One-Pot Synthesis and Evaluation of Antileishmanial Activities of Functionalized S-Alkyl/Aryl Benzothiazole-2-carbothioate Scaffold

Article abstract of DOI:10.1021/acs.joc.6b00113

The synthesis of hitherto unreported S-alkyl/aryl benzothiazole-2-carbothioate is reported from thiols, oxalyl chloride, and 2-aminothiophenols using 10 mol % n-tetrabutylammonium iodide (TBAI) as catalyst in acetonitrile through multicomponent reaction (MCR) strategy. The present protocol favored formation of benzothiazoles and thioesters via simultaneous formation of C-N and C-S bonds in good yields with a wide range of substrates. A few of the synthesized derivatives were evaluated for their antimicrobial activity against the protozoan parasite Leishmania donovani, a causative agent of visceral leishmaniasis (VL). Further, these compounds displayed no toxicity toward macrophage RAW 264.7 cells and are therefore nontoxic and effective antileishmanial leads. In silico docking studies were performed to understand the possible binding site interaction with trypanothione reductase (TryR).

Full text of DOI:10.1021/acs.joc.6b00113

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Article
One-pot Synthesis and Evaluation of Antileishmanial Activities of
Functionalized S-alkyl/aryl benzothiazole-2-carbothioate scaffold
Ajaz A. Dar, Md. Shadab, Suman Khan, Nahid Ali, and Abu Taleb Khan
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b00113 • Publication Date (Web): 21 Mar 2016
Downloaded from http://pubs.acs.org on March 25, 2016
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Oneꢀpot Synthesis and Evaluation of Antileishmanial Activities of
Functionalized Sꢀalkyl/aryl benzothiazoleꢀ2ꢀcarbothioate scaffold
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Ajaz A. Dar,a Md. Shadab,b Suman Khan,c Nahid Ali,b,* Abu T. Khan,a,*,†
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aDepartment of Chemistry, Indian Institute of Technology Guwahati, Guwahati 781 039,
India
bInfectious Diseases and Immunology Division, Indian Institute of Chemical Biology, 4,
Raja S.C. Mullick Road, Kolkata 700 032, India.
cDepartment of Chemistry, Pohang University of Science and Technology, Pohang, 37673,
Republic of South Korea
Eꢀmail: atk@iitg.ernet.in (A.T. Khan), nali@iicb.res.in (Nahid Ali)
Tel.: +91 361 2582305; fax: +91 361 2582349.
†Aliah University, IIA/27, New Town, Kolkataꢀ700 156, West Bengal, India.
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ABSTRACT
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The synthesis of hitherto unreported Sꢀalkyl/aryl benzothiazoleꢀ2ꢀcarbothioate is reported
from thiols, oxalyl chloride and 2ꢀaminothiophenols using 10 mol% nꢀtetrabutylammonium
iodide (TBAI) as catalyst in acetonitrile through multicomponent reaction (MCR) strategy.
The present protocol favoured formation of benzothiazoles and thioesters via simultaneous
formation of C−N and C−S bonds in good yields with a wide range of substrates. Few of
the synthesized derivatives were evaluated for their antimicrobial activity against the
protozoan parasite, Leishmania donovani, a causative agent of visceral leishmaniasis (VL).
Further these compound displayed no toxicity toward macrophage RAW 264.7 cells and
therefore nontoxic and effective antileishmanial leads. In-silico docking studies were
performed to understand the possible binding site interaction with trypanothione reductase
(TryR).
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Keywords: Sꢀalkyl/aryl benzothiazoleꢀ2ꢀcarbothioate, Thiols, nꢀTetrabutylammonium
iodide (TBAI), visceral leishmaniasis (VL), trypanothione reductase (TryR).
INTRODUCTION
Protozoan parasites of the genus Leishmania are causative agents of human leishmaniasis.
It is one of the world’s most neglected diseases in terms of drug development, owing to its
major prevalence among the poorest. It is projected 12 million people worldwide are
infected by over 20 species of leishmania and about 350 million people are at risk of
infection in around 98 countries. Depending upon its species, leishmaniasis manifests as
mild cutaneous lesions caused by Leishmania major, disfiguring mucocutaneous disease
caused by Leishmania braziliensis or visceral form caused by Leishmania donovani which
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is often fatal, if left untreated.1 Currently, organic antimonial drugs are the only
chemotherapeutic agents widely used for its treatment and is generally agreed that new
agents with safe, cheap and more effective profile are urgently needed.
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Benzothiazole derivatives are the most essential heterocyclic compounds found in a variety
of natural products and pharmaceutical agents with a high degree of structural diversity.2
These scaffolds are used as functional materials in ratiometric fluorescent pH indicators,
sensors and bioluminogenic probe and as agrochemicals. Various marine and terrestrial
natural products namely Erythrazoles A and B, isolated from mangrove sediments, are
useful intermediates for dyes and are versatile ligands for catalytic reactions.3 It is well
known that amalgamation of benzothiazoles with other functionalities opens doors to
design novel drug like molecules. Some of substituted benzothiazoles are used as drugs:
riluzole (an anticonvulsant drug), zopolrestat (to treat endotoxinꢀrelated inflammatory
diseases namely sepsis, asthma, and uveitis), 5F203 and PMX 610 (exhibit antitumor
properties). They are also used as imaging agents for Aβ plaques in cerebral amyloid
angiopathy, radioactive βꢀamyloid imagining agents (11CꢀPIB), γꢀaminobutyric acid
(GABAꢀA) inhibitors, inhibitors of fatty acid oxidation, aldose reductase inhibitors (ARIs),
falcipain inhibitors, microsomal triglyceride transfer protein inhibitors (MTP), and fatty
acid amide hydrolase (FAAH) inhibitors (Figure 1).4 Giorgio et al. carried out structure
activity relationships (SARs) of position 2 substitutionꢀbearing 6ꢀnitroꢀ and 6ꢀaminoꢀ
benzothiazoles and their corresponding anthranilic acid derivatives against Leishmania
infantum and Trichomonas vaginalis. They also reported benzothiazole substituted
acridinone derivatives as potent antileishmanial agents with promising selectivity index
(SI). Fazal et. al studied antileishmanial activities of benzothiazole derivatives.
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Benzothiazole derivatives containing electron donating group were reported to enhance
antileishmanial activity. Whereas electron withdrawing group completely made the
compounds inactive.5 Consequently,6 our research group focussed to develop new
antileishmanial agents: benzothiazoleꢀthioester derivatives.
