Synthesis and bioactivity evaluation of new pyrimidinone-5-carbonitriles as potential anticancer and antimicrobial agents

Article abstract of DOI:10.1007/s11164-018-3254-y

New series of pyrimidinone-5-carbonitriles 3a–i, 4a–e, 5a–c, 6 and 7 have been synthesized and explored for their activities as anticancer, antibacterial and antifungal agents. Investigation of the anticancer activity revealed that several newly synthesiz

Full text of DOI:10.1007/s11164-018-3254-y

Res Chem Intermed
https://doi.org/10.1007/s11164-018-3254-y
Synthesis and bioactivity evaluation of new
pyrimidinone‑5‑carbonitriles as potential anticancer
and antimicrobial agents
1
2
3
2,4
Amira A. Helwa · Ehab M. Gedawy · Sahar M. Abou‑Seri · Azza T. Taher
Afaf K. El‑Ansary2
·
Received: 17 September 2017 / Accepted: 8 January 2018
©
Springer Science+Business Media B.V., part of Springer Nature 2018
Abstract New series of pyrimidinone-5-carbonitriles 3a–i, 4a–e, 5a–c, 6 and 7
have been synthesized and explored for their activities as anticancer, antibacterial
and antifungal agents. Investigation of the anticancer activity revealed that several
newly synthesized derivatives displayed potent cytotoxic activity against diꢀer-
ent cancer cells. Among them, compound 3g was the most potent on the MCF-7,
A549 and Caco-2 cell lines (IC = 1.42, 1.98 and 9.50 μM, respectively), as com-
5
0
pared with 5-ꢁuorouracil (IC = 1.71, 10.32 and 20.22 μM, respectively), while
5
0
compound 3f was found especially eꢀective against MCF-7 and Caco-2 cell lines
(
IC = 1.48 and 16.15 μM, respectively). Furthermore, the antimicrobial evalu-
50
ation showed that compounds 3f and 3g have potent antibacterial activity against
Gram-positive bacteria Staphylococcus aureus (MIC = 4 and 8 μg/mL, respectively)
and promising activity against Escherichia coli (IZ. = 19 and 17 mm, respectively).
Meanwhile, compound 4b displayed the highest activity toward Bacillus subtilis
(
MIC = 8 μg/mL). In particular, the results suggested that hydrazone derivatives
*
Amira A. Helwa
amira_atef84@yahoo.com
Azza T. Taher
sahar.shaarawy@pharma.cu.edu.eg
Afaf K. El-Ansary
azza.shalaby@pharma.cu.edu.eg
1
2
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Misr University
for Science and Technology (MUST), 6th of October City, Egypt
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo,
Egypt
3
4
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, October 6 University,
6
th of October City, Egypt
1
3

A. A. Helwa et al.
bearing heterocyclic rings 3f and 3g are good lead compounds for the future design
of more potent anticancer or antimicrobial agents.
Keywords Anticancer · Antibacterial · Antifungal · Pyrimidinone-5-carbonotrile
Introduction
Pyrimidines are considered to be an important chemical synthon of various thera-
peutic eꢂcacies and pharmaceutical utility. They possess diverse pharmacological
activities, the most pronounced of which are anticancer [1–9] and antimicrobial
[
10–14]. Furthermore, various drugs containing a pyrimidine nucleus are in clinical
use as anticancer agents, for example, 5-ꢁuorouracil (5-FU), tegafur and imatinib
(
Gleevec™; Fig. 1) [15, 16]. In addition, pyrimidinone-5-carbonitrile derivatives
I–III were reported as anticancer and antimicrobial agents (Fig. 1) [17–19]. Moreo-
ver, a literature survey revealed that elaborating the pyrimidinone-5-carboitrile scaf-
fold with a hydrazinyl, hydrazide or hydazone moiety in compounds IV–VI con-
ferred promising chemotherapeutic activity as anticancer and antimicrobial agents
(
Fig. 1) [20, 21].
O
O
N
F
F
NH
NH
O
N
O
N
H
O
O
N
N
H
N
H
N
N
N
(Tegafur)
Imatinib (Gleevec)
O
(
5-Fluorouracil)
OH
N
O
NH
N
N
N
N
NH
H
N
O
R
N
S
S
N
S
NO
2
N
(
I)
(II)
F
(III)
2 5
IIIa R= C H
3 2
IIIb R= CH(CH )
Broad spectrum
antiproliferative
Active against Gram
positive bacteria
IIIa: 85% growth inhibition against
Melanoa (MALME-3M)
IIIb: 89, 88% growth inhibition against
Colon cancer (HCT-116), Renal cancer
(
UO-31), respectively
OH
O
O
O
N
N
N
NH
O
Br
NH
NH
NH
N
H
N
NH
2
N
N
H
N
S
N
N
H
O
O
(IV)
O
(V)
(VI)
-
73.9-95.8 % growth inhibition
- Possessesd superior antibacterial activity
against Gram positive bactria S. aureus and
B. subtilis compared to the referance drug
amoxicillin
-
78.3-91.7 % growth inhibition relative to
ampicillin and clotrimazole.
More potent than 5-FU against HePG2
and MCF-7 with IC50 = 25.52 and
3.91mM/l
relative to ampicillin and clotrimazole.
-
More potent than 5-FU against
-
HePG2 and MCF-7 with IC50 = 25.73
and 27.71 mM/l
2
Fig. 1 Pyrimidine derivatives as anticancer and antimicrobial agents
1
3

Synthesis and bioactivity evaluation of new…
In view of the biological signiꢃcance of the pyrimidinone-5-carbonitriles, we
herein report the synthesis and biological evaluation of novel pyrimidinone-5-car-
bonitrile derivatives 3a–i, 4a–e, 5a–c, 6 and 7 as anticancer and antimicrobial
agents. The design of the target compounds was initiated through structural exten-
sion of the lead compound 2 identiꢃed in our lab [19] by diꢀerent arylidine groups
to obtain the hydrazone derivatives 3a–i with an active azomethine (N=CH) proton.
The azomethine group is well-acknowledged to be the antitumor and antimicrobial
pharmacophore for a plethora of hydrazone derivatives [22]. Furthermore, two other
series of pyrimidinone-5-carbonitriles were prepared in which modiꢃcation was
focused on changing the methine spacer in 3a–i by C=O in 4a–e or CH –C=O in
2
5
a–c to study the eꢀect of such structural modiꢃcations on the desired biological
activities. Finally, utilizing the hydrazinyl group in 2 for incorporation of a pharma-
cophoric ring, (pyrazol-1-yl)pyrimidinone 6 and (pyrazolidin-1-yl)pyrimidinone 7
were prepared (Fig. 2).
Experimental part
General
All reagents were commercially available and were used without further puriꢃca-
tion. Melting points were determined on a Stuart apparatus and the values given are
2
2
1
1
1
1+
6SDFHUꢄPRGLILFDWLRQ
1
+
+
1
$U
1
$U
1
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;
1
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ꢀꢇDꢂLꢃ
ꢀꢁDꢂHꢃꢄ; ꢄ& 2
ꢀ
ꢅDꢂFꢃꢄ; ꢄ&+ ꢂ& 2
ꢆ
6WUXFWXUHꢄH[WHQVLRQ
2
1
1+
2
2
1
1
1
1
ꢀ
ꢉꢃ
1+
)
&\FOL]DWLRQ
&+ꢇ
1+ꢆ
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)
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1+
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1
+
1
)
ꢀꢈꢃ
Fig. 2 Design of the target compounds 3a–i, 4a–e, 5a–c, 6 and 7
1
3

