Synthesis of canthardin sulfanilamides and their acid anhydride analogues via a ring-opening reaction of activated aziridines and their associated pharmacological effects

Article abstract of DOI:10.3390/molecules21010100

The cantharidinimide derivatives, 5a-h, including sulfanilamides containing pyrimidyl, pyrazinyl, hydrogen, thiazolyl, and oxazolyl groups were synthesized. Modification of cantharidinimide by means of the reaction of activated aziridine ring opening led to the discovery of a novel class of antitumor compounds. The analogues 10i-k, 11l-n, 12o-p, and 16q-s were obtained from treating cantharidinimide 6 and analogues (7, 8, and 13) with activated aziridines, which produced a series of ring-opened products including normal and abnormal types. Some of these compounds showed cytotoxic effects in vitro against HL-60, Hep3B, MCF7, and MDA-MB-231 cancer cells. The most potent cytostatic compound, N-cantharidinimido-sulfamethazine (5a), exhibited anti-HL-60 and anti-Hep3B cell activities. Two compounds 5g and 5h displayed slight effects on the Hep3B cell line, while the other compounds produced no response in these four cell lines.

Full text of DOI:10.3390/molecules21010100

molecules
Article
Synthesis of Canthardin Sulfanilamides and Their
Acid Anhydride Analogues via a Ring-Opening
Reaction of Activated Aziridines and Their
Associated Pharmacological Effects
Ling-Ling Chiang 1,2, Ing-Jy Tseng 3, Pen-Yuan Lin 4, Shiow-Yunn Sheu 4, Ching-Tung Lin 5,
Yun-Han Hsieh 4, Yi-Jing Lin 4, Hsiao-Ling Chen 4 and Mei-Hsiang Lin 4,
*
Received: 3 December 2015; Accepted: 12 January 2016; Published: 16 January 2016
Academic Editor: Wei-Zhu Zhong
1
School of Respiratory Therapy, Taipei Medical University, Taipei 11031, Taiwan; llchiang@tmu.edu.tw
Chest Medicine Department, Shuang-Ho Hospital, Taipei Medical University, Taipei 11031, Taiwan
Gerontology Health Management, College of Nursing, Taipei Medical University, Taipei 11031, Taiwan;
2
3
Ingjy@tmu.edu.tw
4
Department of Pharmaceutical Sciences, School of Pharmacy, Taipei Medical University, Taipei 11031,
Taiwan; lpy0620@tmu.edu.tw (P.-Y.L.); amel@tmu.edu.tw (S.-Y.S.); jay4273803@hotmail.com (Y.-H.H.);
m301101003@tmu.edu.tw (Y.-J.L.); m301101001@tmu.edu.tw (H.-L.C.)
Department of Chemistry, Tam-Kang University, Danshui 25137, New Taipei City, Taiwan;
5
rocketqueenohya@hotmail.com
*
Correspondence: mhl00001@tmu.edu.tw; Tel.: +886-2-2736-1661 (ext. 6138)
Abstract: The cantharidinimide derivatives, 5a
pyrazinyl, hydrogen, thiazolyl, and oxazolyl groups were synthesized.
cantharidinimide by means of the reaction of activated aziridine ring opening led to the discovery of
a novel class of antitumor compounds. The analogues 10i 11l 12o , and 16q were obtained
from treating cantharidinimide and analogues ( , and 13) with activated aziridines, which
–
h, including sulfanilamides containing pyrimidyl,
Modification of
–
k,
–n
,
–p
–s
6
7, 8
produced a series of ring-opened products including normal and abnormal types. Some of these
compounds showed cytotoxic effects in vitro against HL-60, Hep3B, MCF7, and MDA-MB-231 cancer
cells. The most potent cytostatic compound, N-cantharidinimido-sulfamethazine (5a), exhibited
anti-HL-60 and anti-Hep3B cell activities. Two compounds 5g and 5h displayed slight effects on the
Hep3B cell line, while the other compounds produced no response in these four cell lines.
Keywords: cantharidin; cantharidinimide; methylsulfanylaziridine
1. Introduction
Cantharidin
and has a high vesicant potency [
topical application and is also used as an abortifacient and aphrodisiac [
significant activity against liver tumors and the KB cell line in tissue culture at low concentrations [
The clinical application of cantharidin is limited by its cytotoxic properties [ ]. It is an inhibitor of
the serine/threonine protein phosphatases, PP1 and PP2A, leading to multiple cellular effects such as
DNA damage, cell cycle arrest, and apoptosis [ 10]. Cantharidin induces apoptosis in many human
cancer cells such as pancreatic carcinoma [ ], colon cancer [10], lung cancer [11], melanomas [12], and
bladder cancer [13]. It also induces endoplasmic reticular (ER) stress and autophagic cell death [13 15].
