Synthesis of indole based acetohydrazide analogs: Their in vitro and in silico thymidine phosphorylase studies

Article abstract of DOI:10.1016/j.bioorg.2020.103745

In this study, a series of indole based acetohydrazide derivatives (1–22) were synthesized and characterized by ¹³C NMR, ¹H NMR and HREI-MS. The resulted derivatives were tested for thymidine phosphorylase inhibitory potential. These derivatives inhibited thymidine phosphorylase at different concentration ranging from 1.10 ± 0.10 to 41.10 ± 1.10 μM when compared with the standard 7-Deazaxanthine (IC₅₀ value 38.68 ± 1.12 μM). The compound 8 having OH group at 2, 4 and 6 position was found the most potent among the series with IC₅₀ 1.10 ± 0.10 μM. The structure activity relationships (SAR) has been established for all compounds keeping in the view the role of substitution and the effect of functional group which significantly affect thymidine phosphorylase activity. The nature of binding interactions of the most potent compounds and active sites of the enzymes was confirmed through molecular docking study.

Full text of DOI:10.1016/j.bioorg.2020.103745

Journal Pre-proofs
Synthesis of indole based acetohydrazide analogs: their in vitro and in silico
thymidine phosphorylase studies
Muhammad Taha, Ebaa Ahmed Jassim Aldhamin, Noor Barak Almandil, El
Hassane Anouar, Nizam Uddin, Munther Alomari, Fazal Rahim, Bushra
Adalat, Mohamad Ibrahim, Fasial Nawaz, Naveed Iqbal, Bandar Alghanem,
Abdulelah Altolayyan, Khalid Mohammed Khan
PII:
S0045-2068(20)30045-6
DOI:
https://doi.org/10.1016/j.bioorg.2020.103745
YBIOO 103745
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
7 January 2020
5 March 2020
9 March 2020
Please cite this article as: M. Taha, E. Ahmed Jassim Aldhamin, N. Barak Almandil, E. Hassane Anouar, N.
Uddin, M. Alomari, F. Rahim, B. Adalat, M. Ibrahim, F. Nawaz, N. Iqbal, B. Alghanem, A. Altolayyan, K.
Mohammed Khan, Synthesis of indole based acetohydrazide analogs: their in vitro and in silico thymidine
phosphorylase studies, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103745
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Synthesis of indole based acetohydrazide analogs: their in vitro and in silico thymidine
phosphorylase studies
Muhammad Tahaa, Ebaa Ahmed Jassim Aldhaminb, Noor Barak Almandila, El
Hassane Anouarc, Nizam Uddind, Munther Alomarie, Fazal Rahimf, Bushra Adalatf,
Mohamad Ibrahima, Fasial Nawazg, Naveed Iqbalh, Bandar Alghanemi, Abdulelah
Altolayyani, Khalid Mohammed Khanj
aDepartment of clinical pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University,
P.O. Box 1982, Dammam 31441, Saudi Arabia
bCollege of clinical pharmacy, Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia
c Department of Chemistry, College of Science and Humanities, Prince Sattam bin Abdulaziz University, P.O. Box 83, Al Kharj 11942,
Saudi ArabiadFaculty of Applied Science, UiTM Shah Alam, 40450 Shah Alam, Selangor D.E.,
dDepartment of Chemistry, University of Karachi, Karachi-75270 Pakistan.
eDepartment of Stem Cell Biology, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University,
P.O. Box 1982, Dammam 31441, Saudi Arabia
fDepartment of Chemistry, Hazara University, Mansehra-21300, Khyber Pakhtunkhwa, Pakistan
gDepartment of Chemistry, University of Wah, Quaid Avenue, Wah Cantt 47000, Pakistan
hDepartment of chemistry University of poonch Rawalakot AJK.
iMedical Research Core Facility and Platforms (MRCFP), King Abdullah International Medical Research Center/ King Saud bin Abdulaziz
University for Health Sciences (KSAU-HS), King Abdulaziz Medical City (KAMC), NGHA, Riyadh 11426, Saudi Arabia
jH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270,
Pakistan
Abstract
In this study, a series of indole based acetohydrazide derivatives (1-22) were synthesized and
characterized by 13C-NMR, 1H-NMR and HREI-MS. The resulted derivatives were tested for
thymidine phosphorylase inhibitory potential. These derivatives inhibited thymidine
phosphorylase at different concentration ranging from 1.10 ± 0.10 to 41.10 ± 1.10 µM when
compared with the standard 7-Deazaxanthine (IC50 value 38.68 ± 1.12 µM). The compound 8
having OH group at 2, 4 and 6 position was found the most potent among the series with IC50
1.10 ± 0.10 µM. The structure activity relationships (SAR) has been established for all
compounds keeping in the view the role of substitution and the effect of functional group
which significantly affect thymidine phosphorylase activity. The nature of binding
interactions of the most potent compounds and active sites of the enzymes was confirmed
through molecular docking study.
Keywords: acetohydrazide; Synthesis; Thymidine phosphorylase; SAR; Molecular docking
 Correspondence and reprints
E-mail: E-mail: taha_hej@yahoo.com and mtaha@iau.edu.saTel: 00966502057370
1

1. 0. Introduction
Thymidine phosphorylase (TP) enzyme is a nucleoside metabolism enzyme, which is
a key role in the maintenance of mitochondria, recovery of cells destroyed after pathological
stress [1], and in pyrimidine nucleoside recovery. TP is responsible for the conversion of
thymidine to thymine and deoxyribose phosphate (2-deoxy-D-ribose-1-phosphate), which is
then converted to deoxyribose (2-deoxy-D-ribose) [2, 3]. Deoxy ribose is involved in the
angiogenesis, that stimulates vascular endothelial cell growth factor (VEGF). Resulting in
stimulation of endothelial cells to discharge of metalloproteinase enzyme and to migrate these
cells toward tumour tissues. This migration results in generation of new blood vessels and
cancer metastasis [4]. In addition, the over expression of TP is associated to several
pathological disorders such as rheumatoid arthritis, atherosclerosis and inflammatory diseases
[5]. TP inhibitors are believed to hinder the production of 2-deoxy ribose production and are
therefore potential candidates for stopping cancer growth [6, 7].
The literature reveals several reports on the development of TP inhibitors [11-18],
since the discovery of purine based 7-deazaxanthine as 1st TP inhibitor [8-10]. For example,
the pyrimidine based 5-chloro-6-(1-(2-iminopyrrolidinyl) methyl) uracil hydrochloride (TPI)
(Fig. 1) is considered as the most efficient and potent human TP inhibitor so far and has been
evaluated in clinical trials (phase-II) for the treatment of solid tumour [19].
Indole is naturally occurring heterocycle and its derivatives are widely used in medicinal
chemistry due to their broad-spectrum biological activities [20-33]. Recently, the indole ring
has emerged as important pharmacophore for the cancer therapy [34-26] particularly as
tubulin inhibitors [37]. In addition, the indole derivatives can also act as, hypnotic-sedatives,
anti-depressants, anti-convulsant [38] and antioxidant activities [39, 40].
Our research group have already reported oxadiazole, isoquinoline, piperazine and
benzopyrazine analogues as potential thymidine phosphorylase inhibitors [41-45] Figure 1. In
continuation of our research on enzyme inhibition, we synthesized new indole derivatives and
investigated the effect of varying substitution on phenyl ring as thymidine phosphorylase
inhibitor.
O
O
Cl
HN
H
N
HN
N
O
N
H
NH
+
O
N
H
+
Cl
7DX
TPI
2

Fig. 1. Structure of known TP inhibitors- TPI and 7DX
HO
OH
N
O
NO2
N
Cl
S
HN NH
O
Cl
NH
H
N
HN
Cl
N
H
Amylase Inhibitor

