Development of 3,5-Dinitrobenzylsulfanyl-1,3,4-oxadiazoles and Thiadiazoles as Selective Antitubercular Agents Active Against Replicating and Nonreplicating Mycobacterium tuberculosis

Article abstract of DOI:10.1021/acs.jmedchem.5b00608

Herein, we report the discovery and structure-activity relationships of 5-substituted-2-[(3,5-dinitrobenzyl)sulfanyl]-1,3,4-oxadiazoles and 1,3,4-thiadiazoles as a new class of antituberculosis agents. The majority of these compounds exhibited outstanding in vitro activity against Mycobacterium tuberculosis CNCTC My 331/88 and six multidrug-resistant clinically isolated strains of M. tuberculosis, with minimum inhibitory concentration values as low as 0.03 μM (0.011-0.026 μg/mL). The investigated compounds had a highly selective antimycobacterial effect because they showed no activity against the other bacteria or fungi tested in this study. Furthermore, the investigated compounds exhibited low in vitro toxicities in four proliferating mammalian cell lines and in isolated primary human hepatocytes. Several in vitro genotoxicity assays indicated that the selected compounds have no mutagenic activity. The oxadiazole and thiadiazole derivatives with the most favorable activity/toxicity profiles also showed potency comparable to that of rifampicin against the nonreplicating streptomycin-starved M. tuberculosis 18b-Lux strain, and therefore, these derivatives, are of particular interest.

Full text of DOI:10.1021/acs.jmedchem.5b00608

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Article
Development of 3,5-Dinitrobenzylsulfanyl-1,3,4-Oxadiazoles and
Thiadiazoles as Selective Antitubercular Agents Active Against
Replicating and Nonreplicating Mycobacterium tuberculosis
Galina Karabanovich, Júlia Zemanová, Tomáš Smutný, Rita Székely, Michal Šarkan,
Ivana Centárová, Anthony Vocat, Ivona Pávková, Patrik #onka, Jan N#me#ek,
Ji#ina Stola#íková, Marcela Vejsová, Katerina Vavrova, V#ra Klimešová, Alexandr
Hrabalek, Petr Pavek, Stewart T. Cole, Katarína Mikušová, and Jaroslav Roh
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00608 • Publication Date (Web): 05 Mar 2016
Downloaded from http://pubs.acs.org on March 6, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Development of 3,5ꢀDinitrobenzylsulfanylꢀ1,3,4ꢀ
Oxadiazoles and Thiadiazoles as Selective
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Antitubercular Agents Active Against Replicating
and Nonreplicating Mycobacterium tuberculosis
a
b
a
e
b
Galina Karabanovich, Júlia Zemanová, Tomáš Smutný, Rita Székely, Michal Šarkan, Ivana
b
e
c
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a
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Centárová, Anthony Vocat, Ivona Pávková, Patrik Čonka, Jan Němeček, Jiřina Stolaříková,
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Marcela Vejsová, Kateřina Vávrová, Věra Klimešová, Alexandr Hrabálek, Petr Pávek,
e
b
a,
Stewart T. Cole, Katarína Mikušová, and Jaroslav Roh *
a
Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Heyrovského 1203,
0005 Hradec Králové, Czech Republic
5
b
Comenius University in Bratislava, Faculty of Natural Sciences, Department of Biochemistry,
Mlynská dolina, Ilkovičova 6, 842 15 Bratislava, Slovakia
c
University of Defence, Faculty of Military Health Sciences, Department of Molecular
Pathology and Biology, Třebešská 1575, 50005 Hradec Králové, Czech Republic
d
Regional Institute of Public Health, Department of Bacteriology and Mycology, Partyzánské
náměstí 7, 70200 Ostrava, Czech Republic
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Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, CHꢀ1015 Lausanne,
Switzerland
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ABSTRACT
Herein, we report the discovery and structureꢀactivity relationships of 5ꢀsubstitutedꢀ2ꢀ[(3,5ꢀ
dinitrobenzyl)sulfanyl]ꢀ1,3,4ꢀoxadiazoles and 1,3,4ꢀthiadiazoles as
a
new class of
antituberculosis agents. The majority of these compounds exhibited outstanding in vitro activity
against Mycobacterium tuberculosis CNCTC My 331/88 and six multidrugꢀresistant clinically
isolated strains of M. tuberculosis, with minimum inhibitory concentration values as low as 0.03
µM (0.011ꢀ0.026 µg/mL). The investigated compounds had a highly selective antimycobacterial
effect because they showed no activity against the other bacteria or fungi tested in this study.
Furthermore, the investigated compounds exhibited low in vitro toxicities in four proliferating
mammalian cell lines and in isolated primary human hepatocytes. Several in vitro genotoxicity
assays indicated that the selected compounds have no mutagenic activity. The oxadiazole and
thiadiazole derivatives with the most favorable activity/toxicity profiles also showed potency
comparable to that of rifampicin against the nonreplicating streptomycinꢀstarved M. tuberculosis
18bꢀLux strain, and therefore, these derivatives are of particular interest.
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Introduction
Tuberculosis (TB) is a widespread infectious disease predominantly caused by Mycobacterium
tuberculosis (M.tb.), which can be transmitted through the air as droplets and predominantly
affects the lungs. Although there has been a slow decrease in new TB cases and TBꢀrelated
deaths in recent years, the numbers of TB patients and TBꢀrelated deaths remain extremely high.
According to the World Health Organization, an estimated 9.6 million new TB cases and 1.5
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million TBꢀrelated deaths were reported worldwide in 2014.
The application of firstꢀline drugs (isoniazid (INH), rifampicin (RIF), pyrazinamide, and
ethambutol) is an effective approach for treating TB caused by drugꢀsusceptible M.tb. strains.
This therapy leads to recovery in approximately 90% of patients. To cure drugꢀresistant M.tb.
strains, secondꢀline and thirdꢀline antiꢀTB drugs are required. Streptomycin, kanamycin (KAN),
amikacin, capreomycin, viomycin, fluoroquinolones, paraꢀaminosalicylic acid, cycloserine,
terizidone, ethionamide, prothionamide, thioacetazone, and linezolid are secondꢀline antiꢀTB
drugs, whereas amoxicillin/clavulanic acid, imipenem/cilastatin, clarithromycin, and clofazimine
2
are thirdꢀline antiꢀTB drugs. Unfortunately, the majority of these medications are poorly
tolerated when applied as part of a longꢀterm TB chemotherapy regimen. Furthermore, they are
often ineffective: only 50% of patients with multiꢀdrugꢀresistant TB (MDRꢀTB) have been
1
successfully treated using these drugs. Therefore, both the appearance and increasing spread of
MDRꢀTB and the evolution of extensively drugꢀresistant (XDRꢀTB) strains have become a
serious problem for current TB treatments. In 2014, 3.3% of new TB cases and 20% of
1
previously treated cases were MDRꢀTB; an estimated 9.7% of these cases involved XDRꢀTB.
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4, 5
Despite the introduction of two new compounds (delamanid and bedaquiline ) to the
repertoire of antiꢀTB therapies for drugꢀresistant strains of M.tb., further development of
antimycobacterial compounds with new mechanisms of action and that are active against MDR
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2, 6
and XDRꢀTB is urgently needed.
