Synthesis and in vitro antimycobacterial and antileishmanial activities of hydroquinone-triazole hybrids

Article abstract of DOI:10.1007/s00044-020-02553-0

Infectious diseases such as tuberculosis and leishmaniasis are leading causes of human death. One of the major factors contributing to the poor control of these diseases is primarily the reduced effectiveness of the existing chemotherapies as result of th

Full text of DOI:10.1007/s00044-020-02553-0

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02553-0
ORIGINAL RESEARCH
Synthesis and in vitro antimycobacterial and antileishmanial
activities of hydroquinone-triazole hybrids
Chris-Marie Horn1 Janine Aucamp2 Frans J. Smit2 Ronnett Seldon3 Audrey Jordaan4 Digby F. Warner4,5
David D. N’Da
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Received: 27 February 2020 / Accepted: 27 April 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Infectious diseases such as tuberculosis and leishmaniasis are leading causes of human death. One of the major factors
contributing to the poor control of these diseases is primarily the reduced effectiveness of the existing chemotherapies as
result of the increasing rise of multidrug-resistant strains of their causative agents. This leads to the imperative need to
develop new and effective drugs. In search for such agents, a series of hydroquinone-triazole hybrids was investigated. The
design, synthesis, and biological activities against the human virulent H37Rv strain of Mycobacterium tuberculosis (Mtb)
and Leishmaniasis major (L. major), causative pathogen of human cutaneous leishmaniasis, are herein reported. The hybrids
were synthesized following a two-step process Michael addition and Click chemistry. They were found to be noncytotoxic
toward human kidney embryonic cells but expressed poor cellular antileishmanial and antimycobacterial activities. Hybrid
14, 2‐{4‐[(phenylsulfanyl)methyl]‐1H‐1,2,3‐triazol‐1‐yl}benzene‐1,4‐diol, was the most active among synthesized
molecules, with MIC90 16 and IC50 23 µM against Mtb and L. major parasite, respectively, but had a poor safety profile,
being as toxic to mammalian cells as to mycobacteria and parasites. Thus, compound 14 did not stand as potential anti-
infective hit for further investigation. Future endeavor will focus on the investigation of more rigid and flexible hybrids of
both scaffolds in order to assess the impact a spacer might have on their biological activity.
Graphical Abstract
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02553-0) contains supplementary
material, which is available to authorized users.
* David D. N’Da
University of Cape Town, Cape Town 7700, South Africa
david.nda@nwu.ac.za
4
5
SAMRC/NHLS/UCT Molecular Mycobacteriology Research
Unit, Department of Pathology and Institute of Infectious Disease
and Molecular Medicine, Faculty of Health Sciences, University of
Cape Town, Cape Town 7925, South Africa
1
Pharmaceutical Chemistry, School of Pharmacy, North-West
University, Potchefstroom 2520, South Africa
2
Centre of Excellence for Pharmaceutical Sciences (Pharmacen),
North-West University, Potchefstroom 2520, South Africa
Wellcome Centre for Clinical Infectious Diseases Research in
Africa, University of Cape Town, Cape Town 7925, South Africa
3
SAMRC Drug Discovery and Development Research Unit,

Medicinal Chemistry Research
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Keywords Infectious diseases Drug research Molecular hybridization Quinone Triazole Cyclizations
Introduction
by VL (CDC 2019; WHO 2019g). CL, on the other hand, is
the most common form, results in mutilation and esthetic/
social stigma (as a result of multiple lesions) (WHO 2019g)
while MCL causes partial or complete destruction of soft and
hard tissues of the palate, pharynx, and nose (WHO 2019g).
Treatment of leishmaniasis is limited to the use of a
handful of drugs including antimonials, paromomycin,
miltefosine, pentamidine, and amphotericin B (Aronson
et al. 2017; Torres-Guerrero et al. 2017). These therapies
are administered by either intravenous or intramuscular
(painful) routes, and often not consecutively during the
treatment period due to severe adverse effects. Resistance to
antimonial drugs has been established (Hadighi et al. 2006;
Ponte-Sucre et al. 2017) while emergence of resistance
against miltefosine (Deep et al. 2017; Ponte-Sucre et al.
2017) and amphotericin B (Brotherton et al. 2014; Ponte-
Sucre et al. 2017) is proven in experimental settings on
laboratory strains and field isolates, which may be indica-
tive of the looming loss of these two drugs. Monotherapy of
pentamidine is near discontinuation due to the loss of effi-
cacy and high toxicity while therapy of paromomycin is
associated with hepatotoxicity (Ansari et al. 2017). Thus,
the development of new antileishmanial agents with
reduced adverse effects and less invasive administration
routes is highly desired to treat this infection.
After decades of effective chemotherapeutic treatment,
tuberculosis (TB) still affects and kills millions of people
each year. TB is one of the top ten causes of mortality
worldwide (WHO 2018a) and is the leading cause of death
from a single infectious disease, surpassing human immu-
nodeficiency virus (WHO 2018b). In 2017 alone, ten mil-
lion people contracted TB and 1.6 million fatalities were
registered, of which more than 95% occurred in low- and
middle-income countries (CDC 2018a; WHO 2018a).
Majority of the estimated incidence cases occurred in
South-East Asia (44%), and Africa (25%) (WHO 2018c),
making TB a disease of the developing world.
The use of antitubercular drugs is currently the only
viable option available for the control of TB, as other
control measures (such as the use of Bacillus
Calmette–Guérin vaccine and TB chemoprophylaxis)
appear to be unsatisfactory (du Toit et al. 2006). The
existing antitubercular drugs, although of immense value in
controlling the disease, have several limitations with the
most important shortcoming being the emergence of drug-
resistant TB strains that renders the frontline drugs in par-
ticular, less active (Amir et al. 2014).
Furthermore, these drugs possess excessive adverse side
effects, which are alleviated through additional therapies,
incurring more expenses for patients. Overall, the current
TB treatment regimens, apart from being less effective and
safe, are becoming unaffordable for patients in the devel-
oping countries who need them most. This emphasizes the
search for new, effective, and cheap anti-TB alternatives.
Leishmaniasis, on the other hand, is one of the seven
most important tropical diseases and presents a serious
world health challenge. In 2017 alone, the death toll due to
the most common form of the disease (cutaneous leishma-
niasis (CL)) was 20,000–30,000 while 700,000–1 million
new cases were reported (WHO 2018e). This infectious
disease is widely distributed across 97 countries mainly in
Africa, Asia and Latin America (WHO 2018d). Leishma-
niasis is a vector-borne disease that is transmitted by phle-
botomine sand flies and caused by the obligate intracellular
protozoa of the genus Leishmania (WHO 2019f). There are
three clinical forms of leishmaniasis, namely mucocuta-
neous (MCL), CL (Aleppo boil), and visceral leishmaniasis
(VL, kala azar, black fever) (WHO 2019f). The latter affects
bone marrow, liver, and spleen (to a larger extent), pre-
senting symptoms such as fever, hepatosplenomegaly,
pancytopenia, and weight loss, which result in death if left
untreated. Most leishmaniasis related fatalities are caused
A promising strategy for the discovery of such drugs is
molecular hybridization, which is defined as the covalent
chemical linking of two or more pharmacophores to create a
single new chemical entity, with two structural domains and
biological functions that may act on different targets (dual
drug action) or wherein one part may equipoise the side
effects caused by another part (Kumar et al. 2014a; Smit
et al. 2015). Hybrid molecules may have improved efficacy
and affinity (Viegas-Junior et al. 2007), and are ther-
apeutically and medicinally more effective than individual
components (Smit and N’Da 2014). Structures of few pro-
mising hybrid compounds (Kumar et al. 2014a; Smit and
N’Da 2014; Smit et al. 2015) and hybrid drug candidate,
ferroquine (Mairet-Khedim et al. 2019), are shown in Fig. 1.
1,2,3-Triazole is a five-member N-heterocyclic scaffold
(Ali et al. 2017). Synthetic compounds containing this
moiety have been found with an array of biological prop-
erties inter alia antitubercular (Boechat et al. 2011) due to it
having favorable physicochemical features such as hydro-
gen bonding capability, moderate dipole, rigidity etc.
(Zhang et al. 2017). Some of these compounds, similarly to
isoniazid, exert their antitubercular action by inhibiting
microbial cell wall synthesis through blocking of lipid
biosynthesis (Kumar et al. 2014a; Zhang et al. 2017).