Recently, certain reports indicated that N-cantharidin derivatives have antitumor and antihepatoma
properties [16 19]. Some cantharidinimides showed tumor-inhibitory action in animals, and several
1
(Figure 1), isolated from Mylabris caraganae and various other insects, is a terpenoid
]. It is used to treat piles, warts, ulcers, and molluscum through
]. In clinical studies, it had
].
1
1,2
3,4
5,6
7–
9
–
–
Molecules 2016, 21, 100; doi:10.3390/molecules21010100
www.mdpi.com/journal/molecules

Molecules 2016, 21, 100
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cantharidinimides exhibited inhibitory effects against xanthine oxidase [20]. A number of modified
cantharidin analogues were synthesized and their anticancer activities against a wide range of human
tumor cell lines were evaluated [21–27].
Figure 1. Structures of cantharidin (1), cantharidinimines (2, 3), and cantharidin-sulfanilamides.
In our previous research, we synthesized many cantharidinimides by modification with alkyl,
phenyl, pyridinyl, thiazolyl, and thiadiazolyl groups [28–30]. Those compounds showed certain
degrees of cytotoxic activities against HL-60 (human myeloid leukemia cells), Hep 3B and Hep
G2 (human hepatocarcinoma cells), NUGC (human gastric carcinoma cells), and HONE-1 (human
nasopharyngeal carcinoma cells). Cantharidinimides with better solubility showed greater cytotoxicity
against human hepatoma cell lines. The cantharidinimides with an electron-withdrawing group (R1)
on the aromatic ring (X) exhibited increased cytotoxicity to cancer cell lines. Then, we used this
feature and synthesized different imines to study their cytotoxic effects [31]. These results showed
that cantharidinimine derivatives had greater cytotoxic effects than cantharidinimides. Especially,
cantharidinimine derivative
2 (Figure 1), which has a sulfanyl group, exhibited a 50% inhibitory
concentration (IC50) value of 6 µM against HL-60 cells as did cantharidin (IC50 value of 7.2 µM).
However, cantharidinimides and their analogues also display inhibition of the inducible nitric
oxide (NO) synthase (iNOS) pathway, and they inhibited NO synthase activity by more than 90%. Thus,
they might be advantageous for anticancer therapy [32,33]. Because of this, we synthesized canthardin
sulfanilamides (Figure 1) and tested their cytotoxic effects on human cancer cell lines (HL-60 and
Hep3B), human breast carcinoma cells (MCF7), and human breast carcinoma cells (MDA-MB-231)
since incidence rates occur rapidly and more widely among Asian peoples than in Westerners. Growth
inhibition was evaluated using an MTT assay. In order to find new cantharidinimides and related
imides containing the sulfonamide group, we synthesized sulfonamido-ethyldicarboximide derivatives
(10i–k, 11l–n, 12o–p, and 16q–s) and tested their cytotoxic activities.
2. Results and Discussion
2.1. Chemistry
We prepared effective cantharidin sulfanilamides by heating th˝e reactant cantharidin (
1) and
primary arylamines of sulfanilamide derivatives (4a–h) to ca. 200 C with 3 mL of dry toluene
and 1–2 mL of TEA in a high-pressure sealed tube (Buchi glasuster 0032) to provide compounds
5a in good yields (Scheme 1). The sulfonamide derivatives containing pyrimidine, pyrazine,
hydrogen, thiazole, and oxazole were sulfadiazine, sulfamethazine, sulfisomidine, sulfamerazine,
sulfaquinoxaline, sulfacarbamide, 2-thiazolyl-sulfanilamide, and sulfamethoxazole (4a ). Yields
–
h
–
h
varied 2%–62% and showed a trend compatible with the expected basicity, and the characters of
the bulky quinoxaline and the more-basic carbamide groups influenced compounds 5d (6% yield)
and 5e (2% yield). The results obtained with 5a–c and 5f–h, however, showed the same strong
electron-withdrawing sulfonyl (SO2) group and aromatic amino (NH2) basicity, which were unknown
but really influenced the product yields, and the characters of a long side chain with resonance and
induction effects were considered spontaneously.

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Scheme 1. Synthesis of cantharidin-sulfanilamides 5a–h.
