Thymidine phosphorylase and prostrate cancer cell proliferation inhibitory activities of synthetic 4-hydroxybenzohydrazides: In vitro, kinetic, and in silico studies

Article abstract of DOI:10.1371/journal.pone.0227549

Over-expression of thymidine phosphorylase (TP) plays a key role in many pathological complications, including angiogenesis which leads to cancer cells proliferation. Thus in search of new anticancer agents, a series of 4-hydroxybenzohydrazides (1–29) was

Full text of DOI:10.1371/journal.pone.0227549

RESEARCH ARTICLE
Thymidine phosphorylase and prostrate
cancer cell proliferation inhibitory activities of
synthetic 4-hydroxybenzohydrazides: In vitro,
kinetic, and in silico studies
Sumaira Javaid 1☯*, Syed Muhammad Saad2,3☯, Humaira Zafar1☯, Rizwana Malik1‡
,
ID
Khalid Mohammed Khan2,4☯*, M. Iqbal Choudhary1,2,5☯, Atta-ur Rahman1,2☯
1 Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center of Chemical and
Biological Sciences, University of Karachi, Karachi, Pakistan, 2 H. E. J., Research Institute of Chemistry,
International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan,
3 Department of Chemistry, University of Karachi, Karachi, Pakistan, 4 Department of Clinical Pharmacy,
Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University,
Dammam, Saudi Arabia, 5 Department of Biochemistry, Faculty of Sciences, King Abdulaziz University,
Jeddah, Saudi Arabia
a1111111111
a1111111111
a1111111111
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a1111111111
☯ These authors contributed equally to this work.
‡ These authors also contributed equally to this work.
* sumairajavaid17@yahoo.com (SJ); khalid.khan@iccs.edu (KMK)
OPEN ACCESS
Citation: Javaid S, Saad SM, Zafar H, Malik R,
Khan KM, Choudhary MI, et al. (2020) Thymidine
phosphorylase and prostrate cancer cell
Abstract
Over-expression of thymidine phosphorylase (TP) plays a key role in many pathological
complications, including angiogenesis which leads to cancer cells proliferation. Thus in
search of new anticancer agents, a series of 4-hydroxybenzohydrazides (1–29) was synthe-
sized, and evaluated for in vitro thymidine phosphorylase inhibitory activity. Twenty com-
pounds 1–3, 6–14, 16, 19, 22–24, and 27–29 showed potent to weak TP inhibitory activities
with IC50 values in the range of 6.8 to 229.5 μM, in comparison to the standards i.e. tipiracil
(IC50 = 0.014 ± 0.002 μM) and 7-deazaxanthine (IC50 = 41.0 ± 1.63 μM). Kinetic studies on
selected inhibitors 3, 9, 14, 22, 27, and 29 revealed uncompetitive and non-competitive
modes of inhibition. Molecular docking studies of these inhibitors indicated that they were
able to interact with the amino acid residues present in allosteric site of TP, including
Asp391, Arg388, and Leu389. Antiproliferative (cytotoxic) activities of active compounds
were also evaluated against mouse fibroblast (3T3) and prostate cancer (PC3) cell lines.
Compounds 1, 2, 19, and 22–24 exhibited anti-proliferative activities against PC3 cells with
IC50 values between 6.5 to 10.5 μM, while they were largely non-cytotoxic to 3T3 (mouse
fibroblast) cells proliferation. Present study thus identifies a new class of dual inhibitors of
TP and cancer cell proliferation, which deserves to be further investigated for anti-cancer
drug development.
proliferation inhibitory activities of synthetic 4-
hydroxybenzohydrazides: In vitro, kinetic, and in
silico studies. PLoS ONE 15(1): e0227549. https://
doi.org/10.1371/journal.pone.0227549
Editor: Salvatore V. Pizzo, Duke University School
of Medicine, UNITED STATES
Received: September 26, 2019
Accepted: December 11, 2019
Published: January 27, 2020
Copyright: © 2020 Javaid et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files.
Funding: KMK Research project No. 20-1910
Higher Education Commission (HEC), Pakistan,
https://www.hec.gov.pk/english/pages/home.aspx
The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
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Thymidine phosphorylase inhibitors of synthetic origin
Competing interests: The authors have declared
Introduction
that no competing interests exist.
Thymidine phosphorylase (TP) (EC 2.4.2.4) is an enzyme of pyrimidine salvage pathway, pri-
marily responsible for maintaining nucleotide homeostasis. TP Over-expression has been
reported in several pathological conditions, including rheumatoid arthritis, chronic inflamma-
tory diseases, psoriasis, and tumor angiogenesis. Neoplastic tissues of bladder, gastric, cervical,
lung, colon, prostate, esophageal, and breast cancers show hyperactivity of TP. Over-expres-
sion of TP in tumor cell lines is an indication of its role in angiogenesis [1]. Role of TP as
angiogenic enzyme was proposed to be identical to human platelet derived endothelial cell
growth factor (PD-ECGF), which promotes angiogenesis by facilitating endothelial cell prolif-
eration, and migration [2]. TP Enzyme is present in many cells and tissues, such as platelets,
stromal cells, macrophages, endothelial cells, reticulocytes, glial cells, and ovary [1].
TP Comprises of two identical subunits, with molecular weight between 90 to 110 kDa in
Escherichia coli and mammals, respectively. Each subunit consists of a large α/β domain and a
smaller α-helical domain, separated from each other by a cavity [3]. Active site of TP comprises
of thymidine and phosphate binding sites. In addition to this, it also has a hydrophobic pocket
near the substrate binding sites. In the presence of inorganic phosphate, it catalyzes the revers-
ible cleavage of glycosidic bond of pyrimidine 20-deoxynucleotides to 20-deoxyribose-1-phos-
phate. Deoxyribose-1-phosphate then undergoes dephosphorylation to produce 2-D-
deoxyribose, which is used either as energy source for the cell or secreted out of the cell where
it may act as an angiogenic growth factor [3, 4].
Significant efforts have been focused on the development of TP inhibitors with possible
therapeutic potential since 1960’s. Some compounds were identified with excellent TP inhibi-
tory activity, and tested pre-clinically and clinically. However, only one drug (Lonsurf1) is in
clinical use for the management of metastatic colorectal cancer. This drug was approved by
US-FDA in 2015 and is combination of tipiracil (TP inhibitor) and trifluridine (a cytotoxin).
This drugis associated with adverse side effects which demands the identification of new leads
capable of inhibiting TP enzyme and thereby TP associated pathologies [5]. 7-Deazaxanthine
(7-DX) and 6-amino-5-bromouracil (6A5BU) are widely used as reference compounds for in
vitro studies against the TP enzyme [1, 6].
Our research group has reported several classes of synthetic and natural compounds as TP
inhibitors, such as 3-formylchromones [7], 1,3,4-oxadiazoles [8], 2-arylquinazolin-4(3H)-ones
[9], thymidine esters [10], plant glycosides [11], and salirepin derivatives from Symplocos race-
mosa [12]. In continuation of this, we evaluated 4-hydroxylbenzohydarzide derivatives for
their TP inhibitory activity (Fig 1). Schiff bases and hydrazones are reported to possess a wide
variety of biological properties, such as anticonvulsant, antibacterial, antihypertensive, anti-
inflammatory, antifungal, anticancer, antipyretic, antimicrobial, cytotoxic, anti-HIV, and her-
bicidal activities [13, 14]. All compounds were known previously [15–29], except 7, 16, 27, and
29 which were identified as new analogues.
Twenty-nine derivatives of 4-hydroxybenzohydarzide were subjected to an in vitro spectro-
photometric TP inhibition assay. Some of the most active compounds were then subjected to
kinetic and molecular docking studies in order to determine their mechanism of inhibition of
TP enzyme. TP is particularly reported to be over-expressed in the prostate cancer, therefore,
active compounds against TP were also evaluated for their effect on the proliferation of pros-
tate cancer cells (PC3) using the (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-tetrazolium bro-
mide) MTT colorimetric assay [2, 30, 31]. Interestingly, some of these compounds were also
able to inhibit the PC3 cancer cells proliferation. Present study therefore identifies dual inhibi-
tors of TP, and cancer cell proliferation.
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Thymidine phosphorylase inhibitors of synthetic origin
Fig 1. Skeleton of 4-hydroxybenzohydrazide: 4-hydroxybenzohydrazide derivatives 1–29.
https://doi.org/10.1371/journal.pone.0227549.g001
Material and methods
Enzyme thymidine phosphorylase (E. coli, EC 2.4.2.4), thymidine, Dulbecco’s Modified Eagle
Medium (DMEM), cyclohexamide, and doxorubicin were purchased from Sigma Aldrich,
USA. Standard inhibitors 7-deazaxanthine and tipiracil were purchased from Santa Cruz Bio-
technology Inc., USA. Deionized water used for buffer preparation was collected from Simplic-
ity Water Purification System (Milipore). 0.25% Trypsin EDTA was purchased from Gibco,
Invitrogen, New Zealand, fetal bovine serum (FBS) was purchased from A & E Scientific
(PAA), USA, 0.4% Trypan Blue solution was purchased from Amersco, USA, and 3-
(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from MP
Biomedicals, France. 4-Hydroxybenzohydrazide, substituted benzaldehydes, ethanol and ace-
tic acid were purchased from TCI, Japan. All reagents were of analytical grade and used
directly without purification.
