Design, synthesis, molecular docking and in silico ADMET profile of pyrano[2,3-d]pyrimidine derivatives as antimicrobial and anticancer agents

Article abstract of DOI:10.1016/j.bioorg.2021.105186

Pyrano[2,3-d]pyrimidine derivatives were synthesized by treating cyclic compounds containing active methylene group with aldehyde and malononitrile in butanol. The behavior of pyrano[2,3-d]pyrimidine towards some electrophlies namely triethylorthoformate followed by nitrogen nucleophiles as isobutylamine, urea, phenylthiourea, p-toluidine, o-phenylenediamine, o-aminophenol, 2-amino-4-methyl-pyridine and acetic acid with the aim of obtaining some interesting non-mixed heterocyclic compounds. All synthesized compounds to some extent have shown good antimicrobial activity against different microbial strains that had been extracted by inhibiting cell wall synthesis. Compound 5b showed the highest antibacterial activities against B. subtilis, S. aureus and E. coli. On the other hand compound 5 g exhibited the highest antibacterial and antifungal activities against P. aeruginosa and A. niger respectively. In addition, they explore cytotoxic potentialities against different cell lines via DNA intercalation and Top-II inhibition. The cytotoxic activities clarify the strong inhibitory activity of derivative 5a against HepG2 cells with IC₅₀ = 2.09 μM, while HCT-116 cells were highly susceptible to derivative 5c with IC₅₀ = 2.61 μM, in the meantime, derivative 5f showed pronounced negative impact against MCF-7 (IC₅₀ = 2.43 μM) when compared with other prepared compounds. All derivatives exhibited higher anticancer activities than doxorubicin against the three cell lines except compound 2 against both HepG2 and MCF-7 and compound 5e against HepG2 cell lines. Compounds 5a, 5c and 5f potently intercalate DNA at IC₅₀ values of 26.96, 27.13 and 29.86 μM respectively, which were more potent than doxorubicin (IC₅₀ value of 31.27 μM). Moreover, compounds 5a, 5c and 5f exhibited very good Topoisomerase II inhibitory activities with IC₅₀ values of 0.752, 0.791 and 0.776 μM respectively, that were more potent than that of doxorubicin (IC₅₀ = 0.94 μM). For a great extent, the molecular modeling studies were in agreement with that of in vitro cytotoxicity activity, DNA binding and Top-II inhibition results.

Full text of DOI:10.1016/j.bioorg.2021.105186

Bioorganic Chemistry 115 (2021) 105186
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis, molecular docking and in silico ADMET profile of pyrano
[2,3-d]pyrimidine derivatives as antimicrobial and anticancer agents
,
,c,
Nour E.A. Abd El-Sattara *, Khaled El-Adlb *, Maher A. El-Hashasha, Samir A. Salamad,
Mostafa M. Elhadye
a Department of Chemistry, Faculty of Science, Ain Shams University, Abbassia, Cairo, Egypt
b Pharmaceutical Medicinal Chemistry and Drug Design Department, Faculty of Pharmacy (Boys), Al-Azhar University, Nasr City 11884, Cairo, Egypt
c Pharmaceutical Chemistry Department, Faculty of Pharmacy, Heliopolis University for Sustainable Development, Cairo, Egypt
d Division of Biochemistry, Department of Pharmacology, College of Pharmacy, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
e Department of Biochemistry, Faculty of Science, Ain Shams University, Abbassia, Cairo, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
Pyrano[2,3-d]pyrimidine derivatives were synthesized by treating cyclic compounds containing active methy-
lene group with aldehyde and malononitrile in butanol. The behavior of pyrano[2,3-d]pyrimidine towards some
electrophlies namely triethylorthoformate followed by nitrogen nucleophiles as isobutylamine, urea, phenyl-
thiourea, p-toluidine, o-phenylenediamine, o-aminophenol, 2-amino-4-methyl-pyridine and acetic acid with the
aim of obtaining some interesting non-mixed heterocyclic compounds. All synthesized compounds to some extent
have shown good antimicrobial activity against different microbial strains that had been extracted by inhibiting
cell wall synthesis. Compound 5b showed the highest antibacterial activities against B. subtilis, S. aureus and
E. coli. On the other hand compound 5 g exhibited the highest antibacterial and antifungal activities against
P. aeruginosa and A. niger respectively. In addition, they explore cytotoxic potentialities against different cell
lines via DNA intercalation and Top-II inhibition. The cytotoxic activities clarify the strong inhibitory activity of
Pyranopyrimidine derivatives
Barbituric acid
Antimicrobial
Cytotoxic activities
DNA intercalators
derivative 5a against HepG2 cells with IC50 = 2.09
5c with IC50 = 2.61 M, in the meantime, derivative 5f showed pronounced negative impact against MCF-7 (IC50
= 2.43 M) when compared with other prepared compounds. All derivatives exhibited higher anticancer ac-
μM, while HCT-116 cells were highly susceptible to derivative
μ
μ
tivities than doxorubicin against the three cell lines except compound 2 against both HepG2 and MCF-7 and
compound 5e against HepG2 cell lines. Compounds 5a, 5c and 5f potently intercalate DNA at IC50 values of
26.96, 27.13 and 29.86 µM respectively, which were more potent than doxorubicin (IC50 value of 31.27 µM).
Moreover, compounds 5a, 5c and 5f exhibited very good Topoisomerase II inhibitory activities with IC50 values
of 0.752, 0.791 and 0.776 µM respectively, that were more potent than that of doxorubicin (IC50 = 0.94 µM). For
a great extent, the molecular modeling studies were in agreement with that of in vitro cytotoxicity activity, DNA
binding and Top-II inhibition results.
1. Introduction
diuretic, antiallergic [3], anticoagulant, anticancer, and anti-
anaphylactic activity, etc [4]. Therefore, large efforts have been directed
A variety of important natural products, such as carbohydrates, al-
kaloids [1], polyethers, antibiotics, pheromones, and iridoids [2] are
including pyrano[2,3-d]pyrimidine derivatives as ordinary structural
subunits.
towards the preparation of these derivatives. From this perspective the
present study aimed at preparation of novel derivatives that are still
needed in terms of exploring new antimicrobial and anticancer thera-
peutic benefits for solving unmet clinical needs.
These derivatives have received considerable attention over the past
years due to their wide range of biological activity such as spasmolytic,
In low and middle income countries, infectious diseases are one of
the leading causes of death worldwide, where second line antibacterial
* Corresponding authors at: Pharmaceutical Medicinal Chemistry and Drug Design Department, Faculty of Pharmacy (Boys), Al-Azhar University, Nasr City 11884,
Cairo, Egypt (K. El-Adl).
E-mail addresses: nourel-dinahmed@sci.asu.edu.eg (N.E.A. Abd El-Sattar), eladlkhaled74@yahoo.com, eladlkhaled74@azhar.edu.eg, khaled.eladl@hu.edu.eg
(K. El-Adl).
https://doi.org/10.1016/j.bioorg.2021.105186
Received 20 April 2021; Received in revised form 28 June 2021; Accepted 14 July 2021
Available online 22 July 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

N.E.A. Abd El-Sattar et al.
Bioorganic Chemistry 115 (2021) 105186
drugs against resistant bacteria are generally unavailable or unafford-
able. The emergence of multi-drug resistance in both community and
hospital-acquired infections in upper income countries, has outpaced
development and delivery of new drugs to the clinic [5].
scaffolds are of biological importance as antimicrobial [12,15–17]. The
rational of our pyrano[2,3-d]pyrimidines derivatives as antimicrobial
and anticancer agents was based on the reported research by Bhat et al
[18] that identified antibacterial and antitumor pharmacophore sites of
pyrano[2,3-d]pyrimidines on the basis of POM theory. Moreover, hy-
drolysis of the nitrile group leading to semi closed antibacterial phar-
macophore site. These findings encourage us to design the closed
antibacterial pharmacophore and also obtaining the planner poly-
aromatic system (chromophore) as DNA intercalator and Top II inhibitor
in the same structure.
There is a widespread concern that, without the introduction into
clinical practice of new antibiotics effective against multi-drug resistant
(MDR) pathogens, prevention and treatment of infectious diseases will
become significantly at risk. Where will new antibiotics come from? This
question, posed 8 years ago [6], still remains substantially unanswered.
The hard task in discovery of a new antibacterial agent is finding active
compounds against the desired range of pathogens, including resistant
strains, and effective at dosages compatible with human administration
in the experimental models of infection. Thus, only compounds that
have undergone extensive preclinical characterization can be consid-
ered as promising new antibiotics [7].
1.1.1. As antimicrobial agents
There are many reported Pyrano[2,3-d]pyrimidines as antimicrobial
agents as compounds I-IV [12,15–17] that represented in Fig. 2 . Our
designed derivatives and compounds I-IV share four common essential
structural features [18], i) a planar Pyrano[2,3-d]pyrimidine moiety
It is generally agreed that new classes of antibiotics, i.e. compounds
endowed with a novel mechanism of action were needed to best fight
MDR bacteria which unlikely be affected by existing resistance de-
terminants. Such classes of compounds do not necessarily have to be
new to science; rather their usage for human therapy (and possibly for
agriculture and veterinary medicine) should have been null or minimal.
However, modification of existing drugs still represents the preferred
approach and most compounds under clinical development are
improved versions of approved antibiotics [8].
(chromophore). ii) Aromatic ring at position-5 which para substituted
–
with methoxy group. iii) CN, C = NH and / or C O groups at position-6
–
iv) NH2, NHR and /or NR at position-7. Moreover, different distal hy-
drophobic and / or hydrophilic aliphatic and / or aromatic moieties
substituted with different hydrophobic and / or hydrophilic substituents
were designed in order to examine their effects on antimicrobial and
anticancer activities (Fig. 1).
The number of antibiotics has risen in parallel to the emergence of
resistant microorganisms over the past few decades. Lately, however,
the microorganisms are winning the race owing to the ever-increasing
number of new resistant strains. Due to different factors such as aging
population, immunocompromised patients, bacterial resistance, and
organ transplantation, the need for antibiotics and/or antibacterials will
increase in the future. However, research on the development of new
antibiotics has been decreasing [9].
1.1.2. As DNA intercalators
Anticancer drugs targeting Topo II inhibit the enzymatic activity as a
primary mode of action, and are known as catalytic Topo II inhibitors
[18]. Another type of Topo II targeting drugs that interfere with the
enzyme’s cleavage and rejoining activities by trapping the cleavable
complex and thus increasing the half-life of the transient Topo II cata-
lyzed DNA breaks. The latter type has the most clinically useful anti-
cancer drugs and are normally referred to Topo II poisons because they
convert the Topo II enzyme into a DNA-damaging agent [18]. DNA Topo
II poisons are molecules that intercalate between DNA base pairs. They
have attracted particular attention due to their promising antitumor
activities [19]. Many intercalative Topo II poisons are either used
already as anticancer drugs or still under clinical trials (e.g., doxorubicin
I [20], amsacrine II [21], mitoxantrone III [22] (Fig. 2).
Furthermore, there is a continuous and an urgent need to discover
new antitumor compounds with diverse chemical structures and novel
mechanisms of action due to an alarming increase in the incidence of
new and reemerging cancers and development of resistance to the drugs
in current clinical use and as a result, people have being paying more
attention to searching for new antitumor agents.
DNA interaction studies of small molecules are very important in the
development of new anticancer agents [10,11]. DNA is the primary
intracellular target for anticancer drugs as demonstrated in several
biological experiments; interaction between small molecules and DNA
can cause damage in cancer cells, blocking the division and resulting in
cell death [10,11]. Many heterocyclic derivatives exhibited their anti-
cancer activities by inhibiting DNA [12]. Hence, an additional attempt
was made to understand the possible molecular action mode of these
compounds investigating their interaction with DNA [12,13]. Consid-
ering the promising biological potencies of pyranopyrimidine pharma-
cophores, we designed the synthesis of the title compounds by
employing various substituents on the pharmacophores with a pre-
sumption that their incorporation will produce novel compounds.
There are three types of intercalators. Classical intercalators, inser-
ted their aromatic system between base pairs of DNA duplexes from the
top and bottom of the intercalation site [14]. Groove binding inter-
calators that, after the planar aromatic ring of the drugs is bound by
intercalation, have bulky substituents that must be in one groove. To
date, most evidence supports the location of the bulky groups in the
minor groove [14]. Threading intercalation is a special type of interac-
tion with DNA, in which two groove binding bulky side chains leads to
one sugar unit is oriented to the minor groove and the other to the major
groove [14].
DNA intercalators and Topo II poisons share three common essential
structural features, i) a planar polyaromatic system (chromophore),
binding with DNA [23,24]. ii) Cationic species which interact with the
negatively charged DNA sugar–phosphate backbone. The cationic spe-
cies are basic groups (ex: amino or nitrogen containing heterocyclic
groups) that can be protonated under physiological pH [25]. iii) Groove
binding side chain, which can occupy the minor groove of DNA [26–29]
(Fig. 2).
The highly potent cytotoxic derivatives were further evaluated for
their DNA binding and Topo II inhibition. Finally, a molecular docking
was carried out to examine the binding patterns with the prospective
target, DNA-Topo II complex (PDB-code: 4G0U).
2. Results and discussion
2.1. Chemistry
The required compound 1 was prepared in 95% yield via interaction
of 4-methoxybenzaldhyde with malononitrile and barbituric acid as
three component system in refluxed butanol for three hours. The solid
obtained was filtered and re-crystallized from butanol (Scheme 1). The
infrared spectrum exhibits strong absorption bands at 1691, 2223, 3182
–
–
–
and 3301 attributed to C O, C N and NH respectively. The IR data
–
–
were in agreement with the proposed structure of 7-amino-5-(4-
methoxyphenyl)-2,4-dioxo-2,3,4,5-tetrahydro-1H-pyrano[2,3-d]pyrim-
idine-6-carbonitrile 1 .
1.1. Rationale and structure-based design
The six membered nitrogen-containing heterocyclic compounds as
Interaction of 1 with maleic anhydride in boiling glacial acetic acid
afforded 4-((6-cyano-2,4-dihydroxy-5-(4-methoxyphenyl)-5H-pyrano
fused pyranes and pyrimidines forming Pyrano[2,3-d]pyrimidine
2

