Identification of novel thiourea-stilbene-triazine conjugates as persuasive lymphoid tyrosine phosphatase inhibitors

Article abstract of DOI:10.1002/jhet.4060

A library of novel thiourea-based symmetrical stilbene-triazines (5a-i) was synthesized in an effort to develop new protein tyrosine phosphatase LYP inhibitors. The versatile nature of 2,4,6-trichloro-1,3,5-triazine allows considerable scope for derivatization and hence exploration of structure activity relationships. A convenient and versatile three-step synthetic approach involved the successive replacement of the two chloro groups of 2,4,6-trichloro-1,3,5-triazine by a variety of substituents for structural modification. The newly synthesized derivatives were subjected to tyrosine phosphatase LYP inhibition studies. The results for the in vitro bioassays were promising with the identification of compound 5k and 5l having a 4-methyl and 4-methoxy substituent on phenyl ring, as the lead and selective candidate for LYP inhibition with an IC₅₀ value of 2.1 ± 0.05 μM and 28 ± 3.3 μM, respectively. Moreover, docking studies were carried out to determine the possible interaction sites of thiourea-based stilbene-triazine compounds with Lymphoid Tyrosine Phosphatase. Results of docking computations further ascertained the inhibitory potential of compound 5k and 5l. The results indicated that the compound 5k may serve as a structural model for the design of most potent LYP inhibitors.

Full text of DOI:10.1002/jhet.4060

Received: 16 April 2020
DOI: 10.1002/jhet.4060
Revised: 29 May 2020
Accepted: 5 June 2020
A R T I C L E
Identification of novel thiourea-stilbene-triazine conjugates
as persuasive lymphoid tyrosine phosphatase inhibitors
Iram Batool1 | Farukh Jabeen1 | Nadeem Ahmed Vellore2 | Ghulam Shabir1
Aamer Saeed1
|
1Department of Chemistry, Quaid-i-Azam
Abstract
University, Islamabad, Pakistan
2Huntsman Cancer Institute, University of
Utah, Salt Lake City, Utah
A library of novel thiourea-based symmetrical stilbene-triazines (5a-i) was syn-
thesized in an effort to develop new protein tyrosine phosphatase LYP inhibi-
tors. The versatile nature of 2,4,6-trichloro-1,3,5-triazine allows considerable
scope for derivatization and hence exploration of structure activity relation-
ships. A convenient and versatile three-step synthetic approach involved the
successive replacement of the two chloro groups of 2,4,6-trichloro-
1,3,5-triazine by a variety of substituents for structural modification. The newly
synthesized derivatives were subjected to tyrosine phosphatase LYP inhibition
studies. The results for the in vitro bioassays were promising with the identifi-
cation of compound 5k and 5l having a 4-methyl and 4-methoxy substituent
on phenyl ring, as the lead and selective candidate for LYP inhibition with an
IC50 value of 2.1 0.05 μM and 28 3.3 μM, respectively. Moreover, docking
studies were carried out to determine the possible interaction sites of thiourea-
based stilbene-triazine compounds with Lymphoid Tyrosine Phosphatase.
Results of docking computations further ascertained the inhibitory potential of
compound 5k and 5l. The results indicated that the compound 5k may serve
as a structural model for the design of most potent LYP inhibitors.
Correspondence
Prof Dr Aamer Saeed Department of
Chemistry, Quaid-i-Azam University,
Islamabad, 45320, Pakistan.
Email: asaeed@qau.edu.pk
Present address
Farukh Jabeen, Department of Chemistry
and Biochemistry Laurentian University,
935 Ramsey Lake Road, Sudbury, ON,
P3E 2C6, Canada
Funding information
University of Utah, College of Pharmacy
1 | INTRODUCTION
inhibition,[3]
hedgehog signaling pathway inhibition.[6] Heterobicyclic
nitrogen systems bearing
1,2,4-triazine moiety,[7]
and
anti-inflammatory,[4]
antimicrobial,[5]
Fluorescent brightening agents (FBAs) are dyes that absorb
light in the UV and violet region (340-370 nm) of the elec-
tromagnetic spectrum and re-emit light in the blue region
(typically 420-470 nm). These include five or six membered
heterocycles such as imidazolines, diazoles, triazoles,
benzoxazolines, coumarins, naphthalimides, pyrazines,
and triazines.[1,2] Stilbenes especially diphenyl stilbenes are
the most common structural motif used in these com-
pounds. The extensive pi-systems of these compounds are
associated with the closely spaced electronic energy levels
that allow for energy transitions within the visible range
(eg, n-pi transitions).[2] Moreover, 1,3,5-triazine derivatives
have been found to exhibit the variety of biological applica-
tions such as an antioxidant,[2] protein kinase CK2
a
6-substituted-2-b-galactosyl-1,2,4-triazine,[8]
2,4-diamino-1,3,5-triazine derivatives[9] have been reported
as potent anticancer inhibitors.
Triazine-stilbene hybrids are one of the important clas-
ses of FBAs and are effective for attaining high degrees of
whiteness on polyamide-6.[10] These have been used as
textile auxiliaries or in detergents.[11] The charge-transfer
complexes were also obtained by the interaction between
symmetrically substituted stilbene-triazine derivatives with
chloranilic acid. Stilbene-triazine derivatives comprising
the polymerizable groups have been used for radical poly-
merization of styrene. The photophysical and photochemi-
cal characteristics in aqueous and ethanol solution of
J Heterocyclic Chem. 2020;1–12.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals LLC.
1

2
BATOOL ET AL.
some stilbene-triazine fluorescent brighteners containing
2-hydroxyethylamino groups and their trans-cis isomers in
equilibrium have been studied.[12,13] The cytotoxic activity
of some stilbene-triazine derivatives containing amino acid
groups has also been reported.[14]
The reversible phosphorylation of tyrosine residues
play a key role in regulating numerous cellular functions
including cell division, gene transcription, transport of
materials across the cell membrane, cellular adhesion,
and activation of the immune response.[15] Control of
tyrosine phosphorylation is maintained through protein
tyrosine kinases (PTKs) and protein tyrosine phospha-
tases (PTPs) enzymes. These two enzyme classes operate
in opposition of each other, and the delicate balance of
their activity gives the cell control of the associated path-
ways. Disruption of this activity can impair cell function
and lead to a variety of negative consequences.
The PTP lymphoid tyrosine phosphatase (LYP) is a
105 kDa enzyme found in lymphocytes, where it func-
tions as a negative regulator in T cell receptor (TCR) sig-
naling through dephosphorylation of the kinases Lck,
Fyn, and Zap-70, resulting in reduced T cell activation.[16
Two single nucleotide polymorphism (SNP) mutations
found in LYP are of therapeutic interest as they have
been shown to be associated with changes in disease risk.
