S-substituted 3,5-dinitrophenyl 1,3,4-oxadiazole-2-thiols and tetrazole-5-thiols as highly efficient antitubercular agents

Article abstract of DOI:10.1016/j.ejmech.2016.11.041

Two new classes of antitubercular agents, namely 5-alkylsulfanyl-1-(3,5-dinitrophenyl)-1H-tetrazoles and 2-alkylsulfanyl-5-(3,5-dinitrophenyl)-1,3,4-oxadiazoles, and their structure-activity relationships are described. These compounds possessed excellent activity against Mycobacterium tuberculosis, including the clinically isolated multidrug (MDR) and extensively drug-resistant (XDR) strains, with no cross resistance with first or second-line anti-TB drugs. The minimum inhibitory concentration (MIC) values of the most promising compounds reached 0.03 μM. Furthermore, these compounds had a highly selective antimycobacterial effect because they were completely inactive against 4 gram positive and 4 gram negative bacteria and eight fungal strains and had low in vitro toxicity for four mammalian cell lines, including hepatic cell lines HepG2 and HuH7. Although the structure-activity relationship study showed that the presence of two nitro groups is highly beneficial for antimycobacterial activity, the analogues with a trifluoromethyl group instead of one of the nitro groups maintained a high antimycobacterial activity, which indicates the possibility for further structural optimization of this class of antitubercular agents.

Full text of DOI:10.1016/j.ejmech.2016.11.041

Accepted Manuscript
S-substituted 3,5-dinitrophenyl 1,3,4-oxadiazole-2-thiols and tetrazole-5-thiols as
highly efficient antitubercular agents
Galina Karabanovich, Jan Němeček, Lenka Valášková, Alejandro Carazo, Klára
Konečná, Jiřina Stolaříková, Alexandr Hrabálek, Oto Pavliš, Petr Pávek, Kateřina
Vávrová, Jaroslav Roh, Věra Klimešová
PII:
S0223-5234(16)30978-3
10.1016/j.ejmech.2016.11.041
EJMECH 9074
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 October 2016
Revised Date: 18 November 2016
Accepted Date: 20 November 2016
Please cite this article as: G. Karabanovich, J. Němeček, L. Valášková, A. Carazo, K. Konečná, J.
Stolaříková, A. Hrabálek, O. Pavliš, P. Pávek, K. Vávrová, J. Roh, V. Klimešová, S-substituted 3,5-
dinitrophenyl 1,3,4-oxadiazole-2-thiols and tetrazole-5-thiols as highly efficient antitubercular agents,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.11.041.
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ACCEPTED MANUSCRIPT
S-Substituted 3,5-Dinitrophenyl 1,3,4-Oxadiazole-2-
thiols and Tetrazole-5-thiols as Highly Efficient
Antitubercular Agents
a
a
a
a
a
Galina Karabanovich, Jan Němeček, Lenka Valášková, Alejandro Carazo, Klára Konečná,
b
a
c
a
a
Jiřina Stolaříková, Alexandr Hrabálek, Oto Pavliš, Petr Pávek, Kateřina Vávrová, Jaroslav
a,
a
Roh * and Věra Klimešová
a
Charles University, Faculty of Pharmacy in Hradec Králové, Heyrovského 1203, 50005 Hradec
Králové, Czech Republic
b
Regional Institute of Public Health, Department of Bacteriology and Mycology, Partyzánské
náměstí 7, 70200 Ostrava, Czech Republic
c
Military Health Institute, Biological Defense Department, Techonin, Czech Republic
ABSTRACT
Two new classes of antitubercular agents, namely 5-alkylsulfanyl-1-(3,5-dinitrophenyl)-1H-
tetrazoles and 2-alkylsulfanyl-5-(3,5-dinitrophenyl)-1,3,4-oxadiazoles, and their structure-
activity relationships are described. These compounds possessed excellent activity against
1

ACCEPTED MANUSCRIPT
Mycobacterium tuberculosis, including the clinically isolated multidrug (MDR) and extensively
drug-resistant (XDR) strains, with no cross resistance with first or second-line anti-TB drugs.
The minimum inhibitory concentration (MIC) values of the most promising compounds reached
0
.03 µM. Furthermore, these compounds had a highly selective antimycobacterial effect because
they were completely inactive against 4 gram positive and 4 gram negative bacteria and eight
fungal strains and had low in vitro toxicity for four mammalian cell lines, including hepatic cell
lines HepG2 and HuH7. Although the structure-activity relationship study showed that the
presence of two nitro groups is highly beneficial for antimycobacterial activity, the analogues
with a trifluoromethyl group instead of one of the nitro groups maintained a high
antimycobacterial activity, which indicates the possibility for further structural optimization of
this class of antitubercular agents.
Keywords
Tuberculosis
Antitubercular agent
Mycobacterium tuberculosis
Tetrazole
Oxadiazole
Structure-activity relationships
Introduction
Tuberculosis (TB) is an enormously widespread and dangerous infection disease. Despite the
treatment success rates and progress in reducing the mortality rate since 1990, there were still an
estimated 10.4 million incident cases of TB and 1.8 million deaths from TB in 2015 [1]. A
particular problem is the development and increasing dissemination of multidrug-resistant forms
2

ACCEPTED MANUSCRIPT
of TB (MDR-TB). In 2015, 3.9% of new TB cases and 21% of previously treated cases of TB
were MDR-TB. Moreover, 9.5% of those cases involved the extensively drug-resistant strains of
TB (XDR-TB), which have limited treatment options. Only 50% of patients with MDR-TB were
successfully cured, whereas therapy of TB caused by the drug-susceptible strains of
Mycobacterium tuberculosis (M.tb.) led to a 90% recovery rate. The low recovery rate of patients
with MDR-TB is due to poor adherence to the treatment regimen because of its long duration,
complexity and high cost and the adverse effects of the currently used anti-TB drugs when
applied to a long TB therapy regimen. Furthermore, the wide spread of TB among HIV-positive
patients worsens the TB epidemic situation and increases the death rates. The complicated
diagnose of TB in HIV patients and drug-drug interactions between anti-TB drugs and
antiretroviral drugs decreases the treatment success for both TB and HIV. All of the above
mentioned problems highlight the necessity for the development of new, highly effective, anti-
TB compounds.
Figure 1. New anti-TB compounds for the treatment of MDR-TB.
3

ACCEPTED MANUSCRIPT
In the last 40 years, only two novel anti-TB drugs have been approved for the treatment of
pulmonary MDR-TB, bedaquiline (1) [2, 3] and delamanid (OPC-67683, 2) [4] (Figure 1).
Recently, several experimental anti-TB drugs, which have nitro group dependent
antimycobacterial activities like delamanid, have been intensively studied. PA-824 (3) [5-7],
TBA-354 (4) [8] and their parent compound, CGI-17341 (5) [9], are nitroimidazole-based
compounds that are highly effective against drug-susceptible and drug-resistant TB forms and
are active against both replicating and non-replicating mycobacteria (Figure 2A) [10].
Benzothiazinones BTZ043 (6) [11] and its piperazine derivative PBTZ-169 (7) [12] represent
another class of nitro group containing compounds with excellent in vitro and in vivo
antimycobacterial activity. These compounds act as irreversible inhibitors of
decaprenylphosphoryl-β-D
ribose 2′
oxidase (DprE1), an essential enzyme in arabinan
biosynthesis [13]. A structure–anti-TB activity study of BTZs showed that only one nitro-group
is essential for the efficiency of these compounds, and derivatives with two nitro groups on the
benzothiazinone core showed a lower potency [14]. Other nitro group-containing DprE1
inhibitors are also known: N-substituted 3,5-dinitrobenzamides (8) [15, 16], nitro group
substituted quinoxaline VI-9376 (9) [17] or nitro group substituted triazole 377790 (10) (Figure
2
B) [18].
4

