Identification and development of 2,5-disubstituted oxadiazole as potential candidate for treatment of XDR and MDR tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2011.10.054

Tuberculosis, the infection on the verge of eradication once, is now a great threat to mankind. Emergence of MDR and XDR-TB synergised with HIV and other immune-compressive diseases have increased the life threatening capacities of the disease. A small mo

Full text of DOI:10.1016/j.ejmech.2011.10.054

European Journal of Medicinal Chemistry 47 (2012) 278e282
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Identification and development of 2,5-disubstituted oxadiazole as potential
candidate for treatment of XDR and MDR tuberculosis
,
b
Ravi L. Bakal a , Surendra G. Gattani
*
a Department of Medicinal Chemistry, Wadhwani College of Pharmacy, Dhamangaon Road, Yavatmal, Maharashtra 445001, India
b H.R.Patel Institute of Pharmaceutical Education and Research, Near Karvand Naka, Shirpur Dist., Dhule, Maharashtra 425405, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Tuberculosis, the infection on the verge of eradication once, is now a great threat to mankind. Emergence
of MDR and XDR-TB synergised with HIV and other immune-compressive diseases have increased the life
threatening capacities of the disease. A small molecule has been identified here, which showed potent
anti-tubercular activity. The identified hit compound has also been proved active against nearly 25
clinical isolates comparable with isoniazid.
Received 28 September 2011
Received in revised form
24 October 2011
Accepted 28 October 2011
Available online 6 November 2011
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
MDR-TB
Mycobacterium tuberculosis
Oxadiazole
Clinical isolates
1. Introduction
tuberculosis requires 18e36 months and is associated with an
unacceptable rate of treatment failure and relapse. Consequently,
Mycobacterium tuberculosis (MTB, M. tuberculosis), the etiolog-
ical agent of tuberculosis (TB), is the leading cause of mortality due
to bacterial pathogens, claiming about 2 million lives annually. The
field of anti-tuberculosis drug discovery culminated in the 1960s
with the incorporation of rifampicin and pyrazinamide in the
tuberculosis drug regimen. The use of these two antimicrobials, in
combination with isoniazid, ethambutol and/or streptomycin,
represents a landmark in the treatment of human tuberculosis and
resulted in the implementation of short-course chemotherapy
(SCC), reducing the time of treatment from 18 to 6 months [1e3].
Short-course chemotherapy contributed towards controlling
tuberculosis burden for the next 20 years. Nevertheless, tubercu-
losis cases started to rise again in the 1990s under the pressure of
the HIV pandemic and the emergence of multidrug resistant (MDR)
and extremely drug resistant (XDR) tuberculosis strains. MDR
strains are resistant to at least isoniazid (INH) and rifampicin (RIF),
whereas XDR strains are MDR isolates that are additionally resis-
tant to fluoroquinolones and to one of the three injectable drugs
capreomycin, amikacin and kanamycin. The emergence and
dissemination of MDR and XDR isolates, estimated to account for
more than 400,000 new cases per year, impart new challenges in
tuberculosis control [4]. Indeed, current treatment of drug resistant
developing new compounds active against MDR and XDR tuber-
culosis constitutes a main objective in anti-tuberculosis drug
discovery. In addition, new antimycobacterial agents should ideally
contribute to shorten tuberculosis treatment to 2 months or less
[5,6]. Few promising drug candidates fulfilling these criteria have
been discovered in recent years [7e9]. Mainly, TMC207, which has
been shown to be highly active in proof-of-concept trials, and
shows the potential to shorten the duration of therapy [10,11].
Recently, there are several reports citing oxadiazole as potential
antibacterial and anti-tubercular agent [12e15]. Inspired by the
citations, we decided to design around the heterocycles and eval-
uate the antimycobacterial activity of the same. Nonetheless, given
the number of tuberculosis cases and the rate of emergence of drug
resistance, more compounds are clearly needed to combat and have
a significant impact on the control and spread of tuberculosis. Thus
in continuation with the search of new drug candidate we herein
discuss this report about development of a lead to hit molecule.
2. Results and discussion
2.1. Chemistry
In our attempt to synthesise cost effective drug, oxadiazole was
identified as better target, easy and cheaper to synthesis. The
* Corresponding author. Tel.: þ91 9421831717; fax: þ91 7232 238747.
