Synthesis, in-vitro antibacterial and anticancer screening of novel nicotinonitrile-coumarin hybrids utilizing piperazine citrate

Article abstract of DOI:10.1080/00397911.2020.1743318

An efficient, low-cost, high yield% and eco-friendly synthetic procedure is designed for the synthesis of novel series of nicotinonitrile-coumarin hybrid molecules bearing several aryl and/or heteroaryl moieties. Our strategy includes the synthesis of a novel series of 2-hydroxybenzaldehydes, bearing nicotinonitrile moiety, followed by its Knoevenagel reaction with ethyl acetoacetate in the presence of an organo-base. The above reaction is studied in different reaction conditions. The optimized conditions are performing the reaction in ethanol at 80 °C in the presence of 10 mol% of piperazine citrate (1:1). The in-vitro antibacterial activities of the nicotinonitrile-coumarins were evaluated against different Gram-positive and negative bacterial strains. Moreover, the in-vitro cytotoxicity of nicotinonitrile-coumarins were estimated against different eukaryotic cell lines. Compounds 5a and 5b exhibited the most promising antibacterial agents among the novel series. The structures of novel series of the target nicotinonitrile-coumarin hybrid molecules were confirmed by considering their elemental analyses and spectral data.

Full text of DOI:10.1080/00397911.2020.1743318

Synthetic Communications
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ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: https://www.tandfonline.com/loi/lsyc20
Synthesis, in-vitro antibacterial and anticancer
screening of novel nicotinonitrile-coumarin
hybrids utilizing piperazine citrate
Sherif M. H. Sanad & Ahmed E. M. Mekky
To cite this article: Sherif M. H. Sanad & Ahmed E. M. Mekky (2020): Synthesis, in-vitro
antibacterial and anticancer screening of novel nicotinonitrile-coumarin hybrids utilizing piperazine
citrate, Synthetic Communications, DOI: 10.1080/00397911.2020.1743318
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https://doi.org/10.1080/00397911.2020.1743318
Synthesis, in-vitro antibacterial and anticancer screening
of novel nicotinonitrile-coumarin hybrids utilizing
piperazine citrate
Sherif M. H. Sanad
and Ahmed E. M. Mekky
Chemistry Department, Faculty of Science, Cairo University, Giza, Egypt
ABSTRACT
ARTICLE HISTORY
Received 24 December 2019
An efficient, low-cost, high yield% and eco-friendly synthetic proced-
ure is designed for the synthesis of novel series of nicotinonitrile-
coumarin hybrid molecules bearing several aryl and/or heteroaryl
moieties. Our strategy includes the synthesis of a novel series of 2-
hydroxybenzaldehydes, bearing nicotinonitrile moiety, followed by
its Knoevenagel reaction with ethyl acetoacetate in the presence of
an organo-base. The above reaction is studied in different reaction
conditions. The optimized conditions are performing the reaction in
ethanol at 80 ꢀC in the presence of 10 mol% of piperazine citrate
(1:1). The in-vitro antibacterial activities of the nicotinonitrile-coumar-
ins were evaluated against different Gram-positive and negative
bacterial strains. Moreover, the in-vitro cytotoxicity of nicotinonitrile-
coumarins were estimated against different eukaryotic cell lines.
Compounds 5a and 5b exhibited the most promising antibacterial
agents among the novel series. The structures of novel series of the
target nicotinonitrile-coumarin hybrid molecules were confirmed by
considering their elemental analyses and spectral data.
KEYWORDS
Knoevenagel reaction;
nicotinonitrile-coumarin
hybrids; piperazine citrate;
pyridine-2(1H)-thiones; in-
vitro antibacterial agents;
cytotoxicity
GRAPHICAL ABSTRACT
CONTACT Sherif M. H. Sanad
sherif_hamed1980@yahoo.com
Chemistry Department, Faculty of Science, Cairo
University, Giza, Egypt.
Supplemental data for this article can be accessed on the publisher’s website.
ß 2020 Taylor & Francis Group, LLC

