A General synthetic procedure for 2-chloromethyl-4(3H)-quinazolinone derivatives and their utilization in the preparation of novel anticancer agents with 4-anilinoquinazoline scaffolds

Article abstract of DOI:10.3390/molecules15129473

In our ongoing research on novel anticancer agents with 4-anilinoquinazoline scaffolds, a series of novel 2-chloromethyl-4(3H)- quinazolinones were needed as key intermediates. An improved one-step synthesis of 2-chloromethyl-4(3H)-quinazolinones utilizin

Full text of DOI:10.3390/molecules15129473

Molecules 2010, 15, 9473-9485; doi:10.3390/molecules15129473
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
A General Synthetic Procedure for 2-chloromethyl-4(3H)-
quinazolinone Derivatives and Their Utilization in the
Preparation of Novel Anticancer Agents with
4-Anilinoquinazoline Scaffolds
Hong-Ze Li 1,2,†, Hai-Yun He 1,†, Yuan-Yuan Han 1,†, Xin Gu 3, Lin He 2, Qing-Rong Qi 2,
Ying-Lan Zhao 1 and Li Yang 1,*
1
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China
Medicinal School, Sichuan University, Chengdu 610041, China
2
West China School of Pharmacy, Sichuan University, Chengdu 610041, China
3
Department of Chemistry, Sichuan University, Chengdu 610064, China
†
These authors equally contributed to this work.
* Author to whom correspondence should be addressed; E-Mail: yangli@scu.edu.cn;
Tel.: +86-28-85164063; Fax: +86-28-85164060.
Received: 27 October 2010; in revised form: 9 December 2010 / Accepted: 13 December 2010 /
Published: 22 December 2010
Abstract: In our ongoing research on novel anticancer agents with 4-anilinoquinazoline
scaffolds, a series of novel 2-chloromethyl-4(3H)-quinazolinones were needed as key
intermediates. An improved one-step synthesis of 2-chloromethyl-4(3H)-quinazolinones
utilizing o-anthranilic acids as starting materials was described. Based on it, 2-hydroxy-
methyl-4(3H)-quinazolinones were conveniently prepared in one pot. Moreover, two novel
4-anilinoquinazoline derivatives substituted with chloromethyl groups at the 2-position
were synthesized and showed promising anticancer activity in vitro.
Keywords:
2-chloromethyl-4(3H)-quinazolinones;
anticancer
activity;
4-anilino-quinazoline

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1. Introduction
2-Chloromethyl-4(3H)-quinazolinones are valuable intermediates in the preparations of a wide
range of biologically active compounds such as anticancer agents 1a [1] and 1b [2], anti-inflammatory
agent 1c [3], and hedgehog antagonist 1d [4], etc. In addition, they also represent a class of
functionalized and versatile building blocks, that is, these compounds can be converted into 2-
hydroxymethyl-4(3H)-quinazolinones 1a, 2-formyl-4(3H)-quinazolinones 1e, etc. (Figure 1)
Figure 1. The versatile conversion of 2-chloromethyl-4(3H)-quinazolinones 3.
OMe
Cl
O
N
N
N
OMe
N
N
1b
Cl
N
1c
S
N
N
NH
F
O
N
O
N
O
N
NH
N
NH
R
OH
Cl
X
1a
X=Cl,H
1d
HN
HN
O
3
CF3
O
NH
R
O
N
1e
Our research group has been interested in the design, screening, synthesis and biological evaluation
of novel tumor growth inhibitors and apoptosis inducers as potential anticancer agents [5-8]. In our
ongoing research of novel anticancer agents with 4-anilinoquinazoline scaffold, a series of novel 2-
chloromethyl-4(3H)-quinazolinones 3 are needed as key intermediates (Scheme 3).
Figure 2. The representive hit compounds 1f, 1g and 1h in our screening of anticancer agents.

