Synthesis of alkynylated 1,2,4-oxadiazole/1,2,3-1H-triazole glycoconjugates: Discovering new compounds for use in chemotherapy against lung carcinoma and Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2021.113472

A total of forty-three compounds were synthesized, including thirty-two new ones. Among those compounds, seventeen were selected and tested on human tumor cell lines: PC-3 (prostate adenocarcinoma), HCT-116 (colorectal tumor), NCIH-460 (lung carcinoma), S

Full text of DOI:10.1016/j.ejmech.2021.113472

European Journal of Medicinal Chemistry 220 (2021) 113472
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Synthesis of alkynylated 1,2,4-oxadiazole/1,2,3-1H-triazole
glycoconjugates: Discovering new compounds for use in
chemotherapy against lung carcinoma and Mycobacterium tuberculosis
b
a
ꢀ
Valentina Nascimento Melo de Oliveira , Cybele Flavia do Amaral Moura ,
Aline dos Santos Peixoto c, Vanessa Pinheiro Gonçalves Ferreira d
,
,
e
Heverton Mendes Araújo e, Lilian Maria Lapa Montenegro Pimentel c,
d,
ꢀ
d
d,
Claudia do O Pessoa , Roberto Nicolete e, Janaína Versiani dos Anjos b,
ꢀ
f
f
g
h
ꢀ
a, *
Prem Prakash Sharma , Brijesh Rathi , Lindomar Jose Pena , Patrick Rollin ,
Arnaud Tatibouet , Ronaldo Nascimento de Oliveira
h
€
a
~
Department of Chemistry, Federal Rural University of Pernambuco, Dois Irmaos, 52171-900, Recife, Brazil
b Department of Fundamental Chemistry, Federal University of Pernambuco, 50.740-540, Recife, Brazil
c
~
Department of Immunology, Aggeu Magalhaes Institute, Oswaldo Cruz Foundation, Brazil
d Laboratorio de Oncologia Experimental, Nucleo de Pesquisa Em Desenvolvimento de Medicamentos (NPDM), Universidade Federal Do Ceara, Fortaleza,
Brazil
e Oswaldo Cruz Foundation (Fiocruz), Eusebio, Brazil
f Laboratory for Translational Chemistry and Drug Discovery, Department of Chemistry, Hansraj College University Enclave, University of Delhi, Delhi, India
g Department of Virology, Oswaldo Cruz Foundation, Fiocruz, 50740-465, Recife, PE, Brazil
h Universite D’Orleans et CNRS, ICOA, UMR 7311, BP 6759, F-45067, Orleans, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A total of forty-three compounds were synthesized, including thirty-two new ones. Among those
compounds, seventeen were selected and tested on human tumor cell lines: PC-3 (prostate adenocar-
cinoma), HCT-116 (colorectal tumor), NCIH-460 (lung carcinoma), SKMEL-103 (melanoma) and AGP-01
(gastric tumor). Alkynylated 1,2,4-oxadiazoles 2m, 3g and 3k exhibited antiproliferative activities
against NCIH-460 in culture. Alkynylated N-cyclohexyl-1,2,4-oxadiazoles 3a-m and bis-heterocycle
glucoglycero-1,2,3-triazole-N-cyclohexyl-1,2,4-oxadiazole derivatives 5a-k and 6e11 were evaluated
for their in vitro efficacy towards Mycobacterium tuberculosis (Mtb) H37Ra and H37Rv strains. In general,
glycerosugars conjugated to 1,2,4-oxadiazole via a 1,2,3-triazole linkage (5a, 5e, 5j, 5k, and 7) showed
in vitro inhibitory activity against Mtb (H37Rv). The largest molecules bis-triazoles 10 and 11, proved
inactive against TB. Probably, the absence of the N-cyclohexyl group in compound 8 and 1,2,4-oxadiazole
nucleus in compound 9 were responsible for its low activity. Glucoglycero-triazole-oxadiazole de-
Received 21 January 2021
Received in revised form
8 April 2021
Accepted 9 April 2021
Available online 18 April 2021
Keywords:
Glycerosugar
1,2,3-Triazole
1,2,4-Oxadiazole
Antitubercular activity
Antiproliferative activity
MD simulations
rivatives 5e (10 mM) and 7 (23.9 mM) were the most promising antitubercular compounds, showing a
better selective index than when tested against RAW 264.7 and HepG2 cells. Vero cell were used to
investigate cytotoxicity of compounds 5a, 5h, 5j, 5k, and these compounds showed good cell viability.
Further, in silico studies were performed for most active compounds (5e and 7) with potential drug
targets, DprE1 and InhA of Mtb to understand possible interactions aided with molecular dynamic
simulation (100ns).
© 2021 Elsevier Masson SAS. All rights reserved.
* Corresponding author.
E-mail address: ronaldo.noliveira@ufrpe.br (R. Nascimento de Oliveira).
https://doi.org/10.1016/j.ejmech.2021.113472
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

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V.N. Melo de Oliveira, C. Flavia do Amaral Moura, A.S. Peixoto et al.
European Journal of Medicinal Chemistry 220 (2021) 113472
1. Introduction
spectrum biological activities [19,20].
The 1,2,4-oxadiazole [20] and 1,2,3-triazole [21] are important
small heterocycles used in the design of active molecules, notably
in antituberculosis studies (Fig. 1) [20e22]. Phatak et al. [23]
describe a series of new indanol-1,2,3-triazole derivatives as new
antitubercular agents. Most of these compounds have displayed
good antitubercular activity against M. tuberculosis H37Rv with MIC
Since the 1990s, studies in glycobiology have increased
considerably and aroused the interest of organic chemists for the
development of new synthetic methodologies aiming at the
conjugation between carbohydrates and compounds oaf biological
interest. When such compounds come from non-natural sources,
glycoconjugates are called neo-glycoconjugates [1e3]. This new
approach could not only provide data for studying some cellular
phenomena not yet fully elucidated, but also supply new bioactive
substances [4].
values ranging from 6.25 to ꢀ25
mM, except for one compound with
1.56 M. In addition, a molecular docking study demonstrated that
m
these compounds show affinity with the active site of Mycobacte-
rial enoyl-ACP-reductase (MtbInhA) enzyme.
According to the World Health Organization (WHO) “Tubercu-
losis (TB) is a major yet preventable global health problem, with an
estimated 10 million people developing TB and 1.5 million people
dying every year worldwide, making it the leading infectious dis-
ease cause of death worldwide” [5]. Although therapeutic schemes
are available for the treatment of tuberculosis, the emergence of
resistant strains due to resistance mechanisms has hampered
healing process and increased death cases [6]. These findings show
that the development of new anti-TB agents is imperative [7]. TB is
an infectious disease affecting the lungs that causes death espe-
cially in developing countries [8].
Cancer is the second leading cause of death globally accountable
for human casualties, 8.8 million in 2015 and 9.6 million in 2018 [9].
Lung carcinoma is the leading cause of death by cancer. It is an
aggressive tumor with reduced survival rate, showing a high
prevalence of bone metastasis [10]. Tuberculosis is one of the risk
factors for cancer; numerous studies have been conducted in this
thematic, which suggested a complex relationship between previ-
ous tuberculose infection and the lung cancer. However, data on the
treatment of TB in patients with lung cancer still needs further
investigation [11].
Recently, Thanh et al. disclosed a competitive inhibition of the
M. tuberculosis protein tyrosine phosphatase B (MtbPtpB) by a
pyrano-pyrimidine-(1H-1,2,3-triazole)
(IC50 ¼ 1.56 M) in vitro [22]. Analogously, M. Muthukrishnan et al.
used click chemistry to prepare a library of spirochromone glycero-
1,2,3-triazoles showing strong antimicrobial activity
(IC50 ¼ 0.78 g/mL against H37Rv strain) [24]. Following the same
D
-glucoconjugate
A
m
B
m
approach, Naidu et al. designed various derivatives combining
triazole/indole-piperazin-isoxazole which showed improved anti-
TB activity specifically compound C (MIC ¼ 6.16
37Rv strain and two wild strains isolated from TB patients [25].
The conjugation through formation of an oxadiazole was also per-
formed with 1,2,4-oxadiazole-piperazine-quinoline which
g/
mM) against Mtb
H
D
showed antitubercular potential against Mtb H37Rv (MIC ¼ 0.25
m
mL) [19]. In our group, we recently developed a methodology to
prepare closely related N-cyclohexyl-1,2,4-oxadiazole derivatives E
and F which showed antitumor activity against malignant prostatic
cells (PC3, IC50 ¼ 13.6
mM) and glioblastoma cancer (SNB19,
M) [26]. Moxifloxacin-acetyl-1,2,3-1H-triazole-meth-
IC50 ¼ 21.7
m
yleneisatin hybrids (G) were synthesized by Gao et al. as potential
anti-tubercular agents against drug-susceptible (MTB H37Rv) with
Against multidrug-resistant tuberculosis (MDR-TB), various
azole-type drugs are currently used, such as thioridazine, metro-
nidazole, doxycycline, tigecycline, co-trimoxazole, and a combina-
tion of two antimicrobial agents: trimethoprim (TMP) and
sulfamethoxazole (SMX) [12]. Despite some success of those drugs
against TB, novel chemical entities are still needed in order to
develop new alternative drugs [13,14] with improvred efficacy and
novel modes of action. In the same vein, new anticancer drugs are
developed to support this need based on the libraries of hetero-
cyclic derivatives [15].
MIC ranging from 0.12 to 16 mg/mL. Two compounds proved to be
more active against rifampicin-resistant and multidrug-resistant
M. tuberculosis strains [27].
Glycoconjugates are an important class of biomolecules and
useful scaffolds in drug discovery [28]. Carbohydrate-based het-
erocycles have been recently used as templates to generate a wide
range of bioactive molecules [29,30]. As building block, the carbo-
hydrate moiety allows the synthesis of sugar-based libraries for
pharmacological screening, but it also may act as a multifacet pro-
drug entity, in terms of solubility and site selectivity [30,31]. Having
these different aspects in mind, we have recently conjugated a
carbohydrate-glycerol moiety to access new 1,2,3-triazole gluco-
glycerolipid analogues [32].
In the search for anti-TB compounds based on azo-heterocycles,
Joshi et al. [16] reported the synthesis and antitubercular activity
(MIC 6.25e100
mg/mL) of pyrrolyl derivatives. They found that
some compounds indicate occupation into the hydrophobic pocket
of InhA enzyme, thus corroborating that some compounds
exhibited inhibition activities against InhA. Two lead compounds
displayed a significant activity (6.25 mg/mL) against M. tuberculosis
H37Rv strain.
Among nitrogen-based biocompounds, the 1,2,3-triazole moiety
is an important pharmacophore system which is frequently used as
a building block for the discovery of new biological targets [17]. This
heterocycle scaffold with three nitrogen heteroatoms can be pre-
pared by click chemistry, via copper(I)-catalyzed alkyne-azide
cycloaddition (Cu-AAC) [18]. In general, 1,2,3-triazole derivatives
are stable to hydrolysis under acidic or basic metabolic degradation
conditions; beyond that, triazoles are weakly acidic or basic. 1,2,3-
Triazoles can interact with different biological targets through
hydrogen bonding, non-covalent and van der Waals interactions, as
well as via dipole-dipole interactions.
In medicinal chemistry, hybrid molecules are an interesting
concept, where pharmacophore groups are connected in order to
produce a more biologically active molecule [33]. In this context,
the present work aims at extending our approach to prepare 1,2,3-
triazole/1,2,4-oxadiazole hybrid templates of new bioactive chem-
ical entities [19,31,32,34e36]. In this paper, we plan to synthesize
glucoglycero and 1,2,3-triazole moieties linked to 1,2,4-oxadiazole
via copper-catalyzed alkyne-azide cycloaddition (CuAAC) and
explore their biological activity against tumor cells and
M. tuberculosis (Scheme 1).
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of the alkynylated 1,2,4-oxadiazole derivatives
We have recently described an efficient method to prepare
efficiently 3,5-substituted 1,2,4-oxadiazoles 2a-c,e-h,j-k under
microwave irradiation (Scheme 2) [19]. Meta- and para-nitro-
benzamidoximes were used to prepare the corresponding
The synthesis of oxadiazole derivatives has been explored in
order to expand their biological applications, this heterocycle being
well known as a peptidomimetic moiety (amide bioisoster), besides
esters and carbamates, and this class of compound has a broad-
2

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V.N. Melo de Oliveira, C. Flavia do Amaral Moura, A.S. Peixoto et al.
European Journal of Medicinal Chemistry 220 (2021) 113472
Fig. 1. Recent anti-TB and anticancer compounds containing triazole or oxadiazole scaffolds.
Scheme 1. General strategy for the synthesis of glucoglycero-1,2,3-triazole/1,2,4-oxadiazole derivatives.
oxadiazoles 2d and 2i in reasonable yields, 63 and 76%, respectively.
N-alkylation at the amino group in 2a-m. Optimization of the re-
action was first explored on compound 2e (see Table S1). Following
a standard N-alkylation protocol, 2e was reacted with propargyl
bromide (2 equiv) and K2CO3 (1 equiv) in DMF at room tempera-
ture, but a maximum yield of 31% was reached even after 72 h.
Increasing the amounts of base and halide did not result in better
yields. Finally, using tert-BuOK (1 equiv) as the base with 2 equiv of
propargyl bromide made a decisive improvement after 2e4 h re-
action, the expected N-propargylated derivative 3e was isolated in
93e95% yields.
Library of heterocycles was extended involving ethynyl- (1l) and
propargyloxy- (1m) benzamidoximes to afford the alkynylated
1,2,4-oxadiazole derivatives 2l and 2m in satisfactory yields, 63%
and 70%, respectively. The oxadiazole 2m was obtained in a three-
step procedure starting from 4-hydroxybenzonitrile. Alkylation of
the phenol group using propargyl bromide/K2CO3 in DMF afforded
4-propargyloxybenzonitrile in 95% yield, which was efficiently
converted into the corresponding benzamidoxime in 87% yield
using hydroxylamine in 1:1 ethanol/water [37]. Reaction with
dicyclohexylcarbodiimide (DCC) was carried out in DMF under
microwave irradiation [19] to obtain the corresponding 5-
cyclohexylamino-1,2,4-oxadiazole 2m in 80% yield, and an overall
yield of 66% for the three steps. Compound 2n was obtained in 80%
yield after reflux in acetic anhydride for 2h.
With an optimized protocol in hands, we applied these condi-
tions to prepare
a library of N-propargyl-N-cyclohexyl-1,2,4-
oxadiazole derivatives. Conversion of 2a-m gave the correspond-
ing N-alkynylated 1,2,4-oxadiazoles 3a-m in yields ranging from 63
to 95% after chromatography column purification (Table 1). Among
the derivatives prepared, 3l and 3m were equipped with two
In a diversity-oriented approach, we considered at this stage the
3