E-mail address: nddd2011@gmail.com (R.L. Bakal).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.054

R.L. Bakal, S.G. Gattani / European Journal of Medicinal Chemistry 47 (2012) 278e282
279
Fig. 1. Route of synthesis for the compounds 6ae6p.
synthetic route was followed as reported in Fig. 1 [16]. In brief, the
ester to hydrazide chemotransformation was carried out by using
hydrazine hydrate in ethanol at reflux condition. The hydrazide was
then transformed to thiosemicarbazate by usual CS2, KOH and
ethanol method. The resultant solid was then cyclised using tosyl
chloride and pyridine using THF as solvent. The oxadiazole “parent”
then reacted with various chloro-substituted compounds for
further analog synthesis. These reaction led us to the thio
substituted array of compounds 6aep.
electronegative chlorine at the end intact (6b) have shown
reduced potency but same time its cytotoxicity also declined.
Identical alkyl length to 6a, the 6c without chlorine have shown no
improvement in potency and even comparatively cytotoxic. The
chloroacetyl chloride substitution product, 6d have shown
diminished activity.
Looking closely at 6a, 6i and 6m reveals a small difference of
ether linkage. We wondered if the small ether linkage has some-
thing to offer on the activity part and thus synthesised 6ee6h;
compounds without ether linkage. To our surprise, 6e emerged as
a hit with almost equipotent to isoniazid and without any notable
cytotoxicity. Other compounds 6fe6h have also shown potency but
not in comparison with 6e.
2.2. Anti-tubercular activity
A cellular screen was developed to identify mycobacterial
growth inhibitors. The screen was carried out against Mycobacte-
rium bovis (M. bovis) BCG using intracellular ATP content as
a surrogate marker of bacillary growth. Compound hits with
confirmed activity against M. tuberculosis were chemically clus-
tered to identify any emerging SAR. Our attention was drawn to
a cluster of oxadiazole compounds comprising three compounds,
6a, 6i and 6m (synthesised at our laboratory for another program,
When synthesised analogs from the 6i and 6m series, on
contrary to our expectations, none of the compound was as
promising as 6e. Thus we decided to further evaluate 6e to compare
with standard drug isoniazid (INH).
First we compared 6e with INH for their susceptibilities on 18
clinical isolates (Table 2) of MTB (M. tuberculosis), out of which 16
were pan-susceptible and 2 were mono-rifampin resistant isolates.
We are glad to report that our compound 6e have been shown
almost equipotent to that of INH. Having seen its potential, we
decided to evaluate 6e against 9 multidrug-resistant (MDR) and 2
poly-drug resistant MTB strains (Table 3). We are happy to report
that, compound have shown promising activity against almost all
the resistant strains. The compound 6e is now under further eval-
uation stage, which shall be shortly communicated.
unpublished) with an MIC50 ranging from 0.11 to >0.20
mM
(Table 1). The compounds were bactericidal and the cytotoxic
profile was within an acceptable range. Therefore, a lead optimi-
zation programme was initiated with the goal of achieving potent
anti-tubercular activity.
The program of chemotransformation initiated with compound
6a. An increase in the length of alkyl chain keeping the
Table 1
Preliminary structureeactivity relationship of compounds 6ae6p.
ID
R
R1
MIC50
(m
M)
MBC90
(
mM)
CC50-BHK21 (
mM)
CC50-HepG2 (mM)
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
eCH2eOe(2,4-Cl2eC6H3)
eCH2eOe(2,4-Cl2eC6H3)
eCH2eOe(2,4-Cl2eC6H3)
eCH2eOe(2,4-Cl2eC6H3)
eCH2e(2,4-Cl2eC6H3)
eCH2e(2,4-Cl2eC6H3)
eCH2e(2,4-Cl2eC6H3)
eCH2e(2,4-Cl2eC6H3)
eCH2eOe(3-NO2eC6H4)
eCH2eOe(3-NO2eC6H4)
eCH2eOe(3-NO2eC6H4)
eCH2eOe(3-NO2eC6H4)
eCH2e(3-NO2eC6H4)
eCH2e(3-NO2eC6H4)
eCH2e(3-NO2eC6H4)
eCH2e(3-NO2eC6H4)
e(CH2)2Cl
e(CH2)3Cl
e(CH2)2CH3
eCOCH2Cl
e(CH2)2Cl
e(CH2)3Cl
e(CH2)2CH3
eCOCH2Cl
e(CH2)2Cl
e(CH2)3Cl
e(CH2)2CH3
eCOCH2Cl
e(CH2)2Cl
e(CH2)3Cl
e(CH2)2CH3
eCOCH2Cl
0.11 ꢀ 0.04
0.65 ꢀ 0.11
0.63 ꢀ 0.24
1.25e2.5
2.5e5
2.5e5
n.d.
