Norcantharidin analogues - Synthesis and evaluation of growth inhibition in a panel of selected tumor-cell lines

Article abstract of DOI:10.1139/V07-082

A series of norcantharidin (NCTD) analogues have been synthesized by [3+2]1,3-dipolar cycloaddition reaction of norcantharidin derivatives of substituted aromatic amines with four nitrile oximes. All analogues have been screened for their antiproliferative activity in vitro against a panel of tumor cell lines: KB, SGC-7901, HL60, Bel-7402, HO-8910, and ECA109, producing IC ₅₀ values from 0.36 μmol/L to >100 μmol/L. Compound 9d showed potency for the treatment of hepatoma, with IC₅₀ value to Bel-7402 cell line comparable to that of norcantharidin.

Full text of DOI:10.1139/V07-082

938
Norcantharidin analogues — Synthesis and
evaluation of growth inhibition in a panel of
selected tumor-cell lines
Liping Deng, Li Shen, Jing Zhang, Bo Yang, Qiaojun He, and Yongzhou Hu
Abstract: A series of norcantharidin (NCTD) analogues have been synthesized by [3+2]1,3-dipolar cycloaddition reac-
tion of norcantharidin derivatives of substituted aromatic amines with four nitrile oximes. All analogues have been
screened for their antiproliferative activity in vitro against a panel of tumor cell lines: KB, SGC-7901, HL60, Bel-7402,
HO-8910, and ECA109, producing IC50 values from 0.36 µmol/L to >100 µmol/L. Compound 9d showed potency for
the treatment of hepatoma, with IC50 value to Bel-7402 cell line comparable to that of norcantharidin.
Key words: norcantharidin analogues, isoxazoline, growth inhibition.
Résumé : On a réalisé la synthèse d’une série d’analogues de la norcantharidine en procédant à une réaction de cycloaddi-
tion 1,3-dipolaire [3+2] de dérivés de la norcantharidine d’amines aromatiques substituées avec quatre oximes de nitriles. On
a évalué l’activité comme inhibiteur de croissance in vitro de chacun des analogues contre un ensemble de souches de cellu-
les tumorales: KB, SGC-7901, HL60, Bel-7402, HO-8910 et ECA109 pour lesquelles les valeurs de IC50 varient de 0,36
µmol/L à plus de 100 µmol/L. Le composé 9d présente un grand potentiel pour le traitement de l’hépatome, avec une valeur
de IC50 qui, par comparaison avec celle des cellules Bel-7402, est comparable à celle de la norcantharidine.
Mots-clés : analogues de la norcantharidine, isoxazoline, inhibition de la croissance.
[Traduit par la Rédaction] Deng et al. 944
Introduction
Fig. 1. Chemical structures of Cantharidin and Norcantharidin.
Traditional Chinese medicine is a rich source of potential
anticancer agents. In particular, cantharidin (CAN; exo, exo-
2,3-dimethyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic
acid anhydride; Fig. 1) is the active ingredient of Mylabris,
which has been used in China as a medicinal agent for the
treatment of cancer, particularly hepatoma (1). Although
cantharidin is cytotoxic to cancer cells and stimulatory on
the bone marrow, the renal toxicity of this drug has limited
its application (2). A series of bioactive analogues have been
synthesized in an attempt to increase the utility and to re-
duce the toxicity of cantharidin. Norcantharidin (NCTD;
Fig. 1), the demethylated analogue of cantharidin, has been
used to treat human cancers in China since 1984 (2). NCTD
appeared to improve the awkward side of cantharidin, mak-
ing the drug safer in application. It was recently found to be
capable of inducing apoptosis in human cervical, tongue,
ginival, mucoepidermoid carcinoma, adenocystic carcinoma,
neuroblastoma, bone, leukaemia, ovarian, and colon cancer
O
O
7
7
O
O
O
O
H
CH
3O
O
H
CH3
Cantharidin
Norcantharidin
cell lines (3–5); however, the main application so far is
treating hepatoma. We have referred to all the known
cantharidin SAR data; briefly, no modification of the
bicyclo[2.2.1] skeleton is permissible, the 7-oxa bridge is re-
quired to maintain activity, and the presence of a double
bond (5,6-ene) has little effect on activity (6–10). Replace-
ment of the O-atom (anhydride) with N (as N–H and N–R,
where R = aryl or thiazolyl) allows the development of a
new series of anhydride-modified cantharidin analogues;
some are more potent than CAN or display potency similar
to NCTD (11, 12). On the other hand, it is well-known that
isoxazoline derivatives possess a wide range of pharmaco-
logical activities (13). These observations prompted us to
design a new series of norcantharidin analogues by combin-
ing isoxazoline with norcantharidin derivatives in one single
molecule to improve their antiproliferative activities and to
carry out a structure–activity relationship study. In this pa-
per, we describe the synthetic methodology towards the de-
velopment of a series of norcantharidin analogues and their
antiproliferative activities in vitro against various human
tumor-cell lines.
Received 11 September 2006. Accepted 17 May 2007.
Published on the NRC Research Press Web site at
canjchem.nrc.ca on 3 October 2007.
L. Deng, L. Shen, and Y. Hu.1 ZJU-ENS joint laboratory of
medicinal chemistry, School of Pharmaceutical Sciences,
Zhejiang University, Hangzhou 310058, China.
J. Zhang, B. Yang, and Q. He. Department of
Pharmacology and Toxicology, School of Pharmaceutical
Sciences, Zhejiang University, Hangzhou 310058, China.
1Corresponding author (email: huyz@zjuem.zju.edu.cn).
Can. J. Chem. 85: 938–944 (2007)
doi:10.1139/V07-082
© 2007 NRC Canada

Deng et al.
