Synthesis and cytotoxic activities of 4,5-diarylisoxazoles

Article abstract of DOI:10.1016/j.bmcl.2006.11.023

A series of 4,5-diarylisoxazoles related to combretastatin A-4 (CA-4) were synthesized and evaluated for cytotoxicity against three human cancer cell lines. Among them, compound 6e showed better cytotoxic activity than CA-4 in HeLa and HepG2 cell lines as

Full text of DOI:10.1016/j.bmcl.2006.11.023

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1078–1081
Synthesis and cytotoxic activities of 4,5-diarylisoxazoles
Chang-Ming Sun,a Lee-Gin Lin,b Hsi-Jung Yu,b Chih-Yu Cheng,b Ya-Chuan Tsai,b
Chi-Wei Chu,a Yi-Hui Din,a Yat-Pang Chauc and Ming-Jaw Dona,b,
*
aNational Research Institute of Chinese Medicine, No. 155-1, Section 2, Li-Nung Street, Peitou, Taipei 112, Taiwan, ROC
bDepartment of Chemistry, Chinese Culture University, No. 55, Hwa-Kang Road, Yang-Ming-Shan, Taipei 111, Taiwan, ROC
cInstitute of Anatomy and Cell Biology, School of Medicine, National Yang-Ming University, No. 155, Section 2, Li-Nung Street,
Peitou, Taipei 112, Taiwan, ROC
Received 9 July 2006; revised 12 October 2006; accepted 8 November 2006
Available online 10 November 2006
Abstract—A series of 4,5-diarylisoxazoles related to combretastatin A-4 (CA-4) were synthesized and evaluated for cytotoxicity
against three human cancer cell lines. Among them, compound 6e showed better cytotoxic activity than CA-4 in HeLa and HepG2
cell lines assayed with IC50 value as low as 0.022 and 0.065 nM, respectively.
Ó 2006 Elsevier Ltd. All rights reserved.
Antimitotic agents continue to be an interesting target
for medicinal chemist due to possible clinical application
in cancer chemotherapy. Taxol is one successful exam-
ple. Antimitotic agents inhibit cancer cell proliferation
by interfering with microtubule polymerization or depo-
lymerization process.1,2
tion.9,10 Accordingly,
a
number of cis-restricted
analogues of CA-4 were prepared using 1,2-substituted
five-membered heterocycles such as imidazole,11 oxa-
zole,11 pyrazole,11,12 triazole,12 tetrazole,12 thiazole,12
furanone,13 cyclopentenone,14 oxazolone,15 dioxolane,16
and furazan17 to avoid the stability problem. Many of
them showed potent cytotoxicity against various cancer
cells compared to CA-4. In recent study, isoxazoline
derivatives were reported to possess potent apoptosis-in-
ducing activity in HL60 and in MDR cell lines.18
Combretastatins, naturally occurring stilbenes, were iso-
lated from Combretum caffrum by Pettit group.3,4
Among them, combretastatin A-4 (CA-4) strongly
inhibited the polymerization of tubulin by binding to
the chochicine site and showed most potent cytotoxicity
against a variety of human cancer cell lines including
multiple drug-resistant cancer cell lines.5,6 To date,
many CA-4 analogues and their anticancer activity have
been extensively studied and a water-soluble sodium
phosphate prodrug (CA-4P) is currently evaluated for
clinical applications.7,8
In our efforts to discover active antimitotic agents, we
utilized isoxazole ring to mimic the cis double bond in
CA-4. In this study, a series of 4,5-diarylisoxazoles
(Fig. 1) were prepared and their cytotoxic activity was
also evaluated against human cancer cell lines including
human cervical epitheloid carcinoma (HeLa), human
hepatocellular carcinoma (HepG2), and human ovarian
adenocarcinoma (OVCAR-3).
From the previous comparative studies of the combre-
tastatins it appears that the cis orientation of the two
aromatic rings plays an important key role in cytotoxic-
ity. However, during storage and administration cis-
combretastatin analogues tend to isomerize to trans
forms which show a dramatic decrease in their inhibito-
ry effects on cancer cell growth and tubulin polymeriza-
The starting dithiane (1a) was prepared from 3,4,5-tri-
methoxybenzaldehyde by treatment with 1,3-propane-
dithiol in the presence of aniline hydrochloride. The
dithiane (1a) reacted with n-BuLi and benzyl bromides
(2a–c) at À78 °C to give the alkylated dithiane products
(3a–c), respectively,19 which were then converted to the
corresponding ketone derivatives (4a–c) by reaction with
HgO and HgCl2. Treatment of the ketone derivatives
(4a–c) with N,N-dimethylformamide dimethylacetal
(DMFDMA) under reflux resulted in the corresponding
enamino ketones (5a–c)20 which were then reacted with
Keywords: Antimitotic agent; 4,5-Diarylisoxazole; Cytotoxicity.