1.25e2.5
5e10
5e10
n.d.
1.25e2.5
n.d.
n.d.
n.d.
1.25e2.5
n.d.
3.75 ꢀ 0.35
>50
4.50 ꢀ 0.28
>50
13.75 ꢀ 0.87
19.3 ꢀ 8.0
>20
n.d.
n.d.
0.04 ꢀ 0.01
1.42 ꢀ 0.46
1.19 ꢀ 0.39
9.67 ꢀ 1.45
0.19 ꢀ 0.07
11.72 ꢀ 3.57
8.52 ꢀ 1.33
>20
0.20 ꢀ 0.09
7.48 ꢀ 1.28
6.81 ꢀ 1.08
>20
>50
>50
>50
n.d.
>50
>50
>50
n.d.
17.65 ꢀ 0.68
11.3 ꢀ 5.67
>50
>50
>50
n.d.
>50
n.d.
19.70 ꢀ 3.77
14.7 ꢀ 5.89
>50
>50
n.d.
n.d.
n.d.
>50
n.d.
n.d.
>50
n.d.
n.d.
6p
Isoniazid
0.03 ꢀ 0.01
The inhibitory activity (MIC50) was determined against M. tuberculosis H37Rv. The cidal activity (MBC90) and cytotoxicity (CC50) were determined after 5 days of exposure to
a single dose of compound. Assays were carried out at least two times. MIC50: Minimum Inhibitory Concentration 50%; MBC90: Minimum Bactericidal Concentration 90%, CC50
Cyototoxic concentration 50%. n.d.: not determined.
:

280
R.L. Bakal, S.G. Gattani / European Journal of Medicinal Chemistry 47 (2012) 278e282
Table 2
Signal multiplicities are represented by s (singlet), d (doublet), t
(triplet), ds (double singlet), dd (double doublet), m (multiplet),
and bs (broad singlet). Mass spectra were recorded on a Finnigan
LCQ mass spectrometer. Elemental analysis was performed on
a Heracus CHN-Rapid Analyser. Analysis indicated by the symbols
of the elements of functions was within ꢀ0.4% of the theoretical
values. The purity of the compounds was checked on silica gel
coated Al plates (Merck).
6e drug susceptibilities for MTB (pan-susceptible and mono-rifampin resistant)
clinical isolates.
SN
Strain
MIC (m )
g mlꢂ1
Isoniazid
6e
1
2
3
4
5
6
7
8
H37Rv
TN675*
TN913
0.03
0.03
0.03
0.03
0.06
0.03
0.03
0.03
0.03
0.06
0.06
0.03
0.06
0.03
0.19
0.20
0.08
0.03
0.013
0.015
0.06
0.03
0.06
0.06
0.015
0.06
0.06
0.06
0.25
0.015
0.13
0.06
0.25
0.25
0.13
0.06
TN994*
TN1008
TN1037
TN1040
TN1051
TN1082
TN2351
TN2524
TN3183
TN3979
TN4259
AH9584
BE11677
E8133
4.1.1. Synthesis of 2-(substituted-thio)-5-(substituted-methyl)-
1,3,4-oxadiazole (6ae6p)
Compound 4 (1 mmol) was taken in 5% ethanolic NaOH, and
heated to 70 ꢁC. To the reaction mixture, compound 5 (1.4 mmol)
was added slowly for 10 min. The reaction was then refluxed for
3hr, cooled and extracted with EtOAc. The EtOAc layer was dried
with sodium sulfate and the column purified. (Hexane:EtOAc;
90:10).
9
10
11
12
13
14
15
16
17
18
4.1.1.1. 2-(2-Chloroethylthio)-5-(2,4-dichlorobenzyloxy)-1,3,4-oxadia-
zole (6a). Yield 76%; colorless powder; mp 187 ꢁC; 1H NMR
W4
(500 MHz, CDCl3): d 3.21 (t, 2H, eCH2), 3.78 (t, 2H, eCH2), 5.39 (s, 2H,
Compound 6e and Isoniazid drug susceptibilities were determined on 16 pan-
susceptible and 2 mono-rifampin resistant (asterisk) clinical isolates.
eCH2O), 7. 26 (d,1H, ArH), 7.42 (d,1H, ArH), 7.58 (s,1H, ArH); MS m/z
(%) 341 (Mþ, 100); Anal. Calcd. for C11H9Cl3N2O2S (339.63): C, 38.90;
H, 2.67; Cl, 31.32; N, 8.25; O, 9.42; S, 9.44.
