Synthesis and biological evaluation of 1,2,4-triazole-3-thione and 1,3,4-oxadiazole-2-thione as antimycobacterial agents

Article abstract of DOI:10.1111/cbdd.12939

Resistance among dormant mycobacteria leading to multidrug-resistant and extremely drug-resistant tuberculosis is one of the major threats. Hence, a series of 1,2,4-triazole-3-thione and 1,3,4-oxadiazole-2-thione derivatives (4a–5c) have been synthesized and screened for their antitubercular activity against Mycobacterium tuberculosis H37Ra (H37Ra). The triazolethiones 4b and 4v showed high antitubercular activity (both MIC and IC₅₀) against the dormant H37Ra by in vitro and ex vivo. They were shown to have more specificity toward mycobacteria than other Gram-negative and Gram-positive pathogenic bacteria. The cytotoxicity was almost insignificant up to 100?μg/ml against THP-1, A549, and PANC-1 human cancer cell lines, and solubility was high in aqueous solution, indicating the potential of developing these compounds further as novel therapeutics against tuberculosis infection.

Full text of DOI:10.1111/cbdd.12939

DR. DHIMAN SARKAR (Orcid ID : 0000-0002-3912-1104)
Received Date : 29-Jul-2016
Revised Date : 23-Nov-2016
Accepted Date : 10-Dec-2016
Article type : Research Article
Synthesis and biological evaluation of 1,2,4-triazole-3-thione and 1,3,4-
oxadiazole-2-thione as anti-mycobacterial agents
Amol D. Sonawane1‡, Navnath D. Rode1,Laxman Nawale2, Rohini R. Joshi1*,Ramesh
A. Joshi1,Anjali P. Likhite1, Dhiman Sarkar2*
1Division of Organic Chemistry, CSIR-National Chemical Laboratory, Pune-411008, India.
2Combi-Chem Resource Centre, CSIR-National Chemical Laboratory, Pune-411 008, India.
ABSTRACT
Resistance among dormant mycobacteria leading to multi-drug resistant (MDR) and
extremely-drug resistant(XDR) tuberculosis is one of the major threats. Hence, a series of
1,2,4-triazole-3-thione and 1,3,4-oxadiazole-2-thione derivatives (4a-5c) have been
synthesised and screened for their anti-tubercular activity against Mycobacterium
tuberculosis H37Ra (H37Ra). Thetriazolethiones4b and 4v showed high anti-tubercular
activity (both MIC and IC50) against the dormant H37Ra by in vitro and ex vivo. They were
shown to have more specificity towards mycobacteria than other gram-negative and gram-
positive pathogenic bacteria. The cytotoxicity was almost insignificant up to 100 µg/mL
against THP-1, A549 and PANC-1 human cancer cell lines, and solubility was high in
aqueous solution, indicating the potential of developing these compounds further as novel
therapeutics against tuberculosis infection.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.12939
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∗Corresponding authors: Tel.: +91-20-2590-2400; Fax: +91-20-2590-2629 (D. Sarkar); Tel.:
+91-20-25902283; Fax: +91-20-2590-2629 (R.R. Joshi) E-mail addresses:
d.sarkar@ncl.res.in (D. Sarkar),rr.joshi@ncl.res.in (R. R. Joshi).
‡Present address: Department of Chemistry and Biomolecular Science, Faculty of
Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
Tuberculosis (TB) is a devastating disease of poverty, with 95% of the 1.4 million TB
deaths occurring in the developing world and resulting in about 10 million orphaned children
[1]. Furthermore, the emergence of a drug resistant microorganism responsible for TB,
especially multidrug-resistant one along with the lethal combination of TB and HIV-1
infection, makes this disease one of the greatest global health challenges facing us today
[2,3]. Therefore, there is an urgent need for new anti-tubercular drugs and drug targets to
reduce the time for treatment and to identify agents that will be effective against intracellular
persisting Mycobacterium tuberculosis. Although, several compounds are currently in
advanced phases of clinical trials [4], despite a few safety concerns about its use, bedaquiline
(TMC-207) achieved accelerated approval by the FDA for the treatment of MDR-TB in 2012,
becoming the first new TB drug (from a novel class) since 1966 [5,6].
In this regard, it has been established that heterocyclic compounds play an important role
in designing new class of structural entities for medicinal applications [7,8]. Among
pharmacologically important heterocyclic compounds, 1,2,4-triazole and its derivatives are
attracted considerable attention due to their interesting biological activities [9-21]. These
azoles exert their anti-tubercular activity through inhibition of cytochrome P450 gene
CYP121 (CYP121) by a mechanism in which the heterocyclic nitrogen (N-3 of imidazole or
N-4 of 1,2,4-tiazole) binds to the sixth coordination site of the heme iron atom of the
porphyrin in the substrate-binding site of the enzyme [22,23]. These results prompted us to
take-up the 1,2,4-triazoles motif as an active pharmacophore for further diversification to
exploit its anti TB potential. In this regard, we have reported novel triazole derivatives that
have potent anti-mycobacterial activity owing to their known inhibition of sterol synthesis
[24,25]. In continuation to our research project aimed at the development of anti-tubercular
agents herein, we report the synthesis of 1,2,4-triazolethiones and 1,3,4-oxadiazoles thiones
(compounds 4a-4z and 5a-5c, respectively) and their anti-tubercular activity against the
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dormant and active stages of M. tuberculosis H37Ra (MTB). Furthermore, cytotoxicity
studies in three different human cancer cell lines, THP-1, A549, and Panc-1, were carried out
as well to evaluate their specificity for M.tuberculosis.
Experimental section
Chemistry
The routes employed forsynthesis of substituted 5-aryl-4H-1,2,4-triazole-3-thione
derivatives 4 are outlined in Schemes 1 and 2. The acid chloride 2 was prepared by reacting
aromatic carboxylic acid 1 with thionyl chloride. The thiosemicarbazide was acylated with
acid chloride yielding intermediate 3, which, without isolation, upon treatment with ethanolic
or aqueous potassium hydroxide, cyclised to yield the compound 5-aryl-4H-1,2,4-triazole-3-
thione 4 in moderate yields (Scheme1)[26-28]. Some triazolethiones could not be synthesised
by the above procedure. Instead, they were prepared via ester derivatives as depicted in
Scheme 2. The aroylhydrazides3 were obtained by reaction with hydrazine hydrate in EtOH.