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Figure 1. Structures of bioactive 2ꢀsubstituted benzothiazoles.
Several approaches have been designed for the synthesis of benzothiazole derivatives. Most
of these synthesis of benzothiazole derivatives involves metal catalysis and often are
expensive with tedious work up procedures and metallic toxic wastes as offshoots. Other
protocols mainly includes cyclization of different aromatic thiourea linkages, condensation
of various orthoꢀhaloanilides or their analogues with different thiocyanate and
isothiocyanate, condensation of 2ꢀaminothiophenols and carbon disulfide/sodium
sulfide/acids/acid
chlorides/aldehydes/esters/nitriles/ketones/thioesters
under
mild
conditions.7 2ꢀarylbenzothiazole synthesis was extensively explored as compared to 2ꢀ
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acylbenzothiazoles.8 Introducing an acyl group at the 2ꢀposition of benzothiazoles has been
synthetically challenging. However, the synthesis of Sꢀalkyl/aryl benzothiazoleꢀ2ꢀ
carbothioate are yet to be reported.
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Thioester moiety is another useful and versatile functional group in organic transformations
because of its less resonance stabilization, i.e. very poor overlap of 3p orbital, with the 2p
orbitals of the carbonyl resulting in high reactivity in its ground state. They are key
intermediates used in βꢀlactam synthesis, carbocyclization, peptide coupling, in natural
product synthesis, acyl transfer reactions, functional group transformations, carbonꢀcarbon
bond formation, in biosynthetic reactions and in certain instances as protecting groups.9
The synthesis of thioesters by direct reaction of the corresponding thiols with a suitable
acid chloride10a or acid anhydride10b are well known methods. They have also been
synthesized by transthioesterification of active carboxylic acid derivatives, esters,10c Nꢀ
acylbenzotriazoles10d with thiols in varied reaction conditions and catalysts.10e Recently
tertꢀbutyl protected thiols have been utilized for the preparation of thioesters.10f The N–S
acyl shift, mediated by attaching a thiol auxiliary residue to the peptide backbone has been
applied to peptide thioester syntheses.10g They have also been accessed from thiols and
carbon monoxide by carbonylation of organic substrates catalyzed by transition metals such
as Pt10h or Pd.10i The Dess–Martin periodinane (DMP)ꢀmediated reaction of thiols and
aldehydes10j and coupling of aldehyde with disulfide or thiol via free radical mechanism is
another methods for the synthesis of thioesters.11
Herein, we report an unprecedented synthesis of Sꢀalkyl/aryl benzothiazoleꢀ2ꢀcarbothioate
using oneꢀpot threeꢀcomponent reaction between thiols, oxalyl chloride and 2ꢀ
aminothiophenol using nꢀtetrabutylammonium iodide12 (TBAI) in acetonitrile (Scheme 1)
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and the activity of Sꢀalkyl/aryl benzothiazoleꢀ2ꢀcarbothioate against promastigotes of L.
donovani was evaluated.
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Scheme 1. Synthesis of Sꢀalkyl/aryl benzothiazoleꢀ2ꢀcarbothioate.
RESULT AND DISCUSSION
We started with the reaction of 2ꢀmethylthiophenol and oxalyl chloride with 2ꢀ
aminothiophenol in acetonitrile at room temperature in the absence of catalyst and no
desired product was obtained (Table 1, entry 1). When the reaction mixture was subjected
to heating at 60 °C, the desired product was isolated in 15% yield (Table 1, entry 2). To
increase the efficiency of the reaction, the same reaction was examined using 10 mol% nꢀ
tetrabutylammonium iodide (TBAI) and the desired product was obtained in 75% yield. No
significant increase in yield was noted when the reaction was carried out with 20 mol%
catalyst (Table 1, entry 4). Further the yield of the desired product dropped to 40% (Table
1, entry 5) when the reaction was carried out with 5 mol% catalyst. A lower yield was
observed when the reaction temperatures exceeded 70 °C. Various other salts of iodine
such as sodium iodide, potassium iodide and molecular iodine, as well as other quaternary
ammonium salts namely nꢀtetrabutylammonium chloride (TBACl) and nꢀ
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tetrabutylammonium bromide (TBABr) were examined under similar reaction conditions
(Table 1, entries 6ꢀ10). TBAI was found to provide the best yields. The choice of solvent
was next examined by carrying out reactions in 1,2ꢀdichloroethane, ethanol, methanol,
dichloromethane and tetrahydrofuran. 1,2ꢀdichloroethane furnished the desired product in
comparable yield as with acetonitrile, while other solvents were found ineffective (Table 1,
entries 11ꢀ15).
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Table 1. Optimization for reaction conditionsa
Entry
Catalyst (mol %)
No catalyst
No catalyst
TBAI (10)
TBAI (20)
TBAI (5)
Solvent
CH3CN
CH3CN
CH3CN
CH3CN
CH3CN
CH3CN
CH3CN
Time/h Yield (%)
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7
12
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0
15b
75
77
40
24
28
4.0
4.0
4.0
4.0
4.0
NaI (10)
KI (10)
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I2 (10)
CH3CN
CH3CN
CH3CN
DCE
4.0
4.0
4.0
4.5
6.0
6.0
6.0
6.0
20
30
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TBACl (10)
TBABr (10)
TBAI (10)
TBAI (10)
TBAI (10)
TBAI (10)
TBAI (10)
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40
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72
C2H5OH
CH3OH
DCM
≤10
˂10
˂20
≤15
THF
aAll the reactions were carried out in 1 mmol scale. bThe reaction was carried out at 60 oC.
After optimization of reaction conditions, the substrate scope was explored using various
substituted thiols and substituted 2ꢀaminothiophenols. At first, 2ꢀaminothiophenol (1a) was
treated with a set of aromatic thiols bearing both electronꢀdonating and electronꢀ
withdrawing substituents and the corresponding products were obtained in good yields. The
substrates with 4ꢀmethyl (3a) and 4ꢀmethoxy (3b) substituents underwent reactions to give
Sꢀaryl benzothiazoleꢀ2ꢀcarbothioates 4a and 4b (Table 2) respectively in high yields. The
substrates with 4ꢀchloro (3c), 4ꢀbromo (3d), 4ꢀhydroxyl (3e), and 4ꢀfluoro (3f) groups,
underwent condensation to afford Sꢀaryl benzothiazoleꢀ2ꢀcarbothioates 4c, 4d, 4e and 4f
respectively (65–76% yields, Table 2). Moreover the reaction of para substituted
thiophenol with nitro group did not provide any desired product under the optimized
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condition. Substrates 3g and 3h with 3ꢀmethoxy and 3ꢀchloro substituents furnished the
products 4g and 4h (78% and 72% yields respectively, Table 2). It was observed that
aromatic thiols with electronꢀdonating groups served as efficient reacting partners.
