Synthesis and biological evaluation of 3,4-diaryl-5-aminoisoxazole derivatives

Article abstract of DOI:10.1016/j.bmc.2009.07.040

A series of cis-restricted 3,4-diaryl-5-aminoisoxazoles have been synthesized and evaluated for their biological activities. Among them, compound 11a and 13a displayed potent cytotoxic activities in vitro against five human cancer cell lines with IC₅₀ values in the low micromolar range and two compounds inhibited tubulin polymerization with IC₅₀ value of 1.8, and 2.1 μM, respectively, similar to that of CA-4. Compound 13a could arrest at the G2/M phase of the cell cycle at the concentration of 0.1 and 1.0 μM and induce apoptosis at 0.1-1.0 μM.

Full text of DOI:10.1016/j.bmc.2009.07.040

Bioorganic & Medicinal Chemistry 17 (2009) 6279–6285
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 3,4-diaryl-5-aminoisoxazole derivatives
Tao Liu a, Xiaowu Dong a, Na Xue a, Rui Wu b, Qiaojun He b, Bo Yang b, Yongzhou Hu a,
*
a ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
b Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, 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 cis-restricted 3,4-diaryl-5-aminoisoxazoles have been synthesized and evaluated for their bio-
logical activities. Among them, compound 11a and 13a displayed potent cytotoxic activities in vitro
against five human cancer cell lines with IC50 values in the low micromolar range and two compounds
Received 17 June 2009
Revised 18 July 2009
Accepted 21 July 2009
Available online 23 July 2009
inhibited tubulin polymerization with IC50 value of 1.8, and 2.1
4. Compound 13a could arrest at the G2/M phase of the cell cycle at the concentration of 0.1 and
1.0 M and induce apoptosis at 0.1–1.0 M.
lM, respectively, similar to that of CA-
l
l
Keyword:
3,4-Diaryl-5-aminoisoxazole
Cytotoxic activities
Ó 2009 Elsevier Ltd. All rights reserved.
Tubulin polymerization inhibition
1. Introduction
In our previously study, we reported the synthesis and
evaluation of the cytotoxic activities of vicinal diaryl-substituted
imidazol-2-one derivatives (3, Fig. 1), which can be considered as
cis-restricted analogues of CA-4. Some of them showed potent
in vitro/in vivo antitumor activity, strongly inhibited tumor cell
growth, and caused arrest in the G2/M phase of the cell cycle.10
During our continuing studies on the synthesis of new CA-4
analogs, our attention was still focused on the central heterocyclic
ring. Introducing 5-aminoisoxazole ring as the locked cis-type
bridge may improve the solubility of CA-4 analogs and enhance
the cytotoxic activity. We were also interested to investigate the
biological evaluation of N-acetyl-5-aminoisoxazole derivatives. In
this paper, the design, synthesis, molecular modeling, and
in vitro cytotoxic activities of 3,4-diaryl-5-aminoisoxazole deriva-
tives, as well as the inhibition data of tubulin polymerization and
the effects on the cell cycle are reported.
Microtubules represent an attractive target for antitumor drug
discovery because they play an essential role in cellular functions
including shape maintenance, division and intracellular trans-
port.1,2 Drugs interfering with the microtubule dynamics have
attracted much attention. Among them, combretastatin A-4
(CA-4, 1, Fig. 1), a natural cis-stilbene product, strongly inhibits
the tubulin assembly by binding to the colchicine site and prevents
tubulin polymerization. It also displayed potent cytotoxic activities
against a variety of human cancer cell lines, including multidrug
resistant cell lines.3,4 The corresponding water-soluble disodium
phosphate prodrug of CA-4 (CA-4P, 2, Fig. 1) has been demon-
strated to cause reversible vascular shutdown in established tu-
mors in vivo, consistent with an anti-vascular mechanism of
action.5 These biological effects and the structural simplicity of
CA-4 make this natural product an attractive lead compound in
the development of new antitumor agents.
Extensive studies have been conducted to examine the struc-
ture–activity relationships of variously modified CA-4 analogues.
The 3,4,5-trimethoxyphenyl ring (ring A) and the cis-orientation
between the two aryl rings are essential requirements for the
activity of CA-4.6 However, the cis-stilbenes tend to isomerize to
the more stable trans-forms which show dramatic reduction in
both anti-tubulin and cytotoxic activities. Therefore, many studies
have focused on design of analogs with a locked cis-type bridge,
which was achieved by the introduction of a heterocyclic moiety
between rings A and B.7–9
2. Results and discussion
2.1. Chemistry
The synthetic routs to the target isoxazoles 11a–h, 12a–h and
13a–c are outlined in Scheme 1. The phenylacetonitriles 9a–d were
synthesized following the method described in the literature with
* Corresponding author. Tel./fax: +86 571 88208460.
E-mail address: huyz@zju.edu.cn (Y. Hu).
Figure 1. Structure of CA-4 (1), CA-4P (2), and imidazol-2-one derivatives (3).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.040

6280
T. Liu et al. / Bioorg. Med. Chem. 17 (2009) 6279–6285
Scheme 1. Synthesis of target isoxazoles 11a–h, 12a–h, and 13a–c. Reagents and conditions: (a) CH3OH, H2SO4, reflux, 6 h; (b) KBH4, CH3OH, 0 °C, then rt, 3 h; (c) SOCl2, DCM,
Et3N, 0–5 °C, then rt, 2 h; (d) NaCN, CH3CN, reflux, 4 h; (e) NaH, THF, reflux, 5 h; (f) NH2OHÁHCl, CH3CH2OH, reflux, 2 h; (g) (CH3CO)2O, pyridine, rt, 24 h; (h) 10% Pd-C, THF, rt,
12 h.
minor modifications.11,12 Reduction of benzaldehydes 6a–d with
Table 1
Cytotoxicity of compounds 11a–h, 12a–h and 13a–c against five human cancer cell
lines
KBH4 gave benzyl alcohols 7a–d in high yields. Benzyl alcohols
7a–d were chlorinated by SOCl2, followed by treating with NaCN
to afford phenylacetonitriles 9a–d.
