Development of water-soluble 3,5-dinitrophenyl tetrazole and oxadiazole antitubercular agents

Article abstract of DOI:10.1016/j.bmc.2017.08.010

In this work, four series of tertiary amine-containing derivatives of 3,5-dinitrophenyl tetrazole and oxadiazole antitubercular agents were prepared, and their in vitro antimycobacterial effects were evaluated. We found that the studied compounds showed l

Full text of DOI:10.1016/j.bmc.2017.08.010

Accepted Manuscript
Development of Water-Soluble 3,5-Dinitrophenyl Tetrazole and Oxadiazole
Antitubercular Agents
Jaroslav Roh, Galina Karabanovich, Hana Vl ková, Alejandro Carazo, Jan N me
ek, Pavel Sychra, Lenka Valá šková, Oto Pavliš, Ji ina Stola íková, V ra
Klimešová, Kate ina Vávrová, Petr Pávek, Alexandr Hrabálek
PII:
S0968-0896(17)31113-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.08.010
BMC 13913
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
25 May 2017
1 August 2017
7 August 2017
Please cite this article as: Roh, J., Karabanovich, G., ková, H.V., Carazo, A., me ek, J.N., Sychra, P., Valá šková,
L., Pavliš, O., íková, J.i.S., Klimešová, V.r., Vávrová, K.i., Pávek, P., Hrabálek, A., Development of Water-Soluble
3
,5-Dinitrophenyl Tetrazole and Oxadiazole Antitubercular Agents, Bioorganic & Medicinal Chemistry (2017),
doi: http://dx.doi.org/10.1016/j.bmc.2017.08.010
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Development of Water-Soluble 3,5-Dinitrophenyl
Tetrazole and Oxadiazole Antitubercular Agents
a,
a
a
a
a
Jaroslav Roh, * Galina Karabanovich, Hana Vlčková, Alejandro Carazo, Jan Němeček,
a
a
b
c
a
Pavel Sychra, Lenka Valášková, Oto Pavliš, Jiřina Stolaříková, Věra Klimešová, Kateřina
a
a
a
Vávrová, Petr Pávek, and Alexandr Hrabálek
a
ꢀꢁꢂꢃꢄꢅ ꢆꢇ ꢈꢉꢊ ꢄꢂꢅꢉꢋꢌ
ꢀꢁꢂꢃꢄꢅ
ꢆꢀꢇꢈꢉꢊꢋ, Czech Republic
b
Military Health Institute, Biological Defense Department, Techonin, Czech Republic
c
Regional Institute of Public Health, Department of Bacteriology and Mycology, Partyzánské
náměstí 7, 70200 Ostrava, Czech Republic
ABSTRACT
In this work, four series of tertiary amine-containing derivatives of 3,5-dinitrophenyl tetrazole
and oxadiazole antitubercular agents were prepared, and their in vitro antimycobacterial effects
were evaluated. We found that the studied compounds showed lipophilicity-dependent
antimycobacterial activity. The N-benzylpiperazine derivatives, which had the highest
lipophilicity among all of the series, showed the highest in vitro antimycobacterial activities
against Mycobacterium tuberculosis CNCTC My 331/88 (H Rv), comparable to those of the
37
1

first-line drugs isoniazid and rifampicin. The presence of two tertiary amines in these N-
benzylpiperazine derivatives enabled us to prepare water-soluble dihydrochloride salts,
overcoming the serious drawback of previously described 3,5-dinitrophenyl tetrazole and
oxadiazole lead compounds. The water-soluble 3,5-dinitrophenyl tetrazole and oxadiazole
antitubercular agents described in this work are good candidates for further in vitro and in vivo
pharmacokinetic and pharmacodynamic studies.
Keywords
Tuberculosis; Antitubercular agent; Mycobacterium tuberculosis; Structure-activity
relationships; Lipophilicity; Solubility
Introduction
Tuberculosis (TB) is a widespread infectious disease caused by Mycobacterium tuberculosis
(M.tb.) that took 1.8 million lives in 2015 and led to 10.4 million new cases. The major problem
1
with TB treatment is the occurrence of drug-resistant strains of M.tb. Multidrug-resistant (MDR)
strains are resistant to at least two of the most powerful anti-TB drugs: isoniazid (INH) and
rifampicin (RIF). Extensively drug-resistant (XDR) strains are resistant to all first-line anti-TB
2
drugs and several second-line drugs, such as fluoroquinolones and injectable aminoglycosides.
The treatment of drug-resistant forms of TB usually lasts more than 20 months and has lower
1
cure rates. Thus, the development of new, efficient anti-TB drugs with different modes of
actions is vital for success against TB. Recently, two new anti-TB drugs have been approved for
3
treating pulmonary MDR-TB: nitroimidazol derivative delamanide and diarylquinoline
4
, 5
derivative bedaquiline. Several experimental anti-TB compounds are in the preclinical and
6
clinical phases of development, such as nitro group-containing nitroimidazoles PA-824 or TBA-
2

7
3
54 and the inhibitor of mycobacterial decaprenylphosphoryl-β-D-ribofuranose 2´-oxidase
8
-11
(
DprE1) PBTZ-169 (Figure 1).
Figure 1. Structures of the nitro group-containing anti-TB agents nitroimidazoles PA-824 and
TBA-354 and benzothiazinone PBTZ-169.
In our previous studies, we found that 1-substituted 5-(3,5-dinitrobenzylsulfanyl)tetrazoles (1)
showed good in vitro antimycobacterial activity, in some cases comparable to that of the first-
1
2
line anti-TB drug INH. The structure-activity relationship study revealed the importance of both
,5-dinitrobenzylsulfanyl and tetrazole moieties for high antimycobacterial effects and indicated
that the lipophilicity of substituents on tetrazole can influence the compound’sꢍactivity. Later, the
,5-regioisomers, i.e., 2-substituted 5-(3,5-dinitrobenzylsulfanyl)tetrazoles (2), showed slightly
3
2
1
3
better antimycobacterial effects than did the 1,5-regioisomers 1 and became parent structures
3

