Development of (4-methoxyphenyl)-1H-tetrazol-5-amine regioisomers as a new class of selective antitubercular agents

Article abstract of DOI:10.1016/j.ejmech.2019.111882

A series of halogenated (4-methoxyphenyl)-1H-tetrazol-5-amine regioisomers (1a-9a, 1b-9b) were synthesized from their corresponding thiourea analogues (1–9). The synthesis pathway was confirmed by an X-ray crystallographic studies of 1a, 1b and 5a. Title derivatives were tested for their in vitro antitubercular activity against standard, “wild-type” and atypical mycobacteria. The highest therapeutic potential was attributed to isomeric N-(bromophenyl)tetrazoles 8a and 9a. Their growth-inhibitory effect against multidrug-resistant Mycobacterium tuberculosis Spec. 210 was 8-16-fold stronger than that of the first-line tuberculostatics. Other new tetrazole-derived compounds were also more or equally effective towards that pathogen comparing to the established pharmaceuticals. Among non-tuberculous strains, Mycobacterium scrofulaceum was the most susceptible to the presence of the majority of tetrazole derivatives. The synergistic interaction was found between 9a and streptomycin, as well as the additivity of both 8a and 9a in pairs with isoniazid, rifampicin and ethambutol. None of the studied compounds displayed antibacterial or cytotoxic properties against normal and cancer cell lines, which indicated their highly selective antimycobacterial effects.

Full text of DOI:10.1016/j.ejmech.2019.111882

Journal Pre-proof
Development of (4-methoxyphenyl)-1H-tetrazol-5-amine regioisomers as a new class
of selective antitubercular agents
Daniel Szulczyk, Anna Bielenica, Agnieszka Głogowska, Ewa Augustynowicz-Kopeć,
Michał Dobrowolski, Piotr Roszkowski, Karolina Stępień, Alicja Chrzanowska, Marta
Struga
PII:
S0223-5234(19)31034-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.111882
EJMECH 111882
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 August 2019
Revised Date: 1 November 2019
Accepted Date: 11 November 2019
Please cite this article as: D. Szulczyk, A. Bielenica, A. Głogowska, E. Augustynowicz-Kopeć,
Michał. Dobrowolski, P. Roszkowski, K. Stępień, A. Chrzanowska, M. Struga, Development of (4-
methoxyphenyl)-1H-tetrazol-5-amine regioisomers as a new class of selective antitubercular agents,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111882.
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N
N
N
N
N
N
N
N
Br
HN
HN
O
O
Br
9a
8a
MIC 2 µg/ml (Mtb H37Rv, Mtb Spec. 192, Mtb Spec. 210)
FICI 0.375 µg/ml (9a/Streptomycin synergism; Mtb H37Rv, Mtb Spec. 192, Mtb Spec. 800)
IC50 103.3 – 125.5 µM (normal V79 and HaCaT cell lines)

Research article
Development of (4-methoxyphenyl)-1H-tetrazol-5-amine regioisomers as a new class of
selective antitubercular agents
Daniel Szulczyka, Anna Bielenicaa,*, Agnieszka Głogowskab, Ewa Augustynowicz-Kopećb,
Michał Dobrowolskic, Piotr Roszkowskic, Karolina Stępieńd, Alicja Chrzanowskaa, Marta
Strugaa
a Chair and Department of Biochemistry, Medical University of Warsaw, Warszawa, Poland
bDepartment of Microbiology, National Tuberculosis and Lung Diseases Research Institute,
01-138 Warszawa, Poland
cFaculty of Chemistry, University of Warsaw, 02-093 Wars
zawa, Poland
dDepartment of Pharmaceutical Microbiology, Medical University, 02-007 Warszawa, Poland
Keywords: Mycobacterium tuberculosis; Tetrazole; Antitubercular activity; Tuberculosis;
Regioisomers
*Corresponding author: Anna Bielenica, Chair and Department of Biochemistry, Medical
University of Warsaw, 02-097 Warszawa, Poland, Phone: +48225720693, e-mail:
abielenica@wum.edu.pl
1

Abstract:
A series of halogenated (4-methoxyphenyl)-1H-tetrazol-5-amine regioisomers (1a-9a, 1b-9b)
were synthesized from their corresponding thiourea analogues (1-9). The synthesis pathway
was confirmed by an X-ray crystallographic studies of 1a, 1b and 5a. Title derivatives were
tested for their in vitro antitubercular activity against standard, “wild-type” and atypical
mycobacteria. The highest therapeutic potential was attributed to isomeric N-
(bromophenyl)tetrazoles 8a and 9a. Their growth-inhibitory effect against multidrug-resistant
Mycobacterium tuberculosis Spec. 210 was 8-16-fold stronger than that of the first-line
tuberculostatics. Other new tetrazole-derived compounds were also more or equally effective
towards that pathogen comparing to the established pharmaceuticals. Among non-tuberculous
strains, Mycobacterium scrofulaceum was the most susceptible to the presence of the
majority of tetrazole derivatives. The synergistic interaction was found between 9a and
streptomycin, as well as the additivity of both 8a and 9a in pairs with isoniazid, rifampicin
and ethambutol. None of the studied compounds displayed antibacterial or cytotoxic
properties against normal and cancer cell lines, which indicated their highly selective
antimycobacterial effects.
1. Introduction
Despite recent approaches in diagnostics and treatment opportunities, and general
certain decrease in overall incidences, tuberculosis (TB) remains one of the major global
health diseases. The leading factors of TB incidence are country of birth (such as Asia, Africa,
South America), HIV coinfection, prolonged exposure to a case of sputum smear-positive TB
and use of some immunosuppressants, e.g. anti-tumor necrosis factor (TNF) agents [1].
Among all occurrences, pediatric cases constitute 10% of all incidents of the illness [2].
Effective therapy of TB consists of several bactericidal drugs administrated in a
combination for a dozen of months [3]. First-line treatment of drug-sensitive TB is reliant of a
four-drug cure, comprising of isoniazid, rifampicin, ethambutol and pyrazinamide, applied for
at least six months [1, 3, 4]. However, although efficient in around 90% cases [5], this long
therapy is not always well-tolerated or correctly dosed, and may cause a range of adverse
effects, including hepatitis, anemia, neural or gastrointestinal symptoms [1]. What is more,
there are increasing incidences of multidrug-resistant TB cases (MDR-TB), defined as
resistance to isoniazid and rifampicin, and extensively drug – resistant TB (XDR-TB),
covering MDR isolate combined resistance to fluoroquinolone and one injectable second-line
2

antituberculostatic [1, 3, 6]. Treatment of both M/XDR-TB requires a longer cure (up to 24
months) and more expensive antimicrobials with a higher toxicity risk, as compared to the
drug-susceptible disease. Standards of resistant mycobacteria therapy comprise at least five
agents, including an antibacterial fluoroquinolone, that shorten the length of treatment [1, 7].
Nonetheless, according to WHO details, the recovery rate of XDR-TB is only 26% [8]. It has
been also estimated that one third of the population could be latently infected with TB
(LTBI), and has up to 10% danger of progressing to an active tuberculosis form [9].
Development of shorter and cheaper TB regiments combining new and established
drugs, especially to resistant Mycobacterium isolates, is still the greatest global health
challenge. Over the last dozen of years, many natural or (semi-)synthetic chemicals have been
evaluated against mycobacteria to discover new agents to replace or at least to complement
currently used tuberculostatics. Antitubercular pharmaceuticals of natural origin are these
produced by Streptomyces (e.g. streptomycin, cycloserin, rifampicin) [10]. On the other hand,
marine-derived potential anti-TB compounds include derivatives of various biosynthetic
classes, such as alkaloids, terpenoids, quinolones, pyrenes, sterols or lactones [11]. Despite
many efforts, only two substances have been recently approved for the treatment of TB in the
past four decades: delamanid, a representative nitroimidazole and one of the dihydoquinolines
– diarylquinoline. Other heterocycles developed specifically for the therapy of MDR-TB
infections come from a group of oxazolidinones, macrolidine antibiotics and etylenediamines.
Their mode of action is based on inhibition of cell wall synthesis (nitroimidazoles,
etylenediamines), cell respiration (nitroimidazoles), ATP (dihydoquinolines) or protein
synthesis (oxazolidinones, macrolides) [7].
The large potential of 1H-tetrazol-5-yl derivatives as a new type of antimicrobial [12-
15], antifungal [16], antihypertonic [17-19], cardiotonic [20, 21], anti-inflammatory [22, 23],
analgesic [24] and cytotoxic [25-27] agents was recently proved. Current investigations of
selective, highly efficient 1H-tetrazole-based antituberculars have focused on modification of
the structures of dinitrophenyl derivatives with selanyl-, oxa- or sulfanylalkyl linker [28-31].
Roh et al. [32] synthesized a series of water-soluble 2H-terazole compounds bearing N-
benzylpiperazine moiety with high activity against drug-sensitive and MR strains of bacilli.
New thiazolone piperazinyl-tetrazole derivatives bearing organometallic substituents were
found to be more potent against standard M. tuberculosis H37Rv strain than currently used
antitubercular drugs [33]. It was established that combinations of N-phenyltetrazole
compounds with antibacterials (chloromycin, norfloxacin) or antifungal fluconazole gave
synergistic effects with a lower dosage and broader antimicrobial spectrum than their
3