A. A. Helwa et al.
uncorrected. IR spectra were determined as KBr discs on a Shimadzu IR 435 spec-
trophotometer (Faculty of Pharmacy, Cairo University, Egypt) and the values are
−1 1
represented in cm . H-NMR spectra were recorded on Varian Gemini 300-MHz
and 400-MHz spectrophotometers using TMS as an internal standard, and chemical
shift values were recorded in ppm on a δ scale (Microanalytical Center, Cairo Uni-
1
3
versity, Egypt). C-NMR spectra were recorded on Varian Gemini 75 MHz spectro-
photometer using TMS as internal standard and chemical shift values were recorded
in ppm on a δ scale (Microanalytical Center, Cairo University, Egypt). Mass spec-
tra were recorded on a Hewlett Packard 5988 spectrometer (Microanalytical Center,
Cairo University, Egypt). Elemental analyses were carried out at the Regional
Center for Mycology and Biotechnology, Al-Azhar University, Egypt. Progress of
the reactions was monitored using TLC aluminum sheets precoated with UV ꢁuo-
rescent silica gel (Merck 60F 254) and were visualized using a UV lamp.
6
-(4-Fluorophenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile
(
(
1) and 4-(4-ꢁuorophenyl)-2-hydrazinyl-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
2) were synthesized according to the reported procedures [19, 23].
General procedure of synthesis of compounds (3a–i)
A mixture of compound 2 (0.5 g, 2 mmol), the appropriate aldehyde (2 mmol) and
glacial acetic acid (1 mL) in absolute ethanol (20 mL) was heated under reꢁux for
6
h. The obtained precipitate was ꢃltered, dried and crystallized from ethanol.
2
‑(2‑(4‑Fluorobenzylidene)hydrazinyl)‑4‑(4‑fuorophenyl)‑6‑oxo‑1,6‑dihydropyrimi‑
dine‑5‑carbonitrile (3a)
Dark yellow crystals; (0.35 g, 1.00 mmol), yield: 49.78%; m.p.: 320–322 °C; IR
−
1
1
(
cm ): 3273 (2NH), 3140 (N=CH + ArH), 2218 (C≡N), 1640 (C=O); H-NMR
(
DMSO-d -300 MHz, ppm): 6.80 (d, 2H, J = 9.0 Hz, ArH), 7.10–7.29 (m, 2H,
6
ArH), 7.52 (d, 2H, J = 9.0 Hz, ArH), 7.72–8.08 (m, 2H, ArH), 8.17 (s, 1H, N=CH),
9
.76 (s, 1H, NH, exchangeable by D O), 10.58 (s, 1H, NH, exchangeable by D O);
2 2
Anal. calcd. for C H F N O (351.31): C, 61.54; H, 3.16; N, 19.93. Found: C,
1
8
11
2
5
6
1.62; H, 3.22; N, 20.05.
2
‑(2‑(4‑Chlorobenzylidene)hydrazinyl)‑4‑(4‑fuorophenyl)‑6‑oxo‑1,6‑dihydropyrimi‑
dine‑5‑carbonitrile (3b)
−
1
Yellow crystals; (0.48 g, 1.31 mmol), yield: 64.86%; m.p.: 318–320 °C; IR (cm ):
1
3
367 (2NH), 3138 (N=CH + ArH), 2216 (C≡N), 1666 (C=O); H-NMR (DMSO-
d -300 MHz, ppm): 7.35–7.38 (m, 2H, ArH), 7.50 (d, 2H, J = 8.7 Hz, ArH),
6
7
.91–7.95 (m, 2H, ArH), 8.06 (d, 2H, J = 8.7 Hz, ArH), 8.17 (s, 1H, N=CH), 12.50
(
s, 2H, 2NH, exchangeable by D O); Anal. calcd. for C H ClFN O (367.76): C,
2
18 11
5
5
8.79; H, 3.01; N, 19.04. Found: C, 58.87; H, 3.08; N, 19.18.
1
3

Synthesis and bioactivity evaluation of new…
4
‑(4‑Fluorophenyl)‑2‑(2‑(4‑methylbenzylidene)hydrazinyl)‑6‑oxo‑1,6‑dihydropy‑
rimidine‑5‑carbonitrile (3c)
−
1
Orange crystals; (0.21 g, 0.605 mmol), yield: 30.25%; m.p.: 296–298 °C; IR (cm ):
3
1
2
7
369, 3261 (2NH), 3159 (N=CH + ArH), 2850 (CH aliphatic), 2214 (C≡N),
1
664 (C=O); H-NMR (DMSO-d -300 MHz, ppm): 2.34 (s, 3H, CH ), 7.15 (d,
6
3
H, J = 9.0 Hz, ArH), 7.21–7.26 (m, 2H, ArH), 7.59 (d, 2H, J = 9.0 Hz, ArH),
.89–7.94 (m, 2H, ArH), 8.15 (s, 1H, N=CH), 10.75 (s, 1H, NH, exchangeable
1
3
by D O), 12.15 (s, 1H, NH exchangeable by D O); C-NMR (DMSO-d -75 MHz,
2
2
6
ppm): 20.96, 83.86, 111.01, 117.92, 125.40, 125.98, 127.91, 129.24, 130.18, 131.02,
1
3
32.51,138.10, 139.15, 140.14, 146.64, 147.92, 152.86, 162.04, 169.20; MS (m/z):
+
47.00 (M , 18.39%), 348.00 (M + H, 18.71%), 349.00 (M + 2, 12.26%), 103.00
(
100.00%); Anal. calcd. for C H FN O (347.35): C, 65.70; H, 4.06; N, 20.16.
19 14 5
Found: C, 65.81; H, 4.13; N, 20.24.
4
‑(4‑Fluorophenyl)‑2‑(2‑(4‑methoxybenzylidene)hydrazinyl)‑6‑oxo‑1,6‑dihydropy‑
rimidine‑5‑carbonitrile (3d)
Dark yellow crystals; (0.44 g, 1.21 mmol), yield: 60.52%; m.p.: 204–206 °C; IR
−
1
(
cm ): 3360, 3311 (2NH), 3134 (N=CH + ArH), 2830 (CH aliphatic), 2206
1
(
C≡N), 1662 (C=O); H-NMR (DMSO-d -300 MHz, ppm): 3.79 (s, 3H, OCH ),
6
3
6
.98 (d, 2H, J = 8.7 Hz, ArH), 7.09–7.15 (m, 2H, ArH), 7.64 (d, 2H, J = 8.7 Hz,
ArH), 7.89–7.98 (m, 2H, ArH), 8.13 (s, 1H, N=CH), 10.60 (s, 1H, NH, exchange-
+
able by D O), 10.66 (s, 1H, NH, exchangeable by D O); MS (m/z): 363.10 (M ,
2
2
1
.90%), 364.10 (M + H, 2.39%), 135.05 (100%); Anal. calcd. for C H FN O
19 14 5 2
(
363.35): C, 62.81; H, 3.88; N, 19.27. Found: C, 62.89; H, 3.87; N, 19.42.
4
‑(4‑Fluorophenyl)‑2‑(2‑(3,4,5‑trimethoxybenzylidene)hydrazinyl)‑6‑oxo‑1,6‑dihy‑
dropyrimidine‑5‑carbonitrile (3e)
−
1
Yellow crystals; (0.64 g, 1.51 mmol), yield: 75.29%; m.p.: 256–258 °C; IR (cm ):
3
1
6
358, 3328 (2NH), 3120 (N=CH + ArH), 2939 (CH aliphatic), 2204 (C≡N),
1
660 (C=O); H-NMR (DMSO-d -300 MHz, ppm): 3.70 (s, 3H, OCH ), 3.87 (s,
6
3
H, 2(OCH )) 7.01 (s, 2H, ArH), 7.15 (d, 2H, J = 9.0 Hz, ArH), 7.82 (s, 1H, NH,
3
exchangeable by D O), 7.84 (d, 2H, J = 9.0 Hz, ArH), 8.16 (s, 1H, N = CH), 10.74
2
(
s, 1H, NH, exchangeable by D O); Anal. calcd. for C H FN O (423.40): C,
2
21 18
5
4
5
9.57; H, 4.29; N, 16.54. Found: C, 59.61; H, 4.35; N, 16.69.
4
‑(4‑Fluorophenyl)‑2‑(2‑((5‑methylꢀuran‑2‑yl)methylene)hydrazinyl)‑6‑oxo‑1,6‑di‑
hydropyrimidine‑5‑carbonitrile (3f)
Brown red crystals; (0.41 g, 1.21 mmol), yield: 60.74%; m.p.: 230–232 °C; IR
−
1
(
cm ): 3323 (2NH), 3101 (N=CH + ArH), 2879 (CH aliphatic) 2208 (C≡N), 1660
1
(
C=O); H-NMR (DMSO-d -300 MHz, ppm): 2.34 (s, 3H, CH ), 6.77 (d, 1H, ArH),
6
3
6
.80 (d, 1H,ArH), 7.75–7.82 (m, 4H, ArH), 8.10 (s, 1H, N=CH), 10.20 (s, 1H, NH,
exchangeable by D O), 10.66 (s, 1H, NH, exchangeable by D O); Anal. calcd. for
2
2
1
3