N.E.A. Abd El-Sattar et al.
Bioorganic Chemistry 115 (2021) 105186
Fig. 1. Some new and reported antimicrobial pyrano[2,3-d]pyrimidines and their basic pharmacophoric features.
Fig. 2. Some new and reported DNA intercalators and their basic pharmacophoric features.
3

N.E.A. Abd El-Sattar et al.
Bioorganic Chemistry 115 (2021) 105186
Scheme 1. Synthetic route for preparation of the target derivatives 1-4€.
[2,3-d]pyrimidin-7-yl)amino)-4-oxobut-2-en-oic acid . The infrared
imino of pyrimidine moiety which improve the formation of cyclic
compounds (Scheme 2).
spectrum of the product exhibits strong absorption bands at 1670 and
–
broad band at 3430 attributed to C O and acid OH respectively. This IR
Formation of compounds 5a-g can be explained according to the
following possible general pathway (Scheme 3).
–
pattern appears to be consistent with the assigned structure. On the
other hand, when enaminonitrile 1was submitted to react with boiling
glacial acetic acid, it yielded the acetyl derivative 3. Furthermore when
compound 1was allowed to react with triethylorthroformate , it afforded
4. The IR data was agreed with the proposed structure ethyl N-(6-cyano-
5-(4-methoxyphenyl)-2,4-dioxo-1,3,4,5-tetrahydro-2H-pyrano[2,3-d]
pyrimidin-7-yl)formimidate 4 (Scheme 1).
2.2. Biological activities
2.2.1. Antimicrobial activities
The screening strategy of some newly synthesized derivatives results
presented in Tables 1 and 2 lead to the selection of derivative 5b; that
having strong antagonistic properties against Bacillus subtilis which used
as test organism with MIC value = 0.097 µg/ml. The main reasons for the
inhibition of the treated bacterial cells may be due to the effect of the
selected compound on bacterial cell wall synthesis that resulting in
changes to cell shape and size, induction of cell stress responses and
ultimately cell lysis. This assumption was suggested by the observed
morphological changes that have been shown in the transmission elec-
tron micrograph of the treated cells when compared with control Bacillus
Compound 4 was used as a key start for synthesis of some hetero-
cyclic systems. Interaction of compound 4 with nitrogen nucleophiles
namely, isobutylamine, urea, phenyl thiourea and p-toluidine in boiling
butanol furnished compounds 5a-d respectively. Reaction of compound
4 with binucleophiles namely, o-phenlenediamine, o-aminophenol and
2-amino-4-methylpyridine in boiling butanol yielded 5e-g. It has been
observed that in compounds prepared 5a-g do not have CN in their IR
spectra which indicate that we have cyclic compounds and we have
4

N.E.A. Abd El-Sattar et al.
Bioorganic Chemistry 115 (2021) 105186
Scheme 2. Synthetic route for preparation of the target derivatives 5a-g.
subtilis cells (Fig. 3).
[33–35]. Doxorubicin was included in the experiments as reference
cytotoxic drug. The cytotoxic activities were shown in Table 3.; that
clarify the strong inhibitory activity of derivative 5a against HepG2 cells
This postulate was confirmed by the results of the quantification of
N-acetyl glucosamine (NAG) where the results presented in Fig. 4 indi-
cated the accumulation of NAG in the cytoplasm of the treated cells and
the higher concentration of the compound, the more pronounced effect
was observed at different periods in the treatment. The observed effect
of the targeted compound on NAG accumulation is in agreement with
Bajpai et al [30] who reported that the polymerization of NAG moieties
into peptidoglycan is considered as a target for antimicrobial agents.
Also, Yang et al [31] reported that, inhibition of peptidoglycan biosyn-
thesis enzymes result in disruption of the structural integrity of the cell
wall which leads to bacterial death. The same findings were reported in
the literature by Shine et al [32].
with IC50 = 2.09
μM, while HCT-116 cells were highly susceptible to
derivative 5c with IC50 = 2.61
μ
M, in the meantime, derivative 5f shows
pronounced negative impact against MCF-7 (IC50 = 2.43
μ
M) when
compared with other prepared compounds. All derivatives exhibited
higher anticancer activities than doxorubicin against the three cell lines
except compound 2 against both HepG2 and MCF-7 and compound 5e
against HepG2 cell lines.
3. Selectivity index (SI)
In the meantime, Fig. 5 showed the amino acids profiling that indi-
cate an increase in the content of glycine, alanine and lysine amino acids
in the metabolism solution of derivative 5b treated Bacillus subtilis when
compared with their concentrations in the broth of untreated cells.
These results illustrated the action of the selected compound as inhibitor
of the amino acids incorporation into the cell wall in the process of
peptidoglycan synthesis.
The most potent five derivatives 1, 4, 5a, 5c and 5f were tested for
their cytotoxicity against non-cancer VERO cell lines. The results
revealed that the tested compounds have low toxicity against VERO
normal cells with IC50 values ranging from 25.12 to 36.32 μM. The
cytotoxicity of the tested compounds against the cancer cell lines was
from 2.09 to 5.94 M. One criterion for the anticancer drug to be good is
μ
that it should not affect the non-cancer cells. A molecule could consider
as highly selective if it presented SI value ≥ 5. A molecule with moderate
selectivity presents SI value > 2 while low selectivity is considered if the
SI is lower than 2 [36]. In this study, the selectivity index was calculated
for the best effective cytotoxic derivatives 1, 4, 5a, 5c and 5f by assessing
its cytotoxicity against the non-cancer VERO cells and dividing the ob-
tained IC50 value by the IC50 value of the same compound against cancer
cell lines. Compounds 1, 4, 5a, 5c and 5f are respectively, 6.80, 7.61,
2.2.2. In vitro cytotoxic activities
Anti-proliferative activities of the newly synthesized derivatives
were examined against three human tumor cell lines namely, hepato-
cellular carcinoma (HepG2), colorectal carcinoma (HCT-116) and breast
cancer (MCF-7) using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazo-
lium bromide (MTT) colorimetric assay as described by Mosmann
5

N.E.A. Abd El-Sattar et al.
Bioorganic Chemistry 115 (2021) 105186
Scheme 3. The proposed reaction mechanism for preparation of the target derivatives 5a-g.
3.0.2. In vitro Topoisomerase II inhibitory activity
12.02, 7.54 and 5.19 fold times more toxic in HePG2 cancer cell lines
than in VERO normal cells. Also, compounds 1, 4, 5a, 5c and 5f are
respectively, 10.64, 7.82, 9.17, 13.92 and 8.62 fold times more toxic in
HCT-116 cancer cell lines than in VERO normal cells. Moreover, com-
pounds 1, 4, 5a, 5c and 5f are respectively, 11.09, 4.78, 9.93, 8.77 and
12.35 fold times more toxic in breast cancer cell lines (MCF-7) than in
VERO normal cells. All derivatives exhibited high selectivity against the
tested cancer cell lines where SI values > 5 except compound 4 which
exhibited moderate selectivity against MCF-7 cell lines (SI value = 4.78).
Surveying HepG2 treated cells with scanning electron microscopy
(SEM) revealed distinct morphological alteration when exposed to de-
rivative 5a where the cytoplasmic contents were not recognizable with
changes in cell shape and volume, followed by bleeding of external
surface which are typical characteristics of apoptosis that induced by the
selected compound (Fig. 6).
The most active cytotoxic compounds 5a, 5c and 5f were also
selected to evaluate their inhibitory activities against Topo II enzyme.
Topo II catalytic activity was tested according to the reported procedure
[37,38]. Doxorubicin was also tested using the same procedure as a
positive control (Table 4).
As shown in Table 4, compounds 5a, 5c and 5f exhibited very good
Topoisomerase II inhibitory activities with IC50 values of 0.752 ± 0.07,
0.791 ± 0.07 and 0.776 ± 0.07 µM respectively, that were more potent
than that of doxorubicin (IC50 = 0.94 ± 0.4 µM). For a great extent, the
reported results were in agreement with that of in vitro cytotoxicity ac-
tivity, DNA binding and molecular modeling studies.
3.0.3. Figure/band pattern of DNA binding and Top-II inhibition activities
3.0.1. In vitro DNA/Methyl green assay
The DNA binding activity of the studied compounds were plotted
against their corresponding Top II inhibition in simple linear regression
relationship, the obtained coefficient of R square approves a very strong
correlation between the DNA binding activity and Top-II inhibitory ac-
tivity of the tested compounds (R2 = 0.7854) (Fig. 7)
The most active anti-proliferative derivatives 5a, 5c and 5f were
further evaluated for their DNA-binding affinity which revealed the
ability of these compounds to intercalate DNA. It was carried out by use
of methyl green dye according to the reported procedure described by
Burre et al [37,38]. The results of DNA-binding affinity were obtained as
IC50 values and summarized in Table 4. Doxorubicin, as one of the most
powerful DNA intercalators, was used as a positive control.
3.1. Docking studies as DNA intercalators
Molecular docking investigational study was performed using Mol-
soft software for the synthesized compounds. Amsacrine and doxoru-
bicin were included as references DNA intercalators and Topo II
inhibitors (DNA-Topo II poisons). This study was carried out in order to
gain further insight into the binding modes of the synthesized com-
pounds into the DNA binding site of Topo II (PDB ID: 4G0U) [39]. The
binding free energies (ΔG) were presented in Table 5. All studied ligands
have similar position and orientation inside the DNA binding site of
The tested compounds displayed very good DNA-binding affinities.
Compounds 5a, 5c and 5f potently intercalate DNA at IC50 values of
26.96 ± 1.7, 27.13 ± 1.7 and 29.86 ± 1.7 µM respectively, which were
more potent than doxorubicin (IC50 value of 31.27 ± 1.8).
6

