.) The mixture was heated to
100−120 °C for 8−16 h to complete the reaction. The solution was
diluted with CH2Cl2 (100 mL) and washed once with brine. The
organic layer was separated and dried over anhydrous Na2SO4. After
removal of Na2SO4 by filtration and evaporation of the solvents, the
crude product was purified by column chromatography with CH2Cl2/
MeOH/TEA to give the final products listed below in yield between
20% and 90%.
for the luciferase reporter assay in HCT116 cells. Moreover, in
the cell growth inhibition assays, their IC50 values were 0.6−
0.9 μM in HCT 116 cells, as well as ∼1 μM in SW480 cells. As
such, they represent potential chemical probes for biological
studies of β-catenin/Tcf4 signaling and promising candidates for
developing new anticancer therapeutics that target renewable
cancer stem cells.
EXPERIMENTAL SECTION
■
General Methods. All commercially available chemicals and
solvents were purchased and used as received without further
1
purification. H NMR and 13C NMR spectra were recorded with a
Bruker-BioSpin 300 MHz spectrometer using TMS as an internal
standard. The mass spectra were obtained from a Thermo Q-Tof
micro spectrometer. The HPLC results were generated using a Waters
2489 UV/visible detector and Waters 1525 binary HPLC pump.
The colon cancer cell lines HCT116 and SW480 were obtained
from American Type Culture Collection (ATCC) and cultured in
RPMI 1640 (Hyclone, Thermal Scientific) supplemented with 10%
fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, and
100 μg/mL streptomycin. Cells were maintained in a humidified 5%
CO2 atmosphere at 37 °C.
General Procedures for the Synthesis of the Analogues.
Procedure A. The N-Boc-4-aminobenzylamine 1 (2.2 g, 10 mmol)
and triethylamine (2.8 mL, 20 mmol) were dissolved in CH2Cl2
(40 mL) and cooled in an ice−water bath under a N2 atmosphere, and
then 4-fluorobenzoyl chloride (1.2 mL, 10 mmol) was added. The
reaction mixture was stirred at room temperature for 4 h to complete
the reaction, trifluoroacetic acid (10 mL) was added to the suspension,
and the resulting clear solution was stirred at room temperature
overnight. The solvent was removed, and the residue was taken up in
CH2Cl2 (100 mL) and ice (50 g) and the pH adjusted to >10 with
20% NaOH aqueous solution. The layers were allowed to separate, the
aqueous layer was extracted once with CH2Cl2 (50 mL), and the
combined organic layers were washed once with brine and dried over
anhydrous Na2SO4. Removal of Na2SO4 by filtration and evaporation
of solvents produced N-[4-(aminomethyl)phenyl]-4-fluorobenzamide
HPLC Conditions. Column: Waters XBridge BEH130 C18
4.6 mm × 250 mm × 5 μm; detection: 254 nm; flow rate: 1.0 mL/min;
temperature: 25 °C; injection load: 5 μL; concentration: 0.5 mg/mL; run
time: 25 min; mobile phase A: water (0.01% TFA); mobile phase B: 90%
acetonitrile/water (0.01% TFA); gradient program: time (min): 0 18 20
25; % of mobile phase A: 100, 0, 100, 100; % of mobile phase B: 0, 100, 0,
0; Retention time range: 14−16 min.
4-Fluoro-N-[4-[[(2-(methylamino)quinazolin-4-yl)amino]-
methyl]phenyl]benzamide (17). A preparation of N-[4-[[(2-
chloroquinazolin-4-yl)amino]methyl]phenyl]-4-fluorobenzamide (10)
was made starting from N-Boc-4-aminobenzylamine (1), 4-fluoroben-
zoyl chloride, and 2,4-dichloroquinazoline (39) following procedures
1
3 (2.0 g, 82%), which was used directly in the next step. H NMR
(DMSO-d6) δ 3.70 (s, 2H), 7.35 (m, 4H), 7.67 (d, J = 9.0 Hz, 2H),
8.02 (m, 2H), 10.18 (s, 1H).
Procedure B. The 4-[Boc-amino(methyl)]benzoic acid 40 (2.5 g,
10 mmol), HOBt (1.4 g, 10 mmol), DIC (1.9 mL, 12 mmol), and
DIEA (3.3 mL, 20 mmol) were dissolved in CH2Cl2 (50 mL) and
stirred at room temperature for 1 h under a N2 atmosphere, 4-
fluoroaniline (0.96 mL, 10 mmol) was added, and the reaction mixture
was stirred overnight to complete the reaction. The reaction mixture
was diluted with CH2Cl2 (150 mL) and washed once with brine.
The organic layer was separated and dried over anhydrous Na2SO4.
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A and C. The intermediate product 10 was aminated with
methylamine following procedure D to yield the final product 17.
Starting from 39 (0.2 g, 1 mmol), 190 mg (51% yield) of the final
product was isolated. HPLC purity 96.1%; 1H NMR (DMSO-d6)
δ 2.80 (d, J = 3.0 Hz, 3H), 4.69 (d, J = 6.0 Hz, 2H), 6.45 (d, J = 3.0
Hz, 1H), 7.03 (t, J = 6.0 Hz, 1H), 7.28 (m, 1H), 7.36 (m, 4H), 7.46
(t, J = 6.0 Hz, 1H), 7.69 (d, J = 6.0 Hz, 2H), 8.03 (m, 3H), 8.38 (br s,
1H), 10.24 (s, 1H); 13C NMR (DMSO-d6) δ 28.3, 46.1, 115.5, 115.8,
120.3, 120.7, 123.1, 128.1, 130.7, 130.8, 131.8, 132.6, 135.7, 138.1,
152.5, 160.3, 162.8, 164.7, 166.1; MS (ESI) m/z 402.1.
4-Fluoro-N-[4-[[(2-(dimethylamino)quinazolin-4-yl)amino]-
methyl]phenyl]benzamide (18). The intermediate 10 was
aminated with dimethylamine following procedure D to yield the
final product 18. Starting from 39 (0.2 g, 1 mmol), 120 mg (29%
yield) of the final product was isolated. HPLC purity 95.3%; 1H NMR
(DMSO-d6) δ 3.13 (s, 6H), 4.47 (d, J = 6.0 Hz, 2H), 7.11 (t, J = 6.0
Hz, 1H), 7.32 (m, 5H), 7.54 (t, J = 6.0 Hz, 1H), 7.83 (d, J = 9.0 Hz,
2H), 7.98 (m, 2H), 8.28 (d, J = 9.0 Hz, 1H), 9.06 (t, J = 3.0 Hz, 1H),
9.44 (s, 1H); 13C NMR (DMSO-d6) δ 37.2, 42.7, 110.5, 115.5, 115.8,
120.8, 121.9, 123.4, 125.6, 127.7, 130.4, 133.1, 134.5, 138.8, 152.9,
158.0, 159.3, 162.7, 165.5; MS (ESI) m/z 416.2.
4-Fluoro-N-[4-[[(2-(4-methylpiperazin-1-yl)quinazolin-4-yl)-
amino]methyl]phenyl]benzamide (19). The intermediate 10 was
aminated with N-methylpiperazine following procedure D to yield the
final product 19. Starting from 39 (0.1 g, 0.5 mmol), 150 mg (64%
yield) of the final product was isolated. HPLC purity 97.0%; 1H NMR
(CDCl3) δ 2.30 (s, 3H), 2.43 (br s, 4H), 3.89 (br s, 4H), 4.70 (d, J =
6.0 Hz, 2H), 6.39 (br s, 1H), 7.04 (m, 3H), 7.28 (d, J = 6.0 Hz, 2H),
7.46 (m, 2H), 7.55 (d, J = 6.0 Hz, 2H), 7.60 (m, 1H), 7.87 (m, 2H),
8.36 (s, 1H); 13C NMR (CDCl3) δ 43.9, 44.6, 46.1, 55.0, 110.4, 115.5,
115.8, 120.9, 121.3, 125.1, 128.4, 129.5, 129.6, 132.7, 135.1, 137.0,
158.2, 159.6, 163.1, 164.9, 166.5; MS (ESI) m/z 471.1.
N-[4-[[(2-(3-(Dimethylamino)propylamino)quinazolin-4-yl)-
amino]methyl]phenyl]-4-fluorobenzamide (20). The intermedi-
ate 10 was aminated with N,N-dimethylpropane-1,3-diamine following
procedure D to yield the final product 20. Starting from 39 (0.1 g,
0.5 mmol), 130 mg (57% yield) of the final product was isolated. HPLC
purity 95.1%; 1H NMR (DMSO-d6) δ 1.79 (br s, 2H), 2.43 (s, 6H), 2.69
(br s, 2H), 3.32 (m, 2H), 4.45 (d, J = 6.0 Hz, 2H), 6.87 (br s, 1H), 7.11
(t, J = 6.0 Hz, 1H), 7.30 (m, 5H), 7.54 (t, J = 6.0 Hz, 1H), 7.86 (d, J =
9.0 Hz, 2H), 8.01 (m, 2H), 8.33 (m, 1H), 9.19 (t, J = 3.0 Hz, 1H), 9.47
(br s, 1H); 13C NMR (DMSO-d6) δ 38.7, 42.7, 43.7, 45.8, 55.9, 115.5,
115.8, 120.9, 122.3, 123.6, 127.7, 130.3, 130.4, 131.2, 133.1, 134.7, 138.8,
158.5, 159.3, 162.6, 165.4, 165.9; MS (ESI) m/z 473.1.
4-Fluoro-N-[4-[[(2-(propylamino)quinazolin-4-yl)amino]-
methyl]phenyl]benzamide (21). The intermediate 10 was
aminated with n-propylamine following procedure D to yield the
final product 21. Starting from 39 (0.1 g, 0.5 mmol), 56 mg (26%
yield) of the final product was isolated. HPLC purity 96.3%; 1H NMR
(DMSO-d6) δ 0.88 (t, J = 6.0 Hz, 3H), 1.51 (m, 2H), 3.44 (m, 2H),
4.68 (d, J = 6.0 Hz, 2H), 6.48 (br s, 1H), 6.99 (m, 1H), 7.19 (m, 1H),
7.34 (d, J = 9.0 Hz, 2H), 7.46 (m, 2H), 7.68 (d, J = 9.0 Hz, 2H), 8.02
(m, 3H), 8.34 (br s, 1H), 10.23 (s, 1H); 13C NMR (DMSO-d6) δ 11.9,
22.3, 42.5, 42.9, 115.5, 115.8, 119.9, 120.7, 123.1, 128.0, 130.7, 130.8,
132.4, 132.6, 135.8, 138.0, 159.9, 160.2, 162.8, 164.7, 166.1; MS (ESI)
m/z 430.0.
