Development of a novel class of pyrrolo-[1,2,5]benzothiadiazepine derivatives as potential anti-schistosomal agents

Article abstract of DOI:10.1016/j.bmcl.2013.04.085

Analogues of pyrrolo-[1,2,5]benzothiadiazepine were prepared and evaluated against Schistosoma japonica. The biological data revealed that most benzothiazepine derivatives show anti-schistosomal activity to some extent, while α-chloronation of the title compound and another bioisosteric derivative pyrrolo-[1,2,5]benzodiazepine displayed the most distinct worm killing activity. This study proved that benzodiazepine may serve as a novel structural skeleton for the development of anti-schistosomal agents.

Full text of DOI:10.1016/j.bmcl.2013.04.085

Accepted Manuscript
Development of a Novel Class of Pyrrolo-[1,2,5]benzothiadiazepine Deriva‐
tives as Potential anti-Schistosomal Agents
Jinglei Chen, Weizhen Sun, Jingjing Yang, Huan Sun, Zhixia Wang, Lanlan
Dong, Chunhua Qiao, Chao-ming Xia
PII:
S0960-894X(13)00575-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.04.085
BMCL 20450
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
16 January 2013
24 April 2013
30 April 2013
Please cite this article as: Chen, J., Sun, W., Yang, J., Sun, H., Wang, Z., Dong, L., Qiao, C., Xia, C-m., Development
of a Novel Class of Pyrrolo-[1,2,5]benzothiadiazepine Derivatives as Potential anti-Schistosomal Agents,
Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.2013.04.085
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Development of a Novel Class of Pyrrolo-[1,2,5]benzothiadiazepine Derivatives as
Potential anti-Schistosomal Agents
Jinglei Chena, Weizhen Suna, Jingjing Yanga, Huan Sunb, Zhixia Wanga, Lanlan Dongb,
Chunhua Qiaoa*, Chao-ming Xiab*
a College of Pharmaceutical Science, Soochow University, Suzhou 215123, China
b Department of Parasitology, Soochow University, Suzhou 215123, China
qiaochunhua@suda.edu.cn
xiachaoming@suda.edu.cn
College of Pharmaceutical Science, Soochow University, 199 Ren Ai Road, Suzhou
215123, P. R. China
Fax: +86-512-65882092
Abstract: Analogues of pyrrolo-[1,2,5]benzothiadiazepine were prepared and
evaluated against Schistosoma japonica. The biological data revealed that most
benzothiazepine derivatives show anti-schistosomal activity to some extent, while
α-chloronation of the title compound and another bioisosteric derivative
pyrrolo-[1,2,5]benzodiazepine displayed the most distinct worm killing activity. This
study proved that benzodiazepine may serve as a novel structural skeleton for the
development of anti-schistosomal agents.
Key word: benzothiadiazepine derivatives, anti-schistosomal
Schistosomiasis is one of the most burdensome nevertheless neglected tropical
diseases. The World Health Organization (WHO) estimates that the number of people
that treated for schistosomiasis has risen from 12.4 million in 2006 to 33.5 million in
2010.[1] Over the past 40 years, praziquantel (PZQ, Figure 1) has been used as the
only effective drug for schistosomal disease.[2,3]There are no back-up drugs for the
treatment of schistosomiasis should praziquantel become less effective. Thus, there is
an urgent need to develop newer anti-schistosomal agents.
A benzodiazepine derivative 3-methylclonazepam (Figure 1, compound 2) was