Recently, several nitro groupꢀcontaining inhibitors of
Dꢀribofuranose 2’ꢀoxidase (DprE1), which is involved in
mycobacterial decaprenylphosphorylꢀβꢀ
the synthesis of decaprenylphosphoryl arabinose, the only donor of arabinosyl residues for the
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, 8
biosynthesis of arabinan polymers in mycobacteria, have been extensively studied (Figure 1).
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Figure 1. Structures of selected DprE1 inhibitors, including dinitrobenzamide DNB1, its
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10, 11
12, 13
analogue CT319, trinitroxanthone MTX
and piperazinobenzothiazinone PBTZ 169.
14ꢀ16
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In our previous studies, benzazoles (1)
, Figure 2) bearing 3,5ꢀdinitrobenzylsulfanyl fragments were found to exhibit strong
antimycobacterial activities with minimum inhibitory concentration (MIC) values of 1 µM (0.36ꢀ
.44 µg/mL) against drugꢀsusceptible M.tb. and 0.25 µM (0.09ꢀ0.11 µg/mL) against six
and 1,5ꢀ and 2,5ꢀdisubstituted tetrazoles (2 and
3
0
multidrugꢀresistant clinically isolated strains of M.tb. These results indicate that
antimycobacterial 3,5ꢀdinitrobenzylsulfanyl tetrazoles may not show crossꢀresistance to common
antiꢀTB drugs. Moreover, the benzazole derivatives 1 and 5ꢀ(3,5ꢀdinitrobenzylsulfanyl)tetrazole
derivatives 2 and 3 exhibited selective antimycobacterial effects, as they were ineffective against
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Gramꢀpositive and 4 Gramꢀnegative bacteria and 8 fungal (5 yeasts and 3 molds) strains and
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exhibited low cytotoxicity.
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Figure 2. Dinitrobenzylsulfanyl benzimidazoles, benzoxazoles and benzothiazoles 1 and 5ꢀ(3,5ꢀ
dinitrobenzylsulfanyl)tetrazoles 2 and 3 as antiꢀTB lead compounds.
In this work, we focused on 1,3,4ꢀoxadiazole and 1,3,4ꢀthiadiazole scaffolds as possible
bioisosteric surrogates for 2,5ꢀdisubstituted tetrazole in lead compound 3. Various compounds
based on 1,3,4ꢀoxadiazoles and 1,3,4ꢀthiadiazoles, including 2ꢀalkylsulfanyl derivatives, exhibit
1
9ꢀ23
strong antiꢀTB effects.
An overview of several oxadiazoleꢀ and thiadiazoleꢀbased antiꢀTB
compounds is shown in Figure 3.
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Figure 3. Structures of 1,3,4ꢀoxadiazole (4, 5a, and 6) and 1,3,4ꢀthiadiazole (7 and 8) derivatives
with high antimycobacterial activities. Compound 5b bearing a 3,5ꢀdinitrophenyl moiety
possessed only weak antiꢀTB activity.
Oxadiazole derivative 4 exhibited significant activity with MIC values of 0.1 µM against M.tb.
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H37Rv and 1.10 µM against INHꢀresistant M.tb. Derivatives of 4ꢀ(5ꢀpentadecyl/heptadecylꢀ
1,3,4ꢀoxadiazolꢀ2ꢀyl)pyridine (5a) bearing long, highly lipophilic alkyl chains presented MIC
values of 0.35 µM and 0.65 µM against M.tb. H37Rv and were highly efficient against the drugꢀ
resistant strain CIBIN 112. Note that 4ꢀ(5ꢀ(3,5ꢀdinitrophenyl)ꢀ1,3,4ꢀoxadiazolꢀ2ꢀyl)pyridine (5b)
2
5
had only weak potency.
2ꢀ((5ꢀ(4ꢀMethoxyphenyl)ꢀ1,3,4ꢀoxadiazolꢀ2ꢀyl)sulfanyl)ꢀNꢀ
phenethylacetamide (6) is an example of a highly efficient antiꢀTB derivative of 1,3,4ꢀ
oxadiazoleꢀ5ꢀthiol that showed an IC value of 0.38 µM (0.14 µg/mL). Substituted thiadiazole 7
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with an IC90 of 0.62 µM (0.2 µg/mL) is another example of a highly efficient antiꢀTB
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derivative. Among the substituted 1,3,4ꢀthiadiazoleꢀ5ꢀthiols with general structures based on 8,
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compounds with MIC values as low as 1.1 µM (0.39 µg/mL) were found.
The aims of this work were to synthesize a series of 2ꢀalkyl/arylꢀ5ꢀ(dinitrobenzylsulfanyl)ꢀ
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,3,4ꢀoxadiazoles and 1,3,4ꢀthiadiazoles as analogues of lead compound 3 and to investigate
their structureꢀactivity relationships with respect to antiꢀTB activity/cytotoxicity and selectivity.
Furthermore, several trifluoromethylꢀsubstituted and reduced analogues of the most active
compounds were prepared, and their antimycobacterial activities were studied (Table 1).
Table 1. Nitroꢀsubstituted 1,3,4ꢀoxadiazole and 1,3,4ꢀthiadiazole derivatives studied in this
work.
a
B
c
d
e
f
g
4ꢀClPh
m
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1
1
R
R
R
CH
3
CH
3
(CH
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)
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cyclohexyl
Ph
2ꢀClPh
3ꢀClPh
h
i
j
k
L
2,4ꢀCl
Ph 4ꢀCH
Ph 3,5ꢀ(CH
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Ph 3ꢀCH
OPh
4ꢀCH OPh 3,5ꢀ(CH O) Ph
3 3 2
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3
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q
3ꢀNO
Ph 4ꢀNO
Ph
4ꢀHOPh
4ꢀpyridyl
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Results and discussion
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Synthesis of oxadiazole and thiadiazole derivatives. 2ꢀAlkyl/arylꢀ5ꢀbenzylsulfanylꢀ1,3,4ꢀ
oxadiazoles (9o, 10d, 10g, 10l, 10n, 10o, 10q, 11b-q, 15g, 15i, and 15o) were synthesized
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0
according to the previously reported threeꢀstep approach depicted in Scheme 1. The
corresponding methyl esters (19b-p) were either prepared by Fischer esterification of carboxylic
acid or were commercially available. The reaction of methyl esters 19b-p with hydrazine hydrate
in ethanol led to the formation of the corresponding acyl hydrazides 20b-p, which were further
reacted with carbon disulfide in basic ethanol under reflux to produce 2ꢀsubstitutedꢀ1,3,4ꢀ
oxadiazoleꢀ5ꢀthiols 21b-p. The synthesis of 2ꢀ(pyridinꢀ4ꢀyl)ꢀ1,3,4ꢀoxadiazoleꢀ5ꢀthiol (21q) was
accomplished from INH 20q. The alkylation of oxadiazoles 21b-q in the presence of
tetrabutylammonium bromide (TBAB) as a phaseꢀtransfer catalyst resulted in the formation of
the target nitroꢀsubstituted 2ꢀalkyl/arylꢀ5ꢀbenzylsulfanylꢀ1,3,4ꢀoxadiazoles 9o, 10d, 10g, 10l,
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10n, 10o, 10q, 11b-q, 15g, 15i and 15o in 45ꢀ96% yields.