A. A. Helwa et al.
C H FN O (337.31): C, 60.53; H, 3.59; N, 20.76. Found: C, 60.58; H, 3.64; N,
1
7
12
5
2
2
0.39.
2
‑(2‑((1H‑pyrrol‑2‑yl)methylene)hydrazinyl)‑4‑(4‑fuorophenyl)‑6‑oxo‑1,6‑dihydro‑
pyrimidine‑5‑carbonitrile (3g)
−
1
Brown crystals; (0.42 g, 1.30 mmol), yield: 64.61%; m.p.: > 350 °C; IR (cm ):
1
3
313 (3NH), 3109 (N=CH + ArH), 2193 (C≡N), 1647 (C=O); H-NMR (DMSO-
d -300 MHz, ppm): 6.12 (d, 1H, ArH), 6.42 (d, 1H, ArH), 6.77-6.87 (t, 1H, ArH),
6
7
1
1
3
.07-7.16 (m, 2H, ArH), 7.81–7.91 (m, 2H, ArH), 7.97 (s, 1H, N=CH), 10.47 (s,
H, NH, exchangeable by D O), 11.23 (s, 1H, NH, exchangeable by D O), 11.67 (s,
2
2
H, NH, exchangeable by D O); Anal. calcd. for C H FN O (322.30): C, 59.63; H,
2
16 11
6
.44; N, 26.08. Found: C, 59.80; H, 3.41; N, 26.31.
4
‑(4‑Fluorophenyl)‑2‑(2‑(4‑hydroxybenzylidene)hydrazinyl)‑6‑oxo‑1,6‑dihydropy‑
rimidine‑5‑carbonitrile (3h)
−
1
Yellow crystals; (0.35 g, 1.0 mmol), yield: 50%; m.p.: 322–324 °C; IR (cm ):
1
3
446–3286 (2NH,OH), 3032 (N=CH + ArH), 2216 (C≡N), 1653 (C=O); H-NMR
(
DMSO-d -300 MHz, ppm): 6.80 (d, 2H, J = 8.7 Hz, ArH), 7.10 (d, 2H, J = 8.7 Hz,
6
ArH), 7.52 (d, 2H, J = 8.7 Hz, ArH), 7.86 (d, 2H, J = 8.7 Hz, ArH), 8.08 (s, 1H,
N = CH), 9.77 (s, 1H, OH, exchangeable by D O), 10.01 (s, 1H, NH, exchangeable
2
by D O), 10.58 (s, 1H, NH, exchangeable by D O); Anal. calcd. for C H FN O
2
2
18 12
5
2
(
349.32): C, 61.89; H, 3.46; N, 20.05. Found: C, 62.04; H, 3.44; N, 20.23.
4
‑(4‑Fluorophenyl)‑2‑(2‑(3‑hydroxy‑4‑methoxybenzylidene)
hydrazinyl)‑6‑oxo‑1,6‑dihydropyrimidine‑5‑carbonitrile (3i)
Dark yellow crystals; (0.49 g, 1.29 mmol), yield: 64.47%; m.p.: 240–242 °C; IR
−
1
(
cm ): 3545–3261 (2NH, OH), 3001 (N=CH + ArH), 2906 (CH aliphatic), 2212
1
(
C≡N), 1699 (C=O); H-NMR (DMSO-d -300 MHz, ppm): 3.84 (s, 3H, OCH ),
6
3
6
.80 (d, 2H, ArH), 7.04–7.15 (m, 2H, ArH), 7.29 (s, 1H, ArH), 7.83–7.92 (m, 2H,
ArH), 8.08 (s, 1H, N=CH), 9.37 (s, 1H, OH, exchangeable by D O), 9.55 (s, 1H,
2
NH, exchangeable by D O), 10.55 (s, 1H, NH, exchangeable by D O); Anal. calcd.
2
2
for C H FN O (379.34): C, 60.16; H, 3.72; N, 18.46. Found: C, 60.39; H, 3.76;
1
9
14
5
3
N, 18.73.
General procedure of synthesis of compounds (4a–e)
A mixture of compound 2 (0.5 g, 2 mmol), anhydrous potassium carbonate (0.552 g,
4
mmol) and 4-substituted benzoyl chloride (2.2 mmol) in dry benzene (15 mL) was
heated under reꢁux for 10 h. The reaction mixture was ꢃltered while hot. The resi-
due was washed with water (15 mL), dried and crystallized from ethanol.
1
3