for two new classes of potent antitubercular agents, 2-substituted 5-(3,5-dinitrobenzylsulfanyl)-
1
4
1
,3,4-oxadiazoles (3) and 2-substituted 5-(3,5-dinitrophenyl)-2H-tetrazoles (4)
1
5
(
Figure 2). 3,5-Dinitrobenzylsulfanyl oxadiazoles 3 showed outstanding in vitro
antimycobacterial activity, with minimum inhibitory concentration (MIC) values reaching 0.03
µM against drug-susceptible and drug-resistant M.tb. strains. Furthermore, these compounds
were highly effective against a non-replicating M.tb. strain and showed low cytotoxicity and
1
6
genotoxicity. In contrast to other 3,5-dinitro-substituted antitubercular agents, oxadiazoles 3 act
1
4
in a DprE1-independent pathway. Thus, 3,5-dinitrobenzylsulfanyl oxadiazoles 3 were chosen as
one of the lead structures in this study (Figure 2).
The second lead structures of this study, 2-substituted 5-(3,5-dinitrophenyl)-2H-tetrazoles (4),
are the analogues of tetrazoles 2 without the methylsulfanyl linker between 3,5-dinitrophenyl and
15
tetrazole moieties. These easily accessible compounds had submicromolar MICs against drug-
susceptible and MDR/XDR strains of M.tb. and again showed low cytotoxicity in mammalian
cell lines. Furthermore, these compounds are sulfur-free, which might be beneficial for their
metabolic stability (Figure 2).
The third lead structures, S-substituted 5-(3,5-dinitrophenyl)oxadiazole-2-thiols (5), are the
17
reverse analogues of oxadiazoles 3 (Figure 2). As in the structure of tetrazoles 4, the 3,5-
dinitrophenyl group is attached directly to the heterocycle. 3,5-Dinitrophenyl oxadiazoles 5
showed similarly outstanding in vitro antimycobacterial activity as their parent 3,5-
dinitrobenzylsulfanyl oxadiazoles 3, with MIC values against drug-susceptible and MDR/XDR
M.tb. strains as low as 0.03 µM. Furthermore, oxadiazoles 5 had highly selective
antimycobacterial effects, because they showed low cytotoxicity against mammalian cell lines
1
7
and had no antibacterial or antifungal activities. However, the abovementioned lead structures
suffered from very low aqueous solubility.
4

Figure 2. Antitubercular lead compounds used in this study: 3,5-dinitrobenzylsulfanyl
1
4
15
17
oxadiazoles 3 and 3,5-dinitrophenyl tetrazoles 4 and oxadiazoles 5.
The aim of this study was to prepare derivatives of the lead structures 3, 4 and 5 with increased
aqueous solubility and to study their antimycobacterial activity. A way to improve the aqueous
solubility is to introduce an acidic/basic moiety into the structure and to form a corresponding
salt. As we previously demonstrated that acidic functionality has devastating effects on
12
antimycobacterial activities of the abovementioned lead structures, we decided to introduce a
basic amine moiety into the structure. This approach has been widely used successfully to
1
1, 18-23
increase the aqueous solubility of water-insoluble drugs and bioactive substances.
Therefore, derivatives 6a-d and 7a-d of the lead structure 3, derivatives 8a-d of the lead
structure 4 and derivatives 9a-d of the lead structure 5 with at least one tertiary amine in the
substituent R were prepared, and their in vitro antimycobacterial activities were evaluated
5

(
Figure 3). Compounds with highest in vitro antimycobacterial activity were converted to water-
soluble hydrochloride salts, and their in vitro antitubercular activities were re-evaluated.
Furthermore, the cytotoxicity of these water-soluble hydrochloride salts was assessed using four
mammalian cell lines.
Figure 3. Tertiary amine-containing compounds of series 6, 7, 8 and 9 studied in this work.
Results and discussion
Chemistry. Tertiary amine-containing 2-substituted 5-(3,5-dinitrobenzylsulfanyl)-1,3,4-
oxadiazoles 6a-d and 7a-d were prepared as shown in Scheme 1. Hydrazinolysis of the lactones
1
0a and 10b provided corresponding ω-hydroxyalkanehydrazides 11a and 11b, which were
converted to 5-(ω-hydroxyalkyl)-1,3,4-oxadiazole-2-thiols 12a and 12b, respectively, by the
6

reaction with carbon disulfide under alkaline conditions. Oxadiazole-2-thiols 12a and 12b were
then alkylated using 3,5-dinitrobenzyl chloride, and their hydroxy groups were converted to
bromo groups and finally substituted with N-methylpiperazine, morpholine, piperidine or N-
benzylpiperazine heterocycle (Scheme 1).
Scheme 1. Synthesis of tertiary amine-containing 2-subsituted 5-(3,5-dinitrobenzylsulfanyl)-
1
7
,3,4-oxadiazoles 6a-d and 7a-d. Reagents and conditions: (a) NH NH .H O, EtOH; rt, 24 h,
2
2
2
7% (11a); reflux, 70 h, 80% (11b); (b) CS , KOH, EtOH; rt, 48 h, 43% (12a); reflux, 24 h, 95%
2
(
12b); (c) 3,5-(NO ) BnCl, Et N, MeCN, rt, 6 h, 82% (13a), 72% (13b) (d) PPh , NBS, rt, 3 h,
2
2
3
3
9
0% (14a), 71% (14b); (e) heterocyclic amine, MeCN; for conditions and yields, see
Experimental section.
The 2-substituted 5-(3,5-dinitrophenyl)-2H-tetrazoles 8a-d were prepared via two-step
synthesis from 5-(3,5-dinitrophenyl)-1H-tetrazole (15, Scheme 2). In the first step, tetrazole 15
7

was alkylated using 1,3-dibromopropane, and the predominantly formed 2-regiosomer, 2-(3-
bromopropyl)-5-(3,5-dinitrophenyl)-2H-tetrazole (16), was isolated. The chemical shift of the
tetrazole carbon in the product 16 was above 160 ppm, which is typical for 2-substituted 5-
1
5, 24
aryltetrazoles.
Furthermore, the correlation of the NCH hydrogens (t, 4.96 ppm) in compound
2
1
15
1
6 with three nitrogen atoms was clearly visible in the H- N gHMBC experiment, which would
not be possible if the substituent was attached to position 1. Negligible correlation of the NCH2
hydrogens with the aromatic hydrogens in 1D NOESY experiment further supports the presence
of 2,5-regioisomer (see Supplementary material). The reaction of tetrazole 16 with N-
methylpiperazine, morpholine, piperidine or N-benzylpiperazine gave the final products 8a, 8b,
8
c and 8d, respectively (Scheme 2). 5-(3,5-Dinitrophenyl)-1H-tetrazole (15) was prepared as
1
5
described previously.
Scheme 2. Synthesis of tertiary amine-containing 2-substituted 5-(3,5-dinitrophenyl)-2H-
tetrazoles 8a-d. Reagents and conditions: (a) Br(CH ) Br, Et N, MeCN, reflux, 5 h, 60%; (b)
2
3
3
heterocyclic amine, MeCN; for conditions and yields, see Experimental section.
The synthesis of S-substituted 5-(3,5-dinitrophenyl)-1,3,4-oxadiazole-2-thiols 9a-d started
1
7
with the alkylation of 5-(3,5-dinitrophenyl)-1,3,4-oxadiazole-2-thiol 17 with 1,3-
dibromopropane (Scheme 3). The reaction of the resulting 2-(3-bromopropylsulfanyl)-5-(3,5-
8

dinitrophenyl)-1,3,4-oxadiazole 18 with N-methylpiperazine, morpholine, piperidine or N-
benzylpiperazine heterocycles gave the final products 9a, 9b, 9c and 9d, respectively.
Scheme 3. Synthesis of tertiary amine-containing S-substituted 5-(3,5-dinitrophenyl)oxadiazole-
2
-thiols 9a-d. Reagents and conditions: (a) Br(CH ) Br, Et N, MeCN, 50 °C, 10 h, 61%; (b)
2
3
3
heterocyclic amine, MeCN; for conditions and yields, see Experimental section; (c) 1-(3-
chloropropyl)piperidine hydrochloride, Et N, MeCN, reflux, 12 h, 65%.
3
Finally, the three N-benzylpiperazine derivatives with the highest antimycobacterial activities,
7
d, 8d and 9d, were converted to water-soluble dihydrochloride salts 7e, 8e and 9e, respectively,
by dissolving the compounds in aq. hydrochloric acid and subsequent evaporation of all the
volatiles (Scheme 4).
9