1H-NMR Spectra were recorded on Avance Bruker AM 300 and 400 MHz instruments.
Electron Impact Mass Spectrometric (EI-MS) experiments were run on Finnigan MAT-311A
(Germany) mass spectrometer.
Cell lines
Mouse fibroblast cell line (NIH/3T3 (ATCC1 CRL-1658™)), and prostate cancer line (PC3
(ATCC1 CRL-1435™)) were purchased from American Type Culture Collection (ATCC),
USA. As we have purchased these lines directly from ATCC, therefore we did not perform any
quality control testing procedures.
Thymidine phosphorylase inhibition assay
Since human TP is not commercially available, we used commercially available recombinant
TP (expressed in E. coli) enzyme. Primary sequence of TP is mostly conserved throughout the
evolution and mammalian TP share 39% sequence similarity with TP of E. coli. The mamma-
lian enzyme also shares 65–70% similarity with the active site residues of E. coli TP enzyme
[2].
Assay for TP inhibition was performed spectrophotometrically, following the method of
Bera et al. with some modifications [32]. Enzyme TP catalyzed the reversible phosphorolysis of
thymidine to thymine and 2-deoxyribose-1-phosphate. Reaction mixture contained 150 μL of
potassium phosphate buffer (pH 7.0, 50 mM), 20 μL of enzyme (0.058 U/well), and 10 μL of
test compound (0.5 mM in dimethylsulfoxide). The reaction mixture was incubated for 10
min at 30˚C. 20 μL of substrate (1.5 mM) was then added and change in absorbance was
recorded after every minute for a total time period of 10 minutes at 290 nm in ELISA plate
reader (Spectra Max-340, Molecular Devices, CA, USA). Tipiracil and 7-deazaxanthine were
used as standard inhibitors in this assay.
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Thymidine phosphorylase inhibitors of synthetic origin
We have used the 96-well UV-plates, purchased from Corning (Catalogue No. COSTAR
3635, Lot No. 00912050). In addition to this, we also carried out pre-read (blank reference) on
a solution that does not contain the substrate, and this gave us zero reading. We then added
the substrate (thymidine), and recorded the final readings. Therefore, the chances of getting
erroneous results were minimized.
Anti-proliferative assay
Anti-proliferative activity of active compounds was evaluated by using the standard MTT (3-
[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-tetrazolium bromide) colorimetric assay in 96-well
plate [33].
It is a colorimetric assay that measures the reduction of MTT by mitochondrial enzyme i.e.
succinate dehydrogenase. The MTT enters into the mitochondria of cell, where it is reduced to
an insoluble formazan salt. As reduction of MTT can only occur in metabolically active cells,
the level of activity is actually a measure of the viability of the cells [34].
Mouse fibroblast normal cell line 3T3, and prostate cancer line (PC3) were cultured sepa-
rately in DMEM, supplemented with 5% of FBS, 100 IU/mL of penicillin and 100 μg/mL of
streptomycin, and kept at 37˚C in 5% CO2 incubator. For the preparation of cell culture, 100
μL/well of cell solution (5 x 104 cells/mL) was added into 96-well plate. The plate was incubated
for overnight, and fresh medium was added after the removal of old medium. The test com-
pounds were also added in different concentrations into the plate, and again incubated for 48
h. After the completion of this incubation period, 200 μL MTT (0.5 mg/mL) was added, and
plate was again incubated for 4 h, after this final incubation, 100 μL of DMSO was added to
each well. The level of MTT reduction to formazan was evaluated by change in absorbance at
540 nm for 3T3, and 570 nm for PC3 using a micro-plate reader (Spectra Max plus, Molecular
Devices, CA, USA). The anti-proliferative activity was recorded as concentration of the inhibi-
tor causing 50% growth inhibition (IC50) of cells.
Protocol for kinetic studies
Kinetic studies were carried out to identify the mechanism of inhibition by these compounds.
Inhibitor could bind with the enzyme in multiple ways, such as in competitive, non-competi-
tive, mixed or uncompetitive way. In kinetic assay, the enzyme (0.058 U/200 μL) was incubated
with different concentrations of inhibitor for 10 min at 30˚C. The reaction was then initiated
by adding different concentrations (0.1875–1.5 mM) of substrate (thymidine), and the result-
ing degradation of thymidine was measured continuously at 290 nm for 10 min on ELISA
plate reader. Every experiment was run in triplicate.
Lineweaver-Burk plot was plotted to determine the type of inhibition. This was accom-
plished by plotting the reciprocal of the rate of reaction against the reciprocal of the substrate
concentration. Ki values were determined by secondary re-plot of Lineweaver-Burk plot, and
reconfirmed by Dixon plot.
Molecular docking studies
Molecular docking studies were carried out in order to understand the interaction of inhibi-
tors (ligands) with TP (receptors). The structure of TP was taken from PDB (4LHM) which is
the crystallographic structure of TP in E. coli. Protein was prepared using Protein Preparation
Wizard and OPLS3 force field [35,36] in order to add missing hydrogens. The sulphate ions
were replaced by phosphate so they did not affect the conformation of protein and occupy the
same position as that of sulphate. Ligands were prepared to get the correct ionization and pro-
tonation states using Ligprep module [37] in Maestro Schrӧdinger2018-1.
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Thymidine phosphorylase inhibitors of synthetic origin
Since all the inhibitors showed non- and uncompetitive mode of inhibition in kinetic stud-
ies, site map analysis [38,39] was performed to find out the best allosteric site available in TP.
Five allosteric sites were observed and the one with highest score i.e. 1 was finally selected for
´
´
´
˚
˚
˚
the docking studies of these ligands. The dimensions of grid box were 15 A x 15 A x 15 A from
the mass of centre of each docked ligand and extra precision mode of Glide [40–43] was used
for the docking of ligands.
Statistical analysis
Results obtained for the enzyme inhibitory activity and MTT assay were analysed using Soft-
Max Pro 4.8 software (Molecular Devices, CA, USA) and Microsoft Excel. Percent of enzyme
inhibition, and growth inhibition of cells was calculated using the following formula:
Percent Inhibition ¼ 100 À ðAbsorbance of test=Absorbance of controlÞ x 100
Results were presented as means standard error mean from triplicate (n = 3) observation.
IC50 values were determined by using EZ-FIT, Enzyme kinetics software by Perrella Scientific,
Inc., USA. Grafit 7.0 version was used to determine the kinetics parameters. The software was
purchased from the Erithacus Software Ltd. (Wilmington House, West Sussex RH19 3AU,
UK).
General procedure for the synthesis of compounds 1–29
In a typical procedure, 4-hydroxylbenzohydrazones (1–29) were synthesized by mixing
4-hydroxylbenzohydrazide (1.5 mmol), substituted benzaldehydes (1.5 mmol) in ethanol (20
mL) with a catalytic amount of acetic acid (1 mL). The mixture was refluxed for 3 h, while
progress of the reaction was monitored through thin layer chromatography. After completion
of reaction, the reaction mixture was poured into China dish to let the solvent evaporate slowly
at room temperature to afford crystals of the products. Structures of the compounds were
deduced by using NMR and mass spectroscopic techniques.
4-Hydroxyl-N'-[(E)-(20-hydroxylphenyl)methylidene]benzohydrazide (1). Yield:
85.7%; 1H NMR: (400 MHz, DMSO-d6): δ 11.90 (s, 1H, NH), 11.40 (s, 1H, 20-OH), 10.16
(s, 1H, 4-OH), 8.58 (s, 1H, N = CH), 7.82 (d, 2H, J2,3 = J6,5 = 8.4 Hz, H-2, H-6), 7.50 (dd, 1H,
0
0
0
0
0
0
0
0
J6 ,5 = 8.0 Hz, J6 ,4 = 1.2 Hz, H-6 ), 7.30 (m, 1H, H-4 ), 6.92 (m, 1H, H-5 ), 6.90 (m, 1H, H-3 ),
6.87 (d, 2H, J3,2 = J5,6 = 8.8 Hz, H-3, H-5); EI-MS: m/z (rel. abund. %), 256 (M+, 22), 137 (80),
121 (100), 93 (31); Anal. Calcd for C14H12N2O3: C, 65.62; H, 4.72; N, 10.93; O, 18.73; Found:
C, 65.60; H, 4.75; N, 10.98.
