Design, synthesis, and DNA-binding of N-alkyl(anilino)quinazoline derivatives

Article abstract of DOI:10.1021/jm1009605

New N-alkylanilinoquinazoline derivatives 5, 12, 20, and 22 have been prepared from 4-chloro-6,7-dimethoxyquinazoline 3, 4-chloro-6,7- methylenedioxyquinazoline 19, and commercially available anilines. Differents classes of compounds substituted by an aryloxygroup (6a-c, 16a,b, and 17a,b), (aminophenyl)ureas (12a,b and 13a-f), anilines (4a-m, 20a,b), N-alkyl(aniline) (5a-m, 21a,b, 22a,d), and N-aminoalkyl(aniline) (22e-g) have been synthesized. These molecules were evaluated for their cytotoxic activities and as potential DNA intercalating agents. We studied the strength and mode of binding to DNA of these molecules by DNA melting temperature measurements, fluorescence emission, and circular dichroism. The results of various spectral and gel electrophoresis techniques obtained with the different compounds, in particular compounds 5g and 22f, revealed significant DNA interaction. These experiments confirm that the N-aminoalkyl(anilino)-6,7-dimethoxyquinazoline nucleus is an efficient pharmacophore to trigger binding to DNA, via an intercalative binding process.

Full text of DOI:10.1021/jm1009605

J. Med. Chem. 2010, 53, 8089–8103 8089
DOI: 10.1021/jm1009605
Design, Synthesis, and DNA-Binding of N-Alkyl(anilino)quinazoline Derivatives
Antonio Garofalo,†,‡ Laurence Goossens,†,‡ Brigitte Baldeyrou,†,§, Amelie Lemoine, Severine Ravez,†,‡ Perrine Six,†,‡
†,‡
ꢀ
ꢀ
†,§,
Marie-Helene David-Cordonnier,†,§ Jean-Paul Bonte,†,‡ Patrick Depreux,†,‡ Amelie Lansiaux,
and
ꢀ ꢁ
Jean-Franc-ois Goossens*,†,^
ꢀ
†Univ Lille Nord de France, F-59000 Lille, France, ‡UDSL, ICPAL, EA 4481, F-59006 Lille, France, §INSERM U-837, IRCL,
F-59045 Lille, France, Centre Oscar Lambret, IRCL, IMPRT, F-59045 Lille, France, and ^UDSL, UFR de Pharmacie, EA 4481,
F-59006 Lille, France
Received July 28, 2010
New N-alkylanilinoquinazoline derivatives 5, 12, 20, and 22 have been prepared from 4-chloro-6,
7-dimethoxyquinazoline 3, 4-chloro-6,7-methylenedioxyquinazoline 19, and commercially available
anilines. Differents classes of compounds substituted by an aryloxygroup (6a-c, 16a,b, and 17a,b),
(aminophenyl)ureas (12a,b and 13a-f), anilines (4a-m, 20a,b), N-alkyl(aniline) (5a-m, 21a,b, 22a,d),
and N-aminoalkyl(aniline) (22e-g) have been synthesized. These molecules were evaluated for their
cytotoxic activities and as potential DNA intercalating agents. We studied the strength and mode of
binding to DNA of these molecules by DNA melting temperature measurements, fluorescence emission,
and circular dichroism. The results of various spectral and gel electrophoresis techniques obtained with
the different compounds, in particular compounds 5g and 22f, revealed significant DNA interaction.
These experiments confirm that the N-aminoalkyl(anilino)-6,7-dimethoxyquinazoline nucleus is an
efficient pharmacophore to trigger binding to DNA, via an intercalative binding process.
1. Introduction
tumor cell lines.12,13 In contrast, its N-methyl analogue 1b
retains good antiproliferative activities despite its total ab-
sence of EGFR TK inhibitory effect.14 Many studies have
been performed to define the strength and mode of DNA
binding of both products, and the results have proved that 1b
binds to DNA as a typical intercalating agent. Therefore, this
study demonstrated that the methylation of the anilino nitro-
gen of 1a prevents binding to the EGFR TK but improves the
interaction of the drug with DNA and confers a selectivity for
GC-rich sequences. The addition of the N-methyl substituent
on the anilinoquinazoline nucleus increased significantly the
affinity of the ligand for specific sequences in DNA. The
reduction of the conformational flexibility of this molecule
maintains the anilinoquinazoline chromophore in a specific
conformation favorable for the DNA intercalation and G-C
base pairs recognition.12
The anilinoquinazoline skeleton targets DNA, and its
N-methylated derivative represents a useful chemotype for
the development of new DNA-targeted anticancer agent.
These initial results prompted us to design new quinazoline
derivatives and investigate their biological properties. Here,
we report the synthesis, biological evaluation, and DNA
binding of 55 new compounds. The interaction with DNA
was studied and quantified by melting temperature, fluores-
cence, and circular dichroism experiments. The effects of the
molecules on DNA topoisomerase I activities were evaluated.
The cytotoxicity of the most active compounds was evaluated
using three human tumor cell lines.
Quinazoline-containing derivatives form an important
class of synthetic products and represent an attractive scaffold
for designing anticancer drugs. They have attracted interest
over the past years because of their varied biological activity,
notably as kinase inhibitors.1-4 The 4-anilinoquinazoline
scaffold has led to the development and the marketing of
new series of antitumor agents such as gefitinib, erlotinib, and
lapatinib.
In recent years several DNA-targeting agents consisting of
a polycyclic planar moiety, such as quinoline, quinazoline, or
indole linked to an alkyl or aniline, have been shown to be
more active than kinase inhibitors.5-9 These compounds
interact with double stranded DNA and also present selective
recognition of DNA sequences. They can be classified into
two major categories, intercalating and minor groove bind-
ers.10 Previously, our group has documented the DNA in-
teraction capacity of the tyrosine protein kinase inhibitor 1a
(PD153035)11 and its N-methyl analogue 1b (EBE-A22)12
(Figure 1).
The brominated anilinoquinazoline derivative 1a also ex-
hibits a very high affinity and selectivity for the epidermal
growth factor receptor tyrosine kinase (EGFR TKa) and
presents a remarkable cytotoxicity against several types of
*To whom correspondence should be addressed. Phone: þ33 (0)3 20
96 40 21. Fax: þ33 (0)3 20 96 49 06. E-mail: jean-francois.goossens@
univ-lille2.fr.
a Abbreviations: ATP, adenosine 50-triphosphate; CD, circular di-
chroism; DM, N,N-dimethylformamide; DMSO, dimethylsulfoxide;
EGFR TK, epidermal growth factor receptor tyrosine kinase; EtOAc,
ethyl acetate; EtOH, ethanol; FC, flash chromatography; MeOH,
methanol; rt, room temperature; sc, supercoiled DNA ; Tm, melting
temperature; TLC, thick-layer chromatography.
2. Chemistry
We have synthesized two series of new quinazolin com-
pounds. These products form two groups distinguished by the
r
2010 American Chemical Society
Published on Web 10/29/2010
pubs.acs.org/jmc

8090 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Garofalo et al.
nature of the ether linker at the C-6 and C-7 positions of the
quinazoline core: 6,7-dimethoxy- (4, 5, 6, 12, 13, 16, 17, 22)
and 6,7-methylenedioxyquinazoline (20, 21) (Figure 2).
The synthesis of the 6,7-dimethoxyquinazoline derivatives
is illustrated in Schemes 1-3. The key intermediate 4-chloro-
6,7-dimethoxyquinazoline 3 was prepared according to de-
scribed procedures15 using methyl 2-amino-4,5-dimethoxy-
benzoate as a starting material. Compound 3 engaged in a
nucleophilic substitution reaction in the presence of various
commercial anilines in 2-propanol to obtain the final products
4a-m. Compounds 5a-m were synthesized by methylation
reaction in the presence of iodomethane in N,N-dimethylfor-
mamide from 4a-m with low yield (20-40%). Compound 3
on reaction with phenol derivatives by irradiation at 150 ꢀC in
DMSO gave compounds 6a-c in good yields (Scheme 1).
As depicted in Scheme 2, compounds 8 and 9 were prepared
differently: the intermediate 8 was synthesized using commer-
cial N-methyl-4-nitroaniline, and compound 9 was prepared
in a two-step sequence by nucleophilic substitutionof3, giving
anilinoquinazoline 7 that was finally N-methylated. Catalytic
hydrogenation of the nitro group led to amino derivatives 10
and 11 (87-92% of yields) which were converted by con-
densation with commercial isocyanate in THF/H2O mixture,
in the presence of pyridine, to the corresponding ureas 12a,
b and 13a-f.
Selective reaction of chloride derivative 3 with m- or
p-aminophenol and tetra-n-butylammonium bromide in a
mixture of methyl ethyl ketone and sodium hydroxide pro-
vided the intermediates 1415 and 15 which by condensation
with the corresponding isocyanate in chloroform afforded the
desired ureas 16a,b15 and 17a,b in high yields and short time
reactions (Scheme 3).
Figure 1. Structure of 1a and its N-methyl analogue 1b.
Figure 2. Series of compounds synthesized from 4-chloro-6,7-dimethoxyquinazoline 3 or 4-chloro-6,7-methylenedioxyquinazoline 19.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8091
Scheme 1a
a Reagents and conditions: (a) NaOMe, HCONH2, DMF/MeOH, 110 ꢀC, 85%; (b) POCl3, reflux, 92%; (c) aniline derivatives, (CH3)2CHOH, reflux,
80-95%; (d) CH3I, NaH, DMF, room temperature, 25-40%; (e) phenol derivatives, NaH, DMSO, 150 ꢀC, 100 W, microwave, 59-65%.
Scheme 2a
a Reagents and conditions: (a) N-methyl-4-nitroaniline, (CH3)2CHOH, reflux, 47%; (b) 3-nitroaniline, (CH3)2CHOH, reflux, 92%; (c) CH3I, NaH,
DMF, room temperature, 77%; (d) Raney Ni, H2, MeOH, room temperature, 87% for 10 and 92% for 11; (e) isocyanate derivatives, C5H5N, H2O/THF,
room temperature, 27-65%.
Scheme 3a
a Reagents and conditions: (a) n-Bu4NþBr-, 2-butanone, 20% NaOH, reflux, 80% for 14 (with 4-nitrophenol) and 93% for 15 (with 3-nitrophenol);
(b) isocyanate derivatives, NEt3, CHCl3, room temperature, 25-60%.
The synthesis of aniline derivatives 20a,band 21a,bis outlined
in Scheme 4. For the synthesis of 4-chloro-6,7-methylenedioxy-
quinazoline 19, we employed a synthetic route according to
described procedures16 using piperonilic acid 18 as a starting