2
S. M. H. SANAD
Introduction
Coumarins or 2H-chromen-2-ones are natural occurring heterocyclic derivatives that
used as effective components in inhibition and healing of many diseases. These deriva-
tives act as antimicrobial,[1] anti-inflammatory,[2] anticancer,[3] anti-Alzheimer dis-
ease[4,5] and anti-Parkinson agents.[6] Warfarin and dicoumarol are examples of
chromene derivatives which considered as essential oral anticoagulant drugs.[7] Many
publications reported the synthesis of coumarins. They can be prepared via Wittig,[8]
Heck,[9] Perkin and[10] Pechmann reactions.[11,12] In addition, Reformatsky,[13] and
Knoevenagel[14–16] reactions can be used as efficient synthetic routes for the preparation
of coumarins.
Despite the presence of many attempts to design synthetic routes for the preparation
of coumarin derivatives, still there is a considerable need for developing of efficient,
low-cost, high yield and eco-friendly procedure for the synthesis of such biologically
active heterocyclic derivatives.
Piperazine and its derivatives were used as efficient catalysts in the synthesis of vari-
ous heterocyclic derivatives. Several 2-amino-3-cyano-4H-pyrans derivatives were pre-
pared via the Michael addition reactions which catalyzed by piperazine.[17,18] Moreover,
pyrano[2,3-d]pyrimidinone derivatives were prepared by the one pot reaction of alde-
hydes, malononitrile and (thio)barbituric acid which catalyzed by piperazine-1,4-diium
dihydrogen phosphate.[19] Piperazine-containing polymers can be used in the catalysis
for Knoevenagel condensation reactions.[20,21] Chiral 2,5-dibenzylpiperazine was used in
the catalytic asymmetric Michael addition of aldehydes to nitroalkenes.[22] In addition,
piperazine can be incorporated in many heterogeneous catalysts which used to
catalyze several reactions. For example, KG-60-piperazine catalyzes the synthesis of 2-
aminothiophenes via Gewald three-component reaction.[23] Also, reduced graphene
oxide supported piperazine catalyzes Knoevenagel condensation and Michael addition
reactions as well as Knoevenagel-Michael addition one-pot process.[24]
Piperazine citrate is a well-known drug for its wide applications as an anthelmintic. It
used in the treatment and control as well as prevention of several worm infections as
threadworm, roundworm and pinworm in both humans and animals.[25,26] Piperazine
citrate causes paralysis of the parasites that lead to diseases by their invasion of the host
body. Therefore, it leads to remove the harmful parasites and inhibit the spread of dis-
eases. For example, it helps in the treatment of chicken naturally infected with gastro-
intestinal helminths.[27] Also, piperazine citrate can be used in the treatment of ascaris
lumbricoides infestation.[28] Piperazine citrate also helps in the treatment of bacterial
infections.[29]
Considering the broad biological importance of nicotinonitriles,[30–35] our research
group has exerted many efforts in the field of synthesis of several nicotinonitriles which
have been manifested in the publication of many scientific papers.[14,36–39] The work in
this study is aimed to study the utility of piperazine citrate, as a green catalyst, in the
synthesis of novel series of nicotinonitrile-coumarin hybrid molecules bearing several
aryl and/or heteroaryl moieties. The in-vitro antibacterial activities of the nicotinoni-
trile-coumarin hybrid molecules were evaluated against different Gram-positive and
negative bacterial strains. Moreover, the in-vitro cytotoxicity of nicotinonitrile-coumar-
ins were estimated against different eukaryotic cell lines.

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3
Results and discussion
Chemistry
A simple synthetic route was designed for the preparation of novel nicotinonitrile-
coumarin hybrid molecules using 2-hydroxybenzaldehydes 3, incorporating
5
nicotinonitrile moiety, as building blocks. The reaction of nicotinonitriles 1 with
2-hydroxybenzaldehydes 2 in appropriate reaction conditions afforded a novel series of
2-hydroxybenzaldehyde-nicotinonitrile hybrids 3 via an appropriate linker. Then, these
derivatives reacted with ethyl acetoacetate 4 in the presence of basic conditions to afford
the desired nicotinonitrile-coumarin hybrid molecules 5 (Scheme 1).
Thus, the formation of numerous nicotinonitriles was our first step to our aim. In
this study, we investigate the synthetic potential of pyridine-2(1H)-thiones as effective
examples of nicotinonitriles. The most common synthetic route to pyridine-2(1H)-thi-
one derivatives includes the cyclocondensation of 1,3-dicarbonyl or a,b-unsaturated car-
bonyl compounds with 2-cyanothioacetamide. For example, the reaction of
acetylacetone 6 with 2-cyanothioacetamide 7, in boiling ethanol in presence of piperi-
dine,
afforded
4,6-dimethyl-2-thioxo-1,2-dihydropyridine-3-carbonitrile
1a[40]
(Scheme 2). Also, 6-phenyl-2-thioxo-1,2-dihydropyridine-3-carbonitrile 1b[41] and pyri-
dine-2(1H)-thiones 1c–1j[42–44] were synthesized by the reaction of 2-cyanothioaceta-
mide with cinnamaldehyde 8 or the appropriate chalcones 9c–9j[42,44–48] in basic
Scheme 1. General synthetic approach for the synthesis of nicotinonitrile-coumarin hybrids
molecules 5.
Scheme 2. Synthesis of pyridine-2(1H)-thione derivatives 1a–1j.

