Design and synthesis of some barbituric and 1,3-dimethylbarbituric acid derivatives: A non-classical scaffold for potential PARP1 inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104198

Six series based on barbituric acid 5a-e, 10a-d; thiobarbituric acid 6a-e, 11a-d and 1,3-dimethylbarbituric acid 7a-e, 12a-d were prepared and screened for their in vitro PARP1 inhibition. They revealed promising inhibition at nanomolar level especially compounds 5c, 7b, 7d and 7e (IC₅₀ = 30.51, 41.60, 41.53 and 36.33 nM) with higher potency than olaparib (IC₅₀ = 43.59 nM). Moreover, compounds 5b, 5d, 7a, 12a and 12c exhibited good comparable activity (IC₅₀ = 65.93, 58.90, 66.57, 45.40 and 50.62 nM, respectively). Furthermore, the most active compounds 5c, 7b, 7d, 7e, 12a and 12c against PARP1 in vitro were evaluated in the BRCA1 mutated triple negative breast cancer cell line MDA-MB-436 where 5c and 12c showed higher potency compared to olaparib and result in cell cycle arrest at G2/M phase. 5c and 12c showed apoptotic effects in MDA-MB-436 and potentiated the cytotoxicity of temozolomide in A549 human lung epithelial cancer cell line. Compounds 5c and 12c represent interesting starting points towards PARP1 inhibitors.

Full text of DOI:10.1016/j.bioorg.2020.104198

BioorganicChemistry104(2020)104198
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design and synthesis of some barbituric and 1,3-dimethylbarbituric acid
derivatives: A non-classical scaffold for potential PARP1 inhibitors
⁎
Essam Eldin A. Osmana, , Noura S. Hanafyb, Riham F. Georgea, Samir M. El-Moghazya
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Heliopolis University for Sustainable Development, Cairo 11777, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
Six series based on barbituric acid 5a-e, 10a-d; thiobarbituric acid 6a-e, 11a-d and 1,3-dimethylbarbituric acid
7a-e, 12a-d were prepared and screened for their in vitro PARP1 inhibition. They revealed promising inhibition
at nanomolar level especially compounds 5c, 7b, 7d and 7e (IC50 = 30.51, 41.60, 41.53 and 36.33 nM) with
higher potency than olaparib (IC50 = 43.59 nM). Moreover, compounds 5b, 5d, 7a, 12a and 12c exhibited good
comparable activity (IC50 = 65.93, 58.90, 66.57, 45.40 and 50.62 nM, respectively). Furthermore, the most
active compounds 5c, 7b, 7d, 7e, 12a and 12c against PARP1 in vitro were evaluated in the BRCA1 mutated
triple negative breast cancer cell line MDA-MB-436 where 5c and 12c showed higher potency compared to
olaparib and result in cell cycle arrest at G2/M phase. 5c and 12c showed apoptotic effects in MDA-MB-436 and
potentiated the cytotoxicity of temozolomide in A549 human lung epithelial cancer cell line. Compounds 5c and
12c represent interesting starting points towards PARP1 inhibitors.
Barbituric acid
PARP1 inhibitors
Molecular docking
MDA-MB-436
1. Introduction
inhibition compromises SSB repair and BER, and in cells lacking intact
HR mechanisms (BRCA1/2 mutants), the SSD are then converted into
Poly (ADP-ribose) polymerases (PARPs) is a big family comprised of
18 members involved in a number of cellular processes including sur-
veillance of genome integrity, cell cycle checkpoints, DNA repair sig-
naling pathways, apoptosis, and transcription [1–4]. PARP1 is the most
abundant and well-characterized member among this family [1]. It is an
important component of DNA damage response (DDR) that work to
repair the DNA damage manifested as single strand DNA break (SSB) or
double strand DNA break (DSB) and thus maintaining the cellular
genomic stability [5]. More specifically, PARP1 is a cornerstone in the
base excision repair (BER) with potential involvement in nucleotide
excision repair (NER) mechanisms that provides repairment of SSB in
eukaryotic cells [3,6]. Simply, the impaired DNA activates PARP1 to
cleave its substrate nicotinamide adenine dinucleotide (NAD+) and
transfer ADP-ribose units to nuclear target proteins to recruit BER
components to facilitate DNA repair subsequently [4]. Thus, PARP in-
hibitors were suggested to enhance the cytotoxic effects of alkylating
agents or radiation [7]. Moreover, homologous recombination (HR) and
non-homologous end joining (NHEJ) are the two major mechanisms
that cope with DSBs where the breast cancer–associated genes BRCA1
and BRCA2 are known to play an important role in HR [8–11]. BRCA1/
2 deficient cells display impaired HR and high dependency on the ac-
tivity of PARP leading to subsequent genomic instability [12,13]. PARP
DSB, resulting in cell lethality [10,12–14]. Therefore, PARP inhibitors
lead to what is known as “synthetic lethality” whereby DNA repair
mechanisms are functionally shut down, causing cell death due to ac-
cumulation of genetic damage and PARP inhibitors were found to be
toxic particularly in BRCA1/2 mutated cancer cell lines and human
tumors [12–14]. In addition, BRCA1 mutation seems to be significantly
overlapping in patients with “triple negative” breast cancer (TNBC)
with deficiencies in the expression of estrogen receptor α (ER) and
progesterone receptor (PR), and the HER2 gene [15].
More than 50% of BRCA1 carriers have TNBC. TNBC account for
∼15% of the total diagnosed breast cancer cases with higher likelihood
of recurrence and death [15].
Since nicotinamide (I) and 3-aminobenzamide (II) were identified
as PARP1 inhibitors in the 1980s, great efforts have been devoted to
discovering many structurally diverse PARP1 inhibitors [4,14,16–18].
Olaparib (AZD-2281, III), rucaparib (AG014699, IV) and niraparib
(MK4827, V) have been approved by the FDA for the treatment of
ovarian cancer in patients with or without BRCA mutations during the
period of 2014–2017 and many other PARP1 inhibitors are being
evaluated in different phases of clinical trials [18–21]. All these in-
hibitors are NAD analogues featuring a benzamide structure with
common pharmacophoric features: carboxamide moiety and an
⁎ Corresponding author.
E-mail address: essam.osman@pharma.cu.edu.eg (E. Eldin A. Osman).
https://doi.org/10.1016/j.bioorg.2020.104198
Received 2 June 2020; Received in revised form 26 July 2020; Accepted 17 August 2020
Availableonline28August2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

E. Eldin A. Osman, et al.
BioorganicChemistry104(2020)104198
Fig. 1. Structure of select PARP1 inhibitors along with general pharmacophore for PARP1 inhibitors and the structure of the proposed compounds.
aromatic scaffold substituted with hydrophobic tail. This carboxamide
moiety was either embedded into a lactam ring as in olaparib (III) and
rucaparib (IV) or rotationally constrained by virtue of an in-
tramolecular hydrogen bond forming a “pseudoring” as in niraparib (V)
and veliparib (VI) (Fig. 1) [4]. Literature survey revealed various
scaffolds for promising PARP1 inhibitors comprised of these common
pharmacophoric features [4,18,22–27]. Based on several crystal struc-
tures and molecular modeling studies, the carboxamide functionality in
these inhibitors was engaged in a network of hydrogen bonds with
Gly863 and Ser904 in the nicotinamide binding subpocket [4]. Aro-
matic or unsaturated structures of the inhibitors form π stacking in-
teractions with Tyr907 and in some cases with Tyr896 [4]. The back
wall of the nicotinamide subpocket is lined by Ala898 and Lys903
forming a tight pocket capable of accommodating small substituents
(e.g., CH3, F, Cl) on the aromatic ring of the benzamide structures [4].
In some instances, Glu988 also formed a hydrogen bond with the in-
hibitors either directly or bridged through a conserved water molecule
[4]. Lastly, a large hydrophobic subpocket adjacent to the nicotinamide
binding often referred to as the adenine-ribose binding site is where
most of PARP1 inhibitors take advantage to improve potency, solubi-
lity, and other pharmaceutical properties [4]. Recently, the natural 2,5-
diketopiperazines (cyclic dipeptides) VII, VIII, lacking the common
aromatic moiety in NAD+ competitive PARP1 inhibitors, were shown
to inhibit the enzyme by virtue of the interaction with the key residues
of the PARP1 active site [28]. As demonstrated using docking and
molecular dynamics simulations one of the amide groups of cyclo(L-Ala-
2

E. Eldin A. Osman, et al.
BioorganicChemistry104(2020)104198
Scheme 1. Synthetic pathway for preparation of target compounds 5a-e, 6a-e and 7a-e.
L-Ala) VII and cyclo(L-Ala-D-Ala) VIII forms hydrogen bonds with
Gly863, while the second amide group can form a hydrogen bond with
Glu988, and one methyl group was having hydrophobic contact with
Ala898 (Fig. 1S) [28]. Despite their weak affinity, these diketopiper-
azine inhibitors represent a promising basic structure for the design of
more effective inhibitors of PARP family enzymes [28]. The non-se-
lective PARP1/2 tricyclic benzimidazole, BYK236864, IX is another
example of non-classical inhibitor lacking the lactam moiety and thus
the hydrogen bond donor capability prevalent in so many NAD+
competitive inhibitors [29]. Based on the previous findings, barbituric
acid moiety was postulated as a novel scaffold that could mimic the
diketopiperazines for potential PARP1 inhibition. The diketopiperazine
derivatives VII, VIII and barbituric acid were docked into the X-ray
crystal structure of the active site of PARP1 in complex with olaparib
(PDB: 5DS3) [30] to guide the design process (Fig. 1S-4S). Despite its
slightly lower binding score (S = −4.68894053) compared to the di-
molecular modeling studies and calculation of the major ionized species
at the physiological pH, grafting a phenolic group was predicted to
suppress the ionization of the barbituric acid moiety to its conjugate
base (51.9% vs 82.7%). 5-(4’-hydroxybenzylidene) barbituric acid
docked nicely to the PARP1 active site and showed a relatively different
binding mode of the phenyl ring where it was oriented away from the
adenine binding pocket with its phenoxide ion forming a hydrogen
bond with Met890 backbone NH with a slight decrease in the docking
score compared to the non-phenolic derivative (S = −5.48069048)
(Fig. 6S). 3’-methoxy substitution and auxiliary appendage with alky-
lamino methyl moieties were predicted to restore the binding mode
towards adenosine subpocket with proposed interactions with Leu877,
Arg878, Ile879, and Pro881 (Fig. 7S) providing the bases for the design
of barbituric acid derivatives 5a-e. Moreover, the design of other
compounds relied on structure diversification of 5a-e derivatives to
allow useful structure activity relationship information through firstly
isostere replacement of 2-oxo with sulfur atom in compounds 6a-e since
this may increase the lipophilicity of the compounds and afford better
physicochemical properties. Secondly, substitution of the amide hy-
drogens with methyl groups in 7a-e with the assumption that these
compounds would have higher lipophilicity and would allow to orient
the barbiturate moiety towards the nicotinamide binding pocket and
may participate in hydrophobic bonding interactions with Ala898.
Additionally, a longer tail attached to the aromatic core was introduced
in compounds barbituric acid derivatives 10a-d, thiobarbituric acid
derivatives 11a-d and 1,3-dimethylbarbituric acid derivatives 12a-d to
study the effects of these structural modifications on the biological
activity. All the target compounds were screened for their PARP1 in-
hibitory activity and for their antiproliferative activity in the NCI 60
cell line screen. Furthermore, compounds eliciting promising PARP1
inhibition were screened for their cytotoxicity against the BRCA1 mu-
tated triple negative breast cancer (TNBC) cell line MDA-MB-436. The
ketopiperazine
derivatives
(S = -5.32370043
for
VII
and
S = −5.35989285 for VIII), barbituric acid was forming the key hy-
drogen bonds with Gly863 and Ser904 and interestingly was able to
stack to Tyr907 by virtue of its relatively planner structure (Fig. 4S).
Next, we thought of extending the structure of barbituric acid at C5
which has reactive acidic hydrogen atoms (pKa = 4.01) [31]. Thus, 5-
benzylidene derivative of barbituric acid was expected to increase the
binding affinity through additional hydrophobic and π stacking inter-
actions with Tyr896. Docking of the 5-benzylidene barbituric acid
agreed with the previous hypothesis (S = −5.57461023) (Fig. 5S). An
interesting notion was that the energy minimized structure of 5-ben-
zylidene barbituric acid was predicted computationally to exist majorly
as the barbituric acid mono anion (82.7%) at physiological pH. This
mono anion would result in flipping of the compound in such a way that
the barbiturate moiety would be oriented towards the opposite side of
the binding pocket resulting in decreased inhibitory activity. Based on
3

