Synthesis and biological evaluation of some bicyclic [2-(2,4-dimethylphenylthio)phenyl] aniline and its amide derivatives as potential antitubercular agents

Article abstract of DOI:10.1007/s12039-018-1424-5

Abstract: In the present investigation, a series of bicyclic [2-(2,4-dimethylphenylthio)phenyl] aniline analogues were synthesized and characterized by IR, NMR (¹H and ¹³C) and mass spectra. All newly synthesized 15 compounds were inspected for their in vitro antitubercular activity against Mycobacterium tuberculosis (MTB) H ₃₇Ra in both active and dormant state using an established XTT Reduction Menadione assay (XRMA). The titled compounds exhibited minimum inhibitory concentration (MIC90) ranging from 0.05 to?>30 (μ g/mL). The potent four compounds were further evaluated in THP-1 infection model where they demonstrated significant antitubercular activity. All the ex vivo active were further evaluated for cytotoxic activity against THP-1, MCK-7 and HeLa cell lines in order to check selectivity index. All compounds were further screened against four different bacteria to assess their selectivity towards MTB. These derivatives could be considered as a precursor structure for further design of antituberculosis agent. Graphical Abstract: SYNOPSIS A series of bicyclic [2-(2,4-dimethylphenylthio)phenyl] aniline analogues were synthesized. All newly synthesized 15 compounds were inspected for their in vitro antitubercular activity against Mycobacterium tuberculosis (MTB) H ₃₇Ra in both active and dormant state using an established XTT Reduction Menadione assay (XRMA).[Figure not available: see fulltext.].

Full text of DOI:10.1007/s12039-018-1424-5

J. Chem. Sci. (2018) 130:22
© Indian Academy of Sciences
https://doi.org/10.1007/s12039-018-1424-5
REGULAR ARTICLE
Synthesis and biological evaluation of some bicyclic
[2-(2,4-dimethylphenylthio)phenyl] aniline and its amide
derivatives as potential antitubercular agents
YOGESH PATILa, RAMESH SHINGAREa, SHAKTI CHAKRABORTYb,
RACHANA BORKUTEb, DHIMAN SARKARb and BALAJI MADJEa,∗
aDepartment of Chemistry, Vasantrao Naik Mahavidhyalaya, Aurangabad, Maharashtra 431 003, India
bCombi Chem Bio Resource Centre, National Chemical Laboratory, Pune, Maharashtra 411 008, India
E-mail: drmadjebr@gmail.com
MS received 3 September 2017; revised 29 December 2017; accepted 7 January 2018
Abstract. In the present investigation, a series of bicyclic [2-(2,4-dimethylphenylthio)phenyl] aniline
analogues were synthesized and characterized by IR, NMR (1H and 13C) and mass spectra. All newly synthesized
15 compounds were inspected for their in vitro antitubercular activity against Mycobacterium tuberculosis
(MTB) H37Ra in both active and dormant state using an established XTT Reduction Menadione assay (XRMA).
The titled compounds exhibited minimum inhibitory concentration (MIC90) ranging from 0.05 to >30 (μg/mL).
ThepotentfourcompoundswerefurtherevaluatedinTHP-1infectionmodelwheretheydemonstratedsignificant
antitubercular activity. All the ex vivo active were further evaluated for cytotoxic activity against THP-1, MCK-
7 and HeLa cell lines in order to check selectivity index. All compounds were further screened against four
different bacteria to assess their selectivity towards MTB. These derivatives could be considered as a precursor
structure for further design of antituberculosis agent.
Keywords. [2-(2,4-dimethylphenylthio)phenyl] aniline; antituberculosis activity; cytotoxicity.
1. Introduction
targets. Fatty acid amide,7 pyridine containing amide,8
10
glycine containing amide,9 , β unsaturated amide,
α
and sulphide containing amide11 were found to show
broad spectrum of biological activity. Amide bond may
be prone to hydrolysis in vivo due to the presence of
plasma amidases, leading to rapid clearance.12
Hence, in a search of new compounds sulphide based
amide derivatives were synthesized. These derivatives
screened for Mycobacterium tuberculosis (MTB) and
antimicrobial activity. Furthermore, molecular docking
studies of most active compounds against enoyl-acyl
carrier protein reductase (InhA) enzyme helped in
revealing the potential mode of action through their
interactions.
Tuberculosis (TB) is a contagious disease caused by the
Mycobacterium tuberculosis (MTB). It is one of the top
ten diseases and now ranks alongside Human Immunod-
eficiency Virus (HIV), as the main cause of death from
a single infectious disease.1 One third of the world pop-
ulation is infected with latent tuberculosis has a major
threat to humankind.2 Patient with latent tuberculosis is
in class of high risk may be converted into tuberculosis
at a later stage. Majority of TB cases were reported into
Asian, African and western pacific regions. Two Asian
countries, China and India together accounted 40% of
the total TB cases worldwide.3 Side effects and adverse
drug reactions of current anti-TB drugs coupled with
combination drug regimens and lengthy treatment dura-
tionoftencomplicatethetherapyalongexplodingcostof
the treatment.4 Hence, there is an urgent need to develop
potent and cost effective anti-TB drug.
