Synthesis and antitubercular activity of new N-[5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl]-(nitroheteroaryl)carboxamides

Article abstract of DOI:10.1515/hc-2019-0007

Nitro-substituted heteroaromatic carboxamides 1a-e were synthesized and tested against three Mycobacterium tuberculosis cell lines. The activities can be explained in terms of the distribution of the electronic density across the nitro-substituted heteroaromatic ring attached to the amide group. 1,3,5-Oxadiazole derivatives 1c-e are candidates for the development of novel antitubercular agents. Ongoing studies are focused on exploring the mechanism by which these compounds inhibit M. tuberculosis cell growth.

Full text of DOI:10.1515/hc-2019-0007

Heterocycl. Commun. 2019; 25: 52–59
Research Article
Open Access
Roberto Martínez*, Gladys J. Nieves Zamudio, Gustavo Pretelin-Castillo, Rubén O. Torres-Ochoa,
José L. Medina-Franco, Clara I. Espitia Pinzón, Mayra Silva Miranda, Eugenio Hernández and
Blanca Alanís-Garza
Synthesis and antitubercular activity of new
N-[5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl]-
(nitroheteroaryl)carboxamides
https://doi.org/10.1515/hc-2019-0007
Keywords:
antitubercular
agents;
carboxamides;
Received October 30, 2018; accepted November 19, 2018.
1,3,4-oxadiazoles; synthesis.
Abstract: Nitro-substituted heteroaromatic carboxamides
1a-e were synthesized and tested against three Mycobac-
terium tuberculosis cell lines. The activities can be explai-
ned in terms of the distribution of the electronic density
across the nitro-substituted heteroaromatic ring attached
to the amide group. 1,3,5-Oxadiazole derivatives 1c-e are
candidates for the development of novel antitubercular
agents. Ongoing studies are focused on exploring the
mechanism by which these compounds inhibit M. tuber-
culosis cell growth.
Introduction
Tuberculosis (TB) is a one of the top causes of death world-
wide, and in 2017 the World Health Organization reported
10 million new cases of TB, 1.6 million deaths due to TB
and 0.3 million deaths resulting from co-infections by HIV
[1]. Although the rate of decline in TB was 3.9 % from 2015
to 2017, there were 457,560 new cases of multidrug-resistant
TB (MDR-TB) and 558,000 people with rifampicin-resistant
TB (RR-TB). Therefore, TB poses a challenge to researchers
searching for potent drugs that can control the growth of
the bacillus Mycobacterium tuberculosis while minimi-
zing side effects or the development of drug resistance
[2]. Excellent reviews have been published covering the
literature between 2000 and 2015 about compounds with
activity against M. tuberculosis [3-5]. Notably, Kumar and
co-workers grouped anti-TB compounds into 62 different
molecular frameworks. In their most recent compilation
(2005-2015), they only included compounds exhibiting
minimum inhibitory concentration (MIC) values similar to
or higher than those of standard drugs used in bioassays
[6], with 12 different oxadiazoles meeting this criterion. The
1,3,4-oxadiazole ring system indeed exerts a wide variety of
pharmacological activities such as anti-inflammatory [7],
antiviral [8], antineoplastic [9], FAK inhibitory [10], adul-
ticidal [11] and anti-Alzheimer properties [12]. A review of
the literature up to 2017 includes 440 citations of 1,3,4-oxa-
diazoles with biological activity but only 34 of these cita-
tions describe 1,3,4-oxadiazoles with anti-TB activity [13]. A
5-nitrofuranyl scaffold is present in many derivatives with
diverse biological activities including anti-inflammatory
[14, 15], antibacterial [16], antileishmanial [17] and anti-
tubercular agents [18-21]. It has been proposed that the
* Corresponding author: Roberto Martínez, Instituto de Química,
Universidad Nacional Autónoma de México, Circuito Exterior,
Ciudad Universitaria, 04510, Cd. México, México, e-mail: robmar@
unam.mx
Gladys J. Nieves Zamudio, Gustavo Pretelin-Castillo and Rubén O.
Torres-Ochoa, Instituto de Química, Universidad Nacional Autónoma
de México, Circuito Exterior, Ciudad Universitaria, 04510, Cd.