4-Fluoro-N-[4-[[(2-(ethylamino)quinazolin-4-yl)amino]-
methyl]phenyl]benzamide (22). The intermediate 10 was
aminated with ethylamine following procedure D to yield the final
product 22. Starting from 39 (0.1 g, 0.5 mmol), 71 mg (34% yield) of
the final product was isolated. HPLC purity 95.0%; 1H NMR (DMSO-
d6) δ 1.13 (t, J = 6.0 Hz, 3H), 3.31 (m, 2H), 4.67 (d, J = 6.0 Hz, 2H),
6.46 (br s, 1H), 6.99 (m, 1H), 7.19 (d, J = 6.0 Hz, 1H), 7.34 (m, 3H),
7.46 (m, 1H), 7.67 (d, J = 9.0 Hz, 2H), 8.00 (m, 3H), 8.38 (br s, 1H),
10.23 (s, 1H); 13C NMR (DMSO-d6) δ 14.9, 35.7, 46.1, 115.6, 115.8,
120.2, 120.7, 123.1, 128.0, 130.7, 130.8, 132.4, 132.6, 135.7, 138.0,
159.5, 160.2, 163.1, 164.9, 166.1; MS (ESI) m/z 416.3.
starting from 1, isonicotinoyl chloride, and 39 following procedures A
and C. The intermediate product 11 was aminated with methylamine
following procedure D to yield the final product 23. Starting from 39
(0.1 g, 0.5 mmol), 98 mg (53% yield) of the final product was isolated.
HPLC purity 95.7%; 1H NMR (DMSO-d6) δ 2.78 (d, J = 6.0 Hz, 3H),
4.68 (d, J = 6.0 Hz, 2H), 6.50 (br s, 1H), 7.02 (t, J = 6.0 Hz, 1H),
7.25 (d, J = 6.0 Hz, 1H), 7.37 (d, J = 9.0 Hz, 2H), 7.47 (t, J = 6.0 Hz,
1H), 7.69 (d, J = 9.0 Hz, 2H), 7.84 (d, J = 6.0 Hz, 2H), 8.02 (d,
J = 9.0 Hz, 1H), 8.42 (br s, 1H), 8.75 (d, J = 6.0 Hz, 2H), 10.49
(s, 1H); 13C NMR (DMSO-d6) δ 28.3, 45.9, 120.4, 120.8, 122.0, 123.2,
128.2, 132.7, 136.2, 137.6, 142.3, 150.6, 160.0, 164.2; MS (ESI) m/z
385.2.
N-[4-[[(2-(Dimethylamino)quinazolin-4-yl)amino]methyl]-
phenyl]isonicotinamide (24). The intermediate 11 was aminated
with dimethylamine following procedure D to yield the final product
24. Starting from 39 (0.1 g, 0.5 mmol), 118 mg (61% yield) of the
final product was isolated. HPLC purity 95.9%; 1H NMR (DMSO-d6)
δ 3.08 (s, 6H), 4.68 (d, J = 6.0 Hz, 2H), 7.03 (t, J = 6.0 Hz, 1H), 7.26
(d, J = 6.0 Hz, 1H), 7.37 (d, J = 9.0 Hz, 2H), 7.47 (t, J = 6.0 Hz, 1H),
7.70 (d, J = 9.0 Hz, 2H), 7.83 (d, J = 6.0 Hz, 2H), 8.02 (d, J = 6.0 Hz,
1H), 8.52 (t, J = 3.0 Hz, 1H), 8.76 (d, J = 6.0 Hz, 1H), 10.46 (s, 1H);
13C NMR (DMSO-d6) δ 36.9, 43.8, 110.5, 120.4, 120.8, 121.9, 123.1,
125.3, 128.0, 132.7, 136.4, 137.5, 142.3, 150.7, 152.4, 159.6, 159.8,
164.2; MS (ESI) m/z 399.2.
N-[4-[[(2-(4-Methylpiperazin-1-yl)quinazolin-4-yl)amino]-
methyl]phenyl]isonicotinamide (25). The intermediate 11 was
aminated with N-methylpiperazine following procedure D to yield the
final product 25. Starting from 39 (0.1 g, 0.5 mmol), 105 mg (49%
yield) of the final product was isolated. HPLC purity 95.1%; 1H NMR
(CDCl3) δ 2.28 (s, 3H), 2.41 (br s, 4H), 3.86 (br s, 4H), 4.70 (d, J =
6.0 Hz, 2H), 6.34 (br s, 1H), 7.01 (br, 1H), 7.28 (d, J = 9.0 Hz, 2H),
7.44 (m, 2H), 7.58 (m, 3H), 7.67 (d, J = 6.0 Hz, 2H), 8.62 (d, J = 6.0
Hz, 2H), 9.06 (s, 1H); 13C NMR (CDCl3) δ 43.8, 44.5, 45.8, 55.1,
110.5, 121.1, 121.2, 125.6, 128.4, 132.6, 135.8, 136.6, 141.9, 150.3,
152.1, 158.8, 159.7, 164.2; MS (ESI) m/z 454.1.
N-[4-[[(2-(Methylamino)quinazolin-4-yl)amino]methyl]-
phenyl]-4-[(4-methylpiperazin-1-yl)methyl]benzamide (26). A
preparation of N-[4-[[(2-chloroquinazolin-4-yl)amino]methyl]-
phenyl]-4-[(4-methylpiperazin-1-yl)methyl]benzamide (12) was
made starting from 1, 4-[(4-methylpiperazin-1-yl)methyl]benzoyl
chloride,27 and 39 following procedures A and C. The intermediate
product 12 was aminated with methylamine following procedure D to
yield the final product 26. Starting from 39 (0.1 g, 0.5 mmol), 51 mg
(21% yield) of the final product was isolated. HPLC purity 95.0%; 1H
NMR (DMSO-d6) δ 2.13 (s, 3H), 2.34 (br, 8H), 2.78 (d, J = 6.0 Hz,
3H), 3.50 (s, 2H), 4.66 (d, J = 6.0 Hz, 2H), 6.44 (d, J = 6.0 Hz, 1H),
7.01 (t, J = 6.0 Hz, 1H), 7.23 (m, 1H), 7.33 (d, J = 6.0 Hz, 2H), 7.41
(d, J = 9.0 Hz, 2H), 7.46 (t, J = 6.0 Hz, 1H), 7.69 (d, J = 9.0 Hz, 2H),
7.88 (d, J = 6.0 Hz, 2H), 8.02 (d, J = 6.0 Hz, 1H), 8.36 (br s, 1H),
10.16 (s, 1H); 13C NMR (DMSO-d6) δ 28.3, 43.4, 46.1, 52.9, 55.1,
62.0, 120.3, 120.6, 123.1, 128.0, 128.1, 129.0, 131.9, 132.6, 134.0,
135.6, 138.2, 142.6, 152.4, 160.1, 160.2, 165.7; MS (ESI) m/z 496.2.
N-[4-[[(2-(3-(Dimethylamino)propylamino)quinazolin-4-yl)-
amino]methyl]phenyl]-4-[(4-methylpiperazin-1-yl)methyl]-
benzamide (27). The intermediate 12 was aminated with N,N-
dimethylpropane-1,3-diamine following procedure D to yield the final
product 27. Starting from 39 (0.1 g, 0.5 mmol), 60 mg (21% yield) of
the final product was isolated. HPLC purity 95.4%; 1H NMR (DMSO-
d6) δ 1.62 (br, 2H), 2.11 (s, 6H), 2.12 (s, 3H), 2.33 (br, 8H), 3.49
(s, 2H), 4.20 (br, 4H), 4.67 (d, J = 6.0 Hz, 2H), 7.00 (t, J = 6.0 Hz,
1H), 7.20 (m, 1H), 7.32 (d, J = 9.0 Hz, 2H), 7.40 (d, J = 6.0 Hz, 2H),
9.45 (m, 1H), 7.70 (d, J = 9.0 Hz, 2H), 7.87 (d, J = 6.0 Hz, 2H), 7.99
(d, J = 6.0 Hz, 1H), 10.16 (s, 1H); 13C NMR (DMSO-d6) δ 21.8, 39.5,
45.5, 46.0, 46.1, 52.9, 55.1, 57.4, 62.0, 120.2, 120.6, 123.1, 128.0, 129.0,
132.6, 134.0, 135.5, 138.2, 142.6, 152.3, 159.7, 160.2, 165.7, 172.7; MS
(ESI) m/z 567.3.
4-Fluoro-N-[3-chloro-4-[[(2-(methylamino)quinazolin-4-yl)-
amino]methyl]phenyl]benzamide (28). A mixture of 4-amino-
2-chlorobenzonitrile (7.6 g, 0.05 mol) and LiAlH4 (3.8 g, 0.1 mol) was
suspended in anhydrous THF (50 mL) under a N2 atmosphere and
N-[4-[[(2-(Methylamino)quinazolin-4-yl)amino]methyl]-
phenyl]isonicotinamide (23). A preparation of N-[4-[[(2-chloro-
quinazolin-4-yl)amino]methyl]phenyl]isonicotinamide (11) was made
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1
refluxed for 5 h to complete the reaction. The reaction mixture was
poured carefully onto ice and extracted with CH2Cl2. The organic
layer was washed with brine and dried over Na2SO4. The crude
product was purified by column chromatography with CH2Cl2/MeOH
to give 4-(aminomethyl)-3-chloroaniline 38 (5.3 g, 68%) as a faint
brown oil. Compound 38, TEA (9.5 mL, 2 equiv), and Boc2O (8.1 g,
1.1 equiv) were dissolved in CH2Cl2 (80 mL). The reaction mixture
was stirred at room temperature overnight and then diluted with
CH2Cl2 (200 mL). The organic layer was washed with brine and dried
over anhydrous Na2SO4. The crude product was purified by column
chromatography with hexane/CH2Cl2 to give N-Boc-4-amino-
2-chlorobenzylamine 2 (6.2 g, 72%) as a faint yellow solid. MS (ESI)
m/z 257.0.
A preparation of N-[3-chloro-4-[[(2-chloroquinazolin-4-yl)amino]-
methyl]phenyl]-4-fluorobenzamide (13) was made starting from 2, 4-
fluorobenzoyl chloride, and 39 following procedures A and C. The
intermediate product 13 was aminated with methylamine following
procedure D to yield the final product 28. Starting from 39 (0.1 g,
0.5 mmol), 150 mg (71% yield) of the final product was isolated.
HPLC purity 96.6%; 1H NMR (DMSO-d6) δ 2.76 (d, J = 3.0 Hz, 3H),
4.73 (d, J = 6.0 Hz, 2H), 6.44 (d, J = 3.0 Hz, 1H), 7.03 (t, J = 6.0 Hz,
1H), 7.32 (m, 1H), 7.38 (d, J = 6.0 Hz, 2H), 7.51 (m, 2H), 7.95 (d, J =
6.0 Hz, 2H), 8.02 (m, 2H), 8.36 (br s, 1H), 10.40 (s, 1H); 13C NMR
(DMSO-d6) δ 28.3, 41.5, 115.6, 115.9, 119.2, 120.4, 120.7, 120.9,
123.2, 129.2, 130.8, 130.9, 132.1, 132.4, 132.7, 139.3, 152.5, 160.2,
162.9, 164.9, 166.2; MS (ESI) m/z 435.9.