Treatment of the aroylhydrazide3 under basic conditions (KOH/EtOH) yielded the
corresponding potassium aroyldithiocarbazate. The resulting intermediate 3 was refluxed in
aqueousammonia to obtain compounds 4u-z. Aroylhydrazides3 were reacted with CS2 in
ethanolic KOH followed by acidification with HCl to yield 1,2,4-oxadiazole thiones5a-c.
Homogeneity of the compounds was determined by thin-layer chromatography (TLC), and
the structures of the newly synthesised compounds were characterised by infrared (IR)
spectroscopy, 1H-NMR, 13C-NMR, and mass spectral analysis.
Methods
S1. General procedure for preparation of 4(a-z)
To a cold solution of substituted benzoic acid (1.0 mmol, 1 equiv.) in dry DCM (25 mL) with
catalytic amount of DMF, thionyl chloride (1.5 mmol, 1.5 equiv.) was added drop-wise. The
reaction mixture was refluxed for 4 h. The solvent was removed under reduced pressure. To
the crude residue of the acid chloride in pyridine (25 mL), thiosemicarbazide (1.5 mmol,
1.5equiv.) was added portion-wise over a period of 10 min and then stirred at room
temperature for 16 h. Pyridine was removed under reduced pressure. The crude residue was
dissolved in 10% aqueous KOH (25 mL), and the resulting mixture was heated at 80°C-
100°C for 3 h. After completion of the reaction, the mixture was cooled 0°C to 0°C and
neutralised with 10% HCl. The precipitate was filtered, washed with water, and dried to yield
compounds 4a-t.
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5-(4-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (4a)
Yield: 79%; Mp: 290-293°C; IR (KBr): 539, 618, 672, 725, 839, 883, 985, 1105, 1294, 1459,
1
1557, 1611, 1655, 1908, 2855, 3196, 3306 cm-1; H-NMR (DMSO-D6): 7.57 (d, J = 8.6 Hz,
2H), 7.90 (d, J = 8.6 Hz, 2H); 13C-NMR (DMSO-D6): 127.9, 128.6, 128.7, 131.2, 139.4,
168.0; HRMS: m/z= 212.0049,calcd. C8H6ClN3S, found 212.0044 [M+H]+.
5-(4-nitrophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione(4b)
Yield: 72%; Mp: 238-242°C; IR (KBr): 711, 792, 854, 950, 1020, 1073, 1113, 1171, 1245,
1305, 1341, 1375, 1457, 1515, 1582, 2705, 2747, 2858, 2952, 3350 cm-1; 1H-NMR (DMSO-
D6): 8.16 (d, J = 8.8 Hz, 2H), 8.32 (d, J = 8.8 Hz, 2H); 13C-NMR (DMSO-D6): δ 124.6,
127.0, 129.2, 131.3, 148.8, 167.9; HRMS: m/z= 223.0290, calcd.C8H6N4O2S, found 223.0284
[M+H]+.
5-(4-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione(4c)
Yield: 81%; Mp: 265-268°C; IR (KBr): 547, 617, 735, 843, 1161, 1231, 1316, 1457, 1515,
1
1576, 1610, 2677, 2858, 2922, 3058, 3332 cm-1; H-NMR (DMSO-D6): δ 7.37 (t, J = 8.5 &
17.6 Hz, 2H), 7.93-7.97 (m, 2H), 13.70 (s,1H), 13.87 (s,1H); 13C-NMR (DMSO-D6): δ 115.7,
115.2, 116.4, 116.6, 122.3, 128.3, 128.4, 130.5, 149.6, 162.3, 164.8, 167.2; HRMS: m/z =
196.0345 calcd.C8H6FN3S, found 196.039 [M+H]+.
5-(2-methyl-3-nitrophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (4d)
Yield: 73%; Mp: 235°C; IR (KBr): 554, 611, 725, 803, 867, 975, 1032, 1252, 1307, 1370,
1459, 1527, 1578, 1612, 2858, 2922, 3061, 3384 cm-1 1H-NMR (DMSO-D6): δ 2.97 (s, 3H),
7.95 (d, J= 7.7 Hz, 1H), 8.02 (d, J= 7.6 Hz, 1H), 8.13 (m,1H); 13C-NMR (DMSO-D6): δ
20.03, 124.7, 125.8, 129.9, 130.1, 134.2, 148.94, 149.92, 167.98.
5-(3-methyl-4-nitrophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (4e)
Yield: 81%; Mp: 261-263°C; IR (KBr): 581, 615, 749, 791, 832, 881, 967, 993, 1032, 1067,
1
1098, 1234, 1334, 1375, 1456, 1572, 2855 cm-1; H-NMR (DMSO-D6): δ 2.55 (s, 1H), 7.93
13
(dd, J=8.02 Hz, 1H),8.01 (s, 1H), 8.11 (dd, J =8.02, 1H), 13.89 (s,1H), 14.03 (s,1H); C-
NMR (DMSO-D6): δ 20.2, 124.9, 125.98, 129.99, 130.3, 134.3, 149.07, 150.06, 168.1;
HRMS: m/z= 237.0439,calcd. C9H8N4O2S, found 237.0441 [M+H]+.
5-(4-(trifluoromethyl)phenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (4f)
Yield: 79%; Mp: 283°C; IR (KBr): 552, 599, 699, 763, 850, 940, 966, 1017, 1069, 1122,
1
1182, 1329, 1375, 1455, 1527, 1578, 1497, 2676, 2856 cm-1; H-NMR (DMSO-D6): δ 7.85
(dd, J = 8.1 Hz, 2H), 8.10 (dd, J = 8.1 Hz, 2H), 13.89 (s, 1H), 14.10 (s, 1H); 13C-NMR
(DMSO-D6): δ 122.7, 125.5, 125.8, 126.3, 126.3, 126.7, 127.6, 129.5, 130.4, 130.5, 130.8,
134.8, 142.1, 146.4, 149.3, 166.4, 167.7. HRMS: m/z= 246.0313, calcd.C9H6F3N3S, found
246.0307 [M+H]+.