IC =2.03
P Inhibitor

IC =
µ
  
50
µ
   
50
O
HO
HN
N
N
H
OH
HO
1-22
Synthesized compounds
IC50 = 1.10 ± 0.10 to 41.10 ± 1.10 µM
Standard 7-Deazaxanthine IC50 = 38.68 ± 1.12 µM.
Fig. 2. Rational of the current study
1.1. Result and discussion
1.1.1. Chemistry
Synthesis of indole derivative was carried out by reacting and refluxed methyl 2-(2-
methyl-1H-indol-3-yl) acetate with hydrazine hydrate in methanol for 6hrs, that resulted in 2-
(2-methyl-1H-indol-3-yl)acetohydrazide as intermediate product (I). The reaction completion
was checked by TLC. The intermediate product (I) was then recrystallized from methanol. 2-
(2-methyl-1H-indol-3-yl)acetohydrazide was further treated with varied aldehyde in methanol
for 3-4 hrs, this yielded indole-based acetohydrazide analogues (1-22). To obtained pure
products, the crude product was recrystallized in methanol. Then the reaction completion was
checked by TLC.
3

H
N
H
N2H4.H2O
N
O
R
Methanol
Reflux, 3hrs
H
Methanol
6hrs
N
HN N
O
NH2
H
R
O
O
(I)
O
1-22
Scheme 1. Synthesis of indole-based acetohydrazide analogues
Table 1
Structure of indole-based acetohydrazide analogues and their thymidine phosphorylase
activities.
S. No
R
IC50 ± SEMa S. No
R
IC50 ± SEMa
1
13.20 ±0.30
5.60 ±0.20
12
13
8.20 ±0.20
OH
Cl
NO2
OH
2
19.90 ± 0.40
OH
OH
3
4
3.90 ±0.10
14
15
11.60 ± 0.20
22.20 ± 0.60
HO
O
O
O
36.10 ±0.80
5
1.40 ±0.10
16
38.10 ± 0.90
OH
OH
4

OH
6
7
9.20 ±0.20
17
18
18.40 ± 0.40
6.60 ± 0.20
Cl
HO
18.50 ±0.40
OH
OH
HO
OH
8
1.10 ± 0.10
19
20
41.10 ± 1.10
4.80 ± 0.10
OH
OH
O
9
31.10 ± 0.70
15.80 ± 0.10
22.20 ± 0.50
N
O
O
NO2
10
21
22
N.A.
OH
OH
11
1.20 ± 0.05
NO2
7-Deazaxanthine (7DX)
38.68 ± 1.12 µM
1.1.2. In-vitro thymidine phosphorylase inhibition
A new class of indole-bearing acetohydrazide analogues (1–22) were synthesized and
evaluated for thymidine phosphorylase inhibition. The synthesized analogues showed
admirable inhibitory potentials with IC50 values ranging from 1.10 ± 0.10 to 41.10 ± 1.10
µM, when compared to standard 7-Deazaxanthine (7DX) with IC50 value of 38.65 ± 1.12 µM
(Table 1). The structure–activity relationship (SAR) has been established for all compounds
mainly based on varying substitution on phenyl ring.
5

The compound 8 having OH groups at the 2, 4, 6-position was the most potent among
the series with IC50 value of 1.10 ± 0.10 µM. Compounds having two OH groups at various
position on phenyl ring also showed excellent inhibitory activity for thymidine
phosphorylase, such as, compound 22, 5, 3, 2 and 6 with IC50 of 1.20 ± 0.05, 1.40 ± 0.10,
3.90 ± 0.10, 5.0 ± 0.20 and 6.60 ± 0.20 µM respectively. These results showed that position
of the –OH group on phenyl ring is affecting the biological activity of the compound. The
compounds 7, 12 and 18 having one –OH group at ortho, para and meta-position also
showed a good potency with IC50 values of 18.50± 0.40, 8.20± 0.20 and 6.60± 0.20 µM
respectively. The SAR results shows that the analogues having hydroxyl groups at ortho and
para-positions (12 and 18) possess a higher activity than the analogue having hydroxyl group
on meta-position (7).
The compounds 1, 10 and 11 having –NO2 group on phenyl ring also showed good
inhibition with IC50 values of 13.20 ± 0.30, 15.80 ± 0.10 and 22.20 ± 0.50 µM respectively.
The compounds 13 and 17 having chlorine atom on phenyl ring also exhibited a significant
inhibition activity with IC50 values 19.90± 0.40 and 18.40± 0.40 µM respectively. Compound
20 having both –OH and –CH3O groups on phenyl ring also showed a high inhibitory
potential with IC50 of 4.80 ± 0.10 µM. Replacement of hydroxyl group of compound 5 by
methoxy as in compound 20, results in decreasing the biological activity. Furthermore,
having two methoxy groups on phenyl ring as in compound 21, deactivates the compound
activity towards enzyme phosphorylase. Compound 15 bearing methyl group at ortho-
position displayed a good activity (IC50 = 22.20 ± 0.60 µM), While compound 16 having
methyl group at para-position of phenyl ring has less inhibition activity (IC50= 38.10 ± 0.90
µM) and almost close to the standard (38.68 ± 1.12 µM). In addition, having methyl group on
meta-position as in compound 19 (IC50 = 41.10 ± 1.10 µM), declined the inhibitory potential
of this compound.
1.1.3. Molecular docking
The measured IC50 values of indole acetohydrazide inhibitors are presented in Table1.
The thymidine phosphorylase inhibition of the synthesized derivatives is strongly related to
the type, number and positions of the functional group on the aromatic ring. To understand
the enzyme inhibition activity of the synthesized derivatives, molecular docking study has
been carried out to determine the binding modes of some selected synthesized compounds 2,
4-8, 12, 15, 18 and 22 from one side and the active residues of the thymidine phosphorylase
6

from other side. The selected compounds differ by the number and position of the substituted
hydroxyl groups in the aromatic ring. For instance, compounds 18, 7 and 12 are OH-mono
substituted in ortho, meta and para-positions respectively (Table 2). 18 and 12 show better
activity than 7. The compounds with OH groups in ortho-position to each other (5 and 20)
show better activity than compounds where OH groups are in ortho/meta-position to each
other (2 and 6). Compound 8 with three OH groups showed better activity compared with the
corresponding compound 2 with two OH groups (Table 2). The effect of methoxylation and
methylation on thymidine phosphorylase inhibition is investigated by comparing the IC50
values of 18, 4 and 15. The observed results showed that methoxylation and methylation of
hydroxyl group at ortho-position of 18 induces a decrease of thymidine phosphorylase
inhibition of 4 and 15, respectively (Table 2). Table 2 summarizes the calculated binding
energies of the stable complex's ligand-thymidine phosphorylase, the number of established
intermolecular hydrogen bonding between the synthesized compounds (2, 4-8, 12, 15, 18 and
22) and the active site residues of thymidine phosphorylase.
Table 2
The IC50, docking binding energies, hydrogen bonding and the number of closest residues to
the docked ligands in the active site of the selected indole acetohydrazide derivatives 2, 4-8,
12, 15, 18and 22 within the active binding site of thymidine phosphorylase.
Number of closest
No. of
Compound
Free binding energy
(kcal/mol)
H-Bonds
(HBs)
residues to the
docked ligand in the
active site
IC50 ± SEM
18
7
12
4
15
5
22
2
-7.37
-7.65
-7.04
-6.98
-7.27
-7.10
-6.96
-7.18
-7.49
-7.18
4
4
4
3
3
2
4
5
3
6
5
8
8
7
7
5
6
7
6
8
6.60 ± 0.20
18.50 ±0.40
8.20 ± 0.20
36.10 ±0.80
22.20 ±0.60
1.40 ±0.10
1.20 ± 0.05
5.60 ± 0.20
9.20 ±0.20
1.10 ± 0.10
6
8
All formed complexes between the selected compounds (2, 4-8, 12, 15, 18 and 22) and
the active amino acids of thymidine phosphorylase exhibited negative bending energies,
which is a signpost that the inhibition of thymidine phosphorylase by the selected compounds
is thermodynamically favourable (Table 2). As can be seen from the docking results in Table
7

