Design, synthesis, and molecular docking studies of a conjugated thiadiazole-thiourea scaffold as antituberculosis agents

Article abstract of DOI:10.1248/bpb.b15-00698

In view of the emergence and frequency of multidrug-resistant and extensively drug-resistant tuberculosis and consequences of acquired resistance to clinically used drugs, we undertook the design and synthesis of novel prototypes that possess the advantage of the two pharmacophores of thiourea and 1,3,4-thiadiazole in a single molecular backbone. Three compounds from our series were distinguished from the others by their promising activity profiles against Mycobacterium tuberculosis strain H₃₇Rv. Compounds 11 and 19 were the most active representatives with minimum inhibitory concentration (MIC) values of 10.96 and 11.48 μM, respectively. Compound 15 was shown to inhibit M. tuberculosis strain H37Rv with an MIC value of 17.81 μM. Cytotoxicity results in the Vero cell line showed that these three derivatives had selectivity indices between 1.8 and 8.7. In order to rationalize the biological results of our compounds, molecular docking studies with the enoyl acyl carrier protein reductase (InhA) of M. tuberculosis were performed and compounds 11, 15, and 19 were found to have good docking scores in the range of 7.12 to 7.83 kcal/mol.

Full text of DOI:10.1248/bpb.b15-00698

502
Biol. Pharm. Bull. 39, 502–515 (2016)
Vol. 39, No. 4
Regular Article
Design, Synthesis, and Molecular Docking Studies of a Conjugated
Thiadiazole–Thiourea Scaffold as Antituberculosis Agents
Esra Tatar,a Sevgi Karakuş,a Şükriye Güniz Küçükgüzel,a Sinem Öktem Okullu,b Nihan Ünübol,b
Tanıl Kocagöz,b Erik De Clercq,c Graciela Andrei,c Robert Snoeck,c Christophe Pannecouque,c
Sadık Kalaycı,d Fikrettin Şahin,d Dharmarajan Sriram,e Perumal Yogeeswari,e and
,a
İlkay Küçükgüzel*
aꢀDepartmentꢀofꢀPharmaceuticalꢀChemistry,ꢀFacultyꢀofꢀPharmacy,ꢀMarmaraꢀUniversity;ꢀHaydarpaşa,ꢀİstanbulꢀ
b
34668, Turkey: ꢀDepartmentꢀofꢀMedicalꢀMicrobiology,ꢀSchoolꢀofꢀMedicine,ꢀAcıbademꢀUniversity;ꢀAtaşehir,ꢀİstanbulꢀ
c
34752, Turkey: Rega Institute for Medical Research; KU Leuven, Leuven, Minderbroedersstraat 10, B-3000 Leuven,
d
Belgium: Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University;
İstanbulꢀ34755,ꢀTurkey:ꢀandꢀe Medicinal Chemistry and Drug Discovery Research Laboratory, Pharmacy Group, Birla
Institute of Technology and Science-Pilani; Hyderabad Campus, Jawahar Nagar, Telangana 500078, India.
Received September 8, 2015; accepted December 16, 2015
In view of the emergence and frequency of multidrug-resistant and extensively drug-resistant tuberculo-
sis and consequences of acquired resistance to clinically used drugs, we undertook the design and synthesis
of novel prototypes that possess the advantage of the two pharmacophores of thiourea and 1,3,4-thiadiazole
in a single molecular backbone. Three compounds from our series were distinguished from the others by
their promising activity profiles against Mycobacterium tuberculosis strain H37Rv. Compounds 11 and 19
were the most active representatives with minimum inhibitory concentration (MIC) values of 10.96 and
11.48 µM, respectively. Compound 15 was shown to inhibit M. tuberculosis strain H37Rv with an MIC value of
17.81 µM. Cytotoxicity results in the Vero cell line showed that these three derivatives had selectivity indices
between 1.8 and 8.7. In order to rationalize the biological results of our compounds, molecular docking stud-
ies with the enoyl acyl carrier protein reductase (InhA) of M. tuberculosis were performed and compounds
11, 15, and 19 were found to have good docking scores in the range of ꢀ7.12 to ꢀ7.83kcal/mol.
Key words 1,3,4-thiadiazole; thiourea; antituberculosis activity; cytotoxicity; molecular docking
Tuberculosis has been a well known infection throughout rivative and amikacin, kanamycin or capreomycin is ineffec-
known history of humankind and was called “Captain Among tual.4) In view of the frequency and emergence of MDR and
These Men of Death” in 18th and 19th centuries. From 1882, XDR tuberculosis and consequences of acquired resistance
the date when Mycobacterium tuberculosis was identified as to clinically employed drugs, researchers have persisted in
the etiological agent of tuberculosis, till now scientists have performing synthesis and anti-tuberculosis evaluation of novel
struggled to discover efficient therapies against this contem- compounds bearing various chemical entities.
porary infection.1) Approximately twenty years after WHO
Compounds with thiourea, acylthiourea and thioamide
declared tuberculosis (TB) a “global public health emergency,” moiety have been synthesized as a result of drawing inspira-
an estimated 8.6 million new case and 1.3 million death were tion from second line antituberculosis pro-drugs; ethionamide
noted in WHO’s Global Tuberculosis Report 2013.2) These (ETH), prothionamide, thiacetazone and isoxyl (thiocar-
large numbers of cases and deaths were based upon decreas- lide).5–11) It was already known that, ETH inhibits cell wall
ing efficacy of four first line drugs; isoniazid, rifampicin, biosynthesis of M. tuberculosis. Baulard et al. reported the
ethambutol, pyrazinamide and increasing resistance to at identification of ETH-activator; Rv3854c which was then
least isoniazid and rifampicin which was called multidrug- termed EthA.12) Another noteworthy work, revealed the
resistant (MDR) tuberculosis (TB) MDR-TB treatment com- mechanism of activation of ETH due to corresponding S-
prises of second line antituberculosis drugs; ethionamide, oxide by monooxygenase Rv3854c.13) It was noted that isoxyl
prothionamide, thioacetazone, isoxyl (thiocarlide), amikacin, activation also requires EthA-mediated oxidation.14) Isoxyl has
kanamycin or capreomycin as well as fluoroquinolone deriva- been reported to be non-toxic in isoxyl-treated individulas at
tives (ofloxacin, levofloxacin, moxifloxacin and gatifloxacin). therapeutic doses but its poor solubility in water decreases
Second line antituberculosis drugs are less potent, more toxic its bioavalibility henceforth a new administration route-direct
and more expensive than the first line drugs moreover anti- pulmonary delivery has been suggested due to refurbish-
TB injectable drugs; amikacin, kanamycin or capreomycin ment of old agents for emerging clinical needs.15) All of these
decreases the therapy success rate due to their route of admin- phenomenon maintain undivided interest in thiourea synthe-
istration.3)
sis. As well as thiourea based compounds, new candidates
As a consequence of multiple mutations in specific resis- bearing both heterocycles and thiourea moieties have been
tant-associated genes of Mycobacterium tuberculosis (inhA, shown as promising antituberculosis agents.7,8,10,16) It is known
katG, rpoB, gyrA, rrs, tlyA and eis), extensively drug-resistant that heterocyclic scaffolds possess a leading role in design-
(XDR) TB has arisen. It was shown that treatment of XDR-TB ing novel class of chemotypes as drug candidates. Among
by using isoniazid and rifampicin plus a fluoroquinolone de- them, 1,3,4-thiadiazoles have been reported to possess a wide
*To whom correspondence should be addressed. e-mail: ikucukguzel@marmara.edu.tr
© 2016 The Pharmaceutical Society of Japan

