Synthesis and antimycobacterial activity of novel thiadiazolylhydrazones of 1-substituted indole-3-carboxaldehydes

Article abstract of DOI:10.1111/cbdd.12230

A series of novel thiocarbohydrazones of substituted indoles and their corresponding thiadiazole derivatives were prepared, and their structures were confirmed by different analytical and spectroscopic methods. The derivatives were prepared by a sequential synthetic strategy including substitution at N-1 position of indole ring by various aliphatic and benzylic substituents, followed by condensation with thiocarbohydrazide, and finally cyclization by triethyl orthoformate. The derivatives were tested for their antimycobacterial activity against Mycobacterium bovis BCG, and the results revealed that among the synthesized compounds, thiadiazole derivatives 4e, 4f, 4n, 4p, 4q, and 4t exhibited the highest activity with IC₅₀ value of 3.91 μg/mL. The results indicate that the thiadiazole moiety plays a vital role in exerting antimycobacterial activity. A series of indole-based thiadiazole derivatives with various substitutions at N-1 position of indole ring have been synthesized and tested for their antimycobacterial activity. Compounds 4e (R = butyl), 4f (R = pentyl), 4n (R = 4-fluorobenzyl), 4p (R = 4-chlorobenzyl), 4q (R = 4-bromobenzyl) and 4t (R = 4-methoxybenzyl) were the most potent derivatives with IC₅₀ value of 3.91 μg/mL.

Full text of DOI:10.1111/cbdd.12230

Chem Biol Drug Des 2013
Research Article
Synthesis and Antimycobacterial Activity of Novel
Thiadiazolylhydrazones of 1-Substituted Indole-3-
carboxaldehydes
Kamaleddin Haj Mohammad Ebrahim Tehrani1,
Despite the efforts to control the tuberculosis infection, the
prevalence of tuberculosis (TB) has been growing fast in the
recent years, and annual number of new TB cases is higher
than any other time in history (1). TB has been the cause of
Vida Mashayekhi1, Parisa Azerang2,
Soroush Sardari2, Farzad Kobarfard3,4 and
Kobra Rostamizadeh1,5,
*
1.1 million deaths among HIV-negative and 0.35 million
among HIV-positive patients in 2010 globally (2). The emer-
gence of multidrug-resistant (MDR) and extensively drug-
resistant (XDR) strains of Mycobacterium tuberculosis and
also coinfection of HIV and TB (HIV/TB) make the antituber-
cular therapy more and more challenging (3). This condition
suggests that the discovery of new antitubercular drugs with
high efficacy and unique modes of action is urgently needed.
1Department of Medicinal Chemistry, School of Pharmacy,
Zanjan University of Medical Sciences, Gavazang Road,
Zanjan 45139-56184, Iran
2Drug Design and Bioinformatics Unit, Department of
Medical Biotechnology, Biotechnology Research Center,
Pasteur Institute, Tehran 13164, Iran
3Department of Medicinal Chemistry, School of Pharmacy,
Shahid Beheshti University of Medical Sciences, Vali Asr
Ave. Niayesh Junction, Tehran 14155-6153, Iran
4Phytochemistry Research Center, Shahid Beheshti
University of Medical Sciences, Vali Asr Ave. Niayesh
Junction, Tehran 14155-6153, Iran
In antimycobacterial drug discovery efforts, thiosemicarbaz-
one derivatives have attracted the attention of researchers,
mostly because of the development of the drug thiacetaz-
one as a second-line choice in antituberculosis drug ther-
apy (4). The action mechanism of thiosemicarbazones is
not clearly understood. Studies on thiacetazone suggest
that the drug is metabolized by EthA, the mycobacterial
enzyme that is considered as the activator of thiocarbonyl
antimycobacterial drugs (5–7). Dover et al. (5) have shown
that thiacetazone and two other thiosemicarbazone ana-
logues affect the cell wall biosynthesis by interfering with
the enzyme fatty acid cyclopropane synthase.
5Pharmaceutical Nanotechnology Research Center, Zanjan
University of Medical Sciences, Gavazang Road, Zanjan
45139-56184, Iran
*Corresponding author: Kobra Rostamizadeh,
rostamizadeh@zums.ac.ir and Rostamizadeh@gmail.com
A series of novel thiocarbohydrazones of substituted
indoles and their corresponding thiadiazole derivatives
were prepared, and their structures were confirmed
by different analytical and spectroscopic methods.
The derivatives were prepared by a sequential syn-
thetic strategy including substitution at N-1 position of
indole ring by various aliphatic and benzylic substitu-
ents, followed by condensation with thiocarbohydraz-
ide, and finally cyclization by triethyl orthoformate.
The derivatives were tested for their antimycobacterial
activity against Mycobacterium bovis BCG, and the
results revealed that among the synthesized com-
pounds, thiadiazole derivatives 4e, 4f, 4n, 4p, 4q, and
4t exhibited the highest activity with IC50 value of
3.91 lg/mL. The results indicate that the thiadiazole
moiety plays a vital role in exerting antimycobacterial
activity.
During our studies on thiadiazolylhydrazones of aromatic
aldehydes, we have recently examined the bioactivity of
these derivatives as cyclic analogs of active antimycobac-
terial thiosemicarbazones (Figure 1A). Among these
derivatives, indole-3-carboxaldehyde 1,3,4-thiadiazol-2-yl-
hydrazone showed promising inhibitory activity against
Mycobacterium bovis BCG (MIC = 7.81 lg/mL) (8). We
also found that antimycobacterial activity was lost by sub-
stitution of bromine at C-5 of indole ring. Considering the
possible role of bromine substituent in both terms of size
and lipophilicity, it could be suggested that steric
hindrance and/or lipophilicity are the possible responsible
parameters for the observed bioactivities (Figure 1B).
Key words: antimycobacterial, indole, structure–activity rela-
In continuation of our previous work on the structure–activ-
ity relationship of 1,3,4-thiadiazol-2-ylhydrazone of substi-
tuted indoles and also to discover novel derivatives with
improved antimycobacterial activity, we evaluated the inhib-
itory activity of novel N-1-substituted indole-3-carboxalde-
tionship, thiadiazolylhydrazone, thiosemicarbazones
Received 14 March 2013, revised 18 August 2013 and
accepted for publication 5 September 2013
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12230
1

Haj Mohammad Ebrahim Tehrani et al.
A
B
Figure 1: (A) Thiadiazolylhydrazones as novel analogues of active antitubercular thiosemicarbazones, (B) Indole-derived
thiadiazolylhydrazones tested against Mycobacterium bovis BCG.
hyde thiadiazolylhydrazones in this study. Various aliphatic
and benzylic substituents with diverse physicochemical
characters have been introduced as R group on indole ring
(Figure 2) in the structures of the synthesized derivatives.
(10 mL), and the mixture was stirred for 5 min. Haloak-
anes or halomethyl-substituted aromatic compounds
(5 mmol) were then added to the reaction mixture. After
the disappearance of indole-3-carboxaldehyde as evi-
denced by TLC, the mixture was added to water (40 mL)
and extracted by dichloromethane (3 9 20 mL). The
organic phase was washed with water (20 mL) and brine
(2 9 20 mL), dried over anhydrous sodium sulfate, and
concentrated in a water bath set at 40 °C. The residue
was surrounded by ice water and precipitated by addition
of precooled n-hexane. Compounds 2 g and 2i were iso-
lated as oily liquid. The intermediates thus obtained were
used for the next step without further purification.
Experimental Section
Chemistry
All the reagents were freshly used, and solvents were used
as received. The reaction progress was monitored by thin-
layer chromatography (TLC) using TLC silica gel 60 F254
plates (Merck, Darmstadt, Germany) and chloroform/meth-
anol or ethyl acetate/n-hexane were used as mobile
phase. Melting points were measured by an Electrothermal
9200 apparatus and were uncorrected. The 1H-NMR
spectra were taken on a 400 MHz Bruker Avance DRX
spectrometer in d6-Acetone or d6-DMSO, and TMS was
used as internal standard. FT-IR spectra were obtained by
a Bruker Tensor27 spectrophotometer, and KBr was used
as diluent. ESI-MS spectra were obtained by Agilent 6410
Triple Quad. LC/MS. The derivatives were analyzed for C,
H, N, and S on a Costech model 4010, and the obtained
values were in agreement with the proposed structures
within Æ 0.4% of the theoretical values. The calculated
LogP values of the derivatives were obtained using CHEMBIO-
DRAW 11.0 software (Cambridgesoft, Waltham, MA, USA).
The synthesis and spectral characterization of compounds
2c (9), 2d (10), 2e (11), 2f (10), 2l (12), 2m (13), 2n (14),
2o (15), 2p, 2q, 2r, 2s (13), 2u (16), 3a, and 4a (8) are
available in the literature. Compounds 2b and 2k were pur-
chased from Sigma-Aldrich (Taufkirchen, Germany).
1-Hexylindole-3-carboxaldehyde (2g)
1H-NMR (Acetone-d6) d: 0.88 (3H; t, J = 7.0 Hz; CH3), 1.25–
1.42 (6H; m; CH3–(CH2)3–), 1.93 (2H; quintet, J = 7.0 Hz; N–
CH2–CH2–), 4.35 (2H; t, J = 7.2 Hz; N–CH2–), 7.29 (1H; t,
J = 7.4 Hz; indole C5-H), 7.34 (1H; t, J = 7.4 Hz; indole C6-
H), 7.61 (1H; d, J = 8.0 Hz; indole C7-H), 8.18 (1H; s; indole
C2-H), 8.26 (1H; d, J = 7.6 Hz; indole C4-H), 10.01 (1H; s;
aldehyde H). ESI-MS m/z: 230 (M + H+).
1-(Cyclopropylmethyl)indole-3-carboxaldehyde (2h)
1H-NMR (Acetone-d6) d: 0.51 (2H; m; cyclopropyl C2,3
-
Hɑ), 0.66 (2H; m; cyclopropyl C2,3-Hb), 1.45 (1H; m; cyclo-
propyl C1-H), 4.2 (2H; d, J = 6.8 Hz; N–CH2–), 7.29 (1H;
t, J = 7.4 Hz; indole C5-H), 7.33 (1H; t, J = 7.6 Hz; indole
C6-H), 7.63 (1H; d, J = 8.0 Hz; indole C7-H), 8.27 (2H;
second order d; indole C2,4-H), 10.10 (1H; s; aldehyde H).
ESI-MS m/z: 200 (M + H+).
General procedure for N-alkylation/arylmethylation
of indole-3-carboxaldehyde
Potassium carbonate (690 mg, 5 mmol) and indole-3-car-
boxaldehyde (435 mg, 3 mmol) were added to DMSO
1-(Cyclobutylmethyl)indole-3-carboxaldehyde (2i)
1H-NMR (Acetone-d6) d: 1.91 (2H; m; cyclobutyl CH2),
2.03–2.09 (2H; m; cyclobutyl CH2), 2.94 (1H; m; cyclobutyl
C1-H), 4.33 (2H; d, J = 7.6 Hz; N–CH2), 7.27–7.36 (2H;
m; indole C5,6-H), 7.60 (1H; d, J = 8.0 Hz; indole C7-H),
8.14 (1H; s; indole C2-H), 8.27 (1H; d, J = 7.6 Hz; indole
C4-H), 10.05 (1H; s; N-H). ESI-MS m/z: 214 (M + H+).
1-(Cyclohexylmethyl)indole-3-carboxaldehyde (2j)
1H-NMR (Acetone-d6) d: 1.15 (2H; m; cyclohexyl H), 1.22
(3H; m; cyclohexyl H), 1.64–1.74 (5H; m; cyclohexyl H),
Figure 2: General structures of the newly designed derivatives; R
includes aliphatic and benzylic substituents.
2
Chem Biol Drug Des 2013

