Synthesis of chloro, fluoro, and nitro derivatives of 7-amino-5-aryl-6-cyano-5H-pyrano pyrimidin-2,4-diones using organic catalysts and their antimicrobial and anticancer activities

Article abstract of DOI:10.1002/jhet.3695

Chloro, fluoro, and nitro derivatives of 7-amino-5-aryl-6-cyano-5H-pyrano pyrimidin-2,4-diones were produced by reacting malononitrile, barbituric acid, and aromatic aldehydes together with a DABCO catalyst in an aqueous one-pot reaction. This is the firs

Full text of DOI:10.1002/jhet.3695

Received: 3 March 2019
DOI: 10.1002/jhet.3695
Revised: 10 August 2019
Accepted: 12 August 2019
A R T I C L E
Synthesis of chloro, fluoro, and nitro derivatives of 7‐amino‐
5‐aryl‐6‐cyano‐5H‐pyrano pyrimidin‐2,4‐diones using organic
catalysts and their antimicrobial and anticancer activities
Oluwole S. Aremu | Parvesh Singh | Moganavelli Singh | Chunderika Mocktar |
Neil A. Koorbanally
School of Chemistry and Physics, Life
Abstract
Science and Health Science, University of
Chloro, fluoro, and nitro derivatives of 7‐amino‐5‐aryl‐6‐cyano‐5H‐pyrano
pyrimidin‐2,4‐diones were produced by reacting malononitrile, barbituric acid,
and aromatic aldehydes together with a DABCO catalyst in an aqueous one‐pot
reaction. This is the first report of these compounds being synthesized with
DABCO as a catalyst, which produced the compounds in yields in excess of
90%. The 2,4‐difluoro derivative (11) was novel. The structures of the synthe-
sized compounds were elucidated by means of 1H, 13C, and 2D NMR spectros-
copy. Compound 2 (2‐Cl derivative) had MBC values of <200μM against both
Staphylococcus aureus and MRSA, and the 2‐nitro derivative 5 had an MBC
of 191μM against the Gram–ve Escherichia coli. The synthesized compounds
were also tested for their anticancer activity against a HeLa cell line, where
all the compounds showed better activity (IC50 values between 129μM and
340μM) than 5‐fluorouracil, a commonly known anticancer drug.
KwaZulu‐Natal, Durban 4000, South
Africa
Correspondence
Neil A. Koorbanally, School of Chemistry
and Physics, Life Science and Health
Science, University of KwaZulu‐Natal,
Private Bag X54001, Durban 4000, South
Africa.
Email: koorbanally@ukzn.ac.za
Funding information
National Research Foundation, South
Africa, Grant/Award Numbers: 114817
and 118534
1 | INTRODUCTION
intermediates.[8] Pyrano[2,3‐d]pyrimidines are commonly
formed from barbituric acid, malonitrile, and aldehdyes with
Pyrimidines and its analogues are an important class of
heterocyclic compounds, and their structural skeleton is
a key constituent of nucleic acids, alkaloids, and numer-
ous other pharmacophores with a variety of potent biolog-
ical activities.[1] Pyrano[2,3‐d]pyrimidines consists of a
pyran ring fused together with pyrimidine. As such, their
basic framework consists of both nitrogen and oxygen in
their carbocyclic structure. This pyrimidine‐annulated
derivative is known to possess good pharmacological activ-
ity such as antioxidant, antidiabetic,[2] antimicrobial,[3,4]
anti‐inflammatory, analgesic,[5] moderate anticancer,[3]
antimycobacterial,[6] and antimalarial activity.[7]
a catalyst such as 1,4‐diazabicyclo[2,2,2]octane (DABCO), an
organic catalyst,[9] a mesoporous solid acid catalyst (SBA‐15‐
Pr‐SO3H),[10] a ZnFe2O4 nanocatalyst,[11] a nano‐sawdust‐
OSO3H catalyst,[12] a Mn‐doped ZrO2 catalyst,[13] or
triethylammonium acetate (a green catalyst).[4] The same
compounds were made using arylidenemalonitrile with
barbituric acid under microwave irradiation without a cata-
lyst[14] or using ionic liquid catalysts such as N‐butyl‐N‐
methyl imidazole tetrafluoroborate [BMIm]BF4, N‐ethyl‐N‐
methyl imidazole tetrafluoroborate [EMIm]BF4, or N‐butyl
pyridinium tetrafluoroborate [BPy]BF4.[15] The resultant
pyrimidine diones usually have a substituted phenyl group
at position 5 and reactive nitrile and amino groups at C‐6
and C‐7 on the pyrano[2,3‐d]pyrimidine skeleton.
Pyrimidines are generally synthesized by a Biginelli multi-
component reaction (MCR), a method considered green since
they are fast, solvent free, produces good yields, and avoids
exposing the environment to environmentally harmful
By reacting ethyl 2‐cyanoacetate with barbituric acid
and substituted benzaldehydes in the presence of
J Heterocyclic Chem. 2019;1–9.
wileyonlinelibrary.com/journal/jhet
© 2019 Wiley Periodicals, Inc.
1

2
AREMU ET AL.
DABCO, an ethyl ester group was placed at C‐6 instead of
the nitrile group.[16] Pyrano[2,3‐d]pyrimidines with phe-
nyl groups at both positions 5 and 7 were synthesized
by the reaction of chalcones with barbituric acid in the
presence of acetic acid and P2O5 as a catalyst.[17]
comparable yields using a variety of methods or catalysts
(Table 1), including microwave synthesis without the use
of a catalyst,[14] with the ionic liquids 1‐n‐butyl‐3‐
methylimidazolium tetrafluoroborate and 1‐butylpy‐
ridinium tetrafluoroborate,[15] a Zn [(L) proline] cata-
Other pyrimidines with substituted phenyl groups were
synthesized in a one‐pot reaction with 1‐nitroguanidine,
malonitrile, and substituted benzaldehydes under basic
conditions in a one‐pot reaction with short reaction times,
mild reaction conditions, and excellent yields[18] or with
guanidine nitrate, ethylcyanoacetate, and aldehydes using
piperidine as a catalyst.[19] Spiropyrimidinones were syn-
thesized using urea instead of malonitrile together with
barbituric acid and benzaldehydes in the presence of a
lyst,[24] with glycerol without the use of a catalyst,[25]
[26]
nanocatalysts Fe3O4
and SBA‐15‐Pr‐SO3H (a mesopo-
rous solid acid catalyst),[10] and ethyl cyanoacetate.[27]
DABCO has been used previously for the synthesis of
furan‐2‐yl, pyrrol‐2‐yl and thiophen‐2‐yl derivatives of
pyrano pyrimidin‐2,4‐diones in a one pot reaction.
