Synthesis and anti-viral activities of some 3-(naphthalen-1-ylmethylene)-5- phenylfuran-2(3H)-one candidates

Article abstract of DOI:10.1007/s11164-013-1045-z

3-(Naphthalen-1-ylmethylene)-5-phenylfuran-2(3H)-one 1 was prepared and converted into a variety of heterocyclic systems of synthetic and biological importance. Benzylamine was reacted with furanone 1 to afford compounds 2 and 3 according to the reaction conditions. Butanamide 2 was reacted with thionyl chloride or thiourea to give derivatives 4 and 5, respectively. Compound 3 was reacted with ethyl cyanoacetate to give the corresponding pyrrolopyridine derivative 6. Treatment of 1 with hydrazine hydrate afforded compounds 7 and 8 according to the reaction conditions. Also, compound 1 was reacted with phenyl hydrazine, hydroxyl amine, malononitrile or thiourea to give compounds 9-12, respectively. Cyclization of 7 with ethoxymethylene-malononitrile, ethyl-(ethoxymethylene)cyanoacetate, carbon disulphide or acetylacetone afforded the corresponding compounds 13-16, respectively. Condensation of 7 with p-nitrobenzaldehyde gave the corresponding hydrazone 17, which was treated with thioglycolic acid or chloroacetyl chloride to give compounds 18 and 19, respectively. Also, most of the prepared products were tested for anti-avian influenza virus and revealed promising antiviral activity against H5N1 virus [A/Chicken/Egypt/1/2006 (H5N1)] by determination of both TC ₅₀ and ED ₅₀ and confirmed by plaque reduction assay on MDCK cells. Compounds 7, 8, 11, 12 and 13 showed the highest effect compared with the other tested compounds.

Full text of DOI:10.1007/s11164-013-1045-z

Res Chem Intermed
DOI 10.1007/s11164-013-1045-z
Synthesis and anti-viral activities of some
3-(naphthalen-1-ylmethylene)-5-phenylfuran-2(3H)-one
candidates
•
Eman M. Flefel Waled A. Tantawy
•
•
•
Randa E. Abdel-Mageid Abd El-Galil E. Amr
Rolla Nadeem
Received: 30 December 2012 / Accepted: 11 January 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract 3-(Naphthalen-1-ylmethylene)-5-phenylfuran-2(3H)-one 1 was prepared
and converted into a variety of heterocyclic systems of synthetic and biological
importance. Benzylamine was reacted with furanone 1 to afford compounds 2 and 3
according to the reaction conditions. Butanamide 2 was reacted with thionyl
chloride or thiourea to give derivatives 4 and 5, respectively. Compound 3 was
reacted with ethyl cyanoacetate to give the corresponding pyrrolopyridine derivative
6. Treatment of 1 with hydrazine hydrate afforded compounds 7 and 8 according to
the reaction conditions. Also, compound 1 was reacted with phenyl hydrazine,
hydroxyl amine, malononitrile or thiourea to give compounds 9–12, respectively.
Cyclization of 7 with ethoxymethylene-malononitrile, ethyl-(ethoxymethylene)cy-
anoacetate, carbon disulphide or acetylacetone afforded the corresponding com-
pounds 13–16, respectively. Condensation of 7 with p-nitrobenzaldehyde gave the
corresponding hydrazone 17, which was treated with thioglycolic acid or chloro-
acetyl chloride to give compounds 18 and 19, respectively. Also, most of the pre-
pared products were tested for anti-avian influenza virus and revealed promising
antiviral activity against H5N1 virus [A/Chicken/Egypt/1/2006 (H5N1)] by deter-
mination of both TC50 and ED50 and confirmed by plaque reduction assay on
E. M. Flefel (&) Á W. A. Tantawy Á R. E. Abdel-Mageid
Department of Photochemistry, National Research Centre, Cairo, Egypt
e-mail: emanflefel@yahoo.com
A. E.-G. E. Amr
Pharmaceutical Chemistry Department, Drug Exploration & Development Chair (DEDC),
College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
e-mail: aamr1963@yahoo.com
A. E.-G. E. Amr
Applied Organic Chemistry Department, National Research Center, Dokki, Cairo, Egypt
R. Nadeem
Virology Laboratory, Environmental Research Division, National Research Center, Cairo, Egypt
123

E. M. Flefel et al.
MDCK cells. Compounds 7, 8, 11, 12 and 13 showed the highest effect compared
with the other tested compounds.
Keywords Furanone Á Acid hydrazide Á Anti-avian influenza virus (H5N1)
Introduction
The furanones and other related heterocyclic derivatives exhibit a range of
biological activities, such as anti-inflammatory [1, 2], analgesic and ulcerogenic [3]
antimicrobial [4, 5], antiviral [6], and antibiotic [7–9]. Pyrimidines and fused
pyrimidines, being an integral part of DNA and RNA, play an essential role in
several biological processes and have pharmacological importance, particularly, the
pyrimidine ring can be found in nucleoside antibiotics, antibacterials, and cardio-
vascular drugs, as well as in agrochemical and veterinarian products [10–13]. In
addition, thiosemicarbazide and triazole derivatives have been found of interest with
potent activities including antimicrobial, anti-HIV, analgesic, anticancer and
anticonvulsant [14–17]. Also, some of synthetic thiazoles exhibit a range of
biological activities, such as antitumor, antibiotic, antibacterial, antifungal, and
antiinflammatory [18–20]. In view of these observations, and in continuation of our
previous work in heterocyclic chemistry, we synthesized some new furanone and
pyrimidine derivatives and tested their anti-HSV-1 activities.
