Novel 5-(3-(substituted)-4,5-dihydroisoxazol-5-yl)-2-methoxyphenyl derivatives: Synthesis and anticancer activity

Article abstract of DOI:10.1071/CH07395

Thirty novel 5-(3-(substituted phenyl)-4,5-dihydroisoxazol-5-yl)-2- methoxyphenyl derivatives were synthesized and evaluated for their antitumour activity. The bioassays showed that the 2-fluorobenzoyl derivative 6ai, the 4-trifluoromethylbenzoyl derivati

Full text of DOI:10.1071/CH07395

Full Paper
CSIRO PUBLISHING
Aust. J. Chem. 2008, 61, 864–869
www.publish.csiro.au/journals/ajc
Novel 5-(3-(Substituted)-4,5-dihydroisoxazol-5-yl)-
2-methoxyphenyl Derivatives: Synthesis
and Anticancer Activity
Xin-Hua Liu,A,D Bao-An Song,C Pinaki S. Bhadury,C Hai-Liang Zhu,A,B
Ping Cui,A Ke-Ke Hou,C and Hong-Li XuA
AKey Laboratory of Anhui Educational Department, Anhui University of Technology,
Maanshan, 243002, China.
BState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, 210093, China.
CKey Laboratory of Green Pesticide and Agriculture Bioengineering, Ministry of Education,
Guizhou University, Guiyang, 55002, China.
DCorresponding author. Email: zlp@ahut.edu.cn
Thirty novel 5-(3-(substituted phenyl)-4,5-dihydroisoxazol-5-yl)-2-methoxyphenyl derivatives were synthesized and
evaluated for their antitumour activity. The bioassays showed that the 2-fluorobenzoyl derivative 6ai, the 4-
trifluoromethylbenzoyl derivative 6ah, and the 3-trifluoromethyl isoxazole derivatives (6ch and 6ci) were highly effective
against PC-3 cells. The IC50 values of 6ah and 6ai against PC-3 cells were 1.5 and 1.8 µg mL−1, respectively.
Manuscript received: 16 November 2007.
Final version: 16 April 2008.
Introduction
number of selective inhibitors of cancer cells seem to bear
the characteristic diaryloxazole template. Furthermore, incor-
poration of fluorine into aromatic and heterocyclic moieties is
known to significantly enhance the anticancer activity of the
molecule.[15–19] Fascinated by these findings and our own results
(X. H. Liu, B. Li, H. L. Zhu, unpubl. data),[13] with an aim to
developnewandpotentinhibitorswithanticanceractivity, herein
we designed a series of novel 5-(3-(substituted)-4,5-dihydro-
isoxazol-5-yl)-2-methoxyphenyl derivatives that contain a fluo-
rinatedfunctionality.Thesyntheticroutetothetargetcompounds
is shown in Scheme 1. The structures of the novel compounds
wereconfirmedbyspectroscopicanalysis.Thestructure–activity
relationship (SAR) was studied and the compounds were eval-
uated for their antiproliferative activities against PC-3 (human
prostate cancer) and A431 (human epidermoid carcinoma can-
cer cell) in vitro by the MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide) method. Among them, the
fluorinated benzoyl derivatives 6ah, 6ai, 6ch, and 6ci were
found to be highly effective against PC-3 cells, and 6ah, 6ai,
and 6bh showed effective activities againstA431 cells. Acryloyl
and cinnamoyl derivatives 6af and 6bg showed effective activ-
ities against A431 cells. The IC50 values of the highly active
compounds 6ah, 6ai, 6ch, and 6ci against PC-3 cells were in
the range 1.5–2.2 µg mL−1. The IC50 values of the highly active
compounds 6af, 6ah, 6ai, 6bg, and 6bh against A431 cells were
Oxazoles, typical building blocks of numerous biologically
active naturally occurring and synthetically prepared hetero-
cyclic compounds,[1,2] have gained considerable attention in the
past 10 years.[3–8] They are most commonly obtained by the
Hantzschreaction[9] orthecyclodehydrationofβ-ketoamides,[10]
the dehydrogenation of oxazolines, and other processes such as
Schmidt rearrangements[11] have also been employed. Hetero-
cyclic compounds that incorporate the oxazole moiety exhibit a
wide range of bioactivity, e.g., antitumour growth inhibition in
human colorectal DLD-1 xenograft mouse model (compoundA,
Fig. 1),[12] anticancer activity against human prostate and
epidermoid carcinoma cells (compounds B and C, Fig. 1)
(X. H. Liu, B. Li, H. L. Zhu, unpubl. data),[13] and as potential
radiotracers for imaging COX-2 drug with positron emission
tomography (PET) and as epidermal growth factor receptor
bioprobe (fluorinated compound D, Fig. 1).[14] Thus, a large
O
Me
N
N
MeO
MeO
O
O
N
O
N
N
H
R
O
F
B
MeO
A
OMe
in the range 2.3–2.6 µg mL−1
.
OMe
MeO
N
S
R
O
O
O
Results and Discussion
O
H2N
S
N
CH2F
D
Chemistry
C
R
The synthesis of the title heterocyclic isoxazoles 6 from the start-
ing material, vanillin, is achieved in three steps as depicted in
Scheme1. However, ourattemptedconversionofvanillinintothe
Fig. 1. Typical oxazole derivatives possessing inhibitory activities against
cancer cells.
© CSIRO 2008
10.1071/CH07395
0004-9425/08/110864

Novel Isoxazole Derivatives
865
O
HO
O
H2SO4
NH2OH·HCl
Pyridine
R1
HO
MeO
O
ϩ
CeO2/CNTs
O
R1
Vanillin
4
O
O
O
N
N
R2COCl
O
R2
OH
OMe
R1
R1
OMe
6
5
Scheme 1. Synthetic route to the target compounds 6. R1 = 2,4-dichlorophenyl (a), 2,3,4-trimethoxyphenyl (b),
trifluoromethyl (c). R2 = ethyl (d), n-propyl (e), vinyl (f), styryl (g), 4-(trifluoromethyl)phenyl (h), 2-fluorophenyl (i),
4-chlorophenyl (j), 2-nitrophenyl (k), 2-methylphenyl (l), 2-methoxyphenyl (m).
