Synthesis of new 2-amino-6-(4-hydroxy-2-oxo-chromen-3-yl)-4-aryl nicotine nitrile in eco-friendly media and their antimicrobials and DPPH radical scavenging activities

Article abstract of DOI:10.14233/ajchem.2019.22062

A simple, green, efficient and economical procedure for the synthesis of, 2-amino-6-(4-hydroxy-2-oxo-chromen-3-yl)-4-(aryl) nicotine nitrile (4a-g) by two routes carried in same conditions, have been reported. The new compounds 4a-g were characterized by ¹H NMR, ¹³C NMR, FT-IR and elemental analysis. The synthesized compounds were screened for their antibacterial activities against Gram-positive bacterial strains (Micrococcus luteus LB14110, Staphylococcus aureus ATCC 6538, Listeria monocytogenes ATCC 19117), Gram-negative bacteria (Escherichia coli, Salmonella typhimurium ATCC 14028 and Pseudomonas aeruginosa). The coumarin derivative 4a and 3-acetyl-4-hydroxycouamrin (1) were the most active against two bacteria Staphylococcus aureus ATCC 6538 and Candida albicans respectively. The best minimum inhibitory concentration values were obtained for the compound 4a against Candida albicans (0.0195 g/cm³). In addition, compounds 4a-g were investigated for their antioxidant activities by DPPH (2,2-diphenyl-1-picrylhydrazyl) in which most of them displayed significant antioxidant activities.

Full text of DOI:10.14233/ajchem.2019.22062

Asian Journal of Chemistry; Vol. 31, No. 7 (2019), 1609-1616
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2019.22062
Synthesis of New 2-Amino-6-(4-hydroxy-2-oxo-chromen-3-yl)-4-aryl Nicotine Nitrile in
Eco-Friendly Media and their Antimicrobials and DPPH Radical Scavenging Activities
1
2
3
AZIZA MNASRI , A. CHAKCHOUK-MTIBAA , ABDULLAH SULAIMAN AL-AYED ,
MOSBAH JEMMALI , L. MELLOULI , HALLOUMA BILEL1,4 and NACEUR HAMDI
3
2
1,3,*
1Research Laboratory of Environmental Sciences and Technologies (LR16ES09), Higher Institute of Environmental Sciences and Technology,
University of Carthage, Hammam-Lif, Tunisia
2Laboratory of Microorganisms and Biomolecules of the Center of Biotechnology of Sfax-Tunisia. Road of Sidi Mansour, Km 6 B.P. 1117,
3018 Sfax, Tunisia
3Department of Chemistry, College of Science and Arts, Qassim University, Al-Rass, Kingdom of Saudi Arabia
4Department of Chemistry, College of Science, Jouf University, P.O. Box: 2014, Sakaka, Saudi Arabia
*Corresponding author: Fax: +966 63339351; Tel: +966 556394839; E-mail: naceur.hamdi@isste.rnu.tn
Received: 10 March 2019;
Accepted: 22 April 2019;
Published online: 21 May 2019;
AJC-19424
A simple, green, efficient and economical procedure for the synthesis of, 2-amino-6-(4-hydroxy-2-oxo-chromen-3-yl)-4-(aryl) nicotine
nitrile (4a-g) by two routes carried in same conditions, have been reported. The new compounds 4a-g were characterized by 1H NMR, 13C
NMR, FT-IR and elemental analysis. The synthesized compounds were screened for their antibacterial activities against Gram-positive
bacterial strains (Micrococcus luteus LB14110, Staphylococcus aureusATCC 6538, Listeria monocytogenesATCC 19117), Gram-negative
bacteria (Escherichia coli, Salmonella typhimurium ATCC 14028 and Pseudomonas aeruginosa). The coumarin derivative 4a and 3-
acetyl-4-hydroxycouamrin (1) were the most active against two bacteria Staphylococcus aureus ATCC 6538 and Candida albicans
respectively. The best minimum inhibitory concentration values were obtained for the compound 4a against Candida albicans (0.0195 g/
cm3). In addition, compounds 4a-g were investigated for their antioxidant activities by DPPH (2,2-diphenyl-1-picrylhydrazyl) in which
most of them displayed significant antioxidant activities.
Keywords: 3-Acetyl-4-hydroxycoumarin, Multicomponent reactions, Biological activities.
for developing libraries of medicinal scaffolds. In addition,
water has emerged as a versatile solvent for organic reactions
in the last two decades since it is readily available, inexpensive,
environmentally benign, neutral and a natural solvent [13,14].