Results and discussion
The reaction of 3-((naphthalen-1-yl)methylene)-5-phenylfuran-2(3H)-one 1 with
benzylamine in ethanol at room or at refluxing temperature afforded the correspond-
ing open-chain benzylamide 2 and 2(3H)-pyrrolone 3, respectively. Treatment of
compound 2 with thionyl chloride or thiourea afforded the corresponding isothiazo-
lone 4 and pyrimidine thione 5, respectively. The pyrrolone derivative 3 was reacted
with ethyl cyanoacetate in the presence of ammonium acetate in n-butanol to give the
corresponding pyrrolopyridine derivative 6 (Scheme 1).
Condensation of furanone 1 with hydrazine hydrate in ethanol at room temperature
or at refluxing temperature afforded the corresponding hydrazide 7 and pyridazinone
8, respectively. Treatment of 1 with phenyl hydrazine or hydroxylamine hydrochlo-
ride gave the corresponding compounds 9 and 10, respectively. The reaction of 1 with
malononitrile in glacial acetic acid and sodium acetate afforded the pyran derivative
11. Also, pyrimidinethione 12 was prepared by reacting of compound 1 with thiourea
in the presence of sodium ethoxide (Scheme 2).
Cyclization of the hydrazide derivative 7 with ethoxymethylenemalononitrile or
ethyl(ethoxy methylene)cyanoacetate in refluxing ethanol afforded the corresponding
substituted pyrazole derivatives 13 and 14, respectively. Also, hydrazide 7 was
reacted with carbon disulphide or acetylacetone to give the corresponding oxadiazolyl
butenone 15 and pyrazole 16 derivatives, respectively. Reaction of hydrazide 7 with
p-nitrobenzaldeyde afforded Schiff base 17, which was cyclized with thioglycolic
123

Synthesis and anti-viral activities
O
HO
Ph
Ar
ArCHO
O
PhCH2NH2
EtOH, r.t.
PhCH2NH2
EtOH, reflux
Ph
O
O
1
Ar
O
Ar
O
H
N
Ph
Ph
O
N
Ph
CH2Ph
3
2
CNCH2COOEt
CH3COONH4
n-Butanol
Ar
SOCl2
O
NH2CSNH2
KOH/EtOH
CN
O
Cl
Ph
Ar
N
N
H
Ph
Ar
NH
PhH2C
6
S
O
N
N
N
S
H
CH2Ph
4
PhH2C
Ar =
5
Scheme 1 Synthetic pathway for compounds 2–6
acid or chloroacetyl chloride to give the corresponding compounds 18 and 19,
respectively (Scheme 3).
Antiviral activity
Compounds were dissolved in 10 % DMSO and examined for its cytotoxic effect on
MDCK cell lines using MTT bioassay. Applying a range of concentrations of 5, 10,
20, 40, 80 lg/100 lL (applied medium volume on each well of 96-well cell culture
plate) from each compound in order to determine safe concentrations on cell line.
Results were plotted as O.D versus conc. in order to calculate value of TC50 of each
compound (Table 1).
123

E. M. Flefel et al.
Ph
Ar
Ph
Ar
O
HN
NH2
O
N
N
H
O
8
7
NH2NH2
EtOH, r.t.
NH2NH2
EtOH,reflux
Ph
Ar
Ar
PhNHNH2
NaOEt, reflux
N
Ph
N
O
Ph
O
O
H
1
9
NH2CSNH2
NaOEt
CH2(CN)2
AcOH
NH2OH/
CH3COONa
reflux
CH3COONa
Ar
O
Ph
Ar
Ph
CN
Ph
NH
N
O
O
O
O
10
O
NH2
Ar
N
H
12
S
11
A r =
Scheme 2 Synthetic pathway for compounds 7–12
The results of the MTT bioassay indicated that all examined compounds showed
accepted cytotoxic effects, where the cells start being affected by doses higher than
80 lg/100 lL, except for compounds 2, 10, 12, 13, 14 that showed lower safe
concentrations. According to these results, it was found that a concentration lower
than 40 lg/105 cell/mL could be applied for all compounds under examination
through plaque reduction assay, which was applied to determine the percentage of
virus count reduction as a result of its treatment with compounds under
examination. From the results in Table 2, the percentage reduction of viral counts
resulted from virus treatment by 3 concentrations of 10, 20, and 40 lg/105 cell
123

Synthesis and anti-viral activities
CN
OEt
CN
CN
N
NH2
N
N
CS2, EtOH/NaOH
NH
Ph
Ph
reflux
EtOH
O
O
S
O
O
Ar
Ar
Ph
Ar
15
13
O
COOEt
NH2
HN
NH2
O
H3C
OEt
COOEt
Ar
N
CH3
CH3COCH2COCH3
7
O
N
N
N
CN
EtOH
p-NO2PhCHO
EtOH/AcOH, r.t.
O
H
EtOH, r.t.
Ph
Ar
HN
N
Ph
O
Ph
O
Ar'
16
14
O
O
Ar
17
ClCH2COCl
dioxane
HSCH2COOH, Benzene
stirring, r.t.
Ar
Ar
S
O
O
Ph
N
Ph
N
Cl
N
H
N
H
O
O
O
O
Ar
Ar
19
18
Ar'
=
A r
=
NO2
Scheme 3 Synthetic pathway for compounds 13–19
count/mL treatment of each compound individually in triplicate for each
concentration.