Plausible mechanisms for the reaction are provided in
Bhat et al[16] and Jain et al[9]; however, these mechanisms
are either unclear or have missing steps. A more compre-
hensive mechanism is presented in Figure 2, involving for-
mation of an arylidene intermediate, which is subject to a
Michael reaction by barbituric acid. Cyclisation involving
the hydroxy and cyano groups ultimately lead to the prod-
ucts. In order to test the mechanism, we performed several
experiments with compound 4, keeping the reaction time
constant at 1 hour. This produced a yield of 94% in the
one‐pot reaction. With anhydrous ethanol, the reaction
occurred, but in a very low yield (23%), presumably
because the anhydrous ethanol is not as good a proton
donor as water. We then tried THF and water as a solvent,
and the reaction proceeded similarly to the original reac-
tion in a yield of 92%. We then added the aldehyde to the
reaction, 30 minutes after mixing the malonitrile, DABCO,
and barbituric acid. The yield was lower, 58%. Finally, we
added the malonitrile 30 minutes after mixing the alde-
hyde, DABCO, and barbituric acid. Again, the yield was
lower (42%) than the original reported method.
nanoporous solid acid catalyst (SBA‐Pr‐SO3H).[20]
A
totally different synthesis to pyrano[2,3‐d]pyrimidines
was achieved using a one‐pot three‐component reaction
of 2‐amino‐7‐methyl‐5‐oxo‐4‐phenyl‐4,5‐dihydropyran-
o[4,3‐b]pyran‐3‐carbonitriles, N,N‐dimethylacetaldehyde
dimethyl acetal, and aromatic amines in the presence of
1‐butyl‐3‐methylimidazole hydrogen sulphate (an ionic
liquid) as a catalyst.[21] Another synthetic report makes
use of N,N‐dimethyl‐5‐formylbarbituric acid with
maleimide and phenyl isocyanate under microwave irradi-
ation to afford pyrano[2,3‐d]pyrimidines in good yields.[22]
We herein report the synthesis of a small library of
chloro, nitro, and fluoro 7‐amino‐5‐aryl‐6‐cyano‐5H‐
pyrano[2,3‐d]pyrimidin‐(1H,3H)‐2,4‐diones
using
DABCO as a catalyst together with their antibacterial
and anticancer activity. This paper follows our earlier
report on the synthesis, anticancer, and antibacterial
activity of oxygenated pyrimidin‐2,4‐diones.[23]
We had previously investigated four basic organic cata-
lysts, together with K2CO3 to identify the catalyst among
them that produced the greatest yield in the shortest
amount of time.[23] We studied L‐proline, dibutylamine
(DBA), triethylamine (Et3N), and 1,4‐diazabicyclo [2.2.2]
octane (DABCO) and found that DBA, Et3N, and DABCO
all produced yields of over 70% in 1 hour; however,
DABCO was the best of the three, resulting in 94% in
38 minutes.
2 | RESULTS AND DISCUSSION
2.1 | Chemistry
The pyrimidin‐2,4‐diones (1–11) were synthesized using
malonitrile, barbituric acid, and chloro, fluoro, and nitro
benzaldehydes in ethanol (Figure 1). DABCO was used
as an organic catalyst, producing yields of 90% to 96%
(Table 1). NMR spectroscopy was used to confirm the
structures of the compounds, and the structural elucida-
tion was discussed in our previous publication.[23]
3 | ANTIMICROBIAL ACTIVITY
With the exception of the novel, 2,4‐difluoro derivative
11, all compounds were synthesized previously in
The synthesized compounds were tested for their antimi-
crobial activity against two Gram +ve and three Gram–ve
FIGURE 1 Synthetic scheme for
chloro, fluoro, and nitro pyrano
pyrimidines

AREMU ET AL.