Synthesis and bioactivity evaluation of new…
N′‑(5‑Cyano‑4‑(4‑fuorophenyl)‑6‑oxo‑1,6‑dihydropyrimidin‑2‑yl)benzohydrazide
(4a)
−
1
Yellow crystals; (0.11 g, 0.314 mmol), yield: 15.7%; m.p.: 268–270 °C; IR (cm ):
1
3
388, 3338 (3NH), 3100 (ArH), 2204 (C≡N), 1685 (C=O), 1637 (C=O); H-NMR
(
DMSO-d -300 MHz, ppm): 7.28–7.47 (m, 2H, ArH), 7.49–7.59 (m, 5H, ArH),
6
7
.85–7.92 (m, 3H, ArH + NH), 10.50 (br. s, 2H, 2NH, exchangeable by D O); MS
2
+
(
m/z): 349.00 (M , 5.90%), 350.00 (M + H, 3.00%), 351.00 (M + 2, 1.66%), 105.00
(
100.00%). Anal. calcd. for C H FN O (349.32): C, 61.89, H, 3.46, N, 20.05.
1
8
12
5
2
Found: C, 61.96, H, 3.51, N, 20.17.
N′‑(5‑Cyano‑4‑(4‑fuorophenyl)‑6‑oxo‑1,6‑dihydropyrimidin‑2‑yl)‑4‑fuorobenzohy
drazide (4b)
Dark yellow crystals; (0.15 g, 0.41 mmol), yield: 20.54%; m.p.: > 300 °C; IR
−
1
(
cm ): 3442, 3340 (3NH), 3020 (ArH), 2206 (C≡N), 1660 (C=O), 1631 (C=O);
1
H-NMR (DMSO-d -400 MHz, ppm): 7.35–7.39 (m, 4H, ArH), 7.99–8.02 (m,
6
5
H, ArH + NH), 10.55 (s, 2H, 2NH, exchangeable by D O); MS (m/z): 367.00
2
+
(
M , 34.26%), 368.00 (M + H, 27.78%), 149.00 (100.00%). Anal. calcd. for
C H F N O (367.31): C, 58.86; H, 3.02; N, 19.07. Found: C, 58.97; H, 3.00; N,
1
8
11
2
5
2
1
9.19.
4
‑Chloro‑N′‑(5‑cyano‑4‑(4‑fuorophenyl)‑6‑oxo‑1,6‑dihydropyrimidin‑2‑yl)benzohy‑
drazide (4c)
−
1
Yellow crystals; (0.23 g, 0.599 mmol), yield: 29.87%; m.p.: > 300 °C; IR (cm ):
1
3
446, 3421 (3NH), 3070 (ArH), 2204 (C≡N), 1656 (C=O), 1639 (C=O); H-NMR
(
DMSO-d -400 MHz, ppm): 7.44–7.63 (m, 4H, ArH), 7.74–7.97 (m, 4H, ArH), 8.41
6
(
s, 1H, NH, exchangeable by D O), 10.55 (s, 1H, NH, exchangeable by D O), 10.63
2
2
(
s, 1H, NH, exchangeable by D O); Anal. calcd. for C H ClFN O (383.76): C,
2
18 11
5
2
5
6.33; H, 2.89; N, 18.25. Found: C, 56.57; H, 2.87; N, 18.38.
N′‑(5‑Cyano‑4‑(4‑fuorophenyl)‑6‑oxo‑1,6‑dihydropyrimidin‑2‑yl)‑4‑methylbenzohy
drazide (4d)
−
1
Brown crystals; (0.18 g, 0.49 mmol), yield: 24.65%; m.p.: 220–222 °C; IR (cm ):
3
1
419, 3226 (3NH), 3057 (ArH), 2918 (CH aliphatic), 2206 (C≡N), 1660 (C=O),
1
630 (C=O); H-NMR (DMSO-d -300 MHz, ppm): 2.38 (s, 3H, CH ), 7.32 (d, 2H,
6
3
J = 6.0 Hz, ArH), 7.50 (d, 2H, J = 6.0 Hz, ArH), 7.78–7.86 (m, 4H, ArH), 7.98 (s,
1
H, NH, exchangeable by D O), 10.38 (s, 1H, NH, exchangeable by D O), 11.50 (s,
2 2
1
H, NH, exchangeable by D O); Anal. calcd. for C H FN O (363.35): C, 62.81,
2
19 14
5
2
H, 3.88, N, 19.27. Found: C, 62.81, H, 3.88, N, 19.27.
1
3

A. A. Helwa et al.
N′‑(5‑Cyano‑4‑(4‑fuorophenyl)‑6‑oxo‑1,6‑dihydropyrimidin‑2‑yl)‑4‑methoxybenzo
hydrazide (4e)
−
1
Brown crystals; (0.31 g, 0.82 mmol), yield: 40.78%; m.p.: 296–298 °C; IR (cm ):
1
3
446, 3419 (3NH), 3055 (ArH), 2208 (C≡N), 1656 (C=O), 1639 (C=O); H-NMR
(
DMSO-d -400 MHz, ppm): 3.81 (s, 3H, OCH ), 6.79-7.44 (m, 4H, ArH), 7.70-7.82
6
3
(
m, 4H, ArH), 7.94 (s, 1H, NH, exchangeable by D O), 8.22 (s, 1H, NH, exchange-
2
+
able by D O), 8.80 (s, 1H, NH, exchangeable by D O); MS (m/z): 379.00 (M ,
2
2
9
.92%), 380.00 (M + H, 6.53%), 57.00 (100.00%). Anal. calcd. for C H FN O
19 14 5 3
(
379.34): C, 60.16; H, 3.72; N, 18.46. Found: C, 60.38; H, 3.79; N, 18.60.
General procedure of synthesis of compounds (5a–c)
A mixture of compound 2 (0.5 g, 2 mmol), anhydrous potassium carbonate (0.83 g, 6
mmol) and 2-bromo-4′-substitutedacetophenone (2 mmol) in dry benzene (15 mL)
was heated under reꢁux for 24 h. The reaction mixture was ꢃltered while hot. The
residue was washed twice with water (20 mL), dried and crystallized from methanol.
2
‑(2‑(2‑(4‑Chlorophenyl)‑2‑oxoethyl)hydrazinyl)‑4‑(4‑fuorophenyl)‑6‑oxo‑1,6‑dihy‑
dropyrimidine‑5‑carbonitrile (5a)
−
1
Brown crystals; (0.32 g, 0.81 mmol), yield: 40.5%; m.p.: > 300 °C; IR (cm ):
3
1
388–3421 (3NH), 3086 (ArH), 2964 (CH aliphatic), 2208 (C≡N), 1647 (C=O),
1
631 (C=O); H-NMR (DMSO-d -300 MHz, ppm): 3.67 (s, 2H, CH ), 6.8 (s, 1H,
6
2
NH, exchangeable by D O), 7.30 (d, 2H, J = 8.4 Hz, ArH), 7.42 (d, 2H, J = 9.0 Hz,
2
ArH), 7.62 (d, 2H, J = 9.0 Hz, ArH), 7.79 (d, 2H, J = 8.4 Hz, ArH), 8.60(s, 1H,
NH, exchangeable by D O), 10.15 (s, 1H, NH, exchangeable by D O); MS (m/z):
2
2
+
3
97.05 (M , 3.17%), 399.37 (M + 2, 1.38%), 117.02 (100.00%). Anal. calcd. for
C H ClFN O (397.79): C, 57.37; H, 3.29; N, 17.61. Found: C, 57.58; H, 3.34; N,
1
9
13
5
2
1
7.89.
4
‑(4‑Fluorophenyl)‑6‑oxo‑2‑(2‑(2‑oxo‑2‑(p‑tolyl)ethyl)hydrazinyl)‑1,6‑dihydropy‑
rimidine‑5‑carbonitrile (5b)
Reddish brown crystals; (0.23 g, 0.609 mmol), yield: 30.26%; m.p.: 260 °C (Decom-
−1
posed); IR (cm ): 3471, 3462, 3419 (3NH), 3066 (ArH), 2924, 2856 (CH aliphatic),
1
2
216 (C≡N), 1678 (C=O), 1639 (C=O); H-NMR (DMSO-d -300 MHz, ppm): 2.32
6
(
(
s, 3H, CH ), 3.87 (s, 2H, CH ), 4.20 (s, 1H, NH, exchangeable by D O), 7.22–7.24
3
2
2
m, 4H, ArH), 7.78–7.80 (m, 5H, ArH + NH), 8.30 (s, 1H, NH, exchangeable by
+
D O); MS (m/z): 377.16 (M , 4.68%), 378.12 (M + H, 2.43%), 90.08 (100.00%).
2
Anal. calcd. for C H FN O (377.37): C, 63.65; H, 4.27; N, 18.56. Found: C,
2
0
16
5
2
6
3.81; H, 4.30; N, 18.72.
1
3

