Tetrazole regioisomers in the development of nitro group-containing antitubercular agents

Article abstract of DOI:10.1039/c4md00301b

Tetrazole derivatives containing nitro substituents have been identified as promising antitubercular agents. In this study, the antitubercular potency, selectivity and toxicity of tetrazole 1,5- and 2,5-regioisomers were examined. We prepared a series of 1- and 2-alkyl-5-benzylsulfanyl-2H-tetrazoles and their selenium analogs with various nitro group substitutions. These 1,5- and 2,5-regioisomers were isolated and unambiguously identified using ¹H and/or ¹³C NMR. Among the prepared compounds, 1- and 2-alkyl-5-[(3,5-dinitrobenzyl)sulfanyl]-2H-tetrazole derivatives and their selenium bioisosteres showed the highest antimycobacterial activity, with minimal inhibitory concentration (MIC) values of approximately 1 μM (0.37-0.46 μg mL^-1) against Mycobacterium tuberculosis CNCTC My 331/88. The 2-alkyl regioisomers exhibited consistently higher antimycobacterial activity and lower in vitro toxicity against a mammalian cell line compared to the 1-alkyl isomers. The antimycobacterial activity of the 2-alkyl regioisomers was less influenced by the type of alkyl substituent in contrast to 1-alkyl isomers. Furthermore, the 3,5-dinitrobenzyl moiety per se is not the carrier of mutagenicity. These findings encourage further optimization of the 2-alkyl chain to improve the pharmacokinetic properties and toxicity of 2-alkyl-5-[(3,5-dinitrobenzyl)sulfanyl]-2H-tetrazole lead compounds. This journal is

Full text of DOI:10.1039/c4md00301b

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CONCISE ARTICLE
Tetrazole regioisomers in the development of nitro
group-containing antitubercular agents†
Cite this: DOI: 10.1039/c4md00301b
a
a
Galina Karabanovich, Jaroslav Roh, Ondrej Soukup,b Ivona Pavkova,c
*
ˇ
´
´
Marketa Pasdiorova,b Vojtech Tambor,b Jirina Stolarıkova,d Marcela Vejsova,a
ˇ´
´
´
ˇ
ˇ
´
´
Katerina Vavrova,a Vera Klimesovaa and Alexandr Hrabaleka
ˇ
´
´
ˇ
ˇ
´
´
Tetrazole derivatives containing nitro substituents have been identified as promising antitubercular agents.
In this study, the antitubercular potency, selectivity and toxicity of tetrazole 1,5- and 2,5-regioisomers were
examined. We prepared a series of 1- and 2-alkyl-5-benzylsulfanyl-2H-tetrazoles and their selenium
analogs with various nitro group substitutions. These 1,5- and 2,5-regioisomers were isolated and
1
unambiguously identified using H and/or 13C NMR. Among the prepared compounds, 1- and 2-alkyl-5-
[(3,5-dinitrobenzyl)sulfanyl]-2H-tetrazole derivatives and their selenium bioisosteres showed the highest
antimycobacterial activity, with minimal inhibitory concentration (MIC) values of approximately 1 mM
(0.37–0.46 mg mLꢀ1) against Mycobacterium tuberculosis CNCTC My 331/88. The 2-alkyl regioisomers
exhibited consistently higher antimycobacterial activity and lower in vitro toxicity against a mammalian
cell line compared to the 1-alkyl isomers. The antimycobacterial activity of the 2-alkyl regioisomers was
less influenced by the type of alkyl substituent in contrast to 1-alkyl isomers. Furthermore, the 3,5-
dinitrobenzyl moiety per se is not the carrier of mutagenicity. These findings encourage further
optimization of the 2-alkyl chain to improve the pharmacokinetic properties and toxicity of 2-alkyl-5-
[(3,5-dinitrobenzyl)sulfanyl]-2H-tetrazole lead compounds.
Received 10th July 2014
Accepted 23rd September 2014
DOI: 10.1039/c4md00301b
www.rsc.org/medchemcomm
were developed and two nitroimidazole-based compounds,
PA-8246 and OPC-67683 (delamanid),7 are undergoing clinical
trials.8 Another promising group of nitro group-containing anti-
TB agents are dinitrobenzamides9 and benzothiazinones;10 both
of which are inhibitors of decaprenyl-phosphoribose epimerase
(DprE1), an essential enzyme involved in arabinan biosyn-
thesis.11 Piperazinobenzothiazinone PBTZ 169, a benzothiazi-
Introduction
Tuberculosis (TB) is a widespread infectious disease and
remains a serious global problem that takes millions of lives
each year.1 The emergence and distribution of multi-drug
resistant (MDR-TB) and extensively drug-resistant (XDR-TB)
strains of Mycobacterium tuberculosis (M. tb) have become the
biggest challenges in treatment with current anti-TB drugs.
Current anti-TB drugs also suffer from low tolerability or
adverse effects. Moreover, TB frequently occurs in HIV/AIDS
patients who have a further reduced response to TB treatment.
This establishes the need for the discovery of new highly
efficient antimycobacterial agents (for recent reviews, see ref.