939
Scheme 1. Synthetic route of novel norcantharidin analogues 8a–8f, 9a–9f, 10a–10f, and 11a–11f.
O
O
O
O
O
O
H
O
O
H
1
O
O
Et3N, (Ac)2O
Mn(OAc)2
N
R1
NH2
R1
1
H
O
H
2a–2f
3a–3f
O
O
O
N
Cl
N
H4
chloramine T
R1
Ar-C=NOH
3a–3f
H5
Ar
H1
H3
H2
H6
O
4, 5, 6, 7
8a–8f, 9a–9f, 10a–10f, 11a–11f
OH
OH
OCH3
OCH3
OCH3
O
Ar =
11 to 7
8 to 4
Ar =
9 to 5 Ar =
Cl
10 to 6 Ar =
SCH3
2a, 3a, 8a–11a R1 = H; 2b, 3b, 8b–11b R1 = F; 2c, 3c, 8c–11c R1 = Cl;
2d, 3d, 8d–11d R1 = Br; 2e, 3e, 8e–11e R1 = CH3; 2f, 3f, 8f–11f R1 = OCH3;
played growth inhibition with IC50 values ranging from
0.36 µmol/L to >100 µmol/L. Most norcantharidin analogues
were antiproliferative against HL60 cell line after 72 h treat-
ment and were inactive to the KB and SGC7901 cell lines.
Some norcantharidin analogues exhibited higher growth in-
hibition than NCTD, e.g., 9c was more potent than NCTD
against HL60 cell line, and 11c was more potent than NCTD
against HL60 and HO8910 cell lines. Also, compound 9d
displayed potency against Bel-7402 cell line comparable to
that of NCTD, suggesting that the halogeno group in the aryl
moiety (R1) might be crucial for activity. It is also notewor-
thy that the presence of the methoxyl (such as 8f, 9f, 10f,
and 11f) appeared to have an unfavorable effect.
Results and discussion
Synthesis
Norcantharidin analogues were prepared as shown in
Scheme 1. Treatment of furan with maleic anhydride re-
sulted in 5,6-dehydronorcantharidin 1, then compound 1 re-
acted with substituted phenylamine 2a–2f to give
compounds 3a–3f according to the published procedure (16).
[3+2] Cycloaddition of 3a–3f with four nitrile oximes 4–7 in
the presence of chloramine T yielded target compounds 8a–
8f, 9a–9f, 10a–10f, and 11a–11f. All compounds were tested
as racemic mixtures.
To identify the configuration of the norcantharidin ana-
logues (8a–8f, 9a–9f, 10a–10f, and 11a–11f), we have stud-
The apoptosis-inducing activity of NCTD and its ana-
logues was dose- and time-dependent; for instance, the
antiproliferative activity of compound 9d and cantharidin
against Bel-7402 cell line are shown in Fig. 2.
1
ied selective H–1H COSY spectra and NOESY spectra of
1
the compounds. H–1H COSY spectra showed cross peaks
between H-2 and H-3 and H-5 and H-6. NOESY spectra
showed cross peaks between H-2 and H-6 and H-3 and H-5.
The Diels–Alder adduct 5,6-dehydronorcantharidin of furan
with maleic anhydride have the exo configuration exclu-
sively; the endo isomer has never been reported (14, 15),
Conclusion
1
which, combined with H–1H COSY and NOESY spectra
From the data presented, subtle modifications of
norcantharidin’s skeleton permitted the development of a
new series of analogues with alterations in their antipro-
liferative activity against a panel of tumor-cell lines. The
lead compounds cantharidin and norcantharidin are some-
what more potent against tumor-cell lines, respectively. Of
the synthesized analogues, the notable exception being 9c,
which showed both good potency and selectivity for the
HL60 cell line, and compound 9d displayed moderate po-
tency for the treatment of hepatoma with IC50 values to
Bel7402 cell line comparable to that of norcantharidin. We
are currently working towards the development of more po-
data, give us a reasonable exo-adduct configuration, as we
have proved before similarly (16, 17).
Biological activity
The norcantharidin analogues 8a–8f, 9a–9f, 10a–10f, and
11a–11f were screened for their antiproliferative activity
against a panel of six tumor-cell lines: KB, SGC7901,
HL60, Bel7402, HO8910 and ECA109. CAN and NCTD
were included as internal standards. IC50 values of different
compounds are summarized in Table 1.
As shown in Table 1, the synthesized compounds dis-
© 2007 NRC Canada

940
Can. J. Chem. Vol. 85, 2007
Table 1. Antiproliferative activity of the skeletally modified norcantharidin analogues
(8a–8f, 9a–9f, 10a–10f, and 11a–11f).
IC50 (µmol/L)a, b
Compound
KB
21.9
32.2
>100
SGC7901
HL60
0.1
5
>100
>100
>100
7.2
60.6
>100
51.2
74.4
Bel7402
HO8910
ECA109
58.2
79
1
24
1.28
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
16.5
0.38
47.4
CAN
NCTD
8a
8b
8c
8d
8e
8f
9a
9b
9c
9d
9e
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
37.7
>100
>100
>100
>100
>100
>100
>100
>100
>100
88.3
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.36
1.9
3.4
62.4
87.5
>100
>100
4.9
4.8
20
>100
>100
1.6
92.3
1.6
>100
>100
69.6
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
9f
10a
10b
10c
10d
10e
10f
11a
11b
11c
11d
11e
11f
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
17.8
13
>100
>100
>100
>100
aThe IC50 values represent the concentration that results in a 50% decrease in cell growth after 72 h
incubation.
bThe IC50 values were the mean values of three repeated experiments.
tent and selective analogues of this nature and will report the
outcome of these studies in due course.
it was cooled to room temperature, water (10 mL) was
added to the reaction mixture, and it was extracted with di-
chloromethane (30 mL). The extract was dried over anhyd.
sodium sulfate, concentrated in vacuum, and the residue was
recrystallized from acetone to give the compound 8a. The
synthesis of compounds 8b–8f, 9a–9f, 10a–10f, 11a–11f
were performed using the same method.