2. Experimental
2.1 Materials and methods
Moreover, the literature reveals that sulfides5 and
amides6 are extremely attractive and rewarding research
All reagents, solvents and raw materials are commercially
available used without further purification. Melting points
*
For correspondence
Electronic supplementary material: The online version of this article (https://doi.org/10.1007/s12039-018-1424-5) contains
supplementary material, which is available to authorized users.

22 Page 2 of 7
J. Chem. Sci. (2018) 130:22
◦
υ
were determined by open capillary method and are uncor- M.p.: 97–98 C; IR (Neat) : 733 (o-substituted Ar), 810 (o, p-
rected. IR spectra (neat in cm−1) were recorded using Perkin substituted Ar), 1332 (-NO2), 1518 (-NO2) and 1567 (C=C)
Elmer Spectrum-100 analyzer. Spectrophotometer. NMR (1H cm−1; H NMR (CDCl3): δ 2.36 (s, -CH3), 2.45 (s, -CH3),
1
and13C)spectrawererecordedinCDCl3 andDMSO-d6 using 6.76 (dd, J = 8.0 Hz, Ar-H), 7.17 (dd, J = 7.0 Hz, Ar-H),
a JEOL 400 MHz FT NMR spectrometer; the chemical shifts 7.26 (dd, Ar-2H), 7.36 (m, Ar-H), 7.52 (d, J = 7.0 Hz, Ar-H)
are reported in δ ppm relative to TMS. The following abbre- and 8.31 (dd, J = 8.0 Hz, Ar-H) ppm; 13C NMR (CDCl3): δ
viations were used for spin multiplicity: s = singlet = doublet, 20.34, 21.24, 124.52, 125.91, 126.19, 127.25, 128.35, 132.16,
t = triplet, dd = double doublet, m = multiplet, brs = broad sin- 133.44, 137.12, 139.26, 141.01, 143.06 and 144.85 ppm; MS
glet. Chemical shifts for 13C NMR reported in ppm relative to (EI): m/z calculated for C14H13NO2S [M+H]+: 260.1; found
the solvent peak. Mass spectrometry was carried out using an 260.0.
Agilent LC/MSD Trap 1100 series. The reaction monitoring
and purity of the compounds were checked by thin layer chro-
2.2c Synthesis of 2-(2,4-dimethylphenylthio)benzena-
matography (TLC). Silica gel coated aluminium sheets (Silica
mine (5): In an oven-dried 3-necked round-bottomed flask,
Gel 60 F254). TLC check with hexane/ethyl acetate 9:1.
a mixture of (2, 4-dimethylphenyl) (2-nitrophenyl) sulfane
All the chemicals such as sodium salt XTT and MTT,
(4, 15 g, 58 mmol) and Pd/C (wet, 0.75◦g, 5%) in methanol
DMSO, ampicillin and rifampicin were purchased from
(75 mL) was stirred for 6h at 25–30 C under H2 atmo-
Sigma-Aldrich, USA. Dubos medium was purchased from
sphere (1 atm). Reaction monitored on TLC with hexane/ethyl
DIFCO, USA. Synthesized compounds were dissolved in
acetate 9:1. After completion, reaction mixture was filtered
DMSO and it was used as stock solution (10 mg/mL) for
through a celite pad and filter cake washed with methanol
further biological testing.
(15 mL). The combined filtrate was evaporated in vacuo to
furnish comp◦ound 5. Brown semi solid; yield:13 g, 98%;
υ
M.p.: 34–36 C; IR (Neat) : 750 (o-substituted Ar), 807
2.2 Synthesis
(o, p-substituted Ar), 1608 (C=C), 3369 (-NH2) and 3446
1
(-NH2) cm−1; H NMR (CDCl3): δ 2.26 (s, CH3), 2.39 (s,
2.2a Synthesis of 2, 4 Dimethyl thiophenol (2): In an
oven-dried 3-necked round-bottomed flask, a mixture of 2, 4
dimethyl aniline (1, 10 g, 82 mmol) in aqueous HCl (50 mL,
5 N) at 0–5 ◦C followed by dropwise addition of sodium nitrite
(10.5 g, 120 mmol) in water (30 mL). After 1 h, the diazo-
nium reaction mass was added into potassium ethyl xanthate
(19.5 g, 120 mmol), in water (50 mL) solution. The mixture
CH3), 4.22 (s, NH2), 6.69 (d, J = 8.0 Hz, Ar-H), 6.73–6.80
(m, Ar-2H), 6.85 (d, J = 8.0 Hz, Ar-H), 7.00 (s, Ar-H),
7.19–7.23 (m, Ar-H) and 7.36 (dd, J = 7.0 Hz, Ar-H)
ppm; 13C NMR (CDCl3): δ 19.98, 20.73, 115.01, 115.24,
118.78, 126.49, 127.26, 130.41, 131.13, 131.68, 135.28,
135.71, 136.55 and 148.13 ppm; MS (EI): m/z calculated for
C14H15NS [M+H]+: 230.1; found 230.0.