Scheme 4. Synthesis of dihydrochloride salts 7e, 8e and 9e of N-benzylpiperazine derivatives 7d,
8
d and 9d. Reagents and conditions: (a) HCl, H O, 60 °C
2
The solubilities of dihydrochloride salts 7e, 8e and 9e in pure water at room temperature were
1
7.1 mM (9 mg/mL), 20.9 mM (11 mg/mL) and 22.8 mM (12.5 mg/mL), respectively. As the
solubility of these compounds was pH-dependent, we also measured the solubility in 0.05 M
citrate buffer (pH 3), 0.1 M acetate buffer (pH 5) and phosphate-buffered saline (PBS, pH 7.4)
using HPLC. The results are summarized in Table 1.
Table 1. Solubilities (mM) of dihydrochloride salts 7e, 8e and 9e in selected buffers.
pH 3
pH 5
pH 7.4
PBS
citrate
buffer
acetate
buffer
7
8
9
e
e
e
18.0 ± 0.4
19.2 ± 0.6
21.2 ± 0.4
9.0 ± 1.0
11.6 ± 1.4
14.2 ± 0.6
0.113 ± 0.007
0.051 ± 0.005
0.075 ± 0.005
1
0

In vitro antimycobacterial activity
All tertiary amine-containing compounds of series 6, 7, 8 and 9 were evaluated for their in
vitro antimycobacterial activity against M.tb. CNCTC My 331/88 (H Rv) and the non-
37
tuberculous mycobacterial strains of M. avium CNCTC My 330/88, M. kansasii CNCTC My
35/80 and the clinically isolated M. kansasii 6509/96. The antimycobacterial activity of all
2
compounds was evaluated after 14 days of incubation. The activities are expressed as minimum
inhibitory concentrations (MICs). INH and RIF were used as standard anti-TB drugs (Table 2).
The antimycobacterial activities of all tertiary amine-containing compounds of series 6, 7, 8
and 9 were strongly influenced by their lipophilicity. N-Methylpiperazine derivatives 6a, 7a, 8a
and 9a with very low lipophilicities (log P < 1) showed negligible antimycobacterial activity.
The highest activities in all series showed the most lipophilic N-benzylpiperazine derivatives,
i.e., compounds 6d, 7d, 8d and 9d. The MIC values of these compounds were 2, 1, 1 and 0.125
µM against the M.tb. strain, respectively, which are comparable to the activity of the first-line
anti-TB drug INH. N-Benzylpiperazine-substituted 5-(3,5-dinitrophenyl)-1,3,4-oxadiazole 9d
showed the highest activity among all of the prepared compounds, with MIC values against all
four tested mycobacterial strains better than those of INH and comparable with those of RIF.
Table 2. Calculated log P and antimycobacterial activities of compounds of series 6, 7, 8 and 9
expressed as MICs (µM) after 14 days of incubation.
a
CLog P
M. tuberculosis
M. avium
My 330/88
1000
M. kansasii
M. kansasii
My 331/88
My 235/80
6509/96
6
6
6
a
b
c
- 0.18
0.75
32
8
32
8
32
8
500
1.96
16
250
16
16
1
1

6
7
7
7
7
8
8
8
8
9
9
9
9
d
a
b
c
3.03
0.88
1.81
3.02
4.09
0.64
1.55
2.77
3.84
0.50
1.41
2.62
3.69
2
32
2
2
32
8
250
125
62.5
16
32
8
32
8
16
1
16
2
32
2
d
a
b
c


4

500
62.5
8


16
2


16
d
a
b
c
1
1
32
8
125
125
62.5
2
16
8
 32
16
8
32
1
16
d
0.125
0.5
INH
0.5
>250
32
>250
0.25
4
RIF
0.25
0.125
a
Calculated using ChemDraw Professional 15.0
The most active compounds 7d, 8d and 9d were converted to water-soluble dihydrochloride
salts 7e, 8e and 9e and their in vitro antitubercular effects were re-evaluated. We found that the
antimycobacterial activities of dihydrochloride salts remained excellent (Table 3). Moreover,
dihydrochloride salts 7e, 8e and 9e were highly efficient against clinically isolated MDR/XDR
strains of M.tb. (Table 4). We can expect that in the biological system the equilibrium between
(
di)hydrochloride salt and the free-base forms of compounds of series 6, 7, 8 and 9 will occur
according to pH in given compartments and, thus, that a lipophilic free-base form would be able
to distribute itself through the biological system.
1
2

Table 3. Antimycobacterial activities of dihydrochlorides 7e, 8e and 9e expressed as MICs (µM)
after 14 days of incubation.
M. tuberculosis
M. avium
M. kansasii
M. kansasii
My 331/88
My 330/88
My 235/80
6509/96
7
8
9
e
e
e
2
32
2
2
1
32
1
1
0.125
2
2
1
INH 0.5
RIF 0.25
>250
32
>250
0.25
4
0.125
Table 4. Antimycobacterial activities of dihydrochlorides 7e, 8e and 9e against seven
clinically isolated MDR/XDR-TB strains expressed as MICs (µM) after 14 days of incubation.
MDR/XDR M. tuberculosis strains
Praha 1 Praha 4 Praha 131 9449/2007 234/2005 7357/1998 8666/2010
7
8
9
e
e
e
2
2
2
2
2
2
2
1
1
1
1
1
1
1
0.125
0.25
0.125
>32 (R)
16 (R)
32 (R)
>8 (R)
16 (R)
>8 (R)
0.25 (S)
0.125
>32 (R)
64 (R)
8 (S)
>8 (R)
2 (S)
1 (S)
0.125 (S)
0.125
32 (R)
16 (R)
16 (R)
>8 (R)
0.5 (S)
0.25
0.125
>32 (R)
32 (R)
16 (R)
>8 (R)
8 (R)
2 (S)
2 (R)
Streptomycin 16 (R)
>32 (R)
16 (R)
16 (R)
>8 (R)
>16 (R)
0.5 (S)
>32 (R)
16 (R)
16 (R)
>8 (R)
8 (R)
Isoniazid
16 (R)
32 (R)
>8 (R)
1 (S)
Ethambutol
Rifampicin
Ofloxacin
Gentamicin
Clofazimine
1 (S)
0.25 (S) 1 (S)
0.5 (R) 0.5 (R)
0.125 (S) 0.125 (S)
1
3

