Anthranilic acid-based diamides derivatives incorporating aryl-isoxazoline pharmacophore as potential anticancer agents: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2012.06.001

A series of novel anthranilic diamides derivatives containing aryl-isoxazoline moiety were designed and synthesized as a part of our ongoing search for potential anticancer agents. Their structures were confirmed by ¹H NMR, ¹³C NMR and ESI-MS analyses. The preliminary assays showed that some of the compounds displayed moderate to good antitumor activities against human lung cancer (NCI-H460), hepatocellular liver carcinoma (HepG2), gastric cancer (SGC-7901 and BGC-823) and breast epithelial adenocarcinoma (MCF-7) cell lines at μM level, which might be developed as novel lead scaffold for potential anticancer agents.

Full text of DOI:10.1016/j.ejmech.2012.06.001

European Journal of Medicinal Chemistry 54 (2012) 549e556
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Anthranilic acid-based diamides derivatives incorporating aryl-isoxazoline
pharmacophore as potential anticancer agents: Design, synthesis and biological
evaluation
1
1
*
Liqiao Shi , Rui Hu , Yanhong Wei, Ying Liang, Ziwen Yang, Shaoyong Ke
National Biopesticide Engineering Research Center, Hubei Academy of Agricultural Sciences, No. 8, Nanhu Avenue, Wuhan 430064, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel anthranilic diamides derivatives containing aryl-isoxazoline moiety were designed and
synthesized as a part of our ongoing search for potential anticancer agents. Their structures were
confirmed by 1H NMR, 13C NMR and ESI-MS analyses. The preliminary assays showed that some of the
compounds displayed moderate to good antitumor activities against human lung cancer (NCI-H460),
hepatocellular liver carcinoma (HepG2), gastric cancer (SGC-7901 and BGC-823) and breast epithelial
Received 13 February 2012
Received in revised form
26 May 2012
Accepted 1 June 2012
Available online 12 June 2012
adenocarcinoma (MCF-7) cell lines at
potential anticancer agents.
m
M level, which might be developed as novel lead scaffold for
Keywords:
Ó 2012 Elsevier Masson SAS. All rights reserved.
Anthranilic diamides
Aryl-isoxazoline
Synthesis
Antitumor activity
1. Introduction
pharmacological activities including antitumor (Compds. 1, 2 and 3)
[7,15,16], antifungal [17], anti-inflammatory activities. In addition,
some diamides derivatives are used as Factor Xa inhibitors
(Compds. 4 and 5) [18,19], glycogen phosphorylase inhibitors
(Compd. 6) [20], human sialidase inhibitors (Compd. 7) [21], and
CCK1 receptor antagonists (Compd. 8) [22] etc. The promising
bioactive diversity of this class of diamide compounds urges us to
synthesize and biologically evaluate a series of novel structural
variants of anthranilic diamides derivatives.
On the other hand, aryl-isoxazoline scaffolds (Fig. 2) have
attracted considerable attention for decades and represented an
important bioactive heterocycle [23e26]. Recently, numerous
studies have demonstrated that this pharmacophore correlates
highly with multiple biological activities, and plays an important
role in medicinal [27e34] and agrochemical industry [35e38]. In
addition, the synthesis and evaluation of new molecular scaf-
folds based on privileged structures have been identified as
a key method in drug discovery process [39,40]. Thus, based on
the aforementioned results, we hypothesized that integrating
aryl-isoxazoline moiety in anthranilic diamides scaffold
(Fig. 2) may lead to novel potential anticancer agents with broad
biological activity profiles and improved pharmacokinetic
properties.
Nowadays, cancer has gradually become the leading disease-
related cause of deaths of human population, and which has
also been seriously threatening the health and life of humans for
a long period. Among all the current therapeutic methods,
chemotherapy still remains an important option for cancer
treatment [1e5]. However, the major hindrance to successful
cancer chemotherapy is the development of drug and multidrug-
resistance, in which cancer cells become simultaneously resistant
to different structural types of chemotherapeutic agents [6e9].
Therefore, the discovery and identification of novel highly effec-
tive anticancer agents with special characteristics than the
currently used is an important endeavor in medicinal chemistry
[10e13], and it was necessary to attempt neotype chemical enti-
ties as potential chemotherapeutic agents with an alternative
mechanism of action.
Recently, diamides derivatives received significant attention for
their antitumor properties, especially for those bearing a basic
diamide scaffold and pharmacophores [14]. Various diamides
derivatives (Fig. 1) have been reported for their wide range of
As part of our medicinal chemistry program aimed at the
discovery of potential anticancer agents, we wish to describe
herein the molecule design, convenient synthesis, and biological
* Corresponding author.
E-mail address: keshaoyong@163.com (S. Ke).
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.001

550
L. Shi et al. / European Journal of Medicinal Chemistry 54 (2012) 549e556
R1
NH
R1
S
R
N
O
O
O
N
Cl
R2
O
H3CO
S
I
NH
H
NH
HN
R4
n
NH
N
H
N
Cl
X
R3
R
S
O
O
O
R2
O
Cl
Compd. 1
Compd. 2
Compd. 3
Compd. 4
O
R1
R
O
O
H
Cl
NH
NH
N
R2
O
N
NH
NH
R3
HN
HN
O
N
NH
R1
NH
O
O
R2
O
O
O
Cl
Compd. 5
OH
F
COOH
Compd. 7
Compd. 6
Compd. 8
Fig. 1. Representative active molecules bearing diamides scaffolds.
evaluation of novel series of anthranilic diamides derivatives con-
taining aryl-isoxazoline pharmacophore. We utilized anthranilic
acid scaffold as a key prototype structural unit and planned for the
hybridization of the active pharmacophore to the core structure as
shown in Fig. 2. Therefore, a series of novel diamides derivatives
6aev were designed and synthesized as shown in Scheme 1, and
their antitumor activities against various cancer cell lines (HepG2,
SGC-7901, BGC-823, NCI-H460, MCF-7) were also evaluated by
MTT method.
satisfactory chemical analyses, and the chemical structures of the
synthesized compounds were summarized in Table 1.
2.2. Pharmacology evaluation
The anthranilic diamides 6aev were evaluated for their in vitro
cytotoxic effects against HepG2 (hepatocellular liver carcinoma),
SGC-7901 (gastric cancer), BGC-823 (gastric cancer), NCI-H460
(lung cancer) and MCF-7 (breast epithelial adenocarcinoma) cell
lines by the standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide) assay [41] using 5-FU (5-
Fluorouracil) as a positive control. The results are summarized in
Tables 2 and 3. The IC50 value represents the drug concentration
required to inhibit cell growth by 50%.