Table 1. Yields of 5a obtained under various reaction conditions
A431. The cells were allowed to proliferate in the presence of
tested material for 48 h, and the results are reported in terms of
IC50 values (Table 2). From the IC50 values, it is obvious that
compounds6ah, 6ai, 6ch, and 6ci exhibitthestrongestinhibitory
activities against the PC-3 cell and the values could compare well
with that of the potent 5-fluorouracil serving as a positive con-
trol. Compounds 6af, 6ah, 6ai, and 6bh showed potent activities
against the A431 cell comparable to that of the positive control.
The rationale behind selecting a large number of compounds
bearing different functionalities was to establish a definite SAR
pattern and emphasize the role of fluorine in imparting bioactiv-
ity. The choice of compounds was also governed by availability
of the reagents and ease of preparation of title compounds
under laboratory conditions. Scanning Table 2, we found that
there was a clear SAR against PC-3 cells. Inspection of the
chemical structures of the final compounds (Scheme 1) sug-
gests that the synthesized isoxazoles 6 could be divided into
their 3-trifluoromethylated and 3-aryl derivatives without bear-
ing any fluorine moiety. The influence of a 3-CF3 substituent
on bioactivity is clearly demonstrated by the observed higher
activities against PC-3 cells with the IC50 values in the range
1.9–8.9 µg mL−1. Similarly, it can be derived from the data
presented in Table 2 that the nature of the R2 group in the
title isoxazole also significantly influences the antitumour activ-
ity. With a fluorinated substituent (F or CF3) in the phenyl
ring, the compounds exhibited enhanced bioactivity against the
PC-3 cells (6ah, 6ai, 6bh, 6ch, 6ci) and A431 cells (6ah, 6ai,
6bh, 6bi). The corresponding non-fluorinated ethyl, propyl,
vinyl, styryl, 4-chlorophenyl, 2-nitrophenyl, 2-methylphenyl,
and 2-methoxyphenyl analogues revealed lower cytotoxicity.
Entry
Solvent
Base
Time [h]
Conditions
Yield [%]
1
2
3
4
5
6
7
n-Butanol
n-Butanol
Ethanol
Pyridine
Pyridine
Pyridine
Pyridine
Na2CO3
NaOH
NaOH
—
NaOH
KOH
10
10
10
10
10
6
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
—
—
—
11
31
56
46
KOH
10
intermediate α,β-unsaturated ketone, e.g., (E)-1,1,1-trifluoro-
4-(4-hydroxy-3-methoxyphenyl)but-3-en-2-one 4c under acidic
conditions, by following a literature procedure,[20] did not
yield the expected result. Addition of a catalytic amount
of CeO2/CNTs (carbon nanotube supported cerium dioxide
nanoparticles) to the reaction mixture under reflux conditions
helped to obtain the desired product with 85% yield within 10 h.
The preparation of the intermediate isoxazole 5 is the key
step for the synthesis of the title compounds. In the reported
routes, there are two methods: the first utilizes ene as a start-
ing material, which is first converted into the vinyl nitrate and
then rearranged photochemically to yield theisoxazole;[21] In the
second, an enone is converted into the oxime derivative, which
was rearranged to isoxazole by Pb(OAc)4, I2, or by treatment
with KI.[22] In order to optimize the reaction conditions, prepa-
ration of 5a was carried out under various conditions (Table 1). It
could be observed from the data presented in Table 1 that while
in alcoholic solvents like ethanol and n-butanol (entries 1–3)
practically no product resulted, the use of pyridine significantly
improved the yield, particularly in the presence of an added base
(entries 5–7). With extended reaction time (entry 7), however,
the overall yield was lower, presumably because of the formation
of side reaction products. Under optimal conditions, a yield of
up to 56% (entry 6) could be achieved when a mixture of the
enone 4a, hydroxylamine hydrochloride (1.5 equiv.) and KOH
(1.5 equiv.) was refluxed for 6 h in pyridine.
Conclusions
A series of arylisoxazole derivatives were synthesized and their
SAR were studied against two cancer cell lines. The bioassay
results revealed that incorporation of fluorine into the title com-
pounds played an important role towards the development of
a new class of anticancer drugs. Among the synthesized com-
pounds 6ah, 6ai, 6ch, and 6ci exhibited the strongest inhibitory
activities against PC-3 cells. Compounds 6af, 6bg, 6ai, 6ah,
and 6bh showed effective activities against A431 cells, whereas
3-trifluoromethyl isoxazole derivatives showed higher activi-
ties against PC-3 cells with IC50 values ranging from 1.9 to
Esterification of compound 5a with acyl chloride produced
6a in moderate yield. In order to optimize the reaction condi-
tions, various bases, such as pyridine, triethylamine, potassium
carbonate, NMM (N-methylmorpholine), and DMAP (N,N-
dimethyl-4-aminopyridine) were used. Furthermore, effects of
solvent, reaction time, and molar ratio of the substrates on the
esterification were examined. As a consequence, the best result
was obtained when compound 5a was treated with 1.5 equiv. of
acyl chloride and 1.5 equiv. of pyridine in chloroform for 10 h.
8.9 µg mL−1
.
Experimental
General
Biological Activity
The melting points of the products were determined on an XT-4
binocular microscope (Beijing Tech Instrument Co., China) and
In the screening assay studies, all the 30 compounds were eval-
uated for their cytotoxic activity against two cell lines, PC-3 and
1
are not corrected. H and 13C NMR spectra were recorded on

866
X.-H. Liu et al.