2–5). Recently, several nitro group-containing anti-TB agents
none-derivative,
is
currently
undergoing
preclinical
development.12 In our previous work, we found that 2-(dini-
trobenzylsulfanyl)benzazoles 1 exhibited high in vitro anti-
mycobacterial activities with minimal inhibitory concentrations
(MICs) from 2 to 8 mM against M. tb (Fig. 1).13–16 Hence, to
continue this study, we prepared a series of 1-alkyl/aryl-5-
(dinitrobenzylsulfanyl)-1H-tetrazoles 2 with 1-substituted tetra-
zole in place of the original benzazole moiety (Fig. 1). The
antimycobacterial activities of these compounds conrmed that
a
´
´
´
Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Heyrovskeho
´
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1203, 50005 Hradec Kralove, Czech Republic. E-mail: jaroslav.roh@faf.cuni.cz
b
´
´
´
Biomedical Research Center, University Hospital Hradec Kralove, Sokolska 581,
´
´
50005 Hradec Kralove, Czech Republic
cDepartment of Molecular Pathology and Biology, Faculty of Military Health Sciences,
ˇ
ˇ
´
´
´
University of Defence, Trebesska 1575, 50005 Hradec Kralove, Czech Republic
dRegional Institute of Public Health, Department of Bacteriology and Mycology,
´
´ ´ ˇ ´
Partyzanske namestı 7, 70200 Ostrava, Czech Republic
† Electronic supplementary information (ESI) available: Details of chemical
synthesis and characterization of all the reported compounds. Details of in vitro
antimycobacterial, antibacterial, antifungal, cell proliferation/viability assays
and AMES uctuation test. Tables S1 and S2. See DOI: 10.1039/c4md00301b
Fig. 1 Dinitrobenzyl-bearing benzazole and tetrazole derivatives with
high and selective antimycobacterial activity.
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the presence of two nitro groups on the benzylsulfanyl moiety is
vital for increased antimycobacterial activity. 3,5-Dinitro
substituted tetrazole derivatives of series 2 exhibited higher
activities compared to the 2,4-dinitro analogs, with MIC values
of 1 mM (0.36–0.44 mg mLꢀ1) against M. tb, i.e., values equivalent
to the rst-line anti-TB drug isoniazid, and 0.25–1 mM against
six MDR M. tb strains and with no cross-resistance with
common anti-TB drugs. Moreover, the series of compounds 2
were highly selective for mycobacteria, because they exhibited
no antibacterial or antifungal activity and low toxicity on
selected mammalian cell lines. A structure–activity relationship
study showed that the isosteric replacement of sulfur for the
selenium or oxygen atom had no signicant effect on the anti-
mycobacterial activity of these compounds. In addition, twenty-
three substituents at position 1 of the tetrazole were studied,
and these appeared to inuence the antimycobacterial activity
of the compound through their effects on its lipophilicity.17
As 5-substituted tetrazole forms 1H- and 2H-tautomers (3
and 4, Fig. 2),18–20 1,5- and 2,5-disubstituted tetrazole isomers
can be recognized. Several studies have indicated that the bio-
logical properties of 1,5- and 2,5-tetrazole regioisomers differ,
particularly in their effective concentration levels.21–23 4-Phenyl-
1-(1H-tetrazol-1-yl)-2-butanone (5, Fig. 3), an inhibitor of heme
oxygenase-1 (HO-1), displayed an IC50 value of 2.6 mM, while its
2H-tetrazol-2-yl analog exhibited greater than 3.5 times weaker
inhibitory activity.24 1-Methyl derivatives of cephalosporin 6
Fig. 4 General structure of the studied compounds.
a long carbon chain in position 2 of the tetrazole ring displayed
10-fold higher acyl-CoA:cholesterol O-acyltransferase inhibition
compared to the 1-regioisomers (Fig. 3).27
In this work, we focused on the tetrazole-based lead
compounds 2 and studied how the position of substituent R on
tetrazole inuenced their biological properties, specically,
antimycobacterial activity. Moreover, their reverse analogs, with
a dinitrobenzyl moiety on tetrazole and with substituent R on
sulfur/selenium in position 5 of tetrazole, were prepared to
determine the optimal position of the crucial dinitrobenzyl
fragment. We also examined the effect of isosteric replacement
of sulfur for selenium on the biological properties of the target
substances (Fig. 4).
Results and discussion
Chemistry
displayed a two-fold increase in antibacterial activity against Synthesis of 1-alkyl-5-(alkylselanyl)-1H-tetrazoles (15–19) and
both Gram-positive and Gram-negative bacteria compared to their 2-regioisomers (20–24) was conducted according to
the 2-methyl isomers.25 Conversely, 2,5-disubstituted tetrazoles Scheme 1. The starting alkyl selenocyanates (9–13) were
7
were efficient Pseudomonas aeruginosa quorum-sensing obtained by the reaction of the corresponding alkyl halides with
inhibitors, whereas 1,5-disubstituted analogs had low inhibi- a slight excess of potassium selenocyanate in THF or DMF.
tory activity.26 N-Aryl-N0-tetrazole-substituted ureas 8 containing Subsequently, they were converted into 5-(alkylselanyl)-1H-tet-
razolates by the reaction with sodium azide and triethy-
lammonium chloride in toluene at an elevated temperature.28,29
For the preparation of 5-[(2,4-dinitrobenzyl)selanyl]-1H-tetrazo-
late, the reaction mixture was stirred in THF at room tempera-
ture overnight to avoid the precipitation of selenium, which was
observed using the above-mentioned procedure in toluene.
5-[(3,5-Dinitrobenzyl)selanyl]-1H-tetrazolate was sensitive to
acidication, and the corresponding tetrazole 14 could only be
Fig. 2 1H- and 2H-tautomers of 5-substituted tetrazoles.
prepared in low yield; therefore, we alkylated selanyltetrazolates
in situ under the conditions of phase-transfer catalysis in the
presence of tetrabutylammonium bromide (TBAB). Expectedly,
the alkylation of 5-(alkylselanyl)-1H-tetrazolates led to the
formation of both regioisomers,20 1-alkyl-5-(alkylselanyl)-1H-
tetrazoles (15–19) and 2-alkyl-5-(alkylselanyl)-2H-tetrazoles
(20–24), in moderate yields. 1- (15–19) and 2-isomers (20–24) of
the target selanyltetrazoles were separated and puried by
column chromatography. The ratios of 1- and 2-regioisomers
ranged from 1 : 1 to 1 : 4.