4
S. M. H. SANAD
medium, respectively (Scheme 2, Table 1). Among the ten pyridine-2(1H)-thione deriva-
tives studied in this work, five new pyridine-2(1H)-thiones were prepared. Therefore,
compounds 1c–1e, 1h and 1i, bearing benzo[d][1,3]dioxole or 1,3-diphenyl-1H-pyrazole
moiety, were prepared by the above procedure (see experimental section).
Next step was the synthesis of novel series of 2-hydroxybenzaldehydes 3, incorporat-
ing pyridine moiety. Formerly, pyridine-2(1H)-thione 1a was stirred with an equivalent
amount of potassium hydroxide in ethanol at room temperature for 10 min. Then, an
Table 1. Synthesis of pyridine-2(1H)-thiones 1 and 2-hydroxybenzaldehydes 3.
2-Hydroxy-
Pyridine-2(1H)-thione 1
benzaldehyde 3
Entry
Cpd. 1(3)
R1
R2
Yield%
94
m.p. (oC)
263-265[41]
245-247[42]
Yield%
97
Time (h)
1
2
1
a
65
93
1.5
b
3
4
5
6
70
79
77
80
244-246*
232-234*
218-220*
240-242[43]
92
91
89
86
3
c
d
e
f
2.5
2.5
3
7
8
72
70
75
86
273[44]
84
81
84
84
3
4
g
h
i
254*
9
> 300*
3.5
10
> 300[45]
3
j
ꢁ
New pyridine-2(1H)-thione derivatives.

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5
equivalent amount of 5-(chloromethyl)ꢂ2-hydroxybenzaldehyde 2[49] was added with
continuous stirring. After one hour of stirring, TLC analysis of the reaction mixture
showed the presence of a sole reaction product. The reaction most likely to proceed via
a simple nucleophilic substitution reaction between thiolate anion 10 and benzyl chlor-
ide derivative 2 to give 2-((3-formyl-4-hydroxybenzyl)thio)ꢂ4,6-dimethylnicotinonitrile
3a in 97% yield (Scheme 3). The structure of the isolated product 3a was established by
its elemental analysis and spectral data. The IR spectrum of 3a shows the presence of
OH, CN and CO groups at ύ 3433, 2233 and 1678 cmꢂ1, respectively. Its mass spectrum
1
gave a molecular ion peak at m/z ¼ 298. Its H-NMR spectrum revealed six singlet sig-
nals at d 2.37, 2.52, 4.44, 7.10, 10.22 and 10.66 ppm assigned to two CH3 protons in
addition to SCH2, pyridine-H5, CHO and OH protons, respectively. In addition, it
reveals two doublet signals at d 6.93 and 7.58 ppm and one singlet signal at d 7.75 ppm
corresponding to aromatic H3, H4 and H6 protons, respectively. Its 13C-NMR spectrum
reveals the presence of five signals at d 20.0, 24.6, 32.9, 115.4 and 191.4 ppm corre-
sponding to two CH3, SCH2, CN and C¼O carbons, respectively, in addition to 11 sig-
nals corresponding to pyridine and aromatic carbons (see experimental section).
Similarly, the pyridine-2(1H)-thione derivatives 1b–j were reacted with compound 2
to afford the corresponding 2-hydroxybenzaldehydes 3b–3j, bearing nicotinonitrile moi-
ety in 81–93% yield (Scheme 3, Table 1).
Subsequently, Knoevenagel synthesis of novel coumarin derivatives 5 was carried
out using 2-hydroxybenzaldehydes 3 and ethyl acetoacetate 4. For example, 2-hydroxy-
benzaldehyde derivative 3a was reacted with ethyl acetoacetate 4 in ethanol at reflux in
the presence of piperidine as an organo-base to give the corresponding 2-(((3-acetyl-2-
oxo-2H-chromen-6-yl)methyl)thio)ꢂ4,6-dimethylnicotinonitrile 5a in 45% yield
(Scheme 4; Entry 1, Table 2). The structure of 5a was confirmed via elucidation of its
elemental analysis and spectral data. The structure of the isolated product 5a was estab-
lished by its elemental analysis and spectral data. The IR spectrum of 5a shows the pres-
ence of CN and two CO groups at ύ 2224, 1722, 1678 cmꢂ1, respectively. Its mass
spectrum gave a molecular ion peak at m/z ¼ 364. The mass fragmentation pattern of
Scheme 3. Synthesis of 2-hydroxybenzaldehydes, bearing nicotinonitrile moiety 1a–1j.
Scheme 4. Synthesis of nicotinonitrile-coumarin hybrid molecule 5a.