Medicinal Chemistry Research
Fig. 1 Structures of hybrid
compounds
conditions. Due to this constant interconversion in an aqu-
eous medium, we justified synthesizing HQ-linked 1,2,3-
triazoles. Pharmacological properties of BQ compounds
include anti-inflammatory (Sagnou et al. 2009), anticancer
(Lindsey et al. 2004; Tasdemir et al. 2006), antiviral and
antimalarial (Tasdemir et al. 2006), antimycobacterial (Jyoti
et al. 2016; Tasdemir et al. 2006), and antileishmanial
(Valderrama et al. 2005). Thus, the available literature
suggests that BQ and HQ moieties possess both anti-
mycobacterial and antileishmanial activities.
Scheme 1 Interconversion of benzoquinone (1) to hydroquinone (2)
with conditions favouring the reduction of 1
Several 1,2,3-triazole containing drugs are currently on the
market (Smit et al. 2019).
Quinones have a number of biological activities and,
although the precise mechanism of action is not fully
understood, most of their effects are attributed to redox
cycling and the generation of reactive oxygen species
(ROS) such as superoxide and hydrogen peroxide that
damage cells (Tran et al. 2004). ROS directly combat
infection by signaling cascades to prompt other protective
cellular measures (such as apoptosis) or by causing severe
oxidative stress within cells (Tasdemir et al. 2006). Like-
wise, HQ might affect Mtb and Leishmania by disrupting
intracellular components such as RNA, DNA, internal
proteins, and other organelles.
1,4-Benzoquinone (BQ) is the envisaged partner pharma-
cophore to 1,2,3-triazole investigated in this study. The
reduction of BQ to hydroquinone (HQ) via semiquinone,
occurs spontaneously and by the action of various enzymes in
a biological system (Scheme 1), the same also being valid for
the reverse reaction (HQ ↔ BQ) (McGregor 2007; Netherlands
HCot 2012). In an aqueous solution, 1,4-BQ is reduced to HQ
at a significant rate, further stimulated by the presence of
nicotinamide adenine dinucleotide phosphate hydrogen
(NADPH), and more so in the presence of NADPH and
microsomes (a complete microsomal system). In contrast, the
autoxidation of HQ is slow, even further slower in the presence
of NADPH, and stopped entirely by a complete microsomal
system. Therefore, in an aqueous medium, the main direction
of conversion is the reduction of BQ, with the complete
reduction to HQ taking place within 5 min in a complete
system (NADPH and microsomes) (Souček et al. 2000). HQ
autoxidation in an aqueous medium is pH-dependent, occur-
ring rapidly under alkaline conditions and slowly under acidic
conditions (McGregor 2007; Netherlands HCot 2012).
BQ and HQ are metabolites of each other, with the
interconversion rate dependent on prevailing local
We herein report the synthesis and in vitro anti-
mycobacterial and antileishmanial activities of a small library
of hybrids formed from HQ and 1,2,3-triazole scaffolds.
Materials and methods
Materials
Para-Benzoquinone (p-BQ), sodium azide (NaN3),
β-cyclodextrin, sodium ascorbate (NaAsc), copper ascorbate
(CuAsc), copper sulfate (CuSO4), 1-ethynylcyclohexanol,