2, binding energies of the stable complexes vary slightly from -6.96 to -7.65 kcal/mol. Such
variation is low enough to be considered as potent descriptor in rationalizing the observed
inhibitions. However, the number of hydrogen bonding, its distances and intermolecular
interactions between the docked ligands and the active residues may help to understand the
observed activities for specific cases. For instance, the strong inhibitions of 18 and 12
compared with 7 may refer to the presence of strong hydrogen bond formed between the
carbonyl group and amino acid ARG 171 in the formers and its absence in the latter (Fig. 3).
The slightly higher activity of analogue 18 compared with 12 may refer to the strength of the
hydrogen bond formed between the carbonyl group and amino acid ARG 171 in their
corresponding complexes. Indeed, the intermolecular hydrogen bond formed between the
carbonyl group of 18 and amino acid ARG 171 is of 3.08 Å, while the one formed with 12 is
of 3.08 Å.
7
12
8

18
Fig. 3. 3D (right) and 2D (left) closest interactions between active site residues of
thymidine phosphorylase and synthesized compounds 7, 12, and 18.
Compound 8 shows the higher inhibition toward thymidine phosphorylase with an IC50 of
1.10 µM (Table 2). Its higher activity may be attributed to the number of the substituted OH
groups, and therefor to the number of hydrogen bonding that can be established between
these hydroxyl groups and the active residues of thymidine phosphorylase. In addition to the
hydrogen bonding formed between carbonyl group and amine groups with the active moieties
of the thymidine phosphorylase, the substituted hydroxyl groups involved in the formation of
four hydrogen bond with active moieties of thymidine phosphorylase (Fig. 4).
9

Fig. 4. 3D (right) and 2D (left) closest interactions between active site residues of
thymidine phosphorylase and synthesized compound 8.
The methoxylation and methylation of the synthesized derivatives induces a decrease in
thymidine phosphorylase inhibition. For instance, the substitution of OH group of 18 by
methoxy (4) and methyl (15) groups leads to a decrease of thymidine phosphorylase
inhibition (Table 2). It is obvious from docking results (Table 5 and Figures 3 and 5) that the
methoxylation and methylation of 18 lead to a decrease of number of hydrogen bonding
established between 4 (15) and the active residues of thymidine phosphorylase. Indeed, four
hydrogen bonds are formed between 18 and the active residues of thymidine phosphorylase,
while three hydrogen bonds are formed between 4 (15) and the active residues of thymidine
phosphorylase. Further, the methoxylation and methylation of 18 lead to the destabilization of
the formed complexes (Table 2).
4
15
10

Fig. 5. 3D (right) and 2D (left) closest interactions between active site residues of
thymidine phosphorylase and synthesized compounds 4 and 15.
2.0. Material and method
2.1. General experiment
For NMR analysis JNM−ECS 400 MHz spectrometer was used. Thin layer
chromatography (TLC) analysis were performed using pre-coated silica gel aluminium plate
(Kieselgel 60, 254, E. Merck, Germany). UV at 254 and 365 nm were used to visualize the
chromatogram.
2.2. General procedure for the synthesis of Indole derivatives
Synthesis of indole derivative was carried out by reacting and refluxing 2-(2-methyl-
1H-indol-3-yl) acetate (5mmol) with hydrazine hydrate (5mL) in methanol (15 mL) for 6 hrs,
that resulted in 2-(2-methyl-1H-indol-3-yl) acetohydrazide as intermediate product (I). The
reaction completion was monitored with the help of TLC. The excess methanol and hydrazine
hydrate were evaporated from the product to obtain the crude product, and then recrystallized
in methanol to obtained pure product. Equimolar 2-(2-methyl-1H-indol-3-yl)acetohydrazide
(5 mmol) was further treated with varied aldehyde (5mmol) in methanol (15 mL) for 3-4 hrs
in the presence of acetic acid in catalytic amount, yielded indole-based acetohydrazide
analogues (1-22). The reaction completion was checked by TLC. To obtained pure product
the crude product was recrystallized in methanol.
2.2.1. 2-(2-methyl-1H-indol-3-yl)-N'-(4-nitrobenzylidene)acetohydrazide (1)
Yield: (89%); 1HNMR (400 MHz, DMSO-d6):  11.85 (s, 1H, NH), 11.56 (s, 1H, NH), 8.32
(s, 1H, ArCH=NAr), 8.27 (d, 2H, J = 8.2 Hz), 7.91 (d, 2H, J = 8.2 Hz), 7.54 (t, 1H, J = 7.2
Hz), 7.28 (d, 1H, J = 7.3 Hz), 7.21 (t, 1H, J = 7.3 Hz), 6.99-6.95 (m, 1H), 3.63 (s, 2H, CH2),
2.85 (s, 3H, CH3); 13CNMR (100 MHz, DMSO-d6) δ 168.6, 152.3, 143.1 139.8, 135.0, 129.4,
128.1, 124.7, 124.7, 124.3, 124.3, 123.1, 121.2, 118.9, 113.4, 109.3, 36.7, 18.9. HREI-MS:
m/z calcd for C18H16N4O3, [M]+ 336.1222; Found: 336.1209.
2.2.2. N'-(2,4-dihydroxybenzylidene)-2-(2-methyl-1H-indol-3-yl)acetohydrazide (2)
11

1
Yield: (86%); HNMR (400 MHz, DMSO-d6) δ 11.51 (s, 1H, NH), 11.32 (s, 1H, NH), 10.62
(s, 1H, OH), 9.86 (s, 1H, OH), 8.23 (s, 1H, ArCH=NAr) 7.52-7.48 (m, 2H), 7.26-7.19 (m,
2H), 6.98 (t, 1H, J = 7.2 Hz), 6.31 (dd, 1H, J = 7.2, 2.0 Hz), 6.24 (d, 1H, J = 2.0 Hz), 3.53 (s,
13
2H, CH2), 2.34 (s, 3H, CH3); CNMR (100 MHz, DMSO-d6) δ 168.6, 161.7, 161.3, 145.9,
135.7, 134.1, 129.4, 128.1, 123.1, 121.2, 118.9, 113.4, 111.8, 109.3, 108.0, 107.7, 36.7, 18.9.
HREI-MS: m/z calcd for C18H17N3O3, [M]+ 323.1270; Found: 323.1256.
2.2.3. N'-(3,5-dihydroxybenzylidene)-2-(2-methyl-1H-indol-3-yl)acetohydrazide (3)
1
Yield: (82%); HNMR (400 MHz, DMSO-d6) δ 11.34 (s, 1H, NH), 11.12 (s, 1H, NH), 9.42
(s, 2H, 2xOH), 8.03 (s, 1H, ArCH=NAr) 7.46 (t, 1H, J = 7.4 Hz), 7.21 (d, 1H, J = 7.2 Hz),
6.96-6.91 (m, 2H), 6.51 (s, 1H), 6.22 (s, 2H), 3.51 (s, 2H, CH2), 2.32 (s, 3H, CH3); 13CNMR
(100 MHz, DMSO-d6) δ 168.6, 161.1, 161.1, 146.0, 137.0, 135.7, 129.4, 128.1, 123.1, 121.2,
118.9, 113.4, 109.9,109.9, 109.3, 107.3, 36.7, 18.9. HREI-MS: m/z calcd for C18H17N3O3,
[M]+ 323.1270; Found: 323.1279.
2.2.4. N'-(2-methoxybenzylidene)-2-(2-methyl-1H-indol-3-yl)acetohydrazide (4)
1
Yield: (83%); HNMR (400 MHz, DMSO-d6) δ 11.45 (s, 1H, NH), 11.22 (s, 1H, NH), 8.52
(s, 1H, ArCH=NAr) 7.82 (t, 1H, J = 7.2 Hz), 7.72 (t, 1H, J = 7.2 Hz), 7.49-7.44 (m, 2H),
7.22-7.16 (m, 2H), 7.04-6.98 (m, 2H), 3.78 (s, 3H, OCH3), 3.51 (s, 2H, CH2), 2.32 (s, 3H,
CH3); 13CNMR (100 MHz, DMSO-d6) δ 168.6, 154.4, 145.7, 135.7, 133.2, 132.0, 129.4,
128.1, 123.1, 121.8, 121.2, 118.9, 117.0, 113.4, 111.6, 109.3, 55.7, 36.7, 18.9. HREI-MS:
m/z calcd for C19H19N3O2 [M]+ 321.1477; Found: 321.1463.
2.2.5. N'-(3,4-dihydroxybenzylidene)-2-(2-methyl-1H-indol-3-yl)acetohydrazide (5)
Yield: (81%); 1HNMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H, NH), 10.86 (s, 1H, NH), 10.31
(s, 1H, OH), 10.03 (s, 1H, OH), 8.02 (s, 1H, ArCH=NAr) 7.51-7.46 (m, 2H), 7.20-7.14 (m,
13
2H), 6.97-6.86 (m, 3H), 3.49 (s, 2H, CH2), 2.35 (s, 3H, CH3); CNMR (100 MHz, DMSO-
d6) δ 168.6, 151.1, 147.6, 145.9, 135.7, 131.6, 129.4, 128.1, 124.0, 123.1, 121.2, 119.2,
118.9, 118.4, 113.4, 109.3, 36.7, 18.9. HREI-MS: m/z calcd for C18H17N3O3, [M]+ 323.1270;
Found: 323.1253.
2.2.6. N'-(2,5-dihydroxybenzylidene)-2-(2-methyl-1H-indol-3-yl)acetohydrazide (6)
12