Compounds result in a reduction in viral count of C80 % are considered a
promising ground for emerging a new antiviral drug, from the results, compound 12
showed a reduction in viral count of 88 % at a concentration of 40 lg/105 cell
count/ml with a statistically high significance (Tables 2, 3; Fig. 1).
From Table (2), we indicated that compounds number 7, 8, 11, and 13 showed a
percentage of reduction of more than 50 % when applied in their concentration of
40 lg/105 cell count/mL (Figs. 2, 3).
123

E. M. Flefel et al.
Table 1 Values of TC50 of each
compound using MTT bioassay
Comp. No.
TC50 (lg/mL)
1
2
1050
743.2
1118.1
1473.3
903.1
2450
3
4
5
6
7
983.7
1819.4
658.3
1511.4
284.7
103.5
724.3
1774.9
659.8
1393.6
8
10
11
12
13
14
15
17
19
Value of TC50 derived from the
equation was multiplied by 10 in
order to get concentration of
TC50 effect in 1 mL
Conclusion
There is a increasing work on drug discovery of novel antiviral compounds against
H5N1 virus for its recurrent pandemics, especially after the events of the last two
decades. In the present study, we examined the effect of 16 compounds for their
antiviral activity against rH5N1 (2006) Egyptian strain. Compound 12 showed a
reduction of viral activity by 88 % compared to an untreated viral activity, which is
considered to be a promising effect leading to a focus on such a compound for
further investigation of its potential contribution to drug development. Regarding
compounds 7, 8, 11, and 13, all showed a mild promising effect—if applied
singly—on virus activity that may be of pharmaceutical and clinical interest if
incorporated in drug combinations or subjected to some chemical modifications
through further investigation in order to increase their effect.
Experimental
Melting points were measured using an Electrothermal 9100 digital melting point
apparatus (Bu¨chi, Switzerland) and are uncorrected. IR spectra were recorded on a
Perkin–Elmer 1600 FTIR (Perkin–Elmer, USA) in KBr discs. 1H NMR spectra were
measured on a Jeol 270 MHz spectrometer (Jeol, Japan) and a Bruker Avance
spectrometer (300 MHz) (Bruker, Germany) in DMSO-d6, and chemical shifts were
recorded in d ppm relative to the internal standard TMS. The mass spectra were run
at 70 eV with a Finnigan SSQ 7000 spectrometer (Thermo-electron, Madison, WI,
USA) using EI and the values of m/z are indicated in Dalton. Elemental analyses
were performed on a Perkin–Elmer 2400 analyzer (Perkin–Elmer) and were found
123

Synthesis and anti-viral activities
Table 2 Antiviral activity of compounds applied in 10, 20, and 40 lg/mL showing the resulted per-
centage of reduction
Comp. No.
Conc. lg/mL
Intial viral
count/mL
PFU/mL (9105)
Mean SD
% reduction
1
10
20
40
10
20
40
10
20
40
10
20
40
10
20
40
10
20
40
10
20
40
10
20
40
10
20
40
10
20
40
10
20
40
10
20
40
10
20
40
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
4.87500 0.12
4.55000 0.200
3.90000 0.180
5.85100 0.118
5.57186 0.019
4.82885 0.015
4.64295 0.017
4.48500 0.019
4.27180 0.019
5.20000 0.170
5.13500 0.180
4.98550 0.110
3.61400 0.100
3.51650 0.900
3.43200 0.710
4.61500 0.120
4.33550 0.140
3.97800 0.090
4.26595 0.090
3.859375 0.100
3.05500 0.130
4.87500 0.190
4.06250 0.190
3.04720 0.070
3.82200 0.190
3.68940 0.100
3.51390 0.198
4.55000 0.090
3.68550 0.080
3.00950 0.100
2.08000 0.070
1.82000 0.090
0.78000 0.100
3.50350 0.110
3.52885 0.070
3.00040 0.130
4.68000 0.080
4.42000 0.070
3.90000 0.116
25.00
30.00
40.00
10.00
14.28
25.71
28.57
31.00
34.28
20.00
21.00
23.30
44.40
45.90
47.20
29.00
33.30
38.80
34.37
40.625
53.00
25.00
37.50
53.12
41.20
43.24
45.94
30.00
43.30
53.70
68.00
72.00
88.00
46.10
48.71
53.84
28.00
32.00
40.00
2
3
4
5
6
7
8
10
11
12
13
14
123

E. M. Flefel et al.
% reduction
Table 2 continued
Comp. No.
Conc. lg/mL
Intial viral
count/mL
PFU/mL (9105)
Mean SD
15
17
19
10
20
40
10
20
40
10
20
40
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.5 9 105
6.27250 0.600 3.50
5.80450 0.900 10.70
4.17885 0.150 35.71
6.31150 0.080 2.90
6.12300 0.100 5.80
5.92800 0.100 8.80
5.72000 0.080 12.00
4.74500 0.140 27.00
4.55000 0.125 30.00
Results interpreted by t test, p \ 0.001
PFU Plaque-forming unit, SD standard deviation
Table 3 TC50 and ED50 values
Comp. No.