Amikacin
0.5 (S)
1 (S)
>32 (R)
0.5 (S)
0.5 (S)
1 (S)
2 (S)
S – strain susceptible to given anti-TB drug; R - strain resistant to given anti-TB drug
In vitro cell proliferation/viability assays
To gain basic insight into the toxicity of the most active water-soluble derivatives 7e, 8e and
9
e in mammalian cell lines, their in vitro effects on Caco-2 (Caucasian colon adenocarcinoma),
HepG2 (human hepatocellular carcinoma), A431 (human epidermoid carcinoma) and COS-1
African green monkey kidney) cell lines were evaluated (Table 5). Because the values of IC50
(
were not reached for any compound, 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 the dihydrochloride salts of N-benzylpiperazine derivatives 7e, 8e and 9e had
negligible effects on cell viability after 48 h of treatment, thus demonstrating good selectivity
toward mycobacterial cells.
Table 5. Viability determined by viability cell assay (CellTiter96® Assay) after 48 hours
treatment with the dihydrochloride salts of N-benzylpiperazine derivatives 7e, 8e and 9e in the
Caco-2, HepG2, A431 and COS-1 cell lines. Vehicle-treated control viability was set to 100%.
Caco-2
HepG2
A431
COS-1
IC50
Viability
at 30 µM
IC50
(µM)
Viability
at 30 µM
IC50
(µM)
Viability
at 30 µM
IC50
(µM)
Viability
at 30 µM
(µM)
7
8
9
e
e
e
˃30
˃30
˃30
105
102
121
˃30
˃30
˃30
112
118
121
˃30
˃30
˃30
86
˃30
˃30
˃30
99
100
93
100
121
1
4

Conclusion
In this work, the tertiary amine-containing derivatives of previously described 3,5-dinitrophenyl
1
5
14, 17
tetrazole and oxadiazole
antitubercular lead compounds were prepared, and their
antimycobacterial activities were studied. We found that all of the tertiary amine-containing
compounds showed lipophilicity-dependent antimycobacterial activity. Compounds with low
lipophilicity (log P < 1) practically lost their effects against mycobacteria. Among all of the
series, the N-benzylpiperazine derivatives with the highest lipophilicities, i.e., 6d, 7d, 8d and 9d,
showed the highest antimycobacterial activities. Their MIC values against M.tb. CNCTC My
3
0
31/88 (H Rv) were comparable to those of first-line anti-TB drugs INH and RIF, ranging from
37
.125 to 2 µM. Dihydrochloride salts 7e, 8e and 9e of these N-benzylpiperazine derivatives
showed good activity against drug-susceptible and multidrug-resistant strains of M.tb. and had
good aqueous solubility, thus overcoming the main drawback of previously described 3,5-
dinitrophenyl tetrazole and oxadiazole lead compounds, which suffered from very low solubility
1
4, 17
in aqueous formulations. The water solubility of compounds 7e, 8e and 9e ranged from 17.1
to 22.8 mM (9 - 12.5 mg/mL). Regarding the cytotoxicity, the dihydrochloride salts 7e, 8e and 9e
showed negligible effects on four mammalian cell lines at a concentration of 30 µM. The
structure of N-benzylpiperazine derivatives can be further optimized, mainly in the linker
between tertiary amine and heterocycle and on the benzyl moiety, to reach better selectivity and
toxicity profiles.
To conclude, the dihydrochloride salts 7e, 8e and 9e of the N-benzylpiperazine-containing
compounds 7d, 8d and 9d described in this work can be suitable candidates for further in vitro
and, mainly, in vivo evaluation of their antitubercular activity.
Experimental section
1
5

1
General. The structural identities of the prepared compounds were confirmed by H-NMR and
1
3
C-NMR spectroscopy and HPLC-HRMS experiments. 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
2
54
mm) was used for column chromatography. Melting points were recorded with a Büchi B-545
1
13
apparatus (BUCHI Labortechnik AG, Flawil, Switzerland) and are uncorrected. H and C NMR
spectra were recorded using a Varian Mercury Vx BB 300 or VNMR S500 NMR spectrometer
(
(
Varian, Palo Alto, CA, USA). Chemical shifts were reported as δvalues in parts per million
ppm) and were indirectly referenced to tetramethylsilane (TMS) via the solvent signal. The
elemental analysis was performed on an Automatic Microanalyzer EA1110CE (Fisons
Instruments S.p.A., Milano, Italy). HPLC-HRMS (ESI) experiments were performed using the
HRMS system Acquity UPLC I-class and a Synapt G2Si Q-TOF mass spectrometer (Waters,
Milford, MA, USA). Atmospheric pressure chemical ionization (APCI) was performed using an
Agilent 500 Ion Trap LC/MS (Agilent Technologies, Santa Clara, CA, USA).
General method for the synthesis of tertiary amine-containing 3,5-dinitrobenzylsulfanyl
oxadiazoles 6a-d and 7a-d. N-Methylpiperazine, morpholine, piperidine (1.5 mmol) or N-
benzylpiperazine (1 mmol) was added to a solution of 2-(3-bromopropyl)-5-(3,5-
dinitrobenzylsulfanyl)-1,3,4-oxadiazole 14a or 2-(5-bromopentyl)-5-(3,5-dinitrobenzylsulfanyl)-
1
,3,4-oxadiazole 14b (0.5 mmol) in MeCN (10 mL). Upon completion, as determined by TLC,
the solvent was evaporated, and the crude product was dissolved in EtOAc (20 mL) and washed
with brine (2 × 20 mL). The organic phase was separated, dried over anhydrous Na SO and
2
4
evaporated. The product was purified using column chromatography.
-((3,5-Dinitrobenzyl)sulfanyl)-5-(3-(4-methylpiperazin-1-yl)propyl)-1,3,4-oxadiazole (6a):
The reaction was stirred at rt overnight. The product was purified using column chromatography
2
1
6