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Biol. Pharm. Bull.
503
Fig. 1. Several Drugs on Market Possessing 1,3,4-Thiadiazole Core
range of biological activities including antiproliferative, anti- been found as a potent inhibitor of mycobacterial growth
bacterial, antifungal, and antimycobacterial functions.17–21) (IC90=0.23µg/mL).29) In a previous report, we identified a
Based on reported antitumor and uricogenic activity of thiourea derivative linked with 3-alkylthio-1,2,4-triazole moi-
2-amino-1,3,4-thiadiazole (ATDA, NSC4728); Abdel Rahman ety [D] as an inhibitor of M. tuberculosis H37Rv though it has
and Mohamed22) synthesized 1,3-disubstituted thioureas via a low selectivity.7)
5-(4-bromophenyl)-1,3,4-thiadiazol-2-amine. Some analogues
Considering the findings above and in continuation of our
from this series, that unite 1,3,4-thiadiazol ring with thiourea efforts for the development of anti-infective agents, we under-
moiety, were reported to demonstrate promising IC50 values took the design and synthesis of some novel prototypes which
(2.58–6.47 µm) against A549 (Non-small Cell Lung Cancer) possess advantage of the two pharmacophores of thiourea and
cell line.22) Another work on very similar chemotypes with 1,3,4-thiadiazole in single molecular backbone. Our design
our compounds has reported the synthesis of triazolothiadi- strategy included to combine the antimycobacterial pharma-
azolethiones and thiadiazolothiadiazolimines that were gained cophores (indicated in dashed boxes), a 2-amino/5-aryl sub-
by heterocyclization of N-(4-chlorophenyl)/phenyl-N′-[5-(4- stituted 1,3,4-thiadiazole ring [A, B, C] with aryl/heteroaryl
methoxyphenyl)/(2-chlorophenyl)/phenyl-1,3,4-thiadiazol-2-yl]- thiourea moiety [D, E, F], as illustrated in Fig. 2. During the
thioureas and N-phenyl/(2-chlorophenyl)-N′-(5-phenyl-1,3,4- course of this study, we synthesized and characterized hybrid
thiadiazol-2-yl)thioureas as well as their antifungal activity compounds comprising thiourea and 1,3,4-thiadiazole motifs
against Aspergillus niger and Fusarium oxysporium.23)
and evaluated them for their anti-tuberculosis activity against
Actually, 1,3,4-thiadiazole ring is found in several drugs in M. tuberculosis H37Rv strain, whereas their cytotoxicity pro-
clinical use. Typical examples are Acetazolamide and Meth- file was assayed by using Vero cells.
azolamide (carbonic anhydrase inhibitors); Sulfamethizole and
Cefazolin (antibacterials) and Megazol (treatment of human
African trypanosomiasis) (Fig. 1). We have recently reported
two groups of novel 1,3,4-thiadiazole–4-thiazolidinone hy-
MATERIALS AND METHODS
Synthetic Chemistry All solvents and reagents were
brids which were active against hepatitis C virus (HCV) via obtained from commercial sources and used without purifica-
inhibition of NS5B polymerase.24,25) It is also worth to mention tion. All melting points (°C, uncorrected) were determined
that M. tuberculosis contains three β-carbonic anhydrase (CA) using Kleinfeld SMP-II basic model melting point apparatus.
genes in its genome; Rv1284, Rv3588c and Rv3273 encoding Elemental analyses were obtained using Elementar Analysen-
for mtCA 1, mtCA 2 and mtCA 3, respectively. Inhibitory systeme GmbH varioMICRO CHNS and are consistent with
activity of acetazolamide and methazolamide against mtCA 1 the assigned structures. Infrared spectra were recorded on a
and mtCA 3, with inhibition constants in the submicromolar Shimadzu FTIR 8400S and data are expressed in wavenumber
ranges has been reported.26) This promising result may lead to ν (cm−1). NMR spectra were recorded on Bruker AVANCE-
1
the refurbishment of old carbonic anhydrase inhibitors as new DPX 400 at 400MHz for H-NMR and 100MHz for 13C-NMR
antimycobacterials.
(decoupled), the chemical shifts were expressed in δ (ppm)
In 2011, GlaxoSmithKline has reported a 1,3,4-thiadiazole downfield from tetramethylsilane (TMS) using dimethyl sulf-
derivative containing pyrazole and thiazole linkages. This oxide (DMSO)-d6 as solvent. High resolution (HR) electron
scaffold [A] has been reported to show potent activity towards impact (EI) and FAB-MS was recorded on a Jeol JMS-700 in-
Mtb (minimum inhibitory concentration (MIC)=0.19 µM) via strument. HR electrospray ionization (ESI)-MS was recorded
potent enoyl acyl carrier protein reductase (InhA) inhibition on a ICR Apex-Qe instruments. The liquid chromatographic
(IC50=3nM)27,28) (Fig. 1). Two other reports reveal antituber- system consists of an Agilent technologies 1100 series instru-
culosis properties of 1,3,4-thiadiazole scaffolds [B and C] ment equipped with a quaternary solvent delivery system and
with aryl substitution at C5 position.29,30) On the other hand, a model Agilent series G1315 A photodiode array detector.
antimycobacterial potency of thioureas such as isoxyl [E] is A Rheodyne syringe loading sample injector with a 50µL
well documented. A thiophenyl–thiourea derivative [F] has sample loop was used for the injection of the analytes. Chro-

504
Biol. Pharm. Bull.
Vol. 39, No. 4 (2016)
Fig. 2. Structures of Previously Reported 1,3,4-Thiadiazoles and Disubstituted Thioureas as Inhibitors of M. tuberculosis and the Strategy Employed
for Designing Thiadiazolyl Thioureas
matographic data were collected and processed using Agilent with HCl 5% at 70°C and then recrystallized from appropri-
Chemstataion Plus software. The separation was performed ated solvents.
at ambient temperature by using a reversed phase Kromasil
1-[5-(4-Chlorophenyl)-1,3,4-thiadiazole-2-yl]-3-[2-
5C-18 (4.6×250mm, 5µm particle size) column. All experi- (morpholine-4-yl)ethyl]thiourea (5)
ments were performed in gradient mode. The mobile phase
1H-NMR (400MHz, DMSO-d6) δ: 2.43–2.55 (4H, m),
was prepared by mixing acetonitrile and bidistilled water 3.47–3.85 (8H, m), 7.57 (2H, d, J=8.5Hz), 7.92 (2H, d,
(50:50 v/v during 0–3min, 75:25 v/v during 3–5min, 100:0 J=8.5Hz), 8.47 and 8.61 (1H, brs and brs), 12.37 (1H, brs).
v/v during 5–7min, 100:0 v/v during 7–12min, 75:25 v/v 13C-NMR (100MHz, DMSO-d6) δ: 53.35 and 53.72 (d), 56.47
during 12–15min, 50:50 v/v during 15–18min) and filtered (s), 65.45 (s), 66.85 (s), 128.79 (s), 129.86 (s), 130.07 (s), 135.64
through a 0.45µm pore filter and subsequently degassed by ul- (s), 157.66 (s), 163.96 (s), 180.49 (s). IR, cm−1: 3321, 1654, 1114.
trasonication, prior to use. Solvent delivery was employed at a HR-MS (EI+) m/z: 383.0649 (Calcd for C15H1835ClN5O32S2:
flow rate of 1mL·min−1. Detection of the analytes was carried 383.0641), 349.0879 (Calcd for C15H17N5O32S34S: 349.0874).
out at 254 and 280nm.
HPLC tR (min): 10.45. mp: 247–249°C (acetonitrile).
Synthesis of 1-Aroylthiosemicarbazides 1, 2 were car-
1-[5-(4-Chlorophenyl)-1,3,4-thiadiazole-2-yl]-3-[2-(phenyl)-
ried out according to the procedure given in lit24); 4-chloro- ethyl]thiourea (6)
benzoylthiosemicarbazide 1 (melting point (mp): 214°C lit31):
1H-NMR (400MHz, DMSO-d6) δ: 2.91 (2H, t, J=7.2Hz),
218–220°C), 4-fluorobenzoylthiosemicarbazide 2 (mp: 180°C 3.73–3.78 (2H, m), 7.21–7.35 (5H, m), 7.57 (2H, d, J=8.5Hz),
lit31): 172°C).
7.91 (2H, d, J=8.5Hz), 8.61 (1H, brs), 12.41 (1H, brs).
Synthesis of 2-Amino-5-(4-substituted phenyl)-1,3,4- 13C-NMR (100MHz, DMSO-d6) δ: 34.63 (s), 46.43 (s), 126.97
thiadiazoles 3, 4 were performed according to the litera- (s), 128.58 (s), 128.98 and 129.04 (d), 129.14 and 129.37 (d),
ture method24); 2-amino-5-(4-chlorophenyl)-1,3,4-thiadiazole 129.47 and 129.66 (d), 130.05 (s), 130.97 (s), 135.01 and 135.55
3 (mp: 230°C lit32): 226–227°C), 2-amino-5-(4-fluorophenyl)- (d), 139.65 and 139.98 (d), 180.49 (s). IR, cm−1: 3321, 1641,
1,3,4-thiadiazole 4 (mp: 240°C lit33): 232–234°C).
1112. HR-MS (EI+) m/z: 374.0473 (Calcd for C17H1535ClN432S2:
Synthesis of 1-[5-(4-Chloro/4-fluorophenyl)-1,3,4-thia- 374.0426), 340.0526 (Calcd for C17H1335ClN432S : 340.0549).
diazole-2-yl]-3-substituted Thioureas 5–20 The solution HPLC tR (min): 10.59. mp: 244°C (acetonitrile).
of 5-(4-chloro/4-fluorophenyl)-1,3,4-thiadiazol-2-amine (3, 4)
1-[5-(4-Chlorophenyl)-1,3,4-thiadiazole-2-yl]-3-[2-(4-
in acetonitrile was reacted with equimolar amounts of ap- chlorophenyl)ethyl]thiourea (7)
propriate isothiocyanates at 140°C for 20h. Acetonitrile was
1H-NMR (400MHz, DMSO-d6) δ: 2.84–2.96 (2H, m),
evaporated under vacuo and the solid precipitated was washed 3.75–3.82 (2H, m), 7.21–7.35 (4H, m), 7.57–7.62 (2H, m),