E. Eldin A. Osman, et al.
BioorganicChemistry104(2020)104198
Scheme 2. The synthetic pathway for preparation of compounds 10a-d, 11a-d and 12a-d.
effects of the most active compounds on the cell cycle of MDA-MB-436
cell line was investigated using flow cytometry. Potentiation of anti-
proliferative activity of temozolomide by the most active compounds
was reported and molecular docking study was performed to shed light
on the binding features essential for their activity.
the 2CH3, NCH2 and OCH3 protons, respectively along with the ali-
phatic signals assigned to diethylamino, pyrrolidine-1-yl, piperidin-1-
yl, morpholin-4-yl and 1-methylpiperazin-4-yl protons. In addition, a
singlet signal appeared at 8.13–8.45 ppm assigned to methylidene
proton with the D2O exchangeable singlet signal at 9.70–12.70 ppm
attributed to OH protons.13C NMR of 7c exhibited signals at 23.4, 25.2,
25.5 and 53.5 ppm due to piperidine carbons along with CH2N carbon
at 61.1 ppm in addition to signals appeared at 56.0, 151.6, 159.1 and
164.7 ppm due to OCH3 group and three C=O. Furthermore, 13C NMR
of compound 7d displayed morpholine carbons at 52.7 and 66.5 ppm in
addition to signals at 28.4, 29.1, 56.1 and 61.0 ppm due to two CH3,
OCH3 and NCH2 carbons, respectively. Furthermore, acylation of the
appropriate secondary amine with chloroacetyl chloride was achieved
in dry DCM in the presence of triethylamine as HCl scavenger to obtain
compounds 8a-d as reported [34]. The reaction of the latter with p-
hydroxybenzaldehyde in dry acetonitrile in the presence of potassium
carbonate resulted in formation of 9a-d.[34]. Then, condensation of the
aldehyde derivatives 9a-d with the appropriate barbituric acid 4a-c in
ethanol afforded the target compounds 10a-d, 11a-d and 12a-d, re-
spectively (Scheme 2). The expected structures of compounds 10a-d
were confirmed by IR spectra that displayed characteristic vibration
bands in the range of 3221–3132 and 1743–1661 cm-1attributed to the
NH and C]O, respectively. 1H NMR spectra revealed a singlet signal of
the methylene protons (-O-CH2-C]O) at 4.87–5.00 ppm, in addition to
another aliphatic signals at 1.01–3.60 ppm due to the secondary amine
protons. Also, a singlet signal appeared at 8.19–8.23 ppm corre-
sponding to the methylidene protons(–C=CH) along with two D2O
exchangeable singlet signals assigned to the 2NH group appeared at
9.08–11.27 ppm. 13C NMR of compound 10d exhibited signals at 45.0
and 66.1 ppm due to morpholine carbons along with OCH2 carbon at
66.5 ppm in addition to the signals corresponding to the aromatic and
olefinic carbons at 114.6–156.0 ppm and 4C=O signals at 150.7, 162.6,
164.4 and 165.9 ppm. Additionally, IR spectra of 11a-d demonstrated
the appearance of the characteristic stretching vibration bands of the
amidic NH, C]O and C]S groups at 3190–3116, 1700–1661 and
1226–1215 cm−1, respectively. 1H NMR spectra of compounds 11a-d
revealed aliphatic signals assignable for diethylamino, pyrrolidinyl,
piperidinyl, morpholinyl protons at 1.05–3.61 ppm, together with a
singlet signal at 4.61–5.01 ppm due to OCH2 in addition to the ap-
pearance of aromatic doublet signals at 6.68–8.37 ppm corresponding
to the inserted phenyl moiety along with the singlet signal at
8.20–8.32 ppm assigned to the methylidene proton. In addition to the
appearance of D2O exchangeable singlet signals around
11.35–12.35 ppm corresponding to the two NH protons.13C NMR of
2. Results and discussion
2.1. Chemistry
The steps followed for the synthesis of the key intermediates and the
final compounds are summarized in Schemes 1 and 2. The functiona-
lized aldehydes 2a-e were prepared as reported by Mannich reaction of
vanillin 1 with different secondary amines and 37% formaldehyde [32].
Furthermore, the reaction of urea 3a or thiourea 3b with diethylma-
lonate in ethanol in the presence of sodium ethoxide afforded as re-
ported barbituric 4a or thiobarbituric acid 4b, respectively [33].
Knoevenagel condensation of aldehyde derivatives 2a-e on the active
methylene of 4a-c in ethanol resulted in formation of 5a-e, 6a-e and 7a-
e, respectively (Scheme 1). Microanalyses and spectral data confirmed
the structure of the synthesized compounds. The IR spectra of 5a-e and
6a-e showed the presence of OH, 2NH and C]O stretching band at
3422–3383, 3232–3182 and 1728–1670 cm−1, while in case of com-
pounds 6a-e, an additional band appeared at 1192–1180 due to C]S.
1H NMR charts showed characteristic signals at δ = 1.06–3.80 ppm
assignable for diethylamino, pyrrolidinyl, piperidinyl, morpholinyl,
piperazinyl protons. In addition, two singlets at 3.61–3.95 and
3.80–4.06 ppm attributed to NCH2 and the methoxy protons, respec-
tively along with singlet signals at δ = 7.72–9.48, 9.74–10.04,
11.40–11.47 and 11.53–11.58 ppm attributed to methylidene proton,
OH and 2NH protons, respectively. 13C NMR of compound 5a was also
consistent with the assigned structure where signals appeared at 11.8,
51.2 ppm assigned to ethyl carbons in addition to signals at 54.0,
56.2 ppm attributed to NCH2 and OCH3carbons, respectively along with
signals at 91.8–147.0, 151.2, 164.2 and 165.4 ppm due to aromatic,
olefinic carbons and 3carbonyl groups. Similarly, 13C NMR spectrum of
5d revealed signals at 52.6 and 65.7 ppm attributed to morpholine
carbons in addition to signals at 56.2 and 57.6 ppm due to NCH2 and
OCH3, respectively along with aromatic, olefinic carbons and three
C=O at 91.7–147.3, 150.8 and 164.5 ppm. Moreover, the postulated
structures of compounds 7a-e were proved by the appearance of OH
band and C]O in the IR spectra at 3441–3290 and 1724–1662 cm−1
,
respectively. 1H NMR spectra revealed the presence of three singlet
signals at 2.98–3.43, 3.80–4.06 and 3.89–4.37 ppm corresponding to
4

E. Eldin A. Osman, et al.
BioorganicChemistry104(2020)104198
compound 11a revealed signals at 14.2, 14.6 and 41.1, 41.3 ppm as-
signed to diethyl carbons along with signals at 66.7 ppm due to me-
thylene carbon. Furthermore, 13C NMR of compound 11c displayed
piperidine carbons at 24.1, 25.6, 26.2, 42.9 and 45.6 ppm in addition to
signal at 66.1 ppm due to CH2 carbon.13C NMR of 11d exhibited signals
at 41.0 and 61.5 due to morpholine carbons along with OCH2 carbon at
63.0 ppm. IR spectra of 12a-d revealed the presence of stretching C]O
most active compound among the barbituric acid derivatives 5a-e. The
order of activity was piperidino ˃ morpholino ˃ pyrrolidino ˃ diethy-
lamino ˃ methylpiperazino. It is worth to mention that 5c displayed the
highest activity among all the target compounds (IC50 = 30.51 nM) and
even higher than the reference drug olaparib (IC50 = 43.59 nM). The
thiobarbituric acid derivatives 6a-e displayed much lower potency
compared to their oxygen counterparts 5a-e. The diminished activity of
6a-e might be justified by the weaker hydrogen bonding capability of
the thioxo group (electronic and/or steric features) or the lower solu-
bility of this series. Only the methylpiperazino derivative 6e showed
appreciable activity among this series (IC50 = 72.37 nM) and the order
of activity was methylpiperazino ˃ piperidino ˃ diethylamino ˃ pyrro-
lidino ˃ morpholino. Generally, the 1,3-dimethylbarbituric acid deri-
vatives 7a-e exhibited the highest activity among all the pyrimidinone
compounds. Compounds 7b and 7d were nearly equipotent. Moreover,
compound 7e displayed the highest activity among this series
(IC50 = 36.33 nM). Concerning the 4-alkylaminoacetoxy derivatives
10a-d, 11a-d and 12a-d, the target compounds exhibited a wide range
of PARP1 inhibitory activity with IC50 in the range of 45–667 nM. Re-
garding the 5-benzylidenepyrimidinone derivatives 10a-d (barbituric
acid derivatives) and the 5-bezylidene-2-thioxodihydropyrimidinone
11a-d (thiobarbituric acid derivatives), a comparison between the two
series revealed that 11a-d displayed lower activity than 10a-d series.
Moreover, barbituric acid derivatives 10a-d showed less inhibitory
activity relative to the standard olaparib (IC50 = 43 nM). For the
compounds bearing 1,3-dimethylbarbituric moiety 12a-d, introduction
of two methyl groups on the barbituric acid ring increased the in-
hibitory activity. The order of activity was diethylamino ˃ piperidino ˃
morpholino ˃ pyrrolidino. Moreover, pyrrolidino 12b and morpholino
12d derivatives were nearly equipotent (IC 50 = 91.70 and 90.03 nM,
respectively). It is worth to mention that compound 12a showed higher
potency (IC50 = 45.40 nM) being nearly equivalent to 12c
around 1732–1661 cm−1
.
1H NMR showed the appearance of a sharp
singlet signal around 3.38–3.41 ppm corresponding to the protons of
two methyl groups together with an extra singlet signal at
4.72–4.79 ppm assigned for the methylene group in addition to signals
attributed to the aliphatic secondary amine protons at 1.13–3.74 ppm.
Moreover, three signals appeared in the aromatic region, two doublet
signals at 7.01–7.02 and 8.28–8.30 ppm assigned for aromatic protons
and one singlet signal at 8.49–8.50 ppm due to the methylidene pro-
ton.13C NMR spectrum of 12b revealed signals at 23.8, 25.9, 45.3 and
46.2 ppm attributed to pyrrolidine carbons in addition to signals at 28.3
and 66.2 ppm due to CH3 and CH2 carbons. Moreover, 13C NMR spec-
trum of compound 12c revealed signals at 24.3, 25.5, 26.4, 43.1 and
46.1 ppm assigned to piperidine carbons along with signal at 67.0 ppm
due to methylene carbon in addition to signals at 28.9 and 29.0 ppm
due to two methyl carbons.
2.2. Biological evaluation
2.2.1. PARP1 inhibitory activity and structure activity relationship
All the synthesized compounds were tested in PARP1 inhibitory
assay in vitro using olaparib as a reference standard. The PARP1 in-
hibitory activities as IC50 (nM) against PARP1 are presented in Table 1.
The target pyrimidinones 5a-e, 6a-e, 7a-e exhibited a wide range of
PARP1 inhibitory activity but all in the nanomolar range with IC50
values in the range of 30–265 nM. The piperidine derivative 5c was the
Table 1
In vitro inhibitory activity of the synthesized compounds against PARP1.a
Compd. ID.
5a
R
IC50 (nM)
SD.
4.62
1.41
8.71
1.73
6.03
Compd. ID.
7e
R
IC50 (nM)
SD.
204.35
36.33
0.92
7.23
5b
5c
5d
65.93
30.51
58.90
10a
10b
10c
244.05
197.30
111.62
6.28
2.84
5e
204.72
10d
98.28
2.71
6a
6b
6c
6d
102.26
229.61
96.73
1.57
6.85
11a
11b
11c
11d
131.27
310.08
667.56
136.77
4.13
8.41
17.1
3.85
2.44
6.59
1.82
265.90
6e
72.37
12a
45.40
1.61
7a
7b
7c
66.57
41.60
84.22
2.4
12b
12c
12d
91.70
50.62
90.03
3.25
13.08
4.36
1.26
2.1
7d
41.53
1.35
Olaparib
–
43.59
1.37
a
Values are means from three independent dose–response curves.
5