3
TABLE 1 Yields and melting points of aromatic pyrano [2,3‐d] pyrimidines catalyzed by DABCOa in comparison with other reported
catalysts and methods
Entry
R
Yield
mp, °C
Reference
Yield
Catalyst/method
1
H
90
205‐208
Gao et al[14]
92
84
85
65
Microwavec
ILsd
Zn [(L) proline]
SBA‐Pr‐SO3He
Yu and Wang[15]
Heravi et al[24]
Ziarani et al[20]
2
2‐Cl
93
212‐215
Gao et al[14]
Yu and Wang[15]
Safaei et al[25]
89
94
90
Microwavec
ILsd
Glycerolc
3
4
3‐Cl
4‐Cl
95
94
223‐225
235‐237
Safaei et al[25]
Kidwai et al[26]
89
94
Glycerolc
Fe3O4
e
Gao et al[14]
92
92
90
30
91
97
Microwavec
ILsd
Yu and Wang[15]
Heravi et al[24]
Ziarani et al[20]
Safaei et al[25]
Kidwai et al[26]
Zn [(L) proline]
SBA‐Pr‐SO3He
Glycerolc
e
Fe3O4
5
6
2‐NO2
3‐NO2
96
90
258‐259
256‐258
Safaei et al[25]
92
Glycerolc
Heravi et al[24]
Ziarani et al[20]
Safaei et al[25]
90
80
94
Zn [(L) proline]
SBA‐Pr‐SO3He
Glycerolc
7
4‐NO2
93
225‐228
Gao et al[14]
86
92
90
95
95
Microwavec
Zn [(L) proline]
SBA‐Pr‐SO3He
Glycerolc
Heravi et al[24]
Ziarani et al[20]
Safaei et al[25]
Kidwai et al[26]
e
Fe3O4
8
2‐F
3‐F
4‐F
90
96
92
226‐229
224‐226
228‐230
Commercially availableb
Sharanin and Klokol[27]
9
90
Ethyl cyanoacetate
10
Gao et al[14]
Yu and Wang[15]
91
90
Microwavec
ILsd
11
2,4‐diF
92
250‐252
Novel
‐
aReaction conditions: barbituric acid (1.0 mmol), substituted benzaldehydes (1.0 mmol), and malononitrile (1.1 mmol), EtOH:water (1:1 v/v, 10 mL), room
temp.;
bAkos Consulting and Solutions; Sigma Aldrich;
cno catalyst;
dionic liquids – 1‐n‐butyl‐3‐methylimidazolium tetrafluoroborate and 1‐butylpyridinium tetrafluoroborate;
enanocatalyst
strains of bacteria. The compounds were initially
screened for their antimicrobial potential using the disk
diffusion assay, where compounds showing a mean inhi-
bition zone of greater than 9 mm were selected to deter-
mine their minimum bactericidal concentrations
(MBCs). In general, the compounds were more active
against the Gram +ve Staphylococcus aureus and MRSA
than the Gram –ve strains (Table 2). Compound 2, the
2‐chloro derivative, was active at <200μM for both the
Gram +ve strains. Several of the compounds were also
active at <200μM against MRSA, showing better activity
than ampicillin. Only compound 5, the 2‐nitro derivative
showed activity <200μM against E coli. None of the other
compounds showed any appreciable activity to the Gram
–ve strains.
4 | MTT CYTOTOXICITY ASSAY
The in vitro cytotoxicity levels of pyrimidinones 1‐11
against HeLa cells are summarized in Table 3 below.
Most of the compounds tested showed a dose‐dependent
cytotoxicity profile. Based on the IC50 values, it can be
deduced that all compounds are most active at

4
AREMU ET AL.
FIGURE 2 A plausible mechanism for the one pot reaction of malonitrile, barbituric acid, and benzaldehydes in the presence of DABCO
TABLE 2 Antibacterial activity of the synthesized compounds
Minimum Bactericidal Concentration, μM
Bacterial strains
Compounds
2
3
4
5
8
11
AMP
CIP
Gram + ve
Staphylococcus aureus
MRSA
197
197
394
197
394
394
382
191
208
196
393
56
1.84
7.36
1665
894
Gram −ve
Escherichia coli
Pseudomonas aeruginosa
Klebsiella pneumoniae
1579
1579
394
1579
1579
789
789
191
1665
1665
1665
786
447
1.84
1.84
3.68
789
1528
382
1571
1571
1788
447
1579
Note. Data are reported as the average of duplicate readings
concentrations greater than 100 μg mL−1. The activities of
all compounds were better than 5‐fluorouracil (5‐FU), a
commonly used anticancer drug, with IC50 values ranging
from 129 to 340μM. 5‐FU had an IC50 in the same assay at
480μM. The activities of the compounds could be due to
the pyranopyrimidinone core structure, with aryl groups
at C‐5, to which electron withdrawing groups were
attached. Compounds 3, 4, 7, and 8 were active at lower
concentrations, indicating promise as anticancer agents.
Further studies are however needed before this conclu-
sion can be made.
5 | MOLECULAR DOCKING
STUDIES
In order to support the experimental anticancer activity of
the synthesized compounds and to predict their

AREMU ET AL.
5
TABLE 3 Viabilities (%) of the HeLa cell lines at different concentrations of compounds 1‐11
Cpd
10 μg mL−1
25 μg mL−1
50 μg mL−1
100 μg mL−1
IC50 μg mL−1
IC50 μM
1
95.58 0.02
96.72 0.01
79.11 0.01
72.08 0.02
98.22 0.03
95.81 0.02
86.65 0.02
81.04 0.02
87.88 0.02
88.55 0.02
84.79 0.01
90.28 0.01
78.40 0.03
85.82 0.0005
83.06 0.01
65.64 0.001
52.13 0.02
82.46 0.01
78.36 0.01
73.82 0.02
68.92 0.01
83.25 0.01
69.79 0.01
77.37 0.02
85.15 0.01
58.89 0.03
70.10 0.01
65.40 0.02
53.20 0.02
37.99 0.02
69.79 0.02
69.31 0.02
62.80 0.01
50.87 0.03
69.91 0.01
57.86 0.01
63.67 0.02
71.56 0.01
50.47 0.02
41.35 0.02
40.48 0.01
43.33 0.04
27.84 0.02
46.68 0.02
57.15 0.02
40.64 0.02
42.54 0.03
50.91 0.03
41.19 0.01
52.41 0.02
71.56 0.02
38.00 0.02
84.92
81.34
74.02
40.67
90.86
111.27
78.81
72.54
101.11
96.51
101.2
108.75
62.41
301
257
234
129
278
340
241
242
337
322
318
339
480
2
3
4
5
6
7
8
9
10
11
12
5‐FU
Note. Data are reported as the standard deviation of triplicate readings.
mechanism of action, we docked two representative com-
pounds (RCs) 4 (4‐Cl) and 8 (2F), observed to be potent
under in vitro conditions, into the binding site of human
kinesin protein, Eg5. Inhibitors of human kinesin protein,
through cancer cell line screening resulted in the develop-
ment of new anticancer therapeutic agents.[28,29] Thus,
this enzyme was used in our docking studies. The CDocker
docking method embedded in the Discovery Studio (DS)
was used for all docking simulations. The docking results
obtained suggested both compounds to be strong inhibi-
tors of Eg5 based on the computed binding energy (BE)
data. The most active compound 4 with BE of value
−184.4 kcal mol−1 exhibited a stronger interaction than
its structural analogue 8 (BE = −150.1 kcal mol−1). Both
the RCs exhibited stronger binding affinity for Eg5 relative
to the standard drug, 5‐FU (BE = −116.7 kcal mol−1).