As shown in Table 2, the prepared anthranilic diamides deriva-
tives (6aev) displayed moderate to good antitumor activities
against five human cancer cell lines. In general, the compounds
derived from non-substituted 2-aminobenzoic acid (6qev)
exhibited lower activity than the corresponding compounds from
substituted 2-aminobenzoic acid (Entry 1e16). The compounds
bearing nPr group (6aec) have relatively high activities than the
2. Results and discussion
2.1. Synthesis of anthranilic diamides derivatives
In the present study, a series of novel anthranilic diamides
derivatives were constructed by integrating functionalized aryl-
isoxazoline acid 3aec with substituted 2-aminobenzoic acid 4.
The general method for the preparation of anthranilic diamides
derivatives containing aryl-isoxazoline moiety 6aev is outlined in
Scheme 1.
The easily available aryl-aldehyde was selected as starting
material, which was routinely transferred to the corresponding aryl-
aldoximes 2aec by general condensation reaction. The subsequent
heterocyclization reaction of compound 2aec was treated with a,b-
R1
R1
R1
O
H
N
H
OH
unsaturated acid resulting in 4,5-dihydroisoxazolecarboxic acid
3aec via classical 1,3-dipolar cycloaddition reaction in the presence
of pyridine, and the key heterocyclic acids 3aec can be easily
separated as colorless crystals with high yields. The derived aryl-
isoxazoline acid 3aec can further be conveniently coupled with
substituted 2-aminobenzoic acid 4 to form 4H-benzo[d] [1,3]oxazin-
4-one 5aef in high yields using methanesulfonyl chloride and
pyridine in acetonitrile. The key six-membered lactone intermedi-
ates 5aef can be directly used to transform to corresponding
anthranilic diamides derivatives 6aev via routine aminolysis reac-
tion. All the target anthranilic diamides derivatives 6aev gave
a
b
OH
O
[3+2]
N
O
3a-c
1a-c
2a-c
R2
COOH
NH2
c
R3
4
R5
O
O
O
N
O
N
N
R4
d
R1
R2
HN
R2
R1
R3
N
O R3
6a-v
O
5a-f
a-c
Compd.
Compd.
a-f
Aryl-Isoxazole
a. R1 = 4-F, R2 = Cl, R3 = CH3;
b. R1 = 4-Cl, R2 = Cl, R3 = CH3
a. R1 = 4-F
b. R1 = 4-Cl
O
R1
R2
1
2
3
Incorporate with
Aryl-Isoxazole Het.
O
R1
R2
N
N
c. R1 = 3,4,5-(MeO)3, R2 = Cl, R3 = CH3
d. R1 = 4-F, R2 = R3 = H
c. R1 = 3,4,5-(MeO)3
5
NH
NH
e. R1 = 4-Cl, R2 = R3 = H
R
Pharmacophores Hybridization
Yn
Xn
O
R
1 = 3,4,5-(MeO)3, R2 = R3 = H
N
Xn
f.
O
O
Diamides Scaffold
Scheme 1. Reagents and conditions: a. NH2OH$HCl, CH3COONa, MeOH; b. Acrylic acid,
NCS, Py, CHCl3; c. MeSO2Cl, Py, MeCN; d. Amine, MeCN, rt for 2e5 h.
Fig. 2. Design strategy of anthranilic diamides containing aryl-isoxazoline moiety.

L. Shi et al. / European Journal of Medicinal Chemistry 54 (2012) 549e556
551
Table 1
heterocyclic molecule containing trimethoxyphenyl group might
be an active scaffold, which further confirmed the compound 6i
should be a potential lead molecule for discovery of N-heterocyclic
derivatives as potential drugs.
The chemical structure of anthranilic diamides derivatives 6aev.
Entry Compd. Substituents
Yield (%)a Mp (ꢂC)
No.
R1
R2 R3 R4
R5
Additionally, for comparison, the selective heterocyclic inter-
mediates 5aec have also been tested for their in vitro cytotoxic
effects. On the basis of the results in Table 3, the intermediate 5a
exhibited stronger cytotoxicity than compounds 5b and 5c against
all cell lines, and the inhibitory activity against BGC-823 and NCI-
H460 for compound 5a is up to 72.1 and 64.6% at tested concen-
tration, respectively. In contrast, compound 5c bearing trimethox-
yphenyl group almost lost inhibitory activity at the same
concentration. According to the results from Tables 2 and 3, it can
be figured out that the diamide products have relatively better
activities than the corresponding intermediates, respectively, and
the designed anthranilic diamides derivatives containing aryl-
isoxazoline moiety may be used as potential lead compounds for
optimization of novel anticancer agents.
1
2
3
4
5
6
7
8
6a
6b
6c
6d
6e
6f
6g
6h
6i
4-F
4-Cl
Cl CH3
Cl CH3
H
H
H
H
H
H
H
H
H
CH2CH2CH3 64
CH2CH2CH3 67
CH2CH2CH3 56
222e225
232e234
190e192
223e225
232e235
215e216
245e247
140e142
193e194
145e147
153e155
159e161
170e172
249e251
187e188
225e226
146e148
131e133
198e200
195e197
198e199
222e224
3,4,5-(MeO)3 Cl CH3
4-F
4-Cl
3,4,5-(MeO)3 Cl CH3
4-F
4-Cl
3,4,5-(MeO)3 Cl CH3
4-F
4-Cl
3,4,5-(MeO)3 Cl CH3 CH2CH3 CH2CH3
4-F
4-Cl
3,4,5-(MeO)3 Cl CH3
4-F
4-F
4-Cl
3,4,5-(MeO)3
4-F
4-Cl
Cl CH3
Cl CH3
CH(CH3)2
CH(CH3)2
CH(CH3)2
Cy
58
62
48
56
62
68
58
55
63
54
64
60
42
55
63
52
68
70
54
Cl CH3
Cl CH3
Cy
Cy
9
10
11
12
13
14
15
16
17
18
19
20
21
22
6j
6k
6l
Cl CH3 CH2CH3 CH2CH3
Cl CH3 CH2CH3 CH2CH3
6m
6n
6o
6p
6q
6r
6s
6t
Cl CH3
Cl CH3
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
Ph
Cl CH3
H
H
H
H
H
H
H
H
H
H
H
H
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH3
On the other hand, since some target compounds displayed
relatively higher inhibitory activity against tested cancer cell lines
(Table 2), the selective compounds (6a, 6c, 6i, 6j, 6k, 6m, 6n and 6o)
were further tested for their inhibitory potential as well as IC50
values. As indicated in Table 4, compound 6i showed the strongest
inhibitory effect against MCF-7 and BGC-823, with an IC50 values of
6u
6v
CH3
CH3
3,4,5-(MeO)3
a
Isolated yields.