Antimycobacterial Thiadiazolylhydrazones
1.98 (1H; m; cyclohexyl C1-H), 4.20 (2H; d, J = 7.2 Hz;
N–CH2), 7.28 (1H; t, J = 7.6 Hz; indole C5-H), 7.34 (1H; t,
J = 7.4 Hz; indole C6-H), 7.61 (1H; d, J = 8.0 Hz; indole
C7-H), 8.14 (1H; s; indole C2-H), 8.25 (1H; d, J = 7.6 Hz;
indole C4-H), 10.01 (1H; s; aldehyde H). ESI-MS m/z: 242
(M + H+).
J = 7.2 Hz; indole C5-H), 7.28 (1H; t, J = 7.4 Hz; indole
C6-H), 7.49 (1H; d, J = 8.0 Hz; indole C7-H), 7.81 (1H;
s; imine H), 8.26 (1H; s; indole C2-H), 8.32 (1H; d,
J = 7.2 Hz; indole C7-H), 9.11 and 11.19 (each 1H; s;
NH). IR (KBr) cmÀ1: 3265, 1677, 1610, 1513, 1321,
1240, 1017, 729. ESI-MS m/z: 234 (M + H+). Anal.
Calcd for C11H13N5S (233.29): C, 53.42; H, 5.30; N,
28.32; S, 12.97. Found: C, 53.52; H, 5.28; N, 28.28; S,
13.00.
1-(4-Methoxybenzyl)indole-3-carboxaldehyde (2t)
1H-NMR (Acetone-d6) d: 2.31 (3H; s; OCH3), 5.42 (2H; s;
methylene H), 7.18 (2H; d, J = 8.0 Hz; Ar C3,5-H), 7.24
(2H; d, J = 8.0 Hz; Ar C2,6-H), 7.27–7.31 (2H; m; indole
1-Ethylindole-3-carboxaldehyde
C
5,6-H), 7.55 (1H; d, J = 7.6 Hz; indole C7-H), 8.26 (2H;
thiocarbohydrazone (3c)
m; indole C2,4-H), 10.03 (1H; s; NH). ESI-MS m/z: 266
(M + H+).
Recrystallized from ethanol. Mp 210–213 °C. Yield: 69%.
1H-NMR (DMSO-d6) d: 1.40 (3H; t, J = 7.2 Hz; CH3), 4.23
(2H; quartet, J = 7.2 Hz; CH3–CH2–), 4.84 (2H; br s; NH),
7.18 (1H; t, J = 7.6 Hz; indole C5-H), 7.26 (1H; t,
J = 7.2 Hz; indole C6-H), 7.54 (1H; d, J = 8.4 Hz; indole
C7-H), 7.89 (1H; s; imine H), 8.26 (1H; s; indole C2-H),
8.30 (1H; d, J = 8.0 Hz; indole C4-H), 9.11 and 11.18
(each 1H; s; NH). IR (KBr) cmÀ1: 3286, 1662, 1617, 1516,
1311, 1233, 991, 735. ESI-MS m/z: 248 (M + H+). Anal.
Calcd for C12H15N5S (247.32): C, 55.13; H, 5.79; N,
26.80; S, 12.27. Found: C, 55.03; H, 5.29; N, 26.89; S,
12.29.
1-(Phenylsulfonyl)indole-3-carboxaldehyde (2v)
The title compound was prepared by an alternative
method: Indole-3-carboxaldehyde (435 mg, 3 mmol) was
dissolved in 1 mL of DMF and the flask was surrounded
by an ice water bath. Then, dichloromethane (6 mL) and
triethylamine (456 lL, 3.3 mmol) were added followed by
dropwise addition of benzenesulfonyl chloride (0.53 g,
3.02 mmol) in dichloromethane (3 mL). The mixture was
stirred for 2 h at 0 °C and 10 h at room temperature. The
mixture was then diluted by 15 mL of dichloromethane
and washed with water (2 9 20 mL), saturated solution of
NaHCO3 (2 9 20 mL) and brine (2 9 20 mL). The organic
layer was concentrated at 40 °C and the residue was pre-
cipitated by addition of cold n-hexane. Compound 2t was
thus obtained as white crystals (692 mg, 81%). Mp
157–159 °C [Lit. 157.5–158.5 °C (17)]. ESI-MS m/z: 288
(M + H+).
1-Propylindole-3-carboxaldehyde
thiocarbohydrazone (3d)
Mp 217–220 °C. Yield: 77%. 1H-NMR (DMSO-d6) d:
0.85 (3H; t, J = 7.4; CH3), 1.82 (2H; m; CH3–CH2–),
4.16 (2H; t, J = 7.5 Hz; N–CH2), 4.84 (2H; br s; NH),
7.17 (1H; t, J = 7.2 Hz; indole C5-H), 7.25 (1H; t,
J = 7.2 Hz; indole C6-H), 7.53 (1H; d, J = 8.0 Hz; indole
C7-H), 7.87 (1H; s; imine H), 8.26 (1H; s; indole C2-H),
8.31 (1H; d, J = 7.6 Hz; indole C4-H), 9.12 and 11.19
(each 1H; s; NH). IR (KBr) cmÀ1: 3286, 3174, 1660,
1614, 1505, 1279, 983, 740. ESI-MS m/z: 276 (M + H+).
Anal. Calcd for C13H17N5S (275.37): C, 56.70; H, 6.22;
N, 25.43; S, 11.64. Found: C, 56.81; H, 6.21; N, 25.49;
S, 11.62.
General procedure for the synthesis of
thiocarbohydrazones 3a–v
Thiocarbohydrazide (233 mg, 2.2 mmol) and glacial ace-
tic acid (6 drops) were added to water (6 mL) and the
mixture was heated at 90 °C. After the complete disso-
lution of thiocarbohydrazide, a solution of N-substituted
indole-3-carboxaldehyde derivative (2 mmol) in 15 mL of
absolute ethanol was added dropwise and the mixture
was heated under reflux for further 1 h. The mixture
was then cooled to room temperature and the solids
were filtered, washed with water and n-hexane and
dried to afford the thiocarbohydrazones 3a–v. The prod-
ucts obtained by the above procedure had acceptable
analytical purity, but in some cases, the crudes were
recrystallized from appropriate solvents to improve the
purity.
1-Butylindole-3-carboxaldehyde
thiocarbohydrazone (3e)
Mp 202–205 °C. Yield: 83%. 1H-NMR (DMSO-d6) d: 0.89
(3H; t, J = 7.6 Hz; CH3), 1.26 (2H; sextet, J = 7.6 Hz; –
CH2–CH3), 1.77 (2H; quintet, J = 7.6 Hz; –CH2–CH2–
CH3), 4.19 (2H; t, J = 7.6 Hz; N–CH2–), 4.85 (2H; br s;
NH), 7.18 (1H; t, J = 7.2 Hz; indole C5-H), 7.25 (1H; t,
J = 7.2 Hz; indole C6-H), 7.53 (1H; d, J = 8.0 Hz; indole
C7-H), 7.87 (1H; s; imine H), 8.26 (1H; s; indole C2-H),
8.29 (1H; d, J = 7.6 Hz; indole C4-H), 9.11 and 11.17
(each 1H; s; NH). IR (KBr) cmÀ1: 3291, 3248, 1614,
1505, 1281, 1180, 738. ESI-MS m/z: 290 (M + H+). Anal.
Calcd for C14H19N5S (289.40): C, 58.10; H, 6.62; N,
24.20; S, 11.08. Found: C, 58.08; H, 6.60; N, 24.22; S,
11.06.
1-Methylindole-3-carboxaldehyde
thiocarbohydrazone (3b)
Mp 228–229 °C. Yield: 74%. 1H-NMR (DMSO-d6) d:
3.82 (3H; s; methyl H), 4.85 (2H; s; NH), 7.19 (1H; t,
Chem Biol Drug Des 2013
3