Esterification of benzoic acids 4a–d in methanol catalyzed by
H2SO4 yielded methyl benzoates 5a–d. Reaction of methyl benzo-
Compd
Cytotoxicity (IC50
A549
l
M)a
K562
ECA-109
SMMC-7721
PC-3
11a
11b
11c
11d
11e
11f
11g
11h
12a
12b
12c
12d
12e
12f
12g
12h
13a
13b
13c
CA-4
0.04
1.27
0.64
3.65
NDb
1.40
0.022
NDb
0.38
3.71
3.62
3.97
NDb
8.75
1.00
NDb
0.068
4.29
3.96
0.046
5.84
20.55
>50
>50
>50
1.66
9.82
>50
10.84
17.65
9.53
13.94
>50
4.49
>50
>50
>50
38.25
>50
12.00
>50
>50
3.03
21.12
>50
>50
>50
>50
15.01
>50
>50
>50
1.91
14.88
>50
>50
>50
>50
0.36
>50
2.63
5.40
ates 5a–d with phenylacetonitriles 9a–d resulted in
a-cyanoke-
tones 10a–g, then followed condensation with hydroxylamine
hydrochloride in refluxing alcohol to afford 3,4-diaryl-5-aminois-
oxazoles 11a–g in moderate yields.13 Debenzyl-protecting group
of 11e by hydrogenation afforded phenol 11h. When the 5-ami-
noisoxazoles 11a–c were acetylated with acetic anhydride, it
yielded a mixture of N-acetyl-5-aminoisoxazoles 12a–c and N,N-
diacetyl-5-aminoisoxazoles 13a–c in approximate ratio of 2:1,
which could be separated by flash chromatography. Otherwise,
acetylation of 5-aminoisoxazoles 11d–g at the same condition,
the main products were N-acetyl-5-aminoisoxazoles 12d–g and
only a trace amount of 13d–g were obtained. This is mainly attrib-
uted to compound 11d–g possessing a sterically hindering group,
3,4,5-trimethoxy phenyl ring. Compound 12h was got by removal
of benzyl protecting group of 12e under catalytic hydrogenation
condition.
>50
NDb
>50
21.75
>50
39.76
NDb
>50
46.31
>50
24.63
NDb
>50
10.69
19.84
24.95
21.14
>50
>50
>50
>50
0.43
>50
>50
>50
>50
0.39
>50
>50
NDb
1.36
>50
39.83
4.50
>50
0.42
>50
3.22
a
IC50 values are means of three experiments.
ND: Not Determined.
b
2.2. Biological evaluation
All the prepared compounds were evaluated for their cytotoxic
activities in vitro against five human cancer cell lines (human mye-
loid leukemia cells K562, human esophageal carcinoma cells ECA-
109, human non-small lung cancer cells A549, human hepatocellu-
lar carcinoma cells SMMC-7721, and human prostate carcinoma
cells PC-3). CA-4 was used as a positive control. The results are
summarized in Table 1.
As shown in Table 1, the tested compounds displayed more po-
tent cytotoxic activities against leukemia cells K562 than other cell
lines, with IC50 values in low micromolar range (IC50 0.022–
phenyl)-4-(4-methoxyphenyl)-5-aminoisoxazole, revealed the
highest cytotoxic activities with IC50 values in the range of 0.04–
12.00 lM against all tested cancer cell lines. Transferring the tri-
methoxyphenyl group from 3-position of isoxazole ring to 4-posi-
tion (11a vs 11g), led to a loss of cytotoxic activity against most of
tested cancer cell lines, except to leukemia cells K562. Compound
11a with 4-methoxy group on B ring showed more potent activity
than that of 11b and 11d with 3,4-dimethoxy or 3,4,5-trimethoxy
groups on B ring. Replacement the 4-methoxy group of B ring with
chlorine atom (11c), displayed a decrease in the cytotoxic activity
by one order of magnitude against K562. This result implies that
the methoxy group at the C-4 position of B ring is important for
the cytotoxic activity profile in the tested compounds.
8.75 lM), some of them even in nanomolar range (e.g., 11a, 11g,
and 13a). On the other hand, compounds 11a and 13a presented
significant cytotoxicities against five human cancer cell lines.
Obviously, the cytotoxic potency in 5-aminoisoxazoles series
(11a–h) was highly dependent on the structures of 3- or 4-position
substituents of isoxazole. Compound 11a, 3-(3,4,5-trimethoxy-
In the N-acetyl-5-aminoisoxazoles series, compounds 12a
and 12b showed higher cytotoxic activities than those of their

T. Liu et al. / Bioorg. Med. Chem. 17 (2009) 6279–6285
6281
regioisomers 12g and 12f against tested cancer cell lines. It is also
at the concentration of 0.1 and 1.0
lM arrested at the G2/M phase
proved that 3,4,5-trimethoxyphenylgroup at the 3-position was
essential for cytotoxicity.
of the cell cycle and induced cell apoptosis at 0.1–1.0 lM. Future
progress on related series will be reported in due course.
It is interesting that compound 13a, a N,N-diacetyl derivative of
compound 11a, showed similar or more potent cytotoxic activities
compared with that of 11a as well as CA-4.
4. Experimental
Further study on the tubulin polymerization inhibitory activity
was performed with selected compounds 11a and 13a. Compounds
Melting points were obtained on a B-540 Buchi melting point
apparatus and are uncorrected. 1H NMR spectra was recorded on
a Bruker AM 400 instrument at 400 MHz (chemical shifts are ex-
pressed as d values relative to TMS as internal standard). Mass
spectra (MS) and ESI (positive) were recorded on an Esquire-LC-
00075 spectrometer. Element analysis was obtained on an Eager
300 instrument.