Medicinal Chemistry Research
1-hexyne, 1-heptyne, 1-octyne, 1-decyne, phenylacetylene,
tetrahydro-2-(2-propynyloxy)-2H-pyran, methyl propiolate,
propargyl alcohol, 4-ethynyltoluene, phenyl propargyl sul-
fide, sodium nitrite (NaNO2) magnesium sulfate (MgSO4),
sodium bicarbonate (NaHCO3) were purchased from
Sigma-Aldrich (South Africa). All solvents used, methanol
(MeOH), acetone, tetrahydrofuran (THF), ethyl acetate
(EtOAc), dichloromethane (DCM), hexane, dimethyl sulf-
oxide (DMSO) were purchased from Associated Chemical
Enterprises (South Africa) or from Sigma-Aldrich (South
Africa). All chemicals and reagents were of analytical grade
and were used without further purification. For inert reac-
tions, DCM was distilled over calcium hydride and stored
over 3 Å molecular sieves.
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 to 20 μL of 0.25 mM). The
eluent was monitored at wavelengths of 210, 254, and
300 nm.
Synthesis
2‐Azidobenzene‐1,4‐diol 3
The intermediate was prepared by adopting the literature
reported (Couladouros et al. 1997) method with minor
modifications as illustrated in Scheme 2, step i.
In a single neck round bottom flask, p-BQ 1 (18.6 mmol,
2.0 g, 1 eq.) was dissolved in MeOH (80 mL) upon stirring
at −78 °C (on dry ice), and flushed with argon. NaN3,
(74.0 mmol, 4.8 g, 4.00 eq.) was dissolved in MeOH
(30 mL) and pH of the solution was adjusted to 4 using 1 M
HCl then added portion wise to BQ solution at regular
intervals (5 × 10 min). The resulting mixture was continued
stirring at −78 °C under argon for another 1.5 h to produce
the azide intermediate 3. The progress of the reaction was
monitored using TLC. Upon completion, the solvent was
evaporated and NaHCO3 (50 mL) was added to the residue.
The crude organic layer was successively extracted with
EtOAc (150 mL) and DCM (150 mL). The resulting organic
layers were dried over anhydrous MgSO4, filtered, evapo-
rated to dryness. The resulting residue was purified by
column chromatography on silica gel eluting DCM/MeOH
(9:1, v/v) to yield 3.
General procedures
The 1H and 13C nuclear magnetic resonance (NMR) spectra
were recorded on a Burker AvanceTM III 600 spectrometer
at a frequency of 150 MHz, respectively, in deuterated
DMSO (DMSO-d6). Chemical shifts are reported in parts
per million (ppm) with the residual protons of the solvent as
reference (1H NMR (600 MHz, DMSO-d6) δ 2.5 and 13C
NMR (151 MHz, DMSO-d6) δ 13C 40). The splitting pattern
abbreviations are as follows: s (singlet), d (doublet), dd
(doublet of doublet), t (triplet), q (quartet), p (pentet), and m
(multiplet).
High resolution mass spectrometry (HRMS) was recor-
ded on a Bruker MicroTOF Q II mass spectrometer that had
an atmospheric pressure chemical ionization (APCI) source
set at 200 °C using Bruker Compass DataAnalysis 4.0 soft-
ware. A full scan, ranging between 50–1500 m/z, was
generated at a capillary voltage of 4500 V, an end plate
offset voltage of 500 V, and a collision cell radio frequency
voltage of 100 Vpp.
Fourier Transformed Infrared (FTIR) spectra were
recorded on a Bruker Alpha-P FTIR instrument. Melting
points (m.p.) were determined with a BÜCHI m.p. B-545
instrument and were uncorrected. Thin layer chromato-
graphy (TLC) was performed using silica gel plates
(60F254), acquired from Merck (South Africa).
High performance liquid chromatography (HPLC) ana-
lysis of the final compounds was performed to determine
purity. An Agilent 1100 HPLC system equipped with a
quaternary pump and an Agilent 1100 series diode array
detector were utilized. HPLC-grade acetonitrile (Merck)
and Milli-Q water (Millipore) were used for chromato-
graphy. A Venusil XBP C-18 column (4.60 × 150 mm,
5 μm), with an initial mobile phase (70% Milli-Q water:
30% acetonitrile), was employed at a flow rate of 1 mL/min.
The concentration of acetonitrile in the mobile phase was
linearly increased over a period of 5 min to a final con-
centration of 85%. The time allowed for equilibration
1
Dark brown powder; H NMR (600 MHz, DMSO-d6) δ
9.21 (s, 1H, H-1a), 8.96 (s, 1H, H-4a), 6.67 (d, J = 2.8 Hz,
1H, H-3), 6.41 (dd, J = 8.7, 2.8 Hz, 1H, H-5), 6.33 (d, J =
8.7 Hz, 1H, H-6). 13C NMR (151 MHz, DMSO-d6) δ
150.77 (C-4), 143.08 (C-1), 125.99 (C-2), 117.43 (C-5),
112.80 (C-6), 107.54 (C-3).
Synthesis of hybrids 4–14
Compounds 4–14 were prepared in accordance with the
general procedure (Dixit et al. 2012) depicted in Scheme 2
step ii, and is described as follows:
In a single neck flat bottom flask, alkyne (1.20 eq.) was
mixed in THF (6 mL), MeOH (6 mL), and distilled H2O
(6 mL). β-Cyclodextrin (0.02 eq.), NaAsc (0.20 eq.), 3
(1.00 eq.), and CuSO4 (0.10 eq.) were consecutively added
to the flask. The reaction was left to stir at room temperature
for 28–29 h. The progress of the reaction was monitored
using TLC. Afterwards, the solvent was evaporated, the
product was extracted with hot EtOAc, washed with
NaHCO3, dried over anhydrous MgSO4, and recrystallized
using hot EtOAc and hexane, to produce the desired pure
compound.

Medicinal Chemistry Research
Compd.
R
Compd.
10
R
Yield (%)
Yield (%)
4
5
42
47
69
29
11
6
7
12
46
52
43
13
14
45
23
8
9
70
58
Scheme 2 General reaction procedure of hydroquinone-1,2,3-triazole
hybrids. Reagents and conditions: (i) 1: p-benzoquinone (1.00 eq.),
MeOH, −78 °C, NaN3 (4.00 eq.)/MeOH sol. (pH~4), 1.5 h. (ii) 4–14:
alkyne (1.20 eq.), THF, MeOH, H2O, β-cyclodextrin (0.02 eq.),
sodium ascorbate (0.20 eq.), 3 (1 eq.), and CuSO4 (0.10 eq.), r.t, 28 h
2-(4-Butyl-1H-1,2,3-triazol-1-yl)benzene-1,4-diol 4
22.22 (C-14), 14.19 (C-15); HRMS (APCI) m/z: [M + H]+
234.1233 (Calc. for C12H16N3O2: 234.1198); purity
(HPLC): 96%.
The reaction with 1-hexyne afforded hybrid 4 as brown
crystals; m.p. 159.4–162.4 °C; IR vmax: 3185, 3079, 2955,
2579, 1614, 1533, 1467, 1432, 1407 cm−1
;
1H NMR
2-(4-Pentyl-1H-1,2,3-triazol-1-yl)benzene-1,4-diol 5
(600 MHz, DMSO-d6) δ 9.71 (s, 1H, H-4a), 9.25 (s, 1H, H-
1a), 8.21 (s, 1H, H-11), 7.06 (d, J = 2.8 Hz, 1H, H-3), 6.92
(d, J = 8.9 Hz, 1H, H-6), 6.74 (dd, J = 8.9, 2.8 Hz, 1H, H-
5), 2.69 (t, J = 7.6 Hz, 2H, H-12), 1.68–1.58 (m, 2H, H-13),
The reaction with 1-heptyne gave hybrid 5 as off white
crystals; m.p. 156.3–158.3 °C; IR vmax: 3180, 3080, 3024,
2588, 1614, 1529, 1473, 1439, 1380 cm−1 1H NMR
;
1.41–1.31 (m, 2H, H-14), 0.92 (t, J = 15 Hz, 3H, H-15); 13
C
(600 MHz, DMSO-d6) δ 9.71 (s, 1H, H-4a), 9.24 (s, 1H, H-
1a), 8.21 (s, 1H, H-11), 7.04 (d, J = 2.9 Hz, 1H, H-3), 6.92
(d, J = 8.8 Hz, 1H, H-6), 6.74 (dd, J = 8.8, 2.9 Hz, 1H, H-
5), 2.68 (t, J = 7.6 Hz, 2H, H-12), 1.65 (p, J = 7.6 Hz, 2H,
NMR (151 MHz, DMSO-d6) δ 150.55 (C-1), 147.10 (C-4),
141.88 (C-10), 125.09 (C-2), 123.59 (C-11), 118.33 (C-5),
116.88 (C-3), 111.15 (C-6), 31.59 (C-13), 25.09 (C-12),