3. Conclusion
4.1.1.2. 2-(3-Chloropropylthio)-5-(2,4-dichlorobenzyloxy)-1,3,4-oxadia-
zole (6b). Yield 68%; colorless powder; mp 189 ꢁC; 1H NMR (500 MHz,
Keeping a widespread use of future antimycobacterials, we were
aiming to synthesis a cheaper but better agent for today’s XDR &
MDR tuberculosis. In order to do so, we have zeroed at oxadiazole,
which gave us really tractable small molecules. The evaluation of
the synthesised series revealed a potent compound 6e which was
comparable with Isoniazid against H37Rv. The next step of evalua-
tion surprised us with the effectiveness of 6e against 25 different
isolates. The newer compound has shown promising anti-XDR and
anti-MDR tuberculosis activity. Further attempts to study the tox-
ophore of the compound are on and communicated short as the
outcomes are available.
CDCl3):
d
1.97 (m, 2H, eCH2ꢂ), 3.10 (t, 2H, eCH2), 3.68 (t, 2H, eCH2),
5.40 (s, 2H, eCH2O), 7. 23 (d, 1H, ArH), 7.44 (d, 1H, ArH), 7.56 (s, 1H,
ArH); MS m/z (%) 355 (Mþ, 100); Anal. Calcd. for C12H11Cl3N2O2S
(353.65): C, 40.75; H, 3.14; Cl, 30.07; N, 7.92; O, 9.05; S, 9.07.
4.1.1.3. 2-(2,4-Dichlorobenzyloxy)-5-(propylthio)-1,3,4-oxadiazole
(6c). Yield 82%; colorless powder; mp 212 ꢁC; 1H NMR (500 MHz,
CDCl3):
d
0.90 (t, 3H, eCH3), 1.41 (m, 2H, eCH2ꢂ), 3.12 (t, 2H,
eCH2), 5.38 (s, 2H, eCH2O), 7. 27 (d, 1H, ArH), 7.40 (d, 1H, ArH), 7.54
(s, 1H, ArH); MS m/z (%) 320 (Mþ, 100); Anal. Calcd. for
C12H12Cl2N2O2S (319.21): C, 45.15; H, 3.79; Cl, 22.21; N, 8.78; O,
10.02; S, 10.05.
4. Experimental
4.1.1.4. 5-(2,4-Dichlorobenzyloxy)-1,3,4-oxadiazol-2-yl 2-chloroetha-
nethioate (6d). Yield 68%; colorless powder; mp 208 ꢁC; 1H NMR
4.1. Chemistry
(500 MHz, CDCl3): d 4.49 (s, 2H, eCH2), 5.39 (s, 2H, eCH2O), 7. 24 (d,
1H NMR spectra were recorded on a Bruker Avance 500 MHz
instrument using TMS as internal standard; the chemical shifts (d)
are reported in ppm and coupling constants (J) are given in Hertz.
1H, ArH), 7.41 (d, 1H, ArH), 7.59 (s, 1H, ArH); MS m/z (%) 355 (Mþ,
100); Anal. Calcd. for C11H7Cl3N2O3S (353.61): C, 37.36; H, 2.00; Cl,
30.08; N, 7.92; O, 13.57; S, 9.07.
4.1.1.5. 2-(2-Chloroethylthio)-5-(2,4-dichlorobenzyl)-1,3,4-oxadiazole
Table 3
(6e). Yield 82%; colorless powder; mp 112 ꢁC; 1H NMR (500 MHz,
CDCl3): d 3.21 (t, 2H, eCH2), 3.78 (t, 2H, eCH2), 3.84 (s, 2H, eCH2), 7.
6e drug susceptibilities for MTB (MDR and poly-resistant) clinical isolates.
6e MIC (m )
g mlꢂ1
10 (d, 1H, ArH), 7.27 (d, 1H, ArH), 7.66 (s, 1H, ArH); MS m/z (%) 325
(Mþ, 100); Anal. Calcd. for C11H9Cl3N2OS (323.63): C, 40.82; H, 2.80;
Cl, 32.86; N, 8.66; O, 4.94; S, 9.91.
