Synthesis and antiviral bioactivities of 2-aryl- or 2-methyl-3- (substituted- benzalamino)-4(3H)-quinazolinone derivatives

Article abstract of DOI:10.3390/12122621

A simple and general method has been developed for the synthesis of various 4(3H)-quinazolinone derivatives by the treatment of the appropriate 3-amino-2-aryl-4(3H)-quinazolinone with a substituted benzaldehyde in ethanol. The structures of the compounds were characterized by elemental analysis, IR, ¹H-NMR and ¹³C-NMR spectra. The title 2-aryl- or 2-methyl-3-(substituted-benzalamino)-4(3H)-quinazolinone compounds III-1-III-31 were found to possess moderate to good antiviral activity. Semi-quantitative PCR and Real Time PCR assays were used to ascertain the target of action of compound III-31 against TMV. The studies suggest that III-31 possesses antiviral activity due to induction of up-regulation of PR-1a and PR-5, thereby inhibiting virus proliferation and movement by enhancement of the activity of some defensive enzyme.

Full text of DOI:10.3390/12122621

Molecules 2007, 12, 2621-2642
molecules
ISSN 1420-3049
2007 by MDPI
www.mdpi.org/molecules
©
Full Paper
Synthesis and Antiviral Bioactivities of 2-Aryl- or
-Methyl-3-(substituted- Benzalamino)-4(3H)-quinazolinone
2
Derivatives
Xingwen Gao, Xuejian Cai, Kai Yan, Baoan Song*, Lili Gao and Zhuo Chen
Center for Research and Development of Fine Chemicals, Guizhou University, Key Laboratory of
Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guiyang, 550025, P. R.
China. E-mails: chem3333@yahoo.com.cn; alpsjian@163.com; lk81@163.com; glili1983@sina.com;
gychenzhuo@yahoo. com.cn
*
Author to whom correspondence should be addressed; E-mail: songbaoan22@yahoo.com;
Tel.:(+86) 851 3620521, Fax: (+86) 851 3622211.
Received: 8 November 2007; in revised form: 17 December 2007 / Accepted: 25 December 2007 /
Published: 27 December 2007
Abstract: A simple and general method has been developed for the synthesis of various
4
(3H)-quinazolinone derivatives by the treatment of the appropriate 3-amino-2-aryl-4(3H)-
quinazolinone with a substituted benzaldehyde in ethanol. The structures of the compounds
1
13
were characterized by elemental analysis, IR, H-NMR and C-NMR spectra. The title 2-
aryl- or 2-methyl-3-(substituted-benzalamino)-4(3H)-quinazolinone compounds III-1~III-
3
1 were found to possess moderate to good antiviral activity. Semi-quantitative PCR and
Real Time PCR assays were used to ascertain the target of action of compound III-31
against TMV. The studies suggest that III-31 possesses antiviral activity due to induction
of up-regulation of PR-1a and PR-5, thereby inhibiting virus proliferation and movement
by enhancement of the activity of some defensive enzyme.
Keywords: 4(3H)-Quinazolinone, Schiff base, synthesis, antiviral activity

2
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Molecules 2007, 12
Introduction
4
(3H)-Quinazolinones and their derivatives constitute an important class of heterocyclic
compounds. They occupy an important position in medicinal and pesticide chemistry, presenting a
wide range of bioactivities. As medicines, many of them display antifungal [1], antimicrobial [2], anti-
HIV [3], antitubercular [4], anticancer [5], antiinflammatory [6], anticonvulsant [7], antidepressant [8],
hypolipidemic [9], antiulcer [10], analgesic [11] or immunotropic activities [12] and are also known to
act as thymidyalate synthase [13], poly(ADP-ribose) polymerase (PARP) [14], and protein tyrosine
kinase [15] inhibitors. As pesticides, they are used as insecticides [16], fungicides [17] and antiviral
agents [18] such as TMV , CMV inhibitors. In light of the growing number of applications in recent
years there has been an enormous increase in the interest among biologists and chemists in their
synthesis and bioactivity of quinazoline derivatives. In our previous work in this area we reported that
some of these compounds showed antifungal activities [17]. Nanda and his co-workers synthesized ten
3
-(arylideneamino)-2-phenylquinazoline-4(3H)-ones, which were investigated for their antimicrobial
activity against both Gram-positive (Staphylococcus aureus 6571 and Bacillus subtilis) and Gram-
negative bacteria (Escherichia coli K12 and Shigella dysenteriae 6) using a turbidometric assay
method. It was found that the incorporation of the 3-arylideneamino substituent enhanced the anti-
bacterial activity of the quinazolone system [19]. However, no attention has been paid to the antiviral
activities for 3-(arylideneamino)-2-phenylquinazoline-4(3H)-ones against TMV (tobacco mosaic virus).
For this report we have designed and synthesized a series of 2-aryl- or 2-methyl-3-(substituted-
benzalamino)-4(3H)-quinazolinone derivatives III-1~III-31 and investigated their antiviral
bioactivities. The synthetic route used is shown in Scheme 1. Substitution patterns and optimized
yields are given in Table 3. The structures of these compounds were firmly established by well defined
1
13
IR, H-NMR, C-NMR data and elemental analysis. Preliminary bioassay tests showed that some
compounds displayed in vivo antiviral activity against TMV at 500 μg/mL.
Scheme 1. Synthetic route to the title compounds.
O
O
R
NH2
R
R
CO H
2
80% N H H O
2 4. 2
2
N
R COCl
O
Pyridine
R2
R2
N
Pyridine
NH2
II
I
(
R=H, 6-Br)
(
(
R=H,6-Br)
2
R =Ph,Me, 4-pyridinyl)
2
R =Ph,Me, 4-pyridinyl)
CHO
O
N
R1
R1
R
N
N
R2
III
III-1-III-31)
(

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Molecules 2007, 12
Results and Discussion
Different homologues of 3-amino-2-aryl-4(3H)-quinazolinone II were prepared following a
literature procedure [20]. Reaction conditions were non-homogeneous and the use of an increased
amount of hydrazine hydrate did not afford successful results. The reaction conditions for the synthesis
1
2
of II-1 (R=H, R =H, R =Ph) were optimized in various solvents at different temperatures for different
times and the results are shown in Table 1. An 87 % yield could be obtained when the reaction mixture
was heated in pyridine at 116 °C for 0.5 h.
1
2
Table 1. The effect of reaction conditions on yield of intermediate II-1 (R=H, R =H, R =Ph).
Reaction
Reaction time
Entry
Solvent
Yield (％)
temperature (°C)
(h)
1
2
3
4
5
6
Anhydrous ethanol
Isopropanol
n-Butanol
-
78
3
24
31
35
22
87
89
85
3
3
117
118
116
116
3
Pyridine
0.5
3
Pyridine
In order to optimize the reaction conditions for the title compounds, the synthesis of III-1 (R=H,
R =3, 5-di-chloro，R =Ph) was examined under different conditions. First, the role of the catalyst
HOAc) in accelerating the reaction rate was ascertained. While in the presence of catalyst, a 72%
1
2
(
yield of III-1 was achieved in only 10 h (Table 2, entry 1), a yield of only 52% was obtained with a
prolonged reaction period of 30 h in the absence of the catalyst (Table 2, entry 2). In addition, we also
examined the effects of reaction time. When the reaction time was prolonged further to 20 h, no
improvement (75 %) was obtained (Table 2, entry 3), as compared to that of 10 h (72 %). Also, it could
be observed that the yield was significantly lower at room temperature (Table 2, entry 4). For different
substituted benzaldehydes, under optimal conditions, as depicted in Table 3, III-1-III-31 could be
obtained in 53-89% yields at 78 °C in ethanol in presence of HOAc as the catalyst.
Table 2. The effect of reaction conditions on yield of title compound III-1.
Reaction
Reaction time
Entry
Catalyst
Yield (％)
temperature (°C)
(h)
10
30
20
10
1
2
3
4
HOAc
-
78
78
78
r.t
71.9
52.4
74.8
22.1
HOAc
HOAc