22.4 and 23.3 m
g mLꢀ1, respectively. While the intermediates 5a and
5b were less effective.
compounds containing iPr moiety (6def). Notably, compounds 6i
and 6k (Entries 9 and 11) exhibited significant inhibitory activity
against all tested cell lines with 59.2e76.3 % growth inhibition at
Furthermore, the selective doseeresponse analysis of cell
growth inhibition activity for representative compounds 6i, 6k has
been displayed in Fig. 3, which indicated that the cytotoxic effects
on selective cell lines of target compounds showed obvious
concentration-dependent manner. Compounds 6i exhibited potent
growth inhibitory activities against BGC-823 and HepG2 cell lines
40
mg/mL concentration. Also, it is interesting to note that
compound 6u (Entry 21) showed cytotoxic selectivity for a special
human hepatocellular liver carcinoma cell type, and the inhibition
against HepG2 cell was obviously higher than other tested cell lines
(Entry 21). The results of preliminary antitumor assay indicated the
with the IC50 values of 23.3 ꢁ 4.9 and 34.8 ꢁ 3.7
m ,
g mLꢀ1
respectively.
3. Conclusion
Table 2
Cytotoxic activity (% cell growth inhibition) of the compounds 6aev at 40 mg/mL
concentration against various human cancer cell lines.
In the present study, we have described the molecular design,
synthesis, and biological evaluation of a series of novel anthranilic
diamides derivatives containing aryl-isoxazoline pharmacophore.
Twenty two novel anthranilic diamides derivatives have been
conveniently synthesized, and characterized by 1H NMR, 13C NMR
and MS spectra analyses. The preliminary bioassay results indicated
that some of the compounds displayed moderate to good antitumor
activities against human lung cancer (NCI-H460), hepatocellular
liver carcinoma (HepG2), gastric cancer (SGC-7901 and BGC-823)
and breast epithelial adenocarcinoma (MCF-7) cell lines, which
might be developed as novel lead scaffold for anticancer agents.
Further structural optimization and activity profiles about the
designed novel diamides derivatives are well ongoing in our
laboratory.
Entry Compd. No. Growth-inhibitory properties
HepG2a
SGC-7901a BGC-823a NCI-H460a MCF-7a
1
2
3
4
5
6
7
8
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
29.1 ꢁ 4.7 30.5 ꢁ 5.3 23.5 ꢁ 2.2 20.0 ꢁ 2.3 33.0 ꢁ 0.3
16.7 ꢁ 4.4 14.7 ꢁ 1.7 24.5 ꢁ 4.5 13.6 ꢁ 6.3 22.2 ꢁ 4.3
21.1 ꢁ 7.5 15.4 ꢁ 6.5 28.0 ꢁ 2.7 9.5 ꢁ 0.4 29.1 ꢁ 3.4
13.6 ꢁ 1.4 17.2 ꢁ 1.6
0
0
4.8 ꢁ 1.4
3.9 ꢁ 1.6
9.1 ꢁ 3.9
12.3 ꢁ 0.4 24.2 ꢁ 4.6 38.7 ꢁ 0.6 10.3 ꢁ 2.4
12.9 ꢁ 1.9 11.7 ꢁ 4.0 0.5 ꢁ 0.2
0
10.0 ꢁ 3.4 15.6 ꢁ 2.4 34.3 ꢁ 0.4 33.3 ꢁ 4.0 24.8 ꢁ 2.2
15.6 ꢁ 4.1
3.1 ꢁ 0.5 18.3 ꢁ 2.7 15.1 ꢁ 6.0
9.6 ꢁ 2.1
9
64.0 ꢁ 5.2 60.2 ꢁ 0.7 69.7 ꢁ 3.0 74.0 ꢁ 4.5 62.7 ꢁ 4.4
18.2 ꢁ 6.2 15.8 ꢁ 1.1 36.4 ꢁ 5.5 10.0 ꢁ 1.72 14.4 ꢁ 2.1
59.2 ꢁ 5.3 76.3 ꢁ 1.4 59.6 ꢁ 3.1 63.5 ꢁ 5.1 62.1 ꢁ 2.6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
28.6 ꢁ 7.0 28.3 ꢁ 7.4 27.8 ꢁ 1.7
0
23.5 ꢁ 1.3
26.7 ꢁ 5.4 12.8 ꢁ 0.1 14.6 ꢁ 3.3 42.9 ꢁ 3.8 40.8 ꢁ 3.0
4. Materials and methods
18.7 ꢁ 1.1 24.6 ꢁ 6.6 13.2 ꢁ 1.7 22.6 ꢁ 2.7
0
30.6 ꢁ 3.8 27.3 ꢁ 3.9 34.1 ꢁ 6.9 5.5 ꢁ 3.0 26.1 ꢁ 3.9
5.1 ꢁ 1.3 11.6 ꢁ 2.3 26.4 ꢁ 2.1
10.3 ꢁ 4.1 21.5 ꢁ 0.7
0
0
0
0
0
17.0 ꢁ 3.0
4.1. Instrumentation and chemicals
0
0
0
0
0
5.5 ꢁ 1.3 13.6 ꢁ 2.9 14.6 ꢁ 3.0
All melting points (m.p.) were obtained with a digital model X-5
apparatus and are uncorrected. 1H NMR (600 MHz) and 13C NMR
(100 MHz) spectra were recorded on a Brucker spectrometer with
CDCl3 as the solvent and TMS as the internal standard. Chemical
6s
6t
6u
6v
5-FUb
11.7 ꢁ 8.7
0
0
0
9.1 ꢁ 8.7
21.4 ꢁ 6.0
50.6 ꢁ 11.5 7.8 ꢁ 2.4 13.3 ꢁ 2.5 5.7 ꢁ 2.2 16.5 ꢁ 6.3
31.4 ꢁ 1.2 12.0 ꢁ 5.1 20.8 ꢁ 9.1
0
9.1 ꢁ 3.3
81.3 ꢁ 2.8 80.8 ꢁ 1.4 85.7 ꢁ 4.4 89.7 ꢁ 0.6 80.4 ꢁ 3.7
shifts are reported in
constants nJ are reported in Hz. Standard abbreviations indicating
multiplicity are used as follows: singlet, d doublet,
d (parts per million) values. Coupling
a
Abbreviations: HepG2: human hepatocellular liver carcinoma cell line; SGC-
7901: human gastric cancer cell line; BGC-823: human gastric cancer cell line;
NCI-H460: human large cell lung cancer cell line; MCF-7: human breast adenocar-
cinoma cell line.
s
¼
¼
dd ¼ doublet of doublets, t ¼ triplet, q ¼ quadruplet, m ¼ multiplet
b
Used as a positive control.
and br ¼ broad. Mass spectra were performed on a MicroMass

552
L. Shi et al. / European Journal of Medicinal Chemistry 54 (2012) 549e556
Table 3
Growth-inhibitory properties for the selective intermediates 5aec at 40
m
g/mL concentration against various human cancer cell lines.