For these reasons, water has been used for multicomponent
reaction (MCRs) as well [15,16]. Multicomponent reaction
in water are of outstanding value in organic synthesis and
green chemistry. In the course of our continuing interest on
the synthesis of condensed coumarin derivatives with anti-
inflammatory and antioxidant activities [17,18] we have
extended our research to the synthesis, characterization and
biological evaluation of new bioactive coumarins derivatives
4a-g via an environmentally friendly reaction condition. Here,
we report: (i) the synthesis of novel 2-amino-6-(4-hydroxy-2-
oxo-chromen-3-yl)-4-(aryl) nicotine nitrile (4a-g) which were
obtained by two different routes in the pursuit of finding bio-
INTRODUCTION
Coumarins and their derivatives have attracted conside-
rable attention due to their extensive biological activities,
including antioxidant and anti-inflammatory [1], antiviral [2,3],
anticoagulant [4], antimicrobial [5,6] and anticancer properties
[7,8]. On the other hand, multicomponent reactions have been
successfully employed to generate highly diverse combina-
torial libraries for high-throughput screening of biological and
pharmacological activities [9,10]. This type of reaction becomes
increasingly important in organic and medicinal chemistry
because it allows to obtain highly sophisticated polyfunctional
molecules through simple one-pot procedures [11,12]. Multi-
component reaction protocol with environmentally benign
solvents and catalytic systems is one of the most suitable strategies,
which meets the requirements of green aspects of chemistry
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1610 Mnasri et al.
Asian J. Chem.
logically active compounds that can be further investigated
for their antimicrobial and antioxidant activities, (ii) the
structures of these new coumarins derivatives were characte-
rized by spectroscopic methods (1H and 13C NMR, FT-IR and
elemental analysis, (iii). The obtained compounds were screened
for their ability to inhibit the growth of a number of Gram-
positive and Gram-negative and fungi strains, which have not
been studied in the past. In addition, their antioxidant activities
were evaluated.
(Carom) 132.95 (O-Carom); 157.54 (CO-O); 162.92 (C=C-OH);
176.70 (H3C-CO). Elemental analysis % calcd. (found) for
C11H8O4: C, 64.707 (64.60); H, 3.949 (4.09); N, 8.123 (8.07).
General procedure for the synthesis of 2-amino-6-(4-
hydroxy-2-oxo-chromen-3-yl)-4-(aryl) nicotine nitrile (4):
A reaction mixture of (5 mmol) of 3-acetyl-4-hydroxycou-
marin, (5 mmol) of aromatic aldehyde, malonitrile (5 mmol),
with ammonium acetate (10 mmol) and 10 mL of dichloro-
methane was refluxed for an appropriate time under continuous
agitation. The reaction was followed by (TLC). After comple-
tion of the reaction, the mixture was cooled to room tempe-
rature. The solid obtained was filtered, washed with ice water
and purified by recrystallization in 95 % ethanol.
EXPERIMENTAL
All manipulations were performed using standard
Schlenck techniques under argon atmosphere. Chemicals were
purchased from Sigma Aldrich and used without further puri-
fication. All the solvents were purified and dried by MBraun
SPS 800 solvent purification system. Column chromatography
2-Amino-6-(4-hydroxy-2-oxo-chromen-3-yl)-4-phenyl
nicotine nitrile (4a):Yield: 95 %; m.p.: 120 °C; FT-IR (KBr,
ν
max, cm–1): 3505 (O-H); 3402 (NH2); 1592 (C = C); 1715 (CO
1
lactone), 2215 (CN). 1H NMR (400 MHz, DMSO-d6) in ppm:
δ 7.27-8.18 (m, 12H, 10Harom + aryl-NH2). 13C NMR (100 MHz,
DMSO-d6) in ppm: δ 104,125.7, 124.6, 123.5, 122.4, 112.9,
119.1, 153.5, 152.1, 143.3, 134, 128.7, 128.4, 127.5 (Carom);
116.2 (aryl-CN); 108.8 (aryl-C=C-OH); 136.6 (O-Carom); 159.5
(C=C-OH); 157.9 (NH2-Carom, O-COarom). Elemental analysis
% calcd. (found) for C21H13N3O3: C, 70.980 (70.82); H, 3.687
(3.90); N, 11.825 (11.80).
was performed using silica gel 60 (70-230 mesh). H NMR
and 13C NMR spectra were recorded at 400 and 100 MHz,
respectively. Chemical shifts, δ, are reported in ppm relative
to the internal standard TMS for both 1H and 13C NMR. The
NMR studies were carried out in high-quality 5 mm NMR
tubes. Signals are quoted in parts per million as δ down-field
from tetramethylsilane (δ = 0.00) as an internal standard. IR
spectra were recorded on a 398 spectrophotometer (Perkin-
Elmer, King Saud University, Ryadh, SaudiArabia). Elemental
microanalysis was performed on an Elementar Vario El III
Carlo Erba 1108 elemental analyzer (Rennes, France) and the
values found were within 0.3 % of the theoretical values.
Melting points were determined with Kofler bench at Isste of
Borjcedria (Hammam Lif, University of Carthage, Borj Cedria,
Tunisia).