first reported by Kelly Chibale to show a marked degree of therapeutic and
prophylactic activity against all stages of schistosomiasis caused by Schistosoma
mansoni and S. haematobium.[4] Mechanism of action study indicated that this
compound caused an increase in the influx of extracellular calcium and subsequent
spastic paralysis of the parasite’s musculature by binding to a low affinity
benzodiazepine receptor on the epidermis of the schistosome.[5a,5b] Later on, another
pyrrolo[1,2,5]benzothiadiazepine derivative (Figure 1, compound 3) was revealed to
demonstrate good anti-schistosomal efficacy.[6] It is concealed that cytochalasin D
(CyD) and calcium channel blockers counteracted the activity of compound 3, both
cytochalasin D (CyD) and calcium channel blockers may be involved in the calcium
channels of schistosome.[7a,7b] These results prompted us to deepen our insight into a
novel skeleton of pyrrolo[1,2,5]benzothiadiazepine as therapeutic agents against
schistosomiasis.
O
H
N
O
O
H
H
N
OEt
CH3
N
N
O2N
N
N
S
Cl
O
1. Praziquantel (PZQ)
2. 3-Methylclonazepam 3. Pyrrolo-[1,2,5]benzothiadiazepine
Figure 1. Structures of reported compounds with anti-schistosomal activities
Pyrrolo[1,2,5]benzothiadiazepine 3 was initially synthesized as non-nucleoside
reverse transcriptase inhibitors,[8] but didn’t show appreciable activity against HIV-1.
Following the reported preparation method, compound 3 was synthesized. Its
derivatives SW-1 and SW-2 (Figure 2) were then prepared efficiently using
N-bromosuccinimide (NBS) and N-chlorosuccinimide (NCS) as halogenation
reagents, respectively. Briefly, a mixture of compound 3 and halogenation reagents in
acetonitrile was stirred at -20 ℃ for 8 hours, the reaction mixture was partitioned
between aqueous and organic phase, purification by flash silica gel column afforded
the desired compounds. In both cases, only α-halogenation products were obtained
and nuclear magnetic resonance spectra were analyzed.[9] Introduction of the
electron-withdrawing halogen atom to the electron-rich pyrrole ring was supposed to

increase the chemical structure stability. SW-11[10] was prepared and evaluated
because it possessed the same structural skeleton as compound 3. Given the high
structural
similarity
between
pyrrolo[1,2,5]benzothiadiazepine
5-monooxide
SW-3,[10]
and
pyrrolo[1,2-b][1,2,5]benzothiadiazepine,
and
pyrrolo[1,2-b][1,2,5]benzothiadiazepine 5,5-dioxides,[11] SW-4[12] as well as SW-5[12]
were prepared (Figure 2). Employing isosteric replacement principle, the sulfur atom
in SW-1 was replaced by nitrogen in SW-6, [11] SW-7, [13] SW-8 and SW-9[14] (Figure
2). The synthesis of these compounds were previously reported by us or other groups,
and their biological activity against schistosomiasis was not fully described.[15]
Analogously, one carbon takes the sulfur position provided compound SW-10, the
synthesis of SW-10 was shown in Scheme 1. Briefly, bromination of 1-methyl-2-nitro
benzene by NBS provided 1-(bromomethyl)-2-nitrobenzene, following the reaction
with hexamethylenetetramine in the same reaction pot, a white precipitation was
formed and filtered, the benzylbromide was converted to amine after treating this
reaction
intermediate
with
conc.
HCl
in
aqueous
solution.
1-(2-Nitrobenzyl)-1H-pyrrole was prepared in 80% yield by treating the previous
amine with 2,5-dimethoxy tetrahydrofuran. Next, the nitro group was reduced to
amine using sodium hypophosphite, following the reaction with ethyl
2-ethoxy-2-hydroxyacetate, SW-10 was prepared and its structure was fully
1
characterized.[16] All the prepared compounds were characterized by H, 13C NMR
and HRMS analysis.
O
O
O
EtO
HO
H
N
H
N
H
N
OEt
OEt
H
H
OEt
O
O
N
N
N
N
N
N
S
S
N
S
S
S
O
O
O
O
O
Br
Cl
SW-1
SW-2
SW-3
SW-4
SW-5
EtO
EtO
CH3
O
H
N
H
H
N
N
O
N
O
O
H
N
N
OEt
N
N
N
N
N
N
N
S
N
H
N
N
CH3
CH3
CH3
SW-6
SW-7
SW-8
SW-9
SW-11
SW-10