The SNP G778A produces the R263Q mutant, which
leads to a catalytic site restructuring that is believed to be
the cause of an observed reduction in enzymatic activity
found with this mutant.[17] R263Q has been associated
with a reduced risk for several diseases, including rheu-
matoid arthritis,[18] systemic lupus erythematosus,[17]
and ulcerative colitis.[19] The SNP C1858T results in a
R620W mutation, which occurs in a region responsible
for binding to Csk,[20] a tyrosine kinase, which also acts
as a negative regulator of TCR signaling.[21] It is currently
believed that R620W results in increased TCR signaling
suppression through a gain-of-function, but the impact
this mutation has on LYP activity and the mechanism by
which it functions is currently unclear.[22] In contrast to
the R263Q, R620W has been implicated in an increased
prevalence of a number of diseases including type
1 diabetes,[20] rheumatoid arthritis,[23] systemic lupus
erythematosus,[24] Graves' disease, and others.[25–27]
The apparent relationship between LYP activity and
disease risk suggests that LYP may be a valuable drug tar-
get for inhibitor development. Over the past few five years,
a number of LYP inhibitors have been reported in the lit-
erature. He et al. reported on the discovery of a salicylic
acid based inhibitor (A) showing both excellent potency
and selectivity. The compound was found to have an IC50
value of 0.259 μM against LYP and over 9-fold specificity
against other PTPs.[28] Both bone marrow derived mast
cells and mouse models showed that (A) inhibited the
granulation of mast cells, suggesting that it has in vivo effi-
cacy. The discovery that this compound was found to bind
to both the catalytic site as well as several nearby pockets
was cited as the reason for its effectiveness. Stanford et al.
described a non-competitive inhibitor (B) which does not
use a phosphomimetic in its structure.[29] Mapping studies
suggested a mechanism of inhibition in which (B) binds to
an allosteric binding pocket rather than the catalytic site.
This was supported by experiments, which showed that
both B and C a known catalytic site inhibitor could induce
inhibition simultaneously. The molecule was found to
have moderate potency (IC50 = 5.28 μM) and cell perme-
ability, but only showed selectivity towards HePTP.
Kulkarni et al identified several LYP inhibitors from the
Spectrum Collection, a compound library containing 2000
chemicals.[30] In addition to a number of known PTP
inhibitors, several new inhibitors were identified, includ-
ing epigallocatechin 3,5-digallate (EGCDG), which was
found to be very potent against LYP (IC50 = 50 nM) and
selectivity against CD45 but not PTP-PEST. Incubation at
500 nM with Jurkat T cells resulted in increased Zap-70
phoare potentssphorylation, suggesting that EGCDG can
inhibit LYP in vivo. On the other hand, various triazine
derivatives have been reported as potent inhibitors of
P13K; sulfonyl stilbene derivatives are HIV inhibitors
whilst the bisthioureas are NNRT inhibitors (Figure 1).
Thus, design and synthesis of new molecules incorpo-
rating all the three bioactive components in a single struc-
tural unit leading to a new series of C-2 symmetric novel
molecules as a new entry to potent and selective LYP
inhibitors was intended (Scheme 2).Using an in vitro-LYP
activity assay, these compounds were tested for their effi-
cacy in inhibiting LYP and the potential of this scaffold as
future inhibitors development is discussed.
2 | RESULTS AND DISCUSSION
2.1 | Synthesis of thiourea-based
stilbene-triazine derivatives (5a-l)
The new thiourea-based stilbene-triazine derivatives were
prepared by multistep synthetic pathway as described in
Scheme 1. Synthesis was achieved by reacting
2,4,6-trichloro-1,3,5-triazine with separately synthesized
thioureas (2a-l). This reaction resulted in the formation
of dichlorotriazinyl intermediates (3a-l), which were then
condensed with diaminostilbene disulfonic acid (4) to
afford bis-monochlorotriazines (5a-l) in good yields. The
maintenance of low temperature and pH was found to be
most critical for the successful coupling of 2,4,6-trichloro-
1,3,5-triazine with thioureas. The newly synthesized mol-
ecules contain bisthiourea, stilbene sulfonic acid, and

BATOOL ET AL.
3
FIGURE 1 Some lead inhibitors of (LYP) and bioactive triazines, stilbenes, and bis-thioureas
Cl
N
Cl
N
N
R
H
N
R
R
H
N
H
NH2
Cl
N
N
Cl
Cl
KSCN/Acetone
Aq NH3
Acetone, 0-4 oC
4h
O
S
O
1a-l
O
S
N
N
2a-l
3a-l
Cl
HO3S
NH2
pH=6-6.5
40-45 oC
4h
4
H2N
SO3H
R
H
H
H
HO3S
N
N
N
N
Cl
N
N
N
S
O
O
S
N
N
Cl
N
H
N
N
SO3H
R
H
H
5a-l
SCHEME 1 Synthesis of thiourea-based stilbene-triazine derivatives (5a-l)
triazine scaffolds in a single structural unit. Table 1 lists
the variety of substituents attached to phenyl ring of
these symmetrical molecules.
FTIR analysis data of compounds 5a-l showed the
peaks in the range of 3372 to 3465 cm−1 due to the
stretching vibrations of NH groups and the characteristic

4
BATOOL ET AL.
TABLE 1 Substituents attached to phenyl ring of hybrid molecules (3a-l) and (5a-5L) and their percentage yields
Compound
R
Yield (%)
Compound
R
Yield (%)
3a
3b
3c
3d
3e
3f
H
81
75
69
70
73
78
79
79
77
78
74
72
5a
5b
5c
5d
5e
5f
H
68
71
65
75
70
67
69
72
78
58
79
67
2-OH,3,4-diNO2
3,4,5-tri-OCH3
2-F,3-OCH3,4-Br
3,5-diOH
2-Br
2-OH,3,4-diNO2
3,4,5-tri-OCH3
2-F,3-OCH3,4-Br
3,5-diOH
2-Br
3g
3h
3i
2-CH3
5g
5h
5i
2-CH3
2-OH
2-OH
2-NO2
2-NO2
3j
4-NO2
5j
4-NO2
3k
3l
4-CH3
5 k
5l
4-CH3
4-OCH3
4-OCH3
peaks for S O and C O were observed in the range of
1035 to 1098 cm−1 and 1703 to 1714 cm−1, respectively.