2-Amino-6-(4-hydroxy-2-oxo-chromen-3-yl)-4-(4-
methylphenyl)nicotine nitrile (4b): Yield: 75 %; m.p.: 142
°C; FT-IR (KBr, νmax, cm–1): 3502 (O-H); 3410 (NH2); 1585
(C = C); 1710 (CO lactone), 2215 (CN). 1H NMR (400 MHz,
DMSO-d6) in ppm: δ 2.27 (s, 3H, aryl-CH3); 7.27-8.19 (m,
11H, 9 Harom + aryl-NH 2). 13C NMR (100 MHz, DMSO-d6) in
ppm: δ 24.5 (aryl-CH3); 104.1, 125.7, 124.6, 123.5, 116.2,
153.6, 116.2, 104.1, 134.0, 132.8, 129.0, 127.4 (Carom); 136.2
(O-Carom); 119.1 (aryl-CN); 112.9 (aryl-C=C-OH); 157.9 (NH2-
Carom, O-COarom); 159.5 (C=C-OH). Elemental analysis % calcd.
(found) for C22H15N3O3: C, 71.536 (71.60); H, 4.093 (3.09);
N, 11.376 (11.40).
2-Amino-6-(4-hydroxy-2-oxo-chromen-3-yl)-4-(4-N,N′-
dimethylaminophenyl)nicotine nitrile (4c): Yield: 80 %;
m.p.: 160 °C; FT-IR (KBr, νmax, cm–1): 3441 ((O-H); NH2); 1604
(C = C); 1721 (CO lactone), 2233 (CN). 1H NMR (400 MHz,
DMSO-d6) in ppm: δ 3.10 (s, 6H, aryl-N(CH3)2); 7.30 (s, 2H,
aryl-NH2); 6.83-8.03 (m, 9H, Harom). 13C NMR (100 MHz,
DMSO-d6) in ppm: δ 58.9, 69.1 (aryl-N(CH3)2); 105.1, 134.0,
131.3, 128.6, 125.0, 116.2, 104.1, 150.0, 153.2, 116.7, 134.0
(Carom), 112.2 (aryl-C=C-OH); 119.2 (aryl-CN); 152.4 (O-
Carom); 159.3 (C=C-OH); 158.3 (O-COarom), 153.6 (NH2-Carom).
General procedure for synthesis of 3-acetyl-4-hydroxy-
coumarin (1): In a round bottom flask, 3 g (18.6 mmol) of 4-
hydroxycoumarin, 16 mL of acetic acid (27.9 mmol) (solvent
and reagent at a time) and 5.6 mL (60 mmol) of phosphorus
oxychloride are added. The reaction was refluxed under conti-
nuous stirring for 1 h. Control of the evolution of the reaction
by thin layer chromatography (TLC) (eluent: hexane-ethanol
80:20) revealed the formation of a single compound. At the
end of the reaction, the product was filtered, recrystallized
from ethanol, dried and recovered (Scheme-I).
Yield: 92 %; m.p.: 225 °C; FT-IR (KBr, νmax, cm–1): 3185
1
(O-H); 1700 (CO lactone); H NMR (400 MHz, DMSO-d6)
ppm δ: 2.45 (s, 3H, CO-CH3); 7.15-7.92 (m, 4H, Harom), 17.72
(s, OH). 13C NMR (100 MHz, DMSO-d6) in ppm δ: 32.36
(H3C-CO); 102.32 (aryl-C=C-OH); 115.91, 122.98, 125.66
R
N
O
OH
C
C
CN
Bmim [triflate]
OH
O
CHO
+
+
18h/reflux
N
NH2
R
O
O
N
2
CH3COOHNH3
1
O
3
4
4a
4b
4c
4d
R = : H, : p-CH3, : p-N(CH3)2, : p-NO2,
4e: m-Br, 4f: m-OCH3, 4g: m-OH
Scheme-I: Synthetic protocol of 2-amino-6-(4-hydroxy-2-oxo-chromen-3-yl) -4-aryl nicotine nitrile (4a-g)

Vol. 31, No. 7 (2019) Synthesis of New 2-Amino-6-(4-hydroxy-2-oxo-chromen-3-yl)-4-aryl Nicotine Nitrile in Eco-Friendly Media 1611
Elemental analysis % calcd. (found) for C23H18N4O3: C, 69.336
(69.41); H, 4.554 (4.70); N, 14.062 (14.10).
strains were grown overnight in Luria-Bertani (LB) agar
medium (g/L): peptone 10; yeast extract 5 and NaCl 5 at pH
7.2 under aerobic conditions and constant agitation (200 rpm)
at 30 °C for M. luteus LB14110 and L. monocytogenes ATCC
19117 and at 37 °C for S. aureus ATCC 6538, S. typhimurium
ATCC 14028 and P. aeruginosaATCC 49189 and then diluted
1:100 in LB media and incubated for 5 h under constant
agitation (200 rpm) at the appropriate temperature.
Agar well diffusion method:Agar well diffusion method
was employed for the determination of the antimicrobials
activity of the synthesized compounds according to Güven
[20] with some modifications. Briefly, the synthesized com-
pounds are allowed to diffuse out into the appropriate agar
medium (LB agar medium) and interact in a plate freshly
seeded with a suspension of the indicators microorganisms
(0.1 mL of 108 cells per mL). The plate was incubated at the
appropriate temperature after staying at 4 °C for 2 h. The resul-
ting zones of inhibition will be uniformly circular as there
will be a confluent lawn of growth. The antibacterial activity
was assayed by measuring in millimeters the diameter of the
inhibition zone formed around the well. All tests are assayed
in triplicate and expressed as the average standard deviation
of the measurements.
