Synthesis, biological evaluation and molecular docking study of 1,2,3-1H-triazoles having 4H-pyrano[2,3-d]pyrimidine as potential Mycobacterium tuberculosis protein tyrosine phosphatase B inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.12.009

Some heterocycles, namely 2-amino-4H-pyran-3-carbonitriles, were synthesized in a three-component reaction from substituted benzaldehydes, malononitrile, and ethyl acetoacetate. These heterocycles have been converted subsequently into 4H-pyrano[2,3-d]pyri

Full text of DOI:10.1016/j.bmcl.2018.12.009

Accepted Manuscript
Synthesis, biological evaluation and molecular docking study of 1,2,3-1H-tria-
zoles having 4H-pyrano[2,3-d]pyrimidine as potential Mycobacterium tuber-
culosis protein tyrosine phosphatase B inhibitors
Nguyen Dinh Thanh, Do Son Hai, Nguyen Thi Thu Ha, Do Tien Tung, Cao Thi
Le, Hoang Thi Kim Van, Vu Ngoc Toan, Duong Ngoc Toan, Le Hai Dang
PII:
S0960-894X(18)30945-4
https://doi.org/10.1016/j.bmcl.2018.12.009
BMCL 26178
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 October 2018
26 November 2018
4 December 2018
Please cite this article as: Thanh, N.D., Hai, D.S., Ha, N.T.T., Tung, D.T., Le, C.T., Van, H.T.K., Toan, V.N., Toan,
D.N., Dang, L.H., Synthesis, biological evaluation and molecular docking study of 1,2,3-1H-triazoles having 4H-
pyrano[2,3-d]pyrimidine as potential Mycobacterium tuberculosis protein tyrosine phosphatase B inhibitors,
Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.12.009
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Synthesis, biological evaluation and molecular docking study of 1,2,3-1H-triazoles
having 4H-pyrano[2,3-d]pyrimidine as potential Mycobacterium tuberculosis
protein tyrosine phosphatase B inhibitors
Nguyen Dinh Thanh a*, Do Son Hai a,b, Nguyen Thi Thu Ha a, Do Tien Tung a, Cao Thi Le a, Hoang
Thi Kim Van a,c, Vu Ngoc Toan a,d, Duong Ngoc Toan a,e, Le Hai Dang a,f
a Faculty of Chemistry, VNU University of Science, 19 Le Thanh Tong, Ha Noi, Viet Nam
b Institute of Technique in Chemistry, Biology and Security Documents (Ministry of Public Security),
Cau Giay, Ha Noi, Viet Nam
c Faculty of Chemical Technology, Viet Tri University of Industry, Tien Kien, Lam Thao, Phu Tho,
Viet Nam
d Institute for Chemistry and Materials, Military Institute of Science and Technology (Ministry of
Military), Cau Giay, Ha Noi, Viet Nam
e Faculty of Chemistry, Thai Nguyen University of Education, 20 Luong Ngoc Quyen, Thai Nguyen,
Viet Nam
f Thai Nguyen College of Education, Quang Trung, Thinh Dan, Thai Nguyen, Viet Nam
Abstracts
Some heterocycles, namely 2-amino-4H-pyran-3-carbonitriles, were synthesized in a three-component
reaction from substituted benzaldehydes, malononitrile, and ethyl acetoacetate. These heterocycles
have been converted subsequently into 4H-pyrano[2,3-d]pyrimidine ring by ring-closing reaction with
acetic anhydride in the presence of the concentrated sulfuric acid as catalyst. The successive alkylation
reaction of lactam N−H bond on pyrimidine-4-one ring was carried out using propargylic bromide in
dry acetone in the presence of anhydrous potassium carbonate. The click chemistry of 3-propargyl-4H-
pyrano[2,3-d]pyrimidine compounds has been accomplished by reaction with tetra-O-acetyl-α-D-
1

glucopyranosyl azide using the metal-organic framework Cu@MOF-5 as a catalyst in absolute ethanol.
All the synthesized 1H-1,2,3-triazoles 8a-y were screened for their in vitro Mycobacterium tuberculosis
protein tyrosine phosphatase B (MtbPtpB) inhibition. Kinetic studies of the most active compounds 8v,
8x, and 8y showed their competitive inhibition toward the MtbPtpB enzyme. The detailed structure-
activity relationship (SAR) in vitro and in silico studies suggested that the interaction of Arg63 amino
acids with anion type of para-hydroxyl group via a salt bridge of iminium cation was essential for
strong inhibitory activity against MtbPtpB.
Keywords
2-Amino-4H-pyran-3-carbonitriles; Antitubercular activity; Molecular docking; 4H-Pyrano[2,3-
d]pyrimidine; PtpB.
It’s known that Mycobacteria were bacteria, which caused serious diseases in human and animals.
These bacteria included tuberculosis (M. tuberculosis) and the classic Hansen’s strain of leprosy
(Mycobacterium leprae). Among these bacteria, Mycobacterium tuberculosis caused tuberculosis due
to the lack of proper therapeutic agents for its remedy.1 The morbidity and mortality from tuberculosis
and the emergence of multidrug-resistant (MDR) strains of M. tuberculosis became growing health
problems in the world, particularly in Vietnam.2, 3 Nowadays, Viet Nam is a lower, middle income
country and is one of the 30 highest TB burden countries.4 Tuberculosis (TB) kills nearly 2 million
people annually in the world. The World Health Organization (WHO) declared that TB is as a global
health emergency, which highlights the importance of TB as a major threat to humans.4 The actions of
second-line antituberculosis drugs were applied as the last resort for combating MDR infections when
the first-line protocols were no effective in tuberculosis cases due to resistance problems.3 On the other
hand, drug resistance and patient noncompliance are two key factors that affect the success rate of
2