ACCEPTED MANUSCRIPT
Figure 2. Experimental anti-TB drugs with nitro group dependent antimycobacterial activity:
nitroimidazole-based compounds (A) and DprE1 inhibitors (B).
In our previous work, four classes of highly efficient nitro group containing anti-TB
compounds were developed: 3,5-dinitrobenzylsulfanyl 1H- and 2H-tetrazoles (11 and 12,
respectively) [19, 20], 1,3,4-oxadiazoles (13) and 1,3,4-thiadiazoles (14) (Figure 3) [21]. The
majority of the compounds of series 11 - 14 possessed outstanding and highly selective
antimycobacterial activity. They were highly effective against drug-susceptible M.tb., multidrug-
resistant clinically isolated strains of M.tb. and nonreplicating M.tb. They also displayed low
cytotoxicity and mutagenicity. Despite the structural similarities with the DprE1 inhibitors,
especially with the dinitrobenzamides (8), compounds 11 - 14 did not inhibit DprE1 [21]. The
SAR study showed the necessity of both the 3,5-dinitrobenzylsulfanyl fragment and the
tetrazole/oxadiazole heterocycle for high antimycobacterial efficiency. The compounds with a 3-
5

ACCEPTED MANUSCRIPT
nitro-5-(trifluoromethyl)benzyl group instead of the 3,5-dinitrobenzyl group showed very low in
vitro anti-TB activity [21].
N N
NO2
N N
1 N
NO2
N
S
R
S
N
N
R1
NO2
R = alkyl, aryl
NO2
1
1
R = alkyl
11
12
MIC 1-4 µM
MIC 1-4 µM
N N
NO2
R1
S
X
NO2
R = alkyl, aryl
3, X = O
1
1
14, X = S
MIC 0.03 - 0.06 µM
Figure 3. 3,5-Dinitrobenzylsulfanyl 1H- and 2H-tetrazoles (11 and 12), 1,3,4-oxadiazoles (13)
and 1,3,4-thiadiazoles (14) as anti-TB lead structures.
As a continuation of our work, we were interested in how the direct attachment of a 3,5-
dinitrophenyl group to the heterocycle would affect the antimycobacterial efficiency. Thus, the
aim of this work was to synthesize a series of novel 5-alkylsulfanyl-1-(3,5-dinitrophenyl)-1H-
tetrazoles (20) and 2-alkylsulfanyl-5-(3,5-dinitrophenyl)-1,3,4-oxadiazoles (23) as the reverse
analogues of the anti-TB lead compounds of series 11 and 13 and to investigate their structure-
activity relationships. Furthermore, derivatives with hydroxy, acetoxy, amino and acetamido
groups (25, 26, 29 and 31, respectively) instead of one of the nitro groups as well as derivatives
with one trifluoromethyl group were prepared and studied (Table 1).
Table 1. Nitro-substituted tetrazole and oxadiazole derivatives studied in this work
6

ACCEPTED MANUSCRIPT
1
1
1
1
R
R
R
R
a
b
c
H
d
e
f
Ph
g
h
i
3-ClPh
4-ClPh
j
3,4-Cl
4-BrPh
4-NO Ph
2
Ph
CH
CH
4-CH
OPh
k
l
3
3
2
3
CH
(CH
)
4
2-ClPh
2,4-Cl
Ph
2
2
2
Results and discussion
Chemistry. The synthesis of the 5-alkylsulfanyl-1-(3,5-dinitrophenyl)-1H-tetrazoles (20d,
0h, 20j), which are reverse analogues of the previously studied compounds of series 11, is
2
shown in Scheme 1. 3,5-Dinitrophenyl isothiocyanate (18) was prepared via the reaction of 3,5-
dinitroaniline with thiophosgene in toluene and was converted into 1-(3,5-dinitrophenyl)-1H-
tetrazole-5-thiol (19) via reaction with sodium azide in water at 80 °C. Alkylation of tetrazole 19
in acetonitrile in the presence of triethylamine gave the final products 20d, 20h and 20j in 72 -
9
6% yields.
7

ACCEPTED MANUSCRIPT
a, b
Scheme 1. Synthesis of 5-alkylsulfanyl-1-(3,5-dinitrophenyl)-1H-tetrazoles 20d, 20h and 20j.
a
Reagents and conditions: (a) CSCl , PhCH , 100 °C, 8 h, 51%; (b) NaN , H O, 80 °C, 5 h,
2
3
3
2
1
b
1
1
7%; (c) R CH X, Et N, CH CN, 2 h, 72 - 96%. A list of R groups can be found in Table 1.
2 3 3
2-Alkylsulfanyl-5-(3,5-dinitrophenyl)-1,3,4-oxadiazoles (23a - l, 23x), which are reverse
analogues of the previously studied compounds of series 13, were synthesized as shown in
Scheme 2. The slow addition of hydrazine hydrate to an ice cold suspension of methyl 3,5-
dinitrobenzoate in ethanol resulted in the formation of the corresponding hydrazide 21, which
was further reacted with carbon disulfide and KOH in water at 90 °C. This reaction resulted in
the formation of two main products, 5-(3,5-dinitrophenyl)-1,3,4-oxadiazole-2-thiol (22) in a 40%
yield and 3-nitro-5-(5-sulfanyl-1,3,4-oxadiazol-2-yl)phenol (24) in a 32% yield.
The main product, 5-(3,5-dinitrophenyl)-1,3,4-oxadiazole-2-thiol (22), was converted into 2-
alkylsulfanyl-5-(3,5-dinitrophenyl)-1,3,4-oxadiazoles (23a - l) via the alkylation of oxadiazole-2-
thiol 22 with various alkyl halides in acetonitrile in the presence of triethylamine (Method A) or
under the conditions of phase-transfer catalysis in the presence of tetrabutylammonium bromide
(TBAB) (Method B). The modified Method A was also used for the synthesis of 2-
cyclohexylsulfanyl-5-(3,5-dinitrophenyl)-1,3,4-oxadiazole (23x), which is the only example
1
without the methylene linker between the sulfur atom and the substituent R described in this
work.
8

ACCEPTED MANUSCRIPT
a, b
Scheme 2. Synthesis of 2-alkylsulfanyl-5-(3,5-dinitrophenyl)-1,3,4-oxadiazoles 23a - l, 23x.
O
O
N
N
N
N
O N
O2N
2
R1
O N
O N
2
NH2
2
O
N
SH
c
S
b
a
O
O
H
NO2
NO2
O N
2
O2N
2
1
22
23a-l
d
N
N
O N
2
S
O
O N
2
2
3x
a
Reagents and conditions: (a) 80% NH NH .H O, EtOH, 0 °C - rt, 4 h, 84%; (b) CS , KOH,
2
2
2
2
1
H O, 90 °C, 10 h, 40%; (c) Method A: R CH X, Et N, CH CN, rt, overnight, 20 - 86%; Method
2
2
3
3
1
B: R CH X, NaOH, TBAB, H O/CH Cl , rt, overnight, 52 - 68%; (d) C H Br, Et N, DMF, 80
C, 20 h, 20%. A list of R groups can be found in Table 1.
2
2
2
2
6
11
3
b
1
°
3-Nitro-5-(5-sulfanyl-1,3,4-oxadiazol-2-yl)phenol (24) was alkylated using 3,4-dichlorobenzyl
chloride to give the product 25j in a 90% yield. In a further step, the hydroxy group of 25j was
acetylated to form compound 26j (Scheme 3).
Scheme 3. Synthesis of 3-(5-((3,4-dichlorobenzyl)sulfanyl)-1,3,4-oxadiazol-2-yl)-5-nitrophenol
a
2
5j and its acetyl derivative 26j.
a
2 2 3 3 3
Reagents and conditions: (a) 3,4-Cl PhCH Cl, Et N, CH CN, reflux, 1 h, 90%; (b) CH COCl,
Et N, THF, 5 °C – reflux, 5 h, 52% or CH COCl, DIPEA, CH CN, rt, 48 h, 72%.
3
3
3
9