6
S. M. H. SANAD
Table 2. Optimization of the reaction conditions for products 5a.
Entry
Solvent
Catalyst
Temp. (oC)
Time (h)
Yield (%)
1
2
3
4
5
6
7
8
EtOH
EtOH
Dioxane
EtOH
EtOH
Dioxane
EtOH
Dioxane
DMF
EtOH
Water
EtOH
Piperidine
Piperidine
Piperidine
Et3N
Et3N
80
rt
110
rt
80
110
80
110
150
80
rt
rt
5
8
6
8
5
5
5
6
6
2
8
8
8
45
Traces
26
Traces
38
19
35
21
27
Et3N
Morpholine
Morpholine
Morpholine
Piperazine citrate
Piperazine citrate
Piperazine citrate
Piperazine citrate
9
ꢁ
ꢁ
ꢁ
ꢁ
10
11
12
13
ꢁ
92
Traces
40
Dioxane
110
83
25 mol% of piperazine citrate (1:1) was used.
1
5a was shown in supplemental file. The H-NMR spectrum of 5a revealed five singlet
signals at d 2.33, 2.43, 2.56, 4.55 and 7.08 ppm assigned to two CH3 protons in addition
to COCH3, SCH2 and pyridine-H5 protons, respectively. Moreover, it reveals two doub-
let signals at d 7.35 and 7.41 ppm and two singlet signals at d 7.80 and 8.58 ppm corre-
sponding to four chromene-H8, H7, H5 and H4 protons, respectively. Its 13C-NMR
spectrum reveals the presence of five signals at d 20.0, 24.6, 32.9, 115.5 and 195.2 ppm
corresponding to two CH3, SCH2, CN and CH3C¼O carbons, respectively, in addition
to 14 signals corresponding to pyridine and chromene carbons (see experimen-
tal section).
Then, we investigate the optimized reaction condition for this formation of 5a. We
repeated such reaction using different organo-bases and solvents as shown in Table 2.
The use of piperidine or triethylamine as a catalyst gave only traces of 5a when the
reaction was carried out in ethanol at room temperature for long reaction times
(Entries 2 and 4, Table 2). Then again, 5a was prepared in 19–38% yields using piperi-
dine, triethylamine or morpholine in different solvents such as ethanol, dioxane or
dimethylformamide (DMF) at reflux for long reaction times (Entries 3 and 5–9,
Table 2).
The isolation of 5a in such low yields utilizing the traditional organo-bases stimulated
our interest to investigate catalytic potential of new catalysts. Then, we explored the
using of piperazine citrate (1:1) as an eco-friendly basic catalyst for the formation of 5a.
Therefore, 25 mol% of piperazine citrate was used to catalyze the above reaction in the
presence of water, ethanol or dioxane as a solvent and at different temperatures (Entries
10–13, Table 2). The best yield (95%) was to carry out the reaction in ethanol and at
80 ꢀC for two hours (Entry 10, Table 2). The minimum mol% of piperazine citrate,
required to catalyze the formation of 5a, was studied as shown in Table 3. Different
mol% of piperazine citrate were used (Entries 1–6, Table 3). Obviously, the optimum
conditions for the formation of 5a was (Entry 5, Table 3), in which 10 mol% of pipera-
zine citrate was used as an organo-base in ethanol at 80 ꢀC for two hours.
The optimized conditions were applied to Knoevenagel condensation of 2-hydroxy-
benzaldehydes 3b–j with ethyl acetoacetate 4. A structurally divergent range of novel
nicotinonitrile-coumarin hybrid molecules 5b–5j, were prepared in good yields in
82–90% yields (Scheme 5, Table 4).

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7
Table 3. Optimization of the mol% of piperazine citrate
(1:1). All entries were carried out using ethanol as solv-
ent and at 80 ꢀC.
Entry
Mol%
Time (h)
Yield (%)
1
2
3
4
5
6
7
1
2
5
5
5
4
4
2
2
2
Traces
Traces
39
74
95
94
94
8
10
15
20
Scheme 5. Synthesis of nicotinonitrile-coumarin hybrid molecules 5b–5j.
Table 4. Synthesis of nicotinonitrile-coumarin hybrid molecules 5b–5j using 10 mol% of piperazine
citrate as a catalyst.