*
Corresponding author. Tel.: +886 2 28201999x8411; fax: +886 2
28264276; e-mail: mjdon@nricm.edu.tw
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.023

C.-M. Sun et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1078–1081
1079
N
N
O
O
MeO
MeO
MeO
MeO
R
MeO
OMe
OMe
R
R
MeO
OMe
OMe
OMe
OMe
CA-4, R = OH
CA-4P, R = OPO3Na2
4,5-Diarylisoxazoles
Figure 1. Chemical structures of CA-4, CA-4P, and 4,5-diarylisoxazoles.
hydroxylamine hydrochloride to yield desired 4,5-diary-
lisoxazoles (6a–c), respectively.21 Deprotection of the
benzyl group of 6b by hydrogenation gave 6d. Reduc-
tion of nitro group of 6c using acetic acid and zinc pow-
der yielded 6e (Scheme 1).22
cytotoxicity. Therefore, we chose 3,4,5-trimethoxyphe-
nyl and 4-methoxyphenyl or 3-hydroxy-4-methoxyphe-
nyl as two aromatic rings and first synthesized two
isomers 6a and 6d. Assay results showed that 6a and
6d were active against three cancer cell lines, and 6d
was more potent than 6a. Next, we switched two aro-
matic rings and synthesized two other isomers 6f and
6h. Compounds 6f and 6h showed moderate cytotoxic
activity and less potent than 6a and 6d, which indicated
the 3,4,5-trimethoxyphenyl group near the oxygen atom
of isoxazole ring is important for strong cytotoxicity.
The activity of 6d and 6h was greater than that of 6a
and 6f, respectively, which indicated with the hydroxyl
group in 6d and 6h is better than without the hydroxyl
group in 6a and 6d. This result was in consistence with
other reports.12,14,16 Previous reports also indicated that
replacement of a hydroxyl group with an amino group
exhibited similar or greater activity.13,15,17,25 Thus, 6c
and 6e were prepared for activity comparison. Surpris-
ingly, 6e displayed excellent in vitro cytotoxicity against
three cancer cell lines and showed better cytotoxicities
than CA-4 in HeLa and HepG2 cell lines assayed with
IC50 value as low as 0.022 and 0.065 nM, respectively.
Compounds 6f and 6h were prepared by using the same
synthetic strategy as that for 6a and 6d, which is shown
in Scheme 2 starting from 4-methoxybenzaldehyde (1b),
benzylic derivative of 3-hydroxy-4-methoxybenzalde-
hyde (1c), and 3,4,5-trimethoxybenzyl bromide (2d).
The structures of compounds (6a, c–f, h) were confirmed
by detailed NMR analysis and elemental analysis. The
cytotoxic activity of synthesized 4,5-diarylisoxazoles
was evaluated against human cancer cell lines including
human cervical epitheloid carcinoma (HeLa), human
hepatocellular carcinoma (HepG2), and human ovarian
adenocarcinoma (OVCAR-3). The results are summa-
rized in Table 1. The cell line culture conditions23 and
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide] colorimetric assay for IC50 were carried
out according to the procedures previously described.24
Previous reports indicated that 3,4,5-trimethoxy group
on one of two aromatic rings was essential for strong
The effects of the two most active compounds 6d and 6e
on the cell cycle were measured by flow cytometry
S
O
S
S
MeO
MeO
MeO
MeO
b
a
c
S
R
MeO
MeO
OMe
Br
OMe
OMe
R
MeO
R
OMe
OMe
1a
2a, R = H
3a, R = H
3b, R = OBn
4a, R = H
4b, R = OBn
2b, R = OBn
2c, R = NO2
3c, R = NO2
4c, R = NO2
O
N
O
MeO
MeO
MeO
MeO
NMe2
d
OMe
OMe
R
R
e
OMe
OMe
5a, R = H
5b, R = OBn
5c, R = NO2
6a, R = H
6b, R = OBn
6c, R = NO2
6d, R = OH
6e, R = NH2
f
Scheme 1. Reagents and conditions: (a) n-BuLi, THF, À78 °C, 30 min, then TMEDA, benzyl bromides, 25 °C, overnight; (b) HgO, HgCl2, MeCN/
H2O, rt, 16 h; (c) DMFDMA, reflux, 14 h; (d) NH2OHÆHCl, Na2CO3, MeOH, AcOH, reflux, 2 h; (e) H2, 10% Pd/C, EtOAc, rt, 3 h; (f) Zn, AcOH, rt,
1 h.