separate administration [34]. On the other hand, fluorinated 2-aryltetrazole analogues of
nitroimidazooxazines exerted in vivo a strong antimycobacterial effect, retaining high
microsomal stability [35]. Antitubercular potency was also denoted for 5-phenyltetrazoles
with para-methoxyphenyl substituent on a β-lactam core [36].
In view of above considerations, this work reports the synthesis of a series of halogen-
containing N-[4-(methoxy)phenyl]-1H-tetrazol-5-amine derivatives and the in vitro evaluation
of their antitubercular and cytotoxic properties. The interactions of the most active tetrazoles
with first-line anti-TB drugs were also assessed.
2. Experimental
2.1 Chemistry
2.1.1 General procedure
The reagents were supplied from Alfa Aesar or Sigma Aldrich. Organic solvents
(acetonitrile, DMF, chloroform and methanol) were supplied from POCh (Polskie Odczynniki
Chemiczne). All chemicals were of analytical grade. Before use, dried DMF and acetonitrile
were kept in crown cap bottles over anhydrous phosphorus pentoxide (Carl Roth). The NMR
1
spectra were recorded on a Bruker AVANCE spectrometer operating at 300 MHz for H
NMR and at 75 MHz for 13C NMR. The spectra were measured in DMSO-d6 and are given as
δ values (in ppm) relative to TMS. Mass spectral ESI measurements were carried out on
Waters ZQ Micro-mass instruments with quadruple mass analyzer. The spectra were
performed in the negative ion mode at a declustering potential of 40-60 V. The sample was
previously separated on a UPLC column (C18) using UPLC ACQUITYTM system by Waters
connected with DPA detector. TLC analyses were performed on silica gel plates (Merck
Kiesegel GF254) and visualized using UV light or iodine vapour. Column chromatography was
carried out at atmospheric pressure using Silica Gel 60 (230-400 mesh, Merck).
The X-ray measurements of compounds 1a and 1b were performed at 130 (2) K,
whereas of the derivative 5a at 100 (2) K on a Bruker D8 VENTURE diffractometer with
TRIUMPH monochromator and MoKα fine-focus sealed tube (0.71073 Å). The crystals were
positioned 70 mm (1a), 40 mm (1b) and 34 mm (5a) from the PHOTON II detector with
CPAD technology; 464 frames were measured at 1° intervals with a counting time of 1s, 317
frames were measured at 1.3° intervals with a counting time of 20s, 1106 frames were
measured at 0.5° intervals with a counting time of 20 for 1a, 1b and 5a, respectively.
Data collection, cell refinement and data reduction for 1a, 1b and 5a were carried out
with Bruker SAINT software package [37]. The data were corrected for Lorentz and
4

polarization effects. Absorption correction was applied using the multi-scan method
(SADABS) [38]. The structures were solved with the ShelXT [39] structure solution program
using Intrinsic Phasing and refined with the ShelXL [39] refinement package and Olex2
program [40] using Least Squares minimization. The atomic scattering factors were taken
from the International Tables [41]. Crystal structures and refinement are specified in Table 1.
The structure of 1a is disordered, with fluorine atom occupying two alternative sites with 50%
occupancy each. The figures were generated with Mercury (ver. 4.1.0) [42]. Molecular
geometries for 1a and 1b were optimized at the HF/3-21G* level of theory, using the
Gaussian 03 program [43]. CCDC 1942278-1942280 contains the supplementary
crystallographic data for this paper. These data can be obtained via
http://www.ccdc.cam.ac.uk/conts/retrieving.html.
2.1.2 General procedure for the synthesis of 3-(4-methoxyphenyl)thiourea derivatives (1-9)
A solution of commercially available (4-methoxyphenyl)aniline (0.003 mol, 0.40 g) in
anhydrous acetonitrile (6 ml) was treated with a corresponding isothiocyanate (0.003 mol).
The mixture was stirred at room temperature for 12 h. After that, the solvent was evaporated.
The solid residue was crystallized from acetonitrile or purified by column chromatography
(chloroform: methanol; 9.5:0.5 vol.).
The synthesis of thiourea derivatives 3-6 [44, 45], 7 [46] and 9 [47] under different
conditions was published previously.
1-(2-fluorophenyl)-3-(4-methoxyphenyl)thiourea (1)
1
Yield 52%, white solid, m.p. 165°C. H NMR (300 MHz, DMSO) δ (ppm): 3.75 (s, 3H,
CH3O), 6.94-6.88 (m, 2H), 7.26-7.14 (m, 3H), 7.37-7.30 (m, 2H), 7.63 (t, J = 7.9 Hz, 1H),
9.30 (s, 1H, NH), 9.77 (s, 1H, NH). 13C NMR (DMSO-d6, 75 MHz) δ (ppm): 55.7, 114.2,
116.1 (d, 2JC-F = 20.2 Hz), 124.5 (d, 3JC-F = 3.7 Hz), 126.5, 127.6, 127.6 (d, 2JC-F = 20.2 Hz),
129.0, 132.4, 157.1, 156.8 (d, 1JC-F = 244.5 Hz), 181.2. MS (ESI) calc. for C14H13FN2OS [M ‒
H]‒: 276.33, found: 275.00.
1-(3-fluorophenyl)-3-(4-methoxyphenyl)thiourea (2)
1
Yield 50%, white solid, m.p. 145°C. H NMR (300 MHz, DMSO) δ (ppm): 3.75 (s, 3H,
CH3O), 6.95-6.89 (m, 3H), 7.26-7.22 (m, 1H), 7.38-7.30 (m, 3H), 7.54 (dt, J1 = 2.2 Hz, J2 =
13
2.1 Hz, 1H), 9.75 (d, 2H, J = 9.3 Hz, NH). C NMR (DMSO-d6, 75 MHz) δ (ppm): 55.7,
5

2
2
4
110.3 (d, JC-F = 24.7 Hz), 111.0 (d, JC-F = 20.2 Hz),114.2, 119.3 (d, JC-F = 3.0 Hz), 126.5,
130.3 (d, 3JC-F = 9.7 Hz), 132.4, 141.9 (d, 3JC-F = 10.5 Hz), 157.1, 162.2 (d, 1JC-F = 240.0 Hz),
180.2. MS (ESI) calc. for C14H13FN2OS [M ‒ H]‒: 276.33, found: 275.17.
1-(4-fluorophenyl)-3-(4-methoxyphenyl)thiourea (3)
1
Yield 81%, white solid, m.p. 165°C. H NMR (300 MHz, DMSO) δ (ppm): 3.75 (s, 3H,
CH3O), 6.95-6.90 (m, 2H), 7.39-7.21 (m, 4H), 7.49 (dd, J1 = J2 = 1.5 Hz, 1H), 7.64 (dd, J1 =
J2 = 1.8 Hz, 1H), 9.23 (s, 1H, NH), 9.81 (s, 1H, NH). 13C NMR (DMSO-d6, 75 MHz) δ
2
1
(ppm): 55.7, 114.3, 126.6, 127.6 (d, JC-F = 15.7 Hz), 129.8, 130.2, 131.3 (d, JC-F = 150.0
Hz), 137.0, 157.2, 180.9. MS (ESI) calc. for C14H13FN2OS [M ‒ H]‒: 276.33, found: 275.07.
1-(2-chlorophenyl)-3-(4-methoxyphenyl)thiourea (4)
Yield 67%, white solid, m.p. 128°C. 1H NMR (300 MHz, DMSO) δ (ppm): 9.74 (d, 2H, J =
10.5 Hz, NH), 7.70 (t, 1H, J = 1.95 Hz, Ar-H), 7.41-7.30 (m, 4H, Ar-H), 7.17-7.13 (m, 1H,
Ar-H), 6.94-6.88 (m, 2H, Ar-H), 3.75 (s, 3H, OCH3). 13C NMR (75.4 MHz, DMSO) δ (ppm):
179.82, 156.69, 141.20, 132.40, 131.82, 129.88, 126.03 (2C), 123.76, 122.88, 121.83, 113.75
(2C), 55.21. MS (ESI) calc. for C14H13ClN2OS [M ‒ 2H]‒: 292.78, found: 292.91.
1-(3-chlorophenyl)-3-(4-methoxyphenyl)thiourea (5)
1
Yield 83%, white solid, m.p. 169°C. H NMR (300 MHz, DMSO) δ (ppm): 9.79 (s, 1H, NH),
9.18 (s, 1H, NH), 7.65 (dd, 1H, J1 = 1.5 Hz, J2 = 1.2 Hz, Ar-H), 7.58 (dd, 1H, J1 = 1.5 Hz, J2
= 1.8 Hz, Ar-H), 7.39-7.34 (m, 3H, Ar-H), 7.20-7.14 (m, 1H, Ar-H), 6.95-6.90 (m, 2H, Ar-H),
3.75 (s, 3H, OCH3). 13C NMR (75.4 MHz, DMSO) δ (ppm): 179.87, 156.71, 141.19, 132.42,
131.73, 129.80, 126.09 (2C), 123.69, 122.85, 121.81, 113.73 (2C), 55.26. MS (ESI) calc. for
C14H13ClN2OS [M ‒ 2H]‒: 292.78, found: 292.82.
1-(4-chlorophenyl)-3-(4-methoxyphenyl)thiourea (6)
1
Yield 20%, white solid, m.p. 128°C. H NMR (300 MHz, DMSO) δ (ppm): 9.73 (d, 2H, J =
15.6 Hz, NH), 7.82-7.81 (m, 1H, Ar-H), 7.45-7.40 (m, 1H, Ar-H), 7.34-7.24 (m, 4H, Ar-H),
6.94-6.88 (m, 2H, Ar-H), 3.75 (s, 3H, OCH3). 13C NMR (75.4 MHz, DMSO) δ (ppm): 179.81,
156.70, 141.34, 131.80, 130.17, 126.65, 126.03 (2C), 125.76, 122.29, 120.79, 113.75 (2C),
55.22. MS (ESI) calc. for C14H13ClN2OS [M ‒ 2H]‒: 292.78, found: 292.69.
1-(2-bromophenyl)-3-(4-methoxyphenyl)thiourea (7)
6