In order to find some new cantharidinimides, cantharidin was treated with ammonia and yielded
cantharidinimide ( ) which was reacted with dimethyl or monomethyl-activated aziridins. Thus,
and its imide derivatives ( and ) were reacted with N,N’-dimethylformamide (DMF) in a sealed tube
under reflux for 3 h, after which it was cooled, and then activated aziridine ( ) was added dropwise
and refluxed for another 2 h to yield compounds 10i 11l , and 12o (Scheme 2). In this study,
6
6
7
8
9
–
k,
–
n
–p
the ring opening reaction of N-sulfonyl 2,2-dimethylaziridine by imide nucleophiles (Figure 2a,d,e),
which attacked the unsubstituted carbon, was detected. However, the ring-opening reaction of N-acyl
2,2-dimethylaziridine by various nucleophiles in the absence of an acid was reported to proceed with
a formal attack on the tertiary carbon of the aziridine ring (abnormal opening) [34]. No products of
a “normal opening” (in the SN2 sense) were observed when the R groups were phenyl or biphenyl
(Figure 2). The small amount of normal opening yield (when R was phenyl) was possibly the result
of a displacement in the inversional ground states. It was through the regioselectivity ring-opening
reaction, and the strong preference for normal-opening sulfonyl activation (R group was tosyl) that
simply reflected the steric hindrance of a nucleophilic attack on the tertiary aziridine carbon in the
inversional ground state. A single electron transfer (SET) mechanism was proposed to account for
this unexpected behavior [35
reactions of activated aziridines were slowed down compared to a reaction in the absence of the two
methyl groups, the former clearly more so than the latter, and the white crystalline compounds 10i
,36]. Therefore, we proposed that both normal and abnormal opening
–k
were obtained (Scheme 2). The same method was used with cantharidin analogue compounds to
obtain products 11l–n and 12o–p.
Scheme 2. Synthesis of compounds 10i–k, 11l–n, and 12o–p.

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Figure 2. Regioselectivity in nucleophilic ring opening of N-sulfony and N-acyl activated 2-methylaziridine.
1
Structures of products 10j and 10i followed from the H-NMR spectra of chromatographic
fractions, and the main evidence for included signals of cantharidinimide’s CH3 (position 8 and 9),
A
NCH2 (position 10), and OCH (position 4 and 7) protons at 1.22 (singlet), 3.51 (singlet), and 4.54 ppm
(singlet), respectively (Table 1, Figure 3). These signals indicated the same values of the protons
on two kinds of methyl, OCH, and NCH2 groups of product 10i which might have greater steric
hindrance (compared to 10j, Figure 3B) repulsion between the two pairs of methyl groups, and the
conformation for 10i might have the least electrostatic repulsion between the proton of nitrogen and
the two oxygen atoms of cantharidinimide with their negative partial charge. A downfield shift
of product 10j relative to 10i was observed for these protons. These shifts and coupling constants
(Table 1) became complicated for several possible reasons: the hydrogen bonding bridge enhanced the
electron-withdrawing capacity of the sulfonyl group and shifted the signals downfield from the usual
range, and the three methyl groups might have less steric hindrance to each other. Thus, the structure
became the conformationally fixed type B (Figure 3).
1
Table 1. H chemical shifts (™) of sulfonamide-cantharidinimides 10i and 10j (CDCl3, 500 MHz).
10i
10j
Position
δH (J in Hz)
δH (J in Hz)
4, 7
5, 6
8, 9
10
11
12
15, 19
16, 18
20
21
22
4.54 (s)
1.68–1.70 (m); 1.79–1.81 (m)
4.55 (s)
1.67-1.70 (m); 1.79–1.83 (m)
1.21 (s)
3.38 (dd, 3.8, 3.8); 3.61–3.64 (m)
3.52 (dd, 9.7, 9.7)
4.68 (d, 9)
1.22 (s)
3.51 (s)
-
5.77 (s)
7.72 (d, 8)
7.23 (d, 8)
2.39 (s)
1.14 (s)
1.14 (s)
7.70 (d, 8)
7.27 (d, 8)
2.41 (s)
0.93 (d, 6.7)
-

Molecules 2016, 21, 100
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Figure 3. Conformation of 10i and 10j.
The N-pyrimidyl, N-thiazolyl, N-oxazolylsulfonylcantharidinimides, 5a
means of a pressure technique synthesis, and their analogous derivatives (10i
–
–
h
, were prepared by
k, 11l–m, and 12o–p)
were synthesized via nucleophilic ring opening of methyl- or dimethylbenzenesulfonylaziridine and
methyl- or dimethyltoluenesulfonylaziridine, after reacting with several analogous nucleophilic bases.
Compounds 16q–s were obtained from N-hydroxylimide 13 and activated aziridine by treatment with
NaH base in dry THF for 24 h (Scheme 3). After evaporation, it was purified by silica gel column
chromatography and recrystallized in methanol. From these reactions, it should be noted that the
characters of various bases (Figure 2a–e) strongly confirmed the influence of the positions of the ring
opening of the activated aziridines. The preparative technique, the temperature, the drying solvent,
and the formation of nucleophilic bases were also influenced by other factors that can cause strong
variations in the results. The reaction steps and time were also crucial factors in this formation. The
results of these yields strongly confirmed the influence of the sulfanilamide side chain (5e, 2%, R = H).
Scheme 3. Synthesis of compounds 16q–s.