8092 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Garofalo et al.
Scheme 4a
a Reagents and conditions: (a) (i) SOCl2, MeOH, reflux, 70%; (ii) HNO3, SnCl4, CH2Cl2, -70 ꢀC, 67%; (iii) Raney Ni, H2, MeOH, room temperature,
70%; (iv) NaOMe, HCONH2, DMF/MeOH, 110 ꢀC, 84%; (v) POCl3, reflux, 94%; (b) aniline derivatives, (CH3)2CHOH, reflux, 82-91%; (c) CH3I,
NaH, DMF, room temperature, 38-47%.
Scheme 5a
a Reagents and conditions: (a) R-I or R-Br or R-Cl, NaH, DMF, room temperature, 18-25%.
Drug-DNAcomplex
material. Obtention of the final 4-anilino-6,7-methylenedioxy-
quinazoline derivatives 20a,b and 21a,b was performed accord-
ing to the same synthetic strategy: nucleophilic displacement of
the chlorine atom with various arylamino groups in 2--propanol
gave 20a,b, and N-methylation reaction of these compounds
provided 21a,b.
(ΔTm = Tm
Table 1 using a drug/DNA base pair ratio of 1.
- TmDNAalone) are presented in
ΔTm values for 1b are 8.1 and 12.2 ꢀC for ctDNA and
poly(dAdT)2, respectively. These results are in agreement
with our previous data. For the anilino (4b-m, 20a,b) and
aryloxy (6a-c, 16a,b, 17a,b) series, the low ΔTm values (<5
ꢀC) indicate that these compounds have a weak DNA
affinity. In the N-methyl analogue series (5a-m, 13a-f,
21a,b), the values range from 1.4 to 8.9 ꢀC for ctDNA and
from 3.3 to 15.0 ꢀC for poly(dAdT)2 (except for 12a,b). This
observation indicates that the substitution by a methyl group
leads to compounds that stabilize DNA against heat dena-
turation. The 4-anilino-6,7-methylenedioxyquinazoline ske-
leton (series B), as in 21a,b, is not adapted to stabilize the
DNA duplex structure (ΔTm < 5 ꢀC) unlike the 6,7-di-
methoxyquinazoline core (0 ꢀC < ΔTm< 15 ꢀC) (series A).
Very interesting data were obtained with meta-position of
halogens on the aniline ring. We demonstrated that this
meta-position is more favorable than the para-position.
Bisubstitution by halogens and methyl group (5g-j) was
characterized by the best ΔTm values especially for bromide
(5g) and chloride (5h) with ΔTm values for 5g of 8.4 and 15 ꢀC
for ctDNA and poly(dAdT)2, respectively (Table 1). A larger
ΔTm value was observed for 5g compared to 1b. Then the
bisubstitution was considered as a positive element to re-
inforce DNA interaction. In addition, no significative differ-
ences of ΔTm values were observed by replacement of bromine
From the bromide derivatives 4g, a series of compounds was
synthesized by modulation of the N-methyl group by alkyl-
(22a-d) or cationic side chain (22e-g) to explore putative
additional interaction site (Scheme 5).
3. Results and Discussion
3.1. DNA-Binding Studies. A previous study performed
with 1b had demonstrated the occurrence of an intercalative
binding process upon complex formation with DNA.12
Similarly, we studied the capacity of the new quinazoline
derivatives to bind to the DNA macromolecule. We engaged
different methods to study the binding mode to DNA for the
newly synthesized compounds, with the aim to characterize
the DNA-binding process and measure the affinity of the
compounds for DNA.
The ability of the drugs to protect calf thymus DNA
(ctDNA) or the synthetic AT polynucleotide (poly (dAdT)2)
against thermal denaturation was used as an indicator of
their capacity to bind DNA and to stabilize the DNA double
strand. The variations of the temperature melting (Tm) values

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8093
a
Table 1.
ΔTm (in ꢀC) drug/DNA ratio in BPE buffer
compd
series
X
R
ctDNA
poly(dA-dT)2
1a
1b
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
NH
3-Br
3-Br
0.2
8.1
0.4
12.2
N-CH3
NH
NH
4a (AG14035)
4b
4c
3-Cl
4-Br
1.8
3.9
0.3
0.2
NH
NH
3-F
3-CF3
0.2
0.2
0.4
0.5
4d
4e
NH
NH
4-CF3
3-Me
0.1
0.3
0.5
0.3
4f
4g
NH
NH
3-Br-4-Me
3-Cl-4-Me
3-F-4-Me
3-CF3-4-Me
2,5-Br
0.2
0.3
0.4
0.4
4h
4i
NH
NH
0.2
0.4
0.5
0.5
4j
4k
NH
NH
0.4
0.2
0.5
0.4
4l
3-Cl-4-F
4-Br-2-F
3-Cl
4m
5a
NH
0.1
0.3
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
O
7.7 ( 0.6
6.0 ( 0.8
7.3 ( 0.6
5.8 ( 0.5
6.5 ( 0.6
7.1 ( 0.7
8.4 ( 0.6
8.9 ( 0.5
7.3 ( 0.5
7.8 ( 0.6
1.6
12.4 ( 0.7
10.8 ( 0.6
9.8 ( 0.8
9.5 ( 0.5
9.1 ( 0.6
9.7 ( 0.7
15.0 ( 0.8
13.7 ( 0.6
11.3 ( 0.5
11.0 ( 0.6
3.3
5b
5c
4-Br
3-F
5d
5e
3-CF3
4-CF3
3-Me
5f
5g
3-Br-4-Me
3-Cl-4-Me
3-F-4-Me
3-CF3-4-Me
2,5-Br2
3-Cl-4-F
4-Br-2-F
3-Br
5h
5i
5j
5k
5l
6.1 ( 0.6
1.4
0.7
10.5 ( 0.7
3.3
0.7
5m
6a
6b
6c
O
3-Cl-4-F
4-Br-2-F
1.2
0.8
0.5
0.5
O
12a
12b
13a
13b
13c
13d
13e
13f
16a
16b
17a
17b
20a
20b
21a
21b
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
O
p-NHCONH-(phenyl)
p-NHCONH-(butyl)
m-NHCONH-(phenyl)
m-NHCONH-(4-methoxyphenyl)
m-NHCONH-(3-chloro-4-fluorophenyl)
m-NHCONH-(ethyl)
m-NHCONH-(butyl)
m-NHCONH-(cyclohexyl)
p-NHCONH-(phenyl)
p-NHCONH-(4-methoxyphenyl)
m-NHCONH-(phenyl)
m-NHCONH-(4-methoxyphenyl)
3-Cl-4-F
0.4
0.3
0.6
0.4
6.0 ( 0.5
6.8 ( 0.5
7.7 ( 0.6
5.2 ( 0.6
5.7 ( 0.5
5.7 ( 0.7
0.4
9.9 ( 0.8
11.0 ( 0.6
10.5 ( 0.8
7.9 ( 0.6
8.1 ( 0.7
9.7 ( 0.6
0.5
O
0.3
0.4
0.5
0.5
O
O
NH
0.2
0.3
0.3
0.4
B
NH
N-CH3
N-CH3
4-Br-2-F
3-Cl-4-F
4-Br-2-F
0.3
2.8
0.5
4.6
B
B
3.6
4.2
a Means values are calculated from at least two separate experiments with typical standard deviations of less than (10%.
by chlorine (5a) on the aryl ring, whereas the introduction of F,
CH3, or CF3 apparently reduces the DNA interaction capacity.
Introduction of a bulky substituent (12a,b, 13a-f) de-
creases DNA interaction compared to halogen derivatives.
In this series, the meta-position is also favorable when a urea
group is introduced.
If we consider the compound 5g in series A, its extent of
interaction with DNA is similar to the reference compound as
judged from the Tm measurements. To increase the DNA
interaction, news compounds were synthesized bearing a
N-alkyl or a cationic side chain. The addition of the N-alkyl
substituent (ethyl, propyl, isopropyl) on the anilinoquinazoline

8094 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Garofalo et al.
a
Table 2.
DNA, an isosbestic point at 362 nm was detected with the
different compounds pointing to the existence of a single
binding mode.
To confirm these results and to define more precisely the
binding process, we deployed a spectroscopic method utilizing
polarized light with circular dichroism measurements. Solutions
of DNA in the presence of drugs 5g and 22f were analyzed by
circular dichroism (CD) and compared with reference com-
pounds (1a and 1b). Circular dichroism measurements showed
that a negative band is centered at 350-370 nm with 1b, 5g, and
22f (Figure 4). Such a typical negative CD in the absorption
band of the ligand is consistent with an intercalative DNA
binding mode for these compounds. Under identical conditions
with a low-salt medium (1 mM sodium cacodylate buffer, pH
7.0), the reduced dichroism of 1a showed a 10 nm blue-shifted
negative band. In addition, for 5g, a decrease of the CD band at
330-340 nm is concomitant to an increase of the CD amplitude
at 350-370 nm, with a relatively well resolved isodichroic
crossover. This type of CD behavior may be assigned to
excitonic coupling between adjacent intercalated molecules.
This binding mode was observed with other intercalative
agents.17,18 At low binding ratios, a weak monomeric CD with
the same shape as the absorption curve is observed, while at
higher binding ratios, a strong exciton CD dominates because of
interaction between pairs of molecules. For all tested com-
pounds, a weak negative induced CD was observed, and this
is consistent with an intercalative mode of binding to DNA, in
particular for compound 5g.
3.2. Fluorescence Measurements. Binding affinities for
compounds 1b, 5g, and 22f were quantified by means of
intrinsic fluorescence of the compounds (Table 3). The
fluorescence emission at 436 nm is weak when the drug is
free in solution, but it is significantly enhanced when the drug
is bound to DNA. The intrinsic fluorescence variation
induced by DNA titration allows the determination of the
apparent binding (KD) constant using nonlinear least-
squares analysis (GraphPad Prism software).
The data summarized in Table 3 attest that DNA ligand 5g
is a potent DNA binder, with an apparent binding constant
of the same order as that of the reference 1b. The binding
affinity of 22f is stronger compared to the KD value calcu-
lated for 1b. This molecule provides better stabilization of
the DNA complex, as mentioned above using the Tm mea-
surements. Compound 22f displayed the highest ΔTm value
and the lowest KD value, both indicating a marked duplex
stabilization and a good affinity for DNA. These results led
to the assumption that the introduction of cationic side chain
triggers an additional mode of binding implicating the
phosphate groups of DNA. In the case of 1a, the changes
of the fluorescence emission at 420 nm were very weak.
Under such conditions, attempts to estimate the binding
constant are generally doomed to failure.
3.3. DNA Sequence Selectivity. Sequence-specific DNA
binding properties were determined by the DNase I foot-
printing methodology, using a radiolabeled 265-bp DNA
restriction fragment as a substrate. A complete set of
typical autoradiographs of the sequencing gels used to
fractionate the products of partial digestion of DNA
fragment complexed with the tested compounds is pre-
sented in Figure 5. These quinazoline derivatives strongly
affected the cleavage of the DNA substrates by the nucle-
ase. Visual inspections of the gels suffice to conclude that
the synthesized compounds are sequence-selective bind-
ers, similar to 1b.
a Means values are calculated from three separate experiments.
nucleus (compounds 22a-d) did not improve the DNA inter-
action. However, the introduction of a cationic side chain
(compounds 22e-g) was found to strongly stabilize DNA
(ΔTm from 9.9 to 14 ꢀC (ctDNA) and from 20 to 27.7 ꢀC for
poly(dAdT)2) when compared with the N-methyl analogue 5g
(Table 2). This is most likely attributable to additional electro-
static interactions with DNA phosphodiesters groups.
The binding of four compounds, 1a, 1b, 5g, and 22f was
studied by different methods to evaluate the spectroscopic
changes of anilinoquinazoline chromophore (UV and CD
experiments) or of DNA double helix (ΔTm experiment).
Figure 3 displays UV absorption measurements recorded
upon ctDNA titration into a buffered aqueous solution of
drugs. In both cases, the addition of DNA induces marked
changes of the absorption spectra of 1a, 1b, 5g, and 22f. The
binding to DNA remains more or less unchanged. In the
medium-salt buffer, strong bathochromic and hypochromic
shifts were observed with the three drugs with an absorption
maximum red-shifted from 346 to 367 nm and a 2-fold
decrease absorbance at 346 nm. During the titration with