Yield 64%, white solid, m.p. 190°C. 1H NMR (300 MHz, DMSO) δ (ppm): 9.68 (s, 2H, NH),
7.51-7.43 (m, 4H, Ar-H), 7.34-7.29 (m, 2H, Ar-H), 6.93-6.88 (m, 2H, Ar-H), 3.75 (s, 3H,
OCH3). 13C NMR (75.4 MHz, DMSO) δ (ppm): 179.80, 156.62, 139.04, 131.92, 131.08 (2C),
125.98 (2C), 125.50 (2C), 116.15, 113.70 (2C), 55.21. MS (ESI) calc. for C14H13BrN2OS [M
‒ H]‒: 337.23, found: 336.94.
1-(3-bromophenyl)-3-(4-methoxyphenyl)thiourea (8)
1
Yield 62%, white solid, m.p. 175°C. H NMR (300 MHz, DMSO) δ (ppm): 9.60 (d, 2H, J =
11.1 Hz, NH), 7.48-7.41 (m, 2H, Ar-H), 7.34-7.28 (m, 2H, Ar-H), 7.19-7.11 (m, 2H, Ar-H),
6.93-6.88 (m, 2H, Ar-H), 3.75 (s, 3H, OCH3). 13C NMR (75.4 MHz, DMSO) δ (ppm): 180.23,
159.05 (d), 156.59, 135.83 (d), 131.99, 126.21 (d, 2C), 126.05 (2C), 114.91 (d, 2C), 113.69
(2C), 55.21. MS (ESI) calc. for C14H13BrN2OS [M ‒ H]‒: 337.23, found: 336.56.
1-(4-bromophenyl)-3-(4-methoxyphenyl)thiourea (9)
Yield 65%, white solid, m.p. 179 – 180°C. 1H NMR (300 MHz, DMSO) δ (ppm): 9.67 (s, 2H,
NH), 7.53-7.48 (m, 2H, Ar-H), 7.39-7.29 (m, 4H, Ar-H), 6.94-6.88 (m, 2H, Ar-H), 3.75 (s,
3H, OCH3). 13C NMR (75.4 MHz, DMSO) δ (ppm): 179.89, 156.63, 138.59, 131.91, 128.17
(2C), 128.05, 126.00 (2C), 125.24 (2C), 113.71 (2C), 55.21. MS (ESI) calc. for
C14H13BrN2OS [M ‒ H]‒: 337.23, found: 336.72.
2.1.3 General procedure for the synthesis of N-(halogenophenyl)-1-(4-methoxyphenyl)-1H-
tetrazol-5-amines (1a–9a) and 1-(halogenophenyl)-N-(4-methoxyphenyl)-1H-tetrazol-5-
amines (1b–9b)
A suspension of an appropriate thiourea derivative 1-9 (0.001 mol), sodium azide
(0.00375 mol) and mercury (II) chloride (0.00125 mol) in 5ml of dried DMF was prepared.
Next 2-3 drops of triethylamine were added. The resulting mixture was stirred for maximum 6
h at room temperature or until TLC showed the end of the reaction. The reaction solution was
filtered and then washed with CHCl3. The filtrate was diluted with water and extracted with
CHCl3 (3x15ml). The combined organic fractions were dried over anhydrous MgSO4, filtered
and concentrated under reduced pressure. The resulting residue was purified by silica gel
chromatography (chloroform: methanol; 9.5: 0.5 vol.).
N-(2-fluorophenyl)-1-(4-methoxyphenyl)-1H-tetrazol-5-amine (1a)
7

Yield 61%, white solid, m.p. 108°C. 1H NMR (DMSO-d6, 300 MHz) δ (ppm): 3.84 (s,
13
3H, CH3O), 7.12-7.27 (m, 5H), 7.54-7.60 (m, 2H), 7.61-7.67 (m, 1H), 8.98 (s, 1H, NH). C
2
4
NMR (DMSO-d6, 75 MHz) δ (ppm): 55.6, 114.9, 115.8 (d, J = 18.7 Hz), 123.7 (d, J = 1.5
3
3
2
Hz), 124.5 (d, J = 3.7 Hz), 125.1 (d, J = 7.4 Hz), 125.8, 126.7, 127.5 (d, J = 11.2 Hz),
1
153.0, 154.3 (d, J = 243.8 Hz), 160.1. MS (ESI) calc. for C14H12FN5O [M ‒ H]‒: 285.28,
found: 285.11.
1-(2-fluorophenyl)-N-(4-methoxyphenyl)-1H-tetrazol-5-amine (1b)
1
Yield 39%, white solid, m.p. 136-138°C. H NMR (DMSO-d6, 300 MHz) δ (ppm):
3.73 (s, 3H, CH3O), 6.89-6.95 (m, 2H), 7.45-7.62 (m, 4H), 7.69-7.80 (m, 2H), 9.29 (s, 1H,
13
2
NH). C NMR (DMSO-d6, 75 MHz) δ (ppm): 55.2, 114.0, 117.2 (d, J = 18.0 Hz), 120.2,
2
3
3
120.5 (d, J = 12.8 Hz), 125.7 (d, J = 3.7 Hz), 129.6, 132.5, 133.0 (d, J = 7.5 Hz), 153.4,
1
154.8, 156.8 (d, J = 249.7 Hz). MS (ESI) calc. for C14H12FN5O [M ‒ H]‒: 285.28, found:
285.34.
N-(3-fluorophenyl)-1-(4-methoxyphenyl)-1H-tetrazol-5-amine (2a)
Yield 53%, white solid, m.p. 166°C. 1H NMR (DMSO-d6, 300 MHz) δ (ppm): 3.87 (s,
3H, CH3O), 6.78-6.84 (m, 1H), 7.16-7.21 (m, 2H), 7.30-7.38 (m, 1H), 7.43-7.47 (m, 1H), 7.
56-7.61 (m, 3H), 9.45 (s, 1H, NH). 13C NMR (DMSO-d6, 75 MHz) δ (ppm): 55.7, 104.8 (d, 2J
= 24.8 Hz), 108.3 (d, 2J = 24.0 Hz), 113.9 (d, 4J = 3.0 Hz), 115.0, 125.3, 127.6, 130.4 (d, 3J =
3
1
9.7 Hz), 141.7 (d, J = 11.2 Hz), 152.2, 160.5, 162.4 (d, J = 241.0 Hz). MS (ESI) calc. for
C14H12FN5O [M ‒ H]‒: 285.28, found: 285.09.
1-(3-fluorophenyl)-N-(4-methoxyphenyl)-1H-tetrazol-5-amine (2b)
Yield 47%, white solid, m.p. 138°C. 1H NMR (DMSO-d6, 300 MHz) δ (ppm): 3.73 (s,
13
3H, CH3O), 6.90-6.94 (m, 2H), 7.46-7.54 (m, 4H), 7.63-7.73 (m, 2H), 9.15 (s, 1H, NH). C
NMR (DMSO-d6, 75 MHz) δ (ppm): 55.2, 113.4 (d, 2J = 24.7 Hz), 114.0, 117.0 (d, 2J = 20.2
4
3
3
Hz), 120.5, 122.0 (d, J = 3.7 Hz), 131.7 (d, J = 9.0 Hz), 132.7, 134.3 (d, J = 11.2 Hz),
1
152.7, 154.8, 162.2 (d, J = 244.5 Hz). MS (ESI) calc. for C14H12FN5O [M ‒ H]‒: 285.28,
found: 285.39.
N-(4-fluorophenyl)-1-(4-methoxyphenyl)-1H-tetrazol-5-amine (3a)
1
Yield 72%, white solid, m.p. 156-158°C. H NMR (DMSO-d6, 300 MHz) δ (ppm):
3.86 (s, 3H, CH3O), 7.13-7.21 (m, 4H), 7.55-7.59 (m, 2H), 7.63-7.68 (m, 2H), 9.22 (s, 1H,
8