conventional treatments against TB. Therefore, there is an urgent need to identify novel therapeutic
targets for TB treatment as well as new drugs that could act on them. Tuberculosis infections by drug-
sensitive strains could be successfully treated using the specific remedy, but the appearance of drug-
resistant strains of M. tuberculosis have promoted researches in new drug development, particularly the
search for new drug targets. Protein tyrosine phosphatases (PTPs) are an important class of enzymes.
Together with protein tyrosine kinases (PTKs), they control the level of phosphorylation of their
protein substrate targets. Protein tyrosine phosphorylation plays a critical role in the signal transduction
of many cellular events including immune response, metabolism, growth and gene transcription.5, 6 It is
known that there are two protein tyrosine phosphatases (PtpA and PtpB) secreted by MTB in infected
human macrophages.7 These enzymes have been recognized in MTB’s genome.8 One assumed that
these enzymes are profitable targets for the development of novel therapeutics against TB.9, 10 Although
several reports realize that PtpA and PtpB inhibition by small molecules could affect MTB survival in
the host, thus paving the way for the development of innovative therapeutic strategies.10-12
Recently, the syntheses of pyranopyrimidine derivatives have obtained interest in medicinal chemistry
due to the broad range of their pharmacological activities. Pyrano[2,3-d]pyrimidine is an unsaturated
heterocyclic system which is formed by fusion of two six member pyran and pyrimidine rings together.
This ring system consisted of one oxygen atom at position 8 and two nitrogen atoms at positions 1 and
3, respectively. It is realized that if pyrano[2,3-d]pyrimidine moiety are connected to one certain
molecule, then resultant derivative can enhance its inherent pharmaceutical activity in bioactive
compounds, such as antitumour,13 antitubercular,14 antibacterial,15, 16 antifungal,16 antimicrobial17
activities, and so on. Figure 1 below represented some bioactive compounds. Kamdar et al. showed that
compound A, which was fused with quinoline ring, had antitubercular activity against M. tuberculosis
with MIC = 62.5 μg/mL.14 Sabry et al. indicated that benzopyrano[2,3-d]pyrimidines B with chloro-
and bromo-substituents had inhibitory activities against some typical Gram-negative (and) Gram-
positive bacteria, and fungi with MICs = 14−27, 15−26, and 15−24 μg/mL, respectively.18 According
3

to Dangolani et al., compound C had antioxidant activity as Trolox equivalent antioxidant capacity
(TEAC) with TEAC = 5.237±0.053 μM.19 Paliwal et al. announced that compounds D had good
anticancer and antibacterial activities.20 Kalaria and co-workers showed that fused pyran derivative E
had antimalarial activity with IC50 of 0.062 μg/mL and 92% inhibition against M. tuberculosis H37Rv at
concentration of 100 μg/mL.21
OCH3
HO
OH
R
O
OH
OH
HO
HO
O
N
O
O
OH
NH
HN
CO2Et
NH2
HN
N
OH
O
N
O
N
O
NH2
Et2N
B:
O
N
H
A
R = Cl, Br
C
M. tuberculosis H37Rv,
MIC = 62.5 g/mL
MIC = 14-27 g/mL (Gram-(-) bacteria)
MIC = 15-26 g/mL (Gram-(+) bacteria)
TEAC = 5.237 ± 0.053 M
MIC = 15-24 g/mL (fungi)
C6H5
N N
N
Z
N
O
O
CN
CO2Et
NH2
HN
HN
O
N
O
NH2
O
N
O
H
H
D
E
Z = O, S, NH
Anticancer & antibacterial
Antimalarial, IC50 = 0.062 g/mL
92% Inhibition against M. tuberculosis H37Rv at 100 g/mL
Figure 1. Some pyrano[2,3-d]pyrimidine derivatives having biological activities.
Besides derivatives containing pyrano[2,3-d]pyrimidine ring, 1H-1,2,3-triazoles are also studied for
their biological, therapeutic and industrial potential.22, 23 1H-1,2,3-Triazoles set up an important class of
nitrogen-containing heterocycles in the area of organic and medicinal chemistry. Medicinally, they
showed a broad range of diverse interesting pharmacological properties such as antitubercular,24
anticancer,25 anti-HIV,26 antiallergic,27 antimalarial,28 antifungal,29 and antibacterial30 activities, and so
on. Some 1H-1,2,3-triazoles were potential MtbPtpB inhibitors, such as compound F with significant
cellular activity (IC50 1.27 μM);31 1,2,3-1H-triazole G was the most active representative in the library
4