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8095
Figure 3. UV measurements. CT-DNA titrations of 1a, 1b, 5g, and 22f. The phosphate-DNA/drug ratio increased from 0 to 10 (top to bottom
curves, λ = 346 nm).
A densiometry analysis of the autoradiograph obtained
with the 265 bp fragments from plasmid pBS is presented in
Figure 6. In the presence of drugs, several regions of atten-
uated DNA cleavage can be discerned around positions 43,
71, and 91. All footprintings coincide with the position of
nucleotide sequences with a high GC content. These regions
of drug-induced enhanced cleavage all correspond to AT-
rich sequence, which is attributable to intercalation-induced
perturbations of the double-helical structure of DNA. Plots
show that compounds interact preferentially with sequences
essentially composed of two or more consecutive G-C base
pairs. Like most intercalating drugs, anilinoquinazoline
derivatives strongly discriminate between runs of adenines
or thymines.
indicated no stabilization of the covalent complexes between
DNA and topoisomerase I or II (data not shown).
3.5. In Vitro Antiproliferative Activity. The antiprolifera-
tive activities of the compounds were tested (Table 4) using
three cancer cell lines: PC3 (hormono-independent prostate
cancer), HT-29 (colon cancer), and MCF-7 (breast cancer).
Introduction of a methyl group on the aniline core (5g)
induces a detectable antiproliferative effect more or less iden-
tical at 1b (6.1 and 9.2 μM on HT-29 and MCF-7 cancers
cells, respectively). The data indicate that molecules contain-
ing a cationic side chain exert an antiproliferative effect on
the cell lines tested here, with IC50 values in the micromolar
range. Compound 22f emerges as the most cytotoxic agent in
this series (IC50 = 5.6 μM in HT-29 cell line). 1b has an
inhibitory effect less pronounced than that of the aminoalkyl
derivatives. The introduction of a cationic side chain tends to
favor the antiproliferative activity.
3.4. Topoisomerase I Inhibition. A conventional DNA
relaxation assay was used to access the effects of the com-
pounds on the catalytic activity of human topoisomerase I.
This enzyme introduces transient nicks in DNA at specific
sites, leading to relaxation of the DNA helix. Incubation
of the DNA-topoisomerase I mixture in the absence and
in the presence of the test drugs results in different distribu-
tion of topoisomers which can be revealed by agarose gel
electrophoresis.19 Typical gels are presented in Figure 7. 1a
has practically no effect on the unwinding of DNA mediated
by human topoisomerase I. In contrast, other products, in
particular 5g, induce a shift of the topoisomer distribution,
indicating that these compounds affect the superhelical
density of the plasmid. These topoisomerization assays thus
reveal that these compounds unwind closed circular duplex
DNA. These effects are typical of intercalating agents. These
results suggest also that none of these molecules act as
a poison for human topoisomerase. Cleavage experiments
4. Conclusion
Different conjugated structures were obtained by linking
the planar quinazoline core with different aniline groups.
Previous studies on DNA interaction of the tyrosine protein
kinase inhibitor 1a and its N-methyl analogue 1b led to
synthesis of new molecules which were evaluated as cytotoxic
and DNA intercalating agents. It was shown that the meth-
ylation of the anilino nitrogen of quinazoline derivatives is
essential to obtain an intercalating effect. This conformation
must facilitate the interaction of the drug with DNA and
confers sequence selectivity, compared to the corresponding
anilino or aryloxy derivatives. The affinity of the N-meth-
ylated anilinoquinazoline derivatives for DNA is relatively
weak compared to classical potent intercalators such ethidium

8096 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Garofalo et al.
Figure 4. Circular dichroism measurements. CT-DNA titrations of 1a, 1b, 5g, and 22f. The DNA/drug ratio increased as follows from ratio
0 to 4.
bromide, but nevertheless, the DNA-binding studies con-
firm that the quinazoline core is a suitable carrier, allowing
an effective DNA intercalative process. In this study, an
additional cationic side chain was introduced on the anilino
group substituting the quinazoline core. The designed mole-
cules generally show an increased DNA interaction consis-
tent with the formation of additional electrostatic contacts
between the drug and the DNA receptor. Interestingly,
a satisfactory correlation was observed between the extent
of DNA interaction and the antiproliferative activity. Novel
quinazoline molecules with a superior bioactivity profile
compared to the reference compound 1b have been described,
and the objectiveis thusfullyreached. Further developmentof
these series should include another heterocycle as quinolein or
acridine for more systematic structure-activity studies.
The synthesis of many compounds has been reported: 1b14
and 16a,b.15
5.1.1. 4-Chloro-6,7-dimethoxyquinazoline (3).15 To a solution
of methyl-2-amino-4,5-dimethoxybenzoate (4.0 g, 19.0 mmol) in
N,N-dimethylformamide (40 mL) and methanol (10 mL), for-
mamide (76.0 mmol) and sodium methoxide (54.0 mmol) were
added. The resulting mixture was refluxed for 16 h. After the
reaction was quenched by water (100 mL), the mixture was
neutralized by 1 M HCl solution. The precipitated was collected
by filtration, washed with H2O (30 mL) and Et2O (30 mL), and
dried in vacuo to give quinazolinone 2 as a white solid (82%)
which used directly in the next step. A mixture of 2 (3.0 g,
15.0 mmol) and phosphorus oxychloride (30 mL) was refluxed
for 2 h. After removal of the solvent, the residue was dissolved in
ice-water (50 mL) and the mixture was neutralized by ammo-
nium hydroxide. The precipitate was collected by filtration and
dissolved in CH2Cl2 (100 mL). The organic layer was washed
with a 1 M solution of K2CO3 (3 ꢀ 40 mL), brine (1 ꢀ 40 mL)
and dried over CaCl2, and the solvent was removed under
reduced pressure. Spectroscopic data for compound 3 are in
agreement with the data reported in the literature.15
5. Experimental section
€
5.1. Chemistry. Melting points were determined with a Buchi
535 capillary melting point apparatus and are uncorrected.
Kieselgel 60 F-254 commercial plates were used for analytical
TLC as well as UV light and/or with iodine to follow the course
of the reaction. Flash chromatography (FC) was performed
with silica gel Kieselgel Si 60, 0.063-0.200 mm (Merck). The
structure of each compound was confirmed by IR (Bruker
VECTOR 22 instrument) and by 1H NMR (300 MHz, Bruker
AC300P spectrometer). Chemicals shifts (δ) are reported in ppm
downfield from TMS. J values are in hertz, and the splitting
patterns are designed as follows: s, singlet; d, doublet; t, triplet;
q, quartet; m, multiplet. APCIþ (atmospheric pressure chemical
ionization) mass spectra were obtained on an LC-MS system
Thermo Electon Surveyor MSQ. Purity of the tested com-
pounds was >95% using interpretation of a combination of
LC-MS and NMR data.
5.1.2. General Procedure for Nucleophilic Substitution by
Aniline (4a-m). 4-Chloro-6,7-dimethoxyquinazoline 3 (0.30 g,
1.34 mmol) was dissolved in refluxing 2-propanol (15 mL), and
commercial aniline (1.60 mmol) was added dropwise. After 3 h,
the precipitate was filtered off and washed with 2-propanol
(10 mL) and Et2O (10 mL). Spectroscopic data and melting
point for compounds 4a (AG14035),20 4b,21 4c,21 4d,20 4f,22 and
4l23 are in agreement with those reported in the literature.
4-(4-Trifluoromethylanilino)-6,7-dimethoxyquinazoline Hydro-
chloride (4e). Crystallization from N,N-Dimethylformamide gave
pure 4e asawhitesolid(91%). Mp>250ꢀC. IR cm-1 2467 (NHþ),
1
1320 (CF3), 1076 (C-O-C methoxy). H NMR (DMSO-d6) δ
ppm 11.72 (s, 1H, NHþ), 8.85 (s, 1H, ArH), 8.47 (s, 1H, ArH), 8.03
(d, J = 8.40 Hz, 2H, ArH), 7.82 (d, J = 8.40 Hz, 2H, ArH), 7.40 (s,

