Design, synthesis, in-vitro thymidine phosphorylase inhibition, in-vivo antiangiogenic and in-silico studies of C-6 substituted dihydropyrimidines

Article abstract of DOI:10.1016/j.bioorg.2018.05.026

Thymidine phosphorylase (TP) is an angiogenic enzyme. It plays an important role in angiogenesis, tumour growth, invasion and metastasis. In current research work, we study the effect of structural modification of dihydropyrimidine-2-ones (DHPM-2-ones) on TP inhibition. A series of eighteen new derivatives of 3,4-dihydropyrimidone-2-one were designed and synthesized through the structural modification at C-6 position. All these new derivatives were then assessed for in-vitro inhibition of thymidine phosphorylase (TP) from E. coli. Oxadiazole derivatives 4a-e exhibited excellent TP-inhibition at low micromolar concentration levels better than standard drug 7-deazaxanthine (7-DX). Among all these compounds, 4b was found to be the most potent with IC₅₀ = 1.09 ± 0.004 μM. Anti-angiogenesis potential of representative compounds were also studied in a chorioallantoic membrane (CAM) assay. Here again, compound 4b was found to be the potent anti-angiogenesis compound in a CAM assay. Docking studies were also performed with Molecular Operating Environment (MOE) to further analyse the mode of inhibition of these compounds. Binding mode analysis of the most active inhibitors showed that these are well accommodated into the binding site of enzyme though stable hydrogen bonding and hydrophobic interactions.

Full text of DOI:10.1016/j.bioorg.2018.05.026

Accepted Manuscript
Design, Synthesis, In-Vitro Thymidine Phosphorylase Inhibition, In-Vivo An-
tiangiogenic and In-Silico Studies of C-6 substituted dihydropyrimidines
Fatima Iftikhar, Farhana Yoqoob, Nida Tabassum, Muhammad Saeed Jan,
Abdul Sadiq, Saba Tahir, Tahira Batool, Basit Niaz, Farzana Latif Ansari,
Muhammad Iqbal Chaudhary, Umer Rashid
PII:
S0045-2068(18)30346-8
DOI:
https://doi.org/10.1016/j.bioorg.2018.05.026
YBIOO 2379
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
9 April 2018
24 May 2018
25 May 2018
Please cite this article as: F. Iftikhar, F. Yoqoob, N. Tabassum, M. Saeed Jan, A. Sadiq, S. Tahir, T. Batool, B. Niaz,
F. Latif Ansari, M. Iqbal Chaudhary, U. Rashid, Design, Synthesis, In-Vitro Thymidine Phosphorylase Inhibition,
In-Vivo Antiangiogenic and In-Silico Studies of C-6 substituted dihydropyrimidines, Bioorganic Chemistry (2018),
doi: https://doi.org/10.1016/j.bioorg.2018.05.026
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Design, Synthesis, In-Vitro Thymidine Phosphorylase Inhibition,
In-Vivo Antiangiogenic and In-Silico Studies of C-6 substituted
dihydropyrimidines
Fatima Iftikhara, Farhana Yoqooba, Nida Tabassum b, Muhammad Saeed Jan c ,
Abdul Sadiq c, Saba Tahir d, Tahira Batool e,f, Basit Niaz a,, Farzana Latif Ansari g,
Muhammad Iqbal Chaudhary e and Umer Rashid d, **
a Department of Chemistry, Hazara University, Mansehra 21120, Pakistan
b Department of Pharmacology, Rawalpindi Medical University, Rawalpindi-463000, Pakistan
c Department of Pharmacy, University of Malakand, Chakdara 18000 Dir (L), Pakistan
d Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus 22060, Pakistan
e H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi 75270, Pakistan
f Department of Biotechnology, University of Azad Jammu & Kashmir Bagh, Pakistan
g Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
*Corresponding author at Department of Chemistry, COMSATS University Islamabad, Abbottabad
Campus. E-mail: umerrashid@ciit.net.pk; umer_rashid39@hotmail.com
Corresponding Author Tel. +92 (992) 383591-6
1

Abstract
Thymidine phosphorylase (TP) is an angiogenic enzyme. It plays an important role in
angiogenesis, tumour growth, invasion and metastasis. In current research work, we study the
effect of structural modification of dihydropyrimidine-2-ones (DHPM-2-ones) on TP
inhibition. A series of eighteen new derivatives of 3,4-dihydropyrimidone-2-one were
designed and synthesized through the structural modification at C-6 position. All these new
derivatives were then assessed for in-vitro inhibition of thymidine phosphorylase (TP) from
E. coli. Oxadiazole derivatives 4a-e exhibited excellent TP-inhibition at low micromolar
concentration levels better than standard drug 7-deazaxanthine (7-DX). Among all these
compounds, 4b was found to be the most potent with IC50 = 1.09±0.004 μM. Anti-
angiogenesis potential of representative compounds were also studied in a chorioallantoic
membrane (CAM) assay. Here again, compound 4b was found to be the potent anti-
angiogenesis compound in a CAM assay. Docking studies were also performed with
Molecular Operating Environment (MOE) to further analyse the mode of inhibition of these
compounds. Binding mode analysis of the most active inhibitors showed that these are well
accommodated into the binding site of enzyme though stable hydrogen bonding and
hydrophobic interactions.
Key words: Dihydropyrimine-2-ones; Thymidine phosphorylase; Chorioallantoic
membrane assay; Antiangiogenesis
2

1. Introduction
Thymidine phosphorylase (TP, EC 2.4.2.4), discovered in 1954, is involved in the reversible
catalysis of phosphorylation of thymidine nucleoside to thymine and 2-deoxy-D-ribose-1-
phosphate. 2-Deoxy-D-ribose-1-phosphate then de-phosphorylated to 2-deoxy-D-ribose. This
catabolised 2-deoxy-D-ribose is basically responsible for angiogenesis by its involvement in
the secretion and stimulation of angiogenic factors like vascular endothelial growth factor
(VEGF) and matrix metalloproteinases (MMPs). Angiogenesis is the formation of new blood
vessels in the replicated cell-lines and thus recognized as the key process in the metastasis of
tumour. Human Thymidine phosphorylase (hTP) is identical to platelet-derived endothelial-
cell growth factor (PD-ECGF) or gliostatin (an endothelial cell growth factor derived from
platelets) and this factor is responsible for angiogenesis in human cell lines. If uncontrolled,
then a complex series of events start, eventually ending up in the tumour growth [1-7]. In
addition to its role in cancers, the hyper proliferation due to over-expressed TP have been
also been found in diseases like rheumatoid arthritis and psoriasis, which is again attributed
to its angiogenic activity [8]. These factors are in turn involved in many sub-processes like
proliferation of endothelial cells due to metalloproteinase and formation of new blood vessels
in tumour tissues, thus supporting the cancer metastasis. Moreover, TP not only act on
thymidine but its poor substrate specificity renders it to recognize and catabolise the
deoxyuridine and other related pyrimidine nucleosides. That is why many chemotherapeutics
based on 5-substituted pyrimidine deoxyribonucleoside scaffolds remained in-effective in
vitro despite having real or potential efficacy against tumours [9]. All these facts lead to
conclusion that in vitro angiogenic activities and hence tumour growth can be
inhibited/controlled by inhibiting TP enzyme.
3