ACCEPTED MANUSCRIPT
Furthermore, derivatives with one amino group instead of the original nitro group were
prepared. In this case, methyl 3,5-dinitrobenzoate reacted with hydrazine hydrate in refluxing
ethanol for 4 hours. The elevated temperature led to the formation of acyl hydrazide and the
reduction of one nitro group. 3-Amino-5-nitrobenzohydrazide (27) was isolated in a 63% yield
and was converted into the corresponding 1,3,4-oxadiazole-2-thiol (28) in an 82% yield.
Alkylation of thiol 28 under phase-transfer catalysis conditions led to the formation of target 3-
(
5-alkylsulfanyl-1,3,4-oxadiazol-2-yl)-5-nitroanilines (29d, 29e, 29h, 29k, 29l) in high yields (51
91%) (Scheme 4). The alkylation of the amine group of compound 28 was not observed under
-
these conditions.
Scheme 4. Synthesis of 3-(5-alkylsulfanyl-1,3,4-oxadiazol-2-yl)-5-nitroanilines 29d, 29e, 29h,
a, b
2
9k and 29l.
a
Reagents and conditions: (a) 80% NH NH .H O, EtOH, 4 h, reflux, 63%; (b) CS , KOH,
2
2
2
2
1
b
EtOH, 8 h, reflux, 82%; (c) R CH X, NaOH, TBAB, H O/CH Cl , rt, overnight, 51 - 91%. A
list of R groups can be found in Table 1.
2
2
2
2
1
The synthesis of the acetyl derivatives 31d, 31e, 31h, 31k and 31l is shown in Scheme 5. 5-(3-
Amino-5-nitrophenyl)-1,3,4-oxadiazole-2-thiol (28) was acetylated with acetyl chloride in the
presence of diisopropylethylamine (DIPEA) in acetonitrile. Alkylation of the resulting
oxadiazole-2-thiol 30 in acetonitrile led to the formation of N-(3-(5-alkylsulfanyl-1,3,4-
oxadiazol-2-yl)-5-nitrophenyl)acetamides (31d, 31e, 31h, 31k, 31l) in 52 - 92% yields.
10

ACCEPTED MANUSCRIPT
Scheme 5. Synthesis of N-(3-(5-alkylsulfanyl-1,3,4-oxadiazol-2-yl)-5-nitrophenyl)acetamides
a, b
3
1d, 31e, 31h, 31k and 31l.
a
1
Reagents and conditions: (a) CH COCl, DIPEA, CH CN, rt, 24 h, 81%; (b) R CH X, Et N,
3
3
3
2
3
b
1
CH CN, reflux, 3 - 4 h, 52 - 92%. A list of R groups can be found in Table 1.
Trifluoromethyl group-containing analogues 35d, 35e, 35h and 35j were prepared as shown in
Scheme 6. 3-Nitro-5-(trifluoromethyl)benzoic acid (32a) was prepared from commercially
available 3-(trifluoromethyl)benzoic acid and was converted to methyl 3-nitro-5-
(trifluoromethyl)benzoate (32b) via a standard acid-catalyzed esterification in boiling methanol.
Hydrazinolysis of methyl 3-nitro-5-(trifluoromethyl)benzoate (32b) provided hydrazide 33,
which was then converted to 5-(3-nitro-5-(trifluoromethyl)phenyl)-1,3,4-oxadiazole-2-thiol (34)
via a reaction with carbon disulfide in basic ethanol under reflux conditions. The target 2-
(alkylsulfanyl)-5-(3-nitro-5-(trifluoromethyl)phenyl)-1,3,4-oxadiazoles (35d, 35e, 35h and 35j)
were prepared via alkylation of oxadiazol-2-thiol 34 under phase-transfer catalysis conditions.
Scheme 6. Synthesis of 2-(alkylsulfanyl)-5-(3-nitro-5-(trifluoromethyl)phenyl)-1,3,4-
a, b
oxadiazoles 35d, 35e, 35h and 35j.
11

ACCEPTED MANUSCRIPT
a
Reagents and conditions: (a) H SO , MeOH, 48 h, reflux, 87%; (b) NH NH .H O, EtOH, 48
2
4
2
2
2
1
h, reflux, 43%; (c) CS , KOH, EtOH, 48 h, reflux, 26%; (d) R CH X, NaOH, TBAB,
H O/CH Cl , rt, 4 h, 16 - 54%. A list of R groups can be found in Table 1.
2
2
2
b
1
2
2
In vitro antimycobacterial activity. The results of the in vitro antimycobacterial evaluation
revealed very good activities for the 5-alkylsulfanyl-1-(3,5-dinitrophenyl)-1H-tetrazoles (20d,
2
0h, 20j) and outstanding activities for the 2-alkylsulfanyl-5-(3,5-dinitrophenyl)-1,3,4-
oxadiazoles (23a - l, 23x) against the drug-susceptible M.tb. My 331/88 strain, seven MDR/XDR
strains of M.tb. and against the non-tuberculous strains M. kansasii My 235/80 and clinically
isolated M. kansasii 6509/96 (Table 2, Table S1).
The direct attachment of the 3,5-dinitrophenyl moiety to the tetrazole nitrogen in the
compounds of series 20 led to 2 - 4 times higher antimycobacterial efficiencies against M.tb. and
both strains of M. kansasii compared to the activities of the parent compounds 1-alkyl/aryl-5-
[(3,5-dinitrobenzyl)sulfanyl]-1H-tetrazoles (11). The MIC values of the compounds of series 20
against M.tb. were in the range of 0.25 - 1 µM. The efficiency of the compounds of series 20
against M. avium My 330/88 also increased significantly, and the MIC values against M. avium
were 4 - 32 µM, whereas the MIC values of the parent compounds 11 were in the range of 8 -
2
50 µM.
The antimycobacterial activities of the 1,3,4-oxadiazole reverse analogues 23a - l and 23x
against M.tb. and against both M. kansasii strains were comparable or even better that those of
the parent compounds 13 or the first-line anti-TB drugs. They had MIC values of 0.03 - 0.5 µM.
12

ACCEPTED MANUSCRIPT
The comparison between the antimycobacterial activities of the tetrazole (20) and 1,3,4-
oxadiazole derivatives (23) showed that the antimycobacterial activity of the reported
compounds depended on the heterocycle involved. The 1,3,4-oxadiazole-based compounds of
series 23 showed significantly higher activities than the tetrazole-containing substances of series
2
0. This is the same result observed in our previous study. The 3,5-dinitrobenzylsulfanyl-1,3,4-
oxadiazoles 13 also showed considerably higher antimycobacterial activities compared to the
,5-dinitrobenzylsulfanyltetrazole analogues 11 and 12 [19-21]. The presence of 1,3,4-
3
oxadiazole seems to be highly beneficial for the antimycobacterial activities of anti-TB agents
based on the 3,5-dinitrobenzylsulfanyl moiety (13) and the 3,5-dinitrophenyl moiety (23).
The antimycobacterial activity of 2-alkylsulfanyl-5-(3,5-dinitrophenyl)-1,3,4-oxadiazoles (23a
1
–
l, 23x) was strongly influenced by the lipophilicity of the substituent R . This behavior was
observed in our previous studies [19-21]. The significantly lower efficiency of 2-(3,5-
dinitrophenyl)-5-methylsulfanyl-1,3,4-oxadiazole (23a) compared to other derivatives (23b – l,
2
3x) can be explained by its lower lipophilicity. The loss of antimycobacterial activity in 3,5-
dinitrobenzohydrazide (21) or 2-(3,5-dinitrophenyl)-1,3,4-oxadiazole-5-thiol (22) further
supports this assumption.
The absence of antimycobacterial activity in the hydroxy analogue 25j, its acetyl derivative
2
6j, amino analogues 29d, 29e, 29h, 29k, 29l and their acetyl derivatives 31d, 31e, 31h, 31k, 31l
confirmed our previous observation that both nitro groups are beneficial for a high
antimycobacterial effect. However, in contrast to our previous studies, the compounds of series
3
5 with a trifluoromethyl group instead of one of the nitro groups also possessed
antimycobacterial activities similar to the 3,5-dinitrophenyl derivatives of series 23 (Table 2,
Table S1).
13

