Synthesis and antimycobacterial activity of disubstituted benzyltriazoles

Article abstract of DOI:10.1007/s00044-019-02458-7

The increasing prevalence of multidrug-resistant strains of Mycobacterium tuberculosis (Mtb), the pathogen of human tuberculosis (TB), serves as a strong incentive for the discovery and development of new agents for the treatment of this plight. In search

Full text of DOI:10.1007/s00044-019-02458-7

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02458-7
ORIGINAL RESEARCH
Synthesis and antimycobacterial activity of disubstituted
benzyltriazoles
4,5,6
3
Frans J. Smit1 Ronnett Seldon2 Janine Aucamp3 Audrey Jordaan4 Digby F. Warner
David D. N’Da
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Received: 22 July 2019 / Accepted: 5 October 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
The increasing prevalence of multidrug-resistant strains of Mycobacterium tuberculosis (Mtb), the pathogen of human
tuberculosis (TB), serves as a strong incentive for the discovery and development of new agents for the treatment of this
plight. In search for such drugs, we investigated a series of benzyltriazole derivatives. We herein report the design, synthesis
and biological activity of disubstituted benzyltriazoles against the human virulent H37Rv strain of Mtb as well as the toxicity
on human embryonic kidney (HEK-293) cells. The derivative 21 featuring trifluoromethyl substituent in para position on the
phenyl ring and n-butyl chain in position 4 on the triazole ring was the most active with MIC90 and MIC99 values of 1.73 and
3.2 µM, respectively, in the albumin-free medium. It also displays high selectivity towards bacteria growth inhibition (SI >
58), thus stands as a better hit for further investigation, including lead optimization, DMPK parameters determination and
assessment of its activity in animal models.
Graphical Abstract
Disclaimer: Any opinions, findings and conclusions, or
recommendations expressed in this material are those of the authors
and therefore the NRF does not accept any liability in regard thereto.
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02458-7) contains supplementary
material, which is available to authorized users.
* David D. N’Da
4
SAMRC/NHLS/UCT Molecular Mycobacteriology Research
Unit, Division of Medical Microbiology, Department of
david.nda@nwu.ac.za
Pathology, University of Cape Town, Cape Town, South Africa
1
Pharmaceutical Chemistry, School of Pharmacy, North-West
University, Potchefstroom 2520, South Africa
5
Institute of Infectious Disease and Molecular Medicine, University
of Cape Town, Rondebosch 7701, South Africa
2
3
H3D Drug Discovery and Development Centre,
Potchefstroom 2520, South Africa
6
Wellcome Centre for Infectious Diseases Research in Africa,
University of Cape Town, Rondebosch 7701, South Africa
Centre of Excellence for Pharmaceutical Sciences, North-West
University, Potchefstroom 2520, South Africa

Medicinal Chemistry Research
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Keywords Tuberculosis TB Drug discovery Benzyltriazole Click chemistry
Introduction
However, apart from drug resistance the current anti-TB
drugs possess several limitations, including a high pre-
valence of adverse side effects and the inability to act upon
latent forms of the Mtb bacillus (Amir et al. 2014), leading
to patient non-adherence and ultimately to a discontinuation
of therapy with further implications. Adverse side effects
caused by drug therapy may incur substantial additional
costs due to outpatient visits, additional tests and, in more
serious cases, hospitalisation. Second-line agents often have
more severe effects, are less effective, have worse toxicity
profiles and are more expensive (Kumar et al. 2014a; Yee
et al. 2003). The treatment duration of second-line agents is
often prolonged by 2 years (Kumar et al. 2014a), adding
additional challenges to patient compliance and resulting in
a high risk of treatment failure and relapse (Yee et al. 2003).
An additional factor driving up the TB death toll is co-
infection with HIV. In 2017 TB caused 300,000 deaths
among HIV-positive people (WHO 2018b), while HIV co-
infection accounted for 900,000 new cases to the global TB
morbidity (WHO 2018b). The resistance against mainstay
anti-TB drugs, such as isoniazid, RIF and PYZ that have
been attributed to parasite genetic mutations (Jureen et al.
2008; Seifert et al. 2015; Zaw et al. 2018), have rendered
the disease intractable. Thus, the emergence of MDR strains
of Mtb threatens the global strategy to eradicate the disease
by 2030 (WHO 2018b). Moreover, roughly one-third of the
world’s population carries a latent form of TB—meaning
these individuals have been infected but are not ill and
cannot transmit the disease yet. However, the danger of
passing the infection on to another person can occur
anytime.
Tuberculosis (TB) is a major health challenging problem
worldwide. According to the latest statistics of the World
Health Organization (WHO), TB, alongside human immu-
nodeficiency virus (HIV), malaria, dengue and hepatitis
(WHO 2018a), are the leading infectious diseases causing
death worldwide. In 2017 alone 10 million people con-
tracted TB and 1.6 million fatalities were registered, of
which more than 95% occurred in low- and middle-income
countries (WHO 2018b). The majority of the estimated
incidence cases occurred in South-East Asia (44%), and
Africa (25%) (WHO 2018b), making TB a disease of the
developing world.
TB is a treatable disease. However, its treatment is long
and complex and despite the availability of useful drugs and
regimens, the disease still causes numerous fatalities
annually. The existing antitubercular drugs, although of
immense value in controlling the disease, have several
limitations with the most important being the emergence of
drug-resistant TB strains against the frontline drugs (Amir
et al. 2014). Drug-resistant TB occurs when Mycobacterium
tuberculosis (Mtb), the causative agent of human TB,
becomes resistant to at least one of the first-line anti-TB
drugs. Multidrug-resistant TB (MDR-TB) occurs when Mtb
becomes resistant to the two most effective anti-TB drugs,
isoniazid (INH) and rifampicin (RIF) (CDC 2018a). In
addition, MDR-TB patients with further resistance to any
fluoroquinolone and at least one of the three injectable
second-line drugs (capreomycin, kanamycin or amikacin) is
classified as extensively drug-resistant TB (XDR-TB) (CDC
2018a). In 2017 alone, the WHO revealed an estimate of
558,000 new cases of RIF resistance with 82% suffering
from MDR-TB, of which 8.5% were reported as XDR-TB
cases. To make matters worse, only 55% of MDR-TB cases
were treated successfully during that year (WHO 2018b).
The advent of drug-resistant TB has made the treatment and
cure of TB even more complicated.
Altogether, these facts underscore the need for new anti-
TB drugs to treat all the forms of the diseases. In recent
years efforts have focused on the discovery of new anti-TB
agents with divergent, unique structures and mechanisms of
action different from those of currently used drugs, in order
to treat TB to achieve effective disease control (Sajja et al.
2017).
Drug susceptible TB chemotherapy is a lengthy regimen,
spanning over a 6–9-month period and consisting of a
multidrug therapy that combines four anti-TB drugs,
namely RIF, INH, pyrazinamide (PYZ) and ethambutol
(CDC 2018b). In contrast, the treatment of drug-resistant
TB requires a regimen that consists of at least five effective
TB agents during the intensive phase. The combination
should comprise PYZ and four core second-line TB agents,
namely one fluoroquinolone, one aminoglycoside, one
thionamide (ethionamide or prothionamide), and either
cycloserine or terizidone (WHO 2016).
A well-known privileged nucleus that has drawn much
attention in drug discovery is the triazole core (Dheer et al.
2017). The 1,2,3-triazole is a five-member N-heterocyclic
compound (Ali et al. 2017) that has attracted significant
attention due to the wide range of biological properties of
compounds containing this moiety, including anti-
tubercular (Boechat et al. 2011b), antifungal (Dai et al.
2015), anti-HIV (Mohammed et al. 2016), antimalarial
(Kumar et al. 2014b; Singh et al. 2017) and anti-
inflammatory activity (Shafi et al. 2012). This triazole
scaffold possesses hydrogen bonding capability, moderate