There are evidences where competitive inhibitors of this enzyme have shown blocking of
enzymatic activity hence reduced or terminated angiogenesis at the point of solid tumours.
Since a large number of potential drug candidates based on mono and multi-substrate, largely
pyrimidines or purines-based have been discovered or developed. These drugs were actually
designed in such a way that these can effectively interact with the binding site of thymidine.
The classical inhibitor 6-amino-5-bromouracil (1, 6A5BU, IC50 value of 0.035 μM), is
typically used as the benchmark for the design of more potent inhibitors. 7-deazaxanthine (2,
7-DX, IC50 value= 40 μM) was the first purine derivative to display inhibition of both E. coli
TP and angiogenesis in chorioallantoic membrane. Another compound structurally related to
5-chloro-6-[(2-iminopyrrolidin-1-yl)methyl]uracil (3) is also identified as potent TP inhibitor
(TPI) [3, 10].
1,3,4-oxadiazoles are an important class of heterocyclic compounds exhibiting a broad
spectrum of biological activities in different studies. Shahzad et al. reported the synthesis and
anti-TP activities of a series of oxadiazoles derivatives [11-12]. The two most active
inhibitors of that series were found to be compounds 4 and 5 with IC50 values 14.40 ± 2.45
and 17.60±1.07 μM respectively. Similarly, Schiff base derivatives also showed some good
activities against EcTP. Khan et al reported Schiff based derivative 6 (Figure 1) as a good
inhibitor of EcTP with an IC50 value of 19.77 μM [18]. Bensaber et al reported pyrazol-3-one
Schiff base derivatives of type 7 (Figure 1) as inhibitors of hTP [13].
4

O
O
Cl
O
NH
Br
HN
HN
N
H
O
O
N
H
NH2
N
N
O
N
H
H
H2N
1
2
3
O2N
O
O
NH OMe
O
NH OMe
R
N
N
N
N
O
N
N
N
S
S
O
N
O
H
H3C
Br
4
6
5
7
Figure 1: Potent TP inhibitors (1-3); Some important 1,3,4-oxadiazoles derivatives as TP inhibitors
(4-5); Schiff base derivatives 6-7 as TPI.
The classical TP-inhibitors 1-3 share the typical core of pyrimidine-one’s moiety, therefore,
these are considered as the basic-model for designing new pharmacophores for TP-inhibition.
The motivation behind current research is to design and synthesis potent compounds having
structural similarities with potent TPI 3 (IC50 = 35 nM) [10]. Figure 2 depicts the design
strategy for our new compounds where the points of similarities between the basic cores of
clinically administered drug 3 and our compounds is also highlighted. Hence, in this study a
series of dihydropyrimidine-2-ones (DHPMs-2-ones) based TP-inhibitors have been designed
and synthesized. We selected C-6 position of DHPM-2-one core for the design strategy and
synthesized a variety of TP inhibitors by introducing structural units such as Schiff-base and
1,3-oxadiazoles. All these compounds were synthesized via 6-bromomethylene and 6-
aminomethyl DHPM intermediates.
5

O
Cl
NH
O
X
O
R
NH
N
H
O
N
N
O
H2N
H
Figure 2: Structural similarities between 3 and proposed design strategy of our work.
2. Results and discussion
2.1. Chemistry
Compounds 1a-k (Scheme 1) were obtained as reported previously by our research group
using classical three-component Biginelli reaction under ultrasonic irradiation (USI) [20].
Whereas, compounds 2a-h, 3a-d and 4a-e were synthesized as shown in Schemes 2-5 by the
structural modification of 1f (Scheme 2).
R2
R2
+
O
SnCl2, ACN
CHO
O
R3
NH
+
USI, 70-75 oC
R3
NH2
O
R4
N
R1
O
R4
O
HN
R1
(1a-k)
Scheme 1: Synthesis of DHPM-2-one core (1a-k)
The structural modification of 1f was carried out via allylic bromination at C-6 position [14].
Allylic bromination of 1f resulted in 6-bromo derivative 8 (Scheme 2). The intermediate 8 is
vital to our further studies as it can undergo the nucleophilic substitution reactions to yield a
series of compounds. Initially, a series of diversely substituted eight compounds (2a-h) were
synthesized. Pyrazolone ring containing compound 2a was obtained via Paal-Knorr type of
reaction by condensing compound 9 with ethyl acetoacetate (10). The formation of
6

regioisomers cannot be ruled out here. Compounds 2b-h (Scheme 3) were synthesized by the
reaction of compound 8 with 11-17 in the presence of K2CO3 and DMF.
Cl
Cl
Cl
O
O
O
THF, USI
Br2 / CHCl3
0oC; 12 hr
O
NH
O
NH
O
NH
H3C
N
H
O
N
H
O
NH2-NH2 . H2O
N
O
Br
H
NH
H2N
8
1f
9
O
O
O
O
O
O
O
Cl
10
N
NH
HN
NH
H3C
2a
Scheme 2: Synthesis of pyrazolone ring 2a from 6-bromomethylene derivative (8)
7

Cl
O
O
N
H
NH
HN
O
2b
O
O
Cl
H
O
N
N
H
Cl
HN
H2N
NH
O
2c
O
K2CO3/ DMF
O
O
NH
HN
O
NH
O
11-17
Cl
N
H
O
HN
Br
NH
N
2dO
8
O
N
NH2
N
O
O
H
N
O
HN
O
NH2
Cl
N
H2N
NH
N
H
H
N
NH2
N
14
13
11
2e
12
Cl
O
O
OH
O
R = H
(15)
O
R
NH
= 4-NO2 (16)
= 2-NH2 (17)
HN
O
R
2f-h
Scheme 3: Synthesis of 6-bromomethylene derivatives 2b-h from compound 8
Synthesis of compounds 3a-d and 4a-e (Schemes 4-5) were commenced via the synthesis of
intermediate compound 6-aminomethyl derivative (18). Intermediate 18 was synthesized by
the reaction of ammonium acetate (as a substitute of ammonia) with compound 8 in a good
yield (88 %) [15]. This derivative was further used as precursor for the synthesis of Schiff
base derivatives (3a-e) and 5-substituted-1,3-oxadiazoles (4a-e). Schiff-base derivatives 3a-e
were the result of classical condensation between four different aryl aldehydes and isatin with
amino group of C-6 in acidic medium (Scheme 4). These compounds were identified by the
8