Synthesis and bioactivity evaluation of new…
4
‑(4‑Fluorophenyl)‑2‑(2‑(2‑(4‑methoxyphenyl)‑2‑oxoethyl)hydrazinyl)‑6‑oxo‑1,6‑di‑
hydropyrimidine‑5‑carbonitrile (5c)
Brownish red crystals; (0.20 g, 0.508 mmol), yield: 25.32%; m.p.: 300 °C (Decom-
−1
posed); IR (cm ): 3545, 3462, 3419 (3NH), 3064 (ArH), 2920, 2837 (CH aliphatic),
1
2
3
218 (C≡N), 1670 (C=O), 1637 (C=O); H-NMR (DMSO-d -400 MHz, ppm):
6
.78 (s, 3H, OCH ), 3.82 (s, 2H, CH ), 5.89 (s, 1H, NH exchangeable by D O), 6.79
3
2
2
(
d, 2H, J = 8.0 Hz, ArH), 7.40–7.46 (m, 2H, ArH), 7.69–7.85 (m, 5H, ArH +NH),
8
6
.23 (s, 1H, NH exchangeable by D O); Anal. calcd. for C H FN O (393.37): C,
2 20 16 5 3
1.07; H, 4.10; N, 17.80. Found: C, 61.28; H, 4.16; N, 18.04.
General procedure of synthesis of compounds (6, 7)
A mixture of compound 2 (0.5 g, 2 mmol), and either ethyl acetoacetate (0.38 mL, 3
mmol) or ethyl cyanoacetate (0.23 mL, 2 mmol) in glacial acetic acid (20 mL)
was heated under reꢁux for 8 h. The reaction mixture was ꢃltered while hot and
the ꢃltrate was left to cool; the formed precipitate was ꢃltered, washed with water
(
15 mL), dried and crystallized from ethanol.
4
‑(4‑Fluorophenyl)‑2‑(3‑methyl‑5‑oxo‑4,5‑dihydro‑1H‑pyrazol‑1‑yl)‑6‑oxo‑1,6‑di‑
hydropyrimidine‑5‑carbonitrile (6)
Pale yellow crystals; (0.25 g, 0.803 mmol), yield: 40.3%; m.p.: 298–300 °C; IR
−
1
(
(
(
cm ): 3454 (NH), 3070 (ArH), 2902, 2848 (CH aliphatic), 2225 (C≡N), 1680
1
C=O), 1643 (C=O); H-NMR (DMSO-d -400 MHz, ppm): 2.27 (s, 3H, CH ), 2.51
6
3
s, 2H, CH overlapped), 5.37 (s, 1H, NH exchangeable by D O), 7.47–7.49 (m, 2H,
2
2
ArH), 8.11–8.15 (m, 2H, ArH); Anal. calcd. for C H FN O (311.27): C, 57.88; H,
1
5
10
5
2
3
.24; N, 22.50. Found: C, 58.12; H, 3.29; N, 22.81.
4
‑(4‑Fluorophenyl)‑2‑(5‑imino‑3‑oxopyrazolidin‑1‑yl)‑6‑oxo‑1,6‑dihydropyrimi‑
dine‑5‑carbonitrile (7)
Pale yellow crystals; (0.19 g, 0.608 mmol), yield: 30.6%; m.p.: 240–242 °C; IR
−
1
(
(
(
cm ): 3305–3275 (3NH), 3088 (ArH), 2931 (CH aliphatic), 2222 (C≡N), 1683
1
C=O), 1635 (C=O); H-NMR (DMSO-d -300 MHz, ppm): 1.91 (s, 2H, CH ), 6.76
6
2
d, 2H, J = 9.0 Hz, ArH), 7.80 (d, 2H, J = 9.0 Hz, ArH), 8.31 (s, 1H, NH, exchange-
able by D O), 9.72 (s, 1H, NH, exchangeable by D O), 9.97 (s, 1H, NH, exchange-
2
2
2
+
able by D O). MS (m/z): 312.00 (M , 82.89%), 313.00 (M + H, 81.85%), 200.00
(
100.00%); Anal. calcd. for C H FN O (312.26): C, 53.85; H, 2.91; N, 26.91.
14 9 6 2
Found: C, 53.99; H, 2.89; N, 26.91.
1
3

A. A. Helwa et al.
Biological evaluation
In‑vitro cytotoxicity assessment
Cytotoxic activity screening was performed at Al-Azhar University, Faculty of
Pharmacy, Pharmacology Department. All compounds were evaluated for cytotoxic
activity against MCF-7, HepG2, A549 and Caco-2 cells using XTT assay [24]. 5-FU
was employed as positive control. Exponentially, cells were seeded at a density of
3
5
× 10 cells/well into 96-well plates. For each compound, a set of concentration
range of 1–500 µg/mL was used. Cell viabilities were determined 72 h post-incu-
bation using XTT assay. Each concentration was processed four times then the IC
5
0
value was calculated. MCF-7, HepG2 and A549 cells were maintained in RPMI
1
640 media supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin
and 100 mg/mL streptomycin. Caco-2 cells were maintained in Dulbelcco’s modi-
ꢃed Eagle’s medium (DMEM), supplemented with 10% FCS, 500 U/mL penicillin,
5
00 mg/mL streptomycin, 1% sodium pyruvate and 1% l-glutamine. All cells were
cultivated at 37 C, 5% CO and 95% humidity. The dye was obtained from Sigma-
2
Aldrich (Munich, Germany).
Antimicrobial activity screening
The testing of the antimicrobial activity of all novel derivatives was carried out at
the Department of Microbiology, Faculty of Pharmacy, Misr University for Sci-
ence and Technology using the agar well diꢀusion technique according to Jahang-
irian et al. [25]. The tested strains included: Gram-positive bacteria Staphylococcus
aureus (ATCC 25923), Bacillus subtilis (ATCC CC33) and Gram-negative bacteria
Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and fun-
gus Candida albicans. The diameters of the measured zones showing complete inhi-
bition were recorded to the nearest millimeter.
Determination of the minimum inhibitory concentration (MIC)
MIC values of the most active synthesized compounds were determined against S.
aureus (ATCC 25923) and B. subtilis (ATCC CC33) using ciproꢁoxacin as refer-
ence drug according to microbroth dilution method [26].
Results and discussion
Chemistry
The synthesis of new pyrimidinone-5-carbonitrile derivatives was achieved through
Schemes 1 and 2. The structure of the newly synthesized compounds was conꢃrmed
1
13
by elemental analysis and spectral data (IR, H-NMR, C-NMR and MS). Numer-
ous research works have focused on the synthesis of pyrimidinone derivatives [23,
1
3