Presence of an electronꢀwithdrawing group however gave slightly lower yields.
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The steric factor due to ortho-substituted thiols was found to have less impact on the
present protocol as with 2ꢀmethyl (3i), 2ꢀchloro (3j) and 2ꢀbromo (3k) correspondingly
provided 4i, 4j and 4k in good yields. In addition, thiophenol (3l) and 2ꢀnaphthalenethiol
(3m) also reacted with 2ꢀaminothiophenol to obtain the products 4l and 4m (68% and 70%
yields respectively, Table 2). Benzylthiol (3n) and 2ꢀchlorobenzylthiol (3o) reacted to give
corresponding Sꢀalkylbenzothiazoleꢀ2ꢀcarbothioates 4n and 4o (Table 2) on condensation
with 2ꢀaminothiophenol. Aliphatic thiols such as ethanethiol (3p), propanethiol (3q) and
dodecanethiol (3r) also underwent reaction to give the corresponding Sꢀalkyl
benzothiazoleꢀ2ꢀcarbothioates 4p, 4q and 4r (Table 2), in good yields.
Table 2. Reaction of 2ꢀaminobenzenethiol (1a) with various thiolsa,b
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aConditions: 1a (1 mmol), 2 (1 mmol), 3 (1 mmol), CH3CN (5.0 mL), 60 °C, 4 h. bYield based on 3.
To explore the scope of the reaction, the presence of chloro or methoxy/ethyl groups at 4ꢀ
or 5ꢀposition of the 2ꢀaminothiophenol did not interfere with the formation of the product
i.e., the yields were found to be comparable. However presence of trifluoromethyl at 4ꢀ
position of the 2ꢀaminothiophenol decreased the rate of reaction and eventually the product
formation. Furthermore, the remaining factors for the synthesis of 4s to 4z and 4za to 4zd
(Table 3) were identical as discussed above i.e., the reactions of 2ꢀaminoꢀ4ꢀ
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chlorobenzenethiol with thiophenol produced 4s in 71% yield while substrates bearing
substituted aryl rings with 4ꢀmethyl, 4ꢀchloro, and 4ꢀbromo functionalities produced the
corresponding Sꢀalkylbenzothiazoleꢀ2ꢀcarbothioates 4sꢀv in 73−77% yields. Similarly the
reactions of 2ꢀaminoꢀ4ꢀtrifluoromethylbenzenethiol with thiophenol having 4ꢀmethyl, 4ꢀ
methoxy, 2ꢀchloro, and 2ꢀbromo substituents in the aryl ring underwent reaction to furnish
4wꢀz in 56ꢀ62% yields. Likewise, the reactions of 2ꢀaminoꢀ5ꢀmethoxybenzenethiol with
substrates bearing substituted aryl rings with 4ꢀmethyl, 4ꢀmethoxy, and 4ꢀbromo
functionalities produced the corresponding Sꢀalkylbenzothiazoleꢀ2ꢀcarbothioates 4zaꢀc in
77−80% yields. Finally, the reactions of 2ꢀaminoꢀ5ꢀethylbenzenethiol with 4ꢀ
methylthiophenol produced 4zd in 76% yield.
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Maximum yields were observed when both the substrates possessed electronꢀdonating
groups. Replacement with either an electron neutral or electronꢀwithdrawing substituent
reduced yields. Also drop in the yields were noticed when electronꢀwithdrawing groups
were present on both the reacting substrates.
All the isolated products were confirmed on the basis of their analytical data (1H NMR, 13C
NMR, IR, and HRMS). Additionally, the target compounds 4a and 4o were further
determined by Xꢀray crystallographic analysis (Figure S1).
Table 3. Reaction with substituted 2ꢀaminobenzenethiols with arylthiolsa
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Cl
Cl
O
S
O
S
N
S
N
S
CH3
7
8
4s (71%)
4t (77%)
9
Cl
Cl
O
S
O
S
N
S
N
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S
Cl
Br
4u (73%)
4v (75%)
F3C
F3C
O
S
O
S
N
S
N
S
CH3
OCH3
4w (60%)
4x (62%)
F3C
F3C
O
S
O
S
N
S
N
S
4z (58%)
4y (56%)
Cl
Br
O
S
O
S
N
S
N
S
CH3
Br
OCH3
CH3
H3CO
H3CO
H3CO
H3C
4za (79%)
4zb (80%)
O
S
O
N
S
N
S
S
4zc (77%)
4zd (76%)
aConditions: 1b/1c/1d/1e (1 mmol), 2 (1 mmol), 3 (1 mmol), CH3CN (5.0 mL), 60 °C, 6 h.
To understand the reaction mechanism, we carried out three sets of reaction using
permutation and combination of three substrates (Scheme 2). It was observed that 4ꢀ
methylthiophenol (3a) reacts with oxalyl chloride (2) under the experimental conditions to
give the expected product 5 in 50% yield by nucleophilic substitution reaction along with
some unreacted starting material (Scheme 2, Eq. 1). Similarly, 4ꢀmethylthiophenol (3a)
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was reacted with 2ꢀaminothiophenol (1a), the expected products 6a and 6b were obtained
in equal ratio (Scheme 2, Eq. 2). Likewise, treatment of 2ꢀaminothiophenol with oxalyl
chloride furnished 7, but in lower yield and major starting materials were recovered
(Scheme 2, Eq. 3). On the basis of these results, it can be speculated that present protocol
suppresses the formation of these above byꢀproduct during the course of reaction, thereby
generating the desired product in major yields.
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Scheme 2. Crossover experiments.
The formation of the Sꢀalkyl benzothiazoleꢀ2ꢀcarbothioate products can be proposed as:
TBAI reacts with thiol to generate thiolate anion in the reaction medium. Subsequently, the
anion attacks a side of oxalyl chloride to form thioester moiety intermediate. Finally, 2ꢀ
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aminobenzenethiol in the presence of TBAI can undergo condensation with the in situ
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generated thioester intermediate to form the desired products (Scheme 3).