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8097
a
Table 3.
a The values are averages of three independent experiments. b ND: not
determined. The weak fluorescence properties of 1a could not allow us to
determine an apparent binding constant.
1H, ArH), 4.01 (s, 3H, OCH3), 3.97 (s, 3H, OCH3). LC-MS
(APCIþ), calcd for C17H14F3N3O2, m/z: 350 (M þ H)þ.
4-(3-Bromo-4-methylanilino)-6,7-dimethoxyquinazoline Hy-
drochloride (4g). Crystallization from ethanol 95% gave pure
4g as a white solid (95%). Mp >250 ꢀC. IR cm-1 2465 (NHþ),
1076 (C-O-C methoxy), 1056 (C-Br). 1H NMR (DMSO-d6)
δ ppm 11.42 (s, 1H, NHþ), 8.85 (s, 1H, ArH), 8.32 (s, 1H, ArH),
8.02 (s, 1H, ArH), 7.69 (d, J = 7.40 Hz, 1H, ArH), 7.42 (d, J =
7.40 Hz, 1H, ArH), 7.31 (s, 1H, ArH), 4.00 (s, 3H, OCH3), 3.98
(s, 3H, OCH3), 2.37 (s, 3H, CH3). LC-MS (APCIþ), calcd for
C17H16BrN3O2, m/z: 374 [(M þ H)þ for 79Br] and 376 [(M þ H)þ
for 81Br].
Figure 5. DNase I footprinting assay. Radiolabeled 265 bp DNA
fragment was incubated alone (lane “DNA”) or graded concentra-
tions of the various compounds from 0 to 100 μM (as indicated at
the top of each lane). The G track lanes labeled “G” were used as
markers for guanines to locate the footprint areas and to establish
the scale indicated from 40 to 120 bp. Vertical black lines localize the
sites protected on the gel.
8.41 (s, 1H, ArH), 7.88 (d, J = 2.30 Hz, 1H, ArH), 7.45 (m, 2H,
ArH), 7.34 (s, 1H, ArH), 4.01 (s, 3H, OCH3), 3.97 (s, 3H,
OCH3), 2.34 (s, 3H, CH3). LC-MS (APCIþ), calcd for
C18H16F3N3O2, m/z: 364 (M þ H)þ.
4-(3-Chloro-4-methylanilino)-6,7-dimethoxyquinazoline Hy-
drochloride (4h). Crystallization from N,N-dimethylformamide
gave pure 4h as a white solid (87%). Mp >250 ꢀC. IR cm-1 2466
(NHþ), 1088 (C-Cl), 1078 (C-O-C methoxy). 1H NMR
(DMSO-d6) δ ppm 11.51 (s, 1H, NHþ), 8.82 (s, 1H, ArH),
8.38 (s, 1H, ArH), 7.88 (d, J = 2.10 Hz, 1H, ArH), 7.68 (dd, J =
2.10, 8.50 Hz, 1H, ArH), 7.45 (d, J = 8.50 Hz, 1H, ArH), 7.32 (s,
1H, ArH), 4.02 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 2.35 (s, 3H,
CH3). LC-MS (APCIþ), calcd for C17H16ClN3O2, m/z: 330 [(M
þ H)þ for 35Cl] and 332 [(M þ H)þ for 37Cl].
4-(2,5-Dibromoanilino)-6,7-dimethoxyquinazoline Hydrochlor-
ide (4k). Crystallization from EtOH 95% gave pure 4k as a white
solid (95%). Mp >250 ꢀC. IR cm-1 2466 (NHþ), 1076 (C-O-C
1
methoxy), 1059 (C-Br). H NMR (DMSO-d6) δ ppm 11.71 (s,
1H, NHþ), 8.80 (s, 1H, ArH), 8.29 (s, 1H, ArH), 7.83 (d, J = 2.40
Hz, 1H, ArH), 7.79 (d, J = 8.60 Hz, 1H, ArH), 7.59 (dd, J = 2.40,
8.60 Hz, 1H, ArH), 7.39 (s, 1H, ArH), 4.02 (s, 3H, OCH3), 3.99 (s,
3H, OCH3). LC-MS (APCIþ), calcd for C16H13Br2N3O2, m/z:
438 [(M þ H)þ for 79Br] to 440 [(M þ H)þ for 79Br and 81Br] and
442 [(M þ H)þ for 81Br].
4-(3-Fluoro-4-methylanilino)-6,7-dimethoxyquinazoline Hydro-
chloride (4i). Crystallization from N,N-dimethylformamide gave
pure 4i as a white solid (95%). Mp >250 ꢀC. IR cm-1 2466
(NHþ), 1221 (C-F), 1076 (C-O-C methoxy). 1H NMR
(DMSO-d6) δ ppm 11.51 (s, 1H, NHþ), 8.83 (s, 1H, ArH), 8.38
(s, 1H, ArH), 7.66 (dd, J = 2.10, 7.70 Hz, 1H, ArH), 7.51 (dd, J =
2.00, 8.30 Hz, 1H, ArH), 7.36 (m, 2H, ArH), 4.01 (s, 3H, OCH3),
3.98 (s, 3H, OCH3), 2.28 (s, 3H, CH3). LC-MS (APCIþ), calcd
for C17H16FN3O2, m/z: 314 (M þ H)þ.
4-(4-Bromo-2-fluoroanilino)-6,7-dimethoxyquinazoline Hydro-
chloride (4m). Crystallization from EtOH/cyclohexane gave pure
4m as a white solid (87%). Mp >250 ꢀC. IR cm-1 2467 (NHþ),
1221 (C-F), 1076 (C-O-C methoxy), 1058 (C-Br). 1H NMR
(DMSO-d6) δ ppm 11.69 (s, 1H, NHþ), 8.82 (s, 1H, ArH), 8.31 (s,
1H, ArH), 7.79 (d, 1H, ArH), 7.60-7.50 (m, 2H, ArH), 7.41 (s,
1H, ArH), 4.02 (s, 3H, OCH3), 3.97 (s, 3H, OCH3). LC-MS
(APCIþ), calcd for C16H13BrFN3O2, m/z: 378 [(M þ H)þ for
79Br] and 380 [(M þ H)þ for 81Br].
4-(3-Trifluoromethyl-4-methylanilino)-6,7-dimethoxyquinazo-
line Hydrochloride (4j). Crystallization from N,N-dimethylfor-
mamide gave pure 4j as a white solid (81%). Mp >250 ꢀC. IR
1
2467 cm-1 (NHþ), 1324 (CF3), 1079 (C-O-C methoxy). H
5.1.3. General Procedure for N-Alkylation (5a-m). A mixture
of the N-substituted 6,7-dimethoxyquinazoline 4a-m (0.60 mmol)
NMR (DMSO-d6) δ ppm 11.51 (s, 1H, NHþ), 8.84 (s, 1H, ArH),

8098 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Garofalo et al.
Figure 6. Densitometric analysis. Differential cleavage plots derived from the footprinting assay. Black boxes localize the sites protected on
the gel.
Figure 7. Topoisomerase I relaxation assay. Native supercoiled pUC19 (lane a) was incubated with 6 units of topoisomerase I in the absence
(lane b) or in the presence of test compounds at the indicated concentration: Rel, relaxed; Nck, nicked; Topo, topoisomer products; Sc,
supercoiled.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8099
a
Table 4.
J = 2.10 Hz, 1H, ArH), 7.17 (d, J = 8.60 Hz, 1H, ArH), 6.94 (dd,
J = 2.10, 8.60 Hz, 1H, ArH), 6.91 (s, 1H, ArH), 3.96 (s, 3H,
OCH3), 3.82 (s, 3H, OCH3), 3.61 (s, 3H, NCH3), 2.30 (s, 3H,
CH3). LC-MS (APCIþ), calcd for C18H18BrN3O2, m/z: 388 [(M
þ H)þ for 79Br] and 390 [(M þ H)þ for 81Br].
4-(N-Methyl-3-chloro-4-methylanilino)-6,7-dimethoxyquinazoline
(5h). Crystallization from heptane/ethanol gave pure 5g as a yellow
solid (27%). Mp 195-196 ꢀC. IR cm-1 2955 (N-CH3), 1088
(C-Cl), 1076 (C-O-C methoxy). 1H NMR (DMSO-d6) δ ppm
8.01 (s, 1H, ArH), 7.65 (s, 1H, ArH), 7.19 (d, J = 8.80 Hz, 1H,
ArH), 7.13 (d, J = 2.10 Hz, 1H, ArH), 6.94 (s, 1H, ArH), 6.89 (dd,
J = 2.10, 8.80 Hz, 1H, ArH), 3.95 (s, 3H, OCH3), 3.83 (s, 3H,
OCH3), 3.64 (s, 3H, NCH3), 2.27 (s, 3H, CH3). LC-MS (APCIþ),
calcd for C18H18ClN3O2, m/z: 344 [(M þ H)þ for 35Cl] and 346 [(M
þ H)þ for 37Cl].
4-(N-Methyl-3-fluoro-4-methylanilino)-6,7-dimethoxyquinazoline
(5i). Crystallization from toluene/cyclohexane gave pure 5i as a
yellow solid (30%). Mp 193-194 ꢀC. IR cm-1 2956 (N-CH3), 1224
1
(C-F), 1076 (C-O-C methoxy). H NMR (DMSO-d6) δ ppm
8.02 (s, 1H, ArH), 7.71 (s, 1H, ArH), 7.11 (t, 1H, ArH), 6.92 (s, 1H,
ArH), 6.84 (dd, J = 2.20, 7.90 Hz, 1H, ArH), 6.73 (d, J = 7.90 Hz,
1H, ArH), 3.95 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.65 (s, 3H,
NCH3), 2.19 (s, 3H, CH3). LC-MS (APCIþ), calcd for
C18H18FN3O2, m/z: 328 (M þ H)þ.
a The cell growth rate was evaluated by performing the MTS assay
(readout time: 72 h). Means values ( SD correspond to three indepen-
dent experiments.
4-(N-Methyl-3-trifluoromethyl-4-methylanilino)-6,7-dimethoxy-
quinazoline (5j). Crystallization from H2O/EtOH gave pure 5j as
yellow solid (28%). Mp 204-205 ꢀC. IR cm-1 2955 (N-CH3),
1328 (CF3), 1076 (C-O-C methoxy). 1H NMR (DMSO-d6)
δ ppm 7.96 (s, 1H, ArH), 7.65 (s, 1H, ArH), 7.29 (d, J = 2.00
Hz, 1H, ArH), 7.26 (d, J = 8.40 Hz, 1H, ArH), 7.16 (dd, J = 2.00,
8.40 Hz, 1H, ArH), 6.91 (s, 1H, ArH), 3.95 (s, 3H, OCH3), 3.87 (s,
3H, OCH3), 3.63 (s, 3H, NCH3), 2.38 (s, 3H, CH3). LC-MS
(APCIþ), calcd for C19H18F3N3O2, m/z: 378 (M þ H)þ.
and sodium hydride (60% in oil) (1.20 mmol) in N,N-dimethylfor-
mamide (10 mL) was stirred 5 h at room temperature in a
nitrogen atmosphere. Iodomethane (1.20 mmol) was added, and
the mixture was stirred for 16 h. The reaction was quenched by
water, and then the aqueous solution was extracted with EtOAc
(3 ꢀ 40 mL), washed with a solution of NaHCO3, and dried over
MgSO 4. The solvent was removed under reduced pressure. The
residue was purified by FC (CH2Cl2/MeOH, 9:1) to give a white
or yellow solid. Spectroscopic data and melting points for
compounds 5a, 5d, 5f are in agreement with those reported in
the literature, and they were prepared by nucleophilic substitu-
tion using the commercial N -methylaniline. 24
4-(N-Methyl-2,5-dibromoanilino)-6,7-dimethoxyquinazoline (5k).
Crystallization from toluene/EtOH gave pure 5k as yellow solid
(77%). Mp >250 ꢀC. IR cm-1 2958 (N-CH3), 1078 (C-O-C
methoxy), 1058 (C-Br). 1H NMR (DMSO-d6) δ ppm 7.98 (s, 1H,
ArH), 7.68 (s, 1H, ArH), 7.47 (d, J = 8.40 Hz, 1H, ArH), 7.20 (d,
J = 2.30 Hz, 1H, ArH), 7.02 (dd, J = 2.30, 8.40 Hz, 1H, ArH), 6.91
(s, 1H, ArH), 3.96 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.65 (s, 3H,
NCH3). LC-MS (APCIþ), calcd for C17H16Br2N3O2, m/z: 453
[(M þ H)þfor 79Br] to 455 [(M þ H)þ for 79Br and 81Br] and 457
[(M þ H)þ for 81Br].
4-(N-Methyl-4-bromoanilino)-6,7-dimethoxyquinazoline (5b).
Crystallization from toluene/petroleum ether gave pure 5b as
yellow solid (35%). Mp 205-207 ꢀC. IR cm-1 2957 (N-CH3),
1076 (C-O-C methoxy), 1056 (C-Br). 1H NMR (DMSO-d6)
δ ppm 8.02 (s, 1H, ArH), 7.68 (s, 1H, ArH), 7.37 (d, J = 8.40 Hz,
2H, ArH), 7.02 (d, J = 8.40 Hz, 2H, ArH), 6.92 (s, 1H, ArH),
3.95 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 3.67 (s, 3H, NCH3).
LC-MS (APCIþ), calcd for C17H16BrN3O2, m/z: 374 [(M þ
H)þ for 79Br] and 376 [(M þ H)þ for 81Br].
4-(N-Methyl-3-chloro-4-fluoroanilino)-6,7-dimethoxyquinazoline
(5l). Crystallization from ethanol/cyclohexane gave pure 5l as a
yellow solid (39%). Mp >250 ꢀC. IR cm-1 2957 (N-CH3),
1
1225 (C-F), 1086 (C-Cl), 1077 (C-O-C methoxy). H NMR
(CDCl3) δ ppm 7.88 (s, 1H, ArH), 7.71 (s, 1H, ArH), 7.22 (d, J =
7.90 Hz, 1H, ArH), 7.11 (m, 1H, ArH), 6.95 (dd, J = 2.20, 7.90 Hz,
1H, ArH), 6.58 (s, 1H, ArH), 4.01 (s, 3H, OCH3), 3.97 (s,
3H, OCH3), 3.61 (s, 3H, NCH3). LC-MS (APCIþ), calcd for
C17H15ClFN3O2, m/z: 348 [(M þ H)þ for 35Cl] and 350 [(M þ H)þ
for 37Cl].
4-(N-Methyl-3-fluoroanilino)-6,7-dimethoxyquinazoline (5c).
Crystallization from toluene/petroleum ether gave pure 5c as
yellow solid (29%). Mp 177-178 ꢀC. IR cm-1 2958 (N-CH3),
1
1228 (C-F), 1077 (C-O-C methoxy). H NMR (DMSO-d6)
δ ppm 7.92 (s, 1H, ArH), 3.63 (s, 3H, NCH3), 7.65 (s, 1H, ArH),
7.20 (m, 1H, ArH), 6.91 (s, 1H, ArH), 6.80-6.65 (m, 3H, ArH),
3.97 (s, 3H, OCH3), 3.89 (s, 3H, OCH3). LC-MS (APCIþ),
calcd for C17H16FN3O2, m/z: 314 (M þ H)þ.
4-(N-Methyl-4-bromo-2-fluoroanilino)-6,7-dimethoxyquinazoline
(5m). Crystallization from toluene/cyclohexane gave pure 5m as
yellow solid (29%). Mp >250 ꢀC. IR cm-1 2956 (N-CH3), 1224
(C-F), 1078 (C-O-C methoxy), 1058 (C-Br). 1H NMR
(DMSO-d6) δ ppm 7.92 (s, 1H, ArH), 7.63 (s, 1H, ArH), 7.35
(dd, J = 2.00, 8.40 Hz, 1H, ArH), 7.23 (d, J = 8.40 Hz, 1H, ArH),
6.95 (m, 2H, ArH), 3.98 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.62 (s,
3H, NCH3). LC-MS (APCIþ), calcd for C17H15BrFN3O2, m/z:
392 [(M þ H)þ for 79Br] and 394 [(M þ H)þ for 81Br].
4-(N-Methyl-4-trifluoromethylanilino)-6,7-dimethoxyquinazoline
(5e). Crystallization from toluene/cyclohexane gave pure 5e as a
white solid (31%). Mp 214-215 ꢀC. IR cm-1 2955 (N-CH3),
1
1321 (CF3), 1078 (C-O-C methoxy). H NMR (DMSO-d6)
δ ppm 7.97 (s, 1H, ArH), 7.67 (s, 1H, ArH), 7.53 (d, J = 8.20
Hz, 2H, ArH), 7.11 (d, J = 8.20 Hz, 2H, ArH), 6.91 (s, 1H,
ArH), 3.96 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.64 (s, 3H,
NCH3). LC-MS (APCIþ), calcd for C18H16F3N3O2, m/z: 364
(M þ H)þ.
5.1.4. General Procedure for Nucleophilic Substitution by the
Commercial Phenol (6a-c). 4-Chloro-6,7-dimethoxyquinazo-
line 3 (0.20 g, 0.90 mmol) was added dropwise to a solution of
the commercial phenol (1.07 mmol) and NaH (60% in oil)
(1.60 mmol) in DMSO (4 mL). The mixture was placed into
the cavity of a focused monomode microwave reactor and
irradiated for 10 min at 150 ꢀC (100 W). The reaction was
quenched by water, and the precipitate was collected by filtration
4-(N-Methyl-3-bromo-4-methylanilino)-6,7-dimethoxyquinazo-
line (5g). Crystallization from toluene/cyclohexane gave pure 5g
as a yellow solid (28%). Mp 228-229 ꢀC. IR cm-1 2956
1
(N-CH3), 1076 (C-O-C methoxy), 1056 (C-Br). H NMR
(DMSO-d6) δ ppm 7.98 (s, 1H, ArH), 7.63 (s, 1H, ArH), 7.28 (d,