TC50 (lg/mL)
ED50 (lg/mL)
TI
for promising compounds 7, 8,
11, 12, and 13
7
983.7
1819.4
1511.4
284.7
23.95
35.85
22.27
7.5
41.07
50.75
67.86
37.96
3.95
8
11
12
13
103.5
26.15
Antiviral effect of compound 12
640,000
560,000
480,000
400,000
320,000
240,000
160,000
80,000
0
Control
10
20
40
Conc. (µg/ml)
Fig. 1 Antiviral effect of compound 12 (reduction in plaque count compared to untreated control of
M.O.I of 0.001)
within 0.4 % of the theoretical values (Table 1). Follow-up of the reactions and
checking the purity of the compounds was made by TLC on silica gel-precoated
aluminum sheets (type 60 F254; Merck, Germany). All solvents and chemical
reagents were purchased from Aldrich (Germany).
123

Synthesis and anti-viral activities
Antiviral effect of compounds 7, 8, 11, 13 at 40 µg/mL
640,000
560,000
480,000
400,000
320,000
240,000
160,000
80,000
0
Control
7
8
11
13
Conc. (40µg/ml)
Fig. 2 Antiviral effect of compounds 7, 8, 11, and 13 on viral count when used at 40 lg/mL (reduction
in plaque count compared to untreated control of M.O.I of 0.001)
200
TC50 ED50
180
160
140
120
100
80
60
40
20
0
7
8
11
12
13
Fig. 3 Values of TC50 and ED50 of promising compounds 7, 8, 11, 12, and 13
3-(Naphthalen-1-ylmethylene)-5-phenylfuran-2(3H)-one (1)
A mixture of 3-(benzoyl)propionic acid (1.78 g, 10 mmol) and 1-naphthaldehyde
(1.56 g, 10 mmol) in acetic anhydride (3 mL) was fused for 5 min then a few drops
of triethylamine were added. The reaction mixture was refluxed for 20 min. The
obtained solid was crystallized from methanol/chloroform (1:1) to give compound
1. Yield 62 %; m.p. 148–150 °C; IR (KBr, m, cm-1): 1765 (C=O); 1H NMR
(DMSO-d6, d ppm): 6.8 (s, 1H, furanone ring), 7.49–8.42 (m, 13H, Ar–H?1H, =CH);
MS, m/z (%): 298 (M?, 100). Analysis for C21H14O2 (298.33): calcd. C, 84.54; H, 4.73;
found C, 84.49; H, 4.66.
N-Benzyl-2-(naphthalen-1-ylmethylene)-4 oxo-4-phenylbutanamide (2)
To a solution of 1 (1.5 g, 5 mmol) in ethanol (20 mL), benzylamine (0.54 g,
5 mmol) was added. The reaction mixture was stirred at room temperature
for 30 min. The obtained product was filtered off, washed with benzene,
dried, and crystallized from benzene to give the amide derivative 2. Yield
1
82 %; m.p. 184–186 °C; IR (KBr, m, cm-1): 1675 (C=O), 3248 (NH); H NMR
123

E. M. Flefel et al.
(DMSO-d6, d ppm): 3.29 (s, 2H, CH2–CO), 4.2 (s, 2H, NH–CH2), 6.38–8.44 (m,
18H, Ar–H?1H, =CH), 9.18 (s, 1H, NH, D2O exchangeable); MS, m/z (%): 405
(M?, 40). Analysis for C28H23NO2 (405.49): calcd. C, 82.94; H, 5.72; N, 3.45;
found C, 82.87; H, 5.61; N, 3.31.
1-Benzyl-3-(naphthalen-1-ylmethylene)-5-phenyl-1H-pyrrol-2 (3H)-one (3)
To a solution of 1 (1.5 g, 5 mmol) in ethanol (20 mL), benzylamine (0.54 g, 5 mmol)
was added. The reaction mixture was heated under reflux for 3 h. After cooling, the
formed product was filtered off, washed with ethanol and crystallized from ethanol to
give the pyrrolone 3. Yield 80 %; mp 160–162 °C; IR (KBr, m, cm-1): 1672 (C=O);
1H NMR (DMSO-d6, d ppm): 4.9 (s, 2H, NH–CH2), 6.31 (s, 1H, pyrrol), 7.02–8.22
(m, 17H, Ar–H) 8.62 (s, 1H, =CH), MS, m/z (%): 387 (M?, 100). Analysis for
C28H21NO (387.47): calcd. C, 86.79; H, 5.46; N, 3.61; found C, 86.70; H, 5.39; N,
3.52.
5-Benzoyl-2-bezyl-4-(chloro(naphthalen-1-yl)methyl)isothiazol-3 (2H)-one (4)
To a cold (-5 °C) thionyl chloride (10 mL), compound 2 (2.03 g, 5 mmol) was
added portion wise with stirring. The reaction mixture was heated for 3 h on a water
bath. Chloroform (50 mL) was then added and the reaction mixture was
decomposed with ice-cold saturated sodium carbonate solution and separated. The
organic layer was washed with water and dried over anhydrous calcium chloride.
After evaporation of the solvent, the residue was recrystallized from ethanol to give
compound 4. Yield 72 %; m.p. 150–152 °C; IR (KBr, m, cm-1): 1678, 1594
1
(2 C=O); H NMR (DMSO-d6, d ppm): 4.04 (s, 2H, CH2), 5.83 (s, 1H, methine),
7.12–7.47 (m, 17H, Ar–H); MS, m/z (%): 369 (M?, 100). Analysis for
C28H20ClNO2S (469.98): calcd. C, 71.56; H, 4.29; Cl, 7.54; N, 2.98; S, 6.82;
found C, 70.36; H, 4.18; Cl, 7.39; N, 2.82; S, 6.76.