2-Amino-6-(4-hydroxy-2-oxo-chromen-3-yl)-4-(4-
nitrophenyl)nicotine nitrile (4d):Yield: 85 %; m.p.: 210 °C;
FT-IR (KBr, νmax, cm–1): 3507 (O-H); 3405 (NH2); 1585 (C =
1
C); 1715 (CO lactone), 2215 (CN). H NMR (400 MHz,
DMSO-d6) in ppm: δ 7.28 (s, 2H, aryl-NH2); 7.62-8.40 (m,
9H, Harom). 13C NMR (100 MHz, DMSO-d6) in ppm: δ 85.2,
104.1, 134.0, 134.1, 125.6, 124.4, 148.8, 153.2, 143.4, 144.8,
130.1, 125.6 (Carom) 144.8 (O-Carom); 109.9 (C aryl-C=C-OH);
119.2 (aryl-CN); 177.9 (C=C-OH); 161.7 (O-COarom), 153.6
(NH2-Carom). Elemental analysis % calcd. (found) for C21H12N4O5:
C, 63.002 (63.10); H, 3.021 (3.20); N, 13.995 (13.90).
2-Amino-6-(4-hydroxy-2-oxo-chromen-3-yl)-4-(3-
bromophenyl) nicotine nitrile (4e): Yield: 87 %; m.p.: 160
°C; FT-IR (KBr, νmax, cm–1): 3502 (O-H); 3405 (NH2); 1592
(C = C); 1716 (CO lactone), 2218 (CN). 1H NMR (400 MHz,
DMSO-d6) in ppm: δ 7.31-8.38 (m, 11H, 9Harom + NH2). 13
C
NMR (100 MHz, DMSO-d6) in ppm: δ 100.9, 134.0, 134.1,
125.6, 124.3, 148.8, 104.1, 132.9, 153.1, 125.7 (Carom), 109.3
(aryl-C=C-OH); 119.2 (aryl-CN); 139.3 (O-Carom). 159.3 (O-
COarom), 178.4 (C=C-OH); 153.6 (Br-Carom); 154.0 (NH2-Carom).
Elemental analysis % calcd. (found) for C21H12N3O3Br : C,
58.084 (58.10); H, 2.785 (2.90); N, 9.677 (9.80).
Minimum inhibitory concentration (MIC) were done regar-
ding our previous study [19].
2-Amino-6-(4-hydroxy-2-oxo-chromen-3-yl)-4-(3-
methoxyphenyl) nicotine nitrile (4f): Yield: 87 %; m.p.: 160
°C; FT-IR (KBr, νmax, cm–1): 3510 (O-H); 3437 (NH2); 1604
(C = C); 1710 (CO lactone), 2360 (CN). 1H NMR (400 MHz,
DMSO-d6) in ppm: δ 3.72 (s, 3H, aryl-OCH3); 7.40 (s, 2H,
aryl-NH2); 6.80-7.89 (m, 9H, Harom). 13C NMR (100 MHz,
DMSO-d6) in ppm: δ 55.4 (aryl-CH3); 112.4, 133.3, 130.1,
125.1, 122.9, 120.1, 119.6, 125.7, 117, 97.9, 145.3 (Carom);
97.9 (aryl-C=C-OH); 104.3 (aryl-CN); 160 (C=C-OH); 159.7
(O-COarom), 158.4 (NH2-Carom); 153.9 (O-Carom); 152.6 (H3C-
OCarom). Elemental analysis % calcd. (found) for C22H15N3O4:
C, 68.566 (68.61); H, 3.923 (3.90); N 10.904 (10.82).
2-Amino-6-(4-hydroxy-2-oxo-chromen-3-yl)-4-(3-
hydroxyphenyl) nicotine nitrile (4g):Yield: 95 %; m.p.: 180
°C; FT-IR (KBr, νmax, cm–1): 3501 (O-H); 3408 (NH2); 1588
(C = C); 1731 (CO lactone), 2233 (CN). 1H NMR (400 MHz,
DMSO-d6) in ppm: δ 7.02-8.51 (m, 12 H, 9Harom + aryl-NH2 +
aryl-OH). 13C NMR (100 MHz, DMSO-d6) in ppm: δ 85.3,
116.6, 120.8, 119.8, 117.7, 104.1, 130.5, 116.6, 117.7 (Carom);
92 (aryl-C=C-OH); 115.2 (aryl-CN); 134.3 (O-Carom); 148.8
(NH2-Carom); 177.7 (C=C-OH); 158.0 (O-COarom); 153.2 (HO-
Carom). Elemental analysis % calcd. (found) for C21H13N3O4:
C, 67.922 (67.81); H, 3.53 (3.60); N 11.316 (11.42).