1
In H NMR spectra, characteristic broad signal appeared
at δ 11.22 to 11.39 ppm indicating the presence of NH
protons and the signals for aromatic and olefinic protons
appeared in the range of δ 6.44 to 8.39 ppm. 13C NMR
spectral data showed the most deshielded carbon at δ
179.62 to 175.71 ppm assigned to C S, whereas C O car-
bon resonated in the range of δ 167.89 to 168.81 ppm. Sig-
nals appearing at δ 126.23 to 126.63 ppm were assigned
to olefinic carbons, while those in the range δ 112.25 to
151.89 ppm correspond to aromatic carbon atoms
(Supporting Figures S1-S16).
2.2 | LYP inhibition screening
Dephosphorylation of the compound 6,8-difluoro-
4-methylumbelliferyl phosphate (DiFMUP) to the
fluorophoric molecule 6,8-difluoro-4-methylumbelliferyl
(DiFMU) is an established PTP activity assay.[31] Com-
pound inhibition effectiveness was determined by mea-
suring the extent of dephosphorylation of DiFMUP by
LYP. The synthesized compounds were initially screened
at a concentration of 100 μM. Of the twelve synthesized
compounds, 5b, and 5c were found to be only partially
soluble in DMSO at the required concentrations and were
thus excluded from testing. Additionally, compounds 5g
and 5i were found to have reached maximum fluores-
cence at the first read, preventing determination of inhi-
bition values for these compounds. Results of the initial
screening are presented in bar graph shown in Figure 2.
The figure shows the percentage inhibition at ordinate
(y-axis), while abscissa shows the compounds tested.
FIGURE 2 Percent inhibition of LYP activity by synthesized
compounds in the initial screen. Compounds were tested at a
concentration of 100 μM
Compounds 5k and 5l showed potent inhibition with
reductions of 100% and 95.3% in LYP activity, respec-
tively. Interestingly compound 5g, which contains a
methyl group in the 2 positions as opposed to the 4 posi-
tion as seen in compound 5k, showed a much smaller
percent inhibition, suggesting that a substituent in the
4 position plays a critical role in the function of the mole-
cule. Both compounds 5k and 5l contain non-charged
substituents (methyl and methoxide), but due to the fact

BATOOL ET AL.
5
that the nitro containing compound 5j could not be suc-
cessfully tested it is unknown if or how formal charge at
the 4 position would impact inhibition.
Figure 5A,B represents the docking pose of ligand 5l
inside the active site of LYP in tri and di dimensional
space, respectively. The active site is surrounded by the
key amino acid residues including, Lys32, Ser35, Lys39,
Tyr44, Thr46, Asp62, Ile63, Tyr66, Cys129, Met130,
Tyr132, Glu133, Met134, Lys191, Asn192, Trp193, Ser227,
Ser228, Ala229, Gly230, Cys231, Gly232, Arg233, Thr234,
Arg266, Ser271, Pro270, Gln274, Thr275, Glu277, Gly278.
In our docking model, the carbonyl oxygen in ligands 5l
made close contact with the active site through copious
strong hydrophobic and hydrophilic interactions. The
enhanced affinity of ligand 5l towards the residues was
reflected by their strong interaction with residues such as
Lys32, Lys39, Asp62, Asn192, Ser227, Ser228, Ala229,
Arg233, Ser271, Gly278. Besides being directly involved
in the architecture of the active site, the residue plays a
vital role in positioning other key residues in the active
site appropriately for the catalysis. These residues
established strong interaction with each other through
hydrogen binding, van der Waals and other hydrophobic
and polar interactions. Residues are aligned around the
active site and are available to interact with the ligand
due to these interactions.
Compounds 5k and 5l showed IC50 values below
100 μM and were selected for IC50 testing. IC50 curves for
these compounds are shown in Figure 3. Compound 5k
and 5l are the most active compounds of the series, they
showed good inhibition potential and were selected for
the IC50 testing. Compound 5k found to be most potent
agent pf the series followed by compound 5l. The IC50
value for 5l was calculated to be 28 3.3 μM, while com-
pound 5k was found to be over 13 times more potent
with an IC50 value of 2.1 0.05 μM. To further under-
stand the binding interaction of these compounds, we
used computational docking of compounds to LYP pro-
tein. In particular, both compound 5k and 5l were found
to interact favorably with LYP protein with a docking
score of −9.5 and 10.2 kcal/mol (Scheme 2).
Based on the initial assessment of the binding pose,
we found that the sulfonate group docked at the P-loop
in all the cases providing as an anchoring point for the
flanking regions to interact with the protein (Figure 4). It
is also interesting to note that both compounds occupied
the adjacent binding site (LYP insert loop), while forming
a cation-pi interaction with K39 residue (of the LYP-
insert loop) and the terminal phenyl group of the com-
pound. While, the other end of the compound showed
differential binding interaction. The compound 5k
showed favorable contacts with the conserved WPD loop
while compound 5l interacted with β3 loop. Interestingly,
other compounds, such as 5a, 5d, 5e and 5g also showed
favorable docking score with the LYP protein.
The docking model represents that compounds ligand
5l showed a distinct orientation for interacting with the
active site residues LYP. The ligands anchored in active
site through determinant hydrogen bonding, where all
the three atoms of sulphur trioxide attached to the phenyl
ring of left half of the stilbene molecule showed the most
interesting pattern in anchoring the molecule inside the
active site by establishing 10 strong hydrogen bonding
with Ser228, Ala229, Lys230, Ser227, Cys231, Arg233 as
FIGURE 3 IC50 curves for compounds, A, 5k and, B, 5 L. The abscissa shows the concentration of the compound 5 k and 5 L in μM,
while ordinate represent the percentage inhibition. The IC50 values were calculated to be 2.1 0.05 μM and 28 3.3 μM, respectively

6
BATOOL ET AL.
SCHE ME 2 Structural
architecture of C-2 symmetrical
molecules and effect of
substituent on activity
FIGURE 4 Modeling of thiourea-based stilbene-triazine derivatives on LYP protein. Docked conformation of compound 5k and 5 l are
overlaid on LYP protein. The protein is represented using, A, secondary structure (with P-loop (red), Lyp-insert (blue), β3-loop (orange), and
WPD loop (green) highlighted) and, B, using the van der Waals surface
shown in Figure 5A,B. This part of the molecule got
privileged to be strengthen inside the active site by esta-
blishing further hydrogen bonding through both the
amine of thiourea and carbonyl oxygen with Asp62,
Lys32, Ser271, respectively, in addition to three strong
aromatic hydrogen bonding (Figure 4A) with the Lys39,
and Asp62 by terminal Phenyl ring, which consequently
provided the strong binding to the entire left half of the
molecule. The flanking region made close contacts by
establishing the π-π and π-H interactions with Arg233 as
shown in Figure 5A,B. Moreover, a hydrogen bonding of
amine of thiourea of the flanking region (right half of the
molecule) architected a hydrogen bonding with Asn192,
as displayed in Figure 5A,B, a well required contact,
playing a significant role in anchoring the flanking part
of the molecule inside the active site gorge.