◦
was heated to 40–45 C for 2 h. The compound extracted
with MTBE (2×50 mL) and washed with 10% NaOH solu-
tion (80 mL). Organic layer dried over CaCl2 and evaporated
in vacuo to afford 2, 4dimethyl ethyl xanthate (2a). It was
further refluxed into ethanolic KOH solution (100 mL in 15
gm) for 4–5 h (TLC check with hexane/ethyl acetate 9:1). The
reaction was allowed to cool, quenched with water (400 mL)
and evaporated in vacuo. After acid and base treatment and
2.2d General procedure for the preparation of com-
pound (6a-h): In an oven-dried two neck round-bottomed
flask, a mixture of 2-(2,4-dimethylphenylthio)benzenamine
(5, 8.7 mmol) and sodium hydroxide (17.5 mmol in 2 mL
◦
water) in acetonitrile (10 mL) cooled 0–5 C. Acetyl chlo-
ride (10 mmol in 5 mL) added into the reaction mixture and
allowed to warm at room temperature. Stirred the reaction
mixture for 1–2 h, for completion (TLC check with hex-
ane/ethyl acetate 8:2) and evaporated in vacuo. Reaction mass
quenched with water (15 mL) and solid precipitated out. Fil-
tation followed by recrystallization from ethanol afforded
desired product (6a–h).
vacuum distillation afforded compound 2. Faint yellow liq-
uid; yield: 8.2 g, 72%; B.p.: 207–209 C; IR (Neat) : 806
◦
υ
(o, p-substituted Ar), 1477 (C=C for Ar) and 2567 (-SH sub-
stituted on Ar) cm−1; H NMR (CDCl3): δ 2.27 (s, -CH3),
1
2.30 (s, -CH3), 3.20 (s,-SH), 6.88 (d, J = 8.0 Hz, Ar-H),
6.99 (s, Ar-H) and 7.17 (1H, d, J = 8.0 Hz, Ar-H) ppm;
13C NMR (CDCl3): δ 20.79, 20.97, 126.86, 127.26, 130.27,
131.15, 135.77 and 136.22 ppm; MS (EI): m/z 137agreed for
C8H10S [M-H]+ of compound 2.
2.2d1 N-(2-(2,4-dimethylphenylthio)phenyl) acetamide
◦
(6a): White solid; yield: 95%; M.p.: 42–44 C; IR−(1Neat)
υ
:1575 (C=C), 1678 (C=O), and 3107 (-NH) cm
;
1H
2.2b Synthesis of (2,4-dimethylphenyl)(2-nitrophenyl) NMR (CDCl3): δ 2.08 (s, CH3), 2.28 (s, CH3), 2.39 (s,
sulfane (4): A mixture of 2,4-dimethyl thiophenol (2, 10 g, CH3), 6.72 (d, J = 7.0 Hz, Ar-H), 6.87 (d, J = 7.0 Hz,
72 mmol), 2-chlro nitrobenzene (3, 11.5 g, 72 mmol), and Ar-H), 7.06 (m, Ar-2H), 7.39 (q, J = 8.0 Hz, Ar-2H),
anhydrous potassium carbonate (15 g, 108 mmol) in acetoni- 8.06 (s, NH) and 8.39 (d, J = 8.0 Hz, Ar-H) ppm; 13C
trile (80 mL) was stirred and refluxed for 8–10 h (TLC check NMR (CDCl3): δ 20.16, 20.78, 24.70, 120.93, 121.21, 124.44,
with hexane/ethyl acetate 9:1). The mixture was allowed to 127.66, 128.31, 129.93, 130.51, 131.41, 135.03, 136.66,
cool and filtered. Recrystallized solid in methanol (50 mL) 139.19, 139.25 and 168.24 ppm; MS (EI): m/z calculated for
to afford compound 4. Bright yellow solid; yield:17 g, 92%; C16H17NOS [M+H]+: 272.1; found 272.1.