7-Bezyl-4-(naphthalen-1-yl)-3,4-dihydro-1H-pyrrolo[2,3-d]pyrimidine-2(7H)-
thione (5)
A mixture of 2 (2.03 g, 5 mmol) and thiourea (0.4 g, 5 mmol) in ethanolic
potassium hydroxide (15 mL, 10 %) was refluxed for 6 h. The reaction mixture was
concentrated under reduced pressure, and the residue was triturated with diluted
hydrochloric acid. The solid formed was collected by filtration, washed with water,
dried, and crystallized from ethanol to give 5. Yield 72 %; m.p. 100–102 °C; IR
(KBr, m, cm-1): 3300, 3220 (2NH), 1252 (C=S); 1H NMR (DMSO-d6, d ppm): 4.15
(s, 1H, CH-pyrimidine), 4.39 (2H, methylene), 5.98–6.30 (m, 2H, pyrrole),
7.13–8.14 (m, 12H, Ar–H), 9.20, 10.66 (2s, 2H, 2 NH exchangeable with D2O);
MS, m/z (%): 369 (M?, 100). Analysis for C23H19N3S (369.48): calcd. C, 74.77; H,
5.18; N, 11.37; S, 8.68; found. 74.76; H, 5.10; N, 11.29; S, 8.56.
123

Synthesis and anti-viral activities
1-Bezyl-4-(naphthalen-1-yl)-6-oxo-2-phenyl-6,7-dihydro-1H-pyrrolo[2,3-b]
pyridine-5-carbo-nitrile (6)
A mixture of 3 (0.388 g, 1 mmol), ethyl cyanoacetate (0.113 g, 1 mmol) and ammonium
acetate (0.616 g, 8 mmol) in n-butanol (5 mL) was refluxed for 10 h. The precipitated
solid was filtered off, dried, and crystallized from dioxane to give compound 6. Yield
1
30 %; m.p. 212–214 °C; IR (KBr, m, cm-1): 3430 (NH), 2222 (CN), 1680 (CO); H
NMR (DMSO-d6, d ppm): 5.44 (s, 2H, CH2), 6.00 (s, 1H, pyrrole), 7.60–8.40 (m, 17H,
Ar–H), 13.00 (s, 1H, NH, D2O exchangeable); MS, m/z (%): 451 (M?, 100). Analysis
for C31H21N3O (451.52): calcd. C, 82.46; H, 4.69; N, 9.31; found C, 82.41; H, 4.58; N,
9.22.
2-(Naphthalen-1-ylmethylene)-4-oxo-4-phenylbutanehydrazide (7)
A mixture of 1 (2.98 g, 10 mmol) and hydrazine hydrate (0.6 mL, 12 mmol) in
ethanol (30 mL) was stirred at room temperature for 12 h. The obtained product was
filtered off, washed with ethanol and crystallized from ethanol to give acid
hydrazide derivative 7. Yield 75 %; m.p. 88–90 °C; IR (KBr, m, cm-1): 1,650, 1,685
1
(2 C=O), 3300, 3190, 3110 (NH2?NH); H NMR (DMSO-d6, d ppm): 3.22 (s, 2H,
CH2–CO), 4.49 (s, 2H, NH2, D2O exchangeable), 6.69 (s, 1H, NH–CO, D2O
exchangeable), 7.30–7.98 (m, 12H, Ar–H), 8.96 (s, 1H, =CH); MS, m/z (%): 330
(M?, 100). Analysis for C21H18N2O2 (330.38): calcd. C, 76.34; H, 5.49; N, 8.48;
found C, 76.30; H, 5.50; N, 8.41.
4-(Naphthalen-1-ylmethylene)-6- phenyl pyridazin-3-(2H)-one (8)
Toasolutionof1(0.298 g,1 mmol)inethanol (10 mL), hydrazinehydrate(*0.06 mL,
1.2 mmol) was added. The reaction mixture was refluxed for 3 h, after cooling, the
obtained product was filtered off, washed with cold ethanol, dried, and crystallized from
ethanol to give pyridazinon derivative 8. Yield 86 %; m.p. 164–166 °C; IR (KBr, m,
cm-1):1675(C=O), 3280(NH);1H NMR (DMSO-d6, d ppm): 3.22(s, 2H, CH2),5.18 (s,
1H, pyridazinone), 6.72–8.11 (m, 12H, Ar–H), 8.4 (s, 1H, NH, D2O exchangeable); MS,
m/z (%): 312 (M?, 100). Analysis for C21H16N2O (312.36): calcd. C, 80.75; H, 5.16; N,
8.97; found C, 80.70; H, 5.10; N, 8.79.
4-(Naphthalen-1-ylmethylene)-1, 6- diphenyl-1,2-dihydropyridazin-3(4H)-one (9)
A mixture of 1 (0.298 g, 1 mmol) and phenylhydrazine (0.108 g, 1 mmol) in sodium
ethoxide (10 mL, 1 %) was refluxed for 3 h. The reaction mixture was poured into
water, the obtained solid was filtered off, washed with water, dried, and crystallized
from benzene/petroleum ether (60–80) to give compound 9. Yield 80 %; m.p.
1
120–122 °C; IR (KBr, m, cm-1): 1657 (C=O), 3226 (NH); H NMR (DMSO-d6, d
ppm): 6.1 (s, 1H, H pyridazinone), 6.35–8.4 (m, 15H, Ar–H?ethylene–H), 10.2
(br., 1H, NHCO–, D2O exchangeable), MS, m/z (%):388 (M?, 100). Analysis
for C27H20N2O (388.46): calcd. C, 83.48; H, 5.19; N, 7.21; found C, 83.36; H, 5.00;
N, 7.01.
