Synthesis and anti-proliferative activity evaluation of novel benzo[d][1,3] dioxoles-fused 1,4-thiazepines

Article abstract of DOI:10.1016/j.ejmech.2017.01.021

Benzo[d][1,3]dioxoles 1,4-thiazepines remarkable antitumor activities, benzo[d][1,3]dioxoles-fused 1,4-thiazepines, which combine two biologically active heterocyclic cores, are expected to be of pharmacological interest, We therefore envisaged that integrating 1,4-thiazepine and benzo[d][1,3]dioxole moieties in one molecular platform could potentially produce novel compounds with significant synergistic antitumor properties. A series of novel benzo[d][1,3]dioxoles-fused 1,4-thiazepines, designed via molecular hybridization approach, were synthesized in very good yields using one-pot condensation of 3,4-methylenedioxyaniline, aldehydes, and α-mercaptocarboxylic acids under solvent-free condition. The anti-proliferative activities of all the synthesized compounds were assessed on two different human cancer cell lines (Esophageal squamous cell carcinoma Ec9706 and Eca109), and the results showed that compound 4e showed the best anti-tumor activity with IC₅₀values of 8.23 μM and 16.22 μM against Ec9706 and Eca109 cell lines, respectively, which was 2–3 times more potent than 5-Fluorouracil (IC₅₀= 23.26 μM and 30.25 μM against Ec9706 and Eca109 respectively). These novel benzo[d][1,3]dioxoles fused with bioactive heterocyclic skeletons may find their pharmaceutical applications after further investigations.

Full text of DOI:10.1016/j.ejmech.2017.01.021

Accepted Manuscript
Synthesis and anti-proliferative activity evaluation of novel benzo[d][1,3] dioxoles-
fused 1,4-thiazepines
Liqiang Wu, Xiaojuan Yang, Qiujv Peng, Guangfei Sun
PII:
S0223-5234(17)30029-6
10.1016/j.ejmech.2017.01.021
EJMECH 9172
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 September 2016
Revised Date: 10 January 2017
Accepted Date: 13 January 2017
Please cite this article as: L. Wu, X. Yang, Q. Peng, G. Sun, Synthesis and anti-proliferative activity
evaluation of novel benzo[d][1,3] dioxoles-fused 1,4-thiazepines, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.01.021.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and anti-proliferative activity evaluation of novel benzo[d][1,3]
dioxoles-fused 1,4-thiazepines
Liqiang Wua,* Xiaojuan Yangb Qiujv Penga Guangfei Suna
, , ,
aSchool of Pharmacy, Xinxiang Medical University, Xinxiang 453003, China
bCollege of Chemistry and Chemical Engineering, Xinxiang University, Xinxiang, Henan 453003, China
ABSTRACT
Benzo[d][1,3]dioxoles
1,4-thiazepines
remarkable
antitumor
activities,
benzo[d][1,3]dioxoles-fused 1,4-thiazepines, which combine two biologically active heterocyclic cores,
are expected to be of pharmacological interest, We therefore envisaged that integrating 1,4-thiazepine
and benzo[d][1,3]dioxole moieties in one molecular platform could potentially produce novel
compounds with significant synergistic antitumor properties.
A
series of novel
benzo[d][1,3]dioxoles-fused 1,4-thiazepines, designed via molecular hybridization approach, were
synthesized in very good yields using one-pot condensation of 3,4-methylenedioxyaniline, aldehydes,
and α-mercaptocarboxylic acids under solvent-free condition. The anti-proliferative activities of all the
synthesized compounds were assessed on two different human cancer cell lines (Esophageal squamous
cell carcinoma Ec9706 and Eca109), and the results showed that compound 4e showed the best
anti-tumor activity with IC50 values of 8.23 µM and 16.22 µM against Ec9706 and Eca109 cell lines,
respectively, which was 2-3 times more potent than 5-Fluorouracil (IC50 = 23.26 µM and 30.25 µM
against Ec9706 and Eca109 respectively). These novel benzo[d][1,3]dioxoles fused with bioactive
heterocyclic skeletons may find their pharmaceutical applications after further investigations.
Keywords: Benzo[d][1,3]dioxoles; 1,4-Thiazepines; Molecular hybridization; Anti-proliferative;
Solvent-free
*Corresponding author. Tel.: +86 373 3831652; fax: +86 373 3029879, e-mail: wliq1974@163.com