of MTB PtpB inhibitors,32 with a Ki value of 0.15 μM and IC50 = 0.64 μM (Fig. 2). Some nucleosides
and oligonucleotides bearing 1H-1,2,3-triazole residues with nucleobase have been synthesized and
studied for antitumor,25 antiviral33 and antitubercular34 activities, as 1H-1,2,3-triazole H bearing D-
galactose inhibited bacterial growth by 76% and 78% at concentrations of 5 μg/mL and 25 μg/mL,
respectively (Fig. 2).35
F
H
O
O
S
N
O
N
O
N
N
N
O
N
O
N
CH3
HOOC
N
N
HO
O
G
, Ki 0.15 M
COOH
F
, IC50 1.27 M
IC50 0.64 M
OAc
O
AcO
AcO
N
O
N
N
OAc
N
H
, 76% of bacterial inhibition at 5 g/mL
78% of bacterial inhibition at 25 g/mL
Figure 2. Some structures of selected MtbPtpB inhibitors having 1H-1,2,3-triazole ring.
The connection of these above pharmacophores could bring forth remarkably biological activities or
strengthen their inherent biological activities in new derivatives. They helped to find the bioactive
compounds easily and favorably. On the other hand, pyrano[2,3-d]pyrimidine system have been
synthesized directly by the reaction of aromatic aldehydes, malononitrile or cyanoacetic ester or
chalcones (arylideneacetophenones) with barbituric acid under different catalytic conditions, such as
microwave irradiations, ultrasonic irradiations, solvent free condition and in aqueous medium in the
absence of catalysts.36, 37 In addition, this ring could be synthesized from 2-amino-4H-pyran-3-
carbonitriles by taking advantage of the chemistry of amino and nitrile groups.38 Up to now, the
connection between 4H-pyrano[2,3-d]pyrimidine and D-glucose ring tethering by 1H-1,2,3-triazole was
not touched upon. Therefore, we have designed molecules that contained both pyrano[2,3-d]pyrimidine
and 1H-1,2,3-triazole ring having D-glucose moiety. We reported herein the synthesis of substituted
5

4H-pyrano[2,3-d]pyrimidines bearing propargyl unit and its click chemistry. We further evaluated the
in vitro inhibitory activity of synthesized 1H-1,2,3-triazole-tethered 4H-pyrano[2,3-d]pyrimidines and
D-glucose conjugates against M. tuberculosis protein tyrosine phosphatase PtpB.
The 4H-pyrano[2,3-d]pyrimidines 5a-y were synthesized from corresponding ethyl 6-amino-5-cyano-
2-methyl-4-aryl-4H-pyran-3-carboxylates 4a-y by cyclizing with acetic anhydride (and) using
concentrated sulfuric acid as catalyst. The yields of these products were 62−71% (Scheme 1). In turn,
ethyl 4H-pyran-3-carboxylates 4a-y were prepared by three-component reaction between ethyl
acetoacetate 1, malononitrile 2 and substituted benzaldehydes 3a-y in 63−89% yields. Ammonium
hydroxide (Ammonium solution 25%) or Cu@MOF-5 were used as the catalysts in these reactions. In
general, the latter gave higher yields of 4a-y (63−89% in method A v.s. 78−90% in method B).
Ring-closing reaction with acetic anhydride of 4H-pyrans 4a-y into the corresponding 4H-pyrano[2,3-
d]pyrimidines 5a-y was confirmed by IR and NMR spectra. The ring-closing process was confirmed by
the disappearance of the amino group, with two IR absorption bands in the regions at 3420−3250 cm−1,
and by the appearance of IR absorption band of the amido group of the pyrimidine-4-one ring in the
region at 1672−1643 cm−1. The signal of the 2H integral of an amino group in δ = 7.09−6.86 ppm also
was no longer present, and simultaneously a signal singlet with 1H integral appeared in extreme
downfield region, with δ=12.49−12.47 ppm. The signal of proton H-5 of pyran ring also shifted toward
downfield, at δ=4.79−4.72 ppm when compared with corresponding values of δ=4.5−4.20 ppm in the
initial 4H-pyran 4a-y. Three new signals appeared 13C NMR spectra of these 4H-pyrano[2,3-
d]pyrimidines, belong to the newly carbon atoms that were added to the molecular skeleton. That’s
carbon atom C-2, with δ=160.4−160.3 ppm, methyl group on position 2, with δ=21.4−21.5 ppm, and
amide carbonyl carbon atom at δ=162.4 ppm, derived from the C≡N functional group (with NMR
signal at δ=120.3−119.8 ppm, and no longer appeared in the 13C NMR spectra of 5a-y).
6

N
N
O
Ar
O
Ar
N
a
b
+
+
O
O
O
O
H
(CH3CO)2O
O
NH2
3a-y
2
1
4a-y
O
N
O
Ar
O
O
N
O
Ar
O
c
d
O
NH
O
N
OAc
Br
O
AcO
AcO
N3
5a-y
6a-y
OAc
7
3a-y 4a-y 6a-y 8-a-y
:
,
,
,
OAc
OAc
AcO
a
b
c
d
Ar = H ( ); 4-NO2 ( ); 3-NO2 ( ); 2-NO2 ( );
e
f
g
h
O
2,3-dichloro ( ); 4-Cl ( ); 3-Cl ( ); 2-Cl ( );
O
O
Ar
O
i
j
k
l
m
q
4-Br ( ); 4-Me ( ); 4-iPr ( ); 4-OH ( ); 3-OH ( );
OAc
O
N
N
N
n
o
p
2-OH ( ); 4-OMe ( ); 3-OMe ( ); 2-OMe ( );
N
r
s
t
2,4-NMe2 ( ); 3,5-diMe ( ); 3-OEt-4-OH ( );
N
u
v
3-OMe-4-OH ( ); 3,5-diOMe-4-OH ( );
8a-y
x
y
3-OH-4-OEt ( ); 3-OMe-4-OH-5-NO2 ( ).
Scheme 1. Reagents and conditions: (a) Ammonium solution 25%, 25°C, 3 hrs., or Cu@MOF-5 (10
mol%), 96% EtOH, 50°C, 1 hr; (b) Acetic anhydride, conc. H2SO4 (cat.), 15 min, in refluxing, then
25°C, 24 hrs.; (c) Propargyl bromide, anhydrous potassium carbonate, dry acetone, in refluxing, 4−5
hrs; (d) Cu@MOF-5 (cat.), 79−80°C, abs. EtOH, 4−5 hrs.
Reaction of 4H-pyrano[2,3-d]pyrimidines 5a-y with propargyl bromide in dry acetone in the presence
of anhydrous K2CO3 gave corresponding N-propargyl derivatives 6a-y with the yields of 76−88%. The
N-propargylation reaction of the lactam group of pyrimidine ring was confirmed by the appearance of
two proton signals of the CH2 group, of the terminal alkyne in 1H NMR spectra, and of three carbon-13
signals in the 13C NMR spectra of the propargyl group, −CH2C≡CH. The two protons of the propargyl-
methylene group were not equivalent to each other in the magnetic term. Therefore, their signals split
due to spin-spin coupling in the doublet-doublet signal with vicinal coupling constant J=18.0 Hz. There
is another interaction with the acetylenic proton in faraway with coupling constant J=2.5 Hz. However,
due to the small coupling constants, the signal of this acetylenic proton degenerate into counterfeit
7