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Scheme 3. Proposed Reaction Mechanism.
Antimicrobial Activity. The antileishmanial activity and mammalian cell toxicity of the
16 Sꢀalkyl/aryl benzothiazoleꢀ2ꢀcarbothioate derivatives were investigated. The IC50 and
IC90 values were calculated for each compound (Table 4). The results of statistcal analysis
showed that all the compounds possessed moderate to potent activity against the
promastigote forms of L. donovani. However, toxicity incurred on mammalian macrophage
cell line RAW264 by few of them could limit their use as antileishmanial agents. For
instance, four compounds namely 4s, 4a, 4i, and 4b displayed toxicity towards the
mammalian macrophage cells with IC50 values of 313.7±52.28 ꢁM, 322.9±66.8 ꢁM,
360.5±37.11 ꢁM, and 432.5±82.32 ꢁM, respectively. These IC50 values against
macrophages are near to IC90 values against promastigotes. Therefore, the efficacy of these
compounds is limited due to their toxicity. The compounds 4f, 4x, 4l, 4h, 4e, 4g, 4j, 4m,
and 4n with IC50 values of 191.1±2.78 ꢁM, 200.4±4.31 ꢁM, 202.5±23.45 ꢁM, 207.5±30.89
ꢁM, 210.6±30.34 ꢁM, 212.2±28.82 ꢁM, 215.2±35.33 ꢁM, 236.4±47.09 ꢁM, and
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280.6±64.79 ꢁM, respectively against promastigotes forms could be promising
antileishmanial agents as their toxic effect towards macrophage is negligible at the doses
used against the parasite (as depicted by their IC50 values). Herein, the compounds 4y, 4z
and 4w are found to be most effective against promastigotes with IC50 values of
190.3±14.05 ꢁM, 198.1±19.65 ꢁM, and 199.8±2.370 ꢁM, respectively.
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Table 4. Leishmanicidal activity of compounds against L. donovani promastigotes
Activity
Cytotoxicity
Macrophages
(IC50
L. donovani promastigotes
(IC50
(IC90
(IC90
Compounds
µM±SD)a
µM±SD)a
µM±SD)a
322.9±66.80
432.5±82.32
6803±65.45
765.5±31.62
1775±229.90
µM±SD)a
581.1±120.30
778.4±148.20
12245±117.80
1382±56.94
211.6±17.36
250.1±29.52
210.6±30.34
191.1±2.78
212.2±28.82
207.5±30.89
208.4±20.66
215.2±35.33
202.5±23.45
236.4±47.09
380.9±31.25
450.2±53.14
379±54.610
344.1±5.01
381.9±51.88
373.5±55.61
375.1±37.18
387.3±63.59
364.5±42.21
425.5±84.76
4a
4b
4e
4f
3196±413.80
4g
4h
4i
935.3±234.80 1683±422.70
360.5±37.11
3165±56.65
543.4±52.78
2554±383.10
648.8±66.79
5697±102.0
978.1±95.01
4597±689.50
4j
4l
4m
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280.6±64.79
651.2±90.07
323.9±18.76
359.6±4.26
360.8±7.77
342.6±25.28
356.5±35.37
1745±171.30
3140±308.40
564.6±94.10
4332±145.80
1584±260.60
4n
4s
180±10.42
199.8±2.37
200.4±4.317
190.3±14.05
198.1±19.65
313.7±52.28
2407±114.50
880±144.80
4w
4x
4y
4z
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6338±340.40 11409±612.70
2559±330.70 4607±595.30
an=2
The percent cell viability of L. donovani and macrophages upon treatment with increasing
concentration of these three compounds from 0.00 to 500 ꢁM were also evaluated as shown
in Figure 2 and Figure 3, respectively. The results showed a dose dependent killing of the
promastigotes of L. donovani. Further, at these doses of these compounds, no or
insignificant killing of mammalian macrophages was observed indicating to their potential
as promising antileishmanials.
Figure 2. Leishmanicidal effect of 4y, 4z and 4w on promastigotes of L. donovani. L. donovani
promastigotes were treated for 24 h with increasing concentrations of three different compounds 4y,
4z and 4w and then cell viability was assessed using alarm blue reagent (as described in material
and methods).
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Figure 3. Toxic effect of 4y, 4z and 4w on murine peritoneal macrophages. Peritoneal macrophages
were treated for 24 h with increasing concentrations of these three compounds 4y, 4z and 4w and
cell viability was assessed.
Molecular Docking Study of benzothiazoleꢀ2ꢀcarbothioate derivatives against
Trypanothione Reductase.
The molecular docking of benzothiazole derivatives against Trypanothione Reductase
(TryR) showed that most of the compounds are showing hydrogen bonds with Asn 340 and
Arg 472. Interestingly, some of these derivatives show hydrophobic interaction with Hisꢀ
461’ (protonated histidine) which is a part of catalytic triad (Cysꢀ52ꢀHisꢀ461’ꢀCysꢀ57). The
Hisꢀ461’ forms a πꢀcation interaction with the aryl group of these derivatives (̰∼5Å)
whereas, the Arg 472 is showing πꢀcation interaction with the benzothiazole nucleus (∼3Å)
which may help in stabilizing the ligand in the cavity. The two representative examples 4u
and 4f revealing the mode of interactions are provided in Figure 4. The Gscores of 4u and
4f are ꢀ4.15 and ꢀ3.03 respectively. Table 5 shows the molecular docking scores and
Emodel scores for the 25 compounds. The molecular docking results indicates that the
binding of these designed compounds to trypanothione reductase enzyme is sufficiently
strong, which may play important role against promastigotes.
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Figure 4. Binding model of ligands at the interface of homodimer. Ligands are shown in stick
models (magenta colour). Hydrogen bonding interactions are shown as black dashes and residues
involved in hydrogen bonding or hydrophobic interactions (π−cation) are represented in stick
models (A) Represent the binding mode of 4u and (B) Represent the binding mode of 4f.