N.E.A. Abd El-Sattar et al.
Bioorganic Chemistry 115 (2021) 105186
Table 1
In Vitro antimicrobial potentialities of the synthesized compounds against different test organisms.
Compound
B. subtilis
S. aureus
E. coli
P. aeruginosa
A. niger
C. albicans
ATCC 6633
ATCC 25,923
ATCC 25,922
ATCC 27,853
ATCC 6275
ATCC 10,231
1
17.8 ± 0.25
(++)
17.4 ± 0.81
(++)
15.4 ± 0.81
Nil
10.2 ± 0.62
Nil
(++)
(+)
2
18.0 ± 0.15
(++)
17.1 ± 0.56
(++)
17.2 ± 0.42
(++)
13.3 ± 0.36
(+)
11.8 ± 0.59
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
(+)
3
19.3 ± 0.2
(+)
17.3 ± 0.25
(++)
15.5 ± 0.19
(++)
13.8 ± 0.23
(+)
Nil
4
20.1 ± 0.17
(++)
12.9 ± 0.621
(+)
15.0 ± 0.62
(++)
Nil
Nil
5a
26.0 ± 0.32
(+++)
20.8 ± 0.53
(++)
16.6 ± 0.17
(++)
14.6 ± 0.25
(+)
13.2 ± 0.25
(+)
5b
26.0 ± 0.32
(+++)
21.7 ± 0.77
(++)
18.0 ± 0.46
(++)
10.9 ± 0.72
(+)
13.8 ± 0.32
(+)
5c
15.8 ± 0.12
(+)
13.4 ± 0.42
(+)
13.9 ± 0.25
(+)
11.2 ± 0.34
(+)
12.1 ± 0.17
(+)
5d
16.2 ± 0.19
(+)
15.6 ± 0.67
(+)
17.2 ± 0.44
(++)
Nil
Nil
5e
20.2 ± 0.38
(++)
19.6 ± 0.65
(++)
18.1 ± 0.33
(++)
15.8 ± 0.43
(++)
11.4 ± 0.52
(++)
5f
17.9 ± 0.12
(++)
14.3 ± 0.25
(+)
12.8 ± 0.8
(+)
Nil
Nil
5 g
17.9 ± 0.73
(++)
17.4 ± 0.82
(++)
16.9 ± 0.53
(++)
17.3 ± 0.91
(++)
17.2 ± 0.56
(++)
Ampicillin
Gentamicin
Amphotericin B
32.4 ± 0.58
(+++)
26.1 ± 0.77
(+++)
Nil
Nil
Nil
Nil
Nil
21.3 ± 0.58
(+++)
Nil
17.3 ± 0.63
(++)
Nil
Nil
Nil
Nil
26.3 ± 0.3
(+++)
23.9 ± 0.1
(+++)
Gram-positive bacteria: (+++): Highly active = inhibition zone diameter > 25 (103 µm); (++): Moderately active = inhibition zone diameter = 17–25 (103 µm); (+):
Slightly active = inhibition zone diameter = 9–17 (103 µm). Gram-negative bacteria: (+++): Highly active = inhibition zone diameter > 18 (103 µm); (++):
Moderately active = inhibition zone diameter = 15–18 (103 µm); (+): Slightly active = inhibition zone diameter 12–15 (103 µm). Fungi: (+++): Highly active =
inhibition zone diameter > 20 (103 µm); (++): Moderately active = inhibition zone diameter = 15–20 (103 µm); (+): Slightly active = inhibition zone diameter =
10–15 (103 µm).
Table 2
Minimum inhibitory concentration (expressed as μg/ml) of the synthesized compounds against different test organisms.
Compound
B. subtilis
S. aureus
E. coli
P. aeruginosa
A. niger
C. albicans
ATCC 6633
ATCC 25,923
ATCC 25,922
ATCC 27,853
ATCC 6275
ATCC 10,231
1
1.562
0.781
0.781
0.781
0.097
0.097
3.125
3.125
0.390
1.562
1.562
0.45
3.125
6.250
3.125
25
6.250
1.562
3.125
6.250
3.125
0.781
12.50
1.562
0.781
6.250
1.562
Nil
> 200
12.50
12.50
> 200
12.50
25
100
50
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
Nil
2
3
> 200
> 200
50
4
5a
1.562
0.781
12.50
6.250
1.562
12.50
3.125
8.62
5b
50
5c
25
50
5d
> 200
6.250
> 200
3.125
Nil
> 200
50
5e
5f
> 200
25
5 g
Ampicillin
Gentamicin
Amphotericin B
Nil
Nil
Nil
3.68
15.36
Nil
Nil
Nil
Nil
Nil
Nil
0.781
3.125
DNA-Topo II (Fig. 8).
H-bond with Asparagine786. The N-(3-methoxyphenyl)-meth-
anesulfonamide side chain was oriented into minor groove of DNA and
stabilized by formation of seven H-bonds with the key amino acids (5H-
bonds with Arginine945, one H-bond with Glycine793 and one H-bond
with Serine794) (Fig. 9).
The most favorable pose was selected according to the minimum free
energy of the DNA–ligand complex for analyzing the interaction be-
tween the ligand and the DNA.
The proposed binding mode of the co-crystallized ligand amsacrine
revealed affinity value of ꢀ 101.73 kcal/mol and eight H-bonds. The
planar aromatic system occupied the same hydrophobic pocket formed
by Phenyalanine834, Phenyalanine738, Glutamine742, Leucine799,
Asparagine798, Arginine1025, Leucine787, Asparagine786, Argi-
nine945, Asparagine867, Asparagine795, Glycine868, Alanine869,
Glycine737 and Leucine799. It also stacked between the diphosphate
Cytidine (Dc3, Dc4 and Dc8), diphosphate Guanosine (Dg5 Dg10 and
Dg13) and Adinine monophosphate 6 (Da6). Its amino group formed one
The proposed binding mode of doxorubicin revealed affinity value
of ꢀ 100.31 kcal/mol and ten H-bonds. The planar aromatic system
occupied the hydrophobic pocket formed by Arginine945, Aspara-
gine882, Lucine880, Glutamate870, Alanine869, Glycine868, Aspara-
gine867,
Asparagine795,
Glutamine789,
Asparagine786,
Glutamine742, Lysine739, Phenyalanine738 and Glycine737. It formed
five H-bonds with the key amino acids in the active site (one H-bond
with Asparagine786, two H-bonds with Asparagine795, one H bond
7

N.E.A. Abd El-Sattar et al.
Bioorganic Chemistry 115 (2021) 105186
Fig. 3. Transmission electron micrographs of Bacillus subtilis cells. a) untreated and b) derivative 5b treated cells.
The two arms of 5a is suggesting its action as threading intercalation in
which one arm is oriented to the minor groove and the other to the major
groove (Fig. 11). These interactions of 5a may explain its highest anti-
cancer activity.
The proposed binding mode of 5f exhibited affinity value of ꢀ 98.71
kcal/mol and five H-bonds. The planar aromatic system occupied the
hydrophobic pocket formed by Arginine945, Asparagine882, Gluta-
mate870, Alanine869, Glycine868, Asparagine867, Asparagine795,
Asparagine786, Glutamine742, Lysine739 and Glycine737. It formed
two H-bonds with the key amino acid Asparagine786 and one H-bond
with Glycine868. It also stacked between the diphosphate Cytidine (Dc3,
Dc4 and Dc8) and diphosphate Guanosine (Dg5, Dg10 and Dg13). The 4-
methoxyphenyl arm was oriented into minor groove of DNA and stabi-
lized by formation of one H-bond with Tyrosine908. The 2-hydroxy-
phenyl arm was oriented into major groove of DNA and formed one
H-bond with Asparagine867. The two arms of 5f is suggesting its action
as threading intercalation in which one arm is oriented to the minor
groove and the other to the major groove (Fig. 12). These interactions of
5f may explain its high anticancer activity.
Fig. 4. Percent change for accumulated cytoplasmic NAG of derivative 5b
treated Bacillus subtilis referring to control.
The proposed binding mode of 5c exhibited affinity value of ꢀ 98.11
kcal/mol and five H-bonds. The planar aromatic system occupied the
hydrophobic pocket formed by Arginine945, Asparagine882, Gluta-
mate870, Alanine869, Glycine868, Asparagine867, Asparagine795,
Asparagine786, Glutamine742, Lysine739 and Glycine737. It formed
three H-bond with the key amino acid Asparagine786 in the active site
and one H-bond with Asparagine867. It also stacked between the
diphosphate Cytidine (Dc3, Dc4 and Dc8) and diphosphate Guanosine
(Dg5, Dg10 and Dg13). The 4-methoxyphenyl arm was oriented into
minor groove of DNA and stabilized by formation of one H-bond with
Tyrosine908. The N-phenylmethanethioamide arm was oriented into
major groove of DNA. The two arms of 5c is suggesting its action as
threading intercalation in which one arm is oriented to the minor groove
and the other to the major groove (Fig. 13). These interactions of 5c may
explain its high anticancer activity.
with Arginine945, one H-bond with Asparagine867). It also stacked
between the diphosphate Cytidine (Dc3, Dc4 and Dc8), diphosphate
Guanosine (Dg5, Dg10 and Dg13) and Adinine monophosphate 6 (Da6).
The sugar moiety was oriented into minor groove of DNA and stabilized
by formation of five H-bonds with the key amino acids (two H-bonds
with Arginine945, one H-bonds with Leucine880 and two H-bonds with
Asparagine882) (Fig. 10).
The proposed binding mode of 5a exhibited affinity value of ꢀ 99.15
kcal/mol and five H-bonds. The planar aromatic system occupied the
hydrophobic pocket formed by Arginine945, Asparagine882, Gluta-
mate870, Alanine869, Glycine868, Asparagine867, Asparagine795,
Asparagine786, Glutamine742, Lysine739 and Glycine737. It formed
three H-bond with the key amino acid Asparagine786 in the active site
and one H-bond with Asparagine867. It also stacked between the
diphosphate Cytidine (Dc3, Dc4 and Dc8) and diphosphate Guanosine
(Dg5, Dg10 and Dg13). The 4-methoxyphenyl arm was oriented into
minor groove of DNA and stabilized by formation of one H-bond with
Tyrosine908. The isobutyl arm was oriented into major groove of DNA.
As planned, our small aromatic molecules were concluded to act
through two ways firstly, through non-covalent interaction with the
directly bound proteins to DNA hence inhibit topoisomerase-II enzyme.
The second is through non-covalently binding to double helical
8