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Molecules 2007, 12
Table 3. Substituents and yields of title compounds III-1-III-31 under optimized conditions.
1
2
Compound No.
R
H
R
R
Yields (%)
79
III-1
III-2
3,5-Cl
3-NO
Ph
H
2
-4-Cl
Ph
74
III-3
H
4-CF
3-Cl
3
Ph
72
III-4
H
Ph
71
III-5
H
2,3-Cl
2,6-Cl
3,4-F
Ph
70
III-6
H
Ph
78
III-7
H
Ph
69
III-8
H
3-CF
H
3
Ph
74
III-9
H
4-Pyridinyl
4-Pyridinyl
4-Pyridinyl
4-Pyridinyl
Me
67
III-10
III-11
III-12
III-13
III-14
III-15
III-16
III-17
III-18
III-19
III-20
III-21
III-22
III-23
III-24
III-25
III-26
III-27
III-28
III-29
III-30
III-31
H
2-Cl
4-F
69
H
89
H
2,3-Cl
2-F
89
6-Br
6-Br
6-Br
6-Br
6-Br
6-Br
6-Br
6-Br
6-Br
6-Br
6-Br
6-Br
6-Br
6-Br
6-Br
6-Br
6-Br
6-Br
H
59
3-F
Me
63
4-F
Me
68
2-CF
3-Cl
3
Me
66
Me
63
2,4-Cl
2,6-Cl
3,4-F
Me
72
Me
70
Me
64
2-Cl-5-NO
4-Cl-3-NO
2
Me
70
2
Me
68
2-F-3-CF
3,4-OMe
2,3-OMe
2,5-OMe
3
Me
64
Me
66
Me
61
Me
63
3-NO
2-OH
2
Me
56
Me
53
2,4-OMe
Me
62
5-Cl-3-OH
2,3-Cl
Me
64
Me
87
1
Representative analysis of spectral data: IR and H-NMR of compound III-3.
14 3 3
The IR spectrum of III-3 (molecular formula C22H F N O, m.w. 393.37, structure and carbon
-1
numbering in Figure 1) over the 3060-3032 cm range showed multiple weak absorption peaks
corresponding to Qu-H and Ar-H stretching vibration absorption peaks. The strong absorption at 1671
-
1
-1
cm is due to the C=O stretching vibration and the moderate intensity absorption at 1607 cm
-1
corresponds to a C=N stretching vibration. The 1591~1474 cm absorptions are due to the skeleton
-1
vibration of the aryl and heterocyclic rings. The series of bands in the 1319, 1174, 1125 cm region
-1
correspond to the para C-F stretching of the imine-Ar-CF group, the one at 843 cm is due to a
methylene linked to the benzene ring at the substituted 4-position. The absorption peaks at 766 and
-1
6
96 cm arise due to the phenyl-substituted at the 2-position in the quinazolinone.

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Molecules 2007, 12
Figure 1. The structure and carbon atom numbering of compound III-3
14
1
3 CF3
15
O
4
7
3
8
N
12
5
6
2 N
1
0
9
11
16
2
1
N
1
17
20
19
18
It can be seen from the chemical structure that different pairs of carbons e.g. C-11 and C15, C-12
and C-14, C-17 and C-21, C-18 and C-20 are attached to chemically equivalent protons. The proton
attached at C-5 position appears as a multiplet at δ 7.46-7.58 ppm due to mutual coupling with C4-H
and C6-H. Chemical shift in the aromatic region with a multiplet centered at δ 7.63 ppm corresponds
to C4-H and the multiplet in the region 7.80 - 7.94 ppm arises due to the five aromatic protons present
in the phenyl ring directly attached to the quinazolinone ring. The multiplet at 7.70-7.73 ppm must be
for the two equivalent protons at C-11 and C-15. The relatively downfield multiplet at 8.19-8.26 ppm
which integrates up to 2H corresponds to the protons in the vicinity of trifluoromethyl group, i.e.
attached to C-12 and C-14. The singlet at 9.25 ppm appears due to the proton attached to the imine
carbon at C-9.
1
3
C-NMR of compound III-8
The chemical structure of compound III-8 and the assignment of carbon atoms are shown in Figure
2
. This compound (molecular formula C22
14 3 3
H F N O, m.w. 393.37) contains 22 carbon atoms. Among
1
3
them, there are two equivalent pairs, C-17, C-21 and C-18,C-20. The C chemical shift values of the
2
0 non equivalent carbons are assigned and shown in Table 4.
Figure 2. The structure and carbon atoms assignment of compound III-8.
14
1
5
13
CF3
O
2
4
7
3
8
N
5
6
N
1
0
12
9
11
1
6
22
2
1
N 1
1
7
20
19
1
8
1
3
Table 4. C-NMR analysis of compound III-8.
Chemical shift
Carbon atom No.
(
δ, ppm)
158.4
167.2
121.6
130.3
128.2
135.1
C-1
C-2
C-3
C-4
C-5
C-6

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Molecules 2007, 12
Table 4. Cont.
C-7
123.2
153.7
146.7
135.4
127.4
132.6
129.3
130.5
134.1
130.1
127.8
130.4
131.0
125.4
C-8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-22
Compound III-8 essentially has three parts. The chemical shifts of the quinazolinone ring carbons
vary from δ = 167.2 to 121.6 ppm. The carbon nuclei under the influence of a strong electronegative
environment appear downfield, e.g. the C-2 carbonyl, which is directly linked to the ring nitrogen has a
chemical shift value of δ =167.2 ppm, whereas the C-1, linked to two ring nitrogen atoms, appears at
1
58.4 ppm. The chemical shift of the ring carbon at C-8 is affected by the presence of the directly
attached ring nitrogen atom and appears at δ =153.7 ppm. The imine carbon at C-9, conjugated to the
phenyl ring, records a downfield chemical shift at 146.7 ppm. The carbons of the phenyl ring
conjugated to the imine functionality have chemical shifts in the 127.4 to 135.4 ppm range. The
trifluoromethyl carbon present on the side chain appears at 125.4 ppm, under the strong electron
withdrawing influence exerted by the three fluorine atoms. The carbons of the unsubstituted phenyl
ring directly linked to the quinazolinone ring through the intervening carbon between two ring
nitrogens appear upfield (127.8 ppm-131.0 ppm) compared to those of quinazolinone ring carbons at
C1, C2 and C8. The equivalent C17,C-21 and C-18,C-20 have chemical shift values δ = 127.8 and
1
30.4 ppm respectively.
Antifungal activity
The antiviral bioassay results are given in Table 5. It could be seen that these newly synthesized
derivatives exhibit weak to good antiviral activities. At 500 μg/mL, the title compounds exhibited
weak activities against Fusarium oxysporum, Valsa mali and Gibberella Zeae, which are lower than
that of a hymexazol standard.
Antiviral activity
The in vivo bioassay results against TMV are given in Table 6. Ningnanmycin was used as
reference antiviral agent. It was found that these compounds exhibit certain activities against TMV in
vivo. The compounds III-31, III-16, III-3, III-1 and III-8 have relatively higher curative effect than

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Molecules 2007, 12
the other compounds III-2, III-4, III-5, III-6, III-7, III-9-III-15, III-17 and III-30. The data listed in
Table 6 indicate that antiviral activity depends on the nature of the substituents present in the title
compound. When R was 4-CF , the title compounds III-31 and III-16 showed curative rates of 55 and
3
54%, which was slightly higher than that of reference (54%) against TMV at 500 µg/mL. The other
compounds displayed a slightly lower antiviral activity than that of the reference.
Table 5. Inhibitory effects on phytopathogenic fungi.
Fusarium
Gibberella
Compd (50 μg/mL)
Valsa mali
oxysporum
17.5±0.77
6.14±0.50
1.59±0.68
2.90±0.88
10.2±0.62
5.68±0.91
1.90±0.38
5.68±0.69
4.09±0.51
5.90±0.77
9.54±0.95
8.89±0.37
17.5±3.25
13.9±3.92
﹣0.89±3.22
12.4±3.69
14.2±3.40
12.4±3.80
16.3±3.14
17.0±1.42
20.11±2.99
9.00±3.11
19.91±2.88
19.00±2.77
16.9±1.11
21.1±1.89
26.7±7.55
28.8±3.09
39.7±3.01
29.4±9.99
12.8±1.19
68.7±3.59
zeae
III-1
III-2
11.3±0.29
1.07±0.32
-3.23±0.54
3.78±0.94
6.45±0.52
0.00±0.55
3.09±0.94
2.69±0.94
4.77±0.71
7.12±0.99
6.18±0.26
4.77±0.55
7.55±1.70
5.03±2.06
5.03±1.84
1.41±1.74
3.14±1.7
6.75±0.84
1.23±0.51
2.45±0.37
2.00±0.55
-0.31±1.49
9.51±1.15
5.66±0.30
5.52±0.52
0.90±0.12
1.09±0.33
1.23±0.81
7.98±0.86
-0.81±1.79
0±1.51
III-3
III-4
III-5
III-6
III-7
III-8
III-9
III-10
III-11
III-12
III-13
III-14
III-15
III-16
III-17
III-18
III-19
III-20
III-21
III-22
III-23
III-24
III-25
III-26
III-27
III-28
III-29
III-30
III-31
Hymexazol
-1.35±2.50
1.89±1.59
4.04±1.45
3.50±1.60
2.43±1.54
8.09±1.09
6.78±2.09
3.09±1.01
10.32±4.92
6.77±1.99
4.93±1.09
7.68±0.96
4.99±1.40
7.77±2.10
6.66±1.08
20.5±3.33
3.04±2.19
67.4±2.06
6.60±1.53
11.0±1.31
9.00±1.0
15.22±2.11
24.21±1.99
26.01±6.43
17.70±3.44
19.0±1.22
9.99±0.77
3.97±1.18
34.1±10.1
22.30±1.55
10.4±2.90
3.12±0.84
62.2±1.86