Yield: (83%); Yield: 1HNMR (400 MHz, DMSO-d6) δ 11.57 (s, 1H, NH), 10.82 (s, 1H, NH),
10.78 (s, 1H, OH), 10.24 (s, 1H, OH), 8.72 (s, 1H, ArCH=NAr) 7.49-7.44 (m, 2H), 7.20-7.14
(m, 2H), 7.04-6.94 (m, 2H), 6.73-6.68 (m, 1H), 3.49 (s, 2H, CH2), 2.35 (s, 3H, CH3);
13CNMR (100 MHz, DMSO-d6) δ 168.6, 153.0, 151.0, 145.9, 135.7, 129.4, 128.1, 123.1,
122.2, 122.0, 121.9, 121.2, 119.3, 118.9, 113.4, 109.3, 36.7, 18.9. HREI-MS: m/z calcd for
C18H17N3O3, [M]+ 323.1270; Found: 323.1255.
2.2.7. N'-(3-hydroxybenzylidene)-2-(2-methyl-1H-indol-3-yl)acetohydrazide (7)
1
Yield: (85%); HNMR (400 MHz, DMSO-d6) δ 11.21 (s, 1H, NH), 10.81 (s, 1H, NH), 10.76
(s, 1H, OH), 9.92 (s, 1H, ArCH=NAr) 7.46-7.42 (m, 2H), 7.18-6.85 (m, 6H), 3.52 (s, 2H,
CH2), 2.34 (s, 3H, CH3); 13CNMR (100 MHz, DMSO-d6) δ 168.6, 154.8, 147.0, 139.1, 135.7,
131.2, 129.4, 128.1, 123.7, 123.1, 121.2, 120, 118.9, 117.3, 113.4, 109.3, 36.7, 18.9. HREI-
MS: m/z calcd for C18H17N3O2, [M]+ 307.1321: Found: 307.1319.
2.2.8. 2-(2-methyl-1H-indol-3-yl)-N'-(2,4,6-trihydroxybenzylidene)acetohydrazide (8)
Yield: (87%); 1HNMR (400 MHz, DMSO-d6) δ 11.45 (s, 1H, NH), 10.92 (s, 3H, NH, 2xOH),
9.76 (s, 1H, OH), 8.52 (s, 1H, ArCH=NAr) 7.52 (t, 1H, J = 7.4 Hz), 7.28 (d, 1H, J = 7.2 Hz),
7.09-6.94 (m, 2H), 5.76 (s, 2H), 3.54 (s, 2H, CH2), 2.35 (s, 3H, CH3); 13CNMR (100 MHz,
DMSO-d6) δ 168.6, 161.9, 161.9, 158.9, 144.0, 135.7, 129.4, 128.1, 123.1, 121.2, 118.9,
113.4, 109.3, 107.6, 103.4, 103.4, 36.7, 18.9. HREI-MS: m/z calcd for C18H17N3O4, [M]+
339.1219; Found: 339.1207.
2.2.9. N'-(4-(dimethylamino)benzylidene)-2-(2-methyl-1H-indol-3-yl)acetohydrazide (9)
1
Yield: (86%); HNMR (400 MHz, DMSO-d6) δ 11.06 (s, 1H, NH), 10.96 (s, 1H, NH), 8.32
(s, 1H, ArCH=NAr) 7.82 (d, 2H, J = 7.2 Hz), 7.42-7.34 (m, 3H), 7.12-7.03 (m, 2H), 7.04-
6.98, 6.82 (t, 1H, J = 7.4 Hz), 3.52 (s, 2H, CH2), 2.92 (s, 6H, 2xCH3), 2.33 (s, 3H, CH3);
13CNMR (100 MHz, DMSO-d6) δ 168.6, 155.0, 144.7, 135.7, 130.0, 130.0, 129.4, 128.1,
125.5, 123.1, 121.2, 118.9, 113.6, 113.6, 113.4, 109.3, 41.9, 41.9, 36.7, 18.9. HREI-MS: m/z
calcd for C20H22N4O, [M]+ 334.1794; Found: 334.1781
2.2.10. 2-(2-methyl-1H-indol-3-yl)-N'-(2-nitrobenzylidene)acetohydrazide (10)
1
Yield: (83%); HNMR (400 MHz, DMSO-d6) δ 11.82 (s, 1H, NH), 11.53 (s, 1H, NH), 8.33
(s, 1H, ArCH=NAr) 8.07-7.94 (m, 2H), 7.74 (t, 1H, J = 7.2 Hz), 7.59 (t, 1H, J = 7.1 Hz), 7.39
13

(dd, 1H, J = 7.2, 2.0 Hz), 7.21 (d, 1H, J = 7.1 Hz), 7.02-6.96 (m, 2H), 3.53 (s, 2H, CH2),
2.34 (s, 3H, CH3); 13CNMR (100 MHz, DMSO-d6) δ 168.6, 151.0, 144.7, 137.2, 135.7,
133.3, 131.7, 129.4, 129.0, 128.1, 126.1, 123.1, 121.2, 118.9, 113.4, 109.3, 36.7, 18.9. HREI-
MS: m/z calcd for C18H16N4O3, [M]+ 336.1222; Found: 336.1209.
2.2.11. 2-(2-methyl-1H-indol-3-yl)-N'-(3-nitrobenzylidene)acetohydrazide (11)
1
Yield: (88%); HNMR (400 MHz, DMSO-d6) δ 11.72 (s, 1H, NH), 11.54 (s, 1H, NH), 8.44
(s, 1H, ArCH=NAr), 8.21 (t, 1H, J = 7.3 Hz), 8.09-8.03 (m, 2H), 7.71 (t, 1H, J = 7.2 Hz),
7.47 (t, 1H, J = 7.1 Hz), 7.22 (d, 1H, J = 7.1 Hz), 6.99-6.94 (m, 2H), 3.55 (s, 2H, CH2), 2.36
(s, 3H, CH3); 13CNMR (100 MHz, DMSO-d6) δ 168.6, 151.5, 143.8, 135.7, 133.5, 133.0,
131.0, 129.4, 128.1, 127.8, 123.1, 123.0, 121.2, 118.9, 113.4, 109.3, 36.7, 18.9. HREI-MS:
m/z calcd for C18H16N4O3, [M]+ 336.1222; Found: 336.1207.
2.2.12. N'-(4-hydroxybenzylidene)-2-(2-methyl-1H-indol-3-yl)acetohydrazide (12)
1
Yield: (85%); HNMR (400 MHz, DMSO-d6) δ 11.23 (s, 1H, NH), 11.04 (s, 1H, NH), 9.87
(s, 1H, OH), 8.65 (s, 1H, ArCH=NAr), 7.75 (t, 3H, J = 7.2 Hz), 7.45 (d, 2H, J = 7.2 Hz),
13
7.05-6.95 (m, 3H), 3.61 (s, 2H, CH2), 2.33 (s, 3H, CH3); CNMR (100 MHz, DMSO-d6) δ
168.6, 158.8, 145.0, 135.7, 133.6, 133.6, 129.4, 129.0, 128.1, 123.1, 121.2, 119.8, 119.8,
118.9, 113.4, 109.3, 36.7, 18.9. HREI-MS: m/z calcd for C18H17N3O2, [M]+ 307.1321: Found:
307.1305.
2.2.13. N'-(4-chlorobenzylidene)-2-(2-methyl-1H-indol-3-yl)acetohydrazide (13)
1
Yield: (86%); HNMR (400 MHz, DMSO-d6) δ 11.49 (s, 1H, NH), 11.29 (s, 1H, NH), 8.82
(s, 1H, ArCH=NAr), 8.17 (d, 2H, J = 7.4 Hz), 7.44 (d, 2H, J = 7.4 Hz), 7.45-7.40 (m, 2H),
13
7.12-7.06 (m, 2H), 3.53 (s, 2H, CH2), 2.31 (s, 3H, CH3); CNMR (100 MHz, DMSO-d6) δ
168.6, 145.1, 139.0, 135.7, 132.6, 131.8, 131.8, 129.4, 128.1, 127.4, 127.4, 123.1, 121.2,
118.9, 113.4, 109.3, 36.7, 18.9. HREI-MS: m/z calcd for C18H16ClN3O [M]+ 325.0982,
Found: 325.0968.
2.2.14. methyl-4-((2-(2-(2-methyl-1H-indol-3-yl)acetyl)hydrazono)methyl)benzoate (14)
1
Yield: (81%); HNMR (400 MHz, DMSO-d6) δ 10.82 (s, 1H, NH), 10.76 (s, 1H, NH), 8.76
(s, 1H, ArCH=NAr), 8.44 (d, 2H, J = 7.2 Hz), 7.83 (d, 2H, J = 7.2 Hz), 7.56 (t, 1H, J = 6.8
Hz), 7.28 (t, 1H, J = 6.8 Hz), 7.45-7.40 (m, 2H), 7.05-6.96 (m, 2H), 3.86 (s, 3H, OCH3) 3.54
14