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1. Introduction
With more than 10 million new cases every year, cancer has become one of the most devastating
diseases worldwide. Despite the efforts to discover and develop small molecule anticancer drugs in the
last decade [1], the development of new antitumor agents with improved tumor selectivity, efficiency,
and safety remains desirable.
Benzo[d][1,3]dioxole ring system present in various natural active substances [2-4]. The
existence of a benzo[d][1,3]dioxole structural unit is a key to the pharmacological activities of many
natural and synthetic drugs. A large number of compounds having 1,3-benzodioxole ring system has
been reported to possess different kind of biological activities like anticancer [5], anticonvulsant [6],
anti-inflammatory [7], antidepressant [8]. Recently, benzo[d][1,3]dioxoles include podophyllotoxin [9],
4-aza-podophyllotoxin [10], dicentrine [11], liriodenine [11] and nitidine [12] have been reported to
show remarkable antitumor activities through the inhibition of topoisomerase. Some of these
compounds exhibited topoisomerase II inhibition, leading to the development of etoposide [13], a drug
used for the treatment of cancer.
Heterocycles containing the 1,4-thiazepine moiety are important targets in synthetic and medicinal
chemistry because this fragment is a key moiety in a wide number of natural and synthetic biologically
active agents [14]. Among them, aryl- and heteroaryl- fused derivatives of this heterocycle represent an
important group of compounds with interesting pharmaceutical properties [15]. In particular, different
alkyl derivatives of 3,4-dihydro-5-oxo-1,4-benzothiazepine were described as antitumor agents [16].
Numerous methods for the synthesis of fused 1,4-thiazepine derivatives in recent years have been
reported with respect to their different structures [17-19].

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There has been significant interest in the combination of two pharmacophores on the same scaffold
leading to hybrid molecules [20]. These hybrids combine two active moieties in a single molecule with
the goal of creating a chemical entity that is medically more effective than its individual components.
As a result, benzo[d][1,3]dioxoles-fused 1,4-thiazepines, which combine two biologically active
heterocyclic cores, are expected to be of pharmacological interest, We therefore envisaged that
integrating 1,4-thiazepine and benzo[d][1,3]dioxole moieties in one molecular platform could
potentially produce novel compounds with significant synergistic antitumor properties. These hybrid
molecules are proposed to target the inhibition of DNA topoisomerase (Figure 1).
< Figure 1>
These new benzo[d][1,3] dioxoles-fused 1,4-thiazepines (4a–4r) were prepared via one-pot
three-component cyclocondensation reaction between 3,4-methylenedioxyaniline, aldehydes, and
α-mercaptocarboxylic acids at 110 oC under solvent-free conditions (Scheme 1).
<Scheme 1>
2. Results and Discussion
First, to find the optimal solvent for the synthesis of benzo[d][1,3]dioxoles possessing
1,4-thiazepine scaffolds, we tested the three-component condensation reaction of
3,4-methylenedioxyaniline, benzaldehyde, and 2-mercaptoacetic acid as a simple model system at
o
reflux temperature 110 C or using EtOH, MeOH, H2O, CH2Cl2, CH3COOH and DMF as solvent or
under solvent-free condition. It is shown in Table 1 that the reaction under solvent-free condition gave
the corresponding product 4a in high yield (Table 1, entry 7). Furthermore, the relation between the
yields of the model reaction and temperature was also studied. We carried out the reaction at
temperatures ranging from 25 oC to 120 oC under solvent-free condition (Table 1, entries 7–10),
finding that the best result was obtained at 110 °C under solvent-free condition.

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< Table 1>
Using these optimized reaction conditions, the title compounds 4 were prepared via one-pot
three-component cyclocondensation reaction between 3,4-methylenedioxyaniline, aldehydes, and
o
α-mercaptocarboxylic acids at 110 C under solvent-free conditions. From this approach the novel
benzo[d][1,3]dioxoles-fused 1,4-thiazepines 4 were isolated by chromatography and in good yields
(31–70%) as stable solids (Table 2). Structures of compounds 4 were characterized by spectroscopic
and analytical methods. IR spectrum of 4a showed absorptions at 3157, 1663, 1477 and 1361
cm-1indicating the presence of O=C-NH bond. The high resolution mass spectrum of 4a displayed the
quasi-molecular ion ([M+Na] +) peak at m/z = 322.0512, which was consistent with the 1 : 1 : 1 adduct
of 3,4- methylenedioxyaniline, benzaldehyde, and 2-mercaptoacetic acid with the loss of two water
1
molecule. The H NMR spectrum of 4a showed four singlet was observed (δ = 5.58, 6.36, 6.64, 9.44
ppm) for the H-9, H-4, H-10 and NH group, respectively. There were two doublets at δ = 3.15, 2.91
ppm for CH2 of 1,4-thiazepine, which probably arise from geminal proton-proton coupling of
methylene group. The 13C NMR spectrum of 4a showed characteristic signals at δ = 31.4 (generated by
methylene group of 1,4-thiazepine ), 47.3 ppm (due to the Ar–CH group), and 169.2 ppm (arising from
the carbonyl groups).
< Table 2>
The formation of isomeric systems (Figure 2) is possible in the reaction. The HMBC experiments
can observe typical two-bond and three-bond proton to carbon couplings. It could be observe that the
long-range correlations of C-10 to the protons of H-9, carbonyl carbons C-6 to the protons of CH3, C-9
to the protons of the 4-nitrophenyl in HMBC spectrum of 4o, which was able to prove that the
formation of the benzo[d][1,3]dioxoles-fused 1,4-thiazepine units in the reaction (see Supplementary
data).