8
S. M. H. SANAD
Table 5. The in-vitro antibacterial activities of the novel nicotinonitrile-coumarins against the tested
bacterial strains.
Inhibition zones (mm)
Gram-negative bacteria
Gram-positive bacteria
Compound
E. coli
K. pneumonia
P. aeruginosa
S. aureus
S. mutans
5a
5b
5c
5d
5e
5f
5g
5h
44.2 1.0
38.6 0.6
22.8 0.5
38.9 1.0
37.9 0.7
27.5 0.6
21.2 0.5
22.9 0.6
23.7 0.6
28.7 0.7
25.2 0.6
47.3 1.0
31.5 0.6
19.1 0.5
36.6 1.0
43.2 1.0
21.3 0.6
15.4 0.5
17.8 .5
19.0 0.5
31.7 0.7
22.5 0.6
45.0 1.0
40.8 1.0
21.0 0.5
35.5 0.7
39.8 1.0
29.9 0.7
20.4 0.5
19.9 0.5
25.4 0.6
35.5 1.0
27.3 0.7
38.6 0.7
43.6 1.0
18.5 0.5
33.6 0.6
37.7 0.8
33.3 1.0
15.9 0.5
17.8 0.5
20.8 0.6
27.4 0.7
24.5 0.6
45.4 1.0
40.9 1.0
22.6 0.6
35.8 0.7
40.6 1.0
28.9 0.7
17.8 0.5
19.8 0.5
22.6 0.6
23.8 0.6
26.0 0.5
5i
5j
Ciprofloxacin
Antimicrobial activities were screened in triplicate and the average reading of the zone of inhibition was determined in
mm SEM; Inactive (ꢂve) ¼ (inhibition zone < 8 mm).
Biology
Antibacterial screening
The in-vitro antibacterial activities of the novel nicotinonitrile-coumarin hybrids were
evaluated against three Gram-negative (Escherichia coli, Klebsiella pneumonia and
Pseudomonas aeruginosa) as well as two Gram-positive (Staphylococcus aureus and
Streptococcus mutans) bacterial stains. The values of inhibition zones and MIC were to
determine the strength of antibacterial activities using Ciprofloxacin as a standard
drug.[50–52] The results of inhibition zones and MIC values were listed in Tables 5 and
6, respectively.
The nicotinonitrile-coumarin derivative 5a exhibit the most potent antibacterial activ-
ities with MIC values of 1.9, 3.9, 3.9, 3.9 and 7.8 mg/mL against Klebsiella pneumonia,
Escherichia coli, Pseudomonas aeruginosa, Streptococcus mutans and Staphylococcus aur-
eus bacterial stains, respectively, when compared with Ciprofloxacin. Also, compound
5b exhibited potent antibacterial activities (MIC values ¼ 3.9–15.6 mg/mL) against the
tested bacterial stains.
With regards to nicotinonitrile-coumarin derivatives 5c–5f bearing benzo[d][1,3]diox-
ole moiety, compounds 5d, 5e and 5f exhibited stronger antibacterial activities (MIC
values in the range of 7.8–62.5 mg/mL) when compared with compound 5c (MIC values
in the range of 125–250 mg/mL) against the tested bacterial stains.
With regards to nicotinonitrile-coumarin derivatives 5g–5j, bearing 1,3-diphenyl-1H-
pyrazole moiety, compound 5j exhibited stronger antibacterial activities (MIC values in
the range of 15.6–62.5 mg/mL) when compared with compounds 5h and 5i (MIC values
in the range of 62.5–250 mg/mL) against the tested stains. In addition, compound 5g
exhibited the least antibacterial activities with MIC values in the range of
250–500 mg/mL when compared to Ciprofloxacin.
Cytotoxicity against eukaryotic cells
Cytotoxicity against cancer cell lines. The in-vitro cytotoxic activity of the most potent
nicotinonitrile-coumarins 5a, 5b, 5d, 5e, 5f and 5j was evaluated using neutral red