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5-(3-(trifluoromethyl)phenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione(4g)
Yield: 76%; Mp: 120-122°C;IR (KBr): 653, 699, 746, 811, 915, 979, 1082, 1127, 1173,
1229, 1323, 1369, 1457, 1502, 1574, 1614, 1695, 2586, 2921, 3054 cm-1; 1H-NMR (DMSO-
D6): 7.71-7.91 (m, 2H), 8.22 (d, J = 7.3 Hz, 2H), 8.27 (s, J = 1.3 Hz, 1H), 13.83 (s, 1H),
14.04 (s, 1H);13C-NMR: 122.5, 122.6, 125.4, 125.7, 126.7, 127.3, 129.7, 130.3, 130.6, 132.1,
133.4, 149.2, 166.2, 167.7. HRMS: m/z= 246.0313, calcd. C9H6F3N3SF3S, found 246.0307
[M+H]+.
5-(4-nitro-3-(trifluoromethyl)phenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione(4h)
Yield: 79%; Mp: 138-141°C; 1H-NMR (DMSO-D6): 8.1-8.2 (m, 21), 8.56-8.66 (m, 2H),
13C-NMR: 122.8, 128.09, 128.3, 130.3, 131.8, 134.5, 147.04, 148.9, 149.1, 165.2, 167.8.
5-(4-chloro-2-methylphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione(4i)
Yield: 81%; Mp: 205°C;NMR (DMSO-D6): 2.44 (s, 3H), 7.42 (dd,J = 8.3 & 1.6 Hz, 1H),
13
7.47 (d, J = 1.6 Hz, 1H), 7.61 (d, J = 8.3 Hz, 1H), 13.66(s, 1H), 13.74 (s, 1H) C-NMR:
21.06, 128.8, 130.4, 131.1, 133.7, 136.4, 149.8, 167.07 HRMS: m/z= 226.0206, calcd.
C9H8ClN3S, found 226.0200 [M+H]+.
5 -(2-chloro-5-(trifluoromethyl)phenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione(4j)
Yield: 81%; Mp: 185°C: P1PH NMR (DMSO-D6): 7.80 (d, J = 8.7 Hz, 1H), 7.92 (d, J = 8.7
13
Hz, 1H), 8.12 (s, 1H) C-NMR: 126.4, 128.6, 128.91, 129.3, 131.9, 132.3, 136.96, 147.6,
164.5, 167.5
5-(3-chloro-2-methylphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione(4k)
Yield: 81%; Mp: 177-189°C;IR (KBr): 673, 721, 761, 796, 914, 975, 1019, 1075, 1115,
1158, 1253, 1303, 1380, 1454, 1503, 1574, 1647, 2651, 2857, 3116 cm-1; 1H-NMR (DMSO-
13
D6): 2.52 (s, 3H), 7.27-7.40 (m, 1H), 7.51-7.71 (m, 2H), 13.69 (s, 1H), 13.79 (s, 1H); C-
NMR: 127.7, 127.92, 128.05, 128.96, 131.6, 150.4, 150.6, 167.07; HRMS: m/z=
226.0206,calcd. C9H8ClN3S, found 226.0200 [M+H]+.
5-(5-chloro-2-methylphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (4l)
Yield: 79%; Mp: 230 °C;IR (KBr): 557, 608, 655, 731, 818, 981, 1036, 1073, 1159, 1376,
1
1457, 1598, 1703, 2858, 2923,3163, 3347 cm-1; H-NMR (DMSO-D6): 2.44 (s, 3H), 7.33-
7.55 (m, 2H), 7.73-7.79 (m, 1H), 13.69 (s, 1H), 13.80 (s, 1H); 13C-NMR: 21.06, 128.8, 130.4,
131.1, 133.7, 136.4, 149.8, 167.07; HRMS: m/z = 226.0206, calcd. C9H8ClN3S, found
226.9551 [M+H]+.
5-(2-(trifluoromethyl)phenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione(4m)
Yield: 86%; Mp:245°C;1H-NMR (DMSO-D6): 7.66-7.97 (m, 4H). 13C-NMR: 126.98, 129.8,
131.07, 132.6, 133.02, 133.3, 148.7, 166.8.
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5-(4-methoxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (4n)
Yield: 83%; Mp: 255°C, IR (KBr): 631, 732, 796, 840, 963, 1015, 1078, 1122, 1176, 1253,
1
1300, 1389, 1456, 1519, 1607, 2858, 2923, 3243 cm-1; H-NMR (DMSO-D6): 3.80 (s, 3H),
7.06 (d, J = 8.8 Hz, 2H), 7.86 (d, J = 8.8 Hz, 2H); 13C-NMR: 55.6, 113.95, 114.7, 127.6,
129.6, 161.15. HRMS: m/z= 208.0545, calcd. C9H9N3OS, found 208.0539 [M+H]+.
5-(2-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione(4o)
Yield: 79%; Mp: 205°C; IR (KBr): 559, 608, 663, 731, 765, 968, 1040, 1159, 1304, 1375,
1458, 1595, 1711, 2858, 2923, 3054, 3127, 3331 cm-1; 1H-NMR (DMSO-D6): 7.44-7.68 (m,
4H), 13.81 (s, 1H); 13C-NMR:128.4, 128.7, 132.3, 133.03, 149.8, 151.02, 166.4, 182.6;
HRMS: m/z = 212.0049, calcd. C8H6ClN3SN3S, found 212.0044 [M+H]+.