SN
Strain
Drug resistance
1
2
3
4
5
6
7
8
TN565
TN576
TN702
TN715
TN768
TN772
TN1195
TN1314
TN1618
TN1811
TN2557
R,S,EM,ET,K.Cl
I,R,S,EM,ET,K
I,S,EM,P
I,R,EM,P
I,R,S,EM,ET
I,R,EM
I,S,EM
I,R
I,R,S,EM,ET,CI
I,R,S,EM
I,R,S,EM,CA
0.06
0.06
0.25
0.06
0.06
0.03
0.25
0.03
0.06
0.03
0.13
4.1.1.6. 2-(3-Chloropropylthio)-5-(2,4-dichlorobenzyl)-1,3,4-oxadiazole
(6f). Yield 69%; beige powder; mp 198 ꢁC; 1H NMR (500 MHz, CDCl3):
d
1.97 (m, 2H, eCH2ꢂ), 3.10 (t, 2H, eCH2), 3.68 (t, 2H, eCH2), 3.83 (s,
2H, eCH2), 7.13 (d,1H, ArH), 7.24 (d,1H, ArH), 7.70 (s,1H, ArH); MS m/
z (%) 355 (Mþ,100); Anal. Calcd. for C12H11Cl3N2OS (353.65): C, 40.75;
H, 3.14; Cl, 30.07; N, 7.92; O, 9.05; S, 9.07.
9
10
11
The 6e susceptibilities were also tested on 9 multidrug resistant (MDR) and 2 poly-
resistant MTB strains. (b) Twenty of the twenty-five sensitive and resistant clinical
isolates tested were previously determined to be genetically distinct by IS6110
genotyping. I, isoniazid; R, rifampin; S, streptomycin; EM, ethambutol; ET, ethion-
amide; K, kanamycin; P, pyrazinamide; Cl, ciprofloxacin; CA, capreomycin.
4.1.1.7. 2-(2,4-Dichlorobenzyl)-5-(propylthio)-1,3,4-oxadiazole (6g).
Yield 73%; colorless powder; mp 216 ꢁC; 1H NMR (500 MHz, CDCl3):
d
0.90 (t, 3H, eCH3), 1.41 (m, 2H, eCH2ꢂ), 3.12 (t, 2H, eCH2), 3.79 (s,
2H, eCH2), 7. 15 (d, 1H, ArH), 7.29 (d, 1H, ArH), 7.64 (s, 1H, ArH); MS

R.L. Bakal, S.G. Gattani / European Journal of Medicinal Chemistry 47 (2012) 278e282
281
m/z (%) 320 (Mþ, 100); Anal. Calcd. for C12H12Cl2N2OS (319.21): C,
(Mþ, 100); Anal. Calcd. for C11H8ClN3O4S (313.72): C, 42.11; H, 2.57;
45.15; H, 3.79; Cl, 22.21; N, 8.78; O, 10.02; S, 10.05.
Cl, 11.30; N, 13.39; O, 20.40; S, 10.22.
4.1.1.8. 5-(2,4-Dichlorobenzyl)-1,3,4-oxadiazol-2-yl-2-chloroethane-
4.2. Antimycobacterial activity
thioate (6h). Yield 81%; colorless powder; mp 227 ꢁC; 1H NMR
(500 MHz, CDCl3):
d
3.81(s, 2H, eCH2), 4.50 (s, 2H, eCH2), 7. 11 (d,
4.2.1. Strains and growth conditions
1H, ArH), 7.25 (d, 1H, ArH), 7.68 (s, 1H, ArH); MS m/z (%) 339 (Mþ,
100); Anal. Calcd. for C11H7Cl3N2O2S (337.61): C, 39.13; H, 2.09; Cl,
31.50; N, 8.30; O, 9.48; S, 9.50.
M. tuberculosis H37Rv (ATCC, cat. no. 27294), derivative strains
and clinical isolates were maintained in Middlebrook 7H9 broth
medium supplemented with 0.2% glycerol, 0.05% Tween 80 and
10% ADS supplement. Culture media were supplemented with
hygromycin (50
required.