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V.N. Melo de Oliveira, C. Flavia do Amaral Moura, A.S. Peixoto et al.
European Journal of Medicinal Chemistry 220 (2021) 113472
Scheme 2. Synthesis of 3-aryl-1,2,4-oxadiazole 2a-n.
ethynyl moieties, aiming at further synthesizing bis-triazole
compounds.
the diazirine cation stabilization by p-OCH3 (3k) and p-OCH2C^CH
(3m) substituents, which should favour cleavage to the m/z 164
fragment (Fig. S1).
The structures of synthesized compounds 2a-m and 3a-m were
assigned by ES-HRMS, and 1H and 13C NMR spectral analysis (see
SI). As representative sprectroscopy data, compound 3a displayed
large signals between 1.12 and 2.00 ppm (10H) due to the cyclo-
hexyl CH2 groups; the triplet of triplets at 3.98 ppm (1H, Jax-
2.1.2. Synthesis of glucoglycero-heterocycles based on 1,2,3-triazole
and 1,2,4-oxadiazole
The azidosugar 4 was prepared according to our published
protocol [32]. The 1,3-dipolar cycloaddition protocol was then
applied in reacting 4 with 2l, 2m and 3a-m to furnish a diversity of
glucoglycero-heterocyclic systems.
¼ 11.7 and Jax-eq ¼ 3.5 Hz) is assignable to the a
ax
-neighbouring cyclohexyl CH. The triplet at 2.27 ppm
(J ¼ 2.4 Hz) corresponding to the ethynyl H and the CH2 signal at
d
¼ 4.30 ppm (J ¼ 2.4 Hz) confirmed the N-alkynylated 1,2,4-
In
a first attempt, the reaction was carried out using
oxadiazole structure 3a. The 13C NMR spectrum showed oxadia-
zolic carbon signals (168.5 and 170.9 ppm) and the propargyl
moiety was characterized by signals at 30.5 ppm (CH2) and 71.9 and
79.7 ppm (alkynyl carbons).
CuSO4,5H2O/tert-BuOH/H2O and sodium ascorbate as catalytic
system to convert 3a into 5a; such conditions resulted in low yields
(35%), probably due to the low solubility of the reagents. However,
the yield was increased when tert-butanol was replaced by
dichloromethane, employing a 1:1 (v/v) biphasic mixture of
CH2Cl2:H2O. Under inert atmosphere, the reaction was completed
in 45 min at room temperature and compounds 5a and 5b were
obtained in 78% and 64% yield, respectively (Table 2). N-propargyl-
N-cyclohexyl-1,2,4-oxadiazoles 3c-k were similarly reacted with 4
to afford glucoglycero-heterocycles 5c-k in moderate yields
(47e64%) for meta-derivatives, and good-to-excellent yields
(85e90%) for para-substituted derivatives, while no electron-
withdrawing or donating effects was observed, see Table 2. The
structures of compounds 5a-k were assigned on the basis of their
1H and 13C NMR spectral analysis, and ESI-HRMS. In the 1H NMR
spectrum of compounds 5a-k, the signal of H-1 (anomeric
All the ESI-HRMS of the compounds were dominated by their
protonated species. Although the ESI technique was used for exact
mass determination, some important fragments could be detected.
Compounds 2l and 2m showed the expected MHþ peaks. Addi-
tionally, compound 2l showed MS fragments m/z 268 (base peak)
and m/z 143 (C9H7N2) due to the C5eN4 and N2eO cleavages of the
oxadiazole ring to form a diaziridine cation (Fig. S1). In general,
protonated forms of 2l or 2m undergo dissociation via pathway A.
Compound 2m showed MS fragments m/z 298 (MHþ) and m/z 173
(C10H9N2O, base peak); this last one was the major ion, likely
because the diazirine cation was stabilized by resonance-donating
effect of the p-OCH2C^CH substituent (Fig. S1).
On the other hand, the N-propargylated 1,2,4-oxadiazoles 3a-m
undergo dissociation via pathway B, by which the neutral diazirine
molecule is released, leading to the m/z 164 peak for all the de-
rivatives (Fig. S1). Compounds 3b, 3c, 3f and 3j showed heterolytic
cleavage of the NeC bond on the propargylic moiety to form the
ions m/z 258, 322, 278 and 258, respectively. In addition, for com-
pounds 3k and 3m, an increase of intensity was observed for the m/
z 164 peak. A possible explanation for this abundance is also due to
hydrogen) ranged from
vinylic protons H-2 and H-3 showed
d
¼ 4.54e4.99 ppm as a broad singlet. The
a
chemical shift
d
¼ 5.7e5.9 ppm, with J2,3 ¼ 9.6e10.6 Hz vicinal coupling. H-5
protons appeared at
d
¼ 4.07e4.11 as a multiplet ddd (J5,4; J5,6a
;
J5,6b
)
and remarkable trans diaxial relationship with H-4
-D-
(J5,4 ¼ 9.1e9.9 Hz). These data corroborate with 2,3-dideoxy-
a
erythro-hex-2-enopyranosidic structures. Aditionally, DEPT spec-
troscopy, COSY, and HSQC were used for complete assignment.
4

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European Journal of Medicinal Chemistry 220 (2021) 113472
Table 1
Synthesis of the N-propargyl-1,2,4-oxadiazole derivatives 3a-m.
5

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V.N. Melo de Oliveira, C. Flavia do Amaral Moura, A.S. Peixoto et al.
European Journal of Medicinal Chemistry 220 (2021) 113472
Table 2
Synthesis of glucoglycero-triazole-oxadiazole derivatives 5a-k.
Alkynylated 5-amino-1,2,4-oxadiazoles 2l and 2m were also
reacted with 4 to furnish the new triazole/oxadiazole derivatives 6
and 7 in 91 and 94% yield, respectively (Scheme 3). In the 1H NMR
To prepare differents glucoglycero derivatives, compounds 8
and 9 were synthesized with 69% and 87% of yields, respectively. In
the structure of compound 8 N-cyclohexyl group was replaced by
methyl group at C-5 carbon position of oxadiazole. Compound 9
represent a derivative without 1,2,4-oxadiazole moiety, and with a
long chain of ten-carbons at C-4’ position of the triazole.
spectrum, the NH protons appeared as
a broad doublet at
d
¼ 5.18e5.24 ppm with J ¼ 8.3 Hz. The 1H and 13C NMR, and 2D
NMR correlation spectroscopy were used for
assignment.
a complete
The azido-sugar 4 was condensed with the bis-alkynes 3l and
6

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V.N. Melo de Oliveira, C. Flavia do Amaral Moura, A.S. Peixoto et al.
European Journal of Medicinal Chemistry 220 (2021) 113472
Scheme 3. Synthesis of glucoglycero-triazole derivatives 6e9.
3m to afford in 62 and 67% yield, respectively, two sugar-derived
bis-triazoles 10 and 11 (Scheme 4). The 1H and 13C NMR sprec-
trum revealed the duplicated sugar moieties. In the case of 10 for
instance, anomeric protons appeared as two broad singlets at
We focused our attention on NCIH 460 (lung carcinoma) cells
because this cancer represents one of the major public health
problems, and there are reports linking lung cancer and tubercu-
losis infections [11,38]. Data are presented in Fig. 2 as % inhibition of
proliferation against NCIH-460, showing cell growth inhibition
d
¼ 4.98 and 5.05 ppm and anomeric carbons appeared at
d
¼ 94.6
and 94.7 ppm. The 1H and 13C NMR were used for a complete
assignment.
above 50% at single concentration (25
values in g/mL and M for compounds 2m, 3g and 3k, after 72 h of
treatment as determined by the MTT method. According to Fig. 2
and Table 4, compounds 2m (17.8 M), 3g (3.87 M) and 3k
(3.21 M) expressed the best inhibition of proliferation.
mM) and in Table 5 as IC50
m
m
We have thus synthesized a large library of 43 compounds, from
which a subfamily of glycoconjugates was developed and tested for
their activity against human tumor cell lines as well as for their
antitubercular potential.
m
m
m
Propargylamino derivatives such as pargyline and rasagiline are
known for their activity against human breast tumors [39] and
against Parkinson’s disease [40]. Pargyline is a monoamine oxidase
(MAO) inhibitor, which prevents the activity of lysine-specific
demethylase 1 (LSD1), an overexpressed enzyme in breast cancer.
Another mechanism of action may be related to substances con-
taining a 1,2,4-oxadiazole moiety, that have already been reported
as apoptosis inducers in cancer cell strains. Such compounds act by
activating the caspase pathway, leading to proteolytic degradation
and programmed cell death [41]. Although mechanistic studies
could not be performed so far, it is possible that compounds 3g and
3k might act in the same way.
2.2. Biological evaluation
2.2.1. In vitro antiproliferative activities
Seventeen compounds were selected and tested to verify their
cytotoxic potential (at 25 mM concentration) against five tumor cell
lines: PC-3 (prostate adenocarcinoma), HCT-116 (colorectal tumor),
NCIH 460 (lung carcinoma), SKMEL-103 (melanoma) and AGP-01
(gastric tumor). The compounds were shown to inhibit cell
growth, and we focused on those inhibiting above 50% cell prolif-
eration as being qualified for the IC50 determination assay (50%
inhibitory concentration of cell growth) (Table 3).
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V.N. Melo de Oliveira, C. Flavia do Amaral Moura, A.S. Peixoto et al.
European Journal of Medicinal Chemistry 220 (2021) 113472
Scheme 4. Synthesis of bis-1,2,3-triazoles 10 and 11.
bioactivity screening of compounds and predict potential effect
in vivo [47]. HepG2 cell lines is a well recognized model system,
frequently employed to evaluate liver cell function in vitro. The liver
is the main organ involved in metabolism, then HepG2 liver cell
lines are often used as tools to screen toxicity from test compounds
and may thus serve as a useful model [48].
Table 3
Percentage of inhibition of cellular proliferation of the compounds (25
determined by the MTT method.
mM) after 72 h
Compounds SKMEL-103 AGP-01
HCT-116
PC-3
NCIH-460
2d
2i
32.0 0.09 11.6 0.22 14.1 0.14 9.9 0.25 n.d.
50.7 0.15 53.2 0.03 n.d.
20.3 0.19 10.4 0.14
2l
40.1 0.28 44.4 0.58 60.7 0.01 46.0 0.08 34.0 0.40
62.8 0.50 2.6 0.25 43.3 0.49 64.2 0.42 75.7 0.55
Compounds 2l, 2m and 3a-m showed low activity against
M. tuberculosis (Table 5). However, some derivatives from series 5
and compound 7 produced good antimycobacterial activity against
TB (Mtb H37Rv). Compounds 5e and 7 induced particularly good
2m
3a
3b
3c
3d
3g
3i
3k
5b
5c
5d
5g
5i
28.6 0.10 47.0 0.14 n.d.
23.7 0.54 22.6 0.40
87.7 0.04 94.5 0.04 86.5 0.18 29.4 0.26 40.2 0.38
39.4 0.12 69.1 0.23 65.6 0.22 58.6 0.09 17.9 0.26
44.4 0.39 46.0 0.18 24.8 0.27 7.2 0.02 5.8 0.14
47.3 0.34 50.4 0.56 44.9 0.43 25.4 0.07 50.5 0.86
18.5 0.08 52.5 0.13 37.2 0.26 53.7 0.28 n.d.
effect with MIC ¼ 8
mg/mL (10 mM) and 16 mg/mL (23.9 mM),
respectively, and considering the selective index (Table 6). In gen-
eral, glycerosugars functionalized with 1,2,3-triazole/1,2,4-
oxadiazole moieties were active (5a, 5e, 5j, 5k, and 7); in
contrast, larger molecules such as 10 and 11 proved to be unactive
against TB. These results agree with the importance of finding small
molecules at a lower cost to encourage the development of drugs
for the treatment of tuberculosis [49].
37.3 0.42 57.3 0.35 40.7 0.14 23.7 0.54 69.1 0.12
69.0 0.26 38.9 0.25 69.5 0.04 9.2 0.05 26.1 0.60
52.3 0.05 59.9 0.04 n.d.
45.5 0.43 27.1 0.15
42.2 0.07 44.7 0.12 27.7 0.09 38.4 0.27 n.d.
20.7 0.12 69.4 0.22 44.9 0.43 7.1 0.11 17.7 0.48
30.6 0.06 47.6 0.22 39.1 0.10 28.8 0.08 36.3 0.21
29.8 0.24 59.2 0.37 24.2 0.01 21.6 0.20 24.2 0.24
5k
Compounds 8 and 9 showed low activities, perhaps this may
indicate that the presence of 1,2,4-oxadiazole nucleus and N-
cyclohexyl group were an important fact to increase the anti-TB
activity of glucoglycero-triazole-N-cyclohexyl-oxadiazole de-
rivatives and should be considered in future designs of new
chemical entities with anti-TB potential.
In general, the lipophilicity of a molecule impacts on the ca-
pacity of transmembrane diffusion of a drug, and in case of action
against tuberculosis it can inhibit lipid biosynthesis. To understand
if there are lipophilic effects from the tested compounds, logP was
calculated (see Table 5). We observed, despite moderate-to-low
activity against Mtb, that the series of compounds 3a-m was
active when logP was above 4.5, except for compound 3j; and
negligible activity when logP <4.5. On the other hand, from series 5
no indication of correlation with logP was found.
*The best results are highlighted in bold; n.d. ¼ not determined.
2.2.2. In vitro antimycobacterial activity
In continuation, all new compounds were screened against
M. tuberculosis H37Rv (ATCC 27294) and M. tuberculosis H37Ra
(ATCC 25177) strains. The minimum inhibitory concentration (MIC)
was determined (mg/mL), rifampicin and ethambutol being used as
standard compounds. The MIC results of the synthesized com-
pounds along with reference drugs are reported in Table 5.
Cytotoxicity was initially evaluated against two different
mammalian cell lines (Raw 264.7 and HepG2) by MTT assay [42,43].
In in vitro screening studies of compounds against Mycobacterium
tuberculosis, it is common to use different cell lines, such as Raw
264.7 (ATCC TIB - 71) murine macrophages cells and HepG2 cells, as
demonstrated in different scientific studies [44e46]. Murine
macrophage Raw 264.7 cell line is normaly used for initial
Despite inhibitory activities against Mtb H37Rv observed in
series 5, some of the active compounds presented toxicity in Raw
8

ꢀ
V.N. Melo de Oliveira, C. Flavia do Amaral Moura, A.S. Peixoto et al.
European Journal of Medicinal Chemistry 220 (2021) 113472
Fig. 2. Percentage of inhibition of proliferation of the compounds against NCIH-460 (0.1x106 cells/mL) after 72 h.
Table 4
IC50 values in mg/mL and mM for compounds 2m, 3g and 3k against NCIH-460.
Compounds
Structure
2 m
3g
3k
doxorubicine
IC50
IC50
(
(
m
m
g/mL)
M)
5.30
17.82 1.8
1.16
3.87 0.40
1.00
3.21 0.70
0.08
0.15 0.02
iii) Compound 7 was more potent than compound 6, which may
which may suggest that structure 7 is influenced by confor-
mational factors that can potentiate anti-TB activity;
iv) Compound 8 shown to be inactive; probably caused by the
absence of the N-cyclohexyl group and too low lipophilicity
(logP ¼ 2.34);
264.7 and HepG2 cells. In order to learn more about their cyto-
toxicity, CC50 M) of selected compounds was evaluated for Vero
cells, which are derived from African green monkey kidney. Com-
pounds 5a (606.8 M), 5h (706.3 M), 5j (498.3 M), and 5k
(575.0 M) were shown to have low cytotoxicity in Vero cells. The
(m
m
m
m
m
selective index (7.9e24.7) can be seen in Table S2.
v) Compound 9, glucoglycerol-triazole derivative, was no active
for TB; this can indicate that 1,2,4-oxadiazole moiety is
important for the activity anti-TB;
In recent years, a few articles have made significant strides to-
wards the synthesis of new anti-TB compounds [16,17,21e27,50];
however, there remain important gaps in research on the discovery
of a new chemical entity against a new/old drug target.
vi) Large molecules such as 10 and 11 proved to be inactive
against TB.
2.3. Structure activity relationship (SAR) for anti-TB compounds
From the structure of 1,2,4-oxadiazoles structural modification
were made. First, N-alkylation made available alkynylated 1,2,4-
oxadiazole derivatives with no activity or moderate. Some SAR
observations from anti-TB data can be deduced (Fig. 3).
2.4. Molecular docking
Two proteins mycobacterial Enoyl-[acyl-carrier-protein] reduc-
tase, InhA (PDB ID: 2NSD) and Decaprenylphosphoryl-beta-D-
ribose oxidase, DprE1 (PDB ID: 5OEQ) have recently attracted
enough attention for being potential target for growth inhibition of
the pathogens. Due to the availability of crystal structure for these
two proteins, several reports have demonstrated the potential of
the triazole scaffold to inhibit InhA and DprE1 enzymes [51,52] and
1,2,4-oxadiazole inhibits InhA enzyme [53]. Anti-tubercular activity
displayed by compounds 5e and 7 inspired us to perform in-silico
studies to identify their binding affinity towards InhA and DprE1
protein targets of M. tuberculosis. Both proteins were analyzed for
its stereo-chemical geometry by procheck Ramachandran plot
where DprE1 and InhA has 0% and 0.9% outlier residues, respec-
tively (Table 6: entries 1 and 2; Fig. S2).
i) In the serie 3 compounds with logP >4.5 (3b,c,e,f,h) have
moderate activity anti-TB, except for 3j; and compounds
with logP < 4.5 (3a,d,g,i,k,l,m) were no effective against Mtb.
In general, when we look at Table 5, the data suggest that
these compounds were more potent against TB, when their
logP ranges from 3.9 to 4.9 (exception 2m), but not higher or
lower. On the other hand, logP in this range does not guar-
antee an attractive anti-TB activity, e.g. 5g-i.
ii) Small molecules containing the 1,2,4-oxadiazole scaffold
linked to a triazole-glycerosugar possess anti-TB activity,
various derivatives 5a, 5e, 5j and the compound 7 showing
antimycobacterial activity against Mtb H37Rv;
Molecular docking analysis revealed that compound 5e interacts
9