N'-[(E)-(20,30-Dihydroxylphenyl)methylidene]-4-hydroxylbenzohydrazide (2). Yield:
80.7%; 1HNMR: (400 MHz, DMSO-d6): δ 11.89 (s, 1H, NH), 11.30 (s, 1H, 20-OH), 10.15 (s,
1H, 4-OH), 9.13 (s, 1H, 30-OH), 8.53 (s, 1H, N = CH), 7.82 (d, 2H, J2,3 = J6,5 = 8.4 Hz, H-2,H-
0
0
0
0
0
6), 6.92 (dd, 1H, J6 ,5 = 8.0 Hz,0J6 ,4 = 1.2 Hz, H-6 ), 6.87 (d, 2H, J3,2 = J5,6 = 8.4 Hz, H-3, H-5),
6.84 (d, 1H, J4 ,5 = 8.0 Hz, H-4 ), 6.74 (t, 1H, J5 (4 ,6 ) = 7.6 Hz, H-50); EI-MS: m/z (rel. abund.
%), 272 (M+, 64), 137 (28), 121 (100), 93 (32); Anal. Calcd for C14H12N2O4: C, 61.76; H, 4.44;
N, 10.29; O, 23.51; Found: C, 61.78; H, 4.45; N, 10.35.
0
0
0
0
0
N'-[(E)-(30,40-Dihydroxylphenyl)methylidene]-4-hydroxylbenzohydrazide (3). Yield:
81.7%; 1HNMR: (400 MHz, DMSO-d6): δ 11.36 (s, 1H, NH), 10.05 (br s, 1H, 4-OH), 9.27 (br s,
2H, 40-OH, 30-OH), 8.21 (s, 1H, N = CH), 7.77 (d, 2H, J2,3 = J6,5 = 8.4 Hz, H-2, H-60), 7.21 (s,
1H, H-20), 6.9 (d, 2H, J3,2 = J5,6 = 7.6 Hz, H-3, H-5), 6.84 (d, 1H, J6 ,5 = 8.4 Hz, H-6 ), 6.77 (d,
0
0
+
0
0
0
1H, J5 ,6 = 8.0 Hz, H-5 ); EI-MS: m/z (rel. abund. %), 272 (M , 8), 137 (27), 121 (100), 93 (21);
Anal. Calcd for C14H12N2O4: C, 61.76; H, 4.44; N, 10.29; O, 23.51; Found: C, 61.75; H, 4.40; N,
10.30.
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Thymidine phosphorylase inhibitors of synthetic origin
4-Hydroxyl-N'-[(E)-(20,40,60-trihydroxylphenyl)methylidene]benzohydrazide (4).
Yield: 84.5%; 1HNMR: (400 MHz, DMSO-d6): δ 11.64 (s, 1H, NH), 11.08 (s, 2H, 20-OH, 60-
OH), 10.09 (s, 1H, 4-OH), 9.74 (s, 1H, 40-OH), 8.75 (s, 1H, N = CH), 7.79 (d, 2H, J2,3 = J6,5
=
8.4 Hz, H-2, H-6), 6.85 (d, 2H, J3,2 = J5,6 = 8.4 Hz, H-3, H-5), 5.82 (s, 2H, H-30, H-50); EI-MS:
m/z (rel. abund. %), 288 (M+, 3), 152 (16), 137 (6), 121 (100), 93 (41); Anal. Calcd for
C14H12N2O5: C, 58.33; H, 4.20; N, 9.72; O, 27.75; Found: C, 58.30; H, 4.25; N, 9.73.
4-Hydroxyl-N'-[(E)-(20,30,40-trihydroxylphenyl)methylidene]benzohydrazide (5).
Yield: 75.0%; 1HNMR: (400 MHz, DMSO-d6): δ 11.72 (s, 1H, NH), 11.64 (s, 1H, 20-OH), 10.12
(s, 1H, 4-OH), 9.39 (s, 1H, 40-OH), 8.44 (s, 1H, 30-OH), 8.40 (s, 1H, N = CH), 7.80 (d, 2H,
0
0
J
2,3 = J6,5 = 8.8 Hz, H-2, H-6), 6.86 (d, 2H, J3,2 = J5,6 = 8.8 Hz, H-3, H-5), 6.74 (d, 1H, J5 ,6 = 8.4
+
Hz, H-50), 6.38 (d, 1H, J6 ,5 = 8.4 Hz, H-6 ); EI-MS: m/z (rel. abund. %), 288 (M , 73), 151 (8),
137 (23), 121 (100), 93 (37); Anal. Calcd for C14H12N2O5: C, 58.33; H, 4.20; N, 9.72; O, 27.75;
Found: C, 58.35; H, 4.21; N, 9.74.
0
0
0
4-Hydroxyl-N'-[(E)-(30-hydroxylphenyl)methylidene]benzohydrazide (6). Yield:
84.6%; 1H NMR: (400 MHz, DMSO-d6): δ 11.54 (s, 1H, NH), 10.08 (s, 1H, 4-OH), 9.58 (s, 1H,
30-OH), 8.32 (s, 1H, N = CH), 7.79 (d, 2H, J2,3 = J6,5 = 8.8 Hz, H-2, H-6), 7.25 (t, 1H, J5 (4 ,6 )
0
0
0
=
0
7.6 Hz, H-50), 7.16 (s, 1H, H-20), 7.07 (d, 1H, J6 ,5 = 7.6 Hz, H-6 ), 6.85 (d, 2H, J3,2 = J5,6 = 8.8
0
0
0
0
0
0
0
Hz, H-3, H-5), 6.81 (dd, 1H, J4 ,5 = 8.0 Hz, J4 ,6 = 1.6 Hz, H-4 ); EI-MS: m/z (rel. abund. %),
256 (M+, 8), 163 (2), 137 (89), 121 (100), 93 (41); Anal. Calcd for C14H12N2O3: C, 65.62; H,
4.72; N, 10.93; O, 18.73; Found: C, 65.65; H, 4.76; N, 10.96.
4-Hydroxyl-N'-[(E)-(20,40,50-trihydroxylphenyl)methylidene]benzohydrazide (7).
Yield: 78.7%; 1HNMR: (400 MHz, DMSO-d6): δ 11.57 (s, 1H, NH), 10.73 (s, 1H, 20-OH), 10.09
(s, 1H, 4-OH), 9.50 (s, 1H, 40-OH), 8.51 (s, 1H, 50-OH), 8.38 (s, 1H, N = CH), 7.8 (d, 2H, J2,3
=
J
6,5 = 8.8 Hz, H-2, H-6), 6.85 (d, 2H, J3,2 = J5,6 = 8.8 Hz, H-3, H-5), 6.82 (s, 1H, H-60), 6.31 (s,
1H, H-30); EI-MS: m/z (rel. abund. %), 288 (M+, 7), 152 (5), 151 (15), 137 (24), 121 (100), 93
(11); Anal. Calcd for C14H12N2O5: C, 58.33; H, 4.20; N, 9.72; O, 27.75; Found: C, 58.35; H,
4.23; N, 9.70.
4-Hydroxyl-N'-[(E)-(40-hydroxylphenyl)methylidene]benzohydrazide (8). Yield:
90.0%; 1H NMR: (400 MHz, DMSO-d6): δ 11.40 (s, 1H, NH), 9.96 (br s, 2H, 4-OH, 40-OH),
0
0
0
0
8.30 (s, 1H, N = CH), 7.78 (d, 2H, J2,3 = J6,5 = 8.8 Hz, H-2, H-6), 7.53 (d, 2H, J2 ,3 = J6 ,5 = 8.0
Hz, H-20, H-60), 6.84 (d, 2H, J3,2 = J5,6 = 6.0 Hz, H-3, H-5), 6.82 (d, 2H, J3 ,2 = J5 ,6 = 5.6 Hz, H-
3,H-50); EI-MS: m/z (rel. abund. %), 256 (M+, 13), 137 (70), 121 (100), 93 (23); Anal. Calcd for
C14H12N2O3: C, 65.62; H, 4.72; N, 10.93; O, 18.73; Found: C, 65.64; H, 4.70; N, 10.90.
N'-[(E)-(40-Chlorophenyl)methylidene]-4-hydroxylbenzohydrazide (9). Yield: 89.4%;
0
0
0
0
1HNMR: (400 MHz, DMSO-d6): δ 11.67 (s, 1H, NH), 10.11 (s, 1H, 4-OH), 8.40 (s, 1H,
0
0
0
0
0
N = CH), 7.80 (d, 2H, J2,3 = J6,5 = 8.8 Hz,0H-2, H-6), 7.72 (d, 2H, J2 ,3 = J6 ,5 = 8.8 Hz, H-2 , H-
60), 7.51 (d, 2H, J3 ,2 = J5 ,6 = 8.4 Hz, H-3 , H-5 ), 6.86 (d, 2H, J3,2 = J5,6 = 8.8 Hz, H-3, H-5);
EI-MS: m/z (rel. abund. %), 276 (M++2, 2), 274 (M+, 4), 137 (37), 121 (100), 111 (4), 93 (25);
Anal. Calcd for C14H11ClN2O2: C, 61.21; H, 4.04; Cl, 12.90; N, 10.20; O, 11.65; Found: C,
61.20; H, 4.07; N, 10.20.