4-Fluoro-N-[3-chloro-4-[[(2-(ethylamino)quinazolin-4-yl)-
amino]methyl]phenyl]benzamide (29). The intermediate product
13 was aminated with ethylamine following procedure D to yield the
final product 29. Starting from 39 (0.1 g, 0.5 mmol), 115 mg (52%
yield) of the final product was isolated. HPLC purity 95.1%; 1H NMR
(DMSO-d6) δ 1.03 (t, J = 6.0 Hz, 1H), 3.25 (q, J = 6.0 Hz, 2H), 4.74
(d, J = 6.0 Hz, 2H), 6.52 (br, 1H), 7.03 (t, J = 6.0 Hz, 1H), 7.24
(m, 1H), 7.32 (d, J = 9.0 Hz, 2H), 7.35 (m, 1H), 7.49 (m, 1H), 7.60
(m, 1H), 8.02 (m, 4H), 8.38 (br, 1H), 10.40 (s, 1H); 13C NMR
(DMSO-d6) δ 15.5, 35.7, 41.5, 115.6, 115.9, 119.2, 120.1, 120.3, 120.9,
123.1, 123.2, 129.0, 130.8, 130.9, 132.1, 132.7, 139.3, 159.5, 160.3,
162.9, 164.9, 166.2; MS (ESI) m/z 450.3.
HPLC purity 95.5%; H NMR (CDCl3) δ 2.30 (s, 6H), 2.44 (br s,
4H), 3.86 (br s, 4H), 4.64 (d, J = 3.0 Hz, 2H), 6.38 (br s, 1H), 6.98
(m, 1H), 7.02 (d, J = 9.0 Hz, 2H), 7.15 (m, 4H), 7.46 (m, 3H), 7.57
(d, J = 6.0 Hz, 1H), 7.64 (d, J = 9.0 Hz, 2H); 13C NMR (CDCl3) δ
21.5, 43.7, 44.4, 46.0, 54.9, 110.3, 121.2, 121.3, 121.7, 125.1, 127.1,
128.6, 129.5, 132.7, 135.5, 136.1, 136.4, 143.6, 158.2, 159.6; MS (ESI)
m/z 503.1.
N-[4-[[(2-(Methylamino)quinazolin-4-yl)amino]methyl]-
phenyl]-4-fluorobenzenesulfonamide (33). A preparation of
N-[4-[[(2-chloroquinazolin-4-yl)amino]methyl]phenyl]-4-fluoroben-
zenesulfonamide (15) was made starting from 1, 4-fluorobenzene-
sulfonyl chloride, and 39 following procedures A and C. The inter-
mediate product 15 was aminated with methylamine following
procedure D to yield the final product 33. Starting from 39 (0.1 g,
0.5 mmol), 57 mg (26% yield) of the final product was isolated. HPLC
purity 96.5%; 1H NMR (DMSO-d6) δ 2.39 (s, 3H), 4.57 (d, J =
6.0 Hz, 2H), 6.56 (d, J = 3.0 Hz, 1H), 6.71 (br s, 1H), 6.95 (d, J = 9.0 Hz,
2H), 7.19 (t, J = 3.0 Hz, 1H), 7.27 (m, 1H), 7.35 (m, 4H), 7.81 (br s,
1H), 7.91 (br s, 1H), 8.48 (br s, 1H); 13C NMR (DMSO-d6) δ 23.3,
44.4, 112.8, 119.5, 119.7, 123.4, 123.9, 126.8, 127.0, 129.8, 132.2,
135.8, 136.2, 145.5, 156.5, 158.3, 169.9; MS (ESI) m/z 438.1.
N-[4-[[(2-(Cyclopropylamino)quinazolin-4-yl)amino]methyl]-
phenyl]-4-fluorobenzenesulfonamide (34). The intermediate
product 15 was aminated with cyclopropylamine following procedure
D to yield the final product 34. Starting from 39 (0.1 g, 0.5 mmol),
80 mg (35% yield) of the final product was isolated. HPLC purity
1
95.1%; H NMR (DMSO-d6) δ 1.22 (m, 4H), 4.57 (d, J = 6.0 Hz,
2H), 6.39 (d, J = 3.0 Hz, 1H), 6.95 (m, 1H), 6.99 (d, J = 9.0 Hz, 2H),
7.21 (m, 3H), 7.34 (m, 2H), 7.76 (m, 2H), 7.93 (d, J = 6.0 Hz, 1H),
8.26 (d, J = 6.0 Hz, 1H); 13C NMR (DMSO-d6) δ 7.7, 28.2, 46.2, 116.5,
116.9, 120.2, 120.8, 121.8, 123.1, 128.1, 129.0, 130.0, 132.5, 136.3, 136.6,
152.4, 160.1, 162.9, 166.3; MS (ESI) m/z 464.0.
N-(4-Fluorophenyl)-4-[[(2-(methylamino)quinazolin-4-yl)-
amino]methyl]benzamide (62). The intermediate product 49
(procedure C) was aminated with methylamine following procedure
D to yield the final product 62. Starting from 39 (0.1 g, 0.5 mmol),
140 mg (69% yield) of the final product was isolated. HPLC purity
1
97.0%; H NMR (DMSO-d6) δ 2.74 (d, J = 3.0 Hz, 3H), 4.60 (d, J =
6.0 Hz, 2H), 6.67 (d, J = 3.0 Hz, 1H), 6.99 (m, 1H), 7.03 (d, J = 6.0
Hz, 2H), 7.23 (d, J = 6.0 Hz, 2H), 7.26 (m, 1H), 7.35 (m, 2H), 7.46
(m, 1H), 7.79 (m, 2H), 7.96 (d, J = 9.0 Hz, 1H), 8.33 (br s, 1H); 13C
NMR (DMSO-d6) δ 24.3, 43.1, 116.7, 117.0, 120.5, 120.8, 123.1,
123.4, 125.1, 128.7, 130.0, 130.2, 132.6, 136.4, 152.3, 160.1, 160.6,
163.0, 166.3; MS (ESI) m/z 402.3.
N-[4-[[(2-(Methylamino)quinazolin-4-yl)amino]methyl]-
phenyl]-4-methylbenzenesulfonamide (30). A preparation of
N-[4-[[(2-chloroquinazolin-4-yl)amino]methyl]phenyl]-4-methylben-
zenesulfonamide (14) was made starting from 1, tosyl chloride, and 39
following procedures A and C. The intermediate product 14 was
aminated with methylamine following procedure D to yield the final
product 30. Starting from 39 (0.1 g, 0.5 mmol), 105 mg (51% yield) of
the final product was isolated. HPLC purity 96.1%; 1H NMR (DMSO-
d6) δ 2.29 (s, 3H), 2.74 (d, J = 3.0 Hz, 3H), 4.57 (d, J = 6.0 Hz, 2H),
6.41 (d, J = 6.0 Hz, 1H), 6.96 (m, 1H), 7.01 (d, J = 9.0 Hz, 2H), 7.20
(d, J = 9.0 Hz, 2H), 7.24 (m, 1H), 7.29 (d, J = 9.0 Hz, 2H), 7.44 (t, J =
6.0 Hz, 1H), 7.60 (d, J = 9.0 Hz, 2H), 7.94 (d, J = 9.0 Hz, 1H), 8.28
(br s, 1H); 13C NMR (DMSO-d6) δ 21.3, 28.3, 43.1, 120.4, 123.1,
125.0, 127.1, 128.6, 130.0, 132.6, 136.0, 136.7, 137.2, 143.5, 152.4,
160.2; MS (ESI) m/z 434.2.
N-[4-[[(2-(Dimethylamino)quinazolin-4-yl)amino]methyl]-
phenyl]-4-methylbenzenesulfonamide (31). The intermediate
product 14 was aminated with dimethylamine following procedure
D to yield the final product 31. Starting from 39 (0.1 g, 0.5 mmol),
125 mg (58% yield) of the final product was isolated. HPLC purity
95.2%; 1H NMR (DMSO-d6) δ 2.28 (s, 3H), 3.01 (s, 6H), 4.57 (d, J =
6.0 Hz, 2H), 6.97 (m, 1H), 7.01 (d, J = 6.0 Hz, 2H), 7.20 (d, J = 9.0 Hz,
2H), 7.26 (m, 1H), 7.28 (d, J = 6.0 Hz, 2H), 7.44 (t, J = 6.0 Hz, 1H),
7.60 (d, J = 9.0 Hz, 2H), 7.97 (d, J = 6.0 Hz, 1H), 8.43 (t, J = 3.0 Hz,
1H); 13C NMR (DMSO-d6) δ 21.3, 36.9, 43.6, 110.5, 120.3, 120.4,
123.1, 125.3, 127.1, 128.5, 130.0, 132.6, 136.2, 136.7, 137.1, 143.5,
152.4, 159.5, 159.8; MS (ESI) m/z 448.1.
N-(4-Fluorophenyl)-4-[[(2-(ethylamino)quinazolin-4-yl)-
amino]methyl]benzamide (63). The intermediate product 49 was
aminated with ethylamine following procedure D to yield the final
product 62. Starting from 39 (0.1 g, 0.5 mmol), 150 mg (72% yield) of
the final product was isolated. HPLC purity 96.2%; 1H NMR (DMSO-
d6) δ 1.06 (t, J = 6.0 Hz, 3H), 3.29 (d, J = 6.0 Hz, 2H), 4.78 (d, J =
6.0 Hz, 2H), 6.46 (br s, 1H), 7.02 (t, J = 6.0 Hz, 1H), 7.16 (d, J =
9.0 Hz, 2H), 7.23 (m, 1H), 7.42 (m, 1H), 7.50 (d, J = 6.0 Hz, 2H),
7.77 (m, 2H), 7.90 (d, J = 6.0 Hz, 2H), 8.02 (d, J = 9.0 Hz, 1H), 8.48
(br s, 1H), 10.23 (s, 1H); 13C NMR (DMSO-d6) δ 27.9, 28.3, 43.6,
115.4, 115.7, 120.3, 122.5, 123.1, 125.1, 127.7, 128.0, 132.6, 133.6,
136.0, 144.3, 152.5, 157.0, 160.2, 160.4, 165.8; MS (ESI) m/z 416.3.
N-(4-Fluorophenyl)-4-[[(2-(cyclopropylamino)quinazolin-
4-yl)amino]methyl]benzamide (64). The intermediate product
49 was aminated with cyclopropylamine following procedure D to yield
the final product 64. Starting from 39 (0.1 g, 0.5 mmol), 130 mg (65%
yield) of the final product was isolated. HPLC purity 95.8%; 1H NMR
(DMSO-d6) δ 0.45 (m, 2H), 0.60 (m, 2H), 2.75 (m, 1H), 4.78 (d, J =
6.0 Hz, 2H), 6.88 (d, J = 3.0 Hz, 1H), 7.04 (t, J = 6.0 Hz, 1H), 7.16 (d,
J = 9.0 Hz, 2H), 7.27 (d, J = 9.0 Hz, 1H), 7.50 (m, 3H), 7.77 (m, 2H),
7.89 (d, J = 9.0 Hz, 2H), 8.02 (d, J = 9.0 Hz, 1H), 8.50 (br s, 1H),
10.22 (s, 1H); 13C NMR (DMSO-d6) δ 6.8, 24.3, 43.6, 115.4, 115.7,
120.5, 122.6, 123.1, 125.2, 127.7, 128.0, 132.6, 133.6, 136.0, 144.3,
152.5, 157.0, 160.2, 160.7, 165.7; MS (ESI) m/z 428.3.