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9
Table 6. MIC values of the tested antibacterial nicotinonitrile-coumarins.
MIC (mg/mL)
Gram-negative bacteria
Gram-positive bacteria
S. aureus S. mutans
Compound
E. coli
K. pneumonia
P. aeruginosa
5a
5b
5c
5d
3.9
7.8
125
7.8
1.9
15.6
125
7.8
3.9
7.8
250
15.6
7.8
7.8
3.9
250
15.6
7.8
3.9
7.8
125
15.6
7.8
5e
7.8
3.9
5f
5g
5h
5i
31.2
250
125
62.5
31.2
31.2
62.5
500
250
125
15.6
31.2
31.2
250
250
62.5
15.6
31.2
15.6
500
250
125
31.2
31.2
31.2
500
250
125
62.5
31.2
5j
Ciprofloxacin
Table 7. The cell inhibition% and IC50 values of some novel nicotino-
nitrile-coumarins against each of MCF-7, Caco2 and HEPG2 cell lines
using Doxorubicin as a standard drug.
IC50 in mg/mL (in mM)
Compound
MCF-7
Caco2
HEPG2
5d
44.74 0.4
(81.94)
29.62 0.5
(54.25)
37.87 0.5
(69.36)
5e
33.17 0.3
(59.02)
35.53 0.4
(63.22)
45.94 0.5
(81.74)
5f
34.33 0.4
(63.81)
38.55 0.4
(71.65)
30.59 0.3
(56.86)
5j
32.58 0.4
(49.36)
29.79 0.3
(45.13)
30.81 0.4
(46.68
Doxorubicin
17.58 0.3
(32.37)
25.17 0.4
(54.86)
20.90 0.3
(38.49)
uptake assay against each of the human breast carcinoma cell line (MCF-7), colon can-
cer cell line (Caco2) and liver hepatocellular carcinoma cell line (HEPG2) (see Table 7
and Figures 1–3). The results of cytotoxic activity are expressed as the concentration of
the tested coumarin required to inhibit 50% of cell growth (IC50, mM).[50,53,54] All the
tested coumarins were tested at four different concentrations (5, 12.5, 25 and 50 mg/mL)
in comparison with Doxorubicin was used as a standard drug with IC50 ¼ 17.58 0.3,
25.17 0.4 and 20.90 0.3 mg/mL against MCF-7, Caco2 and HEPG2, respectively
(Table 7).
With regards to compounds 5a and 5b, Figures 1–3 reveals that the viability of
each of MCF-7, Caco2 and HEPG2 cells are greater than 70% (lowest value estab-
lished by the ISO 10993-5[55] to consider a material as non-cytotoxic). Thus, both
compounds 5a and 5b are the least cytotoxic antibacterial agents of the
tested coumarins.
The coumarins 5d, 5e, 5f and 5j exerted cytotoxic effects in dose-dependent manners.
Compounds 5d and 5j exhibited the best cytotoxic activities with IC50 values of
29.62 0.5 and 29.79 0.3 mg/mL against Caco2 cell line, respectively, while compounds
5f and 5j exhibited the best cytotoxic activities with IC50 values of 30.59 0.3 and

10
S. M. H. SANAD
120
100
80
60
40
20
0
Concentration (mg/mL)
0
5
12.5
86.9
77.4
54.5
73.2
62.5
74.7
25
50
5a
5b
5d
5e
5f
100
100
100
100
100
100
85.4
80.3
74.3
95.4
91.5
87.8
90.2
87.6
52.3
42.4
49.7
55.2
94.5
90.4
56.4
37.1
39.8
29.4
5j
Figure 1. Cell viability% after treatment with some novel nicotinonitrile-coumarins and Doxorubicin
for 24 h on the human breast carcinoma cell line (MCF-7).
120
100
80
60
40
20
Concentration (mg/mL)
0
0
5
12.5
92.3
83.6
44.5
62.4
59.3
62.4
25
50
5a
5b
5d
5e
5f
100
100
100
100
100
100
97.5
92.7
67.6
72.7
84.4
82
87.4
85
90.6
89.4
45.2
40.3
52.5
35.9
39.7
57.9
42.7
38.7
5j
Figure 2. Cell viability% after treatment with some novel nicotinonitrile-coumarins and Doxorubicin
for 24 h on the human colon cancer cell line (Caco2).

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11
120
100
80
60
40
20
0
Concentration (mg/mL)
0
5
12.5
25
50
5a
100
100
100
100
100
100
94.2
92.4
84.4
87.6
83.4
80.3
92.1
79.5
66.4
74.7
71.8
59.4
90.4
83.6
44.2
52.1
42.6
50.6
93
5b
5d
5e
5f
5j
94.2
49.2
55.5
32.3
33.4
Figure 3. Cell viability% after treatment with some novel nicotinonitrile-coumarins and Doxorubicin
for 24 h on the human liver hepatocellular carcinoma cell line (HEPG2).
30.81 0.4 mg/mL against HEPG2 cell line, respectively, when compared with
Doxorubicin. The rest of tested compounds exhibited decreased cytotoxic activities
against both Caco2 and HEPG2 cell lines. On the other hand, all the tested coumarins
exhibited dose-dependent cytotoxic activities with IC50 values in the range of
32.58–44.74 mg/mL against MCF-7 cell line (Table 7). In general, the results of cytotoxic
activity of the nicotinonitrile-coumarins against each of MCF-7, Caco2 and HEPG2 cell
lines are consistent with previous publications which reported that nicotinonitriles as
well as coumarins possess promising anticancer activities.[3,56–59]
Cytotoxicity against normal cell line. The cytotoxicity activities of coumarin hybrids
5a, 5b and 5e were examined using the human breast epithelial cell line MCF-10A. All
hybrids were evaluated at four different concentrations (5, 12.5, 25 and 50 mg/mL). For
comparison, Doxorubicin was used as a standard drug.
Figure 4 revealed that Doxorubicin considered as a cytotoxic agent at concentration
12.5 mg/mL or higher, as the viability of the MCF-10A cells became less than 70%.[55]
Compounds 5a and 5b were found to be non-cytotoxic agents to MCF-10A cells at con-
centrations up to 50 mg/mL. Thus, both coumarin hybrids 5a and 5b are less harmful
against MCF-10A cells than Doxorubicin.
Moreover, compound 5e can be considered as toxic to MCF-10A cells at concentration
equal to or higher 25 mg/mL. This concentration is higher than its MIC values against all the
tested strains of bacteria.