Medicinal Chemistry Research
H-13), 1.38–1.23 (m, 4H, H-14 & H-15), 0.88 (J = 16 Hz,
3H, H-16). 13C NMR (151 MHz, DMSO-d6) δ 150.56 (C-
1), 147.16 (C-4), 141.88 (C-1), 125.10 (C-2), 123.57 (C-
11), 118.35 (C-5), 116.89 (C-3), 111.15 (C-6), 31.34 (C-
14), 29.12 (C-12), 25.38 (C-13), 22.34 (C-15), 14.38 (C-
16); HRMS (APCI) m/z: [M + H]+ 248.1411 (Calc. for
C13H18N3O2: 248.1354); purity (HPLC): 97%.
150.58 (C-1), 146.53 (C-4), 142.42 (C-10), 131.05 (C-12),
129.41 (C-2), 128.44 (C-14), 125.80 (C-15), 124.90 (C-13),
123.27 (C-11), 118.36 (C-5), 117.51 (C-6), 111.66 (C-6);
HRMS (APCI) m/z: [M + H]+ 254.0917 (Calc. for
C14H12N3O2: 254.0885); purity (HPLC): 98%.
2-[4-(p-Tolyl)-1H-1,2,3-triazol-1-yl]benzene-1,4-diol 9
2-(4-Hexyl-1H-1,2,3-triazol-1-yl)benzene-1,4-diol 6
The reaction with 4-ethynyltoluene gave hybrid 9 as
brown crystals; m.p. 275.7–276.7 °C; IR vmax: 3188,
The reaction with 1-octyne produced hybrid 6 as brown
crystals; m.p. 157.4–159.5 °C; IR vmax: 3180, 3081, 3020,
2583, 1614, 1472, 1388 cm−1; 1H NMR (600 MHz, DMSO-
d6) δ 9.72 (s, 1H, H-4a), 9.24 (s, 1H, H-1a), 8.21 (s, 1H, H-
11), 7.06 (dd, J = 17.9, 2.9 Hz, 1H, H-3), 6.93 (d, J =
8.8 Hz, 1H, H-6), 6.74 (dd, J = 8.8, 2.9 Hz, 1H, H-5), 2.69
(t, J = 7.6 Hz, 2H, H-12), 1.71–1.57 (m, 2H, H-13),
1.41–1.19 (m, 6H, H-14, -15, H-16), 0.87 (t, J = 6.9 Hz,
3H, H-17). 13C NMR (151 MHz, DMSO-d6) δ 150.55 (C-
1), 147.14 (C-4), 141.87 (C-10), 125.08 (C-2), 123.59 (C-
11), 118.33 (C-5), 116.88 (C-6), 111.13 (C-6), 31.50 (C-
15), 29.42 (C-14), 28.79 (C-12), 25.42 (C-13), 22.53 (C-
16), 14.43 (C-17); HRMS (APCI) m/z: [M + H]+ 262.1561
(Calc. for C14H20N3O2: 262.1511); purity (HPLC): 96%.
3074, 3025, 2581, 1615, 1474 cm−1
;
1H NMR
(600 MHz, DMSO-d6) δ 9.81 (s, 1H, H-4a), 9.31 (s, 1H,
H-1a), 8.85 (s, 1H, H-11), 7.84 (d, J = 8.1 Hz, 2H, H-
13), 7.29 (d, J = 8.1 Hz, 2H, H-14), 7.07 (d, J = 2.9 Hz,
1H, H-3), 6.97 (d, J = 8.8 Hz, 1H, H-6), 6.80 (dd, J =
8.8, 2.9 Hz, 1H, H-5), 2.35 (s, 3H, H-16). 13C NMR
(151 MHz, DMSO-d6) δ 150.55 (C-1), 146.58 (C-4),
142.39 (C-10), 137.75 (C-15), 129.96 (C-12), 128.26 (C-
2), 125.72 (C-14), 124.91 (C-13), 122.84 (C-11), 118.32
(C-5), 117.43 (C-3), 111.63 (C-6), 21.35 (C-16); HRMS
(APCI) m/z: [M + H]+ 268.1096 (Calc. for C15H14N3O2:
268.1041); purity (HPLC): 98%.
Methyl 1-(2,5-dihydroxyphenyl)-1H-1,2,3-triazole-4-
carboxylate 10
2-(4-Octyl-1H-1,2,3-triazol-1-yl)benzene-1,4-diol 7
The reaction with methyl propionate produced hybrid 10 as
The reaction with 1-decyne gave hybrid 7 as off white
crystals; m.p. 146.3–150.3 °C; IR vmax: 3173, 3042, 2958,
fluffy off white crystals; m.p. 238.0–238.4 °C; IR vmax
:
3177, 2953, 1693, 1556, 1475, 1212 cm−1 1H NMR
;
1
2592, 1614, 1477, 1440, 1219 cm−1; H NMR (600 MHz,
(600 MHz, DMSO-d6) δ 9.97 (s, 1H, H-4a), 9.36 (s, 1H, H-
1a), 9.02 (s, 1H, H-11), 7.07 (d, J = 2.9 Hz, 1H, H-3), 6.96
(d, J = 8.9 Hz, 1H, H-6), 6.82 (dd, J = 8.9, 2.8 Hz, 1H, H-
5), 3.88 (s, 3H, H-14). 13C NMR (151 MHz, DMSO-d6) δ
161.17 (C-12), 150.57 (C-1), 142.40 (C-4), 138.87 (C-10),
130.63 (C-2), 124.09 (C-11), 118.39 (C-5), 118.07 (C-3),
111.48 (C-6), 52.39 (C-14); HRMS (APCI) m/z: [M + H]+
236.0668 (Calc. for C10H10N3O4: 236.0627); purity
(HPLC): 96%.
DMSO-d6) δ 9.71 (s, 1H, H-4a), 9.24 (s, 1H, H-1a), 8.21 (s,
1H, H-11), 7.04 (d, J = 2.9 Hz, 1H, H-3), 6.92 (d, J =
8.8 Hz, 1H, H-6), 6.73 (dd, J = 8.8, 2.9 Hz, 1H, H-5), 2.68
(t, J = 7.6 Hz, 2H, H-12), 1.67–1.56 (m, 2H, H-13),
1.38–1.14 (m, 10H, H-14… & H-18), 0.86 (t, J = 7.6 Hz,
3H, H-19). 13C NMR (151 MHz, DMSO-d6) δ 150.55 (C-
1), 147.19 (C-4), 141.86 (C-10), 125.08 (C-2), 123.60 (C-
11), 118.32 (C-5), 116.87 (C-3), 111.12 (C-6), 31.76 (C-
17), 29.46 (C-16), 29.26–29.23 (C-15), 29.20 (C-14), 29.14
(C-12), 25.52–25.33 (C-13), 22.58 (C-18), 14.44 (C-19);
HRMS (APCI) m/z: [M + H]+ 290.1862 (Calc. for
C16H24N3O2: 290.1824); purity (HPLC): 97%.
2-[4-(Hydroxymethyl)-1H-1,2,3-triazol-1-yl]benzene-1,4-diol 11
The reaction with propargyl alcohol gave hybrid 11 as dark
brown crystals; m.p. 228.0–233.6 °C; IR vmax: 3357, 3183,
1
2-(4-Phenyl-1H-1,2,3-triazol-1-yl)benzene-1,4-diol 8
3085, 1615, 1472 cm−1; H NMR (600 MHz, DMSO-d6) δ
9.77 (s, 1H, H-4a), 9.25 (s, 1H, H-1a), 8.33 (s, 1H, H-11),
7.06 (d, J = 8.7 Hz, 1H, H-6), 6.93 (d, J = 2.9 Hz, 1H, H-3),
6.75 (dd, J = 8.7, 2.9 Hz, 1H, H-5), 5.26 (t, J = 5.3 Hz, 1H,
H-13), 4.60 (d, J = 4.8 Hz, 2H, H-12); 13C NMR (151 MHz,
DMSO-d6) δ 150.58 (C-1), 148.16 (C-4), 141.92 (C-10),
124.98 (C-2), 124.49 (C-5), 118.37 (C-11), 117.04 (C-2),
111.17 (C-6), 55.43 (C-12); HRMS (APCI) m/z: [M + H]+
208.0727 (Calc. for C9H10N3O3: 208.0677); purity
(HPLC): 98%.
The reaction with phenylacetylene afforded hybrid 8 as
brown–yellow crystals; m.p. 257.0–261.7 °C; IR vmax: 3194,
3080, 3028, 2584, 1610 cm−1; 1H NMR (600 MHz, DMSO-
d6) δ 9.81 (s, 1H, H-4a), 9.30 (s, 1H, H-1a), 8.91 (s, 1H, H-
11), 7.89 (d, J = 7.7 Hz, 2H, H-13), 7.48 (t, J = 7.7 Hz, 2H,
H-14), 7.37 (t, J = 7.4 Hz, 2H, H-15), 7.08 (d, J = 2.9 Hz,
1H, H-3), 6.98 (d, J = 8.8 Hz, 1H, H-6), 6.81 (dd, J = 8.8,
2.9 Hz, 1H, H-5). 13C NMR (151 MHz, DMSO-d6) δ