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<Figure 2>
The biological activities of this series of benzo[d][1,3]dioxoles-fused 1,4-thiazepines were evaluated
by an anti-proliferative assay, which was carried out in a panel of two human tumor cell lines
comprising Ec9706 and Eca109 by using the MTT method. The results are summarized in Table 3 and
Figure 3, and compared to 5-fluorouracil. It is observed that most of the compounds are significantly
anti- proliferative. It appears from the data that introduction of 3,5-(MeO)2-C6H3 or 3-Br-4-MeO-C6H3
group into the C-9 position was found to be quite favorable for increasing anti-proliferative activity as
seen in compounds 4d, 4e for the two cancer cell line. Among the 18 new compounds screened, 4e has
pronounced activity.
<Table 3>
<Figure 3>
3. Conclusion
In conclusion, the efficient and straightforward preparation of
benzo[d][1,3]dioxoles-fused 1,4-thiazepines was described, using a one-pot multicomponent approach
employing 3,4- methylenedioxyaniline, aldehydes, and α-mercaptocarboxylic acids under
a
series of
solvent-free condition. Subsequent biological assessment pointed out the relevance of a number of
these novel scaffolds in terms of their anti-proliferative activity, in general most of the new compounds
possessed good deal of anti-proliferative activity against Ec9706 and Eca109 cell lines. On the whole
among all the compounds tested, compound 4e exhibited showed the best antitumor activity agains the
two human cell lines. Therefore, these novel benzo[d][1,3]dioxoles fused with bioactive heterocyclic
skeletons may find their pharmaceutical applications after further investigations.
4. Experimental

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4.1. General
IR spectra were determined on FTS-40 infrared spectrometer. NMR spectra were determined on
Bruker AV-400 spectrometer at room temperature using TMS as internal standard. Chemical shifts (d)
are given in ppm and coupling constants (J) in Hz. Elemental analysis were performed by a Vario-III
elemental analyzer. Melting points were determined on a XT-4 binocular microscope and were
uncorrected. Commercially available reagents were used throughout without further purification unless
otherwise stated.
4.2. General procedure for the synthesis of compounds 4
To
a
mixture of 3,4-methylenedioxyaniline (1 mmol) and aldehydes (1 mmol),
α-mercaptocarboxylic acids (1 mmol) was added. The mixture was stirred at 110 °C for an appropriate
time (Table 1). After completion of the reaction (TLC), the reaction mixture was treated with CH2Cl2
(20 mL), filtered and the solvent evaporated in vacuo. Solvent was evaporated and the crude product
puried by silica gel column chromatography using dichloromethane: ethyl acetate (v:v = 5:1) as eluent
to afford the pure product 4.
4.2.1. 9-Phenyl-5,9-dihydro-1,3-dioxa-8-thia-5-aza-cyclo-hepta[f]inden-6-one (4a)
Pale yellow powder, m.p. 213-215 oC, 1H NMR (400 MHz, DMSO-d6) δ: 9.44 (s, 1H), 7.49-7.36 (m,
5H), 6.64 (s, 1H), 6.36 (s, 1H), 6.01 (d, 2H, J = 10.4 Hz), 5.58 (s, 1H), 3.15 (d, 1H, J = 12.0 Hz), 2.91
(d, 1H, J = 12.0 Hz); 13C NMR (100 MHz, DMSO-d6) δ: 169.2, 147.2, 146.0, 138.3, 131.8, 130.0,
129.1, 128.5, 127.6, 107.7, 105.6, 102.2, 47.3, 31.43; IR (KBr): 2877, 1663, 1035, 745 cm-1;
HRMS-ESI (m/z): calcd for C16H13NNaO3S [M+Na]+ 322.0514, found: 322.0512.
4.2.2. 9-(4-Nitro-phenyl)-5,9-dihydro-1,3-dioxa-8-thia-5-aza-cyclo-hepta[f]inden-6-one (4b)
o
1
Yellow powder, m.p.215-216 C, H NMR (400 MHz, DMSO-d6) δ: 9.23 (s, 1H), 8.23 (d, 2H, J =
8.8 Hz), 6.71 (s, 1H), 6.61 (s, 1H), 6.05 (d, 2H, J = 7.2 Hz), 5.66 (s, 1H), 3.08 (d, 1H, J = 12.4 Hz),