loss of two NH2-protons, and appearance of a more de-shielded double bond, characteristic of
an imine formation.
Cl
Cl
Cl
O
O
CH3CO2NH4
DMSO
O
i) RCHO (19-22, 23)
O
NH
O
NH
O
NH
ii) USI, 50 oC, 45-50 min.
N
O
H
N
H
O
N
O
N
23
3e
Br
H
HC
R
NH2
3a-d
8
18
Cl
O
O
R =
O
O
NH
N
H
OCH3
Cl
Br
Cl
N
O
H
23
3a
3b
3c
3d
N
(19-22)
HN
O
3e
Scheme 4: Schiff-base derivatives of compound 18
A series 5-substituted-1,3-oxadiazoles (4a-e, Scheme 5) containing 6-aminomethyl DHPM
derivative (18) and 1,3,4-oxadiazole-2(3H)-thiones (33-37) were synthesized by using
Mannich reaction. Intermediate 34-38, formalin (40 %) and compound 18 were stirred in
ethanolic solution at room temperature to obtain 4a-e.
9

O
O
NH
O
N
NH2-NH2.H2O (80%)
CS2, KOH
Al2O3, USI
NH2
S
C2H5
R
N
R
R
O
H
24-28
34-38
29-33
Cl
O
O
HCHO, EtOH
R=
18
NH
Cl
CH3
OCH3
Br
HN
NH
4a
4b
4d
4c
4e
O
N
N
S
(24-38)
O
R
4a-e
Scheme 5: synthesis of 5-substituted-1,3-oxadiazoles derivatives (4a-e)
2.2. Structure-activity relationship (SAR) around hit 1f and in vitro activity against
EcTP
Initially, a series of diversely substituted 3,4-dihydropyrimidin-ones (1a-k) were synthesized
and tested for their TP-inhibitory activity (Table 1) against EcTP. Unlikely as expected, out
of these eleven compounds, only compound 1f showed above 70% inhibition (Table 1, IC50=
150.5±0.005 μM) when compared to the standard 7-DX. Compound 1a showed above 50%
inhibition. Rest of all the compounds showed less than 50% inhibition i.e. insignificant IC50
values. With an aim to improve the potential of TP inhibition, we carried out structure-
activity relationship (SAR) of this 1f hit. All the new eighteen derivatives of 1f are
categorized into three different sets i.e. 2a-h (Schemes 2-3), 3a-e (Scheme 4) and 4a-e
(Scheme 5). These derivatives have also been assessed for their in vitro enzyme inhibition
potential against 7-DX as standard drug. All the tested compounds along with their IC50
values are shown in Tables 1-4.
10

Table 1: IC50 values of some 3,4-DHPM-ones(1a-k) as thymidine phosphorylase
R2
O
R3
H3C
NH
N
R1
O
(1a-k)
IC50±SEM
(µM)
No.
R1
R2
R3
H
H
H
H
Ph
Ph
OEt
Me
1a
1b
1c
1d
279.0±0.04
NI
NI
NI
3- OMe,4-OH C6H3
2- Me.C6H4
OEt
Me
Ph
1e
1f
Me
H
Me
NI
4-Cl.C6H4
Ph
OEt
OEt
150.5±0.005
NI
1g
Bn
1h
1i
H
H
H
H
Ph
OEt
Me
NI
NI
NI
NI
Ph
1j
3-OH.C6H4
3-OH.C6H4
OEt
Me
1k
7-DX (Standard TP inhibitor)
a NI= No inhibition shown in tested concentration
38.68±0.42
The IC50 values of eight compounds 2a-h is shown in Table 2. A close inspection revealed
that the presence of amide or carboxyl group at C-6 side chain, strongly affects the activities
as evident from the IC50 values of 2a, 2c, 2d and 2h with IC50=53.34±1.05, 38.50±0.85,
25.64±1.10 and 40.65±0.85 μM respectively. DHPM with semicarbazide nucleus (2c)
showed good inhibition with IC50 value of 38.50±0.85 μM which is close to standard 7-DX.
Replacement of amide –NH2 with pyridine ring (2d, using isoniazid 13 as starting material)
11

showed even better inhibition (IC50 =25.64±1.10 μM) than 2c. Compounds 2b, 2f and 2e
showed poor inhibition against target enzyme with an IC50 = 123.64±2.44, 95.25±1.54 and
129.14+2.84 μM respectively. Compound 2g is found to be totally in-active against TP and
showed no inhibition at all (Table 2).
Second series comprises five Schiff-base derivatives (3a-e) of 6-aminomethyl derivative (18)
with four different aldehydes (19-22) and one isatin (23). These all compounds showed poor
inhibition (Table 2), except compound 3e. Compound 3e with IC50 of 32.26±1.20 μM
emerged as active compound of this series. This activity may be attributed to the isatin core,
where, isatin have shown promising anticancer activities in previous studies [16].
Third series belongs to 5-substituted-1,3-oxadizole derivatives (4a-e) of compound 18. IC50
values of all the compounds from this series are tabulated in Table 2. It is clear from these
values that all of these five compounds proved to be stronger TP-inhibitors, as compared to
the standard. Compound 4b being the strongest inhibitor of this series with an
IC50=1.09±0.004 μM. Next more active compound of this series is compound 4c with an
IC50=6.57±0.03 μM. Rest of three also showed excellent inhibition where IC50 values range
from 9.14±0.06- 14.50 ±0.82 μM.
12

Table 2: IC50 values of derivatives of 5-substituted-1,3-0xadiazole derivatives of 3,4-DHPM-ones as
thymidine phosphorylase.
Cl
Cl
O
N
Cl
O
O
O
N
Cl
O
NH
N
O
X
O
NH
N
N
R
N
O
H
O
HN
NH
H
O
NH
N
O
S
H
N
H
O
O
HC
R
HN
O
2a-h
3a-d
3e
4a-e
IC50±SEM
Compound No.
X/R
(µM)
N
O
NH
CH3
53.34±1.05
2a
123.64±2.44
38.50±0.85
2b
2c
HN
O
NH
HN
H2N
O
NH
25.64±1.10
2d
2e
HN
N
N
129.14+2.84
N
O
O
95.25±1.54
NI
2f
O
O
O2N
2g
13