Synthesis and bioactivity evaluation of new…
2
+
2
6
2
1
1
ꢀ
Lꢃ
1+
+
ꢆ1
1+ꢆ
2
1
+
6
)
ꢀꢊꢃ
)
ꢀ
LLꢃ
2
1
2
1
1
1
1
+
1+
ꢀ
LLLꢃ
1
$U
1+ꢆ
1
1
+
+
ꢀꢆꢃ
ꢀ
ꢇDꢂLꢃ
)
)
ꢇDꢀꢁ$Uꢁ ꢁꢂꢃ)&ꢂ+ꢄ
ꢇEꢀꢁ$Uꢁ ꢁꢂꢃ&O&ꢄ+ꢂ
ꢇFꢀꢁ$Uꢁ ꢂꢃ&+ꢆ&ꢄ+ꢂ
ꢇIꢀꢁ$Uꢁ ꢁꢅꢃ&+ꢆ&ꢂ+ꢇ2ꢁ
ꢇJꢀꢁ$Uꢁ ꢁ&ꢂ+ꢂ1
ꢇKꢀꢁ$Uꢁ ꢂꢃ2+&ꢄ+ꢂ
ꢇGꢀꢁ$Uꢁ ꢁꢂꢃ2&+ꢆ&ꢄ+ꢂ
ꢇLꢀꢁ$Uꢁ ꢁꢂꢃ2+ꢃꢆꢃ2&+ꢆ&ꢄ+ꢆ
ꢇHꢀꢁ$Uꢁ ꢁꢆꢈꢂꢈꢅꢃꢉ2&+ꢆꢊꢆ&ꢄ+ꢇ
ꢀLYꢃ
ꢀYꢃ
2
1
2
1
1
1
5
1
+
1+
2
+
+
1
1
1
1
+
+
2
)
ꢀꢁDꢂHꢃ
)
5ꢊ
ꢀ
ꢅDꢂFꢃ
ꢅDꢀꢁ5ꢋꢁ ꢁ&O
ꢁ
ꢁ
ꢁ
Dꢋꢁ5ꢁ ꢁ+ꢁꢁꢁGꢀꢁ5ꢁ ꢁ&+ꢆ
Eꢀꢁ5ꢁ ꢁ)ꢁꢁꢁHꢀꢁ5ꢁ ꢁ2&+ꢆ
Fꢀꢁ5ꢁ ꢁ&O
ꢋꢁ
ꢅEꢀꢁ5 ꢁ&+ꢆ
ꢋꢁ
ꢅFꢀꢁ5 ꢁ2&+ꢆ
Scheme 1 Reagents and conditions: (i) anhydrous K CO /absolute ethanol/reꢁux 5 h/48%; (ii) NH NH /
2
3
2
2
reꢁux 12–14 h/58%; (iii) the appropriate aldehyde/glacial acetic acid/absolute ethanol/reꢁux 6 h/30–75%;
(
iv) 4-substituted benzoyl chloride/anhydrous K CO /dry benzene/reꢁux 10 h/15–40%; (v) 2-bromo-4′-
2
3
substituted acetophenone/anhydrous K CO /dry benzene/reꢁux 24 h/25–40%
2
3
2
2
2
1
1
1
1+
2
1+
1+
1+
2
ꢀLLꢃ
ꢀLꢃ
1+ꢆ
1
1
1
1
1
1
1
+
+1
ꢀꢉꢃ
ꢀꢆꢃ
ꢀꢈꢃ
)
)
)
Scheme 2 Reagents and conditions: (i) CH COCH COOCH CH /glacial acetic acid/reꢁux 8 h/40%; (ii)
3
2
2
3
NCCH COOCH CH /glacial acetic acid/reꢁux 8 h/30%
2
2
3
2
7, 28]. In this context, the starting pyrimidinone-5-carbonitrile 1 was synthesized
in a one-pot, three-component reaction as outlined in Scheme 1, according to the
reported procedure [23]. Furthermore, the synthesis of 4-(4-ꢁuorophenyl)-2-hy-
drazinyl-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (2) has already been described
by the reaction of 1 with excess of hydrazine hydrate [19].
1
3

A. A. Helwa et al.
Preparation of new compounds 3a–i was achieved following a previously reported
procedure for analogous compounds [29–32], in which compound 2 was treated
with the appropriate aldehyde in absolute ethanol in the presence of a catalytic
amount of glacial acetic acid. The structures of 3a–i were proven by the IR spectra,
which revealed the disappearance of the forked peak of NH in the key intermedi-
2
1
ate 2. The H-NMR spectra of compounds 3a‑i showed the appearance of a singlet
signal of one proton around δ 7.97–8.17 ppm, pointing to the presence of CH=N
and two exchangeable singlet signals around δ 9.55–12.50 ppm due to 2NH protons.
1
Moreover, the H-NMR spectra of compounds 3c and 3f showed the appearance of
a singlet signal at δ 2.34 ppm with integration of three protons disclosing the pres-
1
ence of a methyl group. In addition, the H-NMR spectra of compounds 3d and 3i
indicated a singlet signal of three protons around δ 3.8 ppm disclosing the presence
of a methoxy group.
Alternatively, the hydrazinyl intermediate 2 was coupled with substituted benzoyl
chloride or substituted phenacyl bromide in dry benzene using anhydrous potassium
carbonate as an acid binder to aꢀorded hydrazide derivatives 4a–e and 2‑(2-aryl-
2
-oxoethyl)hydrazinyl derivatives 5a–c, respectively (Scheme 1). The IR spectra of
compounds 4a–e and 5a–c are characterized by the appearance of two absorption
−1
bands around 1630–1685 cm , disclosing the presence of two carbonyl groups. In
1
addition, the H-NMR spectra of compounds 4d and 4e displayed a singlet signal of
three protons at δ 2.38 and 3.81 ppm, respectively corresponding to the methyl and
1
methoxy groups. The H-NMR spectra of compounds 5a–c revealed the appearance
of a singlet signal of two protons between δ 3.67-3.87 ppm corresponding to CH
2
1
of oxyethyl spacer. Additionally, the H-NMR spectra of compounds 5b and 5c dis-
closed a singlet signal of three protons at δ 2.32 and 3.78 ppm, respectively pointing
to the presence of methyl and methoxy groups.
On the other hand, cyclocondensation of the hydrazinyl intermediate 2 with ethyl
acetoacetae or ethyl cyanoacetate in glacial acetic acid furnished 2-(3-methyl-5-oxo-
4
,5-dihydro-1H-pyrazol-1-yl)pyrimidine-5-carbonitrile 6 and 2-(5-imino-3-oxopyra-
zolidin-1-yl)pyrimidine-5-carbonitrile 7, respectively (Scheme 2). The IR spectra
−
1
of compounds 6 and 7 showed two carbonyl stretch bands at 1643 and 1680 cm
−1
1
for compound 6 and at 1635 and 1680 cm for compound 7. The H-NMR spec-
trum of compound 6 revealed the appearance of a singlet signal of three protons at
δ 2.27 ppm corresponding to the presence of a methyl group, and a singlet signal of
1
two protons at δ 2.51 ppm disclosing the presence of CH of pyrazole. The H-NMR
2
spectrum of compound 7 showed the presence of three exchangeable singlet signals
each integrated for one proton at δ 8.31 and 9.72 and 9.97 ppm corresponding to
three NH protons along with a singlet signal of two protons at δ 1.91 ppm assigned
for the pyrazolidinone CH .
2
Cytotoxic activity
All the newly synthesized compounds were evaluated for their cytotoxic activity
against four human cancer cell lines; namely MCF-7 (breast), HepG2 (liver), A549
(
lung) and Caco-2 (colon), compared to 5-FU. The results are shown in Table 1 and
1
3

