Probing the high potency of pyrazolyl pyrimidinetriones and thioxopyrimidinediones as selective and efficient non-nucleotide inhibitors of recombinant human ectonucleotidases

Article abstract of DOI:10.1016/j.bioorg.2019.03.067

With the aim to discover novel, efficient and selective inhibitors of human alkaline phosphatase and nucleotide pyrophosphatase enzymes, two new series of pyrazolyl pyrimidinetriones (PPTs) (6a–g) and thioxopyrimidinediones (PTPs) (6h–n) were synthesized in good chemical yields using Knoevenagel condensation reaction between pyrazole carbaldehydes (4a–g) and pharmacologically active N-alkylated pyrimidinetrione (5a) and thioxopyrimidinedione (5b). The inhibition potential of the synthesized hybrid compounds was evaluated against human alkaline phosphatase (h-TNAP and h-IAP) and ectonucleotidase (h-NPP1 and h-NPP3) enzymes. Most of the tested analogs were highly potent with a variable degree of inhibition depending on the functionalized hybrid structure. The detailed structure-activity relationship (SAR) of PPT and PTP derivatives suggested that the compound with unsubstituted phenyl ring from PPT series led to selective and potent inhibition (6a; IC₅₀ = 0.33 ± 0.02 μM) of h-TNAP, whereas compound 6c selectively inhibited h-IAP isozyme with IC₅₀ value of 0.86 ± 0.04 μM. Similarly, compounds 6b and 6h were identified as the lead scaffolds against h-NPP1 and h-NPP3, respectively. The probable binding modes for the most potent inhibitors were elucidated through molecular docking analysis. Structure-activity relationships, mechanism of action, cytotoxic effects and druglikeness properties are also discussed.

Full text of DOI:10.1016/j.bioorg.2019.03.067

Accepted Manuscript
Probing the high potency of pyrazolyl pyrimidinetriones and thioxopyrimidi-
nediones as selective and efficient non-nucleotide inhibitors of recombinant
human ectonucleotidases
Hina Andleeb, Shahid Hameed, Syeda Abida Ejaz, Imtiaz Khan, Sumera Zaib,
Joanna Lecka, Jean Sévigny, Jamshed Iqbal
PII:
S0045-2068(18)30982-9
DOI:
https://doi.org/10.1016/j.bioorg.2019.03.067
YBIOO 2893
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 September 2018
19 March 2019
25 March 2019
Please cite this article as: H. Andleeb, S. Hameed, S. Abida Ejaz, I. Khan, S. Zaib, J. Lecka, J. Sévigny, J. Iqbal,
Probing the high potency of pyrazolyl pyrimidinetriones and thioxopyrimidinediones as selective and efficient non-
nucleotide inhibitors of recombinant human ectonucleotidases, Bioorganic Chemistry (2019), doi: https://doi.org/
10.1016/j.bioorg.2019.03.067
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Probing the high potency of pyrazolyl pyrimidinetriones and thioxopyrimidinediones as
selective and efficient non-nucleotide inhibitors of recombinant human ectonucleotidases
Hina Andleeb,a,b Shahid Hameed,a* Syeda Abida Ejaz,c Imtiaz Khan,a,d,e Sumera Zaib,c Joanna
Lecka,f,g Jean Sévigny,f,g Jamshed Iqbal,c*
aDepartment of Chemistry, Quaid-i-Azam University-45320, Islamabad, Pakistan
bSulaiman Bin Abdullah Aba Al-Khail – Centre for Interdisciplinary Research in Basic Science
(SA-CIRBS), Faculty of Basic and Applied Sciences, International Islamic University, Islamabad,
Pakistan
cCentre for Advanced Drug Research, COMSATS Institute of Information Technology,
Abbottabad-22060, Pakistan
dSchool of Chemistry, The University of Manchester, Oxford Road, Manchester M13 9PL, United
Kingdom
eManchester Institute of Biotechnology, The University of Manchester, 131 Princess Street,
Manchester M1 7DN, United Kingdom
fDépartement de microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université
Laval, Québec, QC, G1V 0A6, Canada
gCentre de Recherche du CHU de Québec – Université Laval, Québec, QC, G1V 4G2, Canada
_________________________________________________________________________
*Corresponding authors. Tel.: +92-51-9064-2133; Fax: +92-51-9064-2241; E-mail:
shameed@qau.edu.pk (S. Hameed); Tel.: +92-992-383591/96; Fax: +92-992-383441; E-mail:
drjamshed@ciit.net.pk (J. Iqbal).
1

Abstract
With the aim to discover novel, efficient and selective inhibitors of human alkaline phosphatase
and nucleotide pyrophosphatase enzymes, two new series of pyrazolyl pyrimidinetriones (PPTs)
(6a‒g) and thioxopyrimidinediones (PTPs) (6h‒n) were synthesized in good chemical yields using
Knoevenagel condensation reaction between pyrazole carbaldehydes (4a‒g) and pharmacologically
active N-alkylated pyrimidinetrione (5a) and thioxopyrimidinedione (5b). The inhibition potential
of the synthesized hybrid compounds was evaluated against human alkaline phosphatase (h-TNAP
and h-IAP) and ectonucleotidase (h-NPP1 and h-NPP3) enzymes. Most of the tested analogs were
highly potent with a variable degree of inhibition depending on the functionalized hybrid structure.
The detailed structure-activity relationship (SAR) of PPT and PTP derivatives suggested that the
compound with unsubstituted phenyl ring from PPT series led to selective and potent inhibition
(6a; IC50 = 0.33 ± 0.02 µM) of h-TNAP, whereas compound 6c selectively inhibited h-IAP
isozyme with IC50 value of 0.86 ± 0.04 µM. Similarly, compounds 6b and 6h were identified as the
lead scaffolds against h-NPP1 and h-NPP3, respectively. The probable binding modes for the most
potent inhibitors were elucidated through molecular docking analysis. Structure-activity
relationships, mechanism of action, cytotoxic effects and druglikeness properties are also discussed.
Keywords: Pyrazole; pyrimidinetriones; thioxopyrimidinediones; recombinant enzymes;
ectonucleotidases; alkaline phosphatatses, biological screening.
1. Introduction
Ecto-nucleotidases (neucleotide metabolizing enzymes) modulate P2 purinergic signaling [1] and
are responsible for maintaining cell function. Overexpression of a number of these ubiquitous ecto-
enzymes has been involved in a variety of disorders [1] including cell activation, adhesion,
apoptosis, proliferation and degenerative neurological and immunological responses. Selective
inhibition of ecto-enzymes is an area that is currently being explored with immense interest and
may prove to result in many beneficial therapeutic implications.
2