O
O
40.65±0.85
2h
NH2
Cl
87.35±3.14
64.64±2.81
ppta
3a
3b
3c
Br
O2N
H3CO
118.0±3.27
32.26±1.20
9.14+0.06
3d
3e
4a
-
Cl
1.09±0.004
6.57±0.03
11.80±0.63
4b
4c
4d
4e
Br
H3CO
H3C
14.50±0.82
7-DX (Standard TP inhibitor)
38.68±0.42
a Compound was precipitated out in the assay medium
2.3. Antiangiogenic studies
The study has been extended to assess the potential of our synthesized compounds on normal
vascular development in chick embryos using chicken chorioallantoic membrane (CAM)
model [17]. CAM is a multifaceted, simple and low cost preclinical in vivo model for tumour
biology and used as alternative to commonly used murine models. It is widely used to study
tumour angiogenesis and different malignancies [18]. The chorioallantoic membrane (CAM)
is a transient respiratory organ of the chick, which lies just beneath the shell membrane [19].
14

Tumour growth substantially depends upon the blood supply and the blood supply depends
upon the number of blood vessels. So, each tumour requires new blood vessels formation
repeatedly due to its significantly rapid growth. Therefore, to inhibit the tumour growth, the
inhibition of neovascularization is one of the targets. We employed CAM assay to evaluate
the potential of our test compounds on inhibition of new blood vessels formation. The
angiogenic inhibitory assay was carried out on fertilized eggs. Dexamethasone and
methotrexate was used as positive control. Some of us reported CAM assay for the antitumor
and anti-angiogenic potentials of crude extracts of some medicinal plants using
dexamethasone as positive control [20]. Dexamethasone has been reported to be anti-
angiogenic and have the ability to inhibit the new vascularization [21]. The highest
angiogenesis inhibition potential was demonstrated by 4b with IC50 value of 11.34 μM
(Table 3). Similarly, in the remaining tested compounds the 4a have also been good activity
with IC50 value of 13.05 μM. It is clear from the mentioned results that all the other samples
will also showed dose dependent response. In a reported CAM assay, 7-DX inhibited
partially the TP-induced angiogenesis [22]. In current experiment, 7-DX was found to have
IC50 value of 9.92±0.65 μM on normal vascular development in chick embryos. Methotrexate
showed highest antiangiogenic properties with IC50 value of 0.85±0.42 μM. The IC50 values
for anti-angiogenesis activity are given in Table 3. Figures of the CAM results are shown in
Figure 3.
The anti-angiogenic activity of the synthesized compound different concentration (1000, 500,
250, 125 and 62.5 µg/ml) were based on the number of blood vessels formed in the CAM of
chick embryo. Table S-1 (in Supporting Information) showed the average number of blood
vessels of each concentration in the CAM of chick embryo using different five concentration
of each compound. The IC50 value was calculated via regression analysis of dose response
15

curve by adding all the concentrations and percent activities (Figure S-1 in Supporting
Information).
Table 3: Results of anti-angiogenic assay of various samples
Samples
IC50 ± SEM
(μM)
2a
63.1±0.55
503.7±0.85
307.1±0.06
56.2±1.41
13.05±0.58
11.34±0.71
27.6±0.90
25.0±1.15
9.92±0.65
30.0±1.05
0.85±0.42
2e
3d
3e
4a
4b
4d
4e
7-DX
Dexamethasone
Methotrexate
16

Figure 3: Representative images of CAM results of anti-angiogenic assay
2.4. Docking studies
Many structures of Thymidine phosphorylase (TP) are now a day’s available, including
Salmonella typhimurium (4YEK) and E. coli (4EAD). Recently, structure of human TP (hTP)
17

has also been solved and available from PDB (1UOU and 2JOF) [23-26]. The IUOU, one of
the reported structure of hTP is basically co-crystallized structure with uracil derivative at a
resolution of 2.1 Å. Omari et al deposited another crystal structure of hTP in PDB (code:
2J0F), at 2.3 Å resolution. This structure is basically a non-trypsinized hTP–thymine complex
crystal structure. Structural analysis of enzyme revealed that thymine, thymidine, and
phosphate binding sites are situated in a 10 Å deep and 8 Å wide cavity like space.
With most of the conserved α-domain, the TP of E. Coli (EcTP) and hTP have almost ∼42%
of sequence similarity overall [24, 27]. This conserved sequence is even similar for the
active-site residues with a single exception for Phe210 in EcTP. In detail the active site of
hTP consists of residues His116, Ser117, Leu148, Arg171, Ser186, Lys190, Arg202, Val208,
Ile214, His216, Lys221 and Val241. Where, Val241in hTP corresponds to Phe210 in EcTP.
His85, His116, Arg171, Ser186, and Lys190 are some of residues of active site that hold key
importance in all the catalytic processes.
Owing to the conserved active site residues of hTP and EcTP, we checked the propensity of
the compounds to bind to hTP. We selected 2J0F and docked the compounds into its active
site. The reliability of the docking algorithm was validated using RMSD method. The co-
crystalized ligand (thymine) was re-docked and the root mean square deviation (RMSD)
between co-crystallized and re-docked conformation was determined. The RMSD value of
<2.0 Å is considered as accurate in predicting binding orientation of ligand. In order to
compare the binding poses of our compounds, we also performed the docking studies on
potent TPIs 2 and 3. The two-dimensional binding pose of 2 and 3 are shown in Figure S-2
in Supporting information.
Visual inspection of the docked poses of all the series of the synthesized compounds into the
active site of 2J0F revealed that phenyl rings of the compounds formed extensive π-π
stacking interactions with His116, a key residue of α/β domain. Similarly, Ser117, Arg202
18

also formed hydrogen bonding interactions with carbonyl oxygens and –N/-NH of the
compounds. Compounds of Series 2 (2a-h) showed variable in vitro TP inhibition data.
Comparison of two active compounds of this series 2c and 2d revealed that semicarbazide
derivative 2c (IC50=38.50±0.85 μM) forms five HB interactions with Ser117, Thr118,
Gly120, Ser144 and Thr154 (Figure 4a). While, isoniazid 2d (IC50=25.64±1.10 μM) forms
five HB interactions and one π-stacking interactions with His116 (Figure 4b). Oxygen atom
of DHPM ring orients itself towards Ser217 and Lys221. Pyridine nitrogen of isoniazid
accepts hydrogen form Ser126 and ring forms π-stacking interactions with His116. The
binding poses of compounds 2a-b are shown in Figure S-2 (Supporting Information). The
poor inhibitor of the series is imidazole derivative 2e (IC50=129.14±2.84 μM) forms only π-
stacking interactions with His116 (Figure S-3 in Supporting Information).
Figure 4: Two-dimensional (2D) ligand interaction map of compound docked into the binding site of
2JOF. a) 2c and b) 2d
We have analysed the docking poses of active compound of Schiff base derivative (3e) and
the most potent TPI studied in present research (4b, IC50 = 1.09±0.004 μM). The
superimposed docking pose of these compounds, TPI and 7-dx is shown in Figure 5a-b.
19