(s, 2H, CH2), 2.32 (s, 3H, CH3); 13CNMR (100 MHz, DMSO-d6) δ 168.6, 165.0, 145.3,
141.1, 135.7, 134.5, 131.3, 131.3, 129.4, 129.0, 129.0, 128.1, 123.1, 121.2, 118.9, 113.4,
109.3, 52.5, 36.7, 18.9. HREI-MS: m/z calcd for C20H19N3O3, [M]+ 349.1426; Found:
349.1412.
2.2.15. 2-(2-methyl-1H-indol-3-yl)-N'-(2-methylbenzylidene)acetohydrazide (15)
1
Yield: (85%); HNMR (400 MHz, DMSO-d6) δ 10.83 (s, 1H, NH), 10.77 (s, 1H, NH), 8.67
(s, 1H, ArCH=NAr), 8.06 (d, 1H, J = 2.0 Hz), 7.62-7.58 (m, 2H), 7.43-7.35 (m, 2H), 7.04-
13
6.97 (m, 3H), 3.52 (s, 2H, CH2), 2.30 (s, 3H, CH3), 2.22 (s, 3H, CH3); CNMR (100 MHz,
DMSO-d6) δ 168.6, 144.6, 137.1, 135.7, 133.0, 131.5, 129.4, 129.2, 128.1, 128.0, 125. 2,
123.1, 121.2, 118.9, 113.4, 109.3, 36.7, 21.6, 18.9. HREI-MS: m/z calcd for C19H19N3O, [M]+
305.1528 Found: 305.1515.
2.2.16. 2-(2-methyl-1H-indol-3-yl)-N'-(4-methylbenzylidene)acetohydrazide (16)
1
Yield: (86%); HNMR (400 MHz, DMSO-d6) δ 10.78 (s, 1H, NH), 10.74 (s, 1H, NH), 8.56
(s, 1H, ArCH=NAr), 7.84 (d, 2H, J = 7.3 Hz), 7.78 (d, 1H, J = 7.4 Hz), 7.46 (d, 2H, J = 7.3
Hz), 7.39 (t, 1H, J = 7.2 Hz), 7.04-6.97 (m, 2H), 3.49(s, 2H, CH2), 2.35 (s, 3H, CH3), 2.32 (s,
3H, CH3); 13CNMR (100 MHz, DMSO-d6) δ 168.6, 144.6, 141.0, 135.7, 131.3, 131.3, 131.0,
129.4, 129.2, 129.2, 128.1, 123.1, 121.2, 118.9, 113.4, 109.3, 36.7, 21.9, 18.9. HREI-MS:
m/z calcd for C19H19N3O, [M]+ 305.1528 Found: 305.1505.
2.2.17. N'-(3-chlorobenzylidene)-2-(2-methyl-1H-indol-3-yl)acetohydrazide (17)
1
Yield: (84%); HNMR (400 MHz, DMSO-d6) δ 10.72 (s, 1H, NH), 10.67 (s, 1H, NH), 8.62
(s, 1H, ArCH=NAr), 7.76 (d, 1H, J = 2.0 Hz), 7.62-7.58 (m, 2H), 7.48-7.42 (m, 2H), 7.17 (t,
1H, J = 7.1 Hz), 6.92-6.86 (m, 2H), 3.51 (s, 2H, CH2), 2.32 (s, 3H, CH3); 13CNMR (100
MHz, DMSO-d6) δ 168.6, 145.4, 139.9, 136.4, 135.7, 132.0, 131.7, 130.4, 130.1, 129.4,
128.1, 123.1, 121.2, 118.9, 113.4, 109.3, 36.7, 18.9. HREI-MS: m/z calcd for C18H16ClN3O
[M]+ 325.0982, Found: 325.0971.
2.2.18. N'-(2-hydroxybenzylidene)-2-(2-methyl-1H-indol-3-yl)acetohydrazide (18)
Yield: (82%); 1HNMR (400 MHz, DMSO-d6) δ 11.64 (s, 1H, NH), 11.31 (s, 1H, NH), 11.28
(s, 1H, OH), 8.34 (s, 1H, ArCH=NAr), 7.86-7.80 (m, 2H), 7.62-7.56 (m, 2H), 7.48-7.42 (m,
13
2H), 7.21-7.15 (m, 4H), 3.53 (s, 2H, CH2), 2.31 (s, 3H, CH3); CNMR (100 MHz, DMSO-
15