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V.N. Melo de Oliveira, C. Flavia do Amaral Moura, A.S. Peixoto et al.
European Journal of Medicinal Chemistry 220 (2021) 113472
Table 5
In vitro antimycobacterial activities of the target compounds 2e11 expressed as MICs (
m
g/mL).
g/mL)
Compd
Mol. Wt
logPa
Strain (MIC,
H37Ra
m
g/mL)
H37Rv
CC50
(
m
SI (CC50/MIC)
RAW
HepG2
(CC50Raw/MIC-H37Rv)
(CC50HepG2/MIC-H37Rv)
264.7
2l
2m
4
267.32
297.15
371.34
281.35
295.38
360.25
326.35
360.25
315.80
299.34
349.35
326.35
295.38
311.38
305.37
335.40
652.69
666.72
697.69
731.59
687.14
670.68
720.69
697.69
666.72
682.72
638.67
668.69
585.57
537.65
1048.06
1078.08
822.94
204.31
3.91
3.46
1.52
4.10
4.52
4.88
4.03
4.91
4.78
4.26
4.99
4.06
4.55
4.16
3.86
4.32
3.99
4.42
3.93
4.80
4.67
4.16
4.89
3.95
4.44
4.05
3.78
4.00
2.34
5.24
e
32
32
32
32
ꢀ64
ꢀ64
32
19.82
38.28
106.3
32.13
25.89
31.77
34.18
15.67
15.12
20.92
38.32
26.74
17.57
27.96
13.11
100.7
28.61
46.38
100.4
39.11
35.54
31.48
64.11
80.22
45.32
86.32
16.29
33.28
111.8
30.37
116.70
>200
80.39
>400
54.20
118.70
>200
108.60
44.38
53.45
45.48
101.80
41.26
41.08
61.89
43.73
91.93
72.54
27.95
>200
19.91
39.20
>200
47.63
46.91
28.80
81.40
>200
36.78
141.20
79.84
99.46
37.67
59.05
11.79
47.02
175.90
>400
0.62
1.29
1.66
0.50
0.80
0.90
0.53
0.48
0.95
0.32
1.20
0.41
0.27
0.46
0.20
1.57
1.78
0.72
1.56
4.89
0.55
0.49
1.00
1.25
2.83
2.70
0.25
2.08
1.74
0.47
1.82
3.12
643.12
400
1.69
3.70
3.12
1.69
1.38
1.67
0.71
3.18
2.58
0.64
1.93
0.68
1.43
1.13
0.43
3.12
1.24
0.61
3.12
5.95
0.73
0.45
1.67
3.12
2.30
4.41
1.24
6.21
0.58
0.92
0.18
0.73
1407.20
400
ꢀ64
ꢀ64
ꢀ64
ꢀ64
ꢀ64
32
32
64
64
3a
3b
3c
3d
3e
3f
3g
3h
3i
32
ꢀ64
32
16
64
32
ꢀ64
ꢀ64
64
ꢀ64
3j
3k
3l
64
ꢀ64
nd
ꢀ64
3m
5a
5b
5d
5e
5f
5g
5h
5i
5j
5k
6
7
8
ꢀ64
64
16
16
ꢀ64
ꢀ64
4
ꢀ64
ꢀ64
8
64
64
ꢀ64
ꢀ64
ꢀ64
8
ꢀ64
ꢀ64
ꢀ64
16
16
32
ꢀ64
ꢀ64
16
16
ꢀ64
64
ꢀ64
ꢀ64
0.062
1
ꢀ64
64
ꢀ64
ꢀ64
0.125
1
9
10
11
RIF
EMB
e
2.67
0.35
The best results are highlighted in bold; RIF: Rifampicin; EMB: Ethambutol.
a
The logP was calculated using the Molinspiration molecular property (http://www.molinspiration.com). The SMILES notation was generated in ACD/ChemSketch (version
12.0) and input in molinspiration program.
Table 6
Number of amino acid residues and their percentage in favored, additional allowed, additional generously allowed, and outlier region of Ramachandran plot for both DprE1 and
InhA proteins in complex with compounds 5e and 7.
Entry
Protein complexes
Most favored region
Additional allowed region
Generously allowed region
Disallowed region
1
2
3
4
5
6
DprE1
InhA
DprE1-5e complex
DprE1-7 complex
InhA-5e complex
InhA-7 complex
325 (91%)
32 (9%)
0 (0.0%)
1 (0.4%)
4 (1.1%)
5 (1.4%)
1 (0.4%)
1 (0.4%)
0 (0.0%)
2 (0.9%)
1 (0.3%)
1 (0.3%)
0 (0.0%)
2 (0.9%)
209 (92.9%)
292 (81.8%)
295 (82.6%)
195 (86.7%)
192 (85.3%)
13 (5.8%)
60 (16.8%)
56 (15.7%)
29 (12.9%)
30 (13.3%)
with binding site residues of DprE1 protein (Dscore, ꢁ5.205 kcal/
mol;
and pi-pi interaction (His_132). In case of InhA-5e complex, com-
pound 5e showed interactions with binding site residues of InhA
complexes displayed good structural geometry without any sig-
nificant outlier residues (Figs. S2cef; Table 6: entries 3e6).
D
G⁰ ꢁ99.55 kcal/mol) via H-bond (Gly_55, Gly_117, Lys_418)
During simulation, compound 5e had average RMSD of 3.02 Å
and 1.44 Å in complexed with InhA and DprE1 proteins, respec-
tively (Figs. S3a and S3b). On other side, compound 7 complexed
with InhA and DprE1 revealed the average RMSD of 2.05 Å and
2.09 Å, respectively (Figs. S3c and S3d). These compounds showed
fluctuations in the acceptable range (3 Å). The histogram plot
depicted in Fig. S4, showed the nature of interaction between
ligand and protein during MD simulation where ligand interacted
to the residues by H-bond (green), salt bridge (pink), water bridge
(blue), and hydrophobic interaction (grey). For InhA-5e complex,
benzyl moiety-maintained pi-pi interaction with Phe_41 for at least
39% of the simulation time (Fig. S4a). The oxygen atom of acetic
acids-maintained H-bond with Ala_22 and Ile_194 by 98% and 73%
protein (Dscore, ꢁ2.094;
D
G⁰ ꢁ108.47 kcal/mol) via H-bond
(Ser_20, Ala_22) as depicted in Fig. 4a and b (Table 7: entries 1 and
2). Likewise, compound 7 interacts with binding site residue of
DprE1 protein (Dscore, ꢁ1.763 kcal/mol;
D
G⁰ ꢁ123.82 kcal/mol)
through H-bonds (Ser_59, Try_60, Gly_117, Lys_418, Tyr_415) and
pi-pi interaction (Tyr_415). Compound 7 showed interactions with
the binding site residue of InhA protein (Dscore, ꢁ3.464 kcal/mol;
D
G⁰ ꢁ87.30 kcal/mol) through H-bond (Ser_20, Met_98) as shown
in Fig. 4c and d (Table 7: entries 3 and 4).
Next, MD simulations were performed to evaluate the stability
of these complexes in he binding site. Post simulations, all the four
10

ꢀ
V.N. Melo de Oliveira, C. Flavia do Amaral Moura, A.S. Peixoto et al.
European Journal of Medicinal Chemistry 220 (2021) 113472
Fig. 3. Structure-activity relationship for anti-TB compounds.
of the simulation time, respectively. For DprE1-5e complex, 1,2,3-
triazole maintained H-bond and pi-pi interaction to the residues
Gly_117 and His_132 by 37% and 34%, respectively (Fig. S4b). The
oxygen atom of acetic acid and nitrogen atoms of N-cyclohexyl-
1,2,4-oxadiazol displayed various interactions as shown in Figs. S4c
and d. Both the compunds 5e and 7 showed interactions with
several common residues in complex with both InhA and DprE1
proteins. While MD simulation process, compound 5e and 7 were
always bounded with 8e10 and 10e12 contacts with binding site
residues of InhA protein (Figs. S5a and S5b). Similarly, compound 5e
and 7 were always bounded with 5e7 and 10e12 contacts with
binding site residues of DprE1 protein during MD simulations
(Figs. S5c and S5d).
anti-TB activity. The largest molecules bis-triazoles 10 and 11,
proved inactive against TB. In contrast, glucoglycero-triazole-
oxadiazoles 5e (10
mM) and 7 (23.9 mM) were the most active
with the best selectivity index. In addition, molecular docking
study for the most active compounds (5e and 7) against
M. tuberculosis was established for Mtb DprE1 and InhA enzymes.
MD simulation carried out at 100 ns supported that both the
compounds 5e and 7 complexed with proteins DprE1 and InhA
have stable interactions. Also, RMSD plots indicated both the
compounds showed fluctuations in the acceptable range, suggest-
ing their stability within the binding pocket of both the proteins.
We thus have found promising hits for the development of new
lead compounds against NCIH-460 and tuberculosis. Further
development of this new family of compounds is ongoing and will
be published in due course.
3. Conclusion
We have developed protocols to prepare a library of thirty new
compounds applying CuAAC and N-alkylation. Among the various
molecules, N-propargyl-1,2,4-oxadiazoles 3g and 3k exhibited the
best antiproliferative activities in vitro against NCIH-460 (lung
carcinoma). Glycerosugars conjugated to 1,2,4-oxadiazole via a
1,2,3-triazole linkage (5a, 5e, 5j and 7) showed in vitro inhibitory
4. Experimental section
4.1. Chemistry
Melting points were determined in a capillary tube and per-
formed with a PFM II BioSan instrument. HRMS analyses were
performed on a Micromass LC-ESI-TOF at the ICOA (Universite
ꢀ
activity on the level IC50 < 24.5
mM against M. tuberculosis (Mtb
ꢀ
H37Rv). In general, the data suggest that these compounds were
more potent against TB, when their logP ranges from 3.9 to 4.9. On
the other hand, logP in this range does not guarantee an attractive
d’Orleans, France). NMR spectra were obtained with Varian Unity
Plus-300 or 400 spectrometers using CDCl3 as solvent and cali-
brated for the solvent signal. Optical rotations were measured using
11

ꢀ
V.N. Melo de Oliveira, C. Flavia do Amaral Moura, A.S. Peixoto et al.
European Journal of Medicinal Chemistry 220 (2021) 113472
process [37]; this arylamidoxime was used as starting material to
prepare the oxadiazole 2m [19].
4.1.1. Synthesis of 3-aryl-5-cyclohexylamino-1,2,4-oxadiazole
derivatives (2a-n)
The literature protocol was applied [19].
4.1.1.1. N-Cyclohexyl-3-(4-ethynylphenyl)-1,2,4-oxadiazol-5-amine
(2l). Yield 63% (168 mg), colorless solid; mp 131e133 ꢂC. Rf 0.7
(Hexane-EtOAc, 7:3). 1H NMR (300 MHz)
d 1.15e1.50; 1.61e1.81;
2.07e2.12 (m, 10H, cyclohexyl), 3.18 (s, 1H, CH), 3.66e3.72 (m, 1H,
CH cyclohexyl), 5.28 (d, 1H, J ¼ 8.2 Hz, NH), 7.55e7.58 (m, 2H, HAr),
7.95e7.98 (m, 2H, HAr). 13C NMR (75 MHz)
d 24.6, 25.3, 33.2, 52.9,
78.9, 83.2, 124.4, 127.1, 127.9, 132.3, 167.8, 170.6. ESI-HRMS calcd for
C
16H17N3O [MþH]þ: 268.1445; found: 268.1444.
4.1.1.2. N-Cyclohexyl-3-[4-(prop-2-yn-1-yloxy)phenyl]-1,2,4-
oxadiazol-5-amine (2m). Yield 70% (208 mg), colorless solid; mp
119e121 ꢂC; Rf 0.6 (Hexane-EtOAc, 7:3). 1H NMR (300 MHz)
d
1.19e1.49; 1.61e1.68; 1.73e1.8; 2.05e2.12 (m, 10H, cyclohexyl),
2.54 (t, 1H, J ¼ 2.3 Hz, CH), 3.65e3.73 (m, 1H, CH cyclohexyl), 4.74
(d, 2H, J ¼ 2.3 Hz, CH2), 5.26 (d, 1H, J ¼ 8.2 Hz, NH), 7.01e7.05 (m,
2H, HAr), 7.92e7.97 (m, 2H, HAr). 13C NMR (75 MHz)
d 24.6, 25.3,
33.2, 52.8, 55.8, 75.8, 78.1, 115.0, 121.1, 128.7, 159.4, 168.0, 170.4. ESI-
HRMS calcd for C17H20N3O2 [MþH]þ: 298.1555; found: 298.1549.
4.1.1.3. Synthesis of 5-methyl-3-[4-(prop-2-yn-1-yloxy)phenyl]-
1,2,4-oxadiazole (2n). In a 25 mL flask, 190 mg (1 mmol) of aryl-
amidoxime and 153 mg (1.5 mmol) of acetic anhydride were
added. The mixture was kept under magnetic stirring, at 140 ꢂC, for
2 h. At the end of the reaction, an acid-base extraction was carried
out, using a saturated solution of NaHCO3 and ethyl acetate. The
product was obtained after crystallization from methanol-water.
Yield 90% (117.5 mg); colorless crystal; mp 53e55 ꢂC; Rf ¼ 0.8
(Hexano/EtOAc, 1:1). 1H NMR (300 MHz):
d
2.55 (t, 1H, J ¼ 2.4 Hz,
C^CH), 2.63 (s, 3H, CH3),4.75 (d, 2H, J ¼ 2.1 Hz, CH2), 7.04e7.09 (m,
2H, HAr), 8.00e8.05 (m, 2H, HAr). 13C NMR (75 MHz):
12.4 (CH3),
d
55.8 (CH2OAr), 75.9 (C^C), 78.0 (C^C), 115.2 (2CAr), 120.2 (CAr),
128.9 (2CAr), 159.7 (CAr), 168.0 (C-oxad), 176.3 (C-oxad).
4.1.2. Synthesis of 3-aryl-N-cyclohexyl-N-(prop-2-yn-1-yl)-1,2,4-
oxadiazol-5-amines (3a-m)
Oxadiazoles 2a-m (0.5 mmol) were dissolved in dry DMF
(0.5 mL), followed by addition of potassium tert-butoxide (1 equiv).
Then, propargyl bromide (2 equiv) was added and the mixture was
stirred at room temperature for 2 h. The reaction mixture was
filtered, washed with dichloromethane-water and the solvents
were evaporated under reduced pressure. The products were pu-
rified by column chromatography using hexane and dichloro-
methane (2: 8).
Fig. 4. 3D and 2D interaction poses of compounds to the binding site residue of target
proteins [InhA (PDB ID: 2NSD); (PDB ID: 5OEQ)]. A) DprE1-5e; B) InhA-5e; C) DprE1-7;
and D) InhA-7.
Table 7
Docking score and DG⁰ (kcal/mol) for molecules 5e and 7.
Entry
Protein
Ligand
D Score (kcal/mol)
DG⁰ (kcal/mol)
1
2
3
4
DprE1
InhA
DprE1
InhA
5e
5e
7
ꢁ5.205
ꢁ2.094
ꢁ1.763
ꢁ3.464
ꢁ99.55
ꢁ108.47
ꢁ123.82
ꢁ87.30
4.1.2.1. N-Cyclohexyl-3-phenyl-N-(prop-2-yn-1-yl)-1,2,4-oxadiazol-
5-amine (3a). Yield 87% (122 mg), colorless solid; mp 112e115 ꢂC;
Rf 0.4 (hexane-CH2Cl2, 1:1). 1H NMR (300 MHz)
d 1.11e1.26,
7
1.34e1.50, 1.65e1.78, 1.87e2.01 (m, 10H, cyclohexyl), 2.27 (t, 1H,
J ¼ 2.4 Hz, CH), 3.98 (dd, 1H, Jax-ax ¼ 11.7 and Jax-eq ¼ 3.5 Hz, NeCH),
4.30 (d, 2H, J ¼ 2.4 Hz, CH2), 7.42e7.48 (m, 3H, HAr), 8.01e8.04 (m,
a Anton Paar Model MCP200 polarimeter (Na-
D
-line 589 nm) with a
2H, HAr). 13C NMR (75 MHz)
d 25.3, 25.7 (2C), 30.5 (2C), 34.5, 59.0,
path length of 1 dm. Purifications were performed by column
chromatography on Merck silica gel 60 (70e230 mesh). The ary-
lamidoximes 1a-m were either prepared according to the con-
ventional protocol or by using ultrasound energy [54]. 4-(Prop-2-
yn-1-yloxy)benzonitrile [RN 33143-80-5] was prepared by con-
ventional alkylation. N0-Hydroxy-4-(prop-2-yn-1-yloxy)benzimi-
damide [RN 2241224-25-7] was synthesized according to literature
71.9, 79.7, 127.3 (2C), 127.8, 128.5 (2C), 130.6, 168.5, 170.9. ESI-HRMS
calcd for C14H19N3O [M ꢁ C3H]þ: 244.1414; found: 244.1414.
4.1.2.2. N-Cyclohexyl-3-(3-methylphenyl)-N-(prop-2-yn-1-yl)-1,2,4-
oxadiazol-5-amine (3b). Yield 70% (104 mg), colorless oil; Rf 0.8
(Hexane-CH2Cl2, 2:8). 1H NMR (300 MHz)
d
1.11e1.26, 1.35e1.50,
1.65e1.78, 1.86e2,01 (m, 10H, cyclohexyl), 2.27 (t, 1H, J ¼ 2.3 Hz,
12