Examination of the best-ranked pose of most active compound of the series 3e, revealed that
isatin nucleus is located close to the key residues Arg202 and Ser217 (Figure 5c). DHPM
ring forms HB interactions with Tyr199 and Thr154. Chlorobenzene ring at 4-position of
DHPM forms π-stacking interactions with His116. These results are in good agreement with
bioactivity data. Oxadiazole ring point towards hydrophobic pocket formed by Val208 and
Ile214. Chlorobenzene ring at 4-position of DHPM forms π-stacking interactions with His116
and chloro-group interacts with Asp114. DHPM ring itself directed towards Ser117, Thr118,
Gly119, Gly120 pocket and forms HB interactions with these residues. Moreover,2-thioxo
group displays interactions with Arg202. On the other hand, 4-chlorophenyl at 5-position of
oxadiazole ring directed towards phosphate binding site (His150 and Gly239) and forms HB
interactions with Gly239 (Figure 5d). It is noted that in all the cases, the same amino acids
are involved in the binding of our compounds and TPIs 2-3.
20

Figure 5: (a-b) Lowest energy superimposed model of 3e (green), 4b (pink), TPI (brown) and 7-
dx (yellow) into the binding site of 2JOF. (c-d) 2D ligand interaction map of compound 3e (c)
and 4b (d) docked into the binding site of 2JOF.
3. Conclusions
Three series of compounds were synthesized by the structural modification of initial DHPM
hit 1f at C-6 position. The resulting 18 compounds were evaluated for their in vitro thymidine
phosphorylase inhibition potential. A number of compounds showed remarkable activity
profile toward TP. Compounds 2c and 2d of series-1 were found more active than standard
drug. For series-2, only 3e was able to inhibit TP with IC50 value of 32.26 μM. Oxadiazole
derivatives 4a-e exhibited excellent affinity with TP and display activity in low micromolar
range (IC50 values in μM). Based on these results, we further investigated the in vivo
antiangiogenic activity of some of the compounds. Compound 4b, an excellent TP inhibitor
of current study, also showed excellent antiangiogenic activity. By using MOE, binding mode
analysis of all the compounds into the active site of human TP was explored and explained
the results of TP inhibition.
4. Materials and methods
All the reagents and solvents were purchased from standard commercial vendors and were
used without any further purification. Isoniazid was taken from Islamabad Pharmaceutical
1
Products (IPP), Islamabad. H and 13C-NMR spectra were recorded in DMSO-d6 on a Bruker
spectrometer at 300 and 75 MHz respectively using tetramethylsilane (TMS) as internal
reference. Chemical shifts are given in δ scale (ppm). Melting points were determined in
open capillaries using Gallenkamp melting point apparatus (MP-D). The progress of all the
reactions was monitored by TLC on 2.0 x 5.0 cm aluminium sheets pre-coated with silica gel
21

60F254 with a layer thickness of 0.25 mm (Merck). LC-MS spectra were obtained using
Agilent technologies 1200 series high performance liquid chromatography comprising of
G1315 DAD (diode array detector) and ion trap LCMS G2445D SL. Elemental analyses were
conducted using a LECO-932 CHNS analyser. Reported results are the mean value of three
individual readings for each element (C, H and N).
4.1: Synthesis of 3,4-dihydropyrimidinones (1a-k)
In a pyrex tube equimolar quantities of variety of aldehydes and di-carbonyl compounds (10
mmol each) along with urea (12 mmol) were mixed in acetonitrile (10 mL). SnCl2.2H2O (10
mol %) was used as catalyst. The reaction mixture was then sonicated in an ultrasonic bath at
o
70-75 C. Progress of the reaction was monitored through TLC. Pure product was obtained
through silica gel chromatography. All the products were characterized by their melting
points, LC/MS and their spectral data. Compounds (1a-k) were already synthesized and
reported earlier by our research group [20-21].
4.2: Ethyl-6-(bromomethyl)-4-(4-chlorophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (8)
To a solution of 30 mmol of dihydropyrimidine (1f, 8.85 g) suspended in 30 ml of CHCl3,
added 20 mmol bromine dropwise through a dropping funnel. The reaction mixture was
stirred for 12 hrs at 4 oC. Solvent was removed under pressure on completion of reaction and
pure products were obtained through column chromatography (silica gel; petroleum
ether:ethyl acetate) [20]. 1H NMR (300 MHz, DMSO-d6): δ 9.22 (s, 1H, NH), 7.77 (s, 1H,
NH), 7.14 (d, 2H J= 7.5 Hz, Ar-H), 7.02 (d, 2H J= 7.5 Hz, Ar-H), 5.14 (d, 1H, J=3.3 Hz,
CH), 4.62 (s, 2H, CH2Br), 3.98 (q, 2H, J=7.2 Hz, OCH2), 1.09 (t, 3H, J=7.2 Hz, OCH2CH3).
13C-NMR (75 MHz, DMSO-d6): δ 166.6 153.1, 147.26, 144.8, 135.1, 128.3, 127.9, 126.9,
126.7, 106.95, 60.61, 52.83, 37.63, 15.0. LCMS : m/z = 373.1 [M+H].
22

4.2.1:
Ethyl-4-(4-chlorophenyl)-6-hydrazinylmethyl)-2-oxo-1,2,3,4-tetrahydro-
pyrimidine-5-carboxylate (9)
Compound 8 (10 mmol) and hydrazine hydrate (10 mmol) were mixed in 20 mL of ethanol.
Then few drops of H2SO4 were added to the reaction mixture. Reaction mixture was then
refluxed for 4-5 hrs. After completion of reaction the solvent was removed under vacuum.
Pure product 9 was obtained through silica gel column chromatography with 10% methanol
(MeOH) in chloroform (CHCl3). Rf =0.39 (CHCl3/MeOH 5:1). Yield 68%. m.p. 208-210°C.
1H NMR (300 MHz, DMSO-d6): δ 9.11 (s, 1H, NH), 7.61 (s, 1H, NH), 7.12 (d, 2H J= 8.4 Hz,
Ar-H), 6.88 (d, 2H J= 8.4 Hz, Ar-H), 5.03 (d, 1H, J=2.7 Hz, CH), 3.97 (q, 2H, J=6.9 Hz,
CH), 3.57 (s, 2H, CH2NH), 3.25 (brs, 2H, NH2), 2.88 (brs, 1H, NH), 1.11 (t, 3H, J=6.9 Hz,
OCH2CH3). 13C-NMR (75 MHz, DMSO-d6): δ 168.7, 153.02, 142.6, 140.07, 135.3, 128.3,
127.9, 126.7, 126.5, 106.4, 59.87, 52.9, 50.09, 15.03. LCMS : m/z = 325.1 [M+H].
4.2.1.1: Synthesis of ethyl-4-(4-chlorophenyl)-6-((3-methyl-5-oxo-2H-pyrazol-1(5H)-
yl)methyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (2a)
2a was prepared by reacting compound 9 (5 mmol) and ethyl acetoacetate (5 mmol). The
reaction mixture was refluxed in acidified methanol. After completion of reaction the solvent
was removed under pressure. The crude solid was recrystallized with ethyl acetate to obtain
1
pure product. Rf = 0.43 (CHCl3/MeOH; 6:1). Yield 73%. m.p. 196-198 °C. H NMR (300
MHz, DMSO-d6): δ 10.86 (s, 1H, NH), 9.11 (br s, 1H, NH), 7.65 (br s, 1H, NH), 7.12 (d, 2H
J= 8.1 Hz, Ar-H), 7.02 (d, 2H J= 8.1 Hz, Ar-H), 5.28 (1H, CO-CH), 5.04 (d, 1H, J=2.7 Hz,
CH), 3.99 (q, 2H, J=7.2 Hz, OCH2), 3.60 (s, 2H, CH2N), 1.91 (s, 3H, C=CCH3), 1.01 (t, 3H,
J=7.2 Hz, OCH2CH3).13C-NMR (75 MHz, DMSO-d6): δ 164.9, 162.7, 154.7, 151.4, 143.3,
23