O
O
N
O
N
R2
R1
R3
Entry
Compd. No.
Substituents
R1
Growth-inhibitory properties
R2
R3
HepG2a
SGC-7901a
BGC-823a
NCI-H460a
MCF-7a
1
2
3
4
5a
5b
4-F
4-Cl
3,4,5-(MeO)3
e
Cl
Cl
Cl
CH3
CH3
CH3
38.9 ꢁ 3.4
52.4 ꢁ 8.9
16.5 ꢁ 5.3
81.3 ꢁ 2.8
42.7 ꢁ 4.4
35.6 ꢁ 7.2
0
80.8 ꢁ 1.4
72.1 ꢁ 2.2
31.5 ꢁ 3.3
0
85.7 ꢁ 4.4
64.6 ꢁ 2.0
58.1 ꢁ 5.6
0
89.7 ꢁ 0.6
54.7 ꢁ 6.4
37.7 ꢁ 5.5
12.4 ꢁ 4.6
80.4 ꢁ 3.7
5c
5-FUb
a
Abbreviations: HepG2: human hepatocellular liver carcinoma cell line; SGC-7901: human gastric cancer cell line; BGC-823: human gastric cancer cell line; NCI-H460:
human large cell lung cancer cell line; MCF-7: human breast adenocarcinoma cell line.
b
Used as a positive control.
Quattro microTM API instrument. Analytical thin-layer chromatog-
raphy (TLC) was carried out on precoated plates, and spots were
visualized with ultraviolet light. All chemicals or reagents used for
syntheses were commercially available, were of AR grade, and were
used as received. Anhydrous CH2Cl2 and CH3CN were dried
according to standard methods [42]. All other solvents and reagents
were analytical reagents and used directly without purification.
4.3. General synthetic procedure for aryl-isoxazoline acid 3aec
A solution of the corresponding aryl-aldoxime (2 mmol) in
CHCl3 (2 mL) was added dropwise to an ice-cooled solution of
CHCl3 (8 mL), acrylic acid (2.3 mmol), pyridine (3 mmol) and
N-chlorosuccinimide (NCS) (0.31 g, 2.3 mmol) under nitrogen for
30 min. The reaction was then stirred at room temperature for
several hours and monitored to the completion by thin-layer
chromatography. The solution was washed with water and dried
with Na2SO4. The filtrate was evaporated under a reduced pressure
and the residue purified by recrystallization from appropriate
solvents. The physicoechemical properties and spectral data for
representative compounds are as follows:
4.2. General synthetic procedure for substituted aldoximes 2aec
To a corresponding substituted benzaldehyde (10 mmol) and
hydroxylamine hydrochloride (12 mmol) was added 25 mL of
ethanol. The reaction mixture was stirred for 10 min and then
sodium acetate (15 mmol) in water (5 mL) was added. The mixture
was heated at 45e50 ꢂC during 3e6 h. After completion of the
reaction, the mixture was cooled and poured into ice water. The
precipitates were filtered off and washed with little ethanol.
The product from filtrate was purified by recrystallization.
Compound 2a. (E)-4-fluorobenzaldehyde oxime: white powder,
yield 94%, m.p. 78e79 ꢂC; Compound 2b. (E)-4-chlorobenzaldehyde
oxime: white powder, yield 90%, m.p.99e100.5 ꢂC; Compound 2c.
(E)-3,4,5-trimethoxybenzaldehyde oxime: white powder, yield
83%, m.p. 94e95 ꢂC.
4.3.1. Compound 3-(4-fluorophenyl)-4,5-dihydroisoxazole-5-
carboxylic acid 3a
This compound was obtained following the above-described
method as buff powder, yield 73%, m.p. 148e151 ꢂC; 1H NMR
(600 MHz, CDCl3):
PheH), 5.28e5.25 (m, 1H, CHeO), 3.74e3.69 (m, 2H, CH2CH).
d
¼ 7.71e7.67 (m, 2H, PheH), 7.17e7.11 (m, 2H,
4.3.2. Compound 3-(4-chlorophenyl)-4,5-dihydroisoxazole-5-
carboxylic acid 3b
This compound was obtained following the above-described
method as buff powder, yield 67%, m.p. 164e167 ꢂC; 1H NMR
Table 4
(600 MHz, CDCl3):
2H, PheH), 5.26e5.23 (m, 1H, CHeO), 3.71e3.69 (m, 2H, CH2CH).
d
¼ 7.61 (d, J ¼ 8.4 Hz, 2H, PheH), 7.41 (d, J ¼ 8.4 Hz,
Selective cytotoxic activity of the compounds against human tumor cells.
a
Entry Compd. No. In vitro cytotoxicity IC50
(m )
g mLꢀ1
HepG2b
SGC-7901b BGC-823b NCI-H460b MCF-7b
4.3.3. Compound 3-(3,4,5-trimethoxyphenyl)-4,5-
dihydroisoxazole-5-carboxylic acid 3c
1
2
3
4
5
6
7
8
6a
6c
6i
6j
6k
6m
6n
6o
5a
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
This compound was obtained following the above-described
method as off-white powder, yield 70%, m.p. 71e73 ꢂC; 1H NMR
34.8 ꢁ 3.7 36.8 ꢁ 3.5 23.3 ꢁ 4.9 24.7 ꢁ 6.2 22.4 ꢁ 3.5
>100 >100 >100 >100 >100
(600 MHz, CDCl3):
CHeO), 3.91 (s, 9H, CH3O), 3.71 (d, J ¼ 9 Hz, 2H, CH2CH).