Medicinal Chemistry Research
Fig. 1 Structures of 1,2,3-
triazole and benzyltriazole
derivatives
dipole character, rigidity and stability under in vivo con-
ditions that all together are responsible for the enhanced
biological properties of compounds containing that scaf-
fold (Zhang et al. 2017). Interestingly, some triazole-
containing compounds and the mainstay anti-TB drug,
INH, share a similar mechanism of action as they inhibit
microbial cell wall synthesis by blocking lipid biosynth-
esis (Kumar et al. 2014a; Zhang et al. 2017), presumably
by targeting the enoyl-acyl carrier protein (PDB ID: 1ZID)
that is essential for the fatty acid (mycolic acids) synthase
system (FAS-II) in mycobacterial cells (Rozwarski et al.
1998). Drugs currently in the market that possess the
1,2,3-triazole moiety include cefatrizine and tazobactam
(antibiotics) (Ali et al. 2017; Zhang et al. 2017), TSAO
(anti-HIV) (Zhang et al. 2017), and carboxyamidotriazole
(CAI) (anticancer) (Fig. 1) (Ali et al. 2017; Zhang et al.
2017). Benzofuran salicylic acid derivative (I-A09), a
synthesized 1,2,3-triazole derivative, is currently the lead
anti-TB agent under clinical evaluation and may be used to
treat TB in the near future (Ali et al. 2017; Zhang et al.
2017). The triazole derivatives A (Labadie et al. 2011) and
B (Boechat et al. 2011a) (Fig. 1) have shown activity
against different pathogenic and opportunistic myco-
bacteria, including M. avium and Mtb.
The benzyl moiety also plays an important role in bio-
chemical and pharmacological processes and is herein
envisaged as a partner to 1,2,3-triazole. Incorporated into a
quinoxaline scaffold it acts as a DNA intercalation switch,
resulting in anticancer properties (Mahata et al. 2016). On
the other hand, when incorporated into polyamine it con-
tributes to the inhibition of Plasmodium falciparum’s
essential polyamine biosynthetic pathway (El Bissati et al.
2019). Benzyl containing compounds display a wide array
of pharmacological activities, including antitubercular
(Cheng et al. 2019; Gallardo-Macias et al. 2019; Zhang et al.
2018), antimicrobial (Belz et al. 2013; Swetha et al. 2019),
antimalarial (Courtens et al. 2018; El Bissati et al. 2019;
Tahghighi et al. 2018) and antifungal (Ballari et al. 2017;
Belz et al. 2013). Clinical pharmacological drugs wherein
the 1,2,3-triazole and benzyl scaffolds are conjugated to
form the benzyltriazole (BZT) moiety include the anticancer
agent CAI and the antiepileptic rufinamide (Fig. 1).
The mycobacterium cell wall is lipophilic in nature
(Suresh et al. 2014); therefore, lipophilicity is an imperative
consideration in the design of active antitubercular mole-
cules. Thus, in the midst of addressing the drug-resistance
issue through increased activity, the 1,2,3-triazole scaffold
was directly anchored to the benzyl moiety in position N-1

Medicinal Chemistry Research
to generate novel BZT derivatives with varying lipophilicity
imparted by the substituents on the rings. We herein report
the synthesis, in vitro antimycobacterial activities as well as
cytotoxicity of these derivatives.
employed at a flow rate of 1 ml/min. The concentration of
ACN in the mobile phase was linearly increased over a
period of 5 min to a final concentration of 85%. The time
allowed for equilibration between runs was 5 min and the
duration of each HPLC run was 15 min. The concentration
of the test compounds injected varied (20 μl of 1 mM–20 μl
of 0.25 mM). The eluent was monitored at wavelengths of
210, 254 and 300 nm.
Material and methods
Materials
Furthermore, the following abbreviations are adopted,
CAr (carbon of aromatic ring), HAr (hydrogen of aromatic
ring), Tz (triazole) and BZT.
Solvents: acetonitrile (ACN), dichloromethane (DCM),
hexane and methanol (MeOH) were obtained from ACE
Chemicals. Reagents, such as benzyl bromides, alkynes,
copper sulfate pentahydrate (CuSO4·5H2O), sodium azide
(NaN3), sodium ascorbate (NaAsc), β-cyclodextrin (β-CD)
and 3-(phenylmercapto)propylene-1, were all acquired from
Sigma-Aldrich South Africa.
Synthesis
Benzyl halide (1 eq.) and the appropriate alkyne (1.2 eq.) were
mixed in a round-bottomed flask. Tetrahydrofuran (THF;
5 ml), MeOH (5 ml) and distilled water (5 ml) were added
consecutively. To this stirring solution were added β-CD
(60 mg, 0.02 eq.), NaAsc (240 mg, 0.2 eq.), NaN3 (230 mg,
1.2 eq.) and CuSO4·5H2O (120 mg, 0.2 eq.) sequentially. The
solution was stirred until a white precipitate formed. If the
solution turned black, an additional portion of sodium
ascorbate (50 mg) was added until the solution turned clear
yellow. The reaction was followed by TLC. After completion,
DCM (50 ml) and saturated sodium bicarbonate (NaHCO3;
50 ml) were added. The organic layer was separated and the
aqueous phase was extracted with DCM (3 × 50 ml). The
combined organic phase was washed with water (3 × 50 ml),
dried over MgSO4, filtered off and rotated to dryness in
vacuo, resulting in a hot oil residue. Hexane (50 ml) was
added to the hot oil and the supernatant was decanted into a
flat bottom flask. This was repeated two times, the combined
extracts cooled at room temperature and then allowed to
crystallize at 0 to −4 °C to afford the target compound.
General procedures
The 1H and 13C nuclear magnetic resonance (NMR) spectra
were recorded on a Bruker Advance™ III 600 spectrometer
at a frequency of 600 and 150.913 MHz, respectively, in
CDCl3-d. Chemical shifts are reported in parts per million δ
(ppm), with the residual protons of the solvent as reference.
The splitting pattern abbreviations are as follows: singlet
(s), doublet (d), doublet of doublet (dd), doublet of triplets
(dt), triplet (t), triplet of doublets (td), triplet of triplets (tt),
quartet of doublets (qd) and multiplet (m).
High resolution mass spectrometry (HRMS) was recor-
ded on a Bruker MicroTOF Q II mass spectrometer,
equipped with an APCI source, set at 200 or 180 °C,
respectively, using Bruker Compass Data Analysis
4.0 software. A full scan from 50–1500 m/z was performed
at a capillary voltage of 4500 V, an end plate offset voltage
of −500 V, with the nebulizer set at 1.6 and 0.4 Bar,
respectively, and a collision cell RF voltage of 100 Vpp.
Infrared (FTIR) spectra were recorded on a Bruker
Alpha-P FTFTIR instrument. Thin layer chromatography
1-benzyl-4-butyl-1H-1,2,3-triazole 1
Light off-white crystals; yield (68%); m.p. 61.5–64.1 °C;
FTIR vmax: 3113 (=C–H, Tz), 3062 (=C–HAr), 1568 (C=C,
1
(TLC) was performed using silica gel plates (60F254
)
Tz), 1492 (N=N) cm−1; H NMR (600 MHz, CDCl3) δ
obtained from Merck (Johannesburg, South Africa). TLC
was utilized to monitor the progress of reactions and
visualization of spots was done under UV-light (254 nm) as
well as iodine. Column chromatography was performed
using silica gel (230–400 mesh, Sigma-Aldrich).
High performance liquid chromatography (HPLC) ana-
lysis of all final compounds were performed to determine
purity. An Agilent 1100 HPLC system, equipped with a
quaternary pump and an Agilent 1100 series diode array
detector, was utilized. HPLC grade acetonitrile (Merck) and
Milli-Q water (Millipore) were used for chromatography. A
Venusil XBP C18 column (4.60 × 150 mm, 5 μm) with an
initial mobile phase (70% Milli-Q water: 30% ACN) was
7.37–7.28 (m, 3H, 2 H-9 and H-5), 7.26–7.12 (m, 3H, 2 H-8
and H-10), 5.46 (s, 2H, H-6), 2.66 (t, J = 7.6 Hz, 2H, H-11),
1.65–1.53 (m, 2H, H-12), 1.38–1.28 (m, 2H, H-13), 0.88 (t,
J = 7.4 Hz, 3H, H-14); 13C NMR (151 MHz, CDCl3) δ
134.92 (C-7), 129.00 (C-9), 128.56 (C-8), 127.92 (C-10),
53.99 (C-6), 31.42 (C-12), 25.34 (C-11), 22.27 (C-13),
13.76 (C-14); HRMS (APCI) m/z: [M+H]+ 216.1507
(Calc. for C13H18N3: 216.1501); purity (HPLC): 96%.
1-benzyl-6-hexyl-1H-1,2,3-triazole 2
Fluffy off-white crystals; yield (87%); m.p 60.3–61.1 °C.
FTIR vmax: 3036 (=C–H, Tz), 2917 (–C–H), 1493 (N=N)