Table 5. Gscore and Emodel for the 25 docked compoundsa
S.No. Compounds Gscore
Emodel S.No. Compounds Gscore
Emodel
ꢀ33.45
ꢀ35.39
ꢀ30.66
ꢀ30.19
ꢀ33.02
ꢀ42.99
ꢀ37.22
ꢀ34.70
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ꢀ3.70
ꢀ3.42
ꢀ3.62
ꢀ3.40
ꢀ3.81
ꢀ3.03
ꢀ3.81
ꢀ3.00
ꢀ44.12
ꢀ31.10
ꢀ32.90
ꢀ33.02
ꢀ35.44
ꢀ35.49
ꢀ36.36
ꢀ31.43
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ꢀ3.54
ꢀ3.58
ꢀ3.84
ꢀ3.66
ꢀ3.58
ꢀ4.72
ꢀ4.15
ꢀ3.57
4a
4b
4c
4d
4e
4f
4n
4o
4p
4q
4s
4t
4g
4h
4u
4v
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ꢀ3.69
ꢀ3.53
ꢀ3.12
ꢀ3.53
ꢀ3.00
ꢀ33.45
ꢀ30.17
ꢀ32.33
ꢀ43.75
ꢀ32.02
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ꢀ3.70
ꢀ3.85
ꢀ4.87
ꢀ3.81
ꢀ48.00
ꢀ40.57
ꢀ44.62
ꢀ32.52
4i
4j
4w
4x
4y
4z
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4k
4l
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4m
aGscore values are listed, for comparision gscore of diarrylpyrrole13a is ꢀ4.27
Lipinsky Rule. The ligands were also checked for amenability to the Lipinsky rule of five,
and the results are summarized in Table 6. The rule states that a molecule likely to be
developed as an orally active drug candidate should show no more than one violation of the
following four criteria: It should not have more than five hydrogen bond donors, it should
not have more than 10 hydrogen bond acceptors, it should not have molecular weight
greater than 500 Da, and it should not have an octanol–water partition coefficient greater
than 5. Molecular properties of all ligands were calculated by molinspiration, and it was
found that all the ligands followed the Lipinsky rule with maximium ligands (except 4m,
4w, 4y and 4z) showed no violation of the above criteria (Table 6). Therefore, these ligands
have a good potential for ensuing development as an oral agents and can be potentially
active drug candidates.
Table 6. Molinspiration Calculation of Properties for the Lipinsky Rulea
Entry
acceptable
range→
nViol natoms miLog P
MW
<500
nON nOHNH nrotb
<5
<10
<5
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0
0
0
0
0
0
0
0
1
0
0
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0
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4.46
4.07
3.53
4.17
4.04
4.66
4.41
4.64
4.01
5.19
4.07
4.66
5.33
4.94
5.51
5.64
285.39
301.39
287.37
289.36
301.39
305.81
285.39
305.81
271.37
321.43
285.39
305.81
353.39
369.39
373.81
418.26
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3
3
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2
2
2
2
2
2
2
2
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2
2
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0
0
0
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4
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3
4
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4
4
4a
4b
4e
4f
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4g
4h
4i
4j
4l
4m
4n
4s
4w
4x
4y
4z
anViol, no. of violations; natoms, no. of atoms; miLog P, molinspiration predicted Log P; MW,
molecular weight; nON, no. of hydrogen bond acceptors; nOHNH, no. of hydrogen bond donors;
and nrotb, no. of rotatable bond.
CONCLUSION
In summary, we have developed a one pot threeꢀcomponent condensation with a variety of
thiols and 2ꢀaminothiophenols with oxalyl chloride in acetonitrile through multicomponent
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reaction (MCR) strategy, in a mild reaction conditions, with high efficiency, tolerance to a
wide range of substrates and easy synthesis of Sꢀalkyl/aryl benzothiazoleꢀ2ꢀcarbothioate
derivatives. The nꢀtetrabutylammonium iodide (TBAI) was found to be particularly suitable
and of importance for the success of this transformation. The protocol involves the
formation of three new bonds i.e. two C−S and one C=N in a highly efficient, atom
economical and without addition of oxidizing or reducing agents or coupling catalysts. In
vitro analysis indicated compounds 4y, 4z, and 4w to be most active among the others.
Docking studies also revealed a possible mode of binding of in the binding pocket of TryR.
Benzothiazoleꢀ2ꢀcarbothioate derivatives can be expected as promising lead molecules for
the advancement of novel drug against leishmania.
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EXPERIMENTAL SECTION
Material and Method for Biological Screening.
Cell Culture and Parasite. L. donovani strain AG83 (MHOM/IN/1983/AG83),
originally isolated from an Indian kalaꢀazar patient was maintained by serial passage in
hamsters. L. donovani amastigotes periodically recovered from the spleens of infected
hamsters were transformed into promastigotes through amastigote culture in M199
supplemented with 10% FCS, 2 mM glutamine, penicillin G (100 U/ml), streptomycin
sulfate (100 ug/ml) at 22°C. Promastigotes were used at the log phase of growth,
approximately 2 to 3 days after subculture. Parasites were kept in culture by weekly
passaging.
In vitro antipromastigote activity. Antiparasitic activity of compounds against L.
donovani (AG83) promastigotes was determined by a quantitative colorimetric assay using
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Alamar blue reagent (Life Technologies). When cells are alive they maintain a reducing
environment within their cytosol. Resazurin/AlamarBlueH (7ꢀHydroxyꢀ3Hꢀphenoxazinꢀ3ꢀ
one 10ꢀoxide), the active ingredient of AlamarBlue reagent, is a nonꢀtoxic, cell permeable
compound that is blue in color and virtually nonꢀfluorescent. Upon entering cells, resazurin
is reduced to resorufin, a compound that is red in colour and highly fluorescent. Viable
cells continuously convert resazurin to resorufin, increasing the overall fluorescence and
colour of the media surrounding cells. This assay has been reported to be more sensitive
than MTT assay when compared for their efficacy in estimating cell viability against a
cancer cell line treated with more than hundred drugs.13b So we have taken this assay for
assessing the cell viability of cells treated with our compounds.