NH). 13C NMR (DMSO-d6, 75 MHz) δ (ppm): 55.7, 115.0, 115.3 (d, 2J = 21.7 Hz), 119.9 (d,
3J = 6.7 Hz), 125.5, 127.5, 136.2 (d, 4J = 2.2 Hz), 152.6, 156.9 (d, 1J = 237.0 Hz), 160.4. MS
(ESI) calc. for C14H12FN5O [M ‒ H]‒: 285.28, found: 285.21.
1-(4-fluorophenyl)-N-(4-methoxyphenyl)-1H-tetrazol-5-amine (3b)
1
Yield 28%, white solid, m.p. 156-158°C. H NMR (DMSO-d6, 300 MHz) δ (ppm):
3.73 (s, 3H, CH3O), 6.88-6.93 (m, 2H), 7.46-7.53 (m, 4H), 7.71-7.75 (m, 2H), 9.07 (s, 1H,
13
2
NH). C NMR (DMSO-d6, 75 MHz) δ (ppm): 55.2, 114.0, 116.8 (d, J = 23.2 Hz), 120.3,
3
4
1
128.5 (d, J = 5.2 Hz), 129.4 (d, J = 2.2 Hz), 132.8, 152.9, 154.7, 158.9 (d, J = 248.2 Hz).
MS (ESI) calc. for C14H12FN5O [M ‒ H]‒: 285.28, found: 285.53.
N-(2-chlorophenyl)-1-(4-methoxyphenyl)-1H-tetrazol-5-amine (4a)
1
Yield 57%, white solid, m.p. 100-102°C. H NMR (DMSO-d6, 300 MHz) δ (ppm):
3.84 (s, 3H, CH3O), 7.13-7.20 (m, 3H), 7.31-7.37 (m, 1H), 7.48 (dd, J1 = 5.1 Hz, J2 = 1.5 Hz,
1H), 7.57-7.63 (m, 2H), 7. 67 (dd, J1 = 5.1 Hz, J2 = 1.5 Hz, 1H), 8.69 (s, 1H, NH). 13C NMR
(DMSO-d6, 75 MHz) δ (ppm): 55.7, 115.0, 123.8, 125.5, 125.7, 126.0, 126.1, 127.9, 129.7,
136.4, 152.8, 160.1. MS (ESI) calc. for C14H12ClN5O [M ‒ H]‒: 301.73, found: 285.85.
1-(2-chlorophenyl)-N-(4-methoxyphenyl)-1H-tetrazol-5-amine (4b)
1
Yield 43%, white solid, m.p. 131-133°C. H NMR (DMSO-d6, 300 MHz) δ (ppm):
3.72 (s, 3H, CH3O), 6.88-6.94 (m, 2H), 7.50-7.56 (m, 2H), 7.60-7.65 (m, 1H), 7.68-7.74 (m,
1H), 7. 78-7.83 (m, 1H), 9.19 (s, 1H, NH). 13C NMR (DMSO-d6, 75 MHz) δ (ppm): 55.2,
114.0, 120.1, 128.7, 130.3, 130.5, 130.6, 131.4, 132.6, 132.7, 153.2, 154.8. MS (ESI) calc. for
C14H12ClN5O [M ‒ H]‒: 301.73, found: 285.68.
N-(3-chlorophenyl)-1-(4-methoxyphenyl)-1H-tetrazol-5-amine (5a)
Yield 63%, white solid, m.p. 208°C. 1H NMR (DMSO-d6, 300 MHz) δ (ppm): 3.87 (s,
3H, CH3O), 7.02-7.06 (m, 1H), 7.16-7.21 (m, 2H), 7.34 (t, J = 8.4 Hz, 1H), 7.55-7.61 (m,
13
3H), 7. 81 (t, J = 2.1 Hz, 1H), 9.42 (s, 1H, NH). C NMR (DMSO-d6, 75 MHz) δ (ppm):
55.7, 115.0, 116.5, 117.4, 121.5, 125.3, 127.7, 130.4, 133.2, 152.1, 160.5. MS (ESI) calc. for
C14H12ClN5O [M ‒ H]‒: 301.73, found: 285.69.
1-(3-chlorophenyl)-N-(4-methoxyphenyl)-1H-tetrazol-5-amine (5b)
9

1
Yield 34%, white solid, m.p. 134-136°C. H NMR (DMSO-d6, 300 MHz) δ (ppm):
3.73 (s, 3H, CH3O), 6.89-6.94 (m, 2H), 7.49-7.54 (m, 2H), 7.63-7.71 (m, 3H), 7.83-7.84 (m,
13
1H), 9.18 (s, 1H, NH). C NMR (DMSO-d6, 75 MHz) δ (ppm): 55.2, 114.0, 120.5, 124.6,
125.9, 130.0, 131.5, 132.7, 133.9, 134.2, 152.8, 154.8. MS (ESI) calc. for C14H12ClN5O [M ‒
H]‒: 301.73, found: 285.81.
N-(4-chlorophenyl)-1-(4-methoxyphenyl)-1H-tetrazol-5-amine (6a)
Yield 69%, white solid, m.p. 161°C. 1H NMR (DMSO-d6, 300 MHz) δ (ppm): 3.86 (s,
3H, CH3O), 7.16-7.21 (m, 2H), 7.34-7.40 (m, 2H), 7.55-7.60 (m, 2H), 7.66-7.71 (m, 2H), 9.36
(s, 1H, NH). 13C NMR (DMSO-d6, 75 MHz) δ (ppm): 55.7, 115.0, 119.6, 125.4, 125.5, 127.6,
128.6, 138.9, 152.3, 160.4. MS (ESI) calc. for C14H12ClN5O [M ‒ H]‒: 301.73, found: 285.77.
1-(4-chlorophenyl)-N-(4-methoxyphenyl)-1H-tetrazol-5-amine (6b)
Yield 31%, white solid, m.p.. 131°C. 1H NMR (DMSO-d6, 300 MHz) δ (ppm): 3.73 (s,
13
3H, CH3O), 6.90-6.93 (m, 2H), 7.49-7.54 (m, 2H), 7.68-7.75 (m, 4H), 9.12 (s, 1H, NH). C
NMR (DMSO-d6, 75 MHz) δ (ppm): 55.2, 114.0, 120.3, 127.7, 129.9, 131.9, 132.8, 134.6,
152.8, 154.7. MS (ESI) calc. for C14H12ClN5O [M ‒ H]‒: 301.73, found: 285.58.
N-(2-bromophenyl)-1-(4-methoxyphenyl)-1H-tetrazol-5-amine (7a)
1
Yield 52%, white solid, m.p. 133-135°C. H NMR (DMSO-d6, 300 MHz) δ (ppm):
3.85 (s, 3H, CH3O), 7.07-7.12 (m, 1H), 7.15-7.20 (m, 2H), 7.36-7.42 (m, 1H), 7.59-7.69 (m,
13
4H), 8.62 (s, 1H, NH). C NMR (DMSO-d6, 75 MHz) δ (ppm): 55.7, 115.0, 117.0, 124.3,
125.6, 126.1, 126.4, 128.5, 132.9, 137.6, 152.9, 160.2. MS (ESI) calc. for C14H12BrN5O [M ‒
H]‒: 346.19, found: 346.28.
1-(2-bromophenyl)-N-(4-methoxyphenyl)-1H-tetrazol-5-amine (7b)
1
Yield 48%, white solid, m.p. 136-138°C. H NMR (DMSO-d6, 300 MHz) δ (ppm):
3.72 (s, 3H, CH3O), 6.88-6.94 (m, 2H), 7.51-7.57 (m, 2H), 7.59-7.69 (m, 2H), 7.75-7.79 (m,
1H), 7.93-7.96 (m, 1H), 9.15 (s, 1H, NH). 13C NMR (DMSO-d6, 75 MHz) δ (ppm): 55.2,
114.0, 120.1, 121.8, 129.3, 130.6, 131.9, 132.6, 132.9, 133.7, 153.1, 154.8. MS (ESI) calc. for
C14H12BrN5O [M ‒ H]‒: 346.19, found: 346.03.
N-(3-bromophenyl)-1-(4-methoxyphenyl)-1H-tetrazol-5-amine (8a)
10