Table 2. Cytotoxic activity of the synthesized compounds against PC-3 and A431 cell linesA
B
B
Compound
IC50 [µg mL−1
]
Compound
IC50 [µg mL−1
]
PC-3
A431
PC-3 A431
6ad
6ae
6af
6ag
6ah
6ai
6aj
6ak
6al
6am
6bd
6be
6bf
6bg
17.8 0.16
13.5 0.01
8.9 0.08
12.5 0.01
1.5 0.02
1.8 0.09
17.1 0.01
24.8 0.19
21.3 0.11
16.4 0.19
48.9 0.08
28.7 0.31
11.2 0.20
13.7 0.18
2.6 0.11
2.0 0.13
20.1 0.32
18.8 0.02
2.5 0.05
2.9 0.11
2.5 0.18
2.6 0.15
19.2 0.03
19.8 0.22
19.0 0.18
17.6 0.20
16.2 0.17
15.4 0.30
13.5 0.04
2.4 0.10
2.3 0.18
2.3 0.21
6bi
6bj
6bk
6bl
6bm
6cd
6ce
6cf
6cg
6ch
6ci
6cj
6ck
6cl
8.4 0.07
22.3 0.11
19.8 0.28
32.5 0.33
22.6 0.08
6.2 0.08
7.1 0.15
4.0 0.06
2.9 0.12
2.2 0.20
1.9 0.16
8.9 0.07
6.1 0.06
7.7 0.01
8.8 0.05
3.3 0.16
29.8 0.12
24.6 0.10
27.2 0.07
35.3 0.18
29.7 0.13
25.8 0.22
27.4 0.12
19.8 0.20
21.3 0.11
24.0 0.08
20.0 0.19
19.6 0.08
25.6 0.10
29.4 0.25
6bh
5-FluorouracilC
6cm
AThe data represent the mean of three experiments in triplicate and were expressed as mean s.d.
BThe IC50 value was defined as the concentration at which 50% survival of cells was observed. The results are listed in the table.
CUsed as a positive control.
a Varian INOVA400 (400 MHz) pulse Fourier-transform NMR
spectrometer in CDCl3 using tetramethylsilane as an internal
standard. 19F NMR spectra were obtained on aVarian EM-360A
spectrometer using CF3COOH (TFA) as an external standard,
positive for downfield shift. Electrospray ionization (ESI) mass
spectra were obtained on a Mariner 5304 mass spectrometer. Ele-
mental analysis was performed on an Elementar Vario-III CHN
analyzer. Column chromatographic operations were performed
on silica gel GF254. The reagents were all of analytical reagent
grade or chemically pure.All solvents were dried, deoxygenated,
and redistilled before use. Compounds 4a and 4b (Scheme 1)
were synthesized according to a previously published report.[23]
4-(3-(2,4-Dichlorophenyl)-4,5-dihydroisoxazol-5-yl)-
2-methoxyphenylate Derivatives 6a
To a solution of 4-(3-(2,4-dichlorophenyl)-4,5-dihydro-
isoxazol-5-yl)-2-methoxyphenol 5a (2 mmol) and pyridine
(3.0 mmol) in dry chloroform (30 mL) at 5◦C was added
acid chloride (3 mmol) in a dropwise fashion over 30 min.
The reaction mixture for the preparation of 6ad–6af was
stirred at 10◦C and the reaction mixture for the preparation
of 6ag–6am was refluxed for 10 h. The solution was then
poured into 20 of mL water. After removal of the aqueous
layer, the chloroform layer was washed with water (10 mL),
5% potassium carbonate solution, and dried over anhydrous
sodium sulfate. The solvent was evaporated and the crude
residue was purified by chromatography on SiO2 (acetone/
petroleum, v/v, 2/1) to give 6ad–6am as colourless solids. Their
spectra are given in the Accessory Publication.
Syntheses
4-(3-(2,4-Dichlorophenyl)-4,5-dihydroisoxazol-5-yl)-
2-methoxyphenol 5a
A mixture of 4a (2 mmol), KOH (3 mmol), and hydroxyl-
amine hydrochloride (3 mmol) were dissolved in pyridine
(20 mL) and the resulting mixture was refluxed for 6 h. The
reaction mixture was acidified with glacial acetic acid (3 mmol),
poured into ice water, and allowed to stand at room temperature
overnight. The product was collected by filtration and recrys-
tallized from alcohol to give compound 5a as yellow crystals
(56%), mp 139–140◦C. δH (400 MHz, DMSO) 3.20 (dd, J 9.2
and 19.3, 1H, H4a), 3.51 (dd, J 11.8 and 19.3, 1H, H4b), 3.75 (s,
3H, OMe), 5.15 (dd, J 9.2 and 11.8, 1H, H5), 5.98 (s, 1H, OH),
6.56–6.67 (m, 3H, ArH), 7.32–7.49 (m, 3H, ArH).
Compounds 5b and 5c were synthesized by the same method.
Compound 5b was obtained as colourless crystals (65%), mp
144–145◦C. δH (400 MHz, DMSO) 3.31 (dd, J 8.8 and 18.8, 1H,
H4a), 3.68 (dd, J 11.3 and 18.8, 1H, H4b), 3.73–3.81 (4s, 12H,
4OMe), 5.17 (dd, J 8.8 and 11.3, 1H, H5), 5.92 (s, 1H, OH),
6.55–6.67 (m, 3H, ArH), 6.85–7.24 (m, 2H, ArH).
Compounds 6bd–6bm and 6cd–6cm were synthesized by the
same method.
Compound 6ad was obtained as colourless crystals (60%),
mp 128–129◦C. δH (CDCl3, 400 MHz) 0.98 (t, J 7.2, 3H, CH3),
2.19 (m, 2H, CH2), 3.16 (dd, J 9.2 and 19.2, 1H, H4a), 3.41 (dd,
J 11.9 and 19.2, 1H, H4b), 3.80 (s, 3H, OMe), 5.22 (dd, J 9.2 and
11.9, 1H, H5), 6.70–6.88 (m, 3H,ArH), 7.30–7.46 (m, 3H,ArH).
δC 9.91, 27.60, 42.13, 56.55, 80.91, 111.98, 120.40, 123.34,
127.82, 131.21, 132.80, 135.95, 136.24, 138.42, 138.86, 139.28,
156.56, 157.75, 173.16. m/z 393.6 (C19H17Cl2NO4, [M + H]+).
(Found: C 58.2, H 4.7, N 3.9. Calc. for C19H17Cl2NO4: C 57.9,
H 4.4, N 3.6%.)
Compound 6ae was obtained as colourless crystals (58%),
mp 133–134◦C. δH (CDCl3, 400 MHz) 0.93 (t, J 7.2, 3H, CH3),
1.55–2.28 (m, 4H, 2CH2), 3.20 (dd, J 9.2 and 19.3, 1H, H4a),
3.46 (dd, J 11.9 and 19.3, 1H, H4b), 3.77 (s, 3H, OMe), 5.20
(dd, J 9.2 and 11.8, 1H, H5), 6.65–6.90 (m, 3H, ArH), 7.28–
7.49 (m, 3H, ArH). δC 13.22, 18.88, 36.36, 42.28, 56.39, 80.97,
112.13, 120.56, 123.30, 127.89, 131.38, 132.65, 135.87, 136.16,
138.40, 138.98, 139.21, 156.67, 157.88, 173.25. m/z 408.7
(C20H19Cl2NO4, [M + H]+). (Found: C 58.6, H 4.6, N 3.1. Calc.
for C20H19Cl2NO4: C 58.8, H 4.7, N 3.4%.)