Haj Mohammad Ebrahim Tehrani et al.
1-Pentylindole-3-carboxaldehyde
thiocarbohydrazone (3f)
994, 735. ESI-MS m/z: 302 (M + H+). Anal. Calcd for
C15H19N5S (301.41): C, 59.77; H, 6.35; N, 23.24; S,
Mp 197–199 °C. Yield: 79%. 1H-NMR (DMSO-d6) d: 0.86
(3H; t, J = 7.0 Hz; CH3), 1.19-1.33 (4H; m; CH2), 1.78
(2H; quintet, J = 7.2 Hz; CH2), 4.19 (2H; t, J = 7.2 Hz;
N–CH2–), 4.87 (2H; br s; NH), 7.17 (1H; t, J = 7.2 Hz;
indole C5-H), 7.25 (1H; t, J = 7.2 Hz; indole C6-H), 7.52
(1H; d, J = 8.0 Hz; indole C7-H), 7.88 (1H; s; imine H),
8.26 (1H; s; indole C2-H), 8.30 (1H; d, J = 8.0 Hz; indole
10.64. Found: C, 59.90; H, 6.36; N, 23.19; S, 10.63.
1-(Cyclohexylmethyl)indole-3-carboxaldehyde
thiocarbohydrazone (3j)
Mp 227–230 °C. Yield: 80%. 1H-NMR (DMSO-d6) d:
0.98–1.16 (5H; m; cyclohexyl H), 1.50-1.65 (5H; m; cyclo-
hexyl H), 1.81 (1H; m; cyclohexyl H), 4.03 (2H; d,
J = 7.2 Hz; N–CH2–), 4.84 (2H; s; NH), 7.16 (1H; t,
J = 7.2 Hz; indole C5-H), 7.25 (1H; t, J = 7.2 Hz; indole
C6-H), 7.53 (1H; d, J = 8.4 Hz; indole C7-H), 7.83 (1H; s;
imine H), 8.25 (1H; s; indole C2-H), 8.29 (1H; d,
J = 8.0 Hz; indole C4-H), 9.11 and 11.18 (each 1H; s;
NH). IR (KBr) cmÀ1: 3277, 1617, 1558, 1508, 1470, 1280,
999, 740, 631. ESI-MS m/z: 330 (M + H+). Anal. Calcd for
C4-H), 9.12 and 11.18 (each 1H; s; NH). IR (KBr) cmÀ1
:
3286, 1614, 1537, 1393, 1177, 996, 743. ESI-MS m/z:
304 (M + H+). Anal. Calcd for C15H21N5S (303.43): C,
59.38; H, 6.98; N, 23.08; S, 10.57. Found: C, 59.17; H,
7.00; N, 23.13; S, 10.59.
1-Hexylindole-3-carboxaldehyde
thiocarbohydrazone (3g)
C17H23N5S (329.46): C, 61.97; H, 7.04; N, 21.26; S, 9.73.
Mp 177–180 °C. Yield: 84%. 1H-NMR (DMSO-d6) d: 0.84
(3H; t, J = 6.8; CH3), 1.26 (6H; m; CH2), 1.78 (2H; m;
CH2), 4.11 (2H; t, J = 7.6 Hz; N–CH2–), 4.87 (2H; br s;
NH), 7.17 (1H; t, J = 7.2 Hz; indole C5-H), 7.25 (1H; t,
J = 7.2 Hz; indole C6-H), 7.52 (1H; d, J = 8.0 Hz; indole
C7-H), 7.87 (1H; s; imine H), 8.26-8.31 (2H; m; indole
Found: C, 62.09; H, 7.04; N, 7.06; S, 7.09.
1-Benzylindole-3-carboxaldehyde
thiocarbohydrazone (3k)
Recrystallized from ethanol. Mp 244 °C (dec.). Yield: 66%.
1H-NMR (DMSO-d6) d: 4.85 (2H; br s; NH), 5.45 (2H; s;
methylene H), 7.17-7.33 (7H; m; indole C5,6-H + 5phenyl
H), 7.51 (1H; d, J = 8.0 Hz; indole C7-H), 8.01 (1H; s;
imine H), 8.29 (1H; s; indole C2-H), 8.32 (1H; d,
J = 7.6 Hz; indole C4-H), 9.14 and 11.19 (each 1H; s;
NH). IR (KBr) cmÀ1: 3291, 3222, 1660, 1612, 1553, 1508,
1468, 1255, 1175, 983, 743. ESI-MS m/z: 324 (M + H+).
Anal. Calcd for C17H17N5S (323.42): C, 63.13; H, 5.30; N,
21.65; S, 9.91. Found: C, 63.05; H, 5.31; N, 21.71; S,
9.89.
C
2,4-H), 9.11 and 11.17 (1H; s; NH). IR (KBr) cmÀ1: 3291,
1604, 1537, 1497, 1247, 1196, 1175, 1004, 740. ESI-MS
m/z: 318 (M + H+). Anal. Calcd for C16H23N5S (317.45): C,
60.54; H, 7.30; N, 22.06; S, 10.10. Found: C, 60.42; H,
7.31; N, 22.10; S, 10.07.
1-(Cyclopropylmethyl)indole-3-carboxaldehyde
thiocarbohydrazone (3h)
Mp 209–212 °C. Yield: 71%. 1H-NMR (DMSO-d6) d: 0.42
(2H; m; cyclopropyl C2,3-Ha), 0.54 (2H; m; cyclopropyl
C2,3-Hb), 1.28 (1H; m; cyclopropyl C1-H), 4.06 (2H; d,
J = 7.2 Hz; N–CH2–), 4.85 (2H; s; NH), 7.17 (1H; t,
J = 7.6 Hz; indole C5-H), 7.26 (1H; t, J = 7.6 Hz; indole
C6-H), 7.57 (1H; d, J = 8.0 Hz; indole C7-H), 7.92 (1H; s;
imine H), 8.27 (1H; s; indole C2-H), 8.30 (1H; d, J = 7.6;
indole C4-H), 9.11 and 11.20 (each 1H; s; NH). IR (KBr)
cmÀ1: 3291, 1620, 1564, 1534, 1510, 1398, 1281, 983,
740. ESI-MS m/z: 288 (M + H+). Anal. Calcd for
1-(2-Fluorobenzyl)indole-3-carboxaldehyde
thiocarbohydrazone (3l)
Recrystallized from 2-propanol. Mp 212 °C (dec.). Yield:
68%. 1H-NMR (DMSO-d6) d: 4.86 (2H; br s; NH), 5.52
(2H; s; methylene H), 7.15-7.28 (5H; m; indole C5,6-H +
Ar
C3,5,6-H), 7.36 (1H; m; Ar C4-H), 7.52 (1H; d,
J = 8.0 Hz; indole C7-H), 7.95 (1H; s; imine H), 8.27 (1H;
s; indole C2-H), 8.33 (1H; d, J = 7.6 Hz; indole C4-H),
9.16 and 11.20 (each 1H; s; NH). IR (KBr) cmÀ1: 3291,
3216, 1617, 1550, 1505, 1273, 1167, 986, 754. ESI-MS
m/z: 342 (M + H+). Anal. Calcd for C17H16FN5S (341.41):
C, 59.81; H, 4.72; N, 20.51; S, 9.39. Found: C, 59.75; H,
4.73; N, 20.59; S, 59.74.
C
14H17N5S (287.38): C, 58.51; H, 5.96; N, 24.37; S,
11.16. Found: C, 58.54; H, 5.97; N, 24.30; S, 11.18.
1-(Cyclobutylmethyl)indole-3-carboxaldehyde
thiocarbohydrazone (3i)
1
Mp 234–236 °C. Yield: 81%. H-NMR (DMSO-d6) d: 1.76-
1.89 (4H; m; cyclobutyl CH2), 1.93-1.99 (2H; m; cyclobutyl
CH2), 2.81 (1H; septet, J = 8.0 Hz; cyclobutyl CH), 4.21
(2H; d, J = 7.6 Hz; N–CH2–), 4.85 (2H; br s; NH), 7.17
(1H; t, J = 7.6 Hz; indole C5-H), 7.25 (1H; t, J = 7.6 Hz;
indole C6-H), 7.54 (1H; d, J = 8.0 Hz; indole C7-H), 7.87
(1H; s; imine H), 8.25 (1H; s; indole C2-H), 8.29 (1H; d,
J = 8.0 Hz; indole C4-H), 9.10 and 11.18 (each 1H; s;
NH). IR (KBr) cmÀ1: 3275, 1617, 1558, 1508, 1396, 1177,
1-(3-Fluorobenzyl)indole-3-carboxaldehyde
thiocarbohydrazone (3m)
Mp 228 °C (dec.). Yield: 70%. H-NMR (DMSO-d6) d: 4.90
(2H; br s; NH), 5.48 (2H; s; methylene H), 7.08-7.13 (3H;
m; Ar H), 7.16-7.25 (2H; m; indole C5,6-H), 7.37 (1H; dd,
J = 14.2 Hz, J = 7.6 Hz; Ar H), 7.52 (1H; d, J = 8.4 Hz;
indole C7-H), 8.03 (1H; s; imine H), 8.29 (1H; s; indole
1
4
Chem Biol Drug Des 2013