11a and 13a, with IC50 = 1.8 and 2.1
polymerization inhibitory activity in comparison with CA-4
(IC50 = 1.2 M) (Table 2).
lM, showed similar tubulin
l
To gain further insight into the mechanisms of action of these
new compounds, the most cytotoxic compound 13a was assayed
for its effect on cell cycle (by flow cytometry). SMMC-7721 cells
were treated with 13a at different concentrations for 24 h.
4.1. Synthesis
In 0.1
l
M 13a treatment group, 41.5% cells were arrested in G2/
M 13a
M phase and 50.4% cells were arrested in S phase, in 1.0
l
4.1.1. Synthesis of a-cyanoketones (10): general procedure
treatment group, 70.0% cells were arrested in G2/M phase and
24.3% cells were arrested in S phase, while in control group,
25.0% and 38.0% cells were observed in G2/M phase and S phase
(Fig. 2). Further research showed that compound 13a, at graded
To a solution of corresponding phenylacetonitrile (1.0 mmol) in
anhydrous THF (10 mL), NaH (2.4 mmol) was added at room tem-
perature. The suspension was stirred for a few minutes, a solution
of methyl benzoate (1.2 mmol) in anhydrous THF (20 mL) was
added dropwise and the mixture was refluxed for 5 h. The reaction
mixture was quenched with water and THF was removed under re-
duced pressure. The residue was acidic with 3 M HCl solution and
extracted with CH2Cl2 (20 mLÂ3). The organic layers were com-
bined, washed with brine (20 mLÂ2), dried over anhydrous sodium
sulfate, and concentrated in vacuo to obtain the crude product.
Purification by crystallization from EtOH afforded the correspond-
concentrations of 0.1 lM, 0.5 lM, and 1.0 lM, induced apoptosis
in SMMC-7721 cells for 48 h, the percentages of apoptotic cells
were 37%, 70%, and 72%, respectively (Fig. 3).14
2.3. Molecular modeling
Molecular modeling studies were performed to investigate the
binding ability of the isoxazoles to the colchicines binding site of
ing a-cyanoketone.
a,b-tubulin. The proposed mechanism of action was supported
by docking studies of compound 13a in the colchicine site of
tubulin by using the reported high-resolution crystal structure of
tubulin-DAMAcolchicine complex. Figure 4 showed the docking
conformation of 13a well overlaps with CA-4 in the crystallized
protein complex. The trimethoxyphenyl moiety (ring A) and 4-
methoxyphenyl moiety (ring B) of isoxazole 13a was positioned
in the hydrophobic pocket between Alab250ꢀAlab316 and Va-
4.1.1.1. 2-(4-Methoxyphenyl)-3-oxo-3-(3,4,5-trimethoxy-
phenyl)propanenitrile (10a). White solid (78%); mp 90–92 °C;
1H NMR (400 MHz, CDCl3) d 3.78 (s, 3H, OCH3), 3.85 (s, 6H,
OCH3Â2), 3.90 (s, 3H, OCH3), 5.56 (s, 1H, HC–CN), 6.91 (d, 2H,
J = 8.8 Hz, Ar–H), 7.18 (s, 2H, Ar–H), 7.35 (d, 2H, J = 8.8 Hz, Ar–H);
MS (ESI) m/z = 342 [M+1]+; Anal. Calcd for C19H19NO5: C, 66.85;
H, 5.61; N, 4.10. Found: C, 66.77; H, 5.69; N, 4.31.
l
a
181ꢀMetb259, respectively. The oxygen atom of 3-methoxy
group of ring A formed a hydrogen bond with the thiol group of
Cysb241, which was also found in CA-4. Besides, the oxygen atom
of isoxazole ring formed one hydrogen bond with the NH of
Alab250 and the oxygen atom of an acetyl group of 5-NH2 formed
4.1.1.2. 2-(3,4-Dimethoxyphenyl)-3-oxo-3-(3,4,5-trimethoxy-
phenyl)propanenitrile (10b). White solid (71%); mp 159–160 °C;
1H NMR (400 MHz, CDCl3) d 3.88 (s, 6H, OCH3Â2), 3.89 (s, 6H,
OCH3Â2), 3.92 (s, 3H, OCH3), 5.48 (s, 1H, HC–CN), 6.88 (d, 1H,
J = 8.4 Hz, Ar–H), 6.92 (s, 1H, Ar–H), 6.99 (d, 1H, J = 8.4 Hz, Ar–H),
7.21 (s, 2H, Ar–H); MS (ESI) m/z = 372 [M+1]+; Anal. Calcd for
C20H21NO6: C, 64.68; H, 5.70; N, 3.77. Found: C, 64.53; H, 5.87;
N, 3.63.
one hydrogen bond with the NH of Asna101. Although 12a had a
similar binding mode as 13a, but it showed weak cytotoxicity,
we think maybe 13a has a more restrictive configuration than
12a because of the diacetyl substitution.
3. Conclusions
4.1.1.3. 2-(4-Chlorophenyl)-3-oxo-3-(3,4,5-trimethoxy-
phenyl)propanenitrile (10c). White solid (80%); mp 148–150 °C;
1H NMR (400 MHz, CDCl3) d 3.65 (s, 6H, OCH3Â2), 3.83 (s, 3H,
OCH3), 5.29 (s, 1H, HC–CN), 6.52 (s, 2H, Ar–H), 7.23 (d, 2H,
J = 8.4 Hz, Ar–H), 7.33 (d, 2H, J = 8.4 Hz, Ar–H); MS (ESI) m/
z = 346 [M+1]+; Anal. Calcd for C18H16ClNO4: C, 62.52; H, 4.66; N,
4.05. Found: C, 62.61; H, 4.54; N, 4.13.
A novel series of 3,4-diaryl-5-aminoisoxazoles bearing struc-
tural similarity to CA-4 have been synthesized and evaluated for
their biological activities. Some of these compounds exhibited po-
tent cytotoxic activities against five human cancer cell lines
in vitro. The most potent compounds 11a and 13a displayed compa-
rable cytotoxic activities with that of CA-4 against the tested cancer
cell lines and they are potent inhibitors of tubulin polymerization.