1
(
mobile phase: CHCl /CH OH, 30:1). Yield: 57% yield as a brown oil. H NMR (500 MHz,
3
3
CDCl ) δ8.96 (t, J = 2.1 Hz, 1H), 8.70 (d, J = 2.1 Hz, 2H), 4.60 (s, 2H), 2.85 (t, J = 7.5 Hz, 2H),
3
1
3
2
1
.60-2.31 (m, 10H), 2.26 (s, 3H), 1.92 (p, J = 7.2 Hz, 2H). C NMR (126 MHz, CDCl ) δ168.75,
3
61.77, 148.51, 141.02, 129.27, 118.28, 56.99, 55.06, 52.96, 45.97, 34.78, 23.51, 23.41. HRMS
+
+
(
ESI+): m/z [M+H] calcd for C H N O S : 423.1445; found: 423.1452.
1
7
23
6
5
2
-((3,5-Dinitrobenzyl)sulfanyl)-5-(3-(morpholine-4-yl)propyl)-1,3,4-oxadiazole (6b): The
reaction mixture was refluxed for 2 h. The product was purified using column chromatography
1
(
mobile phase: CHCl /CH OH, 25:1). Yield: 60% yield as a brown oil. H NMR (500 MHz,
3
3
CDCl ) δ8.96 (t, J = 2.1 Hz, 1H), 8.80 – 8.53 (m, 2H), 4.60 (s, 2H), 3.56 (t, J = 4.7 Hz, 4H), 2.87
3
1
3
(
t, J = 7.3 Hz, 2H), 2.48 – 2.26 (m, 6H), 1.93 (p, J = 7.0 Hz, 2H). C NMR (126 MHz, CDCl ) δ
3
1
68.75, 161.67, 148.51, 141.01, 129.26, 118.29, 66.77, 57.48, 53.40, 34.73, 23.49, 23.11. HRMS
+
+
(
ESI+): m/z [M+H] calcd for C H N O S : 410.1129; found: 410.1131.
1
6
20
5
6
2
-((3,5-Dinitrobenzyl)sulfanyl)-5-(3-(piperidin-1-yl)propyl)-1,3,4-oxadiazole (6c): The
reaction mixture was stirred at rt overnight. The product was purified using column
1
chromatography (mobile phase: Hexan/EtOAc/Et N, 30:10:1). Yield: 74% as a brown oil. H
3
NMR (500 MHz, CDCl ) δ8.96 (t, J = 2.1 Hz, 1H), 8.70 (d, J = 2.1 Hz, 2H), 4.60 (s, 2H), 2.85 (t,
3
J = 7.4 Hz, 2H), 2.46 – 2.26 (m, 6H), 1.94 (p, J = 7.3 Hz, 2H), 1.56 – 1.48 (m, 4H), 1.44 – 1.35
1
3
(
m, 2H). C NMR (126 MHz, CDCl ) δ168.79, 161.75, 148.49, 141.07, 129.29, 118.26, 57.81,
3
+
+
5
4
4.39, 34.77, 25.78, 24.25, 23.54, 23.46. HRMS (ESI+): m/z [M+H] calcd for C H N O S :
1
7
22
5
5
08.1336; found: 408.1331.
-(3-(4-Benzylpiperazin-1-yl)propyl)-5-((3,5-dinitrobenzyl)sulfanyl)-1,3,4-oxadiazole (6d):
The reaction mixture was stirred at rt for 48 h. The product was purified using column
2
1
chromatography (mobile phase: EtOAc/Hexan/Et N, 70:10:2). Yield: 78% as a brown oil. H
3
NMR (500 MHz, CDCl ) δ8.96 (t, J = 2.1 Hz, 1H), 8.70 (d, J = 2.1 Hz, 2H), 7.34 – 7.12 (m,
3
1
7

5
H), 4.59 (s, 2H), 3.48 (s, 2H), 2.85 (t, J = 7.5 Hz, 2H), 2.51-2.37 (m, 10H), 1.98 – 1.87 (m, 2H).
1
3
C NMR (126 MHz, CDCl ) δ168.74, 161.78, 148.50, 141.02, 137.99, 129.27, 129.13, 128.14,
3
+
1
26.98, 118.28, 62.99, 57.03, 53.00 (2C), 34.77, 23.51, 23.40. HRMS (ESI+): m/z [M+H] calcd
+
for C H N O S : 499.1758; found: 499.1761.
23
26
6
5
2
-((3,5-Dinitrobenzyl)sulfanyl)-5-(5-(4-methylpiperazin-1-yl)pentyl)-1,3,4-oxadiazole (7a):
The reaction mixture was stirred at rt overnight. The product was purified using column
1
chromatography (mobile phase: CHCl /CH OH, 40:1). Yield: 77% yield as a brown oil. H NMR
3
3
(
500 MHz, CDCl ) δ8.96 (t, J = 2.1 Hz, 1H), 8.69 (d, J = 2.1 Hz, 2H), 4.59 (s, 2H), 2.80 (t, J =
3
7
1
1
.6 Hz, 2H), 2.63 – 2.38 (m, 8H), 2.35 – 2.29 (m, 2H), 2.28 (s, 3H), 1.77 (p, J = 7.6 Hz, 2H),
13
.58 – 1.48 (m, 2H), 1.42 - 1.35 (m, 2H). C NMR (126 MHz, CDCl ) δ168.70, 161.89, 148.53,
3
41.04, 129.30, 118.32, 58.22, 55.05, 53.14, 45.98, 34.82, 26.89, 26.34, 26.14, 25.32. HRMS
+
+
(
ESI+): m/z [M+H] calcd for C H N O S : 451.1758; found: 451.1759.
1
9
27
6
5
2
-((3,5-Dinitrobenzyl)sulfanyl)-5-(5-(morpholine-4-yl)pentyl)-1,3,4-oxadiazole (7b): The
reaction mixture was refluxed for 2 h. The product was purified using column chromatography
1
(
mobile phase: CHCl /CH OH, 25:1). Yield: 72% as a brown oil. H NMR (500 MHz, CDCl ) δ
3
3
3
8
7
1
1
.96 (t, J = 2.1 Hz, 1H), 8.70 (d, J = 2.1 Hz, 2H), 4.60 (s, 2H), 3.73 – 3.66 (m, 4H), 2.81 (t, J =
.6 Hz, 2H), 2.47 – 2.38 (m, 4H), 2.38 – 2.27 (m, 2H), 1.82 – 1.72 (m, 2H), 1.57 – 1.48 (m, 2H),
1
3
.46 – 1.36 (m, 2H). C NMR (126 MHz, CDCl ) δ168.64, 161.88, 148.51, 141.01, 129.25,
3
+
18.29, 66.84, 58.55, 53.64, 34.80, 26.72, 26.07, 25.88, 25.28. HRMS (ESI+): m/z [M+H] calcd
+
for C H N O S : 438.1442; found: 438.1445.
18
24
5
6
2
-((3,5-Dinitrobenzyl)sulfanyl)-5-(5-(piperidin-1-yl)pentyl)-1,3,4-oxadiazole (7c): The
reaction mixture was stirred at rt overnight. The product was purified using column
1
chromatography (mobile phase: Hexan/EtOAc/Et N, 30:10:1). Yield: 61% as a brown oil. H
3
NMR (500 MHz, CDCl ) δ8.96 (t, J = 2.1 Hz, 1H), 8.70 (d, J = 2.1 Hz, 2H), 4.59 (s, 2H), 2.96 –
3
1
8