d
¼ 6.90 (s, 2H, PheH), 5.23 (t, J ¼ 8.7 Hz, 1H,
37.7 ꢁ 8.8 37.4 ꢁ 5.9 36.5 ꢁ 9.7 37.5 ꢁ 2.4 34.5 ꢁ 2.2
>100
>100
>100
>100
>100
>100
50.3 ꢁ 2.7 >100
>100
>100
>100
67.6 ꢁ 8.6 65.5 ꢁ 4.5 54.1 ꢁ 6.3 >100
4.4. General synthetic procedure for the key intermediates 5aef
9
53.4 ꢁ 6.1 53.4 ꢁ 4.0 34.9 ꢁ 2.9 35.8 ꢁ 3.7 51.9 ꢁ 7.4
10
11
12
5b
5c
43.6 ꢁ 4.9 68.8 ꢁ 2.1 54.4 ꢁ 4.1 41.9 ꢁ 8.4 68.7 ꢁ 6.7
>100
>100
>100
4.5 ꢁ 0.9 2.4 ꢁ 0.5
>100
>100
12.5 ꢁ 2.8
The key intermediates benzoxazinone 5aef could be conve-
niently obtained by the coupling of the prepared aryl-isoxazoline
acid 3aec with substituted anthranilic acids 4. The general proce-
dures are as follows: To a solution of equivalent aryl-isoxazoline
acid 3aec (10 mmol) and anthranilic acids 4 (10 mmol) in aceto-
nitrile (50 mL) was added dropwise pyridine (5 mL) and followed
by sequential addition of methanesulfonyl chloride (3 mL). The
reaction mixture was then stirred at room temperature, and which
5-FUc
4.8 ꢁ 0.7 6.3 ꢁ 0.8
a
IC50 e Compound concentration required to inhibit tumor cell proliferation by
50%.
b
Abbreviations: HepG2 e Human hepatocellular liver carcinoma cell line; SGC-
7901 e Human gastric cancer cell line; BGC-823 e Human gastric cancer cell line;
NCI-H460 Human large cell lung cancer cell line; MCF-7 human breast
e
e
adenocarcinoma cell line.
c
Used as a positive control.

L. Shi et al. / European Journal of Medicinal Chemistry 54 (2012) 549e556
553
BGC-823 Cell Line
HepG2 Cell Line
5-FU
6i
5-FU
6i
100
75
50
25
0
100
75
50
25
0
6k
6k
1
5
10
20
40
80
1
5
10
20
40
80
Concentration (µg/ml)
Concentration (µg/ml)
Fig. 3. Doseeresponse analysis of cell growth inhibition activity for representative compounds 6i, 6k and 5-FU (positive control) against BGC-823cells (left) and HepG2 cells (right).
was monitored by thin layer chromatography. After completion of
the reaction, the mixture was treated with ice water and formed
yellowish solid as crude benzoxazinone 5aef, which was used for
the next reaction without further purification.
9H, OCH3), 3.73 (t, J ¼ 8.4 Hz, 2H, CH2eNH), 3.27e3.23 (m, 2H,
CH2eC]N), 2.22 (s, 3H, CH3ePh), 1.52e1.48 (m, 2H, CH2CH3), 0.86
(t, J ¼ 7.2 Hz, 3H, CH2CH3); 13C NMR (100 MHz, CDCl3):
d 170.19,
166.87, 156.84, 153.44, 153.39, 140.33, 138.03, 134.83, 134.15, 132.41,
132.29, 130.96, 125.00, 123.79, 123.71, 79.24, 60.96, 56.35, 56.29,
41.71, 39.71, 22.70, 18.66, 11.34; ESI-MS: calcd for C24H28ClN3O6
([M þ 1]þ), 490.2; found, 490.6.
4.5. General synthetic procedure for the target compounds 6aev
The typical process of synthesis of novel anthranilic diamides
derivatives containing aryl-isoxazoline 6aev is shown as following:
To a solution of newly prepared benzoxazinone 5aef (1 mmol) in
acetonitrile (10 mL) was added aqueous of methylamine (1 mL)
dropwise at room temperature. The reaction mixture was stirred
and detected by thin layer chromatography. After completion of the
reaction, the solvents were removed by rotary evaporation and the
residual solid is purified by silica gel column-chromatography
(ethyl acetate/petroleum ether) or recrystallization to give target
compounds 6aev as white solid or crystal. Their physicoechemical
properties and the spectra data are as follows:
4.5.4. N-(4-Chloro-2-(isopropylcarbamoyl)-6-methylphenyl)-3-(4-
fluorophenyl)-4,5-dihydroisoxazole-5-carboxamide 6d
This compound was obtained following the above method as
white floccule, yield 58%, m.p. 223.3e225.1 ꢂC; 1H NMR (600 MHz,
CDCl3)
d
9.28 (s, 1H, NH), 7.71 (d, J ¼ 6.0 Hz, 2H, PheH), 7.30 (s, 1H,
PheH), 7.24 (s, 1H, PheH), 7.16 (t, J ¼ 8.4 Hz, 2H, PheH), 5.77 (d,
J ¼ 9.0 Hz, 1H, NH), 5.27e5.24 (m, 1H, CHeO), 4.10e4.08 (m, 1H,
CHeNH), 3.73 (d, J ¼ 6.0 Hz, 2H, CH2eC]N), 2.20 (s, 3H, CH3ePh),
1.19 (d, J ¼ 6.6 Hz, 3H, CH3), 1.07 (d, J ¼ 6.0 Hz, 3H, CH3); ESI-MS:
calcd for C21H21ClFN3O3 ([M þ 1]þ), 418.1; found, 418.5.
4.5.1. N-(4-Chloro-2-methyl-6-(propylcarbamoyl)phenyl)-3-(4-
fluorophenyl)-4,5-dihydroisoxazole-5-carboxamide 6a
4.5.5. N-(4-Chloro-2-(isopropylcarbamoyl)-6-methylphenyl)-3-(4-
chlorophenyl)-4,5-dihydroisoxazole-5-carboxamide 6e
This compound was obtained following the above method as
This compound was obtained following the above method as
white floccule, yield 64%, m.p. 222e225 ꢂC; 1H NMR (600 MHz,
white floccule, yield 62%, m.p. 232e235 ꢂC; 1H NMR (600 MHz,
CDCl3)
d
9.25 (s, 1H, NH), 7.71 (t, J ¼ 8.4 Hz, 2H, PheH), 7.31 (s, 1H,
CDCl3)
d
9.28 (s, 1H, NH), 7.64 (d, J ¼ 8.4 Hz, 2H, PheH), 7.40 (d,
PheH), 7.12 (t, J ¼ 8.4 Hz, 2H, PheH), 5.97 (s, 1H, NH), 5.26 (t,
J ¼ 7.8 Hz, 1H, CHeO), 3.72 (d, J ¼ 7.8 Hz, 2H, CH2eNH), 3.23 (d,
J ¼ 7.8 Hz, 2H, CH2eC]N), 2.21 (s, 3H, CH3ePh), 1.48 (t, J ¼ 7.5 Hz,
2H, CH2CH3), 0.86 (t, J ¼ 7.2 Hz, 3H, CH2CH3); 13C NMR (100 MHz,
J ¼ 9.0 Hz, 2H, PheH), 7.30 (s, 1H, PheH), 7.24 (s, 1H, PheH), 5.77 (d,
J ¼ 7.8 Hz, 1H, NH), 5.28e5.25 (m, 1H, CHeO), 4.10e4.08 (m, 1H,
CHeNH), 3.72 (d, J ¼ 7.8 Hz, 2H, CH2eC]N), 2.20 (s, 3H, CH3ePh),
1.19 (d, J ¼ 7.2 Hz, 3H, CH3), 1.08 (d, J ¼ 6.6 Hz, 3H, CH3); ESI-MS:
calcd for C21H21Cl2N3O3 ([M þ 1]þ), 434.1; found, 434.5.