d6) δ 168.6, 157.0, 147.3, 135.7, 133.7, 129.4, 129.0, 128.1, 126.2, 123.1, 121.2, 120.2,
119.4, 118.9, 113.4, 109.3, 36.7, 18.9. HREI-MS: m/z calcd for C18H17N3O2, [M]+ 307.1321:
Found: 307.1308.
2.2.19. 2-(2-methyl-1H-indol-3-yl)-N'-(3-methylbenzylidene)acetohydrazide (19)
1
Yield: (87%); HNMR (400 MHz, DMSO-d6) δ 10.81 (s, 1H, NH), 10.77 (s, 1H, NH), 8.62
(s, 1H, ArCH=NAr), 7.71-7.66 (m, 2H), 7.51-7.46 (m, 2H), 7.39-7.35 (m, 2H), 7.12-7.05 (m,
2H), 3.52 (s, 2H, CH2), 2.34 (s, 3H, CH3), 2.32 (s, 3H, CH3); 13CNMR (100 MHz, DMSO-d6)
δ 168.6, 144.4, 137.5, 135.7, 134.1, 132.1, 130.0, 129.4, 129.0, 128.1, 127.0, 123.1, 121.2,
118.9, 113.4, 109.3, 36.7, 21.6, 18.9. HREI-MS: m/z calcd for C19H19N3O, [M]+ 305.1528
Found: 305.1515.
2.2.20. N'-(3-hydroxy-4-methoxybenzylidene)-2-(2-methyl-1H-indol-3-yl)acetohydrazide (20)
1
Yield: (85%); HNMR (400 MHz, DMSO-d6) δ 11.24 (s, 1H, NH), 11.06 (s, 1H, NH), 9.47
(s, 1H, OH), 8.48 (s, 1H, ArCH=NAr), 7.86-7.81 (m, 2H), 7.65 (d, 1H, J = 2.0 Hz), 7.57-7.52
(m, 2H), 7.13-7.06 (m, 2H), 3.92 (s, 3H, OCH3), 3.54 (s, 2H, CH2), 2.33 (s, 3H, CH3);
13CNMR (100 MHz, DMSO-d6) δ 168.6, 152.0, 149.5, 147.0, 135.7, 132.0, 129.4, 128.1,
125.4, 123.1, 121.2, 118.9, 116.3, 114.6, 113.4, 109.3, 58.1, 36.7, 18.9. HREI-MS: m/z calcd
for C19H19N3O3, [M]+ 337.1426; Found: 337.1408.
2.2.21. N'-(2,4-dimethoxybenzylidene)-2-(2-methyl-1H-indol-3-yl)acetohydrazide (21)
1
Yield: (82%); HNMR (400 MHz, DMSO-d6) δ 11.16 (s, 1H, NH), 11.12 (s, 1H, NH), 8.44
(s, 1H, ArCH=NAr), 7.78 (d, 1H, J = 7.3 Hz), 7.63 (t, 1H, J = 7.1 Hz), 7.41-7.37 (m, 1H),
7.17 (t, 1H, J = 7.2 Hz), 6.62-6.57 (m, 2H), 3.82 (s, 3H, OCH3), 3.52 (s, 2H, CH2), 2.32 (s,
3H, CH3); 13CNMR (100 MHz, DMSO-d6) δ 168.6, 158.6, 156.2, 147.3, 135.7, 133.0, 129.4,
128.1, 123.1, 121.2, 118.9, 113.4, 111.3, 109.9, 109.3, 106.0, 55.5, 55.5, 36.7, 18.9. HREI-
MS: m/z calcd for C20H21N3O3, [M]+ 351.1583; Found: 351.1571.
2.2.22. -(2,3-dihydroxybenzylidene)-2-(2-methyl-1H-indol-3-yl)acetohydrazide (22)
Yield: (80%); 1HNMR (400 MHz, DMSO-d6) δ 11.71 (s, 1H, NH), 11.03 (s, 1H, NH), 10.81
(s, 1H, OH), 9.19 (s, 1H, OH), 8.37 (s, 1H, ArCH=NAr), 7.78 (d, 1H, J = 7.1 Hz), 7.53 (t,
1H, J = 6.5 Hz), 7.24-7.16 (m, 3H), 6.94-6.89 (m, 2H), 3.51 (s, 2H, CH2), 2.33 (s, 3H, CH3);
16

13CNMR (100 MHz, DMSO-d6) δ 168.6, 153.0, 152.5, 145.9, 135.7, 129.4, 128.1, 125, 124.9,
123.1, 121.7, 121.2, 121.0, 118.9, 113.4, 109.3, 36.7, 18.9. HREI-MS: m/z calcd for
C18H17N3O3, [M]+ 323.1270; Found: 323.1258.
2.3. Molecular docking details
The binding modes between the docked selected synthesized indole acetohydrazide
derivatives and the active residues of thymidine phosphorylase have been explored using
Autodock package [46]. The geometries of thymidine phosphorylase and the original docked
ligand 3'-azido-2'-fluoro-dideoxyuridine were obtained from the RCSB data bank web site
(PDB code 4EAD) [47]. Water molecules were removed; polar hydrogen atoms and Kollman
charge were added to the extracted receptor using the automated tool in AutoDock Tools 4.2.
The active site is identified based on co-crystallized receptor-ligand complex structure of
thymidine phosphorylase. The re-docking of the original ligand 3'-azido-2'-fluoro-
dideoxyuridine into the active site is well reproduced with a RMSD value less than 1.14 Å
and a binding energy of -6.63 kcal/mol. The molecular structures geometries of indole
acetohydrazide derivatives were minimized at Merck molecular force field 94 (MMFF94)
level44. The optimized structures were saved as pdb files. Nonpolar hydrogens were merged,
and rotatable bonds were defined for each docked ligand. Docking studies were performed by
Lamarckian genetic algorithm, with 500 as total number of run for binding site for original
ligand the synthesized derivatives. In each respective run, a population of 150 individuals
with 27000 generations and 250000 energy evaluations were employed. Operator weights for
crossover, mutation, and elitism were set to 0.8, 0.02, and 1, respectively. The binding site
was defined using a grid of 35 × 35 × 35 points each with a grid spacing of 0.375 Å. The
docking calculation have been carried out using an Intel (R) Core (TM) i5-3770 CPU @ 3.40
GHz workstation.
2.4.
In vitro studies on E. coli TP (EcTP)
We utilized commercially accessible recombinant E. coli TP. The E. coli TP (EcTP)
distribute a whole sequence resemblance of ∼42% with human TP. The Thymidine
phosphorylase/PD-ECGF (E. coli) activity was concluded by determining the absorbance at
290 nm spectrophotometrically. In short, overall response combination of 200 μL included
145 μL of potassium phosphate buffer (pH 7.4), 30 μL of enzyme (E. coli) at strength 0.05
and 0.002 U, correspondingly, were incubated with 5 μL of test materials for 10 min at 25 °C
in microplate reader. Following incubation, pre read at 290 nm was carried to determine the
17

absorbance of substrate particles. Substrate (20 μL, 1.5 mM), was dispersed in potassium
phosphate buffer, was instantly added to plate and constantly read after 10, 20, and 30 min in
microplate reader (spectra max, molecular devices, CA, USA). All analysis was completed in
triplicate.
3.0. Conclusions
We have synthesized twenty-two analogues of indole based acetohydrazides (1-22) which
were evaluated against thymidine phosphorylase inhibitory potential. All analogues showed
potent inhibitory potential with IC50 ranging from 1.10 ± 0.10 to 41.10 ± 1.10 µM when
compared with the paradigm 7-Deazaxanthine inhibitor (IC50 value 38.68 ± 1.12 µM). The
compound 8 having 3-OH group at 2, 4 and 6 position was the most potent among the series
with IC50 value of 1.10 ± 0.10. Structure activity relationship was mainly based on varying
substituents on phenyl ring. The molecular docking studies were performed for the most
potent analogues.
Reference
1. M. Toi, M. A. Rahman, H. Bando, L. W. Chow, Thymidine phosphorylase (platelet-
derived endothelial-cell growth factor) in cancer biology and treatment. Lancet Oncol.
6(3) (2005) 158–66.
2. F. Iftikhar, F. Yaqoob, N. Tabassum, M. S. Jaan, A. Sadiq, S. Tahir, T. Batool, B. Niaz,
F.L. Ansari, M.I. Choudhary, U. Rashid, Design, synthesis, in-vitro thymidine
phosphorylase inhibition, in-vivo antiangiogenic and in-silico studies of C-6 substituted
dihydropyrimidines. Bioorg. Chem. 80, (2015), 99-111.
3. A. Bronckaers, F. Gago, J. Balzarini, S. Liekens, The dual role of thymidine
phosphorylase in cancer development and chemotherapy. Med. Res. Rev. 29(6) (2009)
903-953.
4. S. Matsushita, T. Nitanda, T. Furukawa, T. Sumizawa, A. Tani, K. Nishimoto, S.
Akiba, K. Miyadera, M. Fukushima, Y. Yamada, T. Kanzaki, S. Akiyama, The effect of a
thymidine phosphorylase inhibitor on angiogenesis and apoptosis in tumors. Cancer
Res. 59(8) (1999) 1911-1916.
5. A. Bronckaers, F. Gago, J. Balzarini, S. Liekens, A Bron, The dual role of thymidine
phosphorylase in cancer development and chemotherapy. Med. Res. Rev. 29(6) (2009)
903-953.
18