ꢀ
V.N. Melo de Oliveira, C. Flavia do Amaral Moura, A.S. Peixoto et al.
European Journal of Medicinal Chemistry 220 (2021) 113472
CH), 2.42 (s, 3H, CH3), 3.99 (dddd, 1H, Jax-ax ¼ 11.8 Hz and Jax-
4.1.2.8. N-Cyclohexyl-N-(prop-2-yn-1-yl)-3-[4-(trifluoromethyl)
phenyl]-1,2,4-oxadiazol-5-amine (3h). Yield 82% (144 mg), colorless
solid, mp 53e56 ꢂC; Rf 0.81 (Hexane-CH2Cl2, 2:8). 1H NMR
¼ 3.5 Hz, NeCH), 4.30 (d, 2H, J ¼ 2.3 Hz, CH2), 7.26 (d, 1H,
eq
J ¼ 7.6 Hz, Hp-oxadiazole), 7.33 (dd, 1H, J ¼ 7.6 and 7.0 Hz, Hm-oxadiazole),
7.82 (m, 2H, Ho-oxadiazole). 13C NMR (75 MHz)
d
21.3, 25.3, 25.7 (2C),
(300 MHz): d 1.12e1.26; 1.35e1.50; 1.63e1.79; 1.88e2.00 (m, 10H,
30.5 (2C), 34.4, 59.0, 71.9, 79.7, 124.4,127.6,127.8, 128.4,131.4,138.2,
168.6, 170.8. ESI-HRMS calcd for C18H22N3O [MþH]þ: 296.1763;
found: 296.1758.
cyclohexyl), 2.28 (t, 1H, J ¼ 2.4 Hz, CH), 3.99 (dddd, 1H, Jax-
¼ 11.7 Hz and Jax-eq ¼ 3.5 Hz, NeCH), 4.30 (d, 2H, J ¼ 2.4 Hz, CH2),
ax
6.70 (d, 2H, J ¼ 8.2 Hz, HAr), 8.15 (d, 2H, J ¼ 8.2 Hz, HAr). 13C NMR
(75 MHz): d 25.3, 25.7, 30.5, 34.6, 59.2, 72.1, 79.4, 125.5, 125.5, 127.6,
129.3, 131.3, 132.1, 132.5, 167.4, 171.1. ESI-HRMS calcd for
C
4.1.2.3. 3-(3-Bromophenyl)-N-cyclohexyl-N-(prop-2-yn-1-yl)-1,2,4-
18H19F3N3O [MþH]þ: 350.1480; found: 350.1477.
oxadiazol-5-amine (3c). Yield 68% (123 mg), colorless oil; Rf 0.4
(Hexane-CH2Cl2, 1:1). 1H NMR (300 MHz)
d 1.11e1.26, 1.35e1.50,
4.1.2.9. N-Cyclohexyl-3-(4-nitrophenyl)-N-(prop-2-yn-1-yl)-1,2,4-
oxadiazol-5-amine (3i). Yield 93% (152 mg), colorless solid, mp
104e105 ꢂC, Rf ¼ 0.4 (Hexane-CH2Cl2, 1:1). 1H NMR (300 MHz):
1.64e1.78, 1.87e1.99 (m, 10H, cyclohexyl), 2.27 (t, 1H, J ¼ 2.3 Hz,
CH), 3.98 (dddd, 1H, Jax-ax ¼ 11.8 Hz and Jax-eq ¼ 3.5 Hz, NeCH), 4.28
(d, 2H, J ¼ 2.4 Hz, CH2), 7.31 (dd, 1H, J ¼ 8.2 and 7.7 Hz, Hm-oxadiazole),
7.58 (m, 1H, H p-oxadiazole), 7.95 (dd, 1H, J ¼ 8.8 and 1.2 Hz, Ho-
d
1.13e1.27; 1.35e1.49; 1.66e1.79; 1.88e2.00 (m, 10H, cyclohexyl),
2.29 (t, 1H, J ¼ 2.3 Hz, CH), 3.98 (dddd, 1H, Jax-ax ¼ 11.7 Hz and Jax-
oxadiazole), 8.18 (s, 1H, Ho-oxadiazole). 13C NMR (75 MHz):
d 25.3, 25.7
¼ 3.5 Hz, NeCH), 4.30 (d, 2H, J ¼ 2.4 Hz, CH2), 8.20 (m, 2H, HAr),
eq
(2C), 30.5 (2C), 34.5, 59.1, 72.0, 79.5, 122.6, 125.8, 129.8, 130.1, 130.3,
133.6, 167.4, 171,0. ESI-HRMS calcd for C17H19BrN3O [MþH]þ:
360.0711; found: 360.0701.
8.29 (m, 2H, HAr). 13C NMR (75 MHz):
d 25.2, 25.7, 30.5, 34.6, 59.3,
72.2, 79.3, 123.8, 128.2, 133.9, 149.1, 166.9, 171.2. ESI-HRMS calcd for
C
17H19N4O3 [MþH]þ: 327.1457; found: 327.1451.
4.1.2.4. N-Cyclohexyl-3-(3-nitrophenyl)-N-(prop-2-yn-1-yl)-1,2,4-
4.1.2.10. N-Cyclohexyl-3-(4-methylphenyl)-N-(prop-2-yn-1-yl)-
oxadiazol-5-amine (3d). Yield 63% (102 mg), colorless oil, Rf ¼ 0.4
1,2,4-oxadiazol-5-amine (3j). Yield 86% (127 mg), colorless oil, Rf
(Hexane-CH2Cl2, 1:1). 1H NMR (300 MHz):
d 1.12e1.27, 1.36e1.51,
0.6 (Hexane-CH2Cl2 2:8). 1H NMR (300 MHz):
d 1.11e1.26;
1.66e1.79, 1.89e2.01 (m, 10H, cyclohexyl), 2.29 (t, 1H, J ¼ 2.4 Hz,
CH), 4.00 (dddd, 1H, Jax-ax ¼ 11.7 Hz and Jax-eq ¼ 3.5 Hz, NeCH), 4.30
(d, 2H, J ¼ 2.9 Hz, CH2), 7.63 (dd,1H, J ¼ 8.2 and 7.6 Hz, Hm-oxadiazole),
8.30e8.37 (m, 2H, Ho-oxadiazole and Hp-oxadiazole), 8.87 (dd, 1H, J ¼ 2.3
1.34e1.49; 1.64e1.78; 1.87e2.00 (m, 10H, cyclohexyl), 2.26 (t, 1H,
J ¼ 2.3 Hz, CH), 2.40 (s, 3H, CH3), 3.98 (dddd, 1H, Jax-ax ¼ 11.8 Hz and
Jax-eq ¼ 3.6 Hz, NeCH), 4.29 (d, 2H, J ¼ 2,.3 Hz, CH2), 7.24 (d, 2H,
J ¼ 8.2 Hz, HAr), 7.91 (d, 2H, J ¼ 8.2 Hz, HAr). 13C NMR (75 MHz):
and 1.8 Hz, Ho-oxadiazole). 13C NMR (75 MHz):
d 25.2, 25.7 (2C), 30.5
d
21.5, 25.3, 25.7, 29.7, 30.5, 34.4, 59.0, 71.9, 97.3, 125.0, 127.2, 129.2,
170.8. ESI-HRMS calcd for C18H22N3O [MþH]þ: 296.1763; found:
(2C), 34.5, 59.2, 72.1, 110.0, 122.4, 125.2, 129.6, 129.7, 132.9, 148.4,
166.8, 171.2. ESI-HRMS calcd for C17H19N4O3 [MþH]þ: 327.1457;
found: 327.1450.
296.1758.
4.1.2.11. N-Cyclohexyl-3-(4-methoxyphenyl)-N-(prop-2-yn-1-yl)-
1,2,4-oxadiazol-5-amine (3k). Yield 87% (140 mg), colorless solid,
mp 88e89 ꢂC; Rf 0.3 (Hexane-CH2Cl2, 1:1). 1H (300 MHz):
4.1.2.5. 3-(4-Bromophenyl)-N-cyclohexyl-N-(prop-2-yn-1-yl)-1,2,4-
oxadiazol-5-amine (3e). Yield 95% (171 mg), colorless solid; mp
72e75 ꢂC, Rf 0.92 (Hexane-CH2Cl2, 1:9). 1H NMR (300 MHz):
d
1.12e1.26; 1.34e1.49; 1.64e1.78; 1.87e1.99 (m, 10H, cyclohexyl),
2.26 (t, 3H, J ¼ 2.3 Hz, CH), 3.86 (s, 3H, CH3), 3.97 (dddd, 1H, Jax-
d
1.12e1.26; 1.35e1.49; 1.65e1.78; 1.87e2.00 (m, 10H, cyclohexyl),
¼ 11.7 Hz and Jax-eq ¼ 3.5 Hz, NeCH), 4.28 (d, 2H, J ¼ 2.4 Hz, CH2)
ax
2.27 (t, 1H, J ¼ 2.3 Hz, CH), 3.97 (dddd, 1H, Jax-ax ¼ 12.3 Hz and Jax-
6.96 (m, 2H, HAr), 7.96 (m, 2H, HAr). 13C NMR (75 MHz):
d
25.3, 25.8,
¼ 3.5 Hz, NeCH), 4.28 (d, 2H, J ¼ 2.4 Hz, CH2), 7.58 (m, 2H, HAr),
eq
7.90 (m, 2H, HAr). 13C NMR (75 MHz):
d 25.3, 25.7, 30.5, 34.5, 59.1,
30.5, 34.4, 55.3, 59.0, 71.8, 79.8, 113.9, 120.3, 128.8, 161.5, 168.2,
170.7. ESI-HRMS calcd for C18H22N3O2 [MþH]þ: 312.1712; found:
312.1705.
72.0, 79.5, 125.1, 126.8, 128.8, 131.8, 167.8, 171.0. ESI-HRMS calcd for
C
17H19BrN3O [MþH]þ: 360.0711; found: 360.0708.
4.1.2.12. N-cyclohexyl-3-(4-ethynylphenyl)-N-(prop-2-yn-1-yl)-
1,2,4-oxadiazol-5-amine (3l). Yield 89% (136 mg), colorless solid,
mp 87e89 ꢂC; Rf 0.45 (Hexane-CH2Cl2, 1:1). 1H NMR (300 MHz):
4.1.2.6. 3-(4-Chlorophenyl)-N-cyclohexyl-N-(prop-2-yn-1-yl)-1,2,4-
oxadiazol-5-amine (3f). Yield 90% (142 mg), colorless solid, mp
62e64 ꢂC; Rf 0.68 (Hexane-CH2Cl2, 2:8). 1H NMR (300 MHz):
d
1.11e1.26; 1.34e1.49; 1.64e1.77; 1.87e1.99 (m, 10H, cyclohexyl),
d
1.11e1.26; 1.34e1.49; 1.65e1.78; 1.87e2.00 (m, 10H, cyclohexyl),
2.27 (t, 2H, J ¼ 2.4 Hz, CH), 3.18 (dddd, 1H, Jax-ax ¼ 11.7 Hz and Jax-
2.27 (t, 1H, J ¼ 2.4 Hz, CH), 3.97 (dddd, Jax-ax ¼ 11.7 Hz and Jax-
¼ 3.5 Hz, NeCH), 4.28 (d, 2H, J ¼ 2.4 Hz, CH2), 7.56 (d, 2H,
eq
¼ 3.5 Hz, NeCH), 4.28 (d, 2H, J ¼ 2.4 Hz, CH2), 7.41 (m, 2H, HAr),
J ¼ 8.2 Hz, HAr), 7.99 (d, 2H, J ¼ 8.2 Hz, HAr). 13C NMR (75 MHz):
eq
7.96 (m, 2H, HAr). 13C NMR (75 MHz):
d 25.3, 25.8, 30.5, 34.5, 59.1,
d
25.2, 25.7, 30.5, 34.5, 59.1, 72.0, 78.8, 79.5, 83.2, 127.1, 128.1, 132.2,
72.0, 79.6, 128.6, 128.8, 136.7, 167.7, 171.0. ESI-HRMS calcd for
167.8, 170.9. ESI-HRMS calcd for C19H20N3O [MþH]þ: 306.1606;
C
17H19ClN3O [MþH]þ: 316.1217; found: 316.1215.
found: 306.1598.
4.1.2.7. N-Cyclohexyl-3-(4-fluorophenyl)-N-(prop-2-yn-1-yl)-1,2,4-
oxadiazol-5-amine (3g). Yield 83% (124 mg), colorless oil, Rf 0.4
4.1.2.13. N-Cyclohexyl-N-(prop-2-yn-1-yl)-3-[4-(prop-2-yn-1-yloxy)
phenyl]-1,2,4-oxadiazol-5-amine (3m). Yield 80% (134 mg), color-
less solid, mp 82e85 ꢂC; Rf 0.6 (Hexane-CH2Cl2, 2:8). 1H NMR
(Hexane-CH2Cl2, 1:1). 1H NMR (300 MHz):
d 1.11e1.26; 1.34e1.49;
1.64e1.78; 1.87e2.00 (m, 10H, cyclohexyl), 2.27 (t, 1H, J ¼ 2.3 Hz,
CH), 3.97 (dddd, 1H, Jax-ax ¼ 12.3 Hz and Jax-eq ¼ 3.5 Hz, NeCH), 4.28
(d, 2H, J ¼ 2.4 Hz, CH2), 7.12 (m, 2H, HAr), 8.02 (m, 2H, HAr). 13C NMR
(300 MHz): d 1.12e1.26, 1.35e1.48, 1.64e1.78, 1.87e1.99 (m, 10H,
cyclohexyl), 2.26 (t, 1H, J ¼ 2.3 Hz, CH), 2.54 (t, 1H, J ¼ 2.4 Hz, CH),
3.97 (dddd, 1H, Jax-ax ¼ 11.7 Hz and Jax-eq ¼ 3.5 Hz, NeCH), 4.28 (d,
2H, J ¼ 2.4 Hz, CH2), 4.74 (d, 2H, J ¼ 2.4 Hz, CH2), 7.03 (d, 2H,
J ¼ 8.8 Hz, HAr), 7.98 (d, 2H, J ¼ 8.8 Hz, HAr). 13C NMR (75 MHz):
(75 MHz): d 25.3, 25.7, 30.5, 34.5, 59.1, 71.9, 79.6, 115.4e115.7 (d, 2C,
JC-F ¼ 21.9 Hz, CoeAr), 124.02e124.07 (d, 1C, JC-F ¼ 3.5 Hz, Cp-Ar),
129.3e129.4 (d, 2C, JC-F ¼ 9.3 Hz, CmeAr), 165.92e167.95 (d, 1C, JC-
d
25.3, 25.8 (2C), 30.5 (2C), 34.5, 55.8, 59.0, 71.9, 75.8, 78.1, 79.7,
¼ 250.9 Hz, Cipso-Ar), 167.6, 170.9. ESI-HRMS calcd for C17H19FN3O
114.8 (2C), 121.3, 128.8 (3C), 159.4, 168.1. ESI-HRMS calcd for
F
[MþH]þ: 300.1512; found: 300.1507.
C
20H22N3O2 [MþH]þ: 336.1712; found: 336.1704.
13