Vol. 39, No. 4 (2016)
Biol. Pharm. Bull.
505
7.78–7.93 (2H, m), 8.70 (1H, brs), 11.99 (1H, brs). 13C-NMR 129.64 (s), 130.11 (s), 130.46 (s), 134.01 (s), 132.52 (s), 135.99
(100MHz, DMSO-d6) δ: 33.82 (s), 46.09 (s), 128.81 and (s), 139.74 (s), 189.45 (s). IR, cm−1: 3313, 1641, 1176. HR-MS
128.86 (d), 129.81 (s), 131.09 (s), 131.71 (s), 135.67 (s), 138.55 (EI+) m/z: 393.9869 (Calcd for C16H1235Cl2N4S2: 393.9880),
(s), 180.49 (s). IR, cm−1: 3321, 1641, 1112, 1089. HR-MS (EI+) 360.0010 (Calcd for C16H1035Cl2N4S: 360.0003). HPLC tR (min):
m/z: 408.0039 (Calcd for C17H1437Cl2N4S2: 408.0037), 374.0152 10.64. mp: 284–286°C (methanol).
(Calcd for C17H1237ClN4S: 374.0159). HPLC tR (min): 10.58.
mp: 222–223°C (acetonitrile).
1-[5-(4-Chlorophenyl)-1,3,4-thiadiazole-2-yl]-3-(benzoyl)-
thiourea (8)
1-[5-(4-Chlorophenyl)-1,3,4-thiadiazole-2-yl]-3-(2-chloro-5-
trifluoromethylphenyl)thiourea (14)
1H-NMR (400MHz, DMSO-d6) δ: 7.56–7.65 (4H, m),
7.76 (1H, d, J=8.7Hz), 7.87–7.98 (3H, m), 10.33 (1H, brs).
1H-NMR (400MHz, DMSO-d6) δ: 7.56–7.64 (4H, m), 13C-NMR (100MHz, DMSO-d6) δ: 122.42 (s), 124.11 (s),
7.67–7.72 (1H, m), 8.01–8.04 (3H, m), 8.14 (1H, d, J=7.2Hz), 125.10 (s), 126.03 (s), 126.84 (s), 128.44 (s), 129.53 (s),
12.29 (1H, brs), 13.23 and 14.38 (1H, brs and brs). 13C-NMR 130.15 (s), 135.34 (s), 136.12 (s), 138.02 (s), 180.49 (s). IR,
(100MHz, DMSO-d6) δ: 116.99 and 117.28 (d), 127.47 (s), cm−1: 3286, 3204, 1634, 1121. HR-MS (FAB) m/z: 448.9626
129.11 and 129.89 (d), 130.01 (s), 132.06 (s), 133.76 (s), (Calcd for C16H1035Cl2F3N4S2: 448.9670), 414.9787 (Calcd
134.21 (s), 160.03 (s), 161.69 (s), 162.38 (s), 181.33 (s). IR, for C16H835Cl2F3N4S: 414.9793). HPLC tR (min): 11.30. mp:
cm−1: 3321, 1670, 1641, 1176. HR-MS (EI+) m/z: 376.0016 306–309°C (methanol).
(Calcd for C16H1137ClN4OS2: 376.0063), 374.0057 (Calcd for
C16H1135ClN4OS2: 374.0063), 105.0315 (Calcd for C7H5O: trifluoromethylphenyl)thiourea (15)
105.0334), 77.0422 (Calcd for C6H5: 77.0385). HPLC tR (min):
1-[5-(4-Chlorophenyl)-1,3,4-thiadiazole-2-yl]-3-(4-chloro-3-
1H-NMR (400MHz, DMSO-d6) δ: 7.53–7.70 (3H, m), 7.85
10.64. mp: 313–315°C (acetonitrile).
(2H, d, J=8.7Hz), 8.08–8.16 (2H, m), 9.61 (1H, brs), 10.84
1-[5-(4-Chlorophenyl)-1,3,4-thiadiazole-2-yl]-3-(4- (1H, brs). 13C-NMR (100MHz, DMSO-d6) δ: 117.98 (s), 121.26
cyanophenyl)thiourea (10)
and 121.59 (d), 124.3 and 124.92 (d), 125.22 (s), 128.55 and
1H-NMR (400MHz, DMSO-d6) δ: 7.58 (2H, d, J=6.6Hz), 128.84 (d), 129.20 and 129.44 (d), 129.66 and 129.82 (d),
7.37 (2H, d, J=8.7Hz), 7.89 (2H, d, J=10.8Hz), 7.99 (2H, 130.03 (s), 131.01 (s), 132.23 and 132.69 (d), 136.21 (s), 139.77
d, J=8.7Hz), 8.70 (1H, brs), 10.90 (1H, brs). 13C-NMR (s), 154.09 (s), 185.32 (s). IR, cm−1: 3327, 1653, 1136, 1121.
(100MHz, DMSO-d6) δ: 105.42 (s), 119.93 (s), 121.94 (s), HR-MS (FAB) m/z: 448.9673 (Calcd for C16H1035Cl2F3N4S2:
128.96 (s), 129.44 (s), 130.16 (s), 133.52 (s), 136.29 (s), 144.51 448.9670), 414.9826 (Calcd for C16H835Cl2F3N4S: 414.9793).
(s), 154.15 (s), 169.52 (s), 185.52 (s). IR, cm−1: 3311, 2205, 1641, HPLC tR (min): 11.40. mp: 298–302°C (methanol).
1176. HR-MS (EI+) m/z: 371.0027 (Calcd for C16H1035ClN5S2:
1-[5-(4 -Fluorophenyl)-1,3,4 -thiadiazole-2-yl]-3-
371.0066), 337.0181 (Calcd for C16H835ClN5S: 337,0188), benzoylthiourea (16)
252.9544 (Calcd for C9H435ClN3S2: 252.9535), 118.0539 (Calcd
1H-NMR (400MHz, DMSO-d6) δ: 7.36–7.64 (5H, m),
for C7H6N2: 118.0531). HPLC tR (min): 9.68. mp: 263–264°C 8.01–8.11 (4H, m), 12.31 (1H, brs), 13.15 (1H, brs). 13C-NMR
(methanol). (100MHz, DMSO-d6) δ: 116.99 and 117.28 (d), 127.47 (s),
1-[5-(4-Chlorophenyl)-1,3,4-thiadiazole-2-yl]-3-(4- 129.11 and 129.89 (d), 130.01 (s), 132.06 (s), 133.76 (s), 134.21
fluorophenyl)thiourea (11)
(s), 160.03 (s), 161.69 and 162.38 (d), 165.68 (s), 181.33 (s).
1H-NMR (400MHz, DMSO-d6) δ: 7.16 (2H, t, J=8.4Hz), IR, cm−1: 3306, 1672, 1635, 1220, 1153. HR-MS (ESI) m/z:
7.49–7.76 (4H, m), 7.88–7.97 (3H, m,), 10.56 (1H, brs). 359.0433 (Calcd for C16H12FN4OS2: 359.0431). HPLC tR (min):
13C-NMR (100MHz, DMSO-d6) δ: 115.57 and 115.87 (d), 8.75. mp: 262°C (methanol).
128.59 (s), 128.89 (s), 129.64 (s), 130.13 (s), 136.06 (s), 136.62
1-[5-(4-Fluorophenyl)-1,3,4-thiadiazole-2-yl]-3-(4-
(s), 180.49 (s). IR, cm−1: 3327, 1653, 1637, 1229, 1176. HR-MS cyanophenyl)thiourea (18)
(EI+) m/z: 364.0007 (Calcd for C15H1035ClFN4S2: 364.0019),
1H-NMR (400MHz, DMSO-d6) δ: 7.37 (2H, t, J=8.1Hz),
330.0129 (Calcd for C15H835ClFN4S: 330.0142), 252.9545 (Calcd 7.73 (2H, d, J=8.7Hz), 7.93–8.02 (5H, m), 10.88 (1H, s).
for C9H435ClN3S2: 252.9535), 111.0471 (Calcd for C6H6NF: 13C-NMR (100MHz, DMSO-d6) δ: 105.37 (s), 117.08 and
111.0484). HPLC tR (min): 9.95. mp: 283–284°C (methanol).
117.38 (d), 121.92 (s), 127.16 and 127.20 (d), 129.63 and 129.75
1-[5-(4-Chlorophenyl)-1,3,4-thiadiazole-2-yl]-3-(3,5- (d), 133.52 (s), 144.54 (s), 162.58 (s), 165.89 (s), 180.49 (s).
bistrifluoromethylphenyl)thiourea (12)
IR, cm−1: 3313, 2207, 1658, 1234, 1157. HR-MS (ESI) m/z:
1H-NMR (400MHz, DMSO-d6) δ: 7.47–7.52 (2H, m), 356.0437 (Calcd for C16H11FN5S2: 356.0434). HPLC tR (min):
7.57–7.61 (1H, m), 7.72–7.76 (2H, m), 7.84 (1H, s), 7.89–7.95 8.53. mp: 281–283°C (methanol).
(1H, m), 8.17 and 8.44 (1H, s and s), 10.99 and 11.65 (1H, s
1-[5-(4-Fluorophenyl)-1,3,4-thiadiazole-2-yl]-3-(4-
and s). 13C-NMR (100MHz, DMSO-d6) δ: 118.31 (s), 121.94 fluorophenyl)thiourea (19)
(s), 122.36 (s), 125.75 (s), 128.58 (s), 129.86 (s), 130.86 (s),
1H-NMR (400MHz, DMSO-d6) δ: 7.16 (2H, t, J=7.8Hz),
131.50 (s), 134.67 (s), 136.32 (s), 141.76 (s), 155.84 (s), 169.53 7.36 (3H, t, J=8.7Hz), 7.64–7.68 (2H, m), 7.91–8.01 (2H, m),
(s), 189.45 (s). IR, cm−1: 3213, 1630, 1176. HR-MS (EI+) m/z: 10.55 (1H, s). 13C-NMR (100MHz, DMSO-d6) δ: 115.57 and
481.9849 (Calcd for C17H935ClF6N4S2: 481.9861), 447.9963 115.86 (d), 117.03 and 117.33 (d), 127.36 (s), 129.53 and 129.65
(Calcd for C17H735ClF6N4S: 447.9984). HPLC tR (min): 11.54. (d), 136.63 (s), 162.46 (s), 165.76 (s), 180.49 (s). IR, cm−1: 3325,
mp: 296–299°C (methanol).
3201, 1651, 1224, 1155. HR-MS (ESI) m/z: 349.0392 (Calcd for
1-[5-(4-Chlorophenyl)-1,3,4-thiadiazole-2-yl]-3-(5-chloro-2- C15H11F2N4S2: 349.0387). HPLC tR (min): 9.02. mp: 293–295°C
methylphenyl)thiourea (13)
(methanol).
1H-NMR (400MHz, DMSO-d6) δ: 2.15 (3H, s), 7.21–7.36
1-[5-(4-Fluorophenyl)-1,3,4-thiadiazole-2-yl]-3-(4-chloro-3-
(3H, m), 7.55–7.76 (3H, m), 7.86 (2H, d, J=8.4Hz), 10.14 (1H, trifluoromethylphenyl)thiourea (20)
brs). 13C-NMR (100MHz, DMSO-d6) δ: 18.00 (s), 128.92 (s), 1H-NMR (400MHz, DMSO-d6) δ: 7.37–7.63 (4H, m),