8100 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Garofalo et al.
and washed with water (20 mL). The residue was dissolved in
EtOAc (50 mL), and the organic layer was washed with a 1 M
solution of K2CO3, 1 M solution of HCl, and brine and dried over
MgSO4. The solvent was removed under reducedpressureand the
residue was purified by FC (CH2Cl2/EtOAc, 9:1) to give a white
solid. Spectroscopic data and melting point for compound 6a are
in agreement with those reported in the literature.25
4-(3-Chloro-4-fluorophenoxy)-6,7-dimethoxyquinazoline (6b).
Crystallization from heptane gave pure 6b as a white solid (62%).
Mp 204-205 ꢀC. IR cm-1 1223 (C-F), 1202 (CdC-O), 1086
(C-Cl), 1078 (C-O-C methoxy). 1H NMR (DMSO-d6) δ ppm
8.55 (s, 1H, ArH), 7.72 (dd, J= 2.20, 7.80 Hz, 1H, ArH), 7.55-7.50
(m, 2H, ArH), 7.45-7.35 (m, 2H, ArH), 4.02 (s, 3H, OCH3),3.97(s,
3H, OCH3). LC-MS (APCIþ), calcd for C16H12ClFN2O3, m/z:
335 [(M þ H)þ for 35Cl] and [(M þ H)þ for 37Cl].
(N-CH3), 1072 (C-O-C methoxy). 1H NMR (DMSO-d6)
δ ppm 8.84 (s, 1H, ArH), 8.48 (s, 1H, ArH), 7.31 (s, 1H, ArH),
7.12 (m, 1H, ArH), 6.81 (s, 1H, ArH), 6.71 (d, J = 6.80 Hz, 1H,
ArH), 6.52 (d, J = 7.60 Hz, 1H, ArH), 5.31 (s, 2H, NH2), 4.03 (s,
3H, OCH3), 4.01 (s, 3H, OCH3), 3.95 (s, 3H, NCH3). LC-MS
(APCIþ), calcd for C17H18N4O2, m/z: 311 (M þ H)þ.
5.1.9. General Procedure for (N-Methylanilino)urea (12a,b).
To a stirred solution of 10 (0.20 g, 0.63 mmol) and pyridine
(0.16 g, 1.89 mmol) in 15 mL of a mixture H2O/THF (5/5) were
added isocyanate derivatives (1.26 mmol). After 16 h, the
solvent was removed under reduce pressure and the oily residue
was purified by FC (CHCl3/MeOH, 8:2).
N-{4-[N-Methyl-6,7-dimethoxyquinazolin-4-ylamino]phenyl}-
N0-phenylurea (12a). Crystallization from cyclohexane/EtOH
95% gave pure 12a as yellow solid (58%). Mp 110-111 ꢀC.
IR cm-1 3212 (NH), 2958 (N-CH3), 1696 (CdO), 1079
(C-O-C methoxy). 1H NMR (DMSO-d6) δ ppm 8.82 (s, 1H,
NH), 8.71 (s, 1H, NH), 8.58 (s, 1H, ArH), 7.51 (d, J = 8.40 Hz,
2H, ArH), 7.45 (d, J = 8.20 Hz, 2H, ArH), 7.29 (m, 2H, ArH),
7.20 (d, J = 8.40 Hz, 2H, ArH), 7.15 (s, 1H, ArH), 6.96 (m, 1H,
ArH), 6.40 (s, 1H, ArH), 3.85 (s, 3H, OCH3), 3.52 (s, 3H,
OCH3), 3.22 (s, 3H, NCH3). LC-MS (APCIþ), calcd for
C24H23N5O3, m/z: 430 (M þ H)þ.
4-(4-Bromo-2-fluorophenoxy)-6,7-dimethoxyquinazoline (6c).
Crystallization from heptane gave pure 6b as a white solid (59%).
Mp 158-159 ꢀC. IR cm-1 1223 (C-F), 1202 (CdC-O), 1077
(C-O-C methoxy), 1058 (C-Br). 1H NMR (DMSO-d6) δ ppm
8.54 (s, 1H, ArH), 7.80 (dd, J = 2.10, 10.10 Hz, 1H, ArH), 7.51 (s,
1H, ArH), 7.55-7.45 (m, 2H, ArH), 7.37 (s, 1H, ArH), 4.02 (s, 3H,
OCH3), 3.97 (s, 3H, OCH3). LC-MS (APCIþ), calcd for
C16H12BrFN2O3, m/z: 379 [(M þ H)þ for 79Br] and 381 [(M þ
H)þ for 81Br].
N-Butyl-N0-{4-[N-methyl-6,7-dimethoxyquinazolin-4-ylamino]-
phenyl}urea (12b). Crystallization from cyclohexane/EtOH 95%
gave pure 12b as yellow solid (46%). Mp 88 ꢀC. IR cm-1 3212
(NH), 2955 (N-CH3), 1697 (CdO), 1075 (C-O-C methoxy).
1H NMR (DMSO-d6) δ ppm 8.61 (s, 1H, ArH), 8.55 (s, 1H, NH),
7.36 (d, J = 8.20 Hz, 2H, ArH), 7.15 (d, J = 8.20 Hz, 2H, ArH),
7.11 (s, 1H, ArH), 6.36 (s, 1H, ArH), 6.13 (s, 1H, NH), 3.88 (s, 3H,
OCH3), 3.51 (s, 3H, OCH3), 3.21 (s, 3H, NCH3), 3.05 (m, 2H,
CH2CH3), 1.40-1.20 (m, 4H, CH2CH2), 0.98 (t, 3H, CH2CH3).
LC-MS (APCIþ), calcd for C22H27N5O3, m/z: 410 (M þ H)þ.
5.1.5. 4-(3-Nitroanilino)-6,7-dimethoxyquinazoline Hydro-
chloride (7).15 Compound 7 was obtained by the same proce-
dure used for 4a-m. Starting from 3 (3.00 g, 13.35 mmol), a
yellow solid was obtained by precipitation (92%). Spectro-
scopic data and melting point for this compound are in
agreement with those reported in the literature.
5.1.6. 4-(N-Methyl-4-nitroanilino)-6,7-dimethoxyquinazoline
Hydrochloride (8). Compound 8 was obtained by the same
procedure used for 4a-m. Starting from 3 (3.00 g, 13.35 mmol)
and N-methyl-4-nitroaniline (2.64 g, 17.40 mmol), a yellow solid
was obtained (47%). Mp 245-246 ꢀC. IR cm-1 2956 (N-CH3),
5.1.10. General Procedure for (N-Methylanilino)urea (13a-f).
Compounds 13a-f were obtained by the same procedure used
for 12a,b. Starting from 11 (0.20 g, 0.63 mmol) and isocyanate
derivatives (1.26 mmol), the residues were purified by FC
(CHCl3/MeOH, 8:2).
1
1502 (NO2), 1079 (C-O-C methoxy). H NMR (DMSO-d6)
δ ppm 9.11 (s, 1H, ArH), 8.32 (d, J = 7.30 Hz, 2H, ArH), 7.70 (d,
J = 7.30 Hz, 2H, ArH), 7.51 (s, 1H, ArH), 6.31 (s, 1H, ArH),
3.98 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.31 (s, 3H, NCH3).
LC-MS (APCIþ), calcd for C17H16N4O4, m/z: 341 (M þ H)þ.
N-{3-[N-Methyl-6,7-dimethoxyquinazolin-4-ylamino]phenyl}-
N0-phenylurea (13a). Crystallization from EtOH gave pure 13a
as yellow solid (50%). Mp 222-223 ꢀC. IR cm-1 3214 (NH),
2956 (N-CH3), 1695 (CdO), 1079 (C-O-C methoxy). 1H
NMR (DMSO-d6) δ ppm 8.63 (s, 1H, NH), 8.49 (s, 1H, NH),
7.88 (s, 1H, ArH), 7.76 (s, 1H, ArH), 7.50-7.25 (m, 4H, ArH),
7.20-6.90 (m, 5H, ArH), 6.53 (s, 1H, ArH), 3.95 (s, 3H, OCH3),
3.87 (s, 3H, OCH3), 3.65 (s, 3H, NCH3). LC-MS (APCIþ),
calcd for C24H23N5O3, m/z: 430 (M þ H)þ.
5.1.7. 4-(N-Methyl-3-nitroanilino)-6,7-dimethoxyquinazoline
(9). Compound 9 was obtained by the same procedure used
for 5a-m. Starting from 7 (2.00 g, 6.10 mmol), a yellow solid was
synthesized which was recrystallized from CH2Cl2/petroleum
ether (77%). Mp 237-238 ꢀC. IR cm-1 2957 (N-CH3), 1502
(NO2), 1079 (C-O-C methoxy). 1H NMR (DMSO-d6) δ ppm
8.01 (s, 1H, ArH), 7.86 (s, 1H, ArH), 7.75 (m, 1H, ArH), 7.65 (s,
1H, ArH), 7.55 (m, 2H, ArH), 6.91 (s, 1H, ArH), 3.97 (s, 3H,
OCH3), 3.91 (s, 3H, OCH3), 3.65 (s, 3H, NCH3). LC-MS
(APCIþ), calcd for C17H16N4O4, m/z: 341 (M þ H)þ.
N-{3-[N-Methyl-6,7-dimethoxyquinazolin-4-ylamino]phenyl}-
N0-(4-methoxyphenyl)urea (13b). Crystallization from EtOH
gave pure 13b as a white solid (27%). Mp 242-243 ꢀC.
IR cm-1 3211 (NH), 2956 (N-CH3), 1695 (CdO), 1078
(C-O-C methoxy). 1H NMR (DMSO-d6) δ ppm 8.91 (s, 1H,
NH), 8.72 (s, 1H, NH), 8.53 (s, 1H, ArH), 7.50-7.35 (m, 4H,
ArH), 7.20-6.90 (m, 5H, ArH), 6.42 (s, 1H, ArH), 3.98 (s, 3H,
OCH3), 3.95 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 3.62 (s, 3H,
NCH3). LC-MS (APCIþ), calcd for C25H25N5O3, m/z: 444
(M þ H)þ.
5.1.8. General Procedure for Catalytic Hydrogenation of Nitro
Group (10, 11). Compound 8 or 9 (1.50 g, 4.40 mmol) was
dissolved in methanol (50 mL), and Raney nickel (0.4 g) was
added. Mixtures were stirred under hydrogen atmosphere at
room temperature for 16 h. Reaction mixtures were passed
through a plug of Celite, and solvents were evaporated under
reduced pressure. Residues were stirred in CH2Cl2, filtered off,
and washed with petroleum ether to give compounds 10 and 11
as yellow solids.
N-{3-[N-Methyl-6,7-dimethoxyquinazolin-4-ylamino]phenyl}-
N0-(3-chloro-4-fluorophenyl)urea (13c). Crystallization from
EtOH 95% gave pure 13c as yellow solid (65%). Mp 173-
174 ꢀC. IR cm-1 3214 (NH), 2956 (N-CH3), 1697 (CdO), 1223
(C-F), 1086 (C-Cl), 1078 (C-O-C methoxy). 1H NMR
(DMSO-d6) δ ppm 8.87 (s, 1H, NH), 8.61 (s, 1H, NH), 7.91 (s,
1H, ArH), 7.79 (d, J = 4.40 Hz, 1H, ArH), 7.64 (s, 1H, ArH),
7.35-7.25 (m, 2H, ArH), 7.15-7.05 (m, 2H, ArH), 6.98 (d, J =
7.40 Hz, 1H, ArH), 6.87 (s, 1H, ArH), 6.58 (d, J = 7.40 Hz, 1H,
ArH), 3.95 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 3.61 (s, 3H,
NCH3). LC-MS (APCIþ), calcd for C24H21ClFN5O3, m/z: 482
[(M þ H)þ for 35Cl] and 484 [(M þ H)þ for 37Cl].
4-(N-Methyl-4-aminoanilino)-6,7-dimethoxyquinazoline Hy-
drocloride (10). Yield 87%. Mp 228-229 ꢀC. IR cm-1 3325
(NH2), 2959 (N-CH3), 1077 (C-O-C methoxy). 1H NMR
(DMSO-d6) δ ppm 8.89 (s, 1H, ArH), 7.37 (s, 1H, ArH), 7.13
(d, J = 8.40 Hz, 2H, ArH), 6.72 (d, J = 8.40 Hz, 2H, ArH), 6.41
(s, 1H, ArH), 3.89 (s, 3H, OCH3), 3.52 (s, 3H, OCH3), 3.32 (s,
3H, NCH3). LC-MS (APCIþ), calcd for C17H18N4O2, m/z:
311 (M þ H)þ.
4-(N-Methyl-3-aminoanilino)-6,7-dimethoxyquinazoline (11).
Yield 92%. Mp 232-234 ꢀC. IR cm-1 3326 (NH2), 2956