ꢀ
V.N. Melo de Oliveira, C. Flavia do Amaral Moura, A.S. Peixoto et al.
European Journal of Medicinal Chemistry 220 (2021) 113472
4.1.3. Synthesis of 1,2,4-oxadiazole-1,2,3-triazole-glycerosugars
(5a-k)
3H, CH3), 2.09 (s, 3H, CH3), 3.54 (dd, 1H, J ¼ 10.9 and 4.7 Hz, H-3’a),
3.83 (dd, 1H, J ¼ 10.9 and 5.6 Hz, H-3’b), 3.97e4.03 (m, 1H, NeCH,
cyclohexyl), 4.08 (ddd, 1H, J ¼ 9.6, 5.2 and 2.3 Hz, H-5), 4.15 (dd, 1H,
J ¼ 12.4 and 2.3 Hz, H-6a), 4.26 (dd, 1H, J ¼ 12.4 and 5.2 Hz, H-6b),
4.59 (dd, 1H, J ¼ 14.3 and 6.4 Hz, H-1’a), 4.64 (dd, 1H, J ¼ 14.4 and
4.4 Hz, H-1’b), 4.77 (s, 2H, CH2eN), 4.99 (br s, 1H, H-1), 5.28e5.32
(m, 2H, H-20 and H-4), 5.78 (ddd, 1H, J ¼ 10.0, 2.6 and 2.1 Hz, H-2),
5.90 (d, 1H, J ¼ 10.3 Hz, H-3), 7.32 (dd, 1H, J ¼ 8.0 and 7.9 Hz, HAr),
7.60 (ddd, 1H, J ¼ 11.2, 2.0 and 0.8 Hz, HAr), 7.68 (s, 1H, H triaz), 7.94
(app dt, 1H, J ¼ 7.9, 1.2 and 1.2 Hz, HAr), 8.16 (dd, 1H, J ¼ 2.0 and
CuSO4$5H2O (0.09 mmol), sodium ascorbate (0.135 mmol) and
alkyne (3a-k) (0.3 mmol) were mixed in water (1.5 mL) in a Schlenk
flask under argon. This mixture was added into another Schlenk
flask containing 0.2 mmol of the azido-carbohydrate 4 (74.2 mg)
dissolved in 1.5 mL of dichloromethane. The mixture was stirred at
room temperature for 45 min. Then, the solvent was removed, and
the residue was purified by column chromatography using hexane-
ethyl acetate, 1:1.
1.5 Hz, HAr). 13C NMR (75 MHz):
d 20.6 (CH3CO), 20.7 (CH3CO), 20.9
4.1.3.1. N-Cyclohexyl-N-[1-(10-O-(4,6-di-O-acetyl-2,3-dideoxy-
erythro-hex-2-enopyranosyl)-(20-acetyl-30-deoxy-sn-glyceryl-1H-
1,2,3-triazol-4-yl)methyl]-3-phenyl-1,2,4-oxadiazol-5-amine (5a).
¼ þ 30 (c 0.2, CH2Cl2); Rf 0.3
a
-D-
(CH3CO), 25.1 (cyclohexyl), 25.7 (2C, cyclohexyl), 30.8 (2C, cyclo-
hexyl), 40.4 (N-Ccyclohexyl), 50.1 (C-10), 59.4 (CH2N-Ccyclohexyl), 62.8
(C-6), 65.1 (C-4), 66.3 (C-30), 67.1 (C-5), 70.4 (C-20), 94.6 (C-1), 122.6
(C5’triaz), 124.0, 125.6, 126.8 (C-2), 129.7 (CAr), 129.8 (C-3), 130.2
(2CAr), 133.7 (CAr), 144.9 (C4’triaz), 167.3 (Coxad), 169.6 (Coxad), 170.2
(OAc), 170.7 (OAc), 171.2 (OAc). ESI-HRMS calcd for C32H40BrN6O9
[MþH]þ: 731.2040; found: 731.2033.
25
Yield 78% (102 mg), colorless oil, [
(Hexane-EtOAc, 1:1).
a]
D
1H NMR (300 MHz):
d 1.16e1.44, 1.64e1.95 (m, 10H, cyclohexyl),
1.94 (s, 3H, CH3), 2.07 (s, 3H, CH3), 2.08 (s, 3H, CH3), 3.53 (dd, 1H,
J ¼ 10.6 and 4.7 Hz, H-3’a), 3.82 (dd, 1H, J ¼ 10.5 and 5.8 Hz, H-3’b),
3.96e4.02 (m, 1H, NeCH, cyclohexyl), 4.07 (ddd, 1H, J ¼ 9.4, 5.3 and
2.4 Hz, H-5), 4.14 (dd, 1H, J ¼ 12.3 and 2.4 Hz, H-6a), 4.26 (dd, 1H,
J ¼ 12.3 and 5.3 Hz, H-6b), 4.58 (dd, 1H, J ¼ 14.1 and 6.5 Hz, H-1’a),
4.64 (dd, 1H, J ¼ 14.0 and 4.1 Hz, H-1’b), 4.78 (s, 2H, CH2eN), 4.98
(br s, 1H H-1), 5.27e5.31 (m, 2H, H-20 and H-4), 5.78 (app dt, J ¼ 9.8,
3.0 and 3.0 Hz, H-2), 5.89 (br d, 1H, J ¼ 9.7 Hz, H-2), 7.42e7.48 (m,
3H, HAr), 7.71 (s, 1H, H-5’triaz), 8.00e8.03 (m, 2H, HAr). 13C NMR
4.1.3.4. N-Cyclohexyl-N-[1-(10-O-(4,6-di-O-acetyl-2,3-dideoxy-
a-D-
erythro-hex-2-enopyranosyl)-(20-acetyl-30-deoxy-sn-glyceryl-1H-
1,2,3-triazol-4-yl)methyl]-3-(3-nitrophenyl)-1,2,4-oxadiazol-5-amine
25
(5d). Yield 57% (79 mg), colorless oil, [
0.5 (Hexane-EtOAc, 1:1).
a
]
¼ þ 7.5 (c 0.2, CH2Cl2); Rf
D
1H NMR (300 MHz):
d 1.17e1.46, 1.66e1.89 (m, 10H, cyclohexyl),
1.98 (s, 3H, CH3), 2.07 (s, 3H, CH3), 2.09 (s, 3H, CH3), 3.56 (dd, 1H,
J ¼ 11.2 and 4.7 Hz, H-3’a), 3.85 (dd, 1H, J ¼ 11.2 and 5.9 Hz, H-3’b),
3.99e4.03 (m, 1H, CHeN), 4.07 (ddd, 1H, J ¼ 12.3, 5.3 and 2.9 Hz, H-
5), 4.15 (dd, 1H, J ¼ 12.3 and 2.9 Hz, H-6a), 4.25 (dd, 1H, J ¼ 12.4 and
5.3 Hz, H-6b), 4.59 (dd, 1H, J ¼ 14.7 and 7.1 Hz, H-1’a), 4.65 (dd, 1H,
J ¼ 14.7 and 4.7 Hz, H-1’b), 4.79 (s, 2H, CH2eN), 4.99 (br s, 1H, H-1),
5.27e5.32 (m, 2H, H-20 and H-4), 5.79 (app dt, 1H, J ¼ 10.0, 2.3 and
2.3 Hz, H-2), 5.90 (br d, 1H, J ¼ 9.9 Hz, H-3), 7.65 (dd, 1H, J ¼ 7.9 and
8.2 Hz, HAr), 7.69 (s, 1H, Htriaz), 8.31e8.36 (m, 2H, HAr), 8.85e8.86
(75 MHz):
d 20.6 (CH3CO), 20.7 (CH3CO), 20.9 (CH3CO), 25.1
(cyclohexyl), 25.7 (2C, cyclohexyl), 30.8 (2C, cyclohexyl), 40.4 (N-
C
cyclohexyl), 50.1 (C-10), 59.3 (CH2N-Ccyclohexyl), 62.8 (C-6), 65.1 (C-4),
66.3 (C-30), 67.1 (C-5), 70.4 (C-20), 94.6 (C-1), 124.1 (C5’triaz), 126.8
(C-2), 127.1 (2CAr), 127.8 (CAr), 128.6 (2C, CAr), 129.7 (C-3), 130.7
(CAr), 145.0 (C4’triaz), 168.4 (Coxad), 169.6 (Coxad), 170.2 (OAc), 170.7
(OAc), 171.1 (OAc). ESI-HRMS calcd for
653.2935; found: 653.2932.
C
32H41N6O9 [MþH]þ:
(m, 1H, HAr). 13C NMR (75 MHz):
d 20.6 (CH3CO), 20.7 (CH3CO), 20.9
4.1.3.2. N-Cyclohexyl-N-[1-(10-O-(4,6-di-O-acetyl-2,3-dideoxy-
a
-D-
(CH3CO), 25.1 (cyclohexyl), 25.7 (2C, cyclohexyl), 30.8 (2C, cyclo-
hexyl), 40.5 (N-Ccyclohexyl), 50.1 (C-10), 59.6 (CH2N-Ccyclohexyl), 62.8
(C-6), 65.1 (C-4), 66.4 (C-30), 67.2 (C-5), 70.4 (C-20), 94.6 (C-1), 122.3
(C5’triaz), 123.9, 125.2, 126.8 (C-2), 129.7 (CAr), 129.8 (C-3), 132.8
(CAr), 144.6 (CAr), 148.5 (C4’triaz), 166.8 (Coxad), 169.6 (Coxad), 170.2
(OAc), 170.7 (OAc), 171.4 (OAc). ESI-HRMS calcd for C32H40N7O11
[MþH]þ: 698.2786; found: 698.2783.
erythro-hex-2-enopyranosyl)-(20-acetyl-30-deoxy-sn-glyceryl-1H-
1,2,3-triazol-4-yl)methyl]-3-(3-methylphenyl)-1,2,4-oxadiazol-5-
25
amine (5b). Yield 64% (85 mg), colorless oil, [
a
]
¼ þ 42 (c 0.2,
D
CH2Cl2); Rf 0.5 (Hexane-EtOAc, 1:1). 1H NMR (300 MHz):
1.15e1.40, 1.66e1.87 (m, 10H, cyclohexyl),1.94 (s, 3H, CH3), 2.07 (s,
d
3H, CH3), 2.08 (s, 3H, CH3), 2,41 (s, 3H, CH3), 3.53 (dd, 1H, J ¼ 10.6
and 4.7 Hz, H-3’a), 3.83 (dd, 1H, J ¼ 10.6 and 5.3 Hz, H-3’b),
3.95e4.01 (m, 1H, NeCH, cyclohexyl), 4.07 (ddd, 1H, J ¼ 9.1, 5.3 and
2.4 Hz, H-5), 4.14 (dd, 1H, J ¼ 12.4 and 2.4 Hz, H-6a), 4.26 (dd, 1H,
J ¼ 12.3 and 5.3 Hz, H-6b), 4.57 (dd, 1H, J ¼ 14.4 and 7.1 Hz, H-1’a),
4.64 (dd, 1H, J ¼ 14.3 and 4.7 Hz, H-1’b), 4.77 (s, 2H, CH2eN), 4.97
(br s, 1H, H-1), 5.27e5.32 (m, 2H, H-4 and H-20), 5.78 (app dt, 1H,
J ¼ 10.2, 2.4 and 2.4 Hz, H-2), 5.89 (br d, 1H, J ¼ 10.4 Hz, H-3),
7.30e7.32 (m, 1H, HAr), 7.33e7.36 (m, 1H, HAr), 7.70 (s, 1H, Htriaz),
4.1.3.5. N-Cyclohexyl-N-[1-(10-O-(4,6-di-O-acetyl-2,3-dideoxy-
a-D-
erythro-hex-2-enopyranosyl)-(20-acetyl-30-deoxy-sn-glyceryl-1H-
1,2,3-triazol-4-yl)methyl]-3-(4-bromophenyl)-1,2,4-oxadiazol-5-
amine (5e). Yield 90% (132 mg), colorless solid, mp 72e75 ꢂC,
25
[
a
]
¼ þ 25 (c 0.1, CH2Cl2); Rf 0.4 (Hexane-EtOAc, 1:1). 1H NMR
D
(300 MHz):
d 1.15e1.43, 1.66e1.87 (m, 10H, cyclohexyl), 1.95 (s, 3H,
CH3), 2.08 (s, 3H, CH3), 2.09 (s, 3H, CH3), 3.54 (dd, 1H, J ¼ 10.6 and
4.7 Hz, H-3’a), 3.83 (dd, 1H, J ¼ 10.5 and 5.3 Hz, H-3’b), 3.91e4.03
(m, 1H, NeCH, cyclohexyl), 4.08 (ddd, 1H, J ¼ 9.4, 5.3 and 2.3 Hz, H-
5), 4.15 (dd, 1H, J ¼ 12.4 and 2.4 Hz, H-6a), 4.26 (dd, 1H, J ¼ 12.3 and
5.3 Hz, H-6b), 4.57 (dd, 1H, J ¼ 14.1 and 6.4 Hz, H-1’a), 4.64 (dd, 1H,
J ¼ 14.1 and 4.1 Hz, H-1’b), 4.76 (s, 2H, CH2eN), 4.98 (br s, 1H, H-1),
5.25e5.32 (m, 2H, H-4 and H-20), 5.78 (app dt, 1H, J ¼ 9.9, 2.3 and
2.3 Hz, H-2), 5.90 (d, 1H, J ¼ 10.0 Hz, H-3), 7.54e7.60 (m, 2H, HAr),
7.79e7.82 (m, 2H, HAr). 13C NMR (75 MHz):
d 20.6 (CH3CO), 20.7
(CH3CO), 20.9 (CH3CO), 21.3 (CH3), 25.1 (cyclohexyl), 25.7 (2C,
cyclohexyl), 30.8 (2C, cyclohexyl), 40.4 (N-Ccyclohexyl), 50.0 (C-10),
59.2 (CH2N-Ccyclohexyl), 62.8 (C-6), 65.1 (C-4), 66.3 (C-30), 67.1 (C-5),
70.4 (C-20), 94.6 (C-1), 124.1 (C5’triaz), 124.6, 126.8 (C-2), 127.6 (CAr),
128.5 (CAr), 129.7 (C-3), 138.3 (CAr), 145.1 (C4’triaz), 168.6 (Coxad),
169.6 (Coxad), 170.1 (OAc), 170.7 (OAc), 171.1 (OAc). ESI-HRMS calcd
for C33H43N6O9 [MþH]þ: 667.3091; found: 667.3083.
7.76 (s, 1H, Htriaz), 7.87e7.91 (m, 2H, HAr). 13C NMR (75 MHz):
d 20.6
(CH3CO), 20.7 (CH3CO), 20.9 (CH3CO), 25.1 (cyclohexyl), 25.7 (2C,
cyclohexyl), 30.8 (2C, cyclohexyl), 40.4 (N-Ccyclohexyl), 50.0 (C-10),
59.4 (CH2N-Ccyclohexyl), 62.8 (C-6), 65.1 (C-4), 66.3 (C-30), 67.1 (C-5),
70.4 (C-20), 94.6 (C-1), 124.0 (C5’triaz), 125.2, 126.8 (C-2), 128.7
(2CAr), 129.8 (C-3), 131.8 (CAr), 144.9 (C4’triaz), 167.7 (Coxad), 169.6
(Coxad), 170.2 (OAc), 170.7 (OAc), 171.2 (OAc). ESI-HRMS calcd for
4.1.3.3. N-Cyclohexyl-N-[1-(10-O-(4,6-di-O-acetyl-2,3-dideoxy-
a-D-
erythro-hex-2-enopyranosyl)-(20-acetyl-30-deoxy-sn-glyceryl-1H-
1,2,3-triazol-4-yl)methyl]-3-(3-bromophenyl)-1,2,4-oxadiazol-5-
25
amine (5c). Yield 47% (147 mg), colorless oil, [
a
]
¼ þ 10 (c 0.2,
D
CH2Cl2); Rf 0.5 (Hexane-EtOAc, 1:1). 1H NMR (300 MHz):
1.16e1.43, 1.65e1.87 (m, 10H, cyclohexyl),1.96 (s, 3H, CH3), 2.08 (s,
d
C
32H40BrN6O9 [MþH]þ: 731.2040; found: 731.2037.
14