Medicinal Chemistry Research
2-[4-(1-Hydroxycyclohexyl)-1H-1,2,3-triazol-1-yl]benzene-
HRMS (APCI) m/z: [M + H]+ 300.0808 (Calc. for
1,4-diol 12
C15H14N3O2S: 300.0807); purity (HPLC): 92%.
The reaction with 1-ethynylcyclohexanol produced hybrid
Biological evaluation
12 as fluffy off white crystals; m.p. 246.2–247.3 °C; IR vmax
:
3263, 3171, 3074, 3025, 2857, 1614, 1494, 1473 cm−1; H
NMR (600 MHz, DMSO-d6) δ 9.76 (s, 1H, H-4a), 9.25 (s,
1H, H-1a), 8.25 (s, 1H, H-11), 7.09 (d, J = 2.9 Hz, 1H, H-3),
6.93 (d, J = 8.8 Hz, 1H, H-6), 6.74 (dd, J = 8.8, 2.9 Hz, 1H,
H-5), 4.98 (s, 1H, H-12a), 1.96–1.83 (m, 4H, H-13 & H-17),
1.82–1.62 (m, 4H, H-14 & H-16), 1.42–1.24 (m, 2H, H-15).
13C NMR (151 MHz, DMSO-d6) δ 156.15 (C-1), 150.59 (C-
4), 141.68 (C-10), 125.03 (C-2), 122.56 (C-11), 118.42 (C-
5), 116.86 (C-6), 110.93 (C-3), 68.51 (C-12), 38.26 (C-13,
C-17), 25.72 (C-15), 22.10 (C-14, C-16); HRMS (APCI) m/
z: [M + H]+ 276.1334 (Calc. for C14H18N3O3: 276.1303);
purity (HPLC): 97%.
In vitro antimycobacterial assay
1
The minimum inhibitory concentration (MIC) of synthesized
compounds was determined using the standard broth micro-
dilution method, where a 10 mL culture of Mycobacterium
tuberculosis (Mtb) H37Rv (27294) obtained from American
Type Culture Collection (ATCC) (Stringer et al. 2017), was
grown to an optical density (OD600) of 0.6–0.7. The growth
medium comprised Middlebrook 7H9 base (Difco™) supple-
mented with 0.03% (v/v) casitone, 0.4% (v/v) glucose, and
0.05% (v/v) tyloxapol (Sigma-Aldrich) as surfactant (Stringer
et al. 2017). Cultures grown in the medium were diluted 1:500,
prior to inoculation in the MIC assay. The compounds to be
tested were reconstituted to a concentration of 10 mM in
DMSO. Twofold serial dilutions of the test compounds were
prepared across a 96-well microtiter plate, after which, 50 µL of
the diluted Mtb cultures were added to each well in the serial
dilution. Assay controls used were a minimum growth control
(Rifampicin (RIF) at 2 × MIC), and a maximum growth control
(5% v/v DMSO). The microtiter plates were sealed in a sec-
ondary container and incubated at 37 °C with 5% CO2 and
humidification. Relative fluorescence (excitation 485 nM;
emission 520 nM) was measured using a plate reader
(FLUOstar OPTIMA, BMG LABTECH), at day 7 and day 14.
The raw fluorescence data were archived and analysed using
the CDD Vault from Collaborative Drug Discovery, in which
data were normalized to the minimum and maximum inhibition
controls to generate a dose response curve (% inhibition), using
the Levenberg–Marquardt damped least squares method, from
which the MIC90 was calculated (Burlingame, CA www.colla
borativedrug.com). The lowest concentration of drug that
inhibited growth of more than 90% of the bacterial population
was considered the MIC90.
2-{4-{[(Tertahydro-2H-pyran-2-yl)oxy]methyl}-1H-1,2,3-
triazol-1-yl}benzene-1,4-diol 13
The reaction with tetrahydro-2-(2-propynyloxy)-2H-pyran
gave hybrid 13 as brown–red crystals; m.p. 140.1–141.3 °C;
IR vmax: 3179, 3084, 2584, 1720, 1614, 1473, 1019 cm−1
;
1H NMR (600 MHz, DMSO-d6) δ 9.81 (s, 1H, H-4a), 9.29
(s, 1H, H-1a), 8.45 (s, 1H, H-11), 7.05 (d, J = 2.9 Hz, 1H,
H-3), 6.93 (d, J = 8.8 Hz, 1H, H-6), 6.76 (dd, J = 8.8,
2.9 Hz, 1H, H-5), 4.76 (t, J = 8.5 Hz, 2H, H-16), 4.59 (t, J
= 9.0 Hz, 2H, H-12), 3.82 (ddd, J = 11.6, 8.7, 3.1 Hz, 1H,
H-14), 3.56–3.45 (m, 1H, H-19b), 1.78–1.57 (m, 2H, H-17),
1.57–1.41 (m, 2H, H-18). 13C NMR (151 MHz, DMSO-d6)
δ 150.56 (C-1), 144.07 (C-4), 142.04 (C-10), 125.85 (C-2),
124.84 (C-11), 118.33 (C-5), 117.22 (C-6), 111.32 (C-3),
97.69 (C-14), 61.78 (C-16), 59.89 (C-12), 30.55 (C-19),
25.47 (C-17), 19.47 (C-18); HRMS (APCI) m/z: [M + H]+
292.1314 (Calc. for C14H18N3O4: 292.1253); purity
(HPLC): 97%.
2-{4-[(Phenylthio)methyl]-1H-1,2,3-triazol-1-yl}benzene-
1,4-diol 14
In vitro cytotoxicity assay
The reaction with phenyl propargyl sulfide afforded hybrid
The colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide] assay was developed by
Mossman (1983) to measure cytotoxicity and cell proliferation
was adopted. Human embryonal kidney, HEK-293 (ATCC
CRL-1573). cells were cultured in Dulbecco’s modified
Eagle’s medium with high glucose (Hyclone; Separations)
supplemented with 10% fetal bovine serum (Thermofisher
14 as brown–black crystals; m.p. 151.8–153.0 °C; IR vmax
:
3177, 3074, 2595, 1719, 1614 cm−1; H NMR (600 MHz,
DMSO-d6) δ 9.79 (s, 1H, H-4a), 9.26 (s, 1H, H-1a), 8.33 (s,
1H, H-11), 7.42 (d, J = 7.7 Hz, 2H, H-14), 7.33 (t, J =
7.7 Hz, 2H, H-15), 7.20 (t, J = 7.4 Hz, 1H, H-16), 7.05 (d,
J = 2.9 Hz, 1H, H-3), 6.93 (t, J = 8.8 Hz, 1H, 1H, H-6),
1
6.75 (dd, J = 8.8, 2.9 Hz, 1H, H-5), 4.37 (s, 2H, H-12); 13
C
Scientific)
and
1%
L-glutamine
(Lonza),
1%
NMR (151 MHz, DMSO-d6) δ 150.59 (C-1), 143.77 (C-4),
141.78 (C-10), 136.30 (C-13), 129.49 (C-14, C-18), 128.75
(C-15, C-17), 126.42 (C-2), 124.94 (C-16), 124.74 (C-11),
118.40 (C-5), 117.17 (C-6), 110.95 (C-3), 27.58 (C-12);
penicillin–streptomycin (Lonza), amphotericin B (Lonza), and
nonessential amino acids (Lonza). The cells are maintained in a
humidified atmosphere at 37 °C and 5% CO2. For the MTT
assay, 96-well plates (Nunc, Thermofisher Scientific) were