N-[4-[[(2-(4-Methylpiperazin-1-yl)quinazolin-4-yl)amino]-
methyl]phenyl]-4-methylbenzenesulfonamide (32). The inter-
mediate product 14 was aminated with N-methylpiperazine following
procedure D to yield the final product 32. Starting from 39 (0.1 g,
0.5 mmol), 165 mg (68% yield) of the final product was isolated.
N-(4-Fluorophenyl)-4-[[(2-(propylamino)quinazolin-4-yl)-
amino]methyl]benzamide (65). The intermediate product 49 was
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aminated with n-propylamine following procedure D to yield the final
product 65. Starting from 39 (0.1 g, 0.5 mmol), 155 mg (72% yield) of
the final product was isolated. HPLC purity 96.6%; 1H NMR (DMSO-d6)
δ 0.85 (m, 3H), 1.55 (m, 2H), 3.22 (m, 2H), 4.78 (d, J = 6.0 Hz, 2H),
6.49 (br s, 1H), 7.02 (t, J = 6.0 Hz, 1H), 7.18 (d, J = 9.0 Hz, 2H), 7.22
(m, 1H), 7.43 (m, 1H), 7.50 (d, J = 9.0 Hz, 2H), 7.78 (m, 2H), 7.91
(d, J = 9.0 Hz, 2H), 8.02 (d, J = 6.0 Hz, 1H), 8.49 (br s, 1H), 10.24
(s, 1H); 13C NMR (DMSO-d6) δ 11.9, 22.4, 42.5, 42.9, 115.4, 115.7,
120.2, 122.5, 122.6, 123.1, 127.5, 128.0, 132.6, 133.5, 136.0, 144.4,
152.6, 157.1, 159.8, 160.2, 165.7; MS (ESI) m/z 430.3.
N-(4-Fluorophenyl)-4-[[(2-(butylamino)quinazolin-4-yl)-
amino]methyl]benzamide (66). The intermediate product 49 was
aminated with n-butylamine following procedure D to yield the final
product 66. Starting from 39 (0.1 g, 0.5 mmol), 105 mg (49% yield) of
the final product was isolated. HPLC purity 95.5%; 1H NMR (DMSO-
d6) δ 0.85 (m, 3H), 1.30 (m, 2H), 1.45 (m, 3H), 3.24 (m, 2H), 4.78
(d, J = 6.0 Hz, 2H), 6.47 (br s, 1H), 7.02 (m, 1H), 7.18 (m, 3H), 7.40
(m, 1H), 7.49 (d, J = 9.0 Hz, 2H), 7.77 (m, 2H), 7.92 (d, J = 9.0 Hz,
2H), 8.01 (d, J = 9.0 Hz, 1H), 8.49 (br s, 1H), 10.22 (s, 1H); 13C
NMR (DMSO-d6) δ 14.2, 20.2, 31.3, 32.0, 46.1, 115.4, 115.7, 120.2,
122.5, 122.6, 123.1, 127.5, 128.0, 132.7, 133.5, 135.9, 144.4, 152.5,
157.1, 159.8, 160.2, 165.7; MS (ESI) m/z 444.2.
Compound 94 (10 g, 56 mmol) was suspended in POCl3 (60 mL),
and the mixture was stirred under reflux for 3−5 h to complete the
reaction. The volatiles were removed under vacuum, and the residue
was poured onto 200 g ice−water and stirred for 1 h. The resulting
solid was collected and dried under reduced pressure. The crude
product was purified by column chromatography with hexane/CH2Cl2
to give 2,4-dichloro-6-methylquinazoline 95 (6.6 g, 56%) as a white
1
solid. H NMR (DMSO-d6) δ 2.56 (s, 3H), 7.92 (d, J = 9.0 Hz, 1H),
7.99 (d, J = 9.0 Hz, 1H), 8.04 (s, 1H); MS (ESI) m/z 213.0.
A preparation of N-(4-fluorophenyl)-4-[[6-methyl-(2-chloroquina-
zolin-4-yl)amino]methyl]benzamide (50) was made starting from 41
(procedure B) and 95 following procedure C. The intermediate
product 50 was aminated with n-propylamine following procedure D
to yield the final product 70. Starting from 95 (0.11 g, 0.5 mmol),
45 mg (20% yield) of the final product was isolated. HPLC purity
1
97.0%; H NMR (DMSO-d6) δ 0.83 (m, 3H), 1.56 (m, 2H), 2.33
(s, 3H), 3.19 (m, 2H), 4.75 (d, J = 6.0 Hz, 2H), 6.38 (br s, 1H), 7.16
(m, 3H), 7.30 (m, 1H), 7.49 (d, J = 6.0 Hz, 2H), 7.76 (m, 2H), 7.83
(s, 1H), 7.89 (d, J = 2.0 Hz, 2H), 8.41 (br s, 1H), 10.23 (s, 1H); 13C
NMR (DMSO-d6) δ 11.1, 21.2, 23.1, 42.5, 43.0, 115.4, 115.7, 122.3,
122.5, 122.6, 127.6, 128.0, 129.2, 133.5, 134.3, 136.0, 144.4, 150.5, 157.1,
159.3, 159.9, 165.7; MS (ESI) m/z 444.1.
N-(4-Fluorophenyl)-4-[[6-methyl-(2-(allylamino)quinazolin-
4-yl)amino]methyl]benzamide (71). The intermediate product 50
was aminated with allylamine following procedure D to yield the final
product 71. Starting from 95 (0.11 g, 0.5 mmol), 95 mg (43% yield) of
the final product was isolated. HPLC purity 96.4%; 1H NMR (DMSO-
d6) δ 2.34 (s, 3H), 3.90 (br s, 2H), 4.11 (br s, 1H), 4.76 (d, J = 6.0 Hz,
2H), 5.01 (m, 1H), 5.17 (m, 2H), 6.55 (br s, 1H), 7.16 (m, 3H), 7.31
(m, 1H), 7.51 (d, J = 6.0 Hz, 2H), 7.78 (m, 2H), 7.85 (s, 1H), 7.90
(d, J = 2.0 Hz, 2H), 8.46 (br s, 1H), 10.25 (s, 1H); 13C NMR (DMSO-
d6) δ 21.2, 42.9, 43.6, 114.8, 115.4, 115.7, 122.3, 122.6, 124.9, 127.7,
128.0, 129.4, 133.6, 134.3, 136.0, 137.4, 144.4, 150.5, 157.1, 159.1,
159.9, 165.7; MS (ESI) m/z 442.1.
N-(4-Fluorophenyl)-4-[[(2-(allylamino)quinazolin-4-yl)-
amino]methyl]benzamide (67). The intermediate product 49 was
aminated with allylamine following procedure D to yield the final
product 67. Starting from 39 (0.1 g, 0.5 mmol), 98 mg (47% yield) of
the final product was isolated. HPLC purity 95.1%; 1H NMR (DMSO-
d6) δ 3.93 (m, 2H), 4.13 (m, 1H), 4.78 (d, J = 6.0 Hz, 2H), 4.99 (m,
1H), 5.18 (m, 2H), 6.74 (br s, 1H), 7.03 (m, 1H), 7.18 (m, 2H), 7.25
(m, 1H), 7.45 (m, 1H), 7.51 (d, J = 6.0 Hz, 2H), 7.78 (m, 2H), 7.90
(d, J = 6.0 Hz, 1H), 8.05 (m, 1H), 8.61 (br s, 1H), 10.25 (s, 1H); 13C
NMR (DMSO-d6) δ 43.0, 43.5, 114.9, 115.4, 115.7, 122.5, 122.6,
123.2, 124.0, 127.7, 128.1, 132.8, 133.6, 136.0, 137.1, 144.2, 151.9,
157.1, 159.3, 160.3, 165.7; MS (ESI) m/z 428.2.
N-(6-Chloropyridin-3-yl)-4-[[(2-(dimethylamino)-6-methyl-
quinazolin-4-yl)amino]methyl]benzamide (72). A preparation of
N-(6-chloropyridin-3-yl)-4-[[(2-chloro-6-methylquinazolin-4-yl)-
amino]methyl]benzamide (51) was made starting from 40,
6-chloropyridin-3-amine, and 95 following procedures B and C. The
intermediate product 51 was aminated with dimethylamine following
procedure D to yield the final product 72. Starting from 95 (0.1 g,
0.5 mmol), 120 mg (55% yield) of the final product was isolated.
HPLC purity 95.7%; 1H NMR (DMSO-d6) δ 2.34 (s, 3H), 3.03 (s, 6H),
4.75 (d, J = 3.0 Hz, 2H), 7.18 (d, J = 9.0 Hz, 1H), 7.32 (dd, J = 9.0 Hz,
3.0 Hz, 1H), 7.47 (d, J = 9.0 Hz, 1H), 7.53 (d, J = 9.0 Hz, 2H), 7.85
(s, 1H), 7.92 (d, J = 9.0 Hz, 2H), 8.24 (dd, J = 9.0 Hz, 3.0 Hz, 1H), 8.52
(t, J = 6.0 Hz, 1H), 8.78 (d, J = 3.0 Hz, 1H), 10.51 (s, 1H); 13C NMR
(DMSO-d6) δ 21.2, 36.9, 44.1, 110.3, 122.3, 124.4, 125.3, 127.6, 128.2,
129.4, 131.2, 132.8, 134.3, 135.9, 141.9, 144.3, 145.1, 150.6, 159.2, 159.6,
166.2; MS (ESI) m/z 447.1.
N-(4-Fluorophenyl)-4-[[(2-(dimethylamino)quinazolin-4-yl)-
amino]methyl]benzamide (68). The intermediate product 49 was
aminated with dimethylamine following procedure D to yield the final
product 68. Starting from 39 (0.1 g, 0.5 mmol), 125 mg (62% yield) of
the final product was isolated. HPLC purity 96.3%; 1H NMR (DMSO-
d6) δ 3.04 (s, 6H), 4.75 (d, J = 6.0 Hz, 2H), 7.04 (m, 1H), 7.16
(m, 2H), 7.26 (m, 1H), 7.50 (d, J = 6.0 Hz, 2H), 7.76 (m, 2H), 7.88
(d, J = 6.0 Hz, 2H), 8.04 (d, J = 9.0 Hz, 1H), 8.64 (br s, 1H), 10.22 (s,
1H); 13C NMR (DMSO-d6) δ 36.9, 44.0, 110.5, 115.4, 115.7, 120.5,
122.5, 123.1, 125.4, 127.5, 128.0, 132.7, 133.6, 136.0, 144.5, 152.5,
159.5, 159.8, 165.7; MS (ESI) m/z 416.1.
(
)-N-(4-Fluorophenyl)-4-[[(2-(3-methylpiperazin-1-yl)-
quinazolin-4-yl)amino]methyl]benzamide (69). The intermedi-
ate product 49 was aminated with 2-methylpiperazine following
procedure D to yield the final product 69. Starting from 39 (0.1 g,
0.5 mmol), 165 mg (72% yield) of the final product was isolated.