1
Yield 60%, white solid, m.p. 215-216°C. H NMR (DMSO-d6, 300 MHz) δ (ppm):
3.87 (s, 3H, CH3O), 7.15-7.21 (m, 3H), 7.28 (t, J = 8.1 Hz, 1H), 7.55-7.59 (m, 1H), 7.62-7.65
(m, 1H), 7. 95 (t, J = 1.9 Hz, 1H), 9.40 (s, 1H, NH). 13C NMR (DMSO-d6, 75 MHz) δ (ppm):
55.7, 115.0, 116.8, 120.3, 121.6, 124.4, 125.3, 127.7, 130.7, 141.5, 152.1, 160.5. MS (ESI)
calc. for C14H12BrN5O [M ‒ H]‒: 346.19, found: 346.31.
1-(3-bromophenyl)-N-(4-methoxyphenyl)-1H-tetrazol-5-amine (8b)
1
Yield 40%, white solid, m.p. 149-151°C. H NMR (DMSO-d6, 300 MHz) δ (ppm):
3.73 (s, 3H, CH3O), 6.89-6.94 (m, 2H), 7.48-7.53 (m, 2H), 7.57-7.63 (m, 1H), 7.67-7.70 (m,
1H), 7.81-7.85 (m, 1H), 7.94-7.96 (m, 1H), 9.18 (s, 1H, NH). 13C NMR (DMSO-d6, 75 MHz)
δ (ppm): 65.2, 124.0, 130.4, 132.2, 135.0, 138.6, 141.7, 142.7, 142.9, 144.3, 162.8, 164.8. MS
(ESI) calc. for C14H12BrN5O [M ‒ H]‒: 346.19, found: 346.16.
N-(4-bromophenyl)-1-(4-methoxyphenyl)-1H-tetrazol-5-amine (9a)
1
Yield 63%, white solid, m.p. 181-184°C. H NMR (DMSO-d6, 300 MHz) δ (ppm):
3.86 (s, 3H, CH3O), 7.16-7.21 (m, 2H), 7.47-7.52 (m, 2H), 7.55-7.60 (m, 2H), 7.61-7.66 (m,
13
2H), 9.36 (s, 1H, NH). C NMR (DMSO-d6, 75 MHz) δ (ppm): 55.7, 113.4, 115.0, 120.0,
125.4, 127.6, 131.5, 139.3, 152.2, 160.4. MS (ESI) calc. for C14H12BrN5O [M ‒ H]‒: 346.19,
found: 346.42.
1-(4-bromophenyl)-N-(4-methoxyphenyl)-1H-tetrazol-5-amine (9b)
1
Yield 37%, white solid, m.p. 155-158°C. H NMR (DMSO-d6, 300 MHz) δ (ppm):
3.73 (s, 3H, CH3O), 6.89-6.94 (m, 2H), 7.49-7.54 (m, 2H), 7.61-7.66 (m, 2H), 7.83-7.88 (m,
13
2H), 9.13 (s, 1H, NH). C NMR (DMSO-d6, 75 MHz) δ (ppm): 55.2, 114.0, 120.3, 123.1,
127.8, 132.3, 132.8, 132.9, 152.7, 154.8. MS (ESI) calc. for C14H12BrN5O [M ‒ H]‒: 346.19,
found: 346.36.
2.1.4 In vitro antitubercular activity
The starting thiourea compounds 1-9 were examined in vitro for their activity against
the M. tuberculosis H37Rv strain (ATCC 25618) and two ‘wild-type’ strains isolated from
tuberculosis patients: one (Spec. 210) being resistant to
p-aminosalicylic acid (PAS),
isonicotinic acid hydrazide (Isoniazid, INH), ethambutol (EMB) and rifampicin (RMP) and
the other (Spec. 192) being fully sensitive to the administrated tuberculostatics. Investigations
were performed by a classical test-tube method of successive dilutions in Youmans’
11

modification of the Proskauer and Beck liquid medium containing 10% of bovine serum [48,
49]. Bacterial suspensions were prepared from 14 days old cultures of slowly growing strains.
Solutions of compounds in DMSO were tested. Stock solutions contained 10 mg of
compounds in 1 milliliter. Dilutions (in geometric progression) were prepared in Youmans’
medium. The medium containing one of the reference drug (INH, RMP, EMB or SM),
without investigated substances, was used for comparison. Incubation was performed at 37
°C. The MIC values were determined as a minimum concentration inhibiting the growth of
tested tuberculous strains in relation to the probe with no tested compound. The influence of
the compound on the growth of bacteria at a certain concentration, 3.1, 6.25, 12.5, 25, 50 and
100 µg/ml, was evaluated.
The synthesized compounds 1a-9a and 1b-9b were tested in vitro for their
tuberculostatic activity against typical strains (M. tuberculosis H37Rv strain (ATCC 25618),
M. tuberculosis Spec. 192, M. tuberculosis Spec. 210, M. tuberculosis Spec. 800) and atypical
isolates (M. bovis, M.scrofulaceum, M. fortuitum, M. avium, M. kansasii, M. xenopi, M.
intracellulare) using the MABA method (Microplate Alamar Blue Assay method) [50, 51].
Investigations were performed by two-fold serial microdilution method (in 96-well microliter
plates) using Middlebrook 7H9 Broth medium (Beckton Dickinson) containing 10% of
OADC (Beckton Dickinson). The inoculum was prepared from fresh LJ culture in
Middlebrook 7H9 Broth medium with OADC, adjusted to a no. 1 McFarland tube, and diluted
1:20. The stock solution of a tested agent was prepared in DMSO. Each stock solution of a
tested compound was diluted in Middlebrook 7H9 Broth medium with OADC by four-fold
the final highest concentration to be tested. Compounds were diluted serially in a sterile 96-
well microtiter plates using 100µl Middlebrook 7H9 Broth medium with OADC.
Concentrations of tested agents ranged from 0.125 to 512 µg/ml. A growth control containing
no antibiotic and a sterile control without inoculation were also prepared on each plate. The
plates were incubated at 37°C for a week. After the incubation period, 30µl of Alamar blue
solution was added to each well, and the plate was re-incubated for 24 h. The growth was
indicated by the color change from blue to pink. The lowest concentration of a compound that
prevented the color change was noted as its MIC. INH, RMP, SM and EMB as the reference
drugs were used for comparison.
2.1.5 Drug interaction analysis
The combinations of the tested derivative and the reference drug were prepared in
Middlebrook 7H9 medium (Becton-Dickinson, USA) containing 10% of OADC (Becton-
12

Dickinson, USA). Each derivative in a concentration ranging from 1 to 1/32 MIC was mixed
with the reference drug in concentration equal to 1/2 MIC. Simultaneously, each of the
reference drugs (INH, RMP, SM and EMB) in concentrations ranging from 1 to 1/32MIC was
mixed with the tested derivative in concentration equal to 1/2MIC (MIC values of tested
derivatives and reference drugs alone were determined as described above). Then, all samples
(4 ml) were supplemented with an inoculum of tested strains (1°McFarland; 12 µl) and
incubated for 14 days at 37 °C. MIC of the tested derivative in combination with the fixed
concentration of the reference drug (MICD/comb with RD) was defined as the lowest
concentration of the tested derivative combined with the reference drug, which inhibited the
growth of microorganisms in a liquid medium. Similarly, MIC of the reference drug in a
combination with the fixed concentration of the tested derivative (MICRD/comb with D) was
defined as the lowest concentration of the reference drug combined with the tested derivative,
which inhibited the growth of microorganisms in a liquid medium. FICI value was calculated
according to the equation: FICI = (MICD/comb. with RD/MICD alone) + (MICRD/comb. with D/MICRD
alone). The obtained FICI values were then used to determine whether synergism (FICI < 0.5),
additivity (0.5 ≤ FICI ≥ 1), indifference (1 < FICI ≤ 4) or antagonism (FICI > 4) occurred
between the tested agents [52].
2.1.6 Antimicrobial studies
The antimicrobial assays were conducted using reference strains of bacteria derived
from international microbe collections: American Type Culture Collection (ATCC) and
National Collection of Type Culture (NCTC). The group of reference strains included Gram-
positive bacteria rods (Staphylococcus aureus: NCTC 4163, ATCC 6538, Staphylococcus
epidermidis ATCC 12228, ATCC 35984/RP62A) and Gram-negative bacteria isolates
(Pseudomonas aeruginosa NCTC 6749, Escherichia coli ATCC 25922, Klebsiella
pneumoniae ATCC 10031, Proteus vulgaris NCTC 4635). Two “wild-type” strains were also
evaluated (S. epidermidis 1457 and P. aeruginosa NCTC PAO 1).
The antimicrobial activity was expressed by minimum inhibitory concentration (MIC),
according to CLCI reference procedures with some modification. MIC was tested by the
twofold serial microdilution method (in 96-well microtiter plates) on MH II liquid medium.
The final inoculum of all studied bacteria was 106 CFU/ml (colony forming unit per ml). The
stock solution of tested compounds was prepared in DMSO and diluted in sterile medium (to
maximum 1 % of solvent content). The concentrations of compounds varied from 0.03125 to
13