México, México
José L. Medina-Franco, Departamento de Farmacia, Facultad de
Química, Universidad Nacional Autónoma de México, Avenida
Universidad 3000, 04510 Cd. México, México
Clara I. Espitia Pinzón, Instituto de Investigaciones Biomédicas,
Universidad Nacional Autónoma de México, Ciudad Universitaria,
04510, Cd. México, México
Mayra Silva Miranda, Catedrática CONACYT adscrita al
Insituto de Investigaciones Biomédicas, Universidad Nacional
Autónoma de México, Ciudad Universitaria, 04510, Cd. México,
México
Eugenio Hernández, Facultad de Ciencias Químicas, Universidad
Autónoma de Nuevo León, Pedro de Alba s/n, Ciudad
Universitaria, 66400 San Nicolás de los Garza, Nuevo León,
México
Blanca Alanís-Garza, Departamento de Química Analítica, Facultad
de Medicina, Universidad Autónoma de Nuevo León, Madero s/n
Col. Mitras Centro. Monterrey, N. L. México C. P. 64460
Open Access. © 2019 Roberto Martínez et al., published by De Gruyter.ꢀ
Attribution alone 4.0 License.
ꢀThis work is licensed under the Creative Commons
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presence of the nitro group increases the ability of a mole- index (SI>10), MIC (lower than 10 µΜ) and activity against
cule to form hydrogen bonds with receptor [22]. drug-resistant strains, to guide our screening of new
We have recently reported the synthesis of caulerpin, a compounds having potential use as anti-TB drugs. In the
bis-indole alkaloid from the marine alga Caulerpa sp. that present study, we report a new compound active against
displays excellent biological activity [23]. Nevertheless, the M. tuberculosis, selected by carrying out a structural ana-
conventional synthetic approach to medicinal chemistry lysis of a major public database of compounds reported to
is a time-consuming, complicated and expensive process have TB activity. We then used the bioisosterism concept
that produces candidate compounds with low diversity. [26] to generate a list of other possible compounds (Figure
Although this approach is still valuable, it is unable to 1) which we subsequently synthesized and tested as inhi-
fulfill the increasing demand for new drugs. Therefore, bitors of the growth of M. tuberculosis.
experimental drug discovery is currently being aided with
chemo-informatics approaches that are frequently used
to identify active compounds, select candidates, and opti- Results and discussion
mize leads. Chemo-informatic approaches has been promi-
sing over the past few years in finding pharmacologically We conducted a chemotype analysis of compounds in
active compounds across a broad range of therapeutic PubChem [27] with reported activity against M. tuberculo-
areas [24]. Katsuno and co-workers published a compre- sis. To this end, the chemotype methodology developed by
hensive summary of the criteria to consider when develo- Johnson and Xu [28] to identify promising molecular scaf-
ping new drugs against infectious diseases such as TB [25], folds as starting points for optimization was used. In the
and in our work we consider criteria such as selectivity approach of Johnson and Xu, each chemotype is assigned
O
NO2
O
N
N
NH
1a
O
Cl
H
O
NO2
O
S
NO2
N
N
N
N
N
NH
NH
O
O
Cl
Cl
1b
1c
NO2
O
O
N
N
N
N
NH
NH
O
O
Cl
Cl
1d
1e
Figure 1ꢂDesigned compounds based on isosteric modification of lead compound 1a.
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R. Martínez et al.: New nitroheteroarylcarboxamides
a unique alphanumeric identifier of four characters [29]. of mycobacterial infections, including TB, and the anti-TB
The same approach was previously published to identify activities of the synthesized compounds 1a-e were compa-
promising scaffolds for anti-AIDS activity [30]. Based on red with the activity of RIF. As shown in Table 1, RIF shows
the substructure search, the molecular scaffold of com- the MIC100 value of 0.06 µg/mL and is hence more active
pound 1a (compound identifier, CID in PubChem 4225334) than compounds 1a-e. Of compounds 1a-e, 1a shows the
was selected (chemotype identifier BKQ4R). We also com- most activity, with the MIC100 value of 7.8 µg/mL (23.45
pared the newly designed compounds with the reference µΜ), followed by 1b, 1d and 1e, each of them with MIC100
molecule 1a. The high structure similarity suggested that of 15.6 µg/mL, followed by 1c. According to the criteria for
their biological activity would be also comparable (Figure
1). This assumption was made considering that here are
Table 1ꢀAnti-M. tuberculosis effects and cytotoxicity levels of
compounds 1a-e.
no activity cliffs, specifically, no similar compounds with
very different biological activity [31]. Physical and spectro-
scopic data for compound 1a have not yet been published.