E. Eldin A. Osman, et al.
BioorganicChemistry104(2020)104198
Table 2
Table 3
The results of the target compounds in the NCI-60 human tumor cell lines
screen.
The antiproliferative activity (IC50, μM) of the tested compounds against MDA-
MB-436 cancer cell line.a
Compound
NSC Number
% Mean Growth
Delta
Range
5a
805279
805277
805274
805273
805280
805282
805278
805276
805275
805281
814336
814335
814333
814334
814337
821612
821611
821613
821614
821602
821601
821603
821604
821607
821606
821608
821609
103.66
105.95
106.01
106.61
104.68
106.66
105.06
102.96
105.40
106.54
100.45
100.66
99.84
17.94
19.52
17.07
22.05
21.74
16.52
18.03
42.18
18.71
21.24
25.37
73.23
24.39
23.12
20.28
15.47
24.50
16.57
20.13
24.63
18.16
15.58
17.46
15.72
26.77
17.34
18.55
28.65
42.37
32.29
38.62
35.61
31.44
32.47
60.10
29.83
43.14
40.92
86.50
40.71
34.12
33.54
26.42
51.59
33.54
44.19
37.62
29.85
28.22
31.22
28.93
39.35
35.67
36.00
5b
5c
5d
5e
6a
Compound ID
R
IC50 (μM)
SD
6b
6c
5c
7b
7d
3.65
13.2
10.4
0.1
6d
6e
0.3
0.3
7a
7b
7c
7d
101.29
102.32
105.52
104.07
105.24
104.49
100.16
98.75
7e
6.01
0.2
7e
12a
29.8
3.73
6.84
0.8
0.1
0.2
10a
10b
10c
10d
11a
11b
11c
11d
12a
12b
12c
12d
12c
Olaparib
–
a
Values are means from three independent dose–response curves.
100.24
100.71
105.47
99.28
against BRCA1 mutated cell line MDA-MB-436. This assay was per-
formed at VACSERA, Egypt and the results are summarized in Table 3.
MDA-MB-436 cell line is a triple negative breast cancer cell line that
had the 5396 + 1G > A mutation in the splice donor site of exon 20
with two identified transcript lengths [37]. The 5396 + 1G > A mu-
tation has been reported 46 times in the BIC mutation database and is
classified as pathogenic [37]. The in vitro antiproliferative effects of the
tested compounds against MDA-MB-436 cell line were variable despite
their nearly equal IC50 against PARP1 in vitro. Moreover, most of the
tested compounds showed moderate antiproliferative activities against
MDA-MB-436 cell line with IC50 values ranging from 3.65 to 29.8 μM.
Three compounds 5c, 7e and 12c exhibited enhanced antitumor ac-
tivities compared to the standard olaparib (IC50 = 6.84 μM). Com-
pounds 7b and 7d displayed moderate activity and compound 12a
showed weak activity. The most active compound was the barbituric
acid derivative 5c with IC50 = 3.65 μM and the 1,3-dimethylbarbituric
105.54
105.16
(IC50 = 50.62 nM). In summary, the most active compounds were 5c,
7b, 7d, 7e, 12a and 12c. Based on these findings, barbituric acid and
1,3-dimethylbarbituric acid could potentially serve as successful scaf-
folds for potential PARP1 inhibitors. Moreover, belonging of the active
compounds to different series suggested that a wide variety of mole-
cular fragments can be tolerated in the tail portion of the molecule.
2.2.2. NCI-60 human tumor cell lines screen
All the compounds were screened at the National Cancer Institute
(NCI), USA for their antiproliferative activity at a single dose of 10 µM
using sulforhodamine B assay (SRB). The results are shown in Table 2.
All the compounds showed no toxicity in the tested cell lines. This was
not surprising, since none of these cell lines harbor BRCA1/2 mutations
with the exception of non-deleterious BRCA1 mutation in the ovarian
cell lines IGROV-1 and OVCAR8 and four variants of BRCA2 identified
in three microsatellite unstable (MSI+) colon carcinoma cell lines of
the NCI-60 (HCT-116, HCT-15 and KM12) [35,36]. BRCA2 has not been
reported as a cancer gene (either a susceptibility gene or somatically
mutated) in colon cancer and somatic mutations of BRCA2 are generally
very rare [35]. The presence of heterozygous truncating variants of
BRCA2 in HCT-15, KM12, and HCT-116 was suggested to be due to the
microsatellite instability of the cell lines and do not contribute to tu-
morigenesis [35]. Olaparib (NSC: 747856 and 753686) showed almost
no growth inhibition at 10 µM in the NCI-60 human tumor cell lines
screen with an average GI50 of 46.1 µM against the whole panel of the
cell lines and GI50 of 100 µM against the BRCA2 mutated cell lines HCT-
116, HCT-15 and KM12. Furthermore, the activity profile of the tested
compounds in the NCI-60 human tumor cell lines screen is suggestive of
the lack of any nonspecific reactivity.
acid derivative 12c with IC50 = 3.73
0.1 μM. The variability in the
antiproliferative activity of the tested compounds may be attributed to
variable physicochemical properties and solubilities that would affect
membrane permeability.
2.2.4. Cell cycle analysis in MDA-MB-436 cells and apoptotic effects of 5c
and 12c
The role of PARP1 inhibitors in induction of apoptosis is well
documented [38–40]. Flow cytometry using Annexin V/PI double
staining was performed on MDA-MB-436 cells incubated with 5c or 12c
at two different concentrations: 1 µM and the IC50 concentration of each
compound for 48 h. Flow cytometric analysis showed that 5c and 12c
caused cell cycle arrest at G2/M phase with dose-dependent increases in
the G2/M cell population and a significant increase in cells with a pre-
G1 DNA content, suggestive of apoptosis, Fig. 2. This profile was con-
sistent with previous reports demonstrating the same trend for PARP1
inhibitors [40–42]. Compounds 5c and 12c showed a definite con-
centration dependent apoptotic effect where the late apoptotic events
were more significant as the concentration of the compound increased,
Fig. 3.
2.2.3. Antiproliferative activity against BRCA1 mutated cell line MDA-MB-
436
As some PARP inhibitors have been shown to display single agent
activity for tumors lacking BRCA1/2 dependent DNA double-stranded
repair mechanisms [8,10]. Therefore, compounds exhibited promising
PARP1 inhibition comparable to olaparib namely 5c, 7b, 7d, 7e, 12a
and 12c were investigated for their potential antiproliferative activity
2.2.5. Potentiation of temozolomide antiproliferative activity in A549
human lung cancer cells
Previous studies document the role of PARP1 inhibitors in sensi-
tizing BRCA competent cell lines to DNA damaging agents as radiation
and methylating agents [25,43,44]. Thus, compounds 5c and 12c were
6

E. Eldin A. Osman, et al.
BioorganicChemistry104(2020)104198
Fig. 2. 5c and 12c result in G2/M cell cycle arrest and increase Pre-G1 cell population in MDA-MB-436 cells. (A) Flow cytometric cell cycle analysis of MDA-MB-436
cells treated with 5c or 12c. (B) Quantification of the different cell cycle phases of MDA-MB-436 cells treated with 5c and 12c.
further tested for their potential to potentiate the antiproliferative ef-
fects of the DNA methylating agent temozolomide (TMZ) in a non BRCA
mutated cell line using MTT assay, Fig. 4. A549 lung epithelial cancer
cell line with competent DNA repair mechanism was selected based on
previous reports [44,45]. TMZ is an alkylating agent whose cytotoxicity
is based on methylation of N7 in guanine and formation of O6-me-
thylguanine in DNA that induces single strand DNA breaks [44]. A549
cells showed low sensitivity to either olaparib or TMZ when used as a
compared to TMZ alone (IC50 24.2
(0.5 µM) with TMZ resulted in higher IC50 for TMZ
(IC50 = 5.05 0.28 µM) compared to 12c. Finally, the barbituric acid
derivative 5c, potentiated the effect of TMZ in A549 cells
1.36 µM). Combining olaparib
(IC50 = 8.03
0.45 µM) but to a lesser extent compared to either
olaparib or 5c. These results demonstrated that compounds 5c and 12c
at 0.5 µM potentiated the antiproliferative effect of TMZ in A549 cell
line and highlight the potential of 12c for further in-depth analysis.
Fig. 4.
single agent (IC50 = 12.3
While the barbituric acid derivative 5c was even less effective
(IC50 = 33.2 1.86 µM), the 1,3-dimethylbarbituric acid derivative
0.69 and 24.2
1.36 µM, respectively.
2.3. Molecular docking study
12c was the most active against A549 cells (IC50 = 7.94
0.45 µM) as
a single agent. Combination of TMZ with 0.5 µM of 5c, 12c or olaparib
Initially, molecular docking of the target compounds was performed
at human PARP1 catalytic domain to support the design approach by
comparing the binding scores and patterns of the designed compounds
resulted in potentiating TMZ activity. TMZ potency when combined
with 0.5 µM 12c was about 7 times higher (IC50 = 3.64
0.2 µM)
Fig. 3. 5c and 12c induce apoptosis in MDA-MB-436 cells. (A) PI/Annexin double staining for detection of apoptosis and necrosis. (B) Summary of the apoptotic and
necrotic effects of 5c and 12c in MDA-MB-436 cells.
7