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3.02 (d, 1H, J = 12.4 Hz); 13C NMR (100 MHz, DMSO-d6) δ: 169.2, 147.7, 147.6, 146.1, 130.2,
127.23, 123.93, 108.8, 106.1, 102.4, 47.3, 31.0; IR (KBr): 2926, 1666, 1035, 698 cm-1; HRMS-ESI
(m/z): calcd for C16H12N2NaO5S [M+Na]+ 367.0365, found: 367.0360.
4.2.3. 9-(3-Nitro-phenyl)-5,9-dihydro-1,3-dioxa-8-thia-5-aza-cyclo-hepta[f]inden-6-one (4c)
o
1
Yellow powder, m.p. 206-208 C, H NMR (400 MHz, DMSO-d6) δ: 9.24 (s, 1H), 8.33 (s, 1H),
8.19-8.17 (m, 1H), 7.85 (d, 1H, J = 8.0 Hz), 6.74 (s, 1H), 6.61 (s, 6H), 6.05 (d, 2H, J = 9.6 Hz), 5.68 (s,
13
1H), 3.08 (d, 1H, J = 12.4 Hz), 3.04 (d, 1H, J = 12.4 Hz); C NMR (100 MHz, DMSO-d6) δ: 169.2,
148.3, 147.7, 146.1, 142.1, 135.6, 131.9, 130.4, 127.3, 122.9, 108.9, 106.1, 102.3, 46.9, 30.9; IR (KBr):
2889, 1668, 1034, 719 cm-1; HRMS-ESI (m/z): C16H12N2NaO5S [M+Na]+ 367.0365, found: 367.0366.
4.2.4. 9-(3,5-Dimethoxy-phenyl)-5,9-dihydro-1,3-dioxa-8-thia-5-aza-cyclo-hepta[f]inden-6-one (4d)
o
1
Yellow powder, m.p. 205-206 C, H NMR (400 MHz, DMSO-d6) δ: 9.41 (s, 1H), 6.63 (d, 3H, J =
2.4 Hz), 6.48 (d, 2H, J = 11.6 Hz), 6.01 (d, 2H, J = 7.2 Hz), 5.50 (s, 1H), 3.74 (s, 6H), 3.14 (d, 1H, J =
13
12.0 Hz), 2.80 (d, 1H, J = 12.0 Hz); C NMR (100 MHz, DMSO-d6) δ: 169.2, 161.0, 147.3, 146.0,
140.6, 131.7, 127.4, 107.6, 107.5, 105.5, 102.1, 100.1, 55.7, 47.4, 31.2；IR (KBr): 2928, 1671, 1155,
728 cm-1; HRMS-ESI (m/z): calcd for C18H17NNaO5S [M+Na]+ 382.0725, found: 382.0727.
4.2.5. 9-(3-Bromo-4-methoxy-phenyl)-5,9-dihydro-1,3-dioxa-8-thia-5-aza-cyclo-hepta[f]inden-6-one
(4e)
o
1
Yellow powder, m.p. 219-221 C, H NMR (400 MHz, DMSO-d6) δ: 9.36 (s, 1H), 7.65 (d, 1H, J =
2.0 Hz), 7.48-7.46 (m, 1H), 7.15 (d, 2H, J = 8.4 Hz), 6.62 (s, 1H), 6.46 (s, 1H), 6.02 (d, 2H, J = 8.4
13
Hz), 5.53 (s, 1H), 3.87(s, 3H), 3.11 (d, 1H, J = 12.0 Hz), 2.93 (d, 1H, J = 12.0 Hz); C NMR (100
MHz, DMSO-d6) δ: 169.2, 161.0, 147.3, 146.0, 140.6, 131.7, 127.4, 107.6，107.5, 105.5, 102.1, 100.1,
55.7, 47.4, 31.2；IR(KBr):2928, 1671, 1155, 728 cm-1; HRMS-ESI (m/z): calcd for C17H14BrNNaO4S
[M+Na]+ 429.9725, found: 429.9716.