HPLC purity 95.8%; 1H NMR (DMSO-d6) δ 0.97 (d, J = 6.0 Hz, 3H),
2.56 (m, 4H), 2.77 (m, 2H), 4.73 (d, J = 6.0 Hz, 2H), 7.06 (t, J = 6.0 Hz,
1H), 7.16 (m, 2H), 7.26 (d, J = 9.0 Hz, 1H), 7.50 (d, J = 9.0 Hz, 2H),
7.77 (m, 2H), 7.89 (d, J = 9.0 Hz, 2H), 8.04 (d, J = 6.0 Hz, 1H), 8.64
(t, J = 6.0 Hz, 1H), 10.20 (s, 1H); 13C NMR (DMSO-d6) δ 19.5, 44.0,
44.2, 45.5, 50.6, 51.0, 110.9, 115.4, 115.7, 120.8, 122.6, 123.1, 125.4,
127.5, 128.0, 132.8, 133.5, 144.4, 152.3, 157.3, 158.7, 160.0, 165.7; MS
(ESI) m/z 471.2.
N-(6-Chloropyridin-3-yl)-4-[[(2-(propylamino)-6-methylqui-
nazolin-4-yl)amino]methyl]benzamide (73). The intermediate
product 51 was aminated with n-propylamine following procedure D
to yield the final product 73. Starting from 95 (0.1 g, 0.5 mmol), 105 mg
1
(48% yield) of the final product was isolated. HPLC purity 95.9%; H
NMR (DMSO-d6) δ 0.86 (t, J = 6.0 Hz, 3H), 1.55 (m, 2H), 2.34
(s, 3H), 3.24 (m, 2H), 4.77 (d, J = 6.0 Hz, 2H), 7.19 (m, 2H), 7.35 (d,
J = 9.0 Hz, 2H), 7.49 (d, J = 9.0 Hz, 2H), 7.53 (s, 1H), 7.88 (d, J = 6.0
Hz, 2H), 7.95 (d, J = 9.0 Hz, 2H), 8.25 (dd, J = 9.0 Hz, 3.0 Hz, 1H), 8.81
(d, J = 3.0 Hz, 1H), 10.57 (s, 1H); 13C NMR (DMSO-d6) δ 11.9, 21.2,
22.9, 42.9, 45.9, 122.5, 122.8, 124.4, 127.7, 128.2, 129.8, 131.3, 132.8,
134.6, 135.9, 141.9, 144.3, 144.7, 150.2, 159.6, 159.9, 166.2; MS (ESI)
m/z 461.1.
N-(4-Fluorobenzyl)-4-[[(2-(dimethylamino)-6-methylquina-
zolin-4-yl)amino]methyl]benzamide (74). A preparation of N-
(4-fluorobenzyl)-4-[[(2-chloro-6-methylquinazolin-4-yl)amino]methyl]-
benzamide (52) was made starting from 40, 4-fluorobenzylamine, and
95 following procedures B and C. The intermediate product 52 was
aminated with dimethylamine following procedure D to yield the final
product 74. Starting from 95 (0.1 g, 0.5 mmol), 135 mg (63% yield) of
N-(4-Fluorophenyl)-4-[[6-methyl-(2-(propylamino)-
quinazolin-4-yl)amino]methyl]benzamide (70). A mixture of
2-amino-5-methylbenzoic acid (15.1 g, 0.1 mol) and urea (60 g,
1 mol) was heated to 150−180 °C for 5 h. The resulting solid was
then allowed to cool to ∼100 °C, and water (100 mL) was added. The
mixture was then ground and stirred for 20 min. The solid was filtered
out and resuspended in water (200 mL), and NaOH (10 g, 0.25 mol)
was added. The mixture was then heated to 100 °C to give a clear
solution. The solution was acidified to pH ∼ 3 with 12 M HCl, and the
resulting precipitate was collected and dried under reduced pressure to
give 6-methylquinazoline-2,4-dione 94 (15.3 g, 87%) as a tan powder.
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the final product was isolated. HPLC purity 96.3%; 1H NMR (DMSO-
d6) δ 2.33 (s, 3H), 3.02 (s, 6H), 4.43 (d, J = 6.0 Hz, 2H), 4.71 (d, J =
6.0 Hz, 2H), 7.14 (m, 3H), 7.32 (m, 3H), 7.45 (d, J = 9.0 Hz, 2H),
7.83 (d, J = 6.0 Hz, 2H), 7.86 (s, 1H), 8.56 (t, J = 6.0 Hz, 1H), 9.01
(t, J = 6.0 Hz, 1H); 13C NMR (DMSO-d6) δ 21.2, 36.9, 42.3, 44.0,
110.2, 115.2, 115.5, 122.3, 125.1, 127.6, 129.6, 133.0, 134.4, 136.4,
144.1, 150.3, 159.1, 159.5, 162.0, 166.5; MS (ESI) m/z 444.0.
N-(4-Fluorobenzyl)-4-[[(2-(propylamino)-6-methylquinazo-
lin-4-yl)amino]methyl]benzamide (75). The intermediate product
52 was aminated with n-propylamine following procedure D to yield
the final product 75. Starting from 95 (0.1 g, 0.5 mmol), 95 mg (39%
yield) of the final product was isolated. HPLC purity 95.1%; 1H NMR
(DMSO-d6) δ 0.83 (m, 3H), 1.46 (m, 2H), 2.32 (s, 3H), 3.17
(m, 2H), 4.43 (d, J = 6.0 Hz, 2H), 4.73 (d, J = 6.0 Hz, 2H), 6.47 (br s,
1H), 7.13 (m, 3H), 7.33 (m, 3H), 7.43 (d, J = 9.0 Hz, 2H), 7.84 (d, J =
6.0 Hz, 2H), 7.86 (m, 1H), 8.49 (br s, 1H), 9.02 (t, J = 6.0 Hz, 1H);
13C NMR (DMSO-d6) δ 10.1, 21.2, 23.1, 42.3, 42.9, 43.7, 110.4, 115.2,
21.2, 23.0, 34.6, 41.2, 42.9, 43.7, 115.2, 115.5, 122.5, 127.5, 129.6,
130.8, 130.9, 133.4, 134.5, 136.1, 143.5, 158.8, 159.6, 159.9, 162.8,
166.4; MS (ESI) m/z 472.1.
N-(trans-2-Phenylcyclopropyl)-4-[[(2-(dimethylamino)-6-
methylquinazolin-4-yl)amino]methyl]benzamide (80). A prep-
aration of N-(trans-2-phenylcyclopropyl)-4-[[(2-chloro-6-methylqui-
nazolin-4-yl)amino]methyl]benzamide (54) was made starting from
40, trans-2-phenylcyclopropylamine hydrochloride, and 95 following
procedures B and C. The intermediate product 54 was aminated with
dimethylamine following procedure D to yield the final product 80.
Starting from 95 (0.1 g, 0.5 mmol), 140 mg (62% yield) of the final
product was isolated. HPLC purity 96.1%; 1H NMR (DMSO-d6)
δ 1.19 (m, 1H), 1.33 (m, 1H), 2.05 (m, 1H), 2.34 (s, 3H), 3.03 (s, 6H),
4.71 (d, J = 6.0 Hz, 2H), 7.14 (m, 2H), 7.25 (m, 3H), 7.34 (d, J =
9.0 Hz, 1H), 7.44 (d, J = 9.0 Hz, 2H), 7.78 (d, J = 9.0 Hz, 2H), 7.85 (s,
1H), 8.32 (m, 2H); 13C NMR (DMSO-d6) δ 15.7, 21.3, 24.4, 33.6, 37.0,
44.1, 110.2, 122.3, 124.8, 126.0, 126.3, 127.6, 128.6, 129.6, 133.1, 134.4,
141.9, 143.9, 149.9, 158.9, 159.5, 167.6; MS (ESI) m/z 452.1.
N-(trans-2-Phenylcyclopropyl)-4-[[(2-(ethylamino)-6-methyl-
quinazolin-4-yl)amino]methyl]benzamide (81). The intermedi-
ate product 54 was aminated with ethylamine following procedure D
to yield the final product 81. Starting from 95 (0.1 g, 0.5 mmol), 120 mg
115.5, 122.4, 127.6, 129.5, 129.6, 133.0, 134.4, 136.4, 143.9, 149.9,
159.1, 159.9, 162.8, 166.4; MS (ESI) m/z 458.1.
(
)-N-(4-Fluorobenzyl)-4-[[(2-(3-methylpiperazin-1-yl)-
6-methylquinazolin-4-yl)amino]methyl]benzamide (76). The
intermediate product 52 was aminated with ( )-2-methylpiperazine
following procedure D to yield the final product 76. Starting from 95
(0.1 g, 0.5 mmol), 215 mg (87% yield) of the final product was
1
(53% yield) of the final product was isolated. HPLC purity 95.5%; H
NMR (DMSO-d6) δ 1.08 (d, J = 6.0 Hz, 3H), 1.20 (m, 1H), 1.34
(m, 1H), 2.06 (m, 1H), 2.33 (s, 3H), 3.01 (m, 1H), 3.27 (p, J = 6.0 Hz,
2H), 4.73 (d, J = 6.0 Hz, 2H), 6.29 (br s, 1H), 7.15 (m, 4H), 7.26 (m,
2H), 7.43 (d, J = 6.0 Hz, 2H), 7.77 (d, J = 6.0 Hz, 2H), 7.84 (m, 1H),
8.36 (br s, 1H), 8.61 (d, J = 3.0 Hz, 1H); 13C NMR (DMSO-d6) δ 15.6,
15.7, 21.2, 24.4, 33.6, 35.7, 43.6, 111.1, 122.3, 126.0, 126.3, 127.7, 128.0,
128.6, 129.2, 133.1, 134.2, 141.9, 143.9, 150.6, 159.2, 159.9, 167.6; MS
(ESI) m/z 452.1.
N-[1-(4-Fluorobenzoyl)piperidin-4-yl]-4-[[(2-(propylamino)-
6-methylquinazolin-4-yl)amino]methyl]benzamide (82). A
preparation of (4-aminopiperidin-1-yl)(4-fluorophenyl)methanone
(96) was made starting from 4-Boc-aminopiperidine and 4-fluoro-
benzoyl chloride following procedure A. 1H NMR (DMSO-d6) δ 1.67
(br, 4H), 2.92 (m, 4H), 7.24 (m, 2H), 7.41 (m, 2H).
A preparation of N-[1-(4-fluorobenzoyl)piperidin-4-yl]-4-[[(2-
chloro-6-methylquinazolin-4-yl)amino]methyl]benzamide (55) was
made starting from 40, 96, and 95 following procedures B and C.
The intermediate product 55 was aminated with n-propylamine
following procedure D to yield the final product 82. Starting from 95
(0.1 g, 0.5 mmol), 175 mg (64% yield) of the final product was isolated.
HPLC purity 95.8%; 1H NMR (DMSO-d6) δ 0.82 (t, J = 6.0 Hz, 3H),
1.46 (m, 4H), 1.82 (m, 2H), 2.32 (s, 3H), 3.17 (m, 2H), 3.47 (m, 4H),
4.72 (d, J = 3.0 Hz, 2H), 6.33 (br s, 1H), 7.13 (d, J = 6.0 Hz, 1H), 7.26
(m, 3H), 7.42 (d, J = 6.0 Hz, 2H), 7.45 (m, 1H), 7.77 (d, J = 6.0 Hz,
2H), 7.80 (m, 1H), 8.23 (d, J = 6.0 Hz, 1H), 8.34 (br s, 1H); 13C NMR
(DMSO-d6) δ 12.0, 21.2, 23.1, 42.9, 43.6, 46.1, 46.8, 115.7, 116.0, 122.3,
125.0, 127.4, 127.6, 129.0, 129.6, 129.7, 133.0, 133.3, 134.2, 143.9, 150.7,
159.9, 161.3, 164.5, 165.9, 168.6; MS (ESI) m/z 555.2.