Synthesis and bioactivity evaluation of new…
Table 1 IC50 in μM of 5-FU and the test substances against MCF-7, HepG2, A549 and Caco-2 cells
O
O
O
N
N
O
N
N
NH
NH
NH
NH
O
H
N
NH
O
N
Ar
Ar
N
N
H
N
N
H
X
N
N
N
N
HN
F
(3a-i)
F
N
F
(
4a-e), (5a-c)
F
(7)
(
6)
CH
3
Compound no.
Ar
–
4-FC H
4-ClC H4
4-CH C H
4-OCH C H
3 6 4
3,4,5-(OCH ) C H
5-CH C H O
X
IC50 (μMa)
MCF-7
HepG2
A549
Caco-2
5
3
3
3
3
3
3
3
3
3
4
4
4
4
4
5
5
5
6
7
‑FU
a
b
c
d
e
–
–
–
–
–
–
–
–
–
–
1.71 ± 0.10 4.12 ± 0.23
42.19 ± 2.53 2.38 ± 0.09
1.50 ± 0.07 92.31 ± 3.69 93.46 ± 6.95
37.91 ± 1.90 2.35 ± 0.10
39.09 ± 1.76 2.32 ± 0.10
1.53 ± 0.08 2.19 ± 0.12
10.32 ± 0.61
> 100
20.22 ± 0.19
29.75 ± 0.29
17.04 ± 0.15
29.97 ± 0.35
29.22 ± 0.21
23.32 ± 0.14
16.15 ± 0.13
9.50 ± 0.09
22.07 ± 0.24
77.40 ± 1.54
26.08 ± 0.29
30.09 ± 0.37
37.95 ± 0.38
41.70 ± 0.50
30.08 ± 0.33
23.77 ± 0.22
36.87 ± 0.51
24.01 ± 0.30
61.41 ± 1.10
60.10 ± 1.14
6
4
6
2.07 ± 0.16
> 100
99.91 ± 5.94
3
6
4
3
3
6
2
f
1.48 ± 0.09 92.31 ± 5.54 88.14 ± 6.17
1.42 ± 0.09 > 100
3
4
2
g
h
i
a
b
c
d
e
a
b
c
C H N
1.98 ± 0.16
2.00 ± 0.14
> 100
93.12 ± 7.44
92.60 ± 6.38
> 100
4
4
4-OHC H4
4-OH-3-OCH C H
25.22 ± 0.90 3.46 ± 0.08
30.09 ± 1.02 2.31 ± 0.11
1.58 ± 0.10 > 100
49.90 ± 1.10 2.20 ± 0.12
1.61 ± 0.08 2.26 ± 0.12
52.07 ± 3.12 2.28 ± 0.11
37.56 ± 2.10 2.24 ± 0.12
1.52 ± 0.09 > 100
1.62 ± 0.11 2.09 ± 0.15
1.54 ± 0.09 2.05 ± 0.16
60.07 ± 4.03 2.28 ± 0.12
59.94 ± 3.92 > 100
6
3
6
3
C H
C=O
C=O
C=O
C=O
C=O
CH –C=O
CH –C = O
CH –C=O
–
–
6
5
4-FC H
4-ClC H4
4-CH C H
4-OCH C H
4-ClC H4
4-CH C H
4-OCH C H
–
–
6
4
6
> 100
3
6
4
86.21 ± 6.29
2.02 ± 0.16
75.88 ± 5.32
88.14 ± 7.23
2.00 ± 0.08
93.64 ± 7.40
3
6
4
6
2
3
6
4
2
3
6
4
2
aThe values given are mean ± SE of four experiments
represented graphically in Fig. 3. The results indicated that most of compounds pos-
sessed moderate to potent cytotoxic activities against one or more cell lines. In par-
ticular, MCF-7 and HepG2 were found to be more sensitive to the tested compounds
than A549 and Caco-2.
Regarding the MCF-7 cell line, the hydrazone derivatives 3a–i displayed potent
to moderate cytotoxic activity (IC = 1.42–42.19 μM) and their potency was
5
0
aꢀected by the nature and type of substituent on the arylidine moiety. Compounds
bearing a heterocyclic ring-like 5-methylfuran (3f) or pyrrole (3g) were the most
potent compounds in this study (IC = 1.48 and 1.42 μM, respectively). The
5
0
potency was also favored by grafting an electron-withdrawing substituent to the ben-
zylidine-like 4-chlorine atom (3b, IC = 1.50 μM) or the m-methoxy groups (3d,
5
0
IC = 1.53 μM). Meanwhile, an electron-donating substituent on the benzylidine
5
0
1
3