The preparation of compounds 1a-e started with
esterification of 4-chlorobenzoic acid (2, Scheme 1), with
MeOH in the presence of sulfuric acid to provide methyl
4-chlorobenzoate (3). Next, the 4-chlorophenylhydrazide
4 was prepared from 3 [32]. Treatment of 4 with cyanogen
bromide (CNBr) in MeOH afforded 5-(4-chlorophenyl)-1,3,4-
oxadiazol-2-amine (5) in 62% yield [33]. The coupling reac-
tion between 5 and crude acyl chlorides 6a-e was achieved
in the presence of sodium hydride in dry THF to afford the
desired products 1a-e in 30-62% yield [34].
Compound
R
MIC100
MBC IC50, in Vero Selectivity
(µg/mL) (µg/mL)
cells
index (SI)
(µg/mL)
1a
5-NO2C4H2O
7.80
2.00
15.60
31.25
3.90
106.0
36.0
68.3
61.1
13.59
4.61
2.18
3.92
1b
5-NO2C4H2S 15.60
5-NO2C4H3N 31.25
1c
1d
3-NO2C6H4
15.60
15.60
0.06
1e
5-C6H5
31.25
44.1
2.83
Rifampin
-
ND
>1000
>16,666
The activities of compounds 1a-e as inhibitors of the
growth of the M. tuberculosis strains H37Rv and 209 were
evaluated using 250 µg/mL-0.98 µg/mL serial two-fold
dilutions. Rifampin (RIF) is usually used for the treatment
M. tuberculosis H37Rv ATCC 27294 reference strain: M. tuberculosis.
MIC: minimal inhibition concentration determined by using REMA. IC50:
inhibitory concentration at 50% determined by carrying out an MTT
assay in Vero cells. MBC: minimal bactericidal activity. SI = IC50/MIC100
.
O
O
O
NH
i
OMe
OH
ii
NH2
Cl
3
Cl
Cl
2
4
iii
O
N N
O
N N
O
iv
+
NH2
R
R
Cl
O
N
Cl
H
5
Cl
6a-e
1a R=
H
5-NO2C4 2O
1b R= 5-
H
NO2C4 2S
1c R= 5-
H3N
H4
NO2C4
1d R=
3-NO2C6
1e R= H5
C6
Scheme 1ꢂSynthetic route to compounds 1a-e. Reagents and conditions: (i) MeOH, H2SO4, reflux; (ii) NH2NH2, MeOH; (iii) CNBr, MeOH;
(iv) NaH, anhydrous THF, reflux.
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anti-TB hits and leads, the MIC100 value of 1a is not com- on the aromaticity of the ring attached to the amide group,
petent under replicating growth conditions (< 10 µM) but particularly if this ring has low aromaticity (phenyl > thi-
compounds 1b-e demonstrate a greater SI and, therefore, ophene > pyrrole > furan) [36]. The relatively low anti-TB
can be considered as anti-TB leads. Minimal microbicidal activity of the 5-nitropyrrole compound 1c, relative to the
activity measurements showed 1a has the highest bacteri- activities of 1b, 1d and 1e, may be due to the pyrrole-NH
cidal activity, followed by 1d and 1b, and 1e, with 1c being group of 1c forming a hydrogen bonding interaction with
the least active. The values of minimum bactericidal acti- the active site.
vity show that 1a also has the highest bactericidal activity
followed by 1d and 1b, with compounds 1c and 1e being
the least active [35].
Conclusions
Another criterion used to determine whether a com-
pound can be considered as an early lead for an anti-TB Compounds 1a-e were synthesized using two convergent
drug is in vitro activity against M. tuberculosis strains that pathways with the hydrazide 2 as a common intermediate.
are resistant to a single TB drug, such as isoniazid or RIF, The anti-TB activities of compounds 1a-e were evaluated
indicating a new mechanism of action. We also evaluated using three Mycobacterium tuberculosis cell lines. The
the compounds with non-virulent bacteria M. tuberculo- results suggest that the anti-TB activity of compounds 1a-e
sis H37Ra. Non-virulent bacteria are susceptible to lower can be explained in terms of the distribution of electronic
concentrations of drugs than virulent strains. Specifi- density across the ring attached to the amide group. Our
cally, the activities of the synthesized compounds 1a–1e ongoing studies are focused on exploring the mechanism
as inhibitors of the growth of bacteria of the M. tubercu- by which these new compounds inhibit M. tuberculosis
losis strains H37Ra and 209 (RIF resistant-strain) using cell growth.