Antimycobacterial Thiadiazolylhydrazones
C2-H), 8.34 (1H; d, J = 7.2 Hz; indole C4-H), 9.16 and
11.22 (each 1H; s; NH). IR (KBr) cmÀ1: 3296, 3216, 1614,
1561, 1513, 1255, 1175, 983, 780, 746. ESI-MS m/z: 342
(M + H+). Anal. Calcd for C17H16FN5S (341.41): C, 59.81;
H, 4.72; N, 20.51; S, 9.39. Found: C, 59.85; H, 4.72; N,
20.48; S, 9.41.
Ar C3,5-H), 7.47 (1H; d, J = 7.6 Hz; indole C7-H), 7.97
(1H; s; imine H), 8.28 (1H; s; indole C2-H), 8.34 (1H; d,
J = 7.2 Hz; indole C4-H), 9.15 and 11.19 (each 1H; s;
NH). IR (KBr) cmÀ1: 3299, 1604, 1489, 1170, 1008. ESI-
MS m/z: 402, 404 (M + H+). Anal. Calcd for C17H16BrN5S
(402.31): C, 50.75; H, 4.01; N, 17.41; S, 7.97. Found: C,
50.63; H, 4.01; N, 17.38; S, 7.99.
1-(4-Fluorobenzyl)indole-3-carboxaldehyde
thiocarbohydrazone (3n)
Mp 230 °C (dec.). Yield: 78%. H-NMR (DMSO-d6) d: 4.88
1-(3-Methylbenzyl)indole-3-carboxaldehyde
thiocarbohydrazone (3r)
1
(2H; br s; NH), 5.44 (2H; s; methylene H), 7.17-7.25 (4H;
m; indole C5,6-H + Ar C2,6-H), 7.35 (2H; dd, J = 8.8 Hz,
J = 5.2 Hz; Ar C3,5-H), 7.53 (1H; d, J = 8.4 Hz; indole
C7-H), 8.01 (1H; s; imine H), 8.28 (1H; s; indole C2-H),
8.32 (1H; d, J = 7.6 Hz; indole C4-H), 9.14 and 11.20
(each 1H; s; NH). IR (KBr) cmÀ1: 3286, 3179, 1660, 1550,
1510, 1223, 1177, 988, 812, 738. ESI-MS m/z: 342
(M + H+). Anal. Calcd for C17H16FN5S (341.41): C, 59.81;
H, 4.72; N, 20.51; S, 9.39. Found: C, 59.69; H, 4.71; N,
20.55; S, 9.40.
Recrystallized from 2-propanol. Mp 233–236 °C. Yield:
64%. 1H-NMR (DMSO-d6) d: 2.26 (3H; s; methyl H), 4.86
(2H; br s; NH), 5.40 (2H; s; methylene H), 7.06-7.24 (6H;
m; indole C5,6-H + 4Ar H), 7.50 (1H; d, J = 8.4 Hz; indole
C7-H), 7.99 (1H; s; imine H), 8.28 (1H; s; indole C2-H),
8.32 (1H; d, J = 7.6 Hz; indole C4-H), 9.14 and 11.20
(each 1H; s; NH). IR (KBr) cmÀ1: 3288, 3173, 1612, 1513,
1339, 1174, 985, 740. ESI-MS m/z: 338 (M + H+). Anal.
Calcd for C18H19N5S (337.44): C, 64.07; H, 5.68; N,
20.75; S, 9.50. Found: C, 64.19; H, 5.67; N, 20.71; S,
9.52.
1-(2-Chlorobenzyl)indole-3-carboxaldehyde
thiocarbohydrazone (3o)
Mp 243 °C (dec.). Yield: 81%. H-NMR (DMSO-d6) d: 4.86
1-(4-Methylbenzyl)indole-3-carboxaldehyde
thiocarbohydrazone (3s)
1
(2H; br s; NH), 5.56 (2H; s; methylene H), 6.82 (1H; dd,
J = 7.6 Hz, J = 1.6 Hz; Ar C6-H), 7.18-7.28 (3H; m; indole
C5,6-H + Ar C5-H), 7.35 (1H; dt, J = 7.6 Hz, J = 2.0 Hz;
Ar C4-H), 7.44 (1H; d, J = 7.6 Hz; indole C7-H), 7.53 (1H;
dd, J = 8.0 Hz, J = 1.6 Hz; Ar C3-H), 7.92 (1H; s; imine
H), 8.28 (1H; s; indole C2-H), 8.37 (1H; d, J = 7.2 Hz;
indole C4-H), 9.17 and 11.21 (each 1H; s; NH). IR (KBr)
cmÀ1: 3328, 3280, 1614, 1542, 1500, 1271, 1177, 986,
743. ESI-MS m/z: 358, 360 (M + H+). Anal. Calcd for
C17H16ClN5S (357.86): C, 57.06; H, 4.51; N, 19.57; S,
8.96. Found: C, 57.15; H, 4.51; N, 19.55; S, 8.99.
Mp 213–216 °C. Yield: 79%. 1H-NMR (DMSO-d6) d: 2.25
(3H; s; methyl H), 4.87 (2H; br s; NH), 5.39 (2H; s; methy-
lene H), 7.12-7.23 (6H; m; indole C5,6-H + Ar C2,3,5,6-H),
7.50 (1H; d, J = 8.0 Hz; indole C7-H), 7.99 (1H; s; imine
H), 8.27 (1H; s; indole C2-H), 8.31 (1H; d, J = 7.6 Hz;
indole C4-H), 9.14 and 11.19 (each 1H; s; NH). IR (KBr)
cmÀ1: 3291, 3227, 1614, 1513, 1468, 1348, 1169, 988,
746. ESI-MS m/z: 338 (M + H+). Anal. Calcd for
C
18H19N5S (337.44): C, 64.07; H, 5.68; N, 20.75; S, 9.50.
Found: C, 63.95; H, 5.67; N, 20.81; S, 9.49.
1-(4-Methoxybenzyl)indole-3-carboxaldehyde
thiocarbohydrazone (3t)
Mp 214 °C (dec.). Yield: 85%. H-NMR (DMSO-d6) d: 2.25
1-(4-Chlorobenzyl)indole-3-carboxaldehyde
thiocarbohydrazone (3p)
1
Mp 200–203 °C. Yield: 85%. 1H-NMR (DMSO-d6) d: 4.85
(2H; br s; NH), 5.51 (2H; s; methylene H), 7.15–7.27 (4H;
(3H; s; OCH3), 4.87 (2H; br s; NH), 5.39 (2H; s; zmethyl-
ene H), 7.12-7.23 (6H; m; indole C5,6-H + 4Ar H), 7.50
(1H; d, J = 8.4 Hz; indole C7-H), 7.98 (1H; s; imine H),
8.30 (2H; m; indole C2,4-H), 9.14 and 11.20 (each 1H; s;
NH). IR (KBr) cmÀ1: 3291, 3174, 1617, 1540, 1390, 1169,
1017, 991, 799. ESI-MS m/z: 354 (M + H+). Anal. Calcd
for C18H19N5OS (353.44): C, 61.17; H, 5.42; N, 19.81; S,
9.07. Found: C, 61.25; H, 5.42; N, 19.79; S, 9.08.
m; indole C5,6-H
+ Ar C2,6-H), 7.38 (2H; d, J = 7.8 Hz;
Ar C3,5-H), 7.46 (1H; d, J = 7.6 Hz; indole C7-H), 7.94
(1H; s; imine H), 8.27 (1H; s; indole C2-H), 8.35 (1H; d,
J = 7.2 Hz; indole C4-H), 9.16 and 11.21 (each 1H; s;
NH). IR (KBr) cmÀ1: 3318, 3270, 1610, 1514, 1016, 738.
ESI-MS m/z: 358, 360 (M + H+). Anal. Calcd for
C17H16ClN5S (357.86): C, 57.06; H, 4.51; N, 19.57; S,
8.96. Found: C, 57.15; H, 4.50; N, 19.55; S, 8.94.
1-(3-Cyanobenzyl)indole-3-carboxaldehyde
thiocarbohydrazone (3u)
1-(4-Bromobenzyl)indole-3-carboxaldehyde
thiocarbohydrazone (3q)
Mp 246–249 °C. Yield: 79%. 1H-NMR (DMSO-d6) d: 4.86
(2H; br s; NH), 5.48 (2H; s; methylene H), 7.14-7.26 (4H;
Mp 226–229 °C. Yield: 75%. 1H-NMR (DMSO-d6) d: 4.88
(2H; br s; NH), 5.52 (2H; s; methylene H), 7.16-7.26 (2H;
m; indole C5,6-H), 7.53-7.58 (3H; m; indole C7-H + Ar
C5,6-H), 7.76 (1H; td, J = 6.4 Hz, J = 2.0 Hz; Ar C4-H),
m; indole C5,6-H
+
Ar C2,6-H), 7.30 (2H; d, J = 7.8 Hz;
7.82 (1H; s; Ar C2-H), 8.05 (1H; s; imine H), 8.29 (1H; s;
Chem Biol Drug Des 2013
5

Haj Mohammad Ebrahim Tehrani et al.
indole C2-H), 8.34 (1H; d, J = 7.6 Hz; indole C4-H), 9.15
and 11.22 (each 1H; s; NH). IR (KBr) cmÀ1: 3318, 3280,
2225, 1614, 1518, 1252, 1015, 775, 685. ESI-MS m/z:
349 (M + H+). Anal. Calcd for C18H16N6S (348.42): C,
62.05; H, 4.63; N, 24.12; S, 9.20. Found: C, 62.16; H,
4.64; N, 24.06; S, 9.21.
1524, 1449, 1390, 1340, 1185, 1129, 1031, 831, 740.
ESI-MS m/z: 272 (M + H+). Anal. Calcd for C13H13N5S
(271.34): C, 57.54; H, 4.83; N, 25.81; S, 11.82. Found: C,
57.35; H, 4.84; N, 25.85; S, 11.80.
(E)-2-(2-((1-propyl-1H-indol-3-yl)methylene)
hydrazinyl)-1,3,4-thiadiazole (4d)
1-(Phenylsulfonyl)indole-3-carboxaldehyde
thiocarbohydrazone (3v)
Mp 243 °C (dec.). Yield: 68%. H-NMR (DMSO-d6) d: 4.92
Mp 235–238 °C. Yield: 44%. 1H-NMR (DMSO-d6) d: 0.85
(3H; t, J = 7.4 Hz; CH3), 1.80 (2H; sextet, J = 7.2 Hz;
CH3–CH2–), 4.17 (2H; t, J = 6.8 Hz; N–CH2–), 7.21 (1H; t,
J = 7.6 Hz; indole C5-H), 7.27 (1H; t, J = 7.6 Hz; indole
C6-H), 7.56 (1H; d, J = 8.0 Hz; indole C7-H), 7.83 (1H; s;
imine H), 8.17 (1H; d, J = 8.0 Hz; indole C4-H), 8.29 (1H;
s; indole C2-H), 8.77 (1H; s; thiadiazole H), 12.04 (1H; s;
NH). IR (KBr) cmÀ1: 3163, 1665, 1606, 1574, 1470, 1393,
1185, 1095, 828, 751. ESI-MS m/z: 286 (M + H+). Anal.
Calcd for C14H15N5S (285.37): C, 58.92; H, 5.30; N, 24.54;
S, 11.24. Found: C, 58.95; H, 5.28; N, 24.50; S, 11.26.
1
(2H; br s; NH), 7.35 (1H; t, J = 7.2 Hz; indole C5-H), 7.44
(1H; t, J = 7.2 Hz; indole C6-H), 7.62 (2H; t, J = 8.0 Hz;
Ar C3,5-H), 7.72 (1H; dt, J = 7.6 Hz, J = 1.2 Hz; Ar C4-H),
7.96 (1H; d, J = 8.4 Hz; indole C7-H), 8.05 (2H; d,
J = 7.2 Hz; Ar C2,6-H), 8.26 (1H; s; imine H), 8.33 (1H; d,
J = 7.6 Hz; indole C4-H), 8.40 (1H; s; indole C2-H), 9.49
and 11.49 (1H; s; NH). IR (KBr) cmÀ1: 3296, 1617, 1537,
1500, 1446, 1372, 1223, 1172, 980, 751. ESI-MS m/z:
374 (M + H+). Anal. Calcd for C16H15N5O2S2 (373.45): C,
51.46; H, 4.05; N, 18.75; O, 8.57; S, 17.17. Found: C,
51.38; H, 4.04; N, 18.79; S, 17.20.
(E)-2-(2-((1-butyl-1H-indol-3-yl)methylene)
hydrazinyl)-1,3,4-thiadiazole (4e)
1
Mp 223 °C (dec.). Yield: 55%. H-NMR (DMSO-d6) d: 0.89
General procedure for synthesis of
thiadiazolylhydrazones of indole-3-
carboxaldehydes (4a–v)
The suspension of thiocarbohydrazones (1 mmol) in triethyl
orthoformate (10 mL) was heated under reflux for 8 h.
Then the mixture was kept in an ice water bath for 1 h.
The precipitate was filtered and washed with 20 mL of
n-hexane. The thiadiazolylhydrazones 4a–v were thus
obtained.
(3H; t, J = 7.6 Hz; CH3), 1.25 (2H; sextet, J = 7.6 Hz;
–CH2–CH3), 1.75 (2H; quintet, J = 7.2 Hz; N–CH2–CH2–),
4.19 (2H; t, J = 6.8 Hz; N–CH2–), 7.19-7.29 (2H; m; indole
C5,6-H), 7.55 (1H; d, J = 8.0 Hz; indole C7-H), 7.82 (1H; s;
imine H), 8.16 (1H; d, J = 7.6 Hz; indole C4-H), 8.29 (1H;
s; indole C2-H), 8.77 (1H; s; thiadiazole H), 12.03 (1H; s;
NH). IR (KBr) cmÀ1: 3104, 1612, 1585, 1446, 1393, 1183,
1026, 828, 754. ESI-MS m/z: 300 (M + H+). Anal. Calcd
for C15H17N5S (299.39): C, 60.18; H, 5.72; N, 23.39; S,
10.71. Found: C, 60.01; H, 5.73; N, 23.46; S, 10.73.
(E)-2-(2-((1-methyl-1H-indol-3-yl)methylene)
hydrazinyl)-1,3,4-thiadiazole (4b)
Mp 270 °C (dec.). Yield: 45%. H-NMR (DMSO-d6) d: 3.83
(E)-2-(2-((1-pentyl-1H-indol-3-yl)methylene)
hydrazinyl)-1,3,4-thiadiazole (4f)
1
(3H; s; CH3), 7.23 (1H; t, J = 7.2 Hz; indole C5-H), 7.29
(1H; t, J = 7.2 Hz; indole C6-H), 7.52 (1H; d, J = 8.4 Hz;
indole C7-H), 7.78 (1H; s; imine H), 8.16 (1H; d,
J = 8.0 Hz; indole C4-H), 8.28 (1H; s; indole C2-H), 8.77
Mp 186–189 °C. Yield: 59%. 1H-NMR (DMSO-d6) d: 0.84
(3H; t, J = 6.8 Hz; CH3), 1.21–1.32 (4H; m; CH2), 1.79
(2H; quintet, J = 6.8 Hz; CH2), 4.19 (2H; t, J = 6.8 Hz;
N–CH2–), 7.19-7.29 (2H; m; indole C5,6-H), 7.55 (1H; d,
J = 8.0 Hz; indole C7-H), 7.83 (1H; s; imine H), 7.16 (1H;
d, J = 7.6 Hz; indole C4-H), 8.28 (1H; s; indole C2-H),
8.77 (1H; s; thiadiazole H), 12.02 (1H; s; NH). IR (KBr)
cmÀ1: 3115, 1614, 1537, 1452, 1390, 1183, 938, 743.
ESI-MS m/z: 314 (M + H+). Anal. Calcd for C16H19N5S
(313.42): C, 61.31; H, 6.11; N, 22.34; S, 10.23. Found: C,
61.35; H, 6.12; N, 24.30; S, 10.19.
(1H; s; thiadiazole H), 12.02 (1H; s; NH). IR (KBr) cmÀ1
:
3179, 1604, 1532, 1452, 1335, 1196, 1127, 1073, 831,
746. ESI-MS m/z: 258 (M + H+). Anal. Calcd for
C
12H11N5S (257.31): C, 56.01; H, 4.31; N, 27.22; S,
12.46. Found: C, 56.08; H, 4.31; N, 27.18; S, 12.45.
(E)-2-(2-((1-ethyl-1H-indol-3-yl)methylene)
hydrazinyl)-1,3,4-thiadiazole (4c)
1
Mp 233 °C (dec.). Yield: 52%. H-NMR (DMSO-d6) d: 1.39
(E)-2-(2-((1-hexyl-1H-indol-3-yl)methylene)
(3H; t, J = 7.2 Hz; CH3), 4.23 (2H; quartet, J = 7.2 Hz;
N–CH2–), 7.22 (1H; t, J = 7.2 Hz; indole C5-H), 7.27 (1H;
t, J = 7.6 Hz; indole C6-H), 7.56 (1H; d, J = 8.0 Hz; indole
C7-H), 7.85 (1H; s; imine H), 8.16 (1H; d, J = 7.6 Hz;
indole C4-H), 8.29 (1H; s; indole C2-H), 8.78 (1H; s; thi-
adiazole H), 12.04 (1H; s; NH). IR (KBr) cmÀ1: 3211, 1609,
hydrazinyl)-1,3,4-thiadiazole (4g)
Mp 183–186 °C. Yield: 38%. 1H-NMR (DMSO-d6) d: 0.83
(3H; t, J = 6.8 Hz; CH3), 1.24 (6H; m; CH2), 1.77 (2H; m;
CH2), 4.19 (2H; t, J = 7.0 Hz; N–CH2–), 7.21 (1H; t,
J = 7.2 Hz; indole C5-H), 7.27 (1H; t, J = 7.2 Hz; indole
C6-H), 7.55 (1H; d, J = 8.0 Hz; indole C7-H), 7.83 (1H; s;
6
Chem Biol Drug Des 2013