0
0
0
0
0
4-Hydroxyl-N'-{(E)-[40-(methoxy)phenyl]methylidene}benzohydrazide (10). Yield:
77.8%; 1HNMR: (300 MHz, DMSO-d6): δ 11.47 (s, 1H, NH), 10.06 (s, 1H, 4-OH), 8.35 (s,01H,
0
0
0
0
N = CH), 7.79 (d, 2H, J2,3 = J6,5 = 8.7 Hz,0H-2, H-6), 7.65 (d, 2H, J2 ,3 = J6 ,5 = 8.1 Hz, H-2 , H-
0
60), 7.02 (d, 2H, J3 ,2 = J5 ,6 = 8.7 Hz, H-3 , H-5 ), 6.85 (d, 2H, J3,2 = J5,6 = 8.7 Hz, H-3, H-5),
3.79 (s, 3H, 40-OCH3); EI-MS: m/z (rel. abund. %), 270 (M+, 14), 150 (55), 137 (80), 121 (100),
93 (13); Anal. Calcd for C15H14N2O3: C, 66.66; H, 5.22; N, 10.36; O, 17.76; Found: C, 66.65; H,
5.20; N, 10.37.
0
0
0
0
N'-[(E)-(30-Chlorophenyl)methylidene]-4-hydroxylbenzohydrazide (11). Yield: 80.3%;
1HNMR: (400 MHz, DMSO-d6): δ 11.74 (s, 1H, NH), 10.12 (s, 1H, 4-OH), 8.39 (s, 1H, N = CH),
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Thymidine phosphorylase inhibitors of synthetic origin
7.80 (d, 2H, J2,3 = J6,5 = 8.8 Hz, H-2, H-6), 7.74 (s, 1H, H-20), 7.65 (s, 1H, H-60), 7.47 (s, 1H, H-40),
7.46 (s, 1H, H-50), 6.86 (d, 2H, J3,2 = J5,6 = 8.4 Hz, H-3, H-5); EI-MS: m/z (rel. abund. %), 276
(M++2, 3), 274 (M+, 8), 137 (95), 121 (100), 111 (3), 93 (44); Anal. Calcd for C14H11ClN2O2: C,
61.21; H, 4.04; Cl, 12.90; N, 10.20; O, 11.65; Found: C, 61.23; H, 4.05; N, 10.21.
4-Hydroxyl-N'-{(E)-[20-(methoxy)phenyl]methylidene}benzohydrazide (12). Yield:
71.9%; 1HNMR: (400 MHz, DMSO-d6): δ 11.61 (s, 1H, NH), 10.07 (s, 1H, 4-OH), 8.76 (s, 1H,
0
0
0
N = CH), 7.85 (d, 1H, J6 ,5 = 6.8 Hz, H-6 ), 7.81 (d, 2H, J2,3 = J6,5 = 8.8 Hz, H-2, H-6), 7.41 (m,
1H, H-40), 7.10 (d, 1H, J3 ,4 = 8.4 Hz, H-3 ), 7.02 (m, 1H, H-5 ), 6.84 (d, 2H, J3,2 = J5,6 = 8.8 Hz,
H-3, H-5), 3.85 (s, 3H, 20-OCH3); EI-MS: m/z (rel. abund. %), 270 (M+, 14), 137 (100), 121
(100), 119 (68), 93 (67); Anal. Calcd for C15H14N2O3: C, 66.66; H, 5.22; N, 10.36; O, 17.76;
Found: C, 66.68; H, 5.24; N, 10.39.
0
0
0
0
N'-{(E)-[30,40-Bis(methoxy)phenyl]methylidene}-4-hydroxylbenzohydrazide (13).
Yield: 71.7%; 1HNMR: (400 MHz, DMSO-d6): δ 11.49 (s, 1H, NH), 10.08 (br s, 1H, 4-OH),
8.34 (s, 1H, N = CH), 7.79 (d, 2H, J2,3 = J6,5 = 8.4 Hz, H-2, H-6), 7.31 (s, 1H, H-20), 7.17 (d, 1H,
0
0
0
0
0
0
J6 ,5 = 8.0 Hz, H-6 ), 7.02 (d, 1H, J5 ,6 = 8.4 Hz, H-5 ), 6.85 (d, 2H, J3,2 = J5,6 = 8.4 Hz, H-3, H-
5), 3.80 (s, 3H, 40-OCH3), 3.79 (s, 3H, 30-OCH3); EI-MS: m/z (rel. abund. %), 300 (M+, 23), 163
(99), 148 (6), 137 (16), 121 (100), 93 (11); Anal. Calcd for C16H16N2O4: C, 63.99; H, 5.37; N,
9.33; O, 21.31; Found: C, 63.97; H, 5.39; N, 9.34.
N'-[(E)-(40-Bromophenyl)methylidene]-4-hydroxylbenzohydrazide (14). Yield:
81.9%; 1HNMR: (400 MHz, DMSO-d6): δ 11.68 (s, 1H, NH), 10.11 (s, 1H, 4-OH), 8.38 (s, 1H,
N = CH), 7.80 (d, 2H, J2,3 = J6,5 = 8.4 Hz, H-2, H-6), 7.64 (s, 4H, H-20, H-60, H-30, H-50), 6.86
(d, 2H, J3,2 = J5,6 = 8.4 Hz, H-3, H-5); EI-MS: m/z (rel. abund. %), 320 (M++2, 5), 318 (M+, 22),
181 (12), 163 (2), 155 (4), 137 (83), 121 (100), 93 (29); Anal. Calcd for C14H11BrN2O2: C, 52.69;
H, 3.47; Br, 25.04; N, 8.78; O, 10.03; Found: C, 52.67; H, 3.49; N, 8.75.
N'-[(E)-(20,40-Dichlorophenyl)methylidene]-4-hydroxylbenzohydrazide (15). Yield:
65.7%; 1HNMR: (400 MHz, DMSO-d6): δ 11.90 (s, 1H, NH), 10.15 (s, 1H, 4-OH), 8.77 (s, 1H,
0
0
0
N = CH), 8.00 (d, 1H, J6 ,5 = 7.6 Hz, H-6 ), 7.82 (d, 2H, J2,3 = J6,5 = 8.8 Hz, H-2, H-6), 7.70 (d,
0
0
0
0
0
0
0
0
1H, J3 ,5 = 2 Hz, H-3 ), 7.52 (dd, 1H, J5 ,6 = 8.8 Hz, J5 ,3 = 2.0 Hz, H-5 ), 6.86 (d, 2H, J3,2 = J5,6
=
8.8 Hz, H-3, H-5); EI-MS: m/z (rel. abund. %), 312 (M++4, 1), 310 (M++2, 1), 308 (M+, 2), 137
(55), 121 (100), 93 (17); Anal. Calcd for C14H10Cl2N2O2: C, 54.39; H, 3.26; Cl, 22.93; N, 9.06;
O, 10.35; Found: C, 54.37; H, 3.28; N, 9.08.
4-Hydroxyl-N'-[(E)-(20-methylphenyl)methylidene]benzohydrazide (16). Yield:
81.3%; 1HNMR: (400 MHz, DMSO-d6): δ 11.59 (s, 1H, NH), 10.10 (s, 1H, 4-OH), 8.71 (s, 1H,
N = CH), 7.80 (d, 2H, J2,3 = J6,5 = 8.4 Hz, H-2, H-6), 7.78 (s, 1H, H-60), 7.29 (m, 3H, H-40, H-50,
H-30), 6.86 (d, 2H, J3,2 = J5,6 = 8.4 Hz, H-3, H-5), 2.49 (s, 3H, 20-CH3); EI-MS: m/z (rel. abund.
%), 254 (M+, 3), 137 (100), 121 (100), 93 (34); Anal. Calcd for C15H14N2O2: C, 70.85; H, 5.55;
N, 11.02; O, 12.58; Found: C, 70.87; H, 5.58; N, 11.01.
N'-{(E)-[40-(Dimethylamino)phenyl]methylidene}4hydroxylbenzohydrazide (17).