Ecto-nucleotide pyrophosphatases/phosphodiesterases (NPPs) belong to metalloenzyme family and
comprise seven different subtypes (NPP1-7). Among different isozymes, the literature data
explicitly reveals the prominence and applications of only three members, i.e. NPP1-3 [1,2]. Both
NPP1 and NPP3 perform the hydrolysis of pyrophosphates and phosphodiesters in various
nucleotides and their derivatives [2]. In addition, these isozymes are also capable of hydrolyzing
the artificial phosphoric acid esters [2-4]. They either exist as trans-membrane glycoproteins or
secreted proteins [3,4]. NPP1-3 are substrate specific and are widely expressed in various tissues.
They have been associated with multiple physiological functions including extracellular
pyrophosphate levels regulation, nucleotide recycling, stimulating cell motility and modulation of
purinergic receptor signaling [5]. The main substrate of NPPs is adenosine triphosphate (ATP),
which is cleaved to adenosine monophosphate (AMP) and diphosphate (ADP) or pyrophosphate
(ppi) (Fig. 1). Specifically, NPP1 is involved in various biological processes including insulin
receptor signaling, bone mineralization and immune modulation. Its overexpression leads to ectopic
calcification (pathologic deposition of calcium salts in tissues), calcium pyrophosphate dihydrate
crystal deposition (CPPD) and cancer cell proliferation, whereas NPP3 is suggested as a tumor
marker since its expression has been associated with carcinogenesis and cancer cell metastasis [6].
NPP1 inhibitors have potential as novel drugs, e.g. for (immuno)oncology. The blockade of NPP1
can increase the concentration of ATP, however, at the same time it can decrease the concentration
of adenosine by reducing the concentration of its precursor, adenosine monophosphate (Fig. 1).
This may lead to an enhancement of the immune response in the body [7]. To date, the reported
NPP inhibitors have been recognized as non-selective inhibitors of various ecto-nucleotidases [8].
Therefore, selective and potent NPP inhibitors for the treatment of several disorders, i.e. cancer or
chondrocalcinosis caused by calcium pyrophosphate dihydrate crystal deposition disease (CPPD),
are desired to be explored.
3

Fig. 1. Work pathway for NPP1 and TNAP, the pathologies associated with their overexpression
and the effect of inhibitors on their work pathway.
Alkaline phosphatases (APs, EC 3.1.3.1) on the other hand, are well-studied homodimeric metallo-
enzymes that catalyze dephosphorylation of nucleoside phosphates and phosphomonoesters [9,10].
In APs, catalytic active site is situated on the external cell surface via a glycosylphosphatidyl
inositol (GPI) anchor making them ideal drug targets, since potential drug molecules do not
necessarily have to be cell-permeable in order to induce the desired physiological effects [11]. This
isozyme family is broadly categorized into two groups, namely: tissue-nonspecific alkaline
phosphatases (TNAP) and tissue-specific alkaline phosphatases (placental, intestinal and germ
cell). Although the mammalian alkaline phosphatases display low sequence similarity with the E.
coli enzyme (25–30%), the active site containing divalent metals ions remains highly conserved
producing a catalytic activity similar to alkaline phosphatases of E. coli and eukaryotes [12,13].
The overexpression of human alkaline phosphatases in various tumor cells has been shown recently
4

[14]. Increased levels of plasma TNAP were observed in Paget’s disease [15], osteosarcomas [16]
and osteoblastic bone metastasis [17].
Increased circulating expression of alkaline phosphatase has a crucial role in vascular calcification
due to imbalance between inhibitors and promoters of mineralization [18]. Overexpression of
TNAP is associated with high rates of mortality and cardiovascular events via mechanisms that
involve endothelial dysfunction, vascular calcification and inflammation (Fig. 2). Interventional
strategies to lower TNAP activity include direct alkaline phosphatase inhibitors, vitamin D
analogues and calcimimetic molecules [18]. Intestinal alkaline phosphatases (IAPs) regulate the
intestinal lipid absorption, bicarbonate secretion and maintain the pH of duodenal surface along
with detoxification of bacterial lipopolysaccharide (LPS) [19]. High levels of intestinal-type
alkaline phosphatase (IAP) and TNAP, owing to sepsis and acute inflammation, can lead to
endotoxin and nucleotide detoxification and cytokine inactivation [18].
Fig. 2. Pathological effects of overexpression of APs.
Although, the inhibition of TNAP is largely achieved by a limited number of small molecules
including levamisole, theophylline and L-homoarginine, the selectivity issue still persists [20].
Development of selective alkaline phosphatase inhibitors is a potential strategy to reduce vascular
5

calcification and improve cardiovascular outcomes in patients with chronic kidney disease (CKD)
or type 2 diabetes mellitus. The aforementioned therapeutic potential of both ecto-nucleotide
pyrophosphatases and alkaline phosphatase isozymes as prospective drug targets, and the lack of
meaningful structure-activity relationship and enzyme selectivity data for both families, encouraged
us to design fundamental motifs and synthesize new series of unexplored molecules with potent and
selective inhibitory effects.
Among five-membered bioactive heterocycles, pyrazoles have maintained a unique and dominant
position due to their presence as key structural unit in several pharmaceutical drugs, such as
rimonabant and celecoxib [21]. Pyrazoles are also present in many natural products including
pyrazofurin, formycin, oxoformycin B and nostocine [22]. They show a diverse range of biological
properties such as anti-hyperglycemic [23], anti-inflammatory [24], anti-cancer [25a-c], anti-viral
[26a,b] and anti-microbial activities [27]. Recently, pyrazoles have been reported as promising
candidates for the inhibition of ectonucleotidases [28]. Pyrimidinetriones and 2-
thioxopyrimidinediones, on the other hand, are versatile poly-functional molecules containing five
potential metal binding sites (lone pairs on three oxygen and two nitrogen atoms in case of
pyrimidinetriones; while on two oxygen, one sulfur and two nitrogen atoms in case of 2-
thioxopyrimidinediones) and are supposed to be suitable to anchor these metallo-enzymes.
Therefore, we designed novel heterocyclic entities incorporating pyrimidinetriones/
thioxopyrimidinediones and pyrazoles, endowed with several exploitable and diversified functional
groups. Using the pharmacophore integration approach, we successfully coupled the two bioactive
nuclei delivering a unique set of hybrid compounds (Fig. 3), which to the best of our knowledge,
has not been reported previously. We herein report a high yielding synthesis for 1-phenyl-3-
arylpyrazol-4-yl-5-methylene-N,N'-dialkylpyrimidine-2,4,6-triones (PPTs) and 1-phenyl-3-
arylpyrazol-4-yl-5-methylene-N,N-dialkyl-2-thioxopyrimidine-4,6-diones (PTPs) with three
6

potential sites for structural modifications. The notable features of our approach include the
generation of varied structures in good to excellent purified yields from easily accessible starting
materials. In vitro assays for nucleotide pyrophosphatase and alkaline phosphatase inhibition were
performed to analyze the biological potential of the newly synthesized compounds, and the
acquired results were rationalized with the help of molecular docking investigations.
Fig. 3. A pharmacophore integrated approach for the structural design of pyrazolyl
pyrimidinetrione and thioxopyrimidinedione conjugates.
2. Results and discussion
2.1. Chemistry
The practical route to the synthesis of pyrazolyl pyrimidinetrione and thioxopyrimidinedione
hybrid compounds (6a‒n) is illustrated in Scheme 1. The synthesis of key pyrazole fragment
(4a‒g) was achieved in two steps. Initially, the reaction of phenylhydrazine (1) with 4-substituted
acetophenones (2a‒g) afforded phenylhydrazones (3a‒g) under acidic conditions. Subsequently,
the Vilsmeier–Haack formylation reaction of phenylhydrazones (3a‒g) provided a facile access to
7

corresponding 4-carboxaldehyde functionalized pyrazole derivatives (4a‒g). The reaction was
performed using 3 equivalents of Vilsmeier–Haack reagent (DMF‒POCl3), followed by treatment
with cold aqueous base (NaOH or NaHCO3) delivering a diversified series of 3-aryl-1H-pyrazole-
4-carbaldehydes (4a‒g) in good to excellent yields [29].
1-Phenyl-3-aryl-1H-pyrazole-4-carbaldehydes
(4a‒g)
were
coupled
to
N-alkylated
pyrimidinetriones (5a) and thioxopyrimidinediones (5b) under previously developed conditions
[29] of Knoevenagel condensation reaction to deliver the range of 1-phenyl-3-arylpyrazol-4-yl-5-
methylene-N,N'-dialkylpyrimidine-2,4,6-triones (PPTs) (6a‒g) and 1-phenyl-3-arylpyrazol-4-yl-5-
methylene-N,N'-dialkyl-2-thioxopyrimidine-4,6-diones (PTPs) (6h–n). This simple methodology
proved to be quite general: unsubstituted and 4-substituted aryl moieties with both electron-rich
(methyl, methoxy) and electron-poor (fluoro, chloro, bromo, nitro) substituents attached to position
3 of the pyrazole fragment afforded the desired hybrid compounds in good to excellent yields. In
addition, small alkyl groups (methyl and ethyl) attached to nitrogen atom of pyrimidinetrione and
thioxopyrimidinedione components were also well tolerated.
8