Medicinal Chemistry Research
1
cm−1; H NMR (600 MHz, CDCl3) δ 7.37–7.27 (m, 3H, 2
7.13 (q, J = 8.2 Hz, 5H, 2 H-9, 2 H-8, H-5), 5.41 (s, 2H, H-
6), 2.32 (s, 3H, H-10), 1.38–1.26 (m, 2H, H-17), 0.92–0.82
(m, 3H, H-18); 13C NMR (151 MHz, CDCl3) δ 138.46 (C-
10), 131.88 (C-7), 129.63 (C-9), 127.98 (C-5), 54.57–51.95
(C-6), 25.35 (C-11), 22.26 (C-17), 21.09 (C-10′), 13.75
(C-18); HRMS (APCI) m/z: [M+H]+ 230.1650 (calc. for
C14H20N3: 230.1657); purity (HPLC): 95%.
H-9 and H-5), 7.27–7.15 (m, 3H, 2 H-8 and H-10), 5.47 (s,
2H, C-6), 1.37–1.19 (m, 6H, H-13, -14 and 15), 0.86–0.78
(m, 3H, H-16); 13C NMR (151 MHz, CDCl3) δ 134.94 (C-
7), 128.97 (C-9), 128.54 (C-8), 127.90 (C-10), 54.08 (C-6),
31.48 (C-12), 25.69 (C-11), 13.99 (C-16); HRMS (APCI)
m/z: [M+H]+ 244.1825 (calc. for C15H22N3: 244.1814);
purity (HPLC): 95%.
4-hexyl-1-[(4-methylphenyl)methyl]-1H-1,2,3-triazole 7
1-benzyl-4-octyl-1H-1,2,3-triazole 3
Cream coloured powder; yield (63%); m.p.: 65.5–72.0 °C;
FTIR vmax: 3114 (=C–H, Tz), 3063 (=C–H), 1615 (C=C,
Light yellow crystals; yield (74%); m.p. 71.6–72.2 °C; FTIR
vmax: 3063 (=C–H, Tz), 2916 (–C–H), 1493 (N=N), 1456
1
Tz), 1455 (N=N) cm−1; H NMR (600 MHz, CDCl3) δ
1
(N=N) cm−1; H NMR (600 MHz, CDCl3) δ 7.39–7.25 (m,
7.18–7.08 (m, 5H, 5H, 2 H-9, 2 H-8, H-5), 5.42 (s, 2H, H-
6), 2.33 (s, 3H, H-10′), 1.36–1.19 (m, 6H, H-13, -14, -25),
0.84 (t, J = 6.6 Hz, 3H, H-6); 13C NMR (151 MHz, CDCl3)
δ 138.50 (C-10), 131.89 (C-7), 129.64 (C-8), 128.02 (C-9,
-5), 31.50 (C-12), 25.71 (C-11), 22.49 (C-15), 21.10 (C-
10′), 14.00 (C-16); HRMS (APCI) m/z: [M+H]+ 258.1957
(calc. for C16H24N3: 258.1970); purity (HPLC): 93%.
3H, 2 H-9 and H-5), 7.25–7.11 (m, 3H, 2 H-8 and H-10),
5.47 (s, 2H, H-6), 1.35–1.15 (m, 10H, H-13, -14, -15, -16,
-17), 0.84 (t, J = 7.0 Hz, 3H, H-18); 13C NMR (151 MHz,
CDCl3) δ 134.93 (C-7), 128.95 (C-9), 128.53 (C-8), 127.90
(C-10), 54.91–52.73 (C-6), 31.75 (C-12), 25.69 (C-11), 22.58
(C-17), 14.03 (C-18); HRMS (APCI) m/z: [M+H]+ 272.2127
(calc. for C17H26N3: 272.2127); purity (HPLC): 96%.
4-octyl-1-[(4-methylphenyl)methyl]-1H-1,2,3-triazole 8
1-benzyl-4-decyl-1H-1,2,3-triazole 4
White powder; yield (68%); m.p.: 69.5–70.4 °C; FTIR vmax
:
Light yellow crystals; yield (64%); m.p. 80.5–81.2 °C;
FTIR vmax: 3063 (=C–H, Tz), 2915 (–C–H), 1468 (N=N)
3113 (=C–H, Tz), 3064 (=C–HAr), 1615 (C=C, Tz), 1468
(N=N) cm−1; 1H NMR (600 MHz, CDCl3) δ 7.13 (dd, J =
17.9, 7.9 Hz, 5H, 2 H-9, 2 H-8, H-5), 5.42 (s, 2H, H-6),
2.33 (s, 3H, H-10′), 1.38–1.11 (m, 10H, H-13, -14, -15, -16,
-17), 0.84 (t, J = 7.0 Hz, 3H, H-18); 13C NMR (151 MHz,
CDCl3) δ 138.50 (C-10), 131.88 (C-7), 129.63 (C-8),
128.03 (C-9), 31.78 (C-12), 25.71 (C-11), 21.09 (C-10′),
14.06 C-18); HRMS (APCI) m/z: [M+H]+ 286.2260 (calc.
for C18H28N3: 286.2283); purity (HPLC): 96%.
1
cm−1; H NMR (600 MHz, CDCl3) δ 7.37–7.27 (m, 3H, 2
H-9 and H-5), 7.26–7.08 (m, 3H, 2 H-8 and H-10), 5.47 (s,
2H, H-6), 1.35–1.14 (m, 14H, H-13, -14, -15, -16, -17, -18
and -19), 0.85 (t, J = 7.0 Hz, 3H, H-20); 13C NMR
(151 MHz, CDCl3) δ 134.92 (C-7), 129.00 (C-9), 128.58
(C-8), 127.94 (C-10), 54.57–52.51 (C-6), 31.85 (C-12),
25.68 (C-11), 22.64 (C-19), 14.08 (C-20); HRMS (APCI)
m/z: [M+H]+ 300.2449 (calc. for C19H30N3: 300.2440);
purity (HPLC): 97%.
4-decyl-1-[(4-methylphenyl)methyl]-1H-1,2,3-triazole 9
1-benzyl-4-[(phenylsulfanyl)methyl]-1H-1,2,3-triazole 5
Light yellow solid; yield (64%); m.p.: 76.1–77.6 °C; FTIR
vmax: 2915 (=C–H, Tz), 2848, 1514 (C=C, Tz), 1468
1
White crystals, yield (66%); m.p. 78.2–81.7 °C; FTIR vmax
:
(N=N) cm−1; H NMR (600 MHz, CDCl3) δ 7.18 (q, J =
3139 (=C–H, Tz), 1582 (C=C, Tz), 1454 (N=N) cm−1; H
NMR (600 MHz, CDCl3) δ 7.35–7.23 (m, 6H, 2 H-9, 2 H-
14, H-8 and H-5), 7.20 (t, J = 7.4 Hz, 2H, H-13), 7.17–7.11
(m, 3H, H-10, H-15), 5.42 (s, 2H, H-6), 4.19 (s, 2H, H-11);
13C NMR (151 MHz, CDCl3) δ 135.16 (C-7), 134.51 (C-13),
129.80 (C-14, -10), 128.85 (C-8), 127.86 (C-15), 126.49 (C-
6), 28.93 (C-11); HRMS (APCI) m/z: [M+H]+ 282.1057
(calc. for C16H16N3S: 282.1065); purity (HPLC): 91%.
8.2 Hz, 5H, 2 H-9, 2 H-8, H-5), 5.46 (s, 2H, H-6), 2.69 (t,
1
J = 7.7 Hz, 2H, H-11), 2.37 (s, 3H, H-10′), 1.64 (dd, J =
14.5, 7.3 Hz, 2H, H-12), 0.89 (t, J = 7.1 Hz, 3H, H-20); 13
C
NMR (151 MHz, CDCl3) δ 138.53 (C-10), 131.97 (C-7),
129.72 (C-8), 128.05 (C-9), 120.40 (C-5), 53.85 (C-6),
25.75 (C-11), 22.71 (–CH2–CH3), 21.18 (C-10′), 14.15
(C-20); HRMS (APCI) m/z: [M+H]+ 314.2575 (calc. for
C20H32N3: 314.2596); purity (HPLC): 80%.
4-butyl-1-[(4-methylphenyl)methyl]-1H-1,2,3-triazole 6
1-[(4-methylphenyl)methyl]-4-[(phenylsulfanyl)methyl]-1H-
1,2,3-triazole 10
Light yellow powder; yield (75%); m.p.: 64.2–65.3 °C;
FTIR vmax: 3115 (=C–H, Tz), 3063 (=C–HAr), 1616 (C=C,
1
Gold powder; yield (74%); m.p.: 80.9–82.4 °C; H NMR
1
Tz), 1456 (N=N) cm−1; H NMR (600 MHz, CDCl3) δ
(600 MHz, CDCl3) δ 7.30–7.10 (m, H-5, -8, -9, -13, -14, -15),