Article
N-Ethyl-N0-{3-[N-methyl-6,7-dimethoxyquinazolin-4-ylamino]-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8101
3.71(s,3H, OCH3).LC-MS(APCIþ),calcdforC24H22N4O5, m/z:
447 (M þ H)þ.
phenyl}urea (13d). Crystallization from EtOH gave pure 13d as
yellow solid (37%). Mp 236-238 ꢀC. IR cm-1 3212 (NH), 2955
(N-CH3), 1695 (CdO), 1079 (C-O-C methoxy). 1H NMR
(DMSO-d6) δ ppm 8.95 (s, 1H, NH), 8.85 (s, 1H, ArH), 8.31 (s,
1H, ArH), 7.81 (s, 1H, ArH), 7.50-7.30 (m, 2H, ArH), 7.25-7.10
(m, 2H, ArH), 6.41 (s, 1H, NH), 4.06 (s, 3H, OCH3), 4.01 (s, 3H,
OCH3), 3.97 (s, 3H, NCH3), 3.10 (m, 2H, CH2CH3), 1.01 (m, 3H,
CH2CH3). LC-MS (APCIþ), calcd for C20H23N5O3, m/z: 382
(M þ H)þ.
5.1.13. General Procedure for Nucleophilic Substitution by
Aniline (20a,b). Compounds 20a,b were obtained by the same
procedure used for 4a-m. Starting from 19 (0.20 g, 0.96 mmol)
and the commercial aniline (1.15 mmol), the residue was filtered
off, washed with 2-propanol (10 mL), Et2O (10 mL), and
recrystallized.
4-(3-Chloro-4-fluoroanilino)-6,7-methylenedioxyquinazoline
Hydrochloride (20a). Crystallization from MeOH/petroleum
ether gave pure 20a as a white solid (82%). Mp >250 ꢀC. IR
cm-1 2465 (NHþ), 1219 (C-F), 1052 (C-Cl), 925 (C-O-C).
1H NMR (DMSO-d6) δ ppm 11.51 (s, 1H, NHþ), 7.81 (s, 1H,
ArH), 7.55 (s, 1H, ArH), 7.20-7.10 (m, 2H, ArH), 6.95 (dd,
J = 2.20, 8.10 Hz, 1H, ArH), 6.61 (s, 1H, ArH), 6.41 (s, 2H,
CH2). LC-MS (APCIþ), calcd for C15H9ClFN3O2, m/z: 318
[(M þ H)þ for 35Cl] and 320 [(M þ H)þ for 37Cl].
N-Butyl-N0-{3-[N-methyl-6,7-dimethoxyquinazolin-4-ylamino]-
phenyl}urea (13e). Crystallization from cyclohexane/EtOH
95% gave pure 13e as yellow solid (32%). Mp 180-181 ꢀC.
IR cm-1 3213 (NH), 2955 (N-CH3), 1696 (CdO), 1079
1
(C-O-C methoxy). H NMR (DMSO-d6) δ ppm 9.10 (s, 1H,
NH), 8.88 (s, 1H, ArH), 8.35 (s, 1H, ArH), 7.79 (s, 1H, ArH),
7.40-7.15 (m, 4H, ArH), 6.51 (s, 1H, NH), 4.08 (s, 3H, OCH3),
4.02 (s, 3H, OCH3), 3.96 (s, 3H, NCH3), 3.12 (m, 2H, CH2CH3),
1.40-1.25 (m, 4H, CH2CH2), 0.98 (m, 3H, CH2CH3). LC-MS
(APCIþ), calcd for C22H27N5O3, m/z: 410 (M þ H)þ.
4-(4-Bromo-2-fluoroanilino)-6,7-methylenedioxyquinazoline
Hydrochloride (20b). Crystallization from EtOH gave pure 20b
as a yellow solid (91%). Mp >250 ꢀC. IR cm-1 2466 (NHþ),
1219 (C-F), 1056 (C-Br), 928 (C-O-C). 1H NMR (DMSO-
d6) δ ppm 11.60 (s, 1H, NHþ), 8.81 (s, 1H, ArH), 8.39 (s, 1H,
ArH), 7.77 (d, J = 9.30 Hz, 1H, ArH), 7.60-7.50 (m, 2H,
ArH), 7.48 (s, 1H, ArH), 6.39 (s, 2H, CH2). LC-MS (APCIþ),
calcd for C15H9BrFN3O2, m/z: 362 [(M þ H)þ for 79Br] and
364 [(M þ H)þ for 81Br].
N-Cyclohexyl-N0-{3-[N-methyl-6,7-dimethoxyquinazolin-4-
ylamino]phenyl}urea (13f). Crystallization from ethanol gave
pure 13f as yellow solid (42%). Mp 234-235 ꢀC. IR cm-1 3215
(NH), 2957 (N-CH3), 1695 (CdO), 1077 (C-O-C methoxy).
1H NMR (DMSO-d6) δ ppm 8.68 (s, 1H, ArH), 8.49 (s, 1H,
NH), 8.05 (s, 1H, ArH), 7.50 (s, 1H, ArH), 7.25-7.10 (m, 3H,
ArH), 6.89 (s, 1H, ArH), 6.32 (s, 1H, NH), 4.03 (s, 3H, OCH3),
3.97 (s, 3H, OCH3), 3.88 (s, 3H, NCH3), 1.70-1.10 (m, 11H,
CH2CH2). LC-MS (APCIþ), calcd for C24H29N5O3, m/z: 436
(M þ H)þ.
5.1.14. General Procedure for N-Alkylation (21a,b). Com-
pounds 21a,b were obtained by the same procedure used for
5a-m. Starting from quinazoline derivatives 20a,b (0.30 g,
0.94 mmol), the oily residue were purified by FC (CH2Cl2/
MeOH, 9:1).
5.1.11. 3-[(6,7-Dimethoxy-4-quinazolinyl)oxy]aniline (15). A
solution of 3 (2.00 g, 8.90 mmol), 3-aminophenol (1.17 g,
10.7 mmol) and tetra-n-butylammonium bromide (1.43 g,
4.45 mmol) in methylethylketone (20 mL) and 20% solution
of NaOH (10 mL) was refluxed for 30 min. After dilution by
CHCl3 (100 mL) and H2O (20 mL), the organic layer was
washed with water, brine and dried over MgSO4. The solvent
was removed under reduced pressure and then the precipitated
solid was collected by filtration and washed with MeOH (15 mL)
to give 15 as a white solid (93%). Mp 203-204 ꢀC. IR cm-1 3384
4-(N-Methyl-3-chloro-4-fluoroanilino)-6,7-methylenedioxy-
quinazoline (21a). Crystallization from toluene gave pure 21a
as a yellow solid (47%). Mp >250 ꢀC. IR cm-1 2955
(N-CH3), 1219 (C-F), 1086 (C-Cl), 926 (C-O-C). 1H
NMR (DMSO-d6) δ ppm 8.53 (s, 1H, ArH), 8.05 (s, 1H, ArH),
7.71 (s, 1H, ArH), 7.50-7.35 (m, 3H, ArH), 6.32 (s, 2H, CH2),
3.81 (s, 3H, NCH3). LC-MS (APCIþ), calcd for
C
16H11ClFN3O2, m/z: 332 [(M þ H)þ for 35Cl] and 334 [(M
1
þ H)þ for 37Cl].
(NH2), 1205 (CdC-O), 1077 (C-O-C methoxy). H NMR
4-(N-Methyl-4-bromo-2-fluoroanilino)-6,7-methylenediox-
yquinazoline (21b). Crystallization from EtOH gave pure 21b
as yellow solid (38%). Mp 239-240 ꢀC. IR cm-1 2958
(N-CH3), 1220 (C-F), 1056 (C-Br), 925 (C-O-C). 1H
NMR (DMSO-d6) δ ppm 7.92 (s, 1H, ArH), 7.61 (s, 1H,
ArH), 7.37 (m, 1H, ArH), 7.22 (m, 1H, ArH), 7.17 (s, 1H,
ArH), 6.95 (m, 1H, ArH), 6.21 (s, 2H, CH2), 3.60 (s, 3H,
NCH3). LC-MS (APCIþ), calcd for C16H11BrFN3O2, m/z:
376 [(M þ H)þ for 79Br] and 378 [(M þ H)þ for 81Br].
(DMSO-d6) δ ppm 8.53 (s, 1H, ArH), 7.51 (s, 1H, ArH), 7.34 (s,
1H, ArH), 7.08 (m, 1H, ArH), 6.49 (m, 1H, ArH), 6.42 (m, 1H,
ArH), 6.36 (m, 1H, ArH), 5.26 (s, 2H, NH2), 4.02 (s, 3H, OCH3),
3.97 (s, 3H, OCH3). LC-MS (APCIþ), calcd for C16H15N3O3,
m/z: 298 (M þ H)þ.
5.1.12. General Procedure for Urea Derivatives (17a,b). To a
stirred solution of 15 (0.20 g, 0.67 mmol) and NEt3 (0.17 g,
1.68 mmol) in 10 mL of CHCl3 were added phenyl isocyanate
derivatives (0.80 mmol). After 5 h, the solvent was removed
under reduced pressure and the oily residue was evaporated with
EtOH (3 ꢀ 15 mL). The residue was filtered off, washed with
EtOH (5 mL), and recrystallized.
5.1.15. General Procedure for N-Alkylation (22a-g). Com-
pounds 22a-g were obtained by the same procedure used for
5a-m. Starting from 4-(3-bromo-4-methylanilino)-6,7-di-
methoxyquinazoline hydrochloride (4g) (0.30 g, 0.73 mmol)
and halogen derivatives (1.46 mmol), the oily residues were
purified by FC (CH2Cl2/MeOH, 9:1).
N-{3-[6,7-Dimethoxyquinazolin-4-yloxy]phenyl}-N0-phenylur-
ea (17a). Crystallization from EtOH gave pure 17a as a white
solid (68%). Mp >250 ꢀC. IR cm-1 3212 (NH), 1696 (CdO),
1205 (CdC-O), 1077 (C-O-C methoxy). 1H NMR (DMSO-
d6) δ ppm 8.88 (s, 1H, NH), 8.72 (s, 1H, NH), 8.55 (s, 1H, ArH),
7.65 (m, 2H, ArH), 7.60-7.45 (m, 4H, ArH), 7.30-7.20 (m, 3H,
ArH), 7.00-6.90 (m, 2H, ArH), 3.99 (s, 3H, OCH3), 3.95 (s, 3H,
OCH3). LC-MS (APCIþ), calcd for C23H20N4O4, m/z: 417
(M þ H)þ.
4-(N-Ethyl-3-bromo-4-methylanilino)-6,7-dimethoxyquinazo-
line (22a). Starting from iodoethane (116 μL, 1,46 mmol),
compound 22a was obtanied by crystallization from CH2Cl2/
petrolum ether as a yellow solid (13%). Mp 160-161 ꢀC. IR
cm-1 1078 (C-O-C methoxy), 1057 (C-Br). 1H NMR
(DMSO-d6) δ ppm 8.03 (s, 1H, ArH), 7.71 (s, 1H, ArH), 7.28
(d, J = 2.00 Hz, 1H, ArH), 7.18 (d, J = 8.60 Hz, 1H, ArH), 6.96
(dd, J = 2.00, 8.60 Hz, 1H, ArH), 6.92 (s, 1H, ArH), 4.18 (m,
2H, CH2), 3.94 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 2.28 (s, 3H,
CH3), 1.31 (m, 3H, CH3). LC-MS (APCIþ), calcd for
C19H20BrN3O2, m/z: 402 [(M þ H)þ for 79Br] and 404 [(M þ
H)þ for 81Br].
N-{3-[6,7-Dimethoxyquinazolin-4-yloxy]phenyl}-N0-(4-meth-
oxyphenyl)urea (17b). Crystallization from EtOH gave pure 17b as
a white solid (70%). Mp 235 ꢀC. IR cm-1 3214 (NH), 1695 (CdO),
1208 (CdC-O), 1079 (C-O-C methoxy). 1H NMR (DMSO-d6)
δ ppm 8.90 (s, 1H, NH), 8.75 (s, 1H, NH), 8.51 (s, 1H, ArH),
7.71 (s, 1H, ArH), 7.40-7.30 (m, 3H, ArH), 7.09 (m, 1H, ArH),
6.85 (d, J = 8.80 Hz, 2H, ArH), 6.52 (m, 1H, ArH), 6.41 (m, 1H,
ArH), 6.37 (m, 1H, ArH), 4.01 (s, 3H, OCH3), 3.96 (s, 3H, OCH3),
4-(N-Prop-1-yl-3-bromo-4-methylanilino)-6,7-dimethoxyqui-
nazoline (22b). Starting from 1-iodopropane (124 μL, 1,46 mmol),