N.E.A. Abd El-Sattar et al.
Bioorganic Chemistry 115 (2021) 105186
Fig. 5. The amino acid profiles of the metabolism solution of Bacillus subtilis. a) untreated and b) derivative 5b treated cells.
Table 3
In vitro cytotoxic activities of the newly synthesized compounds against HepG2,
HCT-116, MCF-7 and VERO cell lines.
IC50 (µM)a
Tested compounds
HepG2
HCT-116
MCF-7
VERO
1
4.98 ± 0.4
8.51 ± 0.6
8.87 ± 0.6
3.73 ± 0. 3
2.09 ± 0.3
6.97 ± 0.5
4.82 ± 0.3
3.73 ± 0.3
8.21 ± 0.7
5.78 ± 0.6
5.91 ± 0.5
7.94 ± 0.6
3.18 ± 0.2
7.63 ± 0.6
7.99 ± 0.6
3.63 ± 0.3
2.74 ± 0.3
4.99 ± 0.3
2.61 ± 0.2
5.08 ± 0.5
5.91 ± 0.4
3.48 ± 0.4
4.68 ± 0.3
8.07 ± 0.8
3.05 ± 0.3
10.1 ± 0.8
10.65 ± 0.9
5.94 ± 0.5
2.53 ± 0.2
5.38 ± 0.4
4.14 ± 0.4
4.51 ± 0.4
3.94 ± 0.4
2.43 ± 0.2
4.37 ± 0.4
6.75 ± 0.4
33.85 ± 0.30
bNT
2
3
bNT
4
28.40 ± 0.30
25.12 ± 0.30
bNT
5a
5b
5c
36.32 ± 0.30
5d
bNT
5e
bNT
5f
30.01 ± 0.30
5 g
bNT
Doxorubicin
bNT
a
IC50: values are the mean ± S.D. of three separate experiments.
b
NT: Not Tested.
structures of DNA by threading intercalation.
3.2. In silico ADMET profile
In the present study, a computational study of the three most active
compounds (5a, 5c and 5f) was conducted to determine the surface area
and other physicochemical properties according to the directions of
Lipinski’s rule [40]. Lipinski suggested that the absorption of a com-
pound is more likely to be better if the molecule achieve at least three
out of four of the following rules: (i) HB donor groups ≤ 5; (ii) HB
acceptor groups ≤ 10; (iii) M. Wt less than 500; (iv) logP less than 5. In
this study, whereas the reference anticancer agent doxorubicin violates
three of Lipinski’s rules, our derivatives 5a, 5c and 5f obey all Lipinski’s
rules. All the highest active derivatives have a number of hydrogen-
bonding acceptor groups 7 and/or 8 and only 3 and/or 4 hydrogen-
bonding donors. Also, molecular weights are less than 500 and log P
less than 5 and all these values agree with the Lipinski’s rules. Also,
absorption, distribution, metabolism, excretion, and toxicity (ADMET)
profiles of the new synthesized derivatives were preliminary assessed to
Fig. 6. SEM micrograph of surface ultra structural characteristics of HepG2
cells treated with derivative 5a for 24 h.
Table 4
DNA binding affinity and In vitro Topoisomerase II inhibitory activity of some
tested compounds.
Compound
DNA binding IC50 (µM)
Top-II assay IC50 (µM)
5a
26.96 ± 1.7
27.13 ± 1.7
29.86 ± 1.7
31.27 ± 1.8
0.752 ± 0.07
0.791 ± 0.07
0.776 ± 0.07
0.940 ± 0.40
5c
5f
Doxorubicin
9

N.E.A. Abd El-Sattar et al.
Bioorganic Chemistry 115 (2021) 105186
Fig. 7. Figure/band pattern of DNA binding and Top-II inhibition.
analyze their potentials to build up as good medication candidates.
Prediction of ADMET profiles conducted with the aid of pkCSM de-
scriptors algorithm protocol [41].
Table 5
The calculated free energy of binding (ΔG in Kcal/mole) for the ligands.
Compound
ΔG [kcal molꢀ 1
]
Compound
ΔG [kcal molꢀ 1
]
After assessing ADMET profiles of compounds 5a, 5c and 5f
(Table 6), we can suggest that these derivatives have the advantage of
better intestinal absorption in human (86.799–90.96) than doxorubicin
(62.3). This preference may attribute to the superior lipophilicity of our
designed ligands, which would make it easier to go along different
biological membranes [42]. Accordingly, they may have significant
good bioavailability after oral administration. Studying the CNS
permeability, our derivatives 5a, 5c and 5f demonstrated the best ability
to penetrate the CNS (CNS permeability values ꢀ 2.704 to ꢀ 3.802),
while doxorubicin is unable to penetrate (CNS permeability ꢀ 4.307). It
1
ꢀ 77.91
ꢀ 76.15
ꢀ 88.68
ꢀ 88.88
ꢀ 99.15
ꢀ 80.47
ꢀ 98.11
5d
ꢀ 92.47
ꢀ 92.31
ꢀ 98.71
ꢀ 69.82
ꢀ 100.31
ꢀ 101.73
2
5e
3
5f
4
5 g
5a
5b
5c
Doxorubicin
Amsacrine
Fig. 8. Superimposition of the docked compounds and doxorubicin inside the active site of DNA-Topo II.
10

N.E.A. Abd El-Sattar et al.
Bioorganic Chemistry 115 (2021) 105186
Fig. 9. Binding of amsacrine with DNA-Topo II, the hydrogen bonds are represented in blue dashed lines.
Fig. 10. Binding of doxorubicin with DNA-Topo II.
11

N.E.A. Abd El-Sattar et al.
Bioorganic Chemistry 115 (2021) 105186
Fig. 11. Predicted binding mode for 5a with DNA-Topo II.
Fig. 12. Predicted binding mode for 5f with DNA-Topo II.
is also clear that the cytochrome P3A4, the main enzyme involved in
drug metabolism could be inhibit under the effect of compound 5a
while compounds 5c, 5f and doxorubicin couldn’t. The total clearance
is a significant parameter in deciding dose intervals as a tool for
assessment of excretion. Doxorubicin exhibited the highest total clear-
ance value compared to other ligands. On the other hand, the new li-
gands showed lower total clearance values. Thus, doxorubicin, could
excreted quicker, and accordingly require shorter dosing intervals.
Dissimilar to doxorubicin, the new compounds exhibited slower clear-
ance rates, which mean the preference of possible extended dosing
intervals of the novel derivatives. The last parameter examined in the
ADMET profiles of our newly synthesized derivatives is the toxicity. As
displayed in Table 6, all the new ligands and doxorubicin showed hep-
atotoxicity drawback. The oral acute toxic doses of the new compounds
(LD50) are more than the reference drug (2.677–2.972 for our new de-
rivatives compared with 2.408 of doxorubicin). Finally, the lower
Minnow toxicity values of the new derivatives compared with doxoru-
bicin reflect the higher selectivity of 5a, 5c and 5f against cancer cells
over their cytotoxicity against normal cells.
12

N.E.A. Abd El-Sattar et al.
Bioorganic Chemistry 115 (2021) 105186
Fig. 13. Predicted binding mode for 5c with DNA-Topo II.
4. Conclusion
structures of DNA by threading intercalation as represented in docking
studies. For a great extent, the reported results of Top-II inhibition were
in agreement with that of in vitro cytotoxicity activity, DNA binding and
molecular modeling studies. Our derivatives showed good ADMET
profile.
In summary, eleven new pyrano[2,3-d]pyrimidine derivatives have
been designed, synthesized and evaluated for their antimicrobial and
anticancer activities. The antimicrobial activities were examined against
gram positive bacteria, gram negative bacteria and fungi. All synthe-
sized compounds to some extent have shown good antimicrobial activity
against different microbial strains that had been extracted by inhibiting
cell wall synthesis. Compound 5b showed the highest antibacterial ac-
tivities against B. subtilis, S. aureus and E. coli. On the other hand
compound 5 g exhibited the highest antibacterial and antifungal activ-
ities against P. aeruginosa and A. niger respectively. Anticancer activ-
ities were carried out for our derivatives against three human tumor cell
lines hepatocellular carcinoma (HepG2), colorectal carcinoma (HCT-
116) and breast cancer (MCF-7) as DNA intercalators and Top II enzyme
inhibitors. Compound 5a showed the highest activity against HepG2
5. Experimental:
5.1. Chemistry
The melting points were measured using a Gallenkamp electric
melting point apparatus and are uncorrected. The IR spectra were
recorded on a PyeUnicam SP-3–300 infrared spectrophotometer using
potassium bromide (KBr) disks. The 1H NMR spectra experiments were
run at 300 and 400 MHz on a Varian Mercury VX-300 NMR spectrometer
(Varian, U.K.) using tetramethylsilane (TMS) as internal standard (IS) in
deuterated chloroform or deuterated dimethyl sulfoxide (DMSO) and
the chemical shifts (δ) were reported in parts per million (ppm). 13C
NMR spectra were recorded on a Bruker 100 MHZ-NMR spectropho-
tometer at Microanalytical Unit, Faculty of pharmacy, Cairo University.
The mass spectra were recorded using Shimadzu GCMS-QP-1000EX
mass spectrometer (Shimadzu, Japan) at ionization energy of 70 eV
with the source 200 ◦C and an accelerative voltage of 8 Kv and Direct
Probe Controller Inlet part to Single Quadropole mass analyzer in
Thermo Scientific GCMS model ISQ LT using Thermo X-Calibur software
at the Regional Center for Mycology and Biotechnology, Al-Azhar Uni-
versity. All the spectral measurements as well as the elemental analyses
were carried out at the Microanalytical Center, Cairo University and the
Main laboratories for chemical warfare. All the newly synthesized
compounds gave satisfactory elemental analyses. The purity of the
synthesized compounds was checked by TLC.
cells with IC50 = 2.09
to derivative 5c with IC50 = 2.61
showed pronounced negative impact against MCF-7 (IC50 = 2.43
μ
M, while HCT-116 cells were highly susceptible
M, in the meantime, derivative 5f
M)
μ
μ
when compared with other prepared compounds. All derivatives
exhibited higher anticancer activities than doxorubicin against the three
cell lines except compound 2 against both HepG2 and MCF-7 and
compound 5e against HepG2 cell lines. Compounds 5a, 5c and 5f
potently intercalate DNA at IC50 values of 26.96, 27.13 and 29.86 µM
respectively, which were more potent than doxorubicin (IC50 value of
31.27 µM). Moreover, compounds 5a, 5c and 5f exhibited very good
Topoisomerase II inhibitory activities with IC50 values of 0.752, 0.791
and 0.776 µM respectively, that were more potent than that of doxo-
rubicin (IC50 = 0.94 µM). The molecular design was performed to
investigate the binding mode of the proposed compounds with DNA-
Topo II active site. Our small aromatic molecules were concluded to
act through two ways firstly, through non-covalent interaction with the
directly bound proteins to DNA hence inhibit topoisomerase-II enzyme.
The second is through non-covalently binding to double helical
13