O
R
AcOH
Me
N
NH2
EtOH, 70 °C
N
H
N
R
H
Me
(1)
(2ag)
(3ag)
X
N
1. DMF, POCl3
0 70 °C, 3 h
Z
O
2. NaOH(aq)
/
NaHCO3(aq)
N
X
X
Z
X
N
O
O
N
5a; X = Me, Z = O
5b; X = Et, Z = S
N
R
N
N
N
DCM/MeOH (8:1)
EDA (0.2 mmol)
AcOH (2 mmol)
O
CHO
(6an)
(4ag)
R
6a:
6h:
6i:
6j:
6k:
6l:
X = Me, Z = O, R = H
X = Me, Z = O, R = F
X = Me, Z = O, R = Cl
X = Me, Z = O, R = Br
X = Me, Z = O, R = Me
X = Me, Z = O, R = OMe
X = Me, Z = O, R = NO2
X = Et, Z = S, R = H
X = Et, Z = S, R = F
X = Et, Z = S, R = Cl
X = Et, Z = S, R = Br
X = Et, Z = S, R = Me
X = Et, Z = S, R = OMe
X = Et, Z = S, R = NO2
6b:
6c:
6d:
6e:
6f:
6m:
6n:
6g:
Scheme 1. Synthetic pathway to pyrazolyl pyrimidinetriones (PPTs) and thioxopyrimidinediones
(PTPs).
All the synthesized compounds were characterized by readily available analytical methods
1
including FTIR, H and 13C NMR spectroscopy and mass spectrometry. In IR spectra of
compounds 6a-n, the stretching vibrations observed in the range 1623-1563 cm‒1 were attributed to
C=N functionality (pyrazole), while the strong bands for carbonyl stretching frequencies appeared
1
in the region 1710‒1698 cm-1. In H NMR spectra, the most characteristic olefinic proton (=C‒H)
resonated in the range of 8.85–8.28 ppm, whereas the =C‒H proton of the pyrazole ring appeared in
the region 9.96–9.70 ppm. 13C NMR spectra showed diagnostic chemical shifts in the region of
179.81-178.45 ppm due to the presence of C=S in case of thioxopyrimidinediones (6h-n), whereas
9

the C=O functional groups in the synthesized compounds resonated from 165.90 to 158.61 ppm.
Finally, elemental analysis was used to ascertain the purity of the target compounds (6a-n).
2.2. Enzyme inhibition studies and structure-activity relationship analysis
To analyze the biological properties of newly synthesized library of 1-phenyl-3-arylpyrazol-4-yl-5-
methylene-N,N'-dialkylpyrimidine-2,4,6-triones (PPTs) (6a‒g) and 1-phenyl-3-arylpyrazol-4-yl-5-
methylene-N,N'-dialkyl-2-thioxopyrimidine-4,6-diones (PTPs) (6h–n), in vitro inhibitory potency
was measured against h-APs and h-NPPs. The biological inhibition results are summarized in Table
1.
Table 1. Alkaline phosphatase (h-TNAP & h-IAP) and nucleotide pyrophosphatase (h-NPP1 & h-
NPP3) inhibition data for compounds 6a‒n.
h-TNAP
h-IAP
h-NPP1
h-NPP3
Compound
IC50 (µM) ± SEM
0.33 ± 0.02
2.21 ± 0.24
>100
>100
>100
>100
0.61 ± 0.05
>100
1.36 ± 0.31
0.66 ± 0.04
>100
6a
6b
0.86 ± 0.04
1.57 ± 0.32
>100
6c
>100
1.21 ± 0.22
1.86 ± 0.31
>100
2.27 ± 0.11
>100
6d
5.83 ± 0.78
>100
6e
>100
>100
6f
>100
12.7 ± 1.45
>100
>100
2.49 ± 0.28
0.57 ± 0.01
2.03 ± 0.02
>100
6g
2.99 ± 0.18
>100
4.61 ± 0.32
>100
6h
>100
6i
>100
33.6 ± 3.45
>100
1.01 ± 0.03
>100
6j
3.07 ± 0.24
5.26 ± 1.09
>100
6.36 ± 0.18
4.55 ± 0.17
2.86 ± 0.18
2.11 ± 0.13
1.27 ± 0.82
—
6k
6.33 ± 0.34
1.31 ± 0.32
21.2 ± 2.56
—
>100
>100
6l
6m
1.78 ± 0.34
—
20.2 ± 1.21
—
>100
6n
8.67 ± 1.31
—
Suramin
Levamisole
L-Phenylalanine
—
100 ± 3.02
—
—
Most of the derivatives of both the synthesized series exhibited dual inhibition of isozymes of APs
and NPPs, but with more selectivity towards h-APs. Interestingly, in the alkaline phosphatase
10

assay, compounds 6a, 6b, 6e, 6h and 6k with pronounced inhibition against h-TNAP were inactive
against h-IAP (except 6l and 6n) and vice versa. These inhibition results explicitly address the
selectivity issues. The detailed structure-activity relationship of PPT and PTP derivatives against
APs suggested that the unsubstituted phenyl ring in case of PPT led to a potent inhibitor (6a; IC50 =
0.33 ± 0.02 µM) of h-TNAP. Compound 6a exhibited ~ 60-fold stronger inhibition than the
reference drug (levamisole: IC50 = 20.2 ± 1.21 µM). On the other hand, the unsubstituted derivative
of PTPs (6h) exhibited less inhibitory potential towards h-TNAP compared to compound 6a.
Compound 6c was identified as the most potent and selective inhibitor of h-IAP with IC50 value of
0.86 ± 0.04 µM unveiling ~ 116-fold higher inhibition than the reference compound (L-
phenylalanine; IC50 = 100 ± 3.01 µM). The higher inhibitory potential of this compound could be
attributed to the presence of an inductively withdrawing chloro substituent at the para-position of
the phenyl ring attached to the pyrazole core. However, comparatively lower activity (IC50 = 1.57 ±
0.32 µM) was observed for 6d incorporating a bromine atom at the same position. A striking
difference was observed when compound 6c was compared to its thioxo analog (6j) where lower
activity (33.6 ± 3.45 µM) was observed for compound 6j. However, activity was almost restored
(1.31 ± 0.32 µM) when chloro group was replaced by a methoxy substituent in compound 6m. A
similar trend was observed in case of PTP derivatives (6h‒n) where compound 6j showed higher
inhibition (IC50 = 33.6 ± 3.45 µM) compared to its fluoro (6i) and bromo analogs (6k). It was also
observed that compound 6b bearing fluorine substituent at the phenyl ring showed stronger and
selective inhibitory potential towards h-TNAP. In case of compound 6e bearing electron-donating
methyl group, selective inhibition was also observed towards h-TNAP, whereas compound 6g
featuring a polarized nitro group, showed selective and efficient inhibition of h-IAP with an IC50
value of 12.7 ± 1.45 µM. Based on these inhibitory results, it could be reasonably suggested that
the presence of appropriate substituent at aromatic moiety of pyrazole and the coupled
11