Yield: 86.0%; 1HNMR: (300 MHz, DMSO-d6): δ 11.31 (s, 1H, NH), 10.04 (br s, 1H, 4-OH),
0
0
0
0
8.27 (s, 1H, N = CH), 7.78 (d, 2H, J2,3 = J6,5 = 8.4 Hz, H-2, H-6), 7.52 (d, 2H, J2 ,3 = J6 ,5 = 8.4
Hz, H-20, H-60), 6.84 (d, 2H, J3 ,2 = J5 ,6 = 8.7 Hz, H-3 , H-5 ), 6.75 (d, 2H, J3,2 = J5,6 = 8.7 Hz,
H-3, H-5), 2.96 (s, 6H, 40-N(CH3)2); EI-MS: m/z (rel. abund. %), 283 (M+, 89), 163 (73), 146
(100), 137 (7), 121 (85), 93 (27); Anal. Calcd for C16H17N3O2: C, 67.83; H, 6.05; N, 14.83; O,
11.29; Found: C, 67.85; H, 6.07; N, 14.81.
0
0
0
0
0
0
4-Hydroxyl-N'-{(E)-[40-(methylsulfanyl)phenyl]methylidene}benzohydrazide (18).
Yield: 75.2%; 1HNMR: (400 MHz, DMSO-d6): δ 11.56 (s, 1H, NH), 10.08 (s, 1H, 4-OH), 8.36
0
0
0
0
(s, 1H, N = CH), 7.79 (d, 2H, J2,3 = J6,5 = 8.8 Hz, H-2, H-6), 7.63 (d, 2H, J2 ,3 = J6 ,5 = 8.0 Hz,
H-20, H-60), 7.32 (d, 2H, J3 ,2 = J5 ,6 = 8.4 Hz, H-3 , H-5 ), 6.85 (d, 2H, J3,2 = J5,6 = 8.8 Hz, H-3,
0
0
0
0
0
0
H-5), 2.53 (s, 3H, 40-SCH3); EI-MS: m/z (rel. abund. %), 286 (M+, 31), 149 (88), 137 (79), 121
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Thymidine phosphorylase inhibitors of synthetic origin
(100), 93 (74); Anal. Calcd for C15H14N2O2S: C, 62.92; H, 4.93; N, 9.78; O, 11.17; S, 11.20;
Found: C, 62.90; H, 4.91; N, 9.75.
4-Hydroxyl-N’-[(E)-(20-hydroxyl-30-methoxyphenyl)methylidene]benzohydrazide
(19). Yield: 82.2%; 1HNMR: (400 MHz, DMSO-d6): δ 11.86 (br s, 1H, NH), 11.13 (br s, 1H,
20-OH), 10.15 (br s, 1H, 4-OH), 8.59 (s, 1H, N = CH), 7.82 (d, 2H, J2,3 = J6,5 = 8.8 Hz, H-2, H-
0
0
0
0
0
0
0
0
6), 7.10 (dd, 1H, J6 ,5 = 7.6 Hz, J6 ,4 = 0.8 Hz, H-6 ), 7.02 (d, 1H, J4 ,5 = 7.6 Hz, H-4 ), 6.87 (d,
2H, J3,2 = J5,6 = 8.8 Hz, H-3, H-5), 6.82 (m, 1H, H-50), 3.80 (s, 3H, 30-OCH3); EI-MS: m/z (rel.
abund. %), 286 (M+, 61), 149 (23), 137 (52), 121 (100), 93 (38); Anal. Calcd for C15H14N2O4: C,
62.93; H, 4.93; N, 9.79; O, 22.35; Found: C, 62.90; H, 4.95; N, 9.77.
N'-[(E)-(50-Bromo-20-hydroxylphenyl)methylidene]-4-hydroxylbenzohydrazide (20).
Yield: 61.9%; 1HNMR: (300 MHz, DMSO-d6): δ 11.97 (s, 1H, NH), 11.40 (s, 1H, 20-OH),
10.15 (s, 1H, 4-OH), 8.55 (s, 1H, N = CH), 7.82 (d, 2H, J2,3 = J6,5 = 8.4 Hz, H-2, H-6), 7.75
0
0
(s, 1H, H-60), 7.42 (dd, 1H, J4 ,3 = 8.7 Hz, J4 ,6 = 2.1 Hz, H-4 ), 6.90 (m, 1H, H-3 ), 6.87 (d, 2H,
J
0
0
0
0
3,2 = J5,6 = 8.4 Hz, H-3, H-5); EI-MS: m/z (rel. abund. %), 336 (M++2, 34), 334 (M+, 37), 197
(4), 137 (97), 121 (100), 93 (54); Anal. Calcd for C14H11BrN2O3: C, 50.17; H, 3.31; Br, 23.84; N,
8.36; O, 14.32; Found: C, 50.15; H, 3.33; N, 8.35.
N'-[(E)-(50-Chloro-20-hydroxylphenyl)methylidene]-4-hydroxylbenzohydrazide (21).
Yield: 75.6%; 1HNMR: (300 MHz, DMSO-d6): δ 11.98 (s, 1H, NH), 11.39 (s, 1H, 20-OH), 10.16
(s, 1H, 4-OH), 8.56 (s, 1H, N = CH), 7.82 (d, 2H, J2,3 = J6,5 = 8.4 Hz, H-2, H-6), 7.62 (s, 1H, H-
0
0
60), 7.31 (dd, 1H, J4 ,3 = 8.7 Hz, J4 ,6 = 2.7 Hz, H-4 ), 6.95 (d, 1H, J3 ,4 = 8.7 Hz, H-3 ), 6.87 (d,
2H, J3,2 = J5,6 = 8.7 Hz, H-3, H-5); EI-MS: m/z (rel. abund. %), 292 (M++2, 21), 290 (M+, 60),
153 (7), 137 (62), 121 (100), 93 (56); Anal. Calcd for C14H11ClN2O3: C, 57.84; H, 3.81; Cl,
12.19; N, 9.64; O, 16.51; Found: C, 57.81; H, 3.84; N, 9.65.
0
0
0
0
0
0
N'-[(E)-(30,50-Dibromo-20-hydroxylphenyl)methylidene]-4-hydroxylbenzohydrazide
(22). Yield: 57.3%; 1HNMR: (400 MHz, DMSO-d6): δ 12.85 (s, 1H, NH), 12.31 (s, 1H, 20-
OH), 10.22 (s, 1H, 4-OH), 8.48 (s, 1H, N = CH), 7.84 (d, 2H, J2,3 = J6,5 = 8.4 Hz, H-2, H-6),
0
0
0
0
7.80 (d, 1H, J6 ,4 = 2.0 Hz, H-6 ), 7.78 (s, 1H, H-4 ), 6.89 (d, 2H, J3,2 = J5,6 = 8.4 Hz, H-3, H-5);
EI-MS: m/z (rel. abund. %), 416 (M++4, 18), 414 (M++2, 50), 412 (M+, 21), 275 (5), 137 (100),
121 (100), 93 (67); Anal. Calcd for C14H10Br2N2O3: C, 40.61; H, 2.43; Br, 38.60; N, 6.77; O,
11.59; Found: C, 40.63; H, 2.45; N, 6.79.
4-Hydroxyl-N'-{(E)-[20-hydroxyl-50-(methoxy)phenyl]methylidene}benzo-hydrazide
(23). Yield: 69.4%; 1HNMR: (300 MHz, DMSO-d6): δ 11.88 (s, 1H, NH), 10.79 (s, 1H, 20-
OH), 10.23 (br s, 1H, 4-OH), 8.56 (s, 1H, N = CH), 7.820(d, 2H, J2,3 = J6,5 = 8.4 Hz, H-2, H-6),
0
0
0
0
7.08 (d, 1H, J6 ,4 = 2.7 Hz, H-6 ), 6.91 (m, 2H, H-4 , H-3 ), 6.87 (d, 2H, J3,2 = J5,6 = 8.4 Hz, H-3,
H-5), 3.72 (s, 3H, 50-OCH3); EI-MS: m/z (rel. abund. %), 286 (M+, 68), 269 (2), 149 (26), 137
(34), 121 (100), 93 (38); Anal. Calcd for C15H14N2O4: C, 62.93; H, 4.93; N, 9.79; O, 22.35;
Found: C, 62.95; H, 4.92; N, 9.78.
N'-{(E)-[30-Ethoxy-20-hydroxylphenyl]methylidene}-4-hydroxylbenzohydrazide (24).
Yield: 55.9%; 1HNMR: (400 MHz, DMSO-d6): δ 11.87 (s, 1H, NH), 11.14 (s, 1H, 20-OH), 10.15 (s,
0
0
1H, 4-OH), 8.58 (s, 1H, N = CH), 7.82 (d, 2H, J2,3 = J6,5 = 8.4 0Hz, H-2, H-6), 7.09 (d, 1H, J6 ,5 = 8.0
0
Hz, H-60), 7.01 (d, 1H, J4 ,5 = 7.6 Hz, H-4 ), 6.87 (m, 1H, H-5 ), 6.82 (d, 2H, J3,2 = J5,6 = 8.0 Hz, H-
3, H-5), 4.07 (q, 2H, J CH2. CH3 = 13.6 Hz, 6.8 Hz, CH2), 1.35 (t, 3H, J CH3,CH2 = 7.2 Hz, CH3);
EI-MS: m/z (rel. abund. %), 300 (M+, 54), 285 (2), 163 (23), 137 (30), 121 (100), 93 (32); Anal.