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E. M. Flefel et al.
5-(Naphthalen-1-ylmethyl)-3-phenyl-6H-1, 2-oxazin-6-one (10)
A mixture of 1 (0.298 g, 1 mmol), hydroxylamine hydrochloride (0.69 g, 1 mmol)
and sodium acetate (0.2 g, *2 mmol) in ethanol (10 mL) was refluxed for 5 h. The
reaction mixture was evaporated under reduced pressure, the obtained residue was
crystallized from ethanol to give compound 10. Yield 62 %; m.p. 78–80 °C; IR
1
(KBr, m, cm-1): 1761 (C=O), 1618 (ArC=C); H NMR (DMSO-d6, d ppm): 4.37
(s, 2H, CH2 methylene), 7.48–8.14 (m, 13H, Ar–H?1H, =CH); MS, m/z (%): 313
(M?, 100). Analysis for C21H15NO2 (313.35): calcd. C, 80.49; H, 4.82; N, 4.47;
found C, 80.20; H, 4.80; N, 4.33.
6-Amino-4-(naphthalen-1-yl)-2-phenyl-4H-furo[2,3-b]pyran-5-carbonitrile (11)
A mixture 1 (0.298 g, 1 mmol), malononitrile (*0.1 g, *1 mmol) and sodium
acetate (0.2 g, *2 mmol) in glacial acetic acid (5 mL) was heated under reflux for
6 h. After cooling, the obtained product was filtered off, washed with water, dried,
and crystallized from dioxane to give compound 11. Yield 60 %; m.p. 158–160 °C;
1
IR (KBr, m, cm-1): 2210 (CN), 3290–3410 (NH2); H NMR (DMSO-d6, d ppm):
5.22 (s, 1H, pyran), 6.90–8.06 (m, 13H, Ar–H?1H, furan), 9.18 (s, 2H, NH2, D2O
exchangeable), MS, m/z (%): 364 (M?, 100). Analysis for C24H16N2O2 (364.40):
calcd. C, 79.11; H, 4.43; N, 7.69; found C, 79.14; H, 4.40; N, 7.55.
6-(Naphthalen-1-yl)-5-(2-oxo-2-phenylethyl)-2-thioxotetrahydro-pyrimidin-
4(1H)-one (12)
A solution of 1 (0.298 g, 1 mmol) and thiourea (0.1 g, 1 mmol) in sodium ethoxide
(10 mL, 1 %) was heated under reflux for 5 h. The reaction mixture was cooled,
poured into cold water and neutralized with HCl (pH *7). The precipitated solid
was filtered off, washed with water, dried, and crystallized from ethanol to give
compound 12. Yield 80 %; m.p. 118–120 °C; IR (KBr, m, cm-1): 1250 (C=S), 1650,
1
1710 (2 C=O), 3200, 3350 (2 NH); H NMR (DMSO-d6, d ppm): 3.6 (m, 1H,
CH– pyrmidinone),4.26 (d, 1H, CH– pyrmidinone), 5.10 (m, 2H, CH2), 6.96–8.03
(m, 12H, Ar–H), 11.10, 12.00 (2s, 2H, 2NH, D2O exchangeable); MS, m/z (%):374
(M?, 100). Analysis for C22H18N2O2S (374.46): calcd. C, 70.57; H, 4.85; N, 7.48; S,
8.56 found C, 70.51; H, 4.73; N, 7.39; 8.40.
Synthesis of compounds 13 and 14
To a solution of compound 7 (0.33 g, 1 mmol) in absolute ethanol (20 mL),
ethoxymethylene-malononitrile or ethyl-(ethoxymethylene)cyanoacetate (1 mmol)
was added. The reaction mixture was refluxed for 1 h, after cooling, the obtained
product was collected by filtration, dried, and crystallized from ethanol to give
compounds 13 and 14, respectively.
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Synthesis and anti-viral activities
5-Amino-1-(2-naphthalen-1-ylmethylene)-4-oxo-4-phenylbutanoyl)-4,5-
dihydro-1H-pyrazole-4-carbonitrile (13)
Yield 86 %; m.p. 158–160 °C; IR (KBr, m, cm-1): 3408, 3210 (NH2), 2,218 (CN); 1H
NMR (DMSO-d6, d ppm): 3.40 (s, 2H, CH2), 4.34–5.2 (m, 2H, 2 CH2), 6.78–8.08
(m, 14h, Ar–H?ethylene–H?pyrazole–H), 13.26 (s, 2H, NH2, D2O exchangeable);
13C NMR: 38.87 (CH–CN), 39.71 (CH2, aliphatic), 40.12 (C-NH2), 115.59
(C, 1-nitrile), 125.46-128.89 (C–Ar), 134.77 (C, 1-ethylene), 137.16 (CH, 1-ethylene),
160.5, (CH, 1-imine), 165.42 (C, amide), 194 (C, 1-carbonyl); MS, m/z (%): 408 (M?,
42). Analysis for C25H20N4O2 (408.45): calcd. C, 73.51; H, 4.94; N, 13.72; found C,
73.49; H, 5.01; N, 13.64.