250 µg/mL- 0.98 µg/mL serial two-fold dilutions were eva-
luated (Table 2). Also listed in Table 2 are the MIC100 values
of RIF. All compounds are active against M. tuberculosis
Experimental
Melting points were measured in open capillaries using
a Mel-Temp apparatus. H-NMR spectra were recorded in
H37Ra (non-pathogenic) and 209 (resistant strain) with
compound 1a being the most active. This result shows that
compound 1a is an anti-TB hit with a new mechanism of
1
action that should be explored.
DMSO-d6 on a Jeol Eclipse 300 spectrometer (300 MHz).
It can be seen that bioisosteric modifications of the
13C-NMR spectra were recorded at 75 MHz under otherwise
5-nitrofuran moiety of compound 1a to give compounds
similar conditions. IR spectra were obtained in KBr pellets
1b-e preserves anti-TB activity, albeit with inhibitory acti-
using a Magna-IR spectrometer. Mass spectra were recorded
vities less than that of the lead compound 1a. The data for
on a Jeol JEM-AX505HA spectrometer with electronic impact
compounds 1b-e suggest that the anti-TB effect depends
(EI) ionization at 70 eV for low-resolution measurements
and a Jeol AccuTOF DART and Jeol JMS700 (FAB+) instru-
Table 2 REMA-determined MIC100 values of 1a-e against virulent,
nonvirulent and RIF-resistant M. tuberculosis bacteria.
ments, for high-resolution measurements. Flash column
chromatography was carried out on silica gel 60 (230–400
mesh ASTM) from Macherey-Nagel GmbH. Progress of the
reactions was monitored by using TLC. The compounds
were visualized using a dual short-wavelength/long-
wavelength UV lamp or staining with an ethanol solution
of potassium permanganate, vanillin or p-anisylaldehyde.
All reagents were used as purchased from Aldrich.
Compound
R
MIC100
MIC100
MIC100
(µg/mL) in ( µg/mL) in (µg/mL) in
H37Rv ATCC
27294
H37Ra
Mtb-209
(resistant)
1a
5-NO2C4H2O
5-NO2C4H2S
5-NO2C4H3N
3-NO2C6H4
5-C6H5
7.80
15.60
31.25
15.60
15.60
0.06
1-2.00
15.60
7.8
7.8
15.60
7.8
1b
1c
Synthesis of methyl 4-chlorobenzoate (3)
1d
31.30
62.50
0.008
15.60
31.25
>64
1e
A solution of 4-chlorobenzoic acid (2, 33.07 mmol) and
H2SO4 (1 mL) in anhydrous methanol (20 mL) was heated
under reflux for 20 h. After consumption of starting
material (monitored using TLC), the mixture was neutra-
lized with a saturated NaHCO3 solution (2 x 25 mL) and
Rifampin
-
M. tuberculosis H37Rv ATCC 27294 reference strain: Mtb. M. tuberculosis
H37Ra: non-virulent strain. Mtb-209: RIF-resistant clinical isolate of
M. tuberculosis.
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R. Martínez et al.: New nitroheteroarylcarboxamides
extracted with AcOEt (3 x 100 mL). The organic extracts (DMF-d7): δ 113.8, 116.5, 123.6, 127.1, 127.8, 129.8, 136.5, 152.2,
were combined, dried over anhydrous Na2SO4, filtered and 157.8, 162.6, 168.3. HRMS (DART). Calcd for C13H8ClN4O5,
concentrated under reduced pressure to give 3 as a yellow [M+1]+: m/z 335.0183. Found: m/z 335.0186.
oil (yield 80 %). The physical and spectral data of 3 were
in accordance with previously reported data [37].