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4.2.6. 9-(Furan-2-yl)-5,9-dihydro-1,3-dioxa-8-thia-5-aza-cyclo-hepta[f]inden-6-one (4f)
Pale yellow powder, m.p. 218-220 oC, 1H NMR (400 MHz, DMSO-d6) δ: 9.43 (s, 1H), 7.68 (s, 1H),
6.64 (s, 1H), 6.52 (t, 3H，J = 9.6 Hz), 6.04 (d, 2H，J = 5.6 Hz), 5.53 (s, 1H), 3.07 (d, 1H, J = 12.4 Hz),
2.94 (d, 1H, J = 12.4 Hz); 13C NMR (100 MHz, DMSO-d6) δ: 169.1, 151.4, 147.7, 146.1, 143.8, 131.7,
125.7, 111.2, 109.8, 107.5, 102.3, 67.5, 31.1; IR (KBr): 2919, 1672, 1033, 735 cm-1; HRMS-ESI (m/z):
calcd for C14H11NNaO4S [M+Na]+ 312.0306, found: 312.0303.
4.2.7. 9-(Thien-2-yl)-5,9-dihydro-1,3-dioxa-8-thia-5-aza-cyclo-hepta[f]inden-6-one (4g)
Pale yellow powder, m.p. 224-226 oC, 1H NMR (400 MHz, DMSO-d6) δ: 9.42 (s, 1H), 7.56-7.54 (m,
1H), 7.15 (d, 1H, J = 3.2 Hz), 7.08-7.06 (m, 1H), 6.64 (s, 1H), 6.55 (s, 1H), 6.03 (d, 2H, J = 6.4 Hz),
5.79 (s, 1H)，3.14 (d, 1H，J = 12.4 Hz), 2.97 (d, 1H, J = 12.0 Hz); 13C NMR (100 MHz, DMSO-d6) δ:
169.0, 147.5, 146.0, 142.3, 131.7, 127.8, 127.7, 127.6, 126.7, 107.4, 105.7, 102.3, 42.6, 31.7; IR (KBr):
2873, 1666, 1033, 697 cm-1; HRMS-ESI (m/z): calcd for C14H11NNaO3S [M+Na]+ 328.0078, found:
328.0079.
4.2.8. 9-(4-Chloro-phenyl)-5,9-dihydro-1,3-dioxa-8-thia-5-aza-cyclo-hepta[f]inden-6-one (4h)
Pale yellow powder, m.p. 221-223 oC, 1H NMR (400 MHz, DMSO-d6) δ: 9.34 (s, 1H), 7.50-7.44 (m,
4H), 6.62 (s, 1H), 6.49 (s, 1H)，6.04-6.02 (d, 2H, J = 7.6 Hz), 5.56 (s, 1H)，3.11-3.08 (d, 1H, J = 12.0
Hz)，2.96 (d, 1H，J = 12.4 Hz); 13C NMR (100 MHz, DMSO-d6) δ: 169.2, 147.4, 137.9, 132.8, 131.8,
131.1, 129.0, 127.4, 108.1, 105.8, 102.2, 46.8, 31.2; IR (KBr): 2898, 1672, 1037, 780 cm-1;
HRMS-ESI (m/z): calcd for C16H12ClNNaO3S [M+Na]+ 356.0124 , found: 356.0116.
4.2.9. 9-(4-Methoxy-phenyl)-5,9-dihydro-1,3-dioxa-8-thia-5-aza-cyclo-hepta[f]inden-6-one (4i)
Pale yellow powde, m.p. 211-213 oC, 1H NMR (400 MHz, DMSO-d6) δ: 9.42 (s, 1H), 7.41 (d, 2H, J
= 8.8 Hz), 6.98 (d, 2H, J = 8.4 Hz), 6.64 (s, 1H), 6.33 (s, 1H), 6.01 (d, 2H, J = 8.8 Hz), 5.53 (s, 1H),
3.78 (s, 3H), 3.17 (d, 1H，J = 12.0 Hz), 2.86 (d, 1H, J = 12.0 Hz); 13C NMR (100 MHz, DMSO-d6) δ:

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169.3, 159.4, 147.1, 146.0, 131.6, 130.8, 129.7, 127.9, 114.5, 107.5, 105.5, 102.2, 55.6, 46.6, 31.6; IR
(KBr): 2900, 1665, 1033, 795 cm-1; HRMS-ESI (m/z): calcd for C17H16NO4S [M+H]+ 330.0800, found:
330.0797.
4.2.10. 9-(4-Methyl-phenyl)-5,9-dihydro-1,3-dioxa-8-thia-5-aza-cyclo-hepta[f]inden-6-one (4j)
Pale yellow powder, m.p. 247-249 oC, 1H NMR (400 MHz, DMSO-d6) δ: 9.42 (s, 1H), 7.36 (d, 2H,
J = 7.6 Hz), 7.22 (d, 2H, J = 7.6 Hz), 6.64 (s, 1H), 6.34 (s, 1H), 6.00 (d, 2H, J = 9.2 Hz), 5.53 (s, 1H),
3.15 (d, 1H, J = 12.4 Hz), 2.88 (d, 1H, J = 12.0 Hz); 13C NMR (100 MHz, DMSO-d6) δ: 169.2, 147.2,
146.0, 137.8, 135.1, 131.7, 129.7,129.4, 127.8, 107.6, 105.5, 102.2, 47.0, 31.5, 21.2; IR (KBr): 2923,
1674, 1035, 773 cm-1; HRMS-ESI (m/z): calcd for C17H16NO3S [M+H]+ 314.0851 , found: 314.0848.
4.2.11. 9-(3-Methoxy-phenyl)-5,9-dihydro-1,3-dioxa-8-thia-5-aza-cyclo-hepta[f]inden-6-one (4k)
Pale yellow powder, m.p. 198-200 oC, 1H NMR (400 MHz, DMSO-d6) δ: 9.41 (s, 1H), 7.33 (t, 1H，J
= 8.0 Hz), 7.04 (d, 2H, J = 6.8 Hz), 6.92 (t, 1H, J = 6.8 Hz), 6.64 (s, 1H), 6.42(s, 1H), 6.01 (d, 2H, J =
8.4 Hz), 5.54 (s, 1H), 3.76 (s, 3H), 3.14 (d, 1H, J = 12.0 Hz), 2.91 (d, 1H, J = 12.0 Hz); 13C NMR (100
MHz, DMSO-d6) δ: 169.2, 159.9, 147.1, 147.3, 146.0, 139.9, 131.7, 130.2, 127.5, 121.7, 115.1, 113.9,
107.7, 105.6, 102.2, 55.5, 47.3, 31.4; IR (KBr): 2905, 1633, 1034, 764 cm-1; HRMS-ESI (m/z): calcd
for C17H16NO3S [M+H]+ 330.0800, found: 330.0795.
4.2.12. 9-(2-Methyl-phenyl)-5,9-dihydro-1,3-dioxa-8-thia-5-aza-cyclo-hepta[f]inden-6-one (4l)
Pale yellow powder, m.p. 239-241 oC, 1H NMR (400 MHz, DMSO-d6) δ: 9.60 (s, 1H), 7.62 (d, 1H,
J = 7.2 Hz), 7.36-7.24 (m, 3H), 6.69 (s, 1H), 5.99 (d, 3H, J = 5.6 Hz), 5.73(s, 1H), 3.19 (d, 1H, J =
13
12.0 Hz), 2.99-2.88 (m, 1H), 2.01 (d, 3H); C NMR (100 MHz, DMSO-d6) δ: 169.2, 147.3, 146.2,
136.5, 136.0, 131.9, 131.4, 128.9, 128.5, 126.8, 126.7, 106.5, 105.3, 102.2, 43.8, 31.4, 19.2; IR (KBr):
2891, 1665, 1035, 752 cm-1; HRMS-ESI (m/z): calcd for C17H15NNaO3S [M+Na]+ 336.0670 , found:
336.0668.