Medicinal Chemistry Research
5.43 (s, 2H, H-6), 4.22 (s, 2H, H-11), 2.37 (s, 3H, H-10′);
13C NMR (151 MHz, CDCl3) δ 138.66 (C-10), 135.37
(C-7), 126.52 (C-5), 54.05 (C-6), 29.01 (C-11), 21.19
(C-10′); HRMS (APCI) m/z: [M+H]+ 296.1194 (calc. for
C17H18N3S: 296.1221); purity (HPLC): 92%.
14H, H-13, -14, -15, -16, -17, -18, -19), 0.85 (t, J = 7.1 Hz,
H-20); 13C NMR (151 MHz, CDCl3) δ 149.14 (C-4),
133.98 (C-7), 132.19 (C-8), 129.52 (C-9), 122.72 (C-10),
120.43 (C-5), 53.26 (C-6), 31.86 (–CH2–CH2–CH3), 25.65
(C-11), 14.08 (C-20); HRMS (APCI) m/z: [M+H]+
378.1536 (calc. for C19H29BrN3: 378.1545); purity
(HPLC): 97%.
1-[(4-bromophenyl)methyl]-4-butyl-1H-1,2,3-triazole 11
White powder; yield (95%); m.p. 76.4–77.1 °C; FTIR vmax
:
1-[(4-bromophenyl)methyl]-4-[(phenylsulfanyl)methyl]-1H-
1,2,3-triazole 15
2918 (=C–H, Tz), 2853, 1488 (C=C, Tz), 1452
(N=N) cm−1; 1H NMR (600 MHz, CDCl3) δ 7.51–7.41 (m,
2H, H-9), 7.14 (d, J = 27.6 Hz, 1H, H-5), 7.10 (d, J =
8.4 Hz, 2H, H-8), 5.42 (s, 2H, H-6), 1.36–1.27 (m, 2H, H-
12), 0.89 (t, J = 7.4 Hz, 3H, H-14); 13C NMR (151 MHz,
CDCl3) δ 149.07 (C-4), 133.96 (C-7), 132.18 (C-9), 129.53
(C-8), 122.71 (C-10), 120.43 (C-5), 53.26 (C-6), 25.31
(C-11), 22.27 (C-13), 13.76 (C-14); HRMS (APCI) m/z:
[M+H]+ 294.0619 (calc. for C13H17BrN3: 294.0606); purity
(HPLC): 93%.
White powder; yield (85%) m.p. 97.7–98.7 °C; FTIR vmax
:
3152 (=C–H, Tz), 3075 (=C–HAr)1581 (C=C, Tz), 1479
1
(N=N) cm1; H NMR (600 MHz, CDCl3) δ 7.46 (t, J =
10.2 Hz, H-9), 7.27–7.13 (m, 6H, 2 H-13, 2 H-14, H-5, 1 H-
15), 7.01 (d, J = 8.3 Hz, 2H, 2 H-8), 5.38 (s, 2H, H-6), 4.20
(d, J = 12.6 Hz, 2H, H-11); 13C NMR (151 MHz, CDCl3) δ
145.60 (C-4), 133.57 (C-7), 132.22 (C-9), 126.67 (C-10),
122.41 (C-5), 53.39 (C-6), 28.88 (C-11); HRMS (APCI)
m/z: [M+H]+ 360.0153 (calc. for C16H15BrN3S: 360.0170);
purity (HPLC): 100%.
1-[(4-bromophenyl)methyl]-4-hexyl-1H-1,2,3-triazole 12
Beige powder; yield (69%); m.p. 76.3–76.9 °C; FTIR vmax
:
4-butyl-1-[(4-nitrophenyl)methyl]-1H-1,2,3-triazole 16
2922 (=C–H, Tz), 2853, 1488 (C=C, Tz), 1465 (N=N) cm−1;
1H NMR (600 MHz, CDCl3) δ 7.51–7.42 (m, 2H, H-9),
7.16 (s, 1H, H-5), 7.10 (t, J = 5.4 Hz, 2H, H-8), 5.42 (s, 2H,
H-6), 1.61 (dt, J = 15.4, 7.6 Hz, 2H, H-12), 1.36–1.17
(m, 6H, H-13, -14, -15); 13C NMR (151 MHz, CDCl3) δ
149.12 (C-4), 133.97 (C-7), 132.20 (C-8), 129.52 (C-9),
122.73 (C-10), 120.44 (C-5), 53.27 (C-6), 14.01 (C-16);
HRMS (APCI) m/z: [M+H]+ 322.0936 (calc. for
C15H21BrN3: 322.0919); purity (HPLC): 95%.
Off yellow powder; yield (84%) m.p. 63.1–65.9 °C; FTIR
vmax
: 2925 (=C–H, Tz), 1607 (C=C, Tz), 1514
1
(N=N) cm−1; H NMR (600 MHz, CDCl3) δ 8.19 (d, J =
7.6 Hz, 2H, H-9), 7.34 (d, J = 7.8 Hz, 2H, H-8), 5.67 (s, 2H,
H-6), 2.68 (s, 2H, H-11), 1.71 (s, 2 H, 2H, H-12), 1.38 (s,
2H, H-13), 0.97–0.84 (m, 3H, H-14); 13C NMR (151 MHz,
CDCl3) δ 147.96 (C-10), 128.51 (C-9), 124.17 (C-8), 25.22
(C-11), 22.24 (C-12, -13), 13.75 (C-14); HRMS (APCI)
m/z: [M+H]+ 261.1369 (calc. for C13H17N4O2: 261.1352);
purity (HPLC): 97%.
1-[(4-bromophenyl)methyl]-4-octyl-1H-1,2,3-triazole 13
Tan powder; yield (59%); m.p. 72.3–73.3 °C; FTIR vmax
:
4-hexyl-1-[(4-nitrophenyl)methyl]-1H-1,2,3-triazole 17
2916 (=C–H, Tz), 2849, 1488 (C=C, Tz), 1466
(N=N) cm−1; 1H NMR (600 MHz, CDCl3) δ 7.50–7.41 (m,
2H, H-9), 7.17 (d, J = 10.2 Hz, 1H, H-5), 7.10 (d, J =
8.4 Hz, 2H, H-8), 5.42 (s, 2H, H-6), 1.65–1.54 (m, 2H, H-
12), 0.86 (dt, J = 14.1, 7.4 Hz, 3H, H-18); 13C NMR
(151 MHz, CDCl3) δ 149.78 (C-4), 133.99 (C-7), 132.18
(C-8), 129.52 (C-9), 122.71 (C-10), 120.43 (C-5), 53.25 (C-
6), 14.07 (C-18); HRMS (APCI) m/z: [M+H]+ 350.1233
(calc. for C17H25BrN3: 350.1232); purity (HPLC): 96%.
Off yellow powder; yield (78%); m.p. 75.5–75.6 °C; FTIR
vmax: 2905 (C–H), 1606 (C=C) cm−1; 1H NMR (600 MHz,
CDCl3) δ 8.19 (d, J = 7.8 Hz, 2H, H-9), 7.34 (d, J = 8.1 Hz,
2H, H-8), 5.65 (s, 2H, H-6), 2.64 (d, J = 42.6 Hz, 2H,
H-11), 1.71 (s, 2H, H-12), 1.45–1.08 (m, 6H, H-13, -14,
-15), 0.97–0.68 (m, 3H, H-16); 13C NMR (151 MHz,
CDCl3) δ 147.96 (C-10), 128.47 (C-9), 124.17 (C-8), 31.46
(C-14) 28.84 (C-12, -13), 22.47 (C-15), 13.99 (C-16);
HRMS (APCI) m/z: [M+H]+ 289.1667 (calc. for
C15H21N4O2: 289.1665); purity (HPLC): 95%.
1-[(4-bromophenyl)methyl]-4-decyl-1H-1,2,3-triazole 14
Beige powder; yield (81%); m.p. 84.3–85.8 °C; FTIR vmax
:
1-[(4-nitrophenyl)methyl]-4-octyl-1H-1,2,3-triazole 18
3112 (=C–H, Tz), 3062 (=C–HAr), 1590 (C=C, Tz), 1467
(N=N) cm−1; 1H NMR (600 MHz, CDCl3) δ 7.51–7.41 (m,
2H, H-9), 7.16 (s, 1H, H-5), 7.10 (d, J = 8.4 Hz, 2H, H-8),
5.42 (s, 2H, H-6), 1.64–1.54 (m, 2H, H-12), 1.35–1.15 (m,
Off yellow powder; yield (75%); m.p. 76.1–76.8 °C; FTIR
vmax: 3060 (CAr–H), 2919 (=C–H), 1606 (C=C), 1463
1
(N=N) cm−1; H NMR (600 MHz, CDCl3) δ 8.18 (t, J =