Compound 5c was obtained as colourless crystals (85%), mp
94–95◦C. δH (400 MHz, DMSO) 3.50 (dd, J 6.2 and 17.7, 1H,
H4a), 3.77 (s, 3H, OMe), 3.98 (dd, J 10.6 and 17.7, 1H, H4b),
5.41 (dd, J 6.2 and 10.6, 1H, H5), 5.96 (s, 1H, OH), 6.55–6.70
(m, 3H, ArH).

Novel Isoxazole Derivatives
867
Compound 6af was obtained as colourless crystals (48%),
mp 126–127◦C. δH (CDCl3, 400 MHz) 3.09 (dd, J 9.2 and 19.3,
1H, H4a), 3.42 (dd, J 11.9 and 19.3, 1H, H4b), 3.81 (s, 3H, OMe),
5.10 (dd, J 9.2 and 11.9, 1H, H5), 5.84 (dd, J 10.5 and 1.3, 1H),
6.16(dd, J10.4and17.2, 1H), 6.32(dd, J1.3and17.2, 1H), 6.69–
6.92 (m, 3H, ArH), 7.28–7.41 (m, 3H, ArH). δC 41.92, 56.25,
81.23, 111.55, 120.33, 123.17, 127.66, 128.28, 131.15, 132.43,
133.58, 135.69, 135.88, 138.25, 138.60, 139.01, 156.72, 157.68,
164.00. m/z (ESI) 392.6 (C19H15Cl2NO4, [M + H]+). (Found:
C 58.2, H 4.2, N 3.9. Calc. for C19H15Cl2NO4: C 58.2, H 3.9,
N 3.6%.)
Compound 6af was obtained as colourless crystals (55%),
mp 148–150◦C. δH (CDCl3, 400 MHz) 3.17 (dd, J 9.2 and 19.3,
1H, H4a), 3.36 (dd, J 11.9 and 19.3, 1H, H4b), 3.73 (s, 3H, OMe),
5.08 (dd, J 9.2 and 11.9, 1H, H5), 6.57 (d, J 16.0, 1H), 6.66–
6.90 (m, 3H, ArH), 7.06–7.40 (m, 8H, ArH), 7.78 (d, J 16.0,
1H). δC 41.84, 56.22, 81.40, 111.68, 115.98, 120.36, 123.03,
126.66, 127.63, 128.37, 128.99, 131.11, 132.29, 135.46, 135.58,
135.67, 138.10, 138.77, 139.23, 148.52, 156.65, 157.69, 164.44.
m/z (ESI) 468.0 (C25H19Cl2NO4, [M + H]+). (Found: C 64.4,
H 4.4, N 3.2. Calc. for C25H19Cl2NO4: C 64.1, H 4.1, N 3.0%.)
Compound 6ah was obtained as colourless crystals (63%),
mp 150–151◦C. δH (CDCl3, 400 MHz) 3.26 (dd, J 9.2 and 19.3,
1H, H4a), 3.39 (dd, J 11.9 and 19.3, 1H, H4b), 3.75 (s, 3H, OMe),
5.14 (dd, J 9.2 and 11.9, 1H, H5), 6.62–6.67 (m, 3H,ArH), 7.28–
7.46 (m, 3H, ArH), 7.76 (d, J 8.4, 2H), 8.15 (d, J 8.4, 2H, CF3-
ArH). δF (CDCl3,TFA)−118.56. δC 41.68, 56.34, 81.28, 111.77,
120.16, 123.03, 124.48, 125.60, 127.52, 130.99, 131.06, 132.38,
133.88, 135.52, 135.63, 136.52, 138.10, 138.45, 138.88, 156.60,
157.71, 165.66. m/z (ESI) 510.5 (C24H16Cl2F3NO4, [M + H]+).
(Found:C56.7, H3.4, N3.1. Calc. forC24H16Cl2F3NO4:C56.5,
H 3.2, N 2.7%.)
Compound 6ai was obtained as colourless crystals (60%), mp
141–142◦C. δH (CDCl3, 400 MHz) 3.20 (dd, J 9.2 and 19.3, 1H,
H4a), 3.46 (dd, J 11.9 and 19.3, 1H, H4b), 3.77 (s, 3H, OMe),
5.19 (dd, J 9.2 and 11.8, 1H, H5), 6.80–7.00 (m, 3H, ArH),
7.18–8.27 (m, 7H, ArH). δF (CDCl3, TFA) −60.64. δC 41.81,
56.22, 81.18, 111.99, 115.77, 119.23, 120.31, 123.18, 124.56,
127.63, 130.84, 132.12, 132.39, 135.48, 135.61, 136.84, 138.06,
138.40, 138.92, 156.68, 157.70, 161.22, 165.48. m/z (ESI) 460.8
(C23H16Cl2FNO4, [M + H]+). (Found: C 60.3, H 3.2, N 3.0.
Calc. for C23H16Cl2FNO4: C 60.0, H 3.5, N 3.0%.)
Compound 6aj was obtained as colourless crystals (56%),
mp 154–155◦C. δH (CDCl3, 400 MHz) 3.18 (dd, J 9.2 and 19.3,
1H, 4-Ha), 3.41 (dd, J 11.9 and 19.3, 1H, 4-Hb), 3.77 (s, 3H,
OMe), 5.12 (dd, J 9.2 and 11.9, 1H, 5-H), 6.78–7.00 (m, 3H,
ArH), 7.36–8.19 (m, 7H, ArH). δC 41.89, 56.30, 81.29, 111.90,
120.26, 123.30, 127.46, 128.82, 129.26, 130.81, 131.99, 132.18,
135.56, 135.65, 138.06, 138.33, 138.85, 139.81, 156.62, 157.71,
165.59. m/z (ESI) 476.5 (C23H16Cl3NO4, [M + H]+). (Found:
C 58.26, H 3.07, N 2.69. Calc. for C23H16Cl3NO4: C 57.95,
H 3.38, N 2.94%.)