6. F. Focher, S. Spadari, Thymidine phosphorylase: a two-face Janus in anticancer
chemotherapy. Curr. Cancer Drug Tar. 1(2) (2001) 141-153.
7. K. Miyadera, T. Emura, N. Suzuki, S. Akiyama, Y. Yamada, Novel functional antitumor
nucleoside TAS-102, combined from of F3Rhd and its modulator (2): Inhibitory effect of
TPI on tumor-derived angiogenesis and metasis. Proc. Am. Assoc.Cancer Res. 39 (1998)
609.
8. L. Sun, H. Bera, W.K. Chui, Synthesis of pyrazolo [1, 5-a][1, 3, 5] triazine derivatives as
inhibitors of thymidine phosphorylase. Eur. J. Med. Chem. 65 (2013) 1-11.
9. S. Yano, H. Kazuno, T. Sato, N. Suzuki, T. Emura, K. Wierzba, J.I. Yamashita, M.
Fukushima, T. Asao, Synthesis and evaluation of 6-methylene-bridged uracil derivatives.
Part 2: optimization of inhibitors of human thymidine phosphorylase and their selectivity
with uridine phosphorylase. Bioorg. Med. Chem. Lett. 12(13) (2004) 3443-3450.
10. J. Balzarini, A.E. Gamboa, R. Esnouf, S. Liekens, J. Neyts, E. De Clercq, M.J.
Camarasa, M.J. Pérez-Pérez, 7‐Deazaxanthine, a novel prototype inhibitor of thymidine
phosphorylase. FEBS Lett. 438(1-2) (1998) 91-95.
11. H. Ullah, F. Rahim, M. Taha, I. Uddin, A. Wadood, S. A. Shah, R.K. Farooq, M. Nawaz,
Z. Wahab, K. M. Khan, Synthesis, molecular docking study and in vitro thymidine
phosphorylase inhibitory potential of oxadiazole derivatives. Bioorg. Chem. 78 (2018)
58–67.
12. S. A. Shahzad, M. Yar, M. Bajda, B. Jadoon, Z. A. Khan, S. A. Naqvi, A. J. Shaikh, K.
Hayat, A. Mahmmod, N. Mahmood, S. Filipek. Synthesis and biological evaluation of
novel oxadiazole derivatives: A new class of thymidine phosphorylase inhibitors as
potential anti-tumor agents. Bioorg. Med. Chem. 22(3) (2014) 1008–1015.
13.
14. K. Pomeisl, I. Votruba, R. Pohl, Syntheses of pyrimidine acyclic nucleoside phosphonates
as potent inhibitors of thymidine phosphorylase (PD-ECGF) from SD-lymphoma.
Nucleos. Nucleot. Nucl. 26(8-90 (2007) 1025-1028.
15. A. Gbaj, P.N. Edwards, P. Reigan, S. Freeman, M. Jaffar, K.T.J. Douglas, Thymidine
phosphorylase from Escherichia coli: Tight-binding inhibitors as enzyme active-site
titrants. Enzyme Inhib. Med. Chem. 21(1) (2006) 69-73.
16. R. Nencka, I. Votruba, H. Hrebabecky, P. Jansa, E. Tloustova, K. Horska, M.
Masojidkova, A. Holy, A. Discovery of 5-substituted-6-chlorouracils as efficient
inhibitors of human thymidine phosphorylase. J. Med. Chem. 50(24) (2007) 6016-6023.
19

17. M. J. Perez-Perez, E. M. Priego, A. I. Hernandez, M. J. Camarasa, J. Balzarini, S.
Liekens, Thymidine phosphorylase inhibitors: recent developments and potential
therapeutic applications. Mini Rev. Med. Chem. 5(12) (2005) 1113-1123.
18. S. Liekens, J. Balzarini, A. I. De Hernandez, E. Clercq, E. M. Priego, M. J. Camarasa, M.
J. Perez-Perez, Thymidine phosphorylase is noncompetitively inhibited by 5′-O-trityl-
inosine (KIN59) and related compounds. Nucleosides, Nucleos. Nucleot. Nucl. 25(9-11)
(2006) 975-980.
19. E. Casanova, A. I. Hernandez, E. M. Priego, S. Liekens, M. J. Camarasa, J. Balzarini, M.
J. Perez-Perez, 5 ‘-O-Tritylinosine and Analogues as Allosteric Inhibitors of Human
Thymidine Phosphorylase. J. Med. Chem. 49(18) (2006) 5562-5570.
20. O. Talaz, I. Gülçin, S. Göksu, N. Saracoglu, Antioxidant activity of 5, 10-dihydroindeno
[1, 2-b] indoles containing substituents on dihydroindeno part. Bioorg. Med. Chem., 17
(2009) 6583-6589.
21. U.M. Kocyigit, O.N. Aslan, I. Gulcin, Y. Temel, M. Ceylan, Synthesis and Carbonic
Anhydrase Inhibition of Novel 2‐(4‐(Aryl) thiazole‐2‐yl)‐3a, 4, 7, 7a‐tetrahydro‐1H‐4,
7‐methanoisoindole‐1, 3 (2H)‐dione Derivatives. Archiv der Pharmazie, 349 (2016) 955-
963.
22. U.M. Kocyigit, Y. Budak, M.B. Gürdere, Ş. Tekin, T.K. Köprülü, F. Ertürk, K. Özcan, İ.
Gülçin, M. Ceylan, Synthesis, characterization, anticancer, antimicrobial and carbonic
anhydrase inhibition profiles of novel (3aR, 4S, 7R, 7aS)-2-(4-((E)-3-(3-aryl) acryloyl)
phenyl)-3a, 4, 7, 7a-tetrahydro-1H-4, 7-methanoisoindole-1,
derivatives. Bioorg. chem., 70 (2017) 118-125.
3
(2H)-dione
23. N.B. Almandil, M. Taha, M. Gollapalli, F. Rahim, M. Ibrahim, A. Mosaddik. Indole
bearing thiadiazole analogs: synthesis, β-glucuronidase inhibition and molecular docking
study. BMC Chem., 13 (2019) 14.
24. F. Rahim, S. Tariq, M. Taha, H. Ullah, K. Zaman, I. Uddin, A. Wadood et al. New
triazinoindole bearing thiazole/oxazole analogues: Synthesis, α-amylase inhibitory
potential and molecular docking study Bioorg. Chem. 92 (2019) 103284.
25. M. Taha, F.J. Alshamrani, F. Rahim, S. Hayat, H. Ullah, K. Zaman, S. Imran, K.M. Khan,
F. Naz, Synthesis of Novel Triazinoindole-Based Thiourea Hybrid: A Study on α-
Glucosidase Inhibitors and Their Molecular Docking. Molecules, 24 (2019) 3819.
20