Figure 5B displays the interaction of the ligand with
amino acid residues in active site in two dimensional
space. Polar amino acids are shown in pink color circle,
while amino acids basic in character are shown in blue
color. Lipophilic amino acids are shown in green color.
Interactions are shown in dotted lines in Figure 5A while
interactions are shown in both solid and dashed lines in
Figure 5B. Hydrogen bonding is shown in red color
dashed lines, Figure 5A, depicting the orientation of mol-
ecule in three dimensions. The direction of the arrow
shows the donor and acceptor (points toward acceptor).
Aren-aren and aren-H interactions are shown in green

BATOOL ET AL.
7
FIGURE 5 Binding mode of compounds 5k inside the active site of LYP. A, Docking poses of compound 5k in 3D space. Ligands are
shown in ball n stick mode in elemental and cyan color and key residues are shown by stick mode, elemental color, and green color,
respectively. Hydrogen bonding of ligand atoms with residue atoms is shown in red dashed lines. Aromatic hydrogen bonding is shown in
blue colored dashed lines. π -cation interactions are represented by green dashed lines. A, Docking pose of compound 5k inside the active
site in 2 dimensional space
and red solid lines in 2D image (Figure 5B) while these
are shown in green colored dashed lines and cyan colored
dashed lines in tri dimensional space (Figure 5A).
Although docking simulation were useful in under-
standing the interaction of these compounds on LYP pro-
tein; however it was not helpful in explaining some of

8
BATOOL ET AL.
the differences in the binding observed experimentally.
This is due to the fact that these compound exhibit large
molecular size with too many rotatable bonds, which
often present challenges in accurate sampling and scor-
ing of the poses. In future studies, we would incorporate
molecular simulation to understand the induced effect,
these flanking region of the compound exerts on the LYP
protein. Additionally, the exact mechanism by which the
methoxide reduces inhibitor efficiency will likely play a
key role in optimizing future inhibitors in this molecule
class.
DMSO-d6): δ 11.25 (br s, 2H, NH), 7.99-7.93 (m, 2H),
7.53-7.45 (m, 2H), 7.31 (tt, J = 7.5, 2.0 Hz, 1H). 13C NMR
(75 MHz, DMSO-d6): δ 179.62 (C S), 167.10 (C O),
165.75 (C─Cl), 163.15(C N), 134.34-128.10 (Ar─C). Anal.
Calcd for C11H7Cl2N5OS: C, 40.26; H, 2.15; N, 21.34; S,
9.77. Found: C, 40.20; H, 2.22; N, 21.23; S, 9.85. EIMS cal-
culated for C11H7Cl2N5OS (M+•) 327.
2-Hydroxy-3,4-dinitrobenzoic acid derivative of
triazine (3b).
Mol.Wt: 433; Yellow solid, Rf 0.31; FTIR: 3520
(O─H), 3437 (N─H), 1705 (C O), 1603 (C N),1455
(NO2), 1059 (S O), 650 (C─Cl) cm−1; 1H NMR
(300 MHz, DMSO-d6): δ 11.22 (br s, 2H, NH), 8.27 (d,
J = 7.5 Hz, 1H), 7.80 (d, J = 7.5 Hz, 1H). 13C NMR
(75 MHz, DMSO-d6): δ 177.65 (C S), 168.25 (C O),
167.93(C─Cl), 163.15 (C N), 160.91(C─OH), 144.07(C-
3 | EXPERIMENTAL
3.1 | General procedure for the synthesis
of stilbene-triazine derivatives (5a-l)
NO2),
131.76-119.79
(Ar─C).Anal.
Calcd
for
C11H5Cl2N7O6S: C, 30.43; H, 1.16; N, 22.58; S, 7.38.
Found: C, 30.36; H, 1.22; N, 22.51; S, 7.43. EIMS calcu-
lated for C11H5Cl2N7O6S (M+•) 433.
3,4,5-Trimethoxybenzoic acid derivative of tri-
azine (3c).
Mol.Wt: 417; Light yellow solid, Rf 0.40; FTIR: 3422
(N─H), 1708 (C O), 1599 (C N), 1110 (C─O), 1073
(S O), 658 (C─Cl) cm−1; 1H NMR (300 MHz, DMSO-d6):
δ 11.24 (br s, 2H, NH), 7.22 (s, 2H), 3.90 (s, 6H), 3.84 (s,
3H). 13C NMR (75 MHz, DMSO-d6): δ 178.62 (C S),
169.13 (C O), 166.96 (C─Cl), 163.15(C N), 151.96
(C─O), 140.07-107.41 (Ar─C), 60.68 (O-CH3), 56.26 (O-
CH3). Anal. Calcd for C14H13Cl2N5O4S: C, 40.20; H,
3.13; N, 16.74; S, 7.67. Found: C, 40.12; H, 3.20; N,
16.70; S, 7.74.EIMS calculated for C14H13Cl2N5O4S
(M+•) 417.
To a well stirred acetone (10 mL) in round bottomed
flask (50 mL) was added cyanuric chloride (1, 0.38 g,
0.002 mol) at 4ꢀC to 5ꢀC in an ice bath. To this suspen-
sion was added an acetone solution (10 mL) of separately
synthesized thioureas 2a-l (0.002 mol) and stirred the
reaction mixture at temperature 4ꢀC to 5ꢀC for 4 hours
until completion of reaction was observed from TLC (PE:
EA, 4:1). The reaction mixture was allowed to stand at
room temperature for 1 hour, which resulted in settling
down of precipitates of intermediates 3a-l which were
dried at room temperature in the folds of filter paper and
calculated their yields.
The compounds 3a (0.001 mol) was suspended in dis-
tilled water (10 mL) at room temperature in 50 mL round
bottomed flask and started stirring the reaction mixture. The
aqueous solution (10 mL) of 4,4-diaminostilbenedisulfonic
acid (4) (0.0005 mol) was added to the above mixture. The
temperature was gradually increased to 40 to 45ꢀC and con-
tinued stirring the reaction at this temperature by
maintaining pH 6 to 6.5 with HCl. The reaction mixture was
stirred for 4 hours until completion of reaction was observed
from TLC (Chloroform: Methanol, 9:1). In this way bis-
monochlorotriazinyl derivatives 5a was synthesized. The
other derivatives of bis-monochlorotriazine 5b-l were
accomplished by using thiourea derivatives 3b-l. The char-
acteristic spectroscopic data of synthesized compounds 5a-l
and intermediate thioureas 3a-l is as follows.