Scheme 1. Synthesis of nitroꢀsubstituted 2ꢀalkyl/arylꢀ5ꢀbenzylsulfanylꢀ1,3,4ꢀoxadiazoles 9o,
a, b
10d, 10g, 10l, 10n, 10o, 10q, 11b-q, 15g, 15i and 15o.
a
Reagents and conditions: (a) hydrazine hydrate, EtOH, reflux, 4ꢀ48 h, 60ꢀ96%; (b) CS ,
2
2
KOH, EtOH, reflux, 6ꢀ44 h, 61ꢀ97%; (c) R CH Cl, NaOH, tetrabutylammonium bromide
(TBAB), H O/CH Cl , rt, overnight, 45ꢀ96%. A list of R groups (aꢀq) can be found in Table 1.
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ꢀ[(3,5ꢀDinitrobenzyl)sulfanyl]ꢀ5ꢀmethylꢀ1,3,4ꢀoxadiazole (11a) was prepared by reacting 5ꢀ
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[
(3,5ꢀdinitrobenzyl)sulfanyl]ꢀ1Hꢀtetrazole (23) with acetic anhydride (Scheme 2).
The
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starting 5ꢀ[(3,5ꢀdinitrobenzyl)sulfanyl]ꢀ1Hꢀtetrazole (23) was prepared using Koguro’s method.
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Unfortunately, this approach was unsuccessful for phenyl derivative 11d. The reaction of 23
with benzoyl chloride proceeded with low yield, and a mixture of products was formed. Thus,
1
1d was prepared using the standard procedure depicted in Scheme 1.
a
Scheme 2. Synthesis of 2ꢀ[(3,5ꢀdinitrobenzyl)sulfanyl]ꢀ5ꢀmethylꢀ1,3,4ꢀoxadiazole (11a).
a
Reagents and conditions: (a) NaN , Et NHCl, PhCH , 105 °C, 2 h, 68%; (b) (CH CO) O,
3
3
3
3
2
microwave, 90 °C, 10 h, 60%.
Thiadiazole derivatives (12o, 13d, 13g, 13o, 14a, 14d-g, 14i-l, 14n, 14o, 14q, 16g, 16i, and
3
4
16o) were synthesized as shown in Scheme 3. Substituted benzohydrazides 20d-g, 20i-l, 20n,
2
0o and 20q were converted to the corresponding potassium 3ꢀaroyl dithiocarbazates 24d-g, 24i-
l, 24n, 24o and 24q in 72ꢀ96% yields. The dithiocarbazates were then cyclized using
concentrated sulfuric acid to the corresponding 2ꢀarylꢀ1,3,4ꢀthiadiazoleꢀ5ꢀthiols 25d-g, 25i-l,
2
5n, 25o and 25q in 24ꢀ76% yields. In some cases, this reaction resulted in low yields of 25.
3
5
Unfortunately, neutralization of the reaction mixture with aqueous ammonia did not improve
the yields. 2ꢀMethylꢀ1,3,4ꢀthiadiazoleꢀ5ꢀthiol (25a) was commercially available. The alkylation
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of 2ꢀsubstitutedꢀ1,3,4ꢀthiadiazoleꢀ5ꢀthiols 25a, 25d-g, 25i-l, 25n, 25o and 25q in the presence of
TBAB led to the formation of the target nitroꢀsubstituted 2ꢀalkyl/arylꢀ5ꢀbenzylsulfanylꢀ1,3,4ꢀ
thiadiazoles 12o, 13d, 13g, 13o, 14a, 14d-g, 14i-l, 14n, 14o, 14q, 16g, 16i and 16o in 48ꢀ87%
yields.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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32
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 3. Synthesis of nitroꢀsubstituted 2ꢀalkyl/arylꢀ5ꢀbenzylsulfanylꢀ1,3,4ꢀthiadiazoles 12o,
a, b
13d, 13g, 13o, 14a, 14d-g, 14i-l, 14n, 14o, 14q, 16g, 16i and 16o.
a
Reagents and conditions: (a) CS , KOH, EtOH, rt, overnight; (b) H SO , 0 °C – rt, 4 h, 24ꢀ
2
2
4
2
b
1
7
6%; (c) R CH Cl, NaOH, TBAB, H O/CH Cl , rt, overnight, 48ꢀ87%. A list of R groups (aꢀq)
2 2 2 2
can be found in Table 1.
The alkyl and benzyl halides used in the aforementioned approaches are commercially
available, with the exception of 3ꢀnitroꢀ5ꢀtrifluoromethylbenzyl chloride (29), which was
prepared via a threeꢀstep procedure starting from 3ꢀtrifluoromethylbenzoic acid (26) (Scheme 4).
a
Scheme 4. Synthesis of 3ꢀnitroꢀ5ꢀtrifluoromethylbenzyl chloride 29.
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
4
4
4
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and conditions: (a) H SO , HNO , 0 – 40 °C, 4 h, 91%; (b) BH , THF, ꢀ15 °C – rt,
2
4
3
3
2
4 h, 90%; (c) PCl , CHCl , 0 °C – reflux, 24 h, 91%.
5 3
The synthesis of reduced analogues 17o and 18o is depicted in Scheme 5. In the first step, 3,5ꢀ
3
6
dinitrobenzyl chloride was reduced using an excess of tin(II) chloride dihydrate in ethanol. The
addition of 5ꢀ(4ꢀnitrophenyl)ꢀ1,3,4ꢀoxadiazoleꢀ2ꢀthiol (21o) in THF to an aqueous solution of
these partially a fully reduced ammonium salts followed by the addition of sodium carbonate (to
pH 8ꢀ10) resulted in the formation of desired products 17o and 18o.
a
Scheme 5. Synthesis of partially and fully reduced derivatives 17o and 18o.
O N
H2N
X
2
N N
O
NH2
a
b
.
HCl
S
Cl
Cl
O N
2
O N
2
X
1
18o
7o: X = NO ; 11%
2
X = NO or NH2
2
X = NH2;
30%
:
a
Reagents and conditions: (a) SnCl .2H O, EtOH, 10 min; (b) 21o, Na CO , THF, rt,
2
2
2
3
overnight.
In vitro antimycobacterial activity against replicating Mycobacterium spp. All synthesized
compounds were assessed for in vitro antimycobacterial activities against M.tb. (H37Rv) and
against the nonꢀtuberculous mycobacterial species M. avium CNCTC My 330/88, M. kansasii
CNCTC My 235/80 and the clinically isolated M. kansasii 6509/96. Selected compounds were
assessed for in vitro antimycobacterial activities against six clinically isolated multidrugꢀresistant
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strains of M.tb. The antimycobacterial activities of the compounds were evaluated after
incubation at 37 °C for 7/14/21 days for both strains of M. kansasii and after 14/21 days for the
M.tb. and M. avium strains. The antimycobacterial activities are expressed as MICs (IC ), which
9
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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32
33
34
35
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
is the lowest concentration of a compound at which the inhibition of mycobacterial growth
occurred. The firstꢀline antiꢀTB drug INH was used as a reference compound.