5-(2-chloro-5-(trifluoromethyl)phenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione(4p)
Yield: 71%; Mp: 288°C, IR (KBr): 728, 773, 901, 963, 1036, 1083, 1162, 1265, 1308, 1374,
1
1457, 1577, 1600, 1657, 2059, 2663, 2850, 2923, 3057, 3192 cmP-1; H-NMR (DMSO-D6):
13
2.42 (s, 3H), 7.27-7.45 (m, 4H); C-NMR: 21.3, 125.4, 126.6, 129.4, 130.7, 131.8, 137.3,
151.03, 166.9.
5-(p-tolyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione(4q)
Yield: 84%; Mp: 255°C; IR (KBr): 723, 813, 1019, 1075, 1118, 1230, 1304, 1375, 1458,
1
1524, 1580,1610, 1711, 2859, 2923, 3084, 3167, 3265,3365 cm-1; H-NMR (DMSO-D6):
2.33 (s, 3H), 7.30 (d, J = 8.1 Hz, 2H), 7.79 (d, J = 8.1 Hz, 2H), 13.64 (s, 1H), 13.78 (s,
1H);13C-NMR: 21.1, 122.9, 125.8, 129.8, 140.7, 150.5, 166.99;HRMS: m/z = 192.0595,
calcd.C9H9N3S, found 192.0590 [M+H]+.
5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione(4r)
Yield: 77%; Mp: 154°C; IR (KBr):551, 664, 719, 751, 851, 907, 1141, 1262, 1304, 1382,
1
1423, 1468, 1570, 1696, 2548, 2599, 2658, 2707, 2952, 3082 cm-1; H-NMR (DMSO-D6):
6.19-6.39 (m, 2H), 6.57-6.63 (m, 2H); 13C-NMR (DMSO-D6): 124.8, 125.9, 127.92, 130.89,
131.6, 134.4, 149.5, 167.8. HRMS: m/z = 212.0049, calcd.C8H6ClN3S, found 212.0044
[M+H]+.
5-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione(4s)
Yield: 71%; Mp: 265°C; IR (KBr):557, 607, 695, 786, 906, 966, 1031, 1079, 1171, 1260,
1
1375, 1470, 1550, 1598, 1635, 1691, 2922, 3135, 3414 cm-1; H-NMR (DMSO-D6): 7.43-
7.66 (m, 3H), 7.90 (dd, J = 1.8 & 8.4 Hz, 2H), 13.70 (s, 1H), 13.86 (s, 1H). 13C-NMR
(DMSO-D6): 125.97, 126.2, 129.6, 131.1, 150.7, 167.5. HRMS: m/z = 178.0439, calcd.
C8H7N3S, found 178.0433 [M+H]+.
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5-(m-tolyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione(4t)
Yield: 79%; Mp: 280°C; IR (KBr):544, 611, 694, 768, 861, 982, 1165, 1226, 1294, 1375,
1
1457, 1568, 1620, 1668, 2670, 2728, 2858, 2922, 3107 cm-1; H-NMR (DMSO-D6): 7.28-
7.38(m, 2H), 7.72 (t, J = 5.5 & 7.8 Hz, 2H), 13.70 (s, 1H), 13.86 (s, 1H); 13C-NMR (DMSO-
D6): 21.2, 123.1, 125.6, 126.4, 129.2, 131.5, 138.7, 150.5, 167.1. HRMS: m/z = 192.0595,
calcd. C9H9N3S, found 192.0590 [M+H]+.
S2. General procedure for preparation of 4u-z
To a solution of ester (1 mmol, 1 equiv.), hydrazine hydrate (3 mmol, 3.0 equiv.) was added
drop-wise. The mixture was refluxed for 5 h; after completion of the reaction, a solid product
was formed, and the excess solvent was removed under reduced pressure. Potassium
hydroxide (1.5 mmol, 1.5 equiv.) was dissolved in absolute ethanol (25mL); the crude residue
obtained from the previous step, acylhydrazine (1 mmol, 1 equiv.), and carbon disulphide
(1.5 mmol, 1.5 equiv.) were added drop-wise, and the reaction mixture was refluxed for 10h.
After completion of the reaction as monitored by TLC, the solvent was evaporated under
reduced pressure, and the residue was used for next step.To the crude residue, 25% ammonia
solution (25 mL) was added and refluxed for 10 h. The reaction mixture was acidified with
10% HCl solution. The precipitate was filtered, washed with water, and dried to give
compounds 4u-z.
5-(4-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione(4u)
Yield: 76%; Mp: 214°C; IR (KBr): 534, 696, 737, 837, 932, 970, 1071, 1202, 1269, 1347,
1444, 1517, 1608, 1919, 2857, 2923, 3364 cm-1; 1H-NMR (DMSO-D6): 6.94 (dd, J = 8.8 Hz,
2H), 7.71 (dd, J = 8.8, 2H), 10.42 (s, 1H); 13C-NMR (DMSO-D6): 113.3, 116.4, 128.3, 161.1,
161.3, 177.2.
5-(4-aminophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione(4v)
Yield: 78%; Mp: 243°C; IR (KBr): 636, 692, 730, 832, 945, 968, 1066, 1118, 1164, 1295,
1
1366, 1457, 1508, 1614, 2852, 3318, 3346, 3393, 3442 cm-1; H-NMR (DMSO-D6): 6.73
(dd, J= 8.4 Hz, 2H), 7.56 (dd,J = 8.4 Hz, 2H);13C-NMR (DMSO-D6): δ 109.9, 114.7, 127.9,
151.4, 161.6, 176.8; HRMS: m/z = 194.0626, calcd. C8H8N4S, found 194.0621 [M+H]+.
5-(furan-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (4w)
Yield: 72%; Mp: 285°C; IR (KBr): 551, 595, 641, 740, 757, 826, 289, 972, 1019, 1077, 1167,
1375, 1456, 1636, 1778, 2724, 2850 cm-1; 1H-NMR (DMSO-D6): 6.67 (dd, J =1.7 & 3.4 Hz,
13
1H), 7.11 (d,J = 3.4 Hz, 1H), 7.89 (s, 1H), 13.67 (s, 1H), 13.88 (s, 1H); C-NMR (DMSO-
D6): δ 112.1, 112.2, 140.6, 143.3,145.6, 166.7; HRMS: m/z = 194.0626, calcd.for C8H10N4S,
found 194.0621 [M+H]+.