J. Chem. Sci. (2018) 130:22
Page 3 of 7 22
2.2d2 N-(2-(2,4-dimethylphenylthio)phenyl)-2,2,2-trif- Ar-2H), 7.14 (d, J = 6.0 Hz, Ar-2H), 7.21 (t, J = 8.0 Hz,
◦luoroacetamide (6b): White solid; yield: 98%; M.p.: 52–54 Ar-H), 7.37 (t, J = 6.0 Hz, Ar-H), 7.78 (d, J = 8.0 Hz,
C; IR (Neat) : 1576 (C=C), 1651 (C=O), and 3107 (-NH) Ar-H) and 10.26 (s, NH) ppm; 13C NMR (DMSO-d6): δ
υ
cm−1; 1H NMR (CDCl3): δ 2.28 (s, CH3), 2.38 (s, CH3), 6.75 13.76, 19.92, 20.49, 62.69, 123.89, 126.75, 127.77, 128.22,
(d, J = 7.0 Hz, Ar-H), 6.88 (d, J = 7.0 Hz, Ar-H), 7.04 (s, 128.37, 129.02, 131.52, 131.60, 132.12, 135.81, 137.65,
Ar-H), 7.22 (t, J = 7.0 Hz, Ar-H), 7.45 (t, J = 7.0 Hz, Ar- 138.66, 154.96 and 160.17 ppm; MS (EI): m/z calculated for
H), 7.51 (m, Ar-H), 8.35 (d, J = 8.0 Hz, Ar-H) and 8.95 (s, C18H19NO3S [M+H]+: 330.1; found 330.1.
NH) ppm; 13C NMR (CDCl3): δ 20.16, 20.82, 114.12, 116.99
(CF3), 121.16, 121.21, 123.5, 126.44, 127.82, 128.94, 129.45,
2.2d7N-(2-(2,4-dimethylphenylthio)pheny◦l)pivalamide
130.09, 131.64, 135.38, 136.27, 137.31, 137.43 and 154.31,
154.41, 154.6, 154.77 (q, C=O) ppm; MS (EI): m/z calculated
for C16H14F3NOS [M+H]+: 226.1; found 326.0.
(6g): White solid; yield: 88%; M.p.: 38–40 C; IR (Neat)
υ
: 1575 (C=C), 1651 (C=C), 1678 (C=O), and 3125 (-
NH) cm−1; H NMR (CDCl3): δ 1.11 (s, 3CH3), 2.25 (s,
1
CH3), 2.39 (s, CH3), 6.56 (d, J = 8.0 Hz, Ar-H), 6.82 (d,
J = 8.0 Hz, Ar-H), 7.01 (s, Ar-H), 7.09 (m, J = 7.0 Hz
2.2d3 N-(2-(2,4-dimethylphenylthio)phenyl) isobutyra-
mide (6c): White solid; yield: 90%; M.p.: 38–40 ◦C;−I1R
& 1.5 Hz, Ar-H), 7.40–7.44 (m, Ar-H), 7.48 (m, J
=
υ
7.0 Hz & 1.5 Hz, Ar-H) and 8.44–8.49 (m, Ar-2H) ppm; 13C
NMR (CDCl3): δ 19.96, 20.72, 27.20, 39.97, 120.39, 120.62,
124.18, 126.60, 127.66, 130.45, 130.57, 131.27, 135.54),
135.75, 136.04, 139.64 and 176.64 ppm; MS (EI): m/z calcu-
lated for C19H23NOS [M+H]+: 314.4; found 314.3.
(Neat) : 1528 (C=C), 1651 (C=O) and 3125 (-NH) cm
;
1H NMR (CDCl3): δ 1.08 (s, CH3), 1.10 (s, CH3), 2.26 (s,
CH3), 2.36–2.46 (m, CH3 and CH), 6.65 (d, J = 8.0 Hz,
Ar-H), 6.84 (d, J = 8.0 Hz, Ar-H), 7.02 (s, Ar-H), 7.08
(m, Ar-H), 7.38–7.46 (m, Ar-2H), 8.16 (s, NH) and 8.45
(d, J = 8.0 Hz, Ar-H) ppm; 13C NMR (CDCl3): δ 19.26,
20.10, 20.77, 36.92, 120.77, 124.26, 127.52, 127.69, 130.26,
130.59, 131.35, 135.48, 136.14, 136.37, 139.46, 139.50
and 175.07 ppm; MS (EI): m/z calculated for C17H19NOS
[M+H]+: 300.1; found 300.2.
2.2d8 N-(2-(2,4-dimethylphenylthio)phenyl) dibenza-
mide (6h): White solid; yield: 95%; M.p.: 154–156 ◦C;
υ
IR (Neat) : 1580 (C=C), 1660 (C=O) and 3057 (-NH)
cm−1 1H NMR (CDCl3): δ 2.34 (s, 2CH3), 6.71 (d,
;
J = 7.0 Hz, Ar-H), 7.03–7.09 (m, Ar-4H), 7.15 (s, Ar-H),
7.33-7.40 (m, Ar-5H), 7.44 (dd, J = 7.0 Hz, Ar-2H) and
7.91 (m, Ar-4H) ppm; 13C NMR (CDCl3): δ = 20.52, 21.10,
126.06, 126.30, 127.61, 127.89, 128.33, 128.73, 129.10,
129.73, 131.85, 132.44, 134.83, 135.91, 136.76, 136.98,
139.69, 142.07 and 173.26 ppm; MS (EI): m/z calculated for
C28H23NO2S [M+H]+: 437.57; found 437.6.