Calcd for C16H16N2O4: C, 63.99; H, 5.37; N, 9.33; O, 21.31; Found: C, 63.97; H, 5.35; N, 9.35.
4-Hydroxyl-N'-[(E)-1-(20-hydroxylphenyl)ethylidene]benzohydrazide (25). Yield:
73.5%; 1HNMR: (400 MHz, DMSO-d6): δ 13.40 (s, 1H, NH), 11.03 (s, 1H, 20-OH), 100.14 (s,
0
0
0
0
1H, 4-OH), 7.83 (d, 2H, J2,3 = J6,5 = 8.8 Hz, H-2, H-6), 7.62 (d, 1H, J6 ,5 = 7.6 Hz, H-6 ), 7.29 (t,
1H, J4 (5 ,3 ) = 7.2 Hz, H-40), 6.89 (s, 2H, H-30, H-50), 6.88 (d, 2H, J3,2 = J5,6 = 8.4 Hz, H-3, H-5),
2.49 (s, 3H, -CH3); EI-MS: m/z (rel. abund. %), 270 (M+, 75), 255 (42), 177 (2), 150 (54), 121
0
0
0
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Thymidine phosphorylase inhibitors of synthetic origin
(100), 93 (39); Anal. Calcd for C15H14N2O3: C, 66.66; H, 5.22; N, 10.36; O, 17.76; Found: C,
66.68; H, 5.24; N, 10.35.
N'-[(E)-1-(20,40-Dihydroxylphenyl)ethylidene]-4-hydroxylbenzohydrazide (26). Yield:
83.9%; 1HNMR: (400 MHz, DMSO-d6): δ 13.57 (s, 1H, NH), 10.85 (s, 1H, 20-OH), 10.1 (s, 1H,
4-OH), 9.79 (s, 1H, 40-OH), 7.80 (d, 2H, J2,3 = J6,5 = 8.4 Hz, H-2, H-6), 7.42 (d, 1H, J6 ,5 = 8.8
0
0
Hz, H-60), 6.86 (d, 2H, J3,2 = J5,6 = 8.40Hz, H-3, H-5), 6.32 (dd, 1H, J5 ,6 = 8.8 Hz, J5 ,3 = 1.6 Hz,
0
0
0
0
H-50), 6.25 (d, 1H, J3 ,5 = 2.0 Hz, H-3 ), 2.37 (s, 3H, -CH3); EI-MS: m/z (rel. abund. %), 286
(M+, 16), 271 (8), 151 (15), 137 (24), 121 (100), 93 (16); Anal. Calcd for C15H14N2O4: C, 62.93;
H, 4.93; N, 9.79; O, 22.35; Found: C, 62.95; H, 4.91; N, 9.77.
0
0
N'-[(E)-1-(20,60-Dihydroxylphenyl)ethylidene]-4-hydroxylbenzohydrazide (27). Yield:
76.4%; 1HNMR: (300 MHz, DMSO-d6): δ 12.62 (s, 1H, NH), 10.97 (br s, 1H, 20-OH), 10.13
(br s, 1H, 4-OH), 8.87 (s,01H, 60-OH), 7.82 (d, 2H, J2,3 = J6,5 = 8.4 Hz, H-2, H-6), 6.95 (d, 1H,
0
0
0
0
0
0
0
0
J4 ,3 = J4 ,5 = 9.0 Hz, H-4 ), 6.87 (d, 2H, J3,2 = J5,6 = 8.7 Hz, H-3, H-5), 6.72 (d, 2H, J3 (4 ,5 ) = J5
+
(4 ,3 ) = 7.2 Hz, H-30, H-50), 2.38 (s, 3H, -CH3); EI-MS: m/z (rel. abund. %), 286 (M , 63), 271
(5), 121 (100), 93 (22); Anal. Calcd for C15H14N2O4: C, 62.93; H, 4.93; N, 9.79; O, 22.35;
Found: C, 62.92; H, 4.92; N, 9.80.
0
0
N'-[(E)-1-(20,50-Dihydroxylphenyl)ethylidene]-4-hydroxylbenzohydrazide (28). Yield:
79.3%; 1HNMR: (300 MHz, DMSO-d6): δ 12.62 (s, 1H, NH), 10.97 (br s, 1H, 20-OH), 10.15 (br
s, 1H, 4-OH), 8.87 (s, 1H, 50-OH), 7.82 (d, 2H, J2,3 = J6,5 = 8.7 Hz, H-2, H-6), 6.94 (s, 1H, H-60),
0
0
0
0
0
0
6.87 (d, 2H, J3 ,4 = J4 ,3 = 8.4 Hz, H-3 , H-4 ), 6.75 (d, 2H, J3,2 = J5,6 = 8.7 Hz, H-3, H-5), 2.38 (s,
3H, CH3); EI-MS: m/z (rel. abund. %), 286 (M+, 50), 269 (8), 166 (38), 121 (100), 93 (10); Anal.
Calcd for C15H14N2O4: C, 62.93; H, 4.93; N, 9.79; O, 22.35; Found: C, 62.92; H, 4.91; N, 9.76.
N'-[(E)-1-(20,50-Dihydroxylphenyl)propylidene]-4-hydroxylbenzohydrazide (29).
Yield: 49.0%; 1HNMR: (400 MHz, DMSO-d6): δ 12.68 (s, 1H, NH), 11.03 (s, 1H, 20-OH), 10.13
(s, 1H, 4-OH), 8.85 (s, 1H, 50-OH), 7.78 (d, 2H, J2,3 = J6,5 = 8.4 Hz, H-2, H-6), 6.95 (s, 1H, H-
60), 6.87 (d, 2H, J3,2 = J5,6 = 8.8 Hz, H-3, H-5), 6.71 (s, 2H, H-30, H-40), 3.00 (q, 2H, J = 13.6 Hz,
6.8 Hz, CH2), 1.14 (t, 3H, J = 7.2, CH3); EI-MS: m/z (rel. abund. %), 300 (M+, 98), 283 (18), 271
(92), 135 (28), 121 (100), 93 (59); Anal. Calcd for C16H16N2O4: C, 63.99; H, 5.37; N, 9.33; O,
21.31; Found: C, 63.97; H, 5.38; N, 9.30.
Results and discussion
Chemistry
For the study of thymidine phosphorylase inhibitory activity, twenty-nine derivatives of
4-hydroxybenzohydrazides were synthesized by reacting 4-hydroxybenzohydrazide with
substituted benzaldehydes in ethanol, catalyzed by acetic acid (Scheme 1).
Scheme 1. Synthesis reaction: Synthesis of 4-hydroxybenzohydrazides 1–29
The structures of these derivatives were identified by EI-MS, and 1H-NMR spectroscopy, and
comparison with the data reported in literature. To confirm the stereochemical assignment of
iminic bond, NOESY (nuclear overhauser enhancement spectroscopy) spectrum was recorded
for a representative compound 29. Strong NOESY interactions of NH and 20-OH with CH2 of
ethyl group revealed the geometry of the compound to be of (E)-configuration. Absence of
NOESY interaction of NH with 20-OH further confirmed the E stereochemistry (Fig 2).
Enzyme inhibition studies
Twenty-nine analogues of 4-hydroxybenzohydarzides (1–29) were evaluated for TP inhibition,
in comparison to the standards i.e. tipracil (IC50 = 0.014 0.002 μM) and 7-deazaxanthine
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Thymidine phosphorylase inhibitors of synthetic origin
Fig 2. NOESY interaction: Distinctive NOESY interactions to confirm E/Z configuration.
https://doi.org/10.1371/journal.pone.0227549.g002
(IC50 = 41.0 1.63 μM). Among them, twenty compounds (1–3, 6–14, 16, 19, 22–24, and 27–
29) were found to be active against TP. Compound 22 was the most active compound of the
series with IC50 = 6.8 0.7μM, while other compounds showed a moderate inhibition of TP
(Fig 3, Fig 4). In Fig 4 and Tables, SEMa stands for standard error of mean, while N.Ab repre-
sent not active.
Eight derivatives with mono-, di- and tri-hydroxy substitutions were evaluated, and six
compounds 1–3, 6–8 were found to be active against TP with IC50 values of 170.6 to 229.5 μM.
Among them, compound 2 with two OH groups at ortho (C-20) and meta (C-30) positions
showed a moderate TP inhibition (IC50 = 170.6 0.5 μM), followed by compound 6 (IC50
=
180.0 3.0 μM) with a single OH group at meta (C-30) position. Tri-hydroxylated derivatives
4–5 showed 8 and 26% inhibition, respectively (Fig 4).