Medicinal Chemistry Research
prepared with 200 μL of cell suspension (25,000 cells/mL) and
incubated for 24 h. The growth medium was then removed and
the cells treated with: (1) 100 μL of emetine dihydrochloride
(Sigma-Aldrich) solution diluted with growth medium to the
necessary concentrations (positive control); (2) 80 μL of growth
medium 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 contained
growth medium without cells. The treated plates were incu-
bated for 48 h.
Due to light sensitivity of MTT reagent, the assay was
performed in the dark. Sterile-filtered MTT solution (20 μL of
1 mg/mL solution in phosphorate-buffered saline (PBS, Sigma-
Aldrich)) was added to initiate the MTT assay, the plates
covered with aluminum foil and incubated for 4 h. The growth
medium-MTT mixture was then aspirated and 100 μL of 2-
propanol (Sigma-Aldrich) added to dissolve purple formazan
crystals. The contents were gently mixed for 5 min at room
temperature. Absorbance was measured at 560 and 650 nm
using the Thermofisher Scientific GO Multiscan plate reader.
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:
72 h at 26 °C in humidified atmosphere. After incubation,
50 μL of resazurin (Sigma-Aldrich) solution (0.01% in PBS)
was added to each well and the plates were further incubated at
26 °C in the dark for 4 h. Absorbance was measured at 570 and
600 nm using the Thermofisher Scientific GO Multiscan plate
reader. Data analysis was performed for each biological repli-
cate using SkanIt 4.0 Research Edition software. Background
absorbance of resazurin (600 nm) was subtracted from the
absorbance values of resazurin (570 nm). The mean absorbance
calculated and the percentage growth inhibition and cell via-
bility were determined by the following equations:
Growth inhibition % ¼
ꢀ
ꢁ
ðΔ Abs neg: control À Δ Abs blankÞÀ
ðΔ Abs sample À Δ Abs blankÞ
=
ðΔ Abs neg: control À Δ Abs blankÞ Â 100
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 three biological replicates using GraphPad Prism 5
and the mean IC50 of the biological replicates served as the
final IC50 of each compound.
Statistical analysis
Cell viability % ¼ ðΔ Abs sample À Δ Abs blankÞ
=ðΔ Abs neg: control À Δ Abs blankÞ Â 100
For antimycobacterial activity expressed as MIC90 values,
statistical analysis was by ANOVA (95% confidence
interval) for three biological replicates.
For the final IC50 of each compound, the mean IC50 of
three biological replicates were calculated in GraphPad
Prism 5.
The in vitro cytotoxicity and antileishmanial activity, indi-
cated as IC50 values, were derived from nonlinear regression
analysis. The results were represented as mean standard error
of the mean from three biological replicate experiments. Sta-
tistical analysis was performed using SkanIt 4.0 Research
Edition software (Thermofisher Scientific) and Prism
V5 software (GraphPad). All reported data were significant at
p < 0.05.
In vitro antileishmanial assay
Resazurin assay was adopted to assess the antileishmanial
potential of the compounds (Kulshrestha et al. 2013). A brief
description is as follows: Leishmania major (strain IR-173
(MHOM/IR/-173) obtained through BEI Resources, NIAID,
NIH, NR-48816) promastigotes were cultured in M199 with
Hank’s salts and 0.68 mM L-glutamine (Sigma-Aldrich) sup-
plemented with 4.2 mM NaHCO3, 25 mM Hepes (Sigma-
Aldrich), 10% fetal bovine serum (Thermofisher Scientific),
and 50 U/mL Penicillin/Streptomycin solution (Lonza) and the
pH adjusted to 7.3–7.4. The promastigotes were maintained at
26 °C. For the resazurin assay, logarithmic phase promastigotes
(1.25 × 105 cells/mL, final volume 100 μL/well) were seeded in
96-well plates (Nunc, Thermofisher Scientific) in the presence
of (1) 10 μM of compound for activity screening or (2) 12
twofold dilution concentrations of compounds for IC50 deter-
mination. Amphotericin B (AMB), 10 μM (Sigma-Aldrich)
served as the standard drug and growth medium without
parasites served as the blank. The plates were incubated for
Results and discussion
Chemistry
Considering the interconversion in an aqueous medium
between BQ and HQ (Netherlands HCot 2012), the syn-
thetic route adopted occurred in two steps and is depicted in
Scheme 2, was described as follows: in the first step (i), 2-
azidohydroquinone, also referred to as azidoquinol, 3 had
previously been synthesized (88%) from the reduction of
BQ in aqueous acidic medium via Michael addition (Cou-
ladouros et al. 1997). The reaction was performed in
methanolic acid medium (pH 4), which favored the