506
Biol. Pharm. Bull.
Vol. 39, No. 4 (2016)
7.92–7.97 (3H, m), 8.13 (1H, s), 10.84 (1H, s). 13C-NMR (200µg/mL), and then serial two-fold dilutions were made in
(100MHz, DMSO-d6) δ: 31.39 (s), 117.08 and 117.37 (d), order to obtain a concentration range from 6.25 to 200µg/mL,
127.24 (s), 129.61 and 129.73 (d), 132.29 (s), 139.80 (s), 162.56 in 15-mL sterile test tubes containing nutrient broth. The 96-
(s), 207.24 (s). IR, cm−1: 3329, 3225, 1651, 1226, 1159. HR-MS well plates were prepared by dispensing into each well 95µL
(ESI) m/z: 432.9972 (Calcd for C16H11ClF4N4S2: 432.9966). of nutrient broth and 5µL of the inoculum. Two hundred
HPLC tR (min): 10.21. mp: 299–301°C (methanol).
microliters of nutrient broth without inoculum was transferred
Anti-tuberculosis Activity The antituberculosis activ- into the first wells as positive control. Aliquots, (100µL) taken
ity of compounds was tested against M. tuberculosis H37Rv from the 200-µg/mL stock solution, were added to the second
strain. For the MIC determination, the compounds were dis- well.
solved in DMSO and serial two fold dilutions were done in
One hundred microliters from the respective serial dilutions
Middlebrook 7H9 broth with glycerol. Microorganisms were was transferred into 5 consecutive wells. The last well con-
suspended in Middlebrook 7H9 broth to match the turbid- taining 195µL of nutrient broth without compound and 5µL
ity of 0.5 McFarland (1.5×108 cfu/mL) and 1/10 dilution was of the inoculum on each strip was used as negative control.
prepared from this suspension and used as inoculum. The Contents of each well were mixed on plate shaker at 300rpm
tested final concentrations ranged between 512 to 0.5µg/mL. for 20s and then incubated at appropriate temperatures for
To make sure that DMSO did not show any inhibitory activ- 24h. Microbial growth in each medium was determined by
ity, controls prepared with serial dilutions of DMSO were reading the absorbance (Abs) at 630nm using the ELx 800
also tested. Tubes were incubated at 37°C for 24h and then universal microplate reader (Biotek Instrument Inc., Highland
examined for turbidity. MIC was determined if turbidity was Park, VT, U.S.A.) and confirmed by plating 5-µL samples
observed in the positive control tube containing no compound from clear wells on nutrient agar medium. The MIC was de-
and no turbidity in the negative control tube containing no fined as the lowest concentration of the compounds to inhibit
microorganism.34–36)
the growth of microorganisms. Cefepime (Maxipime®, Bristol-
Antimicrobial Activity The antimicrobial activities of Myers Squibb, Istanbul, Turkey) at the concentration range
all compounds were evaluated in the Department of Genetics of 500–7.8µg/mL was prepared in nutrient broth and used as
and Bioengineering, Faculty of Engineering and Architecture, standard drug for positive control.
Yeditepe University. The antibacterial and antifungal activities
In Vitro Antiviral Assays A detailed account of experi-
of the compounds were evaluated against 8 microbial cul- mental data related to in vitro antiviral assays (inhibition of
tures isolates of 6 bacteria and 1 yeast species by micro-well human immunodeficiency virus (HIV)-induced cytopathicity
dilution assay as described below.37) Microorganisms were in MT-4 cells, antiviral assays other than HIV and anti-influ-
provided by the Department of Genetics and Bioengineering, enza assays) were given in literature.38)
Faculty of Engineering and Architecture, Yeditepe University,
Istanbul, Turkey. The microorganisms used were Staphylo-
coccus aureus, Escherichia coli, Bacillus subtilis, Klebsiella
pneumoniae, Pseudomonas aeruginosa, Staphylococcus epi-
RESULTS AND DISCUSSION
Chemistry 4-Chlorobenzoylthiosemicarbazide 1, 4-fluo-
dermidis and Candida albicans. Ampicillin, Cefepime and robenzoylthiosemicarbazide 2, their corresponding 1,3,4-thia-
Amphotericin B were used as the positive sensitivity reference diazoles; 2-amino-5-(4-chlorophenyl)-1,3,4-thiadiazole 3 and
standard for bacteria and yeast.
2-amino-5-(4-fluorophenyl)-1,3,4-thiadiazole 4, were synthe-
Micro-well Dilution Assay The sensitivity of the bacte- sized according to the procedures given in literature.24) Their
rial strains towards the compounds was quantitatively evalu- purity were checked by TLC and HPLC and melting points
ated from the MIC values obtained by the micro-well dilution of compounds 1–4 are found to be in accordance with lit-
method. The inocula of the bacterial strains were prepared erature.31–33) Synthetic route to compounds 5–20 is presented
from 12-h broth cultures and suspensions were adjusted to 0.5 in Chart 1. As the final step, 5-(4-substituted phenyl)-1,3,4-
McFarland standard turbidity. Compounds dissolved in DMSO thiadiazol-2-amines (3, 4) were reacted with appropriate
were first prepared at the highest concentration to be tested isothiocyanates in acetonitrile to yield 1-[5-(4-substituted
Reagents and conditions: (a) acetone/NaHCO3; (b) H2SO4 (conc.), rt; (c) R2-NCS/ACN, reflux.
Chart 1. Synthetic Route to Compounds 1–20