N-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)-5-nitrothio-
phene-2-carboxamide (1b)
Synthesis of 4-chlorobenzohydrazide (4)
Yield 38% of a yellow solid; mp 260-262°C; IR: 3103, 1714
A solution of ester 3 (26.36 mmol) and hydrazine (61.85 cm-1; H NMR (DMSO-d6) δ: 7.68 (2H, d, J = 8 Hz), 7.95 (3H,
mmol) in anhydrous methanol (20 mL) was heated under m), 8.15 (1H, d, J = 4 Hz). 13C NMR (DMSO-d6): δ 124.5, 128.9,
reflux for 12 h. After consumption of the starting mate- 129.4, 130.0, 130.8, 131.4, 137.6, 143.9, 154.2, 159.8, 165.4.
rial (as monitored using TLC), the reaction mixture was HRMS (DART). Calcd for C13H7ClN4O4S, [M]+: m/z 350.9954.
cooled, and the resulting solid was filtered using suction. Found: m/z 350.9957.
1
The crude product was crystallized from hexane to give 4
as a white crystalline solid; yield 82%; mp 162-164°C. The N-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)-5-nitro-
1H-pyrrole-2-carboxamide (1c)
physical and spectral data of 4 were in accordance with
previously reported data [37].
Yield 30% of a yellow solid; mp 278-280°C; IR: 3176, 1635
Synthesis of 5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine (5) cm-1; 1H NMR (DMSO-d6) δ: 6.98 (1H, d, J = 4 Hz), 7.07 (1H,
d, J = 4 Hz), 7.67 (2H, d, J = 8.5 Hz), 7.95 (2H, d, J = 8.5 Hz);
A solution of 4 (5.86 mmol) and CNBr (10.65 mmol) in 13C NMR (DMSO-d6): δ 111.5, 114.6, 122.9, 128.2, 130.1, 132.2,
anhydrous methanol was heated under reflux for 5 h. 136.7, 143.4, 158.8, 159.5, 160.0. HRMS (DART). Calcd for
After consumption of the starting material (as monitored C13H9ClN5O4, [M+1]+: m/z 334.0343. Found: m/z 334.0347.
using TLC), the mixture was neutralized with a saturated
NaHCO3 solution (2 x 25 mL) and the resulting solid was N-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)-3-nitrobenz-
amide (1d)
filtered using suction. The crude product was crystallized
from methanol to give 5 as a white solid; yield 62%; mp
242-244°C. The physical and spectral data of 5 were in Yield 59% of a white solid; mp 264-266°C; IR: 3176, 1635
accordance with previously reported data [37].
cm-1; 1H NMR (DMSO-d6): δ 8.88 (1H, s), 8.49 (1H, d, J = 7.5
Hz), 8.39 (1H, d, J = 7.5 Hz), 7.95 (2H, d, J = 8 Hz), 7.78 (1H,
m), 7.65 (2H, d, J = 8 Hz); 13C NMR (DMSO-d6): δ 122.1, 123.2,
123.7, 127.4, 128.0, 129.7, 130.5, 130.6, 134.8, 135.4, 136.7, 147.9,
General procedure for synthesis of compounds 1a-e
A solution of 5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine 165.6. HRMS (DART). Calcd for C15H10ClN4O4, [M+1]+: m/z
(5, 0.51 mmol) in anhydrous THF (15 mL) was treated with 345.0390. Found: m/z 345.0394.
NaH (1.53 mmol), and the resulting mixture was cooled to
0°C under a nitrogen atmosphere before addition of acyl N-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)benzamide (1e)
chloride 6a-e (0.51 mmol). The mixture was stirred at room
temperature for 15 h, quenched with a saturated NaHCO3 Yield 78% of a white solid; mp 224-226°C; IR: 3050, 1712
solution (30 mL) and extracted with AcOEt (3 x 50 mL). cm-1; H NMR (DMSO-d6) δ: 7.65 – 7.51 (3H, m), 7.69 (2H, d,
1
The organic extracts were combined, dried over anhyd- J = 8.5 Hz), 7.98 (2H, d, J = 8.5 Hz), 8.04 (2H, d, J = 7 Hz),
rous Na2SO4, filtered, and concentrated under reduced 12.2 (1H, br); 13C NMR (DMSO-d6): δ 122.2, 127.8, 128.3, 128.6,
pressure. The residue was purified using flash column 129.6, 132.2, 133.0, 136.4, 158.0, 160.4, 164.9. HRMS (DART).
chromatography on silica gel eluting with hexanes/EtOAc Calcd for C15H11ClN3O2, [M+1]+: m/z 300.0540. Found: m/z
(6:4) to furnish the 1,3,4-oxadiazole 1a-e.
300.0540.