ACCEPTED MANUSCRIPT
Table 2. In vitro antimycobacterial activities of the prepared compounds of series 20 - 23, 25,
2
6, 29, 31 and 35 expressed as MIC (µM) after 14 days of incubation.
M. tuberculosis M. avium
M. kansasii
My 235/80
2
M. kansasii
6509/96
2
My 331/88
0.5
My 330/88
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
0d
0h
0j
1
4
8
0.5
2
1
1
0.25
62
8
0.5
125
62
16
8
125
125
125
8
2
62
125
16
3a
3b
3c
3x
3d
3e
3f
3g
3h
3i
4
0.25
0.03
0.03
0.5
2
2
16
16
4
0.03
0.03
0.5
0.03
0.03
0.5
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.5
4
0.125
0.125
0.06
0.06
0.06
0.03
0.03
0.5
0.25
0.06
0.06
0.03
0.03
0.03
0.03
0.5
4
4
n.d.
250
16
125
2
3j
3k
3l
14

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
5j
>250
1000
250
>250
1000
250
>250
1000
250
250
250
125
125
>1000
>1000
>1000
>500
>500
4
>250
1000
250
250
250
125
125
>1000
>1000
>1000
>500
>500
4
6j
9d
9e
9h
9k
9l
125
250
250
250
125
125
125
125
1d
1e
1h
1k
1l
>1000
>1000
250
>1000
> 500
>1000
>500
>500
>250
>250
>250
>250
>250
>500
>500
1
5d
5e
5h
5j
0.5
4
2
0.5
1
1
0.5
1
1
INH
0.5
>250
4
n.d. – not determined
Selected 5-alkylsulfanyl-1-(3,5-dinitrophenyl)-1H-tetrazoles (20h, 20j), 2-alkylsulfanyl-5-(3,5-
dinitrophenyl)-1,3,4-oxadiazoles (23e, 23f, 23g, 23h, 23j, 23k) and two trifluoromethyl
analogues 35d and 35j were further tested against 7 multidrug-resistant strains of M.tb. (Table 3,
Table S2). All of the compounds exhibited outstanding anti-TB activities with MIC values of
0
.25 - 0.5 µM for the tetrazole derivatives 20h and 20j and 0.03 - 0.06 µM for the 1,3,4-
15

ACCEPTED MANUSCRIPT
oxadiazole derivatives of series 23, and no cross-resistance was observed with either the first or
second-line anti-TB drugs. Trifluoromethyl analogues 35d and 35j had slightly lower but
comparable activities to those of the dinitrophenyl derivatives of series 23.
Table 3. In vitro antimycobacterial activities of selected compounds 20h, 20j, 23e, 23f, 23g,
2
3h, 23j, 23k, 35d and 35j and common anti-TB drugs against MDR strains of M. tb. The results
are expressed as MIC (µM) after 14 days of incubation.
MDR M. tuberculosis strains
Praha 1
0.5
Praha 4
0.5
Praha 131 9449/2007 234/2005
7357/1998 8666/2010
2
2
2
2
2
2
2
2
3
3
0h
0j
0.5
0.5
0.5
0.5
0.5
0.25
0.03
0.06
0.03
0.03
0.03
0.03
1
0.25
0.03
0.03
0.03
0.03
0.03
0.03
1
0.25
0.03
0.06
0.03
0.03
0.03
0.03
1
0.25
0.03
0.06
0.03
0.03
0.03
0.03
2
0.25
0.03
0.06
0.03
0.03
0.03
0.03
1
0.25
0.03
0.06
0.03
0.03
0.03
0.03
1
0.25
0.03
0.06
0.03
0.03
0.03
0.03
1
3e
3f
3g
3h
3j
3k
5d
5j
0.5
0.25
0.5
0.5
0.5 / 0.5
0.5 / 0.5
0.5 / 0.5
Streptomycin 16 (R)
>32 (R)
16 (R)
16 (R)
>8 (R)
>16 (R)
0.5 (S)
>32 (R)
16 (R)
32 (R)
>8 (R)
16 (R)
>8 (R)
>32 (R)
64 (R)
8 (S)
32 (R)
16 (R)
16 (R)
>8 (R)
0.5 (S)
0.25 (S)
>32 (R)
16 (R)
16 (R)
>8 (R)
8 (R)
>32 (R)
32 (R)
16 (R)
>8 (R)
8 (R)
Isoniazid
16 (R)
32 (R)
>8 (R)
1 (S)
Ethambutol
Rifampin
Ofloxacin
Gentamicin
>8 (R)
2 (S)
1 (S)
1 (S)
1 (S)
2 (S)
16

ACCEPTED MANUSCRIPT
Clofazimine
Amikacin
0.5 (R)
0.5 (S)
0.5 (R)
1 (S)
0.25 (S)
>32 (R)
0.125 (S)
0.5 (S)
0.125 (S)
0.5 (S)
0.125 (S)
1 (S)
2 (R)
2 (S)
S – Strain susceptible to the given antibiotic drug
R – Strain resistant to the given antibiotic drug
In vitro antibacterial and antifungal activities. To probe the selectivity of the inhibitory
activities of the studied compounds towards mycobacteria, the effects of selected tetrazole-based
compounds 20h and 20j and oxadiazole-based compounds 23e, 23f, 23h, 23j and 23k against
eight bacterial strains (4 gram positive cocci and 4 gram negative rods) and eight fungal strains
(5 yeasts and 3 molds) were evaluated. The results showed that none of the studied compounds
possessed antibacterial or antifungal activities, which indicated that the antimycobacterial effects
of these compounds were highly selective (Table 4 and 5).
a
Table 4. In vitro antifungal activities of selected compounds expressed as MIC (µM).
Yeast strains
CA
Mold strains
CT
CK
CG
TA
AF
AC
TM
2
4 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 72 h 120h
2
2
2
2
2
2
2
0h
0j
>500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500
>500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500
>500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500
>500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500
>125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125
>125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125
>125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125
3e
3f
3h
3j
3k
17

ACCEPTED MANUSCRIPT
FLU 0.82
AMB 0.54
-
-
1.6
-
-
105
1
-
-
26
-
-
210
-
-
>500 -
0.54
>500 -
105
-
-
0.54
0.54
0.27
-
1
-
0.54
a
CA - Candida albicans ATCC 44859; CT - Candida tropicalis 156; CK - Candida krusei
E28; CG - Candida glabrata 20/I; TA - Trichosporon asahii 1188; AF - Aspergillus fumigatus
31; AC - Absidia corymbifera 272; TM - Trichophyton mentagrophytes 445; FLU –
2
Fluconazol; AMB – Amphotericin B.
a
Table 5. In vitro antibacterial activities of selected compounds expressed as MIC (µM).
Strains of gram positive cocci
SA MRSA
4 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h
Strains of gram negative rods
SE
EF
EC KP
KP-E
PA
2
2
2
2
2
2
2
2
0h
0j
125
62
125
62
125
125
125
125
500
250
500
250
>500 >500 >500 >500 >500 >500 >500 >500 >500 >500
>500 >500 >500 >500 >500 >500 >500 >500 >500 >500
3e
3f
>500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500
>500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500
>500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500 >500
>125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125
>125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125 >125
3h
3j
3k
VAN 0.35
GEN -
-
0.35
-
-
-
0.35
-
-
-
0.7
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0.26
0.26
0.26
1
a
SA - Staphylococcus aureus ATCC 6538; MRSA - methicillin resistant Staphylococcus
aureus HK5996/08; SE - Staphylococcus epidermidis HK6966/08; EF - Enterococcus sp.
HK14365/08; EC - Escherichia coli ATCC 8739; KP - Klebsiella pneumoniae HK11750/08;
KP-E - extended spectrum β-lactamase producing Klebsiella pneumoniae HK14368/08; PA -
Pseudomonas aeruginosa ATCC 9027; VAN – Vancomycin; GEN – Gentamicin.
In vitro effects of the studied compounds on mammalian cell proliferation/viability. We
tested the effects of the most promising compounds on mammalian cell viability using the HuH7
(human hepatocellular carcinoma), A431 (human epidermoid carcinoma), MDCK (Madin-Darby
18