Three derivatives with mono-, and di-methoxy substitutions (compounds 10, 12, 13) were
evaluated, and found to be weak TP inhibitors (IC50 = 183.7 1.5, 185.5 2.0, and 204.1 3.0
μM, respectively). Among them compound 12 with a OCH3 group at ortho position showed a
moderate TP inhibition (IC50 = 183.7 1.5 μM).
Empirical structure-activity relationship (SAR) studies proposed that hydroxy and methoxy
substitutions on phenyl ring play an important role in inducing TP inhibition. These groups
may be involved in hydrogen bonding with the amino acid residues, present at the substrate-
binding site or hydrophobic pocket of TP enzyme.
Four halogen-substituted derivatives were evaluated (compounds 9, 11, 14, 15), and three
were found to inhibit the TP activity with IC50 values between 158.0–160.3 μM. Among these
mono-halogenated derivatives, 9, 11, and 14 showed ability to inhibit the enzyme activity,
while di-halogenated derivative 15 was found to be inactive with 20% inhibition. Based on the
IC50 values, halogens substitution were found to be more favorable in comparison to OH and
OCH3 substitutions. It was thus proposed that halogens might increase the ability of these
compounds to interact via hydrogen bonding with amino acid residues present at substrate
binding-site of TP.
Compound 16 with a methyl group at ortho position showed a moderate TP inhibition
(IC50 = 177.2 0.5 μM), while compounds 17, and 18 with dimethylmine and methylsulfanyl
groups, attached to para position, respectively showed less than 50% inhibition thus regarded
inactive.
Three derivatives with hydroxyl-cum-methoxy substitutions 19, 23, 24 were evaluated, and
all were found to be moderately active against TP enzyme (IC50 values between 172.0–190.3
μM). Compound 19 with OH and OCH3 groups at ortho (C-20) and meta (C-30) positions,
respectively, showed a moderate TP inhibition (IC50 = 181.5 1.6 μM). Switching of hydroxy
group to the other meta (i.e. C-50) position, as in compound 23, slightly increased the inhibi-
tion of TP (IC50 = 172.0 2.7 μM). Replacement of methoxy with an ethoxy group, as in
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Thymidine phosphorylase inhibitors of synthetic origin
Fig 3. In-vitro thymidine phosphorylase inhibitory activities of 4-hydroxylbenzohydarazides (1–29): (A) Percent inhibition, blue color
bars represents active compounds (compounds which showed �50%), while red color represent inactive compounds (compounds which
showed < 50% inhibition) (B) IC50 values of active compounds is given in μM.
https://doi.org/10.1371/journal.pone.0227549.g003
compound 24, slightly decreased the TP inhibitory activity (IC50 = 190.3 0.4 μM). SAR pro-
posed that when hydroxyl and methoxy groups are present adjacent to each other, they lower
the ability of compounds to inhibit enzyme, as inferred from their IC50 values.
Three derivatives with hydroxy-cum-halogen substitutions were also evaluated (compounds
20–22). Among them compound 22 with two bromo groups at meta (C-30 and C-50) positions,
and an OH at ortho (C-20) showed a potent TP inhibitory activity (IC50 = 6.8 0.7 μM), in
comparison to the standard used i.e., 7-deazaxanthine (IC50 = 41.0 1.63 μM). This is an
unusual but reproducible behavior exhibited by this compound, whereas compound 20, which
just lacks a bromo group at meta (C-30) position showed 38.6% inhibition. As TP has hydro-
phobic pocket near the substrate binding sites, it is possible that compound 22 with di-bromo
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Thymidine phosphorylase inhibitors of synthetic origin
Fig 4. In-vitro thymidine phosphorylase inhibitory activities of 4-hydroxylbenzohydarazides (1–29).
https://doi.org/10.1371/journal.pone.0227549.g004
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Thymidine phosphorylase inhibitors of synthetic origin
Table 1. Kinetic studies of most active compounds on thymidine phosphorylase.
Compound
Ki (μM SEM)a
Inhibition Type
Uncompetitive
Uncompetitive
Uncompetitive
Non-competitive
Uncompetitive
Uncompetitive
Non-competitive
3
176.65 0.006
138.0 0.009
80.5 0.002
9
14
22
1.75 0.009
27
29
168.0 0.003
159.05 0.002
45.66 0.0009
7-Deazaxanthine
https://doi.org/10.1371/journal.pone.0227549.t001
substitution may be able to fit more appropriately at the hydrophobic pocket of the TP, which
may not be possible for compound 20. Compound 20 might have a conformation which does
not fit well in the hydrophobic pocket of enzyme.
Derivatizations were also made by replacing the benzylidene group with ethylidine and pro-
pylidine groups (Fig 1), in addition to OH substitutions on phenyl ring. Five derivatives 25–29
were evaluated, and among them compounds 27, 28, and 29 with two OH groups on phenyl
ring, showed a moderate inhibition of TP (IC50 = 176.9 1.6, 183.4 0.9, and 173.0 1.4μM,
respectively). This is consistent with the results we obtained for dihydroxylated derivatives
with benzylidine group (Fig 4).
Kinetic studies
Kinetic study on compounds 3, 9, 14, 27, and 29 revealed that they inhibit the TP in an uncom-
petitive manner (Table 1), as deduced from the Lineweaver-Burk plot. Uncompetitive inhibitors
interact with enzyme only when enzyme-substrate (ES) complex is formed. ES complex forma-
tion was proposed to induce conformational changes in the enzyme which facilitates the bind-
ing of the inhibitor. Uncompetitive inhibitors cause decrease in both Km and Vmax values of the
enzyme (Fig 5). Compound 22 inhibited the enzyme in a non-competitive manner (Fig 6). This
compound, therefore, interacted either with the amino acids of hydrophobic pocket of the
Fig 5. Kinetic mode of compound 27 (A) Depicts Lineweaver-Burk plot of compound 27 in which reciprocal of substrate
concentration (1/S) is plotted on x-axis, while reciprocal of rate of reaction (1/V) is plotted on y-axis in the absence and present of
different concentrations of compound 27. (B) Depicts secondary re-plot of Lineweaver-Burk plot between the slopes (Km/Vmax) of each
line versus different concentrations of compound 27.
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Thymidine phosphorylase inhibitors of synthetic origin
Fig 6. Kinetic mode of compound 22 (A) Depicts Lineweaver-burk plot of compound 22 in which reciprocal of substrate
concentration (1/S) is plotted on x-axis, while reciprocal of rate of reaction (1/V) is plotted on y-axis in the absence and presence of
different concentrations of compound 22. Figure shows that apparent km of the enzyme remains unaffected while the apparent Vmax
decreased. (B) Depicts secondary re-plot of Lineweaver-Burk plot between the slopes (Km/Vmax) of each line versus different
concentrations of compound 22. (C) Depicts Dixon plot of reciprocal of rate of reaction (velocities) versus different concentrations of
compound 22.
https://doi.org/10.1371/journal.pone.0227549.g006
enzyme or at allosteric site of the enzyme. Noncompetitive inhibitors do not affect the Km value
but changes the Vmax value. These compounds, therefore, do not competitively interact with the
thymidine or phosphate-binding sites of TP when thymidine is used as the variable substrate.
Values of dissociation constants (Ki) were determined by secondary re-plot of Lineweaver-Burk
plot, and Dixon plot, these were in the range of 1.75–176 μM (Table 1).
Molecular docking studies
Molecular docking studies on selected inhibitors were performed in order to understand the
ligand-receptor interactions at atomic level. Compounds 3, 9, 14, 22, 27, and 29 were found to
be either uncompetitive or non-competitive inhibitors of TP. They showed binding to an allo-
steric site, located adjacent to the substrate binding site of thymidine phosphorylase. Accord-
ing to the crystal structure of TP (4LHM), it has a small α-helical domain containing residues
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Thymidine phosphorylase inhibitors of synthetic origin
Fig 7. Ribbon representation of TP (PDB:4LHM): It is showing a large α/β domain (red dotted lines) and a small
α-helical domain (blue dotted lines). The inhibitor AZZ (green ball and stick) occupies the nucleoside binding
pocket of TP while the phosphate binding site is in the α/β domain of TP. Compounds 3, 9, 14, 22, 27, and 29 (grey
ball and stick) binds in the α/β domain of TP.
https://doi.org/10.1371/journal.pone.0227549.g007
from 1 to 65, 163 to 193 that makes the thymidine binding site. While, a large α/β domain with
residues 80 to 154, 197 to 440 make the phosphate binding site. These two domains are sepa-
rated by a large cleft, and the movement of these two domains brings the two substrate binding
sites closer for the initiation of the catalytic activity. There are several homologous flexible
loops located at different positions of TP, comprising of 173–178, 206–213, 367–381 amino
acid residues [44,45]. The molecular docking analysis of selected inhibitors showed that most
of them were able to interact with the amino acid residues of large α/β domain (197 to 440)
which play an important role in the catalytic activity of TP (Fig 7).