singlet in δ=3.50−3.35 ppm. On the other hand, the absence of NH signal in extreme downfield region
(δ=12.49−12.47 ppm) also denoted that the propargyl group was attached to the N−H bond.
Click chemistry of N-propargyl derivatives 6a-y of 4H-pyrano[2,3-d]pyrimidines with 2,3,4,6-tetra-O-
acetyl-β-D-glucopyranosyl azide 7 gave corresponding 1H-1,2,3-triazoles 8a-y with the yields of 64-
94%. The processes were catalyzed by Cu@MOF-5. The reaction solvent was absolute ethanol for 4−5
hrs. IR spectra of 8a-y had strong absorption bands in the regions of 1757−1747, 1236−1224, and
1041−1037 cm−1 belonging to C=O acetyl stretching vibrations of acetate group in peracetated D-
glucose moiety. The 13C resonance signals of these group had two regions, with δ = 170.50−168.90
ppm belonging to C=O acetate ester group and δ = 20.98−20.27 ppm belonging to a methyl-acetate
group. Chemical shifts of protons in D-glucose moiety were in the range of δ = 6.38−4.08 ppm. The
carbon atoms of the D-glucopyranose ring had six resonance signals at δ = 84.3−62.2 ppm. The
coupling constants between protons H-2 and H-3 were J = 9.50−9.25 Hz that indicated the β-anomeric
configuration of this glucoside. The 1H-1,2,3-triazole ring (was) specified by the unique proton of the
1H-1,2,3-triazole ring that had a downfield chemical shift at δ = 8.55−8.54 ppm in a singlet. This signal
could be used to identify the formation of the 1H-1,2,3-triazole system. Two C-a and C-b carbon atoms
of this ring had 13C NMR signals in the regions at δ = 123.4−123.3 ppm and δ = 143.2−142.9 ppm,
respectively. The two protons of the CH2O bridged group between the 1,2,3-triazole ring and chromene
ring were not magnetically equivalent, so their signal was located in the regions at δ = 5.34−5.25 and
5.07−5.01 ppm. Carbon atom of this bridged group had a chemical shift at δ = 40.1−39.9 ppm, and this
signal shifted more downfield due to the electronegative influence of oxygen atom and the anisotropic
effect of triazole ring.
All the synthesized compounds 8a-y were screened for their in vitro MtbPtpB inhibition. MtbPtpB is a
virulence factor from Mycobacterium tuberculosis, which was cloned, expressed, and purified as
recently described.7 IC50 values were the average of three independent experiments. The obtained
results are shown in Table 1.
8

Table 1. IC50 values of are shown for MtbPtpBa
Compd. Phenyl ring
IC50, μM
Compd. Phenyl ring
IC50, μM
HO
8a
47.03±1.81
8m
35.95±2.29
O2N
O2N
OH
8b
45.01±2.32
8n
28.96±3.13
8c
33.03±2.53
35.13±2.24
8o
8p
29.02±3.14
28.24±2.63
MeO
MeO
NO2
8d
Cl
OMe
8e
44.02±2.53
8q
35.04±2.61
Cl
Cl
Cl
Me
N
Me
8f
35.3±2.2
8r
8s
32.04±2.11
32.51±2.13
MeO
8g
9.52±1.13
MeO
Cl
EtO
HO
8h
8i
33.21±3.32
n.a.
8t
7.94±0.23
7.51±0.33
2.22±0.23
MeO
HO
8u
8v
Br
MeO
HO
MeO
8j
n.a.
Me
9

Compd. Phenyl ring
IC50, μM
Compd. Phenyl ring
IC50, μM
HO
EtO
8k
n.a.
8x
3.53±0.19
iPr
MeO
HO
O2N
8l
35.34±3.10
8y
1.56±0.21
HO
n.a = not active (no inhibition up to a concentration of 100 μM).
a All IC50 values were calculated from at least three independent measurements.
Amongst twenty-four screened compounds, six 1H-1,2,3-triazoles 8g, 8t, 8u, 8v, 8x, and 8y displayed
inhibitory activity against MtbPtpB with IC50 ranging from 1.56 to 9.52 μM (Table 1). The remaining
compounds with IC50 values >100 μM did not exhibit inhibitory activity. Among the above mentioned
active compounds, the compounds having more potent inhibitory activity against MtbPtpB (with IC50
values <5 μM), 8v, 8x, and 8y, had a hydroxyl group on para-position and methoxy, ethyl groups on
meta-position of the benzene ring. The IC50 values of these compounds were 2.22, 3.53, and 1.56 μM,
respectively. The 1H-1,2,3-triazoles 8v and 8y had the highest inhibitory activity against MtbPtpB. In
these cases, the presence of the second substituent in meta-position helped to increase the activity
methoxy or nitro groups). Especially, the presence of nitro on meta-position in 8y (IC50 = 1.56 μM)
enhanced significantly the inhibitory activity against MtbPtpB of this compound compared to
compound 8v (IC50 = 2.22 μM), whereas the methoxy or nitro substituents located (at) the ortho- or
para-positions made the inhibitory activity decreasing. The situation happened in 1H-1,2,3-triazoles 8d
(IC50 = 35.13 μM) and 8q (IC50 = 35.04 μM). The absence of the hydroxyl group in para-position in
compound 8s also reduced the inhibitory activity (its IC50 value of 32.51 μM).
10