1
isolated. HPLC purity 96.9%; H NMR (DMSO-d6) δ 1.01 (d, J =
6.0 Hz, 3H), 2.34 (s, 3H), 2.56 (m, 4H), 2.71 (m, 2H), 2.84 (m, 1H),
4.43 (d, J = 6.0 Hz, 2H), 4.69 (d, J = 6.0 Hz, 2H), 7.14 (m, 3H), 7.32
(m, 3H), 7.43 (d, J = 9.0 Hz, 2H), 7.84 (d, J = 9.0 Hz, 2H), 7.86 (m,
1H), 8.49 (t, J = 6.0 Hz, 1H), 8.95 (t, J = 6.0 Hz, 1H); 13C NMR
(DMSO-d6) δ 12.0, 19.6, 21.2, 42.3, 44.1, 45.6, 50.6, 51.2, 110.7, 115.2,
115.5, 122.2, 125.3, 127.6, 129.6, 133.0, 134.4, 136.4, 144.1, 150.5,
158.4, 159.7, 163.2, 166.4; MS (ESI) m/z 499.1.
N-(4-Fluorophenethyl)-4-[[(2-(methylamino)-6-methylquina-
zolin-4-yl)amino]methyl]benzamide (77). A preparation of N-
(4-fluorophenethyl)-4-[[(2-chloro-6-methylquinazolin-4-yl)amino]methyl]-
benzamide (53) was made starting from 40, 2-(4-fluorophenyl)ethanamine,
and 95 following procedures B and C. The intermediate product 53
was aminated with methylamine following procedure D to yield the
final product 77. Starting from 95 (0.1 g, 0.5 mmol), 185 mg (85%
yield) of the final product was isolated. HPLC purity 96.0%; 1H NMR
(DMSO-d6) δ 2.33 (s, 3H), 2.76 (d, J = 3.0 Hz, 3H), 2.81 (t, J =
6.0 Hz, 2H), 3.44 (q, J = 6.0 Hz, 2H), 4.72 (d, J = 6.0 Hz, 2H), 7.07
(m, 2H), 7.23 (m, 3H), 7.32 (d, J = 9.0 Hz, 1H), 7.42 (d, J = 9.0 Hz,
2H), 7.75 (d, J = 9.0 Hz, 2H), 7.82 (s, 1H), 8.32 (br s, 1H), 8.44 (t, J =
6.0 Hz, 1H); 13C NMR (DMSO-d6) δ 21.2, 28.3, 34.6, 41.2, 43.6,
111.2, 115.2, 115.5, 122.3, 125.0, 127.5, 129.2, 130.9, 133.4, 134.2,
136.1, 143.7, 150.6, 159.8, 162.8, 166.5; MS (ESI) m/z 444.0.
N-(4-Fluorophenethyl)-4-[[(2-(dimethylamino)-6-methylqui-
nazolin-4-yl)amino]methyl]benzamide (78). The intermediate
product 53 was aminated with dimethylamine following procedure
D to yield the final product 78. Starting from 95 (0.1 g, 0.5 mmol),
140 mg (62% yield) of the final product was isolated. HPLC purity
95.5%; 1H NMR (DMSO-d6) δ 2.34 (s, 3H), 2.81 (t, J = 6.0 Hz, 2H),
3.03 (s, 6H), 3.43 (q, J = 6.0 Hz, 2H), 4.71 (d, J = 6.0 Hz, 2H), 7.07
(m, 2H), 7.22 (m, 3H), 7.32 (dd, J = 9.0 Hz, 3.0 Hz, 1H), 7.43 (d, J =
9.0 Hz, 2H), 7.76 (d, J = 9.0 Hz, 2H), 7.84 (s, 1H), 8.44 (t, J = 6.0 Hz,
1H), 8.48 (t, J = 6.0 Hz, 1H); 13C NMR (DMSO-d6) δ 21.2, 34.6,
36.9, 41.2, 44.0, 110.2, 115.2, 115.5, 122.3, 125.2, 127.5, 129.4, 130.9,
133.4, 134.3, 136.1, 143.9, 150.5, 159.2, 159.6, 162.8, 166.5; MS (ESI)
m/z 458.1.
N-[1-(4-Fluorobenzoyl)piperidin-4-yl]-4-[[(2-(allylamino)-6-
methylquinazolin-4-yl)amino]methyl]benzamide (83). The in-
termediate product 55 was aminated with allylamine following
procedure D to yield the final product 83. Starting from 95 (0.1 g,
0.5 mmol), 130 mg (47% yield) of the final product was isolated.
1
HPLC purity 95.1%; H NMR (DMSO-d6) δ 1.49 (br s, 2H), 1.82
(br s, 2H), 2.33 (s, 3H), 3.10 (m, 4H), 3.54 (m, 4H), 4.73 (d, J = 3.0 Hz,
2H), 5.03 (m, 2H), 5.87 (m, 1H), 6.63 (br s, 1H), 7.26 (m, 4H), 7.42
(m, 3H), 7.78 (d, J = 6.0 Hz, 2H), 7.84 (m, 1H), 8.25 (d, J = 6.0 Hz,
1H), 8.50 (br s, 1H); 13C NMR (DMSO-d6) δ 21.2, 31.5, 43.6, 46.8,
114.9, 115.7, 116.0, 122.4, 127.6, 129.7, 133.0, 134.4, 137.2, 143.7,
158.8, 160.0, 161.3, 164.5, 165.9, 168.6; MS (ESI) m/z 553.1.
N-[1-(4-Fluorobenzyl)piperidin-4-yl]-4-[[(2-(methylamino)-
6-methylquinazolin-4-yl)amino]methyl]benzamide (84). A prep-
aration of 1-(4-fluorobenzyl)piperidin-4-amine (97) was made starting
from 4-Boc-aminopiperidine and 4-fluorobenzyl chloride following
N-(4-Fluorophenethyl)-4-[[(2-(propylamino)-6-methylquina-
zolin-4-yl)amino]methyl]benzamide (79). The intermediate prod-
uct 53 was aminated with n-propylamine following procedure D to
yield the final product 79. Starting from 95 (0.1 g, 0.5 mmol), 90 mg
(38% yield) of the final product was isolated. HPLC purity 96.2%; 1H
NMR (DMSO-d6) δ 0.83 (t, J = 6.0 Hz, 3H), 1.45 (m, 2H), 2.33
(s, 3H), 2.83 (t, J = 6.0 Hz, 2H), 3.19 (m, 2H), 3.44 (q, J = 6.0 Hz,
2H), 4.72 (d, J = 6.0 Hz, 2H), 6.52 (br s, 1H), 7.07 (m, 2H), 7.23 (m,
3H), 7.32 (d, J = 9.0 Hz, 1H), 7.42 (d, J = 6.0 Hz, 2H), 7.75 (d, J =
6.0 Hz, 2H), 7.87 (s, 1H), 8.46(br s, 2H); 13C NMR (DMSO-d6) δ 11.9,
1
procedure A. H NMR (DMSO-d6) δ 1.67 (m, 2H), 1.91 (m, 2H),
2.66 (m, 2H), 2.94 (m, 2H), 3.38 (s, 2H), 7.08 (m, 2H), 7.32 (m, 2H).
1356
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Journal of Medicinal Chemistry
Article
A preparation of N-[1-(4-fluorobenzyl)piperidin-4-yl]-4-[[(2-
chloro-6-methylquinazolin-4-yl)amino]methyl]benzamide (56) was
made starting from 40, 97, and 95 following procedures B and C.
The intermediate product 56 was aminated with methylamine
following procedure D to yield the final product 84. Starting from
95 (0.1 g, 0.5 mmol), 210 mg (82% yield) of the final product was
(DMSO-d6) δ 28.9, 36.9, 41.6, 42.2, 44.0, 110.5, 115.2, 115.5, 120.6,
123.1, 125.1, 127.2, 127.7, 129.4, 132.8, 135.0, 136.2, 141.9, 152.0,
159.4, 159.9, 163.1, 169.4, 174.2; MS (ESI) m/z 541.1.
1-[4-[[(2-(Propylamino)quinazolin-4-yl)amino]methyl]-
benzoyl]-N-(4-fluorobenzyl)piperidine-4-carboxamide (89).
The intermediate product 59 was aminated with n-propylamine
following procedure D to yield the final product 89. Starting from 39
(0.1 g, 0.5 mmol), 160 mg (57% yield) of the final product was iso-
lated. HPLC purity 96.0%; 1H NMR (DMSO-d6) δ 0.82 (t, J = 6.0 Hz,
3H), 1.54 (m, 4H), 1.72 (br, 2H), 2.90 (br, 2H), 3.23 (m, 2H), 3.66
(br, 2H), 4.24 (d, J = 6.0 Hz, 2H), 4.75 (s, 2H), 6.45 (br s, 1H), 7.01
(m, 1H), 7.10 (m, 2H), 7.24 (m, 3H), 7.32 (d, J = 6.0 Hz, 2H), 7.42
(d, J = 6.0 Hz, 2H), 7.49 (m, 1H), 8.03 (d, J = 9.0 Hz, 1H), 8.35 (t, J =
6.0 Hz, 1H), 8.44 (br s, 1H); 13C NMR (DMSO-d6) δ 11.9, 22.4, 23.1,
28.8, 41.6, 42.2, 42.9, 111.4, 115.2, 115.5, 119.9, 123.1, 125.0, 127.1,
127.6, 129.4, 132.6, 135.1, 136.2, 141.9, 152.7, 159.9, 160.3, 163.1,
169.5, 174.2; MS (ESI) m/z 555.2.
1-[4-[[(2-(Dimethylamino)-6-methylquinazolin-4-yl)amino]-
methyl]benzoyl]-N-(4-fluorobenzyl)piperidine-4-carboxamide
(90). A preparation of 1-[4-[[(2-chloro-6-methylquinazolin-4-yl)-
amino]methyl]benzoyl]-N-(4-fluorobenzyl)piperidine-4-carboxamide
(60) was made starting from 40, 98, and 95 following procedures B
and C. The intermediate product 60 was aminated with dimethyl-
amine following procedure D to yield the final product 90. Starting
from 95 (0.1 g, 0.5 mmol), 120 mg (43% yield) of the final product
was isolated. HPLC purity 95.4%; 1H NMR (DMSO-d6) δ 1.53 (m, 2H),
1.72 (br, 2H), 2.35 (s, 3H), 2.90 (br, 2H), 3.08 (s, 6H), 3.31 (br s, 2H),
4.22 (d, J = 6.0 Hz, 3H), 4.73 (d, J = 3.0 Hz, 3H), 7.10 (m, 2H), 7.24
(m, 2H), 7.31 (m, 3H), 7.37 (m, 1H), 7.43 (d, J = 9.0 Hz, 2H), 7.91
(s, 1H), 8.35 (t, J = 6.0 Hz, 1H), 8.79 (br s, 1H); 13C NMR (DMSO-d6)
δ 21.2, 28.9, 37.2, 41.6, 42.2, 44.1, 110.1, 115.2, 115.5, 122.6, 123.6,
127.2, 127.8, 129.4, 130.4, 134.8, 135.1, 136.3, 141.6, 159.4, 159.9, 163.1,
169.4, 174.2; MS (ESI) m/z 555.2.