Figure 2. Design and prepared pyrrolo-[1,2,5]benzothiadiazepine derivatives
NO2
N
N
NO2
CH3
NO2
Br
hexamethylene
tetramine
NBS, BPO
CCl4
Br
N
N
CCl4, 71% in 2 steps
O
OH
H
OEt
NO2
N
OEt
N
K2CO3, Pd
H2O-THF
NH2
N
EtO
1. HCl; 2. NaOH
O
NaH2PO2
93%
3: 2,5-dimethoxy-THF
DCM, 80%
N
EtOH, 67%
SW-10
Scheme 1. Synthesis of compound SW-10.
The newly prepared analogues were evaluated for their ability against adult
Schistosoma japonica in vitro. According to previously described method,[17] 6 male
worms obtained from rat infections were distributed in duplicate tissue culture dishes
(3.5 cm) in Dulbecco’s modified minimum Eagle’s medium (bicarbonate buffered)
supplemented with 20% newborn calf serum, 100 U mL-1 penicillin, 100 μg mL-1
streptomycin and 0.5 μg mL-1 amphotericin B. Cultures were kept at 37℃in an
atmosphere of 5% CO2 in air and were observed under a Leica MZ 12.5
stereomicroscope. Worms of Schistosoma japonica were exposed overnight (16 h) to
various compounds, washed and subsequently cultured in compound-free medium.
Worms were obtained at day 42 infection. Compounds were then added from 5.0 mM
DMSO stock solutions to achieve a concentration of 50 μM. Compound activity was
assessed by percentage of survival and vitality score within 72 hours. The total score
of 6 worms was counted for in vitro test. Data representative of repeated experiments
was shown in Table 1.
Table 1. In vitro activity of pyrrolo-[1,2,5]benzothiadiazepine analogues against adult
Schistosoma japonica.
24 h
48 h
72 h
% worm
survival
Vitality
score
% worm Vitality
survival score
% worm Vitality
survival score
Compd.

SW-1
100
14
100
13
66.7
9
SW-2
SW-3
100
100
13
14
83.3
100
7
33.3
100
2
16
14
100
13
100
16
100
16
SW-4
SW-5
SW-6
SW-7
SW-8
SW-9
SW-10
100
100
100
100
100
100
14
12
14
12
14
12
100
100
100
100
100
100
16
15
11
12
14
9
100
100
14
15
10
10
10
8
83.3
83.3
83.3
83.3
SW-11
PZQ
100
11
3
100
13
100
13
66.7
33.3
2.0
33.3
2.0
Comp 3
Control
100
100
13
18
66.7
100
3.0
18
16.7
100
2
18
The vitality score for each worm is illustrated as: 3 scores: the highest score, as observed in the control
group during the observation period. Worms moved actively and softly, and the body was transparent.
The highest score for 6 tested worms is 3х6=18; 2 scores: worms acted all over the body, but stiffly and
slowly, with the body translucent and shrunken; 1 score: parasites moved partially with opaque and
vacuole appearance. 0 score: the worm remained contracted and show severe tegument damage, did not
resume movements, we could deem it ‘dead’; Compound stock solutions (50.0 mM) were prepared in
100% DMSO immediately before use.
For the compounds tested against adult Schistosoma japonica in vitro, SW-1,
SW-2, SW-7, SW-8, SW-9 and SW-10 show obvious effect within 72 hours. SW-2
reduced worm motility significantly, the worm survival rate is only 33.3%. For
comparison, PZQ demonstrated close worm killing capability as SW-2 in this
experiment. The activity of compound SW-1 was weaker compared to SW-2, the
worm survival percentage is 66.7% in 72 hours. Compounds SW-7, SW-8, SW-9, and
SW-10 exhibits almost the same moderate worm killing capability. Other derivatives
SW-3, SW-4, SW-5 and SW-6 showed only very weak ability to reduce worm

survival rate within 24 hours. It is also interesting to notice that compound 3 killed
most worms within 72 hours, the bromo- and chloro- substituted anologues SW-1 and
SW-2 displayed reduced activity compared to compound 3, but the in vitro worm
killing activity is still the most potent among all tested compounds.
Based on the in vitro biological activity, compounds displayed moderate to good
potency at 50 μM were further evaluated for their in vivo worm killing ability (Table
2). Female ICR mice were infected with ca. 100 Schistosoma japonicum cercariae,
on day 42 post-infection (adult stage), the infected mice were administrated with 3
consecutive days of 200 mg/kg oral doses of the test compounds, which was
suspended in distilled water. Each experimental group used ten mice. At 21 days
post-treatment, animals were sacrificed and dissected to assess total worm reduction
as described in detail.[18 ]
Table 2 Effect of compounds 3, SW-1, SW-2, SW-7, SW-8 SW-9, and SW-10 to mice
harboring 42-day-old adult Schistosoma japonica.
Compounds
Number of detected
worms/mice ( x s )
Total worm reduction (%)
control
Compd 3
SW-1
66 7
63 7
58 5
32 5
43 4
50 6
62 8
17 5
2 2
0
4.5
12.0
51.5
34.8
24.2
6.1
SW-2
SW-7
SW-8
SW-9
SW-10
PZQ
74.2
96.9
The in vivo activity data revealed that among these compounds, SW-2 and SW-10
are the two compounds with significant activity, reduced the worm load more than
50%. Especially compound SW-10, which displayed only week in vitro efficacy,