Medicinal Chemistry Research
7.9 Hz, 2H, H-9), 7.39–7.25 (m, 2H, H-8), 5.60 (s, 2H,
C-6), 2.68 (s, 2H, H-11), 1.63 (s, 2H, H-12), 1.39–1.07 (m,
10H, H-13, -14, -15, -16, -17), 0.83 (t, J = 6.9 Hz, 3H, H-
18); 13C NMR (151 MHz, CDCl3) δ 147.92 (C-10), 142.03
(C-7), 128.40 (C-8), 124.20 (C-9), 53.03 (C-6), 31.77 (C-
16), 25.63 (C-11), 22.59 (C-17), 14.04 (C-18); HRMS
(APCI) m/z: [M+H]+ 317.1990 (calc. for C17H25N4O2:
317.1978); purity (HPLC): 82%.
CDCl3) δ 7.65 (d, J = 7.5 Hz, 2H, H-9), 7.35 (d, J = 7.6 Hz,
2H, H-8), 5.63 (s, 2H, H-6), 2.90–2.50 (m, 2 H, 2H, H-11),
1.77 (d, J = 39.8 Hz, 2H, 2H, H-12), 1.47–1.22 (m, 6H, H-
13, -14, -15), 0.90 (d, J = 6.0 Hz, 3H, H-16); 13C NMR
(151 MHz, CDCl3) δ 138.78 (C-7), 130.95 (d, J = 32.4 Hz,
C-10), 128.18 (C-8), 126.04 (C-9), 123.79 (d, J = 272.2 Hz,
C-10′), 28.57 (C-12), 25.74 (C-11), 14.07 (C-16); HRMS
(APCI) m/z: [M+H]+ 312.1654 (calc. for C16H21F3N3:
312.1688); purity (HPLC): 95%.
4-decyl-1-[(4-nitrophenyl)methyl]-1H-1,2,3-triazole 19
4-octyl-1-{[4-(trifluoromethyl)phenyl]methyl}-1H-1,2,3-
White powder; yield (76%) m.p. 85.9–86.3 °C; FTIR vmax
:
triazole 23
2913 (=C–H), 2848, 1522 (C=C), 1470 (N=N) cm−1; H
NMR (600 MHz, CDCl3) δ 8.19 (d, J = 8.3 Hz, 2H, H-9),
7.35 (d, J = 8.4 Hz, 2H, H-8), 5.60 (s, 2H, H-6), 2.68 (s,
2H, H-11), 1.64 (s, 2H, H-12), 1.37–1.03 (m, 14H, H-13,
-14, -15, -16, -17, -18, -19), 0.84 (t, J = 7.0 Hz, 3H, H-20);
13C NMR (151 MHz, CDCl3) δ 147.93 (C-10), 142.01 (C-
7), 128.40 (C-9), 124.20 (C-8), 53.14 (C-6), 25.64 (C-11),
14.06 (C-20); HRMS (APCI) m/z: [M+H]+ 345.2290 (calc.
for C19H29N4O2: 345.2291); purity (HPLC): 93%.
1
White powder; yield (70%); m.p. 81.9–82.8 °C; FTIR vmax
:
3112 (=C–H, Tz), 3058 (=C–HAr), 1618 (C=C, Tz), 1327
(C–F) cm−1; H NMR (600 MHz, CDCl3) δ 7.63 (d, J =
1
7.7 Hz, 2H, H-9), 7.34 (d, J = 7.7 Hz, 2H, H-8), 5.60 (s, 2H,
H-6), 2.70 (s, 2H, H-11), 1.72 (d, J = 35.8 Hz, 2H, H-12),
1.42–1.13 (m, 10H, H-13, -14, -15, -16, -17), 0.87 (t, J =
7.0 Hz, 3H, H-18); 13C NMR (151 MHz, CDCl3) δ 138.89
(C-7), 130.94 (d, J = 32.6 Hz, C-10), 128.14 (C-8), 126.05
(C-9), 123.80 (d, J = 272.2 Hz, C-10′), 25.76 (C-11), 14.12
(H-18); HRMS (APCI) m/z: [M+H]+ 340.1987 (calc. for
C18H25F3N3: 340.2001); purity (HPLC): 97%.
1-[(4-nitrophenyl)methyl]-4-[(phenylsulfanyl)methyl]-1H-
1,2,3-triazole 20
Yellow crystal; yield (80%); m.p. 74.1-75.0 °C; FTIR vmax
:
4-decyl-1-{[4-(trifluoromethyl)phenyl]methyl}-1H-1,2,3-
triazole 24
1516 (C=C), 1419 (N=N) cm−1 1H NMR (600 MHz,
;
CDCl3) δ 8.16 (d, J = 8.6 Hz, 2H, H-9), 7.35–7.09 (m, 8H,
2 H-8, -13, -14, -15), 5.54 (s, 2H, H-6), 4.21 (s, 2H, H-11);
13C NMR (151 MHz, CDCl3) δ 147.94 (C-10), 141.66 (C-
7), 135.03 (C-12), 124.21 (C-9), 53.02 (H-6), 28.71 (C-11);
HRMS (APCI) m/z: [M+H]+ 327.0927 (calc. for
C16H15N4O2S: 327.0916); purity (HPLC): 96%.
Fine white powder; yield (65%), m.p. 78.6–79.2 °C; FTIR
vmax: 3112 (=C–H, Tz), 3059 (=C–HAr), 1621 (C=C, Tz),
1468 (N=N), 1331 (C-F) cm−1 1H NMR (600 MHz,
;
CDCl3) δ 7.65 (d, J = 7.5 Hz, 2H, H-9), 7.36 (d, J = 7.3 Hz,
2H, H-8), 5.62 (s, 2H, H-6), 2.72 (s, 2H, H-11), 1.75 (d,
J = 34.3 Hz, 2H, H-12), 0.89 (t, J = 7.0 Hz, 3H, H-20); 13
C
4-butyl-1-{[4-(trifluoromethyl)phenyl]methyl}-1H-1,2,3-
triazole 21
NMR (151 MHz, CDCl3) δ 138.87 (C-7), 130.95 (d, J =
32.0 Hz, C-10), 128.16 (C-8), 126.05 (C-9), 123.79 (d, J =
272.3 Hz, C-10′), 25.45 (C-11), 14.14 (C-20); HRMS
(APCI) m/z: [M+H]+ 368.2305 (calc. for C20H29F3N3:
368.2314); purity (HPLC): 90%.
Fine white powder; yield (70%); m.p. 62.7–66.4 °C; FTIR
1
vmax: 2915 (=C–H), 1321 (C–F) cm−1; H NMR (600 MHz,
CDCl3) δ 7.64 (d, J = 7.8 Hz, 2H, H-9), 7.36 (d, J = 7.9 Hz,
2H, H-8), 5.60 (s, 2H, H-6), 2.72 (s, 2H, H-11), 1.69 (s, 2H,
H-12), 1.40 (d, J = 5.4 Hz, 2H, 2H, H-13), 0.94 (t, J = 7.1 Hz,
3H, H-14); 13C NMR (151 MHz, CDCl3) δ 138.90 (C-7),
131.02 (m, C-10), 128.13 (C-8), 126.05 (C-9), 123.80 (d, J =
272.3 Hz, C-10′), 32.17–30.49 (C-12), 25.41 (C-11), 22.34
(C-13), 13.83 (C-14); HRMS (APCI) m/z: [M+H]+ 284.1344
(calc. for C14H17F3N3: 284.1375); purity (HPLC): 96%.
4-[(phenylsulfanyl)methyl]-1-{[4-(trifluoromethyl)phenyl]
methyl}-1H-1,2,3-triazole 25
Off-white powder; yield (65%); m.p. 123.7–126.9 °C; FTIR
vmax: 3122 (=C–H, Tz), 3081 (=C–HAr), 1619 (C=C, Tz),
1424 (N=N), 1326 (C–F) cm1; 1H NMR (600 MHz, CDCl3)
δ 7.60 (d, J = 8.1 Hz, 2H, H-9), 7.32–7.14 (m, 7H, H-8,
-13, -14, -15), 5.52 (s, 2H, H-6), 4.22 (s, 2H, H-11); 13C
NMR (151 MHz, CDCl3) δ 138.55 (C-7), 135.08 (C-12),
130.99 (d, J = 32.6 Hz, C-10), 123.78 (d, J = 272.3 Hz,
C-10′), 53.52 (C-6), 28.88 (C-11); HRMS (APCI) m/z:
[M+H]+ 350.0938 (calc. for C17H15F3N3S: 350.0939);
purity (HPLC): 92%.
4-hexyl-1-{[4-(trifluoromethyl)phenyl]methyl}-1H-1,2,3-
triazole 22
White powder; yield (81%); m.p. 68.5–69.8 °C; FTIR vmax
:
2917 (=C–H), 1325 (C–F) cm−1 1H NMR (600 MHz,
;