CDCl3):
d 170.09, 166.87, 156.07, 138.09, 134.19, 132.50, 130.94,
129.17, 129.00, 124.95, 116.19, 115.97, 79.24, 41.70, 39.69, 22.71,
18.64, 11.32; ESI-MS: calcd for C21H21ClFN3O3 ([M þ 1]þ), 418.1;
found, 418.6.
4.5.6. N-(4-Chloro-2-(isopropylcarbamoyl)-6-methylphenyl)-3-
(3,4,5-trimethoxyphenyl)-4,5-dihydroisoxazole-5-carboxamide 6f
This compound was obtained following the above method as
white floccule, yield 48%, m.p. 215e216 ꢂC; 1H NMR (600 MHz,
4.5.2. N-(4-Chloro-2-methyl-6-(propylcarbamoyl)phenyl)-3-(4-
chlorophenyl)-4,5-dihydroisoxazole-5-carboxamide 6b
CDCl3) d 9.32 (s, 1H, NH), 7.31 (s, 1H, PheH), 7.25 (d, 1H, PheH), 6.93
This compound was obtained following the above method as
(s, 2H, PheH), 5.79 (d, J ¼ 7.8 Hz, 1H, NH), 5.27e5.24 (m, 1H, CHeO),
4.13e4.09 (m, 1H, CHeNH), 3.89 (s, 9H, OCH3), 3.77e3.70 (m, 2H,
CH2eC]N), 2.21 (s, 3H, CH3ePh), 1.19 (d, J ¼ 6.6 Hz, 3H, CH3), 1.09
white floccule, yield 67%, m.p. 231.5e234.2 ꢂC; 1H NMR (600 MHz,
CDCl3)
d
9.26 (s, 1H, NH), 7.65 (d, J ¼ 8.4 Hz, 2H, PheH), 7.41 (d,
J ¼ 9.0 Hz, 2H, PheH), 7.31 (s, 2H, PheH), 5.97 (s,1H, NH), 5.28e5.25
(m, 1H, CHeO), 3.70 (t, J ¼ 5.7 Hz, 2H, CH2eNH), 3.23 (d, J ¼ 8.4 Hz,
2H, CH2eC]N), 2.21 (s, 3H, CH3ePh), 1.51e1.47 (m, 2H, CH2CH3),
0.86 (t, J ¼ 7.5 Hz, 3H, CH2CH3); ESI-MS: calcd for C21H21Cl2N3O3
([M þ 1]þ), 434.1; found, 434.5.
(d, J ¼ 6.0 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3):
d 170.12, 166.14,
156.85, 153.36, 140.32, 138.01, 134.31, 132.32, 132.26, 130.91, 124.96,
123.70,104.46, 79.26, 60.96, 56.28, 42.04, 39.63, 22.49, 22.39, 18.65;
ESI-MS: calcd for C24H28ClN3O6 ([M þ 1]þ), 490.2; found, 490.6.
4.5.7. N-(4-Chloro-2-(cyclohexylcarbamoyl)-6-methylphenyl)-3-
(4-fluorophenyl)-4,5-dihydroisoxazole-5-carboxamide 6g
4.5.3. N-(4-Chloro-2-methyl-6-(propylcarbamoyl)phenyl)-3-(3,4,5-
trimethoxyphenyl)-4,5-dihydroisoxazole-5-carboxamide 6c
This compound was obtained following the above method as
This compound was obtained following the above method as
white floccule, yield 56%, m.p. 245.2e247.0 ꢂC; 1H NMR (600 MHz,
white floccule, yield 56%, m.p. 190.2e192.0 ꢂC; 1H NMR (600 MHz,
CDCl3)
d 9.28 (s, 1H, NH), 7.72e7.69 (m, 2H, PheH), 7.30 (s, 1H,
CDCl3)
d
9.27 (s, 1H, NH), 7.31 (s, 1H, PheH), 7.01 (s, 1H, PheH), 6.94
PheH), 7.24 (s, 1H, PheH), 7.12 (t, J ¼ 8.4 Hz, 2H, PheH), 5.81 (d,
(s, 2H, PheH), 5.98 (s, 1H, NH), 5.27e5.24 (m, 1H, CHeO), 3.89 (s,
J ¼ 8.4 Hz, 1H, NH), 5.27e5.25 (m, 1H, CHeO), 3.78e3.70 (m, 3H,

554
L. Shi et al. / European Journal of Medicinal Chemistry 54 (2012) 549e556
CHeNH and CH2eC]N), 2.20 (s, 3H, CH3ePh), 1.95e1.54 (m, 10H,
(CH2)5); ESI-MS: calcd for C24H25ClFN3O3 ([M þ 1]þ), 458.2; found,
458.6.
4.5.14. N-(4-Chloro-2-methyl-6-(methylcarbamoyl)phenyl)-3-(4-
chlorophenyl)-4,5-dihydroisoxazole-5-carboxamide 6n
This compound was obtained following the above method as
white floccule, yield 64%, m.p. 249.1e250.6 ꢂC; 1H NMR (600 MHz,
4.5.8. N-(4-Chloro-2-(cyclohexylcarbamoyl)-6-methylphenyl)-3-
(4-chlorophenyl)-4,5-dihydroisoxazole-5-carboxamide 6h
CDCl3) d 9.31 (s, 1H, NH), 8.09 (s, 1H, PheH), 7.71e7.41 (m, 6H,
PheH and NH), 5.90 (s, 1H, CHeO), 4.78e4.75 (m, 1H, CH2eC]N),
3.84 (s, 3H, NeCH3), 3.58e3.55 (m, 1H, CH2eC]N), 2.19 (s, 3H,
CH3ePh); ESI-MS: calcd for C19H17Cl2N3O3 ([M þ 1]þ), 406.1;
found, 406.8.
