Structure-activity relationship, cytotoxicity and mode of action of 2-ester-substituted 1,5-benzothiazepines as potent antifungal agents

Article abstract of DOI:10.1002/cjoc.201300316

Our studies examined the structural features responsible for the antifungal activity of 2-ethoxycarbonyl-1,5-benzothiazepine (7a). Three series of 1,5-benzothiazepine derivatives were synthesized and screened for their antifungal activity. The results suggested that the ethoxycarbonyl group at the 2 position and the imine moiety on the seven-membered ring are essential for activity. The most potent of the synthesized analogues (7a, 7b) were further studied by evaluating their cytotoxicity and mode of action (for 7a). The results showed that compounds 7a and 7b were relatively safe for BV₂ cells, but compound 7a interfered with Cryptococcus neoformans cell wall integrity by increasing the chitinase activity. Therefore, compound 7a was considered safe as an antifungal agent for animal cells. Three series of 1,5-benzothiazepine derivatives were synthesized and their antifungal activities were evaluated to determine the structure-activity relationships with respect to the antifungal activity of 2-ester-substituted 1,5-benzothiazepines. The effective antifungal compounds 7a and 7b were further studied for their antifungal activity, cytotoxicity and mechanism of action (for compound 7a). The results provided important information about this class of benzothiazepines. Copyright

Full text of DOI:10.1002/cjoc.201300316

FULL PAPER
DOI: 10.1002/cjoc.201300316
Structure-Activity Relationship, Cytotoxicity and Mode of
Action of 2-Ester-substituted 1,5-Benzothiazepines
as Potent Antifungal Agents
Wang Kang,a Xingqiong Du,b Lanzhi Wang,a Lijuan Hu,a Yuhuan Dong,c Yanqing Bian,*,b and
Yuan Li*,a
a College of Chemistry & Material Science, Hebei Normal University, Shijiazhuang, Hebei 050024, China
b College of Life Sciences, Hebei Normal University, Shijiazhuang, Hebei 050024, China
c Department of Chemistry, Tangshan Normal University, Tangshan, Hebei 063000, China
Our studies examined the structural features responsible for the antifungal activity of 2-ethoxycarbonyl-1,5-
benzothiazepine (7a). Three series of 1,5-benzothiazepine derivatives were synthesized and screened for their anti-
fungal activity. The results suggested that the ethoxycarbonyl group at the 2 position and the imine moiety on the
seven-membered ring are essential for activity. The most potent of the synthesized analogues (7a, 7b) were further
studied by evaluating their cytotoxicity and mode of action (for 7a). The results showed that compounds 7a and 7b
were relatively safe for BV2 cells, but compound 7a interfered with Cryptococcus neoformans cell wall integrity by
increasing the chitinase activity. Therefore, compound 7a was considered safe as an antifungal agent for animal
cells.
Keywords 1,5-benzothiazepines, antifungal agents, antifungal mechanism, cytotoxicity, structure-activity rela-
tionships
Introduction
Based on these findings, we decided to explore the
structural features that are important for its antifungal
properties. Therefore, using 7a as the prototypical
structure, three series of 1,5-benzothiazepine derivatives
were designed, synthesized and evaluated for their anti-
fungal activity against C. neoformans (Figure 1). The
first series of compounds were benzothiazepines bearing
various substituents at the 4 position of the aryl ring
(7a－7f) or various ester groups at the 2 position of the
seven-membered ring (7g－7j) to determine the effects
of these substituents on the antifungal activity. The
1,5-Benzothiazepines exhibit various biological ac-
＋
tivities, such as blocking Ca2 channels,[1-3] CNS inhi-
bition,[4,5] antiplatelet aggregation, and anti-thrombotic
activity;[6] some 1,5-benzothiazepines have become
clinically useful drugs.[7] In recent years, a number of
1,5-benzothiazepine derivatives have been evaluated for
their antibacterial and antifungal activities,[8-15] and
strong efficacy against microorganisms resulted in lead
compounds for further investigation. Cryptococcus
neoformans is an opportunistic fungal pathogen that
may cause meningitis in immunocompromised indi-
viduals. Meningitis is uniformly fatal if untreated. Up to
now, there are few antifungal agents, meanwhile the
drug-resistant strains are emerging.. Therefore, re-
searches on drugs against C. neoformans are of great
significance. During the course of our research on the
antimicrobial activity of 1,5-benzothiazepine derivatives,
we found that 2-ester-substituted 1,5-benzothiazepine
(7a) exhibited excellent antifungal activity against C.
neoformans with a minimum inhibitory concentration
(MIC) and minimum fungicidal concentration (MFC)
similar to or lower than that of the standard antifungal
drug fluconazole.
CO2R2
S
N
Ph R1
7b 7j
O
O
COOH
Ph
CO2C2H5
CO2C2H5
S
N
S
S
N
N
H
Ph
Ph
8a 8e
7a
9a 9f
Figure 1 Structures of 7a and other designed analogues.
*
E-mail: liyuanhbsd@163.com; Tel.: 0086-0311-80787400
Received April 16, 2013; accepted June 25, 2013; published online July 26, 2013.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.2013xxxxx or from the author.
Chin. J. Chem. 2013, 31, 1305—1314 © 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Kang et al.
FULL PAPER
second series of compounds tested were 1,5-benzothi-
azepines with a sulfone unit (8a－8e) to evaluate the
influence of the oxidation state of the sulfur on activity.
The third series of analogues were 2,3,4,5-tetrahedron-
1,5-benzothiazepines 9a－9f without a C＝N double
bond to clarify the role of the imine moiety on the anti-
fungal properties. Furthermore, the most effective anti-
fungal derivatives 7a and 7b were further studied for
their antifungal activities, cytotoxicities and modes of
action (for compound 7a).
anhydrous sodium sulfate as a dehydrating agent. A
Michael addition of o-aminothiophenol 5 to 4-oxo-4-
phenylbut-2-enoate 4a－4j in acetic acid/methanol af-
forded intermediates 6a－6j, which then underwent an
intramolecular cyclization with the loss of water to ob-
tain 7a－7j.
As shown in Scheme 2, compounds 8a－8e were
prepared by oxidation of 7a－7e with 30% hydrogen
peroxide in the presence of catalytic amounts of acetic
acid. Initially, we wanted to synthesize 2-ester-substi-
tuted-1,5-benzothiazepines bearing a sulfone functional-
ity, but the reaction did not generate the expected prod-
ucts because the ester group in the 2 position of the
seven-membered ring was hydrolyzed to the carboxylic
acid during the reaction. This hydrolysis occurred under
various reaction conditions (e.g., catalyst, solvent, tem-
perature, and time), which were investigated in trial ex-
periments. Therefore, we could obtain only the 2-carb-
oxyl-substituted analogues 8a－8e.
Results and Discussion
Synthesis of the target compounds
The synthetic route to compounds 7a－7j is depicted
in Scheme 1. Compounds 1a－1f reacted with maleic
anhydride 2 in the presence of anhydrous aluminum
chloride to obtain 4-oxo-4-arylbutenoic acids 3a－3f.