Experimental section
General
All reagents were of commercial quality and were used as
received (Aldrich). Solvents were dried and purified using
standard techniques. Reactions were monitored by TLC on
aluminum plates coated with silica gel with fluorescent indi-
cator (Merck 60 F254). Melting points were obtained on a
B-540 Büchi melting-point apparatus and are uncorrected.
1H NMR and NOESY spectra were recorded on a Brucker
AM-400 MHz spectrometer with SiMe4 as the internal stan-
dard in DMSO-d6. Mass Spectra were recorded with a
HP5989B analyzer. Elemental analyses were performed on
an EA-1110 instrument.
exo,exo-4,8-Epoxy-3a,4,4a,7a,8,8a-hexahydro-6-phenyl-3-
(3,4,5-trimethoxyphenyl)-pyrrolo[3,4-f]-1,2-benzisoxazole (8a)
Compound 8a was obtained as a colorless solid: yield
94%; mp 284 °C. IR ν (KBr, cm–1): 3062 and 3027 (ArH),
1
1778, 1721 (C=O), 1598 (C=N), 1240 (C–O–C). H NMR
(DMSO-d6) δ: 7.83–7.19 (m, 7H, Ar–H), 5.24 (d, J =
7.9 Hz, 1H, H-5), 4.98 (s, 1H, H-4), 4.75 (s, 1H, H-1), 4.52
(d, J = 7.9 Hz, 1H, H-6), 3.77 (s, 9H, 3 × OCH3), 3.59 (d,
J = 7.9 Hz, 1H, H-3), 3.38 (d, J = 7.9 Hz, 1H, H-2). MS m/z:
450 (M+). Anal. calcd. for C24H22N2O7: C 63.99, H4.92, N
6.22; found: C 63.80, H 4.99, N 6.18.
General procedure for the preparation of the 5,6-
dehydronorcantharidinisoxazoline adducts (8a–8f, 9a–9f,
10a–10f, 11a–11f)
To a solution of 3-acetyl-7-oxa-bicyclo[2.2.1]hept-5-ene-
2-carboxylic acid phenylamide 3a (1 mmol) and 4 (1 mmol)
in ethanol (20 mL) was added chloramine T (1.2 mmol), and
the reaction mixture was refluxed in ethanol for 4–7 h. Then,
exo,exo-6-(4-fluorophenyl)-4,8-epoxy-3a,4,4a,7a,8,8a-
hexahydro-3-(3,4,5-trimethoxyphenyl)-pyrrolo[3,4-f]-1,2-
benzisoxazole (8b)
Compound 8b was obtained as a colorless solid: yield
81%; mp 300 °C (Dec.). IR ν (KBr, cm–1): 3121 and 3010
© 2007 NRC Canada

Deng et al.
941
91%; mp 280 °C. IR ν (KBr, cm–1): 3056 and 3043 (ArH),
Fig. 2. The antiproliferative activity of (a) cantharidin and (b) 9d in
Bel-7402 cell line. Cells were treated with various concentrations of
cantharidin and 9d for 24 h, 48 h, and 72 h. The percent of Bel-
7402 cell line at each time point (post-treatment) is observed by
MTT assay and expressed as the mean D of three independent ex-
periments. Antiproliferative activity induced by cantharidin and 9d
is dose- and time-dependent in Bel-7402 cell line.
1
1780, 1721(C=O), 1567 (C=N), 1243 (C–O–C). H NMR
(DMSO-d6) δ: 7.83–7.25 (m, 6H, Ar–H), 5.24 (d, J =
7.9 Hz, 1H, H-5), 4.98 (s, 1H, H-4), 4.75 (s, 1H, H-1),
4.51(d, J = 7.9 Hz, 1H, H-6), 3.59 (d, J = 7.9 Hz, 1H, H-3),
3.77 (s, 9H, 3 × OCH3), 3.38 (d, J = 7.9 Hz, 1H, H-2). MS
m/z: 528 (M+), 530 (M + 2). Anal. calcd. for C24H21 BrN2O7:
C 54.46, H 4.00, N 5.29; found: C 54.48, H 4.03, N 5.25.
exo,exo-4,8-Epoxy-3a,4,4a,7a,8,8a-hexahydro-3-(3,4,5-
trimethoxyphenyl)-6-(p-tolyl)-pyrrolo[3,4-f]-1,2-
benzisoxazole (8e)
Compound 8e was obtained as a colorless solid: yield
90%; mp 261 °C. IR ν (KBr, cm–1): 3072 and 3060 (ArH),
1
1781, 1721(C=O), 1569 (C=N), 1286 (C–O–C). H NMR
(DMSO-d6) δ: 7.83–7.15 (m, 6H, Ar–H), 5.23 (d, J =
7.9 Hz, 1H, H-5), 4.97 (s, 1H, H-4), 4.75 (s, 1H, H-1), 4.51
(d, J = 7.9 Hz, 1H, H-6), 3.59 (d, J = 7.9 Hz, 1H, H-3), 3.35
(d, J = 7.9 Hz, 1H, H-2), 3.77 (s, 9H, 3 × OCH3), 2.33
(–CH3). MS m/z: 464 (M+). Anal. calcd. for C25H24N2O7: C
64.65, H 5.21, N 6.03; found: C 64.67, H 5.22, N 6.00.