512 µg/ml. The MIC value was the lowest concentration of the tested compound, at which
bacteria growth was no longer observed after 18 h of incubation at 35 ◦C [53].
2.1.7 Cytotoxicity studies
2.1.7.1 Cell culture
Chinese hamster lung fibroblast (V79) and human immortal keratinocytes (HaCaT)
cell lines were purchased from the American Type Culture Collection (ATCC, Rockville,
USA) and cultured in DMEM (Dulbecco’s Modified Eagle’s Medium, Biowest SAS, France).
Cells were seeded in 6 ml medium in a tissue culture flask (50 ml) in a 37 °C/5% CO2
humidified incubator. Medium was supplemented with 10% heat-inactivated fetal bovine
serum (Gibco Life Technologies, USA), penicillin (100 U/ml), streptomycin (100 µg/ml) and
HEPES (20 mM). After reaching 80-90% confluence, cells were passaged using trypsin-
EDTA (Gibco Life Technologies, USA) and seeded in 96-well plates (1 × 104 cells per well)
for MTT assay.
2.1.7.2 MTT assay
The cell viability was assessed by using an enzymatic conversion of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide salt (MTT) to insoluble formazan
crystals by mitochondrial dehydrogenases, occurring in living cells. Cells were cultured in 96-
well plates at a density of lx104 cells per well, and cultured to adhere for 24 hours at 37°C in a
CO2 incubator. After 24 hours of incubation, culture medium was replaced with a fresh
medium, then cells were treated with various concentrations of the tested compounds (ranged
from 140 µM to 20 µM ), and incubated for 72 hours at 37°C in a CO2 incubator. Untreated
cells were used as the control. Subsequently MTT solution (0,5 mg/mL in free-serum
medium) was added and samples were incubated for 4 hours at 37°C in a CO2 incubator. Then
the medium was aspirated and formed formazan crystals were solubilized by adding an
isopropanol - DMSO mixture (1:1 vol). The optical density of the dissolved formazan crystals
was measured using a UVM 340 reader (ASYS Hitech GmbH, Austria) at a wavelength of
570 nm. IC50 value was estimated using CompuSyn version 1.0.
2.1.7.3 LDH assay
Human immortal keratinocyte cell line from adult human skin (HaCaT), Human
melanoma cell line (HTB-140), Human epithelial lung carcinoma cell line (A549) and Human
14

colorectal adenocarcinoma (CaCo-2) were purchased from American Type Culture Collection
(Rockville, USA), and cultured in Dulbecco's Modified Eagle's Medium (DMEM) and Eagle's
Minimum Essential Medium (EMEM), supplemented with 1% antibiotics (penicillin and
streptomycin) and 10% heat-inactivated FBS-fetal bovine serum (Gibco Life Technologies,
USA) at 37°C in a 5% CO2 atmosphere. Cells were passaged using trypsin-EDTA (Gibco Life
Technologies, USA) and cultured in 96-well plates (1×104 cells per well). Experiments were
conducted in DMEM and EMEM with 2% FBS.
The determination was performed after 72 h incubation of cells in 96-well plates with
chosen compounds as described before [25]. LDH Cytotoxicity Assay Kit was used to assess
the activity of lactate dehydrogenase (LDH) released from cytosol of damaged cells to the
supernatant and measured according to the protocol described by the manufacturer (Roche
Diagnostics, Germany). An absorbance was measured at 490 nm using a microplate reader
(Epoch microplate reader, BioTek Inc., USA) equipped with Gen5 software (BioTech
Instruments, Inc., Biokom). Cytotoxicity of compounds was expressed as the LDH release
(%) and counted by the following equation: [(A test sample − A low control)/(A high control
− A low control)] × 100% (A-absorbance); where “low control” were cells in DMEM or
EMEM with 2% FBS without tested compounds, and “high control” were cells incubated in
DMEM or EMEM with 2% FBS and 1% Triton X-100 (100% LDH release).
3. Results and discussion
3.1 Chemistry
Our previous studies concerning the bioactivity of thiourea derivatives showed that 4-
methoxyphenyl moiety is not responsible for the antimicrobial activity [54-57]. For this
reason, a series of various halogenated 1-(4-methoxyphenyl)thioureas 1-9 were synthesized
just for transformation to appropriate 1H-tetrazol-5-amines. These thiourea-derived
compounds formed a group of close structural analogs bearing ortho-, meta- and para-
halogenophenyl terminal fragments. Thus, the structures of the parent thiourea compounds
and, in consequence, their tetrazole counterparts, allow to study the influence of the
differently substituted electron-withdrawing element (fluorine, chlorine or bromine) on their
antimicrobial and cytotoxic properties.
Tetrazoles can be obtained using four main methods [58, 59]. In our specific synthesis,
1,5-disubstituted tetrazoles were generated via oxidative desulfurization of 1,3-disubstituted
15

thioureas, using sodium azide as an external nucleophile. Mercury (II) chloride was used as an
desulfurization agent. The reaction was carried out at room temperature in DMF and in the
presence of triethylamine (Scheme 1).
Assumptions based on the results of preliminary experiments have been confirmed,
since for the designed reaction two isomeric products: the form A (1a-9a) and the form B (1b-
9b), occurred simultaneously. However, one of the regioisomers should be considered as
favorable (1a-9a), since it has been found in higher yields.
According to our knowledge, this is the first report showing simultaneous one-pot
synthesis of isomeric tetrazole-based compounds with good yields, using mentioned reaction
conditions. Our previous findings confirmed that the yield of each product form is directly
related to the steric hindrance caused by substituents. We also presumed that physicochemical
properties of substituents are of secondary importance and need to be evaluated individually.
In this case the moieties attached to amine groups of thiourea are similar, therefore cyclization
via oxidative desulfurization can be conducted in two ways, with the use of the one of the
amine groups of the thiourea arrangement. Favored forms A (1a-9a) were obtained in a yield
range of 52 – 72 %, whereas yields of forms B (1b-9b) equaled 28 – 48 %. The proposed
mechanism of the reaction is shown on Scheme 1S (Supplementary material).
Since the cyclization reaction conducting oxidative desulfurization is well known,
there is no need to provide theoretical fundamentals of the method. We have assumed that the
rate of the preferable form A is related to the occurrence of halides attached to the terminal
phenyl ring. The thiourea nitrogen atom bonding to the 4-methoxyphenyl scaffold should be
considered as more attainable during formation of the tetrazole ring.
[Scheme 1]
3.2 X-ray crystallography studies
The compound 1a crystallizes in a monoclinic system, space group P21/c, with one
molecule in the asymmetric part of the unit cell (Fig. 1). There are four molecules in the unit
cell forming pairs bound by N-H…N hydrogen bonds (2.143Å, see Fig. 1S, Supplementary
material). The substituents are tilted with respect to the tetrazole ring. The angles between
mean planes calculated for atoms forming rings are 87.2° (methoxyphenyl) and 44.5°
(fluorophenyl moiety). Similarly, the derivative 1b crystallizes in a monoclinic system, space
group P21/c, with one molecule in the asymmetric part of the unit cell (Fig. 1). There are four
16