E. Eldin A. Osman, et al.
BioorganicChemistry104(2020)104198
docking we noticed that the nature of the secondary amine affects their
major ionized species predicted at physiological pH and this was ex-
pected to affect the physicochemical properties of these compounds and
consequently their binding interaction with PARP1 and their membrane
permeability and pharmacokinetic profile. Compounds 5a-e, 6a-e and
7a-e contain the phenol and secondary amine moieties that were pre-
dicted to exist majorly as zwitter ions where the secondary amines were
protonated, and the phenol was existing as a phenoxide ion. The bar-
bituric acid moiety in 5a-e were existing as a mixture of non-ionized
acid and the conjugate base nearly in the ratio of 2:1. For 6a-e, no
ionized forms were observed for the thiobarbituric acid given its weak
acidity compared to barbituric acid and for 7a-e as the acidic protons of
barbituric acid were replaced by methyl groups. For compounds 10a-d,
11a-d and 12a-d, the 3ry amides in the tail functionalities were not
protonated since the nitrogen atom in these molecules were involved in
resonance with the adjacent carbonyl group. Barbituric acid and thio-
barbituric acid were > 90% existing as non-ionized forms in 10a-d and
11a-d, respectively. Compounds 12a-d were exclusively existing in
non-ionized forms since they contain no ionizable acidic nor basic
groups. For the docking study, the major ionized form of each com-
pound was considered for performing the docking experiment. Docking
was performed using “Triangle Matcher” placement method, poses
were prioritized based on London dG scoring function, refinement of
the results was done using the default forcefield and the binding score
(S) and amino acids interactions were recorded. Generally, the target
compounds were binding the active site of PARP1 in accordance with
the expected pharmacophore guided binding mode and the top docking
poses of compounds of the most active compounds 5c, 7b, 7d, 7e, 12a
and 12c were analyzed in more detail. Barbituric acid derivatives 5a-e
were generally making the crucial hydrogen bonding interactions with
Gly863 and Ser904. Aromatic stacking with Tyr 907 was preserved in
Fig. 4. 5c and 12c potentiate the effect of DNA methylating agent temozolo-
mide (TMZ) in A549 lung epithelial cancer cells. IC50 values are means from
three independent experiments.
with that of the reference drug Olaparib using Molecular Operating
Environment (MOE 10.2015) software provided by Chemical
Computing Group, Canada [16]. Following the in vitro PARP1 inhibition
assay, the docking results were reinspected to correlate and justify the
biological activity. To ensure the accuracy of the docking protocol,
validation was performed by re-docking of the co-crystallized ligand
Olaparib into the PARP1 catalytic domain. The docking validation re-
sults showed perfect superimposition between the coordinates of Ola-
parib in the X-ray crystal structure and its self-docked pose with root
mean square deviation (RMSD) of 0.514 and docking score (S) of −8.1
Kcal mol−1 (Fig. 8S). During the preparation of the compounds for
Fig. 5. The top docking pose of barbituric acid derivative 5c (green) superimposed on olaparib (magenta) at the active site of PARP1 (PDB: 5DS3)
(S = −7.2477994). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
8

E. Eldin A. Osman, et al.
BioorganicChemistry104(2020)104198
all the top docking poses and the benzylidene stacking to Tyr 896 was
recorded in some poses. The benzylidene moiety and the amine part of
the tail functionality were oriented towards the adenosine binding
subpocket in all the docking poses. Hydrogen bonds to Arg878 were
sometimes observed in addition to other favourable hydrophobic in-
teractions with the side chains of amino acids Leu877, Ile879, Ala880
and Pro881. It is worthy to mention that the interaction of these amine
tails could be optimized by extending the structure through a longer tail
as in compounds 10a-d, 11a-d and 12a-d. The top docking pose of the
barbituric acid piperidine derivative 5c is shown in Fig. 5.
their binding interaction with PARP1. This may affect their membrane
permeability and consequently their cellular effects in MDA-MB-436
cancer cell line.
3. Conclusion
Based on molecular modeling, a series of 5-benzylidene barbituric
acid derivatives were designed and synthesized as potential non clas-
sical PARP1 inhibitors. The synthesized compounds were featuring
barbituric acid in 5a-e and 10a-d, thiobarbituric acid in 6a-e and 11a-d
or 1,3-dimethylbarbituric acid in 7a-e and 12a-d. The benzylidene
moieties were functionalized with 3-methoxy, 4-hydroxy and 5-alky-
laminomethyl groups in 5a-e, 6a-e and 7a-e or 4-alkylaminoacetoxy
groups in 10a-d, 11a-d and 12a-d. The synthesized compounds showed
variable inhibitory activity but all in the nanomolar range with IC50 in
the range of 30.5–667.5 nM against PARP1 in vitro compared to the
reference standard olaparib (IC50 = 43.6 nM). Barbituric derivatives
5a-e and 10a-d showed superior inhibitory activity compared to their
thioxo counterparts 6a-e and 11a-d emphasizing the importance of
hydrogen bonding capability of the carbonyl group in PARP1 inhibitors.
Only the piperidinomethyl derivative 5c (IC50 = 30.5 nM) was more
potent compared to olaparib. Interestingly, the 1,3-dimethylbarbituric
acid derivatives 7a-e were among the most active compounds possibly
due to the hydrophobic interaction of one of its methyl groups with
Ala898 at PARP1 active site. The pyrrolidinomethyl derivative 7b was
more potent and 7d and 7e were equipotent compared to olaparib. The
4-alkylaminoacetoxy derivatives 10a-d and 11a-d were less potent than
olaparib and their 3-methoxy, 4-hydroxy, 5-alkylaminomethyl coun-
terparts 5a-e and 6a-e. The 5-(4-alkylaminoacetoxy-benzylidene)-1,3-
dimethylbarbituric acid derivatives 12a and 12c were more or less
equipotent compared to olaparib. The nature of the amine tail within
the same series of compounds was predicted using molecular modeling
study to affect the ionization states of these derivatives but was not
correlated with the observed in vitro activity as well as their docking
scores. All the compounds showed no antiproliferative activity in the
NCI 60 cell line screening assay at 10 µM and that was in accordance
While most of the top docking poses of compounds 10a-d revealed
the placement of the barbituric acid moiety towards the NAD+ binding
subpocket, some poses were flipped where this moiety was oriented
close to the adenine binding subpocket and this may justify for their
lower binding affinities. The docking results agreed with the fact that
thiobarbituric acid derivatives 6a-e and 11a-d were showing lower
binding affinities to PARP1 in vitro. We correlate this to the weaker
hydrogen bonding capabilities of these derivatives, Fig. 6.
Interestingly, the dimethylbarbituric acid moieties in 7a-e and 12a-
d containing no hydrogen bond donor were having the highest score in
our docking study despite binding in a different manner compared to
their non methylated counterparts 5a-e and 10a-d. Taking compounds
7e, 12a and 12c as an example (Figs. 7–9), the steric hinderance eli-
cited by the methyl groups made the interaction of the carbonyl group
at C2 with Gly863 and/or Ser904 impossible. Alternatively, one of the
carbonyl groups at C4 or C6 were the donor of the recorded hydrogen
bond to Gly863 and/or Ser904. Interestingly, C2 carbonyl was engaged
in some dipole or hydrogen bond interaction with Lys903. Tyr907 was
still forming the stacking interaction with the dimethylbarbituric acid
and one of the methyl groups was found nicely fitting the tight hy-
drophobic pocket close to Ala898 while the other was facing the solvent
front near Glu988. No correlation between the nature of the amine
within the same series and the binding score was observed, suggesting
that the major contributing factor for the differences in the in vitro
binding assay was related to differences in physicochemical properties
of these compounds (ionization and/or solubilities) and consequently
Fig. 6. The top docking pose of the thiobarbi-
turic
acid
derivative
6d
(magenta)
(S = −6.07522392) superimposed on barbituric
acid derivative 5c (green) (S = −7.2477994) at
the active site of PARP1 (PDB: 5DS3). (For in-
terpretation of the references to color in this
figure legend, the reader is referred to the web
version of this article.)
9

E. Eldin A. Osman, et al.
BioorganicChemistry104(2020)104198
Fig. 7. . The top docking pose of the 1,3-dimethylbarbituric acid derivative 7e (green) (S = −7.75939894) superimposed on olaparib (magenta) at the active site of
PARP1 (PDB: 5DS3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
with the absence of the BRCA1/2 mutation in these cell lines. The most
active compounds 5c, 7b, 7d, 7e, 12a and 12c against PARP1 in vitro
were further evaluated in the BRCA1 mutated triple negative breast
cancer cell line MDA-MB-436 where 5c and 12c were twice more po-
tent compared to olaparib and 7e was slightly more potent. 5c and 12c
caused cell cycle arrest at G2/M phase in MDA-MB-436 cell line and
induced apoptosis. These compounds at 0.5 µM potentiated the anti-
proliferative effect of the DNA methylating agent temozolomide in
BRCA1 competent A549 lung epithelial cancer cell line. The different
cellular activity of the tested compounds in MDA-MB-436 cell line de-
spite their nearly PARP1 enzymatic inhibitory activity points out to the
effect of their physicochemical properties as ionization and solubility
on membrane permeability. Further studies will be conducted to opti-
mize these properties in a separate study.
1H NMR and 13C NMR were recorded at Micro-analytical Center,
Faculty of Pharmacy, Cairo University on Bruker spectrophotometer at
400 MHz using tetramethylsilane (TMS) as internal reference. Chemical
shift values (δ) are given in parts per million (ppm). Some spectra were
performed on nmr 400 mercury 400 spectrometer. TLC was performed
on TLC plates UV fluorescent silica gel (60 FZSU) and were visualized
using UV lamp. Compounds 2a-e [32], 4a,b [33], 8a-d [34] and 9a-d
[34] were prepared according to their reported procedures.
4.1.1. General procedure for preparation of 5a-e, 6a-e and 7a-e
A mixture of equimolar amounts of 2a-e and 4a-c (1 mmol) in ab-
solute ethanol (15 ml) was heated under reflux for 2 h. The formed
precipitate was filtered, dried and recrystallized from ethanol.
4.1.1.1. 5-(3-((Diethylamino)methyl)-4-hydroxy-5-methoxybenzylidene)
pyrimidine-2,4,6 (1H,3H,5H)-trione (5a). It was prepared from 2a
(0.24 g, 1 mmol) and 4a (0.13 g, 1 mmol) to give (0.25 g, yield: 70%);
m.p.˃300 °C; IR ν (cm−1): 3417 (OH), 3232 (2NH), 1685,1670 (3C]O);
1H NMR (400 MHz, DMSO‑d6, δ, ppm): 1.06 (t, J = 7.20 Hz, 3H, CH3 of
diethylamine), 1.19 (t, J = 7.20 Hz, 3H, CH3 of diethylamine), 2.60 (q,
J = 7.20 Hz, 2H, CH2 of diethylamine), 2.90 (q, J = 7.20 Hz, 2H, CH2
of diethylamine), 3.74 (s, 2H, NCH2), 3.80 (s, 3H, OCH3), 7.14 (s, 1H,
ArH), 7.31 (s, 1H, ArH), 9.48 (s, 1H, -C]CH–), 9.64 (s, 1H, OH, D2O
exchangeable), 13.46 (s, 2H, NH, D2O exchangeable); 13C NMR
(100 MHz, DMSO‑d6, δ, ppm): 11.8 (CH3 of diethylamine), 51.2 (CH2
of diethylamine), 54.0 (NCH2), 56.2 (OCH3), 91.8, 111.0, 120.8, 125.7,
130.4, 135.3, 143.3, 147.0 (Ar and olefinic Cs), 151.2, 164.2, 165.4
4. Experimental
4.1. Chemistry
Melting points were determined by open capillary tube method
using Electrothermal 9100 melting point apparatus MFB-595-010 M
(Gallen Kamp, London, England) and are uncorrected. Microanalyses
were carried out at The Regional Center for Mycology and
Biotechnology, Al-Azhar University. Infrared spectra were recorded at
Micro Analytical Center, Faculty of pharmacy, Cairo University as po-
tassium bromide discs on Schimadzu FT-IR 8400S spectrophotometer
(Shimadzu, Kyoto, Japan) and expressed in wave number υmax (cm−1).
10