exo,exo-4,8-Epoxy-3a,4,4a,7a,8,8a-hexahydro-6-(4-
methoxyphenyl)-3-(3,4,5-trimethoxypheny)-pyrrolo[3,4-f]-
1,2-benzisoxazole (8f)
Compound 8f was obtained as a colorless solid: yield
91%; mp 279 °C. IR ν (KBr, cm–1): 3051 and 3027 (ArH),
1
1778, 1714 (C=O), 1568 (C=N), 1250 (C–O–C). H NMR
(DMSO-d6) δ: 8.37–7.49 (m, 6H, Ar–H), 5.25 (d, J =
7.9 Hz, 1H, H-5), 5.02 (s, 1H, H-4), 4.80 (s, 1H, H-1), 4.53
(d, J = 7.9 Hz, 1H, H-6), 3.66 (d, J = 7.9 Hz, 1H, H-3), 3.44
(d, J = 7.9 Hz, 1H, H-2), 3.77 (s, 9H, 3 × OCH3), 3.83 (s,
3H, –OCH3). MS m/z: 480 (M+). Anal. calcd. for
C25H24N2O8: C 62.49, H 5.03, N 5.83; found: C 62.51, H
5.02, N 5.81.
1
(ArH), 1777, 1718 (C=O), 1570 (C=N), 1224 (C–O–C). H
exo,exo-4,8-Epoxy-3a,4,4a,7a,8,8a-hexahydro-6-phenyl-3-
(4-chlorophenyl)-pyrrolo[3,4-f]-1,2-benzisoxazole (9a)
Compound 9a was obtained as a colorless solid: yield
95%; mp 300 °C (Dec.). IR ν (KBr, cm–1): 3046 and 3008
NMR (DMSO-d6) δ: 7.83–7.25 (m, 6H, Ar–H), 5.23 (d, J =
7.9 Hz, 1H, H-5), 4.98 (s, 1H, H-4), 4.76 (s, 1H, H-1), 4.51
(d, J = 7.9 Hz, 1H, H-6), 3.59 (d, J = 7.9 Hz, 1H, H-3), 3.77
(s, 9H, 3 × OCH3), 3.38(d, J = 7.9 Hz, 1H, H-2). MS m/z:
468 (M+). Anal. calcd. for C24H21FN2O7: C 61.54, H 4.52, N
5.98; found: C 61.60, H 4.55, N 5.95.
1
(ArH), 1788, 1721 (C=O), 1567 (C=N), 1258 (C–O–C). H
NMR (DMSO-d6) δ: 7.74–7.06 (m, 9H, Ar–H), 5.18(d, J =
7.9 Hz, 1H, H-5), 4.95 (s, 1H, H-4), 4.73 (s, 1H, H-1), 4.48
(d, J = 7.9 Hz, 1H, H-6), 3.59 (d, J = 7.9 Hz, 1H, H-3), 3.36
(d, J = 7.9 Hz, 1H, H-2). MS m/z: 394 (M+). Anal. calcd. for
C21H15ClN2O4: C 63.89, H 3.83, N 7.10; found: C 63.90, H
3.84, N 7.06.
exo,exo-6-(4-Chlorophenyl)-4,8-epoxy-3a,4,4a,7a,8,8a-
hexahydro-3-(3,4,5-trimethoxyphenyl)-pyrrolo[3,4-f]-1,2-
benzisoxazole (8c)
Compound 8c was obtained as a colorless solid: yield
83%; mp 279 °C. IR ν (KBr, cm–1): 3072 and 3029 (ArH),
1
exo,exo-6-(4-Fluorophenyl)-4,8-epoxy-3a,4,4a,7a,8,8a-
hexahydro-3-(4-chlorophenyl)-pyrrolo[3,4-f]-1,2-
benzisoxazole (9b)
1777, 1716 (C=O), 1571 (C=N), 1248 (C–O–C). H NMR
(DMSO-d6) δ: 7.83–7.18 (m, 6H, Ar–H), 5.24 (d, J =
7.9 Hz, 1H, H-5), 4.98 (s, 1H, H-4), 4.76 (s, 1H, H-1), 4.51
(d, J = 7.9 Hz, 1H, H-6), 3.60 (d, J = 7.9 Hz, 1H, H-3), 3.77
(s, 9H, 3 × OCH3), 3.38 (d, J = 7.9 Hz, 1H, H-2). MS m/z:
484 (M+), 486 (M+ + 2). Anal. calcd. for C24H21 ClN2O7: C
59.45, H4.37, N 5.78; found: C 59.51, H 4.38, N 5.75.
Compound 9b was obtained as a colorless solid: yield
82%; mp 310 °C (Dec.). IR ν (KBr, cm–1): 3055 and 3017
1
(ArH), 1780, 1721 (C=O), 1543 (C=N), 1252 (C–O–C). H
NMR (DMSO-d6) δ: 7.75–7.06 (m, 8H, Ar–H), 5.18 (d, J =
7.9 Hz, 1H, H-5), 4.95 (s, 1H, H-4), 4.74 (s, 1H, H-1),
4.47(d, J = 7.9 Hz, 1H, H-6), 3.59 (d, J = 7.9 Hz, 1H, H-3),
3.37(d, J = 7.9 Hz, 1H, H-2). MS m/z: 412 (M+). Anal.
calcd. for C21H14ClFN2O4: C 61.10, H 3.42, N 6.79; found:
C 61.12, H 3.45, N 6.75.
exo,exo-6-(4-Bromophenyl)-4,8-epoxy-3a,4,4a,7a,8,8a-
hexahydro-3-(3,4,5-trimethoxyphenyl)-pyrrolo[3,4-f]-1,2-
benzisoxazole (8d)
Compound 8d was obtained as a colorless solid: yield
© 2007 NRC Canada

942
Can. J. Chem. Vol. 85, 2007
422 (M+). Anal. calcd. for C22H18N2O5S: C 62.55, H 4.29, N
6.63; found: C 62.56, H 4.30, N 6.61.