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Assays were performed in sterile 96ꢀwell plates using 100 µl of logꢀphase promastigotes
adjusted to 2x106 cells/ml. These cells were incubated in the absence (control) and
presence of 3.9 ꢁM, 7.8 ꢁM, 15.6 ꢁM, 31.2 ꢁM, 65.5 ꢁM, 125.0 ꢁM, 250.0 ꢁM, and 500.0
ꢁM of compounds and the equivalent volume of the solvent (DMSO) for 24 h. After
completion of treatment, 10 ꢁl of the resazurin dye (0.01%) was added, and plates were
incubated for a further 4 h at 37 °C. After incubation cells were analyzed in a microplate
reader at a wavelength of 570 nm, using 600 nm as a reference wavelength (normalized to
the 600 nm value). Absorbance in the absence of any compound, or solvent was set as the
100% control. Cell viability was evaluated based on a comparison with untreated control
cells and the inhibitory concentration of the compounds or the solvents that are necessary
to reduce the growth of promastigotes by 50% (IC50 values) and 90% (IC90 values) was
calculated.
Culture of RAW264 macrophage cell line and its cytotoxicity measurement.
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The murine macrophageꢀlike cell line, RAW 264.7 was cultured in RPMI medium
containing 10 percent FBS in tissue culture flasks. For the experiment, cells were detached
in medium using cell scraper. The cells were counted and plated overnight (5x104 cells
/well) in a 96 well culture plate. The cytotoxic effect of compounds on RAW cells was than
evaluated in the absence and presence 3.9 ꢁM, 7.8 ꢁM, 15.6 ꢁM, 31.2 ꢁM, 65.5 ꢁM, 125.0
ꢁM, 250.0 ꢁM and 500.0 ꢁM of compounds and the equivalent volume of the solvent
(DMSO) for 24 h. After completion of treatment, 10 ꢁl AlamarBlue reagent was employed
to determine cytotoxicity as described above. The cell viability was evaluated based on a
comparison with untreated control cells and the concentration of the compounds or the
solvents that are necessary to reduce the viability of macrophages by 50% (IC50 values) and
90% (IC90 values) was calculated.
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Molecular Docking Study. There is no crystal structure yet available for trypanothione
reductase of L. donovani and thus conducted our molecular docking studies (by using Glide
5.8 module in maestro 9.3.)13cꢀe with trypanothione reductase from L. infantum as there is
98% similarity between the trypanothione reductase of L. donovani and L. infantum. The
molecular docking study was carried out using Xꢀray crystal structures of trypanothione
reductase from L. infantum (PDB code: 2jk6, 2.95 Å).14 Protein preparation and ligand
preparation were carried out as per our previous work.6 For the purpose of molecular
docking studies, Xꢀray crystal structure of L. Infantum trypanothione reductase was
selected (PDB ID: 2jk6, resolution: 2.95 Å).14 The crystal structure is in dimeric form and
it is coꢀcrystalized with cofactor FAD. Both the chains were considered for molecular
docking studies because the binding site of trypanothione reductase is at the interface of the
chain A and chain B. Protein preparation was done using Maestro 9.3. Hydrogen atoms
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were added during protein preparation wizard. Threeꢀdimensional structures of these
compounds were then prepared using LigPrep module of maestro implementing
OPLS_2005 force field and ionic states for the ligands at pH values of 7.0 ± 2.0 were
generated. Docking was performed using Glide 5.8 (Gridꢀbased Ligand Docking with
Energetics), with the standard precision (SP) mode to estimate protein–ligand binding
affinities and static intermolecular interactions. The grid was defined 15 Å by considering
all the key amino acids of binding pocket. All the 25 compounds were docked into the
active site of TyR. A maximum of 10 docking poses per ligand were generated in each case
and analyzed further for the binding mode and intermolecular interactions.
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General Experimental Details. Melting point was determined on a melting point
1
apparatus and are uncorrected. IR spectra were recorded on IR spectrophotometer. H and
13C NMR spectra were recorded on 400 MHz and 600 MHz spectrometer TMS as internal
1
reference; chemical shifts (δ scale) are reported in parts per million (ppm). H NMR
Spectra are reported in the order: multiplicity, coupling constant (J value) in hertz (Hz) and
no. of protons; signals were characterized as s (singlet), d (doublet), dd (doublet of
doublets), t (triplet), m (multiplet). Mass spectra were recorded using ESI/APCI mode (Qꢀ
TOF type Mass Analyzer). Column chromatographic separations were performed using
silica gel (60ꢀ120 mesh). Complete crystallographic data of 4a and 4o for the structural
analysis have been deposited with the Cambridge Crystallographic Data Centre, CCDC No.
1430385 and 1430384 respectively. Copies of this information may be obtained free of
charge from the Director, Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK, (fax: +44ꢀ1223ꢀ336033, eꢀmail: deposit@ccdc.cam.ac.uk or via:
www.ccdc.cam.ac.uk).
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General procedure for synthesis of compound 4. To a stirred reaction mixture of thiol
(3) (1.0 mmol), oxalyl chloride (2) (85 ꢀL or 0.085 mL, 1.0 mmol) and catalyst TBAI
(36.9 mg, 0.10 mmol) in acetonitrile (3 mL) at room temperature was added dropwise a
solution of (1) 2ꢀaminobenzenethiol or substituted 2ꢀaminobenzenethiol (1.0 mmol) in
acetonitrile (2 mL), in case of 2ꢀaminoꢀ4ꢀ(trifluoromethyl)benzenethiol hydrochloride the
solution is being neutralised with potassium bicarbonate for 10 min before addition, and
subjected to heating at 60 °C till reaction completion. After the completion of reaction the
solvent was evaporated under reduced pressure and extracted with ethyl acetate (3 × 10
mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under
reduced pressure to yield the crude product, which was purified by column chromatography
on silica gel (60ꢀ120 mesh) to give the pure product (hexane:ethyl acetate).
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Spectral data for all products:
S-p-tolyl benzo[d]thiazole-2-carbothioate (4a): White solid; Rf = 0.73; Yield 78% (223
mg); mp 137ꢀ139 °C. IR (KBr): 3055, 1919, 1810, 1690, 1659, 1616, 1593, 1548, 1482,
1455, 1421, 1399, 1376, 1318, 1281, 1240, 1203, 1180, 1157, 1127, 1104, 1063, 1038,
1018, 947, 880, 868, 853, 806, 758, 731, 700, 673, 636, 617, 540, 480 cmꢀ1. 1H NMR (400
MHz, CDCl3): δ 2.42 (S, 3H), 7.30 (d, J = 7.6 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H), 7.55 (t, J =
8.0 Hz, 1H), 7.62 (t, J = 7.2 Hz, 1H), 7.99 (d, J = 7.6 Hz, 1H), 8.27 (d, J = 8.0 Hz, 1H)
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ppm. C{1H} NMR (100 MHz, CDCl3): δ 21.6, 122.5, 122.9, 125.6, 127.4, 127.9, 130.5
(2C), 135.0 (2C), 136.9, 140.5, 153.1, 164.0, 185.6 ppm. HRMS (ESI): calcd for
C15H11NOS2 [M + H]+: 286.0355; Found 286.0374.