E. Eldin A. Osman, et al.
BioorganicChemistry104(2020)104198
Fig. 8. The top docking pose of the 1,3-dimethylbarbituric acid derivative 12a (green) (S = −7.61911821) superimposed on olaparib (magenta) at the active site of
PARP1 (PDB: 5DS3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
(3C]O); Anal. calcd. for C17H21N3O5; C, 58.78; H, 6.09; N, 12.10;
pyrimidine-2,4,6(1H,3H,5H)-trione (5d). It was prepared from 2d (0.25
gm, 1 mmol) and 4a (0.13 g, 1 mmol) to give (0.29 g, yield: 81%);
m.p.˃300 °C; IR ν (cm−1): 3422 (OH), 3209 (2NH), 1739, 1674 (3C]
O); 1H NMR (400 MHz, DMSO‑d6, δ, ppm): 2.66 (brs, N(CH2)2
morpholine), 3.67 (brs, 4H, O(CH2)2 morpholine), 3.86 (s, 2H,
NCH2), 3.91 (s, 3H, OCH3), 7.80 (s, 1H, ArH), 8.16 (s, 1H, ArH), 8.46
(s, 1H, -C]CH–), 9.98 (s, 1H, OH, D2O exchangeable), 11.07 (br s, 1H,
Found; C, 59.06; H, 6.23; N, 12.36.
4.1.1.2. 5-(4-Hydroxy-3-methoxy-5-(pyrrolidin-1-ylmethyl)benzylidene)
pyrimidine-2,4,6 (1H,3H,5H)-trione (5b). It was prepared from 2b
(0.24 g, 1 mmol) and 4a (0.13 g, 1 mmol) to give (0.25 g, yield: 85%);
m.p.˃300 °C; IR ν (cm−1): 3421 (OH), 3194 (2NH), 1728, 1678 (3C]
O); 1H NMR (400 MHz, DMSO‑d6, δ, ppm): 1.82–1.91 (m, 4H, 2CH2
pyrrolidine), 3.16 (brs, 4H, N(CH2)2 pyrrolidine), 3.95 (s, 2H, NCH2)
4.06 (s, 3H, OCH3), 7.76 (s, 1H, ArH), 7.88 (s, 1H, ArH), 8.58 (s, 1H,
-C]CH–), 10.04 (s, 1H, OH, D2O exchangeable), 10.48 (s, 2H, NH, D2O
exchangeable); Anal. calcd. for C17H19N3O5; C, 59.12; H, 5.55; N,
12.17; Found; C,59.35; H,5.67; N,12.41.
NH, D2O exchangeable), 11.15 (br s, 1H, NH, D2O exchangeable); 13
C
NMR (100 MHz, DMSO‑d6, δ, ppm): 52.6 (N(CH2)2 morpholine), 56.0
(–OCH3), 57.6 (–CH2N), 65.7 (O(CH2)2 morpholine), 91.7, 112.6,
121.3, 122.6, 134.9, 135.8, 143.3, 147.3 (Ar and olefinic Cs), 150.8,
164.5 (3C]O); Anal. calcd. for C17H19N3O6; C, 56.51; H, 5.30; N,
11.63; Found; C, 56.79; H, 5.46; N, 11.80
4.1.1.3. 5-(4-Hydroxy-3-methoxy-5-(piperidin-1-ylmethyl)benzylidene)
pyrimidine-2,4,6(1H,3H,5H)-trione (5c). It was prepared from 2c
(0.25 g, 1 mmol) and 4a (0.13 g, 1 mmol) to give (0.30 g, yield: 83%);
m.p.˃300 °C; IR ν (cm−1): 3421 (OH), 3182 (2NH), 1725, 1716 (3C]
O); 1H NMR (400 MHz, DMSO‑d6, δ, ppm): 1.49–1.69 (m, 6H, 3CH2
piperidine), 2.96 (brs, 4H, N(CH2)2 piperidine), 3.64 (s, 2H, NCH2),
3.98 (s, 3H, OCH3), 7.75 (s, 1H, ArH), 7.93 (s, 1H, ArH), 8.55 (s, 1H,
-C]CH–), 10.02 (s, 1H, OH, D2O exchangeable), 10.59 (s, 2H, NH, D2O
exchangeable); 13C NMR (100 MHz, DMSO‑d6, δ, ppm): 22.2, 23.2, 25.3
(piperidine 3CH2), 52.2 (N(CH2)2 piperidine), 55.2 (–OCH3), 56.5
(–CH2N), 102.8, 115.7, 117.4, 119.9, 142.0, 143.9, 147.2, 149.9 (Ar
and olefinic Cs), 151.1, 153.2, 170.9 (3C]O); MS, m/z [%]: 359.06
[M+, 100]; Anal. calcd. for C18H21N3O5; C, 60.16; H, 5.89; N, 11.69;
Found; C, 60.38; H, 5.97; N, 11.88.
4.1.1.5. 5-(4-Hydroxy-3-methoxy-5-((4-methylpiperazin-1-yl)methyl)
benzylidene) pyrimidine-2,4,6 (1H,3H,5H)-trione (5e). It was prepared
from 2e (0.26 gm, 1 mmol) and 4a (0.13 g, 1 mmol) to give (0.21 g,
yield: 56%); m.p. 268–270 °C; IR ν (cm−1): 3422 (OH), 3197 (2NH),
1701, 1681 (3C]O); 1H NMR (400 MHz, DMSO‑d6, δ, ppm): 2.43 (s,
3H, NCH3), 3.32–3.38 (m, 8H, 2 N(CH2)2 methylpiperazine), 3.74 (s,
2H, NCH2), 3.83 (s, 3H, OCH3), 7.15 (s, 1H, ArH), 7.32 (s, 1H, ArH),
7.72 (s, 1H, -C]CH–), 9.74 (s, 1H, OH, D2O exchangeable), 10.04 (s,
2H, NH, D2O exchangeable); Anal. calcd. for C18H22N4O5; C, 57.75; H,
5.92; N, 14.96; Found; C, 58.01; H, 6.14; N, 14.75.
4.1.1.6. 5-(3-((Diethylamino)methyl)-4-hydroxy-5-methoxybenzylidene)-
2-thioxodihydropyrimidine −4,6(1H,5H)-dione (6a). It was prepared
from 2a (0.24 g, 1 mmol) and 4b (0.14 g, 1 mmol) to give (0.20 g,
yield: 56%); m.p. ˃ 300 °C; IR ν (cm−1): 3421 (OH), 3197 (2NH), 1678
(2C]O), 1184 (C]S); 1H NMR (400 MHz, DMSO‑d6, δ, ppm):
4.1.1.4. 5-(4-Hydroxy-3-methoxy-5-(morpholinomethyl)benzylidene)
11

E. Eldin A. Osman, et al.
BioorganicChemistry104(2020)104198
Fig. 9. The top docking pose of the 1,3-dimethylbarbituric acid derivative 12c (green) (S = −7.69142628) superimposed on olaparib (magenta) at the active site of
PARP1 (PDB: 5DS3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
1.16–1.25 (m, 6H, 2CH3 of diethylamine), 2.94–3.06 (m, 4H, 2CH2 of
diethylamine), 3.70 (s, 2H, NCH2), 4.05 (s, 3H, OCH3), 6.62 (s, 1H,
ArH), 7.83 (s, 1H, ArH), 8.51 (s, 1H, -C]CH–), 10.62 (s, 1H, OH, D2O
exchangeable), 11.45 (s, 1H, NH, D2O exchangeable), 11.58 (s, 1H, NH,
D2O exchangeable); MS, m/z [%]: 361.46 [M+, 8.48]; Anal. calcd. for
C17H21N3O4S; C, 56.18; H, 5.82; N, 11.56; Found; C, 56.36; H, 5.94; N,
11.80.
4.1.1.9. 5-(4-Hydroxy-3-methoxy-5-(morpholinomethyl)benzylidene)-2-
thioxodihydropyrimidine-4,6(1H,5H)-dione (6d). It was prepared from
2d (0.25 g, 1 mmol) and 4b (0.14 g, 1 mmol) to give (0.22 g, yield:
59%); m.p. ˃ 300 °C; IR ν (cm−1): 3402 (OH), 3209 (2NH), 1681 (2C]
O), 1192 (C]S); 1H NMR (400 MHz, DMSO‑d6, δ, ppm): 2.85 (brs, 4H,
N(CH2)2 morpholine), 3.73–3.80 (m, 6H, O(CH2)2morpholine and
NCH2), 3.83 (s, 3H, OCH3), 6.57 (s, 1H, ArH), 7.34 (s, 1H, ArH), 8.34
(s, 1H, -C]CH–), 10.87 (s, 1H, OH, D2O exchangeable), 11.40 (s, 1H,
NH, D2O exchangeable), 11.53 (s, 1H, NH, D2O exchangeable); Anal.
calcd. for C17H19N3O5S; C, 54.10; H, 5.07; N, 11.13; Found; C, 54.32; H,
5.21; N, 11.40.
4.1.1.7. 5-(4-Hydroxy-3-methoxy-5-(pyrrolidin-1-ylmethyl)benzylidene)-
2-thioxodihydropyrimidine −4,6(1H,5H)-dione (6b). It was prepared
from 2b (0.24 g, 1 mmol) and 4b (0.14 g, 1 mmol) to give (0.24 g,
yield: 67%); m.p.˃300 °C; IR ν (cm−1): 3402 (OH), 3186 (2NH), 1678
(2C]O), 1180 (C]S); 1H NMR (400 MHz, DMSO‑d6, δ, ppm): 1.84 (br
s, 4H, 2CH2 pyrrolidine), 3.02 (brs, 4H, N(CH2)2 pyrrolidine), 3.75 (s,
2H, NCH2), 3.99 (s, 3H, OCH3) , 6.60 (s, 1H, ArH), 7.12 (s, 1H, ArH),
8.43 (s, 1H, -C]CH–), 10.92 (s, 1H, OH, D2O exchangeable), 11.47 (s,
2H, NH, D2O exchangeable); Anal. calcd. for C17H19N3O4S; C, 56.50; H,
5.30; N, 11.63; Found; C, 56.82; H, 5.43; N, 11.76.
4.1.1.10. 5-(4-Hydroxy-3-methoxy-5-((4-methylpiperazin-1-yl)methyl)
benzylidene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (6e). It was
prepared from 2e (0.26 g, 1 mmol) and 4b (0.14 g, 1 mmol) to give
(0.24 g, yield: 62%); m.p. 275–277 °C; IR ν (cm−1): 3383 (OH), 3197
(2NH), 1670 (2C]O), 1180 (C]S); 1H NMR (400 MHz, DMSO‑d6, δ,
ppm): 2.43 (m, 3H, CH3), 2.83 (br s, 4H, N(CH2)2methylpiperazine),
3.42 (brs, 4H, N(CH2)2methylpiperazine), 3.61 (s, 2H, NCH2), 3.86 (s,
3H, OCH3), 6.49 (s, 1H, ArH), 7.36 (s, 1H, ArH), 7.81 (s, 1H, -C]CH–),
9.78(s, 1H, OH, D2O exchangeable), 11.43 (s, 1H, NH, D2O
exchangeable), 11.55 (s, 1H, NH, D2O exchangeable); Anal. calcd. for
4.1.1.8. 5-(4-Hydroxy-3-methoxy-5-(piperidin-1-ylmethyl)benzylidene)-2-
thioxodihydropyrimidine-4,6(1H,5H)-dione (6c). It was prepared from
2c (0.25 g, 1 mmol) and 4b (0.14 g, 1 mmol) to give (0.23 g, yield:
60%); m.p. ˃ 300 °C; IR ν (cm−1): 3394 (OH), 3197 (2NH), 1671 (2C]
O), 1180 (C]S); 1H NMR (400 MHz, DMSO‑d6, δ, ppm): 1.47–1.63 (m,
6H, 3CH2 piperidine), 3.04–3.13 (m, 4H, N(CH2)2 piperidine), 3.66 (s,
2H, NCH2), 4.00 (s, 3H, OCH3), 6.58 (s, 1H, ArH), 7.13 (s, 1H, ArH),
8.35 (s, 1H, -C]CH–), 10.88 (s, 1H, OH, D2O exchangeable), 11.41 (s,
1H, NH, D2O exchangeable), 11.56 (s, 1H, NH, D2O exchangeable);
Anal. calcd. for C18H21N3O4S; C, 57.58; H, 5.64; N, 11.19; Found; C,
57.81; H, 5.78; N, 10.95.
C
18H22N4O4S; C, 55.37; H, 5.68; N, 14.35; Found; C, 55.46; H, 5.89; N,
14.52.
4.1.1.11. 5-(3-((Diethylamino)methyl)-4-hydroxy-5-
methoxybenzylidene)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione
(7a). It was prepared from 2a (0.24 g, 1 mmol) and 4c (0.16 g, 1 mmol)
to give (0.29 g, yield: 76%), m.p. ˃ 300 °C; IR ν (cm−1): 3441 (OH),
3051 (CH aromatic), 2978 (CH aliphatic), 1701 (3C]O); 1H NMR
12