Determination of the mode of inhibition of MtbPtpB by the identified inhibitors
The type of inhibition with respect to the substrate p-nitrophenyl phosphate (pNPP) was determined for
the most active compounds (inhibitors) 8x, 8y, and 8v. The obtained kinetic measurements showed that
the inhibitors exhibited degrees of saturation and release of p-nitrophenol following a classic
Michaelis-Menten enzymatic mechanism.39, 40 For compounds 8x, 8y, and 8v, all lines converged at the
y-axis (1/Vmax) in Lineweaver-Burk double-reciprocal plots (Fig. 3), and the slope (KMapp/Vmax) and x-
axis interception (−1/KMapp) according to the inhibitor concentrations. The inhibition constants Ki
obtained for 8x, 8y, and 8v were of 2.16, 1.10, and 1.80 μM, respectively (Table 2). Therefore, the
constant value of Vmax and the increased values of KMapp are consistent with a competitive inhibition
mechanism of the evaluated compounds.39
Table 2. Ki values, IC50/Ki ratio and type of inhibition of the MtbPtpB inhibitors.
Compd. Ki (μM)
IC50 (μM)
2.22±0.23
3.53±0.19
1.56±0.21
IC50/Ki
1.23
Type of inhibition
Competitive
8v
8x
8y
1.80 ± 0.07
2.16 ± 0.14
1.10 ± 0.09
1.63
Competitive
1.42
Competitive
11

12

Figure 3. Competitive inhibitory profile of the most active compounds 8v, 8x, and 8y. Lineweaver-
Burk double-reciprocal plots represented inhibitory profiles of these compounds against MtbPtpB.
Kinetic experiments were conducted in the presence of increasing concentrations of inhibitors: 0 μM
(), 1 μM (), 1.5 μM (), 2 μM (), 3 μM (△), 4 μM (); pNPP was used as substrate in all
experiments.
Based on the results obtained from the enzymatic assays, molecular modeling studies were performed
as a step toward understanding the interaction mode of these compounds as inhibitors. We examined
the docking of (the) highest inhibitory activity compounds 8v, 8x, and 8y. Simultaneously, compounds
more active 8g, 8t, and 8u, which had IC50 <10 μM, were also considered in the molecular docking
study. Crystal structure of M. tuberculosis protein tyrosine phosphatase MtbPtpB, was retrieved from
the RCSB Protein Data Bank (https://www.rcsb.org/structure/2oz5), in complex with the specific
inhibitor OMTS (PDB code: 2OZ5).41 The favorably docked molecules were ranked according to the
XP Glide Score (Table 3). Compound 8y gave a better glide score when compared with other five
compounds for the target protein, with binding score of −12.215 kcal/mol. The important
intermolecular protein-ligand interactions of compound 8y are depicted in Figure 4, and the ones of the
13

remaining compounds were represented in the figures in Section 6 (in Supplementary Data in online
version of this article). There were intermolecular amino acid interactions with compounds 8g, 8t, 8u,
8v, 8x, and 8y. The ligand-amino acid interaction of residue Ser57 always took place in all the ligands.
Both Ser57 and Hid94 had hydrogen-bonding interactions with C=O acetate of D-glucose moiety on
positions 6 and 4, respectively. Residue Phe98 always had π-π interactions with 4H-pyrano[2,3-
d]pyrimidine ring of all the ligands. This suggested that π-π interaction improved the docking scores. In
ligands 8t, 8u and 8v, there was a supplemental π-π interaction of this residue to the benzene ring.
However, this one may seem unimportant in interactions with enzyme because docking score in these
cases decreased insignificantly.
Table 2. Molecular docking analysis of protein target 2OZ5 with selected compounds 8g, 8t, 8u, 8v,
8x, and 8y
Compd. Phenyl ring
Glide
IC50 (μM) Compd. Phenyl ring
Glide
IC50 (μM)
score*
−9.881
score*
−10.220
Cl
EtO
HO
8g
9.52±1.13 8t
7.51±0.33 8v
7.94±0.23
MeO
HO
MeO
HO
8u
8x
−10.006
−10.435
−12.215
2.22±0.23
1.56±0.21
MeO
MeO
HO
EtO
−10.337
3.53±0.19 8y
HO
O2N
* In kcal/mol.
14

Figure 4. Amino acids involved in intermolecular interactions of 1H-1,2,3-triazole 8y.
(a)
(b)
15

o(c)
Figure 5. (a,b) Compound 8y (in red color) in MTB phosphotyrosine phosphatase B protein (PDB ID:
2OZ5). (c) Alignment of compound 8y in the binding pocket (in residue-charge color scheme).
There was a π-cation interaction (electrostatic interaction) via imine group between residue Arg166 and
1H-1,2,3-triazole ring in case of compound 8v. This diminished its docking score (−10.435 kcal/mol).
Residues Ser57, Arg59, Arg63, and Hid94 interacted with compound 8y showing high ligand exposure.
Phe98 had two π-π interactions with the compound 8y, one with pyran ring, and another with
pyrimidine ring. Residue Arg63 interacted electrostatically with anion type of para-hydroxyl group via
a salt bridge of iminium cation and Arg59 interacted with this anion via hydrogen-bonding interaction.
This resulted in compound 8y showing the highest binding energy values of −12.215 kcal/mol. The
electrostatic interaction in compounds 8v and 8y also perfected the docking scores. Compound 8y is
bound to the active site amino acid residues in the pocket region as shown in Figure 5a,b. The pocket
region of 2OZ5 is present in a loop region encompass by alpha helix chains seen in Figure 5b (8y in red
color). The location and orientation of the triazole group were complementary to the surrounding
MtbPtpB side chains, which created a specific binding pocket. These observations indicated that
compound 8y could have an important role in anchoring within the active site of the receptor.
16