Medicinal Chemistry Research
In vitro biological evaluation
(75,000 cells/ml) and incubated for 24 h. The cells were
then treated with: (1) 100 μl of emetine dihydrochloride
solution diluted with growth medium to the necessary
concentrations (positive control); (2) 80 μl of growth med-
ium and 20 μl of solvent (negative control to compensate for
possible solvent effects); (3) 80 μl of growth medium and
20 μl of experimental compound solutions. Blanks con-
tained growth medium without cells. The treated plates
were incubated for 48 h.
To initiate the MTT assay, 20 μl of sterile-filtered MTT
solution (1 mg/ml in PBS) was added and the plates
incubated for 4 h. The growth medium-MTT mixture was
then aspirated and 100 μl of 2-propanol was added to
dissolve purple formazan crystals. Absorbance was mea-
sured at 560 and 650 nm using the Thermofisher Scientific
GO Multiscan plate reader. Due to light sensitivity of
MTT reagent, the assay was performed in the dark. Thus,
the plates were covered with aluminium foil and the
contents gently mixed for 5 min at room temperature. Data
analysis was performed for each biological replicate using
SkanIt 4.0 Research Edition software. Background
absorbance (650 nm) was subtracted from absorbance
values (560 nm), the mean absorbance calculated and the
percentage cell viability was determined by the following
equation:
Antimycobacterial activity assessment
The minimum inhibitory concentration (MIC) was deter-
mined using the standard broth micro dilution method,
where a 10 ml culture of Mtb pMSp12::GFP (Abrahams
et al. 2012; Collins and Franzblau 1997; Collins et al. 1998)
is grown to an optical density (OD600) of 0.6–0.7. Cultures
are diluted, prior to inoculation of assays, as follows:
(1) 1:100 in Gaste-Fe (glycerol–alanine–salts) medium
pH 6.6, supplemented with 0.05% Tween-80 and 1%
glycerol (De Voss et al. 2000; Franzblau et al. 2012).
(2) 1:500 in 7H9 supplemented with 10% albumin
dextrose catalase supplement (ADC), 0.4% glucose
and 0.05% Tween-80 (De Voss et al. 2000; Franzblau
et al. 2012).
The compounds to be tested are reconstituted in
DMSO. Twofold serial dilutions of the test compound are
prepared across a 96-well microtiter plate, after which
50 μl of the diluted Mtb cultures are added to each well in
the serial dilution. The plate layout is a modification of the
method previously described (Ollinger et al. 2013). Assay
controls used are a minimum growth control (RIF at 2 ×
MIC), and a maximum growth control (5% DMSO). The
microtiter plates are sealed in a secondary container and
incubated at 37 °C with 5% CO2 and humidification.
Relative fluorescence (excitation 485 nM; emission
520 nM) is measured using a plate reader (FLUOstar
OPTIMA, BMG LABTECH) at day 7 and day 14. The
raw fluorescence data are archived and analysed using the
CDD Vault from Collaborative Drug Discovery, in which
data are normalised to the minimum and maximum inhi-
bition controls to generate a dose response curve (%
inhibition), using the Levenberg–Marquardt damped
least-squares method, from which the MIC90 is calculated
(Burlingame, CA, www.collaborativedrug.com). The
lowest concentration of drug that inhibits growth of more
than 90% of the bacterial population is considered to be
the MIC90.
Cell viability % ¼ ðΔ Abs sample À Δ Abs blankÞ=
:
ðΔ Abs neg control À Δ Abs blankÞ Â 100
The IC50 and Z-score were determined for each com-
pound’s biological replicate using the SkanIt 4.0 software
and four-parameter logistic (sigmoidal) regression. For the
final IC50 of each compound, the mean IC50 of the biolo-
gical replicates were calculated.
Results and discussion
Chemistry
Click chemistry is a term that initially described reactions
giving high yields and selectivity products by
carbon–hetero bond formation. The word “click” refers to
easily joining molecular building blocks, such as two pieces
of a seat belt buckle. Of the reactions comprising the click
universe, the gold standard is the Huisgen 1,3-dipolar
cycloaddition of alkynes with azides to form 1,4-dis-
ubsituted-1,2,3-triazoles as depicted in Scheme 1. This
copper(I)-catalyzed reaction is mild and very efficient,
requiring no protecting groups and no purification in the
majority of cases (Tornøe et al. 2002). The azide and alkyne
functional groups are largely inert towards biological
molecules and aqueous environments, allowing the use of
Cytotoxicity assay
Human embryonic kidney (HEK-293) cells (ATCC® CRL-
1573™) were cultured in Hyclone Dulbecco’s modified
Eagle’s medium with high glucose supplemented with
10% foetal bovine serum and 1% L-glutamine,
Penicillin–Streptomycin, Amphotericin B and non-essential
amino acids. Cell lines are maintained in a humidified
atmosphere at 37 °C and 5% CO2. For the MTT assay, 96-
well plates were prepared with 200 μl of cell suspension

Medicinal Chemistry Research
the Huisgen 1,3-dipolar cycloaddition in target-guided
synthesis (Manetsch et al. 2004) and activity-based pro-
tein profiling (Speers et al. 2003). The resulting triazole
scaffold has similarities to the ubiquitous amide moiety
found in nature. However, unlike the amide bond, the
triazole ring is rigid and not susceptible to cleavage. In
addition, it is impossible to oxidize or reduce.
A series of BZT derivatives were easily synthesized in a
single step using a literature reported method employing
copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition
(CuAAC) in a THF-MeOH-water (v/v/v, 1:1:1) mixture, an
adaptation of the procedure by Shin et al. (2012) as depicted
in Scheme 2. The reaction is an in situ process involving
two major reactions, SN2 and CuAAC. The reagents include
benzyl bromide (electrophile source), NaN3 (nucleophile
source), copper (II) sulfate (catalyst), sodium ascorbate
(reducing agent), β-CD (phase transfer catalyst) and alkyne
(nucleophile source). The nucleophilic substitution SN2 of
the selected benzyl bromide with NaN3 results in a benzy-
lazide intermediate by adopting the synthetic route descri-
bed by Hodson and Peter (2010). Synchronously, the
benzylazide intermediate undergoes a Huisgen copper
alkyne-azide 1,3-dipolar cycloaddition “click” reaction with
the alkyne over 24 h (Shin et al. 2012). The later reaction is
catalyzed by copper (II) while β-CD serves as phase transfer
catalyst between the lipophilic reagents (benzylazide and
alkyne) and the aqueous phase, thus homogenizing the
medium. It is noteworthy to indicate that the solvent com-
bination (THF–MeOH–H2O; 1:1:1, v/v/v) was identified as
affording the best yields after several solvents, including
DMSO, acetone, water, MeOH and THF, were attempted.
Another note of particular importance is the sequential
Scheme 1 Schematic representation of copper(I)-catalyzed azide/
alkyne click reaction
2
5
1
1
4
2
2
1
2
1
Click chemistry
3
Compd
Compd. R1
R2
R1
R2
.
1
H
n-C4H9
14
"
n-C10H21
2
"
n-C6H13
15
"
3
4
"
"
n-C8H17
n-C10H21
16
17
NO2
"
n-C4H9
n-C6H13
5
6
7
"
CH3
"
18
19
20
"
"
"
n-C8H17
n-C4H9
n-C10H21
n-C6H13
8
9
"
"
n-C8H17
n-C10H21
21
22
CF3
"
n-C4H9
n-C6H13
10
11
12
13
"
Br
"
23
24
25
"
"
"
n-C8H17
n-C4H9
n-C6H13
n-C8H17
n-C10H21
"
Scheme 2 Synthesis of benzyltriazole derivatives