Antimycobacterial Thiadiazolylhydrazones
imine H), 8.16 (1H; d, J = 8.0 Hz; indole C7-H), 8.28 (1H;
s; indole C2-H), 8.77 (1H; s; thiadiazole H), 12.02 (1H; s;
NH). IR (KBr) cmÀ1: 3195, 1612, 1593, 1446, 1175, 1031,
828, 746. ESI-MS m/z: 328 (M + H+). Anal. Calcd for
C17H21N5S (327.45): C, 62.36; H, 6.46; N, 21.39; S, 9.79.
Found: C, 62.21; H, 6.44; N, 21.45; S, 9.80.
C
5,6-H + 5phenyl H), 7.54 (1H; d, J = 7.2 Hz; indole
C7-H), 7.97 (1H; s; imine H), 8.17 (1H; d, J = 7.6 Hz;
indole C4-H), 8.31 (1H; s; indole C2-H), 8.78 (1H; s; thi-
adiazole H), 12.07 (1H; s; NH). IR (KBr) cmÀ1: 3155, 1614,
1596, 1468, 1390, 1175, 1026, 828, 770, 740, 695. ESI-
MS m/z: 334 (M + H+). Anal. Calcd for C18H15N5S
(333.41): C, 64.84; H, 4.53; N, 21.01; S, 9.62. Found: C,
64.95; H, 21.01; N, 20.93; S, 9.61.
(E)-2-(2-((1-(cyclopropylmethyl)-1H-indol-3-yl)
methylene)hydrazinyl)-1,3,4-thiadiazole (4h)
1
Mp 228 °C (dec.). Yield: 47%. H-NMR (DMSO-d6) d: 0.41
(E)-2-(2-((1-(2-fluorobenzyl)-1H-indol-3-yl)
(2H; m; cyclopropyl C2,3-Ha), 0.53 (2H; m; cyclopropyl
methylene)hydrazinyl)-1,3,4-thiadiazole (4l)
C
2,3-Hb), 1.27 (1H; m; cyclopropyl C1-H), 4.07 (1H; d,
Mp 234–237 °C. Yield: 52%. 1H-NMR (DMSO-d6) d: 5.23
(2H; s; methylene H), 7.14 (2H; m; Ar H), 7.20-7.28 (3H;
m; indole C5,6-H + Ar H), 7.33-7.38 (1H; m; Ar H), 7.55
(1H; d, J = 7.6 Hz; indole C7-H), 7.91 (1H; s; imine H),
8.18 (1H; d, J = 7.2 Hz; indole C4-H), 8.31 (1H; s; indole
C2-H), 8.78 (1H; s; thiadiazole H), 12.08 (1H; s; NH). IR
(KBr) cmÀ1: 3195, 1614, 1590, 1449, 1390, 1231, 1167,
834, 751. ESI-MS m/z: 352 (M + H+). Anal. Calcd for
C18H14FN5S (351.40): C, 61.52; H, 4.02; N, 19.93; S,
9.12. Found: C, 61.45; H, 4.03; N, 19.48; S, 9.14.
J = 7.2 Hz; N–CH2–), 7.19-7.29 (2H; m; indole C5,6-H),
7.60 (2H; d, J = 8.0 Hz; indole C7-H), 7.89 (1H; s; imine
H), 8.16 (1H; d, J = 7.6 Hz; indole C4-H), 8.30 (1H; s;
indole C2-H), 8.77 (1H; s; thiadiazole H), 12.04 (1H; s;
NH). IR (KBr) cmÀ1: 3163, 1662, 1612, 1577, 1337, 1188,
1020, 828, 748. ESI-MS m/z: 298 (M + H+). Anal. Calcd
for C15H15N5S (297.38): C, 60.58; H, 5.08; N, 23.55; S,
10.78. Found: C, 60.49; H, 50.07; N, 23.61; S, 10.79.
(E)-2-(2-((1-(cyclobutylmethyl)-1H-indol-3-yl)
methylene)hydrazinyl)-1,3,4-thiadiazole (4i)
(E)-2-(2-((1-(3-fluorobenzyl)-1H-indol-3-yl)
Mp 232 °C (dec.). Yield: 53%. 1H-NMR (DMSO-d6) d:
1.82-1.89 (4H; m; cyclobutyl CH2), 1.92-1.98 (2H; m;
cyclobutyl CH2), 2.79 (1H; m; cyclobutyl CH), 4.21 (2H; d,
J = 7.2 Hz; methylene H), 7.20 (1H; t, J = 7.6 Hz; indole
C5-H), 7.26 (1H; t, J = 7.6 Hz; indole C6-H), 7.57 (1H; d,
J = 8.0 Hz; indole C7-H), 7.82 (1H; s; imine H), 8.15 (1H;
d, J = 7.6 Hz; indole C4-H), 8.28 (1H; s; indole C2-H),
8.77 (1H; s; thiadiazole H), 12.09 (1H; s; NH). IR (KBr)
cmÀ1: 3174, 1612, 1582, 1468, 1177, 1023, 831, 754.
ESI-MS m/z: 312 (M + H+). Anal. Calcd for C16H17N5S
(311.40): C, 61.71; H, 5.50; N, 22.49; S, 10.30. Found: C,
61.75; H, 5.50; N, 22.41; S, 10.37.
methylene)hydrazinyl)-1,3,4-thiadiazole (4m)
Mp 241 °C (dec.). Yield: 57%. H-NMR (DMSO-d6) d: 5.49
1
(2H; s; methylene H), 7.06–7.13 (3H; m; Ar H), 7.21-7.27
(2H; m; indole C5,6-H), 7.34-7.39 (1H; m; Ar H), 7.55 (1H;
d, J = 7.2 Hz; indole C7-H), 7.99 (1H; s; imine H), 8.19
(1H; d, J = 6.8 Hz; indole C4-H), 8.32 (1H; s; indole
C2-H), 8.78 (1H; s; thiadiazole H), 12.09 (1H; s; NH). IR
(KBr) cmÀ1: 3190, 1614, 1596, 1449, 1175, 1028, 772,
743, 679. ESI-MS m/z: 352 (M + H+). Anal. Calcd for
C
18H14FN5S (351.40): C, 61.52; H, 4.02; N, 19.93; S,
9.12. Found: C, 61.63; H, 4.03; N, 19.87; S, 9.15.
(E)-2-(2-((1-(4-fluorobenzyl)-1H-indol-3-yl)
(E)-2-(2-((1-(cyclohexylmethyl)-1H-indol-3-yl)
methylene)hydrazinyl)-1,3,4-thiadiazole (4j)
methylene)hydrazinyl)-1,3,4-thiadiazole (4n)
Mp 243–246 °C. Yield: 48%. 1H-NMR (DMSO-d6) d: 5.45
(2H; s; methylene H), 7.14–7.27 (4H; m; indole C5,6-H + Ar
C2,6-H), 7.32 (2H; t, J = 6.6 Hz; Ar C3,5-H), 7.55 (1H; d,
J = 7.6 Hz; indole C7-H), 7.97 (1H; s; imine H), 8.17 (1H;
d, J = 7.6 Hz; indole C4-H), 8.31 (1H; s; indole C2-H),
8.78 (1H; s; thiadiazole H), 12.07 (1H; s; NH). IR (KBr)
cmÀ1: 3190, 1614, 1590, 1513, 1465, 1388, 1228, 1172,
1031, 834, 746. ESI-MS m/z: 352 (M + H+). Anal. Calcd
for C18H14FN5S (351.40): C, 61.52; H, 4.02; N, 19.93; S,
9.12. Found: C, 61.62; H,4.03; N, 19.86; S, 9.10.
Mp 244 °C (dec.). Yield: 42%. 1H-NMR (DMSO-d6) d:
0.97-1.13 (5H; m; cyclohexyl H), 1.50-1.80 (6H; m; cyclo-
hexyl H), 4.04 (2H; d, J = 7.2 Hz; N–CH2–), 7.18-7.28
(2H; m; indole C5,6-H), 7.55 (1H; d, J = 8.0 Hz; indole
C7-H), 7.77 (1H; s; imine H), 8.16 (1H; d, J = 7.6 Hz;
indole C4-H), 8.28 (1H; s; indole C2-H), 8.77 (1H; s;
thiadiazole H), 12.04 (1H; s; NH). IR (KBr) cmÀ1: 3190,
1614, 1590, 1444, 1396, 1183, 1108, 751. ESI-MS m/z:
340 (M + H+). Anal. Calcd for C18H21N5S (339.46): C,
63.69; H, 6.24; N, 20.63; S, 9.45. Found: C, 63.60; H,
6.25; N, 20.66; S, 9.42.
(E)-2-(2-((1-(2-chlorobenzyl)-1H-indol-3-yl)
methylene)hydrazinyl)-1,3,4-thiadiazole (4o)
Mp 252–255 °C. Yield: 42%. 1H-NMR (DMSO-d6) d: 5.56
(2H; s; methylene H), 6.81 (1H; dd, J = 7.8 Hz,
J = 1.4 Hz; Ar C6-H), 7.25 (3H; m; indole C5,6-H + Ar
C5-H), 7.34 (1H; dt, J = 7.6 Hz, J = 1.6 Hz; Ar C4-H),
(E)-2-(2-((1-benzyl-1H-indol-3-yl)methylene)
hydrazinyl)-1,3,4-thiadiazole (4k)
Mp 242–245 °C. Yield: 61%. 1H-NMR (DMSO-d6) d:
5.47 (2H; s; methylene H), 7.24-7.35 (7H; indole
Chem Biol Drug Des 2013
7