This compound was obtained following the above method as
white floccule, yield 62%, m.p. 140.2e141.5 ꢂC; 1H NMR (600 MHz,
CDCl3)
d
9.21 (s, 1H, NH), 7.93 (s, 1H, PheH), 7.64 (d, J ¼ 8.4 Hz, 2H,
PheH), 7.40 (d, J ¼ 9.0 Hz, 2H, PheH), 7.31 (s, 1H, PheH), 6.06 (bs,
1H, NH), 5.01 (s, 1H, CHeO), 3.86 (m, 1H, NeCH), 3.22 (d, 2H,
CH2eC]N), 2.21 (s, 3H, CH3ePh), 1.94e1.59 (m, 10H, (CH2)5); ESI-
MS: calcd for C24H25Cl2N3O3 ([M þ 1]þ), 474.1; found, 474.6.
4.5.15. N-(4-Chloro-2-methyl-6-(methylcarbamoyl)phenyl)-3-
(3,4,5-trimethoxyphenyl)-4,5-dihydroisoxazole-5-carboxamide 6o
This compound was obtained following the above method as
white floccule, yield 60%, m.p. 186.5e188.0 ꢂC; 1H NMR (600 MHz,
4.5.9. N-(4-Chloro-2-(cyclohexylcarbamoyl)-6-methylphenyl)-3-
(3,4,5-trimethoxyphenyl)-4,5-dihydroisoxazole-5-carboxamide 6i
This compound was obtained following the above method as
white floccule, yield 68%, m.p. 193.3e193.5 ꢂC; 1H NMR (600 MHz,
CDCl3) d 9.38 (s, 1H, NH), 7.29e7.25 (m, 3H, PheH and NH), 7.01 (s,
2H, PheH), 6.22 (s, 1H, CHeO), 5.28e5.25 (m, 1H, CH2eC]N),
3.92e3.83 (m, 10H, CH3O and CH2eC]N), 2.83 (s, 3H, CH3eN),
2.20 (s, 3H, CH3ePh); 13C NMR (100 MHz, CDCl3):
d 170.19,
CDCl3)
d
9.27 (s, 1H, NH), 7.30 (s, 1H, PheH), 7.25 (s, 1H, PheH), 6.93
167.40, 156.87, 153.38, 140.33, 137.87, 133.57, 132.47, 132.21, 131.04,
125.11, 123.68, 104.41, 79.24, 60.97, 56.34, 56.29, 39.74, 26.67,
(s, 2H, PheH), 5.84 (d, J ¼ 7.8 Hz, 1H, NH), 5.27e5.24 (m, 1H, CHeO),
3.89 (s, 9H, OCH3), 3.78e3.67 (m, 3H, NeCH and CH2eC]N), 2.21
(s, 3H, CH3ePh), 1.98e1.52 (m, 10H, (CH2)5); ESI-MS: calcd for
C27H32ClN3O6 ([M þ 1]þ), 530.2; found, 530.9.
18.66; ESI-MS: calcd for C22H24ClN3O6 ([M
found, 462.7.
þ
1]þ), 462.1;
4.5.16. N-(4-Chloro-2-methyl-6-(phenylcarbamoyl)phenyl)-3-(4-
fluorophenyl)-4,5-dihydroisoxazole-5-carboxamide 6p
4.5.10. N-(4-Chloro-2-(diethylcarbamoyl)-6-methylphenyl)-3-(4-
fluorophenyl)-4,5-dihydroisoxazole-5-carboxamide 6j
This compound was obtained following the above method as
This compound was obtained following the above method as
white floccule, yield 42%, m.p. 224.7e226.2 ꢂC; 1H NMR (600 MHz,
light yellow floccule, yield 58%, m.p. 144.9e147.2 ꢂC; 1H NMR
CDCl3) d 8.83 (s, 1H, NH), 7.90 (s, 1H, NH), 7.46e7.35 (m, 7H, PheH),
(600 MHz, CDCl3)
d
8.61 (s, 1H, NH), 7.69 (s, 2H, PheH), 7.25 (s, 1H,
7.24e7.02 (m, 4H, PheH), 5.23 (s, 1H, CHeO), 3.62e3.55 (m, 2H,
CH2eC]N), 2.25 (s, 3H, CH3ePh); ESI-MS: calcd for C24H25ClFN3O3
([M þ 1]þ), 458.2; found, 458.7.
PheH), 7.14e7.11 (m, 2H, PheH), 7.07 (s, 1H, PheH), 5.23 (s, 1H,
CHeO), 3.69 (d, J ¼ 7.8 Hz, 2H, NeCH2), 3.23e3.15 (m, 4H, NeCH2
and CH2eC]N), 2.18 (s, 3H, CH3ePh), 1.10 (s, 3H, CH3), 0.98
(s, 3H, CH3); ESI-MS: calcd for C22H23ClFN3O3 ([M þ 1]þ), 432.1;
found, 432.7.
4.5.17. 3-(4-Fluorophenyl)-N-(2-(isopropylcarbamoyl)phenyl)-4,5-
dihydroisoxazole-5-carboxamide 6q
This compound was obtained following the above method as
4.5.11. N-(4-Chloro-2-(diethylcarbamoyl)-6-methylphenyl)-3-(4-
chlorophenyl)-4,5-dihydroisoxazole-5-carboxamide 6k
white floccule, yield 55%, m.p. 146.0e148.2 ꢂC; 1H NMR (600 MHz,
CDCl3)
d
12.05 (s, 1H, NH), 8.59 (d, J ¼ 8.4 Hz, 1H, PheH), 8.26 (d,
This compound was obtained following the above method as
J ¼ 7.8 Hz, 1H, PheH), 7.65e7.36 (m, 4H, PheH), 5.93 (bs, 1H, NH),
5.30e5.27 (m, 1H, CHeO), 4.37e4.34 (m, 1H, CHeNH), 3.77e3.67
(m, 2H, CH2eC]N), 1.27e1.12 (m, 6H, CH3); ESI-MS: calcd for
C20H20FN3O3 ([M þ 1]þ), 370.1; found, 370.6.
white floccule, yield 55%, m.p. 152.7e155.2 ꢂC; 1H NMR (600 MHz,
CDCl3)
d
8.55 (s, 1H, NH), 7.63 (d, J ¼ 7.2 Hz, 2H, PheH), 7.41 (d,
J ¼ 8.4 Hz, 2H, PheH), 7.25 (s, 1H, PheH), 7.07 (s, 1H, PheH), 5.23 (s,
1H, CHeO), 3.68 (d, J ¼ 7.2 Hz, 2H, NeCH2), 3.25e3.16 (m, 4H,
NeCH2 and CH2eC]N), 2.19 (s, 3H, CH3-Ph), 1.10 (s, 3H, CH3), 0.98
(s, 3H, CH3); ESI-MS: calcd for C22H23Cl2N3O3 ([M þ 1]þ), 448.1;
found, 448.6.