ACCEPTED MANUSCRIPT
canine kidney cells) and HepG2 (human hepatocellular carcinoma) cell lines (Table 6). The data
are presented as a relative viability at a concentration of 30 µM compared to the vehicle-treated
controls (100% viability). The results showed that all the 3,5-dinitrophenyltetrazoles 20h and 20j
and 3,5-dinitrophenyloxadiazoles 23e, 23f, 23g, 23h, 23j and 23k had negligible effects on the
cell viability of these cell lines after 48 h of treatment. The selectivity indices, which were
calculated as the ratio between the IC on the HepG2 cell lines and MIC against M.tb., were
5
0
higher than 60 for the tetrazole-based compounds 20h and 20j and higher than 1000 for the
tested oxadiazole-based compounds 23e, 23f, 23g, 23h, 23j and 23k (Table 6).
Table 6. Viability determined via viability cell assays after 48 hours of treatment with
compounds 20h, 20j, 23e, 23f, 23g, 23h, 23j and 23k for the Huh7, A431, MDCK and HepG2
cell lines and the calculated selectivity indices values. Vehicle-treated control viability was set to
a
1
00%. (CellTiter96® Assay)
Selectivity
index
HuH7
A431
Viability IC50
MDCK
Viability IC50
HepG2
Viability IC50
IC50
Viability IC50 (HepG2) /
(µM)
at 30 µM (µM) at 30 µM (µM) at 30 µM (µM) at 30 µM MIC (M.tb.)
2
2
2
2
2
2
2
2
0h ˃30
0j ˃30
3e ˃30
3f ˃30
3g ˃30
3h ˃30
3j ˃30
3k ˃30
115
137
118
144
158
126
153
143
˃30
˃30
˃30
˃30
˃30
˃30
˃30
˃30
74
˃30
˃30
˃30
˃30
˃30
˃30
˃30
˃30
85
˃30
˃30
˃30
˃30
˃30
˃30
˃30
˃30
134
145
126
129
129
126
125
113
˃60
90
84
˃120
93
119
115
105
110
91
˃1000
˃1000
˃1000
˃1000
˃1000
˃1000
91
105
100
126
99
114
19

ACCEPTED MANUSCRIPT
a
Standard deviations were ˂ 10% of the mean
Conclusions
In this work we found that 5-alkylsulfanyl-1-(3,5-dinitrophenyl)-1H-tetrazoles (20) and 2-
alkylsulfanyl-5-(3,5-dinitrophenyl)-1,3,4-oxadiazoles (23) possessed excellent activity against
replicating M.tb. strains, including the clinically isolated multidrug and extensively drug-
resistant strains. The samples had MIC values as low as 0.03 µM, and they did not show any
cross resistance with first or second-line anti-TB drugs. The studied compounds were designed
as the reverse analogues of the previously studied 1-alkyl-5-[(3,5-dinitrobenzyl)sulfanyl]-1H-
tetrazoles (11) [19] and 2-alkyl/aryl-5-[(3,5-dinitrobenzyl)sulfanyl]-1,3,4-oxadiazoles (13) [21].
Interestingly, the direct attachment of a 3,5-dinitrophenyl moiety to the tetrazole in compounds
2
0 or to the oxadiazole in compounds 23 did not lead to significant changes in the
antimycobacterial activity compared to the previous lead compounds 11 and 13. Furthermore, the
compounds of series 20 and 23 had highly selective antimycobacterial effects because they were
completely inactive against 4 gram positive and 4 gram negative bacterial strains and 8 fungal
strains. The in vitro cytotoxicity experiments revealed that the IC50 values of the studied
compounds against four mammalian cell lines, including hepatic cell lines HepG2 and HuH7,
were higher than 30 µM. Thus, the selectivity indices of the most promising compounds 23e,
2
3f, 23g, 23h, 23j and 23k were higher than 1000. The structure-activity relationship study
revealed that the presence of two nitro groups is highly beneficial for high antimycobacterial
activity. The replacement of a nitro group for a hydroxy group in compound 25j or the reduction
of one nitro group to an amino group in series 29 compounds abolished the antimycobacterial
activities, and acetylation of these hydroxy or amino groups did not restore the activity.
However, in contrast to the previous oxadiazole lead compounds 13, the replacement of one nitro
20

ACCEPTED MANUSCRIPT
group in compounds 23 for another strong electron-withdrawing substituent, a trifluoromethyl
group, did not diminish the activity. The antimycobacterial activities of the trifluoromethyl
analogues 35 were only slightly lower than that of the original compounds 23. The compounds
studied in this work, in particular compounds of series 20, 23 and 35, proved to be highly active
antimycobacterial agents with selective action and warrant further studies on their mechanisms
of action and in vitro/in vivo assessments.
Experimental section
1
General. The structural identity of the prepared compounds was confirmed via H-NMR and
1
3
C-NMR spectroscopy analyses. The purity of all the compounds reported was ≥95% as
determined using elemental analysis. All chemicals used for synthesis were obtained from
Sigma-Aldrich (Schnelldorf, Germany) and were used as received. TLC was performed on
Merck aluminum plates with silica gel 60 F . Merck Kieselgel 60 (0.040 - 0.063 mm) was used
2
54
for column chromatography. Melting points were recorded using a Büchi B-545 apparatus
1
13
(
BUCHI Labortechnik AG, Flawil, Switzerland) and are uncorrected. The H and C NMR
spectra were recorded using Varian Mercury Vx BB 300 or VNMR S500 NMR spectrometers
(Varian, Palo Alto, CA, USA). Chemical shifts were reported as δ values in parts per million
(ppm) and were indirectly referenced to tetramethylsilane (TMS) via the solvent signal. The
elemental analysis was performed on an Automatic Microanalyser EA1110CE (Fisons
Instruments S.p.A., Milano, Italy). HRMS (ESI+, ESI-) experiments were performed using the
Q-Exactive Plus Mass Spectrometer (Thermo Scientific, Bremen, Germany) with direct infusion
into the detector of the sample dissolved in acetonitrile:water, 1:1.
21