1-[4-[[(2-(Propylamino)-6-methylquinazolin-4-yl)amino]-
methyl]benzoyl]-N-(4-fluorobenzyl)piperidine-4-carboxamide
(91). The intermediate product 60 was aminated with n-propylamine
following procedure D to yield the final product 91. Starting from 95
(0.1 g, 0.5 mmol), 65 mg (22% yield) of the final product was isolated.
HPLC purity 96.0%; 1H NMR (DMSO-d6) δ 0.84 (t, J = 6.0 Hz, 3H),
1.52 (m, 4H), 1.71 (br, 2H), 2.37 (s, 3H), 2.90 (br, 2H), 3.26 (m,
2H), 3.55 (br, 2H), 4.22 (d, J = 6.0 Hz, 2H), 4.78 (d, J = 3.0 Hz, 2H),
7.10 (m, 2H), 7.24 (m, 2H), 7.33 (d, J = 9.0 Hz, 2H), 7.43 (d, J =
9.0 Hz, 2H), 7.53 (m, 1H), 7.71 (br, 1H), 8.11 (br s, 1H), 8.37 (t, J =
6.0 Hz, 1H), 8.44 (br s, 1H); 13C NMR (DMSO-d6) δ 11.6, 21.1, 22.6,
28.8, 41.6, 42.1, 42.9, 111.3, 115.2, 115.5, 119.8, 123.0, 125.1, 127.2,
127.8, 129.4, 132.6, 135.4, 136.3, 141.9, 152.7, 160.0, 160.3, 163.1,
169.3, 174.2; MS (ESI) m/z 569.2.
N-[4-[[(3-Cyano-2-(methylamino)quinolin-4-yl)amino]-
methyl]phenyl]-4-fluorobenzamide (104). Phenyl isocyanate
(11 mL, 0.1 mol) was added to a solution of ethyl cyanoacetate
(10.6 mL, 0.1 mol) and TEA (28 mL, 0.4 mol) in DMF (100 mL).
The reaction mixture was stirred at room temperature for 1 h, and then
the solution was added to ice−water (500 g) and acidified to pH ∼ 4
with 12 M HCl. The resulting solid was collected by suction filtration
and dried under reduced pressure to give ethyl 2-cyano-3-oxo-
3-(phenylamino)propanoate 100 (19 g, 82%), which was suspended in
dichlorobenzene (100 mL), refluxed for 6 h, and then cooled to room
temperature. The resulting solid was collected by suction filtration,
washed once with ethanol, and dried under reduced pressure to give 3-
cyano-2,4-dioxo-tetrahydroquinoline 101 (9.2 g, 61%). MS (ESI) m/z
187.0.
1
isolated. HPLC purity 95.6%; H NMR (DMSO-d6) δ 1.56 (m, 2H),
1.75 (m, 2H), 2.31 (s, 3H), 2.75 (m, 4H), 2.90 (d, J = 3.0 Hz, 3H),
3.41 (s, 2H), 3.73 (m, 1H), 4.71 (d, J = 3.0 Hz, 2H), 6.29 (br s, 1H),
7.14 (m, 4H), 7.28 (d, J = 6.0 Hz, 2H), 7.32 (m, 1H), 7.41 (d, J = 6.0
Hz, 2H), 7.69 (br s, 2H), 7.80 (m, 2H), 8.14 (d, J = 6.0 Hz, 1H), 8.33
(br s, 1H); 13C NMR (DMSO-d6) δ 21.2, 27.9, 28.3, 31.9, 47.3, 52.6,
61.6, 115.1, 115.4, 122.2, 125.0, 127.5, 129.0, 130.9, 133.6, 133.9,
134.2, 135.2, 143.6, 150.2, 160.0, 160.4, 165.9; MS (ESI) m/z 513.2.
N-[1-(4-Fluorobenzyl)piperidin-4-yl]-4-[[(2-(propylamino)-6-
methylquinazolin-4-yl)amino]methyl]benzamide (85). The in-
termediate product 56 was aminated with n-propylamine following
procedure D to yield the final product 85. Starting from 95 (0.1 g, 0.5
mmol), 210 mg (78% yield) of the final product was isolated. HPLC
1
purity 96.0%; H NMR (DMSO-d6) δ 0.84 (t, J = 6.0 Hz, 3H), 1.53
(m, 4H), 1.74 (m, 2H), 2.32 (s, 3H), 2.76 (m, 2H), 3.19 (m, 4H), 3.40
(s, 2H), 3.75 (m, 1H), 4.71 (d, J = 6.0 Hz, 2H), 6.34 (br s, 1H), 7.11
(m, 3H), 7.28 (m, 3H), 7.40 (d, J = 9.0 Hz, 2H), 7.77 (d, J = 9.0 Hz,
2H), 7.80 (m, 2H), 8.14 (d, J = 6.0 Hz, 1H), 8.34 (br s, 1H); 13C
NMR (DMSO-d6) δ 12.1, 21.2, 22.4, 23.1, 32.0, 43.0, 47.3, 52.6, 61.7,
115.1, 115.4, 122.4, 125.1, 127.6, 128.9, 131.0, 133.6, 134.0, 134.2,
135.2, 143.7, 151.0, 160.1, 161.4, 166.1; MS (ESI) m/z 541.3.
N-[1-(4-Fluorobenzyl)piperidin-4-yl]-4-[[(2-(dimethylamino)-
6-methylquinazolin-4-yl)amino]methyl]benzamide (86). The
intermediate product 56 was aminated with dimethylamine following
procedure D to yield the final product 86. Starting from 95 (0.1 g,
0.5 mmol), 150 mg (56% yield) of the final product was isolated.
1
HPLC purity 95.3%; H NMR (DMSO-d6) δ 1.55 (m, 2H), 1.74 (m,
2H), 2.33 (s, 3H), 2.72 (m, 4H), 3.02 (s, 6H), 3.41 (s, 2H), 3.74 (m,
1H), 4.70 (d, J = 6.0 Hz, 2H), 7.10 (m, 2H), 7.18 (m, 1H), 7.30 (m,
3H), 7.42 (d, J = 6.0 Hz, 2H), 7.76 (d, J = 6.0 Hz, 2H), 7.84 (s, 1H),
8.12 (d, J = 6.0 Hz, 1H), 8.48 (t, J = 6.0 Hz, 1H); 13C NMR (DMSO-
d6) δ 21.2, 31.9, 36.9, 44.0, 47.3, 52.5, 61.6, 110.3, 115.1, 115.4, 122.3,
125.2, 127.4, 127.6, 129.4, 130.9, 133.6, 134.3, 135.2, 143.8, 150.6,
159.2, 159.5, 163.2, 166.0; MS (ESI) m/z 527.3.
( )-N-[1-(4-Fluorobenzyl)piperidin-4-yl]-4-[[(2-(3-methylpi-
perazin-1-yl)-6-methylquinazolin-4-yl)amino]methyl]-
benzamide (87). The intermediate product 56 was aminated with
( )-2-methylpiperazine following procedure D to yield the final
product 87. Starting from 95 (0.1 g, 0.5 mmol), 140 mg (48% yield) of
the final product was isolated. HPLC purity 95.1%; 1H NMR (DMSO-
d6) δ 0.95 (d, J = 6.0 Hz, 3H), 1.56 (m, 2H), 1.74 (m, 2H), 2.34
(s, 3H), 2.56 (m, 4H), 2.78 (m, 6H), 3.41 (s, 2H), 3.74 (m, 1H), 4.68
(d, J = 3.0 Hz, 2H), 7.10 (m, 2H), 7.17 (m, 1H), 7.30 (m, 3H), 7.42
(d, J = 9.0 Hz, 2H), 7.76 (d, J = 9.0 Hz, 2H), 7.83 (s, 1H), 8.10 (d, J =
6.0 Hz, 1H), 8.48 (t, J = 6.0 Hz, 1H); 13C NMR (DMSO-d6) δ 19.6,
21.2, 31.9, 44.1, 45.6, 47.3, 50.6, 51.1, 51.6, 52.6, 61.6, 110.7, 115.1,
115.4, 122.2, 125.4, 127.4, 127.6, 129.7, 130.9, 133.6, 134.4, 135.2,
143.7, 150.5, 158.4, 160.0, 163.2, 165.9; MS (ESI) m/z 582.3.
1-[4-[[(2-(Dimethylamino)quinazolin-4-yl)amino]methyl]-
benzoyl]-N-(4-fluorobenzyl)piperidine-4-carboxamide (88). A
prepration of N-(4-fluorobenzyl)piperidine-4-carboxamide (98) was
made starting from 1-Boc-piperidine-4-carboxylic acid and 4-fluoroben-
zylamine following procedure B.
A preparation of 1-[4-[[(2-chloroquinazolin-4-yl)amino]methyl]-
benzoyl]-N-(4-fluorobenzyl)piperidine-4-carboxamide (59) was made
starting from 40, 98, and 39 following procedure B and C. The
intermediate product 59 was aminated with dimethylamine following
procedure D to yield the final product 88. Starting from 39 (0.1 g,
0.5 mmol), 135 mg (49% yield) of the final product was isolated.
Compound 101 (4.0 g, 21 mmol) and PCl5 (3.0 g, 14 mmol) were
added to POCl3 (50 mL), and the reaction suspension was refluxed for
4 h. The solvent was removed, and the residue was added into ice−
water (100 g) and stirred for 1 h. The resulting solid was collected by
suction filtration. The crude product was purified by column
chromatography with hexane/CH2Cl2 to give 3-cyano-2,4-dichlor-
1
HPLC purity 95.9%; H NMR (DMSO-d6) δ 1.53 (m, 2H), 1.72 (br,
2H), 2.90 (br, 2H), 3.06 (s, 6H), 3.35 (br, 2H), 4.23 (d, J = 6.0 Hz,
3H), 4.73 (d, J = 3.0 Hz, 3H), 7.10 (m, 3H), 7.24 (m, 2H), 7.32 (d, J =
9.0 Hz, 2H), 7.43 (d, J = 9.0 Hz, 2H), 7.48 (m, 1H), 8.03 (d, J =
9.0 Hz, 1H), 8.34 (t, J = 6.0 Hz, 1H), 8.59 (t, J = 6.0 Hz, 1H); 13C NMR
1
oquinoline 102 (3.3 g, 70%). H NMR (DMSO-d6) δ 7.92 (m, 1H),
8.09 (br s, 2H), 8.28 (d, J = 9.0 Hz, 1H); MS (ESI) m/z 223.0.