4.5.18. 3-(4-Chlorophenyl)-N-(2-(isopropylcarbamoyl)phenyl)-4,5-
dihydroisoxazole-5-carboxamide 6r
This compound was obtained following the above method as
white floccule, yield 63%, m.p. 131.4e132.6 ꢂC; 1H NMR (600 MHz,
4.5.12. N-(4-Chloro-2-(diethylcarbamoyl)-6-methylphenyl)-3-
(3,4,5-trimethoxyphenyl)-4,5-dihydroisoxazole-5-carboxamide 6l
This compound was obtained following the above method as
white floccule, yield 63%, m.p. 159.2e160.7 ꢂC; 1H NMR (600 MHz,
CDCl3)
d
12.05 (s, 1H, NH), 8.59 (d, J ¼ 8.4 Hz, 1H, PheH), 7.64 (d,
J ¼ 8.4 Hz, 2H, PheH), 7.47e7.36 (m, 4H, PheH), 7.12e7.09 (m, 1H,
PheH), 5.94 (bs, 1H, NH), 5.29e5.27 (m, 1H, CHeO), 4.37e4.34 (m,
1H, CHeNH), 3.78e3.67 (m, 2H, CH2eC]N), 1.33e1.24 (m, 6H,
CDCl3)
d
8.58 (s, 1H, NH), 7.26 (s, 1H, PheH), 7.08 (s, 1H, PheH), 6.91
CH3); 13C NMR (100 MHz, CDCl3):
d 169.82, 167.80, 159.63, 156.15,
(s, 2H, PheH), 5.22 (s, 1H, CHeO), 3.89 (s, 9H, OCH3), 3.70 (d,
J ¼ 6.6 Hz, 2H, NeCH2), 3.25e3.15 (m, 4H, NeCH2 and CH2eC]N),
2.20 (s, 3H, CH3ePh), 1.13 (s, 3H, CH3), 0.99 (s, 3H, CH3); ESI-MS:
calcd for C25H30ClN3O6 ([M þ 1]þ), 504.2; found, 504.8.
155.91,133.54,132.84,132.20,129.26,129.02,128.33,128.20,123.76,
79.59, 41.95, 39.49, 22.72; ESI-MS: calcd for C20H20ClN3O3
([M þ 1]þ), 386.1; found, 386.5.
4.5.19. N-(2-(Isopropylcarbamoyl)phenyl)-3-(3,4,5-
4.5.13. N-(4-Chloro-2-methyl-6-(methylcarbamoyl)phenyl)-3-(4-
fluorophenyl)-4,5-dihydroisoxazole-5-carboxamide 6m
trimethoxyphenyl)-4,5-dihydroisoxazole-5-carboxamide 6s
This compound was obtained following the above method as
white floccule, yield 52%, m.p. 198.2e199.8 ꢂC; 1H NMR (600 MHz,
This compound was obtained following the above method as
white floccule, yield 54%, m.p. 169.5e172.0 ꢂC; 1H NMR (600 MHz,
CDCl3)
d 12.08 (s, 1H, NH), 8.59 (s, 1H, PheH), 7.45 (s, 2H, PheH),
CDCl3)
d
9.27 (s, 1H, NH), 8.09 (s, 1H, PheH), 7.78 (s, 2H, PheH), 7.72
7.12 (s, 1H, PheH), 6.95 (s, 2H, PheH), 5.93 (bs, 1H, NH),
5.29e5.26 (m, 1H, CHeO), 4.36e4.34 (m, 1H, CHeNH), 3.87 (s, 9H,
OCH3), 3.80e3.71 (m, 2H, CH2eC]N), 1.27 (d, J ¼ 6.0 Hz, 3H, CH3),
1.24 (d, J ¼ 6.0 Hz, 3H, CH3); ESI-MS: calcd for C23H27N3O6
([M þ 1]þ), 442.2; found, 442.6.
(s, 1H, NH), 7.52 (s, 1H, PheH), 7.16e7.12 (m, 2H, PheH), 5.91e5.88
(m, 1H, CHeO), 4.80e4.75 (m, 1H, CH2eC]N), 3.84 (s, 3H, NeCH3),
3.58e3.53 (m, 1H, CH2eC]N), 2.49 (s, 3H, CH3ePh); ESI-MS: calcd
for C19H17ClFN3O3 ([M þ 1]þ), 390.1; found, 390.6.

L. Shi et al. / European Journal of Medicinal Chemistry 54 (2012) 549e556
555
4.5.20. 3-(4-Fluorophenyl)-N-(2-(methylcarbamoyl)phenyl)-4,5-
Appendix A. Supplementary data
dihydroisoxazole-5-carboxamide 6t
This compound was obtained following the above method as
Supplementary data related to this article can be found online at
http://dx.doi.org/10.1016/j.ejmech.2012.06.001.
white floccule, yield 68%, m.p. 195.2e196.8 ꢂC; 1H NMR (600 MHz,
CDCl3)
d 12.43 (s, 1H, NH), 8.71 (s, 1H, PheH), 8.14 (s, 1H, PheH),
7.66e7.57 (m, 3H, PheH), 7.16e7.06 (m, 3H, PheH), 5.30e5.20 (m,
1H, CHeO), 3.81e3.72 (m, 2H, CH2eC]N), 3.04 (s, 3H, NCH3); ESI-
MS: calcd for C18H16FN3O3 ([M þ 1]þ), 342.1; found, 342.7.
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This compound was obtained following the above method as
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d
12.20 (s, 1H, NH), 8.62 (d, J ¼ 7.8 Hz, 1H, PheH),
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4.5.22. N-(2-(Methylcarbamoyl)phenyl)-3-(3,4,5-
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d
12.23 (s, 1H, NH), 8.63 (d, J ¼ 7.8 Hz, 1H, PheH), 7.47 (t,
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(100 MHz, CDCl3):
d 170.15, 168.91, 156.66, 153.41, 153.30, 140.13,
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to each well. The plate was further incubated for 4 h to allow MTT
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We gratefully acknowledge the support of this work by the
Projects in the Youth Science Foundation of Hubei Academy of
Agricultural Sciences (2011NKYJJ19) and the National Natural
Science Foundation of China (31000867). The authors also grate-
fully acknowledge the partial support from Hubei Biopesticide
Engineering Research Center.
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