ꢀ
V.N. Melo de Oliveira, C. Flavia do Amaral Moura, A.S. Peixoto et al.
European Journal of Medicinal Chemistry 220 (2021) 113472
4.1.3.6. N-Cyclohexyl-N-[1-(10-O-(4,6-di-O-acetyl-2,3-dideoxy-
a
-D-
(cyclohexyl), 25.7 (2C, cyclohexyl), 30.8 (2C, cyclohexyl), 40.5 (N-
erythro-hex-2-enopyranosyl)-(20-acetyl-30-deoxy-sn-glyceryl-1H-
C
cyclohexyl), 50.0 (C-10), 59.5 (CH2N-Ccyclohexyl), 62.8 (C-6), 65.1 (C-4),
1,2,3-triazol-4-yl)methyl]-3-(4-chlorophenyl)-1,2,4-oxadiazol-5-
66.4 (C-30), 67.1 (C-5), 70.4 (C-20), 94.6 (C-1), 122.0 (C5’triaz), 123.9,
125.5 (q, 1C, JC-F ¼ 4.6 Hz, CF3), 126.8 (C-2), 127.5 (2CAr), 129.8 (C-3),
131.3, 132.7, 144.8 (C4’triaz), 167.4 (Coxad), 169.6 (Coxad), 170.1 (OAc),
170.7 (OAc),171.3 (OAc). ESI-HRMS calcd for C33H40F3N6O9 [MþH]þ:
721.2809; found: 721.2799.
25
amine (5f). Yield 89% (122 mg), colorless oil, [
a
]
¼ þ 10 (c 0.1,
D
CH2Cl2); Rf 0.4 (Hexane-EtOAc, 1:1). 1H NMR (300 MHz):
1.16e1.44, 1.67e1.87 (m, 10H, cyclohexyl),1.95 (s, 3H, CH3), 2.08 (s,
d
3H, CH3), 2.09 (s, 3H, CH3), 3.54 (dd, 1H, J ¼ 11.2 and 4.7 Hz, H-3’a),
3.83 (dd, 1H, J ¼ 11.1 and 5.3 Hz, H-3’b), 3.96e4.03 (m, 1H, NeCH,
cyclohexyl), 4.07 (ddd, 1H, J ¼ 9.4, 5.3 and 1.7 Hz, H-5), 4.15 (dd,
1H, J ¼ 12.3 and 1.8 Hz, H-6a), 4.26 (dd, 1H, J ¼ 12.3 and 5.3 Hz, H-
6b), 4.58 (dd, 1H, J ¼ 14.3 and 6.4 Hz, H-1’a), 4.64 (dd, 1H, J ¼ 14.5
and 4.7 Hz, H-1’b), 4.77 (s, 2H, CH2eN), 4.99 (br s, 1H, H-1),
5.26e5.32 (m, 2H, H-4 and H-20), 5.79 (app dt, 1H, J ¼ 10.6, 1.7 and
1.7 Hz, H-2), 5.90 (br d, 1H, J ¼ 10.6 Hz, H-3), 7.42 (d, 2H, J ¼ 8.8 Hz,
4.1.3.9. N-Cyclohexyl-N-[1-(10-O-(4,6-di-O-acetyl-2,3-dideoxy-
a-D-
erythro-hex-2-enopyranosyl)-(20-acetyl-30-deoxy-sn-glyceryl-1H-
1,2,3-triazol-4-yl)methyl]-3-(4-nitrophenyl)-1,2,4-oxadiazol-5-amine
25
(5i). Yield 90% (125 mg), colorless oil, [
a]
¼ þ 10 (c 0.1, CH2Cl2); Rf
D
0.3 (Hexane-EtOAc, 1:1). 1H NMR (300 MHz):
d 1.15e1.45, 1.66e1.88
(m, 10H, cyclohexyl), 1.97 (s, 3H, CH3), 2.07 (s, 3H, CH3), 2.08 (s, 3H,
CH3), 3.56 (dd, 1H, J ¼ 10.9 and 4.7 Hz, H-3’a), 3.84 (dd, 1H, J ¼ 11.0
and 5.3 Hz, H-3’b), 3.97e4.03 (m, 1H, NeCH, cyclohexyl), 4.07 (ddd,
1H, J ¼ 11.7, 5.1 and 2.3 Hz, H-5), 4.15 (dd, 1H, J ¼ 12.1 and 2.3 Hz, H-
6a), 4.25 (dd, 1H, J ¼ 12.0 and 5.3 Hz, H-6b), 4.58 (dd, 1H, J ¼ 14.4
and 7.0 Hz, H-1’a), 4.65 (dd, 1H, J ¼ 14.3 and 4.1 Hz, H-1’b), 4.78 (s,
2H, CH2eN), 4.99 (br s, 1H, H-1), 5.26e5.33 (m, 2H, H-4 and H-20),
5.79 (app dt, 1H, J ¼ 10.0, 2.3 and 2.3 Hz, H-2), 5.90 (d, 1H,
J ¼ 10.0 Hz, H-3), 7.68 (s, 1H, Htriaz), 8.17e8.21 (m, 2H, HAr),
H
Ar), 7.69 (s, 1H, Htriaz), 7.95 (d, 2H, J ¼ 8.8 Hz, HAr). 13C NMR
(75 MHz):
d 20.6 (CH3CO), 20.7 (CH3CO), 20.9 (CH3CO), 25.1
(cyclohexyl), 25.7 (2C, cyclohexyl), 30.8 (2C, cyclohexyl), 40.4 (N-
C
cyclohexyl), 50.1 (C-10), 59.4 (CH2N-Ccyclohexyl), 62.8 (C-6), 65.1 (C-4),
66.3 (C-30), 67.2 (C-5), 70.4 (C-20), 94.6 (C-1), 124.0 (C5’triaz), 126.3,
126.8 (C-2), 128.5 (2CAr), 128.9, 129.8 (C-3), 136.8 (CAr), 144.9
(C4’triaz), 167.6 (Coxad), 169.6 (Coxad), 170.2 (OAc), 170.7 (OAc), 171.2
(OAc). ESI-HRMS calcd for C32H40ClN6O9 [MþH]þ: 687.2545; found:
687.2539.
8.29e8.32 (m, 2H, HAr). 13C NMR (75 MHz):
d 20.6 (CH3CO), 20.7
(CH3CO), 20.9 (CH3CO), 25.1 (cyclohexyl), 25.7 (2C, cyclohexyl), 30.7
(2C, cyclohexyl), 40.5 (N-Ccyclohexyl), 50.1 (C-10), 59.6 (CH2N-Ccyclo-
hexyl), 62.7 (C-6), 65.1 (C-4), 66.4 (C-30), 67.1 (C-5), 70.4 (C-20), 94.6
(C-1), 123.8 (2CAr, C5’triaz), 126.8 (C-2), 128.1 (2CAr), 129.8 (C-3),
133.8 (CAr), 144.8 (C4’triaz), 149.2(CAr), 166.8 (Coxad), 169.6 (Coxad),
170.1 (OAc), 170.7 (OAc), 171.4 (OAc). ESI-HRMS calcd for
4.1.3.7. N-Cyclohexyl-N-[1-(10-O-(4,6-di-O-acetyl-2,3-dideoxy-
a-D-
erythro-hex-2-enopyranosyl)-(20-acetyl-30-deoxy-sn-glyceryl-1H-
1,2,3-triazol-4-yl)methyl]-3-(4-fluorophenyl)-1,2,4-oxadiazol-5-
25
amine (5g). Yield 85% (114 mg), colorless oil, [
a]
¼ þ 22.5 (c 0.2,
D
CH2Cl2); Rf 0.4 (Hexane- EtOAc, 1:1). 1H NMR (300 MHz):
1.16e1.39, 1.67e1.87 (m, 10H, cyclohexyl), 1.95 (s, 3H, CH3), 2.08 (s,
d
C
32H40N7O11 [MþH]þ: 698.2786; found: 698.2781.
3H, CH3), 2.09 (s, 3H, CH3), 3.54 (dd, 1H, J ¼ 11.0 and 4.7 Hz, H-3’a),
3.83 (dd, 1H, J ¼ 10.8 and 5.3 Hz, H-3’b), 3.94e4.03 (m, 1H, NeCH,
cyclohexyl), 4.07 (ddd, 1H, J ¼ 9.9, 5.3 and 2.3 Hz, H-5), 4.15 (dd, 1H,
J ¼ 12.3 and 2.9 Hz, H-6a), 4.26 (dd, 1H, J ¼ 12.3 and 5.3 Hz, H-6b),
4.58 (dd, 1H, J ¼ 14.1 and 6.4 Hz, H-1’a), 4.64 (dd, 1H, J ¼ 14.1 and
4.7 Hz, H-1’b), 4.77 (s, 2H, CH2eN), 4.99 (br s, 1H, H-1), 5.26e5.32
(m, 2H, H-4 and H-20), 5.79 (app dt, 1H, J ¼ 10.0, 2.3 and 2.3 Hz, H-
2), 5.90 (d, 1H, J ¼ 10.3 Hz, H-3), 7.11e7.16 (m, 2H, HAr), 7.68 (s, 1H,
4.1.3.10. N-Cyclohexyl-N-[1-(10-O-(4,6-di-O-acetyl-2,3-dideoxy-
a-D-
erythro-hex-2-enopyranosyl)-(20-acetyl-30-deoxy-sn-glyceryl-1H-
1,2,3-triazol-4-yl)methyl]-3-(4-methylphenyl)-1,2,4-oxadiazol-5-
25
amine (5j). Yield 89% (118 mg), colorless oil, [
a]
¼ þ 40 (c 0.2,
D
CH2Cl2); Rf 0.4 (Hexane-EtOAc, 1:1). 1H NMR (300 MHz):
0.39e0.99, 1.22e1.46 (m, 10H, cyclohexyl), 1.50 (s, 3H, CH3), 1.64
d
(s, 3H, CH3), 1.65 (s, 3H, CH3), 1.97 (s, 3H, CH3), 3.09 (dd, 1H, J ¼ 10.5
and 4.6 Hz, H-3’a), 3.39 ((dd, 1H, J ¼ 10.6 and 5.3 Hz, H-3’b),
3.51e3.60 (m, 1H, CHeN), 3.64 (ddd, 1H, J ¼ 9.2, 5.3 and 2.0 Hz, H-
5), 3.72 (dd, 1H, J ¼ 11.9 and 2.0 Hz, H-6a), 3.82 (dd, 1H, J ¼ 11.9 and
5.3 Hz, H-6’b), 4.13 (dd, 1H, J ¼ 14.5 and 6.6 Hz, H-1’a), 4.20 (dd, 1H,
J ¼ 14.5 and 4.6 Hz, H-1’b), 4.33 (s, 2H, CH2), 4.54 (br s, 1H, H-1),
4.78e4.86 (m, 2H, H-4 and H-20), 5.34 (app dt, 1H, J ¼ 9.8, 2.0 and
2.0 Hz, H-2), 5.46 (d, 1H, J ¼ 10.5 Hz, H-3), 6.80e6.83 (m, 3H, 2HAr
and Htriaz), 7.45e7.47 (m, 2H, HAr). For aromatic part in DMSO‑d6:
H
triaz), 7.99e8.04 (m, 2H, HAr). 13C NMR (75 MHz):
d 20.6 (CH3CO),
20.7 (CH3CO), 20.9 (CH3CO), 25.1 (cyclohexyl), 25.7 (2C, cyclohexyl),
30.8 (2C, cyclohexyl), 40.4 (N-Ccyclohexyl), 50.0 (C-10), 59.4 (CH2N-
C
cyclohexyl), 62.8 (C-6), 65.1 (C-4), 66.3 (C-30), 67.2 (C-5), 70.4 (C-20),
94.6 (C-1), 115.6e115.9 (d, 2C, JC-F ¼ 22.0 Hz, CoeAr), 123.9 (C5’triaz),
124.0 (d, 1C, JC-F ¼ 6.8 Hz, Cp-Ar), 126.8 (C-2), 129.2e129.3 (d, 2C, JC-
¼ 8.1 Hz, CmeAr), 129.8 (C-3), 145.0 (C4’triaz), 162.7e166.0 (d, 1C, JC-
F
¼ 250.8 Hz, Cipso-Ar), 167.6 (Coxad), 169.6 (Coxad), 170.2 (OAc), 170.7
F
(OAc), 171.2 (OAc). ESI-HRMS calcd for C32H40FN6O9 [MþH]þ:
d
7.31 (d, 2H, J ¼ 7.90 Hz, HAr), 7.80 (d, 2H, J ¼ 7.90 Hz, HAr), 8.08 (s,
671.2841; found: 671.2835.
1H, Htriaz). 13C NMR (75 MHz in CDCl3):
d
20.6 (CH3CO), 20.7
(CH3CO), 20.9 (CH3CO), 25.1 (cyclohexyl), 25.7 (2C, cyclohexyl), 30.8
(2C, cyclohexyl), 40.4 (N-Ccyclohexyl), 50.0 (C-10), 59.2 (CH2N-Ccyclo-
hexyl), 62.8 (C-6), 65.1 (C-4), 66.3 (C-30), 67.1 (C-5), 70.4 (C-20), 94.6
(C-1), 124.1 (C5’triaz), 125.0, 126.7 (C-2), 127.1 (2CAr), 129.3 (2CAr),
129.7 (C-3), 141.0 (CAr), 145.1 (C4’triaz), 168.4 (Coxad), 169.6 (Coxad),
170.2 (OAc), 170.7 (OAc), 171.0 (OAc). ESI-HRMS calcd for
4.1.3.8. N-Cyclohexyl-N-[1-(10-O-(4,6-di-O-acetyl-2,3-dideoxy-
a-D-
erythro-hex-2-enopyranosyl)-(20-acetyl-30-deoxy-sn-glyceryl-1H-
1,2,3-triazol-4-yl)methyl]-3-(4-trifluoromethylphenyl)-1,2,4-
25
oxadiazol-5-amine (5h). Yield 87% (125 mg), colorless oil, [
35 (c 0.1, CH2Cl2); Rf 0.5 (Hexane-EtOAc, 1:1).
a
]
D
¼ þ
1H NMR (300 MHz):
d
1.15e1.44, 1.65e1.88 (m, 10H, cyclohexyl),
C
33H43N6O9 [MþH]þ: 667.3091; found: 667.3088.
1.95 (s, 3H, CH3), 2.07 (s, 3H, CH3), 2.08 (s, 3H, CH3), 3.55 (dd, 1H,
J ¼ 10.6 and 4.7 Hz, H-3’a), 3.84 (dd, 1H, J ¼ 10.5 and 5.3 Hz, H-3’b),
3.96e4.03 (m, 1H, NeCH, cyclohexyl), 4.07 (ddd, 1H, J ¼ 9.9, 5.2 and
2.7 Hz, H-5), 4.15 (dd, 1H, J ¼ 12.0 and 2.6 Hz, H-6a), 4.25 (dd, 1H,
J ¼ 12.2 and 5.3 Hz, H-6b), 4.58 (dd, 1H, J ¼ 14.4 and 6.4 Hz, H-1’a),
4.64 (dd,1H, J ¼ 14.4 e 4.7 Hz, H-1’b), 4.78 (s, 2H, CH2eN), 4.98 (br s,
1H, H-1), 5.26e5.31 (m, 2H, H-4 and H-20), 5.78 (app dt, 1H, J ¼ 10.4,
3.0 and 3.0 Hz, H-2), 5.90 (d, 1H, J ¼ 10.2 Hz, H-3), 7.68 (s, 1H, Htriaz),
7.71 (d, 2H, J ¼ 8.2 Hz, HAr), 8.14 (d, 2H, J ¼ 8.2 Hz, HAr). 13C NMR
4.1.3.11. N-Cyclohexyl-N-[1-(10-O-(4,6-di-O-acetyl-2,3-dideoxy-
a-D-
erythro-hex-2-enopyranosyl)-(20-acetyl-30-deoxy-sn-glyceryl-1H-
1,2,3-triazol-4-yl)methyl]-3-(4-methoxyphenyl)-1,2,4-oxadiazol-5-
25
amine (5k). Yield 85% (116 mg), colorless oil, [
a
]
D
¼ þ 35 (c 0.1,
CH2Cl2); Rf 0.3 (Hexane-EtOAc, 1:1). 1H NMR (300 MHz):
1.11e1.43, 1.66e1.87 (m, 10H, cyclohexyl),1.95 (s, 3H, CH3), 2.08 (s,
d
3H, CH3), 2.09 (s, 3H, CH3), 3.53 (dd, 1H, J ¼ 10.5 and 4.7 Hz, H-3’a),
3.83 (dd, 1H, J ¼ 10.6 and 5.3 Hz, H-3’b), 3.86 (s, 3H, OCH3),
3.95e4.03 (m, 1H, CH cyclohexyl), 4.07 (ddd, 1H, J ¼ 9.4, 5.3 and
(75 MHz):
d 20.6 (CH3CO), 20.7 (CH3CO), 20.9 (CH3CO), 25.1
15