The minimum inhibitory concentration (MIC) of the
synthesized compounds was determined in accordance with
NCCLS guideline M7-A6 and M38-P [21]. The test was per-
formed in sterile 96-well microplates with a final volume in
each microplate well of 100 mL. The synthesized compounds
(20 mg/mL) were properly prepared in solution of dimethyl-
sulfoxide/water (1/9; v/v). The inhibitory activity of each
synthesized compound was transferred to each well in order
to obtain a twofold serial dilution of the original sample and
to produce the concentration range of 0.0048-20 mg/mL. To
each test well 10 mL of cell suspension were added to final
inoculum concentrations of 106 CFU/mL for each microorga-
nisms. Positive growth control wells consisted of microorga-
nisms only in their adequate medium. Cells suspension at the
same concentration supplemented with ampicillin was used
as control. The plates were then covered with the sterile plate
covers and incubated at the appropriate temperature of each
microorganism. The MIC was defined as the lowest concentra-
tion of the synthesized compound at which the microorganism
does not demonstrate visible growth after incubation. As an
indicator of microorganism growth, 25 mL of thiazolyl blue
tetrazolium bromide (MTT), indicator solution (0.5 mg/mL)
dissolved in sterile water were added to the wells and incubated
at room temperature for 30 min. This determination was done
in triplicate and obtained results were very similar. The reported
value is the average of the three tests.
Biological analysis was performed according to previous
study [19].
Bacterial strains, media and growth conditions: Bacteria
strains used as indicator microorganisms for the antibacterial
activity assays were: Micrococcus luteus LB14110, Staphylo-
coccus aureus ATCC 6538, Listeria monocytogenes ATCC
19117 Salmonella typhimurium ATCC 14028 and Pseudo-
monas aeruginosa ATCC 49189 were obtained from interna-
tional culture collections (ATCC) and local culture collection
of the Laboratory of Microorganisms and Biomolecules of
the Centre of Biotechnology of Sfax, Tunisia. These bacterial
Antioxidant activity: DPPH radical scavenging activity
were done regarding our previous study [19].
Scavenging of 2,2-diphenyl-1-picrylhydrazyl radical
(DPPH_assay) is the simplest and most widely reported method
for screening antioxidant activity. The procedure involves mea-
surement of decrease in absorbance of DPPH at its absorption
maxima of 517 nm. This assay determines the scavenging of

1612 Mnasri et al.
Asian J. Chem.
TABLE-1
stable radical species according to the method of Kirby and
Schmidt [22] with slight modifications. Briefly, synthesized
compounds were dissolved in dimethylsulfoxide (DMSO)/
water (1/9; v/v) and diluted with ultrapure water at different
concentrations (1, 0.5, 0.250, 0.125, 0.0625, 0.03125 mg/mL).
Then, 500 mL of a 4 % (w/v) solution of DPPH radical in
ethanol was mixed with 500 mL of samples. The mixture was
incubated for 30 min in the dark at room temperature. The
scavenging capacity was determined spectrophotometrically
by monitoring the decrease in absorbance at 517 nm against a
blank. The percentage of antiradical activity (%ArA) had been
calculated as follows:
SYNTHESIS OF 4-PHENYL-1,2-DIHYDRO-6-(4-HYDROXY-2-
OXO-2H-CHROMEN-3-YL)-2-OXOPYRIDIN-3-CARBONITRILE
(4a-g) IN THE PRESENCE OF DIFFERENT SOLVENTS
Entry
Solvent
DCM
DCE
Ethanol
THF
Temp. (°C)
Time (min)
30
Yield (%)a
1
2
3
4
5
6
7
90
128
78
66
100
40
83
80
72
62
95
60
62
30
120
120
30
120
120
Bmim [triflate]
DCM
Toluene
110
Reaction conditions: 3-acetyl-4-hydroxycoumarin (5 mmol),
malonitrile (5 mmol), benzaldehyde (5 mmol), solvent (15 mL), reflux,
ammonium acetate (10 mmol).
Absorbance of control − Absorbance of test sample
ArA (%) =
×100
aIsolated yield of product.
Absorbance of control
All tests are assayed in triplicate and expressed as the
average standard deviation of the measurements.
Also, in order to optimize the ammonium acetate loading,
the model reaction was performed with different amounts of
catalyst at ambient temperature. The results are summarized
in Table-2.
RESULTS AND DISCUSSION
The first efficient one-pot synthesis of 4-aryl-1,2-dihydro-
6-(4-hydroxy-2-oxo-2H-chromen-3-yl)-2-oxopyridin-3-
carbonitriles has been developed from malonitrile, aromatic
aldehyde and 3-acetyl-4-hydroxy coumarin, the approach is
outlined in Scheme-I.