Cell cycle distribution analysis showed that cells exposed to 13a
4.1.1.4. 3-Oxo-2,3-bis(3,4,5-trimethoxyphenyl)propanenitrile
(10d). White solid (68%); mp 174–176 °C; 1H NMR (400 MHz,
CDCl3) d 3.82 (s, 6H, OCH3Â2), 3.83 (s, 6H, OCH3Â2), 3.90 (s, 3H,
OCH3), 5.44 (s, 1H, HC–CN), 6.62 (s, 2H, Ar–H), 7.19 (s, 2H, Ar–H);
MS (ESI) m/z = 402 [M+1]+; Anal. Calcd for C21H23NO7: C, 62.83;
H, 5.78; N, 3.49. Found: C, 62.91; H, 5.62; N, 3.56.
Table 2
Inhibition of tublin polymerization by compound 11a, 13a and CA-4
a
Compd
IC50
(
lM)
11a
13a
CA-4
1.8
2.1
1.2
4.1.1.5. 3-(3-Benzyloxy-4-methoxyphenyl)-3-oxo-2-(3,4,5-tri-
methoxyphenyl) propanenitrile (10e). White solid (54%); mp
135–136 °C; 1H NMR (400 MHz, CDCl3) d 3.83 (s, 9H, OCH3Â3),
a
IC50 values are means of three experiments.

6282
T. Liu et al. / Bioorg. Med. Chem. 17 (2009) 6279–6285
Figure 2. Effect of 13a on the cell cycle as determined by flow cytometry. SMMC-7721 cells were treated with 13a at the concentrations of 0
0.1 M (c), and 1.0 M (d) for 24 h.
lM (control, a), 0.01 lM (b),
l
l
Figure 3. Compound 13a induced apoptosis in SMMC-7721 cell. Cells were treated
without (Control), 1.0 M, 0.5 M (c), and 0.1 M 13a for 48 h, respectively.
Figure 4. Proposed binding mode for compound 13a (in blue), and CA-4 (in green)
in the colchicine site of tubulin.
l
l
l

T. Liu et al. / Bioorg. Med. Chem. 17 (2009) 6279–6285
6283
3.93 (s, 3H, OCH3), 5.15 (s, 2H, CH2), 5.48 (s, 1H, HC–CN), 6.60 (s,
2H, Ar–H), 6.89 (d, 1H, J = 8.4 Hz, Ar–H), 7.32–7.45 (m, 5H, Ar–H),
7.56–7.61 (m, 2H, Ar–H); MS (ESI) m/z = 448 [M+1]+; Anal. Calcd
for C26H25NO6: C, 69.79; H, 5.63; N, 3.13. Found: C, 69.65; H,
5.71; N, 3.33.
(s, 3H, OCH3), 3.88 (s, 3H, OCH3), 4.57 (br s, 2H, NH2), 4.98 (s, 2H,
CH2), 6.47 (s, 2H, Ar–H), 6.83 (d, 1H, J = 8.4 Hz, Ar–H), 7.07 (d,
1H, J = 8.4 Hz, Ar–H), 7.11 (s, 1H, Ar–H), 7.28–7.33 (m, 5H, Ar–H);
MS (ESI) m/z = 463 [M+1]+; Anal. Calcd for C26H26N2O6: C, 67.52;
H, 5.67; N, 6.06. Found: C, 67.27; H, 5.55; N, 6.31.
4.1.1.6. 3-(3,4-Dimethoxyphenyl)-3-oxo-2-(3,4,5-trimethoxy-
phenyl)propanenitrile (10f). White solid (72%); mp 123–124 °C;
1H NMR (400 MHz, CDCl3) d 3.78 (s, 3H, OCH3), 3.80 (s, 6H,
OCH3Â2), 3.86 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 5.53 (s, 1H, HC–
CN), 6.60 (s, 2H, Ar–H), 6.84 (d, 1H, J = 8.8 Hz, Ar–H), 7.47 (s, 1H,
Ar–H), 7.56 (d, 1H, J = 8.8 Hz, Ar–H); MS (ESI) m/z = 372 [M+1]+;
Anal. Calcd for C20H21NO6: C, 64.48; H, 5.70; N, 3.77. Found: C,
64.59; H, 5.59; N, 3.84.
4.1.2.6. 3-(3,4-Dimethoxyphenyl)-4-(3,4,5-trimethoxy-
phenyl)isoxazol-5-amine (11f). Yellow solid (58%); mp 132–
134 °C; 1H NMR (400 MHz, CDCl3) d 3.75 (s, 9H, OCH3Â3), 3.86
(s, 3H, OCH3), 3.88 (s, 3H, OCH3), 4.61 (br s, 2H, NH2), 6.46 (s, 2H,
Ar–H), 6.80 (d, 1H, J = 8.0 Hz, Ar–H), 7.04 (dd, 1H, J = 1.6, 8.0 Hz,
Ar–H), 7.10 (d, 1H, J = 1.6 Hz, Ar–H); MS (ESI) m/z = 387 [M+1]+;
Anal. Calcd for C20H22N2O6: C, 62.17; H, 5.74; N, 7.25. Found: C,
62.31; H, 5.54; N, 7.29.