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4.2.13.
9-(3,4,5-Trimethoxy-phenyl)-5,9-dihydro-1,3-dioxa-8-thia-5-aza-cyclo-hepta[f]inden-6-one
(4m)
Pale yellow powder, m.p. 251-253 oC, 1H NMR (400 MHz, DMSO-d6) δ: 9.44 (s, 1H), 6.80 (s, 2H),
6.64 (s, 1H), 6.49 (s, 1H), 6.01 (d, 2H, J = 7.6 Hz), 5.53 (s, 1H), 3.77(s, 6H), 3.69 (s, 3H), 3.17 (d, 1H,
J = 12.0 Hz), 2.88 (d, 1H，J = 12.0 Hz); 13C NMR (100 MHz, DMSO-d6) δ: 169.3, 153.3, 147.2, 146.0,
137.4, 133.6, 131.6, 127.5, 107.7, 106.9, 105.5, 102.1, 60.5, 47.6, 31.4; IR (KBr): 2892，1667，1034，
753 cm-1; HRMS-ESI (m/z): calcd for C19H19NNaO6S [M+Na]+ 412.0831 , found: 412.0826.
4.2.14. 9-(4-Floro-phenyl)-5,9-dihydro-1,3-dioxa-8-thia-5-aza-cyclo-hepta[f]inden-6-one (4n)
Pale yellow powder, m.p. 228-230 oC, 1H NMR (400 MHz, DMSO-d6) δ: 9.39 (s, 1H), 7.54-7.50 (m,
2H), 7.24 (t, 2H, J = 7.6 Hz), 6.64 (s, 1H), 6.41 (s, 1H), 6.01 (d, 2H, J = 8.4 Hz), 5.58 (s, 1H)，3.13 (d,
1H，J = 12.0 Hz), 2.93 (d, 1H, J = 12.0 Hz); 13C NMR (100 MHz, DMSO-d6) δ: 169.2, 160.8, 147.3,
146.0, 134.7, 131.8, 131.5, 131.4, 127.5, 116.0, 115.8, 107.8, 105.7, 102.2, 46.5, 31.4; IR (KBr): 2892,
1667, 1035, 753 cm-1; HRMS-ESI (m/z): calcd for C16H12FNNaO3S [M+Na]+ 340.0420, found:
340.0413.
4.2.15.
7-Methyl-9-(4-nitro-phenyl)-\5,9-dihydro-1,3-dioxa-8-thia-5-aza-cyclo-hepta[f]inden-6-one
(4o)
Pale yellow powder, m.p. 258-260 oC, 1H NMR (400 MHz, DMSO-d6) δ: 9.63 (s, 1H), 8.29 (d, 2H,
J = 7.6 Hz), 7.79 (d, 2H, J = 8.4 Hz), 6.70 (s, 1H), 6.10 (s, 1H), 5.99 (d, 2H, J = 10.0 Hz), 5.85 (s, 1H),
13
3.49-3.44 (m, 1H), 1.17 (d, 3H, J = 6.4 Hz); C NMR (100 MHz, DMSO-d6) δ: 170.9, 147.7, 147.3,
146.2, 145.0, 131.4, 131.3, 127.0, 124.4, 106.6, 105.8, 102.3, 46.8, 36.5, 13.9; IR (KBr): 2899, 1671,
1034, 800 cm-1; HRMS-ESI (m/z): calcd for C17H14N2NaO5S [M+Na]+ 381.0521 , found: 381.0513.
4.2.16. 7-Methyl-9-(4-chloro-phenyl)-5,9-dihydro-1,3-dioxa-8-thia-5-aza-cyclo-hepta[f]inden-6-one
(4p)