137.3, 134.3, 130.8, 130.6, 126.4 (2C), 101.9, 96.5, 60.1, 54.7, 53.8, 27.1, 14.9; LCMS : m/z
= 391.1 [M+H]. Anal. calcd for C18H19ClN4O4: C 55.32; H 4.90; N 14.34 (found) C 55.28; H
4.92; N 14.32.
4.2.2: Ethyl-6-(bromomethyl)-4-(4-chlorophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate derivatives (2b-h):
Equimolar (2 mmol) mixtures of 6-bromo methylene DHPM (8) and compounds (11-17) each
in 5mL of THF was heated in ultrasonic bath at 40 ºC. Reactions were monitored through
TLC and LCMS. Crude mixtures were then purified by re-crystallization or column
chromatography whichever was applicable.
4.2.3:
Ethyl-6-((benzylamino)methyl)-4-(4-chlorophenyl)-2-oxo-1,2,3,4-tetrahydro-
pyrimidine-5-carboxylate (2b)
2b was prepared according to the general procedure by reacting 8 (2 mmol) and benzyl amine
(2 mmol). Rf =0.51 (n-hexanes/ethyl acetate; 5:1). Yield 73%. m.p. 181-183°C. 1H NMR (300
MHz, DMSO-d6): δ 9.97 (br s, 1H, NH), 8.19 (br s, 1H, NH), 7.21 (d, 2H J= 8.1 Hz, Ar-H),
7.16-1.08 (m, 5H, Ar-H), 7.06 (d, 2H J= 8.1 Hz, Ar-H), 6.01 (brs, 1H, J= 6.9 Hz, NH), 4.97
(d, 1H, J=3.3 Hz, CH), 4.17 (q, 2H, J=7.2 Hz, OCH2), 4.57 (brs 2H, J= 6.9 Hz, CH2NH),
13
3.28 (brs, 2H, J= 6.9 Hz, CH2NH), 1.01 (t, 3H, J=7.2 Hz, OCH2CH3). C-NMR (75 MHz,
DMSO-d6): δ168.62, 152.37, 145.21, 138.1, 137.37, 134.01, 130.9, 130.3, 129.9, 126.2,
126.1, 124.6 (2C), 124.2, 124.1, 102.9, 60.8, 53.8, 52.6, 50.9, 14.6. LCMS : m/z = 400.2
[M+H]. Anal. calcd for C21H22ClN3O3: C 63.08; H 5.55; N 10.51 (found) C 63.05; H 5.54; N
10.49.
4.2.4.
Ethyl-6-((benzylamino)methyl)-4-(4-chlorophenyl)-2-oxo-1,2,3,4-tetrahydro-
pyrimidine-5-carboxylate (2c)
24

2c was prepared according to the general procedure by reacting 8 (2 mmol) and
1
semicarbazide (2 mmol). Rf =0.39 (CHCl3/CH3OH; 3:1). Yield 76 %. m.p. 182-183°C. H
NMR (300 MHz, DMSO-d6): δ 11.33(s, 2H, NH2), 10.68 (brs, 1H, NH), 9.89 (brs, 1H, NH),
8.19 (br s, 1H, NH), 7.18 (d, 2H J= 8.1 Hz, Ar-H), 7.03 (d, 2H J= 8.1 Hz, Ar-H), 6.68 (br s,
1H, NH), 5.14 (d, 1H, J=3.3 Hz, CH), 4.13 (q, 2H, J=7.2 Hz, OCH2), 3.48 (brs, 2H,
CH2NH), 1.19 (t, 3H, J=7.1Hz, OCH2CH3).13C-NMR (75 MHz, DMSO-d6): δ167.87, 155.33,
152.23, 143.86, 137.5, 134.1, 130.9, 130.4, 126.4, 126.2, 103.78, 61.53, 53.21, 51.11, 15.14.
LCMS : m/z = 368.1 [M+H]. Anal. calcd for C15H18ClN5O4: C 48.98; H 4.93; N 19.04
(found) C 49.00; H 4.94; N 19.02.
4.2.5 Ethyl 4-(4-chlorophenyl)-2-oxo-6-{[(pyridin-4-yl)formohydrazido]methyl}-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (2d)
2d was prepared according to the general procedure by reacting 8 (2 mmol) and
isonicotinohydrazide (13) (2 mmol). Rf = 0.56 (n-hexanes/ethyl acetate; 2:1). Yield 83 %.
1
m.p. 202-204°C. H NMR (300 MHz, DMSO-d6): δ 11.11 (brs, 1H, NH), 9.88 (s, 1H, NH),
8.77 (d, 2H, J=7.8 Hz, Ar-H), 8.24 (br s, 1H, NH), 7.99 (d, 2H, J=7.8 Hz, Ar-H), 7.12 (d, 2H
J= 8.1 Hz, Ar-H), 7.04 (d, 2H J= 8.1 Hz, Ar-H), 6.69 (br s, 1H, NH), 5.09 (d, 1H, J=3.0 Hz,
CH), 4.00 (q, 2H, J=6.9 Hz, OCH2), 3.34 (brs, 2H, CH2NH), 0.93 (t, 3H, J=6.9 Hz,
OCH2CH3). 13C-NMR (75 MHz, DMSO-d6): δ 167.6, 166.2, 153.0, 151.5 (2C), 142.9, 141.6,
139.8, 134.3, 126.8 (2C), 126.6, 126.4, 124.1 (2C), 104.6, 62.0, 53.2, 50.6, 14.4. LCMS: m/z
= 430.1 [M+H]. Anal. calcd for C20H20ClN5O4: C 55.88; H 4.69; N 16.29 (found) C 55.86; H
4.70; N 16.32.
4.2.6: Ethyl-6-((1H-imidazol-1-yl)methyl)-4-(4-chlorophenyl)-2-oxo-1,2,3,4-tetrahydro-
pyrimidine-5-carboxylate (2e)
25