The alkylation was preferentially directed to position 2,
which is likely due to steric hindrance of the alkylselanyl
substituent in position 5 of tetrazole. The 1,5- and 2,5-isomeric
1
series were unambiguously identied according to the H and
13C NMR chemical shis of the tetrazole carbon and the
nitrogen-bound methylene group. These signals of 2-alkyl-5-
Fig.
3 Examples of 1,5- and 2,5-disubstituted tetrazole-based
compounds with regioisomer-dependent activity.
(alkylselanyl)-2H-tetrazoles were observed downeld compared
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Scheme 1 Synthesis of 1-alkyl- and 2-alkyl-5-(alkylselanyl)-2H-tetrazoles.
to the 1-isomers (Table 1). Correlations between methylene 5-(alkylsulfanyl)-1H-tetrazoles (29–32) were isolated in moderate
hydrogens in 1,5-isomer 19d in 1D NOESY experiments and the yields (50–68%). Alkylation of the tetrazoles (29–32) was per-
absence of such correlations in 2,5-isomer 24d conrmed their formed in THF or DMF in the presence of KOH or under phase-
regioisomeric identities (see ESI†). Furthermore, all 2-alkyl-5- transfer catalysis conditions (compounds 36a, 36b, 40a, and
(alkylselanyl)-2H-tetrazoles had lower retention (higher values 40b). 1-Alkyl-5-(alkylsulfanyl)-1H-tetrazoles (33–36) and their
of Rf) on silica gel compared to their 1-isomers, indicating that respective 2-alkyl isomers (37–40) were separated by column
the 2-isomers were less polar. In contrast to the 1-alkyl-5- chromatography and were obtained in ratios ranging from
(alkylselanyl)-1H-tetrazole regioisomers,30 a report on 2-alkyl 1 : 1.6 to 1 : 4. The identication of the 1,5- and 2,5-isomers was
regioisomers has not yet been published. Nevertheless, our performed using the 1H and 13C NMR chemical shis of the
results are in agreement with previously published differences tetrazole carbon and the methylene group on the tetrazole
between NMR shis of 1,5- and 2,5-disubstituted tetrazoles.31,32 nitrogen.31,32 These results followed the same rules as in the
The synthetic procedure for 1-alkyl-5-(alkylsulfanyl)-1H-tet- above-mentioned selenium derivatives: the signals of 2-alkyl-5-
razoles (33–36) and 2-alkyl-5-(alkylsulfanyl)-2H-tetrazoles (alkylsulfanyl)-2H-tetrazoles were observed downeld compared
(37–40) is shown in Scheme 2. Unlike selenium analogs, to the 1-isomers (Table 2), and the 2-isomers had lower
Table 1 Selected 13C and 1H (in parentheses) NMR chemical shifts (d, ppm) of 1-alkyl-5-(alkylselanyl)-1H-tetrazole (15–19) and 2-alkyl-5-
(alkylselanyl)-2H-tetrazole (20–24) regioisomeric pairsa
1-Isomer
Ctetr
2-Isomer
Ctetr
1-/2-Isomers
R1-CH2Se
Ph
R2-CH2N
R2-CH2N1
R2-CH2N2
15d/20d
15e/20e
16c/21c
16d/21d
16e/21e
17a/22a
17b/22b
17c/22c
17d/22d
17e/22e
18a/23a
18b/23b
18c/23c
18d/23d
18e/23e
19d/24d
19e/24e
3,5-(NO2)2Ph
2,4-(NO2)2Ph
4-NO2Ph
3,5-(NO2)2Ph
2,4-(NO2)2Ph
H
Ph
4-NO2Ph
3,5-(NO2)2Ph
2,4-(NO2)2Ph
H
Ph
4-NO2Ph
3,5-(NO2)2Ph
2,4-(NO2)2Ph
3,5-(NO2)2Ph
2,4-(NO2)2Ph
147.66
148.32
148.06
n.d.
148.10
147.14
n.d.
n.d.
147.79
n.d.
145.62
146.86
148.01
147.54
148.00
147.74
148.38
50.40 (5.87)
48.98 (6.03)
51.12 (5.74)
50.47 (5.94)
49.13 (6.09)
34.67 (3.98)
52.13 (5.57)
51.21 (5.79)
50.59 (6.00)
49.39 (6.14)
34.12 (3.94)
52.09 (5.57)
50.34 (5.79)
50.52 (5.99)
49.29 (6.12)
50.38 (5.87)
48.90 (6.02)
157.68
157.60
156.75
157.08
156.91
155.86
156.24
156.73
157.07
156.92
154.74
156.18
156.72
155.68
156.82
157.84
157.70
55.55 (6.31)
54.19 (6.48)
56.41 (6.10)
55.62 (6.32)
54.26 (6.47)
40.09 (4.39)
57.52 (5.90)
56.47 (6.12)
55.70 (6.33)
54.36 (6.49)
39.78 (4.35)
57.50 (5.87)
56.50 (6.10)
55.71 (6.32)
54.34 (6.47)
55.52 (6.31)
54.15 (6.47)
4-NO2Ph
2,4-(NO2)2Ph
3,5-(NO2)2Ph
4-MeOPh
a
n.d. not determined because of signal overlap or close proximity with the nitro group-bearing carbons.