exo,exo-6-(4-Chlorophenyl)-4,8-epoxy-3a,4,4a,7a,8,8a-
hexahydro-3-(4-chlorophenyl)-pyrrolo[3,4-f]-1,2-
benzisoxazole (9c)
exo,exo-6-(4-Fluorophenyl)-4,8-epoxy-3a,4,4a,7a,8,8a-
hexahydro-3-(4-methylsulfinylphenyl)-pyrrolo[3,4-f]-1,2-
benzisoxazole (10b)
Compound 9c was obtained as a colorless solid: yield
84%; mp 309 °C (Dec.). IR ν (KBr, cm–1): 3053 and 3027
1
(ArH), 1778, 1713 (C=O), 1550 (C=N), 1280 (C–O–C). H
Compound 10b was obtained as a colorless solid: yield
NMR (DMSO-d6) δ: 7.75–7.06 (m, 8H, Ar–H), 5.18 (d, J =
7.9 Hz, 1H, H-5), 4.95 (s, 1H, H-4), 4.74 (s, 1H, H-1), 4.47
(d, J = 7.9 Hz, 1H, H-6), 3.59 (d, J = 7.9 Hz, 1H, H-3),
3.37(d, J = 7.9 Hz, 1H, H-2). MS m/z: 428 (M+). Anal.
calcd. for C21H14Cl2N2O4: C 58.76, H 3.29, N 6.53; found:
C 58.78, H 3.30, N 6.49.
81%; mp 177 °C. IR ν (KBr, cm–1): 3121 and 3011 (ArH),
1
1778, 1718 (C=O), 1570 (C=N), 1224 (C–O–C). H NMR
(DMSO-d6) δ: 7.83–7.25 (m, 8H, Ar–H), 5.23 (d, J = 7.9 Hz,
1H, H-5), 4.97(s, 1H, H-4), 4.76 (s, 1H, H-1), 4.51 (d, J =
7.9 Hz, 1H, H-6), 3.59 (d, J = 7.9 Hz, 1H, H-3), 3.38 (d, J =
7.9 Hz, 1H, H-2), 3.27 (s, 3H, –SOCH3). MS m/z: 440 (M+).
Anal. calcd. for C22H17 FN2O5S: C 59.99, H 3.89, N 6.36;
found: C 59.98, H 3.91, N 6.35.
exo,exo-6-(4-Bromophenyl)-4,8-epoxy-3a,4,4a,7a,8,8a-
hexahydro-3-(4-chlorophenyl)-pyrrolo[3,4-f]-1,2-
benzisoxazole (9d)
exo,exo-6-(4-Chlorophenyl)-4,8-epoxy-3a,4,4a,7a,8,8a-
hexahydro-3-(4-methylsulfinylphenyl)-pyrrolo[3,4-f]-1,2-
benzisoxazole (10c)
Compound 9d was obtained as a colorless solid: yield
70%; mp 325 °C (Dec.). IR ν (KBr, cm–1): 3053 and 3029
1
(ArH), 1778, 1714 (C=O), 1528 (C=N), 1233 (C–O–C). H
Compound 10c was obtained as a colorless solid: yield
NMR (DMSO-d6) δ: 7.76–7.02 (m, 8H, Ar–H), 5.17 (d, J =
7.9 Hz, 1H, H-5), 4.95 (s, 1H, H-4), 4.74 (s, 1H, H-1), 4.47
(d, J = 7.9 Hz, 1H, H-6), 3.59 (d, J = 7.9 Hz, 1H, H-3), 3.37
(d, J = 7.9 Hz, 1H, H-2). MS m/z: 471(M+), 473 (M+ + 2).
Anal. calcd. for C21H14ClBrN2O4: C 53.25, H 2.98, N 5.91;
found: C 53.27, H 2.32, N 5.89.
83%; mp 152 °C. IR ν (KBr, cm–1): 3072 and 3030 (ArH),
1
1774, 1716 (C=O), 1571 (C=N), 1239 (C–O–C). H NMR
(DMSO-d6) δ: 7.83–7.23 (m, 8H, Ar–H), 5.24 (d, J =
7.9 Hz, 1H, H-5), 4.98 (s, 1H, H-4), 4.77 (s, 1H, H-1), 4.51
(d, J = 7.9 Hz, 1H, H-6), 3.60 (d, J = 7.9 Hz, 1H, H-3), 3.38
(d, J = 7.9 Hz, 1H, H-2), 3.27 (s, 3H, –SOCH3). MS (70 eV)
m/z: 456 (M+), 458 (M+ + 2), 460 (M+ + 4). Anal. calcd. for
C22H17ClN2O5S: C 57.83, H 3.75, N 6.13; found: C 57.84, H
3.77, N 6.12.
exo,exo-4,8-Epoxy-3a,4,4a,7a,8,8a-hexahydro-3-(4-
chlorophenyl)-6-(p-tolyl)-pyrrolo[3,4-f]-1,2-benzisoxazole
(9e)
Compound 9e was obtained as a colorless solid: yield
exo,exo-6-(4-Bromophenyl)-4,8-epoxy-3a,4,4a,7a,8,8a-
hexahydro-3-(4-methylsulfinylphenyl)-pyrrolo[3,4-f]-1,2-
benzisoxazole (10d)
68%; mp 342 °C (Dec.). IR ν (KBr, cm–1): 3058 and 3034
1
(ArH), 1787, 1723 (C=O), 1514 (C=N), 1289 (C–O–C). H
NMR (DMSO-d6) δ: 7.77–7.02 (m, 8H, Ar–H), 5.17 (d, J =
7.9 Hz, 1H, H-5), 4.93 (s, 1H, H-4), 4.72 (s, 1H, H-1), 4.46
(d, J = 7.9 Hz, 1H, H-6), 3.59 (d, J = 7.9 Hz, 1H, H-3), 3.37
(d, J = 7.9 Hz, 1H, H-2), 2.33 (–CH3). MS m/z: 408 (M+).