N.E.A. Abd El-Sattar et al.
Bioorganic Chemistry 115 (2021) 105186
C, 57.62; H, 3.47; N, 17.76.
Table 6
ADMET profile of the most active compounds and doxorubicin.
5.1.2. 4-((6-Cyano-5-(4-methoxyphenyl)-2,4-dioxo-1,3,4,5-tetrahydro-
2H-pyrano[2,3-d]pyrimidin-7-yl)amino)-4-oxobut-2-enoic acid (2)
A solution of 7-amino-5-(4-methoxyphenyl)-2,4-dioxo-1,3,4,5-tetra-
hydro-2H-pyrano[2,3-d]pyrimidine-6-carbonitrile 1 (1.56 g, 5mmole),
maleic anhydride (0.49 g,5 mmol) in glacial acetic acid (20 mL) were
heated under reflux for four hours. The solid separated and collected
after concentration and cooling of the solvent to room temperature,
Parameter
5a
5c
5f
Doxorubicin
Molecular properties
Molecular Weight
LogP
395.419
2.00467
474.502
2.27877
431.408
543.525
0.0013
1.
72,847
Rotatable Bonds
Acceptors
4
3
3
5
7
8
8
12
Donors
3
4
4
6
dried and crystallized from ethanol to give 2 as yellow crystals; m.p:
Surface Area
165.053
196.924
179.445
222.081
268–270 C, yield 90%; IR (KBr) 3430 cm-1 (νOH), 3082 cmꢀ 1
(
νNH),
◦
broad band 2600–3100 cmꢀ 1 for acidic OH, 2166 cmꢀ 1
(νCN), 1706,
Absorption
Water solubility
ꢀ 3.344
ꢀ 3.06
ꢀ 3.036
ꢀ 2.915
1670 cmꢀ 1
(
νC=O); 13C NMR (100 MHz, DMSO‑d6): 22.88, 41.41, 44.51,
56.02, 115.53(2), 118.09, 118.83, 125.79, 128.58, 130.19, 132.87 (2),
134.14, 141.20, 161.81, 164.71, 165.88, 167.69; MS m/z (%): 409.40
(M++1, 43.22%), 271 (37.70%), 145.13 (100%, base beak); Anal.
Calculated for C19H14N4O7 (410.34): C, 55.61; H, 3.44; N, 13.65, Found:
C, 55.75; H, 3.47; N, 13.92.
Caco2 permeability
0.607
0.525
0.648
90.96
0.457
Intestinal abs. (human)
89.002
86.799
62.372
Skin Permeability
ꢀ 2.735
Yes
ꢀ 2.735
Yes
ꢀ 2.735
Yes
ꢀ 2.735
Yes
P-glycoprotein substrate
P-glycoprotein I inhibitor
P-glycoprotein II inhibitor
No
No
No
No
Yes
Yes
Yes
No
5.1.3. N-(6-Cyano-5-(4-methoxyphenyl)-2,4-dioxo-1,3,4,5-tetrahydro-
2H-pyrano[2,3-d]pyrimidin-7-yl)acetamide (3)
Distribution
Volume of distribution at
steady state (VDss) (human)
Fraction unbound (human)
BBB permeability
0.661
ꢀ 0.517
ꢀ 0.823
1.647
Heating and refluxing mixture of 7-amino-5-(4-methoxyphenyl)-2,4-
dioxo-1,3,4,5-tetrahydro-2H-pyrano[2,3-d]pyrimidine-6-carbonitrile 1
(1.56 g, 5 mmol) and glacial acetic acid (20 mL) for four hours . The solid
separated and collected after concentration and cooling of the solvent to
room temperature, dried and crystallized from butanol to give 3 as
0.064
0
0.112
0.215
ꢀ 1.063
ꢀ 1.245
ꢀ 1.241
ꢀ 1.379
CNS permeability
Metabolism
ꢀ 2.862
ꢀ 2.704
ꢀ 3.802
ꢀ 4.307
yellow crystals; m.p: 220–223 ◦C, yield 90%; IR (KBr) 3430 cmꢀ 1
(νOH),
CYP2D6 substrate
CYP3A4 substrate
CYP1A2 inhibitor
No
No
Yes
No
No
Yes
No
No
No
No
No
No
3118 cmꢀ 1
(
νNH), 2221 cmꢀ 1
(
νCN), 1717 cmꢀ 1
(
νC=O); 1H NMR
(DMSO‑d6): δ 1.91 (s, 3H, CH3), 3.66 (s, 3H, OCH3), 5.87 (s, 1H, pyran
H), 6.68–6.71 (d, 2H, Ar-H, J = 8.8 Hz), 6.89–6.92 (d, 2H, Ar-H, J = 8.4
Hz), 9.93 (s, D2O exchangeable 3NH); MS m/z (%): 355.93 (M++1,
20.99%), 354.96 (M+, 32.31%), 335.29 (76.18%), 292.20 (100%, base
beak); Anal. Calculated for C17H14N4O5 (354.10): C, 57.63; H, 3.98; N,
15.81 Found: C, 57.85; H, 3.77; N, 15.62.
CYP2C19 inhibitor
CYP2C9 inhibitor
CYP2D6 inhibitor
CYP3A4 inhibitor
Excretion
No
No
No
Yes
Yes
No
No
No
No
No
No
No
No
No
No
No
Total Clearance
0.625
No
ꢀ 0.266
No
0.404
No
0.987
No
5.1.4. Ethyl N-(6-cyano-5-(4-methoxyphenyl)-2,4-dioxo-1,3,4,5-
tetrahydro-2H-pyrano[2,3-d]pyrimidin-7-yl)formimidate (4)
Renal OCT2 substrate
Toxicity
A solution of 7-amino-5-(4-methoxyphenyl)-2,4-dioxo-1,3,4,5-tetra-
hydro-2H-pyrano[2,3-d]pyrimidine-6-carbonitrile 1 (1.56 g, 5 mmol)
and (30 mL) triethyl orthroformate were heated under reflux for seven
hours . The solid separated and collected after concentration and cooling
of the solvent to room temperature, dried and crystallized from butanol
AMES toxicity
No
No
No
No
Max. tolerated dose (human)
hERG I inhibitor
0.796
No
0.738
No
0.521
No
0.081
No
hERG II inhibitor
Yes
Yes
Yes
Yes
Oral Rat Acute Toxicity (LD50
)
2.677
2.972
2.705
2.408
to give 4as yellow crystals; m.p: 132–134 ◦C, yield 85%; IR (KBr) 3170
1
cmꢀ 1 (NH), 2221 cmꢀ 1 (CN), 1718 cmꢀ 1 (C O); H NMR (DMSO‑d ): δ
Oral Rat Chronic Toxicity
(LOAEL)
1.329
Yes
1.35
Yes
1.305
Yes
3.339
Yes
–
–
1.11 (t, 3H, CH3), 3.35 (q, 2H, CH2), 3.85 (s, 3H, OCH3), 4.54 (s,61H,
pyran H), 7.61–7.66 (d, 2H, ArH, J = 9.6 Hz), 7.81–7.83 (d, 2H, Ar-H, J
= 8.4 Hz), 7.96 (s, 1H, imino), 8.37, 10.62 (2 s, D2O exchangeable 2NH);
MS m/z (%): 370 (M++2, 1.12%), 368 (M+, 11.55%), 285 (25.97%), 71
(100%, base beak); Anal. Calculated for C18H16N4O5 (368.34): C, 58.69;
H, 4.38; N, 15.21 Found: C, 58.85; H, 3.47; N, 14.98.
Hepatotoxicity
Skin Sensitization
T. Pyriformis toxicity
Minnow toxicity
No
No
No
No
0.287
2.575
0.285
0.964
0.285
2.515
0.285
4.412
5.1.1. 7-Amino-5-(4-methoxyphenyl)-2,4-dioxo-1,3,4,5-tetrahydro-2H-
pyrano[2,3-d]pyrimidine-6-carbonitrile (1)
5.1.5. Reaction of ethyl N-(6-cyano-5-(4-methoxyphenyl)-2,4-dioxo-
1,3,4,5-tetrahydro-2H-pyrano[2,3-d]pyrimidin-7-yl)formimidate (4) with
nucleopilic reagents
A mixture of 4-methoxy benzaldehyde (13.6 g. 100 mmol), malo-
nonitrile (6.6 g, 100 mmol) and barbituric acid (12.8 g, 0.1 mol) in
butanol (50 mL) were heated under reflux for three hours. The solid
separated and collected after concentration and cooling of solvent at
room temperature, dried and crystallized from butanol to give 1 as
General procedure
A
mixture of ethyl N-(6-cyano-5-(4-methoxyphenyl)-2,4-dioxo-
1,3,4,5-tetrahydro-2H-pyrano[2,3-d]pyrimidin-7-yl)formimidate
4
yellow crystals; m.p: 255–257 ◦C, yield 95%; IR (KBr) 3301 cm-1doublet
(1.84 g, 5 mmol) and the proposed reagent (5 mmol) in (20 mL) butanol
were heated under reflux for four hours. The solid separated and
collected after concentration and cooling of the solvent to room tem-
perature dried and crystallized from the suitable solvent to give the
corresponding derivatives 5a-h respectively.
1
(
νNH), 3184 cmꢀ 1
(
νNH), 2107 cmꢀ 1
(
νCN), 1715, 1691 cmꢀ 1
(
νC=O); H
NMR (DMSO‑d6): δ 3.85 (s, 3H, OCH3), 4.56 (s, 1H, pyran H), 7.61–7.66
(m, 4H, 2 Ar-H & NH2 (D2O exchangeable)), 7.82–7.84 (d, 2H, 2 Ar-H, J
= 7.6 Hz), 10.23, 10.63 (2 s, D2O exchangeable 2NH); MS m/z (%):
314.58 (M++2, 31.52%), 312.19 (M+, 15.59%), 269.50 (100%, base
beak), 243.91 (19.25%), 188.91 (19.66%), 95.12 (84.71%); Anal.
Calculated for C15H12N4O4 (312.29): C, 57.69; H, 3.87; N, 17.94, Found:
5.1.5.1. 7-(sec-Butyl)-6-imino-5-(4-methoxyphenyl)-1,5,6,7-tetrahydro-
2H-pyrano[2,3-d:6,5-d’]dipyrimidine-2,4(3H)-dione
(5a). Reddish
14