To understand the host‐guest relationship between
ligand and receptor, the docked complexes of both RCs
were further visualized using DS visualizer and are dia-
grammatically represented in Figures 3 and 4. Compound
4 (Figure 3) exhibited two concurrent hydrogen bond
interactions through its protonated amine functionality
(NH2) with Glu116 (1.81 Å) and Gly117 (1.98 Å) amino
acid residues of Eg5. Additionally, an electrostatic inter-
action between 4 and Glu116 including several hydropho-
bic forces (with Pro137, Trp127, Arg119, Tyr211, and
Ala133) were also observed. Similarly, 8 (Figure 4)
interacted with Eg5 through two hydrogen bonds; one
conventional with Arg221 (1.17 Å) through its nitrile
group and another nonconventional with Arg119
(2.80 Å) through the nitrogen atom of the pyrimidine
ring. In addition, two electrostatic interactions (with
FIGURE 3 Docked pose of 4 in the binding site of Eg5.
Hydrophobic and electrostatic interactions are shown as magenta
and red dotted lines, respectively. Conventional hydrogen bonds
are shown as green [Color figure can be viewed at
wileyonlinelibrary.com]
FIGURE 4 Docked pose of 8 in the binding site of Eg5.
Hydrophobic and electrostatic interactions are shown as magenta
and red dotted lines, respectively. Conventional hydrogen bonds are
shown as green, and nonconventional hydrogen bonds are shown as
gray [Color figure can be viewed at wileyonlinelibrary.com]

6
AREMU ET AL.
Glu116 and Arg221) and hydrophobic forces (with
Tyr211, Ala218, and Pro137) were also observed.
7.1.2 | 7‐Amino‐5‐(2‐chlorophenyl)‐6‐
cyano‐5H‐pyrano[2,3‐d]pyrimidin‐(1H,3H)‐
2,4‐dione (2)
White powder (93%); mp 212°C‐215°C; UV λmax (DMSO)
nm (log ε) 259 (2.74); IR (KBr) υmax: 3013 (NH), 2192
6 | CONCLUSION
1
(CN), 1714 (C=O), 1673 (C=O) cm−1; H NMR (DMSO‐
An environmentally friendly synthesis of pyrano [2,3‐d]
pyrimidinone derivatives in a one‐pot reaction using an
organic catalyst, DABCO, produced chloro, fluoro, and
nitro pyrimidin‐2,4‐diones in high yields (>90%). The
synthesized compounds showed good anticancer activity
against HeLa cells. The most active compounds also
showed good binding affinity to the human kinesin pro-
tein, Eg5, which supported our experimental findings.
d6, 400 MHz) δ 12.07 (brs, NH), 11.04 (s, NH), 7.36 (1H,
d, J = 8.0 Hz, H‐6′), 7.21‐7.27 (3H, m, H‐3′,4′,5′), 7.12
(s, NH2‐11), 4.72 (1H, s, H‐5); 13C NMR (DMSO‐d6,
100 MHz) δ 162.2 (C‐4), 157.8 (C‐7), 152.7 (C‐8), 149.5
(C‐2), 140.7 (C‐1′), 132.2 (C‐2′), (130.4, 129.5, 128.4,
127.4 (4CH, C‐3′,4′,5′,6′)), 118.7 (C‐10), 87.5 (C‐9), 56.0
(C‐6), 30.6 (CH, C‐5); EIMS (m/z, rel. int.) 316 (M+)
(14), 281 (31), 207 (100), 273 (14), 189 (15); Anal. calcd.
for C14H9ClN4O3: C: 53.09, H: 2.86, N: 17.69, Found: C:
53.00, H: 2.79, N: 17.60.
7 | EXPERIMENTAL
7.1 | General
7.1.3 | 7‐Amino‐5‐(3‐chlorophenyl)‐6‐
cyano‐5H‐pyrano[2,3‐d]pyrimidin‐(1H,3H)‐
2,4‐dione (3)
Chemicals and reagents were purchased from Sigma
Aldrich. Organic solvents were redistilled and dried
according to standard procedures. NMR spectra were
recorded using a Bruker AvanceIII 400‐MHz spectrome-
ter. Chemical shifts (δ) were reported against
tetramethylsilane (TMS), the internal standard. IR spectra
were obtained on a Perkin Elmer Spectrum 100 FTIR
spectrometer with universal ATR sampling accessory.
An Agilent GC‐MSD apparatus fitted with a DB‐5SIL
MS (30 m × 0.25 mm) fused‐silica capillary column was
White powder (95%); mp 223°C‐225°C; UV λmax (DMSO)
nm (log ε) 259 (2.69); IR (KBr) υmax: 3417 (NH), 3317
(NH), 2192 (CN), 1706 (C=O), 1660 (C=O) cm−1 1H
;
NMR (DMSO‐d6, 400 MHz) δ 12.07 (brs, NH), 11.07 (s,
NH), 7.32 (1H, t, J = 8.4 Hz, H‐5′), 7.26‐7.28 (2H, m, H‐
4′,6′), 7.19 (1H, s, H‐2′), 7.16 (s, NH2‐11), 4.26 (1H, s, H‐
5); 13C NMR (DMSO‐d6, 100 MHz) δ 162.4 (C‐4), 157.6
(C‐7), 152.4 (C‐8), 149.5 (C‐2), 146.6 (C‐1′), 132.8 (C‐3′),
130.1 (C‐4′), 127.2 (CH, C‐6′), 126.7 (CH, C‐2′), 126.1
(CH, C‐5′), 118.9 (C‐10), 87.7 (C‐9), 58.1 (C‐6), 35.4 (CH,
C‐5); EIMS (m/z, rel. int.) 316 (M+) (6), 281 (11), 207
(100), 188 (22), 153 (33); Anal. calcd. for C14H9ClN4O3:
C: 53.09, H: 2.86, N: 17.69, Found: C: 53.10, H: 2.59, N:
17.70.
used for GC‐MS analysis using helium (at 2 mL min−1
)
as a carrier gas. The MS was operated in the EI mode at
70 eV. An ErnstLeitzWetzlar micro hot stage melting
point apparatus was used to record melting points and
are uncorrected.