Medicinal Chemistry Research
reduction of the BQ moiety (Netherlands HCot 2012). In
our study, pure instead of aqueous MeOH was used which
resulted a marginal increase in yield (89%), and the struc-
ture characterization data corroborate this fact.
The viability of this conversion has been tested in bio-
logical media with the glutathione (GSH) catalyzed Michael
addition of hydroxyquinone to generate S-glutathionyl-HQ
reductases (Lam et al. 2012).
In the second step (ii), 3 was reacted with various indi-
vidual alkynes through copper-catalyzed alkyne-azide
cycloaddition (CuCAAC) reaction, or otherwise referred to
as click chemistry, to produce different HQ-1,2,3-triazole
hybrids. The CuCAAC reaction employed was adapted
from a procedure previously reported (Dixit et al. 2012). All
synthesized hybrids were produced in poor to moderate
yields (23–70%) after purification by recrystallization with
hexane. These poor to moderate yields could possibly be
explained by the oxidation of HQ to BQ taking place under
atmospheric oxygen during the workup but only the cor-
responding HQ crystallized out (Couladouros et al. 1997).
Structurally, all hybrids differed by the substituent on C-
10 of the triazole ring.
2200–2100 cm−1 region (Supplementary information).
However, the spectra clearly illustrate the vibration of the
OH functional groups of quinol, indicated as variable broad
or sharp peaks in 3200–3000 cm−1, and a strong stretch,
corresponding with the C = C bond of the triazole moiety in
the 1650–1550 cm−1 range.
Furthermore, HRMS using APCI ion source show the
presence of [M + H]+ fragments in the spectra that con-
firmed the presence of M+ molecular ion of each hybrid.
Estimated hydrophilicity/lipophilicity balance
(CLogP)
In order for the cellular uptake of a drug to occur efficiently
through a biological membrane that drug must be neither to
lipophilic nor to hydrophilic. Indeed, lipophilic drugs show
poor aqueous solubility and tend to be taken up in fatty
globules in the intestine. If they reach the blood stream, they
may be adsorbed into tissue. Their slow release may
exacerbate toxicity such as neurotoxicity. Hydrophilic drugs,
on the other hand, may be excreted directly by the kidneys, or
should they be able to penetrate a cell membrane, become
entrapped in intracellular lipophilic media. Thus, an ideal
drug must possess balanced lipophilic/hydrophilic properties
to both permeate biological membranes and be taken up for
systemic circulation. The n-octanol/water partition coefficient
(LogP) provides a good measure of this balance with values
between 1 and 5 being targeted, and ~1–3 being ideal
(Lipinski et al. 1997). LogP values were estimated using
MarvinSketch 19.4 Software and are gathered in Table 1.
All the hybrids possessed favorable drug-like character-
istics with LogP values within the targeted range hence may
be biologically active. However, the opposite may also occur
on account that biological activity is modulated by many
parameters (Pop et al. 2004) aside from physical properties
such hydrophilicity, lipophilicity, and their balance thereof.
The formation of the target compounds was confirmed
by routine chemical structure elucidation techniques, NMR
(1D), HRMS, and FTIR.
1
The H NMR spectra of all title compounds were thor-
oughly examined for characteristic signals, evidence of the
HQ and triazole scaffolds. The HQ moiety was clearly
1
identified in H spectra by the presence of two singlets at
9.7 and 9.2 ppm, attributed to quinolic protons H-4a and H-
1a, respectively. In addition, two doublets at 6.9 and
7.0 ppm attributed to the resonance of H-3 and H-6,
respectively confirm the presence of the quinolic ring. Fur-
ther, a doublet of doublet in the 6.9–6.7 ppm region,
attributed to the resonance of H-5 proton caused by its
coupling with H-6 (ortho-) and H-3 (meta-coupling), was
also present in the spectra of all hybrids. The triazole scaf-
fold was clearly identified by the singlet ca. 8.2 ppm, which
was assigned to the resonance of the triazolyl proton H-11.
13C NMR spectra further confirmed the quinol moiety in
the structure of the hybrids as evidenced by the presence of
six pronounced peaks in the 150–110 ppm range attributed
to the resonance of aryl ring of HQ. Further evidence of the
total conversion of BQ to HQ is the absence of carbonyl
carbons peaks in the spectra of the hybrids. The 13C spectra,
provided further support of the presence of the triazole with
two singlet peaks ca. 140 and 120 ppm pertaining to the
resonance of triazolyl carbons C-10 and C-11, respectively.
The FTIR spectra of all compounds were also inspected
for the presence of characteristic absorptions, allowing for
the identification of functional groups. The azido (N3) group
of intermediate 3 appeared as small and narrow stretch of
relatively low intensity to be peaked up in the
Biological activities
Before a novel drug can advance to the use in humans, a
series of preclinical studies (viz. a sequence of in vitro
assays followed by a series of in vivo assays) must be
completed (Franzblau et al. 2012). Mycobacterium growth
inhibitory potential of the hybrids was assessed using a
Green Fluorescent Protein-expression assay in a glucose-
based Middlebrook 7H9-CAS broth base as protein free
growth medium for tubercle bacilli.
The MIC of each hybrid that causes 90% inhibition of
H37Rv mycobacteria, expressed as MIC90, is shown in
Table 1, alongside RIF as antitubercular standard. In addi-
tion, HEK-293 cells were used to determine the cytotoxicity
of the compounds, alongside cytotoxic drug emetine as
reference (Table 1).

Medicinal Chemistry Research
Table 1 In vitro biological
activities of synthesized hybrids
and reference drugs
Compd. cLogPa Antimycobacterial activity, SI1 Antileishmanial Activity SI2 Cytotoxicity IC50
d
MIC90 (µM)b
IC50 SD (µM)c
SD (µM) HEK-293
1
0.20 >125
>100
>100
2
0.80 >125
2.76 >125
3.29 >125
3.82 >125
4.88 >125
3.00 >125
3.50 33.84
1.21 >125
−0.12 >125
1.89 >125
1.20 >125
3.03 16.38
3.71 0.075
>100
>100
4
>100
>100
5
18.28 0.33
9.21 0.44
>100
6
2
>100
6
17.5 2.1
71.0 7.7
77.6 7.2
41.9 7.9
>100
7
8
30.26 0.43
36.49 1.23
>100
3
1
9
1
6
10
11
12
13
14
RIF
AMB
EM
>100
>100
71.29 0.65
>100
1
4
>100
>100
22.68 0.74
>100
0.03 0.006
0.01 0.001
EM emetine, AMB amphotericin B
Experiments were run in triplicate and all reported data were significant at p < 0.05
acLogP values calculated using MarvinSketch Version 19.4
bCompounds screened in protein free medium 7H9 GLU CAS against H37Rv strain
cCompounds screened against L. major IR-175 strain
dHEK-293: human embryonic kidney cell line
eSelectivity index (SI), SI1 = IC50 HEK-293/MIC90 H37Rv, SI2 = IC50 HEK-293/MIC50 L. major
The antileishmanial activity of synthesized compounds
was evaluated mainly against L. major strain IR-175, which
communicates CL. The susceptibility of the compounds to
the promastigotes (infective stage) was assessed through
determination of the IC50 values. The half maximal inhibi-
tory concentration (IC50) is a measure of the potency of a
substance in inhibiting a specific biological or biochemical
function. IC50 is a quantitative measure that indicates how
much of a particular inhibitory substance (e.g., drug) is
needed to inhibit, in vitro, a given biological process or
biological component by 50%. In this study, antileishmanial
IC50 values determination was achieved using the resazurin
assay, also known as the AlamarBlue® assay, which
involves the irreversible enzymatic reduction of oxidized
blue resazurin dye to pink, highly fluorescent resorufin by
viable cells (Kulshrestha et al. 2013). The IC50 data of
synthesized hybrids are shown in Table 1, alongside that of
AMB as antileishmanial standard.
with MIC90 values greater than 125 µM. Only compounds 9
and 14 had MIC90 values below 125 µM, and thus were
considered active. Antimycobacterial active hybrid 9 had a
methyl group in para position of the aryl ring. Removal of
that group resulted in the completely loss of activity (8 IC50
> 125 µM). Hybrid 14, on the other hand, had phenylthio-
methyl moiety.
Furthermore, hybrids 10 and 13 had electron with-
drawing and donating group, respectively, and yet were
inactive.
The unique lipid rich (Glickman and Jacobs 2001;
Knechel 2009) cell wall structure of Mycobacterium is
crucial for survival of the pathogen (Knechel 2009). Thus,
lipophilicity is an imperative consideration in the design and
activity of novel antimycobacterial drugs (Suresh et al.
2014). The antimycobacterial activity can therefore be
enhanced by improving the lipophilic property of a com-
pound hence the selection of lipophilic moieties in this
study in order to facilitate diffusion of the compounds
through biomembranes.
The hybrids were screened were screened as solutions.
Antimycobacterial activity
All synthesized hybrids showed favorable drug-like
properties (except 11) having cLogP values in the targeted
range, and thus were expected to be efficiently transported
into the Mycobacterium through passive diffusion. How-
ever, none of them displayed significant activity against
None of the hybrids in the series possessed notable anti-
mycobacterial activity. All n-alkyl chain hybrids were
inactive and so were the precursor scaffolds, p-BQ and HQ