Vol. 39, No. 4 (2016)
Biol. Pharm. Bull.
507
phenyl)-1,3,4-thiadiazole-2-yl]-3-substituted thioureas 5–20. amine form in solid state. Fourier transform-infrared (FT-IR)
The purity of the synthesized compounds were checked by spectra revealed C=S strecthing vibrations at 1112–1176cm−1
TLC and HPLC. Since synthesis procedure and structural region.38)
characterization data of compounds 9 and 17 were already
reported by George et al.,39) spectral data were not provided noted as a singlet or two singlets corresponding to one pro-
for these compounds. ton at the region of 10.14–12.41ppm. An other characteristic
The characteristic N2–H signals of thiourea compounds
HR-MS confirmed the molecular weights and empirical for- signal N1–H was determined either at aromatic region or be-
mulae of compounds 5–20, with less than 5mmu bias between tween 8.13–9.61ppm due to their intra and/or intermolecular
calculated and experimental m/z values of molecular ions. hydrogen bond formation tendency by means of equilibria
Ionization mode was EI for compounds 5–13. Compounds between syn and anti conformations.44,45) In the 1H-NMR
14 and 15 which were analysed using FAB procedure and spectra of compounds 8 and 16 with benzoyl moiety on thio-
they demonstrated MH+ peaks instead of molecular ion (M+) urea structure, N1–H was determined at 12.29 and 12.31ppm,
peaks. Compounds 16–20 were also analysed using another respectively and N2-H was determined at 13.23 and 14.38 and
soft ionization technique; ESI procedure and their MH+ peaks 13.15ppm, respectively. 13C-NMR data of representative deriv-
were determined. The mass of M+ peaks and experimental atives was found to be descriptive for carbon framework and
fragments of compounds 5–13 and MH+ peaks of compounds detection of C=S signal between 180–207ppm was evaluated
14–20 match the mass of corresponding calculated ones. as an evidence for thiourea formation. Meanwhile the aromat-
Compounds 5–13 cleave off H2S forming carbodiimide frag- ic carbon resonances due to phenyl and 1,3,4-thiadiazole rings
ments in accordance with literature.40) Compounds 10 and 11 were also observed in expected regions.24,25,39)
allow unambiguous fragmentations of characteristics product
Antituberculosis Activity All synthesized compounds
ions for thiourea based compounds yielding the common 5–20 were initially screened for their in vitro antituberculosis
2-(4-chlorophenyl)-5-isothiocyanato-1,3,4-thiadiazole fragment activity against M. tuberculosis H37Rv strain. The MIC vs. M.
at m/z 252.9544 and as well as 4-aminobenzonitrile fragment tuberculosis H37Rv was determined by a broth microdilution
at m/z 118.0539 and 4-fluoroaniline fragment at m/z 111.0471, method as previously described (Table 1). Minimum cytotoxic
respectively.8,41)
concentration (MCC) on Vero cells was also determined to
The diagnostic N–H strecthing vibrations of thiourea com- evaluate the selectivity of the synthesized compounds. Antitu-
punds 5–20 appeared in 3128–3361cm−1 region.42) This wide berculosis activity and cytotoxicity results of compounds 5–20
range and varying number of N–H bands may be attributable were compared to isoniazid (INH) and ethambutol which were
to intramolecular hydrogen bonds.43) Since a S–H vibration used as reference drugs (Table 1).
at approximately 2600cm−1 has not been detected, we may
Compounds having either 4-chlorophenyl (compounds
indicate that our thiourea compounds remain in thioketo- 5–15) or 4-fluorophenyl (compounds 16–20) substituents on
Table 1. In Vitro Antimycobacterial Activity of Compounds 5–20 against M. tuberculosis H37Rv
PASS predictionc)
Compd
ID
MIC
(µM)
MCCa)
(µM)
SI
Laboratory code
R1
R2
(MCC/MIC)
Pa
Pi
KUC060101
KUC060102
KUC060103
KUC060104
KUC060105
KUC060106
KUC060107
KUC060108
KUC060109
KUC060110
KUC060111
KUC060113
KUC060114
KUC060115
KUC060116
KUC060118
Isoniazid
5
6
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
2-(Morpholin-1-yl)ethyl
2-Phenylethyl
83.78
170.70
78.17
>100
100
100
>100
≥20
≥20
≥20
20
>1.2
0.6
0.364
0.469
0.468
0.658
0.684
0.521
0.582
0.563
0.638
0.483
0.538
0.578
0.611
0.450
0.610
0.458
0.813
0.926
0.043
0.016
0.016
0.005
0.004
0.010
0.006
0.007
0.005
0.014
0.009
0.007
0.005
0.019
0.005
0.018
0.003
0.002
7
2-(4-Chlorophenyl)ethyl
Benzoyl
1.3
8
170.72
46.13
>0.6
≥0.4
≥0.5
≥1.8
0.6
9
Phenyl
10
11
12
13
14
15
16
17
18
19
20
4-Cyanophenyl
43.02
4-Fluorophenyl
10.96
3,5-Bis(trifluoromethyl)phenyl
5-Chloro-2-methylphenyl
2-Chloro-5-(trifluoromethyl)phenyl
4-Chloro-3-(trifluoromethyl)phenyl
Benzoyl
33.14
80.94
20
0.2
71.22
20
0.3
17.81
100
100
100
100
100
4
5.6
178.56
48.42
0.6
F
Phenyl
2.1
F
4-Cyanophenyl
45.02
2.2
F
4-Fluorophenyl
11.48
8.7
F
4-Chloro-3-(trifluoromethyl)phenyl
36.96
0.1
0.073–1.45
6.12–24.47
NDb)
NDb)
NA
NA
Ethambutol
a) Minimum cytotoxic concentration was determined on Vero cells. b) Both isoniazid and ethambutol were reported to be non-toxic at 62.5µg/mL (equivalent to 455.74µM
for isoniazid and 305.91µM for ethambutol) on Vero cells.46) c) Antituberculosis activity prediction (Pa: probability of activity ; Pi: probability of inactivity).