pyrimidinetrione/thioxopyrimidinedione is the key combination for high profile selective inhibition
of h-IAP and h-TNAP isozymes.
On the other hand, when the structure-activity relationship of PPT and PTP derivatives was studied
towards h-NPPs, it was observed that these derivatives exhibited more inhibitory potential towards
h-NPP1 as compared to h-NPP3. The bioactivity results of these derivatives suggested that the
derivatives 6b from PPT and 6j from PTP series bearing F and Cl atom at 4 position of phenyl ring,
respectively, exhibited maximum inhibitory potential towards h-NPP3. It was also notable to
perceive that compound 6h displayed 2-fold higher inhibition (IC50 = 0.57 ± 0.01 µM) of h-NPP3
compared to suramin (positive control) (IC50 = 1.27 ± 0.08 µM). Compound 6b also revealed ~ 14-
fold higher inhibitory values towards h-NPP1 as compared to standard inhibitor, whereas 2-fold
stronger inhibition of h-NPP3 was observed. Compounds 6e also unveiled selective and efficient
inhibition of h-NPP1 with IC50 values of 1.86 ± 0.31 µM. Moreover, compounds 6a, 6g and 6i and
compounds 6k‒n exhibited relatively lower inhibition of h-NPP3 but with complete selectivity.
In general, the analyzed compounds revealed a remarkable inhibition profile against recombinant
human ectonucleotidases identifying several leads with excellent levels of selectivity.
2.3. Molecular docking studies
The binding mode of the selective and potent inhibitors against h-TNAP, h-IAP, h-NPP1 and h-
NPP3 were docked inside the active site of the respective enzymes. Due to the unavailability of
crystal structures in the protein data bank, the homology models were generated by our research
group [30-32] and molecular docking was performed. As the newly generated models do not
contain any cognate ligand inside the active pocket, therefore, the MOE site finder was used to
search and select the binding of protein. For the validation purpose, the reference compounds used
as standards in biological assays were docked within the binding site of protein before carrying out
further docking. The putative interactions were analyzed and compared. The presence of Zinc ions
12

within active center was important for the interaction and binding of the inhibitors. The putative
binding interactions of standard compounds are depicted in Figures 4a, 5a, 6c and 7c against h-
TNAP, h-IAP, h-NPP1 and h-NPP3, respectively.
The results of biological studies suggested that compound 6a was the potent and selective inhibitor
for h-TNAP, whereas compound 6c emerged as selected and potent inhibitor for h-IAP. Moreover,
for h-NPP3, the most potent compound was found to be 6h. Compound 6b not only showed potent
inhibition against h-NPP1 but also exhibited significant inhibition towards h-NPP3 and, therefore,
was not selective. For the investigation of the binding interactions of potent inhibitor against h-
NPP1, 6j was selected. Molecular docking analyses were performed against all the lead derivatives
in their respective proteins. Binding mode investigation of levamisole [(S)-6-phenyl-2,3,5,6-
tetrahydroimidazo[2,1-b]thiazole] and potent compound (6a) within the active pocket of h-TNAP
presented that His362, His437 and His324 were associated with π-π interactions by the phenyl of
levamisole and 1,3-dimethylpyrimidinetrione ring of the compound. In addition, hydrogen bond
interactions were noticed that may have profound impact on the inhibition potential. The H-
bonding was noticed between basic residues and O atom of the trione moiety of 6a. The amino acid
residues intricate in making H-bonds were His321, Tyr170, Arg151, His434 and His154. Very
interestingly, it was noticed that the interactions with metal ions which are important for the
inhibitory activity were shown by carbonyl oxygen of 6a and Zn ion and their bond distance was
2.28 Å. Similarly, phenyl group established contact with Zn ion with a bond distance of 3.76 Å.
However, levamisole interacted with Zn ion by a bond distance of 3.72 Å. These metal interactions
are supposed to form stable inhibitor-enzyme (EI) complex. The 3D binding interactions of
levamisole and compound 6a are shown in Fig. 5 (a and b, respectively).
13

(a)
(b)
Fig. 5 Putative binding interactions of levamisole (a) and compound 6a (b) within the active pocket
of h-TNAP.
The investigation of the binding interactions of L-phenylalanine and the potent compound 6c inside
the active pocket of h-IAP revealed that the important amino acid residues taking part within the
active pocket were Arg150, His153, His317, His320, Glu321 and Glu108. The π-π interactions
were examined between aryl ring of phenylalanine and amino acid His320. Similar interactions
were also noticed for compound 6c and His320 and His317, when 3D diagrams were analyzed.
His360 and Arg166 along with Tyr169 were intricate in H- bonding and these bindings are shown
in Fig. 6 (a and b). Within the active site of h-IAP, Mg+2 ions along with Zn+2 ions were present.
Taking into account the interactions with metal ions, it was found that the bond distance between
pyrimidine ring in compound 6c and Zn+2 ion was 3.59 Å (Fig. 6b). Both the Zn+2 ions were
playing active role in interactions with phenylalanine, first one at a distance of 2.86 Å, however,
second at 4.09 Å (Fig. 6a), while Mg+2 ion was present at bond distance of 5.18 Å from oxygen
atom of phenylalanine. Most of the interactions were shown by O atom of phenylalanine. On the
other hand, the chlorine atom of the phenyl group in compound 6c was directed towards the amino
acid Arg150 and His153, and 1,3-dimethyl pyrimidinetrione moiety was present in the middle of
14

the active pocket where metal ions were positioned. The interactions and layout of compound 6c
against h-IAP may be the contributing factor for its higher inhibition activity.
(a)
(b)
Fig. 6. Putative binding mode of L-phenylalanine (a) and compound 6c (b) within the active pocket
of h-IAP.
When h-NPP1 was considered, the binding interactions were studied for suramin, the reference
standard, and the potent compound (6b) from the tested series. However, the compound, 6j, was
found as most selective inhibitor of NPP1, therefore the docking studies were also carried out for
this compound. The important amino acid residues taking part in the active site of h-NPP1 were
Lys255, His535, His380, Thr256, Leu290 and Asp218 in addition to Zn+2 ions. The amino acid
residues were important to exhibit the interactions with the compounds, 6b and 6j. In case of 6b, it
was observed that fluorophenyl group was oriented more towards Zn+2 ion in the active pocket and
fluorine atom was at a bond distance of 3.57 Å. The oxygen atoms of suramin were showing
interactions with Zn+2 ions at a bond distance of 5.41 Å and 3.66 Å. Suramin contained 4,6,8-
trisulphonapthalen and 1,3,5-trisulfonic acid, the groups interacted in very good way with active
site amino acids of h-NPP1 and interactions are shown in Fig. 7c. The π-π interactions were noticed
among important residues and trisubstituted pyrazole moiety. Moreover, fluorophenyl part was
15

involved in such interactions as well. Hydrogen bonding was noticed among basic amino acids of
the active pocket of h-NPP1 and 2,4,6-pyrimidinetrione moiety of compound 6b (Fig. 7a). When
the docking results of 6j were taken into account, the 4-chloro substituent attached to phenyl ring
was found oriented towards Trp322 and making π-π interactions. Moreover, Ile421 was also
involved in π-π interactions with 2-thioxodihydropyrimidine-4,6(1H,5H)-dione moiety. Zinc+2 ion
was noticed to show interactions with phenyl group of 1H-pyrazol-4-yl-5-methylene attached to 2-
thioxodihydropyrimidine-4,6(1H,5H)-dione of compound 6j. The interactions are presented in Fig
7b.
(a)
(b)
(c)
16