1080
C.-M. Sun et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1078–1081
S
O
S
S
R
R
R
b
a
c
S
MeO
MeO
MeO
MeO
MeO
Br
MeO
OMe
MeO
OMe
1b, R = H
1c, R = OBn
OMe
OMe
OMe
3d, R = H
3e, R = OBn
4d, R = H
4e, R = OBn
2d
O
N
O
R
R
NMe2
d
MeO
MeO
MeO
OMe
MeO
OMe
e
OMe
OMe
5d, R = H
5e, R = OBn
6f, R = H
6g, R = OBn
6h, R = OH
Scheme 2. Reagents and conditions: (a) n-BuLi, THF, À78 °C, 30 min, then TMEDA, benzyl bromides, 25 °C, overnight; (b) HgO, HgCl2, MeCN/
H2O, rt, 16 h; (c) DMFDMA, reflux, 14 h; (d) NH2OHÆHCl, Na2CO3, MeOH, AcOH, reflux, 2 h; (e) H2, 10% Pd/C, EtOAc, rt, 3 h.
Table 1. Cytotoxicity of synthesized compounds in tumor cell linesa
b
Compound
Cytotoxicity (IC50
,
nM)
HeLa
HepG2
OVCAR-3
6a
6c
4.69
11.6
0.90
0.022
299
4.40
13.7
1.43
0.065
396
10.3
9.6
6d
6e
0.53
0.135
223
6f
6h
29.4
2.75
33.9
0.14
14.8
0.01
CA-4
a HeLa, human cervical epitheloid carcinoma; HepG2, human hepatocellular carcinoma; OVCAR-3, human ovarian adenocarcinoma.
b Drug concentration required to inhibit the cell growth by 50%.
Table 2. Effectsa of compounds 6d, 6e, and CA-4 on cell cycle progression
Compound
G0/G1 (%)
S-phase (%)
G2/M (%)
Control (DMSO)
6d (10 nM)
6e (10 nM)
50.0 3.5
17.1 1.0
2.81 1.3
1.68 0.3
27.0 4.8
31.5 2.8
27.4 2.4
19.0 0.7
23.0 2.1
51.4 3.2
69.8 2.7
79.3 1.0
CA-4 (10 nM)
a All experiments were independently performed three times.
against HepG2 human cancer cells after 24 h. The re-
sults are shown in Table 2. Compounds 6d and 6e
caused significant arrest of the cells at the G2/M phase
relative to the untreated control, consistent with the
behavior of tubulin-binding agents. Compound 6d was
recently reported to exhibit potent antitubulin activity.26
Acknowledgments
The authors thank the National Science Council (NSC
93-2113-M-077-002) of Republic of China and National
Research Institute of Chinese Medicine (NRICM 94-
DMC-006) for financial support.
In conclusion, we have presented here the synthesis and
evaluation of cytotoxicity of a series of 4,5-diarylisoxaz-
oles. Compounds 6a, 6c, 6d, and 6e showed strong
growth inhibitory activities against three human cancer
cell lines, and 6e was the most potent compound in this
series. In addition, 6d and 6e caused G2/M phase arrest
of the cells and are considered to be potential new anti-
mitotic agents for future clinical use.
References and notes
1. Hamel, E. Med. Res. Rev. 1996, 16, 207.
2. Jordan, A.; Hadfield, J. A.; Lawrence, N. J.; McGown, A.
T. Med. Res. Rev. 1998, 18, 259.
3. Pettit, G. R.; Cragg, G. M.; Herald, D. L.; Schmidt, J. M.;
Lohavanijaya, P. Can. J. Chem. 1982, 60, 1374.

C.-M. Sun et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1078–1081
1081
4. Pettit, G. R.; Singh, S. B.; Boyd, M. R.; Hamel, E.; Pettit,
R. K.; Schmidt, J. M.; Hogan, F. J. Med. Chem. 1995, 38,
1666.
5. Lin, C. M.; Ho, H. H.; Pettit, G. R.; Hamel, E.
Biochemistry 1989, 28, 6984.
6. McGown, A. T.; Fox, B. W. Cancer Chemother. Pharma-
col. 1990, 26, 79.