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BioorganicChemistry104(2020)104198
(400 MHz, CDCl3, δ, ppm): 1.42–1.58 (m, 6H, 2CH3 of diethylamine),
3.34 (s, 6H, 2CH3), 3.40–3.46 (m, 4H, 2CH2 of diethylamine), 4.06 (s,
2H, NCH2), 4.37 (s, 3H, OCH3), 7.25 (s, 1H, ArH), 7.74 (s, 1H, ArH),
8.14 (s, 1H, -C]CH–), 12.70 (s, 1H, OH, D2O exchangeable). Anal.
calcd. for C19H25N3O5; C, 60.79; H, 6.71; N, 11.19; Found: C, 60.96; H,
6.89; N, 11.08.
4.1.2.1. N,N-Diethyl-2-(4-((2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene) methyl) phenoxy) acetamide (10a). It was prepared from 4a
(0.13 g, 1 mmol) and 9a (0.24 g, 1 mmol) to give (0.20 g, yield: 59%);
m.p. ˃ 300 °C; IR ν (cm−1): 3132 (2NH), 3060 (CH aromatic),
2974–2935 (CH aliphatic), 1693 (4C]O); 1H NMR (400 MHz,
DMSO‑d6, δ, ppm): 1.01–1.21 (m, 6H, 2CH3 diethylamine), 3.12–3.34
(m, 4H, 2CH2 diethylamine), 4.93 (s, 2H, O-CH2-C]O), 7.06 (d,
J = 8.80 Hz, 2H, ArH), 7.83 (s, 1H, -C]CH–), 8.29 (d, J = 8.80 Hz,
2H, ArH), 9.84 (s, 1H, NH, D2O exchangeable), 9.90 (s, 1H, NH, D2O
exchangeable); Anal. calcd. for C17H19N3O5 C, 59.12; H, 5.55; N, 12.17;
Found; C, 59.36; H, 5.42; N, 12.38.
4.1.1.12. 5-(4-Hydroxy-3-methoxy-5-(pyrrolidin-1-ylmethyl)benzylidene)-
1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione (7b). It was prepared
from 2b (0.24 g, 1 mmol) and 4c (0.16 g, 1 mmol) to give (0.24 g,
yield: 64%); m.p.˃300 °C; IR ν (cm−1): 3390 (OH), 3062 (CH aromatic),
2962 (CH aliphatic), 1682 (3C]O); 1H NMR (400 MHz, CDCl3, δ, ppm):
4.1.2.2. 5-(4-(2-Oxo-2-(pyrrolidin-1-yl)ethoxy)benzylidene)pyrimidine-
2,4,6(1H,3H,5H)-trione (10b).. It was prepared from 4a (0.13 g,
1 mmol) and 9b (0.23 g, 1 mmol) to give (0.22 g, yield: 65%);
m.p.˃300 °C; IR ν (cm−1): 3194 (2NH), 3086 (CH aromatic), 2881
(CH aliphatic), 1720, 1690, 1680, 1666 (4C]O); 1H NMR (400 MHz,
DMSO‑d6, δ, ppm): 1.73–1.91 (m, 4H, 2CH2 pyrrolidine), 3.41–3.46 (m,
4H, N(CH2)2 pyrrolidine), 4.87 (s, 2H, OCH2-C]O), 6.86 (d,
J = 8.80 Hz, 2H, ArH), 8.19 (s, 1H, -C]CH–), 8.31 (d, J = 8.80 Hz,
2H, ArH), 11.09 (s, 1H, NH, D2O exchangeable), 11.22 (s, 1H, NH, D2O
exchangeable); 13C NMR (100 MHz, DMSO‑d6, δ, ppm): 23.8, 45.3, 46.2
(pyrrolidine-Cs), 64.8 (O-CH2-C]O), 115.9, 116.2, 128.8, 132.8,
138.7, 151.1 (Ar and olefinic Cs), 158.5, 163.4, 165.0 169.0 (4C]O);
Anal. calcd. for C17H17N3O5; C, 59.47; H, 4.99; N, 12.24; Found; C,
59.70; H, 4.78; N, 12.19.
1.75–1.82 (m, 4H, 2CH2 pyrrolidine), 2.63–2.80 (m, 4H,
N
(CH2)2pyrrolidine), 2.98 (s, 6H, 2CH3), 3.80 (s, 2H, NCH2), 3.89 (s,
3H, OCH3), 6.36 (s, 1H, ArH), 7.29 (s, 1H, ArH), 8.13 (s, 1H, -C]CH–),
9.70 (s, 1H, OH, D2O exchangeable); MS, m/z [%]: 375.26 [M+, 27.21];
Anal. calcd. for C19H23N3O5; C, 61.12; H, 6.21; N, 11.25; Found; C,
60.98; H, 6.43; N, 11.49.
4.1.1.13. 5-(4-Hydroxy-3-methoxy-5-(piperidin-1-ylmethyl)benzylidene)-
1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione (7c). It was prepared
from 2c (0.25 g, 1 mmol) and 4c (0.16 g, 1 mmol) to give (0.30 g,
yield: 76%); m.p. ˃ 300 °C; IR ν (cm−1): 3441 (OH), 3089 (CH
aromatic), 2954 (CH aliphatic), 1708 (3C]O); 1H NMR (400 MHz,
CDCl3, δ, ppm): 1.70–1.76 (m, 6H, 3CH2 piperidine), 2.68–2.73 (m, 4H,
N(CH2)2 piperidine), 3.40 (s, 6H, 2CH3), 3.90 (s, 2H, NCH2), 3.96 (s,
3H, OCH3), 7.74 (s, 1H, ArH), 8.27 (s, 1H, ArH), 8.41 (s, 1H, -C]CH–),
9.77 (s, 1H, OH, D2O exchangeable); 13C NMR (100 MHz, DMSO‑d6, δ,
ppm): 23.4, 25.2, 25.5 (3CH2 piperidine), 28.5 (2CH3), 53.5 (N(CH2)2
piperidine), 56.0 (–OCH3), 61.1 (–CH2N), 111.6, 117.5, 119.7, 122.8,
132.9, 148.0, 154.7, 158.1 (Ar and olefinic Cs), 151.6, 159.1, 164.7
(3C]O). Anal. calcd. for C20H25N3O5 ; C, 62.00; H, 6.50; N, 10.85;
Found; C, 61.83; H, 6.42; N, 10.69.
4.1.2.3. 5-(4-(2-Oxo-2-(piperidin-1-yl)ethoxy)benzylidene)pyrimidine-
2,4,6(1H,3H,5H)-trione (10c). It was prepared from 4a (0.13 g,
1 mmol) and 9c (0.25 g, 1 mmol) to give (0.28 g, yield: 78%); m.p. ˃
300 °C; IR ν (cm−1): 3221 (2NH), 3086 (CH aromatic), 2939 (CH
aliphatic), 1743, 1690, 1666 (4C]O); 1H NMR (400 MHz, DMSO‑d6, δ,
ppm): 1.12–1.55 (m, 6H, 3CH2 piperidine), 3.35–3.41 (m, 4H, N(CH2)2
piperidine), 4.96 (s, 2H, O-CH2-C]O), 6.99 (d, J = 8.80 Hz, 2H, ArH),
8.23 (s, 1H, -C]CH–), 8.31 (d, J = 8.80 Hz, 2H, ArH), 11.14 (s, 1H,
NH, D2O exchangeable), 11.27 (s, 1H, NH, D2O exchangeable); MS, m/z
[%]: 357.69 [M+, 46.08]; Anal. calcd. for C18H19N3O5; C, 60.50; H,
5.36; N, 11.76; Found; C, 60.73; H, 5.48; N, 11.95.
4.1.1.14. 5-(4-Hydroxy-3-methoxy-5-(morpholinomethyl)benzylidene)-
1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione (7d). It was prepared
from 2d (0.25 g, 1 mmol) and 4c (0.16 g, 1 mmol) to give (0.35 g,
yield: 89%); m.p. ˃ 300 °C; IR ν (cm−1): 3433 (OH), 3060 (CH
aromatic), 2978–2939 (CH aliphatic), 1724, 1662 (3C]O); 1H NMR
(400 MHz, CDCl3, δ, ppm): 2.68 (m, 4H, N(CH2)2 morpholine), 3.41 (s,
3H, CH3), 3.42 (s, 3H, CH3), 3.80 (br s, 4H, O(CH2)2morpholine), 3.87
(s, 2H, NCH2), 3.98 (s, 3H, OCH3), 7.70 (s, 1H, ArH), 8.29 (s, 1H, ArH),
8.43 (s, 1H, -C]CH–), 9.09 (s, 1H, OH, D2O exchangeable); 13C NMR
(100 MHz, DMSO‑d6, δ, ppm): 28.4, 29.1 (2CH3), 52.7 (N(CH2)2
morpholine), 56.1 (–OCH3), 61.0 (NCH2), 66.5 (O(CH2)2 morpholine),
113.4, 117.6, 119.9, 124.3, 131.9, 147.6, 154.1, 159.0 (Ar and olefinic
Cs), 151.5, 161.2, 163.4 (3C]O); Anal. calcd. for C19H23N3O6; C,
58.60; H, 5.95; N, 10.79; Found; C, 58.54; H, 6.17; N, 10.58.
4.1.2.4. 5-(4-(2-Morpholino-2-oxoethoxy)benzylidene)pyrimidine-
2,4,6(1H,3H,5H)-trione (10d). It was prepared from 4a (0.13 g,
1 mmol) and 9d (0.25 g, 1 mmol) to give (0.32 g, yield: 90%); m.p. ˃
300 °C; IR
ν
(cm−1): 3194, 3132 (2NH), 3062 (CH aromatic),
2966–2924 (CH aliphatic), 1739, 1697, 1661 (4C]O); 1H NMR
(400 MHz, DMSO‑d6, δ, ppm): 3.44 (brs, 4H, N(CH2)2 morpholine),
3.55–3.60 (m, 4H, O(CH2)2 morpholine), 5.00 (s, 2H, O-CH2-C]O),
7.01 (d, J = 9.20 Hz, 2H, ArH), 8.23 (s, 1H, -C]CH–), 8.31 (d,
J = 8.80 Hz, 2H, ArH), 11.14 (s, 1H, NH, D2O exchangeable), 11.27
(s, 1H, NH, D2O exchangeable); 13C NMR (100 MHz, DMSO‑d6, δ, ppm):
45.0 (N(CH2)2 morpholine), 66.1 (O(CH2)2 morpholine), 66.5 (O-CH2-
C]O), 114.6, 116.0, 125.8, 137.6, 138.8, 155.3, 156.0 (Ar and olefinic
Cs), 150.7, 162.6, 164.4, 165.9 (4C]O); Anal. calcd. for C17H17N3O6;
C, 56.82; H, 4.77; N, 11.69; Found; C, 56.88; H, 4.91; N, 11.78.
4.1.1.15. 5-(4-Hydroxy-3-methoxy-5-((4-methylpiperazin-1-yl)methyl)
benzylidene) −1,3-dimethyl pyrimidine-2,4,6(1H,3H,5H)-trione (7e). It
was prepared from 2e (0.26 g, 1 mmol) and 4c (0.16 g, 1 mmol) to give
(0.34 g, yield: 85%); m.p. ˃ 300 °C; IR ν (cm−1): 3290 (OH), 3001 (CH
aromatic), 2958–2939 (CH aliphatic), 1693, 1666 (3C]O); 1H NMR
(400 MHz, CDCl3, δ, ppm): 2.40 (s, 3H, CH3), 2.74 (brs, 4H, N(CH2)2
methylpiperazine), 3.33–3.34 (m, 4H, N(CH2)2 methylpiperazine), 3.41
(s, 3H, CH3), 3.43 (s, 3H, CH3), 3.86 (s, 2H, NCH2), 3.98 (s, 3H, OCH3),
7.69 (s, 1H, ArH), 8.30 (m, 1H, ArH), 8.45 (s, 1H, -C]CH–), 9.79 (s,
1H, OH, D2O exchangeable); Anal. calcd. for C20H26N4O6; C, 59.69; H,
6.51; N, 13.92; Found; C, 59.91; H, 6.43; N, 14.05.
4.1.2.5. 2-(4-((4,6-Dioxo-2-thioxotetrahydropyrimidin-5(2H)ylidene)
methyl) phenoxy)-N,N-diethylacetamide (11a). It was prepared from 4b
(0.14 g, 1 mmol) and 9a (0.24 g, 1 mmol) to give (0.26 g, yield: 71%);
m.p.˃300 °C; IR ν (cm−1): 3190 (2NH), 3097 (CH aromatic), 2978,
2939 (CH aliphatic), 1690, 1650 (3C]O), 1219 (C]S); 1H NMR
(400 MHz, DMSO‑d6, δ, ppm): 1.05–1.23 (m, 6H, 2CH3 diethylamine),
3.14–3.30 (m, 4H, 2CH2 diethylamine), 4.61 (s, 2H, O-CH2-C]O), 6.68
(d, J = 8.80 Hz, 2H, ArH), 6.84 (d, J = 8.80 Hz, 2H, ArH), 8.35 (s, 1H,
-C]CH-C-), 11.39 (s, 1H, NH, D2O exchangeable), 11.52 (s, 1H, NH,
D2O exchangeable); 13C NMR (100 MHz, DMSO‑d6, δ, ppm): 14.2, 14.6
(2CH3 diethylamine), 41.1, 41.3 (2CH2 diethylamine), 66.7 (O-CH2-C]
O), 96.4, 114.2, 127.8, 138.0, 156.1 (Ar and olefinic Cs), 164.0, 166.9
4.1.2. General procedure for preparation of 10a-d, 11a-d and 12a-d
A mixture of 4a-c and 9a-d (1 mmol) in absolute ethanol (15 ml)
was heated under reflux for 2hrs. The formed precipitate was filtered,
dried and recrystallized from ethanol.
13