2e was prepared according to the general procedure by reacting 8 (2 mmol) and 1H-imidazole
1
(2 mmol). Rf =0.47 (n-hexanes/ethyl acetate; 2:1); Yield 81 %. m.p. 208-210 °C. H NMR
(300 MHz, DMSO-d6): δ 9.68 (s, 1H, NH), 8.10 (s, 1H, NH), 7.64 (s, 1H, -N-HC=N), 7.19
(d, 2H J= 8.1 Hz, Ar-H), 7.03 (d, 2H J= 8.1 Hz, Ar-H), 6.98 (m, 2H, imidazole-H), 5.09 (d,
1H, J= 3.3 Hz, CH), 4.39 (s, 2H, CH2N), 4.16 (q, 2H, J=7.1 Hz, OCH2), 1.27 (t, 3H,
J=7.1Hz, OCH2CH3). 13C-NMR (75 MHz, DMSO-d6): δ 169.13, 152.6, 142.89, 141.37,
134.9, 134.3, 126.8, 126.7, 126.4 (2C), 126.09, 118.71, 104.93, 61.48, 53.01, 50.17, 15.11.
LCMS : m/z = 361.1 [M+H]. Anal. calcd for C17H17ClN4O3; C, 56.59; H, 4.75; N, 15.53
(found) C 56.56; H 4.76; N 15.55.
4.2.7:
Ethyl-6-(benzoyloxymethyl)-4-(4-chlorophenyl)-2-oxo-1,2,3,4-tetrahydro-
pyrimidine-5-carboxylate (2f)
2f was prepared according to the general procedure by reacting 8 (2 mmol) and benzoic acid
1
(2 mmol). Rf = 0.52 (n-hexanes/ethyl acetate; 5:1) . Yield 86 %. m.p. 199-200 °C H NMR
(300 MHz, DMSO-d6): δ 9.476 (s, 1H, NH), 8.011 (s, 1H, NH), 7.99 (d, 2H J= 8.1 Hz, Ar-
H), 7.891-7.583 (m, 1H, Ar-H), 7.55 (d, 2H J= 8.1 Hz, Ar-H), 7.39-7.25 (m, 4H, Ar-H), 5.13
(d, 1H, J=3.3 Hz, CH), 5.10 (s, 2H, CH2O), 3.99 (q, 2H, J=7.2 Hz, OCH2), 1.050 (t, 3H,
J=7.2 Hz, OCH2CH3). 13C-NMR (75 MHz, DMSO-d6): 169.6, 168.18, 152.6, 145.79, 139.6,
132.7, 132.03, 131.97, 130.1, 129.4 (2C), 128.1 (2C), 126.8, 126.5, 106.3, 104.1, 67.79, 62.2,
50.4, 15.19. LCMS : m/z = 415.1 [M+H]. Anal. calcd for C21H19ClN2O5: C 60.80; H 4.62; N
6.75 (found) C 60.76; H 4.64; N 6.77.
4.2.8:
Ethyl-4-(4-chlorophenyl)-6-((4-nitrobenzoyloxy)methyl)-2-oxo-1,2,3,4-tetra-
hydropyrimidine-5-carboxylate (2g)
2g was prepared according to the general procedure by reacting 8 (2 mmol) and 4-nitro
benzoic acid (2 mmol). Rf = 0.48 (n-hexanes/ethyl acetate; 5:1). Yield 81%. m.p. 207-209°C.
26

1H NMR (300 MHz, DMSO-d6): δ 9.98 (s, 1H, NH), 9.81 (s, 1H, NH), 8.33 (d, 2H J= 8.7 Hz,
Ar-H), 8.17 (d, 2H, J= 8.7 Hz, Ar-H), 7.19 (d, 2H J= 7.8 Hz, Ar-H), 7.03 (d, 2H J= 7.8 Hz,
Ar-H), 5.17 (d, 1H, J=3.3 Hz, CH), 5.08 (s, 2H, CH2O), 4.17 (q, 2H, J=7.5 Hz, OCH2), 1.33
(t, 3H, J=7.5 Hz, OCH2CH3).13C-NMR (75 MHz, DMSO-d6): 168.6, 167.9, 154.1, 153.3,
146.36, 139.89, 139.1, 133.4 (2C), 132.6, 130.1 (2C), 129.15, 127.9, 127.3, 126.9, 105.9,
69.01, 62.3, 50.8, 15.21. LCMS: m/z = 460.1. Anal. calcd for C21H18ClN3O7: C, 54.85; H,
3.95; N, 9.14 (found) 54.84; H, 3.97; N, 9.12.
4.2.9.
Ethyl-6-((2-aminobenzoyloxy)methyl)-4-(4-chlorophenyl)-2-oxo-1,2,3,4-tetra-
hydropyrimidine-5-carboxylate (2h)
2h was prepared according to the general procedure by reacting 8 (2 mmol) and 2-
aminobenzoic acid (2 mmol). Rf =0.51 (n-hexanes/ethyl acetate; 5:1). Yield 79%. m.p. 211-
1
213 °C. H NMR (300 MHz, DMSO-d6): δ 10.1 (s, 1H, NH), 9.87 (s, 1H, NH), 7.91 (dd, J=
8.4 Hz, J= 1.5 Hz, 1H, Ar-H), 7.32 (m, 1H, Ar-H), 7.16 (d, 2H J= 8.1 Hz, Ar-H), 7.06 (d, 2H
J= 8.1 Hz, Ar-H), 6.76 (t, 2H, J= 7.5 Hz, Ar-H), 5.84 (s, 2H, NH2), 5.16 (d, 1H, J=3.3 Hz,
CH), 5.10 (s, 2H, CH2O), 4.14 (q, 2H, J=7.2 Hz, OCH2), 1.22 (t, 3H, J=7.2 Hz, OCH2CH3).
13C-NMR (75 MHz, DMSO-d6): 169.1, 168.29, 153.5, 152.8, 146.4, 143.7, 142.91, 135.2,
129.9, 126.79 (2C), 126.6, 126.4, 120.6, 118.42, 108.8, 104.6, 67.87, 62.6, 50.31, 15.17.
LCMS : m/z = 430.1. Anal. calcd for C21H20ClN3O5: C 58.68; H 4.69; N 9.78 (found) C
56.65; H 4.70; N 9.80.
4.3: Synthesis of ethyl-6-(aminomethyl)-4-(4-chlorophenyl)-2-oxo-1,2,3,4-tetrahydro
pyrimidine-5-carboxylate (18)
A solution of 6-bromo DHPM (15 mmol) in DMSO (10 mL), was made to react with 30
mmol of ammonium acetate in an ultrasonic bath at 45-50 °C for 35 minutes. Pure product
was obtained through silica gel chromatography. Rf = 0.56 (n-hexanes/ethyl acetate; 5:2).
27