A. A. Helwa et al.
Dꢀ&Ͳϳ
,ĞƉ'Ϯ
ꢁϱϰϵ
ꢀĂĐŽͲϮ
ϭϮϬ
ϭϬϬ
ϴϬ
ϲϬ
ϰϬ
ϮϬ
Ϭ
ϱͲ&h ϯĂ ϯď ϯĐ ϯĚ ϯĞ ϯĨ ϯŐ ϯŚ ϯŝ ϰĂ ϰď ϰĐ ϰĚ ϰĞ ϱĂ ϱď ϱĐ
ϲ
ϳ
Fig. 3 IC50 in μM of 5-FU and the test substances against MCF-7, HepG2, A549 and Caco-2 cells
as 4-methyl (3c), 4-methoxy (3d) or 4-hydroxy (3h and 3i) group resulted in com-
pounds having moderate cytotoxic activity with an IC range = 25.22–39.09 μM.
5
0
On the other hand, changing the methine linker in the hydrazone derivatives 3a–d
into C=O in 4b–e produced compounds with similar level of cytotoxicity (compare
compound 3a ≠ 4b, 3b ≠ 4c, 3c ≠ 4d and 3d ≠ 4e), whereas, replacing the methine
linker in the hydrazone derivatives 3c and 3d (IC = 37.91 and 39.09 μM, respec-
5
0
tively) by CH –C=O in 5b and 5c resulted in about a 25-fold increase in potency
2
(
IC = 1.62 and 1.54 μM, respectively). Furthermore, direct attachment of func-
5
0
tionalized pyrazole or a pyrazolidine ring to the pyrimidinone-5-carbonitrile scaf-
fold in 6 and 7 led to a mediocre cytotoxic eꢀect.
The majority of the compounds showed potent cytotoxic activity against the
HepG2 cell line with IC ranging from 2.05 to 3.46 μM. In contrast to the MCF-7
5
0
cell line, only compounds with heterocyclic moieties 3f, 3g, 7 and those with an
(
un)/4-chlorosubstituted terminal phenyl ring, compounds 4a, 3b and 5a, exerted
weak inhibitory eꢀect against HepG2 (IC > 90 μM).
5
0
With respect to the less sensitive cell lines, ꢃve compounds, 3c, 3g, 3h, 5a and
6
, showed potent cytotoxic activity against A549 with IC values between 1.98 and
5
0
2
.07 μM. While all the tested compounds exhibited moderate to weak anti-prolifer-
ative activity against Caco-2, only compounds 3b, 3f and 3g were more potent than
5
-FU (IC = 17.04, 16.15, 9.50 and 20.22 μM, respectively).
50
Antimicrobial activity
The new compounds were further evaluated for their antimicrobial activity against
Gram-positive bacteria (S. aureus ATCC 25923 and B. subtilis ATCC CC33),
Gram-negative bacteria (E. coli ATCC 25922 and P. aeruginosa ATCC 27853) and
fungus (Candida albican). The diameters of the zones showing complete inhibition
1
3

Synthesis and bioactivity evaluation of new…
of the microorganism growth were recorded to the nearest millimeter. The mean of
the inhibition zone (IZ) is tabulated in Table 2.
The results revealed that, the target compounds had no signiꢃcant activity toward
Gram-negative bacteria and Candida albican, except for compounds 3f and 3g,
which showed good activity against E.coli with an IZ of 19 and 17 mm, respectively.
On the other hand, the majority of compounds possessed potent to good antibacte-
rial activity against Gram-positive bacteria (S. aureus and B. subtilis). Compounds
3
f, 3g and 6 showed the highest activity against S. aureus with IZ = 29, 25 and
2
2 mm, respectively, compared with 22 mm for the reference drug ciproꢁoxacin.
Moreover compounds 3c, 3e, 4b and 4c demonstrated good activities against the
same microorganism (IZ = 16–19 mm). As for B. subtilis, compounds 3f, 3g, 4b,
4
c, 4d, 5b and 6 registered promising antibacterial activity (IZ rang = 21–17 mm)
in comparison with ciproꢁoxacin (IZ = 24 mm). Accordingly, the quantitative assay
of the antimicrobial activity was performed for the active compounds (3c, 3e, 3f, 3g,
4
b, 4c and 6) with S. aureus and (3f, 3g, 4b, 4c, 4d, 5b and 6) with B. subtilis using
ciproꢁoxacin as the reference drug in order to establish the MIC; the results are
Table 2 The preliminary antimicrobial and antifungal screening test for the prepared compounds using
ciproꢁoxacin and ꢁuconazole® as references, and DMSO as the control
Microorganism
Compound no.
S. aureus (mm)
B. subtilis (mm)
E. coli
mm)
P. aeruginosa Candida
(
(mm)
albicans
(
mm)
3
3
3
3
3
3
3
3
3
4
4
4
4
4
5
5
5
6
7
a
b
c
d
e
f
12
–
19
–
17
29
25
–
–
–
18
16
–
12
–
12
–
–
–
–
–
–
–
–
–
–
–
–
–
–
19
19
13
15
–
20
21
17
–
13
17
14
17
–
19
17
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
g
h
i
–
–
–
–
–
–
–
–
–
–
a
b
c
d
e
a
b
c
–
14
14
–
22
–
–
–
–
–
–
–
–
–
–
28
–
–
23
–
24
Ciproꢀoxacin
Fluconazole
DMSO
22
24
–
–
–
–
1
3

A. A. Helwa et al.
Table 3 Minimum inhibitory
concentration (MIC) values in
µg/mL of the tested compounds
against S. aureus and B. subtilis
Compound no.
S. aureus
B. subtilis
3
3
3
3
4
4
4
5
6
c
e
f
g
b
c
d
b
16
16
4
ND
ND
16
16
8
32
32
32
32
1
8
16
32
ND
ND
8
Ciproꢀoxacin
2
ND not determined
S. aureus
B. subtilis
3
3
2
2
1
1
5
0
5
0
5
0
5
0
Fig. 4 Minimum inhibitory concentration (MIC) values (µg/mL) of the tested compound against S.
aureus and B. subtilis
shown in Table 3 and Fig. 4. Analysis of the results revealed that compounds bear-
ing terminal heterocyclic moieties such as 3f, 3g and 6 (MIC = 4, 8 and 8 μg/mL,
respectively) were the most potent antibacterial agents against S. aureus, whereas
the hydrazone derivatives with 4-methylbenzylidine (3c) and 4-methoxybenzylidine
(
3e) elicited reduced antibacterial eꢀect (MIC = 16 μg/mL). On the other hand, the
ꢁ
uorobenzohydrazide derivative (4b) exhibited the highest inhibition eꢀect toward
B. subtilis (MIC = 8 μg/mL). Otherwise, the rest of the tested compounds were less
active toward B. subtilis than S. aureus (MIC = 16–32 μg/mL).
1
3

Synthesis and bioactivity evaluation of new…
Conclusion
The present study reports the synthesis of a novel series of pyrimidinone-5-carbon-
itriles 3a–i, 4a–e, 5a–c, 6 and 7 as potential anticancer and antimicrobial agents.
Several newly synthesized derivatives displayed potent cytotoxic activity against
diꢀerent cancer cells. Among the evaluated compounds, the hydrazone derivatives
having a heterocyclic ring (3f, Ar = 5-methylfurane and 3g, Ar = pyrrole) dis-
played a potent and broad spectrum of antitumor activity. Compound 3g was the
most potent on the MCF-7, A549 and Caco-2 cell lines with IC = 1.42, 1.98 and
5
0
9
.50 μM, respectively, as compared with 5-FU (IC = 1.71, 10.32 and 20.22 μM,
50
respectively), while compound 3f was found especially eꢀective against MCF-7 and
Caco-2 cell lines with IC = 1.48 and 16.15 μM, respectively. Moreover the same
5
0
compounds showed the highest antibacterial activity against the resistant G+ve S.
aureus with promising inhibition eꢀect toward G+ve B. subtilis and G−ve E.coli.
In brief, compounds 3f and 3g represent good leads for optimization and design of
more potent chemotherapeutic agents with anticancer and antimicrobial activities.
Acknowledgements The authors would like to express their gratitude to Dr. Mohammed Fathy, Depart-
ment of Pharmacology, Faculty of Pharmacy, Al-Azhar University for performing the cytotoxic activity
of the synthesized compounds. The authors would like to express their sincere thanks to the Depart-
ment of Microbiology, Misr University for Science and Technology, for carrying out the antimicrobial
screening.
Compliance with ethical standards
Conꢀict of interest The authors have declared no conꢁict of interest.
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