2-Flouro-3-methoxy-4-bromobenzoic acid deriva-
tive of triazine (3d).
Mol.Wt: 453; Light yellow solid, Rf 0.36 FTIR: 3445
(N─H), 1710 (C O), 1601 (C N), 1234 (C─F), 1115
(C─O), 1072 (S O), 652 (C─Cl) cm−1; 1H NMR
(300 MHz, DMSO-d6): δ 11.29 (br s, 2H, NH), 7.69 (d,
J = 7.5 Hz, 1H), 7.38 (d, J = 7.5 Hz, 1H), 3.94 (s, 3H). 13
C
NMR (75 MHz, DMSO-d6): δ 177.87 (C S), 168.22
(C O), 166.22 (C─Cl), 166.16 (C─F), 163.15 (C N),
154.00-116.91 (Ar-C), 60.80 (O-CH3).Anal. Calcd for
C12H7BrCl2FN5O2S:C, 31.67; H, 1.55; N, 15.39; S, 7.04.
Found:C, 31.62; H, 1.61; N, 15.30; S, 7.14. EIMS calcu-
lated for C12H7BrCl2FN5O2S (M+•) 453.
3,5-Dihydroxybenzoic acid derivative of stilbene-
triazine (3e).
3.2 | Benzoic acid derivative of
triazine (3a)
Mol.Wt: 359; Light yellow solid, Rf 0.29; FTIR: 3510
(O─H), 3395 (N─H), 1716 (C O), 1602 (C N), 1108
(C─O), 1070 (S O), 654 (C─Cl) cm−1; 1H NMR
(300 MHz, DMSO-d6): δ 11.27 (br s, 2H, NH), 6.84 (s,
2H), 6.57 (s, 2H), 6.48 (s, 1H). 13C NMR (75 MHz, DMSO-
Mol.Wt: 326.9; Light yellow solid, Rf 0.31 FTIR: 3427
(N─H), 1712 (C O), 1596 (C N), 1038 (S O),
658 (C─Cl), 595 (C─Br) cm−1 1H NMR (300 MHz,
;

BATOOL ET AL.
9
d6): δ 178.85 (C S), 168.17 (C O), 166.96 (C─Cl), 163.74
(C N), 158.46-105.87 (Ar-C). Anal. Calcd for
C11H7Cl2N5O3S: C, 36.68; H, 1.96; N, 19.45; S, 8.90.
Found: C, 36.64; H, 1.99; N, 19.40; S, 8.98.EIMS calcu-
lated for C11H7Cl2N5O3S (M+•) 359.
C11H6Cl2N6O3S: C, 35.41; H, 1.62; N, 22.52; S, 8.59.
Found: C, 35.36; H, 1.66; N, 22.48; S, 8.65.EIMS calcu-
lated for C11H6Cl2N6O3S (M+•) 372.
4-Nitrobenzoic acid derivative of triazine (3j).
Mol.Wt: 372; Yellow solid, Rf 0.31; FTIR: 3438
(N─H), 1715 (C O), 1607 (C N), 1480 (NO2), 1071
(S O), 651 (C─Cl) cm−1; 1H NMR (300 MHz, DMSO-d6):
δ 11.20 (br s, 2H, NH), 8.28 (d, J = 7.8 Hz, 2H), 7.96 (d,
J = 7.8 Hz, 2H). 13C NMR (75 MHz, DMSO-d6): δ 176.92
(C S), 167.34 (C O), 165.75 (C─Cl), 163.15 (C N),
148.40-123.99 (Ar─C). Anal. Calcd for C11H6Cl2N6O3S: C,
35.41; H, 1.62; N, 22.52; S, 8.59. Found: C, 35.35; H,
1.67; N, 22.48; S, 8.64.EIMS calculated for C11H6Cl2N6O3S
(M+•) 372.
4-Methylbenzoic acid derivative of triazine (3k).
Mol.Wt: 341; Light yellow solid, Rf 0.40; FTIR: 3460
(N-H), 1705 (C O), 1598 (C N), 1420 (C─H), 1080
(S O). 650 (C─Cl) cm−1; 1H NMR (300 MHz, DMSO-d6):
δ 11.24 (br s, 2H, NH), 7.95 (d, J = 7.9 Hz, 2H), 7.38 (d,
J = 7.9 Hz, 2H), 2.30 (s, 3H). 13C NMR (75 MHz, DMSO-
d6): δ 175.71 (C S), 168.55 (C O), 165.75 (C-Cl), 163.15
(C N), 140.36 to 128.32 (Ar─C), 21.42 (CH3). Anal. Calcd
for C12H9Cl2N5OS: C, 42.12; H, 2.65; N, 20.47; S, 9.37.
Found: C, 42.06; H, 2.69; N, 20.40; S, 9.44.EIMS calcu-
lated for C12H9Cl2N5OS (M+•) 341.
4-Methoxybenzoic acid derivative of triazine (3l).
Mol.Wt: 357; Light yellow solid, Rf 0.34; FTIR: 3413
(N─H), 1710 (C O), 1598 (C N), 1108 (C─O), 1067
(S O), 658 (C─Cl) cm−1; 1H NMR (300 MHz, DMSO-d6):
δ 11.22 (br s, 1H, NH), 11.12 (br s, 1H, NH), 8.04 (d,
J = 7.7 Hz, 2H), 7.06 (d, J = 7.7 Hz, 2H), 3.80 (s, 3H). 13C
NMR (75 MHz, DMSO-d6): δ 176.62 (C S), 1167.93
(C O), 165.75 (C─Cl), 163.15 (C N), 162.21-114.06 (Ar-
C), 55.35 (O-CH3). Anal. Calcd for C12H9Cl2N5O2S: C,
40.24; H, 2.53; N, 19.55; S, 8.95. Found: C, 40.18; H,
2.58; N, 19.47; S, 9.02.EIMS calculated for C12H9Cl2N5O2S
(M+•) 357.
2-Bromobenzoic acid derivative of triazine (3f).
Mol.Wt: 405; Light yellow solid, Rf 0.33 FTIR: 3429
(N-H), 1711 (C O), 1590 (C N), 1078 (S O),
648 (C─Cl), 593 (C─Br) cm−1 1H NMR (300 MHz,
;
DMSO-d6): δ 11.28 (br s, 2H, NH), 7.82 (dd, J = 7.4,
2.1 Hz, 1H), 7.72 (dd, J = 7.4, 2.1 Hz, 1H), 7.39 (td,
J = 7.5, 2.1 Hz, 1H), 7.33 (td, J = 7.5, 2.0 Hz, 1H).13C
NMR (75 MHz, DMSO-d6): δ 177.59 (C S), 168.20
(C O), 166.30 (C─Cl), 164.35 (C N), 137.17-118.58
(Ar─C). Anal. Calcd for C11H6BrCl2N5OS: C, 32.46; H,
1.49; N, 17.20; S, 7.88. Found: C, 32.43; H, 1.53; N,
17.14; S, 7.95.EIMS calculated for C11H6BrCl2N5OS
(M+•) 405.