2.2. Pharmacology
Cytotoxicities of the cantharidin-sulfanilamide derivatives, 5a–h, were tested by suppressing
the growth of the HL-60, Hep3B, MCF7, and MDA-MB-231 human carcinoma cells using MTT cell
viability assays. Some compounds showed inhibitory effects on HL-60 and Hep3B cells. Cantharidin
1
was more toxic and exhibited greater cytotoxicity, while the synthesized cantharidinimides were
less toxic but also exhibited inhibitory effects (Table 2). In this study, compound 5a, which had
two methyl groups on the pyrimidyl ring, displayed strong inhibitory effects on the HL-60 and
Hep3B cell lines, as did cantharidin. N-Cantharidinimido-sulfamethazine 5a was less toxic compared
to N-cantharidinimino-sulfamethazine
2 against HL-60. This result confirmed our previous study.

Molecules 2016, 21, 100
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Compounds 5b and 5c also had methyl groups, but, unfortunately, no activities were detected against
either of the two cell lines as compounds 5h and 5d, thus the methyl group positions highly influenced
the results. Compound 5g with an oxazolyl group exhibited medium cytotoxicity against the Hep3B
cell line, but compound 5f, although with a thiazolyl group, displayed no inhibitory effects on either
cell line. Aziridine side chains contained imide derivatives, and analogues 10i
16q
against the HL-60, MCF7, and MDA-MB-231 cell lines.
–
k
,
11l
–
n
,
12o
–
p
, and
–s exhibited weak inhibitory effects against the Hep3B cell line but displayed very low effects
Table 2. Cytotoxicity effect of
12o , and 16q
breast adenocarcinoma (MCF7 and MDA-MB-231) cell lines.
1
, cantharidin-sulfanilamides (5a
–
h
), and imide analogues (10i
–
k
,
11l
–
n
,
–
p
–s) in human hepatocellular carcinoma (Hep3B), myeloid leukemia (HL-60), and human
IC50 (µM) a
Cpd No.
HL-60
Hep3B
MCF7
MDA-MB-231
1
5a
5b
5c
5d
5e
5f
5g
5h
10i
10j
10k
11l
11m
11n
12o
12p
16q
16r
16s
5.6
38.2
3.4
9
-
-
>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
>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
17
80
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
a
IC50 was calculated after 48 h of continuous drug exposure, values are the mean of three to
four experiments with coefficients of variation of 5%–10%.
The cytotoxicity of cantharidin was investigated in both the NCI and Oncotest cell line panels [37].
It showed profound activity at low micromolar concentrations (log10IC50 values of 6.980 to 5.009 M),
´
´
and revealed extreme cytotoxicity toward cell lines of diverse tumor types. According to a COMPARE
analysis [38] of microarray-based transcriptome-wide mRNA expressions, 21 genes were found to
be significantly correlated with the response of 60 tumor cell lines to cantharidin. Some researchers
screened their synthetic compounds against human cancer cell lines and used a COMPARE analysis
to evaluate the anticancer activity and predict the probable mechanism [27,39–42]. Therefore, the
following studies examined the possible mechanism using a standard NCI-60 test and COMPARE
analysis of 5a.
3. Experimental Section
3.1. Synthesis
3.1.1. General Experimental Procedures
General procedures were followed for the reaction of ammonia with cantharidine and the other
acid anhydride. These compounds were prepared according to similar procedures, and the reactions
took place in high-pressure tubes (Büchi Glasuster 0032, Zürich, Switzerland). Cantharidin or acid

Molecules 2016, 21, 100
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anhydrides were added to a tube contain˝ing 3 mL of dry toluene and triethylamine (TEA), and the
solution was stirred and heated to ca. 200 C. After being stirred for 2 h, the mixture was evaporated,
and the residual mass was purified by column chromatography on silica gel and recrystallized
from methanol.
All temperatures are reported in degrees centigrade. Melting points were determined with a
20
Büchi B-545 melting point apparatus (Büchi, Flawil, Switzerland) and were uncorrected. rαsD values
were recorded on a JASCO P-1020 Digital Polarimeter (JASCO, Tokyo, Japan). Infrared spectra were
recorded on a Perkin-Elmer Model 882 (Thermo Fisher Scientific Inc., New York, NY, USA) and Nicolet
1
510 pet and Thermo Mattson IR 300 (Thermo Fisher Scientific Inc.) spectrophotometers. H-nuclear
magnetic resonance (NMR) spectra (in CDCl3 unless otherwise stated) were recorded at 500 MHz on a
Bruker Advance DRX and AM-500 FT (Bruker Co., Bremen, Germany). Mass spectra were obtained
on a JOEL JMSHX 110 FAB-MS (Joel, Tokyo, Japan) spectrometer and MAT-95XL HRMS (Finnigan,
München, Germany).