The α,β-unsaturated compounds 4a－4j were prepared
by reacting acids 3a－3f with the corresponding alco-
hols under reflux in a benzene-alcohol solution using
concentrated sulfuric acid as a catalyst. However,
tert-butyl ester analogue 4j could not be prepared using
the above method; therefore, it was obtained by reacting
3a with tert-butyl alcohol at room temperature using
The third series of analogues 9a－9f were synthe-
sized by reducing the C＝N bond of compounds 7a－7f,
as illustrated in Scheme 2. The C＝N double bond was
reduced at pH 4 with sodium cyanoborohydride in an
HCl-methanol solution, the bromocresol green was used
as a pH indicator. As reported in the literature,[16] so-
Scheme 1 Synthesis of compounds 7a－7j
R1
O
O
R2OH, C6H6, H2SO4(cat.)
AlCl3, CH2Cl2
0 oC, 5 h
CO2H
+
O
reflux 12 h
R1
SH
NH
O
3a 3f
1a 1f
2
S
NH2
O
2, CH3OH, HOAc (cat.)
0oC, 5 12 h
CH3OH, HOAc (cat.)
0 oC, 5 12 h
CO2R2
5
CO2R2
R1
R1
O
4a 4j
6a 6j
R2O2C
S
a: R2= C2H5, R1= H;
c: R2= C2H5, R1= Cl;
e: R2= C2H5, R1= CH3;
g: R1= H, R2= CH3;
b: R2= C2H5, R1= F;
R1
d: R2= C2H5, R1= Br;
f: R2= C2H5, R1= OCH3;
h: R1= H, R2= (CH2)2CH3;
j: R1= H, R2= C(CH3)3;
N
i: R1= H, R2= CH(CH3)2;
7a 7j
Scheme 2 Synthesis of compounds 8a－8e and 9a－9f
CO2C2H5
S
O
O
CO2C2H5
CO2H
S
S
H2O2 (30%)
HOAc, r.t.
HCl-CH3OH, NaBH3(CN)
r.t., 3 h.
N
H
N
N
R1
R1
9a 9f
R1
8a 8e
7a 7f
a: R1 = H;
d: R1 = Br;
b: R1 = F;
e: R1 = CH3;
c: R1 = Cl;
f: R1 = OCH3
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Cytotoxicity and Mode of Action of 2-Ester-substituted 1,5-Benzothiazepines
dium cyanoborohydride is a pH dependent, selective
reducing reagent; however, the ester groups in 7a－7f
were inert to this reagent.
showed that all of the sulfone-bearing analogues were
weakly active or inactive against the microorganisms
tested. These results indicated that replacement of the
sulfur atom and/or ethoxycarbonyl group with a sulfone
functionality and/or carboxyl group led to a marked de-
crease in activity (compare 8a and 8b with 7a and 7b,
respectively). Possible reasons for the decreased bio-
activity in 8a－8e include the transformation of the es-
ters into carboxyl groups and/or changes in the sulfur
oxidation state, which will be studied further.
All of the synthesized analogues of 7a were purified
by recrystallization with methanol as a solvent or by
chromatography on a silica gel column, and their struc-
1
tures were confirmed by elemental analysis, IR, H
NMR, 13C NMR and MS/HRMS.
In vitro antifungal activities of the synthesized com-
pounds
To determine the importance of the imine moiety,
2,3,4,5-tetrahydro-1,5-benzothiazepines 9a － 9f were
prepared by reducing the imine functionalities of 7a－7f.
Surprisingly, compounds 9a and 9b completely lost
their activity compared to 7a and 7b, and analogues
9c－9f were also completely inactive (Table 2). These
findings suggested that the imine moiety on the seven-
membered ring in these benzothiazepines is required for
activity.
The synthesized compounds 7a－7j were tested
against two strains of C. neoformans (one ATCC 34874
and another clinical isolate) using the disk diffusion
method with DMSO as a control. As shown in Table 1,
compounds 7c－7f with electron-donating or electron-
withdrawing groups on the phenyl rings at the position 4
of the seven-membered ring were basically inactive, but
the fluorophenyl-substituted 1,5-benzothiazepine (7b)
exhibited good antifungal activity (average inhibition
zone diameter, 19.3 mm). These findings suggested that
the electronic effect caused by substituents on the
phenyl rings was not important for the bioactivity of the
target compounds; however, the appropriate atom (H or
F) on the phenyl ring was required. The data in Table 1
also showed that replacement of the 2-ethoxycarbonyl
group with other ester groups (2-methoxycarbonyl 7g,
2-propoxycarbonyl 7h, 2-isopropoxycarbonyl 7i, 2-tert-
butyloxycarbonyl 7j) caused a marked decrease in anti-
fungal activity. These results demonstrated that the
2-ethoxycarbonyl group was essential for the biological
activity of this family of compounds.
Table 2 Zone of growth inhibition of compounds 8a－8e, 9a－
9f (mm)a
C. neoformans 1#
Compd R1
C. neoformans (ATCC)
(clinical isolate)
11.3±0.3
6.0±0.1
6.0±0.2
6.0±0.1
12.0±0.3
6.0±0.1
6.0±0.2
6.0±0.1
6.0±0.2
6.0±0.1
6.0±0.1
6.0±0.1
8a
H
9.5±0.1
10.4±0.3
6.0±0.1
10.5±0.2
8b
F
8c
Cl
Br
8d
8e
CH3 12.1±0.4
9a
H
6.0±0.2
6.0±0.3
6.0±0.2
6.0±0.1
9b
F
Table 1 Zone of growth inhibition for compounds 7a－7j
(mm)a
9c
Cl
Br
9d
C. neformans C. neoformans 1#
Compd R1
R2
9e
CH3 6.0±0.2
OCH3 6.0±0.2
(ATCC)
(clinical isolate)
22.8±0.2
20.5±0.1
6.0±0.3
9f
controlb
7a
7b
7c
7d
7e
7f
H
CH2CH3
CH2CH3
CH2CH3
CH2CH3
23.5±0.5
18.1±0.2
6.0±0.2
6.0±0.2
6.0±0.3
11.7±0.3
6.0±0.2
12.7±0.3
12.9±0.4
6.0±0.3
6.0±0.1
－
6.0±0.1
F
a All values were measured after 24 h of incubation at 37 ℃ with
200 μg of the tested compound/disk. b An equivalent volume of
solvent (DMSO) was added as a control.
Cl
Br
6.0±0.2
CH3 CH2CH3
OCH3 CH2CH3
6.0±0.3
Compounds 7a and 7b, which had prominent anti-
fungal activities, were subjected to further pharmacol-
ogical evaluation, including determining the dose de-
pendence of the antifungal activity, MIC, MIC80, and
MFC against five strains of C. neoformans. The data in
Table 3 indicated that compounds 7a and 7b showed
dose-dependent activity when evaluated in the concen-
tration range of 12.5 to 200.0 μg/disk. At 12.5 μg/disk,
the two compounds still exhibited moderate to high ac-
tivity. The MIC, MIC80 and MFC values of 7a, 7b are
shown in Table 4 and the standard drug fluconazole was
used as positive control. The data showed that com-
pounds 7a (MIC80, 0.5－4.0 μg/mL) and 7b (MIC80,
0.5－2.0 μg/mL) had MIC80 values very similar to that
of fluconazole (MIC80, 2.0－4.0 μg/mL). In contrast, the
6.0±0.3
7g
7h
7i
H
H
H
H
－
CH3
15.2±0.4
6.0±0.3
(CH2)2CH3
CH(CH3)2
C(CH3)3
－
6.0±0.4
7j
controlb
6.0±0.2
6.0±0.0
a
All values were measured after 24 h of incubation at 37 ℃
with 200 μg of the tested compound/disk. b 1 equiv. volume of
solvent (DMSO) was added as a control.