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S-(4-methoxyphenyl) benzo[d]thiazole-2-carbothioate (4b): White solid; Rf = 0.53; Yield
80% (242 mg); mp 150ꢀ152 °C. IR (KBr): 2959, 2837, 2048, 1965, 1929, 1893, 1801,
1692, 1673, 1612, 1592, 1573, 1550, 1497, 1480, 1455, 1439, 1419, 1408, 1316, 1294,
1253, 1203, 1185, 1174, 1123, 1109, 1061, 1033, 964, 948, 879, 859, 835, 808, 763, 731,
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701, 674, 641, 619, 579, 545, 499 cmꢀ1. H NMR (400 MHz, CDCl3): δ 3.86 (s, 3H), 7.01
(d, J = 8.8 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 7.55 (t, J = 8.0 Hz, 1H), 7.61 (t, J = 7.6 Hz,
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1H), 7.98 (d, J = 8.0 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H) ppm. C{1H} NMR (100 MHz,
CDCl3): δ 55.5, 115.2 (2C), 116.8, 122.4, 125.5, 127.3, 127.8, 136.5 (2C), 136.8, 153.0,
161.2, 163.9, 185.9 ppm. HRMS (ESI): calcd for C15H11NO2S2 [M + H]+: 302.0304; Found
302.0306.
S-(4-chlorophenyl) benzo[d]thiazole-2-carbothioate (4c): White solid; Rf = 0.71; Yield
74% (227 mg); mp 161ꢀ163 °C. IR (KBr): 3422, 3088, 2925, 2853, 1959, 1909, 1793,
1690, 1670, 1612, 1573, 1552, 1487, 1475, 1457, 1419, 1389, 1318, 1267, 1203, 1164,
1125, 1090, 1012, 941, 878, 861, 815, 758, 747, 726, 703, 669, 619, 529, 484 cmꢀ1. H
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NMR (400 MHz, CDCl3): δ 7.48 (dd, J1 = 7.6 Hz & J2 = 8.4 Hz, 4H), 7.56 (t, J = 7.6 Hz,
1H), 7.62 (t, J = 7.6 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 8.26 (d, J = 8.0 Hz, 1H) ppm.
13C{1H} NMR (100 MHz, CDCl3): δ 122.6, 125.1, 125.7, 127.6, 128.1, 129.9 (2C), 136.3
(2C), 136.7, 137.0, 153.1, 163.5, 184.8 ppm. HRMS (ESI): calcd for C14H8ClNOS2 [M +
H]+: 305.9809; Found 305.9810.
S-(4-bromophenyl) benzo[d]thiazole-2-carbothioate (4d): White solid; Rf = 0.68; Yield
76% (267 mg); mp 191ꢀ193 °C. IR (KBr): 3085, 2923, 2853, 1912, 1671, 1567, 1551,
1486, 1472, 1454, 1419, 1384, 1317, 1202, 1162, 1090, 1066, 1009, 885, 877, 844, 815,
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758, 726, 703, 670, 619, 548, 477 cmꢀ1. 1H NMR (400 MHz, CDCl3): δ 7.44 (d, J = 8.4 Hz,
2H), 7.57 (t, J = 8.0 Hz, 1H), 7.62 (d, J = 7.6 Hz, 3H), 7.80 (d, J = 7.6 Hz, 1H), 8.26 (d, J =
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8.0 Hz, 1H) ppm. C{1H} NMR (100 MHz, CDCl3): δ 122.6, 125.0, 125.7 (2C), 127.6,
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128.1, 132.8 (2C), 136.5 (2C), 137.0, 153.1, 163.5, 184.7 ppm. HRMS (ESI): calcd for
C14H8BrNOS2 [M + H]+: 351.9282; Found 351.9291.
S-(4-hydroxyphenyl) benzo[d]thiazole-2-carbothioate (4e): White solid; Rf = 0.14; Yield
70% (202 mg); mp 191ꢀ192 °C. IR (KBr): 3452, 3059, 2923, 2848, 1650, 1624, 1597,
1581, 1550, 1497, 1480, 1455, 1429, 1417, 1337, 1317, 1269, 1201, 1168, 1098, 1062,
1010, 949, 880, 830, 816, 761, 731, 700, 671, 637, 624, 564, 509 cmꢀ1. 1H NMR (400 MHz,
CDCl3): δ 4.34 (s, 1H, OH), 6.81 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 8.4 Hz, 2H), 7.39 (t, J =
7.2 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H)
ppm. 13C{1H} NMR (100 MHz, CDCl3 + DMSO): δ 114.5, 116.7 (2C), 122.2, 125.1,
127.1, 127.6, 136.3 (2C), 136.4, 152.7, 159.2, 163.6, 185.9 ppm. HRMS (ESI): calcd for
C14H9NO2S2 [M + H]+: 288.0147; Found 288.0149.
S-(4-fluorophenyl) benzo[d]thiazole-2-carbothioate (4f): White solid; Rf = 0.64; Yield
65% (189 mg); mp 139ꢀ141 °C. IR (KBr): 3428, 3093, 3058, 1891, 1664, 1588, 1553,
1488, 1456, 1397, 1317, 1293, 1223, 1205, 1160, 1124, 1088, 1064, 1012, 941, 879, 860,
820, 758, 726, 696, 670, 635, 619, 542, 499 cmꢀ1. 1H NMR (400 MHz, CDCl3): δ 7.18 (t, J
= 8.8 Hz, 2H), 7.54 (t, J = 7.6 Hz, 3H), 7.61 (t, J = 7.6 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H),
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8.25 (d, J = 8.4 Hz, 1H) ppm. C{1H} NMR (100 MHz, CDCl3): δ 116.8, 117.0, 121.8,
121.8, 122.5, 125.7, 127.5, 128.1, 136.9, 137.1, 137.2, 153.1, 162.8, 163.5, 165.3, 185.3
ppm. HRMS (ESI): calcd for C14H8FNOS2 [M + H]+: 290.0104; Found 290.0106.