7.1.4 | 7‐Amino‐5‐(4‐chlorophenyl)‐6‐
cyano‐5H‐pyrano[2,3‐d]pyrimidin‐(1H,3H)‐
2,4‐dione (4)
7.1.1 | Synthesis of aromatic substituted
pyrano [2,3‐d] pyrimidinones (1‐11)
Substituted aromatic benzaldehydes (1.0 mmol each),
malononitrile (396 mg, 1.0 mmol), barbituric acid
(640 mg, 1.0 mmol each), and 10 mol% DABCO
(30.45 mg, 0.271 mmol) were added to 20‐mL aqueous
ethanol and the reaction mixture stirred for 1 hour at
room temperature. The progress of the reaction was mon-
itored by TLC. Upon precipitation, the products were
filtered and, thereafter, recrystallized in ethanol
before being dried at the vacuum pump. The NMR data
of 7‐amino‐5‐phenyl‐6‐cyano‐5H‐pyrano[2,3‐d]pyrimidin
‐(1H,3H)‐2,4‐dione (1) is reported in Aremu et al.[23]
White powder (94%); mp 235°C‐237°C; UV λmax (DMSO)
nm (log ε) 258 (2.68); IR (KBr) υmax: 3383 (NH2), 3186
(NH), 2197 (CN), 1717 (C=O), 1672 (C=O) cm−1 1H
;
NMR (DMSO‐d6, 400 MHz) δ 12.06 (brs, NH), 11.05 (s,
NH), 7.33 (2H, d J = 8.4 Hz, H‐3′/5′), 7.23 (2H, d,
J = 8.4 Hz, H‐2′/6′), 7.13 (s, NH2‐11), 4.24 (1H, s, H‐5);
13C NMR (DMSO‐d6, 100 MHz) δ 162.4 (C‐4), 157.6 (C‐
7), 152.3 (C‐8), 149.5 (C‐2), 143.1 (C‐1′), 131.2 (C‐4′),
129.3 (2CH, C‐2′/6′), 128.2 (2CH, C‐3′/5′), 119.0 (C‐10),
88.0 (C‐9), 58.3 (C‐6), 35.1 (CH, C‐5); EIMS (m/z, rel.
int.) 316 (M+) (15), 281 (33), 207 (100), 188 (25), 153

AREMU ET AL.
7
(23); Anal. calcd. for C14H9ClN4O3: C: 53.09, H: 2.66, N:
17.65, Found: C: 53.09, H: 2.86, N: 17.69.
J = 8.7 Hz, H‐2′/6′), 7.25 (s, NH2‐11), 4.41 (1H, s, H‐5);
13C NMR (DMSO‐d6, 100 MHz) δ 162.4 (C‐4), 157.7
(C‐7), 152.6 (C‐8), 151.7 (C‐4′), 149.5 (C‐2), 146.4 (C‐1′),
128.8 (2CH, C‐2′/6′), 123.5 (2CH, C‐3′/5′), 118.8 (C‐10),
87.4 (C‐9), 57.4 (C‐6), 35.6 (CH, C‐5); EIMS (m/z, rel.
int.): 327 (M+) (27), 207 (100); Anal. calcd. for
C14H9N5O5: C: 51.38, H: 2.77, N: 21.40, Found: C:
50.93, H: 2.37, N: 21.16.
7.1.5 | 7‐Amino‐5‐(2‐nitrophenyl)‐6‐cyano‐
5H‐pyrano[2,3‐d]pyrimidin‐(1H,3H)‐2,4‐
dione (5)
Off‐white powder (96%); mp 258°C‐259°C; UV λmax
(DMSO) nm (log ε) 260 (2.71); IR (KBr) υmax: 3365
1
(NH2), 2198 (CN), 1697 (C=O), 1618 (C=O) cm−1; H
NMR (DMSO‐d6, 400 MHz) δ 12.14 (brs, NH), 11.06 (s,
NH), 7.82 (1H, d, J = 8.3 Hz, H‐6′), 7.65 (1H, dd,
J = 8.1, 7.6 Hz, H‐4′), 7.49 (1H, d, J = 7.6 Hz, H‐3′),
7.45 (1H, dd, J = 8.3, 8.1 Hz, H‐5′), 7.27 (s, NH2‐11),
5.04 (1H, s, H‐5); 13C NMR (DMSO‐d6, 100 MHz) δ
162.4 (C‐4), 158.4 (C‐7), 152.3 (C‐8), 149.4 (C‐2), 149.2
(C‐2′), 138.1 (C‐1′), 133.3 (CH, C‐6′), 130.7 (CH, C‐4′),
128.0 (CH, C‐3′), 123.7 (CH, C‐5′), 118.6 (C‐10), 87.9 (C‐
9), 56.7 (C‐6), 30.3 (CH, C‐5); EIMS (m/z, rel. int.) 327
(M+) (14), 298 (32), 207 (100); Anal. calcd. for
C14H9N5O5: C: 51.38, H: 2.77, N: 21.40, Found: C: 51.04,
H: 2.47, N: 21.36.