Haj Mohammad Ebrahim Tehrani et al.
7.48 (1H; d, J = 7.6 Hz; indole C7-H), 7.53 (1H; dd,
J = 1.0 Hz; Ar C3-H), 7.89 (1H; s; imine H), 8.21 (1H; d,
J = 7.6 Hz; indole C4-H), 8.31 (1H; s; indole C2-H), 8.79
J = 6.8 Hz; indole C7-H), 7.95 (1H; s; imine H), 8.16 (1H;
d, J = 7.2 Hz; indole C4-H), 8.30 (1H; s; indole C2-H),
8.78 (1H; s; thiadiazole H), 12.06 (1H; s; NH). IR (KBr)
cmÀ1: 3195, 1614, 1590, 1446, 1390, 1169, 1031, 834,
775. ESI-MS m/z: 348 (M + H+). Anal. Calcd for
(1H; s; thiadiazole H), 12.09 (1H; s; NH). IR (KBr) cmÀ1
:
3185, 1612, 1590, 1468, 1390, 1175, 1119, 831, 746.
ESI-MS m/z: 368, 370 (M + H+). Anal. Calcd for
C18H14ClN5S (367.86): C, 57.06; H, 4.51; N, 19.57; S,
8.96. Found: C, 57.26; H, 4.52; N, 19.51; S, 8.95.
C
19H17N5S (347.44): C, 65.68; H, 4.93; N, 20.16; S, 9.23.
Found: C, 65.57; H, 4.93; N, 20.20; S, 9.26.
(E)-2-(2-((1-(4-methoxybenzyl)-1H-indol-3-yl)
methylene)hydrazinyl)-1,3,4-thiadiazole (4t)
Mp 240–243 °C. Yield: 35%. 1H-NMR (DMSO-d6) d: 2.25
(3H; s; OCH3), 5.41 (2H; s; methylene H), 7.12-7.25 (6H;
m; indole C5,6-H + 4Ar H), 7.52 (1H; d, J = 7.6 Hz; indole
C7-H), 7.95 (1H; s; imine H), 8.16 (1H; d, J = 7.6 Hz;
indole C4-H), 8.30 (1H; s; indole C2-H), 8.77 (1H; s;
thiadiazole H), 12.05 (1H; s; NH). IR (KBr) cmÀ1: 3184,
1612, 1465, 1390, 1169, 1119, 831, 746. ESI-MS m/z:
364 (M + H+). Anal. Calcd for C19H17N5OS (363.44): C,
62.79; H, 4.71; N, 19.27; S, 8.82. Found: 62.61; H, 4.71;
N, 19.33; S, 8.85.
(E)-2-(2-((1-(4-chlorobenzyl)-1H-indol-3-yl)
methylene)hydrazinyl)-1,3,4-thiadiazole (4p)
1
Mp 232 °C (dec.). Yield: 48%. H-NMR (DMSO-d6) d: 5.51
(2H; s; methylene H), 7.19-7.30 (4H; m; indole C5,6-H +
Ar C2,6-H), 7.41 (2H; d, J = 7.8 Hz; Ar C3,5-H), 7.50 (1H;
d, J = 7.6 Hz; indole C7-H), 7.91 (1H; s; imine H), 8.20
(1H; d, J = 7.6 Hz; indole C4-H), 8.30 (1H; s; indole
C2-H), 8.78 (1H; s; thiadiazole H), 12.01 (1H; s; NH). IR
(KBr) cmÀ1: 3101, 1591, 1445, 1389, 1170, 745. ESI-MS
m/z: 368, 370 (M + H+). Anal. Calcd for C18H14ClN5S
(367.86): C, 57.06; H, 4.51; N, 19.57; S, 8.96. Found: C,
57.12; H, 4.52; N, 19.51; S, 8.97.
(E)-3-((3-((2-(1,3,4-thiadiazol-2-yl)hydrazono)
methyl)-1H-indol-1-yl)methyl)benzonitrile (4u)
Mp 250–253 °C. Yield: 59%. 1H-NMR (DMSO-d6) d: 5.54
(2H; s; methylene H), 7.20-7.30 (2H; m; indole C5,6-H),
(E)-2-(2-((1-(4-bromobenzyl)-1H-indol-3-yl)
methylene)hydrazinyl)-1,3,4-thiadiazole (4q)
Mp 190–193 °C. Yield: 55%. 1H-NMR (DMSO-d6) d: 5.47
(2H; s; methylene H), 7.18-7.29 (4H; m; indole C5,6-H + Ar
C2,6-H), 7.33 (2H; d, J = 7.8 Hz; Ar C3,5-H), 7.52 (1H; d,
J = 7.6 Hz; indole C7-H), 7.94 (1H; s; imine H), 8.19 (1H;
d, J = 7.6 Hz; indole C4-H), 8.31 (1H; s; indole C2-H),
8.80 (1H; s; thiadiazole H), 12.10 (1H; s; NH). IR (KBr)
cmÀ1: 1654, 1530, 1171, 741. ESI-MS m/z: 412, 414
(M + H+). Anal. Calcd for C18H14BrN5S (412.31): C, 52.43;
H, 3.42; N, 16.99; S, 7.78. Found: C, 52.29; H, 3.41; N,
17.02; S, 7.76.
7.52-7.60 (3H; m; indole C7-H
+ Ar C5,6-H), 7.75-7.80
(2H; m; Ar C2,4-H), 8.01 (1H; s; imine H), 8.18 (1H; d,
J = 7.2 Hz; indole C4-H), 8.31 (1H; s; indole C2-H), 8.78
(1H; s; thiadiazole H), 12.09 (1H; s; NH). IR (KBr) cmÀ1
:
3152, 2231, 1612, 1468, 1390, 1172, 1026, 828, 796,
748, 682. ESI-MS m/z: 359 (M + H+). Anal. Calcd for
C19H14N6S (358.42): C, 63.67; H, 3.94; N, 23.45; S, 8.95.
Found: C, 63.51; H, 3.93; N, 23.52; S, 8.94.
(E)-2-(2-((1-(phenylsulfonyl)-1H-indol-3-yl)
(E)-2-(2-((1-(3-methylbenzyl)-1H-indol-3-yl)
methylene)hydrazinyl)-1,3,4-thiadiazole (4r)
methylene)hydrazinyl)-1,3,4-thiadiazole (4v)
Mp 272–275 °C. Yield: 65%. 1H-NMR (DMSO-d6) d: 7.43
(2H; m; indole C5,6-H), 7.62 (2H; t, J = 7.6 Hz; Ar C3,5-H),
7.72 (1H; t, J = 7.2 Hz; Ar C4-H), 8.03 (3H; m; Ar C2,6-H +
indole C7-H), 8.21 (1H; d, J = 7.6 Hz; indole C4-H), 8.32
(1H; s; imine H), 8.35 (1H; s; indole C2-H), 8.82 (1H; s;
thiadiazole H), 12.48 (1H; s; NH). IR (KBr) cmÀ1: 3195,
1614, 1590, 1446, 1177, 1129, 986, 746. ESI-MS m/z:
384 (M + H+). Anal. Calcd for C17H13N5O2S2 (383.45): C,
53.25; H, 3.42; N, 18.26; S, 16.72. Found: C, 53.41; H,
3.43; N, 18.21; S, 16.70.
Mp 216–219 °C. Yield: 39%. 1H-NMR (DMSO-d6) d: 2.25
(3H; s; CH3), 5.42 (2H; s; methylene H), 7.03-7.10 (3H; m;
Ar H), 7.19-7.26 (3H; m; indole C5,6-H + Ar H), 7.53 (1H;
d, J = 7.2 Hz; indole C7-H), 7.96 (1H; s; imine H), 8.17
(1H; d, J = 7.2 Hz; indole C4-H), 8.31 (1H; s; indole
C2-H), 8.78 (1H; s; thiadiazole H), 12.07 (1H; s; NH). IR
(KBr) cmÀ1: 1614, 1590, 1468, 1390, 1175, 1028, 834,
748. ESI-MS m/z: 348 (M + H+). Anal. Calcd for
C
19H17N5S (347.44): C, 65.68; H, 4.93; N, 20.16; S, 9.23.
Found: C, 56.51; H, 4.92; N, 20.22; S, 9.24.
In vitro evaluation of antimycobacterial activity
The inhibitory activity of the synthesized derivatives was
evaluated against Mycobacterium bovis BCG (1173P2) by
broth microtiter dilution method. The detailed procedure
has been described in our previous works (8,18). Etham-
butol and DMSO were used as positive and negative
controls, respectively.
(E)-2-(2-((1-(4-methylbenzyl)-1H-indol-3-yl)
methylene)hydrazinyl)-1,3,4-thiadiazole (4s)
1
Mp 254 °C (dec.). Yield: 46%. H-NMR (DMSO-d6) d: 2.25
(3H; s; CH3), 5.40 (2H; s; methylene H), 7.13 (2H; d,
J = 8.4 Hz; Ar C3,5-H), 7.17 (2H; d, J = 8.4 Hz; Ar
C
2,4-H), 7.20-7.25 (2H; m; indole C5,6-H), 7.52 (1H; d,
8
Chem Biol Drug Des 2013