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Molecules 2007, 12
Table 6. The curative effects of the new compounds III against TMV in vivo.
Agents
Ningnanmycin III-1
III-2
50
III-3
500
III-4
500
III-5
500
Concentration (mg/L)
Inhibition rate (%)
Agents
500
500
53.5
III-6
500
51.1
III-7
500
25.3
III-8
500
52.9
III-9
500
44.7
III-10
500
37.9
III-11
500
Concentration (mg/L)
Inhibition rate (%)
Agents
37.4
III-12
500
49.8
III-13
500
50.4
III-14
500
27.1
9.30
50.4
III-17
500
III-15 III-16
Concentration (mg/L)
Inhibition rate (%)
Agents
500
500
22.1
III-18
500
23.4
III-19
500
36.6
III-20
500
40.4
54.0
42.8
III-23
500
III-21 III-22
Concentration (mg/L)
Inhibition rate (%)
Agents
500
500
45.6
III-24
500
41.4
III-25
500
24.6
III-26
500
26.1
34.8
39.9
III-29
500
III-27 III-28
Concentration (mg/L)
Inhibition rate (%)
Agents
500
500
43.2
III-30
500
40.0
III-31
500
29.9
41.0
41.8
38.9
Concentration (mg/L)
Inhibition rate (%)
44.6
55.4
Product of PCR in PR-1a and PR-5 and its sequence identification
By following RT-PCR, the PCR product produced in 35 cycles was resolved on a 1.5% agarose gel.
The purified products of PCR were sequenced by model ABI 310 DNA sequencer and were identified
as the target gene by BLAST searching in Genbank (Figure 3).
Gene expression analysis of PR-1a and PR-5 in III-31-treated tobacco leaf
In vitro synthesized single-stranded cDNA from RNA samples were isolated from leaves of water-
treated tobacco and the TMV-treated tobacco and the III-31-and TMV-treated tobacco. The differential
expression analysis of the PR-1a and PR-5 gene was determined by semi-quantitative PCR and the
relative quantification real-time PCR analysis. The mRNAs of PR-1a and PR-5 gene accumulated to
detectable levels in III-31-and TMV-treated tobacco leaf, while no-detectable levels were reached in
water-treated tobacco and the TMV-treated tobacco. The mRNA content of III-31-treated tobacco leaf
th
for PR-1a gene started to increase after five days and reached a peak at the end of the 5 day before
falling to the normal level. In contrast, in TMV-treated tobacco leaf, no significant increase in the
levels of gene expression was noticed (Figures 4-A, 5-A). The expression levels of PR-5 gene in III-
th
3
1-treated tobacco rapidly increased and reached a peak within 5 day after the inoculation and then
started to decrease gradually. As depicted in Figure 4-B and Figure 5-B, III-31 treated tobacco leaves
showed significant enhancement in the levels of gene expression as compared to TMV-treated tobacco

2
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Molecules 2007, 12
th
leaves within 5 day after the inoculation. In contrast, in TMV-treated tobacco leaf, no significant
increase in the levels of gene expression was observed.
Figure 3. The clone of PR-1a and PR-5 genes

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Molecules 2007, 12
Figure 4. The real time PCR effects of PR-1a gene.
Conclusions
In summary, the presented new method of preparation of 4(3H)-quinazolinone Schiff base
derivatives from appropriate 3-amino-2-aryl-4(3H)-quinazolinones and substituted benzaldehydes in
ethanol is convenient, rapid and gives moderate yields. It was also found that the title compounds III-
3
1, III-1, III-3, III-8, III-16 displayed good antiviral activity. Semi-quantitative PCR and real time
PCR assay were evaluated to ascertain the target of action of compound III-31 against TMV. The
studies suggest that III-31 possesses antiviral activity by induction up-regulation of PR-1a, PR-5
thereby inhibiting proliferation and movement of virus, by enhancing activity of some defensive
enzyme.

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Molecules 2007, 12
Figure 5. The real time PCR effects of PR-5 gene
Experimental
General
The melting points of the products were determined on a XT-4 binocular microscope (Beijing Tech
Instrument Co., China) and were not corrected. The IR spectra were recorded on a Bruker VECTOR
1
13
2
2 spectrometer on KBr disks. H- and C-NMR experiments (solvent DMSO-d
6
) were recorded at
500 and 125 MHz, respectively, on a JEOL-ECX 500 NMR spectrometer at room temperature using
TMS as an internal standard. Elemental analysis was performed on an Elementar Vario-III CHN
analyzer. All reagents employed were of analytical reagent grade or chemically pure. All solvents were
dried, deoxygenated and redistilled before use.
2
Preparation of 2-aryl-4(3H)-3,1-benzoxazinone I (R=H, R =Ph)
To a stirred solution of anthranilic acid (0.05 mole) in pyridine (60 mL), Benzoyl chloride (0.05
mole) was added dropwise, maintaining the temperature near 0-5 °C for 1 hour. The reaction mixture
was stirred for another 2 hours at room temperature until a solid product was formed. The reaction
mixture was neutralized with saturated sodium bicarbonate solution and the pale yellow solid which
separated was filtered, washed with water and recrystallised from ethanol. Yield 83 %, mp. 113-