7. Pettit, G. R.; Temple, C., Jr.; Narayanan, V. L.; Varma,
R.; Simpson, M. J.; Boyd, M. R.; Rener, G. A.; Bansal, N.
Anticancer Drug Des. 1995, 10, 299.
8. Chaplin, D. J.; Pettit, G. R.; Hill, S. A. Anticancer Res.
1999, 19, 189.
9. Cushman, M.; Nagarathnam, D.; Gopal, D.; Chakraborti,
A. K.; Lin, C. M.; Hamel, E. J. Med. Chem. 1991, 34,
2579.
10. Cushman, M.; Nagarathnam, D.; Gopal, D.; He, H.-M.;
Lin, C. M.; Hamel, E. J. Med. Chem. 1992, 35, 2293.
11. Wang, L.; Woods, K. W.; Li, Q.; Barr, K. J.; McCroskey,
R. W.; Hannick, S. M.; Gherke, L.; Credo, R. B.; Hui, Y.-
H.; Marsh, K.; Warner, R.; Lee, J. Y.; Zielinski-Mozng,
N.; Frost, D.; Rosenberg, S. H.; Sham, H. L. J. Med.
Chem. 2002, 45, 1697.
12. Ohsumi, K.; Hatanaka, T.; Fujita, K.; Nakagawa, R.;
Fukuda, Y.; Nihei, Y.; Suga, Y.; Morinaga, Y.; Akiyama,
Y.; Tsuji, T. Bioorg. Med. Chem. Lett. 1998, 8, 3153.
13. Kim, Y.; Nam, N.-H.; You, Y.-J.; Ahn, B.-Z. Bioorg.
Med. Chem. Lett. 2002, 12, 719.
14. Nam, N.-H.; Kim, Y.; You, Y.-J.; Hong, D.-H.; Kim, H.-
M.; Ahn, B.-Z. Bioorg. Med. Chem. Lett. 2002, 12, 1955.
15. Nam, N.-H.; Kim, Y.; You, Y.-J.; Hong, D.-H.; Kim, H.-
M.; Ahn, B.-Z. Bioorg. Med. Chem. Lett. 2001, 11, 3073.
16. Shirai, R.; Takayama, H.; Nishikawa, A.; Koiso, Y.;
Hashimoto, Y. Bioorg. Med. Chem. Lett. 1998, 8, 1997.
17. Tron, G. C.; Pagliai, F.; Grosso, E. D.; Genazzani, A. A.;
Sorba, G. J. Med. Chem. 2005, 48, 3260.
18. Simoni, D.; Grisolia, G.; Giannini, G.; Roberti, M.;
Rondanin, R.; Piccagli, L.; Baruchello, R.; Rossi, M.;
Romagnoli, R.; Invidiata, F. P.; Grimaudo, S.; Jung, M.
K.; Hamel, E.; Gebbia, N.; Crosta, L.; Abbadessa, V.; Di
Cristina, A.; Dusonchet, L.; Meli, M.; Tolomeo, M. J.
Med. Chem. 2005, 48, 723.
EtOAc in hexane to give 3c as a pale yellow solid (1.02 g,
45%). To a solution of 3c (910 mg, 2.0 mmol) in CH3CN/
H2O (50 mL, 85:15) were added HgO (866 mg, 4.0 mmol)
and HgCl2 (1.19 g, 4.4 mmol) at rt. After stirring for 16 h,
the mixture was filtered through Celite and the filtrate was
concentrated in vacuo. Water was added and the resulting
mixture was extracted three times with EtOAc. The
combined organic layer was washed with brine, dried over
anhydrous Na2SO4, filtered, and evaporated. The residue
was chromatographed over silica gel eluting with 20%
EtOAc in hexane to give 4c as a pale yellow solid (370 mg,
51%). A solution of 4c (580 mg, 1.61 mmol) in DMFDMA
(10 mL) was then refluxed under N2 for 14 h. After
cooling, the reaction mixture was concentrated in vacuo.