12
S. M. H. SANAD
100
90
80
70
60
50
40
30
20
10
0
5
12.5
87.4
88.6
75.6
65.2
25
50
5a
5b
5e
95.2
92.5
86.6
89.4
93.5
85.3
67.3
34.7
90.4
83.7
59.6
18.9
Doxorubicin
Concentration (µg/mL)
Figure 4. Cell viability after treatment with 5a, 5b, 5e and Doxorubicin for 24 h on MCF-10A cell line.
Table 8. Comparison between the physicochemical parameters of orally active compounds and
these of 5a, 5b and 5e.
Parameter
Orally active cpd
5a
5b
5e
Molecular weight
H-bond donors
ꢃ500
ꢃ5
364.4
0
5
3.87
0
412.4
0
5
4.78
0
562.6
0
8
6.1
2
ꢁ
ꢁꢁ
H-bond acceptors
ꢃ10
ꢃ5
ꢁꢁꢁ
CLog p
No. of violations
–
ꢁ
ꢁꢁ
ꢁꢁꢁ
No. of O or N atoms with one or more hydrogen atoms; No. of O or N atoms;
calculated log p (CLog p) is esti-
mated via ChemDraw Professional 16.0.0.82.[60]
Cytocompatibility and not cytotoxicity is essential for an antibacterial agent since it
would be in contact with the infected tissues and their neighboring eukaryotic cells.
Both compounds 5a and 5b are the best antibacterial agents. Both are non-cytotoxic,
against all the tested eukaryotic cells, at concentrations higher than their MIC values
against different tested bacterial strains. Moreover, coumarin 5e is another promising
antibacterial agent. It can be considered nontoxic to all the tested cell lines at concen-
tration equal to or lower 12.5 mg/mL (its cell viability% greater than 70%). This concen-
tration is lower than its MIC values (3.9–7.8 mg/mL) against all the tested bacterial
strains. These results revealed that 5a, 5 b and 5e are less harmful against eukaryotic
cells than Doxorubicin.
Drug-like
Lipinski stated the Rule of five as an easy tool to predict the drug likeness. Rule of five
defines four important physicochemical parameters orally active compounds required
for an orally active compound as follows: its molecular weight, its number of hydrogen
bond donors, its number of hydrogen bond acceptors and its octanol-water partition
coefficient log P (Table 8).[61,62] These parameters are related to acceptable aqueous
solubility and intestinal permeability and include the initial steps in oral bioavailability.

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SYNTHETIC COMMUNICATIONSV
13
The most potent nicotinonitrile-coumarins 5a, 5b and 5e were examined against the
mentioned physicochemical parameters. Both 5a and 5b are considered as drug-like as
both fulfill all the mentioned physicochemical parameters. On the other hand, 5e ful-
filled only two parameters as its H-bond donors are less than five as well as its H-bond
acceptors are less than ten. However, it has two violations as its molecular weight more
than 500 g/mole and its Clog P value is greater than 5 (see Table 8).
Conclusion
An efficient and eco-friendly synthetic procedure is designed for the synthesis of
novel series of nicotinonitrile-coumarin hybrid molecules bearing several aryl and/or
heteroaryl moieties. The proposed strategy includes the alkylation reaction of the
appropriate pyridine-2(1H)-thiones with benzyl chloride derivative in the presence of
mild base to prepare a novel series of 2-hydroxybenzaldehydes, bearing nicotinoni-
trile moiety followed by their Knoevenagel reaction with ethyl acetoacetate in ethanol
at 80 ꢀC in the presence of 10 mol% of piperazine citrate (1:1). The structures of
novel series of 2-hydroxybenzaldehydes as well as the target nicotinonitrile-coumarin
hybrid molecules were confirmed by considering their elemental analyses and spectral
data.
Experimental
All organic solvents were acquired from commercial sources and used as received unless
otherwise stated. Piperazine citrate (1:1) was acquired from Boc Sciences (CAS 14396-
16-8). All other chemicals were acquired from Merck and used without further purifica-
tion. The melting points were measured on a Stuart melting point apparatus and are
uncorrected. IR spectra were recorded on a Smart iTR, which is an ultra-high-perform-
ance, versatile Attenuated Total Reflectance (ATR) sampling accessory on the Nicolet
iS10 FT-IR spectrometer. 1H-NMR spectra were recorded on Varian Mercury at
300 MHz spectrophotometer using TMS as an internal standard and DMSO-d6 as solv-
ent and chemical shifts were expressed as d ppm units. Elemental analyses were carried
out on a EuroVector instrument C, H, N analyzer EA3000 Series. Mass spectra were
recorded on a GC-MS-QP1000EX spectrometer using inlet type at 70 eV.
General procedure for the synthesis of 2-((3-formyl-4-hydroxybenzyl)thio)
nicotinonitrile derivatives 3
A mixture of pyridine-2(1H)-thiones 1 (5 mmol) and potassium hydroxide (5 mmol) in
ethanol (15 mL) was stirred at room temperature for 10 minutes. Then, 5-
(chloromethyl)ꢂ2-hydroxybenzaldehyde 2 was added and the stirring was continued.
The reaction time was monitored by TLC analyses of the reaction mixture. The product
was collected by filtration, washed with each of water and cold ethanol, dried and
recrystallized from the proper solvent.