2
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Molecules 2007, 12
-1
1
1
15 °C (lit [20] 114 °C); IR: ν 3030 (C-H, Ar-H), 1721 (C=O), 1595 (C=N), 1180 (C-O) cm ; H-
NMR: δ 7.61 (m, 9H, Ar-H, quinazolinone-H).
2
Preparation of 3-amino-2-aryl-4(3H)-quinazolinone II-1 (R=H, R =Ph)
To a stirred solution of I (0.05 mole) in pyridine (20 mL), 80% N H •H O (0.15 mole) was added.
2
4
2
The reaction mixture was stirred and refluxed for 20 minutes at 117 °C. After cooling, the crude
product was obtained by filtration and recrystallized from ethanol to afford II as a white solid, yield
8
8%; m.p. 177-178 °C (lit [21] 178-179 °C); IR: ν 3448 (NH ), 3030 (C-H, Ar-H), 1685 (C=O), 1598
2
-1
1
(
2
C=N) cm ; H-NMR: δ 7.92 (m, 9H, Ar-H, quinazolinone-H), 5.67 (s, 2H, 3-quinazolinone-NH ),
13
7.48-8.19 (m, 9H, Ar-H, quinazolinone-H); C-NMR: δ 161.7, 156.3, 147.2, 135.4, 134.8, 130.1,
1
30.0, 127.9, 127.3, 126.6, 120.6.
Preparation of Schiff base derivatives III-1~III-31
To a solution of the appropriate substituted benzaldehyde (1.0 mmol) in ethanol (15 mL), were
added the 3-amino-2-aryl-4(3H)-quinazolinone (1.0 mmol) and a few drops of acetic acid (0.05 mmol).
The reaction mixture was refluxed for 3-24h and the course of the reaction was monitored by TLC
[
petroleum ether/ethyl acetate (V/V=1:2)] to its completion. The reaction mixture was cooled. The
crude product was recrystallized from 95% ethanol to give title compounds III-1~III-31.
2
-Phenyl-3-(3,5-di-chlorobenzalamino-4(3H)-quinazolinone (III-1). White solid, yield 79%; m.p.
-1
2
18~220 °C; IR (cm ): ν 3088 (quinazolinone-H, Ar-H), 1678 (C=O), 1622, 1590 (C=N), 1551-1489
(
C=C, benzene and quinazolinone rings), 875, 775, 688 (1,3,5-substituted benzene), 748, 700 (mono
1
substituted benzene); H-NMR: δ 7.45-8.25 (m, 12H, quinazolinone-H, Ar-H), 9.35 (s, 1H, N=CH);
1
3
C-NMR: δ 170.0, 158.3, 153.6, 146.9, 135.2, 135.1, 133.2, 132.8, 130.4, 130.3, 129.6, 129.1, 128.2,
28.1, 127.7, 127.3, 121.6; Anal. calcd. for C21 O (394.26): C, 63.98%; H, 3.32%; N, 10.66%.
Found: C, 63.86%;, H, 3.22%; N, 10.58%.
1
2 3
H13Cl N
2
-Phenyl-3-(3-nitro-4-chlorobenzalamino)-4(3H)-quinazolinone (III-2). White solid, yield 74%; m.p.
-1
2
09~211 °C; IR (cm ): ν 3057 (quinazolinone-H, Ar-H), 1681 (C=O), 1605, 1589 (C=N), 1568-1445
(
(
C=C, benzene and quinazolinone rings), 889, 830 (1,2,4-trisubstituted benzene), 768, 692
1
monosubstituted benzene); H-NMR: δ 7.28-8.26 (m, 12H, quinazolinone-H and Ar-H), 9.31 (s, 1H,
1
3
N=CH); C-NMR: δ 163.3, 162.7, 161.2, 158.4, 146.8, 135.5 (J
3
= 8.8 Hz), 135.2 (J
= 11.3 Hz), 127.9, 127.8, 127.7, 127.4, 127.3, 125.8,
= 8.8 Hz); Anal. calcd. for C21 (404.85): C, 42.25%;
3
= 6.5 Hz), 134.8,
1
30.3 (J
2
= 12.5 Hz), 130.1, 130.0, 128.2 (J
= 10.0 Hz), 117.0 (J
3
1
21.6, 120.6 (J
2
2
4 3
H13CIN O
H, 3.21%; N, 13.83%. Found: C, 42.30%; H, 3.15%; N,13.75%.
2
1
1
-Phenyl-3-(4-trifluoromethylbenzalamino)-4(3H)-quinazolinone (III-3). White solid, yield 72%; m.p.
-1
79.4~181.0 °C; IR (cm ): ν 3061 (quinazolinone-H, Ar-H), 1672 (C=O), 1605, 1591 (C=N), 1564-
445 (C=C, benzene and quinazolinone rings), 843 (1,4-disubstituted benzene), 766, 696
1
(
monosubstituted benzene); H-NMR: δ 5.68 (s, 2H), 7.42-8.28 (m, 13H, quinazolinone-H and Ar-H),
1
3
9
3
.48 (s, 1H, N=CH); C-NMR: δ 164.4, 157.8, 153.0, 146.1, 134.9, 134.6 (J = 3.8 Hz), 133.9, 130.2,

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1
29.9, 129.7 (J
2 2
= 12.5 Hz), 127.8, 127.6 (J = 12.5 Hz), 127.1, 126.8, 121.0; Anal. Calcd. for
22 14 3 3
C H F N O (393.37): C, 67.17%; H, 3.59%; N, 10.68%. Found: C, 67.41%; H, 4.09%; N, 10.86%.
2
1
1
6
1
1
C
-Phenyl-3-(3-chlorobenzalamino)-4(3H)-quinazolinone (III-4). White solid, yield 71%; m.p.
-1
90.0~192.1 °C; IR (cm ): ν 3059 (quinazolinone-H, Ar-H), 1680 (C=O), 1605, 1584 (C=N),
559,1474 (C=C, benzene and quinazolinone rings), 903, 775, 690 (1,3-disubstituted benzene), 765,
1
92 (monosubstituted benzene); H-NMR: δ 7.45-8.26 (m, 13H, quinazolinone-H and Ar-H), 9.12 (s,
1
3
2
H, N=CH); C-NMR: δ 167.2, 157.7, 153.0, 146.1, 134.7, 134.4 (J = 7.5 Hz), 133.7, 132.1, 131.0,
29.8, 129.6, 127.6 (J = 7.2 Hz), 127.5, , 127.1 (J = 5.0 Hz), 126.7, 120.8; Anal. Calcd. for
2
2
21
H14ClN
3
O (359.82): C, 70.10%; H, 3.92%; N, 11.68%. Found: C, 69.99%; H, 4.19%; N, 11.78%.
2
-Phenyl-3-(2,3-dichlorobenzalamino)-4(3H)-quinazolinone (III-5). White solid, yield 70%; m.p.
-1
2
07~208 °C; IR (cm ): ν 3061 (quinazolinone-H, Ar-H), 1684 (C=O), 1603, 1587 (C=N), 1551-1445
(
(
C=C, benzene and quinazolinone rings), 764, 696 (1,2,3-trisubstituted benzene), 768.0, 690.5
1
monosubstituted benzene); H-NMR: δ 7.42-8.28 (m, 13H, quinazolinone-H and Ar-H), 9.48 (s, 1H,
1
3
N=CH); C-NMR: δ 164.4, 157.8, 153.0, 146.1, 134.9, 134.6 (J
3
= 3.8 Hz), 133.9, 130.2, 129.9, 129.7
= 12.5 Hz), 127.1, 126.8, 121.0; Anal. Calcd. for C21
394.26): C, 63.98%; H, 3.32%; N, 10.66%. Found: C, 63.94%; H, 3.18%; N, 10.83%.
(
(
J
2
= 12.5 Hz), 127.8, 127.6 (J
2
2 3
H13Cl N O
2
-Phenyl-3-(2,6-dichlorobenzalamino)-4(3H)-quinazolinone (III-6). White solid, yield 78%; m.p. 179-
-1
1
80.3 °C; IR (cm ): ν 3055 (quinazolinone-H, Ar-H), 1680 (C=O), 1607, 1587 (C=N), 1566-1472
(
(
C=C, benzene and quinazolinone rings), 770, 690 (1,2,3-trisubstituted benzene), 766, 690
1
monosubstituted benzene); H-NMR: δ 7.43-8.28 (m, 12H, quinazolinone-H and Ar-H), 9.37 (s, 1H,
1
3
N=CH); C-NMR: δ 166.1, 158.1, 153.8, 146.7, 135.3, 135.0, 133.4, 130.2, 130.1, 130.0, 129.3, 128.3,
28.1, 127.8, 127.5, 121.8; Anal. Calcd. for C21 O (394.26): C, 63.98%; H, 3.32%; N, 10.66%.
Found: C, 63.66%;, H, 3.62%; N, 10.58%.
1
2 3
H13Cl N
2
1
1
-Phenyl-3-(3,4-difluorobenzalamino)-4(3H)-quinazolinone (III-7). White needles, yield 69%; m.p.
-1
37.6~139.6 °C; IR (cm ): ν 3057 (quinazolinone-H, Ar-H), 1678 (C=O), 1603, 1591 (C=N), 1566-
445 (C=C, benzene and quinazolinone rings), 881, 830 (1,2,4-trisubstituted benzene), 770, 692
1
(
monosubstituted benzene); H-NMR δ: 7.45-8.25 (m, 13H, quinazolinone-H and Ar-H), 9.11 (s, 1H,
1
3
N=CH); C-NMR δ: 170.0, 161.7, 158.3, 156.3, 153.6, 151.4, 147.2, 146.7, 135.4 (J = 13.8 Hz),
2
1
1
34.9 (J
2
= 28.9 Hz), 130.6-130.0 (m), 128.2-127.8 (m), 127.3 (J = 7.5 Hz),
2
= 10.0 Hz), 126.9 (J
3
26.6, 121.5, 120.6, 119.1 (J
2
= 13.8 Hz), 117.3 (J = 17.6 Hz); Anal. Calcd. for C21H F N O (361.35):
2
13 2 3
C, 69.80%; H, 3.63%; N, 11.63%. Found: C, 69.38%; H, 3.28%; N, 11.09%.
2
-Phenyl-3-(3-trifluoromethylbenzalamino)-4(3H)-quinazolinone (III-8). White solid, yield 74%; m.p.
-1
1
84~186 °C; IR (cm ): ν 3060 (quinazolinone-H, Ar-H), 1670 (C=O), 1603, 1590 (C=N), 1564, 1445
(
C=C, benzene and quinazolinone rings), 903 (1,3-disubstituted benzene), 766, 696 (mono- substituted
1
benzene); H-NMR: δ 5.60 (s, 2H, ), 7.38-8.19 (m, 13H, quinazolinone-H and Ar-H), 9.40 (s, 1H,
1
3
N=CH); C-NMR: δ 158.4, 157.8, 153.0, 146.1, 134.9, 134.6 (J = 3.8 Hz), 133.9, 130.2, 129.9, 129.7
3
(
(
J
2
= 12.5 Hz), 127.8, 127.6 (J
2 14 3 3
= 12.5 Hz), 127.1, 126.8, 121.0; Anal. calcd. for C22H F N O
393.37): C, 67.17%; H, 3.59%; N, 10.68%. Found: C, 67.22%; H, 3.60%; N, 10.80%.