Ethyl-5-amino-1-(2-[naphthalen-1-ylmethylene)-4-oxo-4-phenylbutanoyl]-4,5-
dihydro-1H-pyrazole-4-carboxylate (14)
Yield 80 %; m.p. 114–116 °C; IR (KBr, m, cm-1): 3465, 3350 (NH2), 1,685 (CO, ester);
1H NMR (DMSO-d6, d ppm): 1.37 (t, J = 6.9 Hz, 3H, CH3), 4.34 (q, J = 7.5 Hz, 2H,
CH2), 4.5–5.4 (m, 2H, 2 CH), 6.70 (s, 1H, pyrazole), 7.3–8.36 (m, 13H, Ar–H?1H
ethylene), 10.65 (s, 2H, NH2, D2O exchangeable); MS, m/z (%): 455 (M?, 38). Analysis
for C27H25N3O4 (455.51): calcd. C, 71.19; H, 5.53; N, 9.22; found C, 71.20; H, 5.44;
N, 9.10.
4-(Naphthalen-1-yl)-1-phenyl-3-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)
but-3-en-1-one (15)
To a solution of hydrazide 7 (0.33 g, 1 mmol) in alcoholic sodium hydroxide
(15 mL, 1 %), carbon disulphide (3 mL) was added. The reaction mixture was
refluxed for 2 h, cooled at room temperature and then poured into ice-cold
water, and acidified with HCl. The precipitated solid was filtered off, washed with
water, dried, and crystallized from ethanol to give compound 15. Yield 76 %;
mp 124–126 °C; IR (KBr, m, cm-1): 3215 (NH), 1665 (CO), 1254 (C=S); 1H NMR
(DMSO-d6, d ppm): 4.30 (s, 2H, CH2), 7.37–8.00 (m, 12H, Ar–H), 8.8 (s, 1H, =CH),
13.27 (s, 1H, NH, D2O exchangeable); MS, m/z (%):372 (M?, 36). Analysis for
C22H16N2O2S (372.44): calcd. C, 70.95; H, 4.33; N, 7.52; S, 8.61, found 71.00; H,
4.28; N, 7.40; S, 8.56.
1-(3,5-Dimethyl-1H-pyrazol-1-yl)-2-(naphthalen-1-ylmethylene)-4-
phenylbutane-1,4-dione (16)
A mixture of 7 (0.33 g, 1 mmol) and acetylacetone (0.11 g, 1 mmol) in absolute
ethanol (10 mL) was stirred for 10 h at room temperature. The solvent was
evaporated under reduced pressure to dryness, the formed precipitate was
crystallized from ethanol to give compound 16. Yield 62 %; m.p. 138–140 °C;
1
IR (KBr, m, cm-1): 1655,1688 (2 C=O); H NMR (DMSO-d6, d ppm): 1.9, 2.4 (2s,
6H, 2CH3), 4.3 (s, 2H, CH2), 6.9 (s, 1H, pyrazole), 7.30–8.29 (m, 13H, Ar–H?1H,
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E. M. Flefel et al.
ethylene); MS, m/z (%): 394 (M?, 41). Analysis for C26H22N2O2 (394.47): calcd. C,
79.16; H, 5.62; N, 7.10, found C, 78.94; H, 5.43; N, 6.98.
2-(Naphthalen-1-ylmethylene)-N-(4-nitrobezylidene)-4-oxo-4-
phenylbutanehydrazide (17)
To a solution of hydrazide 7 (3.3 g, 10 mmol) in absolute ethanol (30 mL) containing
a few drops of acetic acid, p-nitrobenzaldehyde (1.51 g, 10 mmol) was added. The
reaction mixture was stirred at room temperature for 10 h. After cooling, the solid
formed was filtered off and crystallized from ethanol/water to give compound 17.
Yield 70 %; m.p. 238–240 °C; IR (KBr, m, cm-1): 3350 (NH), 1595(C=N),
1655,1688 (2 C=O); 1H NMR (DMSO-d6, d ppm): 3.95 (s, 2H, CH2), 7.33–8.39(m,
17H, Ar–H?1H, ethylene), 8.68 (s, 1H, CH=N), 10.25 (s, 1H, NH, D2O
exchangeable); MS, m/z (%): 463 (M?, 39). Analysis for C28H21N3O4 (463.48):
calcd. C, 72.56; H, 4.57; N, 9.07; found C, 72.57; H, 4.48; N, 8.98.
2-(Naphthalen-1-ylmethylene)-N-(2-(4-nitrophenyl)-4-oxothiazolidin-3-yl)-4-
oxo-4-phenyl butanamide (18)
A solution of 17 (1.7 g, 5 mmol) in dry benzene (20 mL), thioglycolic acid (2.5 ml,
5 mmol) was added dropwisely during 10 min, with stirring. The reaction mixture
was stirred overnight at room temperature, and then evaporated of the solvent under
reduced pressure, the residue was triturated with petroleum ether. The obtained
product was filtered off and crystallized from ethanol to give compound 18. Yield
65 %; m.p. 152–154 °C; IR (KBr, m, cm-1): 3400 (NH), 1690 (CO, ester); 1H NMR
(DMSO-d6, d ppm): 1.86 (s, 1H, CH–N of thiazolidine ring), 3.20 (m, 2H, CH2,
aliphatic), 3.48 (s, 2H, thiazolidine ring), 7.38–8.26 (m, 17H, Ar–H?1H ethylene),
9.17 (s, 1H, NH, D2O exchangeable); MS, m/z (%):537 (M?, 61). Analysis for
C30H23N3O5S (537.59): calcd. C, 67.03; H, 4.31; N, 7.82; S, 5.96 found C, 67.00; H,
4.32; N, 7.73; S, 5.80.