demonstrated high in vivo capability and note worthily reduced worm burden in mice,
whether or not this remarked potency is derived from the possible metabolite should
be further investigated. Other evaluated compounds show marginal activity (SW-1,
SW-7 and SW-8), and SW-9 is almost inactive. Although compound 3 almost lost its
in vivo worm killing capability, its chlorination derivative SW-2 exhibited fairly
potent efficacy to reduce mice worm burden.
The structural activity relationship of pyrrolo-[1,2,5]benzothiazepine anologues
revealed that bromination (SW-1) or chlorination (SW-2) of the pyrole ring would
attenuate the parent compound antischistosomal activity both in vitro and in vivo. The
oxidized sulfinyl and sulfonyl analogues, SW-3, SW-4 and SW-5 displayed reduced
efficacy, suggesting the oxygen atom exerted an unfavorable effect for the compound
worm killing ability. Replacement of sulfur atom by nitrogen (SW-6, SW-7, SW-8
and SW-9) would retain the compounds capability in vitro; Meanwhile, this
displacement did not endowed the compounds high worm killing capability in vivo.
Finally, the pyrrolo-[1,2,5]benzodiazepine analogue SW-10 exhibited the highest
worm reducing capability in vivo. Considering its relative low in vitro efficacy, it is
reasonable to believe that its good in vivo worm reducing ability may come from the
metabolite form of SW-10. Therefore, further study of compound SW-10 will be
investigated.
In summary, we have identified pyrrolo-[1,2,5]benzothiadiazepine derivatives
with activities against Schistosoma japonica. Although none of these analogues
displayed activity equal to PZQ, the structural activity relationship revealed that
chlorination of the pyrrol ring in the parent compound displayed elevated worm
killing capability; Another pyrrolo-[1,2,5]benzodiazepine skeleton also displayed
marked in vivo worm reducing efficacy. Therefore, more analogues with this skeleton
is worthy of further exploration in the near future.
Acknowledgments
This project is funded by the Priority Academic Program Development of Jiangsu
Higher Education Institutions.

References and notes
1. World Health Organization Media center/ Schistosomiasis/ fact sheets N15 /
January 2012 / http://www.who.int/mediacentre/factsheets/fs115/en/
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Lett., 2008, 18, 2333.
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202; b: Bennett, J. L. J. Parasitol. 1980, 66, 742
6. Xin L. Induced Resistance of Schistosoma japonicum with Administration of
Praziquantel-pressure and Study of Anti-schistosomal Effect of Five Kinds of
Diazepam Derivatives, Master Degree Thesis, 2009.
7. a: Pica-Mattoccia L., Ruppel A., Xia C.M. Parasitol., 2008, 135, 47; b:
Pica-Mattoccia L., Valle C., Basso A. Exp. Parasitol., 2006, 115, 344.
1
8. SW-1: H NMR (400 MHz, CDCl3 ) δ 7.25–7.20 ( m, 1H ), 7.05 ( t, J = 8.4 Hz,
1H ), 6.58– 6.52 ( m, 2H ), 6.21 ( d, J = 4.4 Hz, 1H ), 6.17 ( d, J = 3.9 Hz, 1H ),
5.96 ( d, J = 3.8 Hz, 1H ), 5.04 ( d, J = 4.2 Hz, 1H ), 4.43–4.30 ( m, 2H ), 1.33 ( t,
J = 7.1 Hz, 3H ). 13C NMR (101 MHz, CDCl3) δ 170.62, 147.20, 136.95, 132.57,
131.70, 118.17, 118.00, 115.84, 111.58, 108.69, 108.14, 62.62, 54.83, 14.26.
HRMS [ESI]+ cacul. for [M+H]+: 352.9954; found: 352.9966, error 4.53 ppm.
SW-2: 1H NMR (400 MHz, CDCl3) δ 7.22 ( d, J = 7.6 Hz, 1H ), 7.06 ( t, J = 7.7
Hz, 1H ), 6.56 ( t, J = 7.9 Hz, 2H ), 6.18 (d, J = 5.1 Hz, 1H), 6.06 ( d, J = 3.9 Hz,
1H ), 5.93 ( d, J = 3.8 Hz, 1H ), 5.04 ( d, J = 4.9 Hz, 1H ), 4.49–4.22 ( m, 2H ),
1.34 ( t, J = 7.1 Hz, 3H ). 13C NMR ( 101 MHz, CDCl3 ) δ 170.60, 147.28, 135.43,
132.58, 131.70, 121.96, 118.21, 118.00, 115.83, 107.44, 107.05, 62.63, 54.62,
14.27. HRMS [ESI]+ cacul. for [M+H]+: 309.0459; found: 309.0461, error 0.64
ppm.
9. Artico, M.; Silvestri, R.; Pagnozzi, E.; Stefancich, G.; Massa, S.; Loi, A.G.;
Putzolu, M.; Corrias, S.; Spiga, M.G.; La Colla, P. Bioorg. Med. Chem., 1996, 4,