4.1.1.7. 3-(4-Methoxyphenyl)-3-oxo-2-(3,4,5-trimethoxy-
phenyl)propanenitrile (10g). White solid (72%); mp 94–96 °C;
1H NMR (400 MHz, CDCl3) d 3.83 (s, 3H, OCH3), 3.85 (s, 6H,
OCH3Â2), 3.87 (s, 3H, OCH3), 5.48 (s, 1H, HC–CN), 6.62 (s, 2H, Ar–
H), 6.94 (d, 2H, J = 8.8 Hz, Ar–H), 7.95 (d, 2H, J = 8.8 Hz, Ar–H);
MS (ESI) m/z = 342 [M+1]+; Anal. Calcd for C19H19NO5: C, 66.85;
H, 5.61; N, 4.10. Found: C, 66.98; H, 5.54; N, 4.24.
4.1.2.7. 3-(3-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)iso-
xazol-5-amine (11g). Yellow solid (63%); mp 146–148 °C; 1H
NMR (400 MHz, CDCl3) d 3.72 (s, 6H, OCH3Â2), 3.82 (s, 3H,
OCH3), 3.88 (s, 3H, OCH3), 4.60 (br s, 2H, NH2), 6.41 (s, 2H, Ar–H),
6.87 (d, 2H, J = 8.8 Hz, Ar–H), 7.44 (d, 2H, J = 8.8 Hz, Ar–H); MS
(ESI) m/z = 357 [M+1]+; Anal. Calcd for C19H20N2O5: C, 64.04; H,
5.66; N, 7.86. Found: C, 64.22; H, 5.52; N, 7.75.
4.1.2. Synthesis of 3,4-diaryl-5-aminoisoxazole derivative (11):
general procedure
NH2OH.HCl (1.2 mmol) was added to a solution of the corre-
sponding a-cyanoketone (1.0 mmol) in EtOH (5 mL), and the reac-
tion was refluxed for 2–3 h. The reaction mixture was cooled, the
solvent was evaporated off, and the residue was recrystallized from
EtOH/H2O to give the corresponding 3,4-diaryl-5-aminoisoxazole.
4.1.2.8. 3-(3-Hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxy-
phenyl)isoxazol-5-amine (11h). To a solution of 11e (463 mg,
1.0 mmol) in THF (25 mL), 10% Pd/C (106 mg, 0.1 mmol) was added
and the reaction mixture was stirred at room temperature under
hydrogen for 12 h. The suspension was filtered on a Celite layer
and concentrated in vacuo. The residue was purified by flash chro-
matography using pertoleum/EtOAc (1:2) to afford 11h. Yellow so-
lid (36%); mp 131–133 °C; 1H NMR (400 MHz, CDCl3) d 3.86 (s, 6H,
OCH3Â2), 3.92 (s, 6H, OCH3Â2), 6.49 (s, 2H, Ar–H), 6.76–6.77 (m,
2H, Ar–H), 7.39–7.40 (m, 1H, Ar–H); MS (ESI) m/z = 373 [M+1]+;
Anal. Calcd for C19H20N2O6: C, 61.28; H, 5.41; N, 7.52. Found: C,
61.43; H, 5.61; N, 7.37.
4.1.2.1. 4-(4-Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)iso-
xazol-5-amine (11a). White solid (65%); mp 157–159 °C; 1H NMR
(400 MHz, CDCl3) d 3.68 (s, 6H, OCH3Â2), 3.83 (s, 3H, OCH3), 3.86
(s, 3H, OCH3), 4.51 (br s, 2H, NH2), 6.72 (s, 2H, Ar–H), 6.94 (d, 2H,
J = 8.8 Hz, Ar–H), 7.19 (d, 2H, J = 8.8 Hz, Ar–H); MS (ESI) m/
z = 357 [M+1]+; Anal. Calcd for C19H20N2O5: C, 64.04; H, 5.66; N,
7.86. Found: C, 64.23; H, 5.42; N, 7.98.
4.1.3. Synthesis of mono- and di-acetylisoxazoles (12, 13):
general procedure
Acetic anhydride (5 mmol) was added to a solution of the 5-ami-
neisoxazole derivative (2 mmol) in pyridine (0.3 mL). After stirred
for 24 h at room temperature, the reaction was poured into EtOAc,
washed with 2 M HCl, 5% NaHCO3 and brine, and the organic layers
were dried over anhydrous Na2SO4, filtered, and evaporated in va-
cuo. The crude product was purified by chromatography (petroleum
ether/EtOAc 4:1–1:2) to give mono- and diacetylisoxazole.
4.1.2.2. 4-(3,4-Dimethoxyphenyl)-3-(3,4,5-trimethoxy-
phenyl)isoxazol-5-amine (11b). Yellow solid (59%); mp 159–
160 °C; 1H NMR (400 MHz, CDCl3) d 3.68 (s, 6H, OCH3Â2), 3.76
(s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 4.57 (br s,
2H, NH2), 6.73 (d, 1H, J = 2.0 Hz, Ar–H), 6.75 (s, 2H, Ar–H), 6.84
(dd, 1H, J = 2.0, 8.0 Hz, Ar–H), 6.91 (d, 1H, J = 8.0 Hz, Ar–H); MS
(ESI) m/z = 387 [M+1]+; Anal. Calcd for C20H22N2O6: C, 62.17; H,
5.74; N, 7.25. Found: C, 62.28; H, 5.62; N, 7.35.
4.1.3.1. N-(4-(4-Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-
isoxazol-5-yl)-acetamide (12a) and N-acetyl-N-(4-(4-methoxy-
phenyl)-3-(3,4,5-trimethoxy-phenyl)isoxazol-5-yl)acetamide
(13a). Compound 12a, yellow solid (62%); mp 129–131 °C; 1H NMR
(400 MHz, CDCl3) d 2.19 (s, 3H, O@C–CH3), 3.64 (s, 6H, OCH3Â2),
3.80 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 6.68 (s, 2H, Ar–H), 6.92 (d,
2H, J = 8.4 Hz, Ar–H), 7.16 (d, 2H, J = 8.4 Hz, Ar–H), 7.49 (br s, 1H,
O@C–NH); MS (ESI) m/z = 399 [M+1]+; Anal. Calcd for C21H22N2O6:
C, 63.31; H, 5.57; N, 7.03. Found: C, 63.05; H, 5.75; N, 7.23.