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2
-(4-Pyridinyl)-3-(benzalamino)-4(3H)-quinazolinone (III-9). White solid, yield 67%; m.p.
-1
2
25~226 °C; IR (cm ): ν 3042 (ArH), 1682 (C=O), 1607, 1585 (C=N), 1566-1452 (C=C, benzene,
quinazolinone and pyridine rings), 847 (4-substituted pyridine), 770, 691 (monosubstituted benzene);
1
H-NMR: δ 7.51-7.73 (t, 9H, Ar-H, Qu-H), 7.82 (d, 2H, 3-Py-H, J = 10 Hz), 7.93 (t, 2H, 2-Py-H, J =
1
3
1
1
0 Hz), 9.11 (s, 1H, Qu-N=CH-Ar); C-NMR: δ 169.5, 158.2, 150.0, 146.6, 135.3, 133.3, 132.7, 129.7,
29.1 (C-4, C-12, C-14), 128.8, 128.2, 127.4, 124.3, 122.0 (C-3, C-17, C-20); Anal. calcd.for
C H N O (326.36): C 73.61%, H 4.32%, N 17.17%. Found: C 73.54%, H 4.48%, N 17.24%.
2
0
14
4
2
-(4-Pyridinyl)-3-(2-chlorobenzalamino)-4(3H)-quinazolinone (III-10). White solid, yield 69%; m.p.
-
1
2
48.7~250.2 °C; IR (cm ): ν 3046 (ArH), 1682 (C=O), 1611, 1584 (C=N), 1564-1458 (C=C, benzene,
quinazolinone and pyridine rings), 835, 725 (4-substituted pyridine), 764 (1,2-disubstituted benzene);
1
H-NMR: δ 6.56 (d, 1H, J = 15 Hz), 7.37-7.87 (m, 8H, Ar-H, Qu-H), 7.91 (d, 2H, 3-Py-H, J = 10 Hz),
1
3
8
1
1
6
.14 (d, 2H, 2-Py-H), 10.22 (s, 1H, Qu-N=CH-Ar); C-NMR: δ 169.2, 163.8, 162.0 (J
58.8, 152.0, 147.8, 135.3, 132.1 (J = 8.8 Hz), 130.0 (J = 28.0 Hz), 129.4, 127.9, 127.3, 127.0, 125.7,
23.6 (J = 10.0 Hz), 121.2, 120.1, 116.7 (J = 21.5 Hz); Anal. calcd. for C H FN O (344.35): C
3
= 10.0 Hz),
3
2
4
2
20 13
4
9.76%, H 3.81%, N 16.27%. Found: C 69.59%, H 3.75%, N 16.39%.
2
-(4-Pyridinyl)-3-(4-fluorobenzalamino)-4(3H)-quinazolinone (III-11). White solid, yield 89%; m.p.
-1
2
03~204 °C; IR (cm ) ν: 3080 (ArH), 1678 (C=O), 1611, 1597 (C=N), 1570-1447 (C=C, benzene,
quinazolinone and pyridine rings), 839 (1,4-disubstituted benzene), 825, 726 (4-subsituted pyridine);
1
H-NMR: δ 7.36-7.82 (m, 8H, Ar-H, Qu-H), 8.27 (d, 2H, 3-Py-H, J = 5 Hz), 8.70 (d, 2H, 2-Py-H, J = 5
1
3
Hz), 9.11 (s, 1H, Qu-N=CH-Ar); C-NMR: δ 168.1, 166.0, 164.0, 158.2, 151.8, 150.0, 146.5, 142.6,
35.4, 131.7 (J = 8.8 Hz), 129.4, 128.3 (J = 2.5 Hz), 127.4, 124.3, 121.9, 117.1 (J = 21.3 Hz); Anal
calcd for C20 O (344.35): C 69.76%, H 3.81%, N 16.27%. Found: C 69.73% H 4.00% N 16.40%.
1
3
4
2
H13FN
4
2
-(4-Pyridinyl)-3-(2, 3-dichlorobenzalamino)-4(3H)-quinazolinone (III-12). White solid, yield 89%;
-1
m.p. 201~203°C; IR (cm ): ν 3060 (ArH), 1681 (C=O), 1614 (C=N), 1577-1447 (C=C, benzene,
quinazolinone and pyridine rings), 826, 727 (4-subsituted pyridine), 790, 696 (1,2,3-trisubstituted
1
13
benzene); H-NMR: δ 7.48-8.27 (m, 11H, Ar-H, Py-H, Qu-H), 9.56(s, 1H, Qu-N=CH-Ar); C-NMR: δ
1
64.6, 158.4, 153.7, 146.6, 135.4, 135.2, 134.4, 133.4, 133.0, 130.4, 130.3, 129.3, 128.3, 128.1, 127.8,
27.5, 126.7, 121.6.
1
6
-Bromo-2-methyl-3-(2-fluorobenzalamino)-4(3H)-quinazolinone (III-13). White solid, yield 59%;
-1
m.p. 179~181 °C; IR (cm ): ν 3059 (Qu-H, Ar-H), 1680 (C=O), 1614, 1595 (C=N), 1571-1452 (C=C,
1
benzene and quinazolinone rings), 760 (1,2-disubstituted benzene); 675 (Qu-Br), H-NMR: δ 2.51 (s,
1
3
3
1
1
3
H, 2-Qu-CH
3
), 7.42-8.23 (m, 7H, Qu-H, Ar-H), 9.20 (s, 1H, Qu-N=CH-Ar); C-NMR: δ 163.3 (J =
5 Hz), 161.3, 157.0 154.6, 146.7, 137.8, 135.7 (J = 10 Hz), 129.7, 129.2 (J = 10 Hz), 128.3, 125.9,
3
23.3, 120.4, 119.2, 117.0 (J = 15 Hz), 22.8; Anal. calcd. for C16H11BrFN O (360.19): C 53.36% H
.08% N 11.67%. Found: C 53.51%, H 3.28%, N 11.70%.
6
-Bromo-2-methyl-3-(3-fluorobenzalamino)-4(3H)-quinazolinone (III-14). White solid, yield 63%;
-1
m.p. 212~214 °C; IR (cm ): ν 3032 (Qu-H, Ar-H), 1670 (C=O), 1616, 1502 (C=N), 1581-1449 (C=C,
1
benzene and quinazolinone rings), 874, 777, 695 (1,3-disubstituted benzene); 675 (Qu-Br); H-NMR: δ