3.1.2. Test Samples
Chinese blister beetles were extracted with a water–ethanol (1:1) solution, filtered through celite,
purified by chromatography on silica gel, and then recrystallized with ethanol to give cantharidin
1.
Compounds 5a 10i 11l 12o , and 16q were prepared from cantharidin, acid anhydride,
–
h,
–
k,
–
n,
–
p
–s
and primary amines in the presence of TEA in toluene in high-pressure tubes. The mass spectra of all
compounds were measured, and 1H-NMR was also used for testing.
3.1.3. Physical Data of Cantharidinimide Derivatives and Analogues
˝
N-Cantharidinimido-sulfamethazine (5a): 62% yield, m.p. 195–196 C. IR (KBr): 1709 (amide) cm´1
;
1H-NMR:
δ
1.25 (6H, s), 1.75–1.77 (2H, m), 1.86–1.87 (2H, m), 2.05 (6H, s), 4.69 (2H, t, J = 2.3, 2.6 Hz),
6.64 (1H, s, pyrimidinyl H), 7.52 (2H, d, J = 8.6 Hz, phenyl H), 8.26 (2H, d, J = 8.7 Hz, phenyl H);
FAB-MS m/z (rel. int.): 457 [M + H]+ (100), 154, 124; HRMS, calcd. for C22H25N4O5S [M + H]+ 457.1546,
found 457.1547.
˝
N-Cantharidinimido-sulfisomidine (5b): 49% yield, m.p. 163–164 C. IR (KBr): 1710 (amide) cm´1
;
1H-NMR:
δ
1.26 (6H, s), 1.75–1.77 (2H, m), 1.78–1.80 (2H, m), 2.54 (3H, s), 2.67 (3H, s), 4.69 (2H, t,
J = 2.3 Hz), 6.9 (1H, s, pyrimidinyl H), 7.55 (2H, d, J = 8.4 Hz, phenyl H), 8.06 (2H, d, J = 8.4 Hz, phenyl
H); FAB-MS m/z (rel. int.) 457 [M + H]+ (22), 279, 154; HRMS (FAB+), calcd. for C20H22N3O6S 457.1546,
found 457.1549.
˝
N-Cantharidinimido-sulfamerazine (5c): 10% yield, m.p. 254–256 C. IR (KBr): 1710 (amide) cm´1
;
1H-NMR:
δ
1.25 (6H, s), 1.74–1.76 (2H, m), 1.84–1.87 (2H, m), 2.42 (3H, s), 4.68 (2H, t, J = 2.3 Hz),
6.80 (1H, d, J = 5.2 Hz, pyrimidinyl H), 7.52 (2H, d, J = 8.5 Hz, phenyl H), 8.24 (2H, d, J = 8.6 Hz,
phenyl H), 8.42 (1H, d, J = 5.1 Hz, pyrimidinyl H); HRMS (FAB+), calcd for C21H23N4O5S 443.1311,
found 443.1313.
˝
N-Cantharidinimido-sulfaquinoxaline (5d): 6% yield, m.p. 253–255 C. IR (KBr):1704 (amide) cm´1
;
1H-NMR:
δ
1.20 (6H, s), 1.67–1.71 (2H, m), 1.80–1.84 (2H, m), 4.63 (2H, t, J = 2.8, 2.4 Hz), 7.45–7.48
(3H, m, H-6’ peak overlap, J = 8.5 Hz), 7.58 (2H, t-like, 2H), 7.89 (1H, d, J = 8.2 Hz, H-8’), 8.14 (2H, d,
J = 8.6 Hz), 8.59 (1H, s). HRMS (FAB+), calcd. for C24H22N4O5S 478.1305, found 478.1307.
˝
´1
;
N-Cantharidinimido-sulfacarbamide (5e): 2% yield, m.p. 115–116 C. IR (KBr): 1776, 1706 (amide) cm
1H-NMR (DMSO-d6): δ 1.28 (6H, s), 1.69–1.70 (2H, m), 1.95–1.96 (2H, m), 4.63 (2H, t-like), 7.54 (2H, d,
J = 8.6 Hz), 8.02 (2H, d, J = 8.4 Hz); HRMS, calcd. for C16H18N2O5S 350.0894, found 350.0895.
˝
N-Cantharidinimido-N’-(2-thiazolyl)-sulfanilamide (5f): 10% yield, m.p. 125–127 C. IR (KBr): 1706 (amide)
cm´1; 1H-NMR:
1.25 (6H, s), 1.74–1.76 (2H, m), 1.85-1.87 (2H, m), 4.68 (2H, s), 6.54 (1H, d, J = 4.5 Hz,
thiazolyl N-CH), 7.12 (1H, d, J = 4.5 Hz, thiazolyl S-CH), 7.47 (2H, d, J = 8.2 Hz, phenyl H), 8.02 (2H,
δ

Molecules 2016, 21, 100
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d, J = 8.5 Hz, phenyl H); FAB-MS m/z (rel, int): 434 [M + H]+ (50), 154 (100); HRMS, calcd. for
C19H20N3O5S2 434.0844, found 434.0847.