Fig. 7. Putative binding mode of compound 6b (a), 6j (b) and suramin (c) inside the active pocket
of h-NPP1.
For h-NPP3, the potent compound was 6h, however, compound 6b also showed significant
inhibition. Therefore, the docking analysis were performed for both the inhibitors and binding
poses and interactions were analyzed and compared with the binding shown by suramin inside the
active pocket (Fig. 8). The observation of metal interactions showed that oxygen group of suramin
was located at 5.37 Å bond distance, while oxygen atom of compound 6b was at a distance of 4.88
Å from Zinc+2 ion. However, phenyl ring of the potent compound was at a distance of 3.31 Å from
Zn+2 ion. The strong metallic interaction may be possible justification for the potential inhibitory
effect exhibited by compound 6h. 3D binding mode investigation showed that compound 6h was
well-occupied within the active pocket of h-NPP3, resulting in strong H bonds. Another reason that
may contribute towards the inhibitory profile of compound 6h may be due to the presence of 1,3-
diethyl group attached to 2-thioxodihydropyrimidine-4,6-(1H,5H)-dione functionality, as it was
noticed that the moiety is showing interactions with most of the amino acids in active site of
enzyme. Both the compounds, 6b and 6h, depicted strong hydrogen bonds in addition to π-π
interactions with the important residues of h-NPP3 (Fig. 8). This type of binding interactions plays
a vital role in the orientation of 3D coordinates of inhibitors/ligands within the active site and
increase the stability/strength of enzyme-ligand complex, therefore, providing comprehensive
structural insight of ligands within the active pocket of enzyme.
The details of ligand-proteins interactions, residues forming hydrogen bonding and π-π interactions
within the active site of target proteins and their colors for all the docking interactions are provided
in Table S1.
17

(b)
(a)
(c)
Fig. 8. Putative binding interactions of compound 6b (a), compound 6h (b), and suramin (c) inside
the active site of h-NPP3.
2.4.1. HYDE assessment of selective compounds
HYDE visual affinity was performed for the first 20 ranked docked conformers inside the active
pocket of the homology models of human TNAP, IAP, NPP1 and NPP3 to depict the optimization
18

of ligands. It correctly gives information for the ligand efficiency in addition to selectivity towards
the most potent compound (Figures S1-S4). The binding energy and docking score by FlexX for the
selective derivatives are given in Table 2. The FlexX docking score presented that the potent
compound has lower energy scores as compared to non-selective inhibitors. Moreover, the binding
free energies ΔG showed that the potent inhibitors exhibited higher affinity towards their respective
targets. Compound 6a interacted with the h-TNAP in a very efficient manner in comparison to 6c
and gave favorable contributions. Similarly, 6c showed higher affinity than 6a towards h-IAP. The
same trend was observed for the most potent inhibitors of h-NPP1 and h-NPP3, 6j and 6h,
respectively. The selective compounds showed significant efficiency towards the protein as
compared to other compounds.
Table 2. Ligand efficiency scores and the ranks after HYDE visual inspection.
Code Docking score by FlexX for top pose Free energy of bindingΔG (kJ mol‒1)
h-TNAP
-18.36
-15.53
‒17
‒9
6a
6c
h-IAP
h-NPP1
h-NPP3
-14.11
-16.42
6
‒4
6a
6c
-18.42
-19.68
‒12
‒17
6h
6j
-20.58
-17.43
‒19
‒11
6h
6j
2.4. Mechanism of inhibition
In order to explore the mode of inhibition, the kinetic studies were performed for compounds 6a,
6c, 6b and 6h, the most potent inhibitors of human TNAP, IAP, NPP1 and NPP3, respectively. The
Lineweaver-Burk plot for compound 6a revealed an un-competitive mode of inhibition, whereas
19

compounds 6c, 6b and 6h showed a competitive mode of inhibition by illustrating the same y-
intercept for uninhibited and inhibited enzymes (Fig. 4). Furthermore, Michaelis-Menten kinetic
parameters; Km and Vmax for h-TNAP, h-IAP, h-NPP1 and h-NPP3 inhibition were also determined
in the presence and absence of inhibitor 6a, 6c, 6b and 6d, respectively. In case of 6c, the maximal
velocity (Vmax) was found in decreasing order at different concentrations of 6c, whereas the
Michaelis constant (Km) was found almost constant (Figure 4a). On the other hand, in case of
compounds 6c, 6b and 6h, the maximal velocity (Vmax) was noted as constant although different
concentrations of respective inhibitors were used, whereas, the Michaelis constant (Km) was noticed
to increase (Figure 4b, 4c and 4d).
(a)
(b)
(c)
(d)
20

Fig. 4 Lineweaver-Burk plot for h-TNAP and h-IAP inhibition by compounds 6a (a); and 6c (b)
where S is concentration of substrate CDP-star® (μM). Lineweaver-Burk plot for h-NPP1 and h-
NPP3 inhibition by compound 6b (c); and 6h (d) where S is concentration of substrate p-Nph-TMP
(μM).
2.5. Anticancer activity
Anticancer activity in terms of cytotoxicity of pyrazolyl pyrimidinetrione and
thioxopyrimidinedione derivatives (6a‒n) against three different cancer cell lines, including breast
cancer (MCF-7), myelogenous leukemia (K-562) and HeLa cell lines in comparison to normal cells
(baby hamster kidney cell line; BHK-21) was determined using MTT assay. The cytotoxicity after
24 h treatment with 100 µM concentration of respective derivative was assessed in these
experiments. Carboplatin was used as a standard drug. The obtained results are expressed as a
percentage of growth inhibition with reference to untreated controlled cells and are presented in
Table 3.
Table 3. Cytotoxic effects of hybrid compounds (6a‒n).
MCF-7
K-562
% inhibition ± SEM*
HeLa
BHK-21
Compound
13.8 ± 0.56
11.3 ± 0.37
15.7 ± 0.98
9.77 ± 0.45
16.4 ± 0.23
5.70 ± 0.17
8.45 ± 0.33
7.81 ± 0.78
10.1 ± 0.27
24.1 ± 0.89
27.6 ± 1.25
25.2 ± 2.56
14.9 ± 0.89
12.6 ± 0.65
18.4 ± 0.45
72.7 ± 2.41
85.4 ± 3.18
59.1 ± 1.44
62.7 ± 1.79
70.9 ± 1.64
61.1 ± 2.11
44.6 ± 1.21
57.1 ± 1.32
69.9 ± 2.49
51.2 ± 2.16
53.2 ± 1.33
50.7 ± 2.94
59.4 ± 2.68
58.7 ± 1.82
90.7 ± 1.26
92.2 ± 2.57
45.1 ± 1.25
19.6 ± 1.06
45.1 ± 1.54
27.5 ± 1.23
17.6 ± 0.78
86.3 ± 2.15
56.9 ± 1.56
84.3 ± 2.71
90.2 ± 3.26
83.2 ± 2.97
56.9 ± 0.77
68.6 ± 1.39
31.4 ± 0.78
82.8 ± 1.78
68.7 ± 2.16
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
77.1 ± 3.27
82.1 ± 3.68
72.4 ± 3.36
48.1 ± 2.46
11.1 ± 1.65
71.8 ± 3.26
73.6 ± 2.78
64.1 ± 1.68
61.4 ± 2.69
73.7 ± 1.79
69.5 ± 2.34
78.4 ± 3.88
66.7 ± 1.78
85.2 ± 1.57
6n
Carboplatin
* The percentage of growth inhibition was expressed in relation to untreated control cells as Means ± SEM values.
21