508
Biol. Pharm. Bull.
Vol. 39, No. 4 (2016)
1,3,4-thiadiazole ring were found to be active in varying lev- a micro well dilution method (Table 2). Compounds 12 and
els. Three compounds (11, 15, and 19) from this series can be 14 were found as the most active compounds with the MIC
distinguished from others by their promising activity profiles values of 0.19 and 0.23µg/µL against B. subtilis. The other
by their MIC values comparable to ethambutol. Compounds compounds were not found active against specifically and sig-
11 and 19 were appreciated as the most active representa- nificantly active against bacteria mentioned above.
tives with the MIC values of 10.96 and 11.48µM, respectively,
Depending on antituberculosis and antibacterial activity
whereas compound 19 was confirmed to possess higher selec- evaluation results we may propose that our compounds are
tivity than compound 11. Compound 15 was shown to inhibit selectively active against M. tuberculosis H37Rv strain.
M. tuberculosis H37Rv strain with the MIC values of 17.81µM.
Antiviral Activity and Cytotoxicity Studies Thiourea
Except for 11, 15 and 19, antituberculosis activity of the other derivatives 5–20 were also evaluated for their broad spectrum
compounds were observed between 33.14–178.56µM. The antiviral activity against a wide range of DNA and RNA
compounds 9, 10, 12, 17, 18, 20 with MIC values between viruses. For each compound, the MIC and the minimal cyto-
33.14 and 48.42µM are assumed to possess mild antituberculo- toxic concentration (MCC) or the 50% cytotoxic concentration
sis activity; and the compounds 6–8, 13, 14, 16 with MIC val- (CC50) were obtained. Anti-HIV activity and cytotoxicity data
ues greater than 70µM are considered as weakly active against were obtained with the synthesized compounds 5–20 using
M. tuberculosis H37Rv strain.
the strains HIV-1 (IIIB) and HIV-2 (ROD) in an MT-4/3-(4,5-
The nature of the substituents on either 1,3,4-thiadiazole dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
(R1) and thiourea (R2) was found to affect the activity of the based assay. None of the synthesized compounds showed any
compounds 5–20. Introduction of 4-fluorophenyl substitution specific activity against HIV-1 (IIIB) or HIV-2 (strain ROD) in
on N3 of thiourea moiety yielded the most selective activ- MT-4 cells. The compounds were also evaluated for in vitro
ity (compound 19). Replacing 4-fluorophenyl substitution on antiviral activity against herpes simplex virus [HSV-1 (strain
N3 of thiourea moiety by 4-chloro-3-trifluoromethylphenyl KOS), thymidine kinase deficient (TK−) strain of HSV-1 resis-
moiety (compound 20) reduced the activity by 3.2-fold as tant to acyclovir (ACVr), HSV-2 (G)], vaccinia virus (VV) and
compared to lead 19. 5-(4-Chlorophenyl)-1,3,4-thiadiazole and vesicular stomatitis virus (VSV) in HEL cell cultures ; VSV,
5-(4-fluorophenyl)-1,3,4-thiadiazole were introduced to N1 of Coxsackie virus B4 and respiratory syncytial virus (RSV) in
thiourea moiety in order to obtain the optimal substitution pat- HeLa cell cultures ; Parainfluenza-3 virus, Reovirus-1, Sind-
tern, consequently 5-(4-fluorophenyl)-1,3,4-thiadiazole moiety bis virus, Coxsackie virus B4 and Punta Toro virus in Vero
seemed to contribute more to the antituberculosis activity than cell cultures; influenza A (H1N1, H3N2) influenza B virus
the others.
in Madin–Darby canine kidney (MDCK) cell cultures; feline
Active compounds 15 and 19 were observed to be non-toxic coronavirus (FIPV) and feline herpes virus in CRFK cell
at 100µM, whereas compound 11 showed toxicity at ≥20µM. cultures.
Standard drugs, isoniazid and ethambutol were reported to be
As a result of antiviral screening of 5–20, none of the
non-toxic up to 62.5µg/mL (equivalent to 455.7µM for isonia- evaluated compounds showed specific antiviral effects (i.e.,
zid and 305.91µM for ethambutol) on Vero cells.46) In another minimal antivirally effective concentration >5-fold lower than
work, IC50 values of isoniazid and ethambutol were reported minimal cytotoxic concentration) against any of the viruses
to be higher than 455.7 and 225.4µM, respectively.47) Thus, examined (data not shown).
isoniazid and ethambutol appeared as safer antituberculosis
agents compared to compounds 11, 15 and 19.
Prediction of Drug-Likeness and Absorption, Distribu-
tion, Metabolism, and Excretion (ADME) Properties of
Antibacterial Activity The antibacterial activity of com- 5–20 In the development of new drug candidates intended
pounds 5–20 except for 6 and 11 was tested against Staphylo- for oral use, oral bioavailability and proper drug delivery
coccus aureus, Escherichia coli, Bacillus subtilis, Klebsiella are considered to play an important role.48) About one-third
pneumoniae, Pseudomonas aeruginosa, Staphylococcus epi- of drug candidates fail during development due to their poor
dermidis and Candida albicans. The MIC was determined by pharmacokinetic profiles.49) As an ADME property, a low and
Table 2. In Vitro Antibacterial and Antifungal Activity of Compounds 5–20 (MIC in µg/µL)
Compd ID
E. coli
S. aureus
B. subtilis
K. pneumoniae
P. aeruginosa
S. epidermidis
C. albicans
5
7
1.29
1.74
0.64
1.74
1.29
1.74
1.29
>3.48
1.53
1.29
1.74
1.29
1.74
1.29
1.74
8
>3.05
1.35
1.53
1.53
1.53
1.53
1.53
9
2.70
1.35
1.35
1.35
1.35
0.68
10
12
13
14
15
16
17
18
19
20
3.73
3.73
3.73
3.73
3.73
3.73
3.73
1.49
0.74
0.19
1.49
1.49
0.74
1.49
1.68
1.68
1.68
1.68
3.35
1.68
3.35
1.80
1.80
0.23
3.60
0.90
0.90
1.80
1.73
3.73
0.86
3.45
1.73
3.73
1.73
1.86
3.73
3.73
3.73
1.86
0.93
3.73
1.78
>3.55
>2.90
>3.48
>2.90
>3.55
>2.90
>3.48
>2.90
3.55
1.78
>3.55
>2.90
>3.48
>2.90
1.78
>2.90
>3.48
>2.90
>2.90
>3.48
>2.90
>2.90
>3.48
>2.90
>2.90
>3.48
>2.90