8102 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Garofalo et al.
compound 22b was obtanied by crystallization from CH2Cl2/
petrolum ether as a yellow solid (20%). Mp 143-144 ꢀC.
IR cm-1 1078 (C-O-C methoxy), 1057 (C-Br). 1H NMR
(DMSO-d6) δ ppm 8.01 (s, 1H, ArH), 7.67 (s, 1H, ArH), 7.35 (d,
J = 2.00 Hz, 1H, ArH), 7.18 (d, J = 8.20 Hz, 1H, ArH), 6.96 (dd,
J = 2.00, 8.20 Hz, 1H, ArH), 6.91 (s, 1H, ArH), 4.11 (m, 2H, CH2),
3.95 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 2.28 (s, 3H, CH3), 1.72 (m,
2H, CH2), 0.91 (m, 3H, CH3). LC-MS (APCIþ), calcd for
C20H22BrN3O2, m/z: 416 [(M þ H)þ for 79Br] and 418 [(M þ
H)þ for 81Br].
calcd for C24H29BrN4O2, m/z: 485 [(M þ H)þ for 79Br] and 487
[(M þ H)þ for 81Br].
5.2. DNA-Binding Methods. 5.2.1. Absorption Spectroscopy
and Melting Experiments Studies. Absorption spectra and melt-
ing curves were determined using an Uvikon XL spectrophot-
ometer coupled to a thermosystem. Titrations of the drug with
DNA, covering a large range of DNA-phosphate/drug ratios
(P/D), were performed by adding aliquots of a concentrated CT-
DNA or poly(dA-dT)2 solution to a drug solution at constant
ligand concentration (20 μM). For each series of Tm measure-
ments (λ = 260 nm), 10 samples were placed in a thermostati-
cally controlled cell-holder, and the quartz cuvettes (10 mm path
length) were Peltier heated. The measurements were performed
in BPE buffer, pH 7.1 (6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM
EDTA) . The temperature inside the cuvette was measured with
a platinum probe; it was increased over the range 20-100 ꢀC
with a heating rate of 1 ꢀC/min. The “melting” temperature Tm,
deduced from melting curve, was taken as the midpoint of the
hyperchromic transition. DNA titrations were performed in the
same medium, salt. To 1 mL of drug solution at 20 μM were
added aliquots of a concentrated calf thymus DNA solution.
5.2.2. Fluorescence Titration Experiments. Fluorescence titra-
tion data were recorded at room temperature using SPEX
fluorometer Fluorolog. Excitation was at 360 nm, and fluores-
cence emission was monitored over the range 380-520 nm.
Samples used for titration experiments were prepared separately
at a constant drug concentration of 10 μM and at DNA
concentrations ranging from 0.01 to 1 mM. Fluorescence titra-
tion data were fitted directly to get apparent binding constants
using a fitting function incorporated into Prism 3.0 defined by
4-(N-Prop-2-yl-3-bromo-4-methylanilino)-6,7-dimethoxyquina-
zoline (22c). Starting from 2-iodopropane (124 μL, 1,46 mmol),
compound 22c was obtanied by crystallization from CH2Cl2/petro-
lum ether as a yellow solid (17%). Mp 174-175 ꢀC. IR cm-1 1077
(C-O-C methoxy), 1058 (C-Br). 1H NMR (DMSO-d6) δ ppm
8.36 (s, 1H, ArH), 7.79 (s, 1H, ArH), 7.42 (d, J = 2.00 Hz, 1H,
ArH), 7.28 (d, J = 8.40 Hz, 1H, ArH), 7.18 (s, 1H, ArH), 7.02 (dd,
J = 2.00, 8.40 Hz, 1H, ArH), 4.98 (m, 1H, CH(CH3)2), 3.95 (s, 3H,
OCH3), 3.88 (s, 3H, OCH3), 2.31 (s, 3H, CH3), 1.49 (m, 6H,
CH(CH3)2). LC-MS (APCIþ), calcd for C20H22BrN3O2, m/z:
416 [(M þ H)þ for 79Br] and 418 [(M þ H)þ for 81Br].
4-(N-Benzyl-3-bromo-4-methylanilino)-6,7-dimethoxyquinazo-
line (22d). Starting from benzyl bromide (174 μL, 1,46 mmol),
compound 22d was obtanied by crystallization from heptane as a
yellow solid (18%). Mp 138-140 ꢀC. IR cm-1 1078 (C-O-C
1
methoxy), 1057 (C-Br). H NMR (DMSO-d6) δ ppm 8.28 (s,
1H, ArH), 7.63 (s, 1H, ArH), 7.40-7.25 (m, 6H, ArH), 7.20 (d,
J = 8.20 Hz, 1H, ArH), 6.96 (dd, J = 2.00, 8.20 Hz, 1H, ArH),
6.81 (s, 1H, ArH), 5.38 (s, 2H, CH2), 3.81 (s, 3H, OCH3), 3.71 (s,
3H, OCH3), 2.30 (s, 3H, CH3). LC-MS (APCIþ), calcd for
C24H22BrN3O2, m/z: 464 [(M þ H)þ for 79Br] and 466 [(M þ H)þ
for 81Br].
K
D = [ligand][DNA]/[ligand-DNA].
5.2.3. Circular Dichroism. CD spectra were recorded on a
Jasco J-810 spectrometer. Solutions of drugs, nucleic acids, and
their complexes (1 mL in a 1 mM sodium cacodylate buffer,
pH 7.0) were scanned in 10 mm quartz cuvettes. Measurements
were made by progressive dilution of drug-DNA complex at
a high P/D (phosphate/drug) ratio with a pure ligand solution to
yield the desired drug/DNA ratio. Four scans were accumulated
and automatically averaged. Then 1 mL of drug solution at
50 μM was successively diluted with increased volumes of DNA
at 5 mM.
4-(N-(2-Dimethylaminoethyl)-3-bromo-4-methylanilino)-6,7-
dimethoxyquinazoline (22e). Starting from 2-dimethylami-
noethyl chloride hydrochloride (210 mg, 1,46 mmol), compound
22e was obtanied by crystallization from CH2Cl2/petrolum
ether as a yellow solid (18%). Mp 107-109 ꢀC. IR cm-1 1078
(C-O-C methoxy), 1057 (C-Br). 1H NMR (DMSO-d6) δ ppm
8.04 (s, 1H, ArH), 7.72 (s, 1H, ArH), 7.37 (d, J = 2.10 Hz, 1H,
ArH), 7.22 (d, J = 8.40 Hz, 1H, ArH), 7.01 (dd, J = 2.10, 8.40
Hz, 1H, ArH), 6.97 (s, 1H, ArH), 4.24 (m, 2H, CH2), 3.95 (s, 3H,
OCH3), 3.86 (s, 3H, OCH3), 2.57 (m, 2H, CH2), 2.31 (s, 3H,
CH3), 2.19 (m, 6H, CH3). LC-MS (APCIþ), calcd for
C21H25BrN4O2, m/z: 445 [(M þ H)þ for 79Br] and 447 [(M þ
H)þ for 81Br].
5.2.4. DNase I Footprinting. Experiments were performed
essentially as previously described.26 Briefly, reactions were
conducted in a total volume of 10 μL. Samples (3 μL) of
the labeled DNA fragments were incubated with 5 μL of the buf-
fered solution containing the ligand at the appropriate concen-
tration. After 30 min of incubation at 37 ꢀC to ensure equilibra-
tion of the binding reaction, the digestion was initiated by the
addition of 2 μL of a DNase I solution whose concentration was
adjusted to yield a final enzyme concentration of about 0.01
unit/mL in the reaction mixture. After 3 min, the reaction was
stopped by freeze-drying. Samples were lyophilized and resus-
pended in 5 μL of an 80% formamide solution containing
tracking dyes. The DNA samples were then heated at 90 ꢀC for
4 min and chilled in ice for 4 min prior to electrophoresis.
4-(N-(2-Diethylaminoethyl)-3-bromo-4-methylanilino)-6,7-di-
methoxyquinazoline (22f). Starting from 2-diethylaminoethyl
chloride hydrochloride (251 mg, 1,46 mmol), compound 22f
was obtanied by crystallization from CH2Cl2/petrolum ether as
a yellow solid (11%). Mp 115-116 ꢀC. IR cm-1 1078 (C-O-C
1
methoxy), 1057 (C-Br). H NMR (DMSO-d6) δ ppm 7.92 (s,
1H, ArH), 7.68 (s, 1H, ArH), 7.30 (d, J = 2.10 Hz, 1H, ArH),
7.18 (d, J = 8.40 Hz, 1H, ArH), 7.01 (dd, J = 2.10, 8.40 Hz, 1H,
ArH), 6.95 (s, 1H, ArH), 4.13 (m, 2H, CH2), 3.95 (s, 3H, OCH3),
3.82 (s, 3H, OCH3), 2.63 (m, 2H, CH2), 2.41 (m, 4H, CH2CH3),
2.29 (s, 3H, CH3), 0.82 (m, 6H, CH2CH3). LC-MS (APCIþ),
calcd for C23H29BrN4O2, m/z: 473 [(M þ H)þ for 79Br] and 475
[(M þ H)þ for 81Br].
5.2.5. DNA Topoisomerase I Relaxation Assay. Supercoiled
pUC19 DNA (130 ng) was incubated with 4 units of recombi-
nant human topoisomerase I (TopoGen Inc.) at 37 ꢀC for 45 mn
in relaxation buffer (50 mM Tris, pH 7.8, 50 mM KCl, 10 mM
MgCl2, 1 mM dithiothreitol, 1 mM EDTA) in the presence of
varying concentrations of the drug under study. Reactions were
terminated by adding SDS to 0.25% and proteinase K to 250 μg/
mL. DNA samples were then added to the electrophoresis drye
mixture (3/l) and electrophoresed in a 1% agarose gel at room
temperature for 2 h at 120 V. Gels were stained with ethidium
bromide (1 μg/mL), washed, and photographed under UV light.
Similar experiments were performed using ethidium-containing
agarose gels.
4-(N-(2-Piperidin-1-ylethyl)-3-bromo-4-methylanilino)-6,7-di-
methoxyquinazoline (22g). Starting from 2-chloroethylpiperi-
dine hydrochloride (269 mg, 1,46 mmol), compound 22g was
obtained by crystallization from CH2Cl2/petrolum ether as a
yellow solid (20%). Mp 139-140 ꢀC. IR cm-1 1078 (C-O-C
1
methoxy), 1059 (C-Br). H NMR (DMSO-d6) δ ppm 7.94 (s,
1H, ArH), 7.68 (s, 1H, ArH), 7.30 (d, J = 2.10 Hz, 1H, ArH),
7.19 (d, J = 8.40 Hz, 1H, ArH), 6.98 (dd, J = 2.00, 8.40 Hz, 1H,
ArH), 6.95 (s, 1H, ArH), 4.20 (m, 2H, CH2), 3.95 (s, 3H, OCH3),
3.82 (s, 3H, OCH3), 2.55 (m, 2H, CH2), 2.39 (m, 4H, CH2CH2),
2.29 (s, 3H, CH3), 1.41 (m, 6H, CH2CH2). LC-MS (APCIþ),
5.3. Cell Culture and Cell Proliferation Assay. Human pros-
tate cancer cells PC3, breast cancer cell line MCF7, and colon