Medicinal Chemistry Research
Mtb. This suggests that lipophilicity did not modulate the
antimycobacterial activity of the hybrids, which is in
accordance with previous findings (Zhang et al. 2017)
BQ 1, HQ 2, and the hybrids (exception of 6–9) in
general showed no toxicity to the HEK-293 cells. Hybrids
6–9 displayed moderate to mild toxicity (10 < IC50 < 50 µM)
(Liu et al. 2017), which may be attributed to their relatively
higher lipophilicity with cLogP values, varying in the 3–5
range. In the n-alkyl chain substituted sub-series, the short
chain containing hybrids 4 (n = 4) and 5 (n = 5) were found
to be noncytotoxic. However, a further increase in chain
length resulted in a cytotoxicity decrease, with 6 (6C;
MIC50 17.5 μM) being more cytotoxic than 7 (8C; MIC50
71.0 μM). Hybrids 6 and 7 possessed moderate and mild
toxicities, respectively.
selective antiparasitic action (SI 2). The 5C side chain
hybrid 5, on the other hand, had twice lower activity (IC50
18.28 µM) and was noncytotoxic to HEK-293 cells (IC50
>
100 µM) but was found to be comparatively selective (SI 6)
toward the parasites. Neither of them stood as hit antil-
eishmanial, not only on account of cellular potency (IC50
<
10 µM) and selectivity index (SI < 10) but also on patho-
genesis consideration as L. major promastigote is herein
investigated instead of L. donavani amastigote as recom-
mended by Katsuno et al. (2015).
Conclusions
A small library of HQ-1,2,3-triazole hybrids was synthe-
sized in poor to moderate yield following a two-step process
that involves acid-catalyzed Michael and copper-catalyzed
azide-alkyne [3 + 2] cycloaddition. The anti-infective
properties namely antimycobacterial and antileishmanial
activities of the hybrids were evaluated in vitro against
human virulent Mtb H37Rv and L. major promastigotes,
respectively. The cytotoxicity of the compounds was also
assessed using HEK-293 cells. Though all hybrids showed
good drug-like properties, they were found to be mostly
inactive. The most active hybrid, 14 featuring phenylthio-
methyl moiety, although nontoxic to mammalian cells,
possessed moderate activities against bacteria (MIC90
16.38 μM) and parasite (IC50 22.68 μM) and poor patho-
genic selective action (SI 4–6). Thus, neither antitubercular
nor antileishmanial hit was discovered during this study.
A common structural characteristic of the hybrids of
this library is rigidity. Thus, more compounds will be
needed to determine if rigidity indeed governs the bio-
logical performance of these hybrids. Future work will
also focus on the investigation of flexible hybrids con-
taining the two pharmacophores in comparison with
rigid ones.
The most antimycobacterial active hybrid 14 (MIC90
16.38 μM) showed no toxicity to HEK-293 cells, but had a
poor selectivity toward the bacteria (SI = 6). All together,
these biological features disqualify this hybrid as a potential
antimycobacterial hit (Katsuno et al. 2015).
Antileishmanial activity
Leishmania spp. have two developmental forms, promasti-
gote and amastigote. The former is responsible for the
infective stage while the latter accounts for the clinical
stage, and is thus liable for the clinical manifestations of the
disease. In this study, a systematic approach, which consists
of evaluating the compounds in primary promastigote
screen then select potential hits for further intracellular
antiamastigote activity (intra-macrophage) screening was
adopted. The former screen targeting the free living and
easily cultured promastigote while the latter targets the
clinically relevant but more difficult to culture intra-
macrophage amastigote. Furthermore, promastigote screen,
although more suitable for automation, fails to identify all
active compounds and leads to numerous false positive hits
(De Muylder et al. 2011). Indeed, only 4% antipromastigote
hits are confirmed in antiamastigote (Siqueira-Neto et al.
2012) screening, which infers that an antiamastigote hit is a
potential antipromastigote hit while the opposite may not be
certain (De Muylder et al. 2011).
Acknowledgements The authors thank Dr D. Otto for NMR and Dr
JHL Jordaan for MS analyses. The following reagent was obtained
through BEI Resources, NIAID, NIH: Leishmania major, Strain IR-
173 (MHOM/IR/-173), NR-48816.
Half of the hybrids showed activity against the promas-
tigotes. Thus, the parasites were more susceptible to the
hybrids than were the bacteria. However, the observed
antiparasitic activity may not be fully intrinsic but possibly
also stemming from systemic toxicity for some of the
compounds. Indeed, those that were moderately to weakly
toxic (20 < IC50 < 80 µM) on the mammalian cell line (Liu
et al. 2017), had poor selectivity indexes ranging from 1 to
6. The best performer was hybrid 6, 2-(4-Hexyl-1H-1,2,3-
triazol-1-yl)benzene-1,4-diol, with IC50 9.21 µM, featured
6C side chain on the triazole ring, and displayed poorly
Funding This work was funded by South African National Research
Foundation Grant to DDN (UID 98937 and 115349) and the North-
West University. All TB screening work in the MMRU (UCT) is
supported by the South African Medical Research Council (SAMRC)
with funds from the South African National Treasury under its Eco-
nomic Competitiveness and Support Package through the Strategic
Health Innovation Partnerships (SHIP) initiative (to DFW).
Author contributions Conceptualization: DDN; methodology: [C-
MH, JA, FJS, RS, AJ, and DFW; formal analysis and investigation:
DDN, JA, and DFW; writing original draft preparation: C-MH; writ-
ing, reviewing and editing: DDN; funding acquisition: DDN and
DFW; resources: DDN and DFW; supervision: DDN.

Medicinal Chemistry Research
Compliance with ethical standards
Iranian cutaneous leishmaniasis due to drug-resistant Leishmania
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extract of Artemisia capillaris containing ursolic acid and
Conflict of interest The authors declare that they have no conflict of
interest.
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jurisdictional claims in published maps and institutional affiliations.
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