2
1
1
.68 (m, 2H), 2.35 (s, 4H), 2.30 – 2.22 (m, 2H), 1.77 (p, J = 7.6 Hz, 2H), 1.61 – 1.48 (m, 6H),
13
.47 – 1.32 (m, 4H). C NMR (126 MHz, CDCl ) δ168.74, 161.83, 148.50, 141.02, 129.27,
3
18.29, 59.08, 54.60, 34.79, 27.01, 26.39, 26.16, 25.91, 25.30, 24.40. HRMS (ESI+): m/z
+
+
[
M+H] calcd for C H N O S : 436.1649; found: 436.1656.
1
9
26
5
5
2
-(5-(4-Benzylpiperazin-1-yl)pentyl)-5-((3,5-dinitrobenzyl)sulfanyl)-1,3,4-oxadiazole (7d):
The reaction was stirred at rt for 48 h. The product was purified using column chromatography
1
(
mobile phase: EtOAc/Hexan/Et N, 70:10:2). Yield: 84% as a brown oil. H NMR (500 MHz,
3
Acetone-d ) δ8.89-8.86 (m, 3H), 7.37 – 7.27 (m, 4H), 7.27 – 7.20 (m, 1H), 4.84 (s, 2H), 3.47 (s,
6
2
H), 2.83 (t, J = 7.5 Hz, 2H), 2.51 – 2.32 (m, 8H), 2.28 (dd, J = 7.6, 6.5 Hz, 2H), 1.74 (p, J = 7.6
13
Hz, 2H), 1.58 – 1.44 (m, 2H), 1.45 – 1.34 (m, 2H). C NMR (126 MHz, Acetone-d ) δ169.31,
6
1
5
5
62.94, 149.33, 143.24, 139.71, 130.40, 129.63, 128.88, 127.60, 118.63, 63.47, 58.73, 54.05,
+
+
3.97, 35.19, 27.32, 27.02, 26.79, 25.64. HRMS (ESI+): m/z [M+H] calcd for C H N O S :
2
5
31
6
5
27.2071; found: 527.2083.
-(3,5-Dinitrophenyl)-2-(3-(4-methylpiperazin-1-yl)propyl)-2H-tetrazole (8a):
5
Bromopropyltetrazole 16 (0.1 g, 0.28 mmol), potassium carbonate (0.039 g, 0.28 mmol) and N-
methylpiperazine (0.040 mL, 0.36 mmol) were mixed in MeCN (10 mL) and the reaction
mixture was refluxed for 5 h. Upon cooling, the solids were filtered off and the residue was
evaporated under vacuum. The product was isolated using column chromatography (mobile
1
phase: CHCl /MeOH, 17:1). Yield: 93% as a yellow oil. H NMR (500 MHz, DMSO) δ9.05 (d, J
3
=
2.1 Hz, 2H), 8.95 (t, J = 2.1 Hz, 1H), 4.84 (t, J = 6.9 Hz, 2H), 2.39 – 2.30 (m, 6H), 2.28 – 2.22
1
3
(
m, 4H), 2.17 (p, J = 6.7 Hz, 2H), 2.11 (s, 3H). C NMR (126 MHz, DMSO) δ161.06, 148.89,
+
1
29.79, 126.04, 119.89, 54.74, 54.37, 52.56, 51.89, 45.73, 25.92. HRMS (ESI+): m/z [M+H]
+
calcd for C H N O : 377.1680; found: 377.1682.
15
21
8
4
1
9

5
-(3,5-Dinitrophenyl)-2-(3-(morpholine-4-yl)propyl)-2H-tetrazole (8b): Bromopropyltetrazole
1
6 (0.1 g, 0.28 mmol), potassium carbonate (0.039 g, 0.28 mmol) and morpholine (0.031 mL,
0
.36 mmol) were mixed in MeCN (10 mL), and the reaction mixture was refluxed for 5 h. Upon
cooling, the solids were filtered off and the residue was evaporated under vacuum. The product
was isolated using column chromatography (mobile phase: EtOAc). Yield: 90% as a yellow oil.
1
H NMR (500 MHz, DMSO) δ9.06 (d, J= 2.1 Hz, 2H), 8.95 (t, J = 2.1 Hz, 1H), 4.86 (t, J = 6.9
Hz, 2H), 3.53 (t, J = 4.7 Hz, 4H), 2.41 (t, J = 6.7 Hz, 2H), 2.36 (t, J = 4.7 Hz, 4H), 2.24 – 2.17
1
3
(
m, 2H). C NMR (126 MHz, DMSO) δ160.87, 148.80, 129.84, 125.82, 119.45, 66.01, 54.61,
+
+
5
3
3.00, 51.66, 25.41. HRMS (ESI+): m/z [M+H] calcd for C H N O : 364.1364; found:
1
4
18
7
5
64.1362.
-(3,5-Dinitrophenyl)-2-(3-(piperidin-1-yl)propyl)-2H-tetrazole (8c): Bromopropyltetrazole 16
100 mg, 0.28 mmol) and piperidine (0.055 mL, 0.56 mmol) were dissolved in MeCN (10 mL)
5
(
and the reaction mixture was refluxed for 4 h. Upon completion, as determined by TLC, the
solvent was evaporated under reduced pressure and the product was purified using column
chromatography (mobile phase: CHCl /MeOH, 50:1). Yield: 78% as a dark yellow solid, mp 83-
3
1
8
4 °C. H NMR (500 MHz, CDCl ) δ9.32 (d, J = 2.1 Hz, 2H), 9.13 (t, J = 2.1 Hz, 1H), 4.81 (t, J
3
=
7.1 Hz, 2H), 2.45 – 2.35 (m, 6H), 2.32 – 2.23 (m, 2H), 1.61 – 1.52 (m, 4H), 1.47 – 1.40 (m,
1
3
2
2
3
H). C NMR (126 MHz, CDCl ) δ161.47, 149.04, 131.23, 126.53, 119.63, 55.48, 54.54, 52.28,
3
+
+
6.71, 25.96, 24.32. HRMS (ESI+): m/z [M+H] calcd for C H N O : 362.1571; found:
1
5
20
7
4
62.1581.
-(3-(4-Benzylpiperazin-1-yl)propyl)-5-(3,5-dinitrophenyl)-2H-tetrazole (8d):
2
Bromopropyltetrazole 16 (1.834 g, 5.14 mmol) and N-benzylpiperazine (1.810 g, 10.27 mmol)
were dissolved in MeCN (100 mL), and the reaction mixture was refluxed for 13 h. Upon
cooling, the solvent was evaporated under vacuum, and the residue was dissolved in EtOAc (80
2
0

mL) and washed with 10% aq. KHCO (3 × 30 mL). The organic phase was dried over
3
anhydrous Na SO and evaporated under vacuum. The product was isolated using column
2
4
1
chromatography (mobile phase: CHCl /MeOH, 19:1). Yield: 76% as yellow oil. H NMR (500
3
MHz, DMSO-d ) δ9.04 (d, J = 2.1 Hz, 2H), 8.95 (t, J = 2.1 Hz, 1H), 7.31 – 7.19 (m, 5H), 4.83 (t,
6
13
J = 6.8 Hz, 2H), 3.39 (s, 2H), 2.46 – 2.21 (m, 10H), 2.15 (p, J = 6.8 Hz, 2H). C NMR (126
MHz, DMSO-d ) δ161.18, 148.99, 138.31, 129.90, 128.91, 128.24, 126.99, 126.16, 120.02,
6
+
+
6
4
2.23, 54.53, 52.81, 52.74, 52.01, 26.03. HRMS (ESI+): m/z [M+H] calcd for C H N O :
2
1
25
8
4
53.1993; found: 453.2003.
-(3,5-Dinitrophenyl)-5-((3-(4-methylpiperazin-1-yl)propyl)sulfanyl)-1,3,4-oxadiazole (9a):
Bromopropylsulfanyl oxadiazole 18 (200 mg, 0.514 mmol) and N-methylpiperazine (0.115 mL,
.03 mmol) were dissolved in MeCN (15 mL), and the reaction mixture was refluxed for 4 h.
2
1
Upon completion, as determined by TLC, the solvent was evaporated under reduced pressure and
the product was purified using column chromatography (mobile phase: CHCl /MeOH, 19:1).
3
1
Yield: 55% yield as a brownish oil. H NMR (500 MHz, CDCl ) δ9.18 – 9.16 (m, 1H), 9.14 (d, J
3
=
2.1 Hz, 2H), 3.43 (t, J = 7.1 Hz, 2H), 2.77 – 2.61 (m, 8H), 2.58 (t, J = 6.8 Hz, 2H), 2.43 (s,
1
3
3
1
H), 2.08 (p, J = 6.9 Hz, 2H). C NMR (126 MHz, CDCl ) δ167.25, 162.07, 149.09, 126.96,
3
+
26.08, 120.66, 56.14, 54.47, 51.88, 45.12, 30.69, 26.08. HRMS (ESI+): m/z [M+H] calcd for
+
C H N O S : 409.1289; found: 409.1293.
1
6
21
6
5
2
-(3,5-Dinitrophenyl)-5-((3-(morpholine-4-yl)propyl)sulfanyl)-1,3,4-oxadiazole (9b):
Bromopropylsulfanyl oxadiazole 18 (200 mg, 0.514 mmol) and morpholine (0.089 mL, 1.03
mmol) were dissolved in MeCN (15 mL), and the reaction mixture was refluxed for 13 h. As a
significant amount of the starting material 18 was detected in the reaction mixture, another
portion of morpholine (0.089 mL, 1.03 mmol) was added. After an additional 6 hours of reflux,
the solvent was evaporated under reduced pressure and the product was purified using column
2
1