Molecules 2010, 15
9475
Although a few methods for the synthesis of 2-chloromethyl-4(3H)-quinazolinones 3a have been
reported (Scheme 1), these methods (pathways A, B, C and D) all suffer from limitations. For example,
only a few examples were reported without any systematic study of pathway A [9], the excessive
amount usage of chloroacetonitrile (ca. 10 eq.) used under acid conditions in pathway B [10], the
tedious route via oxidative annulation in pathway C [11] and the need for microwave irradition of
pathway D [12]. Although pathway A has features that suggest high efficiency (only one step needed),
mild reaction conditions, simple workup and purification, etc., there has been no systematic study of
this method. Thus, it is significant and attractive to develop a general procedure for the synthesis of 2-
chloromethyl-4(3H)-quinazolinones 3.
Scheme 1. The reported routes toward 3a.
O
O
OH
NH2
NH2
NH2
2a
4
O
O
CN
NH2
OR
NH2
NH
Pathway B
Pathway C
Cl
N
6
5
3a
R=methyl or ethyl
Herein, we wish to describe an improved synthetic procedure for 2-chloromethyl-4(3H)-
quinazolinones 3 starting from o-anthranilic acids 2 (pathway A), and a convenient experimental
protocol for the direct conversion of 3 to 2-hydroxymethyl-4(3H)-quinazolinones in one flask, as well
as their utilization in the preparations of novel anticancer agents.
2. Results and Discussion
2.1. Chemistry
Initial studies showed that many o-anthranilic acid substrates such as 2b gave poor yields (up to
52%) via pathway A under literature conditions, although unsubstituted o-anthranilic acid 2a gave a
satisfactory yield (up to 88%). Therefore, 2b was choosed as a model substrate for optimization of the
reaction conditions and we set out to investigate the conditions for the condensation of 2b with
chloroacetonitrile (11) under different conditions. The results are shown in Table 1. The screening
showed that the amount of chloroacetonitrile used was important for yield, and we were delighted to
find that increasing the amount of chloroacetonitrile (from 1.0 to 3.0 equiv.) gave a much better yield
(77%), as shown in entry 3.

Molecules 2010, 15
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Table 1. The conditions optimization for substrate 2b.
Entry
Temp (°C)
Substrates ratio a (2b:11)
Time (h)
Yield (%)
1
2
3
4
5
6
25
-
-
-
-
1:1
1:2
1:3
1:3
1:4
1:3
2
2
2
2
4
2
52
68
77
67b
78
76
50
Note: a All reaction run at 5 mmol scale; b The solvent is ethanol.
To demonstrate the generality of our procedure, we next investigated the condensation of a variety
of o-anthranilic acids 2 with chloroacetonitrile under the optimized conditions (1.0 equiv. anthranilic
acid 2 and 3.0 equiv. chloroacetonitrile were reacted in methanol for 2 h at 25 °C). The results are
presented in Table 2.
Table 2. The synthesis of 2-chloromethyl-4(3H)-quinazolinones 3.
Entry
Substrate
Product
Yield (%)
O
1
88
NH
Cl
N
3a
3b
3ca
3d
2a
2b
2c
2d
2
3
4
76
64
84

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9477
Table 2. Cont.
O
N
5
6
7
75
77
72
65
58
NH
Cl
Br
F
3e
3f
2e
O
O
F
OH
NH
Cl
NH2
N
O
2f
O
OH
NH2
NH
Cl
F
F
N
3g
3ha
2g
2h
8
9
O
N
NH
Cl
2i
3i
O
N
10
11
70
40
16
78
NH
Cl
F3C
2j
3j
O
HO
OH
3ka
NH2
O
2k
2l
12
13
O2N
OH
3la
NH2
O
N
O
O
NH
Cl
3m
2m
a The product was a new compound.
As shown in Table 2, unsubstituted o-anthranilic acid 2a (entry1) gave the best yield (88%).
Reactions of substrates containing electron-withdrawing groups (entries 6-7, 10) and electron-donating
groups (entry 13) on the phenyl ring gave comparable yields within the 70-78% range. Moreover, a
slightly reduced yield was observed when the substitutent was adjacent to the amino and carboxylic
groups (entries 3, 8-9). However, substrates with a hydroxyl and nitro group on the phenyl ring (entry
11, 12) gave only 40% and 16% yield, respectively, due to poor solubility in methanol.