ꢀ
V.N. Melo de Oliveira, C. Flavia do Amaral Moura, A.S. Peixoto et al.
European Journal of Medicinal Chemistry 220 (2021) 113472
2.4 Hz, H-5), 4.15 (dd, 1H, J ¼ 12.3 and 2.9 Hz, H-6a), 4.26 (dd, 1H,
J ¼ 12.4 and 5.9 Hz, H-6b), 4.58 (dd, 1H, J ¼ 14.7 and 7.0 Hz, H-1’a),
4.64 (dd, 1H, J ¼ 14.7 and 4.7 Hz, H-1’b), 4.76 (s, 2H, CH2eN), 4.98
(br s, 1H, H-1), 5.28e5.31 (m, 2H, H-4 and H-20), 5.78 (app dt, 1H,
J ¼ 10.0, 2.3 and 2.3 Hz, H-3), 5.89 (d, 1H, J ¼ 10.5 Hz, H-2), 6.95 (d,
2H, J ¼ 8.8 Hz, HAr), 7.72 (s, 1H, Htriaz), 7.95 (d, 2H, J ¼ 8.9 Hz, HAr).
alkyne-oxadiazole 2n were used.
Yield 64% (198 mg), colorless oil, [
0.3 (Hexane-EtOAc, 1:1). 1H NMR (400 MHz):
25
a]
¼ þ 45 (c 0.1, CH2Cl2), Rf
D
d
2.03 (s, 3H, CH3),
2.08 (s, 3H, CH3), 2.10 (s, 3H, CH3), 2.63 (s, 3H, CH3-oxadiazole), 3.58
(dd, 1H, J ¼ 11.0 and 5.9 Hz, H-1’a), 3.84 (dd, 1H, J ¼ 11.0 and 5.9 Hz,
H-1’b), 4.09 (ddd, 1H, J ¼ 9.8, 5.1 and 2.3 Hz, H-5), 4.18 (dd, 1H,
J ¼ 12.1 and 2.3 Hz, H-6a), 4.27 (dd, 1H, J ¼ 12.1 and 5.1 Hz, H-6b),
4.84 (dd, 1H, J ¼ 14.5 and 6.2 Hz, H-3’a), 4.69 (dd, 1H, J ¼ 14.1 and
4.3 Hz, H-3’b), 5.00 (br s, 1H, H-1), 5.29 (s, 2H, CH2), 5.30e5.34 (m,
2H, H-4 and H-20), 5.81 (ddd, 1H, J ¼ 10.2, 2.7 and 1.9 Hz, H-2), 5.92
(br d, 1H, J ¼ 10.2 Hz, H-3), 7.07 (app dt, 2H, J ¼ 9.0 and 1.7 Hz, HAr),
7.66 (s, 1H, Htriaz), 8.00 (app dt, 2H, J ¼ 9.0 and 1.9 Hz, HAr). 13C NMR
13C NMR (75 MHz):
d 20.6 (CH3CO), 20.7 (CH3CO), 20.9 (CH3CO),
25.1 (cyclohexyl), 25.7 (2C, cyclohexyl), 30.8 (2C, cyclohexyl), 40.4
(N-Ccyclohexyl), 50.0 (C-10), 55.3 (OCH3), 59.2 (CH2N-Ccyclohexyl), 62.8
(C-6), 65.1 (C-4), 66.3 (C-30), 67.1 (C-5), 70.4 (C-20), 94.6 (C-1), 114.0
(2CAr), 120.2 (C5’triaz), 126.8 (C-2), 128.7 (2CAr), 129.7 (C-3), 161.6
(C4’triaz), 168.2 (Coxad), 169.6 (Coxad), 170.2 (OAc), 170.7 (OAc), 171.0
(OAc). ESI-HRMS calcd for C33H43N6O10 [MþH]þ: 683.3041; found:
683.3035.
(100 MHz): d 12.3 (CH3), 20.7 (CH3CO), 20.8 (CH3CO), 20.9 (CH3CO),
50.1 (C-10), 62.0 (CH2OAr), 62.8 (C-6), 65.1 (C-4), 66.3 (C-30), 67.2 (C-
5), 70.4 (C-20), 94.6 (C-1), 115.0 (2CAr), 119.9 (CAr), 123.6 (C5’triaz),
126.8 (C-2), 129.0 (2CAr), 129.8 (C-3), 144.0 (C4’triaz), 160.3 (CAr),
167.9 (Coxad), 169.7 (OAc),170.2 (OAc),170.7 (OAc),176.3 (Coxad). ESI-
HRMS calcd for C27H32N5O10 [MþH]þ: 586.2143; found: 586.2143.
4.1.3.12. N-Cyclohexyl-3-[4e10-O-(4,6-di-O-acetyl-2,3-dideoxy-
a-D-
erythro-hex-2-enopyranosyl)-(20-acetyl-30-deoxy-sn-glyceryl-1H-
1,2,3-triazol-4-yl)phenyl]-1,2,4-oxadiazol-5-amine (6). Yield 91%
25
(110 mg), colorless solid, mp 135e138 ꢂC, [
a
]
¼ þ 9.5 (c 0.1,
D
CH2Cl2); Rf 0.2 (Hexane-EtOAc, 1:1). 1H NMR (300 MHz):
1.18e1.51, 1.63e1.82 (m, 10H, cyclohexyl), 2.07 (s, 3H, CH3), 2.10 (s,
4.1.3. 15 10-O-(4,6-di-O-acetyl-2,3-dideoxy-
a-D-erythro-hex-2- eno-
d
piranosyl)-(20-acetyl-30-deoxy-[4-(n-decil)]-1,2,3-triazol-1-yl-sn-
25
6H, 2CH3), 3.64 (dd, 1H, J ¼ 10.6 and 4.7 Hz, H-3’a), 3.70e3.76 (m,
1H, NeCH cyclohexyl), 3.89 (dd, 1H, J ¼ 10.6 and 5.9 Hz, H-3’b), 4.11
(ddd, 1H, J ¼ 9.4, 5.3 and 2.4 Hz, H-5), 4.19 (dd, 1H, J ¼ 12.3 and
2.4 Hz, H-6a), 4.27 (dd, 1H, J ¼ 12.3 and 5.3 Hz, H-6b), 4.69 (dd, 1H,
J ¼ 14.1 and 8.9 Hz, H-1’a), 4.75 (dd, 1H, J ¼ 14.1 and 4.7 Hz, H-1’b),
5.04 (br s, 1H, H-1), 5.24 (d, 1H, J ¼ 8.2 Hz, NeH), 5.30e5.42 (m, 2H,
H-4 and H-20), 5.84 (app dt, 1H, J ¼ 10.6, 2.4 and 2.4 Hz, H-2), 5.94
(d, 1H, J ¼ 10.6 Hz, H-3), 7.89 (s, 1H, Htriaz), 7.91 (d, 2H, J ¼ 8.2 Hz,
glycerol (9). Yield 87%, colorless oil, [
a]
¼ þ 34 (c 0.1, CH2Cl2), Rf
D
0.53 (Hexano/EtOAcO, 1:1). 1H NMR (400 MHz):
d 0.88 (t, 3H,
J ¼ 7.1 Hz, CH3), 1.26e1.32 (m, 12H, 6 CH2), 1.64e1.67 (m, 4H, 2 x
CH2), 2.07 (s, 3H, CH3), 2.09 (s, 3H, CH3), 2.10 (s, 3H, CH3), 2.71 (t, 2H,
J ¼ 7.8 Hz, CH2), 3.58 (dd, 1H, J ¼ 10.6 and 4.7 Hz, H-1’a), 3.86 (dd,
1H, J ¼ 10.6 and 5.1 Hz, H-1’b), 4.10 (ddd, 1H, J ¼ 9.8, 4.7 and 2.4 Hz,
H-5), 4.18 (dd, 1H, J ¼ 12.1 and 2.7 Hz, H-6a), 4.27 (dd, 1H, J ¼ 12.2
and 5.1 Hz, H-6b), 4.57 (dd, 1H, J ¼ 14.4 and 6.3 Hz, H-3’a), 4.64 (dd,
1H, J ¼ 14.5 and 4.7 Hz, H-3’b), 5.02 (br s, 1H, H-1), 5.29e5.33 (m,
2H, H-20 and H-4), 5.83 (ddd, 1H, J ¼ 10.2, 2.6 and 2.0 Hz, H-2), 5.93
(br d, 1H, J ¼ 10.2 Hz, H-3), 7.30 (s, 1H, Htriaz). 13C NMR (100 MHz):
H
Ar), 8.30 (d, 2H, J ¼ 8.2 Hz, HAr). 13C NMR (75 MHz):
d 20.7 (CH3CO),
20.8 (CH3CO), 20.9 (CH3CO), 24.6 (2C, cyclohexyl), 25.3 (cyclohexyl),
33.2 (2C, cyclohexyl), 50.1 (C-10), 52.9 (N-Ccyclohexyl), 62.8 (C-6), 65.1
(C-4), 66.4 (C-30), 67.2 (C-5), 70.5 (C-20), 94.7 (C-1), 120.8 (C5’triaz),
125.8 (2CAr), 126.9 (C-2), 127.5, 127.7 (2CAr), 129.8 (C-3), 132.5, 147.5
(C4’triaz), 168.0 (Coxad), 169.8 (Coxad), 170.2 (OAc), 170.6 (OAc), 170.7
(OAc). ESI-HRMS calcd for C31H39N6O9 [MþH]þ: 639.2778; found:
639.2774.
d
14.1 (CH3), 20.7 (CH3CO), 20.8 (CH3CO), 20.9 (CH3CO), 22.6 (CH2),
25.6 (CH2), 29.2 (CH2), 29.3 (CH2), 29.4 (CH2), 29.4 (CH2), 29.5 (CH2),
29.6 (CH2), 31.9 (CH2-triaz), 49.8 (C-10), 62.8 (C-6), 65.1 (C-4), 66.5
(C-30), 67.2 (C-5), 70.7 (C-20), 94.7 (C-1), 121.3 (C5’triaz), 127.0 (C-2),
129.8 (C-3), 148.7 (C4’triaz), 169.8 (OAc), 170.2 (OAc), 170.7 (OAc).
ESI-HRMS calcd for
538.3124.
C
27H43N3O8 [MþH]þ: 538.3123; found:
4.1.3.13. N-Cyclohexyl-3-[4e10-O-(4,6-di-O-acetyl-2,3-dideoxy-
erythro-hex-2-enopyranosyl)-(20-acetyl-30-deoxy-sn-glyceryl-1H-
1,2,3-triazol-4-ylmethoxy)phenyl]-1,2,4-oxadiazol-5-amine (7).
a-D-
4.1.4. Synthesis of bis-1H-1,2,3-triazole/1,2,4-oxadiazoles (10 and
11)
25
Yield 94% (126 mg), colorless oil, [
a
]
¼ þ 35 (c 0.1, CH2Cl2);
D
Rf ¼ 0.3 (Hexane-EtOAc, 1:1). 1H NMR (300 MHz):
d
1.18e1.48,
CuSO4$5H2O (0.12 mmol), sodium ascorbate (0.18 mmol) and
bis-alkyne (3l or 3m) (0.2 mmol) were mixed in water (1.5 mL) in a
Schlenk flask under argon. This mixture was added into another
Schlenk flask containing 0.5 mmol of the azido-carbohydrate 4
dissolved in 2 mL of dichloromethane. The mixture was stirred at
room temperature for 45 min. Then, the solvent was removed, and
the residue was purified by column chromatography using ethyl
acetate.
1.63e1.80 (m, 10H, cyclohexyl), 2.03 (s, 3H, CH3), 2.09 (s, 3H, CH3),
2.10 (s, 3H, CH3), 3.58 (dd, 1H, J ¼ 10.8 and 4.9 Hz, H-3’a), 3.63e3.72
(m, 1H, CHeN), 3.84 (dd, 1H, J ¼ 10.9 and 5.9 Hz, H-3’b), 4.09 (ddd,
1H, J ¼ 9.4, 5.0 and 2.4 Hz, H-5), 4.18 (dd, 1H, J ¼ 12.0 and 2.7 Hz, H-
6a), 4.27 (dd, 1H, J ¼ 12.1 and 5.3 Hz, H-6b), 4.64 (dd, 1H, J ¼ 14.4
and 6.2 Hz, H-1’a), 4.68 (dd, 1H, J ¼ 14.4 and 4.7 Hz, H-1’b), 5.00 (br
s, 1H, H-1), 5.18 (d, 1H, J ¼ 8.3 Hz, NeH), 5.28 (s, 2H, CH2O),
5.30e5.35 (m, 2H, H-20 and H-4), 5.81 (app dt, 1H, J ¼ 10.3, 2.6 and
2.6 Hz, H-2), 5.92 (d, 1H, J ¼ 10.3 Hz, H-3), 7.01e7.04 (m, 2H, HAr),
4.1.4.1. N-Cyclohexyl-N-[4,4’-[(1,4-phenylene)-bis(10-O-(4,6-di-O-
7.65 (s, 1H, Htriaz), 7.91e7.94 (m, 2H, HAr). 13C NMR (75 MHz):
d
20.8
acetyl-2,3-dideoxy-
deoxy)-di-(1H-1,2,3-triazol-1-yl)]methyl-sn-glyceryl-1,2,4-oxadiazol-
a
-D-erythro-hex-2-enopyranosyl)-(20-acetyl-30-
(2xCH3CO), 20.9 (CH3CO), 24.6 (2C, cyclohexyl), 25.3 (cyclohexyl),
33.2 (2C, cyclohexyl), 50.1 (C-10), 52.8 (N-Ccyclohexyl), 62.0 (CH2OAr),
62.8 (C-6), 65.1 (C-4), 66.3 (C-30), 67.2 (C-5), 70.4 (C-20), 94.6 (C-1),
114.8 (2CAr), 120.9 (C5’triaz), 123.6 (2CAr), 126.9 (C-2), 128.8 (2CAr),
129.8 (C-3), 144.1 (CAr), 160.0 (C4’triaz), 168.0 (Coxad), 169.8 (Coxad),
170.2 (OAc), 170.4 (OAc), 170.7 (OAc). ESI-HRMS calcd for
25
5-amine (10). Yield 62% (130 mg), colorless oil, [
a
]
D
¼ þ 20 (c 0.1,
CH2Cl2); Rf 0.6 (EtOAc). 1H NMR (300 MHz):
d
1.16e1.43, 1.55e1.88
(m, 10H, cyclohexyl), 1.96 (s, 3H, CH3), 2.07e2.11 (s, 15H, 5xCH3),
3.54 (dd, 1H, J ¼ 10.6 and 4.1 Hz, H-3’a1), 3.64 (dd, 1H, J ¼ 10.6 and
4.5 Hz, H-3’a2), 3.83 (dd, 1H, J ¼ 10.6 and 5.3 Hz, H-3’b1), 3.89 (dd,
1H, J ¼ 10.5 and 5.3 Hz, H-3’b2), 3.98e4.03 (m, 1H, NeCH), 4.08
(ddd, 1H, J ¼ 10.0, 5.3 and 2.3 Hz, H-5a), 4.13 (ddd, 1H, J ¼ 9.4, 4.1
and 2.3 Hz, H-5b), 4.16e4.17 (m, 1H, H-6a), 4.23e4.30 (m, 3H, H-6a0,
H-6b and H-6b0), 4.58 (dd, 1H, J ¼ 14.1 and 6.5 Hz, H-1’a1), 4.64 (dd,
1H, J ¼ 14.1 and 4.7, H-1’a2), 4.71e4.74 (m, 2H, H-1’b1 and H-1’b2),
4.78 (br s, 2H, CH2eN), 4.98 (br s, 1H, H-1a), 5.05 (br s, 1H, H-1b),
C
32H41N6O10 [MþH]þ: 669.2884; found: 669.2877.
4.1.3.14. 10-O-(4,6-di-O-acetyl-2,3-dideoxy-
a-D-erythro-hex-2- eno-
piranosyl)-(20-acetyl-30-deoxy-[4-(5-methyl-3-[4-(methyloxy)
phenyl]-1,2,4- oxadiazolyl)]-1H-1,2,3-triazol-1-yl-sn-glycerol (8).
197 mg (0.53 mmol) of azido-sugar 4 and 170 mg (0.79 mmol) of
16