14
S. M. H. SANAD
2-((3-Formyl-4-hydroxybenzyl)thio)-4,6-dimethylnicotinonitrile (3a)
Colorless solid; m.p. 114–116 ꢀC (ethanol, 97%); IR (t cmꢂ1): 3433 (OH), 2233 (CN),
1
1678 (CO); H-NMR (DMSO-d6): d 2.37 (s, 3H, CH3), 2.52 (s, 3H, CH3), 4.44 (s, 2H,
SCH2), 6.93 (d, 1H, Ar-H3), 7.10 (s, 1H, pyridine-H5), 7.58 (d, 1H, Ar-H4), 7.75 (s, 1H,
Ar-H6), 10.22 (s, 1H, CHO), 10.66 (s, 1H, OH); 13C-NMR (DMSO-d6): d 20.0, 24.6 (2
CH3), 32.9 (SCH2), 104.2 (pyridine-C3), 115.4 (CN), 117.8 (Ar-C), 120.9 (pyridine-C5),
122.3, 129.2, 129.8, 137.6 (Ar-C), 152.9 (pyridine-C4), 160.3 (pyridine-C6), 160.7 (Ar-C),
161.8 (pyridine-C2), 191.4 (C¼O); MS (m/z): 298 (Mþ, 84.2%), 297 (100%), 281
(22.3%), 269 (14%), 136 (4.6%); Anal. for C16H14N2O2S: C, 64.41; H, 4.73; N, 9.39;
found: C, 64.69; H, 4.88; N, 9.24%.
General procedure for the synthesis of 2-(((3-acetyl-2-oxo-2H-chromen-6-
yl)methyl)thio)nicotinonitrile derivatives 5
A mixture of 2-hydroxybenzaldehydes 3 (5 mmol) and ethyl acetoacetate 4 (5 mmol) in
ethanol (15 mL) in the presence of piperazine citrate (10 mol%) was heated at 80 ꢀC.
The reaction time was monitored by TLC analyses of the reaction mixture. The reaction
mixture was cooled, filtrated, washed with each of water and cold ethanol and the reac-
tion product was recrystallized from the proper solvent.
2-(((3-Acetyl-2-oxo-2H-chromen-6-yl)methyl)thio)-4,6-dimethylnicotinonitrile (5a)
Pale yellow solid; m.p. 140–142 ꢀC (dioxane/ethanol, 95%); IR (t cmꢂ1): 2224 (CN),
1
1722, 1678 (2 CO); H-NMR (DMSO-d6): d 2.33 (s, 3H, CH3), 2.43 (s, 3H, CH3), 2.56
(s, 3H, COCH3), 4.55 (s, 2H, SCH2), 7.08 (s, 1H, pyridine-H5), 7.35 (d, 1H, chromene-
H8), 7.41 (d, 1H, chromene-H7), 7.80 (s, 1H, chromene-H5), 8.58 (s, 1H, chromene-
H4); 13C-NMR (DMSO-d6): d 20.0, 24.6 (2 CH3), 32.9 (SCH2), 104.3 (pyridine-C3),
115.5 (CN), 116.7, 118.1 (chromene-C8, C4a), 120.7 (pyridine-C5), 125.0, 130.5, 134.9,
135.2 (chromene-C3, C5, C4, C6), 146.8 (chromene-C7), 152.9 (pyridine-C4), 158.0
(chromene-C8a), 160.5 (pyridine-C6), 161.8 (pyridine-C2), 162.0 (chromene-CO), 195.2
(C¼O); MS (m/z): 364 (Mþ, 69.5%), 349 (12.2%), 321 (100%), 293 (7.2%), 268 (1.5%);
Anal. for C20H16N2O3S: C, 65.92; H, 4.43; N, 7.69; found: C, 65.76; H, 4.51; N, 7.74%.
ORCID
Sherif M. H. Sanad
Ahmed E. M. Mekky
https://orcid.org/0000-0002-0186-6418
https://orcid.org/0000-0002-8226-010X
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