When pyrazolyl pyrimidinetriones/thioxopyrimidinediones (6a-n) were tested against normal cell
lines (baby hamster kidey cell line; BHK-21), it was observed that PPT and PTP derivatives are
less effective against normal cells showing low effect on growth reduction, hence can be good
candidates as anticancer agents. When tested against cancer cell lines, it was observed that
pyrazolyl pyrimidinetriones/thioxopyrimidinediones (6a-n) displayed promising anticancer activity
in terms of cytotoxicity. With regard to MCF-7 cell lines, pyrazolyl pyrimidinetriones (6a-g) were
found to have higher cytotoxic activity as compared to their thioxo analogs (6h-n). Compounds 6a,
6b and 6e showed higher cytotoxicity than the rest of the candidates of the series. However, their
cytotoxicity was relatively lower as compared to carboplatin. Intriguingly, compound 6b (the most
active inhibitor against NPP1) showed highest anticancer activity against breast cancer cell line.
Anticancer activity with regard to chronic myelogenous leukemia cell lines (k-562) revealed that
compounds 6a and 6g from PPTs and compounds 6i, 6j and 6k from PTPs displayed promising
activity with reference to the standard. Interestingly, compound 6a (the most active inhibitor
against TNAP) showed promising activity against K-562 cells. Furthermore, a good cytotoxic
profile was obtained when compounds were tested against HeLa cell lines exhibiting good to
moderate activity. It was found that compound 6c showed high % growth inhibition against Hela
Cells. The most active anticancer compounds were pyrimidinetriones, 6a, 6c and 6b bearing H, F
and Cl substituents on the phenyl ring of pyrazole moiety, causing 92, 82 and 85% reduction in the
cell growth of K-562, HeLa and MCF-7, respectively, at 100 μM concentration (Fig. 9). It may be
derived from the above discussion that the observed effects were dependent on the nature of
substituent on aryl ring of pyrimidine nucleus.
22

(a)
(b)
100
80
60
40
20
0
6i
6l
6f
6j
6k
6c
6e
6a
6g
6b
6d
6h
6n
6m
carboplatin
(c)
(d)
Fig. 9 Cytotoxic potential of compounds 6a‒n against MCF-7 (a); K-562 (b); HeLa in comparison
to normal cells (c); and BHK-21 cells (d). Cytotoxicity was quantified spectrophotometrically using
an MTT assay.
These most active compounds were further selected for the determination of IC50 values against
MCF-7, K-562 and HeLa cells (Table 4). The IC50 values showed that compound 6b bearing
pyrimidinetrione moiety and a fluoro substituent on the phenyl ring of pyrazole moiety possesses
significantly higher cytotoxic activity towards MCF-7 cells. Similarly, compounds 6a (bearing
pyrimidinetrione moiety and 1,3-diphenyl ring on pyrazole nucleus) exhibited maximum cytotoxic
activity towards K-562, while compound 6c (bearing 1-phenyl-3(4-chorophenyl moiety on
pyrazolyl ring and a pyrimidinetrione nucleus) exhibited maximum cytotoxic activity against HeLa
cells.
23

Table 4. Cytotoxic effect of the most potent compounds (6a-c) on different cell lines.
MCF-7
K-562
IC50 ± SEM*
>100
HeLa
Compound
1.35 ± 0.03
4.78 ± 0.66
5.11 ± 0.67
3.91 ± 0.32
8.41 ± 0.89
7.09 ± 0.77
1.24 ± 0.04
5.13 ± 0.45
6b
1.66 ± 0.01
>100
6a
6c
1.8 ± 0.78
Carboplatin
*IC50 denotes compound concentrations that result in a 50% decrease in the cell number compared to non-treated
controls and were derived from 24 h treatment. Results from three independent experiments are presented.
3. Conclusions
In summary, a pharmacophore integration methodology leading to a diverse range of pyrazole
coupled pyrimidinetriones (6a‒g) and thioxopyrimidinediones (6h‒n) was successfully established.
The synthesized derivatives were investigated for the inhibitory potency against human
recombinant alkaline phosphatases (TNAP and IAP) and nucleotide pyrophosphatases (NPP1 and
NPP3). In vitro biological assay results explicitly predicted the selective and potent inhibition of
both target enzymes. Among the screened derivatives, compound 6a caused the selective inhibition
of h-TNAP with ~ 60-fold higher efficacy than the standard, whereas compound 6c selectively
inhibited h-IAP isozyme with IC50 value of 0.86 ± 0.04 µM. Most interestingly, compounds 6b and
6h appeared as lead scaffolds against h-NPP1 and h-NPP3 enzymes, respectively. The docking
analysis revealed that these molecules establish hydrogen bonds as well as several other
electrostatic interactions with important amino acid residues. The Lineweaver-Burk plot for potent
compound 6a demonstrated an un-competitive mode of inhibition, whereas compounds 6c, 6b and
6h showed a competitive mode of inhibition. Moreover, compounds 6a, 6b and 6c caused 92, 85
and 82% reduction in the cell growth of MCF-7, K-562 and HeLa cell lines, respectively, at 100
μM concentration. Collectively, the newly developed hybrid structural scaffolds provide a valuable
platform for the enhancement in the activity profile and to generate lead molecules for drug
discovery.
24

4. Experimental section
4.1. Substrates and reagents
The acetophenones and glacial acetic acid were obtained from Sigma-Aldrich (Steinheim,
Germany). Ethylenediamine was purchased from Merck (Darmstadt, Germany). The reagents used
were of analytical grade. Ethanol, chloroform and methanol were provided by Lab Scan (Patuman,
Bangkok). Ethyl acetate was the product of Riedel de Haen (Seezle, Germany). All solvents used
were dried under nitrogen pressure by passage through activated alumina columns.
4.2. Instrumentation
Thin layer chromatography (TLC) was performed on Merck 0.2mm DF 60F254 precoated
aluminium plates. The compounds were visualized by exposure to UV light at 254 nm. Melting
points were recorded using Gallenkamp melting point apparatus (MP-D) in open capillaries and are
uncorrected. FTIR spectra were recorded using attenuated total reflectance (ATR) facility on a
Thermoscientific Fourier Transform Infra-Red Spectrophotometer (USA) model nicolet 6700. The
elemental analysis was performed using Leco CHNS-932 Elemental Analyzer (USA). NMR spectra
1
13
were acquired on Bruker DQX400 at room temperature. H and C NMR spectra were referenced
to tetramethylsilane via the residual protonated solvent (1H) or the solvent itself (13C). All chemical
shifts are reported in parts per million (ppm). For CDCl3, the shifts are referenced to 7.26 ppm for
1H NMR spectroscopy and 77.16 ppm for 13C NMR spectroscopy. High-resolution mass spectra
were recorded on a Micromass LCT electrospray ionization mass spectrometer operating at a
resolution of 5000 full width half height.
4.3. General procedure for the synthesis of phenyl-3-aryl-1H-pyrazole-4-carbaldehydes (4a‒g)
Synthesis of phenyl-3-aryl-1H-pyrazole-4-carbaldehydes (4a‒g) reported in our previous work [29].
To a stirred solution of phenyl hydrazine 1 (12 mmol, 1.29g or 1.18mL) in absolute ethanol (20 mL)
was added acetophenone 2 (10 mmol) and glacial acetic acid (0.3 mL). The reaction mixture was
kept on stirring at 70 °C. After completion of the reaction (as monitored by TLC), the excess solvent
25