7.1.8 | 7‐Amino‐5‐(2‐fluorophenyl)‐6‐
cyano‐5H‐pyrano[2,3‐d]pyrimidin‐(1H,3H)‐
2,4‐dione (8)
White powder (90%); mp 226‐229°C; UV λmax (DMSO)
nm (log ε) 260 (2.93); IR (KBr) υmax: 3421 (NH), 3303
(NH), 2203 (CN), 1716 (C=O), 1692 (C=O) cm−1 1H
;
NMR (DMSO‐d6, 400 MHz) δ 12.11 (brs, NH), 11.07 (s,
NH), 7.23‐7.28 (2H, m, H‐4′,6′), 7.14 (2H, s, NH2‐11),
7.07‐7.12 (2H, m H‐3′,5′), 4.50 (1H, s, H‐5); 13C NMR
(DMSO‐d6, 100 MHz)
δ
162.3 (C‐4), 160.1 (d,
J = 243.8 Hz, C‐2′), 157.9 (C‐7), 152.6 (C‐8), 149.5 (C‐2),
130.6 (d, J = 11.8 Hz, CH, C‐4′), 129.8 (d, J = 3.8 Hz,
C‐1′), 128.7 (d, J = 8.4 Hz, CH, C‐6′), 124.4 (d,
7.1.6 | 7‐Amino‐5‐(3‐nitrophenyl)‐6‐cyano‐
5H‐pyrano[2,3‐d]pyrimidin‐(1H,3H)‐2,4‐
dione (6)
J
= 3.0 Hz, CH, C‐5′), 118.9 (C‐10), 115.3 (d,
J = 21.7 Hz, CH, C‐3′), 87.2 (C‐9), 57.3 (C‐6), 30.6 (CH,
C‐5); EIMS (m/z, rel. int.): 300 (M+) (25), 281 (35), 226
(35), 207 (100), 167 (40), 159 (42); Anal. calcd. for
C14H9FN4O3: C: 56.00, H: 3.02, N: 18.66, Found: C:
56.10, H: 2.97, N: 18.02.
Off‐white powder (90%); mp 256°C‐258°C; UV λmax
(DMSO) nm (log ε) 262 (2.77); IR (KBr) υmax: 3414
(NH), 3202 (NH), 2192 (CN), 1707 (C=O), 1687 (C=O)
1
cm−1; H NMR (DMSO‐d6, 400 MHz) δ 12.15 (brs, NH),
11.09 (s, NH), 8.08 (1H, d, J = 8.1 Hz, H‐6′), 8.06 (1H,
brs, H‐2′), 7.74 (1H, d, J = 7.7, H‐4′), 7.60 (1H, dd,
J = 8.1, 7.7 Hz, H‐5′), 7.26 (s, NH2‐11), 4.47 (1H, s, H‐
5); 13C NMR (DMSO‐d6, 100 MHz) δ 162.5 (C‐4), 157.8
(C‐7), 152.5 (C‐8), 149.5 (C‐2), 147.7 (C‐1′), 146.4 (C‐3′),
134.7 (C‐2′), 129.8 (CH, C‐6′), 122.0 (CH, C‐4′), 121.9
(CH, C‐5′), 118.8 (C‐10), 87.4 (C‐9), 57.6 (C‐6), 35.4 (CH,
C‐5); EIMS (m/z, rel. int.): 327 (M+) (15), 281 (40), 207
(100); Anal. calcd. For C14H9N5O5: C: 51.38, H: 2.77, N:
21.40, Found: C: 51.01, H: 2.46, N: 21.24.
7.1.9 | 7‐Amino‐5‐(3‐fluorophenyl)‐6‐
cyano‐5H‐pyrano[2,3‐d]pyrimidin‐(1H,3H)‐
2,4‐dione (9)
White powder (96% yield); mp 224°C‐226°C; UV λmax
(DMSO) nm (log ε) 270 (2.93); IR (KBr) υmax: 3376
(NH), 3186 (NH), 2197 (CN), 1717 (C=O), 1674 (C=O)
1
cm−1; H NMR (DMSO‐d6, 400 MHz) δ 12.06 (brs, NH),
11.06 (s, NH), 7.30‐7.36 (1H, m, H‐5′), 7.14 (s, NH2),
7.01‐7.07 (3H, m, H‐2′,4′,6′), 4.27 (1H, s, H‐5); 13C NMR
(DMSO‐d6, 100 MHz)
δ
162.4 (C‐4), 162.1 (d,
7.1.7 | 7‐Amino‐5‐(4‐nitrophenyl)‐6‐cyano‐
5H‐pyrano[2,3‐d]pyrimidin‐(1H,3H)‐2,4‐
dione (7)
J = 241.9 Hz, C‐3′), 157.6 (C‐7), 152.4 (C‐8), 149.5 (C‐2),
147.0 (d, J = 6.3 Hz, C‐1′), 130.1 (d, J = 8.1 Hz, CH, C‐
5′), 123.4 (CH, C‐6′), 119.0 (C‐10), 114.0 (d, J = 21.5 Hz,
CH, C‐2′), 113.5 (d, J = 20.9 Hz, CH, C‐4′), 87.8 (C‐9),
58.2 (C‐6), 35.4 (CH, C‐5); EIMS (m/z, rel. int.): 300
(M+) (18), 281 (41), 207 (100), 172 (74); Anal. calcd. for
C14H9FN4O3: C: 56.00, H: 3.02, N: 18.66, Found: C:
55.97, H: 2.85, N: 18.39.
White powder (93%); mp 225–228°C; UV λmax (DMSO)
nm (log ε) 260 (2.84); IR (KBr) υmax: 3186 (NH), 2196
(CN), 1720 (C=O), 1671 (C=O) cm−1
;
1H NMR
(DMSO‐d6, 400 MHz) δ 12.15 (brs, NH), 11.10 (s, NH),
8.15 (2H, d, J = 8.7 Hz, H‐3′/5′), 7.52 (2H, d,

8
AREMU ET AL.
>9 mm and their MBC values determined. To calculate
the MBC values, the compounds were dissolved in DMSO
(4.0 mg mL−1) and serially diluted in Mueller‐Hinton
broth (Oxoid, England) in Eppendorf tubes. The method
according to Moodley et al[31] was used.