26. E.H. Anouar, M.E. Moustapha, M. Taha, M.H. Geesi, Z.R. Farag, F. Rahim. N.B.
Almandil, R.K. Farooq, M. Nawaz, A. Mosaddik, Synthesis, Molecular Docking and β-
Glucuronidase Inhibitory Potential of Indole Base Oxadiazole Derivatives. Molecules 24
(2019) 963.
27. M. Taha, S. Imran, F. Rahim, A. Wadood, K.M. Khan. Oxindole based oxadiazole hybrid
analogs: Novel α-glucosidase inhibitors, Bioorg. Chem. 76 (2018) 273-280.
28. M. Taha, M.S. Baharudin, N.H. Ismail, S. Imran, M. N. Khan, F. Rahim, M. Selvaraj, S.
Chigurupati, M. Nawaz, F. Qureshi, S. Vijayabalan. Synthesis, α-amylase inhibitory
potential and molecular docking study of indole derivatives. Bioorg. Chem. 80 (2018) 80,
36-42.
29. T. Noreen, M. Taha, S. Imran, S. Chigurupati, F. Rahim, M. Selvaraj, N.H. Ismail, J.I.
Mohammad, H. Ullah, F. Nawaz, M. Irshad, M. Ali. Synthesis of alpha amylase inhibitors
based on privileged indole scaffold. Bioorg. Chem., 72 (2017) 248-255.
30. M.S. Baharudin, M. Taha, S. Imran, N.H. Ismail, F. Rahim, M. T. Javed, K.M. Khan, M.
Ali. Synthesis of indole analogs as potent β-glucuronidase inhibitors. Bioorg. Chem. 72
(2017) 323-332.
31. S. Imran, M. Taha, M. Selvaraj, N.H. Ismail, S. Chigurupati, J.I. Mohammad. Synthesis
and biological evaluation of indole derivatives as α-amylase inhibitor. Bioorg. Chem., 73
(2017) 121-128.
32. M. Taha, F. Rahim, S. Imran, N.H. Ismail, H. Ullah, M. Selvaraj, M.T. Javid, U. Salar, M.
Ali, K.M. Khan. Synthesis, α-Glucosidase Inhibitory Activity and In Silico Study of Tris-
Indole Hybrid Scaffold with Oxadiazole Ring: As Potential Leads for the Management of
Type-II Diabetes Mellitus. Bioorg. Chem. 74 (2017) 30-40.
33. M. Taha, N.H. Ismail, A. Khan, S.A.A. Shah, A. Anwar, S.A. Halim, M. Q. Fatmi, S.
Imran, F. Rahim K.M. Khan. Synthesis of Novel Derivatives of Oxindole, their Urease
Inhibition and Molecular Docking Studies. Bioorg. Med Chem Lett., 25 (2015) 3285-
3289.
34. V. A. McNally, M. Rajabi, A. Gbaj, I. J. Stratford, P. N. Edwards, K. T. Douglas, R. A.
Bryce, M. Jaffar, S. J. Freeman, Design, synthesis and enzymatic evaluation of
6‐bridged imidazolyluracil derivatives as inhibitors of human thymidine phosphorylase. J.
Pharm. Pharmacol. 59(4) (2007) 537-547.
21

35. B. Parrino, A. Carbone, G. Di Vita, C. Ciancimino, A. Attanzio, V. Spanò, A.
Montalbano, P. Barraja, L. Tesoriere, M. Livrea P. Diana, G. Cirrincione, 3-[4-(1H-Indol-
3-yl)-1, 3-thiazol-2-yl]-1H-pyrrolo [2, 3-b] pyridines, nortopsentin analogues with
antiproliferative activity. Mar. Drugs 13(4) (2015) 1901–1924.
36. B. Parrino, A. Attanzio, V. Spanò, S. Cascioferro, A. Montalbano, P. Barraja, L.
Tesoriere, P. Diana, G. Cirrincione, A. Carbone, Synthesis, antitumor activity and
CDK1 inhibiton of new thiazole nortopsentin analogues. Eur. J. Med. Chem. 138
(2017) 371–383.
37. V. Spanò, A. Attanzio, S. Cascioferro, A. Carbone, A. Montalbano, P. Barraja, L.
Tesoriere, G. Cirrincione, P. Diana, B. Parrino, Synthesis and antitumor activity of new
thiazole nortopsentin analogs.Mar. Drugs 14 (12) (2016) 226.
38. N.K. Kaushik, N. Kaushik, P. Attri, N. Kumar, C.H. Kim, A.K. Verma, E.H. Choi,
Biomedical importance of indoles, Molecules 18(6) (2013) 6620–6662.
39. T. Saini, S. Kumar, B. Narasimhan, Central nervous system activities of indole
derivatives: an overview. Cent. Nerv. Syst. Agents. Med. Chem. 16(1) (2015) 19–28.
40. B.G.M. Youssif, M.H. Abdelrahman, A.H. Abdelazeem, H.M. Ibrahim, O.I. Salem, M.F.
Mohamed, L. Treambleau, S.N. Bukhari, Design, synthesis, mechanistic and
histopathological studies of small-molecules of novel indole-2-carboxamides and
pyrazino [1, 2-a] indol-1 (2H)-ones as potential anticancer agents effecting the reactive
oxygen species production. Eur. J. Med. Chem. 146 (2018) 260–273.
41. S. Suzen, P. Bozkaya, T. Coban, D. Nebiogu, Investigation of the in vitro antioxidant
behaviour of some 2-phenylindole derivatives: discussion on possible antioxidant
mechanisms and comparison with melatonin. J. Enzym. Inhib. Med. Chem. 21(4) (2006)
405–411.
42. M. T. Javid, F. Rahim, M. Taha, M. Nawaz, A. Wadood, M. Ali, A. Mosaddik, S.A.
Shah, R.K. Farooq, Synthesis, SAR elucidations and molecular docking study of newly
designed isatin based oxadiazole analogs as potent inhibitors of thymidine phosphorylase
Bioorg. Chem. 79 (2018) 323-333.
43. K. Zaman, F. Rahim, M. Taha, A. Wadood, S. A. A. Shah, M. Gollapalli, F. Ullah, A.
Ahmed, Synthesis, thymidine phosphorylase, angiogenic inhibition and molecular
docking study of isoquinoline derivatives. Bioorg. Chem. 89 (2019) 102999.
22

44. I. Uddin, M. Taha, F. Rahim, A. Wadood, Synthesis and molecular docking study of
piperazine derivatives as potent inhibitor of thymidine phosphorylase. Bioorg. Chem. 78
(2018) 324-331.
45. M. Taha, N. H. Ismail, S. Imran, F. Rahim, A. Wadood, L. M. R. Al Muqarrabun, K. M.
Khan, M. Ghufran, M. Ali, In silico binding analysis and SAR elucidations of newly
designed benzopyrazine analogs as potent inhibitors of thymidine phosphorylase. Bioorg.
Chem, 68 (2016) 80-89.
46. G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.J.
Olson, AutoDock4 and AutoDockTools4: Automated docking with selective receptor
flexibility. J. Comput. Chem. 30(16) (2009):2785-2791.
47. hiygoygo Thymidine phosphorylase from E. coli with 3'-azido-2'-fluoro-dideoxyuridine,
http://www.rcsb.org/structure/4EAD.Thymidine phosphorylase from E.coli with 3'-azido-
2'-fluoro-dideoxyuridine, http://www.rcsb.org/structure/4EAD.
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Highlights:
 Synthesis of indole analogues
 In vitro thymidine phosphorylase studies
 Identification of a new thymidine phosphorylase
 Structure Activity Relationship established
 Molecular docking
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Synthesis of indole based acetohydrazide analogs: their in vitro and in silico thymidine
phosphorylase studies
Muhammad Tahaa, Ebaa Ahmed Jassim Aldhaminb, Noor Barak Almandila, El
Hassane Anouarc, Nizam Uddind, Munther Alomarie, Fazal Rahimf, Bushra Adalatf,
Mohamad Ibrahima, Fasial Nawazg, Naveed Iqbalh, Bandar Alghanemi, Abdulelah
Altolayyani, Khalid Mohammed KhanJ
aDepartment of clinical pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University,
P.O. Box 1982, Dammam 31441, Saudi Arabia
bCollege of clinical pharmacy, Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia
c Department of Chemistry, College of Science and Humanities, Prince Sattam bin Abdulaziz University, P.O. Box 83, Al Kharj 11942,
Saudi ArabiadFaculty of Applied Science, UiTM Shah Alam, 40450 Shah Alam, Selangor D.E.,
dDepartment of Chemistry, University of Karachi, Karachi-75270 Pakistan.
eDepartment of Stem Cell Biology, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University,
P.O. Box 1982, Dammam 31441, Saudi Arabia
fDepartment of Chemistry, Hazara University, Mansehra-21300, Khyber Pakhtunkhwa, Pakistan
gDepartment of Chemistry, University of Wah, Quaid Avenue, Wah Cantt 47000, Pakistan
hDepartment of chemistry University of poonch Rawalakot AJK.
iMedical Research Core Facility and Platforms (MRCFP), King Abdullah International Medical Research Center/ King Saud bin Abdulaziz
University for Health Sciences (KSAU-HS), King Abdulaziz Medical City (KAMC), NGHA, Riyadh 11426, Saudi Arabia
jH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270,
Pakistan
7
 Correspondence and reprints
E-mail: E-mail: taha_hej@yahoo.com and mtaha@iau.edu.saTel: 00966502057370
25

Dear editor
Greeting!
We have no conflict of interest
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