2.2d4 N-(2-(2,4-dimethylphenylthio)phenyl) propiona-
◦
mide (6d): White solid; yield: 92%; M.p.: 46–48 C;−I1R
υ
(Neat) : 1575 (C=C), 1678 (C=O) and 3125 (-NH) cm
;
1HNMR(DMSO-d6):δ 1.06(t, CH3), 2.20–2.31(m, CH3 and
CH2), 6.90 (d, J = 7.0 Hz, Ar-H), 7.0 (d, J = 7.0 Hz, Ar-H),
7.07–7.10 (m, Ar-2H), 7.15 (s, Ar-H), 7.30 (t, J = 8.0 Hz,
Ar-H), 7.37 (m, Ar-H), and 9.30 (s, NH) ppm; 13C NMR
(DMSO-d6): δ 9.76, 20.05, 20.77, 29.02, 31.16, 126.03,
127.17, 127.82, 129.58, 130.23, 131.62, 132.83, 135.90,
136.63, 138.04, 139.55, 141.68 and 175.50 ppm; MS (EI):
m/z calculated for C17H19NOS [M+H]+: 286.1; found 286.2.
2.2e General procedure for the preparation of com-
pound (6i-o): To a mixture of 2-(2,4-dimethylphenylthio)
benzenamine (5, 8.7 mmol) and acid derivative (10 mmol) in
DMF (10 mL) at 0–5 ◦C, was added dropwise coupling agent
solution CDI (17.5 mmol in 5 mL DMF) The reaction mass,
allowed to warm at room temperature and stirred for an 1 h
(TLC check with hexane/ethyl acetate 9:1). Quenched reac-
tion mass with water (15 mL) and filtered to afford desired
product (6i-o).
2.2d5 N-(2-(2,4-dimethylphenylthio)phenyl) pentana-
mide (6e): White solid; yield: 90%; M.p.: 56–58 ◦C;−I1R
υ
(Neat) :1651 (C=C), 1678 (C=O) and 3125 (-NH) cm
;
1H NMR (CDCl3): δ 0.85 (t, J = 7.0 Hz, CH3), 1.27 (m,
J = 7.0 Hz, CH2), 1.52 (m, CH2), 2.24 (t, CH2), 2.26 (s,
CH3), 2.39 (s, CH3), 6.67 (d, J = 8.0 Hz, Ar-H), 6.85 (d,
J = 8.0 Hz, Ar-H), 7.02 (s, Ar-H), 7.08 (m, J = 8.0 Hz,
Ar-H), 7.38–7.45 (m, Ar-2H), 8.08 (s, NH) and 8.43 (d,
J = 8.0 Hz, Ar-H) ppm; 13C NMR (CDCl3): δ 13.69,
20.14, 20.78, 22.20, 27.49, 37.80, 120.84, 124.31, 127.71,
127.78, 130.18, 130.64, 131.39, 131.42, 135.39, 136.32,
136.46, 139.39 and 171.38 ppm; MS (EI): m/z calculated for
C19H23NOS [M+H]+: 314.2; found 314.3.
2.2e1 N-(2-(2,4-dimethylphenylthio)phenyl)-2-phenyl-
acetamide (6i): White solid; yield: 82%; M.p.: 78–80 ◦C;−I1R
υ
(Neat) : 1521 (C=C), 1677 (C=O) and 3245 (-NH) cm
;
1H NMR (DMSO-d6): δ 2.22 (s, CH3), 2.27 (s, CH3), 3.67
(s, CH2), 6.96 (m, Ar-3H), 7.10 (t, J = 7.0 Hz, Ar-H), 7.14
(s, Ar-H), 7.28–7.22 (m, Ar-6H), 7.61 (d, J = 7.0 Hz, Ar-
H) and 9.47 (s, NH) ppm; 13C NMR (DMSO-d6): δ 20.09,
20.75, 42.99, 125.25, 126.21, 126.83, 127.78, 127.89, 128.54,
128.86, 129.26, 129.38, 131.13, 131.74, 131.96, 135.72,
136.93, 137.88, 139.08 and 169.63 ppm; MS (EI): m/z calcu-
lated for C22H21NOS [M+H]+: 348.1; found 348.1.