In summary, a series of novel 4H-pyrano[2,3-d]pyrimidine bearing D-glucose moiety tethered 1,2,3-
triazole derivatives were synthesized via an easy and convenient synthetic protocol starting from
substituted benzaldehydes, ethyl acetoacetate, and malononitrile. The synthesis of 24 analogs 8a-y
accomplished in four-step sequences using click chemistry in the key step. Their structures were fully
characterized by IR, NMR and mass spectral data. The in vitro inhibition evaluation on MtbPtpB study
of all the compounds revealed six compounds found to be active against M. tuberculosis PtpB.
Amongst six 1H-1,2,3-triazoles 8g, 8t, 8u, 8v, 8x, and 8y displayed inhibitory activity against MtbPtpB
with IC50 ranging from 1.56 to 9.52 μM, compound 8y is the most potent compound in vitro with an
IC50 value of 1.56 μM. Kinetic studies of the most active compounds 8v, 8x, and 8y showed the
competitive inhibition of toward the MtbPtpB enzyme. Cross-docking studies revealed compound 8y to
be more effective against Mycobacterium tuberculosis protein tyrosine phosphatase. Docking results
indicated that Ser57, Arg59, Hid94, and Phe98 amino acids in the binding pocket as potential ligand
binding hot-spot residues.
Acknowledgements
This research is funded by Vietnam National Foundation for Science and Technology Development
(NAFOSTED) under grant number 104.01-2017.02.
Supplementary data
Supplementary data (experimental section, IR, 1H NMR, 13C NMR and ESI-Mass spectral data for the
synthesized compounds), and also ligand-docking results associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl...
17

References
1. Dye C. Global epidemiology of tuberculosis. Lancet. 2006;367(9514):938−940.
https://doi.org/10.1016/S0140-6736(06)68384-0.
2. Beyer P, Moorthy V, Paulin S, et al. The drugs don't work: WHO's role in advancing new
antibiotics. Lancet. 2018;392(10144):264−266. https://doi.org/10.1016/S0140-6736(18)31570-8.
3. Zignol M, Dean AS, Falzon D, et al. Twenty Years of Global Surveillance of Antituberculosis-
Drug Resistance. New Eng J Med. 2016;375(11):1081−1089.
https://doi.org/10.1056/NEJMsr1512438.
4. WHO. Global tuberculosis report 2017. 2017.
http://www.who.int/tb/publications/global_report/en/.
5. Bialy L, Waldmann H. Inhibitors of Protein Tyrosine Phosphatases: Next-Generation Drugs?
Angew Chem Int Ed. 2005;44(25):3814−3839. https://doi.org/10.1002/anie.200461517.
6. Lim WA, Pawson T. Phosphotyrosine Signaling: Evolving a New Cellular Communication
System. Cell. 2010;142(5):661−667. https://doi.org/10.1016/j.cell.2010.08.023.
7. Koul A, Herget T, Klebl B, Ullrich A. Interplay between mycobacteria and host signalling
pathways. Nat Rev Microbiol. 2004;2:189−202. https://doi.org/10.1038/nrmicro840.
8. Cole ST, Brosch R, Parkhill J, et al. Deciphering the biology of Mycobacterium tuberculosis from
the complete genome sequence. Nature. 1998;393:537−544. https://doi.org/10.1038/31159.
9. Caselli A, Paoli P, Santi A, et al. Low molecular weight protein tyrosine phosphatase: Multifaceted
functions of an evolutionarily conserved enzyme. Biochim Biophys Acta Proteins Proteom.
2016;1864(10):1339−1355. https://doi.org/10.1016/j.bbapap.2016.07.001.
10. Mascarello A, Chiaradia-Delatorre LD, Mori M, Terenzi H, Botta B. Mycobacterium tuberculosis-
Secreted Tyrosine Phosphatases as Targets Against Tuberculosis: Exploring Natural Sources in
Searching for New Drugs. Curr Pharm Des. 2016;22(12):1561−1569.
http://dx.doi.org/10.2174/1381612822666160112130539.
18