4.1.1.9. 2-(2-Chloroethylthio)-5-(3-nitrobenzyloxy)-1,3,4-oxadiazole
m m
g mlꢂ1) or kanamycin (20 g mlꢂ1) when
(6i). Yield 53%; colorless powder; mp 248 ꢁC; 1H NMR (500 MHz,
CDCl3): d 3.21 (t, 2H, eCH2), 3.78 (t, 2H, eCH2), 5.40 (s, 2H, eCH2O),
7.57e7.64 (m, 2H, ArH), 8.07 (d, 1H, ArH), 8.11 (s, 1H, ArH); MS m/z
(%) 317 (Mþ, 100); Anal. Calcd. for C11H10ClN3O4S (315.73): C, 41.84;
H, 3.19; Cl, 11.23; N, 13.31; O, 20.27; S, 10.16.
4.2.2. High-throughput cell-based screen
M. bovis BCG was cultured to an OD600 of 0.5e0.6 in complete
7H9 broth medium. In preparation for 1536-well dispensing, the
culture was diluted to an OD600 of 0.01 using complete 7H9 media.
4.1.1.10. 2-(3-Chloropropylthio)-5-(3-nitrobenzyloxy)-1,3,4-oxadiazole -
A volume of 4 ml of complete 7H9 media was dispensed into a white,
(6j). Yield 60%; colorless powder; mp 237 ꢁC; 1H NMR (500 MHz,
solid bottom 1536-well plate using a custom Bottle Valve liquid
dispenser (GNF). A volume of 100 nl of test compound in DMSO
(1 mM) was then transferred into each assay plates using a custom
CDCl3):
d
1.97 (m, 2H, eCH2ꢂ), 3.10 (t, 2H, eCH2), 3.68 (t, 2H,
eCH2), 5.40 (s, 2H, eCH2O), 7.58e7.63 (m, 2H, ArH), 8.05 (d, 1H,
ArH), 8.10 (s, 1H, ArH); MS m/z (%) 331 (Mþ, 100); Anal. Calcd. for
C12H12ClN3O4S (329.76): C, 43.71; H, 3.67; Cl, 10.75; N, 12.74; O,
19.41; S, 9.72.
1536 Pintool (GNF). Diluted culture (4
to the assay plates using a Bottle Valve liquid dispenser (final OD600
in 8
l is 0.005). The plates were incubated at 37 ꢁC for 48 h. Growth
ml) was subsequently added
m
was assessed by measuring ATP levels using the BacTiter-Glo
Microbial Cell Viability Assay (Promega). Luminescence was
measured using a ViewLux plate reader.
4.1.1.11. 2-(3-Nitrobenzyloxy)-5-(propylthio)-1,3,4-oxadiazole (6k).
Yield 68%; colorless powder; mp 227 ꢁC; 1H NMR (500 MHz, CDCl3):
d
0.90 (t, 3H, eCH3), 1.41 (m, 2H, eCH2ꢂ), 3.12 (t, 2H, eCH2), 5.38 (s,
2H, eCH2O), 7.57e7.65 (m, 2H, ArH), 8.04 (d, 1H, ArH), 8.11 (s, 1H,
ArH); MS m/z (%) 296 (Mþ, 100); Anal. Calcd. for C12H13N3O4S
(295.31): C, 48.81; H, 4.44; N, 14.23; O, 21.67; S, 10.86.
4.2.3. MIC50 determination
MIC50 were determined as previously described, with slight
modifications [17]. Briefly, compounds dissolved in 90% DMSO
were twofold serial-diluted in duplicates and spotted by mosquito
HTS (TTP LabTech) to 384-well clear plates, resulting in 10 dilu-
4.1.1.12. 5-(3-Nitrobenzyloxy)-1,3,4-oxadiazol-2-yl 2-chloroethane-
thioate (6l). Yield 56%; colorless powder; mp 208 ꢁC; 1H NMR
tions of each compound. A volume of 50 ml of M. tuberculosis
(500 MHz, CDCl3):
d
4.49 (s, 2H, eCH2), 5.39 (s, 2H, eCH2O),
culture (final OD600 of 0.02) was added to each well, and the assay
plates were incubated at 37 ꢁC for 5 days. OD600 values were
recorded using a SpectraMax M2 spectrophotometer, and MIC50
curves were plotted using GraphPad Prism 5 software. Under the
assay setting, MIC50 values, which fall in the linear part of the
7.56e7.64 (m, 2H, ArH), 8.06 (d, 1H, ArH), 8.15 (s, 1H, ArH); MS m/z
(%) 331 (Mþ, 100); Anal. Calcd. for C11H8ClN3O5S (329.72): C, 40.07;
H, 2.45; Cl, 10.75; N, 12.74; O, 24.26; S, 9.73.