J 9.2 and 19.3, 1H, 4-Ha), 3.40 (dd, J 11.9 and 19.3, 1H, 4-Hb),
3.73 (s, 3H, OMe), 5.15 (dd, J 9.2 and 11.8, 1H, 5-H), 6.75–
7.00 (m, 3H, ArH), 7.21–8.08 (m, 7H, ArH). δC 17.78, 41.76,
56.19, 81.25, 111.90, 120.20, 123.11, 126.01, 127.41, 129.28,
130.58, 130.82, 130.88, 132.27, 134.19, 135.58, 135.70, 138.00,
138.31, 138.96, 139.69, 156.52, 157.60, 165.57. m/z (ESI) 456.6
(C24H19Cl2NO4, [M + H]+). (Found: C 63.42, H 4.09, N 3.34.
Calc. for C24H19Cl2NO4: C 63.17, H 4.20, N 3.07%.)
Compound 6am was obtained as colourless crystals (50%),
mp 154–155◦C. δH (CDCl3, 400 MHz) 3.11 (dd, J 9.2 and 19.3,
1H, 4-Ha), 3.35 (dd, J 11.9 and 19.3, 1H, 4-Hb), 3.73–3.78
(2s, 6H, 2OMe), 5.10 (dd, J 9.2 and 11.8, 1H, 5-H), 6.77–
6.98 (m, 3H, ArH), 7.44–8.06 (m, 7H, ArH). δC 41.90, 56.18,
56.25, 81.16, 111.87, 114.15, 114.51, 120.20, 121.27, 123.13,
127.46, 130.79, 131.68, 132.50, 135.42, 135.78, 135.90, 138.00,
138.21, 138.86, 156.61, 157.74, 161.89, 165.57. m/z (ESI) 472.5
(C24H19Cl2NO5, [M + H]+). (Found: C 61.31, H 4.40, N 3.24.
Calc. for C24H19Cl2NO5: C 61.03, H 4.05, N 2.97%.)
Compound 6bd was obtained as colourless crystals (64%),
mp 136–137◦C. δH (CDCl3, 400 MHz) 1.12 (t, J 7.2, 3H,
CH3), 2.31 (m, 2H, CH2), 3.40 (dd, J 8.8 and 18.9, 1H, 4-Ha),
3.67 (dd, J 11.3 and 18.9, 1H, 4-Hb), 3.73–3.81 (4s, 12H,
4OMe), 5.10 (dd, J 8.8 and 11.3, 1H, 5-H), 6.22–7.22 (m, 5H,
ArH). δC 10.10, 27.48, 43.89, 55.98, 56.55, 56.69, 56.88, 81.87,
108.43, 111.15, 112.36, 120.58, 123.65, 124.46, 138.66, 139.43,
140.22, 152.44, 153.89, 156.22, 157.80, 172.88. m/z (ESI) 415.0
(C22H25NO7, [M + H]+). (Found: C 63.45, H 6.35, N 3.12. Calc.
for C22H25NO7: C 63.60, H 6.07, N 3.37%.)
Compound 6be was obtained as colourless crystals (66%),
mp 140–141◦C. δH (CDCl3, 400 MHz) 0.99 (t, J 7.2, 3H, CH3),
1.70–2.39 (m, 4H, 2CH2), 3.41 (dd, J 8.8 and 18.9, 1H, 4-Ha),
3.70 (dd, J 11.3 and 18.9, 1H, 4-Hb), 3.77–3.82 (4s, 12H, 4OMe),
5.07 (dd, J 8.8 and 11.3, 1H, 5-H), 6.32–7.30 (m, 5H, ArH). δC
14.21, 18.99, 36.33, 43.66, 56.12, 56.76, 56.98, 57.11, 81.32,
108.10, 111.38, 112.10, 120.88, 123.80, 124.11, 138.42, 139.40,
139.98, 152.69, 154.10, 156.59, 157.46, 173.31. m/z (ESI) 429.7
(C23H27NO7, [M + H]+). (Found: C 64.20, H 6.61, N 3.44. Calc.
for C23H27NO7: C 64.32, H 6.34, N 3.26%.)
Compound 6bf was obtained as colourless crystals (53%),
mp 137–138◦C. δH (CDCl3, 400 MHz) 3.42 (dd, J 8.8 and
18.8, 1H, 4-Ha), 3.69 (dd, J 11.3 and 18.8, 1H, 4-Hb), 3.73–
3.81 (4s, 12H, 4OMe), 5.17 (dd, J 8.8 and 18.8, 1H, 5-H),
5.77 (dd, J 1.4 and 10.5, 1H), 6.21 (dd, J 10.4 and 17.2,
1H), 6.41 (dd, J 1.4 and 17.2, 1H), 6.44–7.28 (m, 5H, ArH).
δC 43.98, 56.28, 56.64, 56.77, 57.01, 81.18, 108.36, 111.01,
112.12, 120.86, 123.28, 124.11, 127.98, 133.59, 138.12, 138.99,
140.11, 152.55, 153.78, 156.87, 158.00, 165.22. m/z (ESI) 413.0
(C22H23NO7, [M + H]+). (Found: C 64.19, H 5.89, N 3.10. Calc.
for C22H23NO7: C 63.91, H 5.61, N 3.39%.)
Compound 6bg was obtained as colourless crystals (61%),
mp 156–158◦C. δH (CDCl3, 400 MHz) 3.37 (dd, J 8.8 and 18.9,
1H, 4-Ha), 3.71 (dd, J 11.3 and 18.9, 1H, 4-Hb), 3.76–3.80 (4s,
12H, 4OMe), 4.99 (dd, J 8.8 and 11.3, 1H, 5-H), 6.56–7.38 (m,
11H,ArH and =CH), 7.83 (d, J 16.0, 1H). δC 43.50, 56.31, 56.69,
56.77, 57.01, 81.28, 108.23, 111.18, 112.24, 115.88, 120.44,
123.21, 123.96, 127.11, 128.95, 129.58, 135.78, 138.21, 139.06,
140.10, 148.77, 152.36, 153.22, 156.69, 158.22, 164.75. m/z
(ESI) 490.1 (C28H27NO7, [M + H]+). (Found: C 68.82, H 5.28,
N 3.12. Calc. for C28H27NO7: C 68.70, H 5.56, N 2.86%.)