Compound 13a, yellow solid (27%); mp 119–121 °C; 1H NMR
(400 MHz, CDCl3) d 2.34 (s, 6H, O@C–CH3Â2), 3.68 (s, 6H, OCH3Â2),
3.85 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 6.75 (s, 2H, Ar–H), 6.94 (d,
2H, J = 8.8 Hz, Ar–H), 7.12 (d, 2H, J = 8.8 Hz, Ar–H); MS (ESI) m/
z = 441 [M+1]+; Anal. Calcd for C23H24N2O7: C, 62.72; H, 5.49; N,
6.36. Found: C, 62.54; H, 5.56; N, 6.24.
4.1.2.3. 4-(4-Chlorophenyl)-3-(3,4,5-trimethoxyphenyl)iso-
xazol-5-amine (11c). Yellow solid (70%); mp 170–172 °C; 1H
NMR (400 MHz, CDCl3) d 2.84 (br s, 2H, NH2), 3.68 (s, 6H, OCH3Â2),
3.84 (s, 3H, OCH3), 6.64 (s, 2H, Ar–H), 7.18 (d, 2H, J = 8.8 Hz, Ar–H),
7.34 (d, 2H, J = 8.8 Hz, Ar–H); MS (ESI) m/z = 387 [M+1]+; Anal.
Calcd for C20H22N2O6: C, 62.17; H, 5.74; N, 7.25. Found: C, 62.28;
H, 5.62; N, 7.35.
4.1.2.4. 3,4-Bis(3,4,5-trimethoxyphenyl)isoxazol-5-amine
(11d). Yellow solid (47%); mp 160–162 °C; 1H NMR (400 MHz,
CDCl3) d 3.70 (s, 6H, OCH3Â2), 3.78 (s, 6H, OCH3Â2), 3.86 (s, 6H,
OCH3Â2), 4.60 (br s, 2H, NH2), 6.47 (s, 2H, Ar–H), 6.76 (s, 2H, Ar–
H); MS (ESI) m/z = 417 [M+1]+; Anal. Calcd for C21H24N2O7: C,
60.57; H, 5.81; N, 6.73. Found: C, 60.25; H, 5.93; N, 6.38.
4.1.3.2. N-(4-(3,4-Dimethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-
isoxazol-5-yl)-acetamide (12b) and N-acetyl-N-(4-(3,4-dime-
thoxyphenyl)-3-(3,4,5-trimethoxyphenyl)isoxazol-5-yl)acetam-
ide (13b). Compound 12b, white solid (63%); mp 137–139 °C; 1H
4.1.2.5. 3-(3-Benzyloxy-4-methoxyphenyl)-4-(3,4,5-trimethoxy-
phenyl)isoxazol-5-amine (11e). Yellow solid (48%); mp 156–
158 °C; 1H NMR (400 MHz, CDCl3) d 3.74 (s, 6H, OCH3Â2), 3.87

6284
T. Liu et al. / Bioorg. Med. Chem. 17 (2009) 6279–6285
NMR (400 MHz, CDCl3) d 2.20 (s, 3H, O@C–CH3), 3.66 (s, 6H,
OCH3Â2), 3.75 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.89 (s, 3H,
OCH3), 6.72 (s, 2H, Ar–H), 6.76 (d, 1H, J = 2.0 Hz, Ar–H), 6.82 (dd,
1H, J = 2.0, 8.4 Hz, Ar–H), 6.89 (d, 1H, J = 8.4 Hz, Ar–H), 7.69 (br s,
1H, O@C–NH); MS (ESI) m/z = 429 [M+1]+; Anal. Calcd for
C22H24N2O7: C, 61.67; H, 5.65; N, 6.54. Found: C, 61.88; H, 5.42; N,
6.76.
4.1.3.8. N-(3-(3-Hydroxy-4-methoxyphenyl)-4-(3,4,5-trime-
thoxyphenyl)isoxazol-5-yl)acetamide (12h). Light yellow solid
(46%); mp 238–240 °C; 1H NMR (400 MHz, CDCl3) d 2.45 (s, 3H,
O@C–CH3), 3.62 (s, 6H, OCH3Â2), 3.77 (s, 6H, OCH3Â2), 6.20 (s, 2H,
Ar–H), 6.51–6.53 (m, 2H, Ar–H), 6.69 (s, 1H, Ar–H), 7.86 (s, 1H,
O@C–NH); MS (ESI) m/z = 415 [M+1]+; Anal. Calcd For C21H22N2O7:
C, 60.86; H, 5.35; N, 6.76. Found: C, 60.76; H, 5.21; N, 6.89.
Compound 13b, white solid (25%); mp 139–140 °C; 1H NMR
(400 MHz, CDCl3) d 2.21 (s, 6H, O@C–CH3Â2), 3.67 (s, 6H, OCH3Â2),
3.76 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 6.74 (s,
2H, Ar–H), 6.78 (d, 1H, J = 2.0 Hz, Ar–H), 6.85 (dd, 1H, J = 2.0,
8.4 Hz, Ar–H), 6.91 (d, 1H, J = 8.4 Hz, Ar–H); MS (ESI) m/z = 471
[M+1]+; Anal. Calcd for C24H26N2O8: C, 61.27; H, 5.57; N, 5.95.
Found: C, 61.54; H, 5.32; N, 5.69.