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5-(pyridin-3-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (4y)
Yield: 82%; Mp: 229°C; IR (KBr): 544, 630, 705, 810, 926, 1026, 1072, 1154, 1191, 1365,
1
1460, 1529, 1603, 2362, 2715, 2857, 2924 cm-1; H-NMR (DMSO-D6): 7.62 (dd, J = 4.9 &
7.9 Hz, 1H), 7.24 (dt, J = 1.9 & 8.9 Hz, 1H), 7.77 (d, J= 3.8 Hz, 1H), 9.01 (s, 1H); 13C-NMR
(DMSO-D6): 118.7, 123.9, 133.4, 146.3, 152.1, 158.4, 177.1.
S3. General procedure for preparation of 5(a-b)
To a solution of ester (1 mmol, 1 equiv.), hydrazine hydrate (3 mmol, 3.0 equiv.) was added
drop-wise. The mixture was refluxed for 5 h; after completion of the reaction, a solid product
was formed, and the excess solvent was removed under reduced pressure. Potassium
hydroxide (1.5 mmol, 1.5 equiv.) was dissolved in absolute ethanol (25 mL). The crude
residue obtained from previous step, acylhydrazine(1 mmol, 1 equiv.),and carbon disulfide
(1.5 mmol, 1.5 equiv.) were added drop-wise,and the reaction mixture was refluxed for 10
h.After completion of the reaction as monitored by TLC, the residue was poured in ice-cold
water and acidified with 10% HCl. The precipitate was filtered, washed with water, and dried
to give compound 5.
5-(4-chlorophenyl)-2,3-dihydro-1,3,4-oxadiazole-2-thione(5a)
Yield: 76%; Mp: 215°C; IR (KBr): 615, 671, 770, 835, 882, 983, 1102, 1168, 1359, 1467,
1
1608, 2717, 2845, 2924, 3192, 3303, 3544 cm-1; H-NMR (DMSO-D6): 7.6 (d, J = 8.2 Hz,
13
2H), 7.89 (d, J = 8.1 Hz, 2H); C-NMR (DMSO-D6): 121.95, 128.4, 130.1, 137.5, 160.2,
177.98; MS (EI): m/z = 235[M++ Na].
5-(4-fluorophenyl)-1,3,4-oxadiazole-2-thione (5b)
1
Yield: 72%; Mp: 282°C; H-NMR: 7.43 (t, J = 8.8 & 17.9 Hz, 2H), 7.93 (m, J= 8.8 & 17.9
Hz, 2H). 13C-NMR (DMSO-D6): 117.1, 117.4, 119.7, 129.3, 129.4, 160.3, 163.4, 165.98,
177.96.
Biology
5-aryl-4H-1,2,4-triazole-3-thiones 4a-z and 5a-c were tested for their in vitro effect against
M. tuberculosis H37Ra (strainATCC 25177), which is susceptible to control drugs
(rifampicin, isoniazid, ethambutol, and pyrazinamide). In vitro Activity against MTB was
determined using the XTT Reduction Menadione Assay (XRMA) and ex vivo in a THP1
infection modelby NR assay as per the protocol described previously [29-31].The minimum
inhibitory concentration (MIC; in µg/mL) was recorded as the lowest concentration highest
dilution of the compounds control drug that completely inhibited the growth of
Mycobacterium cultures.
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In vitro cytotoxicity of thiones(4a-z and 5a-c) against THP-1, A549, and Panc-1 human
cell lines was determined using the MTT assay as described previously [32,33].Briefly, the
cells were seeded into a 96-well plate at a density of 1×104 per well for 24 h, followed by
drug treatment for 48 h. Next, 5 mg/mL MTT were added to the medium, and the cells were
incubated for 4 h at 37°C and 5% CO2. After removing the culture medium, acidified
isopropanol (0.04N HCl) was added. The absorbance was measured using a Molecular Probes
plate reader at 570 nm. The percentage of cytotoxicity was calculated using the formula:
% cytotoxicity = [(control –test) / (control – blank)] × 100,
Where,
Control= cell growth in medium without thiones, but with DMSO,
Test= cell growth in presence of thiones,
Blank= culture medium without cells.
Each experiment was performed in triplicate, and the quantitative value was expressed as the
average standard deviation.
Determining the solubility of acompound in water has become anessential early measurement
in the drug discovery process. In this regard, the aqueous solubility of 5-aryl-4H-1,2,4-
triazole-3-thiones 4b, 4c, 4f, 4j, 4t, 4v, and 4z were measured according to instructions
provided by Millipore using the 96-well plate format protocol (Figure 4).
Data were collected using a Molecular Devices SPECTRAmax® Plus microplate
spectrometer. The ratio of filtrate vs. standard absorbance was calculated to quantify the
aqueous solubility using the formula below:
∑
(
)
ꢀꢁ: ( ꢂꢃꢄꢅꢆꢇꢈ,ꢆꢉꢈ,ꢊꢈꢈ,ꢊꢆꢈ,ꢊꢋꢈ,ꢊꢇꢈꢌꢍ) ꢎ ꢂꢃꢄꢅꢉꢈꢈꢌꢍ ꢁꢏꢐꢅꢑꢄꢅꢒ≈1.0
(∑
) (
)
ꢂꢃꢄꢅꢆꢇꢈ,ꢆꢉꢈ,ꢊꢈꢈ,ꢊꢆꢈ,ꢊꢋꢈ,ꢊꢇꢈ ꢎ ꢂꢃꢄꢅꢉꢈꢈꢌꢍ ꢓꢅꢄꢌꢔꢄꢑꢔ
Then, Aqueous Solubility ≥ 500 μM
∑
(
)
ꢀꢁ: ( ꢂꢃꢄꢅꢆꢇꢈ,ꢆꢉꢈ,ꢊꢈꢈ,ꢊꢆꢈ,ꢊꢋꢈ,ꢊꢇꢈꢌꢍ) ꢎ ꢂꢃꢄꢅꢉꢈꢈꢌꢍ ꢁꢏꢐꢅꢑꢄꢅꢒ≤ 0.5
(∑
) (
)
ꢂꢃꢄꢅꢆꢇꢈ,ꢆꢉꢈ,ꢊꢈꢈ,ꢊꢆꢈ,ꢊꢋꢈ,ꢊꢇꢈ ꢎ ꢂꢃꢄꢅꢉꢈꢈꢌꢍ ꢓꢅꢄꢌꢔꢄꢑꢔ
Then, Aqueous Solubility ≤ 100 μM
∑
(
)
ꢀꢁ: ( ꢂꢃꢄꢅꢆꢇꢈ,ꢆꢉꢈ,ꢊꢈꢈ,ꢊꢆꢈ,ꢊꢋꢈ,ꢊꢇꢈꢌꢍ) ꢎ ꢂꢃꢄꢅꢉꢈꢈꢌꢍ ꢁꢏꢐꢅꢑꢄꢅꢒ<1.0 and >5.0
(∑
) (
)
ꢂꢃꢄꢅꢆꢇꢈ,ꢆꢉꢈ,ꢊꢈꢈ,ꢊꢆꢈ,ꢊꢋꢈ,ꢊꢇꢈ ꢎ ꢂꢃꢄꢅꢉꢈꢈꢌꢍ ꢓꢅꢄꢌꢔꢄꢑꢔ
Then, 100 μM<Aqueous Solubility <500 μM.