The results of the in vitro antimycobacterial evaluation revealed that almost all compounds
bearing the 3,5ꢀdinitrobenzylsulfanyl moiety (11b-q and 14d-g, 14i-l, 14n, 14o, and 14q)
exhibited excellent activity against drugꢀsusceptible M.tb. strains and against nonꢀtuberculous M.
kansasii strains (Table 2).
Table 2. In vitro antimycobacterial activities of the target compounds from series 9-18 expressed
as MICs (ꢁM).
M. tuberculosis
My 331/88
14 / 21 days
250 / 250
2 / 4
M. avium
M. kansasii
My 235/80
7 / 14 / 21 days
250 / 250 / 250
4 / 8 / 16
M. kansasii
My 330/88
6509/96
9
1
1
1
1
1
1
1
1
o
250 / 250
>16 / >16
8 / 16
62 / 62 / 62
2 / 8 / 8
0d
0g
0l
2 / 2
2 / 4 / 4
4 / 8 / 8
0.5 / 1
4 / 4
2 / 8 / 16
2 / 8 / 16
0n
0o
0q
1a
1b
16 / 16
32 / 32
8 / 16 / 32
16 / 16 / 32
2 / 4 / 8
2 / 2
>125 / >125
62 / 62
2 / 4 / 8
8 / 8
16 / 32 / 32
4 / 8 / 16
32 / 32 / 32
16 / 16 / 32
0.25 / 0.25 / 0.25
4 / 8
500 / 500
4 / 8
0.25 / 0.25
0.125 / 0.25 / 0.25
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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5
5
5
5
5
5
5
5
5
5
6
1
1
1
1
1
1c
1d
1e
1f
≤0.03 / ≤0.03
0.06 / 0.06
0.125 / 0.125
≤0.03 / 0.06
0.125 / 0.125
≤0.03 / 0.06
≤0.03 / ≤0.03
0.06 / 0.06
0.125 / 0.125
0.125 / 0.125
0.125 / 0.125
0.125 / 0.125
0.125 / 0.125
1 / 1
n.d.
0.06 / 0.125 / 0.25
0.06 / 0.25 / 0.25
0.5 / 1 / 2
16 / 32
0.5 / 1 / 1
16 / 32
0.125 / 0.25 / 0.5
0.125 / 0.25 / 0.5
0.125 / 0.25 / 0.25
0.125 / 0.5 / 0.5
0.06 / 0.06 / 0.06
0.25 / 0.5 / 1
0.125 / 0.25 / 0.5
0.125 / 0.25 / 0.25
0.125 / 0.25 / 0.5
0.25 / 0.5 / 0.5
0.25 / 0.25 / 0.5
4 / 8 / 16
0.125 / 0.5 / 1
0.125 / 0.5 / 1
0.125 / 0.25 / 0.5
0.125 / 0.25 / 0.5
0.06 / 0.06 / 0.125
0.5 / 1 / 2
16 / 16
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1g
>125 / >125
16 / 32
11h
11i
11j
8 / 16
>32 / >32
16 / 16
1
1
1
1
1
1
1
1
1
1k
1l
0.125 / 0.5 / 1
0.125 / 0.25 / 0.5
0.125 / 0.5 / 1
0.5 / 1 / 2
16 / 32
1m
1n
1o
1p
1q
2o
3d
16 / 16
16 / 16
>125 / >125
62 / 62
0.25 / 0.5 / 1
4 / 8 / 16
0.5 / 0.5
62 / 62
1 / 2 / 2
2 / 2 / 4
62 / 62
250 / 250
8 / 32
250 / 250 / 250
1 / 4 / 4
62 / 62 / 62
1 / 2 / 4
8 / 16
13g
13o
14a
2 / 4
4 / 8
1 / 2 / 4
1 / 2 / 4
16 / 32
250 / 250
125 / 125
8 / 16
1 / 4 / 8
1 / 4 / 8
4 / 4
4 / 4 / 8
8 / 8 / 16
1
1
1
1
1
1
4d
4e
4f
4g
4i
0.06 / 0.125
0.125 / 0.125
0.125 / 0.125
0.06 / 0.06
0.125 / 0.125
0.125 / 0.125
0.25 / 0.5 / 1
0.5 / 1 / 1
0.25 / 0.5 / 1
0.5 / 1 / 1
16 / >16
8 / 16
0.25 / 1 / 1
0.25 / 0.5 / 0.5
0.125 / 0.25 / 0.5
0.5 / 1 / 2
8 / 16
0.125 / 0.5 / 0.5
0,5 / 0,5 / 1
>125 / >125
>16 / >16
4j
0.25 / 1 / 1
0.25 / 0.5 / 0.5
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1
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1
1
4k
≤0.03 / 0.06
0.125 / 0.125
0.125 / 0.125
0.125 / 0.125
0.125 / 0.125
>32 / >32
>32 / >32
32 / 32
8 / 16
0.25 / 0.5 / 0.5
0.25 / 1 / 1
0.25 / 0.5 / 0.5
0.25 / 1 / 1
4l
8 / 16
4n
4o
4q
>16 / >16
>16 / >16
>125 / >125
>500 / >500
>500 / >500
>500 / >500
>500 / >500
>500 / >500
>500 / >500
250 / 250
0.25 / 1 / 1
0.5 / 1 / 1
0.25 / 0.5 / 1
0.25 / 0.5 / 1
0.5 / 1 / 1
10
11
12
13
14
15
16
17
18
19
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22
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25
26
27
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46
47
48
49
50
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56
57
58
59
60
0.5 / 1 / 1
15g
15i
>32 / >32 / >32
>32 / >32 / >32
16 / 32 / 32
>32 / >32 / >32
>32 / >32 / >32
16 / 32 / 32
15o
1
1
1
1
1
6g
6i
>32 / >32
>32 / >32
>32 / >32
125 / 125
>32 / >32 / >32
>32 / >32 / >32
>32 / >32 / >32
250 / 250 / 250
250 / 250 / 250
>250 / >250 / >250
>32 / >32 / >32
>32 / >32 / >32
>32 / >32 / >32
250 / 250 / 250
250 / 250 / 250
4 / 4 / 4
6o
7o
8o
250 / 250
250 / 250
INH
0.5 / 1
>250 / >250
The results confirmed that dinitrobenzylsulfanyl substitution is necessary for high
17, 18
antimycobacterial activity, which is consistent with our previous observations.
The 4ꢀ
nitrobenzyl derivatives 9o and 12o exhibited the lowest antiꢀTB effects in all cases. The
positions of the two nitro groups substantially impacted the antimycobacterial activities of the
prepared compounds. The antimycobacterial potencies of the 3,5ꢀdinitroꢀsubstituted derivatives
of series 11 and 14 were found to be considerably higher than those of the 2,4ꢀdinitroꢀsubstituted
derivatives of series 10 and 13.