2.2d6 Ethyl(2-(2,4-dimethylphenylthio)phenylcarbam-
oyl)formate (6f): White solid; yield: 90%; M.p.: 50–52 ◦C;
−1
υ
IR(Neat) :1639(C=C), 1651(C=O)and3125(-NH)cm
;
1H NMR (DMSO-d6): δ 1.29 (t, J = 7.0 Hz, CH3), 2.25 (s, 2.2e2 (2-(2,4-dimethylphenylthio)phenylcarbamoyl)
CH3), 2.28 (s, CH3), 4.29 (q, J = 7.0 Hz, CH2), 6.99 (m, (phenyl)methylacetate (6j):Whitesolid;yield:80%;M.p.:

22 Page 4 of 7
J. Chem. Sci. (2018) 130:22
◦
υ
86–88 C; IR (Neat) : 1516 (C=C), 1578 (C=C), 1693 (d, J = 7.0 Hz, Ar-H), 7.14 (s, Ar-H), 7.16–7.19 (m, Ar-2H),
(C=O), 1744 (C=O) and 3333 (-NH) cm−1
;
1H NMR 7.26 (t, J = 6.0 Hz, Ar-H), 7.42 (d, J = 6.0 Hz, Ar-H), 8.99
(CDCl3): δ = 2.06 (s, CH3), 2.29 (s, CH3), 2.39 (s, CH3), 6.12 (s, Ar-H) and 9.87 (s, -NH) ppm; 13C NMR (DMSO-d6): δ
(s, CH), 6.53 (d, J = 8.0 Hz, Ar-H), 6.83 (d, J = 8.0 Hz, 12.02, 20.0, 20.55, 111.53, 126.60, 127.07, 127.61, 127.74,
Ar-H), 7.04 (s, Ar-H), 7.12 (m, Ar-H), 7.28 (m, Ar-6H), 7.39– 128.46, 129.12, 131.53, 133.24, 133.70, 135.16, 138.28,
7.44 (m, Ar-H), 7.48 (m, Ar-H), 8.49 (d, J = 8.0 Hz, Ar-H), 140.06, 149.01, 159.37 and 172.60 ppm; MS (EI): m/z calcu-
and 9.09 (s, -NH) ppm; 13C NMR (CDCl3): δ 20.01, 20.59, lated for C19H18N2O2S [M+H]+: 339.1; found 339.0.
20.79, 75.81, 120.43, 125.0, 126.66, 127.26, 127.80, 128.70,
2.2e7 N-(2-(2,4-dimethylphenylthio)phenyl)-5-chlorot-
128.97, 130.59, 130.65, 131.39, 135.05, 135.52, 135.95,
136.14, 136.20, 138.91, 166.37and168.72ppm;MS(EI):m/z
calculated for C24H23NO3S [M+H]+: 406.1; found 406.1.
hiophene-2-carboxamide (6o): White solid; yield: 90%;
◦
υ
M.p.: 74–76 C; IR (Neat) : 1536 (C=C), 1633 (C=O) and
3250 (-NH) cm−1; H NMR (DMSO-d6): δ 2.22 (s, CH3),
1
2.26 (s, CH3), 6.85 (d, J = 7.0 Hz, Ar-H), 7.01 (s, Ar-H),
7.14 (d, J = 7.0 Hz, Ar-H), 7.19 (m, Ar-2H), 7.25 (m,
Ar-2H), 7.41 (d, J = 8.0 Hz, Ar-H), 7.86 (d, J = 4.0 Hz,
Ar-H) and 10.20 (s, -NH) ppm; 13C NMR (DMSO-d6): δ
20.03, 20.58, 126.54, 127.24, 127.74, 127.78, 128.21, 128.33,
128.87, 129.14, 131.56, 133.78, 133.85, 133.98, 134.97,
138.42, 140.24, and 159.07 ppm; MS (EI): m/z calculated for
C19H16ClNOS2[M]+: 373.8; found 373.6, 375.5 (Table 1).
2.2e3 N-(2-(2,4-dimethylphenylthio)phenyl)-5-methyl-
pyrazine-2-carboxamide (6k): White solid; yield: 85%;
◦
υ
M.p.: 100–102 C; IR (Neat) : 1576 (C=C), 1687 (C=O)
and 3289 (-NH) cm−1; H NMR (CDCl3): δ 2.22 (s, CH3),
1
2.46 (s, CH3), 2.66 (s, CH3), 6.82 (m, Ar-2H), 6.99 (s, Ar-
H), 7.11–7.15 (m, Ar-H), 7.46 (m, Ar-2H), 8.39 (s, Ar-H),
8.63 (d, J = 8.0 Hz, Ar-H), 9.30 (s, Ar-H) and 10.65
(s, -NH) ppm; 13C NMR (CDCl3): δ 20.27, 20.77, 21.80,
120.68, 122.80, 124.73, 127.48, 128.96, 129.85, 130.66,
131.17, 135.32, 136.60, 137.21, 138.55, 141.88, 142.14,
143.41, 157.22 and 161.10 ppm; MS (EI): m/z calculated for
C20H19N3OS [M+H]+: 350.1; found 349.9.