ACCEPTED MANUSCRIPT
3,5-Dinitrophenyl isothiocyanate (18): Thiophosgene (2.51 g, 1.76 mL, 0.022 mol) was added
dropwise to a solution of 3,5-dinitroaniline (2 g, 0.011 mol) in toluene (20 mL). The mixture was
stirred under a nitrogen atmosphere at 100 °C for 8 hours. Upon completion, the reaction mixture
was cooled to room temperature (rt) and neutralized with a saturated solution of K CO . The
2
3
bilayer mixture was intensively stirred for 24 hours at rt. The organic phase was separated, dried
with anhydrous sodium sulfate and evaporated under reduced pressure. The product was purified
using column chromatography (Hexane/EtOAc, 10:1). Yield: 51% as an orange solid; mp 39 –
1
13
4
1 °C. H NMR (300 MHz, CDCl
MHz, CDCl ) δ 149.03, 143.45, 135.44, 125.63, 116.56. Anal. Calcd for C
H, 1.34; N, 18.66; O, 28.42; S, 14.24. Found: C, 37.50; H, 1.17; N, 18.62; S, 14.36.
-(3,5-Dinitrophenyl)-1H-tetrazole-5-thiol (19): The suspension of 3,5-dinitrophenyl
3
) δ 8.91 (t, J = 2.1 Hz, 1H), 8.38 – 8.33 (m, 2H), C NMR (75
3
7 3 3 4
H N O
S: C, 37.34;
1
isothiocyanate (18) (1.25 g, 5.55 mmol) and sodium azide (0.54 g, 8.33 mmol) in water (25 mL)
was stirred at 80 °C for 5 hours. Upon completion, the reaction mixture was cooled down to rt,
washed with EtOAc (2 × 25 mL) and then acidified with HCl to pH 1 - 2. The precipitated
3
product was filtered, washed with water, dried and recrystallized from CH OH. Yield: 17% as a
1
yellow solid; mp: 134 – 136 °C. H NMR (300 MHz, DMSO) δ 9.33 (d, J = 2.1 Hz, 2H), 8.93 (t,
13
J = 2.1 Hz, 1H). C NMR (75 MHz, DMSO) δ 164.54, 148.26, 135.66, 124.15, 119.04. Anal.
Calcd for C H N O S: C, 31.35; H, 1.50; N, 31.33; S, 11.95. Found: C, 31.33; H, 1.67; N, 31.29;
7
4
6
4
S, 11.82.
General procedure for the synthesis of 5-alkylsulfanyl-1-(3,5-dinitrophenyl)-1H-
tetrazoles (20d, 20h, 20j): An alkylating agent (0.51 mmol) was added to a solution of 1-(3,5-
dinitrophenyl)-1H-tetrazole-5-thiol (19) (0.15 g, 0.56 mmol) and triethylamine (0.062 g, 0.61
3
mmol)) in CH CN (5 mL). The reaction mixture was refluxed for 2 hours. Upon completion, the
22

ACCEPTED MANUSCRIPT
solvent was evaporated under reduced pressure. The crude product was dissolved in EtOAc (15
mL), washed with 5% aq. Na CO (1 × 10 mL) and water (2 × 15 mL), and the organic layer was
2
3
dried over anhydrous sodium sulfate and evaporated. The pure product was obtained using
column chromatography (hexane/EtOAc, 10:1).
5
-Benzylsulfanyl-1-(3,5-dinitrophenyl)-1H-tetrazole (20d): Benzyl bromide was used as an
1
alkylating agent. Yield: 96% as a light yellow solid; mp 117 - 119 °C. H NMR (500 MHz,
acetone) δ 9.13 (td, J = 2.1, 0.6 Hz, 1H), 8.93 (dd, J = 2.0, 0.7 Hz, 2H), 7.46 – 7.41 (m, 2H), 7.34
1
3
–
1
7.25 (m, 3H), 4.67 (s, 2H). C NMR (126 MHz, acetone) δ 155.41, 149.97, 136.86, 136.14,
30.05, 129.55, 128.87, 125.66, 120.59, 38.68. Anal. Calcd for C14 S: C, 46.93; H, 2.81;
10 6 4
H N O
N, 23.45; S, 8.95. Found: C, 47.31; H, 3.18; N, 23.59; S, 8.62.
5
-[(4-Chlorobenzyl)sulfanyl]-1-(3,5-dinitrophenyl)-1H-tetrazole (20h): 4-Chlorobenzyl chloride
1
was used as an alkylating agent. Yield: 82% as a light yellow solid; mp 100 - 102 °C. H NMR
(500 MHz, acetone) δ 9.13 (td, J = 2.1, 0.7 Hz, 1H), 8.96 (dd, J = 2.1, 0.7 Hz, 2H), 7.49 (d, J =
1
3
8
1
.3 Hz, 2H), 7.34 (d, J = 8.3 Hz, 2H), 4.68 (s, 2H). C NMR (126 MHz, acetone) δ 155.28,
49.98, 136.14, 134.23, 131.85, 129.52, 125.61, 120.60, 37.67. Anal. Calcd for C14 ClN S:
H
9
6 4
O
C, 42.81; H, 2.31; N, 21.40; S, 8.16. Found: C, 42.46; H, 2.4; N, 21.68; S, 8.19.
5
-[(3,4-Dichlorobenzyl)sulfanyl]-1-(3,5-dinitrophenyl)-1H-tetrazole (20j): 3,4-Dichlorobenzyl
1
chloride was used as an alkylating agent. Yield: 72% as a light yellow solid; mp 97 - 99 °C. H
NMR (500 MHz, acetone) δ 9.14 (t, J = 2.1 Hz, 1H), 8.98 (d, J = 2.1 Hz, 2H), 7.73 (d, J = 2.1
13
Hz, 1H), 7.52 (d, J = 8.3 Hz, 1H), 7.48 (dd, J = 8.3, 2.1 Hz, 1H), 4.70 (s, 2H). C NMR (126
MHz, acetone) δ 155.20, 150.00, 138.50, 136.10, 132.62, 132.18, 132.15, 131.51, 130.25,
1
25.62, 120.65, 36.94. Anal. Calcd for C14
8 2 6 4
H Cl N O S: C, 39.36; H, 1.89; N, 19.67; S, 7.50.
Found: C, 39.52; H, 2.06; N, 19.87; S, 7.63.
23

ACCEPTED MANUSCRIPT
3
,5-Dinitrobenzohydrazide (21) [22]: Methyl 3,5-dinitrobenzoate was obtained in a 98% yield by
refluxing 3,5-dinitrobenzoyl chloride in MeOH for 1 hour. Upon cooling, the precipitated
product was filtered and washed with 1% NaOH. In the next step, hydrazine hydrate (80%, 0.94
g, 0.91 mL, 15 mmol) was added dropwise into the solution of methyl 3,5-dinitrobenzoate (1.13
g, 5 mmol) in EtOH (20 mL) at 0 °C. The reaction mixture was stirred for 4 hours at rt. Upon
completion, the precipitated product was filtered and washed with EtOH (10 mL) and Et O (10
2
mL). The product was used without further purification. Yield: 84% as a yellowish solid; mp 153
1
-
154 °C (lit.[22] mp 155 - 157 °C). H NMR (300 MHz, DMSO) δ 10.50 (s, 1H, NH), 9.00 (d, J
1
3
=
2
2.1 Hz, 2H), 8.93 (t, J = 2.1 Hz, 1H), 4.74 (s, 2H, NH ). C NMR (75 MHz, DMSO) δ 161.50,
1
48.38, 136.09, 127.36, 120.84. Anal. Calcd for C H N O : C, 37.18; H, 2.67; N, 24.77. Found:
7 6 4 5
C, 37.42; H, 2.68; N, 24.32.
5
-(3,5-Dinitrophenyl)-1,3,4-oxadiazole-2-thiol (22) [23]: Carbon disulfide (0.7 g, 0.56 mL, 9.2
mmol) was added to a solution of 3,5-dinitrobenzohydrazide (21) (0.7 g, 3.1 mmol) and KOH
0.21 g, 3.75 mmol) in water (15 mL). The reaction mixture was heated at 90 °C for 10 hours.
(
The product precipitated from the reaction mixture with some side products, and 1 M NaOH (3
mL) was added to the reaction mixture. The reaction mixture was stirred for 10 min and filtered.
The aqueous solution was then acidified with HCl to pH 2 - 3 and extracted with EtOAc (2 × 20
mL). The organic layers were combined, washed with water (2 × 15 mL) and dried over
anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product
was purified using column chromatography (mobile phase: CHCl /hexane/CH COOH, 24:6:1, Rf
3
3
1
=
0.26). Yield: 40% as a yellow solid; mp 203 - 210 °C. H NMR (300 MHz, acetone) δ 9.12 (t,
13
J = 2.1 Hz, 1H), 9.02 (d, J = 2.1 Hz, 2H). C NMR (75 MHz, acetone) δ 179.37, 158.54, 150.13,
24