Antimycobacterial Thiadiazolylhydrazones
Cytotoxicity assay
general, indole-3-carboxaldehyde was first stirred with
potassium carbonate and then commercially available alkyl
halides or arylmethyl halides were then added to the mix-
ture. Addition of water followed by extraction by dichlo-
romethane and finally evaporation of solvent furnished the
desired intermediates as sufficiently pure solids or in some
cases oily liquids. However, in case of the preparation of
1-(phenylsulfonyl)indole-3-carboxaldehyde (2v) from benze-
nesulfonyl chloride, the above procedure did not lead to
the desired product in good yield, and a substantial
amount of starting indole-3-carboxaldehyde was recovered
following the typical workup. Therefore, this derivative was
prepared by a modified method in which triethylamine in
dichloromethane was used as the base, and the reaction
was carried out under a controlled temperature (see
Experimental Section).
Cytotoxicity of the synthesized derivatives was evaluated
on Fibroblast L929 cell line by MTT [3-(4,5-dimethylthiazol-
2-yl-2,5-tetrazolium bromide)] method. The cells were
grown in RPMI1640 medium at 37 °C under 5% CO2 sup-
plemented with 10% heat-inactivated fetal bovine serum
(FBS), 100 U/mL penicillin, and 100 lg/mL streptomycin.
The cells were then seeded into 96-well plates at a con-
centration of 6000 cells/well and incubated for 24 h. The
medium was then discarded, and different concentrations
of test compounds in complete medium were added to
each well. After further incubation for 24 h at 37 °C, the
medium was discarded and 100 lL MTT (2 mg/mL) was
added to the wells and incubated for 3 h at 37 °C. The
produced formazan crystals were dissolved in 100 lL of
DMSO. Plates were incubated for 20 min at 37 °C, and
the optical densities were read at 570 nm with a reference
wavelength of 630 nm as background using a spectropho-
tometer plate reader (Infiniteꢀ M200; TECAN, Morrisville,
NC, USA). DMSO was used as the solvent of the test com-
pounds, and its final concentration was < 0.2%. IC50 was
calculated by GRAPHPAD PRISM 5.04 (GraphPad Software, La
Jolla, CA, USA). All the tests were performed in triplicates.
The second step was a Schiff base formation reaction
between the intermediate aldehydes and thiocarbohydraz-
ide. To avoid the reaction of thiocarbohydrazide with two
equivalents of aldehydes, the ethanolic solution of alde-
hyde was added dropwise to excess amounts of the hot
aqueous solution of thiocarbohydrazide. After addition of
the first drops, the thiocarbohydrazone product precipi-
tated out, and the products were obtained in good yields.
Analysis of the thiocarbohydrazone products by
spectroscopic methods did not indicate the formation of
bis-hydrazone by-product. In the next step, thiocarbohyd-
razone derivatives 3a–v were reacted with triethyl
orthoformate and converted to the corresponding thia-
diazolylhydrazones by the proposed mechanism disclosed
in Figure 3. The reaction proceeded to completion within
5–8 h, and the products were isolated in moderate to
good yield with acceptable analytical purity.
Results and Discussion
Chemistry
The synthetic route is disclosed in Scheme 1. The final
derivatives were prepared in a three-step synthetic meth-
odology. The first step was introduction of a diverse set of
substituents to N-1 position of indole-3-carboxaldehyde by
a nucleophilic substitution reaction. According to the previ-
ous reports, a variety of bases such as sodium hydride,
sodium/potassium hydroxide, and potassium carbonate in
polar aprotic solvents such as dimethylsulphoxide, N,
N-dimethylformamide, and acetone have been used to
perform this reaction (19–23). The reaction has also been
conducted in aprotic solvents such as dichloromethane
using sodium hydroxide and an ammonium salt as phase
transfer catalyst (24). After examining the above-mentioned
methods, we found that using potassium carbonate in
DMSO was the most convenient method by which we
obtained the intermediates 2a–v with satisfactory yields. In
The final derivatives were analyzed by spectroscopic
methods including IR, NMR, mass spectrometry, and also
elemental analysis. In complete agreement with the pro-
posed structures, the obtained spectroscopic data con-
firmed the formation of the desired derivatives in excellent
purity. The 1H-NMR spectra of aldehyde intermediates
2a–v showed
a characteristic pattern for indole ring
hydrogens. Two sets of triplets at approximately 7.3 ppm,
two sets of doublets at approximately 7.5 and 8.2 ppm,
Scheme 1: The synthetic pathway; reagents and conditions: (a) K2CO3, appropriate halides, DMSO; (b) thiocarbohydrazide, H2O/ethanol,
AcOH; (c) HC(OEt)3, reflux.
Chem Biol Drug Des 2013
9

Haj Mohammad Ebrahim Tehrani et al.
Figure 3: The proposed
mechanism for the formation of
thiadiazole derivatives 4a–v.
and a singlet at approximately 8.3 ppm were assigned to
indole ring hydrogens, whereas a singlet at approximately
10.0 ppm was characteristic of aldehyde hydrogen. The
substituents on N-1 were readily distinguishable in
1H-NMR spectra by their chemical shifts and multiplicity
patterns. In the 1H-NMR spectra of the thiocarbohydraz-
one derivatives 3a–v, appearances of imine hydrogen at
~ 8.0 ppm replacing the aldehyde hydrogen and three
D2O-exchangeable singlets were indicative of the intro-
duced thiocarbohydrazone moiety. Conversion of thiocar-
bohydrazone to thiadiazole moiety was confirmed by the
disappearance of the three D2O-exchangeable hydrogens
in 1H-NMR spectrum and the presence of a new singlet
at approximately 8.8, which was characteristic of thiadiaz-
ole C5-H. All the synthesized derivatives were analyzed
by electrospray ionization mass spectrometry (ESI-MS),
and their molecular mass was detected as M + 1 and/or
M + 23 related to hydrogen and sodium adducts of the
intact molecules, respectively.
Among the thiadiazole derivatives, the butyl- and pentyl-
substituted derivatives 4e and 4f showed the highest
activity
in
the
aliphatic
substituted
subgroup
(MIC = 3.91 lg/mL). Equipotent to the latter two deriva-
tives, compounds 4n (R = p-fluorobenzyl), 4p (p-chlorob-
enzyl), 4q (p-bromobenzyl), and 4t (p-methoxybenzyl) were
also among the most potent ones. In addition, the inhibi-
tory activity of cyclopropyl and o-chlorobenzyl derivatives
4 h and 4o is still acceptable (MIC = 7.81 lg/mL).
Table 1: Antimycobacterial activity of thiocarbohydrazone deriva-
tives 3a–v
H
N
H
N
NH2
N
S
3a-v
N
R
Antimycobacterial activity,
MIC (lg/mL)
24 h/48 h
Biological activity
Derivative
R
The inhibitory activity of the synthesized derivatives was
assessed against Mycobacterium bovis BCG using broth
microtiter dilution method. Tables 1 and 2 show the MIC
values of the synthesized derivatives. Bioactivity of com-
pounds 3a–v was also evaluated as they could be viewed
as non-cyclized analogues of thiadiazole derivatives 4a–v
and provide some clues on the structure–activity relation-
ship of the thiadiazole moiety. As evident in Table 1, com-
3a
3b
H
250/250
Methyl
Ethyl
Propyl
Butyl
Pentyl
Hexyl
Cyclopropylmethyl
Cyclobutylmethyl
Cyclohexylmethyl
Benzyl
2-Fluorobenzyl
3-Fluorobenzyl
4-Fluorobenzyl
2-Chlorobenzyl
4-Chlorobenzyl
4-Bromobenzyl
3-Methylbenzyl
4-Methylbenzyl
4-Methoxybenzyl
3-Cyanobenzyl
Phenylsulfonyl
>500/>500
>500/>500
>500/>500
>500/>500
>500/>500
>500/>500
>500/>500
>500/>500
>500/>500
>500/>500
>500/>500
>500/>500
>500/>500
500/>500
>500/>500
>500/>500
>500/>500
>500/>500
>500/>500
>500/>500
>500/>500
6.5/6.5% (v/v)
0.75/0.75
3c
3d
3e
3f
3 g
3 h
3i
pounds 3a–v did not show
a significant activity at
concentration of 500 lg/mL. Interestingly, the cyclized
analogues 4a–v exhibited promising antimycobacterial
activity. This trend may suggest a pharmacophoric charac-
ter for the thiadiazole moiety, as its substitution by the
non-cyclized thiocarbohydrazide counterpart (as in 3a–v)
leads to the loss of bioactivity. It is noteworthy that the
thiadiazole ring has been employed by the other groups to
develop active antimycobacterial small molecules (25). As
an example, Rollas and coworkers reported some active
antimycobacterial derivatives by considering 2-amino-
1,3,4-thiadiazole as the core structure (26,27). In this
regard, the present study confirms the potential pharma-
cophoric character of thiadiazole ring in antimycobacterial
drug development.
3j
3k
3 l
3 m
3n
3o
3p
3q
3r
3s
3t
3u
3v
DMSO
Ethambutol
10
Chem Biol Drug Des 2013

Antimycobacterial Thiadiazolylhydrazones
Table 2: Antimycobacterial activity and cytotoxicity of thiadiazole derivatives 4a–v
N
N
HN
N
S
4a-v
N
R
Antimycobacterial activity, MIC (lg/mL)
24 h/48 h
Cytotoxicitya IC50 (lg/mL)
24 h
Derivative
R
CLogPb
4a
4b
H
Methyl
Ethyl
Propyl
Butyl
Pentyl
Hexyl
Cyclopropylmethyl
Cyclobutylmethyl
Cyclohexylmethyl
Benzyl
2-Fluorobenzyl
3-Fluorobenzyl
4-Fluorobenzyl
2-Chlorobenzyl
4-Chlorobenzyl
4-Bromobenzyl
3-Methylbenzyl
4-Methylbenzyl
4-Methoxybenzyl
3-Cyanobenzyl
Phenylsulfonyl
7.81/7.81c
15.63/62.5
125/250
62.68
2.43
2.67
3.01
3.49
3.91
4.33
4.75
3.39
3.81
4.65
4.40
4.56
4.56
4.56
4.96
4.96
5.23
4.89
4.89
4.28
4.44
3.88
>100
>100
>100
>100
4c
4d
250/250
4e
4f
3.91/3.91
3.91/3.91
15.63/15.63
7.81/7.81
31.25/31.25
15.63/15.63
125/250
32.92
4 g
4 h
4i
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
4j
4k
4 l
125/250
250/500
4 m
4n
3.91/3.91
7.81/31.25
3.91/3.91
3.91/3.91
15.63/62.5
62.5/125
3.91/3.91
125/250
250/250
4o
4p
4q
4r
4s
4t
4u
4v
DMSO
Ethambutol
6.5/6.5% (v/v)
0.75/0.75
aAssayed against fibroblast L929 cell line.
bCalculated by ChemBioDraw 11.0 software.
cDetermined in our previous study (8).
Analysis of the activity data reveals that among the ali-
phatic-chain-substituted thiadiazoles 4b–g, the activity is
the highest when R is butyl or pentyl. Interestingly, reduc-
ing the chain size to propyl or its expansion to hexyl leads
to the derivatives with lower activity. A similar trend was
observed in cycloalkyl series 4 h–j, where cyclopropyl
derivative 4 h exhibited the best MIC. This suggests that
the chain/ring size and lipophilicity are the possible param-
eters that might be at least partly responsible for the
observed bioactivities (see Table 2 for CLogP values).
However, among the benzylic derivatives 4k–u, some
other parameters rather than lipophilicity appear to deter-
mine the bioactivity of the subgroup. The most active ben-
zylic derivatives including compounds 4n, 4p, 4q, and 4t
bear a heteroatom (halogens or oxygen) at the para posi-
tion of their aromatic phenyl ring. The necessity of the
para-heteroatom substituent is reconfirmed by the obser-
vation that its replacement by methyl group (as in 4q)
leads to loss of activity. In addition, changing the location
of fluorine to ortho or meta positions of phenyl ring (as in
4 l and 4 m) also results in poor activity. Likewise, para-
chloro-substituted 4p showed a higher activity than its
ortho-chloro-substituted analogue 4o (MIC = 3.91 and
7.81 lg/mL, respectively). These together suggest that
oxygen and halogens may pose an important effect on the
bioactivity of derivatives 4n, 4p, 4q, and 4t by altering the
electronic map of the molecule, and their best location on
the phenyl ring is at para position.
Determining the cytotoxicity of the investigational bioactive
molecules is crucial to their fate in lead identification and
the subsequent phases of drug discovery process. Taking
this fact into consideration, the active thiadiazole deriva-
tives (4a–v) were evaluated for their toxicity against fibro-
blast L929 as a normal cell line. As shown in Table 2, the
majority of the derivatives were non-toxic, and their IC50
values were above the 100 lM concentration. However,
compounds 4a and 4f showed toxicity on the tested cell
line with IC50 values of 62.68 and 32.92, respectively.
Based on the activity data, it could be concluded that the
Chem Biol Drug Des 2013
11