In the present study, we evaluated the antimicrobial and anti-proliferative activity for a
set of thiones. In a preliminary screening, the anti-tubercular and antimicrobial activities of
the thiones were assessed at concentrations of 30, 10, and 3 μg/mL. Then, dose-dependent
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inhibition assays were performed at concentrations of 100, 30, 10, 3, 1, 0.3, 0.1, and 0.03
μg/mL to determine the MIC values (Table S1).
All of the triazolethione compounds (4a-z and 5a-c) tested in present study exhibited
anti-tubercular activities against the dormant and active phases of MTB. The MIC values
against the dormant stage ranged from 0.46to 30 μg/mL, while the values against the active
stage ranged from 9.05 to 30 μg/mL. The IC50values against the dormant stage ranged from
<0.03 to 26.54 μg/mL, while the values against the active stage ranged from <0.03 to 30
μg/mL(refer table S1). The triazole compounds 4b, 4c, 4f, 4j, 4t, 4v, and 4z showed the
highest anti-tubercular activities (both MIC and IC50; Table S1) against the dormant phase of
MTB. The other substituted triazole compounds also had significant anti-tubercular activity
(both MIC and IC50) against the dormant phase. Although the triazole thione compounds 4b,
4c, 4f, 4j, 4t, 4v, and 4z showed MIC values of <5 μg/mL against the dormant phase of MTB,
they exhibited lower potencies than the standards (rifampicinand isoniazid) used as control
drugs. Compound 4v was found to be more active than the other compounds with an MIC
value of 0.46μg/mL against the dormant phage of MTB. Since these triazolethione
compounds are efficacious against intracellular M. tuberculosis, they have the potential to be
developed as anti-tubercular drug candidates.
The synthesised compounds 4a-z and 5a-c were assayed further for their cytotoxic
activity in three different human cancer cell lines, THP-1, A549, and Panc-1, after 48-h
exposure to the tested compounds using the MTT assay [34,35].As shown in Figure 2, the
data reveal that all 5-aryl-4H-1,2,4-triazole-3-thiones tested show low cytotoxicity (0 to 100
µg/mL) in human cancer cell lines. The anti-tubercular activities against the dormant phase of
MTB and the non-toxic properties in THP-1, A549, and Panc-1 cell lines of 4a, 4b, 4c, 4f, 4j,
4t, 4v, and 4z demonstrate that these compounds can be used as potential dormant stage
inhibitors.
The aqueous solubility of triazolethione derivatives (4a-z and 5a-c) was measured using
the Millipore 96-well plate format protocol (PC2445EN00). The solubility of the thione
compounds was calculated according to the US pharmacopeia definition of solubility. The
solubility of thiones 4a-f was >500 to 1280 μM and that of 4g-5c was >1280 to 3200 μM,
while the solubility of testosterone and furosemide was 365 μM and 500 μM, respectively.
The solubility results demonstrate that these synthetic thione compounds are highly soluble in
aqueous solution for oral drugs. The solubility values were somewhat reproducible. In case of
the reference compounds, the solubility values also were reproducible and not significantly
different from those reported in the literature.(Figure 5)
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Our SAR investigation began with preparation of a series of analogues in which the 3
position of a 1, 2, 4 triazole thione was fixed as aromatic substituent. The structure activity
relationship (SAR) of the molecular diversity is summarized in Table 1. The MIC values
against M. tuberculosis H37Ra were obtained in the range of 0.46 - > 100 μg/ml. The
presence of versatile substituent’s on 3 position of a 1, 2, 4 triazole thione skeleton was
significantly affected on ant-tubercular activity. Thione para substituted -NH2 (electron
donating) and -NO2 (electron withdrawing) analogs were found to be considerably potent at
MIC 0.46 and 0.64 μg/mL. Next; the influence of hydrophilic or lipophilic variants on the
parent structure was probed. The thione analogs in which aromatic ring constituted with
different electron withdrawing groups, compounds 4c, 4e, 4f, 4g, 4h, 4i, 4j, 4q, 4t, 4z and 5c
displayed superior activity with MIC values < 16 and > 1 μg/mL. The aromatic ring
containing para Chloro (4 –Cl) substituent at , for instance 4a, 4k, 4l, 4r and 5a were found to
be weakly actives with MIC > 30 μg/mL. In the same context, 2-substituted aromatic ring of
different analogs, 4d, 4m, 4n, 4o, 4p were also found to be weakly actives with MIC > 30
μg/mL. Also, few analogs such as 4w, 4x, 4y and 4z thione substitution with hetroatomic
aromatic ring were less active (MIC > 30 μg/mL). The compound 5a and 5b with oxadiazole
thione at 4-substitution exhibited almost very less anti-TB activity. This trend has clearly
indicated that not only the presence of -NO2 and -NH2 but its site-selectivity in the particular
ring is also essential to generate a ‘lead’ molecule.