4.2. Biology
4.2.1. Tubulin polymerization assay15
Tubulin polymerization assays were conducted with reagents as
described by the manufacturer (Cytoskeleton, Inc.). In brief, com-
pound 11a and 13a with variable concentrations (3
lM, 0.75 lM,
0.1875 M) were incubated with purified bovine tubulin and buf-
l
4.1.3.3. N-(4-(4-Chlorophenyl)-3-(3,4,5-trimethoxyphenyl)iso-
xazol-5-yl)acetamide (12c) and N-acetyl-N-(4-(4-chlorophenyl)-
3-(3,4,5-trimethoxyphenyl)isoxazol-5-yl)acetamide
fer containing 20% glycerol, 1 mM GTP, 80 mM PIPES (pH 6.9),
2.0 mM MgCl2, and 0.5 mM EGTA at 37 °C and the effect of com-
pound 11a and 13a on tubulin polymerization was monitored
kinetically using a fluorescent plate reader. The IC50 value is de-
fined as the concentration of product which inhibits the rate of
polymerization by 50%.
(13c). Compound 12c, white solid (57%); mp 162–164 °C; 1H NMR
(400 MHz, CDCl3) d 2.18 (s, 3H, O@C–CH3), 3.66 (s, 6H, OCH3Â2),
3.86 (s, 3H, OCH3), 6.63 (s, 2H, Ar–H), 7.20 (d, 2H, J = 8.8 Hz, Ar–
H), 7.37 (d, 2H, J = 8.8 Hz, Ar–H), 7.90 (s, 1H, O@C–NH); MS (ESI)
m/z = 403 [M+1]+; Anal. Calcd for C20H19ClN2O5: C, 59.63; H,
4.75; N, 6.95. Found: C, 59.43; H, 4.63; N, 6.86.
4.2.2. Cytotoxicity assay16
The cytotoxic activity in vitro against several cancer cell lines
was measured by MTT assay. These cancer cell lines included hu-
man leukemia (K562), human esophageal carcinoma (ECA-109),
human lung carcinoma (A-549), human liver carcinoma (SMMC-
7721), and human prostate carcinoma (PC-3). MTT solution
Compound 13c, white solid (21%); mp 165–166 °C; 1H NMR
(400 MHz, CDCl3) d 2.21 (s, 6H, O@C–CH3Â2), 3.67 (s, 6H, OCH3Â2),
3.86 (s, 3H, OCH3), 6.63 (s, 2H, Ar–H), 7.20 (d, 2H, J = 8.8 Hz, Ar–H),
7.39 (d, 2H, J = 8.8 Hz, Ar–H); MS (ESI) m/z = 445 [M+1]+; Anal.
Calcd for C22H21ClN2O6: C, 59.40; H, 4.76; N, 6.30. Found: C,
59.12; H, 4.62; N, 6.56.
(10.0 lL/well) in RPMI-1640 (Sigma, St. Louis, MO) was added after
cells were treated with drug for 48 h, and cells were incubated for a
further 4 h at 37 °C. The purple formazan crystals were dissolved in
4.1.3.4. N-(3,4-Bis(3,4,5-trimethoxyphenyl)isoxazol-5-yl)acet-
amide (12d). Yellow solid (55%); mp 160–162 °C; 1H NMR
(400 MHz, CDCl3) d 2.24 (s, 3H, O@C–CH3), 3.67 (s, 6H, OCH3Â2),
3.75 (s, 6H, OCH3Â2), 3.85 (s, 6H, OCH3Â2), 6.48 (s, 2H, Ar–H),
6.73 (s, 2H, Ar–H), 7.81 (br s, 1H, O@C–NH); MS (ESI) m/z = 459
[M+1]+; Anal. Calcd for C23H26N2O3: C, 60.26; H, 5.72; N, 6.11.
Found: C, 60.43; H, 5.51; N, 6.47.
100.0 ll DMSO. After 5 min, the plates were read on an automated
microplate spectrophotometer (Bio-Tek Instruments, Winooski,
VT) at 570 nm. Assays were performed in triplicate in three inde-
pendent experiments. The concentration required for 50% inhibi-
tion of cell viability (IC50) was calculated using the software
‘Dose-Effect Analysis with Microcomputers’. In all of these experi-
ments, three replicate wells were used to determine each point.
4.1.3.5. N-(3-(3-(Benzyloxy)-4-methoxyphenyl)-4-(3,4,5-trime-
thoxyphenyl)-isoxazol-5-yl)acetamide (12e). White solid (50%);
mp 178–180 °C; 1H NMR (400 MHz, CDCl3) d 2.21 (s, 3H, OCH3),
3.72 (s, 6H, OCH3Â2), 3.87 (s, 6H, OCH3Â2), 4.96 (s, 2H, CH2), 6.41
(s, 2H, Ar–H), 6.82 (d, 1H, J = 8.4 Hz, Ar–H), 7.04 (d, 1H, J = 8.4 Hz,
Ar–H), 7.08 (s, 1H, Ar–H), 7.26–7.32 (m, 5H, Ar–H), 7.83 (s, 1H,
O@C–NH); MS (ESI) m/z = 505 [M+1]+; Anal. Calcd for C28H28N2O7:
C, 66.66; H, 5.59; N, 5.55. Found: C, 66.45; H, 5.76; N, 5.33.
4.2.3. Flow cytometry analysis17
For flow cytometry analysis, SMMC-7721 cells (5 Â 104/mL)
were treated with DMSO, CA-4 and graded concentrations of 13a
(0.01–1.0
fixed in 75% ethanol at À20 °C. The cell pellet was resuspended
in 100.0 L of PBS containing 200.0 mg/mL RNase (Amersco, Solon,
lM) for 24 h. Cells were washed twice with PBS and
l
OH), then incubated at 37 °C for 0.5 h. After incubation, the cells
were stained with 20.0 mL/L propidium iodide (PI, Sigma, St. Louis,
MO) at 4 °C for 15 min. The fluorescence of cell was measured with
FACSCalibur (Becton–Dickinson, Lincoln Park, NJ).