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Scheme 2 Synthesis of 1-alkyl and 2-alkyl-5-(alkylsulfanyl)-2H-tetrazoles.
Table 2 Selected 13C and 1H (in parentheses) NMR chemical shifts (d, ppm) of 1-alkyl-5-(alkylsulfanyl)-1H-tetrazole (33–36) and 2-alkyl-5-
(alkylsulfanyl)-2H-tetrazole (37–40) regioisomeric pairs
1-Isomer
Ctetr
2-Isomer
Ctetr
1-/2-Isomers
R1-CH2S
R2-CH2N
R2-CH2N1
R2-CH2N2
33d/37d
34c/38c
34d/38d
35a/39a
35b/39b
35c/39c
35d/39d
36a/40a
36b/40b
36c/40c
36d/40d
36f/40f
Ph
4-NO2Ph
3,5-(NO2)2Ph
4-NO2Ph
3,5-(NO2)2Ph
H
154.65
154.10
154.31
153.77
153.72
154.15
154.38
152.33
153.06
153.91
153.30
153.58
152.15
49.80 (5.89)
50.54 (5.74)
49.92 (5.95)
34.77 (3.94)
51.51 (5.53)
50.61 (5.75)
49.94 (5.96)
35.37 (3.92)
50.51 (5.54)
50.61 (5.77)
49.11 (5.97)
50.78 (5.57)
50.00 (5.35)
165.02
164.06
164.37
162.96
163.35
163.83
163.28
162.02
163.34
163.83
163.25
163.52
163.70
55.61 (6.27)
56.45 (6.06)
55.69 (6.27)
40.14 (4.33)
57.57 (5.83)
56.53 (6.05)
54.90 (6.26)
39.82 (4.30)
57.54 (5.83)
56.51 (6.05)
54.92 (6.27)
56.74 (5.85)
56.13 (5.89)
2,4-(NO2)2Ph
3,5-(NO2)2Ph
Ph
4-NO2Ph
3,5-(NO2)2Ph
H
Ph
4-NO2Ph
3,5-(NO2)2Ph
4-ClPh
36g/40g
3,4-Cl2Ph
retention (higher values of Rf) on silica compared to the
The antimycobacterial activities of the most potent
1-isomers. The regioisomeric identities were conrmed by 1D compounds in the series of 1- and 2-isomers of selanyltetrazoles
NOESY experiments of compounds 33d and 37d, which showed (15–24) and sulfanyltetrazoles (33–40) were 1 mM against M. tb,
correlations between methylene hydrogens in 1,5-regioisomer which is equivalent to the rst-line anti-TB drug isoniazid
33d only (see ESI†).
(INH), and 1–2 mM against both INH-resistant and INH-
susceptible M. kansasii. These results support our previous
observations that 3,5-dinitrobenzylsulfanyl/selanyl derivatives
In vitro antimycobacterial activity
had
signicantly
higher
activities
than
2,4-dini-
trobenzylsulfanyl/selanyl derivatives and both nitro groups are
necessary for the high antimycobacterial efficiency of
the studied compounds. Hence, the most active substances
were 1-alkyl-5-[(3,5-dinitrobenzyl)selanyl]-1H-tetrazoles (18a–e),
In vitro antimycobacterial activities of the synthesized
compounds were evaluated against M. tb CNCTC My 331/88 and
non-tuberculous mycobacteria – M. avium CNCTC My 330/88,
M. kansasii CNCTC My 235/80 and M. kansasii 6509/96. All
strains were obtained from the Czech National Collection of
Type Cultures (CNCTC), with the exception of M. kansasii 6509/
96, which was a clinical isolate. The activities of the compounds
were determined in the Sula semisynthetic medium. Minimum
inhibitory concentrations (MICs), i.e., the lowest concentrations
that inhibit the visible growth of mycobacteria, were deter-
2-alkyl-5-[(3,5-dinitrobenzyl)selanyl]-2H-tetrazoles
(23a–e),
1-alkyl-5-[(3,5-dinitrobenzyl)sulfanyl]-1H-tetrazoles (36a–d, 36f,
36g) and 2-alkyl-5-[(3,5-dinitrobenzyl)sulfanyl]-2H-tetrazoles
(40a–d, 40f, 40g). Considering the position of the alkyl substit-
uent, the 2-isomer series (23 and 40) exhibited higher activity
than the 1-isomer series (18 and 36). Interestingly, the antitu-
bercular activities of the 2-isomers 23a–e, 40a–d, 40f and 40g
reached MIC values of 1–2 mM regardless of the substituent on
the tetrazole cycle. Conversely, the activities of the corre-
sponding 1-isomers 18a–e, 36a–d, 36f and 36g, with MIC values
ranging from 1 to 8 mM, appeared to be more inuenced by the
ꢁ
mined aer incubation at 37 C for 7, 14, and 21 days for both
strains of M. kansasii and aer 14 and 21 days for M. tb and M.
avium. The values of MICs are expressed in mM and are pre-
sented in Tables 3 and 4. Isoniazid (INH) was used as a proto-
type drug.