N.E.A. Abd El-Sattar et al.
Bioorganic Chemistry 115 (2021) 105186
brown crystallized from dimethyl formamide (DMF), m.p 258 ◦C, yield
2.38%), 380.16 (67.36%), 379.23 (80.04%), 140.06 (100%, base beak);
Anal. Calculated for C22H17N5O5 (431.41): C, 61.25; H, 3.97; N, 16.23
Found: C, 61.01; H, 4.02; N, 17.40.
74%; IR (KBr) 3200 and 3123 cmꢀ 1
(
ν
NH), 1689 cmꢀ 1
(
ν
C = O); 1H NMR
(DMSO‑d6): δ 0.90 (t, 3H, CH3), 1.82 (m, 2H, CH2), 2.62 (d, 3H, CH3 J =
6.0 Hz), 3.32 (s, 3H, OCH3), 3.77 (m, 2H, 2CH), 6.84–6.93 (d, 2H, ArH,
J = 7.1 Hz), 6.94–7.05 (d, 2H, ArH, J = 7.1 Hz), 8.09 (s, 1H, of py-
rimidine), 8.92, 8.94, 8.96 (s, D2O exchangeable 3NH); MS m/z (%),
398.21 (M++3, 4.84%), 392.19 (7.79%), 380.17 (26.10%), 300.08
(44.92%), 89.09 (72.71), 67.07 (90.87), 62.06 (100%, base beak); Anal.
Calculated for C20H21N5O4 (395.42): C, 60.75; H, 5.35; N, 17.71, Found:
C, 60.51; H, 5.11; N, 17.98.
5.1.5.7. 6-Imino-5-(4-methoxyphenyl)-7-(4-methylpyridin-2-yl)-1,5,6,7-
tetrahydro-2H-pyrano[2,3-d:6,5-d’]dipyrimidine-2,4(3H)-dione (5 g).
Brown crystals crystallized from DMF, decompose at 260 ◦C, yield 60%;
IR (KBr) 3220, 3184 cmꢀ 1
(
νNH), 1671 cmꢀ 1
(
νC=O); 1H NMR
(DMSO‑d6): δ 2.61 (s, 3H, CH3), 3.87 (s, 3H, OCH3), 5.82 (s, 1H, pyran
H), 6.72–7.33 (m, 5H, ArH). 7.24 (s, 1H, of pyrimidine), 8.35–8.37 (d,
2H, ArH, J = 7.6 Hz), 10.14, 11.16, 11.29 (3 s, D2O exchangeable 3NH);
MS m/z (%): 431.35 (M++1, 0.46%), 155.07 (43.25%), 108.11
(74.71%), 41.07 (100%, base beak); Anal. Calculated for C22H17N5O5
(430.14): C, 61.39; H, 4.22; N, 19.53, Found: C, 61.10; H, 4.42; N, 19.81.
5.1.5.2. 6-Imino-5-(4-methoxyphenyl)-2,4-dioxo-1,3,4,5-tetrahydro-2H-
pyrano[2,3-d:6,5-d’]dipyrimidine-7(6H)-carboxamide (5b). Yellow crys-
tals crystallized from DMF, m.p: over 300 ◦C, yield 74%; IR (KBr) 3317,
doublet at 3201 and 3167 cmꢀ 1
(νNH), broad band 3000–3500 for enol-
keto tautomerization, 1729 and 1670 cmꢀ 1
(
νC=O); 1H NMR (DMSO‑d6):
6. Biological assay
δ 3.32 (s, 3H, OCH3), 5.39 (s, 1H, pyran H), 6.79–6.80 (m, 2H, ArH),
6.93–6.97 (m, 2H, ArH), 7.12 (s, 1H, of pyrimidine), 10.73–10.74 (5 s,
D2O exchangeable 3NH & NH2); MS m/z (%), 385.32 (M++3, 1.52%),
382.62 (M+, 1.15%), 167.09 (43.13), 60.05 (100%, base beak); Anal.
Calculated for C17H14N6O5 (382.10): C, 53.41; H, 3.69; N, 21.98, Found:
C, 53.21; H, 3.52; N, 22.10.
6.1. Antimicrobial screening of the tested compounds
The synthesized derivatives were evaluated in vitro for their anti-
microbial efficacy against 6 microbial strains purchased from the
American type culture collection (ATCC; Manassas, VA, U.S.A.). The
microbes were 2 Gram-positive strains (Bacillus subtilis ATCC® 6633TM
,
and Staphylococcus aureus ATCC® 25,923 TM), 2 Gram-negative strains
(Escherichia coli ATCC® 25,922 TM, and Pseudomonas aeruginosa ATCC®
27,853 TM) and 2 fungal strains (Aspergillus niger ATCC® 6275 TM, and
Candida albicans ATCC® 10,231 TM). The tested bacterial and fungal
strains were cultivated in Nutrient broth/agar and Sabouraud dextrose
broth/agar respectively (Oxoid TM, Basingstoke, Hampshire, U.K.).
The microbial broth culture was incubated at 37 ◦C for bacteria and
at 25 ◦C for fungi until the turbidity of the 0.5 McFarland standards was
achieved. To obtain turbidity comparable to that of the required
McFarland standards, the turbidity of the actively growing broth culture
was adjusted with sterile broth to have an optical density of 0.1 at 600
nm. This resulted in a suspension containing microbes approximately 2
× 108 colony forming units per milliliter (CFU/mL). Aiming to obtain
homogenous microbial growth; Nutrient/Sabouraud dextrose agar was
melted, cooled to 45 ◦C, inoculated by the microbial strain using spore
suspension technique and poured into Petri dishes to get a uniform
thickness of 6 mm. Once the medium had solidified, six wells were then
carefully cut out of the agar using a sterile cork borer. The antimicrobial
activities of the prepared compounds were tested using the agar well
5.1.5.3. 6-Imino-5-(4-methoxyphenyl)-2,4-dioxo-N-phenyl-1,3,4,5-tetra-
hydro-2H-pyrano[2,3-d:6,5-d’]dipyrimidine-7(6H)-carbothioamide (5c).
Yellow crystals crystallized from DMF, m.p: over 300 ◦C, yield 74%; IR
(KBr) 3357, 3201 and 3178 cmꢀ 1
(νNH), broad band 3000–3500 for enol-
keto tautomerization , 1680 cmꢀ 1
(
νC=O); 1H NMR (DMSO‑d6): δ 3.67 (s,
3H, OCH3), 5.8 (s, 1H, pyran H), 6.66–7.42 (m, 9H, ArH), 8.01 (s, 1H, of
pyrimidine), 9.15, 9.47, 10.02, 10.54 (4 s, D2O exchangeable 4 NH);
Anal. Calculated for C23H18N6O4S (474.5): C, 58.22; H, 3.82; N, 17.71
Found: C, 58.01; H, 4.02; N, 17.50.
5.1.5.4. 6-Imino-5-(4-methoxyphenyl)-7-(p-tolyl)-1,5,6,7-tetrahydro-2H-
pyrano[2,3-d:6,5-d’]dipyrimidine-2,4(3H)-dione (5d). Pale yellow crys-
tals crystallized from DMF, m.p.: over 300 ◦C, yield 60%; IR (KBr)
3423and 3205 cmꢀ 1
(νNH), broad band 2800–3300 for enol-keto tau-
tomerization, 1683 cmꢀ 1
(
νC=O). 1H NMR (DMSO‑d6): δ 2.36 (s, 3H,
CH3), 3.67 (s, 3H, OCH3), 5.86 (s, 1H, pyran H), 6.43–6.46 (d, 2H, ArH,
J = 8.7 Hz), 6.89–6.92 (d, 2H, ArH, J = 8.7 Hz), 7.21–7.24 (d, 2H, ArH,
J = 8.1 Hz), 7.37–7.40 (d, 2H, ArH, J = 7.8 Hz), 8.49 (s, 1H, of py-
rimidine), 8.75, 9.86, 10.01 (3 s, D2O exchangeable 3NH). MS m/z (%):
432.20 (M++3, 0.64%), 431.22 (M++2, 3.39%), 428.19 (1.29%),
412.19 (12.75%), 401.18 (19.76%), 206.28 (100%, base beak); Anal.
Calculated for C23H19N5O4 (429.14): C, 64.33; H, 4.46; N, 16.31 Found:
C, 64.01; H, 4.32; N, 16.50.
diffusion method. 100 μl of each compound solution (100 µg/mL) was
introduced into the corresponding well using a sterile micro-pipette.
Ampicillin, Gentamicin and Amphotericin B were used as standard
reference antimicrobial drugs for Gram-positive bacteria, Gram-
negative bacteria, and fungal strains respectively. Afterwards the petri
dishes were incubated for 24 h at 37◦ C (bacteria) or for 7 days at 25◦ C
(fungi). The antimicrobial effectiveness of each compound was deter-
mined by measuring the diameter of inhibition zone in mm. Experiments
were triplicate and the results were reported as the mean of inhibition
zone diameter in mm ± standard deviation (S.D.). The results classify
the tested compounds into inactive, mildly active, moderately active, or
highly active candidate according to the standards of the Clinical and
Laboratory Standards Institute.
5.1.5.5. 7-(2-Aminophenyl)-6-imino-5-(4-methoxyphenyl)-1,5,6,7-tetra-
hydro-2H-pyrano[2,3-d:6,5-d’]dipyrimidine-2,4(3H)-dione (5e). Yellow
crystals crystallized from DMF, m.p: 290 ◦C, yield 60%; IR (KBr)
3380,3348, 3210 and 3184 cmꢀ 1
keto tautomerization, 1682 cmꢀ 1
(νNH), broad band 2800–3300 for enol-
(
νC=O); 1H NMR (DMSO‑d6): δ 3.84 (s,
3H, OCH3), 5.10 (s, 2H, NH2, D2O exchangeable), 5.86 (s, 1H, pyran H),
6.08–7.11 (d, 2H, ArH, J = 9.0 Hz), 7.18–7.19 (m, 2H, ArH), 7.53–7.59
(m, 2H, ArH), 8.04–8.09 (m, 2H, ArH), 8.19 (s, 1H, of pyrimidine), 9.42,
10.92, 11.56 (3 s, D2O exchangeable 3NH); MS m/z (%): 433.42 (M++3,
0.64%), 430.53 (M+, 0.27%), 255.23 (18.65%), 57.09 (100%, base
beak); Anal. Calculated for C22H18N6O4 (430.14): C, 61.39; H, 4.22; N,
19.53 Found: C, 61.01; H, 4.02; N, 19.70.
6.1.1. MIC determination
The minimum inhibitory concentration (MIC) was defined as the
least concentration of the antimicrobial agent that able to inhibit mi-
crobial growth. According to the results of initial screening, the syn-
thesized compounds that have pronounced antimicrobial potentialities
were selected for MIC determinations using the broth macro-dilution
method.
5.1.5.6. 7-(2-Hydroxyphenyl)-6-imino-5-(4-methoxyphenyl)-1,5,6,7-tet-
rahydro-2H-pyrano[2,3-d:6,5-d’]dipyrimidine-2,4(3H)-dione (5f). Pale
Yellow crystals crystallized from DMF, m.p: 270–272 ◦C, yield 60%; IR
A stock solution (100
μg/mL) of each test compound was prepared
(KBr) broad band 3100–3300 cmꢀ 1
(
νOH), 3365, 3200 cmꢀ 1
(νNH), 1684
and a serial dilution was carried out to obtain the concentrations range
cmꢀ 1
(
νC=O); MS m/z (%): 434.26 (M++3, 12.33%), 432.63 (M++1,
15