Molecules 2010, 15
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After the substrate extension, we next turned to synthesizing 2-hydroxymethyl-4(3H)-quinazolinone
(7), a reported anticancer agent [1], with our procedure. To improve the operational efficiency, we
speculated that the synthesis of 7 might be achieved in one flask without isolation and purification of
3a. Gratifyingly, this one-flask procedure proceeded successfully and 7 was prepared from 2a in two
steps in one pot with 60% overall yield together with the virtue of simple purification (Scheme 2). It is
noteworthy that the literature route needed three steps and gave 30% overall yield [1].
Scheme 2. The one-pot synthetic route toward 2-hydroxymethyl-4(3H)-quinazolinone (7).
2.2. Anticancer activity
4-Anilinoquinazolines constitute an important class of protein kinase inhibitors approved by the US
Food and Drug Administration (FDA) as marked drugs. Three of them with 4-anilinoquinazoline
scaffolds (Figure 3), namely erolotinib (Tarceva®), gefitinib (Iressa®), lapatinib (Tykerb®) have
achieved great clinical success [13]. The anticancer structure-activity relationships (SARs) of 4-
anilinoquinazoline derivatives have been intensively studied, but the 2-position SAR has seldom been
explored [2], so as part of our ongoing anticancer research on 4-anilinoquinazoline derivatives we
focused on 2-position modification as shown in Scheme 3.
Figure 3. The structure of erolotinib, gefitinib and lapatinib.
Erlotinib
Gefitinib
Lapatinib
Compounds 9 and 10, with 2-chloromethyl substituted 4-anilinoquinazoline scaffolds, were
synthesized via chloration and condensation with aniline derivatives [2] starting from 2-chloromethyl-
4(3H)-quinazolinone 3g as shown in Scheme 3.

Molecules 2010, 15
9479
Scheme 3. The synthetic route of 9 and 10.
Reagents and conditions: (a) POCl3, DIPEA, toluene, 80 °C, 4 h, 80% yield; (b) aniline derivatives,
IPA, 80 °C, 1-2 h, 80%.
The antiproliferative activities of compounds 9 and 10 were examined in three different types of
cancer cell lines including human hepatoma HepG2, breast cancer cell line MDA-MB-468, and
colorectal cancer cell line HCT-116 using the MTT assay with gefitinib as the positive control. The
experimental data are summarized in Table 3.
Table 3. The anti-proliferative activities of compounds 9 and 10 against various cancer cell lines.
IC50 (µM) a
MDA-MB-468
Compound
HepG2
HCT-116
3.8
3.2
12.4
9
4.3
3.2
20
10
6.4
20
160
a The cytotoxicity effects of compounds on various cancer cell lines were determined by the MTT
assay. The results were expressed as the IC50, and were the means calculated from three
independent experiments.

Molecules 2010, 15
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Compounds 9 and 10 show a broad-spectrum anti-cancer activity and low micromolar inhibition
potency. It is noteworthy that the IC50 for HCT-116 cells was 12.4-20 µM, compared with an IC50 of
160 µM for gefitinib, the positive control. Besides the viability assay, the morphological changes were
also studied under a contrast phase microscope as shown in Figure 4. Taking compound 9 for example,
the obvious morphological changes of HepG2 cells were observed compared with the control (Figure
4a), after the treatment with compound 9 for 24 h (Figures 4b-4d). The results suggested that
compound 9 might induce HepG2 cell death, because the cell shrinkage and rounding are common
events in many cell death processes including both apoptosis and necrosis.
Figure 4. Morphology image of the HepG2 liver cancer cells treated with the control and
compound 9 (2.5-20 µM) for 24 h. (4a) the cells treated with DMSO 0.1% (v/v) as a
vehicle control; (4b) 2.5 µM; (4c) 10 µM; and (4d) 20 µM.
3. Experimental
3.1. General
All solvents and reagents were analytical grade pure and used without further purification. All
1
melting points were determined on electric melting point apparatus and were uncorrected. H-NMR
and 13C-NMR spectra were recorded on a Bruker Avance (Varian Unity Inova) 400 MHz spectrometer
using TMS as internal reference chemical shift in δ, ppm. Low resolution ESI-MS spectra were carried
out on a Waters triquadrupole mass spectrometer. High resolution Mass spectra were recorded on a
Waters Q-TOF Premier mass spectrometer.
3.2. Typical procedure for the preparation of 2-chloromethylquinazolinones 3a-3m
To a flask containing sodium (23 mg, 1 mmol) was added anhydrous MeOH (5 mL), then
chloroacetonitrile (0.95 mL, 15 mmol) with a syringe via a rubber septum, and the solution was stirred
at ambient temperature for about 40 min under nitrogen. A solution of appropriate o-aminobenzoic
acid 2 (5 mmol) in anhydrous MeOH (25 mL) was then added. The reaction mixture was stirred at this