3. Results and Discussion
3.1 Chemistry
2.2e4 N-(2-(2,4-dimethylphenylthio)phenyl)furan-2-
Synthesis of the target compounds 6a–o were achieved
through the straight pathway illustrated in Scheme 1
◦carboxamide (6l): White solid; yield: 88%; M.p.: 120–122
υ
C; IR (Neat) : 1578 (C=C), 1651 (C=C), 1675 (C=O),
1702 (C=O), and 3309 (-NH) cm−1; 1H NMR (DMSO-d6): starting from commercially available 2,4 dimethyl ani-
line (1). In the first step, Leuckart reaction13,14 allows the
δ 2.25 (s, CH3), 2.28 (s, CH3), 6.67 (s, Ar-H), 6.96 (s, Ar-
H), 7.03 (m, Ar-2H), 7.08 (d, J = 8.0 Hz, Ar-H), 7.15 (t,
preparation of 2, 4 dimethyl thiophenol (2). It involved
J = 7.0 Hz, Ar-H), 7.29 (m, Ar-2H), 7.71 (t, J = 8.0 Hz,
formation of an aryl diazonium salt by using sodium
nitrite in acidic condition. Further reaction with potas-
siumalkyl xanthate produced 2, 4 dimethyl aryl xanthate
Ar-H), 7.88 (s, Ar-H) and 9.75 (s, -NH) ppm; 13C NMR
(DMSO-d6): δ 19.92, 20.48, 112.28, 114.94, 125.21, 126.27,
127.67, 127.72, 128.99, 129.87, 131.01, 131.46, 132.24,
(2a), in situ basic hydrolysis produced 2, 4 dimethyl
136.14, 137.77, 139.12, 145.74, 147.14 and 156.0 ppm; MS
thiophenol (2). This compound was purified by vacuum
(EI): m/z calculated for C18H15NO2S [M+H]+: 324.1; found
distillation and identity confirmed by spectral data. The
324.3.
IR spectra suggest –SH characteristic band 2567 cm−1,
1H NMR suggest four singlet at δ 2.27, 2.30, 3.20 and
2.2e5 2-(2-(2,4-dimethylphenylthio)phenylcarbamoyl)
6.99 belongs to aromatic C-2 and C-4 methyl sub-
stitution, D2O exchangeable –SH and aromatic C-3
◦benzoic acid (6m): White solid; yield: 87%; M.p.: 126–128
υ
C; IR (Neat) : 1513 (C=C), 1690 (C=O), 1702 (C=O),
and 3301 (-NH) cm−1 1H NMR (DMSO-d6): δ 2.24 (s, respectively. Remaining two δ 6.88 and 7.17 belong to
;
CH3), 2.29 (s, CH3), 6.88 (d, J = 7.0 Hz, Ar-H), 7.03 (d,
J = 7.0 Hz, Ar-H), 7.15 (m, Ar-3H), 7.28 (t, J = 6.0 Hz, Ar-
H), 7.42 (d, J = 6.0 Hz, Ar-H) 7.56–7.64 (m, Ar-3H), 7.86 (d,
J = 7.0 Hz, Ar-H), 9.86 (s, -NH) and 13.06 (s, -COOH) ppm;
13C NMR (DMSO-d6): δ 20.10, 20.80, 115.3, 118.91, 125.25,
126.21, 126.83, 127.8, 128.7, 128.86, 129.26, 129.38, 131.13,
131.74, 131.96, 135.72, 136.93, 137.88, 139.08, 167.46 and
169.88 ppm; MS (EI): m/z (%) 378 (M++ 1, 100).
C-5 and C-6 doublet with ortho coupling J = 8.0 Hz.
Further nucleophilic substitution on 2-chloro nitro-
benzene (3) with 2, 4 dimethyl thiophenol (2) yielded (2,
4-Dimethylphenyl)(2-nitrophenyl)sulfane (4).15,16 This
on further catalytic hydrogenation produced [2-(2,4-
dimethylphenylthio)phenyl] aniline (5).17 The inter-
mediate amine compound formed amide bond with
different acyl chloride or acid derivatives in presence
of inorganic base or coupling agent with suitable sol-
vent to get desired compounds 6a–o with moderate to
good yields. The structures of all newly synthesized
compounds 6a–o were confirmed by IR, NMR, Mass
2.2e6 N-(2-(2,4-dimethylphenylthio)phenyl)-3-methyli-
soxazole-4-carboxamide (6n): White solid; yield: 85%;
◦
υ
M.p.: 80–82 C; IR (Neat) : 1578 (C=C), 1702 (C=O) and
3028 (-NH) cm−1; H NMR (DMSO-d6): δ 2.22 (s, CH3),
1
2.27 (s, CH3), 2.65 (s, CH3), 6.87 (d, J = 7.0 Hz, Ar-H), 7.01 spectral analyses and were in full agreement with

J. Chem. Sci. (2018) 130:22
Page 5 of 7 22
Table 1. Physical data of bicyclic [2-(2,4-dimethylphenylthio)phenyl]
aniline derivatives (6a–o).