The highest activities against M.tb. were exhibited by the 1,3,4ꢀoxadiazole derivatives 11c,
ꢀ1
11d, 11f, 11h, 11i and 11j, with MIC values of 0.03ꢀ0.06 µM (approx. 0.011ꢀ0.026 µg.mL ),
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and by the 1,3,4ꢀthiadiazole derivatives 14d and 14k, with MIC values of 0.03ꢀ0.125 µM
ꢀ1
(
approx. 0.012ꢀ0.06 µg.mL ). Nevertheless, almost all of the 3,5ꢀdinitrobenzylsulfanyl
derivatives of series 11 and 14 that were prepared and studied in this work possessed
significantly higher activities than current firstꢀline antiꢀTB drugs. Furthermore, selected
compounds bearing 3,5ꢀdinitrobenzylsulfanyl groups (11d, 11l, 11o, 14l, 14g and 14o) exhibited
outstanding activities against all six MDR strains of M.tb., with MIC values of 0.03ꢀ0.5 µM and
no crossꢀresistance with any firstꢀ or secondꢀline antiꢀTB drugs (Table 3).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. In vitro antimycobacterial activities of compounds 11d, 11l, 11o, 14g, 14l and 14o and
of selected common antiꢀTB drugs against MDR strains of M.tb. The results are expressed as
MICs (ꢁM) after 14/21 days of incubation for the compounds and after 14 days of incubation for
the antiꢀTB drugs.
MDR M. tuberculosis strains
Praha
131
234/2005
9449/2007 8666/2010 Praha 1
Praha 4
11d
≤0.03 / 0.06
0.06 / 0.125
0.06 / 0.125
0.5 / 0.5
0.06 / 0.06
0.06 / 0.125
0.06 / 0.125
0.25 / 0.5
0.125 / 0.25
0.06 / 0.06
>32 (R)
≤0.03 / 0.06
0.06 / 0.06
0.06 / 0.06
0.25 / 0.5
0.06 / 0.125
≤0.03 / 0.06
>32 (R)
0.06 / 0.06
≤0.03 / 0.06 ≤0.03 / 0.06
1
1
1
1
1
1l
0.06 / 0.125 0.06 / 0.125 0.06 / 0.125
0.06 / 0.125 0.06 / 0.125 0.06 / 0.125
1o
4g
4l
0.25 / 0.5
0.25 / 0.5
0.5 / 0.5
0.125 / 0.25
0.06 / 0.06
0.125 / 0.25 0.125 / 0.25 0.125 / 0.25
4o
0.06 / 0.06
16 (R)
0.06 / 0.06
>32 (R)
16 (R)
0.06 / 0.06
Streptomycin 32 (R)
>
32 (R)
6 (R)
2 (R)
1
3
Isoniazid
16 (R)
16 (R)
>8 (R)
64 (R)
32 (R)
16 (R)
Ethambutol
Rifampin
8 (S)
16 (R)
32 (R)
16 (R)
>8 (R)
>8 (R)
>8 (R)
>8 (R)
>8 (R)
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0.5 (S)
2 (S)
8 (R)
2 (S)
2 (R)
2 (S)
1 (S)
>16 (R)
0.5 (S)
0.5 (R)
1 (S)
1
6 (R)
8 (R)
.25 (S)
32 (R)
Ofloxacin
0.25 (S)
0.125 (S)
0.5 (S)
1 (S)
1 (S)
>
Gentamicin
Clofazimine
Amikacin
0.125 (S)
0.5 (S)
0.5 (R)
0.5 (S)
0
>
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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32
33
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39
40
41
42
43
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46
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48
49
50
51
52
53
54
55
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59
60
S – Strain susceptible to the given antibiotic drug
R – Strain resistant to the given antibiotic drug
1
Substituent R in position 2 on the 1,3,4ꢀoxadiazole and 1,3,4ꢀthiadiazole cycles appears to
have a negligible influence on the antimycobacterial activities of the investigated compounds.
1
However, the R substituent determines the physicochemical properties of the investigated
compounds, as the remainder of the molecules remains unchanged. The low activity of 2ꢀ[(3,5ꢀ
dinitrobenzyl)sulfanyl]ꢀ5ꢀmethylꢀ1,3,4ꢀoxadiazole (11a) and its thiadiazole analogue (14a)
against all four mycobacterial strains can be explained by the low lipophilicity of the methyl
1
substituent R . The slightly lower activity of hydroxyphenyl derivative 11p compared to the
other derivatives of series 11 can be attributed to the same phenomenon.
The 2ꢀalkyl/arylꢀ5ꢀ[(3,5ꢀdinitrobenzyl)sulfanyl]ꢀ1,3,4ꢀoxadiazoles of series 11 and 1,3,4ꢀ
thiadiazoles of series 14 exhibited only moderate activities against highly resilient M. avium
(
MIC 4ꢀ62 µM). Nevertheless, clinically isolated M. kansasii 6509/96 and INHꢀresistant M.
kansasii My 235/80 were susceptible to compounds from series 11 and 14. Almost all
compounds from both series were effective against these strains, with MIC values of 0.125ꢀ1
µM. The compounds from series 10 and 13 again appeared to be considerably less active than
their 3,5ꢀdinitro analogues.
The loss of the antimycobacterial activities of the partially and fully reduced derivatives 17o
and 18o showed that the presence of both nitro groups is crucial for the high efficacies of the
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
investigated compounds from series 11 and 14. Because the lead compounds 11 and 14 showed
9
10, 11
structural similarities with the known DprE1 inhibitors DNB1 and MTX,
we replaced one
nitro group with a trifluoromethyl group, such as in the structures of the DprE1 inhibitors PBTZ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
10
1
69 and CT319. However, trifluoromethyl analogues 15 and 16 were found to be inactive.
In vitro antimycobacterial activity against dormant mycobacteria. The antimycobacterial
activities of two selected compounds, 11i and 14g, were further evaluated against nonreplicating
streptomycin (STR)ꢀstarved M.tb. strain 18bꢀLux (SS18bꢀLux) using a luciferase assay (Figure
37
4
) and a standard resazurin reduction microplate assay (Figure S2). Both compounds showed
equal or even better potency on the latent model SS18b than RIF. Furthermore, the SS18bꢀLux
assay also enabled us to monitor the timeꢀdependent potency of the compounds. Here, we can
conclude that 11i and 14g act on the dormant mycobacteria in a doseꢀdependent rather than timeꢀ
dependent manner.
Figure 4. Assessment of compounds 11i, 14g and RIF in the luciferaseꢀbased assay with SS18bꢀ
Lux. Relative luminescence ꢀ luminescence values normalized to the untreated control values.