Anal. calcd. for C22H17ClN2O4: C 64.63, H 4.19, N 6.85;
found: C 64.65, H 4.23, N 6.85.
Compound 10d was obtained as a colorless solid: yield
91%; mp 244 °C. IR ν (KBr, cm–1): 3063, 3041 (ArH),
1
1779, 1721 (C=O), 1567 (C=N), 1240 (C–O–C). HNMR
(DMSO-d6) δ: 7.83–7.25 (m, 8H, Ar–H), 5.24 (d, J =
7.9 Hz, 1H, H-5), 4.98 (s, 1H, H-4), 4.76 (s, 1H, H-1), 4.51
(d, J = 7.9 Hz, 1H, H-6), 3.59 (d, J = 7.9 Hz, 1H, H-3), 3.38
(d, J = 7.9 Hz, 1H, H-2), 3.27 (s, 3H, –SOCH3). MS m/z:
500 (M+), 502 (M+ + 2), 503 (M+ + 3), 504 (M+ + 4). Anal.
calcd. for C22H17BrN2O5S: C 52.70, H 3.42, N 5.59; found:
C 52.71, H 3.44, N 5.56.
exo,exo-4,8-Epoxy-3a,4,4a,7a,8,8a-hexahydro-3-(4-
chlorophenyl)-6-(4-methoxyphenyl)-pyrrolo[3,4-f]-1,2-
benzisoxazole (9f)
Compound 9f was obtained as a colorless solid: yield
58%; mp 315 °C(Dec.). IR ν (KBr, cm–1): 3057 and 3021
exo,exo-4,8-Epoxy-3a,4,4a,7a,8,8a-hexahydro-3-(4-
methylsulfinylphenyl)-6-(p-tolyl)-pyrrolo[3,4-f]-1,2-
benzisoxazole (10e)
1
(ArH), 1786, 1718 (C=O), 1516 (C=N), 1258 (C–O–C). H
NMR (DMSO-d6) δ: 7.86–7.22 (m, 8H, Ar–H), 5.19 (d, J =
7.9 Hz, 1H, H-5), 4.72 (s, 1H, H-1), 4.46 (d, J = 7.9 Hz, 1H,
H-6), 3.56 (d, J = 7.9 Hz, 1H, H-3), 3.33 (d, J = 7.9 Hz, 1H,
H-2), 3.83 (s, 3H, –OCH3). MS m/z: 424 (M+). Anal. calcd.
for C22H17ClN2O5: C 62.20, H 4.03, N 6.59; found: C 62.23,
H 4.03, N 6.58.
Compound 10e was obtained as a colorless solid: yield
71%; mp 341 °C (Dec.). IR ν (KBr, cm–1): 3069, 3035
1
(ArH), 1781, 1721 (C=O), 1568 (C=N), 1256 (C–O–C). H
NMR (DMSO-d6) δ: 7.83–7.19 (m, 6H, Ar–H), 5.23 (d, J =
7.9 Hz, 1H, H-5), 4.97 (s, 1H, H-4), 4.75 (s, 1H, H-1), 4.51
(d, J = 7.9 Hz, 1H, H-6), 3.59 (d, J = 7.9 Hz, 1H, H-3), 3.35
(d, J = 7.9 Hz, 1H, H-2), 3.27 (s, 3H, –SOCH3), 2.33
(–CH3). MS m/z: 436 (M+), 437 (M+ + 1). Anal. calcd. for
C23H20N2O5S: C 63.29, H 4.62, N 6.42; found: C 63.28,
H 4.64, N 6.41.
exo,exo-4,8-Epoxy-3a,4,4a,7a,8,8a-hexahydro-6-phenyl-3-
(4-methylsulfinylphenyl)-pyrrolo[3,4-f]-1,2-benzisoxazole (10a)
Compound 10a was obtained as a colorless solid: yield
95%; mp 310 °C (Dec.). IR ν (KBr, cm–1): 3062, 3028
1
(ArH), 1778, 1722 (C=O), 1597 (C=N), 1239 (C–O–C). H
NMR (DMSO-d6) δ: 7.83–7.20 (m, 9H, Ar–H), 5.24 (d, J =
7.9 Hz, 1H, H-5), 4.98 (s, 1H, H-4), 4.75 (s, 1H, H-1), 4.52
(d, J = 7.2 Hz, 1H, H-6), 3.59 (d, J = 7.9 Hz, 1H, H-3), 3.38
(d, J = 7.9 Hz, 1H, H-2), 3.27 (s, 3H, –SOCH3). MS m/z:
exo,exo-4, 8-Epoxy-3a,4,4a,7a,8,8a-hexahydro-6-(4-
methoxyphenyl)-3-(4-methylsulfinylphenyl)-pyrrolo[3,4-f]-
1,2-benzisoxazole (10f)
Compound 10f was obtained as a colorless solid: yield
© 2007 NRC Canada

Deng et al.