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Molecules 2007, 12
1
3
2
.53 (s, 3H, 2-Qu-CH ), 7.50－8.23 (m, 7H, Ar-H, Qu-H), 9.04 (s, 1H, Qu-N=CH-Ar); C-NMR: δ
3
168.6, 163.9, 162.0, 157.0, 154.7, 145.8, 137.9, 135.1 (J = 10.0 Hz), 132.0 (J = 10.0 Hz), 129.8, 126.0,
1
23.2, 1203 (J = 25.0 Hz), 119.3, 115.0 (J = 25.0 Hz), 22.8; Anal. calcd. for C H BrFN O (360.19):
1
6
11
3
C 53.36%, H 3.08%, N 11.67%. Found: C 53.03%, H 3.49%, N 11.75%.
6
-Bromo-2-methyl-3-(4-fluorobenzalamino)-4(3H)-quinazolinone (III-15). White solid, yield 68%;
-1
m.p. 228.4~229.0 °C; IR (cm ): ν 3032 (Qu-H, Ar-H), 1674 (C=O), 1610, 1589 (C=N), 1568-1474
1
(
C=C, benzene and quinazolinone rings), 860, 815 (1,2,4-trsubstituted benzene), 675 (Qu-Br); H-
1
3
NMR: δ 2.57 (s, 3H, 2-Qu-CH ), 7.64-8.24 (m, 5H, Ar-H), 9.42 (s, 1H, Qu-N=CH-Ar); C-NMR: δ
3
1
2
3
63.2, 158.2 154.1, 146.6, 136.4, 135.1, 130.1. 129.9, 129.8, 129.6, 128.9, 128.8, 127.3, 127.0, 121.6,
2.9; Anal. calcd. for C16
.29%, N 11.52%
3
H11BrFN O (360.19): C 53.36%, H 3.08%, N 11.67%. Found: C 53.35%, H
6
-Bromo-2-methyl-3-(2-trifluoromethylbenzalamino)-4(3H)-quinazolinone (III-16). White solid, yield
-1
6
6 %; m.p. 196~198 °C; IR (cm ): ν 3032 (Qu-H, Ar-H), 2980 (Qu-CH ), 1678 (C=O), 1616, 1602
3
(
C=N), 1568-1469 (C=C, benzene and quinazolinone rings), 772 (1,2-disubstituted benzene); 675 (Qu-
1
13
Br), H-NMR: δ 2.57 (s, 3H, 2-Qu-CH
NMR: δ 164.2, 161.1, 158.2 146.5, 136.4, 135.1, 130.1. 131.3, 129.8, 129.6, 128.9, 128.8, 127.3, 117.0,
15.6, 22.9; Anal. calcd. for C17 O (410.20): C 49.78%, H 2.70%, N 10.24%. Found: C
3
), 7.64-8.24 (m, 5H, Ar-H), 9.42 (s, 1H, Qu-N=CH-Ar); C-
1
3 3
H11BrF N
49.27%, H 3.07%, N 10.26%.
6
-Bromo-2-methyl-3-(3-chlorobenzalamino)-4(3H)-quinazolinone (III-17). White solid, yield 63%;
1
m.p. 212~214 °C; H-NMR: δ 2.57 (s, 3H, 2-Qu-CH
3
), 7.64-8.24 (m, 5H, Ar-H), 9.42 (s, 1H, Qu-
1
3
N=CH-Ar); C-NMR: δ 163.2, 158.2 154.1, 146.6, 136.4, 135.1, 130.1. 129.9, 129.8, 129.6, 128.9,
28.8, 127.3, 127.0, 121.6, 22.9; Anal. calcd. for C16 O (376.64): C 51.02%, H 2.94%, N
1
H11BrClN
3
11.16%; found: C 51.11%, H 2.88%, N 11.25%.
6
-Bromo-2-methyl-3-(2,4-dichlorobenzalamino)-4(3H)-quinazolinone (III-18). White solid, yield 72%;
-1
m.p. 212.3~214.4 °C; IR (cm ): ν 3061 (Qu-H, Ar-H), 1688 (C=O), 1605, 1587 (C=N), 1575-1468
1
(
C=C, benzene and quinazolinone rings), 868, 827 (1,2,4-trisubstituted benzene); 675 (Qu-Br), H-
1
3
3
NMR: δ 2.55 (s, 3H, 2-Qu-CH ), 7.60-8.24 (m, 6H, Ar-H, Qu-H), 9.39 (s, 1H, Qu-N=CH-Ar); C-
NMR: δ 164.0, 157.3, 154.8, 145.7. 147.0138.6, 137.8, 130.5, 129.8, 129.7, 129.4, 129.3, 129.0, 123.3,
119.3, 22.9; Anal. calcd. for C H BrCl N O (411.09): C 46.75%, H 2.45%, N 10.22%. Found: C
16 10 2 3
46.68%, H 2.30%, N 10.22%.
6
-Bromo-2-methyl-3-(2,6-dichlorobenzalamino)-4(3H)-quinazolinone (III-19). White solid, yield 70%;
-1
m.p. 222~224 °C; IR (cm ): ν 3066 (Qu-H, Ar-H), 1678 (C=O), 1597 (C=N), 1579-1443 (C=C,
1
benzene and quinazolinone rings), 781, 710 (1,2,3-trisubstituted benzene), 675 (Qu-Br); H-NMR: δ
1
3
2
1
.50 (s, 3H, 2-Qu-CH
65.9, 157.0, 154.6, 145.7, 138.0, 135.0, 133.7, 130.2, 129.7, 129.4, 129.3, 123.4, 119.4, 23.4; Anal.
O (411.09): C 46.75%, H 2.45%, N 10.22%. Found: C 46.60%, H 2.60%, N
3
), 7.45-8.95 (m, 6H, Ar-H, Qu-H), 9.37 (s, 1H, Qu-N=CH-Ar); C-NMR: δ
calcd. for C16
0.29%.
2 3
H10BrCl N
1