was evaporated to yield crude solid, which was filtered, washed with cold ethanol, dried and
recrystallized from ethanol to yield the corresponding hydrazone (3).
The appropriate acetophenone phenyl hydrazone (1.0 mmol) was added to a cold solution of
dimethylformamide (DMF) (4 mL) and phosphorus oxychloride (0.5 g, 3.0 mmol) and the resulting
mixture heated at 70 °C for 3 h. The mixture was cooled to room temperature and poured onto
crushed ice, neutralized with an aqueous solution of sodium hydroxide (for compounds 4a, 4e and
4f) or sodium hydrogen carbonate (for compounds 4b-d and 4g). The precipitated solid was filtered
and purified by flash column chromatography (20% EtOAc in n-hexane) in case of compounds 4a,
4e and 4f, while recrystallized from ethanol to afford the rest of pyrazole-4-carbaldehydes (4b-d and
4g).
4.4. General procedure for the synthesis of 1-phenyl-3-arylpyrazol-4-yl-5-methylene-N,N'-
dialkylpyrimidine-2,4,6-triones (PPTs) (6a‒g) and 1-phenyl-3-arylpyrazol-4-yl-5-methylene-
N,N'-dialkyl-2-thioxopyrimidine-4,6-diones (PTPs) (6h–n)
To a stirred solution of 1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione or 1,3-dimethyl-2-
thioxodihydropyrimidine-4,6-dione (1.0 mmol) in chloroform/methanol (8:1; 20 mL), glacial acetic
acid (2 mmol) was added followed by ethylenediamine (0.2 mmol). The resulting mixture was
stirred for 15 minutes followed by the addition of 3-(4-substituted phenyl)-1-phenyl-1H-pyrazole-4-
carbaldehyde (4a–g) (1.2 mmol) at ambient temperature. After completion of the reaction (TLC;
30% acetone in n-hexane), excess solvent was removed in vacuo to afford a bright yellow solid. The
crude solid was washed with water, dried and recrystallized (chloroform/ethanol) to yield the desired
compounds (6a-n).
4.4.1.
5-((1,3-Diphenyl-1H-pyrazol-4-yl)methylene)-1,3-dimethylpyrimidine-2,4,6-
(1H,3H,5H)trione (6a). Pale yellow solid (84%); m.p.: 198–200 °C; Rf: 0.42 (30% acetone in n-
hexane); FTIR (ATR, cm‒1): 3025 (=C-H), 2944, 2813 (2×C-H), 1690 (C=O), 1564 (C=N), 1570,
1
1513 (2×C=C); H NMR (400 MHz, Chloroform-d): δ 9.83 (1H, s, C-Hpyrazolyl), 8.28 (1H, s, =C-
26

Holefinic), 8.00–7.92 (2H, m, Ar-H), 7.62 (7H, m, Ar-H), 7.54–7.46 (1H, m, Ar-H), 3.28 (3H, s,
CH3), 3.22 (3H, s, CH3); 13C NMR (101 MHz, Chloroform-d): δ 162.79 (C=O), 161.75 (C=O),
158.61 (C=O), 151.53, 144.88, 138.97, 134.84, 131.17, 130.41, 129.99, 129.92, 129.40, 128.61,
120.16, 115.72, 114.55, 29.00 (CH3), 28.43 (CH3); HRMS (ESI+ve) exact mass calculated for
C22H18N4O3 [M+H]+: 386.4001; Found: 386.4006; Analysis calculated: C 68.38, H 4.70, N 14.50;
Found: C 68.45, H 4.74, N 15.43.
4.4.2. 5-((3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-1,3-dimethylpyrimidine-
2,4,6-(1H,3H,5H)-trione (6b). Pale yellow solid (89%); m.p.: 202–204 °C; Rf: 0.49 (30% acetone
in n-hexane); FTIR (ATR, cm‒1): 3014 (=C-H), 2940, 2811 (2×C-H), 1662 (C=O), 1563 (C=N),
1
1569, 1507 (2×C=C); H NMR (400 MHz, Chloroform-d): δ 9.81 (1H, s, C-Hpyrazolyl), 8.48 (1H, s,
=C-Holefinic), 7.87–7.79 (2H, m, Ar-H), 7.60–7.52 (2H, m, Ar-H), 7.51–7.42 (2H, m, Ar-H), 7.39–
7.30 (1H, m, Ar-H), 7.22–7.13 (2H, m, Ar-H), 3.37 (3H, s, CH3), 3.33 (3H, s, CH3); 13C NMR (101
MHz, Chloroform-d): δ 165.79 (C=O), 164.61 (C=O), 163.07 (C=O), 159.41 (d, 1J = 249.4 Hz, C-
F), 149.80, 147.52, 135.08, 131.61, 131.53, 129.67, 128.19, 121.34, 120.08, 116.22, 116.01, 28.92
(CH3), 28.27 (CH3); HRMS (ESI+ve) exact mass calculated for C22H17FN4O3 [M+H]+: 404.3918;
Found: 404.3918; Analysis calculated: C 65.34; H 4.24, N 13.85; Found: C 65.39, H 4.27, N 13.90.
4.4.3. 5-((3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-1,3-dimethylpyrimidine-
2,4,6-(1H,3H,5H)-trione (6c). Brownish orange solid (80%); m.p.: 197–199 °C; Rf: 0.52 (30%
acetone in n-hexane); FTIR (ATR, cm‒1): 3016 (=C-H), 2933, 2843 (2×C-H), 1695 (C=O), 1617
(C=N), 1563, 1511 (2×C=C); 1H NMR (400 MHz, Chloroform-d): δ 9.88 (1H, s, C-Hpyrazolyl), 8.54
(1H, s, =C-Holefinic), 7.93–7.86 (2H, m, Ar-H), 7.67–7.58 (2H, m, Ar-H), 7.58–7.49 (2H, m, Ar-H),
7.46–7.37 (1H, m, Ar-H), 7.29–7.19 (2H, m, Ar-H), 3.44 (3H, s, CH3), 3.40 (3H, s, CH3); 13C NMR
(101 MHz, Chloroform-d): δ 163.54 (C=O), 162.37 (C=O), 159.12(C=O), 152.13, 147.98, 139.69,
135.87, 132.94, 131.94, 130.61, 130.43, 128.99, 124.87, 120.81, 116.69, 113.65, 29.68 (CH3),
27