943
67%; mp 304 °C (Dec.). IR ν (KBr, cm–1): 3051, 3027
exo,exo-4,8-Epoxy-3a,4,4a,7a,8,8a-hexahydro-3-(1,3-
diolphenyl)-6-(p-tolyl)-pyrrolo[3,4-f]-1,2-benzisoxazole
(11e)
1
(ArH), 1778, 1717 (C=O), 1568 (C=N), 1246 (C–O–C). H
NMR (DMSO-d6) δ: 8.37–7.49 (m, 6H, Ar–H), 5.25 (d, J =
7.9 Hz, 1H, H-5), 5.01 (s, 1H, H-4), 4.79 (s, 1H, H-1), 4.53
(d, J = 7.9 Hz, 1H, H-6), 3.66 (d, J = 7.9 Hz, 1H, H-3), 3.43
(d, J = 7.9 Hz, 1H, H-2), 3.83 (s, 3H, –OCH3), 3.27 (s, 3H,
–SOCH3). MS m/z: 452 (M+), 453 (M+ + 1). Anal. calcd. for
C23H20N2O6S: C 61.05, H 4.46, N 6.19; found: C 61.03, H
4.46, N 6.18.
Compound 11e was obtained as a colorless solid: yield
68%; mp 176 °C. IR ν (KBr, cm–1): 3058, 3034 (ArH),
1
1780, 1723 (C=O), 1544 (C=N), 1282 (C–O–C). H NMR
(DMSO-d6) δ: 7.77–7.02 (m, 7H, Ar–H), 5.25 (s, 2H, OH),
5.17 (d, J = 7.9 Hz, 1H, H-5), 4.94 (s, 1H, H-4), 4.72 (s, 1H,
H-1), 4.46 (d, J = 7.9 Hz, 1H, H-6), 3.59 (d, J = 7.9 Hz, 1H,
H-3), 3.37 (d, J = 7.9 Hz, 1H, H-2), 2.33 (–CH3). MS m/z:
406 (M+), 408 (M+ + 2). Anal. calcd. for C22H18N2O6: C
65.02, H 4.46, N 6.89; found: C 65.01, H 4.47, N 6.87.
exo,exo-4,8-Epoxy-3a,4,4a,7a,8,8a-hexahydro-6-phenyl-3-
(1,3-diolphenyl)-pyrrolo[3,4-f]-1,2-benzisoxazole (11a)
Compound 11a was obtained as a colorless solid: yield
95%; mp 183 °C. IR ν (KBr, cm–1): 3047, 3018 (ArH),
exo,exo-4,8-Epoxy-3a,4,4a,7a,8,8a-hexahydro-3-(1,3-
diolphenyl)-6-(4-methoxyphenyl)-pyrrolo[3,4-f]-1,2-
benzisoxazole (11f)
1
1782, 1723 (C=O), 1564 (C=N), 1248 (C–O–C). H NMR
(DMSO-d6) δ: 7.74–7.07 (m, 8H, Ar–H), 5.25 (s, 2H, OH),
5.18 (d, J = 7.9 Hz, 1H, H-5), 4.96 (s, 1H, H-4), 4.73 (s, 1H,
H-1), 4.48 (d, J = 7.9 Hz, 1H, H-6), 3.59 (d, J = 7.9 Hz, 1H,
H-3), 3.35 (d, J = 7.9 Hz, 1H, H-2). MS m/z: 392 (M+).
Anal. calcd. for C21H16N2O6: C 64.28, H 4.11, N 7.14;
found: C 64.27, H 4.13, N 7.13.
Compound 11f was obtained as a colorless solid: yield
58%; mp 185 °C. IR ν (KBr, cm–1): 3057, 3021 (ArH),
1
1786, 1719(C=O), 1536 (C=N), 1238 (C–O–C). H NMR
(DMSO-d6) δ: 7.86–7.22 (m, 7H, Ar–H), 5.25 (s, 2H, OH),
5.25 (d, J = 7.9 Hz, 1H, H-5), 4.97 (s, 1H, H-4), 4.73 (s, 1H,
H-1), 4.46 (d, J = 7.9 Hz, 1H, H-6), 3.83 (s, 3H, –OCH3),
3.56 (d, J = 7.9 Hz, 1H, H-3), 3.33 (d, J = 7.9 Hz, 1H, H-2).
MS m/z: 422 (M+), 424 (M+ + 2). Anal. calcd. for
C22H18N2O7: C 62.56, H 4.30, N 6.63; found: C 62.55, H
4.32, N 6.62.
exo,exo-6-(4-fluorophenyl)-4,8-epoxy-3a,4,4a,7a,8,8a-
hexahydro-3-(1,3-diolphenyl)-pyrrolo[3,4-f]-1,2-
benzisoxazole (11b)
Compound 11b was obtained as a colorless solid: yield
82%; mp 176 °C. IR ν (KBr, cm–1): 3054, 3016 (ArH),
Cell culture and antiproliferative assays
1
1780, 1720 (C=O), 1544 (C=N), 1252 (C–O–C). H NMR
Stock solutions of drugs were prepared as follows (and
stored at –20 °C): Cantharidin (Biomol, USA) as a
12 mmol/L solution in dimethylsulphoxide (DMSO);
norcantharidin and cantharidin analogues as 10 mmol/L so-
lutions in phosphate buffered saline. All cell lines were cul-
tured at 37 °C in 5% CO2.
(DMSO-d6) δ: 7.75–7.06 (m, 7H, Ar–H), 5.25 (s, 2H, OH),
5.18 (d, J = 7.9 Hz, 1H, H-5), 4.9 (s, 1H, H-4), 4.74 (s, 1H,
H-1), 4.47 (d, J = 7.9 Hz, 1H, H-6), 3.59 (d, J = 7.9 Hz, 1H,
H-3), 3.37 (d, J = 7.9 Hz, 1H, H-2). MS m/z: 410 (M+).
Anal. calcd. for C21H15FN2O6: C 61.47, H 3.68, N 6.83;
found: C 61.46, H 3.69, N 6.81.