The synthesis and biological evaluation of a series of 5-aryl-4H-1,2,4-triazole-3-thiones 4a-z
and 5a-c as anti-tubercular agents have been discussed, and all were found to be active
against the dormant stage of Mycobacterium tuberculosis H37Ra. The compounds 4b, 4c, 4f,
4j, 4t, 4v, and 4z were highly active. Compounds 4b and 4v displayed the most potent
overall anti-tubercular activity against dormant Mycobacterium tuberculosis H37Ra in vitro
(MIC <0.7 μg/mL and IC50<0.03 μg/mL). From the present study, compounds 4b and 4v
were found to have low cytotoxicity (up to100 µg/mL) in THP-1, A549, and PANC-1 human
cancer cell lines and are highly soluble in aqueous solution. SAR of this work demonstrates
that compounds 4b and 4v may be the best candidates for further investigation as potential
drugs in the search for new, safe and effective anti-tubercular drugs.
Acknowledgment
This work was financially supported by CSIR, New Delhi, India, (Project Code:
BSC0103, CSC0406). Authors also are thankful to Dr. P. R. Rajamohanan, NCL, Pune for
his help on spectral analysis. Authors are also thankful to Professor Mamoru Koketsu, Gifu
university, Japan for his help on spectra analysis.
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Supplementary data
Supplementary data (experimental details, Proton and Carbon NMRs of new compounds
and Tables of biological activity) associated with this article can be found, in the online
version, at http://
References
[1] Global Tuberculosis Control: WHO Report. (2013)World Health Organization: Geneva,
Switzerland.(2011)
[2] CorbettE.L., Watt C.J., Walker N., Maher D., Williams B.G., RaviglioneM.C., Dye
C.(2003)The Growing Burden of Tuberculosis Global Trends and Interactions With the HIV
Epidemic. Arch. Intern. Med;163:1009.
[3] Long R. (2000)Drug-resistant tuberculosis. Can. Med. Assoc. J.;163: 425.
[4] ZumlaA., NahidP., Cole S.T. (2013)Advances in the development of new tuberculosis
drugs and treatment regimens. Nature Rev. Drug Discovery.;12: 388−404.
[5] Wong E.B., Cohen K.A., BishaiW.R. (2013)Rising to the challenge: new therapies for
tuberculosis. Trends Microbiol.;21: 493−501.
[6] FieldS. K., Clin. (2013)Safety and efficacy of delamanid in the treatment of multidrug-
resistant tuberculosis (MDR-TB)Med. Insights: Ther.;5: 137−149.
[7] Sachs G., Shin J.M., HowdenC.W. (2006)Review article: the clinical pharmacology of
proton pump inhibitors. Pharmacol.Ther.;23: 2-8.
[8] GomtsyanA., Chem. (2012)Heterocycles in drugs and drugs discovery. Heterocycl.
Compd.;48: 8-10.
[9] EswaranS., AdhikariA.V., ShettyN.S., (2009)Synthesis and antimicrobial activities of
novel quinoline derivatives carrying 1,2,4-triazole moiety. Eur. J. Med. Chem.;44: 4637.
[10] DemirbasA., SahinD., DemirbasN., KaraogluS.A. (2009) Synthesis of some new 1,3,4-
thiadiazol-2-ylmethyl-1,2,4-triazole derivatives and investigation of their antimicrobial
activities. Eur. J. Med. Chem.;44:2896-2903.
[11] Pal D.K., Singh V., PandeyD.D., MauryaR.K.(2014)Synthesis, characterization and
Antimicrobial evaluation of some 1,2,4-triazole derivatives. Int. J. Pharm. Pharm. Sci.;6: 213-
216.
[12] KucukguzelI., KucukguzelS.G., RollasS., KirazM. (2001) Some 3-Thioxo/Alkylthio-
1,2,4-triazoles with a Substituted Thiourea Moiety as Possible Antimycobacterials. Bioorg.
Med. Chem. Lett.;11:1703.
This article is protected by copyright. All rights reserved.

[13]KandemirliF., ShvetsN., UnsalanS., KucukguzelI., RollasS., KovalishynV., DimogloA.
(2006)The Structure - Antituberculosis Activity Relationships Study in a Series of 5-(4-
Aminophenyl)-4-Substituted-2,4-Dihydro-3h-1,2,4-Triazole- 3-Thione Derivatives.
Combined Electronic-Topological and Neural Networks Approach. Med Chem.;2:415-422.
[14] Garcia M.A., SantamariaS.M., CachoM., de la LlaveF.M., Julian M., Martinez A. ,
Pascual-Teresa B.D., Ramos A.(2005)Synthesis, Biological Evaluation, and Three-
A
Dimensional Quantitative Structure−Activity Relationship Study of Small-Molecule Positive
Modulators of Adrenomedullin. J. Med. Chem.;48:4068–4075.
[15] NavidpourL., ShafaroodiH., AbdiK., AminiM., GhahremaniM.H., DehpourA.R.,
ShafieeA.(2006)Design, synthesis, and biological evaluation of substituted 3-alkylthio-4,5-
diaryl-4H-1,2,4-triazoles as selective COX-2 inhibitors. Bioorg.Med. Chem.;14: 2507.
[16]Amir M., ShikhaK.(2004)Synthesis and anti-inflammatory, analgesic, ulcerogenic and
lipid peroxidation activities of some new 2-[(2,6-dichloroanilino) phenyl]acetic acid
derivatives. Eur. J. Med. Chem.;39: 535.
[17] KumidhaD., Leonard J.T., MuthumaniM., ChidhambaranathanN., KalavathiT.
(2013)Synthesis and evaluation of some 1,2,4-Triazole derivatives as anticonvulsant, anti-
inflammatory and antimicrobial agents. Asian J. Pharm. Clin.;6:5-8.