2-Methylbenzoic acid derivative of triazine (3g).
Mol.Wt: 341; Light yellow solid, Rf 0.38 FTIR: 3411
(N─H), 1705 (C O), 1595 (C N), 1440 (C─H), 1055
(S O), 662 (C─Cl) cm−1; 1H NMR (300 MHz, DMSO-d6):
δ 11.22 (br s, 2H, NH), 7.56 (dd, J = 7.2, 2.2 Hz, 1H),
7.51-7.44 (m, 1H), 7.38 (dd, J = 8.2, 6.3 Hz, 2H), 2.39 (d,
J = 1.0 Hz, 3H). 13C NMR (75 MHz, DMSO-d6): δ 177.47
(C S), 168.15 (C O), 167.99 (C─Cl), 162.27(C N),
137.76-127.02 (Ar─C), 19.98 (CH3). Anal. Calcd for
C12H9Cl2N5OS: C, 42.12; H, 2.65; N, 20.47; S, 9.37.
Found: C, 42.06; H, 2.69; N, 20.44; S, 9.40.EIMS calcu-
lated for C12H9Cl2N5OS (M+•) 341.
2-Hydroxybenzoic acid derivative of tri-
azine (3h).
Mol.Wt: 343; Light yellow solid, Rf 0.33; FTIR: 3505
(O─H), 3370 (N─H), 1710 (C O), 1610 (C N), 1105
(C─O), 1072 (S O), 656 (C─Cl) cm−1
;
1H NMR
(300 MHz, DMSO-d6): δ 11.30 (br s, 2H, NH), 7.73 (dd,
J = 7.5, 2.0 Hz, 1H), 7.43 (td, J = 7.5, 2.0 Hz, 1H), 7.04
(dd, J = 7.4, 2.1 Hz, 1H), 6.93 (td, J = 7.5, 2.0 Hz, 1H),
5.73 (s, 1H). 13C NMR (75 MHz, DMSO-d6): δ 178.76
(C S), 168.29 (C O), 167.96 (C-Cl), 163.15 (C N),
159.59-117.18 (Ar-C). Anal. Calcd for C11H7Cl2N5O2S: C,
38.39; H, 2.05; N, 20.35; S, 9.32. Found: C, 38.33; H,
2.00; N, 20.29; S, 9.40.EIMS calculated for C11H7Cl2N5O2S
(M+•) 343.
Benzoic acid derivative of stilbene-triazine (5a).
Mol.Wt: 997.8; Light yellow solid, Yield 68%; Rf 0.21
1
FTIR: 3414 (N-H), 1708 (C O), 1035 (S O) cm−1; H
NMR (300 MHz, DMSO-d6): δ 11.14 (br s, 3H, NH),
7.91-7.21 (Ar-H), 7.09 (d, 1H, CH CH); 13C NMR
(75 MHz, DMSO-d6): δ 177.36 (C S), 168.81 (C O),
164.37 (C─Cl), 146.82-113.12 (Ar-C), 126.42 (CH CH).
Anal. Calcd for C36H24Cl2N12Na2O8S4: C, 43.33; H,
2.42; N, 16.85; S, 12.85. Found: C, 43.41; H, 2.40; N,
16.79; S, 12.90. EIMS calculated for C36H24Cl2N12Na2O8S4
(M+•) 997.
2-Nitrobenzoic acid derivative of triazine (3i).
Mol.Wt: 372; Yellow solid, Rf 0.36; FTIR: 3421 (N-H),
1703 (C O), 1592 (C N), 1485 (NO2), 1094 (S O), 663
(C─Cl) cm−1 1H NMR (300 MHz, DMSO-d6): δ 11.33
;
(br s, 2H, NH), 8.43 (dd, J = 7.4, 2.0 Hz, 1H), 7.95 (dd,
J = 7.5, 2.0 Hz, 1H), 7.84 (td, J = 7.5, 2.0 Hz, 1H), 7.72
(td, J = 7.4, 2.0 Hz, 1H). 13C NMR (75 MHz, DMSO-d6): δ
179.53 (C S), 167.87 (C O), 165.20 (C─Cl), 163.15
(C N), 146.66-125.42 (Ar-C). Anal. Calcd for
2-Hydroxy-3,4-dinitrobenzoic acid derivative of
stilbene-triazine (5b).
Mol.Wt: 1209.8; Deep yellow solid, Yield 71%; Rf 0.19
1
FTIR: 3428 (N─H), 1703 (C O), 1049 (S O) cm−1; H
NMR (300 MHz, DMSO-d6): δ 11.15 (br s, 3H, NH),

10
BATOOL ET AL.
8.28-7.63 (Ar-H), 7.09 (d, 1H, CH CH), 5.13 (s, 1H, OH);
13C NMR (75 MHz, DMSO-d6): δ 177.15 (C S), 167.08
(C O), 165.65 (C─Cl), 159.27 (C─OH), 148.09 (C─NO2),
139.95-108.30 (Ar─C), 126.21 (CH CH). Anal. Calcd for
C36H20Cl2N16Na2O18S4: C, 35.74; H, 1.67; N, 18.52; S,
10.60. Found: C, 35.67; H, 1.62; N, 18.58; S, 10.51. EIMS
calculated for C36H20Cl2N16Na2O18S4 (M+•) 1209.
3,4,5-Trimethoxybenzoic acid derivative of
stilbene-triazine (5c).
C36H22Br2Cl2N12Na2O8S4: C, 37.42; H, 1.92; N, 14.54; S,
11.10. Found: C, 37.55; H, 1.96; N, 14.49; S, 11.21. EIMS cal-
culated for C36H22Br2Cl2N12Na2O8S4 (M+•) 1155.
2-Methylbenzoic acid derivative of stilbene-
triazine (5g).
Mol.Wt: 1025.8; Light yellow solid, Yield 69%; Rf 0.17
FTIR: 3405 (N─H), 1705 (C O), 1051 (S O) cm−1; 1H NMR
(300 MHz, DMSO-d6): δ 12.02 (br s, 3H, NH), 7.88-7.48
(Ar─H), 7.09 (d, 1H, CH CH), 2.92 (s, 3H, CH3); 13C NMR
(75 MHz, DMSO-d6): δ 178.14 (C S), 170.78 (C O),
163.14 (C─Cl), 154.13-102.10 (Ar─C), 136.56 (C─CH3),
126.13 (CH CH), 18.29 (CH3). Anal. Calcd for
C38H28Cl2N12Na2O8S4: C, 44.49; H, 2.75; N, 16.38; S, 12.50.