In vitro antibacterial and antifungal activities. To obtain basic insights regarding the
selectivity of the inhibitory effects of the investigated compounds, the MIC values of compounds
10d, 10g, 10l, 10o, 11b, 11d, 11g-i, 11k, 11n, 11o, 13g, 13o, 14g, 14l, and 14o against 8
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bacterial strains and 8 fungal strains (5 yeasts and 3 molds) were evaluated (Tables S1 and S2,
see the Supporting Information). Interestingly, none of the compounds bearing both 3,5ꢀ
dinitrobenzyl and 2,4ꢀdinitrobenzyl moieties exhibited antibacterial or antifungal activities (MIC
values from >125 to >500 ꢁM, depending on solubility), indicating that the molecular target of
these compounds is exclusively mycobacterial.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
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59
60
In vitro effects of the investigated compounds on cell proliferation/viability and
genotoxicity/mutagenicity. First, we tested the effects of compounds 10d, 10l, 10o, 11d, 11f,
11h, 11i, 11k, 11l, 11o, 14d, 14f, 14g, 14k, 14l, and 14o on cellular viability using HuH7
(human hepatocellular carcinoma), HeLa (human cervical epithelioid carcinoma) and MDCKIIꢀ
MDR1 (MadinꢀDarby canine kidney cells permanently expressing human efflux transporter
MDR1) cell lines (Table 4). The data are presented as relative viability at a concentration of 20
µM compared to control vehicleꢀtreated samples (100% viability). The results suggest that the
3,5ꢀdinitrobenzylsulfanyl derivatives 11d, 11f, 11h, 11i, 11k, 11l, 11o, 14d, 14f, 14g, 14k, 14l
and 14o, each of which possessed excellent antiꢀTB activity, produced limited negative effects
on cellular viability in these three mammalian cell lines after 24 h of treatment at a concentration
of 20 µM. Interestingly, compounds 11o and 14o, each bearing a third nitro group, produced no
toxic effects even after 48 h of treatment. The remaining compounds, however, affected viability
in at least one cell line each after 48 h of treatment. The 2,4ꢀdinitrobenzylsulfanyl derivatives
10d, 10l and 10o produced insignificant negative effects on cellular viability following both 24 h
and 48 h of treatment.
Table 4. Cell viability in three different mammalian cell lines determined by a
proliferation/viability cell assay (MTS assay) after treatments with test compounds for 24 h and
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8 h. Vehicle (DMSO) and background (Triton Xꢀ100 0.9%; v/v, toxic control) controls of cell
a
viability were set to be 100% and 0%, respectively.
HuH7
4 h treatment
HeLa
MDCKII-MDR1
24 h treatment
0
1
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0
2
48 h treatment
24 h treatment
48 h treatment
48 h treatment
IC50 viability at IC50 viability at IC50 viability
IC50 viability at IC50 viability at IC50 viability at
(µM) 20 µM
(µM) 20 µM
>20 92.8 ±12.0 >20 97.7 ±6.2 >20 112.9 ±15.0 >20 69.6 ±6.8
>20 77.0 ±7.3
10o >20 107.3 ±10.0 >20 99.6 ±11.3 >20 90.1 ±6.3 >20 117.0 ±24.2 >20 85.2 ±8.7
(µM) at 20 µM (µM) 20 µM
(µM) 20 µM
(µM) 20 µM
1
0d >20 99.5 ±5.6
>20 73.3 ±6.6
10l >20 85.1 ±8.6
>20 89.9 ±5.8 >20 105.5 ±7.8 >20 87.9 ±15.1 >20 78.5 ±16.3
>20 119.8 ±19.0
>20 73.8 ±10.9 >20 88.3 ±7.4 n.d. 33.2 ±10.1 >20 72.6 ±14.3 n.d. 42.4 ±9.9
1
1
1
1
1d >20 99.3 ±5.5
1f >20 97.9 ±5.2
1h >20 95.7 ±5.7
n.d. 45.9 ±11.9 >20 81.9 ±5.9 n.d. 34.6 ±8.0
n.d. 37.7 ±8.5 >20 76.4 ±4.9 n.d. 39.9 ±9.4
>20 93.3 ±19.5 >20 57.9 ±30.1
>20 105.9 ±8.6 n.d. 27.9 ±20.5
1i >20 100.9 ±7.0 >20 53.1 ±8.6
20 n.d.
1l >20 91.6 ±6.8 n.d. 34.1 ±5.4
1o >20 101.0 ±6.4 >20 111.3 ±18.7 >20 93.6 ±8.9 >20 108.7 ±10.8 >20 82.0 ±10.5 >20 97.4 ±11.9
>20 87.2 ±6.9 >20 118.1 ±20.9 >20 98.0 ±5.5
n.d. 48.5 ±15.7
7
4.2
>
>20
16.4 ±3.9
>20
1
1
1
1k
89.6 ±2.3
47.0 ±8.4
±14.0
4.1
104.7 ±8.0 n.d. 10.5 ±4.5
>20 83.9 ±8.4 n.d. 37.3 ±5.7
>20 80.6 ±17.1 10.6 14.5 ±5.9
14d >20 84.3 ±8.0
14f >20 90.7 ±7.5
n.d. 36.3 ±10.7 >20 78.5 ±9.6 2.4
n.d. 32.8 ±9.2 >20 78.6 ±4.3 2.6
15.9 ±4.7
16.8 ±3.3
>20 97.8 ±8.5
3.1
7.3 ±11.4
>20 107.6 ±6.3 n.d. 48.6 ±42.7
1
1
1
1
4g >20 94.0 ±7.0
4k >20 86.3 ±7.1
4l >20 95.4 ±4.9
4o >20 99.5 ±8.4
n.d. 43.4 ±18.5 >20 92.8 ±7.4 >20 107.5 ±26.1 >20 101.6 ±43.8 >20 91.4 ±7.9
n.d. 33.0 ±6.3
>20 84.2 ±9.3
>20 97.9 ±5.0
>20 71.2 ±3.2 4.0
>20 88.0 ±5.9 4.4
17.5 ±3.7
27.1 ±2.9
>20 100.3 ±6.5 6.4
18.5 ±15.6
>20 101.4 ±6.8 >20 60.0 ±17.0
>20 89.2 ±5.2 >20 120.0 ±29.3 >20 75.0 ±15.2 >20 103.8 ±11.3
1
±5.4
01.7
>
20
>20
>20
>20 100.4 ±8.1 >20
>20
RIF
98.3 ±25.1
52.7 ±18.4
103.1 ±9.1
105.6 ±7.5
a
n.d. not determined
In addition to these three model proliferating cell lines, we also studied the effects of compounds
1
1h, 11i, 11o, 14g and 14k on cell viability in primary human hepatocytes (Table 5).
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Importantly, none of the studied compounds were cytotoxic to hepatocytes after 48 h of
treatment at a concentration of 20 µM. These results indicate that neither the compounds
themselves nor their potential metabolites were cytotoxic to primary human hepatocytes.
Furthermore, compounds 11i and 14g showed no toxic effect on HepG2 cells at the highest
concentration tested (50 µM). Thus, the selectivity indices of these two compounds are higher
than 500.
10
11
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Table 5. Cell viabilities of isolated primary human hepatocytes and HepG2 cells after a 48 h
treatment with test compounds at a concentration of 20 µM for primary human hepatocytes and
50 µM for HepG2 cells.
Primary human hepatocytes HepG2
48 h treatment
IC50
48 h treatment
IC50
Viability at 20 µM
Viability at 50 µM
(µM)
(µM)
1
1h >20 81.1 ±5.8
1
1i >20 95.1 ±8.6
1
1o >20 106.9 ±1.5
1
4g >20 101.8 ±16.4
1
4k >20 120.6 ±5.9
>50 95.5 ±1.3
>50 98.5 ±0.98
RIF >20 123.2 ±3.0
In our previous study, we showed that the 3,5ꢀdinitrobenzylsulfanyl moiety, which is crucial
1
8
for the high antiꢀTB activities of our compounds, is not a mutagenic carrier. To further explore
how this moiety contributes to the mutagenic effects caused by selected compounds, a 96ꢀwell
microꢀplate "fluctuation" version of the classical reverse mutation Salmonella typhimurium
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Ames Test was performed. We found that compounds 11f, 11i, 11k, 14d, 14g and 14k produced
no mutagenic effects on either strain TA98 or TA100 when used at a concentration of 30 µM.