Molecules 2010, 15
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temperature for about 2 h under nitrogen. The precipitate was collected by filtration, washed with
MeOH (8 mL), H2O (8 mL) and MeOH (5 mL), respectively, and then dried under vacuum at room
temperature to obtain the corresponding 2-chloromethylquinazolinone derivatives 3a-3l. The following
title compounds were prepared as described above:
2-Chloromethylquinazolin-4(3H)-one (3a). Prepared from o-anthranilic acid (2a) as a white solid; m.p.
249-250 °C; 1H-NMR (DMSO-d6): δ 12.59 (br s, 1H), 8.12 (dd, J = 1.2, 7.2 Hz, 1H), 7.84 (dd, J = 1.2,
7.2 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.56 (t, J = 8.0 Hz, 1H), 4.55 (s, 2H); 13C-NMR (DMSO-d6): δ
161.79, 152.62, 148.47, 134.88, 127.52, 126.14, 121.52, 106.89, 43.53; ESI-MS: m/z 195.03 (M+H+).
2-Chloromethyl-7-chloroquinazolin-4(3H)-one (3b). Prepared from 7-chloro-2-aminobenzoic acid (2b)
1
as a white solid; m.p. 232-234 °C; H-NMR (DMSO-d6): δ 12.72 (br s, 1H), 8.10 (d, J = 8 Hz, 1H),
13
7.75 (s, 1H), 7.58 (d, J = 8 Hz, 1H), 4.55 (s, 2H); C-NMR (DMSO-d6): δ 161.15, 154.13, 149.53,
139.44, 128.10, 127.68, 126.64, 120.30, 43.29; ESI-MS: m/z 227.05 (M-H+).
2-Chloromethyl-5-chloroquinazolin-4(3H)-one (3c). Prepared from 6-chloro-2-aminobenzoic acid (2c)
1
as a white solid; m.p. 220-221 °C; H-NMR (DMSO-d6): δ 12.63 (br s, 1H), 7.74 (t, J = 8.0 Hz, 1H),
13
7.60 (d, J = 8.0 Hz, 1H), 7.54 (d, J = 8.0 Hz, 1H), 4.51 (s, 2H); C-NMR (DMSO-d6): δ 159.87,
153.35, 150.91, 134.56, 132.78, 129.76, 127.00, 118.42, 42.97; ESI-MS: m/z 227.08 (M-H+).
2-Chloromethyl-6-bromoquinazolin-4(3H)-one (3d). Prepared from 5-bromo-2-aminobenzoic acid (2d)
as a white solid; m.p. 240-242 °C; 1H-NMR (DMSO-d6): δ 12.80 (br s, 1H), 8.19 (d, J = 2.0 Hz, 1H),
7.98 (dd, J = 2.4, 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 4.55 (s, 2H); 13C-NMR (DMSO-d6): δ 160.62,
153.21, 147.37, 137.63, 129.80, 128.23, 123.07, 119.97, 43.34; ESI-MS: m/z 271.01 (M-H+).
2-Chloromethyl-7-bromoquinazolin-4(3H)-one (3e). Prepared from 4-bromo-2-aminobenzoic acid (2e)
as a white solid; m.p. 238-240 °C; 1H-NMR (DMSO-d6): δ 12.77 (br s, 1H), 8.02 (d, J = 8.0 Hz, 1H),
13
7.91 (s, 1H), 7.72 (d, J = 8.0 Hz, 1H), 4.55 (s, 2H); C-NMR (DMSO-d6): δ 161.24, 154.01, 149.56,
130.42, 129.67, 128.38, 128.10, 120.55, 43.28; ESI-MS: m/z 270.99 (M-H+).
2-Chloromethyl-6-fluoroquinazolin-4(3H)-one (3f). Prepared from 5-fluoro-2-aminobenzoic acid (2f)
as a white solid; m.p. 239-241 °C; 1H-NMR (DMSO-d6): δ 12.75 (br s, 1H), 7.76 (m, J = 8.0 Hz, 3H),
4.56 (s, 2H); 13C-NMR (DMSO-d6): δ 161.81, 161.17, 159.36, 152.06, 145.27, 130.35, 123.36, 123.00,
122.80, 110.91, 43.40; ESI-MS: m/z 211.06 (M-H+).
2-Chloromethyl-7-fluoroquinazolin-4(3H)-one (3g). Prepared from 7-fluoro-2-aminobenzoic acid (2g)
as a white solid; m.p. 247-249 °C; 1H-NMR (DMSO-d6): δ 12.72 (br s, 1H), 8.18 (dd, J = 2.4, 8.0 Hz,
1H), 7.48 (dd, J = 2.0, 8.0 Hz, 1H), 7.42 (dd, J = 2.4, 8.0 Hz, 1H), 4.55 (s, 2H); 13C-NMR (DMSO-d6):
δ 167.19, 164.70, 161.04, 154.07, 150.58, 129.15, 118.45, 115.83, 112.57, 43.25; ESI-MS: m/z 213.03
(M+H+).