[18] HouY.P., Sun J., Pang Z.H., LvP.C., Li D.D., Yan L., Zhang H.J., ZhengE.X., Zhao J.,
Zhu H.L.(2011)Synthesis and antitumor activity of 1,2,4-triazoles having 1,4-benzodioxan
fragment as a novel class of potent methionine aminopeptidase type II inhibitors. Bioorg.
Med. Chem.;19: 5948.
[19]Lin R., Connolly P.J., Huang S., Wetter S.K., Lu Y., Murray W.V., Emanuel S.L.
GruningerR.H., Fuentes-PesqueraA.R., RuggC.A., Middleton S.A., JolliffeL.K. (2005)1-
Acyl-1H-[1,2,4]triazole-3,5-diamine analogues as novel and potent anticancer cyclin-
dependent kinase inhibitors: synthesis and evaluation of biological activities. J. Med.
Chem.; 48: 4208-4211.
[20] SaadH.A., Osman N.A., MoustafaA.H. (2011)Synthesis and Analgesic Activity of Some
New
Pyrazoles and Triazoles Bearing a 6,8-Dibromo-2-methylquinazoline Moiety.
Molecules; 16: 10187-10201.
[21] ShashikalaD.K., RamaiahM., VanitaG.K., VeenaK., VaidyaV.P. (2011)Synthesis and
analgesic activity of triazolothiadiazoles and triazolothiadiazines encompassing 3–
nitronaphtho[2,1-b]furan. Chem. Pharm. Res.;3: 445-451.
This article is protected by copyright. All rights reserved.

[22] BellamineA., LepeshevaG.I., WatermanM.R.(2004)Fluconazole binding and sterol
demethylation in three CYP51 isoforms indicate differences in active site topology.J. Lipid
Res.;45: 2000–2007.
[23] DunfordA.J., McLean K.J., SabriM., Harriet E.S., HeyesD.J., ScruttonN.S., Munro
A.W., (2007)Rapid P450 Heme Iron Reduction by Laser Photoexcitation of Mycobacterium
tuberculosis CYP121 and CYP51B1: Analysis of CO complexation reactions and
reversibility of the P450/P420 equilibrium. J. Biol. Chem.;282: 24816-24824.
[24] SarkarD., DeshpandeS.R., MaybhateS.P., LikhiteA.P., SarkarS., Khan A.,
ChaudhryP.M., ChavanS.R.A method of screening anti-tubercular compounds. Patent no:
WO-2012123971 A2.
[25] SarkarD., LikhiteA.P., Joshi R.A., Joshi R.R., KhedkarV. (2014)Novel Anti-tubercular
agents. NF-0233.
[26] GolovlyovaS.M., MoskvichevY.A., AlovE.M., KobylinskyD.B., ErmolaevaV.V.(2001)
Synthesis of novel five-membered nitrogen containing heterocyclic compounds from
derivatives of arylsulfonyl- and aryl thioacetic acid and –propionoc acid. Chem.
Heterocycl.Compd.;37:1102.
[27] LabanauskasL., UdrenaiteE., GaidelisP., BrukstusA.(2004)Synthesis of 5-(2-,3- and 4-
methoxyphenyl)-4H-1,2,4-triazole-3-thiol
activityFarmaco.; 59:255.
derivatives
exhibiting
anti-inflammatory
[28] OzturkS., AkkurtM., CansizA.,KoparirM.,SekerciM.,Heinemann F.W. (2004)4-(4-
Chlorophenyl)-3-(furan-2-yl)-1H-1,2,4-triazole-5(4H)-thione. Acta.Cryst.; E60: O425.
[29] SarkarD., Singh U.(2011)A novel screening method based on menadione mediated rapid
reduction of tetrazolium salt for testing of anti-mycobacterial agents. J. Microbiol.
Methods;84:202.
[30] Khan A., SarkarS., SarkarD.(2008)Bactericidal activity of 2-nitroimidazole against the
active replicating stage of Mycobacterium bovis BCG and Mycobacterium tuberculosis with
intracellular efficacy in THP-1 macrophages. Int. J. Antimicrob. Agents; 32: 40.
[31] SarkarS., SarkarD. (2012)Potential use of nitrate reductase as a biomarker for the
identification of active and dormant inhibitors of Mycobacterium tuberculosis in a THP1
infection model. J. Bio Mol. Screen.; 17:966.
[32] CiapettiG., CenniE., PratelliL., PizzoferratoA. (1993)II vifro evaluation of
cell/biomaterial teraction by MTI’ assay. Biomaterials;14: 359-364.
[33] MosmannT. (1983)Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays. J. Immunol. Meth.;65:55-63.
This article is protected by copyright. All rights reserved.

[34] DzoyemJ.P., Guru S.K., PiemeC.A., KueteV., Sharma A., Khan I.A., SaxenaA.K.,
VishwakarmaR.A. (2013)Cytotoxic and antimicrobial activity of selected Cameroonian edible plants.
BMC Complement.Altern. Med.;13: 78.
[35] Alley M.C., ScudieroD.A., Monks A., HurseyM.L., Czerwinski M.J., Fine D.L., Abbott
B.J., Mayo J.G., Shoemaker R.H., Boyd M.R. (1988)Evaluation of a Soluble Tetrazolium
/Formazan Assay for Cell Growth and Drug Sensitivity in Culture Using Human and Other
Tumor Cell Lines. Cancer Res.;48: 589.
Figures
Figure 1 In vitro: Dose-dependent inhibition of 4b (a) on dormant Mycobacterium
tuberculosis H37Ra compared with Rifampicin (b) as standard drug.
Figure 2 In vitro: Dose-dependent inhibition of 4v (a) on active Mycobacterium tuberculosis
H37Ra compared with Rifampicin (b) as standard drug.
Figure 3 Ex vivo: Dose-dependent inhibition of 4b and 4v (a) on dormant Mycobacterium
tuberculosis H37Ra compared with Rifampicin (b) as standard drug.
Figure 4 Schematic representation of the aqueous solubility screening assay.
Figure 5 Percentage cytotoxicity of thiones at 100μg/mL against human cell lines
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This article is protected by copyright. All rights reserved.

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