4.1.3.6. N-(3-(3,4-Dimethoxyphenyl)-4-(3,4,5-trimethoxy-
phenyl)isoxazol-5-yl)-acetamide (12f). White solid (49%); mp
168–170 °C; 1H NMR (400 MHz, CDCl3) d 2.25 (s, 3H, O@C–CH3),
3.73 (s, 9H, OCH3Â3), 3.86 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 6.44
(s, 2H, Ar–H), 6.79 (d, 1H, J = 8.0 Hz, Ar–H), 7.01 (dd, 1H, J = 1.6,
8.0 Hz, Ar–H), 7.07 (d, 1H, J = 1.6 Hz, Ar–H), 7.45 (br s, 1H, O@C–
NH); MS (ESI) m/z = 429 [M+1]+; Anal. Calcd for C22H24N2O7: C,
61.67; H, 5.65; N, 6.54. Found: C, 61.43; H, 5.78; N, 6.65.
4.3. Molecular modeling18
Tubulin-DAMAcolchicine crystal structure (PDB ID: 1SA0) was
chosen as a template. The functional A, B chains of were kept, polar
hydrogens were added, and CHARMm force field was employed.
Binding sphere (45.4407, 41.1246, 34.6219, 9) was selected from
the active site using the binding site tools. For all tested compounds,
hydrogens were added and CHARMm force fields were employed.
Each of the compounds was minimized by Dreiding Minimize tool.
CDOCKER (Discovery Studio 2.1) was used for the docking simula-
tion. For each compound, the docking parameters were as follows:
Top Hits: 25; Random Conformations: 10; Random Conformations
Dynamics Steps: 1000; Grid Extension: 8.0; Random Dynamics
Time Step: 0.002. Final docked conformations were scored by Calcu-
lated Cdocker-Energy and Cdocker interaction Energy.
4.1.3.7. N-(3-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-
isoxazol-5-yl)-acetamide (12g). Yellow solid (65%); mp 175–
177 °C; 1H NMR (400 MHz, CDCl3) d 2.21 (s, 3H, O@C–CH3), 3.70
(s, 6H, OCH3Â2), 3.80 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 6.41 (s,
2H, Ar–H), 6.85 (d, 2H, J = 8.4 Hz, Ar–H), 7.39 (d, 2H, J = 8.4 Hz,
Ar–H), 7.88 (br s, 1H, O@C–NH); MS (ESI) m/z = 399 [M+1]+; Anal.
Calcd for C21H22N2O6: C, 63.31; H, 5.57; N, 7.03. Found: C, 63.23;
H, 5.68; N, 7.05.

T. Liu et al. / Bioorg. Med. Chem. 17 (2009) 6279–6285
6285
9. Odlo, K.; Hentzen, J.; Chabert, J. F.; Ducki, S.; Gani, A. B. S. M. O.; Sylte, I.; Skrede,
M.; Flørenes, V. A.; Hansen, T. V. Bioorg. Med. Chem. 2008, 16, 4829.
10. Xue, N.; Yang, X.-C.; Wu, R.; Chen, J.; He, Q.-J.; Yang, B.; Lu, X.-Y.; Hu, Y.-Z.
Bioorg. Med. Chem. 2008, 16, 2550.
11. Zhong, R.-Q.; Zhou, Y.; Zhang, X.-J.; Wang, Z.-X.; Ling, H.-Z. Chin. J. Pharm. 2005,
36, 328.
12. Sundaresan, A. K.; Ramamurthy, V. Org. Lett. 2007, 9, 3575.
13. Logemann, G. Chem. Ber. 1954, 87, 1175.
14. Wu, R.;Ding, W.-J.; Liu, T.; Zhu, H.; Hu, Y.-Z.; Yang, B.; He, Q.-J. Cancer Lett., inpress.
15. Chen, J.; Lou, J.-S.; Liu, T.; Wu, R.; Dong, X.; He, Q.-J.; Yang, B.; Hu, Y.-Z. Arch.
Pharm. Chem. Life Sci. 2009, 342, 165.
16. Carmichael, J.; DeGraff, W. G.; Gadzar, A. F.; Minna, J. D.; Mitchell, J. B. Cancer
Res. 1987, 47, 936.
17. Yang, B.; Reynolds, C. P. Clin. Cancer Res. 2005, 11, 2774.
18. Ravelli, R. B.; Gigant, B.; Curmi, P. A.; Jourdain, I.; Lachkar, S.; Sobel, A.;
Knossow, M. Nature 2004, 428, 198.
References and notes
1. (a) Bhattacharayya, B.; Panda, D.; Gupta, S.; Banerjee, M. Med. Res. Rev. 2008,
28, 155; (b) Attard, G.; Greystoke, A.; Kaye, S.; De Bono, J. Pathol. Biol. 2006,
54, 72.
2. (a) Li, Q.; Sham, H. L. Exp. Opin. Ther. Pat. 2002, 12, 1663; (b) Jordan, M. A. Curr.
Med. Chem. 2002, 2, 1.
3. Lin, C. M.; Ho, H. H.; Pettit, G. R.; Hamel, E. Biochemistry 1989, 28, 6984.
4. Pettit, G. R.; Singh, S. G.; Boyd, M. R.; Hamel, E.; Pettit, R. K.; Schmidt, J. M.;
Hogan, F. J. Med. Chem. 1995, 38, 1666.
5. Tozer, G. M.; Kanthou, C.; Baguley, B. C. Nat. Rev. Cancer 2005, 5, 423.
6. Tron, G. C.; Pirali, T.; Sorba, G.; Pagliali, F.; Busacca, S.; Genazzani, A. A. J. Med.
Chem. 2006, 49, 3033.
7. Wu, M.-J.; Sun, Q.-M.; Yang, C.-H.; Chen, D.-D.; Ding, J.; Chen, Y.; Lin, L.-P.; Xie,
Y.-Y. Bioorg. Med. Chem. Lett. 2007, 17, 869.
8. Bellina, F.;Cauteruccio,S.;Monti,S.;Rossi,R.Bioorg. Med.Chem.Lett. 2006, 16, 5757.