TABLE-2
EFFECT OF CATALYST AMOUNT ON THE CONDENSATION
OF BENZALDEHYDE, ETHYL CYANOACETATE AND 3-
ACETYL-4-HYDROXYCOUMARINE (1) IN Bmim[triflate]a
Entry
Catalyst
NH4OAc
NH4OAc
NH4OAc
NH4OAc
NH4OAc
NH4OAc
No catalyst
Mol (%)
Time (h)
Yield (%)b
3-Acetyl-4-hydroxycoumarin (1)was synthesized by acety-
lation of the 4-hydroxycoumarin, by the method of Dholakia
et al. [23] using glacial acetic acid as acetylating agent in the
presence of POCl3. The reaction was rapid, without involving
any kind of competition from the intramolecular condensation
of 4-hydroxycoumarin [24].
1
2
3
4
5
6
7
5
5
10
3
3
60
85
82
95
92
88
10
10
15
20
25
30
-
3
10 h 20 min
10 h 20 min
1
This compound 1 was characterized by IR, H and 13C
aReaction conditions: 3-acetyl-4-hydroxycoumarin (5 mmol),
malonitrile (5 mmol), benzaldehyde (5 mmol), solvent (15 mL), reflux.
bIsolated yield of product.
NMR. The IR spectra of compound 1 revealed a strong band
at 3185 cm–1confirming the presence of OH group and showed
band in the region of 1700 cm–1 which is the characteristic for
C=O of coumarin. The 1H NMR shows that aromatic proton
arrow as a multiplet between 7.15 and 7.92 ppm. A singlet at
2.45 ppm was assigned to methylic proton whilst the OH signal
appeared at 17.72 ppm. This very high value of the chemical
shift might be explained by only an intermolecular hydrogen
bond [25].
In order to seek optimal solvent and optimal amounts of
catalyst, we run a model reaction by stirring equimolecular
amounts of 3-acetyl-4-hydroxycoumarin (1) with malonitrile
and benzaldehyde in the presence of ammonium acetate. The
model reaction was explored using different solvents such
as Bmim[triflate], DCM, DCE, ethanol, tetrahydrofuran and
toluene in the presence of ammonium acetate. The results are
summarized in Table-1.
It was found that polarity of solvent and the presence of
ammonium acetate play an important role for the success of
the reaction. The results indicated that solvents were also
affected on the yield of of the products (Table-1). In the organic
solvents such as dichloromethane, THF, ethanol, or toluene, the
yield of 4 were lower and longer reaction times were required.
It was observed that among all solvents and media, the
best result was obtained when Bmim[triflate] was chosen in the
presence of ammonium acetate at 90 °C. The desired product
was obtained in excellent yield and high purity.
It was found that, when the reaction was carried out in
the presence of 5 mol % of catalyst, 60 % of yield was obtained.
As we increase the percentage of the catalyst to 10, 15 and 20
mol %, the yields were also found to be increased up to 85,
82 and 95 %, respectively, but beyond 20 mol % there is no
significant improvement of the rate as well as yield of the
reaction and further increase in the quantity of catalyst did
not show appreciable improvement in the yield of product.
Thus, 20 mol % of catalyst was chosen as maximum quantity
of the catalyst for the reaction.
To explore the scope and generality of this multicom-
ponent reaction (MCR), we tried to construct a library of
2-amino-6-(4-hydroxy-2-oxo-chromen-3-yl)-4-aryl nicotine
nitrile (4a-g) derivatives using the optimized reaction condi-
tions, the scope of the method was investigated with a series
of substituted aromatic aldehydes (Scheme-I).
A variety of aryl aldehydes containing different substi-
tuents (e.g. –NO2, –OH, –Br, –OCH3, –OMe, -p-N(CH3)2
reacted in this MCR to provide excellent product yields (75–
95 %). The aromatic aldehydes carrying both electron with-
drawing (Entries 1-5) and electron-donating functional groups
under-went successful condensation with ethyl cyanoacetate
and 4-hydroxycoumarin in the presence of catalytic amount
of ammonium acetate in Bmim[triflate] at reflux to afford the

Vol. 31, No. 7 (2019) Synthesis of New 2-Amino-6-(4-hydroxy-2-oxo-chromen-3-yl)-4-aryl Nicotine Nitrile in Eco-Friendly Media 1613
corresponding products 4a-g in good yields. moreover, the
electronic effects of the substituent were not observed.
In IR spectra, bands in the region 3510-3414 cm–1 were
attributed to the OH group of 4a-g seriesand bands in the region
1625-1520 cm–1 were attributed to symmetric and asymmetric
frequency of the NO2 group of the compound 4d. Bands at
1731-1710 cm–1 obtained from the lactone ring of coumarin
CO. 1585-1604 cm–1 stretching frequencies correspond to the
C=C groups of coumarins derivatives. In 1HNMR spectra, the
singlet between δ 7.27–7.40 ppm corresponds to the presence
of the NH2 group of the compounds 4a-g. The absence of
methylic proton signal at δ = 2.45 ppm and the presence of
the signal of NH2 at support the formation of compounds 4a-
g. In 13C NMR, signal at δ 119.2 assigned to CN, signal at δ
168.2–169.2 confirmed lactone carbonyl of 4c and the signal
at δ 3.10 ppm due to N(CH3)2. Other signals are in good agree-
ment with the target compounds in accordance with the litera-
ture data for other 4-hydroxycoumarin derivatives [26-33].