N-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)-5-nitrofuran- In vitro antimycobacterial assay
2-carboxamide (1a)
Stock solutions of all compounds were prepared in 100%
Yield 30% of a yellow solid; mp 250-252°C; IR: 3139, 1607 dimethyl sulfoxide (DMSO) at 10 µg/mL. For the REMA
cm-1; 1H NMR (DMF-d7): δ 7.40 (1H, d, J = 4 Hz), 7.64 (2H, d, assay, compounds were diluted in 7H9 medium without
J = 8 Hz), 7.74 (1H, d, J = 4 Hz), 7.94 (2H, d, J = 8 Hz); 13C NMR tyloxapol. For reference drugs, stocks of 64 µg/mL were
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prepared and filtered through a membrane with pore dia- volume) (Sigma Aldrich) were added to each well and
meters of 0.22 mm (Millipore; Darmstadt, Germany). All the plates were incubated for two more days. Visual ins-
working solutions were kept refrigerated at -200 C until pection was used to determine the colors of the contents
they were evaluated.
of each well, with blue interpreted as no growth, pink
as growth, and MIC as the last concentration in which
blue color were observed. The minimum inhibitory con-
centration (MIC) is defined as the minimum concentra-
The cytotoxicity assay
The assay was carried out using Vero cell lines (kidney of tion of tested compound to which there was not higher
African green monkey) from ATCC. The cells were cultured shift from resazurin (blue) to resorufin (pink) than that
in RPMI 1640 medium supplemented with 10% FBS and generated by control of a 1:100 dilution of the bacterial
nonessential amino acids.
inoculum [36].
Cytotoxicity assay
Minimal bactericidal concentration
Ten-thousand Vero cells were placed in a 96 well-plate The MBC values were determined for the well
and incubated for 24 h in 100 µL of RPMI medium. After (s)+compound that did produce a shift in color and were
incubation, the plate was washed and new fresh medium then re-incubated in fresh medium. A volume of 5 µL of
with the compound at a various concentration was added. this well (s) was added to 195 µL of fresh medium and
Each tested compound was incubated for 48h at 37°C in incubated for carrying out the REMA assay as described
a 5% CO2 atmosphere. Then, a volume of 10 µL of MTT above. MBC corresponds to the minimum concentration of
(5 µg/mL in sterile PBS) was added to each well and incu- tested compound that does not produce a shift in cultures
bation was continued for another 4 h. The medium was reincubated in fresh medium [36].
removed and a volume of 100 µL of DMSO was added to
solubilize the formazan. Absorbance was determined at a Selectivity indexes
wavelength of 570 nm and cytotoxicity was calculated as
% toxicity = (1-(ABS problem/ABS control))*100. Controls The SI values were obtained using the formula SI = IC50
were cells without treatment [38].
in Vero cells and the MIC100 values were determined using
REMA.
M. tuberculosis culture conditions
Acknowledgements: Financial support from the DGAPA
and UNAM (projects PAPIIT IN208015 and NUATEI-
IIB-UNAM) is gratefully acknowledged. We also thank
R. Patiño, A. Peña, E. Huerta, B. Quiroz, L. Velasco,
J. Pérez, and E. Segura Salinas for technical support.
Gladys J. Nieves Zamudio (273584) and Gustavo Pretelín
Castillo (308250) are recipients of CONACYT Graduate
Scholarships.
M. tuberculosis, H37Rv, H37Ra and 209 strains were culti-
vated in a 7H9-glycerol-10% ADC-0.01% tyloxapol medium
at 37°C until an O.D. of 0.4 at a wavelength of 600 nm was
reached. Working bacteria-solutions were obtained by
dilution 1:25 in 7H9-ADC 10%.
Antimicrobial susceptibility test using the resazurin
microtiter assay (REMA)
References
The assay used here was previously described by Collins
and Franzablau [39]. Briefly, the outer wells of a 96-well
plate were each filled with 200 µL of sterile PBS to prevent
dehydration from occurring during the long-duration
(8-day) incubation. RIF was used as a reference drug
(16 -0.001 µg/mL serial two-fold dilutions) in each plate,
and controls of DMSO, DMSO+Mtb, medium, media+Mtb,
and compound only to validate the plate were also inclu-
ded. Compounds were evaluated at various concentra-
tions from 0.98 µg/mL to 250 µg/mL, and in triplicate in
independent assay experiments. Plates were incubated
for six days; then, 30 µL of 0.01% resazurin (weight/
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