In order to explore the influence of the sulfur oxida-
tion state on the antifungal activity, the sulfur atom on
the seven-membered ring was converted to a sulfone, as
shown in compounds 8a－8e. The zone data in Table 2
Chin. J. Chem. 2013, 31, 1305—1314
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Table 3 Zone of growth inhibition of compounds 7a and 7b at 200.0－12.5 μg/disk (mm) a
7a
7b
Fungal strainb
200.0
100.0
50.0
25.0
12.5
200.0
100.0
50.0
25.0
12.5
1
2
3
4
5
23.5±0.5 22.4±0.1 21.2±0.3 19.8±0.2 16.9±0.3 18.1±0.2 18.4±0.1 19.8±0.2 17.2±0.1 15.5±0.3
22.8±0.2 23.9±0.2 20.8±0.2 21.4±0.4 20.7±0.5 20.5±0.1 19.1±0.2 19.3±0.4 23.2±0.3 20.3±0.2
29.1±0.5 25.5±0.4 22.6±0.4 18.7±0.3 15.2±0.2 28.6±0.3 26.2±0.4 21.7±0.3 19.2±0.2 15.1±0.4
27.2±0.2 25.0±0.5 20.4±0.4 17.6±0.1 15.3±0.1 27.4±0.2 25.8±0.1 22.7±0.3 19.8±0.1 14.4±0.2
25.8±0.3 22.2±0.3 18.6±0.3 15.2±0.3 10.4±0.2 29.3±0.4 28.1±0.2 22.3±0.2 18.1±0.2 13.7±0.3
a All values were measured after 24 h of incubation at 37 ℃. An equivalent volume of solvent (DMSO) was added as a control, and the
zone of growth inhibition of the control was 6.0±0.1. b 1: C. neoformans (ATCC), 2－5: C. neoformans clinically isolated, 2: C. neofor-
mans 1#, 3: C. neoformans 2#, 4: C. neoformans 3#, and 5: C. neoformans 4#.
Table 4 MIC, MIC80 and MFC values for compounds 7a, 7b and fluconazole (μg/mL)
7a
7b
Fluconazole
Fungal straina
MIC
2.5
4.5
1.0
2.0
2.0
MIC80
2.0
MFC
10.0
7.5
MIC
4.0
1.0
1.5
2.0
2.5
MIC80
2.0
MFC
8.0
MIC
MIC80
2.0
MFC
1
2
3
4
5
＞128.0
＞128.0
＞128.0
＞128.0
＞128.0
＞128.0
＞128.0
＞128.0
＞128.0
＞128.0
4.0
0.5
15.0
12.5
13.0
13.0
2.0
0.5
10.0
10.0
11.5
0.5
3.0
1.5
0.5
4.0
0.5
0.5
4.0
a 1: C. neoformans (ATCC), 2－5: C. neoformans clinically isolated, 2: C. neoformans 1#, 3: C. neoformans 2#, 4: C. neoformans 3#, 5: C.
neoformans 4#. All values were the average of three trials, and the standard deviations were zero.
MIC and MFC values for 7a (MIC, 1.0－4.5 μg/mL;
MFC, 7.5－11.5 μg/mL) and 7b (MIC, 1.0－4.0 μg/mL;
MFC, 8.0－15.0 μg/mL) were much lower than those of
fluconazole (MIC, ＞128 μg/mL; MFC, ＞128 μg/mL)
for all C. neoformans tested. Fluconazole and other tri-
azole antifungals are main choice for the clinical treat-
ment of the systemic fungal infections, but the obvious
side effect has been found for them. As a result, new and
safe antifungal agents are still needed. The compounds
7a and 7b, with good antifungal activity and different
structural features from those of fluconazole, will be-
come a new kind of promising antifungal compounds
for further optimization.
Figure 2 Effects of compounds 7a, 7b and fluconazole on the
survival of cells.
Cytotoxicities of compounds 7a and 7b
The cytotoxicities of potential agents 7a and 7b were
evaluated using a BV2 cell line and a 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. The cytotoxicity of DMSO was used as a control.
The relative survival rate of the cells significantly de-
creased with compound 7a at concentrations ≥200.0
μg/mL and with compound 7b at concentrations
≥100.0 μg/mL (p＜0.01) (Figure 2). This result indi-
cated that compounds 7a and 7b were relatively safe for
animal cells at concentrations less than or equal to 100.0
and 50.0 μg/mL, respectively, and the safe concentration
of fluconazole for animal cells was less than or equal to
200.0 μg/mL.
7a was further examined by testing the cell wall integ-
rity and chitinase activity, and performing membrane
permeability assays. The fungal strain C. neoformans
(ATCC) was used.
Cell wall integrity assay One of the defining char-
acteristics of fungi is the structure and composition of
their cell walls. Consequently, a cell wall integrity assay
was performed to determine whether compound 7a in-
terfered with cell wall function. When cell wall integrity
is compromised, the rapid thermal expansion of the cells
most likely results in increased cell death. The effect of
compound 7a on the cell wall function is shown in Fig-
ure 3. The number of viable cells in the treated group
decreased 10-fold after 40 s of exposure at 65 ℃, indi-
cating increased sensitivity of the treated cells to heat
shock. Therefore, compound 7a interfered with fungal
Mode of action of compound 7a
Compound 7a was safer than compound 7b in ani-
mal cells. Therefore, the mode of action of compound
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Cytotoxicity and Mode of Action of 2-Ester-substituted 1,5-Benzothiazepines
Figure 3 Effect of compound 7a on cell wall integrity.
Figure 5 Effect of compound 7a on membrane permeability.
cell wall integrity and function.
Chitinase activity assay The chitinase activity of
cells was measured after treatment with compound 7a or
1 equiv. volume of DMSO (control group) for 0, 4, 8, 12,
24, and 32 h. The purpose was to study the influence of
compound 7a on fungal cell wall integrity. Figure 4
showed that chitinase activity significantly increased
after treatment with compound 7a for 12, 24, and 32 h,
but did not significantly change in the control group
(p＜0.01). These results indicated that compound 7a
impaired the cell wall integrity by increasing cell wall
chitinase activity.
rivatives were synthesized and their antifungal activities
were evaluated to determine the structure-activity rela-
tionships with respect to the anti-fungal activity of
2-ester-substituted 1,5-benzothiazepines. The effective
antifungal compounds 7a and 7b were further studied
for their antifungal activity, cytotoxicity and the mecha-
nism of action (for compound 7a).
Preliminary structure-activity relationship studies
showed that the ethoxycarbonyl group at the position 2
and the imine moiety on the seven-membered ring in
this class of benzothiazepines were critical to their bio-
logical activity; the presence of an H or F atom on the
phenyl ring at position 4 was also important. The effect
of the sulfur oxidation state on the antifungal properties
requires further investigation. Of the tested compounds,
7a and 7b were the most promising anti-C. neoformans
compounds, with excellent antifungal properties and
low cytotoxicities. Mode of action assays indicated that
the reactive site for 7a as an antifungal agent was the
cell wall and not the cell membrane. Compound 7a may
also be a safe antifungal drug for humans and animals.