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Table 3 In vitro antimycobacterial activities of selanyltetrazoles 15–24 expressed as MICs (mM)a
M. kansasii
M. kansasii
M. tb My 331/88
M. avium My 330/88
My 235/80
6509/96
R1
R2
14/21 days
7/14/21 days
1,5-Regioisomers
15d
15e
16c
16d
16e
17a
17b
17c
17d
17e
18a
18b
18c
18d
18e
19d
19e
20d
20e
21c
21d
21e
22a
22b
22c
22d
22e
23a
23b
23c
23d
23e
24d
24e
Ph
3,5-(NO2)2Ph
2,4-(NO2)2Ph
4-NO2Ph
3,5-(NO2)2Ph
2,4-(NO2)2Ph
H
Ph
4-NO2Ph
3,5-(NO2)2Ph
2,4-(NO2)2Ph
H
8/8
1/2
62/125
8/8
2/4
32/32
8/16
16/16
16/16
8/16
8/8
1/2
2/4
4/8
4/8
16/16
4/8
8/8
32/32
16/32
8/16/32
32/62/62
62/125/125
16/32/32
32/62/62
62/125/125
16/32/125
16/32/32
16/32/62
32/62/62
8/32/32
2/4/4
4/16/32
16/62/62
8/32/62
16/32/62
16/32/62
8/16/32
8/16/16
32/125/125
32/62/62
1/2/2
32/125/125
16/16/32
16/32/32
8/16/32
16/32/62
2/4/8
1/1/1
1/2/4
2/4/8
4/8/16
16/32/32
16/62/125
32/62/62
32/32/62
32/62/62
62/125/250
16/62/125
32/62/62
32/62/62
32/62/62
16/32/32
4/8/8
4-NO2Ph
250/250
250/250
16/32
250/250
250/250
62/62
250/250
32/32
500/500
32/32
125/125
125/125
125/125
125/125
125/125
16/16
2,4-(NO2)2Ph
3,5-(NO2)2Ph
Ph
4-NO2Ph
4/8/16
3,5-(NO2)2Ph
2,4-(NO2)2Ph
3,5-(NO2)2Ph
2,4-(NO2)2Ph
3,5-(NO2)2Ph
2,4-(NO2)2Ph
4-NO2Ph
3,5-(NO2)2Ph
2,4-(NO2)2Ph
H
Ph
4-NO2Ph
3,5-(NO2)2Ph
2,4-(NO2)2Ph
H
16/62/62
4/16/32
16/32/62
4/16/32
16/32/32
8/16/32
62/125/125
32/62/62
2/4/8
16/62/125
32/32/62
32/62/62
16/32/62
32/62/62
2/4/4
1/1/2
2/4/4
2/4/8
2/8/16
16/32/32
n.d.
4/4/4
4-CH3OPh
Ph
2,5-Regioisomers
1/1
4/8
4-NO2Ph
62/62
32/32
2/2
16/16
16/16
32/32
16/16
2/4
1/2
1/1
1/1
1/1
250/250
250/250
8/16
62/62
62/62
125/125
250/250
16/32
250/250
16/32
125/125
125/125
125/125
16/32
2,4-(NO2)2Ph
3,5-(NO2)2Ph
Ph
4-NO2Ph
3,5-(NO2)2Ph
2,4-(NO2)2Ph
3,5-(NO2)2Ph
2,4-(NO2)2Ph
2/2
4/8
n.d.
0.5/1
4-CH3OPh
INH
8/16/16
n.d.
250/250/250
n.d.
250/250
a
n.d. not determined.
type of substituent on the tetrazole than their corresponding 2-
Nitro group-containing anti-TB agents display various
isomers. The combination of a 3,5-dinitrobenzyl substituent on mechanisms of antimycobacterial action such as inhibition of
sulfur/selenium and a dinitrobenzyl substituent in position 1 of DprE1 (dinitrobenzamides, benzothiazinones),11 inhibition of
tetrazole (18d, 18e, and 36d) decreased antimycobacterial mycolic acid biosynthesis or NO poisoning of cytochrome c
activity compared to substances with a 3,5-dinitrobenzyl oxidase (PA-824, delamanid).33 Although the most potent
substituent on sulfur/selenium and a 1-benzyl (18b, 36b) or 1-(4- compounds 23a–e, 40a–d, 40f and 40g contain 3,5-dinitro-
nitrobenzyl) substituent (18c, 36c) on tetrazole. The MIC values phenyl fragments, such as the DprE1 inhibiting dinitro-
of benzylselanyl/3,5-dinitrobenzyl and 3,5-dinitrobenzylselanyl/ benzamides, their actual mechanism of action remains to be
benzyl reverse analog pairs 15d/18b and 20d/23b and their elucidated.
respective sulfanyl reverse analog pairs 33d/36b and 37d/40b
showed that the position of the 3,5-dinitrobenzyl substituent on
In vitro antibacterial and antifungal activity
sulfur/selenium is benecial, while the 3,5-dinitrobenzyl
substituted tetrazole moiety was generally unfavorable for
antimycobacterial activity. Isomers 15e and 20e bearing a 2,4-
dinitrobenzyl moiety on tetrazole were exceptions and exhibited
surprisingly high antimycobacterial activity. However, this
observation is likely connected with the high non-selective
toxicity of 2,4-dinitrobenzyl derivatives (see below).
The selectivities of the antimycobacterial effect of compounds
18b, 23b, 36b, 40b and 40d, which exhibited promising anti-
mycobacterial activity, were evaluated by determining the MIC
values against 8 bacterial and 8 fungal strains. All compounds
showed no antibacterial and no antifungal activity (Tables S1
and S2, see ESI†).
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Table 4 In vitro antimycobacterial activities of sulfanyltetrazoles 33–40 expressed as MICs (mM)a
M. kansasii
M. kansasii
M. tb My 331/88
M. avium My 330/88
My 235/80
6509/96
R1
R2
14/21 days
7/14/21 days
1,5-Regioisomers
33d
34c
34d
35a
35b
35c
35d
36a
36b
36c
36d
36f
36g
37d
38c
38d
39a
39b
39c
39d
40a
40b
40c
40d
40f
Ph
4-NO2Ph
3,5-(NO2)2Ph
4-NO2Ph
3,5-(NO2)2Ph
H
8/8
500/500
8/8
16/16
8/8
4/4
4/4
4/8
1/2
2/4
4/8
2/4
2/2
16/16
250/250
8/8
4/8
4/4
n.d.