˝
N-Cantharidinimido-sulfamethoxazole (5g): 35% yield; m.p. 211–213 C. IR (KBr): 1710 (C=O) cm´1
;
1H-NMR (acetone-d6):
δ 1.58 (6H, s), 2.25–2.26 (4H, m), 5.02 (2H, t, J = 2.8 Hz), 2.78 (3H, s), 6.69 (1H, s),
8.01 (2H, t, J = 6.9 Hz), 8.48 (2H, d, J = 6.7, 1.9 Hz); FAB-MS m/z (rel, int): 432 [M + H]+ (100); HRMS
calcd. for C20H22N3O6S 432.1229, found 432.1226.
N-Cantharidinimido-sulfadiazine (5h): 27% yield; 262–263 ˝C; IR (KBr): 1710 (amide) cm´1; 1H-NMR
(acetone-d6):
δ 1.27 (6H, s), 1.64–1.66 (4H, m), 4.57 (2H, t, J = 2.8 Hz), 7.06 (1H, t, J = 4.7 Hz), 7.54 (2H, t,
J = 4.3 Hz), 8.23 (2H, t, J = 4.3 Hz), 8.50 (2H, d, J = 5.0 Hz); FAB-MS m/z (rel, int) 429 [M + H]+ (100);
HRMS calcd. for C20H21N4O5S 429.1233, found 429.1229.
N-(2,2-Dimethylethyl-p-toluolsulfonamide)cantharidinimide (10i): 14% yield, 154–155 ˝C. IR (KBr): 1147
1
(S(=O)2), 1401 (S(=O)2), 1691 (C=O), 3264 (NH) cm´1; H-NMR (see Table 1); HRMS, calcd. for
C21H28N2O5S 420.1719, found 420.1718.
N-(2-Methylethyl-p-toluolsulfonamide)cantharidinimi´de1(10j); 21% yield, m.p. 223–225 ˝C. IR (KBr): 1139
20
(S(=O)2), 1397 (S(=O)2), 1691 (C=O), 3244 (NH) cm
;
rαsD
´
5.6 (c 0.5, CHCl3); 1H-NMR (see Table 1);
HRMS, calcd. for C20H26N2O5S 406.1562, found 406.1560.
N-(2,2-Dimethylethyl-benzosulfonamide)cantharidinimide (10k): 22% yield, m.p. 155-156 C. IR (KBr): 1152
(S(=O)2), 1400 (S(=O)2), 1695 (C=O), 3253 (NH) cm´1; 1H-NMR:
1.13 (6H, s), 1.23 (6H, s), 1.68–1.70
˝
δ
(2H, m), 1.78–1.81 (2H, m), 3.52 (2H, s), 4.53 (2H, t, J = 2.4 Hz), 5.88 (1H, s), 7.44 (2H, t, J = 7.4 Hz), 7.49
(1H, t, J = 7.3, 7.2 Hz), 7.84 (2H, d, J = 7.5 Hz); HRMS, calcd. for C20H26N2O5S 406.1562, found 406.1564.
N-[2,2-Dimethylethylamino]-1-naphtharinimido-4-methylsulfanilamide (11l): 10% yield, m.p. 247–248 ˝C
(MeOH). IR (KBr): 1151 (S(=O)2), 1384 (S(=O)2), 1715 (C=O), 3284 cm´1; 1H-NMR (CDCl3, 500 Hz):
δ
1.28 (6H, s), 2.30 (3H, s), 3.89 (2H, s), 5.69 (1H, s), 7.10 (2H, d, J = 8.1 Hz), 7.67 (2H, d, J = 8.2 Hz),
7.78 (2H, dt-like, J = 4.0, 7.0, 8.5 Hz), 7.88 (2H, dt-like, J = 3.1, 5.3, 6.8 Hz); HRMS (EI, 80 ev), calcd.
C19H20N2O4S 372.1144, found 372.1143.
˝
N-[2-Methylethylamino]-1-naphtharinimido-4-methylsulfanilamide (11m): 73% yield, m.p. 200–202 C
1
(MeOH). IR (KBr): 1135 (S(=O)2), 1397 (S(=O)2), 1711 (C=O), 3313 cm´1; H-NMR:
δ 1.28 (3H, d,
J = 6.4 Hz), 2.09 (3H, s), 3.52 (2H, dt-like, J = 4.5, 10.1, 14.4 Hz), 3.68 (1H, d, J = 7.6 Hz), 4.88 (1H, d,
J = 7.9 Hz), 6.86 (2H, d, J = 8.0 Hz), 7.53 (2H, d, J = 8.1 Hz), 7.71 (4H, dt-like, J = 5.4, 9.0, 10.2 Hz); HRMS
(EI, 80 ev), calcd. C18H18N2O4S 358.0987, found 358.0986.