29.03 (CH3); HRMS (ESI+ve) exact mass calculated for C22H17ClN4O3 [M+H]+: 420.8484; Found:
420.8489; Analysis calculated: C 62.79, H 8.42, N 13.31; Found: C 62.85, H 4.10, N 13.28.
4.4.4. 5-((3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-1,3-dimethylpyrimidine-
2,4,6-(1H,3H,5H)-trione (6d). Pale yellow solid (88%); m.p.: 210–212 °C; Rf: 0.62 (30% acetone
in n-hexane); FTIR (ATR, cm‒1): 3018 (=C-H), 2924, 2813 (2×C-H), 1680 (C=O), 1610 (C=N),
1
1565, 1512 (2×C=C); H NMR (400 MHz, Chloroform-d): δ 9.80 (1H, s, C-Hpyrazolyl), 8.45 (1H, s,
=C-Holefinic), 7.88–7.77 (2H, m, Ar-H), 7.65–7.57 (2H, m, Ar-H), 7.51–7.40 (4H, m, Ar-H), 7.39–
13
7.30 (1H, m, Ar-H), 3.36 (3H, s, CH3) 3.32 (3H, s, CH3); C NMR (101 MHz, Chloroform-d): δ
162.79 (C=O), 161.62 (C=O), 158.36 (C=O), 151.38, 147.23, 138.93, 135.12, 132.19, 131.19,
129.85, 129.68, 128.23, 124.11, 120.05, 115.93, 112.89, 28.93 (CH3), 28.28 (CH3); HRMS
(ESI+ve) exact mass calculated for C22H17BrN4O3 [M+H]+: 465.2911; Found: 465.2914; Analysis
calculated: C 56.79, H 3.68, N 12.04; Found: C 56.83, H 3.74, N 12.09.
4.4.5.
1,3-Dimethyl-5-((1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)methylene)pyrimidine-2,4,6-
(1H,3H,5H)-trione (6e). Pale yellow solid (82%); m.p.: 223–225 °C; Rf: 0.66 (30% acetone in n-
hexane); IR (ATR, cm‒1): 3019 (=C-H), 2938, 2809 (2×C-H), 1699 (C=O), 1602 (C=N), 1578,
1
1523 (2×C=C); H NMR (400 MHz, Chloroform-d): δ 9.70 (1H, s, C-Hpyrazolyl), 8.53 (1H, s, =C-
Holefinic), 7.87–7.79 (2H, m, Ar-H), 7.49–7.40 (4H, m, Ar-H), 7.37–7.24 (3H, m, Ar-H), 3.35 (3H, s,
CH3), 3.32 (3H, s, CH3), 2.36 (3H, s, CH3); 13C NMR (101 MHz, Chloroform-d): δ 162.91 (C=O),
161.73 (C=O), 159.77 (C=O), 151.48, 148.21, 139.52, 139.07, 134.99, 129.66, 129.61, 124.30,
128.03, 127.93, 120.06, 116.12, 112.38, 28.88 (CH3), 28.24 (CH3), 21.40 (CH3); HRMS (ESI+ve)
exact mass calculated for C23H20N4O3 [M+H]+: 400.4207; Found: 400.4202; Analysis calculated: C
68.99, H 5.03, N 13.99; Found: C 69.10, H 5.09, N 13.92.
4.4.6. 5-((3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-1,3-dimethylpyrimidine-
2,4,6-(1H,3H,5H)-trione (6f). Bright yellow solid (89%); m.p.: 215–217 °C; Rf: 0.68 (30%
acetone in n-hexane); FTIR (ATR, cm‒1): 3020 (=C-H), 2949, 2816 (2×C-H), 1685 (C=O), 1620
28

(C=N), 1574, 1538 (2×C=C); 1H NMR (400 MHz, Chloroform-d): δ 9.80 (1H, s, C-Hpyrazolyl), 8.54
(1H, s, =C-Holefinic), 7.87–7.79 (2H, m, Ar-H), 7.56–7.41 (4H, m, Ar-H), 7.38–7.29 (1H, m, Ar-H),
7.04–6.96 (2H, m, Ar-H), 3.81 (3H, s, OCH3), 3.36 (3H, s, CH3), 3.34 (3H, s, CH3); 13C NMR (101
MHz, Chloroform-d): δ 162.97 (C=O), 160.74 (C=O), 159.67 (C=O), 148.26, 139.07, 135.01,
132.67, 131.03, 129.61, 128.04, 123.27, 124.55, 120.07, 116.07, 114.50, 113.45, 55.40 (OCH3),
28.90 (CH3), 28.25 (CH3). HRMS (ESI+ve) exact mass calculated for C23H20N4O4 [M+H]+:
416.4263; Found: 416.4263; Analysis calculated: C 66.34, H 4.84, N 13.45; Found: C 66.39, H
4.87, N 13.50.
4.4.7.
1,3-Dimethyl-5-((3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)pyramidine-
2,4,6-(1H,3H,5H)-trione (6g). Pale yellow solid (90%): m.p.: 227–229 °C; Rf: 0.61 (30% acetone
in n-hexane); FTIR (ATR, cm‒1): 3020 (=C-H), 2940, 2825 (2×C-H), 1689 (C=O), 1623 (C=N),
1
1576, 1516 (2×C=C); H NMR (400 MHz, Chloroform-d): δ 9.87 (1H, s, C-Hpyrazolyl), 8.52 (1H, s,
=C-Holefinic), 7.95–7.84 (2H, m, Ar-H), 7.72–7.64 (2H, m, Ar-H), 7.58–7.47 (4H, m, Ar-H), 7.46–
13
7.37 (1H, m, Ar-H), 3.42 (3H, s, CH3) 3.41 (3H, s, CH3); C NMR (101 MHz, Chloroform-d): δ
164.23 (C=O), 163.06 (C=O), 159.80 (C=O), 152.82, 148.67, 140.37, 136.56, 133.63, 132.63,
131.29, 131.12, 129.67, 125.55, 121.50, 117.37, 114.34, 30.37 (CH3), 29.72 (CH3); HRMS
(ESI+ve) exact mass calculated for C22H17N5O5 [M+H]+: 431.4011; Found: 431.4015; Analysis
calculated: C 61.25, H 3.97, N 16.23; Found: C 61.31, H 4.03, N 16.21.
4.4.8.
5-((1,3-Diphenyl-1H-pyrazol-4-yl)methylene)-1,3-diethyl-2-thioxodihydropyrimidine-
4,6-(1H,5H)-dione (6h). Bright yellow solid (89%); m.p.: 193–195 °C; Rf: 0.62 (30% acetone in n-
hexane); FTIR (ATR, cm‒1): 3011 (=C-H), 2938, 2811 (2×C-H), 1699 (C=O), 1616 (C=N), 1578,
1
1509 (2×C=C); H NMR (400 MHz, Chloroform-d): δ 9.86 (1H, s, C-Hpyrazolyl), 8.54 (1H, s, =C-
Holefinic), 7.88–7.80 (2H, m, Ar-H), 7.61–7.53 (2H, m, Ar-H), 7.52–7.41 (5H, m, Ar-H), 7.41–7.29
(1H, m, Ar-H), 4.46 (2H, q, J = 7.0 Hz, CH2), 4.53 (2H, q, J = 7.0 Hz, CH2), 1.30 (3H, t, J = 7.0
Hz, CH3), 1.22 (3H, t, J = 7.0 Hz, CH3); 13C NMR (101 MHz, Chloroform-d): δ 178.50 (C=S),
29