Medicinal Chemistry Research
addition of reagents. The current sequence was adopted
after several other sequences failed to deliver the target
compounds. In those attempts, a quick formation of a black
solution occurred, indicative of the oxidation of the copper
(II) to copper (I) due to fast addition of reagent, incorrect
adding sequence or inappropriate thermal conditions. This
reaction could be reverted if detected soon enough with the
addition of a portion of the reducing agent, sodium ascor-
bate. The target compounds 1–25 were obtained as 1,4-
disubstituted-1,2,3-triazoles and were isolated in moderate
to excellent yields (60–95%) after purification by crystal-
lization in hexane.
Reagents and conditions: alkyne (1.2 eq.), THF/MeOH/
H2O (1/1/1; v/v/v), β-CD (0.02 eq.), NaAsc (0.2 eq.), NaN3
(1.2 eq.), CuSO4·5H2O (0.2 eq.), rt, 10–24 h.
In order to characterize the novel compounds, analytical
instruments and techniques were used as described in the
general procedures. Compounds with similar substitutions
showed similar signal patterns on their NMR and IR spec-
tra. The 1H NMR spectra of all title compounds were
thoroughly examined for characteristic peaks, evidence of
the phenyl and triazole rings.
the identification of functional groups. Evidence of the
triazolyl moiety was confirmed with bends of C=C and
N=N bonds ca. 1600 and 1450 cm−1, respectively, while
vibration of the H-C= bond gave a stretch at around
3100 cm−1. Of note, vibrations of C–F bonds of the tri-
fluoromethyl substituent resulted in rockings around
1300 cm−1 in the spectra of compounds 21–25.
Physiochemical properties
Transmembrane transport gives a good indication of the
biological properties, such as oral bioavailability and cel-
lular uptake, and pharmacological activity of a compound
(Gombar and Enslein 1996). An ideal drug must possess
well-equipoised hydrophilic/lipophilic properties, so as to
efficiently permeate biological membranes and be absorbed
into the systemic circulation (Lipinski et al. 1997). The n-
octanol/water partition coefficient (cLogP) is a key para-
meter used in the measurement of the hydrophilicity and
lipophilicity of a chemical, allowing one to predict the
transport characteristics of a substance across biological
membranes through passive diffusion (Gombar and Enslein
1996). The cLogP values between 1 and 5 are often tar-
geted, with values between 1 and 3 being ideal (Lipinski
et al. 1997). Thus, the lipophilic substituent R2 (n-alkyl or
methylenediphenyl) side chains were anchored to the tria-
zole ring to assess the influence of lipophilicity on the
pharmacological effect of the compounds.
For compounds 1–5, which are unsubstituted on para
position of the phenyl ring, peaks of the five aromatic
protons and that of the triazolyl proton (H-5) overlapped as
a multiplet in the 7.4–7.15 ppm region. In addition, the
singlet ca. 5.40 ppm was assigned to the two methylene
protons of benzyl moiety, while the terminal CH3 in all the
aliphatic chains (1–4) appeared as a multiplet or a triplet
The substituent R1 (H, CH3, Br, NO2 and CF3), on the
other hand, were introduced on the phenyl ring to judge the
impact that electronic effects may have on the anti-
mycobacterial activity.
3
with J ~ 0.9 Hz as a result of the coupling with the two
protons of the neighbouring/preceding methylene group. In
compound 5, an additional singlet ca. 4.19 ppm was
attributed to the protons (H-11) of the methylene group
linked to the thiophenyl moiety, while further peaks of the
aromatic protons of that moiety were found in the above-
Most BZT derivatives, with the exception of long n-alkyl
side chain 8, 9, 13, 14, 19, 23 and 24, showed good drug-
like properties with cLogP values within the targeted range.
However, these above-mentioned threshold cLogP value
compounds may still be active because biological activity is
dependent on many parameters apart from physicochemical
properties (Pop et al. 2004). This is better illustrated with
the most recent drugs introduced in the market, bedaquiline
(cLogP 7.25) and delamanid (cLogP 6.14), which have
cLogP values above the safe threshold of 5 (Machado et al.
2018).
1
mentioned chemical shift range. In the H NMR spectra of
compounds 6–10 (p-methyl substituted), peaks of the aro-
matic protons appeared as a multiplet in the region of
7.13–7.08 ppm. Methylene protons (H-6) of benzyl moiety
gave a singlet around 5.4 ppm, while protons of the
methylene linked to the thiophenyl group appeared as a
singlet at 4.2 ppm in compound 10. The p-methyl protons
(H-10′) were assigned to the singlet around 2.3 ppm, while
the alkyl chain terminal CH3 gave a triplet in the
0.9–0.8 ppm region for compounds 6–9. All above-
Biological activities
1
mentioned characteristic peaks were also found in the H
spectra of 11–25.
For a novel drug to advance to human use, a chain of
preclinical studies, including in vitro assays and in vivo
testing, need to be concluded.
The in vitro mycobacterial growth inhibitory potential of
the synthesized compounds was assessed using the standard
microplate green fluorescent protein (GFP) assay against
In 13C spectra, the phenyl carbons gave peaks in the
130–125 ppm region, while the resonance of triazolyl car-
bons (C-4 and C-5) resulted in singlets in the 128–125 ppm
region.
The FTIR-spectra of all compounds were also inspected
for the presence of characteristic absorptions, allowing for
Mtb H37Rv, using
a reporter mutant constitutively

Medicinal Chemistry Research
Table 1 In vitro biological
activities of synthesized
benzyltriazoles
Antimycobacterial activity (μM)
Cytotoxicity, Selectivity
IC50 (µM)d
HEK-293
index
SIe
Cpd Chain
length (C)
cLogPa MIC90
MIC99
MIC90
[GAST/Fe]b [GAST/Fe]b [7H9/ADC]c
1
4
3.5
4.4
5.2
6.1
3.9
4.0
4.9
5.8
6.7
4.4
4.2
5.0
6.1
6.9
4.6
3.4
4.3
5.2
6.1
3.8
4.3
5.2
6.1
7.1
4.8
−0.7
3.6
7.76
29.6
>125
>125
20
7.7
62.5
>100
>100
>13
>3
2
6
32.5
121.9
>125
>125
>125
>125
>125
>125
>125
>125
58.5
3
8
>125
>125
38.3
4
10
5
>100
>100
>100
>100
>5
6
4
19.6
10.2
29.9
>125
10.3
2.78
nd
>125
12.4
>5
7
6
>10
>3
8
8
31.2
9
10
>125
17.3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
INH
RIF
Em
>100
>100
>100
>100
>10
>36
4
3.82
6
nd
71.6
8
35.7
>125
>125
64.8
66.6
>125
>125
>125
1.73
86.3
>125
>125
>125
0.041
0.001
40.1
>125
>125
>125
>125
66.12
>125
>125
82.03
125
>3
10
>125
>125
>125
>125
>125
>125
>125
3.19
4
>100
>100
>2
>2
6
8
10
4
>100
>100
>58
>1
6
>125
>125
>125
>125
0.046
0.001
3.906
>125
>125
>125
>125
1.06
8
10
0.004
125
0.2
acLogP values calculated using MarvinSketch Version 17.28
bCompounds screened in medium: protein-free GAST-Fe
cCompounds screened in medium: protein-rich 7H9 GLU ADC
dHuman embryonic kidney cell line from ECACC
eSelectivity index, SI=IC50 HEK-293/ MIC90 H37Rv, Em (emetine)
expressing GFP as described previously (Wilson et al.
2017) in two different media namely the Middlebrook 7H9
Broth base, supplemented with glucose (GLU) and enriched
with ADC (albumin BSA-dextrose-catalyse), and GAST-Fe
(glycerol–alanine–salts), BSA free (Soares de Melo et al.
2015). The assay was performed using the media to facil-
itate observation of culture-conditions mediating anti-
mycobacterial activity.
The minimum concentrations of the compounds that
inhibit the growth of 90 and 99% of mycobacteria,
expressed as MIC90 and MIC99, respectively, are summar-
ized in Table 1 alongside those of INH and RIF anti-
tubercular standards.
Furthermore, HEK-293 cells were used to determine the
cytotoxicity of the compounds alongside the cytotoxic drug
emetine as reference.
Half of the synthesized compounds were active in
GAST/Fe medium, while the overwhelming majority (72%)
were found to be inactive in 7H9/ADC medium. Even for
potent compounds in both media, the activity was more
pronounced in GAST/Fe than in 7H9/ADC. In particular,
compound 11 was found with a 20-fold activity difference,
while 1 was 8 times more active in the albumin-free med-
ium instead. This could be due to the lack and presence of
albumin in GAST/Fe and 7H9/ADC media, respectively.
Indeed, serum albumins are the major soluble protein