Yield 88%. m.p. 212-214 °C. 1H NMR (300 MHz, DMSO-d6): δ 9.79 (br s, 1H, NH), 8.09 (br
s, 1H, NH), 7.16 (d, 2H J= 7.8 Hz, Ar-H), 7.00 (d, 2H J= 7.8 Hz, Ar-H), 6.28 (brs, 2H, NH2),
5.07 (d, 1H, J=3.3 Hz, CH), 4.10 (q, 2H, J=7.2 Hz, OCH2), 3.66 (brs, 2H, CH2NH2), 1.12 (t,
3H, J=7.2 Hz, OCH2CH3). 13C-NMR (75 MHz, DMSO-d6): δ168.3, 153.07, 141.8, 139.6,
134.6, 128.1, 127.9, 126.63 (2C), 106.7, 60.59, 50.12, 42.8, 14.99. LCMS : m/z = 310.2.
Anal. calcd for C14H16ClN3O3: C 54.29; H 5.21; N 13.57 (found) C 54.31; H 5.22; N 13.54.
Synthesis of 6-aminomethyl DHPM-derivatives (3a-e):
Compounds 3a-e was synthesized according to the general reported procedure. Compound
(18, 0.010 mol) was refluxed in ethanol with five different aldehydes (0.010 mol) in acidic
medium. Few drops of glacial acetic acid were used as catalyst. After completion of reaction
the solvent was evaporated and pure products were obtained through re-crystallization from
ethanol. All the products were then fully characterized on the basis of physical and spectral
parameters.
4.3.1: (E)-ethyl-6-((4-chlorobenzylideneamino)methyl)-4-(4-chlorophenyl)-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (3a)
3a was prepared according to the general procedure by reacting 18 (2 mmol) and 4-
chlorobenzaldehyde (2 mmol). Rf 0.38 (n-hexanes/ethyl acetate; 5:1). Yield 81 %. m.p. 193-
1
195 °C. H NMR (300 MHz, DMSO-d6): δ 9.11 (br s, 1H, NH), 8.31 (s, 1H, CH=N-), 7.97
(br s, 1H, NH), 7.47 (d, 2H J= 8.4 Hz, Ar-H), 7.34 (d, 2H J= 8.4 Hz, Ar-H), 7.04 (d, 2H, J=
8.4 Hz, Ar-H), 6.65 (d, 2H J= 8.4 Hz, Ar-H), 5.03 (d, 1H, J=3.0 Hz, CH), 4.36 (s, 2H,
13
CH2N=C), 3.97 (q, 2H, J=6.9 Hz, OCH2), 1.11 (t, 3H, J=6.9 Hz, OCH2CH3). C-NMR (75
MHz, DMSO-d6): δ 168.3, 162.2, 153.1, 141.6, 140.6 (2C), 139.7 (2C), 138.2, 134.4, 131.3,
28

131.1, 129.3, 128.0, 127.4, 127.2, 105.95, 60.5, 52.9, 50.1, 14.4. LCMS : m/z = 432.1. Anal.
calcd for C21H19Cl2N3O3: C 58.34; H 4.43; N 9.72 (found) C 58.38; H 4.41; N 9.70.
4.3.2: (E)-ethyl 6-((4-bromobenzylideneamino)methyl)-4-(4-chlorophenyl)-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (3b)
3b was prepared according to the general procedure by reacting 18 (2 mmol) and 4-
bromobenzaldehyde (2 mmol). Rf 0.46 (n-hexanes/ethyl acetate; 5:1). Yield 86 %. m.p. 201-
203°C. 1H NMR (300 MHz, DMSO-d6): δ 9.79 (br s, 1H, NH), 8.34 (s, 1H, CH=N-), 8.10 (br
s, 1H, NH ), 7.71 (d, 2H J= 8.4 Hz, Ar-H), 7.39 (d, 2H J= 8.4 Hz, Ar-H), 7.21 (d, 2H J= 8.1
Hz, Ar-H), 7.03 (d, 2H J= 8.1 Hz, Ar-H), 5.19 (d, 1H, J=3.3 Hz, CH), 4.56 (s, 2H, CH2N=C),
4.13 (q, 2H, J=7.1 Hz, OCH2), 1.27 (t, 3H, J=7.1 Hz, OCH2CH3). 13C-NMR (75 MHz,
DMSO-d6): δ 168.6, 161.9, 153.0, 142.01, 141.9, 141.6, 140.03, 134.6, 132.6, 132.4, 131.2,
130.3, 129.0, 128.78, 128.72, 128.11, 105.83, 60.71, 52.6, 50.11, 14.69. LCMS : m/z =
476.03. Anal. calcd for C21H19BrClN3O3: C, 52.90; H, 4.02; N, 8.81. (found) C 52.87; H
4.03; N 8.84.
4.3.3: (E)-ethyl-6-((4-nitrobenzylideneamino)methyl)-4-(4-chlorophenyl)-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (3c)
3c was prepared according to the general procedure by reacting 18 (2 mmol) and 4-
nitrobenzaldehyde (2 mmol). Rf 0.56 (n-hexanes/ethyl acetate; 5:1). Yield 88 %. m.p. 198-
201°C. 1H NMR (300 MHz, DMSO-d6): δ 9.83 (br s, 1H, NH), 8.41 (d, 2H J= 8.1 Hz, Ar-H),
8.29 (s, 1H, CH=N-), 8.11 (br s, 1H, NH), 7.79 (d, 2H J= 8.1 Hz, Ar-H), 7.18 (d, 2H J= 7.8
Hz, Ar-H), 7.01 (d, 2H J= 7.8 Hz, Ar-H), 5.04 (d, 1H, J=3.3 Hz, CH), 4.37 (s, 2H, CH2N=C),
4.17 (q, 2H, J=7.2 Hz, OCH2), 1.05 (t, 3H, J=7.2 Hz, OCH2CH3). 13C-NMR (75 MHz,
DMSO-d6): δ 169.9, 162.5, 153.5, 151.7, 148.1, 142.9, 142.2, 142.1, 140.03, 132.7, 132.5,
132.4, 131.89, 129.0, 128.9, 128.2, 105.7, 60.67, 53.7, 50.09, 15.1. LCMS: m/z = 443.1
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