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BioorganicChemistry104(2020)104198
(3C]O), 173.05 (C]S); Anal. calcd. for C17H19N3O4S; C, 56.50; H,
23.8, 25.9 (2CH2pyrrolidine), 28.3 (2CH3), 45.3, 46.2 (N(CH2)2
pyrrolidine), 66.2 (O-CH2-C]O), 110.0, 114.8, 115.5, 130.1, 132.3,
137.3 (Ar and olefinic Cs), 156.7, 163.5, 165.0, 166.0 (4C]O); Anal.
calcd. for C19H21N3O5; C, 61.45; H, 5.70; N, 11.31; Found; C, 61.38; H,
5.87; N, 11.43.
5.30; N, 11.63; Found; C, 56.39; H, 5.48; N, 11.76.
4.1.2.6. 5-(4-(2-Oxo-2-(pyrrolidin-1-yl)ethoxy)benzylidene)-2-
thioxodihydro- pyrimidine-4,6(1H,5H)-dione (11b). It was prepared
from 4b (0.14 g, 1 mmol) and 9b (0.23 g, 1 mmol) to give (0.27 g,
yield: 75%); m.p.˃300 °C; IR ν (cm−1): 3113 (2NH), 3062 (CH
aromatic), 2970–2927(CH aliphatic), 1697, 1651 (3C]O), 1215 (C]
S); 1H NMR (400 MHz, DMSO‑d6, δ, ppm): 1.79–1.92 (m, 4H, 2CH2
pyrrolidine), 3.30–3.46 (m, 4H, N(CH2)2 pyrrolidine), 4.89 (s, 2H, O-
CH2-C]O), 7.07 (d, J = 8.80 Hz, 2H, ArH), 8.25 (s, 1H, -C]CH–), 8.37
(d, J = 8.80 Hz, 2H, ArH), 12.26 (s, 1H, NH, D2O exchangeable), 12.35
(s, 1H, NH, D2O exchangeable); Anal. calcd. for C17H17N3O4S; C, 56.81;
H, 4.77; N, 11.69; Found; C, 57.05; H, 4.95; N, 11.48.
4.1.2.11. 1,3-Dimethyl-5-(4-(2-oxo-2-(piperidin-1-yl)ethoxy)benzylidene)
pyrimidine-2,4,6 (1H,3H,5H)-trione (12c). It was prepared from 4c
(0.16 g, 1 mmol) and 9c (0.25 g, 1 mmol) to give (0.32 g, yield: 82%);
m.p. ˃ 300 °C; IR ν (cm−1): 3066 (CH aromatic), 2939 (CH aliphatic),
1728, 1666 (4C]O); 1H NMR (400 MHz, CDCl3, δ, ppm): 1.57–1.66 (m,
6H, 3CH2 piperidine), 3.38 (s, 3H, N-CH3), 3.41 (s, 3H, N-CH3),
3.48–3.54 (m, 4H, N(CH2)2 piperidine), 4.78 (s, 2H, O-CH2-C]O),
7.02 (d, J = 8.80 Hz, 2H, ArH), 8.29 (d, J = 8.80 Hz, 2H, ArH), 8.50 (s,
1H, -C]CH–); 13C NMR (100 MHz, DMSO‑d6, δ , ppm): 24.3, 25.5, 26.4
(3CH2 piperidine), 28.9, 29.0 (2CH3), 43.1, 46.1 (N(CH2)2 piperidine),
67.0 (O-CH2-C]O), 114.6, 115.9, 124.2, 137.6, 138.7, 158.0, 158.8 (Ar
and olefinic Cs), 151.4, 160.8, 162.6, 164.9 (4C]O); Anal. calcd. for
4.1.2.7. 5-(4-(2-Oxo-2-(piperidin-1-yl)ethoxy)benzylidene)-2-
thioxodihydro- pyrimidine-4,6(1H,5H)-dione (11c). It was prepared from
4b (0.14 g, 1 mmol) and 9c (0.25 g, 1 mmol) to give (0.31 g, yield:
84%); m.p.˃300 °C; IR ν (cm−1): 3174 (2NH), 3009 (CH aromatic),
2939 (CH aliphatic), 1675 (3C]O), 1223 (C]S); 1H NMR (400 MHz,
DMSO‑d6, δ, ppm): 1.32–1.50 (m, 6H, 3CH2 piperidine), 3.25–3.31 (m,
4H, N(CH2)2 piperidine), 4.63 (s, 2H, O-CH2-C]O), 6.68 (d,
J = 8.80 Hz, 2H, ArH), 6.84 (d, J = 8.80 Hz, 2H, ArH), 8.20 (s, 1H,
-C]CH–) 11.39 (s, 1H, NH, D2O exchangeable), 11.52 (s, 1H, NH, D2O
exchangeable); 13C NMR (100 MHz, DMSO‑d6, δ, ppm): 24.1, 25.6, 26.2
(3CH2 piperidine), 42.9, 45.6 (N(CH2)2 piperidine), 66.1 (O-CH2-C]
O), 114.1, 115.5, 127.8, 130.1, 132.3, 151.1 (Ar and olefinic Cs), 163.5,
165.6, 166.6 (3C]O), 192.6 (C]S); MS, m/z [%]: 373.18 [M+, 2.40];
Anal. calcd. for C18H19N3O4S; C, 57.90; H, 5.13; N, 11.25; Found; C,
58.18; H, 5.32; N, 11.49.
C
20H23N3O5; C, 62.33; H, 6.02; N, 10.90; Found; C, 62.19; H, 6.29; N,
11.13.
4.1.2.12. 1,3-Dimethyl-5-(4-(2-morpholino-2-oxoethoxy)benzylidene)
pyrimidine-2,4,6(1H,3H,5H)-trione (12d). It was prepared from 4c
(0.16 g, 1 mmol) and 9d (0.25 g, 1 mmol) to give (0.36 g, yield: 92%);
m.p. ˃ 300 °C; IR ν (cm−1): 3066–3047 (CH aromatic), 2962 (CH
aliphatic), 1724, 1666 (4C]O); 1H NMR (400 MHz, CDCl3, δ, ppm):
3.39 (s, 3H, N-CH3), 3.41 (s, 3H, N-CH3), 3.58–3.60 (m, 4H, N(CH2)2
morpholine), 3.67–3.71 (m, 4H, O(CH2)2 morpholine), 4.79 (s, 2H, O-
CH2-C]O), 7.02 (d, J = 8.80 Hz, 2H, ArH), 8.28 (d, J = 8.80 Hz, 2H,
ArH), 8.50 (s, 1H, -C = CH–); MS, m/z [%]: 387.00 [M+, 45.65]; Anal.
calcd. for C19H21N3O6; C, 58.91; H, 5.46; N, 10.85; Found; C, 58.74; H,
5.41; N, 10.98.
4.1.2.8. 5-(4-(2-Morpholino-2-oxoethoxy)benzylidene)-2-
thioxodihydropyrimidine-4,6(1H,5H)-dione (11d). It was prepared from
4b (0.14 g, 1 mmol) and 9d (0.25 g, 1 mmol) to give (0.34 g, yield:
89%); m.p.˃300 °C; IR ν (cm−1): 3116 (2NH), 3074 (CH aromatic),
2970–2901 (CH aliphatic), 1700, 1661 (3C]O), 1226 (C]S); 1H NMR
(400 MHz, DMSO‑d6, δ, ppm): 3.43 (brs, 4H, N(CH2)2 morpholine),
3.55–3.61 (m, 4H, O(CH2)2 morpholine), 5.01 (s, 2H, O-CH2-C]O),
7.03 (d, J = 8.80 Hz, 2H, ArH), 8.24 (s, 1H, -C]CH-C-), 8.36 (d,
J = 8.80 Hz, 2H, ArH), 12.26 (s, 1H, NH, D2O exchangeable), 12.35 (s,
1H, NH, D2O exchangeable); 13C NMR (100 MHz, DMSO‑d6, δ, ppm):
41.0 (N(CH2)2morpholine), 61.5 (O(CH2)2morpholine), 63.0 (O-CH2-
C]O), 111.3, 111.9, 128.0, 128.5, 159.1 (Ar and olefinic Cs), 162.0,
166.6 (3C]O), 188.3 (C]S); Anal. calcd. for C17H17N3O5S; C, 54.39;
H, 4.56; N, 11.19; Found; C, 54.62; H, 4.70; N, 11.38.
4.2. Biological evaluation
4.2.1. PARP1 inhibitory activity assay
PARP1 enzyme inhibition activity was measured for the tested
compounds using
a colorimetric 96-well PARP1 assay kit (BPS
Bioscience) (catalog no. 80580), according to the manufacturer's pro-
tocol. Briefly, the histone mixture was diluted 1:5 with 1× PBS, 50 μl of
histone solution was added to each well and incubated at 4 °C over-
night. The plate was washed three times using 200 μl PBST buffer (1×
PBS containing 0.05% Tween-20) per well. Liquid was removed from
the wells by tapping the strip wells on clean paper towels. To each well,
200 μl of blocking buffer was added, followed by 60–90 min. incubation
at room temperature. Then 25 μl of PARP master mixture (consisting of
(2.5 μl 10× PARP buffer + 2.5 μl 10× PARP Assay mixture + 5 μl ac-
tivated DNA + 15 μl distilled water) was added to each well. AZD2281
(Olaparib) was used as a positive control. 5 μl of Inhibitor solution of
each well labeled as “Test Inhibitor” was added. For the “Positive
Control” and “Blank”, 5 μl of the same solution without inhibitor was
added. 1x PARP buffer was prepared by adding 1 part of 10x PARP
buffer to 9 parts H2O (v/v), 20 μl of 1x PARP buffer was added to the
wells designated as “Blank”. The amount of PARP1 required for the
assay was then calculated. The reaction was initiated by adding 20 μl of
diluted PARP1 enzyme to the wells designated “Positive Control” and
“Test Inhibitor Control”. The strip wells were incubated at room tem-
perature for 1 h. The strip wells were then washed three times with
200 μl PBST buffer. Then, 50 μl of 50 times diluted Streptavidin-HRP
with blocking buffer was added to each well, and the strips were further
incubated at room temperature for 30 min. After washing the wells
three times with 200 μl PBST buffer, HRP colorimetric substrate was
added to each well and the plate was incubated at the room tempera-
ture until a blue color is developed in the positive control well. Then
reaction was quenched with 100 ml/well of 2 M sulfuric acid, and ab-
sorbance at 450 nm was determined. Carrier solvents were assayed as
4.1.2.9. 2-(4-((1,3-Dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl) phenoxy)-N,N-diethylacetamide (12a). It was prepared
from 4c (0.16 g, 1 mmol) and 9a (0.24 g, 1 mmol) to give (0.26 g, yield:
71%); m.p.
˃
300 °C; IR
ν
(cm−1): 3082–3047 (CH aromatic),
2970–2935 (CH aliphatic), 1728, 1666 (4C]O); 1H NMR (400 MHz,
CDCl3, δ, ppm): 1.13–1.25 (m, 6H, 2CH3 of diethylamine), 3.31–3.42
(m, 10H, 2 N-CH3 + 2CH2 of diethylamine), 4.78 (s, 2H, O-CH2-C]O),
7.02 (d, J = 8.80 Hz, 2H, ArH), 8.30 (d, J = 8.80 Hz, 2H, ArH), 8.50 (s,
1H, -C]CH-C-); Anal. calcd. for C19H23N3O5; C, 61.12; H, 6.21; N,
11.25; Found; C, 61.40; H, 6.34; N, 11.49.
4.1.2.10. 1,3-Dimethyl-5-(4-(2-oxo-2-(pyrrolidin-1-yl)ethoxy)
benzylidene) pyrimidine-2,4,6 (1H,3H,5H)-trione (12b). It was prepared
from 4c (0.16 g, 1 mmol) and 9b (0.23 g, 1 mmol) to give (0.28 g, yield:
75%); m.p.˃300 °C; IR ν (cm−1): 3078 (CH aromatic), 2951 (CH
aliphatic), 1732, 1680, 1661 (4C]O); 1H NMR (400 MHz, CDCl3, δ,
ppm): 1.87–1.99 (m, 4H, 2CH2 pyrrolidine), 3.38 (s, 3H, N-CH3), 3.41
(s, 3H, N-CH3), 3.51–3.53 (m, 4H, N(CH2)2 pyrrolidine), 4.72 (s, 2H, O-
CH2-C]O), 7.01 (d, J = 8.80 Hz, 2H, ArH), 8.28 (d, J = 8.80 Hz, 2H,
ArH), 8.49 (s, 1H, -C]CH-C-); 13C NMR (100 MHz, DMSO‑d6, δ, ppm):
14