N.E.A. Abd El-Sattar et al.
Bioorganic Chemistry 115 (2021) 105186
covering from 100
μ
g/ mL to 0.195
μ
g/mL. The various concentrations
detecting the DNA-binding affinity and relative binding strength. IC50
values were determined using the GraphPadPrism 5.0 software. The
reaction was performed as follows.
of the test compounds were introduced into culture tubes containing 2
mL of the standardized microbial suspensions. The culture tubes were
◦
◦
incubated at 37 C for 24 h (bacterial strains) or at 25 C for 7 days
(fungal strains) and then microbial growth were evaluated as manifested
by turbidity. All experiments were performed in triplicate and the last
cultivated tube without turbidity and remained clear was representing
A mixture of Calf thymus DNA (10 mg) and methyl green (20 mg)
(Sigma– Aldrich) in 100 mL of 0.05 M Tris-HCl buffer (pH 7.5) con-
taining 7.5 mM MgSO4. Then, the mixture was stirred for 24 h at 37 ◦C.
The test samples were dissolved in ethanol and dispensed into wells of a
96-well microtiter tray at concentration of 10,100 and 1000 µM. From
each well, the excess solvent was removed under vacuum followed by
addition of 200 µl of the DNA/methyl green solution. The test samples
were incubated in a dark at ambient temperature. After 24 h, the
absorbance of each sample was determined at 642.5–645 nm. Readings
were corrected for initial absorbance and normalized as the percentage
of the untreated DNA/methyl green absorbance value.
the MIC that expressed in
of MIC in g/mL ± S.D.
μg/mL. The results were reported as the mean
μ
6.1.2. Effect of target compound on the bacterial cell wall
The negative impact of the 5b derivative on bacterial cell wall syn-
thesis was examined using transmission electron microscope JEOL-JEM
1200 EXII and confirmed by assessment of the accumulated cytoplasmic
fraction of N-acetylglucosamine (NAG) using colorimetric assessment
according to the method described by Lewy & McAllan [43]. In the same
time, differences in the accumulation of the amino acids in the meta-
bolism solution of control and treated bacterial cells were examined
using HPLC-Pico-Tag after acid hydrolysis of samples using 6 N HCl for
24 h at 110 OC. The samples were injected in amino acid analyzer after
amino acids derivatization using phenylisothiocyanate. The amino acids
were quantified by comparison of peak area with those corresponding
amino acids standard solutions using the Spectra Physics Data System
Program.
6.2.3. In vitro Topoisomerase II inhibitory activity
In this test, Topo II drug screening kit (TopoGEN, Inc., Columbus)
was utilized to determine the Topo II activity according to the reported
method [37,38]. Doxorubicin was used as a reference drug in this test.
A typical enzyme reaction contained a mixture of human Topo II (2
µl), substrate super coiled pHot1 DNA (0.25 µg), 50 µg/ml test com-
pound (2 µl), and assay buffer (4 µl). The reaction started upon incu-
bation of the mixture in 37 ◦C for 30 min. The reaction was terminated
by addition of 10% sodium dodecylsulphate (2 µl) and proteinase K (50
µg/ml) at 37 ◦C for 15 min. followed by incubation for 15 min at 37 ◦C.
Then, the DNA was run on 1% agarose gel in BioRad gel electrophoresis
system for 1–2 h followed by staining with GelRedTM stain for 2 h and
destained for 15 min with TAE buffer. The gel was imaged via BioRad’s
Gel DocTMEZ system. Both supercoiled and linear strands DNA were
incorporated in the gel as markers for DNA-Topo II intercalators. The
results of IC50 values were calculated using the GraphPad Prism version
5.0. Each reaction was performed in duplicate, and at least three inde-
pendent determinations of each IC50 were made.
6.2. Anticancer activities of the tested compounds
6.2.1. In vitro anti-proliferative activity
Cancer cells from different cancer cell lines hepatocellular carcinoma
(HepG2), colorectal carcinoma (HCT-116) and breast cancer (MCF-7),
were purchased from American type Cell Culture collection (ATCC,
Manassas, USA) and grown on the appropriate growth medium Roswell
Park Memorial Institute medium (RPMI 1640) supplemented with 100
mg/ mL of streptomycin, 100 units/ mL of penicillin and 10% of heat-
inactivated fetal bovine serum in a humidified, 5% (v/v) CO2 atmo-
sphere at 37 ◦C Cytotoxicity assay by 3-[4,5-dimethylthiazole-2-yl]-2,5-
diphenyltetrazolium bromide (MTT).
6.2.4. Possible mode of cytotoxic impact of the selected compound
The morphological changes appeared on HepG2 after treatment by
derivative 5a was monitored using scanning electron microscope. After
the end of the treatment as previously described, the samples were fixed
in 2.5% glutaraldehyde and dehydrated by serial dilution of ethanol
using an automatic tissue processor (Leica EM TP). Then, the samples
were dried using CO2. The dried cells were coated by gold sputter coater
(SPI- Module). Finally, the samples were viewed and photographed by
JSM-5500 LV scanning electron microscopy by using high vacuum
mode.
Exponentially growing cells from different cancer cell lines were
trypsinized, counted and seeded at the appropriate densities
(2000–1000 cells/0.33 cm2 well) into 96-well microtiter plates. Cells
then were incubated in a humidified atmosphere at 37˚C for 24 h. Then,
cells were exposed to different concentrations of compounds (0.1, 10,
100 and 1000 µM) for 72 h. Then the viability of treated cells was
determined using MTT technique as follow. Cells were incubated with
200 μl of 5% MTT solution/well (Sigma Aldrich, MO) and were allowed
6.3. Docking studies
to metabolize the dye into colored-insoluble formazan crystals for 2 h.
The remaining MTT solution were discarded from the wells and the
formazan crystals were dissolved in 200 µl/well acidified isopropanol
for 30 min, covered with aluminum foil and with continuous shaking
using a MaxQ 2000 plate shaker (Thermo Fisher Scientific Inc, MI) at
room temperature. The colorimetric assay was measured and recorded
at absorbance of 570 nm using a Stat FaxR 4200 plate reader (Awareness
Technology, Inc., FL). The cell viability were expressed as percentage of
control and the concentration that induces 50% of maximum inhibition
of cell proliferation (IC50) were determined using Graph Pad Prism
version 5 software (Graph Pad software Inc, CA) [32–34].
In the present work, all the target compounds were subjected to
docking study to explore their binding mode towards DNA-Top II. All
modeling experiments were performed using molsoft program, which
provides a unique set of tools for the modeling of protein/ligand in-
teractions. It predicts how small flexible molecule such as substrates or
drug candidates bind to a protein of known 3D structure represented by
grid interaction potentials (http://www.molsoft.com/icm_pro.html).
Each experiment used the biological target DNA-Top II downloaded
from the Brookhaven Protein Databank (http://www.rcsb.
org/pdb/explore/ explore.do?structureId = 4G0U). In order to qualify
the docking results in terms of accuracy of the predicted binding con-
formations in comparison with the experimental procedure, the reported
DNA-Top II inhibitor drug doxorubicin was used as reference ligand.
6.2.2. In vitro DNA/Methyl green assay
Doxorubicin as DNA intercalator was used as positive control. In this
test, methyl green dye can bind DNA to form colored reversible complex
of DNA/methyl green. These complexes still stable at neutral pH. Upon
addition of intercalating agents, the methyl green is displaced from DNA
with addition of H2O molecule to the dye resulting in formation of the
colorless carbinol, leading to a dramatic decrease in spectrophotometric
absorbance [37,38]. ΔA value (the difference between DNA/methyl
green complex and free cabinol) provides the simplest means for
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
16