7.1.10 | 7‐Amino‐5‐(4‐fluorophenyl)‐6‐
cyano‐5H‐pyrano[2,3‐d]pyrimidin‐(1H,3H)‐
2,4‐dione (10)
White powder (92% yield); mp 228°C‐230°C; UV λmax
(DMSO) nm (log ε) 260 (2.93); IR (KBr) υmax: 3194
(NH), 2197 (CN), 1723 (C=O), 1677 (C=O) cm−1 1H
;
NMR (DMSO‐d6, 400 MHz) δ 12.06 (brs, NH), 11.05 (s,
NH), 7.25 (2H, dd, J = 8.6, 5.6 Hz, H‐2′/6′), 7.10 (2H, t,
J = 8.6 Hz, H‐3′/5′), 7.10 (s, NH), 4.26 (1H, s, H‐5); 13C
NMR (DMSO‐d6, 100 MHz) δ 162.4 (C‐4), 161.0 (d,
J = 240.8 Hz, C‐4′), 157.5 (C‐7), 152.2 (C‐8), 149.5 (C‐2),
140.1 (d, J = 2.8 Hz, C‐1′), 129.1 (d, J = 8.1 Hz, 2CH, C‐
2′/6′), 119.1 (C‐10), 114.8 (d, J = 21.2 Hz, 2CH, C‐3′/5′),
88.3 (C‐9), 58.7 (C‐6), 35.0 (CH, C‐5); EIMS (m/z, rel.
int.): 300 (M+) (10), 281 (13), 266 (19), 233 (19), 207
(100); Anal. calcd. for C14H9FN4O3: C: 56.00, H: 3.02, N:
18.66, Found C: 56.22, H: 2.65, N: 18.14.
7.3 | Cytotoxicity tests by the MTT assay
Human cervical cancer (HeLa) cells were purchased from
Highveld Biological (Pty) Ltd. (Lyndhurst, RSA). Cells
were grown, seeded, and incubated according to our pre-
vious method.[23] Compounds 1‐11 (initially dissolved in
DMSO at a concentration of 1 mg mL−1) were then added
in triplicate to the cells (containing 100 μL of fresh
medium) to a final concentration of 10, 25, 50, and
100 μg mL−1 and incubated for 48 hours at 37°C. 5‐FU
was used as a standard positive control. The MTT assay
was adapted from Mosmann et al[32] and Gichumbi
et al.[33] Tests were conducted in triplicate, and the IC50
values (concentration at which 50% cell death was
achieved) were determined using Microsoft Excel 2010.
7.1.11 | 7‐Amino‐5‐(2,4‐difluorophenyl)‐6‐
cyano‐5H‐pyrano[2,3‐d]pyrimidin‐(1H,3H)‐
2,4‐dione (11)
White powder (92% yield); mp 250°C‐252°C; UV λmax
(DMSO) nm (log ε) 265 (2.98); IR (KBr) υmax: 3395
(NH), 3306 (NH), 2195 (CN), 1718 (C=O), 1674 (C=O)
7.4 | Molecular docking
1
cm−1; H NMR (DMSO‐d6, 400 MHz) δ 12.10 (brs, NH),
11.08 (s, NH), 7.32 (ddd, J = 8.6, 8.6, 6.3, H‐6′), 7.15
(1H, ddd, J = 11.8, 9.4, 2.6, H‐3′), 6.99 (1H, ddd,
J = 8.4, 8.4, 2.4 Hz, H‐5′), 7.18 (s, NH2), 4.49 (s, H‐5);
13C NMR (DMSO‐d6, 100 MHz) δ 162.3 (C‐4), 161.1 (dd,
J = 243.0, 12.4 Hz, C‐2′*), 160.1 (dd, J = 246.5, 12.3 Hz,
C‐4′*), 157.9 (C‐7), 152.6 (C‐8), 149.5 (C‐2), 131.1(dd,
J = 9.6, 5.6 Hz, C‐6′), 127.1 (dd, J = 12.3, 3.6 Hz, C‐1′),
118.9 (C‐10), 111.4 (d, J = 20.8 Hz, CH, C‐5′), 103.6 (t,
J = 25.9 Hz, CH, C‐3′), 87.0 (C‐9), 57.0 (C‐6), 29.6 (CH,
C‐5); EIMS (m/z, rel. int.): 318 (M+) (15), 207 (100); Anal.
calcd. for C14H8F2N4O3: C: 52.84, H: 2.53, N: 17.61,
Found: C: 51.98, H: 2.39, N: 17.47. * assignments can be
interchanged.
Different isomers of the RCs at physiological pH were
generated using “Prepare Ligands” and energetically min-
imized in DS using CHARMm force field. The isomer
with the lowest CHARMm energy was selected for
docking. The crystal structure of Human Eg5 protein
(pdb id: 2X7C) was downloaded from the protein data
bank (http://www.rcsb.org). Only the B‐chain of the pro-
tein was considered while the native ligand, (s)‐Enastron
(KZ91367), and associated water molecules were
removed. Initially, the protonation state of the protein
was determined at physiological pH followed by its mini-
mization. The “Prepare Protein” module in DS was used
to build any missing loops/chains and determine the pro-
tonation state of each amino acid of the protein.
The shake algorithm was used to constrain the
hydrogen atoms of the protein during minimization.
Before docking, a binding sphere (diameter 6.34 Å) with
coordinates 16.8 (X), 14.4 (Y), and − 30.9 (Z) was gener-
ated using DS. Docking simulations were conducted
using the CDocker docking program[34] by keeping the
position of the protein fixed while allowing the ligand
to flex. A total of 10 poses were generated for each
compound and ranked according to the scoring function
(‐CDocker energy). The best pose was selected for BE
calculations.
7.2 | Antibacterial assay
The following bacterial strains were used: Gram +ve
S aureus ATCC 25923 and methicillin resistant S aureus
ATCC BAA‐1683 (MRSA), Gram −ve Escherichia coli
ATCC 25922, Pseudomonas aeruginosa ATCC 27853,
and Klebsiella pneumonia ATCC 314588. The standard
antibiotics ciprofloxacin and ampicillin were used as con-
trols for comparison. The method in Andrews[30] was
adapted with modification according to Aremu et al.[23]
Compounds 2‐5, 8, and 11 showed zones of inhibition

AREMU ET AL.
9
[17] M. M. Rahman, S. M. Ahmed, S. M. A. H. Siddiki, M. E. Halim,
K. Akhter, M. G. Ahmed, U. K. R. Romman, Dhakar. Univ. J.
Sci. 2013, 61, 167.