Compound 6bh was obtained as colourless crystals (68%),
mp 158–159◦C. δH (CDCl3, 400 MHz) 3.40 (dd, J 8.8 and 18.9,
1H, 4-Ha), 3.69 (dd, J 11.3 and 18.9, 1H, 4-Hb), 3.75–3.80
(4s, 12H, 4OMe), 5.21 (dd, J 8.8 and 11.3, 1H, 5-H), 6.42–
7.21 (m, 5H, ArH), 7.78 (d, J 8.4, 2H), 8.22 (d, J 8.4, 2H,
Compound 6ak was obtained as colourless crystals (65%),
mp 160–161◦C. δH (CDCl3, 400 MHz) 3.24 (dd, J 9.2 and 19.3,
1H, 4-Ha), 3.49 (dd, J 11.9 and 19.3, 1H, 4-Hb), 3.80 (s, 3H,
OMe), 5.18 (dd, J 9.2 and 11.9, 1H, 5-H), 6.78–7.03 (m, 3H,
ArH), 7.82–8.41 (m, 7H, ArH). δC 41.69, 56.30, 81.25, 112.03,
120.28, 121.33, 123.11, 126.08, 127.44, 130.80, 131.54, 132.28,
135.18, 135.29, 135.64, 135.76, 138.01, 138.33, 138.85, 150.28,
156.55, 157.68, 165.50. m/z (ESI) 487.6 (C23H16Cl2N2O6,
[M + H]+). (Found: C 56.55, H 3.00, N 6.04. Calc. for
C23H16Cl2N2O6: C 56.69, H 3.31, N 5.75%.)
Compound 6al was obtained as colourless crystals (55%), mp
150–151◦C. δH (CDCl3, 400 MHz) 2.32 (s, 3H, Me), 3.17 (dd,

868
X.-H. Liu et al.
CF3-ArH). δF (CDCl3, TFA) −118.00. δC 43.45, 56.27, 56.66,
56.72, 57.00, 81.32, 108.29, 110.96, 112.20, 120.34, 123.17,
124.25, 124.89, 125.63, 131.25, 134.18, 136.95, 138.25, 139.03,
139.80, 152.33, 153.89, 157.21, 158.09, 165.88. m/z (ESI) 531.6
(C27H24F3NO7, [M + H]+). (Found: C 61.34, H 4.77, N 2.49.
Calc. for C27H24F3NO7: C 61.02, H 4.55, N 2.64%.)
m/z (ESI) 317.8 (C14H14F3NO4, [M + H]+). (Found: C 53.32,
H 4.26, N 4.18. Calc. for C14H14F3NO4: C 53.00, H 4.45,
N 4.41%.)
Compound 6cewasobtainedascolourlesscrystals(56%), mp
98–100◦C. δH (CDCl3, 400 MHz) 1.07 (t, J 7.2, 3H, CH3), 1.77–
2.40 (m, 4H, 2CH2), 3.55 (dd, J 6.2 and 16.8, 1H, 4-Ha), 3.81 (s,
3H, OMe), 3.94 (dd, J 10.6 and 16.8, 1H, 4-Hb), 5.28 (dd, J 6.2
and 10.6, 1H, 5-H), 6.78–7.05 (m, 3H, ArH). δF (CDCl3, TFA)
−123.2. δC 13.95, 19.12, 36.42, 44.14, 56.40, 81.77, 112.41,
120.60, 122.88, 123.55, 138.35, 139.27, 158.44, 165.30, 173.00.
m/z (ESI) 331.0 (C15H16F3NO4, [M + H]+). Found: C 54.66,
H 4.90, N 4.11. Calc. for C15H16F3NO4: C 54.38, H 4.87,
N 4.23%.)
Compound 6cf was obtained as colourless crystals (51%),
mp 94–95◦C. δH (CDCl3, 400 MHz) 3.60 (dd, J 6.2 and 16.8,
1H, 4-Ha), 3.77 (s, 3H, OMe), 3.90 (dd, J 10.6 and 16.8, 1H,
4-Hb), 5.30 (dd, J 6.2 and 10.5, 1H, 5-H), 5.82 (dd, J 1.3 and
10.6, 1H), 6.13 (dd, J 10.4 and 17.2, 1H), 6.46 (dd, J 1.3 and
17.2, 1H), 6.81–7.10 (m, 3H, ArH). δF (CDCl3, TFA) −122.4.
δC 44.48, 56.45, 81.77, 112.23, 120.74, 122.45, 123.20, 128.33,
133.59, 138.24, 138.95, 158.20, 165.29, 165.66. m/z (ESI) 315.5
(C14H12F3NO4, [M + H]+). (Found: C 53.25, H 3.80, N 4.65.
Calc. for C14H12F3NO4: C 53.34, H 3.84, N 4.44%.)
Compound 6cg was obtained as colourless crystals (58%),
mp 113–114◦C. δH (CDCl3, 400 MHz) 3.61 (dd, J 6.2 and 16.8,
1H, 4-Ha), 3.77 (s, 3H, OMe), 3.95 (dd, J 10.6 and 16.8, 1H,
4-Hb), 5.29 (dd, J 6.2 and 10.6, 1H, 5-H), 6.44 (d, J 16.0, 1H),
6.74–7.00 (m, 3H,ArH), 7.11–7.35 (m, 5H,ArH), 7.80 (d, J 16.0,
1H). δF (CDCl3, TFA) −123.3. δC 44.07, 56.40, 81.35, 112.31,
116.22, 120.65, 122.36, 123.40, 127.29, 128.78, 129.50, 135.99,
138.32, 139.21, 148.65, 158.34, 164.79, 164.98. m/z (ESI) 391.8
(C20H16F3NO4, [M + H]+). (Found: C 61.20, H 4.22, N 3.77.
Calc. for C20H16F3NO4: C 61.38, H 4.12, N 3.58%.)