Molecules 2010, 15
9482
2-Chloromethyl-5-ethylquinazolin-4(3H)-one (3h). Prepared from 6-ethyl-2-aminobenzoic acid (2h) as
a white solid; m.p. 221-222 °C; 1H-NMR (DMSO-d6): δ 12.34 (br s, 1H), 7.69 (t, J = 8.0 Hz, 1H), 7.49
(d, J = 8.0 Hz, 1H), 7.30 (d, J = 8.0 Hz, 1H), 4.52 (s, 2H), 3.24 (q, J = 7.2 Hz, 2H), 1.19 (t, J = 7.2 Hz,
3H); 13C-NMR (DMSO-d6): δ 162.01, 152.11, 150.19, 140.63, 134.09, 128.54, 125.80, 119.03,
43.14，27.87, 16.46; ESI-MS: m/z 223.11 (M+H+).
2-Chloromethyl-8-methylquinazolin-4(3H)-one (3i). Prepared from 3-methyl-2-aminobenzoic acid (2i)
as a white solid; m.p. 246-248 °C; 1H-NMR (DMSO-d6): δ 12.59 (br s, 1H), 7.96 (d, J = 7.6 Hz, 1H),
7.69 (d, J = 7.2 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 4.56 (s, 2H); 13C-NMR (DMSO-d6): 162.05, 151.40,
146.88, 135.76, 135.28, 126.95, 123.75, 121.46, 43.83, 17.39; ESI-MS: m/z 207.05 (M-H+).
2-Chloromethyl-7-trifluoromethylquinazolin-4(3H)-one (3j). Prepared from 4-trifluoromethyl-2-
1
aminobenzoic acid (2j) as a white solid; m.p. 195-197 °C; H-NMR (DMSO-d6): δ 12.90 (br s, 1H),
8.31 (d, J = 8.0 Hz, 1H), 8.01 (s, 1H), 7.84 (d, J = 8.0 Hz, 1H), 4.58 (s, 2H); 13C-NMR (DMSO-d6): δ
168.95, 161.04, 154.38, 151.61, 148.50, 134.30, 127.81, 124.61, 124.34, 71.62, 43.24; ESI-MS: m/z
261.01 (M-H+).
2-Chloromethyl-6-hydroxyquinazolin-4(3H)-one (3k). Prepared from 5-hydroxy-2-aminobenzoic acid
(2k) as a white solid; m.p. > 250 °C; 1H-NMR (DMSO-d6): δ 12.40 (br s, 1H), 10.52 (s, 1H), 7.54 (d,
13
J = 8.4 Hz, 1H), 7.40 (d, J = 2.4 Hz, 1H), 7.26 (dd, J = 2.4, 8.4 Hz, 1H), 4.51 (s, 2H); C-NMR
(DMSO-d6): δ 161.59, 156.29, 149.20, 141.52, 129.21, 124.25, 122.51, 109.17, 43.70; ESI-MS: m/z
209.02 (M-H+).
2-Chloromethyl-6-nitroquinazolin-4(3H)-one (3l). Prepared from 5-nitro-2-aminobenzoic acid (2l) as a
white solid; m.p. 240-241 °C; 1H-NMR (DMSO-d6): δ 13.10 (br s, 1H), 8.79 (d, J = 2.0 Hz, 1H), 8.55
13
(dd, J = 2.4, 8.0 Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H), 4.60 (s, 2H); C NMR (DMSO-d6): δ 161.07,
156.31, 152.64, 145.50, 129.33, 122.31, 121.67, 43.25; ESI-MS: m/z 240.11 (M+H+).
2-Chloromethyl-6,7-dimethoxyquinazolin-4(3H)-one (3m). Prepared from 2-amino-4,5-dimethoxy-
benzoic acid (2m) as a white solid; m.p. 240-241 °C; 1H-NMR (DMSO-d6): δ 12.30 (br s, 1H), 8.68 (s,
13
1H), 8.34 (s, 1H), 4.54 (s, 2H), 3.97 (s, 3H), 3.83(s, 3H); C-NMR (DMSO-d6): δ 162.04, 157.31,
153.65, 146.54, 128.73, 120.37, 118.87, 56.12, 54.25, 43.15; ESI-MS: m/z 255.01 (M+H+).
3.3. One-pot synthesis of 2-hydroxymethylquiazolin-4(3H)-one (7)
To a flask containing sodium (23 mg, 1 mmol) was added anhydrous MeOH (5 mL), then
chloroacetonitrile (0.95 mL, 15 mmol) with a syringe via a rubber septum, and the solution was stirred
at ambient temperature for about 40 min under nitrogen. A solution of o-aminobenzoic acid (686 mg,
5 mmol) in anhydrous MeOH (25 mL) was then added. The reaction mixture was stirred at this
temperature for about 2 h under nitrogen, the execess MeOH was removed by evaporation in vaccum,
and the residue was added aqueous NaOH solution (2 mol/L, 10 mL), the mixture was stirred under
reflux for 1 h. After cooling to room temperature, the reaction mixture was neutralized with dil. HCl