11. Dutta NK, He R, Pinn ML, et al. Mycobacterial Protein Tyrosine Phosphatases A and B Inhibitors
Augment the Bactericidal Activity of the Standard Anti-tuberculosis Regimen. ACS Infect Dis.
2016;2(3):231−239. https://doi.org/10.1021/acsinfecdis.5b00133.
12. Wong D, Bach H, Sun J, Hmama Z, Av-Gay Y. Mycobacterium tuberculosis protein tyrosine
phosphatase (PtpA) excludes host vacuolar-H+–ATPase to inhibit phagosome acidification. Proc
Natl Acad Sci U S A. 2011;108(48):19371−19376. https://doi.org/10.1073/pnas.1109201108.
13. Valderrama JA, Colonelli P, Vásquez D, González MF, Rodríguez JA, Theoduloz C. Studies on
quinones. Part 44: Novel angucyclinone N-heterocyclic analogues endowed with antitumoral
activity. Biorg Med Chem. 2008;16(24):10172−10181. https://doi.org/10.1016/j.bmc.2008.10.064.
14. Kamdar NR, Haveliwala DD, Mistry PT, Patel SK. Design, synthesis and in vitro evaluation of
antitubercular and antimicrobial activity of some novel pyranopyrimidines. Eur J Med Chem.
2010;45(11):5056−5063. https://doi.org/10.1016/j.ejmech.2010.08.014.
15. Vekariya RH, Patel KD, Vekariya MK, et al. Synthesis of pyrazolo[4ʹ,3ʹ:5,6]pyrano[2,3-
d]pyrimidine derivatives and their antimicrobial, antimalarial and antituberculosis evaluation.
Indian J. Chem. 2018;57B:997−1005. http://nopr.niscair.res.in/handle/123456789/44744
16. Bhat AR, Shalla AH, Dongre RS. Microwave assisted one-pot catalyst free green synthesis of new
methyl-7-amino-4-oxo-5-phenyl-2-thioxo-2,3,4,5-tetrahydro-1H-pyrano[2,3-d]pyrimidine-6-
carboxylates as potent in vitro antibacterial and antifungal activity. J Adv Res. 2015;6(6):941−948.
https://doi.org/10.1016/j.jare.2014.10.007.
17. Shamroukh AH, Zaki MEA, Morsy EMH, Abdel-Motti FM, Abdel-Megeid FME. Synthesis,
Isomerization, and Antimicrobial Evaluation of Some Pyrazolopyranotriazolopyrimidine
Derivatives. Arch Pharm. 2007;340(7):345−351. https://doi.org/10.1002/ardp.200700007.
18. Sabry NM, Mohamed HM, Khattab ESAEH, Motlaq SS, El-Agrody AM. Synthesis of 4H-
chromene, coumarin, 12H-chromeno[2,3-d]pyrimidine derivatives and some of their antimicrobial
19

and cytotoxicity activities. Eur J Med Chem. 2011;46(2):765−772.
https://doi.org/10.1016/j.ejmech.2010.12.015.
19. Khajeh Dangolani S, Panahi F, Tavaf Z, Nourisefat M, Yousefi R, Khalafi-Nezhad A. Synthesis
and Antioxidant Activity Evaluation of Some Novel Aminocarbonitrile Derivatives Incorporating
Carbohydrate Moieties. ACS Omega. 2018;3(8):10341−10350.
https://doi.org/10.1021/acsomega.8b01124.
20. Paliwal PK, Jetti SR, Jain S. Green approach towards the facile synthesis of
dihydropyrano(c)chromene and pyrano[2,3-d]pyrimidine derivatives and their biological
evaluation. Med Chem Res. 2013;22(6):2984−2990. https://doi.org/10.1007/s00044-012-0288-3.
21. Kalaria PN, Satasia SP, Raval DK. Synthesis, characterization and biological screening of novel 5-
imidazopyrazole incorporated fused pyran motifs under microwave irradiation. New J Chem.
2014;38(4):1512−1521. https://doi.org/10.1039/C3NJ01327H.
22. Agalave SG, Maujan SR, Pore VS. Click Chemistry: 1,2,3-Triazoles as Pharmacophores. Chem
Asian J. 2011;6(10):2696−2718. https://doi.org/10.1002/asia.201100432.
23. Dheer D, Singh V, Shankar R. Medicinal attributes of 1,2,3-triazoles: Current developments.
Bioorg Chem. 2017;71:30−54. https://doi.org/10.1016/j.bioorg.2017.01.010.
24. Shaikh MH, Subhedar DD, Nawale L, et al. 1,2,3-Triazole derivatives as antitubercular agents:
synthesis, biological evaluation and molecular docking study. MedChemComm. 2015;6(6):1104-
1116. 10.1039/C5MD00057B.
25. Yu J-L, Wu Q-P, Zhang Q-S, Liu Y-H, Li Y-Z, Zhou Z-M. Synthesis and antitumor activity of
novel 2′,3′-dideoxy-2′,3′-diethanethionucleosides bearing 1,2,3-triazole residues. Bioorg Med
Chem Lett. 2010;20(1):240−243. https://doi.org/10.1016/j.bmcl.2009.10.127.
26. Giffin MJ, Heaslet H, Brik A, et al. A Copper(I)-Catalyzed 1,2,3-Triazole Azide−Alkyne Click
Compound Is a Potent Inhibitor of a Multidrug-Resistant HIV-1 Protease Variant. J Med Chem.
2008;51(20):6263−6270. https://doi.org/10.1021/jm800149m.
20