Found: C, 44.53; H, 2.80; N, 16.44; S, 12.45. EIMS calculated
for C38H28Cl2N12Na2O8S4 (M+•) 1025.
Mol.Wt: 1178; Light yellow solid, Yield 65%; Rf 0.20
1
FTIR: 3412 (N-H), 1709 (C O), 1062 (S O) cm−1; H
NMR (300 MHz, DMSO-d6): δ 11.32 (br s, 3H, NH),
8.70-7.80 (Ar-H), 7.09 (d, 1H, CH CH), 3.75 (s, 9H,
OCH3); 13C NMR (75 MHz, DMSO-d6): δ 178.28 (C S),
168.18 (C O), 161.35 (C─Cl), 150.97-149.43 (C-OCH3),
143.59-108.15 (Ar-C), 126.44 (CH CH), 57.69-57.22
(OCH3). Anal. Calcd for C42H36Cl2N12Na2O14S4: C,
42.82; H, 3.08; N, 14.27; S, 10.89. Found: C, 42.73; H,
3.14; N, 14.32; S, 10.92. EIMS calculated for
C42H36Cl2N12Na2O14S4 (M+•) 1178.
2-Hydroxybenzoic acid derivative of stilbene-
triazine (5h).
Mol.Wt: 1029.8; Light yellow solid, Yield 72%; Rf 0.22
1
FTIR: 3372 (N─H), 1714 (C O), 1075 (S O) cm−1; H
NMR (300 MHz, DMSO-d6): δ 11.24 (br s, 3H, NH),
7.90-7.22 (Ar-H), 7.09 (d, 1H, CH CH), 5.82 (s, 1H, OH);
13C NMR (75 MHz, DMSO-d6): δ 176.75 (C S), 168.26
(C O), 160.14 (C─OH), 158.41 (C─Cl), 146.93-110.12
2-Flouro-3-methoxy-4-bromobenzoic acid deriva-
tive of stilbene-triazine (5d).
Mol.Wt: 1251.6; Light yellow solid, Yield 75%; Rf 0.16
1
FTIR: 3441 (N─H), 1711 (C O), 1072 (S O) cm−1; H
(Ar─C),
126.30
(CH CH).
Anal.
Calcd
for
NMR (300 MHz, DMSO-d6): δ 11.66 (br s, 3H, NH),
7.90-7.28 (Ar-H), 7.09 (d, 1H, CH CH), 3.36 (s, 3H,
OCH3); 13C NMR (75 MHz, DMSO-d6): δ 177.97 (C S),
168.53 (C O), 160.11 (C─Cl), 152.02 (C─OCH3), 148.51
(C─F), 136.30-105.26 (Ar─C), 111.67 (C─Br), 55.62
(OCH3). Anal. Calcd for C38H24Br2Cl2F2N12Na2O10S4: C,
36.47; H, 1.93; N, 13.43; S, 10.25. Found: C, 36.52; H,
1.99; N, 13.49; S, 10.19. EIMS calculated for
C38H24Br2Cl2F2N12Na2O10S4 (M+•) 1251.
C36H24Cl2N12Na2O10S4: C, 41.99; H, 2.35; N, 16.32; S,
12.45. Found: C, 41.89; H, 2.39; N, 16.28; S, 12.39. EIMS
calculated for C36H24Cl2N12Na2O10S4 (M+•) 1029.
2-Nitrobenzoic acid derivative of stilbene-
triazine (5i).
Mol.Wt: 1087.8; Yellow solid, Yield 78%; Rf 0.19 FTIR:
3415 (N-H), 1702 (C O), 1098 (S O) cm−1; 1H NMR
(300 MHz, DMSO-d6): δ 11.80 (br s, 3H, NH), 7.78-7.23 (Ar-
H), 7.09 (d, 1H, CH CH); 13C NMR (75 MHz, DMSO-d6): δ
178.02 (C S), 168.67 (C O), 167.95 (C-Cl), 148.80 (C-NO2),
145.00-105.97 (Ar-C), 126.10 (CH CH). Anal. Calcd for
C36H22Cl2N14Na2O12S4: C, 39.75; H, 2.04; N, 18.03; S, 11.79.
Found: C, 39.70; H, 2.11; N, 18.23; S, 11.84. EIMS calculated
for C36H22Cl2N14Na2O12S4 (M+•) 1087.
3,5-Dihydroxybenzoic acid derivative of stilbene-
triazine (5e).
Mol.Wt: 1061.8; Light yellow solid, Yield 70%; Rf 0.18
1
FTIR: 3398 (N-H), 1712 (C O), 1077 (S O) cm−1; H
NMR (300 MHz, DMSO-d6): δ 11.17 (br s, 3H, NH),
7.82-7.28 (Ar-H), 7.09 (d, 1H, CH CH), 5.45 (s, 1H, OH),
5.39 (s, 1H, OH); 13C NMR (75 MHz, DMSO-d6): δ 177.84
(C S), 168.05 (C O), 163.10 (C─Cl), 161.31 (C─OH),
153.35-103.33 (Ar─C), 126.11 (CH CH). Anal. Calcd for
C36H24Cl2N12Na2O12S4: C, 40.72; H, 2.28; N, 15.83; S,
12.08. Found: C, 40.66; H, 2.35; N, 15.78; S, 12.16. EIMS
calculated for C36H24Cl2N12Na2O12S4 (M+•) 1061.
2-Bromobenzoic acid derivative of stilbene-
triazine (5f).
Mol.Wt: 1151.81; Light yellow solid, Yield 67%; Rf 0.23
FTIR: 3424 (N-H), 1710 (C O), 1068 (S O) cm−1; 1H NMR
(300 MHz, DMSO-d6): δ 11.21 (br s, 3H, NH), 7.68-6.50 (Ar-
H), 7.09 (d, 1H, CH CH); 13C NMR (75 MHz, DMSO-d6): δ
178.66 (C S), 171.51 (C O), 168.26 (C─Cl), 150.41-113.17
(Ar-C), 126.47 (CH CH), 120.18 (C─Br). Anal. Calcd for
4-Nitrobenzoic acid derivative of stilbene-
triazine (5j).