4/4
2/4
1/1
1/2
1/2
1/2
1/1
0.5/1
125/125
250/250
250/250
125/500
250/250
62/62
32/62/62
250/250/250
4/8/16
125/500/500
8/32/62
32/62/62
32/62/62
4/16/32
2/8/8
1/4/4
4/8/8
2/2/4
2/4/4
16/32/32
250/250/250
4/8/16
32/125/125
16/32/62
n.d.
4/16/32
4/8/8
8/16/16
2/2/4
16/32/32
250/250/250
16/16/32
125/500/500
16/32/62
32/62/62
16/32/62
16/32/32
2/4/4
2,4-(NO2)2Ph
3,5-(NO2)2Ph
Ph
4-NO2Ph
3,5-(NO2)2Ph
H
16/32
500/1000
250/250
250/250
250/250
125/125
125/125
125/125
250/250
250/250
125/125
32/62
Ph
4-NO2Ph
3,5-(NO2)2Ph
4-ClPh
3,4-Cl2Ph
3,5-(NO2)2Ph
4-NO2Ph
3,5-(NO2)2Ph
H
2/8/16
8/16/32
4/8/8
2/4/8
2,5-Regioisomers
Ph
4-NO2Ph
16/32/32
250/250/250
16/16/32
32/125/125
8/16/32
n.d.
8/16/16
8/16/16
4/4/8
2/8/8
2/4/8
16/16/32
16/32/32
4/4/4
2,4-(NO2)2Ph
3,5-(NO2)2Ph
Ph
4-NO2Ph
3,5-(NO2)2Ph
H
n.d.
250/250
125/250
62/125
250/250
250/250
16/16
Ph
4-NO2Ph
3,5-(NO2)2Ph
4-Cl
1/2/2
4/8/16
8/16/32
250/250/250
40g
3,4-Cl2Ph
125/125
250/250
INH
a
n.d. not determined.
groups present. Interestingly, introduction of a chlorine atom to
the molecule decreased its toxicity (as in 36b and 36f or 40b and
40f); however, this effect did not show a clear dependence on
the number of chlorine atoms, as observed in compounds 36g
and 40g.
In vitro cell proliferation/viability assays
To further probe the selectivity of the studied compounds,
their effects on the viability of mammalian cells were evalu-
ated. We were also interested in how the type and position of
the substituents on tetrazole or on the sulfur/selenium atom
and the presence of either sulfur or selenium in position 5 of
tetrazole would inuence the overall toxicity of the studied
compounds. Therefore, the effects of ve 1,5- and 2,5-
regioisomeric pairs of the selenium derivatives, 15d/20d,
17b/22b, 18b/23b, 18c/23c and 18d/23d, ve pairs of the
sulfur regioisomers, 33d/37d, 36b/40b, 36c/40c, 36f/40f and
36g/40g, and compounds 39b and 40d on the viability of the
Chinese hamster ovary (CHO–K1) cell line were evaluated
(Table 5).
The resulting IC50 values indicated that there is no signi-
cant difference in the toxicity between sulfur or selenium
derivatives. The 2,4-dinitro derivatives (17b, 22b and 39b) were
highly toxic regardless of their substitution (IC50 values of
8.5–22 mM). The toxicities of the 1-isomers were either similar to
or higher than the 2-isomers; this is observed in the IC50 values
of the isomeric pairs 18b/23b, 18c/23c, 18d/23d, 36c/40c and
36f/40f. The most toxic compound, 18d, with an IC50 value of
6.5 mM has four nitro groups in its structure; however, there is
no clear correlation between toxicity and the number of nitro
Table 5 Viability of CHO–K1 cells (IC50 expressed in mM ꢂ SEM)
determined by proliferation/viability cell assays after a 24 h treatment
with test compoundsa
1-Isomer
IC50
2-Isomer
IC50
15d
17b
18b
18c
18d
33d
35b
36b
36c
36d
36f
85 ꢂ 21
8.5 ꢂ 0.2
71 ꢂ 34
27 ꢂ 14
6.5 ꢂ 0.2
115 ꢂ 18
n.d.
59 ꢂ 11
163 ꢂ 40
n.d.
20d
22b
23b
23c
23d
37d
39b
40b
40c
40d
40f
118 ꢂ 19
22 ꢂ 2
136 ꢂ 6
205 ꢂ 29
40 ꢂ 9
148 ꢂ 11
21 ꢂ 1.4
41 ꢂ 6
243 ꢂ 6
133 ꢂ 5
301 ꢂ 9
150 ꢂ 34
94 ꢂ 16
36g
140 ꢂ 9
40g
a
n.d. not determined.
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optimize the ADME properties and toxicity of these compounds
Ames uctuation test
while maintaining the antimycobacterial efficiency.
The mutagenic activity of regioisomeric pairs 18b/23b, 36b/40b,
36c/40c and 36f/40f was detected using the 96-well micro-plate
version of the Salmonella typhimurium Ames Test. At 50 mM we
found highly variable potencies of the tested compounds to
induce reverse mutation on the S. typhimurium strain TA98 and
no mutagenicity on the strain TA100, with no apparent struc-
ture–mutagenicity relationships. Importantly, compound 36b
did not induce any mutation in both strains. This indicates that
the 3,5-dinitrobenzyl moiety is not generally connected with
frame shi or base-exchange mutagenicity (see ESI†).