ꢀ
V.N. Melo de Oliveira, C. Flavia do Amaral Moura, A.S. Peixoto et al.
European Journal of Medicinal Chemistry 220 (2021) 113472
5.26e5.42 (m, 4H, H-4a, H-4b, H-20 and H-2’’), 5.78 (app dt, 1H,
J ¼ 10.6, 1.7 and 1.7 Hz, H-2a), 5.85 (app dt, 1H, J ¼ 10.5, 2.3 and
2.3 Hz, H-2b), 5.89 (d, 1H, J ¼ 10.7 Hz, H-3), 5.95 (d, 1H, J ¼ 10.5 Hz,
H-3), 7.71 (s, 1H, H-5’triaz), 7.90 (s, 1H, H-5’’triaz), 7.92 (d, 2H,
J ¼ 8.8 Hz, HAr), 8.09 (d, 2H, J ¼ 8.8 Hz, HAr). 13C NMR (75 MHz):
conversion
of
tetrazolium
3-(4,5-dimethyl-2-thiazol)-2,5-
diphenyl-2-H-bromide (MTT) salt to formazan from mitochon-
drial enzymes present only in metabolically active cells [56]. The
cytotoxic study by the MTT method allows to easily define the
cytotoxicity, but not the mechanism of action [57]. Tumor cells
were previously maintained in 75 cm2 cell culture flasks and
further seeded in 96-well plates at concentrations 0.3 x 106 cells/
mL for SKMEL-103 and AGP-01; 0.1x106 cells/mL for PC-3 and
NCIH-460 and 0.7 x 105 cells/mL for the HCT-116. The cells were
d
20.6 (CH3CO), 20.7 (2 x CH3CO), 20.8 (CH3CO), 20.9 (2 x CH3CO),
25.1 (C-cyclohexyl), 25.7 (2C-cyclohexyl), 30.8 (2C, cyclohexyl),
40.4 (N-Ccyclohexyl), 50.0 and 50.1 (2 x C-10), 59.4 (CH2N-Ccyclohexyl),
62.8 (2 x C-6), 65.0 and 65.1 (2 x C-4), 66.3 (2 x C-30), 67.1 and 67.2
(2 x C-5), 70.4 and 70.5 (2 x C-20), 94.6 and 94.7 (2 x C-1), 120.9
(C5’triaz),124.1 (C5’triaz), 125.8 (2CAr), 126.8 and 126.9 (2 x C-2),127.6
(CAr), 127.7 (2CAr), 129.7 and 129.8 (2 x C-3), 132.6 (CAr), 145.0 and
147.3 (2 x C4’triaz), 168.0 (Coxad), 169.6 (Coxad), 169.9 (OAc), 170.2 (2 x
OAc), 170.6 (OAc), 170.7 (OAc), 171.1 (OAc). ESI-HRMS calcd for
incubated with the compounds at a single concentration of 25 mM
for 72 h in a 5% CO2 incubator at 37 ꢂC. At the end of this, they were
centrifuged (400ꢃg for 5 min) and the supernatant was removed.
Then 100 mL of 1% MTT (tetrazolium salt) solution was added, and
the plates were incubated for 3 h. The absorbance was read after
dissolution of the precipitate in 150
plate spectrophotometer.
C
49H62N9O17 [MþH]þ: 1048.4264; found: 1048.4268.
mL of pure DMSO in a 570 nm
4.1.4.2. N-Cyclohexyl-N-[4,4’-[(1,4-phenylene)-bis(10-O-(4,6-di-O-
acetyl-2,3-dideoxy-
-D-erythro-hex-2-enopyranosyl)-(20-acetyl-30-
Statistical analysis: The absorbances obtained were used to
calculate the IC50 values by nonlinear regression employing
appropriate statistical software. Statistical significance was calcu-
lated by Analysis of Variance (ANOVA) followed by Tukey’s test,
with a significance level of p < 0.05.
Cytotoxicity using Vero cells: Vero cells were grown in Dulbecco’s
modified Eagle’s medium (DMEM) (Gibco, Carlsbad, CA) supple-
mented with 10% inactivated fetal bovine serum (FBS) (Gibco),
a
deoxy)-di-(1H-1,2,3-triazol-1-yl)]methoxyphenyl-sn-glyceryl-1,2,4-
oxadiazol-5-amine (11). Yield 67% (144,5 mg), colorless oil,
25
[
d
a
]
¼ þ 35 (c 0.2, CH2Cl2); Rf 0.5 (EtOAc). 1H NMR (300 MHz):
D
1.15e1.43, 166e1.86 (m, 10H, cyclohexyl), 1.95 (s, 3H, CH3), 2.03 (s,
3H, CH3), 2.07 (s, 3H, CH3), 2.08 (s, 3H, CH3), 2.08 (s, 3H, CH3), 2.09
(s, 3H, CH3), 3.53 (dd, 1H, J ¼ 10.6 and 4.7 Hz, H-3’a1), 3.59 (dd, 1H,
J ¼ 10.6 and 4.7 Hz, H-3’a2), 3.82 (dd,1H, J ¼ 5.2 and 2.3 Hz, H-3’b1),
3.85 (dd, 1H, J ¼ 5.9 and 1.8 Hz, H-3’b2), 3.95e4.02 (m, 1H, NeCH,
cyclohexyl), 4.05e4.30 (m, 6H, H-5a, H-5b, H-6a, H-6a0, H-6b and
H6b0), 4.54e462 (m, 2H, H-1’a1 and H-1’a2), 4.65e4.71 (m, 2H, H-
1’b1 and H-1’b2), 4.76 (s, 2H, NeCH2), 4.98 (br s, 1H, H-1a), 5.01 (br
s, 1H, H-1b), 5.27 (br s, 2H, CH2O), 5.31e5.33 (m, 4H, H-4a, H-4b, H-
20 and H-2’’), 5.76e5.83 (m, 2H, H-2a and H-2b), 5.87e5.95 (m, 2H,
H-3ꢂ and H-3b), 7.03 (d, 2H, J ¼ 8.8 Hz, HAr), 7.68 (s, 1H, Htriaz), 7.69
2 mM L-glutamine (Gibco) and 100 U/mL penicillin/streptomycin
(Gibco). The toxicity of the synthesized compounds was evaluated
on growing cells using the MTT method (Sigma, St. Louis, USA) as
previously described [58]. Briefly, Vero cells (1 ꢃ 104 cells/well)
were plated in 96-well microplates 24 h in advance and thentreated
with various concentrations of the compounds (800, 400, 200, 100,
50
m
M) in max. 0.8% of DMSO. After 5 days of incubation at 37 ꢂC,
L of
culture medium was removed and was replenished with 50
m
(s, 1H, Htriaz), 7.94 (d, 2H, J ¼ 8.8 Hz, HAr). 13C NMR (75 MHz):
d
20.6
MTT solution (1 mg/mL) per well and the microplate was incubated
for additional 4 h. MTT formazan crystals were solubilised by
adding dimethylsulfoxide (DMSO) and the optical density at
570 nm (OD570) was determined by spectrophotometry using a
(CH3CO), 20.7 (2 x CH3CO), 20.8 (CH3CO), 20.9 (2 x CH3CO), 25.1 (C-
cyclohexyl), 25.7 (2C-cyclohexyl), 30.8 (2C, cyclohexyl), 40.4 (N-
C
cyclohexyl), 50.0 and 50.1 (2 x C-10), 59.3 (CH2N-Ccyclohexyl), 62.0
(CH2OAr), 62.8 (2 x C-6), 65.1 (2 x C-4), 66.3 (2 x C-30), 67.1 and 67.2
(2 x C-5), 70.4 (2 x C-20), 94.5 and 94.6 (2 x C-1), 114.8 (2CAr), 120.9
(C5’triaz), 123.6 (CAr), 124.0 (C5’triaz), 126.8 (2 x C-2), 128.8 (2CAr),
129.7 and 129.8 (2 x C-3), 145.1 (CAr), 160.1 (2 x C4’triaz), 168.0
(Coxad), 169.6 (Coxad), 169.7 (OAc), 170.2 (2 x OAc), 170.7 (2 x OAc),
171.0 (OAc). ESI-HRMS calcd for C50H64N9O18 [MþH]þ: 1078.4369;
found: 1078.4364.
BioTekTM
ELx800TM
96
well
plate
reader
(Bio-
TekInstrumentsInc.,Winooski,VT). Cell viability was calculated by
subtracting the OD at 570 nm of treated cells from untreated con-
trol cells. The cytotoxic concentration for 50% of the cell culture
(CC50) was defined as the concentration of compound that caused a
50% reduction in absorbance.
4.2.2. In vitro antitubercular activity evaluation
4.2. Biological evaluation
Reference Strain: Mycobacterium tuberculosis H37Ra (ATCC 25177)
and H37Rv (ATCC 27294) from American Type Culture Collection
(Manassas, VA) were used.
4.2.1. Cytotoxicity studies
Cells: Human tumor cell lines PC-3 (prostate adenocarcinoma),
HCT-116 (colorectal tumor), NCIH 460 (lung carcinoma), SKMEL-
103 (melanoma) and AGP-01 (gastric tumor) were used. The cells
were ceded by the National Cancer Institute (USA) and grown in
RPMI 1640 medium (PC-3, HCT-116, and NCIH-460) and in Dul-
becco’s Modified Eagle Medium (DMEM) (SKMEL-103 and AGP-01),
both supplemented with 10% of bovine fetal serum and 1% of an-
tibiotics, kept in the incubator at 37 ꢂC and atmosphere containing
5% of CO2.
Commercial drugs and chemicals: Rifampicin (RIF), ethambutol
(EMB) (Sigma-Aldrich NV/SA, Bornem, Belgium) solutions were
prepared at concentrations of 1 mg/mL in distilled water and
10 mg/mL in methanol, respectively, filter sterilized and frozen
until used.
Determination of Minimum Inhibitory Concentration (MIC): The
in vitro assay for the determination of minimum inhibitory con-
centration (MIC) was performed in duplicate by Resazurin Micro-
titer Assay Plate (REMA) as described previously by Palomino et al.
[59] with modifications. RIF and EMB solutions were thawed and
diluted in 7H9eS medium. Serial two-fold dilutions of each drug in
Compounds: All samples were diluted in sterile pure DMSO at
the stock concentration of 10 mM/1 mL, following laboratory
standard conditions.
100
mL of 7H9eS medium were prepared directly in 96-well plates
Method: MTT cytotoxicity analysis is one of the methods used in
the United States National Cancer Institute (NCI) screening pro-
gram, which tests more than 10,000 samples each year [55]. It is a
fast, sensitive, and inexpensive method, first described by Moss-
man (1983). This method is able to analyze the viability and
metabolic state of the cell. It is a colorimetric analysis based on the
at concentrations of 2.0 to 0.0625
mL for EMB. Serial two-fold dilutions of the triazole derivatives
solutions were performed directly on the microplate, with a con-
centration ranging from 64 mg/mL to 0.5 mg/mL. Growth controls
containing no antibiotic and sterility controls without inoculation
were also included. The inoculum used was prepared from fresh
m
g/mL for RIF and 16.0 to 0.5 g/
m
17

ꢀ
V.N. Melo de Oliveira, C. Flavia do Amaral Moura, A.S. Peixoto et al.
European Journal of Medicinal Chemistry 220 (2021) 113472
€
Lowenstein Jensen medium in 7H9eS medium adjusted to a Mc-
4.3.4. Molecular docking of designed chemical library
Farland tube no. 1 and then diluted 1:20 and 100
were covered, sealed in plastic bags, and incubated at 37 ꢂC under
normal atmosphere. After 9 days of incubation, 30 L of resazurin
m
L. The plates
Site specific molecular flexible docking was performed on glide
platform against DprE1 and InhA protein of Mycobacterium tuber-
culosis [67]. These molecules were docked at extra precision and
m
solution was added to each well, incubated overnight at 37 ꢂC, and
assessed for color development. Resazurin sodium salt powder
(Acros Organic N$V., Geel, Belgium) was prepared at 0.01% (wt/vol)
in distilled water and filter sterilized. A change from blue to pink
indicates reduction of resazurin and therefore bacterial growth. The
MIC was defined as the lowest drug concentration that prevented
this color change. The cut-off points for susceptibility/resistance to
analyzed for docking score. The DG⁰ were also calculated by using
prime mmgbsa for both compounds [63].
4.3.5. Molecular dynamics (MD) simulation
Molecular dynamic simulation was done to validate docking
observations for both molecules in complex with both proteins. The
simulation was done for 100ns time period on Desmond software
to visualize the binding and conformational stability of both mol-
ecules with in binding pocket [68]. The complex was solvated TIP3P
water model prior to simulation where 0.15 M NaCl added to mimic
a physiological ionic concentration.
antimicobacterial strains were 0.5 and 5
respectively [60].
mg/mL for RIF and EMB,
Cytotoxicity test: Raw 264.7 (ATCC TIB - 71) macrophages e
HepG2 cells were used to assess cell cytotoxicity by MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide,
Sigma, USA]. The cell line was cultured in DMEM medium, at 37 ꢂC
in a humidified atmosphere of 5% CO2. The culture medium was
changed after 2e3 days and subcultured when cell population
density reached to 70e80% confluence. Macrofages 105 cells/well
were added in 96-well plates and incubated for 24 h (37 ꢂC and 5%
CO2). Compounds were then added in different concentrations
(1e100 mg/mL) and incubated for 48 h. Untreated, rifampicin (RIF),
ethambutol (EMB) were employed as controls. Experiments were
performed in duplicate. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-2H-tetrazolium bromide, 5 mg/mL in PBS) was added to
each well and incubated again for 2 h. Culture medium and non
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgment
The authors are grateful to National Council for Scientific and
Technological Development - CNPq [grant numbers 448082/2014-
4, PQ 301308/2017-9] and LABEX SYNORG [grant number ANR-11-
LABX-0029] for financial support. This study was financed in part
reduced MTT were removed and 100 mL of DMSO were added. The
amount of formazan was determined by measuring the absorbance
at 570 nm. The negative control was obtained in wells containing
only culture medium and cells (without treatment). Cytotoxic
concentration for 50% inhibition of viability (CC50) was also calcu-
lated by regression analysis using GraphPad Prism software. The
selectivity index (SI) was determined as the ratio of CC50 values
(cell lines) and inhibitory concentration for 50% bacterial, MIC (SI ¼
IC50/MIC); since SI ꢀ 10 or greater indicates when the test com-
pound can be applied at concentration ten times or more above the
MIC value without presenting cytotoxicity [42,43].
~
by the Coordenaçao de Aperfeiçoamento de Pessoal de Nível Su-
perior e Brasil - CAPES finance code 001. We are indebted with
Daniele Mendes Felix by the cytotoxicity evaluated in Vero cell for
four compounds. Our thanks are also extended to Analytical Cen-
ters CENAPESQ-UFRPE and DQF-UFPE. BR and PPS acknowledge the
administration of Hansraj College, University of Delhi for infra-
structural support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113472.
4.3. Molecular docking study
4.3.1. Homology modelling and structure validation
References
The crystal structure of DprE1 and InhA proteins were down-
loaded from RCSB database (www.rcsb.org). The stereo-chemical
geometry of residues was measured by Ramachandran map (By
Procheck) [61]. The computational work was performed using
Schrodinger software (Schrodinger release 2020-1 license dated
November 20, 2020).
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