Mol.Wt: 1087.8; Yellow solid, Yield 58%; Rf 0.21 FTIR:
3430 (N-H), 1701 (C O), 1079 (S O) cm−1; 1H NMR
(300 MHz, DMSO-d6): δ 11.31 (br s, 3H, NH), 8.04-7.84 (Ar-
H), 7.09 (d, 1H, CH CH); 13C NMR (75 MHz, DMSO-d6): δ
176.33 (C S), 169.87 (C O), 168.20 (C-Cl), 151.81 (C-NO2),
138.44-105.24 (Ar-C), 126.37 (CH CH). Anal. Calcd for
C36H22Cl2N14Na2O12S4: C, 39.75; H, 2.04; N, 18.03; S, 11.79.
Found: C, 39.81; H, 2.12; N, 18.26; S, 11.86. EIMS calculated
for C36H22Cl2N14Na2O12S4 (M+•) 1088.
4-Methylbenzoic acid derivative of stilbene-
triazine (5 k).
Mol.Wt: 1025.8; Light yellow solid, Yield 79%; Rf 0.15
1
FTIR: 3465 (N-H), 1706 (C O), 1085 (S O) cm−1; H

BATOOL ET AL.
11
NMR (300 MHz, DMSO-d6): δ 11.36 (br s, 3H, NH),
7.70-7.14 (Ar-H), 7.09 (d, 1H, CH CH), 2.79 (s, 3H, CH3);
13C NMR (75 MHz, DMSO-d6): δ 178.53 (C S), 169.16
(C O), 166.20 (C─Cl), 163.33-100.19 (Ar─C), 143.30
(C─CH3), 126.63 (CH CH), 24.32 (CH3). Anal. Calcd for
C38H28Cl2N12Na2O8S4: C, 44.49; H, 2.75; N, 16.38; S,
12.50. Found: C, 44.40; H, 2.67; N, 16.42; S, 12.59. EIMS
calculated for C38H28Cl2N12Na2O8S4 (M+•) 1025.
4-Methoxybenzoic acid derivative of stilbene-
triazine (5 L).
3.4 | Computational modeling study
Crystal structure of LYP protein was obtained from RCSB
(PDB entry 3OLR)[32,33] and chain A was utilized for
modeling purpose. The crystal structure was (resolution
2.5 Å) solved in complex with phosphotyrosine residue,
thereby providing a suitable starting point for computa-
tional studies. The protein was prepared by adding miss-
ing hydrogens and optimized using Protein Prep module
of the Schrodinger program.[34,35] A grid was generated
for docking purposes by placing a box of length 20 Å in
each XYZ direction centering at the phosphotyrosine resi-
due. The ligands (compound 5a, 5d, 5e, 5g, 5k, & 5l)
were prepared using ligprep module of the Schrodinger
program. Glide standard precision (Glide-SP) module
was used for docking simulations.[35]
Mol.Wt: 1057.8; Light yellow solid, Yield 67%; Rf 0.24
1
FTIR: 3418 (N-H), 1707 (C O), 1069 (S O) cm−1; H
NMR (300 MHz, DMSO-d6): δ 11.12 (br s, 3H, NH),
7.94-7.36 (Ar-H), 7.09 (d, 1H, CH CH), 3.70 (s, 3H,
OCH3); 13C NMR (75 MHz, DMSO-d6): δ 176.43 (C S),
164.24 (C O), 159.64 (C-OCH3), 153.68 (C-Cl),
145.18-110.60 (Ar-C), 126.39 (CH CH), 54.97 (OCH3).
Anal. Calcd for C38H28Cl2N12Na2O10S4: C, 43.14; H,
2.67; N, 15.89; S, 12.12. Found: C, 43.20; H, 2.58; Cl,
6.77; N, 15.95; S, 12.07. EIMS calculated for
C38H28Cl2N12Na2O10S4 (M+•) 1057.
4 | CONCLUSIONS
A series of molecules incorporating thiourea- stilbene-
triazines moieties (5a-i) was synthesized as a new entry
to protein tyrosine phosphatase LYP inhibitors. To ascer-
tain the possible influence of inhibitory potential of com-
pounds 5a to 5l molecular docking was performed. On
the bases of docking computations, compound 5k and 5l
showed good binding mode. Thus, we suggest that the
electrostatic and hydrophobic interactions exist between
the targets and compounds 5l are well corroborated by
the experimental data. Docking simulation also eluci-
dated the valuable information to understand the binding
pattern and inhibitory potential and contribute to the
molecular design of novel synthetic inhibitors to inhibit
the activities of LYP to quite a good level. These results
further demonstrate that the compound 5l can serve as
the potential candidate for hit to lead generation and
design of novel antibacterial and anti-fungal agents.
3.3 | LYP inhibition assay
LYP assays were performed in black 96-well microplates
in a total volume of 100 μL using Molecular Devices
Spectramax M5 plate reader. Unless otherwise noted, all
solutions were prepared using a Bis-Tris (50 mM, pH 6.5)
buffer containing 100 mM NaCl and 0.01% Brij35. Inhibi-
tor compound and TCEP solutions were prepared using
DMSO. Each well contained 3.58 nM LYP, 100 μM tris
(2-carboxyethyl)phosphine (TCEP), 5 μM 6,8-difluoro-
4-methylumbelliferyl phosphate (DiFMUP), and either
100 μM of inhibitor (initial screen), 1-400 μM inhibitor
(IC50 determination), or DMSO, resulting in a final
DMSO concentration of 6.1% (v/v). Prior to testing,
35.8 nM LYP was activated by incubation with 1 mM
TCEP on ice for 30 minutes. After addition to an appro-
priate amount of buffer, inhibitor compounds were added
and allowed to incubate for 30 minutes at room tempera-
ture. Reactions were initiated by addition of DiFMUP to
each. The enzymatic turnover of DiFMUP to the fluores-
cent compound DiFMU was used to determine enzyme
activity, with fluorescence measured every 60 seconds
over a 30 minute period (λex = 350 nm, λem = 455 nm).
Percent inhibition was calculated by averaging the initial
enzyme activities for each inhibitor and normalizing
them against a set of DMSO control wells. The IC50 value
of each inhibitor was determined using plots of initial
enzyme activity against inhibitor concentration. Initial
screen experiments were run in duplicate while IC50 test-
ing was run in triplicates.
ACKNOWLEDGEMENTS
We are grateful to Dr. Amy M. Barrios at the University
of Utah, College of Pharmacy for assistance with the
enzyme inhibition assays.
ORCID
Aamer Saeed https://orcid.org/0000-0002-7112-9296
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SUPPORTING INFORMATION
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in the Supporting Information section at the end of this
article.
How to cite this article: Batool I, Jabeen F,
Vellore NA, Shabir G, Saeed A. Identification of
novel thiourea-stilbene-triazine conjugates as
persuasive lymphoid tyrosine phosphatase
inhibitors. J Heterocyclic Chem. 2020;1–12. https://
doi.org/10.1002/jhet.4060