Antiproliferative activity of different compounds was de-
termined by MTT method. Cells in logarithmic growth were
plated in triplicate in 100 µL medium at a density of 3 000–
5 000 cells/well in 96-well plates. When the cells were in
logarithmic growth, 100 µL medium with or without the test
compounds were added to each well. After 72 h, MTT solu-
tion (5.0 mg/mL) was added (10.0 µL/well), and plates were
incubated for a further 4 h at 37 °C. The purple formazan
crystals were dissolved in 100.0 µL DMSO. After 5 min, the
plates were read on an automated microplate spectro-
photometer at 570 nm. Concentration of drug that inhibited
50% of cells (IC50) was calculated using the software of
Dose–Effect analysis with Microcomputers.
exo,exo-6-(4-Chlorophenyl)-4,8-epoxy-3a,4,4a,7a,8,8a-
hexahydro-3-(1,3-diolphenyl)-pyrrolo[3,4-f]-1,2-
benzisoxazole (11c)
Compound 11c was obtained as a colorless solid: yield
84%; mp 191 °C. IR ν (KBr, cm–1): 3053, 3027 (ArH),
1
1775, 1715 (C=O), 1550 (C=N), 1266 (C–O–C). H NMR
(DMSO-d6) δ: 7.75–7.06 (m, 7H, Ar–H), 5.25 (s, 2H, OH),
5.18 (d, J = 7.2 Hz, 1H, H-5), 4.95 (s, 1H, H-4), 4.73 (s,1H,
H-1), 4.47 (d, J = 7.9 Hz, 1H, H-6), 3.59 (d, J = 7.9 Hz, 1H,
H-3), 3.37 (d, J = 7.9 Hz, 1H, H-2). MS m/z: 426 (M+), 428
(M+ + 2). Anal. calcd. for C21H15ClN2O6: C 59.10, H 3.54,
N 6.56; found: C 59.11, H 3.55, N 6.53.
References
exo,exo-6-(4-Bromophenyl)-4,8-epoxy-3a,4,4a,7a,8,8a-
hexahydro-3-(1,3-diolphenyl)-pyrrolo[3,4-f]-1,2-
benzisoxazole (11d)
1. J.E. Carrel and T. Eisner. Science (Washington, D.C.), 183,
755 (1974).
2. G.S. Wang. J. Ethnopharmacol. 26, 147 (1989).
3. F. Peng, Y.Q. Wei, L. Tien, L. Yang, X. Zhao, and Y. Lu. J.
Cancer Res. Clin. Oncol. 128, 223 (2002).
Compound 11d was obtained as a colorless solid: yield
70%; mp 199 °C. IR ν (KBr, cm–1): 3053, 3028 (ArH),
1
1778, 1714 (C=O), 1538 (C=N), 1244 (C–O–C). H NMR
(DMSO-d6) δ: 7.76–7.02 (m, 7H, Ar–H), 5.25 (s, 2H, OH),
5.17 (d, J = 7.2 Hz, 1H, H-5), 4.96 (s, 1H, H-4), 4.74 (s, 1H,
H-1), 4.47 (d, J = 7.9 Hz, 1H, H-6), 3.59 (d, J = 7.9 Hz, 1H,
H-3), 3.37 (d, J = 7.9 Hz, 1H, H-2). MS m/z: 470 (M+), 472
(M+ + 2), 474 (M+ + 4). Anal. calcd. for C21H15BrN2O6: C
53.52, H 3.21, N 5.94; found: C 53.52, H 3.23, N 5.92.
4. Y.N. Chen, J.C. Chen, S.C. Yin, G.S. Wang, W. Tsauer, S.F.
Hsu,and S.L.Hsu. Int. J. Cancer. 100, 158 (2002).
5. Z.X. Sun, Q.W. Ma, T.D. Zhao, Y.L. Wei, G.S. Wang,and J.S.
Li. World J. Gastroenterol. 6, 263 (2000).
6. A. McCluskey, S.P. Ackland, M.C. Bowyer, M.L. Baldwin, J.
Garner,and C.C. Walkom. Bioorg. Chem. 31, 68 (2003).
© 2007 NRC Canada

944
7. A. McCluskey, A.T.R. Sim, and J.A. Sakoff. J. Med. Chem.
Can. J. Chem. Vol. 85, 2007
12. F.L. Liu, T. jiang, D.S. Zuo, X. Qin, and X.L. Zhan. Chin. J.
Org. chem. 22, 761 (2002). (In Chinese).
45, 1151(2002).
8. A. McCluskey, C. Taylor, R.J. Quinn, M. Suganuma, and
H.Fujiki. Bioorg. Med. Chem. Lett. 6, 1025 (1996).
9. A. McCluskey, M.A. Keane, L.M. Mudgee, A.T.R. Sim, J.
Sakoff, and R.J. Quinn. Eur. J. Med. Chem. 35, 957 (2000).
10. A. McCluskey, M.C. Bowyer, C. Walkom, S.P. Ackland,
E.Gardiner, and J.A. Sakoff. Bioorg. Med. Chem. Lett. 11,
2941 (2001).
13. J.M. Corral, M. Gordaliza, M.A. Castro, H.B. Broughton, and
A.S. Feliciano. Tetrahedron, 53, 6555 (1997).
14. V.J. Ram, U.K. Singha, and P.Y. Guru. Eur. J. Med. Chem. 25,
533 (1990).
15. R.B. Woodward and B.J. Harold. J. Am. Chem. Soc. 70, 1161
(1948).
16. L.P. Deng, F.M. Liu, and H.Y. Wang. J. Heterocycl. Chem. 42,
13 (2005).
17. L.P. Deng and Y.Z. Hu. J. Heterocycl. Chem. 43, 1715 (2006).
11. P.Y. Lin, S.J. Shi, H.L. Shu, H.F. Chen, C.C. Lin, P.C.
Liu,and L.F. Wang. Bioorg. Chem. 28, 266 (2000).
© 2007 NRC Canada