ACCEPTED MANUSCRIPT
1
27.02, 126.69, 121.91. Anal. Calcd for C
Found: C, 36.11; H, 1.90; N, 20.52; S, 11.63.
-Nitro-5-(5-sulfanyl-1,3,4-oxadiazol-2-yl)phenol (24) was isolated as a by-product from a
previous reaction (mobile phase: CHCl /hexane/CH COOH, 24:6:1, Rf = 0.13). Yield: 32% as a
8 4 4 5
H N O S: C, 35.83, H, 1.50; N, 20.89; S, 11.96.
3
3
3
1
yellow solid; mp 195 - 197 °C, H NMR (500 MHz, DMSO) δ 14.82 (s, 1H), 7.62 (t, J = 1.8 Hz,
1
3
1
1
H), 7.54 (t, J = 2.1 Hz, 1H), 7.41 (t, J = 1.9 Hz, 1H). C NMR (126 MHz, DMSO) δ 177.64,
59.53, 151.10, 149.39, 124.31, 115.88, 110.06, 106.65. Anal. Calcd for C S: C, 40.17;
8
5 3 4
H N O
H, 2.11; N, 17.57; S, 13.4. Found: C, 40.63; H, 2.03; N, 17.32; S, 13.59.
General procedure for the synthesis of the 2-alkylsulfanyl-5-(3,5-dinitrophenyl)-1,3,4-
oxadiazoles (23a - l): Method A: An alkylating agent (0.51 mmol) was added to a solution of 5-
(3,5-dinitrophenyl)-1,3,4-oxadiazole-2-thiol (22) (0.15 g, 0.56 mmol) and triethylamine (0.062 g,
0
.61 mmol) in CH CN (7 mL). The reaction mixture was stirred overnight at rt. Upon reaction
3
completion (as determined by TLC), the solvent was evaporated under reduced pressure. The
crude product was dissolved in EtOAc (15 mL), and the organic solution was washed with 1%
NaOH (15 mL) and water (2 × 15 mL) and dried over anhydrous sodium sulfate. The solvent was
evaporated under reduced pressure, and the product was purified via crystallization or column
chromatography.
Method B: The alkylating agent (0.51 mmol) and TBAB (0.025 mmol) solution in CH Cl (5
2
2
mL) was added to the solution of 5-(3,5-dinitrophenyl)-1,3,4-oxadiazole-2-thiol (22) (0.15 g,
.56 mmol) and NaOH (0.024 g, 0.61 mmol) in water (5 mL). The reaction mixture was gently
stirred overnight at rt. Upon completion, the organic layer was separated, washed with water (2 ×
mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced
pressure, and the product was purified via crystallization or column chromatography.
0
7
25

ACCEPTED MANUSCRIPT
5
-(3,5-Dinitrophenyl)-2-methylsulfanyl-1,3,4-oxadiazole (23a): Dimethyl sulfate was used as an
alkylating agent. The product was prepared via Method B and was purified using column
chromatography (mobile phase: hexane/EtOAc, 3:1, Rf = 0.41). Yield: 60% as a white solid; mp
1
13
1
24 - 127 °C. H NMR (500 MHz, acetone) δ 9.10 (s, 3H), 2.86 (s, 3H). C NMR (126 MHz,
acetone) δ 167.82, 163.57, 150.13, 127.73, 126.90, 121.53, 14.72. Anal. Calcd for C H N O S:
9
6
4
5
C, 38.30; H, 2.14; N, 19.85; S, 11.36. Found: C, 38.87; H, 2.35; N, 19.96; S, 11.22. The
formation of 5-(3,5-dinitrophenyl)-3-methyl-1,3,4-oxadiazole-2(3H)-thione was also observed
(mobile phase: hexane/EtOAc, 3:1, R f = 0.56) [24]. The compound was obtained in a 10% yield.
1
H NMR (500 MHz, acetone) δ 9.12 (t, J = 2.1 Hz, 1H), 9.01 (d, J = 2.1 Hz, 2H), 3.82 (s, 3H).
1
3
C NMR (126 MHz, acetone) δ 176.80, 155.52, 149.32, 125.88, 125.79, 36.13.
5
-(3,5-Dinitrophenyl)-2-propylsulfanyl-1,3,4-oxadiazole (23b): Propyl bromide was used as an
alkylating agent. The product was prepared via Method A and was purified using column
chromatography (mobile phase: hexane/EtOAc, 6:1). Yield: 40% as a white solid; mp 70 - 72 °C.
1
H NMR (300 MHz, acetone) δ 9.10 (s, 3H), 3.39 (t, J = 7.2 Hz, 2H), 1.97 – 1.83 (m, 2H), 1.07
1
3
(
t, J = 7.3 Hz, 3H). C NMR (75 MHz, acetone) δ 167.20, 163.49, 150.10, 127.73, 126.91,
21.52, 34.97, 23.48, 13.20. Anal. Calcd for C11 S: C, 42.58; H, 3.25; N, 18.06; S, 10.33.
Found: C, 43.12; H, 3.35; N, 17.86; S, 10.58.
-(3,5-Dinitrophenyl)-2-hexylsulfanyl-1,3,4-oxadiazole (23c): Hexyl bromide was used as an
1
10 4 5
H N O
5
alkylating agent. The product was prepared via Method A and was purified using column
chromatography (mobile phase: hexane/EtOAc, 7:1). Yield: 66% as a white solid; mp 72 - 75 °C.
1
H NMR (500 MHz, acetone) δ 9.12 (s, 3H), 3.47 – 3.38 (m, 2H), 1.96 – 1.83 (m, 2H), 1.57 –
1
3
1
.47 (m, 4H), 1.37 – 1.35 (m, 2H), 0.93 – 0.88 (m, 3H). C NMR (126 MHz, acetone) δ 176.98,
26

ACCEPTED MANUSCRIPT
1
73.23, 159.86, 137.49, 136.64, 131.26, 42.87, 41.63, 39.20, 38.56, 32.86, 23.95. Anal. Calcd for
C H N O S: C, 47.72; H, 4.58; N, 15.90; S, 9.10. Found: C, 48.1; H, 4.42; N, 15.53; S, 9.42.
14
16
4
5
2
-Benzylsulfanyl-5-(3,5-Dinitrophenyl)-1,3,4-oxadiazole (23d): Benzyl bromide was used as an
alkylating agent. The product was prepared via Method B and was purified using column
chromatography (mobile phase: hexane/EtOAc, 3:1). Yield: 68% as a yellow solid; mp 155 - 157
1
°
C (with decomposition). H NMR (300 MHz, DMSO) δ 8.97 (t, J = 2.1 Hz, 1H), 8.94 (d, J = 2.1
1
3
Hz, 2H), 7.55 - 7.47 (m, 2H), 7.39 - 7.27 (m, 3H), 4.64 (s, 2H); C NMR (75 MHz, DMSO) δ
1
65.41, 162.77, 148.88, 136.67, 129.29, 128.78, 128.02, 126.38, 125.92, 121.14, 36.08. Anal.
Calcd for C15
S, 9.12.
10 4 5
H N O S: C, 50.28; H, 2.81; N, 15.64; S, 8.95. Found: C, 49.91, H, 2.74; N, 15.37;
5
-(3,5-Dinitrophenyl)-2-[(4-methoxybenzyl)sulfanyl]-1,3,4-oxadiazole (23e): 4-Methoxybenzyl
chloride was used as an alkylating agent. The product was prepared via Method A and was
1
purified by crystallization (EtOH/H O). Yield: 84% as a yellowish solid; mp 142 - 145 °C. H
2
NMR (500 MHz, DMSO) δ 8.97 (t, J = 2.1 Hz, 1H), 8.94 (d, J = 2.1 Hz, 2H), 7.42 (d, J = 8.6
1
3
Hz, 2H), 6.90 (d, J = 8.6 Hz, 2H), 4.59 (s, 2H), 3.72 (s, 3H). C NMR (126 MHz, DMSO) δ
65.50, 162.71, 159.07, 148.87, 130.61, 128.27, 126.35, 125.92, 121.10, 114.15, 55.25, 35.82.
Anal. Calcd for C H N O S: C, 49.48; H, 3.11; N, 14.43; S, 8.26. Found: C, 49.62: H, 3.28; N,
1
1
6
12
4
6
1
4.35; S, 8.64.
-[(2-Chlorobenzyl)sulfanyl]-5-(3,5-dinitrophenyl)-1,3,4-oxadiazole (23f): 2-Chlorobenzyl
2
chloride was used as an alkylating agent. The product was prepared via Method A and was
1
purified by crystallization (EtOH/H O). Yield: 86% as a yellowish solid; mp 137 - 139 °C. H
2
NMR (500 MHz, DMSO) δ 8.99 - 8.97 (m, 1H), 8.95 - 8.94 (m, 2H), 7.68 - 7.65 (m, 1H), 7.53 -
1
3
7
.49 (m, 1H), 7.39 - 7.32 (m, 2H), 4.72 (s, 2H). C NMR (126 MHz, DMSO) δ 164.86, 162.98,
27