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Pale yellow powder, m.p. 244-245 oC, 1H NMR (400 MHz, DMSO-d6) δ: 9.59 (s, 1H), 7.55-7.49 (m,
4H), 6.68 (s, 1H), 6.13 (s, 1H), 5.99 (d, 2H, J = 7.6 Hz), 5.68 (s, 1H)，3.45-3.40 (m, 1H), 1.15 (d, 3H, J
13
= 6.4 Hz); C NMR (100 MHz, DMSO-d6) δ: 171.1, 147.2, 146.1, 136.3, 133.3, 131.8, 131.2, 129.3,
127.7, 106.6, 105.8, 102.2, 46.7, 36.5, 13.9; IR (KBr): 2893，1668，1036，788 cm-1; HRMS-ESI (m/z):
calcd for C17H15ClNO3S [M+H]+ 348.0461 , found: 348.0462.
4.2.17. 7-Methyl-9-(4-methyl-phenyl)-5,9-dihydro-1,3-dioxa-8-thia-5-aza-cyclo-hepta[f]inden-6-one
(4q)
Pale yellow powder, m.p. 229-231 oC, 1H NMR (400 MHz, DMSO-d6) δ: 9.56 (s, 1H), 7.37 (d, 2H，
J = 8.0 Hz), 6.67 (s, 1H), 6.16 (s, 1H), 5.98 (d, 2H, J = 7.6 Hz), 5.60 (s, 1H), 3.43-3.48 (m, 1H), 2.34
(s, 3H), 1.15 (d, 3H, J = 6.4 Hz); 13C NMR (100 MHz, DMSO-d6) δ: 171.2, 147.0, 146.0, 138.2, 134.1,
131.1, 129.9, 129.8, 128.3, 106.7, 105.7, 102.1, 47.3, 36.4, 21.2, 13.9; IR (KBr): 2893; 1671, 1075,
786 cm-1; HRMS-ESI (m/z): calcd for C18H18NO3S [M+H]+ 328.1007 , found: 328.1007.
4.2.18. 7-Methyl-9-(3-nitro-phenyl)-5,9-dihydro-1,3-dioxa-8-thia-5-aza-cyclo-hepta[f]inden-6-one (4r)
o
1
Yellow powder, m.p. 235-237 C, H NMR (400 MHz, DMSO-d6) δ: 9.61 (s, 1H), 8.37 (s, 1H),
8.26-8.24 (m, 1H), 7.95 (d, 1H，J = 7.6 Hz), 7.73 (t, 2H, J = 8.0 Hz), 6.70 (s, 1H), 6.16 (s, 1H), 5.99 (d,
2H, J = 9.2 Hz), 5.90 (s, 1H), 3.50-3.45 (m, 1H), 1.17 (d, 3H, J = 6.4 Hz); 13C NMR (100 MHz,
DMSO-d6) δ: 170.9, 148.5, 147.3, 146.2, 139.5, 136.6, 131.4, 130.9, 127.2, 124.6, 123.8, 106.8, 105.8,
102.2, 46.5, 36.5, 13.9; IR (KBr): 2897, 1674, 1039, 734 cm-1; HRMS-ESI (m/z): calcd for
C17H15N2O5S [M+H]+ 359.0702 , found: 359.0698.
4.3. Anti-proliferative assay
Ec9706 cells and Eca109 cells were maintained in DMEM (Gibco, Invitrogen Corporation, NY,
USA) medium supplemented with 10% FBS, streptomycin (100 µg/mL) and penicillin (100 units/mL)
and incubated at 37 oC, 5% CO2. HCT116 cells (5000 cells/well) and HepG2 cells (10000 cells/well)

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were seeded into 96-well plates and incubated at 37 oC in 5%CO2/95% air condition. Serially twofold
diluted test compound solutions of each drug were added 24 h later, and the cells were incubated for
the next 48 h. The final concentrations of compounds in the sample wells ranged from 5 µM to 160
µM. After 48 h, 20 mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, 5
mg/mL) was added to each well and the cells were incubated for an additional 4 h. Then, 100 µL
DMSO were added into each well for dissolving the intracellular formazan crystals. Optical density at
570 nm of each plate was measured with a tunable microplate reader. Each group was in triplicate
samples and each drug was divided into at least 5 concentrations (5 µM, 10 µM, 20 µM, 40 µM and 80
µM or 10 µM, 20 µM, 40 µM, 80 µM and 160 µM). The percentage of absorbance from the
sample-treated cells compared to that of the vehicle control (treated with DMSO) was calculated. The
resulting cytotoxic acitvities were expressed as IC50 values and IC50 values were determined by
analysis software (Microsoft Excel).
Acknowledgements
This work is financially supported by NSFC-Henan Joint Fund (No. U1604164), Key Scientific
Research Projects in Universities of Henan Province (No. 16A350010, 15A350011) and Scientfic
Research Fund of Xinxiang Medical University (No. 2013QN130).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
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5005.
Captions
Figure 1. Design of benzo[d][1,3]dioxoles-fused 1,4-thiazepines.
Scheme 1.
Table 1. Reaction condition optimization for the synthesis 4a
Table 2. Synthesis of benzo[d][1,3] dioxoles-fused 1,4-thiazepines
Figure 2. Compound and two possible isomeric systems of 4o.
Table 3. Anti-proliferative activities of compounds 4.
Figure 3. IC50 curve of compound 4