N-[2,2-Dimethylethylamino]-1-naphtharinimidosulfanilamide (11n): 34% yield, m.p. 202–203 ˝C (MeOH).
IR (KBr): 1155 (S(=O)2), 1392 (S(=O)2), 1715 (C=O), 3276 cm´1; 1H-NMR:
δ 1.29 (6H, s), 3.67 (2H, s),
5.78 (1H, s), 7.31 (2H, t, J = 7.7 Hz), 7.39 (1H, t, J = 7.5 Hz), 7.76 (2H, qd, J = 7.5, 5.5, 3.1, 3.1 Hz), 7.81
(2H, dd-like J = 7.7, 1.1), 7.88 (2H, dd, J = 3.1, 5.4 Hz); HRMS (EI, 80 ev), calcd. C18H18N2O4S 358.0987,
found 358.0989.
N-[3-(1,3-Dioxo-1,3,3a,4,7,7a-hexahydro-isoindol-2-yl)-2,2-dimethylethyl]-4-methyl-benzenesulfonamide (12o):
22% yield, m.p. 133–134 ˝C (MeOH). IR (KBr): 1143 (S(=O)2), 1397 (S(=O)2), 1707 (C=O), 3272 (NH)
cm´1; 1H-NMR:
δ 1.20 (6H, s), 2.22 (2H, dd, J = 4.2, 4.6 Hz), 2.40 (3H, s), 2.57 (2H, dd, J = 1.6, 1.6 Hz),
3.10 (2H, t, J = 2.8 Hz), 3.47 (2H, s), 5.79 (2H, t, J = 3.1Hz), 5.85 (1H, s), 7.25 (2H, d, J = 9.4 Hz), 7.71
(2H, d, J = 8.2 Hz); HRMS (EI, 80 ev), calcd. C19H24N2O4S 376.1457, found 376.1458.
N-[3-(1,3-Dioxo-1,3,3a,4,7,7a-hexahydro-isoindol-2-yl)-2-methylethyl]-4-methyl-benzenesulfonamide (12p):
22% yield, m.p. 145–146 ˝C (MeOH). IR (KBr): 1160 (S(=O)2), 1401 (S(=O)2), 1695 (C=O), 3280 (NH)
1
cm´1; H-NMR:
δ 1.00 (3H, d, J = 6.6 Hz), 2.23 (2H, dd, J = 5.3, 15.5 Hz), 2.41 (3H, s), 2.54 (2H, d,
J = 18.1 Hz), 2.96 (1H, dd, J =2.7, 2.9 Hz), 3.01 (1H, dd, J = 2.7, 10.7 Hz), 3.31 (1H, dd, J = 3.7, 13.7 Hz),
3.49 (1H, dt-like, J = 5.9, 9.4, 10.9 Hz), 3.61 (1H, s), 4.78 (1H, d, J = 8.4 Hz), 5.88 (2H, t, J = 2.2 Hz),

Molecules 2016, 21, 100
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7.28 (2H, d, J = 8.1 Hz), 7.70 (2H, d, J = 8.1 Hz); HRMS (EI, 80 ev), calcd. for C18H22N2O4S 362.1300,
found 362.1298.
exo-N-Hydroxyl-7-oxabicyclo[2.2.1]hept-5-ene-2-phenylsulfonamido-1-methylethyl-2,3-dicarboximide (16q):
13% yield, m.p. 127–133 ˝C (MeOH). IR (KBr): 1709 (C=O) cm´1; 1H-NMR:
δ 1.34 (3H, d, J = 3.3 Hz),
2.71 (2H, s), 2.99–3.03 (2H, m, N-CH2), 4.23–4.24 (1H, m, O-CH), 5.02 (1H, s), 5.19 (1H, s), 6.47 (2H, s),
7.49–7.55 (3H, m), 7.85–7.86 (2H, m); FAB-MS m/z (rel. int. %): 379 [M + H]+ (15), 311 (48), 270 (34),
70 (75), 56 (100).