Figure 4 Effect of compound 7a on cell wall chitinase activity.
Experimental
Membrane permeability assay Interference of
cell membrane function results in membrane permeabil-
ity changes. Hence, using a conductivity meter, the rela-
tive cell membrane permeability rate was measured after
treatment with compound 7a or an equivalent volume of
DMSO for 0, 5, 10, 30, 60, 180, and 360 min. The rela-
tive permeability rate was found to be similar to that of
the control group. With longer treatment times, the rela-
tive permeability of the treatment and control groups
increased at the same rate (Figure 5). This result indi-
cated that compound 7a did not affect membrane per-
meability, and that the reactive site of compound 7a was
not on the cell membrane.
Chemistry
Melting points (℃) were determined using an X-4
digital melting point apparatus. Elemental analysis was
performed using a Vario ELIII elemental analyzer
without correction. Infrared spectra (in KBr pellets)
were recorded using a Vertex 70 FT-IR spectrometer. 1H
NMR and 13C NMR spectra were recorded using an
AVIII500Q Bruker spectrometer. Chemical shifts (δ)
were reported relative to SiMe4 as the internal standard
when measured in CDCl3 or DMSO-d6 solution. Mass
spectra were measured using a Xevo mass spectrometer,
utilizing electron ionization (EI) at an ionizing energy of
70 eV. High-resolution mass spectra were measured
using an apex Ultra mass spectrometer, utilizing elec-
trospray ionization (ESI). All reagents and solvents were
dried and distilled according to standard procedures.
Conclusions
In this study, three series of 1,5-benzothiazepine de-
Chin. J. Chem. 2013, 31, 1305—1314
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General method for preparation of 2,3-dihydro-2-
alkoxycarbonyl-4-aryl-1,5-benzothiazepines 7a－7j
163.8, 165.8, 168.0, 170.2; IR (KBr) ν: 1726, 1601 cm−1.
HRMS-ESI m/z: [M＋H]＋ calcd for C18H16FNO2S:
330.09640, found 330.09234.
A suspension of anhydrous AlCl3 (12.0 g, 90 mmol)
and a substituted benzene 1a－1f (45 mmol) in dry
CH2Cl2 (150 mL) was cooled to 0 ℃, and maleic anhy-
dride 2 (29.4 g, 30 mmol) in dry CH2Cl2 (40 mL) was
added. The reaction mixture was stirred in an ice-bath (0
℃) for 5 h. After completion of the reaction (determined
by TLC), a cold aqueous solution of 4 mol/L HCl (100
mL) was added to decompose unreacted AlCl3. The
mixture was poured into ice water to precipitate out the
crude product, which then was filtered and added to an
aqueous, saturated solution of NaHCO3. The resulting
mixture was refluxed for 1 h and then filtered to remove
the insoluble impurities. Next, the filtrate was acidified
with concentrated HCl to crystallize the product, which
was filtered and dried to produce compounds 3a－3f.
To a solution of 4-oxo-4-arylbutenoic acid 3a－3f
(100 mmol) in anhydrous benzene (100 mL), an alcohol
(300 mmol) and a catalytic amount of concentrated
H2SO4 were added. The reaction mixture was refluxed
for 12 h with stirring. After cooling, the organic layer
was washed with a cold, aqueous, saturated solution of
NaHCO3; an aqueous, saturated solution of NaCl; and
water. The organic layer was dried and evaporated under
reduced pressure, and the crude products 4a－4j were
purified by crystallization from ether.
2-Ethoxycarbonyl-4-(4-chlorophenyl)-2,3-dihy-
dro-1,5-benzothiazepine (7c) Yield 2.76 g, 80%; m.p.
87－88 ℃; 1H NMR (500 MHz, CDCl3) δ: 1.29 (t, J＝
7.0 Hz, 3H, -C-CH3), 4.21 (q, J＝7.0 Hz, 2H, -CO-
CH2-), 3.11 (dd, J＝11.5, 13.5 Hz, 1H, -C-H), 3.15 (dd,
J＝6.0, 13.5 Hz, 1H, -C-H), 4.31 (dd, J＝6.0, 11.5 Hz,
1H, -S-C-H), 7.09－7.91 (m, 8H, Ph-H); 13C NMR (125
MHz, CDCl3) δ: 14.0, 18.4, 31.4, 56.0, 58.4, 61.7, 121.0,
124.9, 125.5, 128.7, 129.0, 130.5, 135.5, 135.8, 137.5,
152.3, 168.1, 170.1; IR (KBr) ν: 1720, 1611 cm−1; MS
(70 eV) m/z: 345.93 (M ＋ H) ＋ . Anal. calcd for
C18H16ClNO2S: C 62.57, H 4.65, N 4.40; found C 62.51,
H 4.66, N 4.05.
2-Ethoxycarbonyl-4-(4-bromophenyl)-2,3-dihy-
dro-1,5-benzothiazepine (7d) Yield 2.95 g, 76%; m.p.
89－91 ℃; 1H NMR (500 MHz, CDCl3) δ: 1.29 (t, J＝
7.0 Hz, 3H, -C-CH3), 4.21 (q, J＝7.0 Hz, 2H, -CO-
CH2-), 3.12 (dd, J＝12.0, 13.0 Hz, 1H, -C-H), 3.16 (dd,
J＝6.0, 13.0 Hz, 1H, -C-H), 4.31 (dd, J＝6.0, 12.0 Hz,
1H, -S-C-H), 7.09－7.99 (m, 8H, Ph-H); 13C NMR (125
MHz, CDCl3) δ: 14.0, 31.3, 56.0, 61.63, 121.0, 124.9,
125.5, 126.0, 128.8, 130.5, 131.9, 135.5, 136.3, 152.3,
168.1, 170.1; IR (KBr) ν: 1720, 1609 cm−1; MS (70 eV)
m/z: 389.82 (M＋H)＋. Anal. calcd for C18H16BrNO2S: C
55.35, H 4.22, N 3.54; found C 55.39, H 4.13, N 3.59.
2-Ethoxycarbonyl-4-(4-methylphenyl)-2,3-dihy-
dro-1,5-benzothiazepine (7e) Yield 2.92 g, 90%; m.p.
86－88 ℃; 1H NMR (500 MHz, CDCl3) δ: 1.29 (t, J＝
7.0 Hz, 3H, -C-CH3), 2.43 (s, 3H, Ph-CH3), 4.21 (q, J＝
7.0 Hz, 2H, -CO-CH2-), 3.11 (dd, J＝13.0, 12.5 Hz, 1H,
-C-H), 3.22 (dd, J＝5.0, 13.0 Hz, 1H, -C-H), 4.31 (dd,
J＝5.0, 12.5 Hz, 1H, -S-C-H), 7.08－7.96 (m, 8H,
Ph-H); 13C NMR (125 MHz, CDCl3) δ: 14.0, 20.1, 40.4,
44.6, 57.7, 61.3, 119.5, 120.3, 128.8, 126.6, 128.2,
129.5, 132.8, 137.5, 140.4, 148.9, 170.9; IR (KBr) ν:
1720, 1607 cm−1; MS (70 eV) m/z: 325.97 (M＋H)＋.