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OH
H
N
O
O
O
O
O
O
O
O
N
O
CH3
H
O
H3CO
H3CO
OCH3
OCH3
H3CO
OCH3
OCH3
OCH3
Dicentrine
4-Aza-podophyllotoxin
Podophyllotoxin
O
O
O
N
O
H3CO
H3CO
O
N+
R1
S
O
O
Molecular
Hybridization
R2
N
H
Liriodenine
Nitidine
O
Benzo[d][1,3] dioxoles-fused 1,4-thiazepines
Benzo[d][1,3]dioxoles
(Topoisomerase inhibitors)
O
NH
S
3,4-Dihydro-5-oxo-1,4-benzothiazepine
(Antitumor acitivitiy)
Figure 1. Design of benzo[d][1,3]dioxoles-fused 1,4-thiazepines.

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1
S
R2
O
O
O
O
+
R2
+
neat, 110 oC
OH
R1CHO
HS
N
H
NH2
O
O
1
2
3
4
Scheme 1.

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Table 1. Reaction condition optimization for the synthesis 4a
Entry
Solvent
Temperature Time/ h
Yield/ %a
1
2
EtOH
reflux
reflux
reflux
reflux
110
6
8
31
19
5
MeOH
3
H2O
6
4
CH2Cl2
6
0
5
CH3COOH
4
36
35
43
0
6
DMF
110
3
7
-
-
-
-
110
3
8
25
10
4
9
100
31
43
10
120
3
a Isolated yield.

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Table 2. Synthesis of benzo[d][1,3] dioxoles-fused 1,4-thiazepines
Product
4a
4b
4c
R1
C6H5
R2
H
Time/ h
Yield/ %a
43
3
4
4
3
3
4
3
3
3
3
3
3
3
3
5
3
4
4
4-NO2-C6H4
3-NO2-C6H4
3,5-(MeO)2-C6H3
3-Br-4-MeO-C6H3
C4H3O
H
31
H
45
H
47
4d
4e
H
45
H
36
4f
C4H3S
H
70
4g
4h
4i
4-Cl-C6H4
H
58
4-MeO-C6H4
4-CH3-C6H4
3-MeO-C6H4
2-CH3-C6H4
3,4,5-(MeO)3-C6H2
4-F-C6H4
H
50
H
58
4j
H
38
4k
4l
H
57
H
47
4m
4n
4o
4p
4q
4r
H
42
4-NO2-C6H4
4-Cl-C6H4
CH3
CH3
CH3
CH3
35
47
4-CH3-C6H4
3-NO2-C6H4
53
39
a Isolated yield.

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O2N
O2N
S
S
O
N
NO2
S
O
O
O
N
O
H
O
O
N
H
O
O
4o
Two possible isomeric systems of 4o
Figure 2. Compound and two possible isomeric systems of 4o.

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Table 3. Anti-proliferative activities of compounds 4.
IC50 (µM)
Compound
Ec9706
Eca109
43.19 ± 2.23
24.86 ± 0.69
>200
38.66 ± 2.54
42.28 ± 3.01
>200
4a
4b
4c
11.68 ± 1.66
8.23± 1.16
30.33 ± 1.30
69.60 ± 4.03
34.41 ± 4.36
16.40 ± 1.20
17.10 ± 1.30
54.65 ± 5.04
33.54 ± 1.42
70.93 ± 5.48
105.52 ± 13.43
34.71 ± 1.73
>200
30.29 ± 1.38
16.22 ± 1.22
41.95 ± 2.55
95.45 ± 6.01
36.22 ± 5.02
33.02 ± 3.51
27.71 ± 2.44
49.64 ± 5.14
55.48 ± 4.95
130.12 ± 14.87
>200
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
50.44 ± 6.12
>200
4o
4p
>200
>200
4q
88.39 ± 8.04
23.26± 1.32
106.3 ± 11.22
30.25 ± 1.99
4r
5-Fluorouracil

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Figure 3. IC50 curve of compound 4

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Highlights
►We designed and synthesized a series of new benzo[d][1,3]dioxoles-fused
1,4-thiazepines.
► All the synthesized compounds good to potent anti-proliferative activities.
►Compound 4e was found to be the most active compound.