The formation of compound 4 could be explained by
the reaction sequence in Scheme-II. First, condensation of
H
O
C
H
O
CH
C
C
C
C
N
N
O
OH
N
N
NH4
NH4
O
OH
O
O
O
OH
O
H
OAc
C
H
H2
CH2
R
O
O
OH
OH
O
O
OH
O
O
HOAc
C
H
C
R
R
H2
O
O
H
O
-H2O
NH4
,H
N
N
N
N
C
C
C
C
OH
OH
O
C
H
OH
O
O
C
C
H
R
H
R
O
O
(i2)
(i1)
NH3
NH2
R
CN
H
OH
OH
O
N
O
N
C
OH
O
R
O
O
HN
NH2
(i3)
NH2
NH2
NH2
aromatisation
CN
CN
CN
OH
O
OH
O
N
O
HN
OH
O
N
-H2O
OH
R
O
O
4
Scheme-II: A proposed mechanism for the three-component synthesis of 4-aryl-1,2-dihydro-6-(4-hydroxy-2-oxo-2H-chromen-3-yl)-2-
oxopyridin-3-carbonitriles (4a-g)

1614 Mnasri et al.
Asian J. Chem.
3-acetyl-4-hydroxycoumarin (1)with arylaldehydes is proposed
to give intermediate (i1), then malonitrile react with inter-
mediate (i1) to give intermediate (i2) occurs to provide the
intermediate (i3) which undergoes isomerization to form the
target 4-aryl-1,2-dihydro-6-(4-hydroxy-2-oxo-2H-chromen-3-
yl)-2-oxopyridin-3-carbonitriles (4a-g).
In second path, we tried to synthetize coumarins deriva-
tives 4 with another route. The 3-acetyl-4-hydroxycoumarin
(1) condensed with arylaldehydsin the presence of piperidine
gaves intermediate (i), then this precursor was refluxed with
malonitrile and Bmim[triflate] to achieve targeted molecule 4
(Scheme-III).
The molecular structures and purity of the newly synthe-
sized compounds were identified by NMR (1H and 13C), FT-
IR and elemental analysis (CHN). In FT-IR spectra, absorption
bands attributed to symmetric and asymmetric stretching vibra-
tions of amino groups appeared within ν = 3456-3428 and
3326-3281 cm–1, as well as stretching vibrations of nitro groups
were recorded within ν = 1543-1514 and 1331-1318 cm–1.
The presence of nitrile groups was deduced both from IR bonds
at ν = 2228-2206 cm–1.
In summary, both synthetic routes successfully provided
a strategy for synthesis of synthesized 2-amino-6-(4-hydroxy-
2-oxo-chromen-3-yl)-4-(aryl) nicotinenitrile 4a-g.
In first route, the 2-amino-6-(4-hydroxy-2-oxo-chromen-
3-yl)-4-(aryl) nicotine nitrile 4a-g is available and the starting
material is low-cost and can be obtained easily. This synthetic
route not only saves the time but lowers the cost for manu-
facture. The second route is longer. For these reasons, it is not
suitable for large scale preparation. The second route is based
on the materials commercially available, the reaction involves
2 steps. The total yield was up to 20 %.
Antioxidant activity of synthesized 2-amino-6-(4-
hydroxy-2-oxo-chromen-3-yl)-4-(aryl)nicotinenitrile
(4a-g): The in vitro antioxidant activities of the coumarins
derivatives 4 were screened using the DPPH (1,1-diphenyl-2-
picrilhydrazyl). The results obtained from the two approaches
were consistent
L-Ascorbic acid and BHT were used as the standard anti-
oxidant. The results are expressed as mg/mL The activity results
of the newly synthesized compounds are represented in Fig. 1.
We found that most of the compounds showed considerable
free radical scavenging activity.
The analysis of the results shows that the profiles of anti-
radical activity obtained reveal that most of the synthetic
products tested have an antiradical activity. The three products
4a, 4d and 4e have a medium antioxidant activity, while the
product 4f has a very significant activity and close to the anti-
radical activity of the two synthetic antioxidants used as refe-
rences (gallic acid and BHT).