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8103
cancer cell line HT29 were grown at 37 ꢀC in a humidified
atmosphere containing 5% CO2, respectively, in RPMI-1640
medium (Sigma), MEM (Sigma), and DMEM (Gibco) supple-
mented with 10% fetal bovine serum, glutamine (2 mM),
penicillin (100 IU/mL), and streptomycin (100 μg/mL).
In the cell proliferation assay, cells were plated in triplicate on
96-well plates (3.103 cells per well) and incubated for 72 h. The
cell medium was changed to serum-free medium, and the cells
were starved for 24 h for culture synchronization. Cells were
then incubated in culture medium that contained various con-
centrations of tested compounds, each dissolved in less than
0.1% DMSO. After 72 h, cell growth was estimated by the
colorimetric MTT test.
(11) Bridges, A. J.; Cody, D. R.; Zhou, H.; McMichael, A.; Fry, D. W.
Enantioselective inhibition of the epidermal growth factor receptor
tyrosine kinase by 4-(R-phenethylamino)quinazolines. Bioorg.
Med. Chem. 1995, 3, 1651–1656.
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(12) Goossens, J. F.; Bouey-Bencteux, E.; Houssin, R.; Henichart, J. P.;
Colson, P.; Houssier, C.; Laine, W.; Baldeyrou, B.; Bailly, C. DNA
interaction of the tyrosine protein kinase inhibitor PD153035 and
its N-methyl analogue. Biochemistry 2001, 40, 4663–4671.
(13) Bridges, A. J.; Zhou, H.; Cody, D. R.; Rewcastle, G. W.; McMi-
chael, A.; Hollis Showalter, H. D.; Fry, D. W.; Kraker, A. J.;
Denny, W. A. Tyrosine kinase inhibitors. 8. An unusually
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Acknowledgment. The authors are grateful to the institu-
ꢀ
tions that support our laboratory: Universite Lille Nord de
France and the “Ligue Nationale contre le Cancer, Comite du
ꢀ
(15) Furuta, T.; Sakai, T.; Senga, T.; Osawa, T.; Kubo, K.; Shimizu, T.;
Suzuki, R.; Yoshino, T.; Endo, M.; Miwa, A. Identification of
potent and selective inhibitors of PDGF receptor autophosphor-
ylation. J. Med. Chem. 2006, 49, 2186–2192.
^
Pas-de Calais”, the PRIM, Pole de Recherche Interdiscipli-
naire du Medicament (Region Nord-Pas-de-Calais, Ministere
ꢀ
ꢀ
ꢁ
ꢀ ꢀ ꢀ ꢁ
Delegue a la Recherche et aux Nouvelles Technologies,
European Union (FEDER)).
(16) Matsuno, K.; Ushiki, J.; Seishi, T.; Ichimura, M.; Giese, N. A.; Yu,
J. C.; Takahashi, S.; Oda, S.; Nomoto, Y. Potent and selective
inhibitors of platelet-derived growth factor receptor phosphory-
lation. 3. Replacement of quinazoline moiety and improvement
of metabolic polymorphism of 4-[4-(N-substituted (thio)
carbamoyl)-1-piperazinyl]-6,7-dimethoxyquinazoline derivatives.
J. Med. Chem. 2003, 46, 4910–4925.
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