Molecules 2010, 15
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solution, the precipitates were filtered, washed with cold MeOH to give the title compound 7 as a
white solid, yield: 60%; m.p. 278-279 °C; 1H-NMR (DMSO-d6): δ 11.94 (br s, 1H), 8.11 (d, J = 8 Hz,
1H), 7.82 (t , J = 7.2 Hz, 1H), 7.64 (d, J = 8 Hz, 1H), 7.51 (d, J = 7.2 Hz, 1H), 5.60 (s, 1H), 4.39 (s,
1H); 13C-NMR (DMSO-d6): δ 161.75, 157.05, 148.84, 143.60, 134.67, 127.16, 126.14, 121.54, 61.86;
ESI-MS: m/z 176.92 (M+H+).
3.4. General procedure for the preparation of 4-anilinoquinazoline derivatives 9 and 10
A mixture of 2-chloromethyl-7-fluoroquinazolin-4(3H)-one (3g, 2.551 g, 12 mmol) in anhydrous
toluene (60 mL) and DIPEA (4.2 mL, 24 mmol) contained in a 100 mL flask equipped with a
condenser and a drying tube was refluxed for 40 min. After cooling to room temperature and the
addition of POCl3 (2.2 mL, 24 mmol), the mixture was heated at 80 °C for 4 h. After cooling to room
temperature, the mixture was diluted with ethyl acetate (100 mL), washed successively with water,
saturated NaHCO3 and brine, the organic layer was dried by MgSO4, filtered and concentrated to
obtain the intermediate 8. The mixture of intermediate 8 (480 mg, 2 mmol) and appropriate aniline
(2.4 mmol) in isopropanol (20 mL) was stirred at 60 °C for 2-3 h and cooled to room temperature, the
precipitates were collected by filtration, washed with cold isopropanol, and dried at room temperature
for 24 h under vacuum to provide the corresponding product 9 or 10.
N-(3-chloro-4-fluorophenyl)-2-(chloromethyl)-7-fluoro-3,4-dihydroquinazolin-4-amine hydrochloride
1
(9). Yellow solid, yield: 73%; m.p. 204-206 °C; H-NMR (DMSO-d6): δ 11.31 (br s, 1H), 9.02 (t,
J = 7.2 Hz, 1H), 8.28 (dd, J = 2.4, 7.2 Hz, 1H), 7.93 (m, 1H), 7.74 (m, 2H), 7.52 (t, J = 8.8 Hz, 1H),
4.85 (s, 2H); 13C-NMR (DMSO-d6): δ 167.07, 164.53, 160.26, 158.88, 156.20, 153.76, 134.56,
128.88, 125.81, 124.48, 119.37, 117.87, 110.32, 107.00, 43.81; ESI-MS: m/z 340.07 (M+H+).
N-(3-bromophenyl)-2-(chloromethyl)-7-fluoro-3,4-dihydroquinazolin-4-amine hydrochloride (10).
1
Straw yellow solid, yield: 75%; m.p. 208-210 °C; H-NMR (DMSO-d6): δ 10.88 (br, s, 1H), 8.92 (t,
J = 7.6 Hz, 1H), 8.32 (s, 1H), 7.96 (m, 1H), 7.74 (dd, J = 2.4, 7.6 Hz, 2H), 7.42 (d, J = 7.6 Hz, 2H),
4.82 (s, 2H); 13C-NMR (DMSO-d6): δ 167.20, 164.66, 160.33, 159.07, 139.05, 130.78, 129.06,
128.81, 126.60, 122.87, 121.50, 118.02, 110.51, 107.06, 43.86; ESI-MS: m/z 366.03 (M+H+).
3.5. In vitro assay for cytotoxic activity (MTT assay)
Materials
Methylthiazolyldiphenyl-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) were purchased
from Sigma (St. Louis, MO, USA). HepG2 (ATCC Accession No. HB-8065), MDA-MB-468 (ATCC
Accession No. HTB-132) and HCT-116 (ATCC Accession No. CCL-247) were obtained from
American Type Culture Collection (ATCC, Rockville, MD, USA) and were grown as monolayers in
Dulbecco’s modified Eagle medium or RPMI 1640 medium (Gibco BRL, Grand Island, N.Y., USA).
These cells were supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Auckland,
N.Z.), 100 units/mL penicillin, 100 units/mL streptomycin, at 37 °C, 95% relative humidity, under 5%