1
chromatography (mobile phase: EtOAc). Yield: 58% as a yellow solid, mp 115-117 °C. H NMR
(
500 MHz, CDCl ) δ9.18 – 9.16 (m, 1H), 9.15 (d, J = 2.1 Hz, 2H), 3.75 – 3.70 (m, 4H), 3.45 (t, J
3
1
3
=
7.1 Hz, 2H), 2.53 (t, J = 6.7 Hz, 2H), 2.50 – 2.45 (m, 4H), 2.08 (p, J = 6.8 Hz, 2H). C NMR
(
126 MHz, CDCl ) δ167.35, 162.06, 149.12, 127.00, 126.08, 120.66, 66.92, 56.91, 53.60, 30.77,
3
+
+
2
5.88. HRMS (ESI+): m/z [M+H] calcd for C H N O S : 396.0972; found: 396.0975.
15
18
5
6
2
-(3,5-Dinitrophenyl)-5-((3-(piperidin-1-yl)propyl)sulfanyl)-1,3,4-oxadiazole (9c):
Bromopropylsulfanyl oxadiazole 18 (0.2 g, 0.75 mmol), 1-(3-chloropropyl)piperidine
hydrochloride (0.148 g, 0.75 mmol) and Et N (0.15 g, 0.2 mL, 1.5 mmol) were dissolved in
3
MeCN (10 mL), and the reaction mixture was refluxed for 12 h. Upon completion, the solvent
was evaporated, and the residue was partitioned between EtOAc (20 mL) and 10% aq. K CO (20
2
3
mL). The aqueous phase was then extracted with EtOAc (2 × 20 mL), the combined organic
extracts were dried over anhydrous Na SO and evaporated. The product was purified using
2
4
column chromatography (mobile phase: Hexane/EtOAc/Et N, 75:25:1). Yield: 65% as a yellow
3
1
oil. H NMR (500 MHz, Acetone-d ) δ9.12 (s, 3H), 3.48 (t, J = 7.1 Hz, 2H), 2.50 (t, J = 6.6 Hz,
6
1
3
2
H), 2.42 (br s, 4H), 2.09 – 2.04 (m, 2H), 1.60 – 1.54 (m, 4H), 1.48-1.40 (m, 2H). C NMR (126
MHz, Acetone-d ) δ167.55, 163.41, 150.10, 127.76, 126.87, 121.48, 57.73, 55.14, 31.43, 27.24,
6
+
+
2
6.68, 25.13. HRMS (ESI+): m/z [M+H] calcd for C H N O S : 394.1180; found: 394.1178.
1
6
20
5
5
2
-((3-(4-Benzylpiperazin-1-yl)propyl)sulfanyl)-5-(3,5-dinitrophenyl)-1,3,4-oxadiazole (9d):
Bromopropylsulfanyl oxadiazole 18 (0.277 g, 0.71 mmol) and N-benzylpiperazine (0.251 g, 1.42
mmol) were dissolved in MeCN (20 mL), and the reaction mixture was refluxed for 13 h. Upon
completion, the solvent was evaporated, and the residue was dissolved in EtOAc (40 mL) and
washed with 10% aq. KHCO (3 × 30 mL). The organic phase was dried over anhydrous Na SO
3
2
4
and evaporated under reduced pressure. The product was purified using column chromatography
1
(
mobile phase: Hexane/EtOAc/Et N, 66:33:1). Yield: 81% as a yellowish oil. H NMR (500
3
2
2

MHz, Acetone-d ) δ9.13 – 9.11 (m, 3H), 7.34 – 7.29 (m, 4H), 7.25 – 7.21 (m, 1H), 3.50 – 3.45
6
1
3
(
m, 4H), 2.54 – 2.38 (m, 10H), 2.09 – 2.03 (m, 2H). C NMR (126 MHz, Acetone-d ) δ167.49,
6
1
5
4
63.40, 150.08, 139.61, 129.62, 128.88, 127.74, 127.62, 126.86, 121.48, 63.42, 57.03, 53.93,
+
+
3.89, 31.35, 27.23. HRMS (ESI+): m/z [M+H] calcd for C H N O S : 485.1602; found:
2
2
25
6
5
85.1613.
General method for the synthesis of dihydrochlorides 7e, 8e and 9e: N-Benzylpiperazine-
containing derivative 7d, 8d or 9d (0.1 g) was mixed with water (10 mL) and conc. HCl (3
equiv.). The reaction mixture was heated to 60 °C until a clear solution was obtained. Then, all
of the volatiles were evaporated under reduced pressure, and the residue was dried under vacuum
over P O for 48 h. These reactions gave quantitative yields of products 7e, 8e and 9e.
2
5
2
-(5-(4-Benzylpiperazin-1-yl)pentyl)-5-((3,5-dinitrobenzyl)sulfanyl)-1,3,4-oxadiazole
1
dihydrochloride (7e): Obtained as a yellowish solid, mp 201-202 °C. H NMR (500 MHz, D O) δ
2
8
3
.78 (t, J = 2.2 Hz, 1H), 8.55 (d, J = 2.1 Hz, 2H), 7.46 – 7.34 (s, 5H), 4.48 (s, 2H), 4.35 (s, 2H),
.53 (br s, 8H), 3.18 – 3.09 (m, 2H), 2.72 (t, J = 7.4 Hz, 2H), 1.72 – 1.54 (m, 4H), 1.32 – 1.18
1
3
(
m, 2H). C NMR (126 MHz, D O) δ169.65, 163.64, 148.09, 141.16, 131.12, 130.53, 129.50,
2
1
29.37, 127.48, 118.27, 60.44, 56.50, 48.50, 48.03, 34.68, 24.71, 24.66, 24.24, 22.80. MS
+
(
APCI+): m/z (%) = 527.4 (100), 528.2 (25) [M+H] .
2
-(3-(4-Benzylpiperazin-1-yl)propyl)-5-(3,5-dinitrophenyl)-2H-tetrazole dihydrochloride (8e):
1
Obtained as a yellowish solid, mp 258-259 °C. H NMR (500 MHz, D O) δ9.15 (d, J = 2.1 Hz,
2
2
H), 9.10 (t, J = 2.1 Hz, 1H), 7.45 – 7.38 (m, 5H), 4.86 (t, J = 6.6 Hz, 2H), 4.35 (s, 2H), 3.53 (br
13
s, 8H), 3.35 – 3.29 (m, 2H), 2.56 – 2.44 (m, 2H). C NMR (126 MHz, D O) δ161.98, 148.80,
2
1
31.13, 130.53, 129.59, 129.39, 127.57, 127.08, 120.59, 60.47, 53.73, 50.53, 48.88, 48.30, 23.26.
+
MS (APCI+): m/z (%) = 453.4 (100), 454.3 (20) [M+H] .
2
3