Vol. 39, No. 4 (2016)
Biol. Pharm. Bull.
509
Table 3. Predicted ADME, Lipinski Parameters and Molecular Properties of the Synthesized Compounds 5–20a)
Compd ID
MW
Volume
TPSA
%ABS
n-ROTB
n-ON
n-OHNH
miLogP
n Violations
5
6
381.958
374.922
409.367
374.878
346.868
371.878
364.858
482.862
395.34
323.608
309.065
322.601
294.445
275.462
292.321
280.393
338.057
305.559
320.295
320.295
285.841
266.857
283.717
271.788
311.691
53.076
49.838
49.838
66.909
49.838
73.630
49.838
49.838
49.838
49.838
49.838
66.909
49.838
73.63
91
92
92
86
92
84
92
92
92
92
92
86
92
84
92
92
7
7
7
5
5
5
5
7
5
6
6
5
5
5
5
6
5
4
4
5
4
5
4
4
4
4
4
5
4
5
4
4
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
4.237
5.103
5.781
4.333
4.360
4.115
4.523
6.078
5.414
5.861
5.861
3.818
3.845
3.600
4.009
5.347
0
1
1
0
0
0
0
1
1
1
1
0
0
0
0
1
7
8
9
10
11
12
13
14
15
16
17
18
19
20
449.31
449.31
358.423
330.413
355.423
348.403
432.855
49.838
49.838
a) % ABS: Percentage of absorption, TPSA: topological polar surface area, n-ON: number of hydrogen bond acceptors, n-OHNH: number of hydrogen bond donors, n-
ROTB: number of rotatable bonds. Calculations were performed using Molinspiration online property calculation toolkit (http://www.molinspiration.com).
variable bioavailability is the major reason to quit further de- were estimated by Osiris Property Explorer.55,56) The predicted
velopment of the drug candidate.50) Therefore, a computational risks include mutagenic, tumorigenic, irritant and reproductive
study for prediction of ADME properties of the molecules was toxicity (Table 4). The property predictor detects fragments
performed by determination of lipophilicity, topological polar within a given molecule as an indicator for a potential toxic-
surface area (TPSA), absorption (% ABS) and simple molecu- ity risk. Toxicity risk alerts are used as indicators showing
lar descriptors used by Lipinski in formulating his “rule of that the investigated structure may be harmful concerning
five.”51) Calculations were performed using Molinspiration on- the specified toxicity risk. From the findings given in Table
line property calculation toolkit.52) Table 3 represents a calcu- 4, it can be claimed that the target compounds 5–20 are
lated percentage of absorption (% ABS), topological polar sur- expected to be free of mutagenic, tumorigenic, irritating (ex-
face area (TPSA) and Lipinski parameters of the compounds cept 14) effects and reproductive toxicity (except 8 and 16).
5–20. Percentage of absorption (% ABS) was estimated using Water solubility of a drug candidate significantly influences
the equation: % ABS=109−(0.345×TPSA), according to Zhao its absorption and distribution characteristics. According to
et al.53) TPSA was also calculated using Molinspiration online the Osiris database, more than 80% of the traded drugs have
property calculation toolkit according to the fragment-based predicted solubility values greater than −4. As shown in Table
method of Ertl et al.54) Polar surface area, together with lipo- 4, the synthesized thioureas 5–20 exhibited solubility values
philicity, is an important property of a molecule in transport between –2.95 and −6.48.
across biological membranes. Too high TPSA values give rise
Drug-likeness can be described as a complicated balance
to a poor bioavailability and absorption of a drug. According of diverse molecular properties and structural characteristics
to the above criterions, calculated percentages of absorption indicating whether a given molecule is similar to the common
for compounds 5–20 ranged between 84 and 92%.
drugs or not.57) Osiris property explorer was also utilized to
Number of hydrogen bond donors was constant for all of calculate the fragment-based drug-likeness of the synthesized
the compounds, and number of hydrogen bond acceptors compounds. A positive value demonstrates that the designed
varied 4 to 5. Investigation of Lipinski parameters of the compound contains predominatly fragments which are com-
synthesized compounds showed that all heterocyclic thiourea monly available in traded drugs.
derivatives of 2-amino-1,3,4-thiadiazoles, might be considered
Results presented in Table 4 indicates that majority of
as drug-like candidates for novel anti-tuberculosis agents, as 1,3,4-thiadiazolyl thioureas 5–20 have positive drug-likeness
they obeyed the ruleꢀ ofꢀ five without violating more than one values. The drug score unites porperties such as drug-like-
of them.
ness, cLog P, solubility, molecular weight, and toxicity risks
There were no direct correlations observed between simple in one useful parameter that may be used to estimate the
molecular properties such as logP and anti-tuberculosis activ- compound’s whole potential to qualify for a drug.55) A drug
ity. Nevertheless, it was notable that two most active deriva- score of 0.5 or higher makes the compound a promising lead
tives (11 and 19) among compounds 5–20 with 4-fluorophenyl for further development to reach safe and efficient drugs. The
substitution at R2 position, had good calculated absorption overall drug score values for the thioureas 5–20 were calcu-
values and zero violations to Lipinski rule of five.
lated and compared to those of thiacetazone and isoxyl (Table
Osiris Calculations
4). Compounds 5, 6, 9, 11, 17 and 19 possess good drug score
Prediction of Toxicity, Solubility, Drug-Likeness and Drug values. Of these, compounds 11 and 19 had also good drug-
Score for Compounds 5–20
likeness values and antituberculosis activity.
Potential toxicity, solubility, and drug-like properties (solu-
bility, drug-likeness, and drug score) of the thioureas 5–20
PASS-Assisted Antituberculosis Activity Prediction
The experimental testing of dozens of millions of organic

510
Biol. Pharm. Bull.
Vol. 39, No. 4 (2016)
Drug score
Table 4. Estimation of Toxicity, Solubility, Drug-Likeness and Drug Score for Thioureas 5–20
Toxicity risksa)
Compound
Solubility
Drug-likeness
Mutagenicity
Tumorigenicity
Irritation
Reproductive
5
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
++
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
++
−
−
−
−
−
−
−
+
−
−
−
++
−
−
−
−
−
−
−
++
−
−
−
−
−
–
–2.95
−4.90
−5.63
−4.91
−4.97
−5.74
−5.28
−6.52
−6.05
−6.48
−6.48
−4.49
−4.55
−5.32
−4.86
−6.06
−3.24
−5.76
5.43
4.62
0.82
0.55
0.42
0.35
0.55
0.26
0.51
0.14
0.39
0.10
0.16
0.39
0.54
0.29
0.57
0.19
0.53
0.26
6
7
4.48
8
4.58
9
2.26
10
−3.10
2.49
11
12
−18.50
3.48
13
14
−4.23
−4.16
2.90
15
16
17
0.53
18
19
−4.83
1.93
20
−4.49
4.23
Thiacetazone
Isoxyl
1.23
a) −: low risk; +: moderate risk; ++: high risk.
compounds for thousands biological activities is obviously It is also worth emphasizing that the probability Pa primarily
unachievable, mandating the need for computer methods for projects the resemblance of the structure of a given molecule
the search and optimization of new pharmacologically active to the structures of the most characteristic active molecules in
compounds.58) Structure-based drug design and ligand-based the corresponding subset of the training set. Thus, as a rule,
drug design are the two computer-based approaches of drug there is no direct correlation of the Pa values with quantitative
design which are currently being used.59) Anti-tuberculosis activity characteristics. Essentially, each selection is always a
activity of our thiourea based compounds 5–20 was predicted compromise between the preferred novelty of tested molecule
by using the online version of PASS (Prediction of Activity and risk to obtain the negative result in biological screening.
Spectra for Substances) program.60) PASS online is a software
Another important aspect of interpreting the prediction
that enables to predict biological activity profile of drug-like results is related to novelty of the analyzed compound. If we
organic compounds (with a molecular mass range between limit ourselves only to activity types predicted with the high-
50 and 1250Da) according to their chemical structures for est values of Pa, the compounds selected by the prediction
more than 4000 types of biological activity including phar- may prove to be analogs of known pharmacological agents.
macological effects, mechanisms of actions, interactions with For example, when Pa>0.7, the chances of finding experimen-
biotransformation enzymes, side effects and toxic properties. tal activity are rather high but the compounds found may be
The prediction is based on analysis of the structure–activity close structural analogs of known drugs. If we select in the
relationships in the training set containing information on the range 0.5<Pa<0.7, the chances for detecting experimental
structure and biological activity of more than 300000 organic activity will be lower but the compounds will be less similar
compounds.61) The chemical structure is represented by the set to known pharmaceutical agents. For Pi<Pa<0.5, the chances
of descriptors of multilevel neighborhoods of atoms (MNA).62) of detecting experimental activity will be even lower but if
The average prediction accuracy calculated by the leave-one- the prediction is confirmed, the compound found may prove
out cross-validation procedure for the whole training set and a parent compound for a new chemical class for the biological
for all represented in it types of biological activity is about activity examined.61)
95%.63)
There are many examples of successful use of PASS ap-
The PASS online program provides ‘two parameters as a proach for finding new pharmacological agents.63–69) Antitu-
list of predicted types of activity: the probability “to be ac- berculosis activity were predicted for our compound set 5–20
tive” (Pa) and the probability “to be inactive” (Pi), which vary within the range of 0.364<Pa<0.684. Although no direct
from zero to one.’ The possibility of experimentally determin- correlation were observed between Pa values and MIC values,
ing a certain kind of activity increases with rising value of Pa it was notable that most active three compounds had good Pa
and decreasing value of Pi.61) In analyzing a predicted activity scores (Pa>0.5). As expected, INH and ethambutol gave high
spectrum, if those types of activity are selected, for which Pa values in the same prediction model.
Pa>0.9, the expected probability to find inactive compounds
Molecular Modeling The recent reports on most promis-
in the selected set is very low, but we risk missing about ing 1,3,4-thiadiazole derivatives with highly potent activity
90% of actually active compounds.61) If only compounds with towards Mtb have revealed the target mycobacterial enoyl acyl
Pa>0.8 are chosen, the probability to find inactive compounds carrier protein reductase (InhA) due to assessment of InhA
is still low, but about 80% of active compounds are missed etc. inhibition values of the mentioned compounds.24,25) We were