Haj Mohammad Ebrahim Tehrani et al.
benzylic-substituted thiadiazoles (i.e., compounds 4n, 4p,
4q, and 4t) exhibited potent and selective antimycobacteri-
al activity.
4. Schaaf H.S., Seddon J.A., Caminero J.A. (2011) Sec-
ond-Line Antituberculosis Drugs: Current Knowledge,
Recent Research Findings and Controversies. In:
Donald P.R., van Helden P.D., editors. “Progress in
Respiratory Research” Vol. 40: Antituberculosis Che-
motherapy. Switzerland: Karger Medical and Scientific
Publishers; p. 92–93.
Conclusion
In the present study, 44 derivatives in two groups of thio-
carbohydrazones of substituted indole-3-carboxaldehyde
and their corresponding thiadiazole derivatives have been
prepared and characterized by different analytical and
spectroscopic methods. The antimycobacterial activity of
the synthesized derivatives was assessed against Myco-
bacterium bovis BCG, and some of them exhibited satis-
factory activity. Of the tested compounds, six derivatives
including compounds 4e, 4f, 4n, 4p, 4q, and 4t showed
higher activity than compound 4a, which was considered
as a lead compound in the current study. Furthermore, in
contrast to the poor bioactivity of thiocarbohydrazones
3a-v, thiadiazole derivatives proved to be potent inhibitors
of Mycobacterium bovis BCG, which implies the essential
role of thiadiazole moiety for exerting antimycobacterial
activity. Interestingly, the most potent benzylic-substituted
thiadiazoles (including 4n, 4p, 4q, and 4t) possess a
heteroatom at the para position of their aromatic ring. The
cytotoxicity of the thiadiazoles 4a–v was evaluated using
fibroblast L929 cell line. The low toxicity (if any) of the
above-mentioned benzylic-substituted derivatives suggests
that these thiadiazoles are selective inhibitors of mycobac-
terial micro-organisms. Further lead optimization studies
are currently ongoing in our laboratory based on the struc-
ture–activity relationship data obtained in the present
work.
5. Dover L.G., Alahari A., Gratraud P., Gomes J.M.,
Bhowruth V.E. (2007) EthA, a common activator of
thiocarbamide-containing drugs acting on different
mycobacterial targets. Antimicrob Agents Chemo-
ther;51:1055–1063.
6. Qian L., Ortiz de Montellano P.R. (2006) Oxidative acti-
vation of thiacetazone by the Mycobacterium tubercu-
losis flavin monooxygenase EtaA and human FMO1
and FMO3. Chem Res Toxicol;19:443–449.
7. DeBarber A.E., Mdluli K., Bosman M., Bekker L.G.,
Barry C.E. (2000) Ethionamide activation and sensitivity
in multidrug-resistant Mycobacterium tuberculosis.
Proc Natl Acad Sci USA;97:9677–9682.
8. Haj Mohammad Ebrahim Tehrani K., Sardari S., Mash-
ayekhi V., Esfahani Zadeh M., Azerang P., Kobarfard F.
(2013) One pot synthesis and biological activity evalua-
tion of novel Schiff bases derived from 2-hydrazinyl-
1,3,4-thiadiazole. Chem Pharm Bull;61:160–166.
9. Borgne M.L., Marchand P., Duflos M., Delevoye-Seiller
B., Piessard-Robert S., Baut G.L., Hartmann R.W.,
Palzer M. (1997) Synthesis and in vitro evaluation of
3-(1-azolylmethyl)-1H-indoles and 3-(1-azolyl-1-phe-
nylmethyl)-1H-indoles as inhibitors of P450 arom. Arch
Pharm;330:141–145.
10. Glennon R.A., Chaurasia C., Titeler M. (1990) Binding
of indolylalkylamines at 5-HT2 serotonin receptors:
examination of a hydrophobic binding region. J Med
Chem;33:2777–2784.
Acknowledgment
11. Marcin L.R., Higgins M.A., Zusi F.C., Zhang Y., Dee
M.F., Parker M.F., Muckelbauer J.K. et al. (2011)
Synthesis and SAR of indole-and 7-azaindole-1,3-di-
carboxamide hydroxyethylamine inhibitors of BACE-1.
Bioorg Med Chem Lett;21:537–541.
We are most grateful for the continuing financial support of
this research project by Zanjan University of Medical Sci-
ences (Grant No. A-12-227-6).
12. Penthala N.R., Yerramreddy T.R., Crooks P.A. (2011)
Synthesis and in vitro screening of novel N-benzyl
aplysinopsin analogs as potential anticancer agents.
Bioorg Med Chem Lett;21:1411–1413.
Conflict of Interest
The authors have no conflict of interest to declare.
13. Wu S., Wang L., Guo W., Liu X., Liu J., Wei X., Fang
B. (2011) Analogues and derivatives of oncrasin-1, a
novel inhibitor of the C-terminal domain of RNA poly-
References
merase II and their antitumor activities.
J Med
Chem;54:2668–2679.
1. Dye C., Williams B.G. (2010) The population dynamics
and control of tuberculosis. Science;328:856–861.
2. World Health Organization (2011) Global Tuberculosis
Control: WHO Report 2011. Geneva: World Health
Organization.
14. Battaglia S., Boldrini E., Settimo F.D., Dondio G.,
Motta C.L., Marini A.M., Primofiore G. (1999) Indole
amide derivatives: synthesis, structure–activity relation-
ships and molecular modelling studies of a new series
of histamine H1-receptor antagonists. Eur
J Med
3. World Health Organization (2010) Multidrug and exten-
sively drug-resistant TB (M/XDR-TB): 2010 global
report on surveillance and response. Geneva: World
Health Organization.
Chem;34:93–105.
15. Cavrini V., Gatti R., Roveri P., Cesaroni M.R., Mazzoni
A., Fiorentini C. (1984) Syntheses and in vitro antimy-
12
Chem Biol Drug Des 2013

Antimycobacterial Thiadiazolylhydrazones
cotic activities of 1-benzyl-3-(1-imidazolylmethyl)
indoles. Arch Pharm;317:662–668.
epihalogenohydrins. Tandem oxirane-opening—1,3-
oxazole-closure process. Tetrahedron;67:8775–8779.
23. Na Y.M., Le Borgne M., Pagniez F., Le Baut G., Le
Pape P. (2003) Synthesis and antifungal activity of new
1-halogenobenzyl-3-imidazolylmethylindole derivatives.
Eur J Med Chem;38:75–87.
24. Reddy Y.T., Reddy P.N., Koduru S., Damodaran C.,
Crooks P.A. (2010) Aplysinopsin analogs: synthesis
and anti-proliferative activity of substituted (Z)-5-(N-
benzylindol-3-ylmethylene)imidazolidine-2,4-diones.
Bioorg Med Chem;18:3570–3574.
16. Ewing W.R., Becker M.R., Manetta V.E., Davis R.S.,
Pauls H.W., Mason H., Choi-Sledeski Y.M. et al.
(1999) Design and structure–activity relationships of
potent and selective inhibitors of blood coagulation
factor Xa. J Med Chem;42:3557–3571.
17. Tholander J., Bergman J. (1999) Syntheses of 6-substi-
tuted indolo[3,2-b]carblazoles, including 6-formylindolo
[3,2-b]carblazoles, an extremely efficient ligand for the
TCDD (Ah) receptor. Tetrahedron;55:6243–6260.
18. Rezayan A.H., Azerang P., Sardari S., Sarvary A.
(2012) Synthesis and biological evaluation of coumarin
derivatives as inhibitors of Mycobacterium bovis (BCG).
Chem Biol Drug Des;80:929–936.
25. Jain A.K., Sharma S., Vaidya A., Ravichandran V.,
Agrawal R.K. (2013) 1,3,4-thiadiazole and its deriva-
tives: a review on recent progress in biological activi-
ties. Chem Biol Drug Des;81:557–576.
19. Szmuzkovicz J. (1963) Belg. Patent 621047.
20. Heaney H., Ley S.V. (1974) 1-Benzylindole. Org
Synth;54:58.
21. Zahran M.A.H., Ibrahim A.M. (2009) Synthesis and cel-
lular cytotoxicities of new N-substituted indole-3-carb-
26. Karakus S., Rollas S. (2002) Synthesis and antituber-
culosis activity of new N-phenyl-N′-[4-(5-alkyl/
arylamino-1,3,4-thiadiazole-2-yl)phenyl]thioureas. Far-
maco;57:577–581.
27. Oruc E.E., Rollas S., Kandemirli F., Shvets N., Dimoglo
A.S. (2004) 1,3,4-thiadiazole derivatives. Synthesis,
structure elucidation, and structure-antituberculosis
activity relationship investigation. J Med Chem;47:
6760–6767.
aldehyde and their indolylchalcones.
J
Chem
Sci;121:455–462.
22. Suzdalev K.F., Den’kina S.V., Borodkin G.S., Tkachev
V.V., Kiskin M.A., Kletsky M.E., Burov O.N. (2011)
Reaction of 2-chloroindole-3-carbaldehyde with
Chem Biol Drug Des 2013
13