ACCEPTED MANUSCRIPT
1
48.89, 133.93, 133.55, 131.89, 130.26, 129.84, 127.66, 126.42, 125.89, 121.20, 34.44. Anal.
Calcd for C H ClN O S: C, 45.87; H, 2.31; N, 14.26; S, 8.16. Found: C, 46.06; H, 2.48; N,
1
5
9
4
5
1
4.28; S, 8.29.
-[(3-Chlorobenzyl)sulfanyl]-5-(3,5-dinitrophenyl)-1,3,4-oxadiazole (23g): 3-Chlorobenzyl
2
chloride was used as an alkylating agent. The product was prepared via Method A and was
purified using column chromatography (mobile phase: hexane/EtOAc, 4:1). Yield: 74% as a
1
yellowish solid; mp 112 - 114 °C. H NMR (500 MHz, DMSO) δ 8.97 (t, J = 2.1 Hz, 1H), 8.94
(d, J = 2.2 Hz, 2H), 7.62 – 7.60 (m, 1H), 7.50- 7.47 (m, 1H), 7.40 - 7.34 (m, 2H), 4.64 (s, 2H).
1
3
C NMR (126 MHz, DMSO) δ 165.20, 162.87, 148.86, 139.47, 133.19, 130.56, 129.12, 127.99,
1
1
2
27.93, 126.38, 125.91, 121.15, 35.23. Anal. Calcd for C H ClN O S: C, 45.87; H, 2.31; N,
15 9 4 5
4.26; S, 8.16. Found: C, 46.41; H, 2.54; N, 13.94; S, 8.11.
-[(4-Chlorobenzyl)sulfanyl]-5-(3,5-dinitrophenyl)-1,3,4-oxadiazole (23h): 4-Chlorobenzyl
chloride was used as an alkylating agent. The product was prepared via Method A and was
1
purified by crystallization (CH CN/H O). Yield: 79% as a light beige solid; mp 163 - 168 °C. H
3
2
NMR (500 MHz, DMSO) δ 8.97 (t, J = 2.1 Hz, 1H), 8.93 (d, J = 2.1 Hz, 2H), 7.54 (d, J = 8.3
1
3
Hz, 2H), 7.41 (d, J = 8.3 Hz, 2H), 4.63 (s, 2H). C NMR (126 MHz, DMSO) δ 165.23, 162.83,
48.87, 136.00, 132.63, 131.17, 128.71, 126.38, 125.90, 121.14, 35.24. Anal. Calcd for
C H ClN O S: C, 45.87; H, 2.31; N, 14.26; S, 8.16. Found: C, 46.56; H, 2.54; N, 14.53; S, 8.19.
1
15
9
4
5
2
-[(2,4-Dichlorobenzyl)sulfanyl]-5-(3,5-dinitrophenyl)-1,3,4-oxadiazole
(23i):
2,4-
Dichlorobenzyl chloride was used as an alkylating agent. The product was prepared via Method
A and was purified by crystallization (EtOH/H O). Yield: 63% as a white solid; mp 129 - 130
2
1
°
C. H NMR (500 MHz, acetone) δ 9.14 – 9.10 (m, 3H), 7.78 (d, J = 8.3 Hz, 1H), 7.60 (d, J = 2.2
1
3
Hz, 1H), 7.42 (dd, J = 8.3, 2.2 Hz, 1H), 4.77 (s, 2H). C NMR (126 MHz, acetone) δ 166.00,
28

ACCEPTED MANUSCRIPT
1
63.95, 150.12, 135.74, 135.26, 134.02, 133.75, 130.18, 128.39, 127.58, 127.02, 121.69, 34.55.
Anal. Calcd for C H Cl N O S: C, 42.17; H, 1.89; C, 13.11; N, 7.50. Found: C, 41.8; H, 1.91;
15
8
2
4
5
N, 12.79; S, 7.37.
-[(3,4-Dichlorobenzyl)sulfanyl]-5-(3,5-dinitrophenyl)-1,3,4-oxadiazole
2
(23j):
3,4-
Dichlorobenzyl chloride was used as an alkylating agent. The product was prepared via Method
A and was purified using column chromatography (mobile phase: hexane/EtOAc, 4:1). Yield:
1
8
8
5% as a white solid; mp 132 - 134 °C. H NMR (500 MHz, DMSO) δ 8.97 (t, J = 2.1 Hz, 1H),
.93 (d, J = 2.1 Hz, 2H), 7.81 (d, J = 2.0 Hz, 1H), 7.61 (d, J = 8.3 Hz, 1H), 7.51 (dd, J = 8.3, 2.0
1
3
Hz, 1H), 4.63 (s, 2H). C NMR (126 MHz, DMSO) δ 165.09, 162.90, 148.86, 138.32, 131.28,
1
31.14, 130.83, 130.60, 129.66, 126.38, 125.91, 121.16, 34.61. Anal. Calcd for C H Cl N O S:
15 8 2 4 5
C, 42.17; H, 1.89; N, 13.11; S, 7.50. Found: 42.71; H, 2.25; N, 12.86; S, 8.25.
2
-[(4-Bromobenzyl)sulfanyl]-5-(3,5-dinitrophenyl)-1,3,4-oxadiazole (23k): 4-Bromobenzyl
bromide was used as an alkylating agent. The product was prepared via Method A and was
purified using column chromatography (mobile phase: hexane/EtOAc, 4:1). Yield: 74% as a
1
white solid; mp 188 - 189 °C. H NMR (300 MHz, DMSO) δ 8.97 (t, J = 2.1 Hz, 1H), 8.93 (d, J
1
3
=
2.1 Hz, 2H), 7.55 (d, J = 8.5 Hz, 2H), 7.47 (d, J = 8.5 Hz, 2H), 4.62 (s, 2H). C NMR (75
MHz, DMSO) δ 165.25, 162.87, 148.89, 136.47, 131.66, 131.54, 126.42, 125.90, 121.21, 121.18,
3
2
5
5.29. Anal. Calcd for C H BrN O S: C, 41.21; H, 2.07; N, 12.81; S, 7.33. Found: C, 41.15; H,
15 9 4 5
.23; N, 12.82; S, 7.58.
-(3,5-Dinitrophenyl)-2-[(4-nitrobenzyl)sulfanyl]-1,3,4-oxadiazole (23l): 4-Nitrobenzyl chloride
was used as an alkylating agent. The product was prepared via Method B and was purified using
column chromatography (mobile phase: hexane/EtOAc, 5:1). Yield: 52 % as a white solid; mp
1
1
87 - 188 °C. H NMR (300 MHz, DMSO) δ 8.98 - 8.96 (m, 1H), 8.92 – 8.90 (m, 2H), 8.22 (d, J
29