Reverse mutations were generated in at least one strain by compounds 11o and 14o, both of
which harbor additional nitro groups in their molecular structures, as well as by the 2,4ꢀ
dinitrosubstituted derivative 10d (Table 6). These results confirm that the 3,5ꢀdinitrobenzyl
moiety is not generally connected with either frame shift or baseꢀexchange mutagenicity and that
the investigated compounds bearing two nitro groups possess low mutagenicity.
0
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0
TM
On the basis of in silico ADMETox predictions (using ADMET Predictor , Simulation Plus
Inc., Lancaster, CA), compound 14g was selected for further genotoxicity studies. An in vitro
mammalian cell micronucleus test was performed according to OECD guideline No. 487, and an
in vitro mammalian chromosome aberration test was performed according to OECD guideline
No. 473. Compound 14g produced no genotoxic effects on human peripheral blood lymphocytes
in either of the experiments, both with and without metabolic activation, at concentrations of 0.6ꢀ
12 mM (250ꢀ5000 µg/mL).
Table 6. Evaluation of the mutagenicity of compounds 10d, 11d, 11f, 11i, 11k, 11o, 14d, 14g,
14k and 14o via an Ames fluctuation assay performed on Salmonella typhimurium TA100 and
a
TA98 strains at a concentration of 30 µM.
Ames fluctuation assay
TA100
+
TA98
n.d.
+
Sodium azide
2
-nitrofluorene n.d.
0d
n.d.
1
+
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1d
ꢀ
+
1f
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
1i
ꢀ
1k
1o
ꢀ
10
11
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+
ꢀ
14d
14g
14k
ꢀ
ꢀ
14o
+
a
˗
negative mutagenicity; + positive mutagenicity; n.d. not determined. For all positive
compounds, the statistical significance related to mutagenicity was set at 0.001.
Docking studies. The selectivity of lead compounds of series 11 and 14 toward mycobacteria
suggests the presence of a unique molecular target in this genus. Because they both possess
9
structural similarities to 3,5ꢀdinitrobenzamides (Figure 1), a class of efficient DprE1 inhibitors,
3
8, 39
a docking experiment using DprE1
was performed with AutoDock Vina. The region selected
for docking was represented by a box measuring 20 Å × 20 Å × 20 Å, which was centered at the
N5 atom of the flavin group of the cofactor in DprE1. First, a 3ꢀnitroꢀNꢀ[(1R)ꢀ1ꢀphenylethyl]ꢀ5ꢀ
(
trifluoromethyl) benzamide inhibitor that had been coꢀcrystallized with DprE1 was docked into
the enzyme to evaluate whether AutoDock Vina had the ability to reproduce its binding mode.
The best scoring docked binding mode occupied the same space as the ligand in the crystal
structure with an RMSD of 0.5 Å, confirming that AutoDock Vina is suitable for correctly
identifying binding modes in this system. The best binding modes predicted for compounds 11i
and 14g were similar (Figure S1), with favorable binding energies of ꢀ9.2 and ꢀ8.6 kcal/mol,
respectively.
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Evaluation of the effects of the investigated compounds on DprE1. The inhibition of DprE1
14
by compounds 11i and 14g was assessed by examining the incorporation of P[ C]RPP into
1
4
[
C]decaprenylphosphoryl arabinose and its precursors accomplished by membrane and cell
0
1
2
3
4
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0
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40
envelope enzyme fractions from M. smegmatis mc 155, as previously described. TLC analyses
followed by autoradiography revealed that neither 11i nor 14g affected the biosynthesis of
arabinan lipid precursors when used at concentrations of up to 0.6 mM (250 ꢁg/mL) in the
1
4
reaction mixture (Figure 5A). In addition, we performed [ C]ꢀacetate metabolic labeling of a
model nonꢀpathogenic strain M.tb. H37Ra grown in the presence of these compounds, along with
12
the cultures treated with benzothiazinone BTZꢀ043. Based on our experience, the suitable
concentration of the compounds for examining their specific effects on metabolism is the one
that results in the continued growth of the cultures after the addition of the compound and only
14
modest (up to 40%) growth inhibition of the treated cultures compared to the control. For [ C]ꢀ
acetate metabolic labeling, we chose concentrations of 0.2 ꢁg/mL and 0.5 ꢁg/mL for 11i and 14g
and of 0.2 ꢁg/mL for BTZꢀ043. Growth inhibition after 24 h of the drug treatment followed by
2
4 h of radiolabeling was approximately 12% for 14g (0.5 ꢁg/mL), 36% for 11i (0.5 ꢁg/mL) and
38% for BTZꢀ043 (0.2 ꢁg/mL) (Table S4). Analysis of the lipids extracted from the radiolabeled
cells showed the typical profile of trehalose dimycolate (TDM) accumulation for BTZꢀ043,
similar to the effects observed for ethambutol, which inhibits the synthesis of mycobacterial
4
1
arabinans. This phenotype was not observed in lipids extracted from cells treated with the
investigated compounds (Figure 5B). These data suggest that despite favorable docking of 11i
and 14g to the DprE1 structure, this enzyme is not the target of these compounds.
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Figure 5. Investigation of the effects of 14g and 11i on DprE1. A Monitoring of cellꢀfree
synthesis of decaprenylphosphoryl arabinose in the presence of 14g and 11i. Reactions with
1
4
P[ C]RPP substrate were performed as described in the Experimental Procedure. A total of 25%
of the extracted radiolabeled products were separated on a silica gel TLC plate, which was
developed in CHCl /CH OH/concentrated NH OH/1
M
ammonium acetate/H O
2
3
3
4
(180:140:9:9:23, v/v). The plate was exposed to autoradiography film at ꢀ80 °C for 3 days.
DPPR: decaprenylphosphoryl riboseꢀ5ꢀphosphate, DPR: decaprenylphosphoryl ribose, DPA:
14
decaprenylphosphoryl arabinose. B TLC analysis of the lipids from [ C]ꢀacetateꢀlabeled M.tb.