Acknowledgements
This work was supported by the Czech Science Foundation
project 14-08423S and MH CZ – DRO (UHHK, 00179906). G.K.
thanks Charles University in Prague (SVV 260 062).
Notes and references
1 World Health Organization, Global tuberculosis report, 2012,
http://www.who.int/tb.
2 A. Zumla, P. Nahid and S. T. Cole, Nat. Rev. Drug Discovery,
2013, 12, 388–404.
Conclusions
In this study, a series of nitro group-containing regioisomeric
1-alkyl- and 2-alkyl-5-(alkylsulfanyl)-2H-tetrazoles and their
selenium bioisosteres were prepared and characterized. All
1-alkyl and 2-alkyl regioisomers were isolated and unambigu-
3 Beena and D. S. Rawat, Med. Res. Rev., 2013, 33, 693–764.
4 M. Yan and S. T. Ma, ChemMedChem, 2012, 7, 2063–2075.
5 C. Lienhardt, M. Raviglione, M. Spigelman, R. Hafner,
E. Jaramillo, M. Hoelscher, A. Zumla and J. Gheuens, J.
Infect. Dis., 2012, 205, S241–S249.
6 A. J. Lenaerts, V. Gruppo, K. S. Marietta, C. M. Johnson,
D. K. Driscoll, N. M. Tompkins, J. D. Rose, R. C. Reynolds
and I. M. Orme, Antimicrob. Agents Chemother., 2005, 49,
2294–2301.
7 M. Matsumoto, H. Hashizume, T. Tomishige, M. Kawasaki,
H. Tsubouchi, H. Sasaki, Y. Shimokawa and M. Komatsu,
PLoS Med., 2006, 3, 2131–2144.
8 M. T. Gler, V. Skripconoka, E. Sanchez-Garavito, H. P. Xiao,
J. L. Cabrera-Rivero, D. E. Vargas-Vasquez, M. Q. Gao,
M. Awad, S. K. Park, T. S. Shim, G. Y. Suh, M. Danilovits,
H. Ogata, A. Kurve, J. Chang, K. Suzuki, T. Tupasi,
W. J. Koh, B. Seaworth, L. J. Geiter and C. D. Wells, N.
Engl. J. Med., 2012, 366, 2151–2160.
1
ously identied by the H and 13C NMR chemical shis of the
methylene group adjacent to the tetrazole nitrogen and the 13
C
NMR chemical shi of the tetrazole carbon. The regioisomeric
identities were conrmed by NOESY experiments.
Antimycobacterial evaluation indicated that 1-alkyl- (36a–d,
36f, 36g) and 2-alkyl-5-[(3,5-dinitrobenzyl)sulfanyl]-2H-tetra-
zoles (40a–d, 40f, 40g) and their selenium analogs (18a–e,
23a–e) exhibited promising in vitro antimycobacterial activity
against M. tb CNCTC My 331/88, with MIC values as low as 1 mM.
These derivatives also showed high activities against non-
tuberculous M. kansasii 6509/96 and INH-resistant M. kansasii
CNCTC My 235/80, with MIC values similar to or slightly lower
than that of M. tb. Furthermore, the antimycobacterial effects of
these compounds were found to be highly specic, because they
showed no antibacterial or antifungal activity and low cytotox-
icity in a mammalian cell line. Interestingly, no differences in
these biological properties between sulfur and selenium bio-
isosteres were found. We also found that the 3,5-dinitrobenzyl
moiety per se is not the carrier of mutagenicity.
9 T. Christophe, M. Jackson, H. K. Jeon, D. Fenistein,
M. Contreras-Dominguez, J. Kim, A. Genovesio,
J. P. Carralot, F. Ewann, E. H. Kim, S. Y. Lee, S. Kang,
M. J. Seo, E. J. Park, H. Skovierova, H. Pham, G. Riccardi,
J. Y. Nam, L. Marsollier, M. Kempf, M. L. Joly-Guillou,
T. Oh, W. K. Shin, Z. No, U. Nehrbass, R. Brosch, S. T. Cole
and P. Brodin, PLoS Pathog., 2009, 5, e1000645.
The structure–activity relationship study showed that the
position of the 3,5-dinitrobenzyl substituent on the sulfur/
selenium atom in position 5 of tetrazole is benecial, as the 10 V. Makarov, G. Manina, K. Mikusova, U. Mollmann,
reverse analogs, i.e., 1- or 2-(3,5-dinitrobenzyl)tetrazole deriva-
tives, exhibited signicantly lower antimycobacterial activities.
O. Ryabova, B. Saint-Joanis, N. Dhar, M. R. Pasca,
S. Buroni, A. P. Lucarelli, A. Milano, E. De Rossi,
M. Belanova, A. Bobovska, P. Dianiskova, J. Kordulakova,
C. Sala, E. Fullam, P. Schneider, J. D. McKinney, P. Brodin,
T. Christophe, S. Waddell, P. Butcher, J. Albrethsen,
I. Rosenkrands, R. Brosch, V. Nandi, S. Bharath,
S. Gaonkar, R. K. Shandil, V. Balasubramanian,
T. Balganesh, S. Tyagi, J. Grosset, G. Riccardi and
S. T. Cole, Science, 2009, 324, 801–804.
Derivatives bearing
a 2,4-dinitrobenzyl moiety generally
exhibited lower antimycobacterial activities and higher in vitro
cytotoxicity compared to the 3,5-dinitrobenzyl derivatives.