molecules in the unit cell forming pairs bound by N-H…N hydrogen bonds (2.060 Å, see Fig.
1S, Supplementary material), but arranged a bit unlike 1a. Additionally, the molecules from
adjacent pairs form dimers by C-H…O bonds (2.696 Å). This motif in turn forces substituents
to be tilted differently with respect to the tetrazole ring. The angles between mean planes
denoted for atoms building rings are 35.8° (methoxyphenyl) and 63.7° (fluorophenyl moiety).
On the other hand, the product 5a crystallizes in a monoclinic system, space group P21, with
one molecule in the asymmetric part of the unit cell (Fig.1). There are two molecules in the
unit cell (Fig. 1S, Supplementary material). They are weakly bound by C-H…N bonds (2.640
Å, see Fig. 2S, Supplementary material). Despite its structural similarity to 1a, the
substituents are tilted with respect to the tetrazole ring in a completely different way (their
superpositions are shown in Fig. 2). The angles between mean planes calculated for atoms
forming rings are 49.0° (methoxyphenyl) and 17.7° (chlorophenyl moiety). The tetrazole ring
forms short contacts with the substituents C-H…N (2.567Å, 2.626 Å, chlorophenyl) and C-
H…N (2.747 Å, methoxyphenyl) in the crystal lattice. Additionally, the substituents from
adjacent molecules are bound by C-H…O hydrogen bonds (2.532 Å, Fig. 2S, Supplementary
material). The calculations at HF/3-21G* level of theory showed that the optimized geometry
of 1a has lower energy than 1b by 14.4 kcal/mol. This result indicates that the form A of
compounds synthesized with higher yields should be considered as more stable.
[Fig.1.]
[Fig.2.]
3.3 In vitro antimycobacterial activity
All prepared compounds were tested for their in vitro antitubercular potency against
M. tuberculosis H37Rv strain and three “wild-type” mycobacteria isolated from tuberculosis
patients: multidrug-resistant Spec. 210 (with resistance to p-aminosalicylic acid (PAS), INH,
EMB and RMP), Spec. 192, fully susceptible to established tuberculostatics, and INH-
monoresistant Spec. 800. The tests included also the group of non-tuberculous bacilli (M.
scrofulaceum, M. fortuitum, M. avium, M. kansasii, M. xenopi, M. intracellulare). The
antimycobacterial activities were expressed as minimum inhibitory concentrations (MICs),
with the first-line anti-TB drugs: isoniazid (INH), rifampicin (RMP), streptomycin (SM) and
ethambutol (EMB) used as standards.
The preliminary studies of antimycobacterial activity of parent 1-(halogenophenyl)-3-
(4-methoxyphenyl)thiourea derivatives revealed that their tuberculostatic potency varied from
17

moderate to weak (Table 2). Although none of the new thioureas reached the level of the
references potential, 2-chloro- (4) and 4-bromophenylthiourea (9) exhibited the highest
inhibitory effect against M. tuberculosis 192 strain, with MICs equaled 25 µg/ml. Compounds
4, 6-8 exerted the same activity level towards the 210 isolate as the standards: RMP and
EMB. It was noticed that the presence of bromine at any position of the phenyl ring
(derivatives 7-9) or the substitution of ortho- and para- position with chlorine (4, 6),
promoted antituberculostatic activity of studied phenylthiourea compounds.
The rationally designed structures of tetrazole compounds 1a-9a and 1b-9b allowed to
study how the type of their structural isomerism influenced biological properties, specifically
antimycobacterial activity. Thus, the multidrug-resistant M. tuberculosis 210 pathogen was
found to be the most susceptible to the presence of synthesized 1,5-regioisomers of tetrazole
(Table 3). All of them, except of the derivative 7a, were more effective towards the bacilli
than established pharmaceuticals. N-(bromophenyl)tetrazole-5-amines (8a, 9a), with MIC
values of 2 µg/ml, exerted the highest activity, being up to 16-fold more potent than the
references RMP and EMB, and 8 times more active comparing to INH and SM. Additionally,
other fluorophenyl- (2a, 3b) and chlorophenyl-based (4a, 4b-6b) tetrazoles expressed 2-4-fold
higher inhibitory effect than the standard tuberculostatics. Remaining compounds of the
series, N-substituted (1a, 6a) and 1-substituted (1b, 2b, 7b-9b) were equally active against
mycobacteria as INH and SM (MIC = 16 µg/ml). The MIC values of halogen derivatives 3a
and 5a were the same as these denoted for RMP and EMB (32 µg/ml). Moreover, the activity
profile of two isomeric bromophenyltetrazoles 8a and 9a towards M. tuberculosis H37R and
drug-sensitive Spec.192 strains was comparable to three tuberculostatics used: RMP, SM and
EMB. The MIC for all these agents equaled 1-2 µg/ml. One-quarter of the referential EMB
potency (i.e. 8 µg/ml) was established for tetrazoles derived from the N-substituted series (1a,
4a), and predominantly 1-substituted group (3b-6b). Other synthesized compounds presented
moderate antitubercular activity, with MIC parameters corresponding to 16-64 µg/ml. None of
the derivatives was as active as the most powerful INH. In contrary, INH-monoresistant M.
tuberculosis Spec. 800 appeared the least sensitive to the treatment with new tetrazoles,
comparing to the reference drugs. Selected halogen compounds (8a, 9a, 3b-6b) were effective
just at MIC values of 128 µg/ml. Considering the structure-activity relationship, one can
notice, that the antimycobacterial potency towards standard and “wild-type” pathogens
depends on: 1) the type of positional isomer tested (N-halogenophenyls more effective than 1-
substituted compounds); 2) the character of halogen in the benzene ring (the bioactivity
18

decreases in a line: bromo ˃˃ chloro˃ fluoro); and 3) the place of its substitution (meta- and
para- isomers more effective than ortho-).
Among atypical bacilli, M. scrofulaceum strain was the most sensitive to exposition to
the tested tetrazoles (Table 4). The derivative of 1-(3-chlorophenyl)-1H-tetrazol-5-amine (5b)
revealed the highest effectiveness (MIC = 2 µg/ml) and was 32 time more active than the
standard INH. Halogenophenyl-substituted compounds 2a, 2b, 6b exposed fourfold of
isoniazid activity, whereas tetrazoles 3a, 4a, 1b, 4b, 8b were similarly effective as the
reference. The product 3b, bearing 4-chlorophenyl moiety, was equally potent to INH. A
strong inhibitory effect of N-halogenophenyl derivatives 3a, 6a, 2a against M. intracellulare
strain was also observed; they were several fold more or equally potent as the referential INH.
Newly synthesized compounds were much more less effective against pathogens such as M.
avium, M. kansasii and M. xenopii than antimycobacterials used; their MIC ranged from 64 to
512 µg/ml. On the contrary, only 2-chlorophenyl derivative 4b had the same activity as INH
towards M. fortuitum species, however only at the concentration of 128 µg/ml. To sum up, 1-
halogenophenyl-substituted isomers were the most efficient against evaluated non-tuberculous
mycobacteria. They were preferentially substituted with fluorine or chlorine at rather meta-
and para- than ortho- position of the benzene ring. For these types of bacilli, an introduction
of bromine element and the substitution of primary amine group with halogenophenyl core
decreased the bioactivity.
As it was shown in Table 3, calculated logP values of all synthesized compounds were
lower than 5, that intend them for eventual oral administration (see also Table 1S,
Supplementary material). The lipophilicity factors of poorly active thiourea derivatives (1-9)
were in the range of 3.23-3.61, whereas their cyclic, bioactive tetrazole analogues (1a-9a, 1b-
9b) appeared to be more hydrophilic with logP values varied from 2.69 to 3.07. Compounds
4a, 8a and 9a, the most effective against typical M. tuberculosis strains, possessed the highest
logP indexes among tested N-substituted regioisomers. On the other hand, 1-substituted
tetrazoles, marked as the most potent towards standard and “wild-type” (3b-6b) or non-
tuberculous (5b, 6b) mycobacteria, were characterized by middle-ranged lipophilicity (2.71-
2.97). Finally, heterocycles 2a, 3a with the lowest logP values, exerted the strongest
inhibitory effect against atypical bacilli.
[Table 2]
[Table 3]
[Table 4]
19