ACKNOWLEDGMENTS
This research was supported by the National Research
Foundation, South Africa, grant nos. 118534 (Competitive
Grant for Rated Researchers) and 114817 (Incentive
Funding for Rated Researchers).
[18] S. Xia, S. Yin, S. Tao, Y. Shi, L. Rong, X. Wei, Z. Zong, Res.
Chem. Intermed. 2012, 38, 2435.
[19] A. Bhatewara, S. R. Jetti, T. Kadre, P. Paliwal, S. Jain, Arch.
Appl. Sci. Res. 2012, 4, 1274.
[20] G. M. Ziarani, S. Asadi, S. Faramarzi, M. Amanlou, Iranian. J.
Pharm. Res. 2005, 14, 1105.
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
[21] L. Suresh, Y. Poornachandra, S. Kanakaraju, C. G. Kumar, G.
V. P. Chandramouli, Org. Biomol. Chem. 2015, 13, 7294.
[22] I. Devi, H. N. Borah, P. J. Bhuyan, Tetrahedron. Lett. 2004, 45,
ORCID
2405.
[23] O. S. Aremu, K. Gopaul, P. Kadam, M. Singh, C. Mocktar, P.
Singh, N. A. Koorbanally, Anticancer. Agents. Med. Chem.
2017, 17, 719.
Neil A. Koorbanally
4021
https://orcid.org/0000-0002-6214-
[24] M. M. Heravi, A. Ghods, K. Bakhtiari, F. Derikvand, Synth.
Commun. 2010, 40, 1927.
REFERENCES AND NOTES
[25] H. R. Safaei, M. Shekouhy, S. Rahmanpur, A. Shirinfeshan,
[1] O. O. Ajani, J. T. Isaac, T. F. Owoeye, A. A. Akinsiku, Int. J.
Biol. Chem. 2015, 9, 148.
Green. Chem. 2012, 14, 1696.
[26] M. Kidwai, A. Jain, S. Bhardwaj, Mol. Divers. 2012, 16, 121.
[2] A. Yousefi, R. Yousefi, F. Panahi, S. Sarikhani, A. R. Zolghadr,
A. Bahaoddini, A. Khalafi‐Nezhad, Int. J. Biol. Macromol. 2015,
78, 46.
[27] Y. A. Sharanin, G. V. Klokol, Russian. J. Org. Chem. 1984, 20,
2448.
[28] T. U. Mayer, T. M. Kapoor, S. J. Haggarty, R. W. King, S. L.
[3] N. R. Mohamed, M. M. T. El‐Saidi, Y. M. Ali, M. H. Elnagdi,
Bioorg. Med. Chem. Lett. 2007, 15, 6227.
Schreiber, T. J. Mitchison, Science 1999, 286, 971.
[29] S. DeBonis, D. A. Skoufias, L. Lebeau, R. Lopez, G. Robin, R. L.
Margolis, R. H. Wade, F. Kozielski, Mol. Cancer. Ther. 2004, 3,
1079.
[4] P. K. Paliwal, S. R. Jetti, S. Jain, Med Chem Res 2013, 22, 2984.
[5] H. H. Kadry, Med. Chem. Res. 2014, 23, 5269.
[6] M. L. Read, M. Braendvang, P. O. Miranda, L. L. Gundersen,
[30] J. A. Andrews, J Antimicrob. Chemother. 2001, 48, 5.
Bioorg. Med. Chem. 2010, 18, 3885.
[31] T. Moodley, M. Momin, C. Mocktar, C. Kannigadu, N. A.
[7] A. A. Joshi, S. S. Narkhede, C. L. Viswanathan, Bioorg Med
Chem Lett 2005, 15, 73.
Koorbanally, Magn. Reson. Chem. 2016, 54, 610.
[32] T. Mosmann, J. Immunol. Methods. 1983, 65, 55.
[8] R. P. Gore, A. P. Rajput, Drug. Invention. Today. 2013, 5, 148.
[33] J. M. Gichumbi, B. Omondi, G. Lazarus, M. Singh, N. Shaikh,
H. Y. Chenia, H. B. Friedrich, Z. Anorg. Allg. Chem. 2017,
643, 699.
[9] S. Jain, P. K. Paliwal, G. N. Babu, A. Bhatewara, J. Saudi.
Chem. Soc. 2014, 18, 535.
[10] G. M. Ziarani, S. Faramarzi, S. Asadi, A. Badiei, R. Bazl, M.
[34] G. Wu, D. H. Robertson, C. L. Brooks, M. Vieth, J. Comput.
Chem. 2013, 24, 1549.
Amanlou, DARU. J. Pharm. Sci. 2013, 21, 3.
[11] A. Khazaei, A. Ranjbaran, F. Abbasi, M. Khazaei, A. R.
Moosavi‐Zare, RSC. Adv. 2015, 5, 13643.
[12] B. Sadeghi, M. Bouslik, M. R. Shishehbore, J. Iranian. Chem.
Soc. 2015, 12, 1801.
How to cite this article: Aremu OS, Singh P,
Singh M, Mocktar C, Koorbanally NA. Synthesis of
chloro, fluoro, and nitro derivatives of 7‐amino‐5‐
aryl‐6‐cyano‐5H‐pyrano pyrimidin‐2,4‐diones using
organic catalysts and their antimicrobial and
anticancer activities. J Heterocyclic Chem. 2019;1–9.
https://doi.org/10.1002/jhet.3695
[13] S. N. Maddila, S. Maddila, W. E. van Zyl, S. B. Jonnalagadda,
RSC. Adv. 2015, 5, 37360.
[14] Y. Gao, S. Tu, T. Li, X. Zhang, S. Zhu, F. Fang, D. Shi, Synth.
Commun. 2004, 34, 1295.
[15] J. Yu, H. Wang, Synth. Commun. 2005, 35, 3133.
[16] A. R. Bhat, A. H. Shalla, R. S. Dongre, J. Saudi. Chem. Soc.
2017, 21, S305.