N.E.A. Abd El-Sattar et al.
Bioorganic Chemistry 115 (2021) 105186
[18] Ajmal R. Bhat, Rajendra S. Dongre, Faisal A. Almalki, Malika Berredjem,
Mohamed Aissaoui, Rachid Touzani, Taibi Ben Hadda, Mohammad S. Akhter,
Synthesis, biological activity and POM/DFT/docking analyses of annulated pyrano
[2,3-d]pyrimidine derivatives: Identification of antibacterial and antitumor
pharmacophore sites, Bioorg. Chem. 106 (2021) 104480, https://doi.org/10.1016/
j.bioorg.2020.104480.
Acknowledgments
The authors greatly thank Taif University for providing funds for this
study through Taif University Researchers Supporting Project number
(TURSP-2020/52), Taif University, Taif, Saudi Arabia.
[19] J. L. Delgado, et al., Topoisomerases as anticancer targets. Biochem J. 475(2) 2018
373-398. doi: 10.1042/BCJ20160583. PMID: 29363591; PMCID: PMC6110615.
[20] R. Martinez, L. Chacon-Garcia, The search of DNA-intercalators as antitumoral
drugs: what it worked and what did not work, Curr. Med. Chem. 12 (2005)
127–151.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105186.
[21] L F Liu, DNA topoisomerase poisons as antitumor drugs, Annu. Rev. Biochem. 58
(1) (1989) 351–375.
[22] Adriana Chilin, Giovanni Marzaro, Christine Marzano, Lisa Dalla Via, Maria
Grazia Ferlin, Giovanni Pastorini, Adriano Guiotto, Synthesis and antitumor
activity of novel amsacrine analogs: the critical role of the acridine moiety in
determining their biological activity, Bioorg. Med. Chem. 17 (2) (2009) 523–529.
[23] T.D. Shenkenberg, D.D. Von Hoff, Mitoxantrone: a new anticancer drug with
significant clinical activity, Ann. Intern. Med. 105 (1986) 67–81.
[24] D.E. Graves, L.M. Velea, Intercalative binding of small molecules to nucleic acids,
Curr. Org. Chem. 4 (2000) 915–929.
References
[1] M. Xin, L. Zhang, H. Shen, J. Wen, C. Tu, Z. Liu, L. Cheng, X. Zhao, Design,
synthesis, and biological study of 6,7-dihydro-5H-pyrano[2,3-d]pyrimidine
derivatives as novel hedgehog signaling pathway inhibitors, Med. Chem. Res. 23
(8) (2014) 3784–3792, https://doi.org/10.1007/s00044-014-0959-3.
[2] P.K. Paliwal, S.R. Jetti, S. Jain, Green approach towards the facile synthesis of
dihydropyrano(c)chromene and pyrano[2,3-d]pyrimidine derivatives and their
biological evaluation, Med. Chem. Res. 22 (6) (2013) 2984–2990, https://doi.org/
10.1007/s00044-012-0288-3.
[25] Federico Gago, Stacking interactions and intercalative DNA binding, Methods 14
(3) (1998) 277–292.
´
[26] C. Avendano, J. Menendez, DNA Intercalators and topoisomerase inhibitors,
Medicinal Chemistry of Anticancer Drugs, 2nd edition,, Elsevier, 2008,
pp. 199–228.
[3] K. Yalagala, S. Maddila, S. Rana, S.N. Maddila, S. Kalva, A.A. Skelton, S.
B. Jonnalagadda, Synthesis, antimicrobial activity and molecular docking studies
of pyrano[2,3-d]pyrimidine formimidate derivatives, Res. Chem. Intermed. 42 (4)
(2016) 3763–3774, https://doi.org/10.1007/s11164-015-2243-7.
[4] Y.M. Litvinov, A.M. Shestopalov, Convenient selective synthesis of pyrano[2,3-d]
pyrimidines, Russ. Chem. Bull. 57 (10) (2008) 2223–2226, https://doi.org/
10.1007/s11172-008-0308-0.
[27] G. Minotti, et al., Anthracyclines: molecular advances and pharmacologic
developments in antitumor activity and cardiotoxicity, Pharmacol. Rev. 56 (2004)
185–229.
[28] J. Gallego, et al., Structure–affinity relationships for the binding of actinomycin D
to DNA, J. Comput. Aided Mol. Des. 11 (1997) 114–128.
[29] S.A. Bailey, et al., Binding of actinomycin D to the T (G) nT motif of double-
stranded DNA: determination of the guanine requirement in nonclassical, non-GpC
binding sites, Biochemistry 33 (1994) 11493–11500.
[5] G. Devasahayam, W.M. Scheld, P.S. Hoffman, Newer antibacterial drugs for a new
century, Expert Opin. Invest. Drugs 19 (2) (2010) 215–234, https://doi.org/
10.1517/13543780903505092. PMID: 20053150; PMCID: PMC2831224.
[6] C. Walsh, Where will new antibiotics come from? Nat. Rev. Microbiol. 1 (1) (2003)
65–70, https://doi.org/10.1038/nrmicro727. PMID: 15040181.
[30] V.K. Bajpai, S.M. Al-Reza, U.K. Choi, J.H. Lee, S.C. Kang, Chemical composition,
antibacterial and antioxidant activities of leaf essential oil and extracts of
Metasequioa glyptostroboides Miki ex Hu, Food Chem. Toxicol. 47 (8) (2009)
1876–1883, https://doi.org/10.1016/j.fct.2009.04.043. Epub 2009 May 6 PMID:
19426779.
[7] S. Donadio, S. Maffioli, P. Monciardini, M. Sosio, D. Jabes, Sources of novel
antibiotics–aside the common roads, Appl. Microbiol. Biotechnol. 88 (6) (2010)
1261–1267, https://doi.org/10.1007/s00253-010-2877-8. Epub 2010 Sep 24
PMID: 20865256.
[31] Y. Yang, A. Severin, R. Chopra, G. Krishnamurthy, G. Singh, W. Hu, D. Keeney,
K. Svenson, P.J. Petersen, P. Labthavikul, D.M. Shlaes, B.A. Rasmussen, A.A. Failli,
J.S. Shumsky, K.M. Kutterer, A. Gilbert, T.S. Mansour, 3,5-dioxopyrazolidines,
novel inhibitors of UDP-N- acetylenolpyruvylglucosamine reductase (MurB) with
activity against gram-positive bacteria, Antimicrob. Agents Chemother. 50 (2)
(2006) 556–564, https://doi.org/10.1128/AAC.50.2.556-564.2006. PMID:
16436710; PMCID: PMC1366903.
[8] S. Donadio, S. Maffioli, P. Monciardini, M. Sosio, D. Jabes, Antibiotic discovery in
the twenty-first century: current trends and future perspectives, J. Antibiot.
(Tokyo) 63 (8) (2010) 423–430, https://doi.org/10.1038/ja.2010.62. Epub 2010
Jun 16 PMID: 20551985.
[9] T. Villa, P. Veiga-Crespo, Enzybiotics: Antibiotic Enzymes as Drugs and
Therapeutics, John Wiley & Sons Inc, Canada, 2010, pp. 1–58.
[32] S.Y. Shin, V.K. Bajpai, H.R. Kim, S.C. Kang, Antibacterial activity of bioconverted
eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) against foodborne
pathogenic bacteria, Int. J. Food Microbiol. 113 (2) (2007) 233–236, https://doi.
org/10.1016/j.ijfoodmicro.2006.05.020.
[10] K. El-Adl, A.-G.-A. El-Helby, H. Sakr, A. Elwan, Design, synthesis, molecular
docking and anti-proliferative evaluations of [1,2,4]Triazolo[4,3-a]quinoxaline
and [1,2,4]triazolo[4,3-a]quinoxaline-1-thiol derived DNA intercalators: Design,
synthesis, molecular docking, in silico ADMET profile and antiproliferative
evaluations, New J Chemistry (2020), https://doi.org/10.1039/D0NJ02990D.
[11] Ibrahim. H. Eissa, Ahmed. M. Metwaly, Amany Belal, Ahmed. B. M. Mehany, Rezk
R. Ayyad, Khaled El-Adl, Hazem. A. Mahdy, Mohammed. S. Taghour, Kamal M. A.
El-Gamal, Mohamad E. El-Sawah, Souad A. Elmetwally, Mostafa. A. Elhendawy,
Mohamed. M. Radwan, Mahmoud. A. ElSohly. Discovery and anti-proliferative
evaluation of new quinoxalines as potential DNA intercalators and topoisomerase II
inhibitors. Archiv der Pharmazie, 2019;352:1900123. DOI:10.1002/
ardp.201900123.
[33] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1–2) (1983)
55–63.
[34] D.A. Scudiero, et al., Evaluation of a soluble tetrazolium/formazan assay for cell
growth and drug sensitivity in culture using human and other tumor cell lines,
Cancer Res. 48 (17) (1988) 4827–4833.
[35] F.M. Freimoser, C.A. Jakob, M. Aebi, U. Tuor, The MTT [3-(4,5-Dimethylthiazol-2-
yl)-2,5-Diphenyltetrazolium Bromide] Assay Is a Fast and Reliable Method for
Colorimetric Determination of Fungal Cell Densities, Appl. Environ. Microbiol. 65
(8) (1999) 3727–3729.
[12] Boda SK, Pishka V, Lakshmi PVA, Chinde S, Grover P. 1,2,3-Triazole Tagged 3H-
Pyrano[2,3-d]pyrimidine-6-carboxylate Derivatives: Synthesis, in Vitro
Cytotoxicity, Molecular Docking and DNA Interaction Studies. Chem Biodivers.
2018 Jun;15(6):e18000101. doi: 10.1002/cbdv.201800101. Epub 2018 May 23.
PMID: 29660245.
´
[36] Mariana Franzoni Maioral, Camila do Nascimento Bodack, Natalia
´
Marceli Stefanes, Alisson Bigolin, Alessandra Mascarello, Louise
´
Domeneghini Chiaradia-Delatorre, Rosendo Augusto Yunes, Ricardo Jose Nunes,
´
Maria Claudia Santos-Silva, Cytotoxic effect of a novel naphthylchalcone against
multiple cancer cells focusing on hematologic malignancies, Biochimie 140 (2017)
48–57, https://doi.org/10.1016/j.biochi.2017.06.004.
´
´
ˇ
´
ˇ ´
[13] V. Markovic, N. Debeljak, T. Stanojkovic, B. Kolundzija, D. Sladic, M. Vujcic,
´
´
´
ˇ ´
´
´
B. Janovic, N. Tanic, M. Perovic, V. Tesic, J. Antic, M.D. Joksovic, Anthraquinone-
chalcone hybrids: synthesis, preliminary antiproliferative evaluation and DNA-
interaction studies, Eur. J. Med. Chem. 7 (89) (2015) 401–410, https://doi.org/
10.1016/j.ejmech.2014.10.055. Epub 2014 Oct 18 PMID: 25462255.
[14] K. El-Adl, M.K. Ibrahim, M.S.I. Alesawy, I.H. Eissa, [1,2,4]Triazolo[4,3-c]
quinazoline and bis([1,2,4]triazolo)[4,3-a:4’,3’-c]quinazoline derived DNA
intercalators: Design, synthesis, in silico ADMET profile, molecular docking and
anti-proliferative evaluation studies, Bioorg. Med. Chem. 30 (2021), 115958,
https://doi.org/10.1016/j.bmc.2020.115958. Epub 2020 Dec 21 PMID: 33360576.
[15] H.M. Aly, M.M. Kamal, Efficient one-pot preparation of novel fused chromeno[2,3-
d]pyrimidine and pyrano[2,3-d]pyrimidine derivatives, Eur. J. Med. Chem. 47 (1)
(2012) 18–23, https://doi.org/10.1016/j.ejmech.2011.09.040.
[37] N.S. Burres, et al., A colorimetric microassay for the detection of agents that
interact with DNA, J. Nat. Prod. 55 (1992) 1582–1587.
[38] K. El-Adl, A.-G.A. El-Helby, H. Sakr, A. Elwan, Design, synthesis, molecular
docking and anti-proliferative evaluations of [1,2,4]triazolo[4,3-a]quinoxaline
derivatives as DNA intercalators and Topoisomerase II inhibitors, Bioorg. Chem.
105 (2020), 104399.
[39] C.-C. Wu, T.-K. Li, L. Farh, L.-Y. Lin, T.-S. Lin, Y.-J. Yu, T.-J. Yen, C.-W. Chiang, N.-
L. Chan, Structural basis of type II topoisomerase inhibition by the anticancer drug
etoposide, Science 333 (6041) (2011) 459–462, https://doi.org/10.1126/science:
1204117.
[40] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Experimental and
computational approaches to estimate solubility and permeability in drug
discovery and development settings, Adv. Drug Deliv. Rev. 23 (1–3) (1997) 3–25,
https://doi.org/10.1016/S0169-409X(96)00423-1.
[16] Majid Ghashang, Syed Sheik Mansoor, Krishnamoorthy Aswin,
Pentafluorophenylammonium triflate (PFPAT) catalyzed facile construction of
substituted chromeno[2,3-d]pyrimidinone derivatives and their antimicrobial
activity, J. Adv. Res. 5 (2) (2014) 209–218, https://doi.org/10.1016/j.
jare.2013.03.003.
[41] D.E.V. Pires, T.L. Blundell, D.B. Ascher, pkCSM: Predicting Small-Molecule
Pharmacokinetic and Toxicity Properties Using Graph-Based Signatures, J. Med.
[17] K. Yalagala, S. Maddila, S. Rana, et al., Synthesis, antimicrobial activity and
molecular docking studies of pyrano[2,3-d]pyrimidine formimidate derivatives,
Res. Chem. Intermed. 42 (2016) 3763–3774, https://doi.org/10.1007/s11164-
015-2243-7.
17

N.E.A. Abd El-Sattar et al.
Bioorganic Chemistry 115 (2021) 105186
Chem.. 58 (9) (2015) 4066–4072, https://doi.org/10.1021/acs.
jmedchem.5b00104.
Based Formulation, PLoS ONE 8 (10) (2013), e68237, https://doi.org/10.1371/
journal.pone.0068237.
[42] R. Beig, A. Agbaria, Dahan, Oral Delivery of Lipophilic Drugs: The Tradeoff
between Solubility Increase and Permeability Decrease When Using Cyclodextrin-
[43] G. Lewy and A. McAllan, Colorimetric methods for free and acetylated
hexosamines: In Methods in Enzymology. vol.III, Academic Press Inc., New York.
USA, 1959, pp. 58-66.
18