Anal. calcd for C19H19NO2S: C 70.13, H 5.89, N 4.22;
found C 70.12, H 5.88, N 4.30.
o-Aminothiophenol 5 (1.25 g, 10 mmol) in dry
methanol (10 mL) was added to a solution of 4a－4j (10
mmol) in dry methanol (20 mL) using acetic acid as a
catalyst. The reaction mixture was stirred at 0 ℃ for
5－12 h, then concentrated under reduced pressure to
obtain the crude product. The product was either crys-
tallized with methanol or purified by chromatography
on a silica gel column with petroleum ether/ethyl acetate
(6∶4, V/V) as the eluent to obtain the corresponding
product 7a－7j.
2-Ethoxycarbonyl-4-phenyl-2,3-dihydro-1,5-
benzothiazepine (7a) Yield 2.44 g, 80%; m.p. 99－
1
101 ℃; H NMR (500 MHz, CDCl3) δ: 1.29 (t, J＝7.3
Hz, 3H, -C-CH3), 4.21 (q, J＝7.3 Hz, 2H, -CO-CH2-),
3.13 (dd, J＝13.0, 12.5 Hz, 1H, -C-H), 3.23 (dd, J＝5.0,
13.0 Hz, 1H, -C-H), 4.33 (dd, J＝5.0, 12.5 Hz, 1H,
-S-C-H), 7.11－8.05 (m, 9H, Ph-H); 13C NMR (125
MHz, CDCl3) δ: 14.0, 31.4, 55.9, 61.5, 121.0, 124.8,
125.2, 127.2, 128.7, 130.4, 131.2, 135.4, 137.4, 152.6,
169.2, 170.2; IR (KBr) ν: 1728, 1612 cm−1; MS (70 eV)
m/z: 312.50 (M＋H)＋. Anal. calcd for C18H17NO2S: C
69.44, H 5.52, N 4.50; found C 69.43, H 5.50, N 4.50.
2-Ethoxycarbonyl-4-(4-fluorophenyl)-2,3-dihy-
dro-1,5-benzothiazepine (7b) Yield 2.79 g, 85%; m.p.
2-Ethoxycarbonyl-4-(4-methoxyphenyl)-2,3-dihy-
dro-1,5-benzothiazepine (7f) Yield 1.97 g, 58%; m.p.
76－77 ℃; 1H NMR (500 MHz, CDCl3) δ: 1.23 (t, J＝
7.0 Hz, 3H, -C-CH3), 3.82 (s, 3H, Ph-OCH3), 4.14 (q,
J＝7.0 Hz, 2H, -CO-CH2-), 3.03 (dd, J＝13.0, 12.5 Hz,
1H, -C-H), 3.12 (dd, J＝5.0, 13.0 Hz, 1H, -C-H), 4.24
(dd, J＝5.0, 12.5 Hz, 1H, -S-C-H), 6.91－7.94 (m, 8H,
Ph-H); 13C NMR (125 MHz, CDCl3) δ: 14.1, 37.1, 37.2,
55.5, 55.6, 61.7, 113.8, 117.0, 123.9, 127.3, 128.1,
130.5, 135.6, 163.8, 167.2, 170.1, 194.1; IR (KBr) ν:
1720, 1601 cm−1; MS (70 eV) m/z: 341.85 (M＋H)＋.
Anal. calcd for C19H19NO3S: C 63.07, H 5.87, N 3.60;
found C 63.44, H 5.61, N 4.01.
1
110－111 ℃; H NMR (500 MHz, CDCl3) δ: 1.29 (t,
J＝7.0 Hz, 3H, -C-CH3), 4.21 (q, J＝7.0 Hz, 2H,
-CO-CH2-), 3.12 (dd, J＝12.0, 13.5 Hz, 1H, -C-H), 3.17
(dd, J＝6.0, 13.5 Hz, 1H, -C-H), 4.31 (dd, J＝6.0, 12.0
Hz, 1H, -S-C-H), 7.08－8.06 (m, 8H, Ph-H); 13C NMR
(125 MHz, CDCl3) δ: 14.0, 31.4, 55.9, 61.6, 115.7,
115.8, 124.9, 125.3, 125.4, 129.5, 129.6, 130.5, 133.4,
2-Methoxycarbonyl-4-phenyl-2,3-dihydro-1,5-
benzothiazepine (7g) Yield 1.19 g, 40%; m.p. 69－70
1
℃; H NMR (500 MHz, CDCl3 ) δ: 3.77 (s, 3H, -CO-
CH3), 3.13 (dd, J＝13.5, 12.5 Hz, 1H, -C-H), 3.24 (dd,
1310
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Chin. J. Chem. 2013, 31, 1305—1314

Cytotoxicity and Mode of Action of 2-Ester-substituted 1,5-Benzothiazepines
J＝5.5, 13.5 Hz, 1H, -C-H), 4.36 (dd, J＝5.5, 12.5 Hz,
1H, -S-C-H), 7.09－8.04 (m, 9H, Ph-H); 13C NMR (125
MHz, CDCl3) δ: 37.1, 37.5, 117.0, 120.1, 124.0, 127.4,
128.2, 128.7, 133.5, 135.9, 136.3, 169.0, 195.6; IR (KBr)
ν: 1734, 1612 cm−1; MS (70 eV) m/z: 298.04 (M＋H)＋.
Anal. calcd for C17H15NO2S: C 63.73, H 4.99, N 4.34;
found C 63.86, H 5.08, N 4.71.
crude residue. This residue was purified by a silica gel
column using petroleum ether/ethyl acetate/acetic acid
(8∶2∶1, V/V/V) as the eluent.
4-Phenyl-2,3-dihydro-1,5-benzothiazepine-2-
carboxylic acid 1,1-dioxide (8a) Yield 0.16 g, 52%;
1
m.p. 208－210 ℃; H NMR (500 MHz, DMSO-d6) δ:
3.61 (dd, J＝3.5, 18.0 Hz, 1H, -C-H), 3.88 (dd, J＝8.5,
17.8 Hz, 1H, -C-H), 5.28 (dd, J＝3.5, 8.5 Hz, 1H,
-S-C-H), 11.41 (s, 1H, -CO-OH), 7.25－8.10 (m, 9H,
Ph-H); 13C NMR (125 MHz, DMSO-d6) δ: 29.0, 60.2,
118.7, 123.5, 123.8, 125.3, 128.3, 128.8, 133.7, 135.2,
135.8, 163.5, 194.6; IR (KBr) ν: 1679, 1579, 1168, 1158
cm−1; MS (70 eV) m/z: 315.94 (M＋H)＋. Anal. calcd for
C16H13NO4S: C 59.83, H 4.25, N 4.18; found C 60.24, H
4.16, N 4.44.
2-Propoxycarbonyl-4-phenyl-2,3-dihydro-1,5-
benzothiazepine (7h) Yield 1.62 g, 50%; m.p. 55－57
1
℃; H NMR (500 MHz, CDCl3) δ: 0.97 (t, J＝7.5 Hz,
3H, -C-CH3), 1.65－1.72 (m, 2H, -C-CH2-), 4.07－4.16
(m, 2H, -CO-CH2-), 3.13 (dd, J＝13.0, 12.5 Hz, 1H,
-C-H), 3.24 (dd, J＝5.5, 13.0 Hz, 1H, -C-H), 4.35 (dd,
J＝5.5, 12.5 Hz, 1H, -S-C-H), 7.09－8.05 (m, 9H,
Ph-H); 13C NMR (125 MHz, CDCl3) δ: 10.3, 21.9, 37.1,
55.9, 67.2, 117.3, 123.8, 127.3, 127.9, 128.2, 128.6,
128.8, 133.4, 135.5, 167.7, 170.3, 195.7; IR (KBr) ν:
1728, 1611 cm−1; MS (70 eV) m/z: 325.87 (M＋H)＋.