2
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6
-Bromo-2-methyl-3-(3,4-difluorobenzalamino)-4(3H)-quinazolinone (III-20). White solid, yield 64%;
-1
m.p. 187~189 °C; IR (cm ): ν 3030 (Qu-H, Ar-H), 1678 (C=O), 1605, 1585 (C=N), 1517, 1470 (C=C,
1
benzene and quinazolinone rings), 881, 829 (1,2,4-trisubstituted benzene), 675 (Qu-Br), H-NMR: δ
13
2
1
3
.50 (s, 3H, 2-Qu-CH ), 7.61-8.23 (m, 6H, Qu-H, Ar-H), 9.02 (s, 1H, Qu-N=CH-Ar); C-NMR: δ
67.6, 156.9, 154.8, 151.3, 145.8, 142.0, 137.9, 129.8, 129.1, 127.5, 123.1, 119.3, 119.0, 117.5, 22.8.
6
-Bromo-2-methyl-3-(2-chloro-5-nitrobenzalamino)-4(3H)-quinazolinone (III-21). White solid, yield
-1
7
0%; m.p. 277~279 °C; IR (cm ): ν 3076 (Qu-H, Ar-H), 1682 (C=O), 1610, 1599 (C=N), 1568-1447
(
C=C, benzene and quinazolinone rings), 1529, 1348 (Ar-NO ), 880, 833 (1,2,4-trisubstituted benzene),
2
1
6
75 (Qu-Br); H-NMR: δ 2.59 (s, 3H, 2-Qu-CH
3
), 7.62-8.87 (m, 6H, Ar-H, Qu-H), 9.55 (s, 1H, Qu-
1
3
N=CH-Ar); C-NMR: δ 163.0, 155.7, 154.7, 147.4, 145.4, 141.5, 140.1, 137.9, 132.6, 129.8, 129.4,
123.4, 128.4, 122.9, 119.6, 22.7; Anal. calcd. for C H BrClN O (421.64): C 45.58%, H 2.39%, N
16 10 4 3
13.29%. Found: C 45.75%, H 2.46%, N 13.58%.
6
-Bromo-2-methyl-3-(4-chloro-3-nitrobenzalamino)-4(3H)-quinazolinone (III-22). White solid, yield
-1
6
8%; m.p. 230~232 °C; IR (cm ): ν 3062 (Qu-H, Ar-H), 1668 (C=O), 1612, 1597 (C=N), 1469 (C=C,
benzene and quinazolinone rings), 1537, 1355 (Ar-NO
2
), 870, 829 (1,2,4-trisubstituted benzene); 673
1
(
Qu-Br); H-NMR: δ 2.55 (s, 3H, 2-Qu-CH
3
), 7.61-8.66 (m, 6H, Ar-H, Qu-H), 9.20 (s, 1H, Qu-N=CH-
1
3
Ar); C-NMR: δ 166.1, 157.0, 154.8, 148.6, 145.7, 138.0, 133.7, 133.3, 133.2, 129.8, 129.3, 129.2,
25.8, 123.1, 119.4, 22.9; Anal. calcd. for C16 (421.64): C 45.58%, H 2.39%, N 13.29%;
found: C 45.62%, H 2.17%, N 13.46%.
1
4 3
H10BrClN O
6
-Bromo-2-methyl-3-(2-fluoro-3-trifluoromethylbenzalamino)-4(3H)-quinazolinone (III-23). White
-1
solid, yield 64%; m.p. 230.7~232.0 °C; IR (cm ) ν: 3012 (Qu-H, Ar-H), 1670 (C=O), 1614, 1605
(
C=N), 1566, 1431 (C=C, benzene and quinazolinone rings), 762, 696 (1,2,3-trisubstituted benzene);
1
6
75 (Qu-Br), H-NMR: δ 2.53 (s, 3H, 2-Qu-CH
3
), 7.61-8.39 (m, 6H, Ar-H, Qu-H), 9.02 (s, 1H, Qu-
1
3
N=CH-Ar); C-NMR: δ 168.5, 156.8, 154.7, 145.8, 137.9, 134.9, 134.5, 133.0, 131.7, 129.8, 129.1,
1
28.5, 128.2, 124.3, 123.1, 119.3, 22.8.
6
-Bromo-2-methyl-3-(3,4-dimethoxybenzalamino)-4(3H)-quinazolinone (III-24). White solid, yield
-1
6
3
6%; m.p. 216~218 °C; IR (cm ): ν 3060 (Qu-H, Ar-H), 2934, 2837 (ArO-CH ), 1674 (C=O), 1599
(
C=N), 1571-1466 (C=C, benzene and quinazolinone rings), 1271, 1024 (Ar-O-Me), 880, 833 (1,2,4-
1
trisubstituted benzene); H-NMR: δ 2.51 (s, 3H, 2-Qu-CH ), 7.14-8.37 (m, 6H, Qu-H, Ar-H), 8.78 (s,
3
1
3
1
1
5
H, Qu-N=CH-Ar); C-NMR: δ 170.2, 156.9, 154.7, 153.5, 149.7, 145.9, 139.9, 137.6, 129.7, 129.0,
25.2, 124.0, 123.2, 119.1, 112.0, 110.0, 56.2, 22.8; Anal. calcd. for C18
3.75%, H 4.01%, N 10.45%; found: C 53.67%, H 4.17%, N 10.56%.
3 3
H16BrN O (402.25): C
6
6
1
-Bromo-2-methyl-3-(2,3-dimethoxybenzalamino)-4(3H)-quinazolinone (III-25). White solid, yield
-
1
3 3
1%; m.p. 194.5~196.5 °C; IR (cm ): ν 3060 (Qu-H, Ar-H), 2965, 2935, 2837 (Ar-CH , ArO-CH ),
684 (C=O), 1614, 1597 (C=N), 1577-1468 (C=C, benzene and quinazolinone rings), 1275, 1088 (Ar-
1
O-Me), 746, 711 (1,2,3-trisubstituted benzene), 675 (Qu-Br); H-NMR: δ 2.51 (s, 3H, 2-Qu-CH
3
),
1
3
7.26-8.24 (m, 6H, Qu-H, Ar-H), 9.13 (s, 1H, Qu-N=CH-Ar); C-NMR: δ 165.8, 157.0, 154.7, 153.3,
150.4, 145.8, 137.7, 129.7, 129.2, 126.2, 125.2, 123.3, 119.1, 118.3, 117.6, 62.4, 56.5, 22.8; Anal.

2
637
Molecules 2007, 12
calcd. for C18
H
16BrN O (402.25): C 53.75%, H 4.01%, N 10.45%; found: C 53.94%, H 4.22%, N
3 3
1
0.35%.
6
6
1
-Bromo-2-methyl-3-(2,5-dimethoxybenzalamino)-4(3H)-quinazolinone (III-26). White solid, yield
-1
3%; m.p. 224~226 °C; IR (cm ): ν 3060 (Qu-H, Ar-H), 2943, 2833 (Ar-CH , ArO-CH ), 1682 (C=O),
3
3
604 (C=N), 1575-1445 (C=C, benzene and quinazolinone rings), 1223, 1049 (Ar-O-Me), 875, 800
1
(
1,2,4-trisubstituted benzene); 675 (Qu-Br); H-NMR: δ 2.51 (s, 3H, 2-Qu-CH ), 3.35-3.84 (m, 6H,
3
1
3
3
ArO-CH ), 6.98-8.36 (m, 6H, Ar-H, Qu-H), 9.13 (s, 1H, Qu-N=CH-Ar); C-NMR: δ 166.0, 164.9,
1
2
3
56.8, 154.8, 154.4, 153.7, 145.8, 137.6, 129.7, 129.2, 121.6, 121.0, 119.0, 114.5, 110.4, 56.9, 56.1,
2.8; Anal. calcd. for C H BrN O (402.25): C 53.75%, H 4.01%, N 10.45%; found: C 53.67%, H
1
8
16
3
3
.89%, N 10.32%.
6
-Bromo-2-methyl-3-(3-nitrobenzalamino)-4(3H)-quinazolinone (III-27). White solid, yield 56%; m.p.
-1
2
27~230 °C; IR (cm ): ν 3086 (Qu-H, Ar-H), 1670 (C=O), 1602 (C=N), 1535 1352 (Ar-NO ), 1470
2
1
(
C=C, benzene and quinazolinone rings), 843, 770, 700 (1,3-disubstituted benzene); 675 (Qu-Br); H-
1
3
3
NMR: δ 2.56 (s, 3H, 2-Qu-CH ), 7.63-8.77 (m, 7H, Ar-H, Qu-H), 9.23 (s, 1H, Qu-N=CH-Ar); C-
NMR: δ 167.8, 156.8, 154.7, 148.9, 145.8, 138.0, 135.2, 134.5, 131.5, 129.8, 129.2, 127.5, 123.5,
1
23.1, 119.4, 22.9; Anal. calcd. for C16
9.42%, H 2.75%, N 14.32%.
4 3
H11BrN O (387.20): C 49.63%, H 2.86%, N 14.47%. Found: C
4
6
-Bromo-2-methyl-3-(2-hydroxybenzalamino)-4(3H)-quinazolinone (III-28). White solid, yield 53%;
-1
m.p. 212.4~212.9 °C; IR (cm ): ν 3364 (Ar-OH), 3072 (Qu-H, Ar-H), 1682 (C=O), 160 (C=N), 1464,
1
447 (C=C, benzene and quinazolinone rings), 1273 (Ar-O-H), 750 (1,2-disubstituted benzene); 675
1
(
Qu-Br), H-NMR: δ 2.50 (s, 3H, 2-Qu-CH
3
), 6.97-8.22 (m, 7H, Qu-H, Ar-H), 9.10 (s, 1H, Qu-
1
3
N=CH-Ar), 10.56 (Ar-OH); C-NMR: δ 167.6, 159.3, 157.0, 154.6, 145.9, 140.0, 137.6, 135.1, 129.7,
1
29.1, 128.6, 123.3, 120.2, 118.7, 117.3, 22.8; Anal. calcd. for C16
.38%, N 11.73%. Found: C 53.79%, H 3.66%, N 11.72%.
3 2
H12BrN O (358.20); C 53.65%, H
3
6
-Bromo-2-methyl-3-(2,4-dimethoxybenzalamino)-4(3H)-quinazolinone (III-29). White solid, yield
-1
6
3 3
2 %; m.p. 216~218 °C; IR (cm ): ν 3032 (Qu-H, Ar-H), 2968, 2941, 2833 (Ar-CH , ArO-CH ), 1674
(
C=O), 1599 (C=N), 1570-1468 (C=C, benzene and quinazolinone rings), 1277, 1107 (Ar-O-Me), 876,
1
8
6
1
13 (1,2,4-trisubstituted benzene); 675 (Qu-Br); H-NMR: δ 2.49 (s, 3H, 2-Qu-CH
3
), 3.88 (Ar-O-Me),
13
.58-8.20 (m, 6H, Qu-H, Ar-H), 8.97 (s, 1H, Qu-N=CH-Ar); C-NMR: δ 165.5, 165.0, 161.6, 157.1,
54.8, 145.9, 137.5, 129.7, 129.1, 128.7, 123.3, 119.0, 113.4, 107.6, 98.7, 56.5, 56.3, 22.8; Anal. calcd.
for C18
H16BrN
3
O
3
(402.25): C 53.75%, H 4.01%, N 10.45%. Found: C 53.99%, H 4.19%, N 10.27%.
6
-Bromo-2-methyl-3-(5-chloro-2-hydroxybenzalamino)-4(3H)-quinazolinone (III-30). Yellow solid,
-1
yield 64 %; m.p. 273.2~225.0 °C; IR (cm ): ν 3064 (Qu-H, Ar-H), 1682 (C=O), 1610 (C=N), 1474
1
(
C=C, benzene and quinazolinone rings), 860, 815 (1,2,4-trisubstituted benzene); 665 (Qu-Br); H-
1
3
3
NMR: δ 2.50 (s, 3H, 2-Qu-CH ), 7.49-8.21 (m, 6H, Qu-H, Ar-H), 9.11 (s, 1H, Qu-N=CH-Ar); C-
NMR: δ 165.1, 157.9, 157.0, 154.7, 145.8, 137.7, 134.4, 129.7, 129.1, 126.7, 123.9, 123.2, 120.3,
1
19.2, 119.1, 22.9; Anal. calcd. for C H BrClN O (392.64): C 48.94%, H 2.82%, N 10.70%. Found:
1
6
11
3
2
C 48.80%, H 2.96%, N 10.75%.