3.4 Drug interaction analysis
The recommended tuberculosis treatment is based on a combination of several
pharmaceuticals administrated for months, in order to kill mycobacteria in their various stages
of the illness progress [6, 34, 60, 61]. Since the multidrug resistance to bacilli has become a
problem, the use of new combinations including established anti-TB drugs and newly
synthesized compounds needs to be assessed. For this aim, we have evaluated the potency of
the most promising derivatives 8a and 9a in a combined therapy with one of the four
reference tuberculostatics. Calculated FICI values (Tables 5-8) allowed to determine
interactions of mixed substances as synergism (FICI ˂ 0.5), additivity (0.5 ≤ FICI ≤ 1),
indifference (1 ˂ FICI ≤ 4) or antagonism (FICI ˃ 4) [52].
Synergistic effect among the derivative 9a and SM was observed in both INH-
susceptible M. tuberculosis H37Rv and Spec. 192 strains, as well as in INH-monoresistant 800
species. In all cases, the SM/9a combination showed a fourfold decrease in MIC value of the
drug, when ½ MIC of tetrazole was applied. On the other hand, the RMP/8a, SM/8a and
EMB/8a additive interactions against M. tuberculosis H37Rv and 192 pathogens were denoted.
Addition of the mentioned tetrazole allowed to reduce the tuberculostatic dose, expressed as
MIC, 2 or even 4 times. The additivity between the compound 8a and RMP towards
multidrug-resistant 210 strain was also suggested. What is more, the combination of 8a with
each of the conventionally used drug was additive in inhibition of the growth of INH-
monoresistant M. tuberculosis 800 pathogen. As for previously mentioned isolates, the
effective MIC value of the tuberculostatic could be up to four-fold lower, when it was paired
with that tetrazole derivative. Similarly, the additive interaction of the isomer 9a with RMP or
EMB against M. tuberculosis H37Rv and Spec. 192 isolates was detected. Against the multi-
resistant 210 species, for all drugs, except for EMB, additivity was denoted, with ½ MIC of
the compound 9a added. Interestingly, INH/9a and EMB/9a pairs exerted the additive
inhibitory effect, when applied toward monoresistant Spec. 800 bacillus. Only some
combinations of tested tetrazoles against both resistant isolates revealed indifference. Since
the effect of pairing of investigated derivatives with INH was antagonistic towards both drug-
sensitive mycobacteria strains (M. tuberculosis H37Rv and Spec. 192), they must be excluded
from a combination treatment. However, the established antagonism, along with very high
MICs values of evaluated tetrazoles against INH-monoresistant M. tuberculosis Spec. 800
suggest that INH and derivatives 8a, 9a could hit the same molecular target.
20

INH, the multi-targeted lead antituberculostatic, acts as a prodrug that is activated in a
cell by the mycobacterial bifunctional catalase-peroxidase (KatG) [62]. Generated INH-
derived free radicals form covalent adducts with NADH or NADPH, capable to disrupt the
synthesis of both mycolic and nucleic acids [62, 63]. INH-NAD connections are powerful
inhibitors of InhA, an enoyl acyl carrier protein reductase, involved in the formation of
mycolic acids, major lipid components of the mycobacterial cells [64]. In turn, INH-NADP
adducts restrain the activity of MabA, an NADPH-dependent β-ketoacyl-ACP reductase, also
engaged in the biosynthesis of long-chain fatty acids. On the other hand, complexes of INH
with NADP+ were described as inhibitors of dihydrofolate reductase (DHFR), the key enzyme
necessary for the folate biosynthesis pathway in the microbial cells [62]. Among mentioned
molecular goals, direct InhA- targeted ligands, especially omitting the KatG-activating step,
are considered as the most promising to combat tuberculosis [65].
Numerous polycyclic molecules with nitrogen-based heterocyclic rings, structurally
related to INH and substituted tetrazoles presented in our study, have been found as effective
InhA inhibitors. Guardia et al. described a series of halogenated benzamides bearing 1H-
pyrazolyl and pyridin-2-yl fragments with micromolar activities against InhA [66], whereas
the group of Shaikh found potent inhibitors among derivatives of carbazole, substituted with
thioxothiazolidine-4-one moiety [67]. Additionally, piperazine indoleformamides, pyrazoles,
pyrrolidine carboxamides, imidazopiperidines, thiadiazoles, triazoles, thiazoles and proline-
derived hybrids were also identified as potent enoyl-ACP-reductase inhibitors [64, 68, 69].
Among them, halogen-containing pyrazol-3-yl- and pyridin-2-yl-thiadiazol-2-amines used in
the low nanomolar range, acted as direct, reversible InhA inhibitors. The presence of both a
(hetero)aryl moiety substituted with fluorine, chlorine and/or bromine, and nitrogen-rich rings
have promoted inhibitory properties to the mycobacterial InhA.
The presented in vitro studies of antimycobacterial combinations of the title 1H-
tetrazol-5-amines are a useful first step to predict their possible mechanism of action and to
estimate their effectiveness in further clinical researches.
[Table 5]
[Table 6]
[Table 7]
[Table 8]
3.5 Antibacterial activity
21

In order to check the selectivity of the inhibitory potential of the studied derivatives
against M. tuberculosis strains, the effect of thiourea-based compounds 1-9 and their
tetrazoles towards 10 bacterial isolates (5 Gram-positive rods and 5 Gram-negative cocci) was
examined (Table 9). None of the starting 4-methoxyphenylthioureas (1-9) possessed
antibacterial activity. Only some tetrazole-containing regioisomers (2a-4a, 6a, 1b, 3b, 4b)
exerted slight growth-inhibitory potency towards S. epidermidis and S. aureus strains, with
MIC values ranging from 32 to 64 µM. Other synthesized tetrazoles were inactive towards
representative bacterial isolates (MIC ≥ 128 µM). The obtained results indicated that the
antimycobacterial profile of derivatives presented in this study is highly selective.
[Table 9]
3.6 Cytotoxicity evaluation
To get information concerning toxicity of newly synthesized tetrazoles (1a-9a, 1b-9b)
in mammalian cells, their in vitro effects on two normal cell lines, V79 (Chinese hamster lung
fibroblast) and HaCaT (human immortal keratinocyte) were evaluated (Table 10). The results
showed that compounds, applied at the concentration of 100 µM for 72 h, had negligible
impact on the viability of non-neoplastic cells. The IC50 values of all derivatives were higher
than 100 µM, that demonstrated their good safety and selectivity towards mycobacterial cells.
What is more, they were found to be more than several dozen times less cytotoxic than the
reference doxorubicin against both tested cell lines.
To confirm the safety of the most promising antitubercular agents (8a, 9a), the LDH
cytotoxicity assay on cancer cell lines: HTB-140 (human melanoma), A549 (human epithelial
lung carcinoma), CaCo-2 (Caucasian colon adenocarcinoma), as well as on normal HaCaT
cell line was performed (Fig.3.). Studied tetrazoles were used at the concentration of 100 µM,
because of their high IC50 value. The level of cell damage in normal HaCaT cells caused by
the presence of 8a and 9a was low, and accounted for 4.88 and 7.25 %, respectively. Both
derivatives expressed also low cytotoxic influence on tumor cell lines. Although the LDH
release was the strongest for 8a in HTB-140 and CaCo-2 cells, it was still not significant, and
did not exceed 17%.
22

The obtained cytotoxicity data confirmed a minor influence of 1H-tetrazole derivatives
on the growth and viability of normal and pathogenic cells.
[Table 10]
[Fig.3.]
4. Conclusions
In this work a set of isomeric (4-methoxyphenyl)-1H-tetrazol-5-amines, bearing
differentially substituted halogen atom on the terminal benzene ring, was synthesized from
their thiourea counterparts. These structural modifications led to an increase of hydrophilicity
and activity of compounds against standard and drug-resistant M. tuberculosis strains. Among
studied regioisomers, N-substituted halogenophenyl derivatives were found to be more
effective than 1-substituted compounds. What is more, the bioactivity was the highest for
bromophenyl agents, substituted at meta- or para- position.
Newly synthesized tetrazole-based derivatives exerted also a promising potential in the
combined anti-TB therapy. The synergistic effect of 9a/SM combination against three M.
tuberculosis strains was denoted, and also the additivity of both 8a and 9a compounds when
paired with INH, RMP or EMB was described. The antagonism of tetrazoles-INH
combinations towards both drug-sensitive isolates (M. tuberculosis H37Rv and Spec. 192), and
the high resistance of Spec. 800 to these compounds suggest a possible inhibitory effect of
newly synthesized derivatives against InhA. Regarding the cytotoxicity, the synthesized
tetrazoles showed negligible impact on two normal and three cancer cell lines at a
concentration of 100 µM, and did not noticeably inhibit the growth of Gram-positive and
Gram-negative bacteria.
Although further changes in chemical structure of tetrazole-derived compounds are
needed in order to develop their antimycobacterial profile and therapeutic safety, selective
substances, such as 8a and 9a, are the most promising for the combined human mycobacteria
treatment.
Acknowledgements
Research subject was carried out with the use of CePT infrastructure financed by the
European Union – the European Regional Development Fund within the Operational
Programme “Innovative economy for 2007-2013”. Authors thanks to Katarzyna Chojnowska
23

and Magdalena Błaszczuk from Students' Scientific Society at Department of Pharmaceutical
Microbiology, Faculty of Pharmacy, Medical University of Warsaw, for performing of
preliminary antibacterial studies.
Conflict of interest
The authors declare no conflict of interest.
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