Anal. calcd for C19H19NO2S: C 69.78, H 5.80, N 4.28;
found C 70.12, H 5.88, N 4.30.
4-(4-Fluorophenyl)-2,3-dihydro-1,5-benzothiaze-
pine-2-carboxylic acid 1,1-dioxide (8b) Yield 0.18 g,
1
55%; m.p. 237－238 ℃; H NMR (500 MHz, DMSO-
d6) δ: 3.61 (dd, J＝3.5, 17.5 Hz, 1H, -C-H), 3.87 (dd,
J＝8.5, 18.0 Hz, 1H, -C-H), 5.28 (dd, J＝3.5, 8.5 Hz,
1H, -S-C-H), 11.41 (s, 1H, -CO-OH), 7.24－8.20 (m,
8H, Ph-H); 13C NMR (125 MHz, DMSO-d6) δ: 29.0,
60.2, 115.8, 115.9, 118.7, 123.5, 123.8, 125.3, 131.4,
132.6, 132.6, 135.8, 163.5, 164.3, 166.3, 193.3; IR (KBr)
2-Isopropoxycarbonyl-4-phenyl-2,3-dihydro-1,5-
benzothiazepine (7i) Yield 1.78 g, 55%; m.p. 70－71
1
℃; H NMR (500 MHz, CDCl3) δ: 1.13 (d, J＝6.5 Hz,
3H, -C-CH3), 1.20 (d, J＝6.0 Hz, 3H, -C-CH3), 4.94－
4.99 (m, 1H, -CO-CH-), 3.36 (dd, J＝5.5, 18.0 Hz, 1H,
-C-H), 3.64 (dd, J＝9.0, 18.0 Hz, 1H, -C-H), 4.12 (dd,
J＝5.5, 9.0 Hz, 1H, -S-C-H), 6.64－7.93 (m, 9H, Ph-H);
13C NMR (125 MHz, CDCl3) δ: 21.4, 21.6, 37.2, 37.5,
56.0, 117.3, 119.6, 123.9, 127.3, 127.9, 128.2, 128.6,
133.4, 136.0, 136.4, 167.8, 195.8; IR (KBr) ν: 1680,
1614 cm−1; MS (70 eV) m/z: 325.87 (M＋H)＋. Anal.
calcd for C19H19NO2S: C 69.95, H 5.90, N 4.17; found
C 70.12, H 5.88, N 4.30.
ν: 1693, 1596, 1172, 1160 cm−1. HRMS-ESI m/z: [M＋
＋
Na]
356.03548.
4-(4-Chlorophenyl)-2,3-dihydro-1,5-benzothiaze-
calcd for C16H12FNO4S: 356.03633, found
pine-2-carboxylic acid 1,1-dioxide (8c) Yield 0.16 g,
1
45%; m.p. 230－232 ℃; H NMR (500 MHz, DMSO-
d6) δ: 3.61 (dd, J＝3.5, 18 Hz, 1H, -C-H), 3.86 (dd, J＝
8.5, 17.8 Hz, 1H, -C-H), 5.28 (dd, J＝3.5, 8.5 Hz, 1H,
-S-C-H), 11.40 (s, 1H, -CO-OH), 7.24－8.12 (m, 8H,
Ph-H); 13C NMR (125 MHz, DMSO-d6) δ: 29.0, 30.7,
60.2, 118.7, 123.5, 123.8, 125.3, 129.0, 130.3, 134.5,
135.2, 135.8, 138.6, 163.5, 193.9; IR (KBr) ν: 1692,
1592, 1172, 1157 cm−1; MS (70 eV) m/z: 350.03 (M＋
H)＋. Anal. calcd for C16H12ClNO4S: C 54.90, H 3.51, N
3.84; found C 54.94, H 3.46, N 4.00.
2-tert-Butyloxycarbonyl-4-phenyl-2,3-dihydro-
1,5-benzothiazepine (7j) Yield 2.57 g, 76%; m.p.
1
101－103 ℃; H NMR (500 MHz, CDCl3) δ: 1.47 (s,
9H, -C-(CH3)3), 3.07 (dd, J＝12.5, 13.5 Hz, 1H, -C-H),
3.17 (dd, J＝5.5, 13.5 Hz, 1H, -C-H), 4.23 (dd, J＝5.5,
12.5 Hz, 1H, -S-C-H), 7.07－8.04 (m, 9H, Ph-H); 13C
NMR (125 MHz, CDCl3) δ: 27.8, 31.6, 57.1, 81.9, 121.3,
124.7, 125.2, 127.3, 128.7, 130.3, 131.2, 135.5, 137.5,
152.5, 169.3, 169.6; IR (KBr) ν: 1718, 1607 cm−1; MS
(70 eV) m/z: 340.32 (M ＋ H) ＋ . Anal. calcd for
C20H21NO2S: C 70.80, H 6.33, N 4.03; found C 70.77, H
6.24, N 4.13.
4-(4-Bromophenyl)-2,3-dihydro-1,5-benzothiaze-
pine-2-carboxylic acid 1,1-dioxide (8d) Yield 0.16 g,
1
40%; m.p. 234－236 ℃; H NMR (500 MHz, DMSO-
d6) δ: 3.60 (dd, J＝3.5, 17.8 Hz, 1H, -C-H), 3.85 (dd,
J＝8.5, 17.8 Hz, 1H, -C-H), 5.26 (dd, J＝3.5, 8.3 Hz,
1H, -S-C-H), 11.39 (s, 1H, -CO-OH), 7.24－8.03 (m,
8H, Ph-H); 13C NMR (125 MHz, DMSO-d6) δ: 21.2,
28.8, 60.1, 118.7, 123.5, 123.7, 125.3, 128.5, 129.4,
135.2, 135.8, 144.2, 163.5, 194.0; IR (KBr) ν: 1720,
1587, 1171, 1154 cm−1; MS (70 eV) m/z: 393.84 (M＋
H)＋. Anal. calcd for C16H12BrNO4S: C 48.78, H 3.30, N
3.38; found C 48.74, H 3.07, N 3.55.
General method for preparation of sulfonate-con-
taining 1,5-benzothiazepine derivatives 8a－8e
Aqueous hydrogen peroxide (30%, 3 mL) was added
to a solution of 2,3-dihydro-2-ethoxycarbonyl-4-aryl-
1,5-benzothiazepine 7a－7e (1 mmol) in acetic acid (20
mL). The reaction mixture was stirred at room tempera-
ture until the starting material was consumed (deter-
mined by TLC). A small amount of MnO2 was added to
eliminate unreacted H2O2. The reaction mixture was
then filtered and extracted with diethyl ether (10 mL×
3). The combined organic phases were dried with anhy-
drous MgSO4 and evaporated in vacuo to afford the
4-(4-Methylphenyl)-2,3-dihydro-1,5-benzothiaze-
pine-2-carboxylic acid 1,1-dioxide (8e) Yield 0.16 g,
1
50%; m.p. 232－234 ℃; H NMR (500 MHz, DMSO-
d6) δ: 2.41 (s, 3H, Ph-CH3), 3.56 (dd, J＝3.5, 17.8 Hz,
1H, -C-H), 3.84 (dd, J＝8.5, 17.8 Hz, 1H, -C-H), 5.24
(dd, J＝3.5, 8.3 Hz, 1H, -S-C-H), 11.39 (s, 1H, -CO-
OH), 7.25－8.00 (m, 8H, Ph-H); 13C NMR (125 MHz,
Chin. J. Chem. 2013, 31, 1305—1314
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www.cjc.wiley-vch.de
1311

Kang et al.