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Journal of Medicinal Chemistry
Article
A preparation of N-[4-[[(3-cyano-2-chloroquinolin-4-yl)amino]-
methyl]phenyl]-4-fluorobenzamide (103) was made from 3 and 102
following procedure C. The intermediate product 103 was aminated
with methylamine following procedure D to yield the final product
104. Starting from 102 (0.11 g, 0.5 mmol), 120 mg (56% yield) of the
final product was isolated. HPLC purity 95.2%; 1H NMR (DMSO-d6)
δ 2.84 (d, J = 6.0 Hz, 3H), 4.98 (d, J = 6.0 Hz, 2H), 6.35 (d, J =
3.0 Hz, 1H), 7.15 (m, 1H), 7.28 (d, J = 9.0 Hz, 2H), 7.34 (s, 1H), 7.39
(m, 1H), 7.51 (m, 1H), 7.70 (d, J = 9.0 Hz, 2H), 7.99 (m, 2H), 8.17
(d, J = 9.0 Hz, 1H), 8.23 (t, J = 6.0 Hz, 1H), 10.24 (s, 1H); 13C NMR
(DMSO-d6) δ 28.7, 46.4, 72.1, 114.9, 115.6, 115.9, 118.9, 120.9, 121.5,
122.1, 127.1, 127.4, 130.8, 132.0, 134.6, 138.3, 148.9, 154.4, 156.9,
162.9, 164.8, 166.1; MS (ESI) m/z 426.1.
N-[4-[[(3-Cyano-2-(propylamino)quinolin-4-yl)amino]-
methyl]phenyl]-4-fluorobenzamide (105). The intermediate
product 103 was aminated with n-propylamine following procedure
D to yield the final product 105. Starting from 102 (0.11 g, 0.5 mmol),
75 mg (33% yield) of the final product was isolated. HPLC purity
95.7%; 1H NMR (DMSO-d6) δ 0.86 (t, J = 6.0 Hz, 3H), 1.52 (m, 2H),
3.03 (m, 2H), 4.98 (d, J = 6.0 Hz, 2H), 6.16 (br s, 1H), 7.15 (m, 1H),
7.32 (d, J = 9.0 Hz, 2H), 7.36 (m, 1H),7.70 (d, J = 9.0 Hz, 2H), 8.01
(m, 2H), 8.21 (m, 2H), 10.25 (s, 1H); 13C NMR (DMSO-d6) δ 11.9,
22.6, 42.9, 46.4, 72.0, 114.9, 115.6, 115.9, 119.0, 121.0, 122.1, 127.4,
127.5, 129.0, 130.8, 132.0, 134.6, 138.4, 146.6, 128.9, 154.4, 156.3,
164.8, 166.1; MS (ESI) m/z 454.1.
N-[4-[[(2-(Methylamino)-5-methylpyrimidin-4-yl)amino]-
methyl]phenyl]-4-fluorobenzamide (111). A preparation of N-[4-
[[(2-chloro-5-methylpyrimidin-4-yl)amino]methyl]phenyl]-4-fluoro-
benzamide (109) was made from 3 and 2,4-dichloro-5-methylpyrimidine
(107) following procedure C. The intermediate product 109 was
aminated with methylamine following procedure D to yield the final
product 111. Starting from 107 (0.16 g, 1 mmol), 76 mg (21% yield)
of the final product was isolated. HPLC purity 96.6%; 1H NMR
(DMSO-d6) δ 1.88 (s, 3H), 2.80 (d, J = 3.0 Hz, 3H), 4.52 (d, J =
6.0 Hz, 2H), 6.18 (m, 1H), 7.30 (d, J = 9.0 Hz, 4H), 7.46 (d, J =
9.0 Hz, 2H), 7.65 (d, J = 9.0 Hz, 2H), 8.03 (d, J = 9.0 Hz, 2H), 10.22
(s, 1H); 13C NMR (DMSO-d6) δ 13.4, 28.3, 43.3, 115.4, 115.7, 120.8,
127.8, 129.7, 130.9, 136.4, 137.9, 152.9, 161.4, 162.1, 164.6, 166.1; MS
(ESI) m/z 366.0.
N-[4-[[(2-(Ethylamino)-5-methylpyrimidin-4-yl)amino]-
methyl]phenyl]-4-fluorobenzamide (112). The intermediate
product 109 was aminated with ethylamine following procedure D
to yield the final product 112. Starting from 107 (0.16 g, 1 mmol),
95 mg (25% yield) of the final product was isolated. HPLC purity
96.0%; 1H NMR (DMSO-d6) δ 1.03 (t, J = 6.0 Hz, 3H), 1.87 (s, 3H),
3.33 (p, J = 6.0 Hz, 2H), 4.51 (d, J = 6.0 Hz, 2H), 6.15 (br s, 1H), 6.99
(br s, 1H), 7.28 (d, J = 9.0 Hz, 2H), 7.35 (m, 2H), 7.46 (m, 2H), 7.67
(d, J = 9.0 Hz, 2H), 8.03 (m, 2H), 10.24 (s, 1H); 13C NMR (DMSO-
d6) δ 13.4, 15.5, 35.8, 45.9, 115.5, 115.8, 120.7, 127.9, 130.8, 131.7,
136.6, 137.8, 153.2, 153.9, 161.4, 164.7, 166.1; MS (ESI) m/z 380.3.
N-[4-[[(5-Bromo-2-(methylamino)pyrimidin-4-yl)amino]-
methyl]phenyl]-4-fluorobenzamide (113). A preparation of N-[4-
[[(5-bromo-2-chloropyrimidin-4-yl)amino]methyl]phenyl]-4-fluoro-
benzamide (110) was made from 3 and 5-bromo-2,4-dichloropyrimidine
(108) following procedure C. The intermediate product 110 was
aminated with methylamine following procedure D to yield the final
product 113. Starting from 108 (0.23 g, 1 mmol), 110 mg (26% yield)
of the final product was isolated. HPLC purity 95.8%; 1H NMR
(DMSO-d6) δ 2.70 (d, J = 3.0 Hz, 3H), 4.51 (d, J = 6.0 Hz, 2H), 6.63
(br s, 1H), 7.33 (m, 4H), 7.68 (d, J = 6.0 Hz, 2H), 7.83 (br s, 1H), 8.01
(m, 2H), 10.22 (s, 1H); 13C NMR (DMSO-d6) δ 28.4, 43.5, 115.5,
115.8, 120.7, 128.1, 130.8, 131.8, 135.9, 138.1, 156.3, 158.2, 161.7, 164.7,
166.1; MS (ESI) m/z 431.9.
At 4 h post-transfection, fresh medium containing various concen-
trations of candidate compounds was added to the cells. Luciferase
activities were determined 24 h after compound addition using a “Firefly
Luciferase Assay Kit” (Biotium, Inc. Hayward, CA). The luciferase
activities were normalized to the activities of β-galactosidase and were
expressed as a percentage of the control luciferase activity.
Colon Carcinoma Cell Growth Inhibition Assay. The colon cell
growth inhibition assay is an in vitro assay that helps to determine if
the lead compounds inhibit the β-catenin/Tcf4 signaling pathway.
Inhibition is inferred by observation of an inhibition of growth of
colon cell lines at concentrations similar to the IC50 in the reporter
assay. A compound of interest was considered inhibitory when growth
inhibition occurred at the concentrations similar to the IC50 in the
reporter assay. The assay readout was based on Promega’s Cell Titer-
blue cell viability assay, which is designed to provide a homogeneous
fluorometric method for estimating the number of viable cells present
in multiwell plates. This homogeneous method is based on the ability
of living cells to convert a redox dye (resazurin) into a fluorescent end
product (resorufin). Viable cells retain the ability to reduce resazurin
into resorufin. Nonviable cells rapidly lose metabolic capacity, do not
reduce the indicator dye, and thus do not generate a fluorescent signal.
Both HCT116 and SW480 cell lines were grown in RPMI 1640 + 10%
FBS + penicillin/streptomycin. 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 compounds for 3 days, at which time the cells were
lysed with Promega’s Cell Titer-blue reagent. The resulting lumine-
scence was read on a Synergy 2 reader.
Clonogenic Assays. HCT116 cells were mixed in RPMI 1640 +
10% FBS + penicillin/streptomycin and plated at 300 cells/well onto
six-well plates, 8 h prior to treatments with or without inhibitors
(1.5 μM highest final concentration). Cells were incubated for 9 days,
and colonies were counted after being fixed with 10% acetic acid in
methanol and stained with 0.05% Crystal violet solution.
CoMFA Calculation. CoMFA study was performed using SYBYL-X
suite software (Tripos, Inc.). The conformation of compound 78
with the lowest free energy from confirmation searching was used as
the template, and the other 18 selected compounds were superposed
with the 4-benzylamine-pyrimidine core. The molecules were placed
into a three-dimension cubic lattice with 0.2 nm spacing. The
minimum-σ (column filtering) was set to 8.36 kJ/mol to improve the
signal-to-noise ratio by omitting those lattice points, whose energy
variation was below this threshold. The method of partial least-squares
(PLS) implemented in the Quantitative Structure−Activity Relation-
ship (QSAR) module of SYBYL was used to construct and validate the
models. The CoMFA descriptors were used as independent variables,
and pIC50 values were used as dependent variables. Each CoMFA
descriptor column of a QSAR Molecular Spreadsheet contains the
magnitudes of the steric (van der Waals) and the electrostatic
(Coulombic) fields exerted by the atoms in the tabulated molecules on
an sp3 carbon with a +1.0 net charge as the probe. Steric and electrostatic
energy cutoffs were set at 125.4 kJ/mol. Cross-validation was performed
with the leave-one-out procedure. The optimum number of the
components, N, retained for final PLS analyses was defined as the one
that yielded the highest crossvalidated q2. The robustness of the models
was internally evaluated by calculating the r2, s, and F test values from the
training set.
AUTHOR INFORMATION
■
Corresponding Author
*Telephone: +1 315 464 7952. Fax: +1 315 464 8041. E-mail:
anji@upstate.edu.
Luciferase Reporter Gene Assay. The HCT116 colorectal
cancer cell line has a deletion mutation in β-catenin (WT/Δ45S) and
thus contains a constitutively activated β-catenin/Tcf4 signaling pathway.
This can be monitored by a luciferase reporter with Tcf4 binding sites in
its promoter. Briefly, 1 × 104 HCT116 cells/well (in 96-well plates) were
cotransfected with 50 ng rof eporter vector TOP-FLASH (containing
Tcf4 binding sites) and 50 ng of pTK-β-gal (served as an internal control).
ACKNOWLEDGMENTS
■
This work was supported by grants from the Connolly
Endowment/Hendricks Fund and the LUNGevity Foundation.
1358
dx.doi.org/10.1021/jm201494a | J. Med. Chem. 2012, 55, 1346−1359

Journal of Medicinal Chemistry
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ABBREVIATIONS USED
■
Tcf4, T-cell transcription factor 4; Wnt, wingless int-1; APC,
adenomatous polyposis coli; Tcf, T-cell factor; LEF1, lymphoid
enhancer-binding factor 1; PK, pharmacokinetic; CoMFA,
comparative molecular field analysis; SAR, structure−activity
relationship; HTS, high-throughput screening; PLS, partial
least-squares; QSAR, quantitative structure−activity relation-
ship; HPLC, high-performance liquid chromatography; NMR,
nuclear magnetic resonance; ESI-MS, electrospray ionization
mass spectrometry; THF, tetrahydrofuran; DMSO, dimethyl
sulfoxide; DIC, N,N′-diisopropylcarbodiimide; HOBt, N-
hydroxybenzotrizole; TFA, trifluoroacetic acid; TEA, triethyl-
amine; DIEA, N,N-diisopropylethylamine
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