E. Eldin A. Osman, et al.
BioorganicChemistry104(2020)104198
negative controls. All assays were performed in triplicate. To determine
the IC50 value for each inhibitor, the average absorbance of each in-
hibitor concentration was plotted against the log of the concentration of
each respective inhibitor and the IC50 value for each plot was obtained
using computer-assisted non-linear regression analyses. Data presented
are the results of at least two independent experiments done in tripli-
cate.
4.3. Molecular docking study
The parameters of Amber10:EHT forcefield in MOE was selected for
potential setup. Amber10:EHT forcefield uses the following parameters:
AMBER parameters for proteins and nucleic acids (ff10), EHT para-
meters for small molecules, AM1-BCC charges are expected for small
molecules and Group II ion and Group VIII parameters from OPLS-AA.
The X-ray crystal structure of Olaparib in complex with PARP1 (PDB:
5DS3) was downloaded from Protein Data Bank available at http://
www.rcsb.org/pdb in PDB format [30].The protein–ligand complex was
prepared by removal of water of crystallization, two sulphate ions and
one polyethylene glycol (PEG) that were not involved in the binding.
Protonation of the crystal structure was performed, where hydrogen
atoms were added at their standard geometry, the partial charges were
computed, and the system was optimized using “QuickPrep” prepara-
tion protocol in MOE. Isolation of the active site and recognition of the
involved amino acids was carried out using “Site View” tool. The 2D
interaction diagram of the co-crystallized ligand (Olaparib) and PARP1
was isolated using “Ligand Interactions” tool in MOE. The compounds
were built using ChemBioDraw Ultra 19.0 and their SMILES were
copied to MOE. 3D Protonation of the compounds was carried out using
the precise mode in “Protonate” tool and the most prevalent ionized
form was selected for subsequent steps. Energy minimization of the
structures using 10:EHT forcefield and a gradient of 0.05 was applied
using “Energy Minimize” tool. The partial charges were automatically
calculated for each molecule. Conformational analysis was run using
the default settings for systematic search. The least energy conformer of
each molecule was saved to another data base to be docked into the
catalytic domain of PARP1. Docking of the least energy conformers was
performed by definition of the receptor atoms as (receptor) and site of
placement was defined as (ligand atoms). The database containing the
least energy conformers of the target compounds was used as the input
for the docking procedure. Placement method was adjusted to (triangle
matcher). Rescoring method was adjusted to (London dG). Finally, the
docking poses were examined for protein ligand-interactions and the
docking scores were recorded.
4.2.1.1. NCI-60 human tumor cell lines screen. All the compounds were
selected for NCI-60 Human Tumor Cell Lines Screen by the
Developmental Therapeutics Program (DTP) at the National Cancer
Institute (NCI), Maryland, USA. The compounds were supplied as dry
powder and a single concentration is tested in all 60 cell lines at a single
dose of 10 µM solution in dimethyl sulfoxide (DMSO) using
Sulforhodamine B assay. Briefly, cells are seeded in 96 well plates at
an appropriate density and incubated for 1 day. After 1 day, some of the
plates are processed to determine the density time zero. To the
remaining plates, compounds are added at 10 µM concentration.
Plates are incubated a further 2 days, then fixed and stained with
sulphorhodamine B. Growth inhibition is calculated relative to cells
without drug treatment and the time zero control. The use of a time
zero control allows the determination of cell kill as well as net growth
inhibition.
4.2.1.2. Antiproliferative activity against BRCA mutated cell line MDA-MB-
436. Antiproliferative activity of 5c, 7b, 7d, 7e, 12a and 12c in MDA-
MB-436 cell line was measured spectrophotometrically using in vitro
MTT based toxicology assay kit (catalog no. M-5655, M-8910) (Sigma
Aldrich), according to the manufacturer’s protocol. Briefly, cells were
platted (cells density 1.2 – 1.8 × 10,000 cells/well) in a volume of
100 µl complete growth medium + 100 μl of the tested compound per
well in a 96-well plate for 48 h before the MTT assay. The cultures were
removed from incubator into laminar flow hood then each vial of MTT
[M-5655] should be reconstituted with 3 ml of medium or balanced salt
solution without phenol red and serum. Reconstituted MTT was added
in an amount equal to 10% of the culture medium volume. Cultures
were returned to incubator for 2–4 h. After the incubation period,
cultures were removed from incubator and the resulting formazan
crystals were dissolved by adding an amount of MTT solubilization
solution [M-8910] equal to the original culture medium volume.
Dissolution is enhanced by gentle mixing in a gyratory shaker. Then
the absorbance is measured spectrophotometrically at a wavelength of
450 nm. The absorbance of multiwell plates was measured at 690 nm
and subtracted from the 450 nm measurement. To determine the IC50
value for each inhibitor, the average absorbance of each inhibitor
concentration was plotted against the log of the concentration of each
respective inhibitor and the IC50 value for each plot was obtained using
computer-assisted non-linear regression analyses. Data presented are
the results of at least two independent experiments done in triplicate.
The results of these studies are presented as mean IC50
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgement
The authors are thankful for the members of the Developmental
Therapeutics Program (DTP) at the National cancer Institute (NCI),
Maryland, USA for performing the NCI-60 Human Tumor Cell Lines
Screen.
Appendix A. Supplementary material
(μM)
standard deviation (SD).
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104198.
4.2.1.3. Cell cycle analysis in MDA-MB-436 cells and apoptotic
effects. Annexin V-FITC Apoptosis Detection Kit (BioVision) was used
to determine the effect of 5c and 12c on the cell cycle profile of MDA-
MB-436 according to the manufacturer procedure. BD FACSCalibur
flow cytometer was used for detection of the results at 48 h.
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