FULL PAPER
DMSO-d6) δ: 29.0, 30.7, 60.2, 118.7, 123.5, 123.8,
125.3, 127.9, 130.4, 131.9, 135.2, 135.8, 163.5, 194.1;
IR (KBr) ν: 1720, 1587, 1171, 1154 cm−1; MS (70 eV)
m/z: 330.00 (M＋H)＋. Anal. calcd for C17H15NO4S: C
61.62, H 4.64, N 4.11; found C 61.99, H 4.59, N 4.25.
2.21－2.26 (m, 1H, -C-H), 2.37－2.43 (m, 1H, -C-H),
4.92－4.93 (m, 1H, -N-C-H), 5.47 (d, J＝3.5 Hz, 1H,
-N-H), 6.72－7.59 (m, 8H, Ph-H); 13C NMR (125 MHz,
CDCl3) δ: 14.1, 39.9, 43.9, 57.6, 61.5, 120.1, 121.0,
121.8, 128.2, 128.8, 129.1, 129.2, 133.3, 133.7, 170.9;
IR (KBr) ν: 3335, 1722, 1585 cm−1; MS (70 eV) m/z:
347.9 (M＋H)＋. Anal. calcd for C18H18ClNO2S: C 62.14,
H 5.31, N 3.91; found C 62.15, H 5.22, N 4.03.
General method for preparation of 2-ethoxycar-
bonyl-4-aryl-2,3,4,5-tetrahydro-1,5-benzothiazepines
9a－9f
2-Ethoxycarbonyl-4-(4-bromophenyl)-2,3,4,5-
tetrahydro-1,5-benzothiazepine (9d) Yield 0.34 g,
A trace of bromocresol green in a 2 mol/L HCl-
methanol solution was added to a solution of 2,3-
dihydro-2-ethoxycarbonyl-4-aryl-1,5-benzothiazepine
7a－7f (0.11 mmol) in dry methanol (4 mL). When the
solution turned yellow, NaBH3CN (0.7 g, 0.23 mmol)
was added. The mixture was then stirred under a N2 at-
mosphere at room temperature for 3 h. During the reac-
tion, a HCl-methanol solution was added dropwise to
maintain the yellow color. After completion of the reac-
tion (determined by TLC), the mixture was poured into
a saturated NaHCO3 solution (5 mL), and the organic
layer was separated. The aqueous layer was extracted
with three 5 mL portions of ethyl acetate. The combined
organic phases were dried with anhydrous MgSO4 and
evaporated in vacuo to obtain the crude product. The
crude mass was purified by crystallizing from methanol
or by chromatography using petroleum ether/ethyl ace-
tate (5∶1, V/V) as the eluent.
1
80%; m.p. 87－88 ℃; H NMR (500 MHz, DMSO-d6)
δ: 1.16 (t, J＝7.0 Hz, 3H, -C-CH3), 4.08 (q, J＝7.0 Hz,
2H, -CO-CH2-), 3.89 (dd, J＝4.5, 10.0 Hz, 1H, -S-C-H),
2.21－2.26 (m, 1H, -C-H), 2.37－2.43 (m, 1H, -C-H),
4.90－4.92 (m, 1H, -N-C-H), 5.48 (d, J＝3.5 Hz, 1H,
-N-H), 6.72－7.60 (m, 8H, Ph-H); 13C NMR (125 MHz,
CDCl3) δ: 14.1, 39.8, 43.8, 57.4, 61.4, 119.9, 120.7,
121.5, 121.7, 128.5, 128.7, 132.0, 133.2, 142.4, 148.7,
170.9; IR (KBr) ν: 3328, 1721, 1584 cm−1; MS (70 eV)
m/z: 391.86 (M＋H)＋. Anal. calcd for C18H18BrNO2S: C
54.90, H 4.67, N 3.42; found C 55.11, H 4.62, N 3.57.
2-Ethoxycarbonyl-4-(4-methylphenyl)-2,3,4,5-
tetrahydro-1,5-benzothiazepine (9e) Yield 0.34 g,
1
95%; m.p. 80－81 ℃; H NMR (500 MHz, DMSO-d6)
δ: 1.16 (t, J＝7.0 Hz, 3H, -C-CH3), 2.31 (s, 3H, Ph-CH3),
4.05－4.10 (m, 3H, -CO-CH2-, -S-C-H), 2.18－2.23 (m,
1H, -C-H), 2.37－2.42 (m, 1H, -C-H), 4.93－4.95 (m,
1H, -N-C-H), 5.32 (d, J＝2.5 Hz, 1H, -N-H), 6.69－
7.41 (m, 8H, Ph-H); 13C NMR (125 MHz, CDCl3) δ:
14.0, 21.0, 40.4, 44.6, 57.7, 61.3, 119.5, 120.3, 120.8,
126.6, 128.2, 129.5, 132.8, 137.5, 140.4, 148.9, 170.9;
IR (KBr) ν: 3371, 1724, 1585 cm−1; MS (70 eV) m/z:
327.90 (M＋H)＋. Anal. calcd for C19H21NO2S: C 69.38,
H 6.43, N 4.09; found C 69.69, H 6.46, N 4.28.
2-Ethoxycarbonyl-4-phenyl-2,3,4,5-tetrahydro-
1,5-benzothiazepine (9a) Yield 0.32 g, 93%; m.p.
1
85－86 ℃; H NMR (500 MHz, DMSO-d6) δ: 1.16 (t,
J＝7.0 Hz, 3H, -C-CH3), 4.03－4.10 (m, 3H, -CO-CH2-,
-S-C-H), 2.21－2.26 (m, 1H, -C-H), 2.38－2.44 (m, 1H,
-C-H), 4.96－4.98 (m, 1H, -N-C-H), 5.38 (d, J＝1.5 Hz,
1H, -N-H), 6.70－7.53 (m, 9H, Ph-H); 13C NMR (125
MHz, CDCl3) δ: 14.1, 40.4, 44.5, 58.2, 61.4, 119.7,
120.6, 121.2, 126.8, 127.9, 128.4, 129.0, 133.0, 143.4,
149.0, 171.0; IR (KBr) ν: 3393, 1723, 1587 cm−1; MS
(70 eV) m/z: 314.28 (M ＋ H) ＋ . Anal. calcd for
C18H19NO2S: C 68.48, H 6.13, N 4.44; found C 68.98, H
6.11, N 4.47.