2
-((3-(4-Benzylpiperazin-1-yl)propyl)sulfanyl)-5-(3,5-dinitrophenyl)-1,3,4-oxadiazole
1
dihydrochloride (9e): Obtained as a yellowish solid, mp 246-248 °C. H NMR (500 MHz, D O) δ
2
9
3
1
.17 – 9.15 (m, 1H), 9.06 – 9.03 (m, 2H), 7.52 – 7.31 (m, 5H), 4.35 – 4.28 (m, 2H), 3.50 (s, 8H),
13
.40 – 3.26 (m, 4H), 2.33 – 2.19 (m, 2H). C NMR (126 MHz, D O) δ166.84, 163.33, 148.86,
2
31.09, 130.46, 130.42, 129.36, 126.85, 125.67, 121.71, 60.50, 55.08, 48.93, 48.34, 28.64, 23.69.
+
MS (APCI+): m/z (%) = 485.4 (100), 486.1 (22) [M+H] .
In vitro antimycobacterial assay. The in vitro antimycobacterial activity of the studied
compounds was evaluated against M.tb. CNCTC My 331/88, M. kansasii CNCTC My 235/80
and M. avium CNCTC My 330/88 from the Czech National Collection of Type Cultures
(
CNCTC). Additionally, the in vitro antimycobacterial activity of the studied compounds was
evaluated against the clinically isolated strain M. kansasii 6509/96 and clinically isolated
MDR/XDR strains M.tb. 7357/1998, M.tb. 234/2005, M.tb. 9449/2007, M.tb. 8666/2010, M.tb.
Praha 1, M.tb. Praha 4 and M.tb. Praha 131. Basic suspensions of the mycobacterial strains were
prepared according to a 1.0 McFarland standard. From the basic suspension, subsequent dilutions
-3
-5
-4
of each strain were made: M. tuberculosis, 10 ; M. avium, 10 ; and M. kansasii, 10 . The
appropriate dilutions of the strains were prepared, and 0.1 mL was added to each well of the
microtiter plates containing the compounds. The activities of the compounds were determined
viaꢍtheꢍmicꢇomethodꢍfoꢇꢍtheꢍdeteꢇminationꢍofꢍtheꢍminimumꢍinhibitoꢇyꢍconcentꢇationꢍinꢍŠula'sꢍ
semisynthetic medium (SEVAC, Prague). The compounds were dissolved in dimethyl sulfoxide
and added to the medium at concentrations of 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06 and 0.03
µM for M.tb. and M. kansasii strains and at concentrations of 1000, 500, 250, 125, 64, 32, 16, 8,
4
, 2, and 1 µM for the M. avium strain. The MICs, i.e., the lowest concentration of a substance at
which mycobacterial growth inhibition occurred (the concentration that inhibited >99% of the
2
4

mycobacterial population), were determined after incubation at 37 °C for 14 days. Isoniazid
INH) and rifampicin (RIF) were used as the prototype drugs.
(
Cell proliferation/viability assay. The CellTiter 96® AQueousOne Solution Cell Proliferation
Assay (Promega, Madison, WI, USA) was performed to evaluate in vitro effects of compounds
7
e, 8e and 9e in the four mammalian cell lines. The method is based on the colorimetric method
of MTS bioreduction into colored formazan via viable cells. The formazan production is thus
proportional to the number of viable cells. All experiments were conducted according to the
manufacturer's protocol. Briefly, the cells were treated with the test compounds or vehicle alone
(
DMSO) for 48 h in Opti-MEM, reduced serum medium (Life Technologies) to avoid potential
binding of test compounds onto the serum proteins. At the end of the treatment, 20 µL of the
MTS reagent was added directly to each culture well and further cultivated for 4 h. Finally, the
absorbance of the converted formazan was recorded at 490 nm using a plate reader (BioTec
Synergy 2, Winooski, VT, USA). In the toxic control, SDS (10% v/v) was added to the cells 45
min before the addition of the MTS reagent. The relative viability of the cells treated either by
the vehicle (DMSO, 0.1%) or SDS was set to be 100% or 0%, respectively. The results were
expressed as the relative cell viability at a 30 µM concentration. All experiments were performed
in triplicates and repeated at least three times.
Solubility in Aqueous Media. Compound 7e, 8e or 9e (15 mg) was shaken in 0.05 M citrate
buffer (pH 3, 1 mL), 0.1 M acetate buffer (pH 5, 1 mL) or PBS (pH 7.4, 5 mL) at rt for 2 h. The
suspensions were decanted and the solutions were filtered through 0.22-µm PTFE filters. Clear
solutions in citrate buffer and acetate buffer were diluted 500-times with water/acetonitrile 1:1
(
v/v) prior to analysis. All samples were analyzed using a Shimadzu Prominence HPLC
instrument (Shimadzu, Kyoto, Japan) consisting of LC-20AD pumps with a DGU-20A3
2
5

degasser, an SIL-20A HT autosampler, a CTO-20AC column oven, an SPD-M20A diode array
detector and a CBM-20A communication module. The data were analyzed using LCsolutions
1
.22 software. All experiments were repeated at least 4 times.
Compounds 7e, 8e and 9e were analyzed using a Discovery® HS C18 150-4.6 mm column
with 5-μm particles (Supelco Analytical, Sigma-Aldrich, Schnelldorf, Germany) at 30 °C. The
mobile phase, composed of water/acetonitrile 1:1 (v/v) at a flow rate of 2.5 mL/min, was used.
The samples were monitored at 235 nm for 7e and 229 nm for 8e and 9e. The retention times of
7
e, 8e and 9e were 6.1, 5.9 and 5.5 min, respectively. The calibration curves were linear in the
range of 1 – 100 µM.
SUPPLEMENTARY MATERIAL
Experimental protocols and characterization data of compounds 11a, 11b, 12a, 12b, 13a, 13b,
1
4a, 14b, 16 and 18. NMR a HRMS spectra of the final compounds.
AUTHOR INFORMATION
Corresponding Author
J. Roh. email: jaroslav.roh@faf.cuni.cz.
ACKNOWLEDGMENTS
This work was supported by the Charles University Grant Agency project 361215 and Charles
University project SVV 260 401.
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Graphical abstract
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