Entry Compound
R
Yields (%) Melting points (oC)
1
2
3
4
5
6
7
8
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
Methyl
Trifluoro methyl
2-propane
Ethyl
Butyl
Ethyl Oxalate
tButyl
Dibenzoyl
95
98
90
92
90
90
88
95
82
80
85
88
87
85
90
42-44
52-54
38-40
46-48
56-58
50-52
38-40
154-156
78-80
86-88
100-102
120-122
126-128
80-82
9
Phenyl
10
11
12
13
14
15
O-acetyl mandelic
4-methlyl Pyrazine
2-Furan
Phthalic acid
3-methyl Isoxazole
5-chloroThiophene
74-76
Cl
NH2
(b)
(a)
SH
S
O
NO2
+
S
(1)
(2)
(3)
(2a)
(c)
R
NO2
NH2
HN
O
d
( )
(e)
S
S
S
(6a-o)
(5)
(4)
Scheme 1. Synthetic route for the preparation of title compounds 6a–o. Reagents and conditions: (a)
NaNO2, HCl: water, potassium xhanthate, 25–30 ◦C, (b) KOH, Ethanol, 76–78 ◦C, 72%; (c) K2CO3, Ace-
tonitrile, 78–82 ◦C, 92%; (d) Pd/C, H2, methanol, 25–30 ◦C, 98%; (e) Acyl chloride derivative, NaOH, ACN,
5 − 30◦C or acid derivative, CDI, DMF, 5–30 ◦C, 80–98%.
proposed structures. The IR spectra of synthesized com- using an established XTT Reduction Menadione Assay
pound 6a–o showed band 3050–3107 cm−1and 1660– (XRMA). Among the synthesized compounds, 6a, 6b,
1675 cm−1corresponding to –NH and >C=O stretching 6d, 6g, 6j, 6l, 6m and 6n displayed activity towards
of amide. In 1H NMR aromatic protons were observed at M. tuberculosis H37Ra (Dormant) value less than
δ 6.5–8.0ppm. Characteristicpeakaround160–170ppm 30 μg/mL. The results of the screening are tabulated
in 13C NMR confirmed the presence of >C=O group.
in Table S1.
Active compounds from in vitro screening were
screenedinTHP-1infectionmodel. Compoundsexhibit-
ing MIC90 less than 10 μg/mL was considered as
potent compound. Compound 6a, 6j, 6n showed excel-
lent activity in infection models (Table S2). Moreover,
compound 6n showed significant activity with MIC90
6.04 and 2.72 μg/mL respectively in the active and dor-
mant infection models.
3.2 Biological evaluation
In a standard primary screening, all the newly syn-
thesized compounds (6a–o) were tested for their in
vitro antitubercular activity against M. tuberculosis
H37Ra (ATCC 25177) at 3 μg/mL concentrations

22 Page 6 of 7
J. Chem. Sci. (2018) 130:22
After performing antitubercular screening, the active Supplementary Information (SI)
compounds from ex-vivo infection model were evalu-
All additional information pertaining to molecular biological
ated for cytotoxicity on three human cancerous cell lines
THP-1, HeLa, MCF-7 using MTT assay and IC50 values
were determined. The results are recorded in Table S4.
It has been noticed that compounds 6j and 6m showed
GI90 values >100 μg/mL in all the cancer cell lines.
Further, the selectivity of the compounds (6a, 6j
and 6n) towards human cell lines against MTB was
determined via their selectivity index (Table S5). The
selectivity index reflects the quantity of the compound
that is active against MTB but non-toxic towards host
cells. A higher selectivity index indicates that the com-
pound can be used as a therapeutic agent. It was found
that 6n have a selectivity index of ≥10 at In vitro and
Ex vivo model of MTB when compared to both HeLa
and THP-1 cells.
activity, docking study, ADME study and spectral data for
the characterization of compounds are given in the supporting
information. Supplementary Information is available at www.
ias.ac.in/chemsci.
Acknowledgements
Authors are thankful to the Department of Science & Technol-
ogy, New Delhi, India for financial support under Fast Track
Young Scientist Schemes and are grateful to the Principal and
Head, Department of Chemistry for providing research facil-
ities.
Compounds 6a-o were further screened against
(Gram positive B. subtilis and S. aureus, Gram negative
E. coli and P. aeroginosa) at 3 μg/mL concentration, to
assess their selectivity towards MTB. The antimicrobial
activity is summarized in Table S3. None of the com-
pounds showed significant activity towards any of the
screened bacterial strain.
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4. Conclusion
Insummary, aseriesofbicyclic[2-(2,4-dimethylphenyl-
thio)phenyl] aniline and its amide derivatives containing
aliphatic, aromatic and heterocyclic moieties were syn-
thesized, characterized and evaluated for antituberculo-
sis activity. Most of the compounds displayed good anti-
tuberculosis activity. Among all tested compounds, 6j
(active state MIC90: 24.9 μg/mL, dormant state MIC90:
5.78 μg/mL) and 6n (active state MIC90: 6.04 μg/mL,
dormant state MIC90: 2.72 μg/mL) demonstrated sig-
nificant inhibition against all the strains tested. It may be
helpful for further screening, designing and developing
more potent antituberculosis agents.

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