Antimicrobial activity of 2-amino-6-(4-hydroxy-2-oxo-
chromen-3-yl)-4-(aryl) nicotine nitrile (4a-g): The in vitro
antibacterial activity of the samples 4a-g was assessed against
Escherichia coli, Staphylococcus aureus, Pseudomonas
aeruginosa, Salmonella typhimurium, Listeria monocytogenes,
Micrococcus luteus and Candida albicans, respectively. The
different bacterial species used Gram-positive bacteria:
Micrococcus luteus LB14110, Staphylococcus aureus ATCC
6538, Listeria monocytogenes ATCC 19117, Gram-negative
bacteria: Escherichia coli, Salmonella typhimurium ATCC
H
O
N
OH
O
O
O
H
OH
O
O
H
H
R
N
OH
O
O
O
C H
H2
CH2
O
C
H2
O
H
R
N
H
OH
O
O
OH
O
OH
O
O
O
CH2
OH
C
H2
O
C
H
-H2O
O
O
R
R
(i)
CH2(CN)2
NH2
CN
Bmim[triflate]
OH
N
O
O
R
4
Scheme-III: Synthesis of coumarins derivatives 4 by two steps

Vol. 31, No. 7 (2019) Synthesis of New 2-Amino-6-(4-hydroxy-2-oxo-chromen-3-yl)-4-aryl Nicotine Nitrile in Eco-Friendly Media 1615
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
4a
4d
Gallic acid
BHT
Gallic acid
BHT
Concentration (mg/mL)
Concentration (mg/mL)
100
100
90
80
70
60
50
40
30
20
10
0
90
80
70
60
50
40
30
20
10
0
4f
4e
Gallic acid
BHT
Gallic acid
BHT
Concentration (mg/mL)
Concentration (mg/mL)
Fig. 1. Scavenging activity of 2-amino-6-(4-hydroxy-2-oxo-chromen-3-yl)-4-(aryl)nicotine nitrile (4) on DPPH radical
14028 and Pseudomonas aeruginosa and the fungus Candida
albicans were provided by the Laboratory of Microorganisms
and Biomolecules of the Biotechnology Center of Sfax. These
species kept on nutrient agar, are reactivated on agar medium
24 h before performing the antimicrobial tests.
As a first approach, we used the traditional method of
detecting antimicrobial activities (well method). The synthe-
sized products are prepared with DMSO at an initial concen-
tration of 20 mg/mL. All the results of this study are shown in
Fig. 2.
bacteria compared to the unique structure of Gram-positive
membrane.
From Fig. 2, compound 4a was effective against Candida
albicans and against Staphylococcus aureus ATCC 6538, this
compound has moderate activity. However, compound 4e
synthesized and tested in this study shows significant activity
against Pseudomonas aeruginosa ATCC 49189.
Determination of minimum inhibitory concentration
(MIC): Minimum inhibitory concentration (MIC) values were
determined using the standard broth micro-dilution technique
recommended by the Clinical and Laboratory Standard Insti-
tute [34,35]. The MIC results showed that all tested coumarin
peptide salts (Table-3) had weak antimicrobial effects against
E. coli and good to moderate effects against S. aureus.
45
Micrococcus luteus
40
35
30
25
20
15
10
5
Listeria monocytogenes
Staphylococcus aureus
Salmonella typhimurium
Pseudomonas aeruginosa
Escherichia coli
TABLE-3
MIC VALUES OF PRODUCTS TESTED
AGAINST TWO BACTERIA AND ONE FUNGUS
Candida albicans
Microorganisms
Ampicilline Fluconazole
3
4a
S. aureus
P. aeruginosa
C. albicans
0.6250
0.3125
0,1562
1.2500
1.2500
0.0195
0.04
0.04
–
–
–
0
0.00125
3
4a
4b
4c
4d
Compounds
Fig. 2. Antibacterial activity of the prepared compounds 4a-g
4e
4f
4g AMC
According to the data reported in Table-3, the derivatives
were ordered based on the spread of inhibitory properties and
the MIC values as follows:
It is found that the activity of the synthesized compounds
depends on their concentration and the bacterial strain tested.
Gram-positive bacteria were more sensitive to the antimicrobial
properties of synthesized compounds than Gram-negative
bacteria. This effect can be attributed to the great complexity
of the membrane-containing cell envelope in Gram-negative
4b > 4e > 4d > 4c
We evaluated the antimicrobial activity of the two syn-
thetic products 3 and 4a with the highest activity against two
bacteria (S. aureus and P. aeruginosa) and against a fungus
(Candida albicans) by the determination of the MIC in a liquid

1616 Mnasri et al.
Asian J. Chem.
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49189: 0.3125 and 1.25 µg/mL, respectively for products 3
and 4a.
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In conclusion, a series of 2-amino-6-(4-hydroxy-2-oxo-
chromen-3-yl)-4-(aryl) nicotine nitrile were designed, synthe-
sized using two routes and evaluated for their antimicrobial
and antioxidant activities. The obvious advantages of the
methods are: operational simplicity, high atom economy and
excellent yields. The preparations of the new products are sup-
ported by elemental analyses, IR and 1H NMR and 13C NMR.
The compounds 4a-g were investigated for antioxidant acti-
vities by DPPH (2,2-diphenyl-1-picrylhydrazyl). Furthermore,
compounds 4a-g were evaluated for antimicrobial activity by
well method and determination of minimum inhibitory concen-
tration (MIC). These compounds may serve as lead compounds
for further development into novel antimicrobial and anti-
oxidant agents. In addition the obtained compounds 4 we be
used as new materials for hydrogen storage.
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The authors gratefully acknowledge Qassim University
represented by the Deanship of Scientific Research on the
material support for this research under the number 5272-
alrasscac-2018-1-14-S during the academic year 1440AH/
2018AD.
CONFLICT OF INTEREST
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