Vol. 39, No. 4 (2016)
Biol. Pharm. Bull.
511
Fig. 3. Superimposition of Docked Pose of the Reported Reference Inhibitor (Yellow) to the Original Pose of the Reference Inhibitor (Red)
Fig. 4. Binding Analysis of Compounds 11, 15 and 19 in the Active Site of InhA Protein
also inspired by the experimental MIC data that are generally lected active (compounds 11, 15, 19) and inactive derivatives
in consistent with the InhA inhibitory activities of 1,3,4-thia- (compounds 6 and 17) with InhA of M. tuberculosis.
diazole derivatives to perform molecular docking study.24,25)
The crystal structure of MTB InhA complexed with refer-
In order to further rationalize the biological results of our ence inhibitor 1-cyclohexyl-N-(3,5-dichlorophenyl)-5-oxopyr-
compounds, we perform molecular docking studies on se- rolidine-3-carboxamide (PDB:2H7M) having resolution of

512
Biol. Pharm. Bull.
Vol. 39, No. 4 (2016)
Fig. 5. Binding Analysis of Compounds 6 and 17 in the Active Site of InhA Protein
1.62Å was selected and docking results obtained with Glide, of hydrogen bonds with hydroxyl group of NAD+ along with
version 5.7, Schrodinger, LLC, New York, NY, 2012. Analysis hydrophobic interactions with the active site were predicted to
of the crystal structure of 2H7M revealed that the reference be the most crucial factors affecting the inhibitory potency of
inhibitor in the InhA active site formed hydrogen-bonding these compounds.
network between Tyr158, enzyme active site residues, and the
Binding Analysis of Inactive Compounds (6 and 17)
Ligand binding analysis of one of the less active derivatives
oxidized form of nicotinamide adenine dinucleotide (NAD+)
cofactor that probably served as the key feature that governed from this subset (6 and 17) showed hydrophobic interactions
the orientation of the compound within the active site. Dual with some hydrophobic amino acid residues. However, the
hydrogen bonding network was involved with the oxygen orientation in the active site cavity of InhA pushed the chloro
atom on the pyrrolidine carbonyl group, InhA catalytic resi- group in compound 6 and phenyl group in compound 17 away
due Tyr158, and the NAD+. This hydrogen bonding network from the cavity, which might be the reason for its lesser activ-
seemed to be a conserved feature among all the InhA-inhibi- ity. In silico analysis of both the compounds indicated that the
tor complexes identified so far. The reference 1-cyclohexyl-N- molecule oriented in a different manner than that of other ac-
(3,5-dichlorophenyl)-5-oxopyrrolidine-3-carboxamide was re- tive derivatives and failed to demonstrate any interaction with
docked with the active site residues of the MTB InhA to Tyr158 residue as well as with NAD+ resulting in less activity
validate the active site cavity. The ligand exhibited highest in M. tuberculosis strains (Fig. 5). This is well supported by
Glide score of −8.02kcal/mol and was found in the vicinity of the low docking score of −5.57 and −6.20 kcal/mol.
amino acids Tyr158, Phe149, Met199, Ile215, Pro156, Leu218,
Met155, Ala211, Ile202, Met103, Leu207, Ala157, Met161,
Phe97, Met98, Gly96, Gly104 and Lys165 residues. The re-
docking results showed that the compound exhibited similar
CONCLUSION
As a result of antiviral, antibacterial and antituberculosis
interactions as that of the original crystal structure with activity screening results, it can be concluded that 1,3,4-thia-
root-mean-square deviation (RMSD) of 0.87Å suggesting reli- diazolyl thiourea derivatives 5–20 designed in this study
ability of the docking method. The superimposition of Glide had selective action on M. tuberculosis H37Rv. Cytotoxicity
docked conformation of co-crystal with co-crystal of 2H7M is results on Vero cell line showed that three derivatives which
shown in Fig. 3.
showed the highest activity had selectivity indices between
Binding Analysis of Active Compounds (11, 15 and 19)
1.8 and 8.7. Compounds 11, 15 and 19 have been identified as
The compounds 11, 15 and 19 with chloro and fluoro group useful leads for further development in terms of better solubil-
at R1 position and 4-fluorophenyl, 4-chloro-3-(trifluoromethyl)- ity, ADME properties and toxicity profile. Molecular docking
phenyl and 4-fluorophenyl groups at R2 substituent’s, showed studies on selected active compounds 11, 15 and 19 displayed
good MIC. All the three compounds were docked in the bind- good docking scores in the range of −7.12 to −7.83kcal/mol.
ing site of InhA protein and were displayed good docking Studies on optimized new sets of thiadiazolyl thiourea deriva-
scores in the range of −7.12 to −7.83 kcal/mol. The predicted tives are in progress at our laboratories.
binding pose of the active compounds (11, 15 and 19) sug-
gested that the observed potency may be due to the extensive
Acknowledgments This work was supported by the
hydrophobic interactions predicted to be formed with the side Research Fund of Marmara University, project number:
chains of Met199, Leu218, Met155, Pro156, Ala157, Ile202, SAG-BGS-290506-0073 (approved: 29.05.2006—completed:
Met103, Phe149 and Met161 along with hydrogen bonding 29.05.2009). This work was partly presented in oral presenta-
interactions with the ribose hydroxyl group of NAD+ (Fig. 4). tion form at the Third International Meeting on Pharmacy &
Among these three compounds, only compound 15 [R1: Cl; Pharmaceutical Sciences (IMPPS-3), İstanbul, Turkey, 9–12
R2: 4-chloro-3-(trifluoromethyl)phenyl] was observed to inter- June 2010 and it was also presented in poster presentation
act with side chain of Tyr158 via hydrogen bonding between form at the Fourth International Meeting on Pharmacy &
nitrogen atom at 3rd position of 1,3,4-thiadiazole and phenolic Pharmaceutical Sciences (IMPPS-4), İstanbul, Turkey, 18–21
group of tyrosine. Apart from these interactions, all the three September 2014. The authors are grateful to Dr. Jürgen Gross
compounds were further stabilized by Pi–Pi interactions. from the Institute of Organic Chemistry, University of Hei-
From the docking results, it was evident that the formation delberg, for his generous help on obtaining HR-MS (FAB and

Vol. 39, No. 4 (2016)
Biol. Pharm. Bull.
513
ed-2,4-dihydro-3H-1,2,4-triazole-3-thione derivatives. A combined
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ESI) analyses in this project.
Conflict of Interest The authors declare no conflict of
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