Molecules 2010, 15
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CO2. The cytotoxicity effects of these compounds on various cancer cells were determined by the
MTT assay, and the results were expressed as the IC50, that were means calculated from three
independent experiments. Briefly, cells (2000/well) were seeded in 96-well plates and cultured for
24 h, followed by treatment with the compounds for 48 h. Ten microliters of 10 mg/mL MTT was
added per well and incubated for another 2.5 h at 37 °C. Then the supernatant fluid was removed and
150 μL/well DMSO was added for 15-20 minutes. The absorbance (OD) of each well was measured at
570 nm, using a SpectraMAX M5 microplate spectrophotometer (Molecular Devices).
4. Conclusions
In conclusion, an improved and efficient procedure for the synthesis of 2-chloromethyl-4(3H)-
quinazolinones 3 has been described. The results indicated that: 1) increasing the amount of
chloroacetonitrile improved the yields remarkably; 2) the convenient one-pot process toward
2-hydroxymethyl-4(3H)-quinazolinone allowed the two-step reaction could be done in a single
reaction vessel, without isolation and purification the intermediate other than a simple filtration and
washing. Moreover, two 4-anilinoquinazoline analogs substituted with chloromethyl groups at the 2-
position were synthesized and showed promising anticancer activity in vitro, which deserves
further studies.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (NSFC-81000982).
We thank the Analytical and Testing Center of Sichuan University for NMR measurements.
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Sample Availability: Samples of the compounds 3a-3m, 9 and 10 are available from the authors.
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