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S-(3-methoxyphenyl) benzo[d]thiazole-2-carbothioate (4g): White solid; Rf = 0.50; Yield
78% (236 mg); mp 108ꢀ110 °C. IR (KBr): 3056, 3001, 2943, 2839, 1922, 1861, 1801,
1692, 1665, 1615, 1590, 1550, 1481, 1463, 1454, 1439, 1421, 1346, 1318, 1276, 1255,
1201, 1185, 1160, 1127, 1106, 1072, 1063, 1028, 991, 947, 896, 881, 872, 863, 809, 786,
758, 731, 705, 687, 675, 625, 558 cmꢀ1. 1H NMR (400 MHz, CDCl3): δ 3.84 (s, 3H), 7.03
(d, J = 8.0 Hz, 1H), 7.13 (s, 1H), 7.17 (d, J = 7.6 Hz, 1H), 7.40 (t, J = 8.0 Hz, 1H), 7.56 (t,
J = 7.2 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 8.00 (d, J = 7.6 Hz, 1H), 8.26 (d, J = 8.0 Hz, 1H)
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ppm. C{1H} NMR (100 MHz, CDCl3): δ 55.6, 116.4, 120.0, 122.5, 125.6, 127.2, 127.4,
127.5, 128.0, 130.3, 136.9, 153.1, 160.2, 163.9, 185.1 ppm. HRMS (ESI): calcd for
C15H11NO2S2 [M + H]+: 302.0304; Found 302.0313.
S-(3-chlorophenyl) benzo[d]thiazole-2-carbothioate (4h): White solid; Rf = 0.64; Yield
72% (221 mg); mp 132ꢀ133 °C. IR (KBr): 3422, 3055, 2961, 1798, 1669, 1612, 1575,
1566, 1488, 1457, 1399, 1316, 1261, 1207, 1162, 1082, 1070, 1024, 995, 947, 881, 844,
820, 820, 776, 757, 726, 703, 678, 661, 623 cmꢀ1. 1H NMR (400 MHz, CDCl3): δ 7.40ꢀ7.48
(m, 3H), 7.57 (t, J = 8.0 Hz, 2H), 7.63 (t, J = 7.6 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 8.27 (d,
J = 8.4 Hz, 1H) ppm. 13C{1H} NMR (100 MHz, CDCl3): δ 122.6, 125.7, 127.6, 128.2,
128.3, 130.4, 130.6, 133.2, 134.8, 135.2, 137.0, 153.0, 163.4, 184.6 ppm. HRMS (ESI):
calcd for C14H8ClNOS2 [M + H]+: 305.9809; Found 305.9827.
S-o-tolyl benzo[d]thiazole-2-carbothioate (4i): White solid; Rf = 0.62; Yield 72% (206
mg); mp 65ꢀ67 °C. IR (KBr): 3053, 2925, 1918, 1798, 1697, 1662, 1627, 1589, 1550, 1482,
1456, 1419, 1318, 1281, 1209, 1125, 1060, 1037, 1013, 945, 879, 857, 814, 804, 755, 730,
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703, 675, 669, 619, 551 cmꢀ1. H NMR (400 MHz, CDCl3): δ 2.45 (s, 3H), 7.29 (t, J = 7.2
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Hz, 1H), 7.40 (t, J = 7.2 Hz, 2H), 7.56 (t, J = 8.0 Hz, 2H), 7.62 (t, J = 7.2 Hz, 1H), 8.00 (d,
J = 7.6 Hz, 1H), 8.28 (d, J = 8.0 Hz, 1H) ppm. 13C{1H} NMR (100 MHz, CDCl3): δ 21.0,
122.5, 125.6, 125.9, 127.0, 127.4, 127.9, 130.8, 131.1, 136.3, 136.9, 142.9, 153.1, 164.0,
184.6 ppm. HRMS (ESI): calcd for C15H11NOS2 [M + H]+: 286.0355; Found 286.0369.
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S-(2-chlorophenyl) benzo[d]thiazole-2-carbothioate (4j): White solid; Rf = 0.64; Yield
70% (215 mg); mp 144ꢀ146 °C. IR (KBr): 2924, 2850, 1957, 1921, 1692, 1673, 1612,
1573, 1551, 1485, 1450, 1433, 1419, 1382, 1319, 1249, 1208, 1115, 1064, 1034, 982, 950,
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CDCl3): δ 7.37 (t, J = 7.6 Hz, 1H), 7.45 (t, J = 8.0 Hz, 1H), 7.54ꢀ7.62 (m, 3H), 7.66 (d, J =
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7.2 Hz, 1H), 7.99 (d, J = 7.6 Hz, 1H), 8.27 (d, J = 8.4 Hz, 1H) ppm. C{1H} NMR (100
MHz, CDCl3): δ 122.6, 125.7, 126.2, 127.5, 127.7, 128.1, 130.7, 131.9, 137.0, 137.6,
139.5, 153.1, 163.4, 183.5 ppm. HRMS (ESI): calcd for C14H8ClNOS2 [M + H]+:
305.9809; Found 305.9829.
S-(2-bromophenyl) benzo[d]thiazole-2-carbothioate (4k): White solid; Rf = 0.63; Yield
73% (256 mg); mp 167ꢀ169 °C. IR (KBr): 2926, 2856, 1740, 1691, 1672, 1610, 1485,
1457, 1447, 1429, 1415, 1384, 1319, 1258, 1208, 1109, 1064, 1018, 923, 879, 851, 808,
759, 749, 726, 697, 669, 646, 616 cmꢀ1. 1H NMR (400 MHz, CDCl3): δ 7.36 (t, J = 8.0 Hz,
1H), 7.43 (t, J = 7.6 Hz, 1H), 7.57 (t, J = 8.0 Hz, 1H), 7.63 (d, J = 7.6 Hz, 1H), 7.68 (d, J =
7.6 Hz, 1H), 7.78 (d, J = 7.6 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 8.28 (d, J = 8.0 Hz, 1H)
ppm. 13C{1H} NMR (100 MHz, CDCl3): δ 122.6, 125.8, 127.6, 128.1, 128.4, 128.5, 130.2,
131.9, 134.0, 137.0, 137.6, 153.1, 163.4, 183.6 ppm. HRMS (ESI): calcd for C14H8BrNOS2
[M + H]+: 351.9282; Found 351.9285.
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