Compound 6ch was obtained as colourless crystals (60%),
mp 115–116◦C. δH (CDCl3, 400 MHz) 3.58 (dd, J 6.2 and 16.8,
1H, 4-Ha), 3.79 (s, 3H, OMe), 3.92 (dd, J 10.6 and 16.8, 1H,
4-Hb), 5.24 (dd, J 6.1 and 10.6, 1H, 5-H), 6.88–7.11 (m, 3H,
ArH), 7.82 (d, J 8.4, 2H, CF3-ArH), 8.26 (d, J 8.4, 2H, CF3-
ArH). δF (CDCl3,TFA) −118.6, −122.7. δC 44.90, 56.40, 81.63,
112.44, 120.30, 122.65, 123.31, 124.85, 125.69, 131.31, 134.08,
136.89, 138.37, 139.22, 158.25, 165.20, 165.83. m/z (ESI) 433.3
(C19H13F6NO4, [M + H]+). (Found: C 56.46, H 2.85, N 3.41.
Calc. for C19H13F6NO4: C 56.27, H 3.02, N 3.23%.)
Compound 6ci was obtained as colourless crystals (50%), mp
111–112◦C. δH (CDCl3, 400 MHz) 3.56 (dd, J 6.2 and 16.8, 1H,
4-Ha), 3.77 (s, 3H, OMe), 3.91 (dd, J 10.6 and 16.8, 1H, 4-Hb),
5.27 (dd, J 6.2 and 10.6, 1H, 5-H), 6.81–8.24 (m, 7H, ArH). δF
(CDCl3,TFA) −62.00, −123.10. δC 45.01, 56.41, 81.17, 112.33,
115.67, 119.23, 120.40, 122.55, 123.62, 124.96, 132.20, 136.17,
138.43, 139.00, 157.93, 161.32, 165.14, 165.92. m/z (ESI) 383.1
(C18H13F4NO4, [M + H]+). (Found: C 56.62, H 3.41, N 3.29.
Calc. for C18H13F4NO4: C 56.40, H 3.42, N 3.65%.)
Compound 6cj was obtained as colourless crystals (53%), mp
118–120◦C. δH (CDCl3, 400 MHz) 3.59 (dd, J 6.2 and 16.8, 1H,
4-Ha), 3.83 (s, 3H, OMe), 3.99 (dd, J 10.6 and 16.8, 1H, 4-Hb),
5.26 (dd, J 6.2 and 10.6, 1H, 5-H), 6.78–7.06 (m, 3H,ArH), 7.56–
8.16 (m, 4H, ArH). δF (CDCl3, TFA) −123.30. δC 44.22, 56.21,
81.32, 112.46, 120.79, 122.30, 123.21, 128.95, 129.36, 132.45,
138.20, 139.12, 140.23, 158.16, 164.87, 165.76. m/z (ESI) 400.2
(C18H13ClF3NO4, [M + H]+). (Found: C 54.30, H 3.04, N 3.43.
Calc. for C18H13ClF3NO4: C 54.08, H 3.28, N 3.50%.)
Compound 6ck was obtained as colourless crystals (52%),
mp 125–126◦C. δH (CDCl3, 400 MHz) 3.65 (dd, J 6.2 and 16.8,
1H, 4-Ha), 3.77 (s, 3H, OMe), 3.98 (dd, J 10.6 and 16.8, 1H,
Compound 6bi was obtained as colourless crystals (54%),
mp 154–155◦C. δH (CDCl3, 400 MHz) 3.43 (dd, J 8.8 and 18.9,
1H, 4-Ha), 3.67 (dd, J 11.3 and 18.9, 1H, 4-Hb), 3.73–3.82 (4 × s,
12H, 4OMe), 5.00 (dd, J 8.8 and 11.3, 1H, 5-H), 6.50–8.29 (m,
9H, ArH). δF (CDCl3, TFA) −61.22. δC 43.76, 56.29, 56.87,
56.95, 57.32, 81.30, 108.19, 110.78, 112.23, 115.90, 119.40,
120.52, 123.39, 124.51, 125.17, 132.25, 136.31, 138.56, 139.22,
139.78, 152.16, 153.82, 156.95, 157.77, 161.30, 165.85. m/z
(ESI) 481.9 (C26H24FNO7, [M + H]+). (Found: C 65.11, H 5.31,
N 3.09. Calc. for C26H24FNO7: C 64.86, H 5.02, N 2.91%.)
Compound 6bj was obtained as colourless crystals (50%),
mp 161–162◦C. δH (CDCl3, 400 MHz) 3.39 (dd, J 8.8 and 18.9,
1H, 4-Ha), 3.68 (dd, J 11.3 and 18.9, 1H, 4-Hb), 3.73–3.78
(4s, 12H, 4OMe), 5.20 (dd, J 8.8 and 11.3, 1H, 5-H), 6.58–
7.19 (m, 5H, ArH), 7.51–8.18 (m, 4H, ArH). δC 43.19, 56.38,
56.70, 56.81, 57.22, 81.40, 108.26, 110.96, 112.33, 120.44,
123.35, 124.16, 128.89, 129.29, 132.36, 138.11, 139.35, 140.06,
140.37, 152.44, 153.80, 156.94, 158.01, 165.88. m/z (ESI) 498.3
(C26H24ClNO7, [M + H]+). (Found: C 63.01, H 4.60, N 3.13.
Calc. for C26H24ClNO7: C 62.72, H 4.86, N 2.81%.)
Compound 6bk was obtained as colourless crystals (60%),
mp 165–167◦C. δH (CDCl3, 400 MHz) 3.42 (dd, J 8.8 and 18.9,
1H, 4-Ha), 3.70 (dd, J 11.3 and 18.9, 1H, 4-Hb), 3.72–3.80 (4s,
12H, 4OMe), 5.11 (dd, J 8.8 and 11.3, 1H, 5-H), 6.58–7.09 (m,
5H, ArH), 7.89–8.52 (m, 4H, ArH). δC 43.88, 56.36, 56.77,
56.81, 57.01, 81.40, 107.95, 111.16, 112.43, 120.25, 121.47,
123.20, 124.12, 126.45, 131.80, 135.56, 135.74, 138.18, 138.99,
140.04, 150.65, 152.55, 153.78, 156.83, 157.95, 165.96. m/z
(ESI) 508.7 (C26H24N2O9, [M + H]+). (Found: C 61.68, H 5.09,
N 5.20. Calc. for C26H24N2O9: C 61.41, H 4.76, N 5.51%.)