2-Ethoxycarbonyl-4-(4-methoxyphenyl)-2,3,4,5-
tetrahydro-1,5-benzothiazepine (9f) Yield 0.27 g,
1
72%; m.p. 86－87 ℃; H NMR (500 MHz, DMSO-d6)
δ: 1.16 (t, J＝7.0 Hz, 3H, -C-CH3), 3.76 (s, 3H, Ph-
OCH3), 4.06－4.10 (m, 3H, -CO-CH2-, -S-C-H), 2.17－
2.22 (m, 1H, -C-H), 2.37－2.42 (m, 1H, -C-H), 4.94－
4.96 (m, 1H, -N-C-H), 5.29 (d, J＝2.5 Hz, 1H, -N-H),
6.68－7.44 (m, 8H, Ph-H); 13C NMR (125 MHz, CDCl3)
δ: 14.1, 40.4, 44.6, 55.2, 57.4, 61.3, 114.2, 119.5, 120.3,
120.8, 127.9, 128.3, 132.8, 135.5, 148.9, 159.1, 171.0;
IR (KBr) ν: 3365, 1728, 1581 cm−1; MS (70 eV) m/z:
343.86 (M＋H)＋. Anal. calcd for C19H21NO3S: C 66.18,
H 6.21, N 3.95; found C 66.45, H 6.16, N 4.08.
2-Ethoxycarbonyl-4-(4-fluorophenyl)-2,3,4,5-
tetrahydro-1,5-benzothiazepine (9b) Yield 0.35 g,
1
95%; m.p. 92－93 ℃; H NMR (500 MHz, DMSO-d6)
δ: 1.16 (t, J＝7.0 Hz, 3H, -C-CH3), 4.08 (q, J＝7.0 Hz,
2H, -CO-CH2-), 3.97 (dd, J＝4.5, 10.0 Hz, 1H, -S-C-H),
2.20－2.25 (m, 1H, -C-H), 2.38－2.43 (m, 1H, -C-H),
4.96－4.98 (m, 1H, -N-C-H), 5.43 (d, J＝3.0 Hz, 1H,
-N-H), 6.71－7.60 (m, 8H, Ph-H); 13C NMR (125 MHz,
CDCl3) δ: 14.0, 40.1, 44.0, 57.2, 61.3, 115.6, 115.8,
119.8, 120.5, 121.2, 128.3, 128.4, 128.5, 133.0, 139.2,
148.8, 161.1, 163.1, 170.9; IR (KBr) ν: 3402, 1736,
1587 cm−1. HRMS-ESI m/z: [M ＋ H] ＋ calcd for
C18H18FNO2S: 332.11150, found 332.10808.
Biological evaluations
Cytotoxicity and chitinase activity assays were per-
formed using an Epoch microplate spectrophotometer
(BioTek, USA). Membrane permeability was measured
using a DDS-307 conductivity meter.
2-Ethoxycarbonyl-4-(4-chlorophenyl)-2,3,4,5-
tetrahydro-1,5-benzothiazepine (9c) Yield 0.34 g,
90%; m.p. 88－90 ℃; H NMR (500 MHz, DMSO-d6)
δ: 1.16 (t, J＝7.0 Hz, 3H, -C-CH3), 4.07 (q, J＝7.0 Hz,
2H, -CO-CH2-), 3.90 (dd, J＝4.5, 10.0 Hz, 1H, -S-C-H),
Evaluation of antifungal activity
1
Inhibition zones were measured by the Espinel-
Ingroff disk diffusion method[17] at 12.5－200.0 μg of
the tested compound/disk, and the inhibition zones of an
1312
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© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2013, 31, 1305—1314

Cytotoxicity and Mode of Action of 2-Ester-substituted 1,5-Benzothiazepines
equivalent volume of solvent (DMSO) were measured
as a control. The diameter of the disk was 6 mm. Fungi
were grown in yeast peptone dextrose (YPD) liquid nu-
trient medium at 30 ℃, and the cultures were shaken at
200 r/min for 36 h. When the mid-log phase was
reached, the fungi concentration was adjusted to (1－3)
×106 colony forming units (CFU)/mL for the tests.
MIC assays were performed in sterile 96-well plates
using the method described by Sarmiento.[18] MIC was
recorded as the compound concentration that produced
100% growth reduction. MIC80 was recorded as the
concentration that produced 80% growth reduction
compared to wells with no compound present. The MIC
assays were repeated three times.
water bath for 0, 30, and 40 s. Cell viability was esti-
mated by drop-plate assays using a 10-fold dilution se-
ries. About 10 µL of the treated culture was added to
YPD nutrient agar and incubated at 30 ℃ for 72 h.
Chitinase activity Fungus cells were grown in
YPD liquid nutrient medium at 30 ℃ for 24 h. Com-
pound 7a or an equivalent volume of DMSO was then
added. Cells were harvested at 0, 4, 8, 12, 24, and 32 h.
They were washed and ground on ice until at least 95%
of cells were disrupted. Chitinase activity was measured
in mixed membrane fractions as previously described.[19]
One unit of activity was defined as the amount of en-
zyme mixture that liberated 1 μmol of reducing sugar
per minute.
MFC assays were also conducted in sterile 96-well
plates with different compound concentrations inocu-
lated with 200 μL of fungi suspension (2 × 103
CFU/mL). The plates were incubated for 36 h at 30 ℃,
and growth was visually observed. Approximately 200
μL of fungi suspension from the wells that did not show
growth were plated on nutrient agar. The MFC is the
concentration at which fungi failed to grow in the liquid
nutrient medium and nutrient agar inoculated with 200
μL of suspension. The MFC assays were repeated three
times.
Membrane permeability assay Membrane per-
meability was measured using a conductivity meter. The
cells in mid-log phase were harvested, thrice washed
with sterile distilled water, and resuspended in the same
buffer to (1－3)×106 CFU/mL. Compound 7a or an
equivalent volume of DMSO was then added. The mix-
ture was incubated at 30 ℃, and the conductivity was
measured at 0, 5, 10, 30, 60, 180, and 360 min. The
mixture was boiled, and the conductivity was measured
again. The relative permeability rate was calculated.
Statistical analysis
Evaluation of cytotoxicity
Values were recorded as the means ±SD. All data
were analyzed by the Student’s t-test. Differences were
considered statistically significant when p＜0.01.
The BV2 cells were cultured at 37 ℃ in 96-well
plates at a density of 4×104 cells per well with 5% CO2
in Dulbecco’s Modified Eagle Medium (Invitrogen,
GIBCO) supplemented with 10% fetal bovine serum
(Invitrogen) for 72 h. Compounds 7a, 7b and flucona-
zole were added to each well at final concentrations of
10.0, 20.0, 50.0, 100.0, 200.0, and 500.0 μg/mL. An
equivalent volume of DMSO without the compound was
added as a control. The cells were incubated for 48 h.
Subsequently, 10 μL of aqueous MTT solution (5.0
mg/mL) and the mixture were incubated at 37 ℃ for 4
h. The MTT solution was carefully decanted off, and
100 μL of DMSO was added to each well. The color
was measured with an Epoch microplate spectropho-
tometer at 490 nm with the reference filter set to 620 nm.
All MTT assays were repeated three times. Each meas-
urement contained six parallel treatments (wells).
relative survival rate＝(treatment A490/negative con-
trol A620)×100%
Acknowledgement
The authors gratefully acknowledge the financial
support of the National Natural Science Foundation of
China (No. 20972040).
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Chin. J. Chem. 2013, 31, 1305—1314