Compound 5 with dihydroxyl groups at meta and para positions were found to interact
with Leu389 and Gln244 via H-bonds (Fig 8). Compounds 9, 14, and 22 showed slightly
different docked poses, in comparison to compound 5. For instance, the OH group of phe-
nyl ring in compound 9 was able to form H-bonds with Asp391, and Arg388 (Fig 9). Com-
pound 14 was able to form H-bond interactions with Asp391 (Fig 10). Similarly, compound
22 was also able to interact with Asp391, Arg388, and Leu389 via H-bonds (Fig 11).
Compounds 27, and 29 possess CH3 and C2H5, respectively, as R2 substituents in addition
to the hydroxyl group substituted phenyl ring. These alkyl groups further changed the
docked poses of compounds 27, and 29. For instance, in compound 27 the carboxyl group
of hydrazide was found to be involved in interacting with Arg271 and the OH group of phe-
nyl ring interacted with water molecule via H-bond (Fig 12). The ortho substituted hydroxyl
group in compound 29 interacted with carboxyl group of Leu389 via H-bond (Fig 13),
while the meta substituted OH group interacted with the side chain of Arg271 via H-bond.
Anti-proliferative studies
Compounds found to be active against TP enzyme were then subjected to MTT proliferation
assay. Rapid proliferation is the property inherent to cancerous/ tumor cells. TP is reported to
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Thymidine phosphorylase inhibitors of synthetic origin
Fig 8. 3D Representation of docked pose of compound 3 (IC50 = 229.5 2.1 μM). The two OH groups showed H-
bonding with Leu389 and Gln244 (yellow dotted lines).
https://doi.org/10.1371/journal.pone.0227549.g008
Fig 9. 3D Representation of docked pose of compound 9 (IC50 = 159.0 0.4 μM): It is showing interaction with
the Asp391, and Arg388 via H-bonds (yellow and blue dotted lines).
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Thymidine phosphorylase inhibitors of synthetic origin
Fig 10. 3D Representation of docked pose of compound 14 (IC50 = 158.0 1.0 μM). It is showing interaction with
Asp391 via H-bonds (yellow and blue dotted lines).
https://doi.org/10.1371/journal.pone.0227549.g010
Fig 11. 3D Representation of docked pose of compound 22 (IC50 = 6.8 0.7 μM). The OH group is interacting with
Asp391 via H-bond (yellow dotted lines), while the hydrogens of phenyl ring are interacting with Leu389 via H-bonds
(blue dotted lines).
https://doi.org/10.1371/journal.pone.0227549.g011
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Thymidine phosphorylase inhibitors of synthetic origin
Fig 12. 3D Representation view of docked pose of compound 27 (IC50 = 176.9 1.6 μM). The carboxyl group of
hydrazide is interacting with Arg271 via H-bond (yellow dotted lines), while the ortho substituted OH group is
interacting with Pro270 via H-bond (blue dotted lines).
https://doi.org/10.1371/journal.pone.0227549.g012
Fig 13. 3D Representation of docked pose of compound 29 (IC50 = 173.0 1.4 μM). The ortho substituted OH
group is interacting with the Leu389 via H-bond (yellow dotted lines), and the phenyl ring hydrogen is interacting
with Leu389 via H-bond (blue dotted lines). The meta substituted OH group is interacting with Arg271 via H-bond.
https://doi.org/10.1371/journal.pone.0227549.g013
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Thymidine phosphorylase inhibitors of synthetic origin
Table 2. In-vitro anti-proliferative activities of active compounds.
Compound
IC50 Standard Deviation (μM)
3T3 Cell Line
PC3 Cell Line
7.5 0.4
7.6 0.4
>30
1
13.1 0.3
23.0 1.0
23.5 1.1
>30
2
3
6
>30
7
3.8 0.4
>30
>30
8
>30
9
>30
>30
10
>30
>30
11
>30
>30
12
>30
>30
13
>30
>30
14
>30
>30
16
>30
>30
19
23.6 0.2
9.2 0.1
15.3 0.9
9.4 0.3
26.7 1.0
20.2 0.9
19.5 0.9
0.26 0.1 μM
-
9.6 0.3
8.3 0.9
10.5 0.5
6.5 0.2
>30
22
23
24
27
28
>30
29
>30
Standard Cyclohexamide)
Standard (Doxorubicin)
-
0.91 0.1 μM
https://doi.org/10.1371/journal.pone.0227549.t002
be highly expressed in prostate cancer. Therefore, the compounds active against TP were eval-
uated for their anti-proliferative effect on prostate cancer (PC3) cells, and cytotoxic effect on
mouse fibroblast (3T3) cells [2, 30, 31].
Compounds 1, 2, 3, 7, 19, 22–24, and 27–29 were found cytotoxic towards the proliferation
of 3T3 cells with IC50 values in between 3.8–26.7 μM, in comparison to the reference com-
pound i.e. cyclohexamide (IC50 = 0.26 0.1 μM). Compounds 1, 2, 19, and 22–24 inhibited
the proliferation of PC3 cells with IC50 values in between 6.5–10.5 μM, in comparison to the
reference drug i.e. doxorubicin (IC50 = 0.91 0.1 μM) (Table 2).
IC50 Values of compounds 1, 2, 19, and 22–24 for cytotoxicity activity is lower in cancerous
cells (IC50 = 7.5 0.4, 7.6 0.4, 9.6 0.3, 10.5 0.5, 6.5 0.2, and 8.3 0.9 μM, respectively)
than on normal cells (IC50 = 13.1 0.3, 23.0 1.0, 23.6 0.2, 15.3 0.9, 9.4 0.3, and 9.2 0.1
μM, respectively). These compounds therefore possess dual characteristics as they can inhibit
the TP enzymatic activity, and proliferation of PC3 cells. Their dual inhibitory potential
deserves to be studied further for anticancer activity.
Compounds which showed anti-proliferation activity only towards 3T3 cell lines (such as 3,
7, 27–29) were cytotoxic to normal cells and therefore need to be investigated again for above-
mentioned activities after structural modification.
Conclusion
Role of TP in inducing angiogenesis and tumor growth makes it an important target for the
discovery of anti-angiogenic (anti-cancer) agents. In this regards, a series of 4-hydroxybenzo-
hydrazides (1–29) was evaluated for its in-vitro inhibitory activity against angiogenic enzyme
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Thymidine phosphorylase inhibitors of synthetic origin
TP. Twenty derivatives were found to inhibit the TP enzymatic activity. Among them, com-
pound 22 showed a several fold more potent TP inhibitory activity (IC50 = 6.8 0.7 μM),
when compared to the standard, 7-deazaxanthine (IC50 = 41.0 1.63 μM). In mechanistic
studies, it showed a non-competitive mode of inhibition (Ki = 1.75 0.009 μM). Compound
22 apparently interact with Arg271 and Pro270 of enzyme via H-bonds. Furthermore, it also
showed a good anti-proliferative (cytotoxic) activity against prostate cancer (PC3) cell line.
Present study thus identifies a new class of inhibitors against TP enzyme, and cancer cells pro-
liferation. This class can be investigated further for anti-cancer studies at in-vivo level.
Supporting information
S1 Fig.
(TIF)
Acknowledgments
The authors are thankful to the Searle, Pakistan for financial support (APC charges) through
project entitled as Discovery of New Therapeutic Application of Existing Drugs by Reposition-
ing and Purposing Approach.
Author Contributions
Conceptualization: Sumaira Javaid, Syed Muhammad Saad, Khalid Mohammed Khan, M.
Iqbal Choudhary, Atta-ur Rahman.
Data curation: Sumaira Javaid, M. Iqbal Choudhary.
Formal analysis: Sumaira Javaid, Syed Muhammad Saad, Khalid Mohammed Khan, M. Iqbal
Choudhary.
Investigation: Sumaira Javaid, Syed Muhammad Saad, Khalid Mohammed Khan, Atta-ur
Rahman.
Methodology: Sumaira Javaid, Syed Muhammad Saad, Humaira Zafar, Rizwana Malik, Khalid
Mohammed Khan, M. Iqbal Choudhary, Atta-ur Rahman.
Project administration: Sumaira Javaid, Khalid Mohammed Khan, M. Iqbal Choudhary,
Atta-ur Rahman.
Supervision: Khalid Mohammed Khan, M. Iqbal Choudhary, Atta-ur Rahman.
Validation: Sumaira Javaid, Khalid Mohammed Khan, M. Iqbal Choudhary.
Writing – original draft: Sumaira Javaid, Syed Muhammad Saad, Humaira Zafar, Khalid
Mohammed Khan, M. Iqbal Choudhary.
Writing – review & editing: Sumaira Javaid, Syed Muhammad Saad, Humaira Zafar, Khalid
Mohammed Khan, M. Iqbal Choudhary, Atta-ur Rahman.
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