Compound 6bl was obtained as colourless crystals (52%), mp
156–157◦C. δH (CDCl3, 400 MHz) 2.41 (s, 3H, Me), 3.40 (dd,
J 8.8 and 18.9, 1H, 4-Ha), 3.51 (dd, J 11.3 and 18.9, 1H, 4-Hb),
3.73–3.81 (4s, 12H, 4OMe), 5.20 (dd, J 8.8 and 11.3, 1H, 5-H),
6.55–7.18 (m, 5H, ArH), 7.32–8.15 (m, 4H, ArH). δC 18.10,
43.85, 56.11, 56.77, 56.83, 57.21, 81.26, 108.32, 111.25, 112.21,
120.36, 123.22, 123.86, 126.17, 129.65, 130.78, 130.97, 134.32,
138.20, 139.23, 140.11, 140.75, 152.22, 153.69, 156.82, 157.68,
165.80. m/z (ESI) 477.3 (C27H27NO7, [M + H]+). (Found:
C 67.88, H 5.45, N 3.12. Calc. for C27H27NO7: C 67.91, H
5.70, N 2.93%.)
Compound 6bm was obtained as colourless crystals (52%),
mp 161–162◦C. δH (CDCl3, 400 MHz) 3.38 (dd, J 8.8 and 18.9,
1H, 4-Ha), 3.69 (dd, J 11.3 and 18.9, 1H, 4-Hb), 3.73–3.78 (5s,
15H, 5OMe), 5.13 (dd, J 8.8 and 11.3, 1H, 5-H), 6.57–6.98 (m,
7H, ArH), 7.51–8.15 (m, 2H, ArH). δC 43.86, 56.11, 56.20,
56.88, 56.97, 57.41, 81.31, 108.52, 111.12, 112.47, 114.35,
114.81, 120.33, 121.67, 123.21, 124.26, 131.85, 135.40, 138.22,
138.95, 140.01, 152.36, 153.77, 156.80, 157.94, 162.19, 165.99.
m/z (ESI) 494.0 (C27H27NO8, [M + H]+). (Found: C 66.01, H
5.28, N 3.17. Calc. for C27H27NO8: C 65.71, H 5.51, N 2.84%.)
Compound 6cd was obtained as colourless crystals (60%),
mp 96–97◦C. δH (CDCl3, 400 MHz) 1.18 (t, J 7.2, 3H, CH3),
2.40 (m, 2H, CH2), 3.59 (dd, J 6.2 and 16.8, 1H, 4-Ha), 3.79
(s, 3H, OMe), 3.90 (dd, J 10.6 and 16.8, 1H, 4-Hb), 5.26 (dd, J
6.2 and 10.6, 1H, 5-H), 6.82–7.03 (m, 3H, ArH). δF (CDCl3,
TFA) −122.6. δC 10.33, 27.79, 44.01, 56.45, 81.65, 112.30,
120.66, 122.69, 123.78, 138.46, 139.13, 158.21, 165.22, 173.12.

Novel Isoxazole Derivatives
869
4-Hb), 5.28 (dd, J 6.2 and 10.6, 1H, 5-H), 6.86–7.03 (m, 3H,
ArH), 7.88–8.60 (m, 4H, ArH). δF (CDCl3, TFA) −122.82. δC
45.00, 56.60, 81.71, 111.43, 120.15, 121.76, 122.62, 123.42,
126.20, 131.65, 135.33, 135.51, 138.36, 139.40, 150.77, 157.86,
165.19, 165.81. m/z (ESI) 409.7 (C18H13F3N2O6, [M + H]+).
(Found: C 52.72, H 3.41, N 7.11. Calc. for C18H13F3N2O6:
C 52.69, H 3.19, N 6.83%.)
Compound 6cl was obtained as colourless crystals (50%),
mp 114–115◦C. δH (CDCl3, 400 MHz) 2.38 (s, 3H, Me), 3.56
(dd, J 6.2 and 16.8, 1H, 4-Ha), 3.78 (s, 3H, OMe), 3.96 (dd,
J 10.6 and 16.8, 1H, 4-Hb), 5.26 (dd, J 6.2 and 10.6, 1H,
5-H), 6.77–7.00 (m, 3H, ArH), 7.30–8.11 (m, 4H, ArH). δF
(CDCl3, TFA) −124.01. δC 18.42, 45.10, 56.35, 81.62, 112.35,
120.50, 122.52, 123.47, 126.31, 129.49, 130.70, 131.12, 134.40,
138.28, 139.11, 140.55, 157.88, 165.21, 165.95. m/z (ESI) 379.9
(C19H16F3NO4, [M + H]+). (Found: C 60.41, H 4.29, N 3.91.
Calc. for C19H16F3NO4: C 60.16, H 4.25, N 3.69%.)
Compound 6cm was obtained as colourless crystals (55%),
mp 120–122◦C. δH (CDCl3, 400 MHz) 3.55 (dd, J 6.2 and
16.8, 1H, 4-Ha), 3.75–3.78 (2s, 6H, 2OMe), 3.93 (dd, J
10.6 and 16.8, 1H, 4-Hb), 5.28 (dd, J 6.2 and 10.6, 1H,
5-H), 6.82–7.05 (m, 5H, ArH), 7.58–8.12 (m, 2H, ArH). δF
(CDCl3, TFA) −123.51. δC 44.78, 56.28, 56.36, 81.45, 112.35,
114.46, 114.95, 120.38, 121.54, 122.46, 123.30, 131.80, 135.51,
138.29, 139.32, 157.90, 162.23, 165.00, 165.86. m/z (ESI) 395.8
(C19H16F3NO5, [M + H]+). (Found: C 58.03, H 4.35, N 3.20.
Calc. for C19H16F3NO5: C 57.72, H 4.08, N 3.54%.)
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Cytotoxic Assay
The cytotoxicity evaluation was conducted by using a modi-
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96-well culture plate. The subsequent incubation was performed
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cytotoxicity assessment. Tested samples at pre-set concentra-
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Acknowledgements
The authors thank the Anhui Natural Science Foundation, Anhui Province
(Grant no. 070416274X) for providing financial support for the project and
to thank the Student Research Train Project of Anhui Technology University
(no. 08021). The work was also supported by the opening foundation of
the Key Laboratory of Green Pesticide and Agricultural Bioengineering,
Ministry of Education, Guizhou University, grant no. 2008GDGP0105.