Medicinal Chemistry Research
constituents of the circulatory system, accounting for 4%
weight per volume (w/v). They have the ability to reversibly
bind a large variety of exogenous compounds, including
fatty acids, amino acids, drugs and pharmaceuticals (Dockal
et al. 2000; Ran et al. 2007; Tian et al. 2003). Plasma
protein binding (PB) forms an integral part of distribution
and bioavailability for numerous medications currently in
clinical use; however, binding becomes problematic where
it is extensive (Kandagal et al. 2006; Seetharamappa and
Kamat 2004). The influence of PB on pharmacological
effects is dependent on the biological setting. In vitro
binding to albumin would reduce the amount of compound
available to exert an effect on the mycobacterial cell and, in
theory, reduce the observed MIC of compounds. In vivo,
however, the impact of the albumin binding on MIC of
compounds would depend on the extent and nature of the
binding of the drug to albumin within the host.
The majority of compounds were inactive in 7H9/ADC,
which may suggest an irreversible binding to BSA and the
culprit would be the triazole, possibly binding to protein
and other macromolecules (Massarotti et al. 2014) through
its H-bonding acceptors (N-2 and N-3) or donor (H-5) sites
(Fig. 1).
Albumin binding properties may also provide some
insight into the possible pharmacokinetic behaviour of
compounds. Binding and dissociation of compounds from
plasma proteins is a dynamic process with only the unbound
fraction of the drug available to exert an effect. In a clinical
setting, the dosage of a drug is calculated to ensure that, at
any point in time, sufficient free drug is available to have
the required pharmacological effect. The same principle
would apply to side effects or toxicity and thus, plasma PB
becomes an important consideration, particularly in highly
bound drugs.
In light of the inactivity of most compounds in 7H9/
ADC, the MIC values obtained in GAST-Fe medium were
utilized for the purposes of determining structure activity
relationships (SARs). Comparison of the activities in regard
to the n-alkyl side chain length reveals that the short chain
(4C and 6C) compounds were the most active compounds
in GAST/Fe. However, 8 with its 8 carbons chain was also
found to be active in the medium with MIC90 29.9 µM.
Overall, the long chains (8C and 10C) derivatives were
inactive regardless of the medium. The limited number of
active compounds did not allow for a rigorous stand on the
impact of the variation of chain in relation to resulting
activity. However, the optimum chain, inducing maximum
activity among active compounds in GAST/Fe, seems to be
4C as can be gleaned from the activities of 1 vs. 2; 16 vs.
17; and 21 vs. 22.
values of 20 and 10.3 µM, respectively, in GAST/Fe.
However, this activity was lost in 7H9/ADC medium. The
remaining compounds, 15, 20 and 25 of this sub-series,
were completely inactive irrespective of the assay medium.
Furthermore, consideration of the lipophilicity shows the
most active compounds to be drug-likeable with cLogP
values in the target range (1–5). Due to the lipophilic nature
of the mycobacterium cell wall, one would have expected
more activity from the more lipophilic compounds (Fan
et al. 2018), but the opposite was observed in this study.
Similarly, to the impact of the chain length, a realistic
conclusion of the effect of lipophilicity on the activity could
not be drawn due to the narrow array of active compounds.
The substituents R1 on the phenyl ring were chosen in
order to assess the influence that the electronic effects may
have on the antimycobacterial activity. To ease the analysis,
the compounds are divided in sub-series based on the
identity and nature of R1. Thus, sub-series 1 comprises 1–5
with R1 (H); sub-series 2, 6–10 (CH3), sub-series 3, 11–15
(Br); sub-series 4, 15–20 (NO2) and sub-series 5, 21–25
(CF3). Sub-series 1 is neutral, 2 is electron donating due to
CH3 being an electron donating group (EDG) and 3–5 are
electron withdrawing, accounting on the fact that Br, NO2
and CF3 are electron withdrawing groups (EWGs) with
NO2, CF3 and Br in order of decreasing EW. Sub-series 2
comprised four active compounds in GAST/Fe with MIC90
values varying in the 10–30 µM range, while the others
contain only 2 active compounds. For 4C chain compounds,
the decreasing order of activity in GAST/Fe was 21 (CF3),
11 (Br), 1 (H), 6 (CH3) and 16 (NO2), and 7 (CH3), 2 (H),
17 (NO2) and 22 (CF3) for 6C side chain compounds. It
could be noticed that within a sub-series the activity
decreased as the chain length increased; however, no spe-
cific sub-series stood out. Thus, neither the electronic effect
of substituents on the phenyl ring nor the length of the side
chain of the triazole moiety dictated the activity of the
compounds. Thus, structural specificity rather than the
physical features (lipophilicity/chain length and electronic
effect) governed the activity of these BZT derivatives.
Individual compounds, such as 21 and 11 featuring n-butyl
chain and bearing CF3 and Br EWGs, respectively, were
identified as the most active compounds in the protein-free
GAST/Fe medium. In protein-rich 7H9/ADC medium,
however, 21 possessed weak activity, while 11 was com-
pletely inactive.
Furthermore, the charge distribution of organic mole-
cules is influenced by the presence of halogen atoms. These
can also affect binding interactions with biological systems
(Zhang et al. 2017). Fluorine has an atomic radius com-
parable to hydrogen with higher electronegativity than other
halogens and, hence, is known to have an effect on con-
formation, charge distribution, molecular interaction and
pharmacokinetics (Gillis et al. 2015; O’Hagan 2008). The
Thiophenyl-containing derivatives presented a mixed
activity profile. Derivatives 5 and 10, which bore phenyl
and p-Me phenyl, respectively, were active with MIC90

Medicinal Chemistry Research
short chain (4C) halogen substituted compounds 11 and 21
were more active than their alkyl substituted counterparts 1
and 6.
the active compounds was not determined, and will require
further work to elucidate.
For SAR purposes, only the MIC90 values in GAST/Fe
were considered. No realistic SAR could be drawn from this
study due to the narrow array of active compounds. How-
ever, structural specificity of individual compounds rather
than physicochemical properties in the series guided the
activity. Compounds 21 and 11, featuring n-butyl side chain
with trifluoromethyl and methyl substituent in para position
on the phenyl ring, respectively, stood out as the most active
with micromolar activities, although less potent than the
standards, INH and RIF. Bioisomerism was also on display
as the replacement of hydrogen in p-methyl of compound 6
with fluorine generated the p-trifluoromethyl containing hit
21 that was 11 times more active than 6.
These were also selective in their medium-dependent
antibacterial actions as seen from the antimycobacterial
assay. Both promising compounds had good selectivity
towards mycobacteria, with SI values greater than 10, and
thus stand as validated hits based on cellular potency and
cytotoxicity criteria to be further investigated in the search
for new antitubercular regimens. It will be interesting to
assess the synergism of these antimycobacterial promising
BZT derivatives in combination with higher PB affinity
compounds using the same pathway of action.
Bioisosterism was also assessed. Indeed, a bioisostere is
a molecule resulting from the exchange of an atom or of a
group of atoms with an alternative, broadly similar atom or
group of atoms. The objective of bioisosterism is the
rational modification of a lead compound into safer and
more clinically effective agents. The bioisosteric replace-
ment can attenuate toxicity, modify activity of the lead and/
or alter pharmacokinetics of the lead.
The ability of a group of bioisosteres to elicit similar
biological activity has been attributed to common physi-
cochemical properties. Thus, specific physicochemical
effects, such as electronegativity, steric size and lipophili-
city, were quantitated to correlate these values to the
observed biological activity. The substitution of hydrogen
by fluorine is one of the more commonly employed
monovalent isosteric replacements. Steric parameters for
hydrogen and fluorine are similar with their van der Waal’s
radii being 1.2 and 1.35 Å, respectively (Leroux et al.
2008). Compound 6 is 4-Me (EDG) substituted, while 21
contained a larger 4-CF3 (EWG). Both compounds have
similar lipophilicity (cLogP ~ 4.0), but 21 was 11-fold more
antimycobacterially active than 6 in the albumin-free med-
ium. Thus, the replacement of hydrogen with fluorine
resulted in a safer hit (IC50 > 100 µM) by virtue of its
greater electronegativity, while other parameters, such as
steric size and lipophilicity, are maintained. This confirms
fluorine as a bioisostere of hydrogen.
Acknowledgements This work was funded by a South African
National Research Foundation Grant to DDN’Da (UID 76443). The
South African Medical Research Council is gratefully acknowledged
for financial support of the antimycobacterial screening assays (SHIP-
MRC grant to DFW). The authors thank Dr D. Otto for NMR analysis
and Dr JHL Jordaan for MS analysis. Isoniazid was generously
donated by Aspen Pharmacare (Port Elizabeth, South Africa).
Overall, no synthesized compound was found to possess
potency comparable or higher than any of the reference
drugs of the study. However, both 21 and 11 are anti-
mycobacterially promising hits with MIC90 < 10 µM (Kat-
suno et al. 2015). They were found to be nontoxic to the
mammalian cells with IC50 > 100 µM as compared with
0.2 µM of emetine.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Conclusions
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