27. Buckle DR, Rockell CJM, Smith H, Spicer BA. Studies on 1,2,3,-triazoles. 10. Synthesis and
antiallergic properties of 9-oxo-1H,9H-benzothiopyrano[2,3-d]-1,2,3-triazoles and their S-oxides. J
Med Chem. 1984;27(2):223−227. https://doi.org/10.1021/jm00368a021.
28. Guantai EM, Ncokazi K, Egan TJ, et al. Design, synthesis and in vitro antimalarial evaluation of
triazole-linked chalcone and dienone hybrid compounds. Biorg Med Chem.
2010;18(23):8243−8256. https://doi.org/10.1016/j.bmc.2010.10.009.
29. Sangshetti JN, Nagawade RR, Shinde DB. Synthesis of novel 3-(1-(1-substituted piperidin-4-yl)-
1H-1,2,3-triazol-4-yl)-1,2,4-oxadiazol-5(4H)-one as antifungal agents. Bioorg Med Chem Lett.
2009;19(13):3564−3567. https://doi.org/10.1016/j.bmcl.2009.04.134.
30. Pokrovskaya V, Belakhov V, Hainrichson M, Yaron S, Baasov T. Design, Synthesis, and
Evaluation of Novel Fluoroquinolone−Aminoglycoside Hybrid Antibiotics. J Med Chem.
2009;52(8):2243−2254. http://doi.org/10.1021/jm900028n.
31. Zhou B, He Y, Zhang X, et al. Targeting mycobacterium protein tyrosine phosphatase B for
antituberculosis agents. Proc Natl Acad Sci U S A. 2010;107(10):4573−4578.
https://doi.org/10.1073/pnas.0909133107.
32. Tan LP, Wu H, Yang P-Y, et al. High-Throughput Discovery of Mycobacterium tuberculosis
Protein Tyrosine Phosphatase B (MptpB) Inhibitors Using Click Chemistry. Org Lett.
2009;11(22):5102-5105. https://doi.org/10.1021/ol9023419.
33. Chittepu P, Sirivolu VR, Seela F. Nucleosides and oligonucleotides containing 1,2,3-triazole
residues with nucleobase tethers: Synthesis via the azide-alkyne ‘click’ reaction. Biorg Med Chem.
2008;16(18):8427−8439. https://doi.org/10.1016/j.bmc.2008.08.026.
34. Gupte A, Boshoff HI, Wilson DJ, et al. Inhibition of Siderophore Biosynthesis by 2-Triazole
Substituted Analogues of 5′-O-[N-(Salicyl)sulfamoyl]adenosine: Antibacterial Nucleosides
Effective against Mycobacterium tuberculosis. J Med Chem. 2008;51(23):7495−7507.
https://doi.org/10.1021/jm8008037.
21

35. Kumar KK, Seenivasan SP, Kumar V, Das TM. Synthesis of quinoline coupled [1,2,3]-triazoles as
a promising class of anti-tuberculosis agents. Carbohydr Res. 2011;346(14):2084−2090.
https://doi.org/10.1016/j.carres.2011.06.028.
36. Bhat AR, Dongre RS, Naikoo GA, Hassan IU, Ara T. Proficient synthesis of bioactive annulated
pyrimidine derivatives: A review. J Taibah Univ Sci. 2017;11(6):1047−1069.
https://doi.org/10.1016/j.jtusci.2017.05.005.
37. Mollashahi E, Nikraftar M. Nano-SiO2 catalyzed three-component preparations of pyrano[2,3-
d]pyrimidines, 4H-chromenes, and dihydropyrano[3,2-c]chromenes. J Saudi Chem Soc.
2018;22(1):42−48. https://doi.org/10.1016/j.jscs.2017.06.003.
38. Boda SK, Pishka V, Lakshmi PVA, Chinde S, Grover P. 1,2,3-Triazole Tagged 3H-Pyrano[2,3-
d]pyrimidine-6-carboxylate Derivatives: Synthesis, in Vitro Cytotoxicity, Molecular Docking and
DNA Interaction Studies. Chem Biodivers. 2018;15(6):e18000101.
https://doi.org/10.1002/cbdv.201800101.
39. Copeland RA. Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide for Medicinal
Chemists and Pharmacologists. Second Edition ed.: John Wiley and Sons; 2013:25−95.
40. Guo X-L, Shen K, Wang F, Lawrence DS, Zhang Z-Y. Probing the Molecular Basis for Potent and
Selective Protein-tyrosine Phosphatase 1B Inhibition. J Biol Chem. 2002;277(43):41014−41022.
https://doi.org/10.1074/jbc.M207347200.
41. Grundner C, Perrin D, Hooft van Huijsduijnen R, et al. Structural Basis for Selective Inhibition of
Mycobacterium tuberculosis Protein Tyrosine Phosphatase PtpB. Structure. 2007;15(4):499−509.
https://doi.org/10.1016/j.str.2007.03.003.
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24

Figure 3. Competitive inhibitory profile of the most active compounds 8v, 8x, and 8y. Lineweaver-
Burk double-reciprocal plots represented inhibitory profiles of these compounds against MtbPtpB.
Kinetic experiments were conducted in the presence of increasing concentrations of inhibitors: 0 μM
(), 1 μM (), 1.5 μM (), 2 μM (), 3 μM (△), 4 μM (); pNPP was used as substrate in all
experiments.
25

Figure 4. Amino acids involved in intermolecular interactions of 1H-1,2,3-triazole 8y.
26

(a)
(b)
o(c)
Figure 5. (a,b) Compound 8y (in red color) in MTB phosphotyrosine phosphatase B protein (PDB ID:
2OZ5). (c) Alignment of compound 8y in the binding pocket (in residue-charge color scheme).
Highlights
 Novel thirty-four 1H-1,2,3-triazole-tethered 4H-chromene−D-glucose conjugates
 8g, 8t, 8u, 8v, 8x, and 8y: inhibitory activity IC50=1.56−9.52 μM against MtbPtpB
 Ser57, Arg59, Hid94, and Phe98 residues of 2OZ5 created the specific binding pocket
27

Graphic abstract
OAc
OAc
AcO
O
O
N
O
Ar
O
O
N
O
Ar
O
O
N
OAc
O
N
N
N
N
6a-y
8a-y
(64-94%)
OAc
O
AcO
AcO
8g 8t 8u 8v 8x 8y
:
IC50 = 1.56 - 9.52 M
(inhibitory activity against MtbPtpB)
,
,
,
,
,
N3
OAc
7
8y
Docking on 2OZ5 enzyme of ligand
28