Without purification, the resulting crude product 5c was
dissolved in MeOH/H2O (12 mL, 2:1), and NH2OHÆHCl
(120 mg, 1.77 mmol) and Na2CO3 (95 mg, 0.90 mmol)
were added to the solution at rt. The mixture was adjusted
to pH 4–5 by addition of acetic acid and then refluxed for
2 h. After cooling, the mixture was adjusted to pH 8 by
addition of 25% NH4OH and extracted three times with
CH2Cl2. The combined organic layer was washed with
brine, dried over anhydrous Na2SO4, filtered, and evap-
orated. The residue was chromatographed over silica gel
eluting with 20% EtOAc in hexane to afford 6c as a yellow
solid (320 mg, 51%): mp 120–122 °C; IR (KBr) mmax 2991,
2959, 2935, 2831, 1580, 1535, 1469, 1358, 1245, 1127,
1007 cmÀ1 1H NMR (CDCl3, 500 MHz) d 3.74 (s, 6H),
;
3.87 (s, 3H), 3.98 (s, 3H), 6.83 (s, 2H), 7.12 (d, J = 8.5 Hz,
1H), 7.56 (dd, J = 8.5, 2.0 Hz, 1H), 7.91 (d, J = 2.0 Hz,
1H), 8.34 (s, 1H); EI-MS m/z (%) 386 (100) [M+], 371 (26),
343 (12), 324 (10); Anal. Calcd for C19H18N2O7: C, 59.07;
H, 4.70; N, 7.25. Found: C, 58.97; H, 4.63, N, 7.25. To a
solution of 6c (116 mg, 0.30 mmol) in acetic acid (25 mL)
was added zinc powder (4.70 g) at rt. After stirring for 1 h,
the reaction mixture was filtered through Celite and the
filtrate was concentrated in vacuo. Water was added and
the resulting mixture was extracted three times with
CHCl3. The combined organic layer was washed with
saturated NaHCO3, brine, dried over anhydrous Na2SO4,
filtered, and evaporated. The residue was chromato-
graphed over silica gel eluting with 30% EtOAc in hexane
to give 6e as a yellow solid (46 mg, 43%): mp 82–83 °C; IR
(KBr) mmax 3439, 3344, 3222, 3101, 2991, 2933, 2837, 1632,
´
´
´
19. Medarde, M.; Pelaez-Lamamie de Clairac, R.; Lopez, J.
L.; San Feliciano, A. J. Nat. Prod. 1994, 57, 1136.
´
20. SanMartın, R.; Martınez de Marigorta, E.; Domınguez, E.
´
´
Tetrahedron 1994, 50, 2255.
´
21. Domınguez, E.; Ibeas, E.; Martınez de Marigorta, E.;
1578, 1511, 1468, 1418, 1234, 1125, 1000, 845 cmÀ1 1H
;
´
Palacios, J. K.; SanMartın, R. J. Org. Chem. 1996, 61,
5435.
NMR (CDCl3, 500 MHz) d 3.72 (s, 6H), 3.85 (s, 3H), 3.86
(s, 3H), 6.80–6.82 (m, 3H), 6.92 (s, 2H), 8.26 (s, 1H); EI-
MS m/z (%) 356 (100) [M+], 195 (47), 173 (10); Anal. Calcd
for C19H20N2O5: C, 64.04; H, 5.66; N, 7.86. Found: C,
64.05; H, 5.88, N, 7.65.
´
22. Typical procedure for the synthesis of 6e. To a solution of
1a (1.43 g, 5.0 mmol) in dry THF (40 mL) was added n-
BuLi (3.4 mL, 1.6 M in hexane) at À78 °C under N2. After
stirring for 30 min, a solution of 2c (1.23 g, 5.0 mmol) in
THF (10 mL) and TMEDA (0.75 mL) were added at
À78 °C, and the reaction mixture was allowed to stir at
À25 °C overnight. Saturated NH4Cl was then added and
the mixture was extracted three times with EtOAc. The
combined organic layer was washed with brine, dried over
anhydrous Na2SO4, filtered, and evaporated. The residue
was chromatographed over silica gel eluting with 20%
23. Sun, C.-M.; Syu, W.-J.; Huang, Y.-T.; Chen, C.-C.; Ou,
J.-C. J. Nat. Prod. 1997, 60, 382.
24. Shen, C.-C.; Syu, W.-J.; Li, S.-Y.; Lin, C.-H.; Lee, G.-H.;
Sun, C.-M. J. Nat. Prod. 2002, 65, 1857.
25. Pinney, K. G.; Mejia, M. P.; Villalobos, V. M.; Rosen-
quist, B. E.; Pettit, G. R.; Verdier-Pinard, P.; Hamel, E.
Bioorg. Med. Chem. 2000, 8, 2417.
26. Kaffy, J.; Pontikis, R.; Carrez, D.; Croisy, A.; Monneret,
C.; Florent, J.-C. Bioorg. Med. Chem. 2006, 14, 4067.