2
638
Molecules 2007, 12
2
2
1
2
1
-Methyl-3-(2,3-dichlorobenzalamino)-4(3H)-quinazolinone (III-31). White needles, yield 87%; m.p.
-1
09~210 °C; IR (cm ) ν: 3034 (Qu-H, Ar-H), 2982 (Ar-CH
3
), 1680 (C=O), 1612, 1593 (C=N), 1550-
1
474 (C=C, benzene and quinazolinone rings), 766, 691 (1,2,3-trisubstituted benzene); H-NMR: δ
13
3
.51 (s, 3H, 2-Qu-CH ), 7.53-8.19 (m, 7H, Qu-H, Ar-H), 9.42 (s, 1H, Qu-N=CH-Ar); C-NMR: δ
64.0, 158.2, 154.1, 146.7, 135.1, 134.6, 133.4, 133.0, 129.4, 127.4, 127.3, 127.2, 127.0, 121.6, 22.9;
Anal. calcd. for C H Cl N O (332.19): C 57.85%, H 3.34%, N 12.65%. Found: C 57.78%, H 3.52%,
1
6
11
2
3
N 12.53%.
Bioassays: Antifungal Bioassays
The antifungal activity of all synthesized compounds III-1-III-31 was tested against three
pathogenic fungi, namely Fusarium oxysporum, Gibberella zeae, and Valsa mali, by the poison plate
technique [22]. Compounds III-1-III-31 were dissolved in acetone (10 mL) before mixing with Potato
Dextrose Agar (PDA, 90 mL). The final concentration of compounds III-1-III-31 in the medium was
fixed at 500 μg/mL. Three kinds of fungi were incubated in PDA at 25±1 °C for 5 days to get new
mycelium for antifungal assay, then a mycelia disk of approximately 0.45 cm diameter cut from the
culture medium was picked up with a sterilized inoculation needle and inoculated in the center of PDA
plate. The inoculated plates were incubated at 25±1°C for 5 days. Acetone in sterilized distilled water
served as control, while hymexazole was used as positive control For each treatment, three replicates
were carried out. The radial growth of the fungal colonies was measured on the sixth day and the data
were statistically analyzed. The in vitro inhibiting effects of the test compounds on the fungi were
A − B
calculated by the formula CV =
, where A represents the diameter of fungi growth on untreated
A
PDA, B represents the diameter of fungi on treated PDA, and CV represents the rate of inhibition.
Antiviral Bioassays
The leaves of Nicotiana tabacum. L growing at the same ages were selected. The tobacco mosaic
-
3
virus with the concentration of 6×10 mg/mL was dipped and inoculated on the whole leaves. Then the
leaves were washed with water and dried. The compound solution was smeared on the left side and the
solvent was smeared on the right side for control. The local lesion numbers were then recorded 3-4
days after inoculation [23]. For each compound, three times of repetition were conducted to ensure the
reliability of the results.
av local lesion numbers of control (not treated with compound) − av local lesion numbers smeared with drugs
Inhibition rate (%) =
av local lesion numbers without drugs
Isolation of total RNA from tobacco leaf
Trizol kit was used according to the standard protocol for total RNA isolation. Prior to RT-PCR, the
total RNA samples were treated with DNase I for 10 min and quantified by spectrophotometry and
identified by agarose gel electrophoresis [24].

2
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Molecules 2007, 12
Reagents
RNA isolation kit, MMLV reverse transcriptase and RT-PCR kit were purchased from TAKARA
Biotechnology (Dalian) CO., LTD. DNA Marker pUC/Msp I was purchased from MBI Company, Ltd.
Primer was designed with Beacon Designer software. Oligo (dT)18 and primers were synthesized by
Shanghai Sangon Biotechnology Company, Ltd.
RT-PCR assay
cDNA was synthesized with oligo (dT)18 complementary at the 3’ end of mRNA as a primer. Total
RNA (1μg) was used for temple of first-strand cDNA synthesis using extend reverse transcriptase.
o
Reverse transcription was carried out at 37 C for 1 h. The single-stranded DNA mixture was used as
template in PCR. The primers for PCR amplification are shown in Figure 3. The PCR were performed
in Tris-HCl buffer (10 mM, pH 8.3), KCl (50 mM), MgCl
2
(1.8-2.0 μl), dNTPs (0.02 μM), primers
(
0.04 μM), DNA polymerase (1 U). PCR amplification steps consisted of a preliminary denaturation
o
o
o
step at 94 C for 1 min, followed by 35 cycles at 94 C for 40 seconds, at 58 C for 40 seconds and at
o
7
0
2 C for 50 seconds on icycle of BioRad. PCR products were separated on 1.5% agarose gel in
.5×TBE buffer and visualized under UV light after staining with ethidium bromide [25]
Products of PCR sequencing
The purified PCR products were sequenced on an automated DNA sequencer by model ABI 310
DNA sequencer. The sequencer analysis was carried out using the chromas223 software and BLAST
network services at the National Center for Biotechnology information (NCBI) Genbank.
Semi-quantitative PCR for gene expression
In order to assess relative expression levels of target-gene in water-treated tobacco and (R)-4p- and
TMV-treated tobacco and tobacco by inoculated TMV only, semi-quantity PCR consisting of 20 cycles
(
within the logarithmic range of amplification of gene) with putting primer of β-actin serving as an
internal reference gene was employed for the study. The amplified products were analyzed on a 1.5%
agarose gel by the method of F. Mohamed [26].
The relative quantification real-time PCR for expression of the target gene
The relative quantification real-time PCR was carried out with iCycler IQ according to
manufacturer’s protocol with primer of β-actin serving as an internal reference gene. Precautions were
taken to ascertain reliable quantitative results: Log-linear dilution curves were performed with primers
for the target gene as well as with primers for the β-actin. Reactions performed without reverse
transcriptase or without template did not result in any product. Reactions were conducted in 50 μL
final volumes with IQ Supermix (25 μL) containing SYBR Green and cDNA as template (2μg), and
o
primer mixture (40 pM) for each gene. PCR cycles were as follows: 2 min at 94 C, followed by 40
o
o
o
cycles each of 40 seconds duration at 94 C, 40 seconds at 58 C, and 50 seconds at 72 C. By

2
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Molecules 2007, 12
following PCR, 110 steps for melt curve analysis were completed in 10 seconds at temperature ranging
o
o
from 40 C to 95 C. The amplification efficiency was 95-99% for PR-1a and PR-5 gene, respectively
The figure of standard curve are not shown). Each target gene peak was assigned an arbitrary
quantitative value correlated to the β-actin gene peak, according to the formula:
(
T
ΔΔC =ΔCT (test) - ΔCT (calibrator)
C being the cycle threshold. Rates of stimulation of RNA expression were calculated from the ΔC
T
T
values at various time points [27].
Acknowledgements
This work was financially supported by the National Nature Science Foundation of China (Grant
No. 20562003).
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Sample Availability: Samples of the compounds are available from authors.
2007 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.
©