H37Ra grown in the presence of 14g, 11i or DprE1 inhibitor BTZꢀ043. Bacteria were preꢀtreated
1
4
with the drugs for 24 h, followed by 24 h of radiolabeling with [ C]ꢀacetate. Lipids were
extracted and dissolved in CHCl /CH OH/concentrated NH OH/H O (65:25:0.5:3.6, v/v) in the
3
3
4
2
ratio of 150 ꢁL/OD600 0.5. Five microliters of each sample was loaded on a silica gel TLC plate
and separated in CHCl /CH OH/H O (20:4:0.5, v/v). The plate was exposed to autoradiography
3
3
2
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film at ꢀ80 °C for 3 days. TDM: Trehalose dimycolate, TMM: Trehalose monomycolate, PE:
Phosphatidylethanolamine, PIMs: Phosphatidylinositol mannosides
1
4
14
Radiolabeling of 11i- and 14g-treated M.tb. H37Ra with [ C]-uracil and [ C]-
methionine. To examine the potential effects of the studied drugs on the synthesis of nucleic
acids and proteins, M.tb. H37Ra cultures were preꢀtreated for 1 h with 1 ꢁg/mL 11i and 14g
0
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14
followed by 24 h of incubation with [ C]ꢀuracil and [ C]ꢀmethionine as tracers. RIF (0.05
ꢁ
ꢁ
g/mL) was used as a control drug for the inhibition of the biosynthesis of RNA, and KAN (5
g/mL) served as a control for the inhibition of proteosynthesis. Under these experimental
conditions, the growth inhibition by 11i was comparable to that of RIF and KAN; the effect of
4g was less pronounced (Figure 6A, Table S5). Although the presence of KAN in the culture
1
1
4
strongly inhibited the incorporation of [ C]ꢀmethionine into the cells, there was no defect in the
14
[
C]ꢀuracil radiolabeling of the KANꢀtreated mycobacteria, confirming the known mode of
1
4
14
action of this drug. In contrast, RIF reduced the incorporation of both [ C]ꢀuracil and [ C]ꢀ
14
methionine by 68% and 61%, respectively (Figure 6B, Table S5). [ C]ꢀmethionine radiolabeling
was very likely disturbed by RIF due to secondary effects of RNA synthesis inhibition on
proteosynthesis. Similar trends were observed for compound 11i, which caused a 50% inhibition
14
14
of [ C]ꢀuracil incorporation into the cells and a 37% inhibition of [ C]ꢀmethionine labeling. The
incorporation of both tracers was also inhibited by compound 14g, although to a lesser extent –
14
14
13% for [ C]ꢀuracil and 20% for [ C]ꢀmethionine, probably due to the lower growth inhibition
caused by 14g compared to 11i (Figure 6, Table S5). The analogous effects observed for RIF and
the investigated compounds imply that 14g and 11i could affect the synthesis of nucleic acids.
This finding is consistent with their high activity against the nonreplicating M.tb. SS18bꢀLux
strain.
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1
4
14
Figure 6. Metabolic labeling of M.tb. H37Ra with [ C]ꢀuracil and [ C]ꢀmethionine. A Relative
growth of the cultures treated with the investigated compounds in the following concentrations:
14g – 1 ꢁg/mL (2.5 ꢁM), 11i – 1 ꢁg/mL (2.7 ꢁM), RIF – 0.05 ꢁg/mL (0.06 ꢁM), and KAN – 5
14
14
ꢁ
g/mL (10 ꢁM). B Relative incorporation of [ C]ꢀuracil and [ C]ꢀmethionine into the cells from
drugꢀtreated cultures described in panel A normalized to the same OD600 and expressed as a
percentage of incorporation in untreated controls. The data in both panels represent the average
values of triplicate samples with standard deviations. * indicates statistical significance against
control at p < 0.05 (twoꢀway ANOVA with Dunnett's multiple comparisons test)
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Chemical stability, human microsomal stability and solubility of compounds 11i and 14g
in aqueous media. Solutions of compounds 11i and 14g at concentrations of 100 µM in DMSO
were stored at rt for 1 month. HPLC analysis showed no decomposition of either compound.
Furthermore, compound 14g was heated in DMSO (4 mg/mL) at 60 °C for 3 days under openꢀ
0
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1
vessel conditions. After evaporation, the residue was subjected to H NMR, and the spectrum
was the same as that of pure compound 14g.
Moreover, the stability of compounds 11i and 14g in DMSO was indirectly confirmed via the
in vitro evaluation of their MICs against M.tb. Both compounds were dissolved in DMSO, and
one aliquot of each solution was used to determine its MIC against M.tb. A second aliquot of
each solution was stored at rt for 1 month and then used in the same way. Both experiments
produced the same MIC values.
Because the in silico prediction indicates that the sulfur atom adjacent to the heterocycle in 11i
and 14g is highly susceptible to metabolization by the CYP2D6 and CYP3A4 enzymes of
cytochrome P450 (software ACD/IꢀLab, ACD Lab.), we performed a microsomal stability assay.
Microsomes can be used to determine the in vitro intrinsic clearance (CL ) of a compound,
int
which can be scaled up to the in vivo situation and used in the prediction of human clearance.
Based on CL , compounds can be categorized into lowꢀ, mediumꢀ or highꢀclearance
int
4
2
compounds.
The CLint for 14g was determined to be 29 µL/min/mg protein, and the CLint for 11i was
determined to be 86 µL/min/mg protein. Thus, these compounds can be classified as compounds
with medium and high clearance with respect to phase I metabolic deactivation.
All compounds of series 11 and 14 described in this work possess high lipophilicity. To
determine their aqueous solubility, 20 mM stock solutions of compounds 11i and 14g in DMSO
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Journal of Medicinal Chemistry
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were prepared and added to water to obtain final DMSO concentrations (v/v) of 1% (200 ꢁM of
the investigated compound), 0.1% (20 ꢁM of compound) and 0.01% (2 ꢁM of compound). These
aqueous samples of compounds 11i and 14g were slightly opalescent but highly stable (except
for the highest concentration of 200 ꢁM), as shown by HPLC analysis. To determine the actual
solubility, the opalescent solutions were centrifuged at 10000 rpm for 10 min, and the clear
supernatants were analyzed. The solubilities of 11i and 14g in water with 0.01% ꢀ 1% DMSO
ranged from 0.50 ± 0.05 ꢁM to 0.76 ± 0.09 ꢁM and from 0.54 ± 0.03 ꢁM to 3.44 ± 0.74 ꢁM,
respectively. To determine the solubility in water without DMSO, both compounds were shaken
with distilled water for 24 h, the suspensions were centrifuged at 10000 rpm for 10 min, and the
clear supernatants were analyzed. The solubilities of 11i and 14g in water were 0.067 ± 0.008
ꢁM and 0.030 ± 0.009 ꢁM, respectively.
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11
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Conclusions
In this work, we found that 5ꢀsubstituted 2ꢀ[(3,5ꢀdinitrobenzyl)sulfanyl]ꢀ1,3,4ꢀoxadiazoles (11)
and 1,3,4ꢀthiadiazoles (14) possess excellent activity against drugꢀsusceptible and multidrugꢀ
resistant M.tb., with no cross resistance with firstꢀ and secondꢀline antiꢀTB drugs. The MIC
values of the most active compounds against M.tb. were as low as 0.03 µM (approx. 0.011
µg/mL). Furthermore, these compounds exhibited excellent activity against the nonreplicating
M.tb. strain SS18bꢀLux. Our SAR study determined that 3,5ꢀdinitro substitution has a crucial
role in antimycobacterial activity: any changes to the positions or numbers of nitro groups led to
a significant decrease in antimycobacterial activity. The 2,4ꢀdinitrobenzylsulfanyl derivatives 10
and 13 possessed 10ꢀ to 100ꢀfold lower antimycobacterial effects, and the 4ꢀnitrobenzylsulfanyl
analogues 9o and 12o practically lost activity. Because the relatively high variability of
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