4.1.1.13. 2-(2-Chloroethylthio)-5-(3-nitrobenzyl)-1,3,4-oxadiazole
inhibition curve, are more robust and reproducible than MIC90.
(6m). Yield 63%; beige powder; mp 215 ꢁC; 1H NMR (500 MHz,
Therefore, only MIC50 values are reported. Clinical isolates used in
drug susceptibility testing were strain typed by IS6110 analysis as
described [18].
CDCl3):
d 3.20 (t, 2H, eCH2), 3.76 (t, 2H, eCH2), 3.84 (s, 2H, eCH2),
7.60e7.64 (m, 2H, ArH), 8.03 (d, 1H, ArH), 8.14 (s, 1H, ArH); MS m/z
(%) 301 (Mþ, 100); Anal. Calcd. for C11H10ClN3O3S (299.73): C,
44.08; H, 3.36; Cl, 11.83; N, 14.02; O, 16.01; S, 10.70.
4.2.4. Cytotoxicity
Cytotoxicity was tested against cell lines HepG2 (ATCC, cat. no.
HB-8065) and BHK21 (ATCC, cat. no. CCL-10) in 96-well micro-
plates. The cells were seeded at a density of 105 cells per well,
incubated at 37 ꢁC for 24 h and exposed to twofold serial-diluted
compounds for 3 days. Cell viability was monitored using the Cell
Proliferation Kit II (Invitrogen).
4.1.1.14. 2-(3-Chloropropylthio)-5-(3-nitrobenzyl)-1,3,4-oxadiazole
(6n). Yield 84%; beige powder; mp 235 ꢁC; 1H NMR (500 MHz,
CDCl3):
d
1.95 (m, 2H, eCH2ꢂ), 3.14 (t, 2H, eCH2), 3.62 (t, 2H,
eCH2), 3.80 (s, 2H, eCH2), 7.59e7.65 (m, 2H, ArH), 8.01 (d, 1H, ArH),
8.17 (s, 1H, ArH); MS m/z (%) 315 (Mþ, 100); Anal. Calcd. for
C12H12ClN3O3S (313.76): C, 45.94; H, 3.85; Cl, 11.30; N, 13.39; O,
15.30; S, 10.22.
4.2.5. Determination of intracellular ATP levels
The intracellular ATP level was quantified as previously
4.1.1.15. 2-(3-Nitrobenzyl)-5-(propylthio)-1,3,4-oxadiazole
(6o).
described [19]. Briefly, 25 ml of M. tuberculosis culture was mixed
Yield 80%; colorless powder; mp 242 ꢁC; 1H NMR (500 MHz, CDCl3):
with an equal volume of freshly prepared BacTiter-Glo reagent
in white 384 flat-bottom plates and incubated in the dark for
5 min. Luminescence was measured using a Tecan Safire2 plate
reader.
d
0.96 (t, 3H, eCH3), 1.43 (m, 2H, eCH2ꢂ), 3.16 (t, 2H, eCH2), 3.78 (s,
2H, eCH2), 7.56e7.63 (m, 2H, ArH), 8.08 (d, 1H, ArH), 8.16 (s, 1H,
ArH); MS m/z (%) 280 (Mþ, 100); Anal. Calcd. for C12H13N3O3S
(279.31): C, 51.60; H, 4.69; N, 15.04; O, 17.18; S, 11.48.
4.2.6. Drug preparation
4.1.1.16. 5-(3-Nitrobenzyl)-1,3,4-oxadiazol-2-yl 2-chloroethanethio-
Unless specified, all the compounds were obtained from Sigma
and were prepared in sterile de-ionized water. The experimental
compounds were prepared in dimethyl sulphoxide (Sigma) for
in vitro drug susceptibility testing.
ate (6p). Yield 64%; colorless powder; mp 236 ꢁC; 1H NMR
(500 MHz, CDCl3):
d 3.81 (s, 2H, eCH2), 4.50 (s, 2H, eCH2), 7.59e62
(m, 2H, ArH), 8.07 (d, 1H, ArH), 8.12 (s, 1H, ArH); MS m/z (%) 315

282
R.L. Bakal, S.G. Gattani / European Journal of Medicinal Chemistry 47 (2012) 278e282
[10] A.H. Diacon, A. Pym, M. Grobusch, R. Patientia, R. Rustomjee, L. Page-Shipp,
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