Considering the position of the alkyl substituent on tetrazole,
2-alkyl-5-[(3,5-dinitrobenzyl)sulfanyl]-2H-tetrazoles and their
selenium analogs showed higher antimycobacterial activity
against M. tb and lower cytotoxicity compared to the 1-alkyl
isomers. Consequently, 2-alkyl-5-[(3,5-dinitrobenzyl)sulfanyl]- 11 C. Trefzer, H. Skovierova, S. Buroni, A. Bobovska, S. Nenci,
2H-tetrazoles (40) are new lead antimycobacterial compounds
because they are superior to 1-alkyl derivatives in their anti-
mycobacterial effect and exhibit lower cytotoxicity. Moreover,
E. Molteni, F. Pojer, M. R. Pasca, V. Makarov, S. T. Cole,
G. Riccardi, K. Mikusova and K. Johnsson, J. Am. Chem.
Soc., 2012, 134, 912–915.
the antimycobacterial activity of the 1-alkyl isomers was more 12 V. Makarov, B. Lechartier, M. Zhang, J. Neres, A. M. van der
inuenced by the type of alkyl substituent than were the 2-alkyl
isomers. Thus, variation of the 2-alkyl substituent may further
Sar, S. A. Raadsen, R. C. Hartkoorn, O. B. Ryabova, A. Vocat,
L. A. Decosterd, N. Widmer, T. Buclin, W. Bitter, K. Andries,
This journal is © The Royal Society of Chemistry 2014
Med. Chem. Commun.

View Article Online
MedChemComm
Concise Article
F. Pojer, P. J. Dyson and S. T. Cole, EMBO Mol. Med., 2014, 6, 24 G. Roman, M. N. Rahman, D. Vukomanovic, Z. C. Jia,
372–383.
K. Nakatsu and W. A. Szarek, Chem. Biol. Drug Des., 2010,
75, 68–90.
13 Z. Kazimierczuk, M. Andrzejewska, J. Kaustova and
V. Klimesova, Eur. J. Med. Chem., 2005, 40, 203–208.
14 V. Klimesova, J. Koci, M. Pour, J. Stachel, K. Waisser and
J. Kaustova, Eur. J. Med. Chem., 2002, 37, 409–418.
15 J. Koci, V. Klimesova, K. Waisser, J. Kaustova, H. M. Dahse
and U. Mollmann, Bioorg. Med. Chem. Lett., 2002, 12, 3275–
3278.
16 V. Klimesova, J. Koci, K. Palat, J. Stolarikova, H. M. Dahse
and U. Mollmann, Med. Chem., 2012, 8, 281–292.
17 G. Karabanovich, J. Roh, T. Smutn´y, J. Nemecek, P. Vicherek,
25 M. Kume, T. Kubota, Y. Kimura, H. Nakashimizu and
K. Motokawa, J. Pharm. Soc. Jpn., 1992, 112, 622–637.
26 U. Muh, M. Schuster, R. Heim, A. Singh, E. R. Olson and
E. P. Greenberg, Antimicrob. Agents Chemother., 2006, 50,
3674–3679.
27 A. D. White, M. W. Creswell, A. W. Chucholowski,
C. J. Blankley, M. W. Wilson, R. F. Bousley,
A. D. Essenburg, K. L. Hamelehle, B. R. Krause,
R. L. Staneld, M. A. Dominick and M. Neub, J. Med.
Chem., 1996, 39, 4382–4395.
ˇ
ˇ
ˇ´
´
´
´
´
´
´
J. Stolarıkova, M. Vejsova, I. Duova, K. Vavrova, P. Pavek,
ˇ
´
´
V. Klimesova and A. Hrabalek, Eur. J. Med. Chem., 2014, 82, 28 H. Ozkan, S. Yavuz, A. Disli, Y. Yildirir and L. Turker,
324–340. Heteroat. Chem., 2007, 18, 255–258.
18 G. I. Koldobskii and R. B. Kharbash, Russ. J. Org. Chem., 29 A. Disli and M. Salman, Russ. J. Org. Chem., 2009, 45, 151–
2003, 39, 453–470.
153.
19 G. I. Koldobskii, Russ. J. Org. Chem., 2006, 42, 469–486.
20 J. Roh, K. Vavrova and A. Hrabalek, Eur. J. Org. Chem., 2012,
6101–6118.
30 G. Karabanovich, J. Roh, Z. Padelkova, Z. Novak, K. Vavrova
and A. Hrabalek, Tetrahedron, 2013, 69, 8798–8808.
31 R. E. Trifonov and V. A. Ostrovskii, Russ. J. Org. Chem., 2006,
42, 1585–1605.
21 G. Ortar, M. G. Cascio, A. S. Moriello, M. Camalli, E. Morera,
M. Nalli and V. Di Marzo, Eur. J. Med. Chem., 2008, 43, 62–72. 32 N. N. Sveshnikov and J. H. Nelson, Magn. Reson. Chem., 1997,
22 M. Sabbah, F. Fontaine, L. Grand, M. Boukraa, M. L. Efrit, 35, 209–212.
A. Doutheau, L. Soulere and Y. Queneau, Bioorg. Med. 33 U. Manjunatha, H. I. Boshoff and C. E. Barry, Commun.
Chem., 2012, 20, 4727–4736.
Integr. Biol., 2009, 2, 215–218.
23 J. Garfunkle, C. Ezzili, T. J. Rayl, D. G. Hochstatter, I. Hwang
and D. L. Boger, J. Med. Chem., 2008, 51, 4392–4403.
Med. Chem. Commun.
This journal is © The Royal Society of Chemistry 2014