exo-N-Hydroxyl-7-oxabicyclo[2.2.1]hept-5-ene-2-phenylsulfonamido-2-methylethyl-2,3-dicarboximide (16r):
9% yield; 174–176 ˝C (MeOH); IR (KBr): 1690 (C=O) 3327 cm´1; 1H-NMR:
δ 1.23 (3H, d, J = 2.8 Hz),
2.74 (2H, dd, J = 5.4, 7.3 Hz), 3.88 (2H, td, J = 2.8, 4.2, 8.2 Hz), 3.51 (1H, dd, J = 3.1, 4.4 Hz), 5.24 (2H, d,
J = 4.9 Hz), 6.50 (2H, s), 7.49 (3H, dt-like, J = 5.8, 6.0, 11.3 Hz), 7.84 (2H, dt-like, J = 5.4, 6.0, 9.9 Hz);
FAB-MS m/z (rel. int. %): 379 [M + H]+ (3), 135 (70), 74 (75), 197 (35).
exo-N-Hydroxyl-7-oxabicyclo[2.2.1]hept-5-ene-2-[(2’,5’-dichlorophenyl)sulfonamidoethyl]-2,3-dicarboximide
˝
16s): 48% yield, m.p. 212-216 C (MeOH). IR (KBr): 1780 cm´1; 1H-NMR:
δ 2.77 (2H, s), 3.21 (2H, dd,
(
J = 5.5, 10.1 Hz), 4.09 (2H, t, J = 9.5 Hz), 5.25 (2H, s), 6.50 (2H, s), 6.40 (1H, t, 11.9 Hz), 7.44 (2H, s),
8.04 (1H, s); FAB-MS m/z (rel. int. %): 433 [M + H]+ (4), 155 (30), 365 (10); HRMS (FAB+), calcd. for
C16H15N2O6Cl2S 433.0029, found 433.0005.
3.2. Antineoplastic Bioassays
3.2.1. Cell Culture
Media and sera for cell culture were purchased from Gibco/BRL (Grand Island, NY, USA). Most
chemicals were purchased from Sigma Chemical (St. Louis, MO, USA). The human hepatoma (Hep3B)
and myeloid leukemia (HL-60) cell lines were obtained from the American Type Culture Collection
(ATCC; Rockville, MD, USA). The human breast cancer cell lines, MDA-MB-231 and MCF-7, were
purchased from the cell bank of the National Health Research Institute (Miaoli, Taiwan). These cells
were maintained as monolayers in Dulbecco’s modified Eagle’s medium (DMEM) containing 10%
heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin, 100 µg/mL streptomycin, 100 µM
nonessential amino acids, and 1 mM glutamine in a controlled atmosphere of 5% CO2 and 95% air
˝
at 37 C.
3.2.2. MTT Assay for Cellular Viability
Cells were seeded into 96-well plates and allowed to adhere for 24 h before drugs were introduced.
Following a 48-h incubation, drugs and media were removed, and each well was treated with 100
of 500 g/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in culture medium.
Following a 4-h incubation period to allow the metabolism of MTT by mitochondrial dehydrogenases
of viable cells to form an insoluble formazan product, the plates were centrifuged at 450 g for 10 min,
and supernatants were removed and replaced with 100 L DMSO. The plates were shaken to maximize
µL
µ
ˆ
µ
solubilization of the formazan crystals. The absorbance, as a measure of viable cell numbers, was read
the following day in a model MA310 automated EIA plate reader (Whittaker M. A. Bioproducts, Inc.,
Walkersville, MD, USA) at a wavelength of 550 nm. IC50 values were obtained by a linear regression
analysis of the percent absorbance vs. the log of the drug concentration. It was previously shown that
viable cell numbers are correlated with the optical density as determined in the MTT assay.
4. Conclusions
We synthesized cantharidinimide derivatives, 5a–h, and their analogues 10i–k, 11l–n, 12o–p,
and 16q . The most potent cytostatic compound, N-cantharidinimido-sulfamethazine (5a), exhibited
–
s
anti-HL-60 and anti-Hep3B cell activities. Two compounds, 5g and 5h, displayed slight effects on the
Hep3B cell line, while the other compounds produced no response in these four cell lines. Although

Molecules 2016, 21, 100
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they exhibited very low effects on human carcinoma cells, we observed that these compounds contained
sulfamoyl moieties. When studying the structure-activity relationship, we found that these compounds
had very similar chemical structures to some diuretics, antidiabetic drugs, and hypoglycemic agents.
Thus, their pharmacological effects should be tested in the future.
Acknowledgments: We thank Shwu-Huey Wang for help with the mass and NMR spectra and data calculation.
The authors are indebted to Helmut Stamm (who recently passed away, School of Pharmacy, University of
Heidelberg, Germany) for techniques of synthetic activated aziridines.
Author Contributions: Pen-Yuan Lin, Shiow-Yunn Sheu, and Mei-Hsiang Lin conceived and designed the
experiments; Yun-Han Hsieh, Yi-Jing Lin, and Hsiao-Ling Chen performed the experiments and analyzed the
data; Ling-Ling Chiang, Ing-Jy Tseng, and Ching-Tung Lin wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 10i–k, 11l–n, 12o–p, and 16q–s are available from
the authors.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons by Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).