837.
10. Wei-zhen, S., Si-jie Q., Chao-ming X., Chunhua, Q. Huaxue Yanjiu Yu Yingyong,
2011, 23, 1008.
11. Silvestri, R.; Marfe, G.; Artico, M.; La Regina, G.; Lavecchia, A.; Novellino, E.;
Morgante, M.; Di Stefano, C.; Catalano, G.; Filomeni, G.; Abruzzese, E.; Ciriolo,
M. R.; Russo, M. A.; Amadori, S.; Cirilli, R.; La Torre, F.; Sinibaldi Salimei, P. J.
Med. Chem., 2006, 49, 5840.
12. Marfe, G.; Di Stefano, C.; Silvestri, R.; Abruzzese, E.; Catalano, G.; Di Renzo, L.;
Filomeni, G.; Giorda, E.; La Regina, G.; Morgante, E.; Ciriolo, M.; Russo, M.;
Amadori, S.; Sinibaldi-Salimei, P. BMC Cancer 2007, 7:207.
13. Stefancich, G.; Silvestri, R. J. Heterocyclic. Chem. 1989, 26, 745.
14. Stefancich, G.; Artico, M.; Corelli, F. Massa, S. Synthesis 1983, 9, 757.
15. Wei-zhen S. Synthesis and Evaluation of Benzodiazepine Derivatives as
anti-Schistosoma Compounds. Master Degree Thesis, 2011
1
16. SW-10: H NMR (400 MHz, CDCl3) δ 7.05 (t, J = 7.7 Hz, 1H), 6.99 (d, J = 7.6
Hz, 1H), 6.68 (s, 1H), 6.66 – 6.59 (m, 2H), 6.04 (d, J = 3.0 Hz, 1H), 5.96 (s, 1H),
5.54 (s, 1H), 5.42 (d, J = 15.5 Hz, 1H), 4.87 (d, J = 15.5 Hz, 1H), 4.77 (s, 1H),
4.35 (p, J = 7.0 Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
171.11, 144.78, 129.52, 129.03, 128.47, 121.34, 120.57, 118.42, 118.39, 106.58,
106.40, 62.12, 54.30, 51.44, 14.25. HRMS:[ESI]+ cacul. for [M+H]+: 255.1128;
found: 255.1127, error 0.39 ppm.
17. Duan, W.W; Qiu, S.J.; Zhao, Y.; Sun, H.; Qiao, C.H. Bioorg. Med. Chem. Lett.
2012, 22, 1587.
18. Pica-Mattoccia, L.; Valle, C.; Basso, A.; Troiani, A. R.; Vigorosi, F.; Liberti, P.;
Festucci, A.; Cioli, D. Exp. Parasitol. 2007, 115, 344.

Graphical Abstract
O
H
OEt
N
N
X
O
X = S, C , NH, NHCH3,
with anti-schistosomal activity
S O
or
S
O