N-(3-Chloro-2-(4-nitrophenyl)-4-oxoazetidin-1-yl)-2-(naphthalen-1-
ylmethylene)-4-oxo-4-phenylbutanamide (19)
To a solution of 17 (1.7 g, 5 mmol) in dioxane (20 mL) containing of a few drops of
triethylamine, chloroacetyl chloride (0.07 g, 5 mmol) was added with stirring at
room temperature during 1 h. The reaction mixture was stirred overnight at the
same temperature. The formed precipitate was filtered off and crystallized from
dioxane to give compound 19. Yield 68 %; m.p. 206–208 °C; IR (KBr, m, cm-1):
3430 (NH), 2920 (CH of alicyclic ring), 1670 (CO), 649 (C–Cl); 1H NMR (DMSO-
d6, d ppm): 3.18 (m, 2H, CH2, aliphatic), 3.56 (s, 1H, methane-H), 3.86 (s, 1H,
CH–Cl), 6.74 (s, 1H, ethylene), 7.29–8.32 (m, 16H, Ar–H), 9.76 (s, 1H, NH, D2O
exchangeable); MS, m/z (%): 539 (M?, 21). Analysis for C30H22ClN3O5 (539.97):
calcd. C, 66.73; H, 4.11; Cl, 6.57; N, 7.78, found C, 66.69; H, 4.02; Cl, 6.53; N,
7.62.
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Synthesis and anti-viral activities
Antiviral assay
Influenza viruses belong to the family of Orthomyxoviridae which is defined by
viruses possess a negative sense, single-stranded and segmented RNA genome. It is
well known that influenza viruses cause highly contagious respiratory disease with
potentially fatal outcomes.
Influenza viruses remain a constant health threat, and major efforts have been
directed at discovering effective antivirals over the past several decades.
Fortunately, there are now four US Food and Drug Administration (FDA) drugs
available for use for human: amantadine, rimantadine, oseltamivir and zanamivir.
From the fact that viruses are obligate intracellular parasites, antiviral agents must
be capable of selectively inhibiting viral functions without damaging the host [21],
and amantadine or rimantadine are useful in the therapy of illness caused by
influenza H1N1, H2N2, and H3N2 viruses. And even though the highly pathogenic
H5N1 viruses are resistant to such antivirals [22], it is still sensitive to both
zanamivir and oseltamivir which are well tolerated in animal models and in human
[23, 24], but resistance to the latter antiviral is now being observed clinically [24]
that leads to the importance of searching for new antiviral agents.
Materials and methods
Virus and cells
Avian influenza A virus (A/chicken/Egypt/1/2006 (H5N1)), was used to prepare low
pathogenic rH5N1 by plasmid-based reverse genetics. The prepared low pathogenic
form of H5N1 was tittered on Madin-Darby canine kidney (MDCK) at a multiplicity
of infection (M.O.I) of 0.001. The MDCK cell line was kindly provided by Dr.
Richard Webby at St. Jude Children’s Research Hospital, Memphis, TN, USA.
MDCK cells were routinely passaged in Dulbecco’s modified Eagle medium
(DMEM) supplemented with 10 % fetal bovine serum (FBS) and 1 % antibiotic–
antimycotic mixture (penicillin–streptomycin–amphotericin B).
Compounds preparation for biological assays
Compounds under examination were dissolved in 10 % dimethyl sulphoxide
(DMSO) in ddH2O to a concentration of 10 mg ml-1 stock samples used for further
dilutions according to the assay applied.
Cytotoxicity assay (MTT assay)
The cytotoxic activity of the prepared compounds were tested in MDCK cell line by
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
method [25] with minor modification. Briefly, the cells were seeded in 96-well
cell culture plates (100 lL/well at a density of 105 cells mL-1) and treated with
concentrations of 5, 10, 20, 40, and 80 lg/100 lL of the sample solutions. At 24 h,
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E. M. Flefel et al.
after washing with sterile phosphate buffer (PBS) three times, MTT solution (20 lL
of 5 mg mL-1) was added to each well and incubated at 37 °C for 4 h. After the
medium had been aspirated, the formed formazan crystals were dissolved with
200 lL of acidified isopropanol (0.04 M HCl in absolute isopropanol). An
absorbance of formazan was detected by a dual wavelength UV spectrometer at
540 nm with 620 nm reference wavelength. The percentage of cytotoxicity
compared to the untreated cells was determined with the following equation.
Absorbance of cell without treatment À Absorbance of cell with treatment
% cytotoxicity =
 100
Absorbance of cell without treatment
And 50 % cytotoxicity (TC50) for each compound was calculated from the
equation derived from the plot of its standard curve of percentage of cytotoxicity
versus sample concentration.
Antiviral activity and therapeutic index
The antiviral activity of the compounds was determined using plaque reduction
assay. In a six-well cell culture plate, confluent MDCK cells were infected with a
pre-incubated mixture of: 100 lL of avian influenza H5N1 virus (MOI of 0.001),
100 lL of DMEM [containing 2 % FBS and 1 mg mL-1 of L-1-tosyl-amido-2-
phenylethyl chloromethyl ketone (TCPK)] and different concentrations of each
compound (10, 20, and 40 mg mL-1). Inoculated plates were incubated for 1 h at
37 °C in 5 % CO2 to allow virus adsorption, followed by the addition of 2 mL of
agarose overlayer in 29 DMEM containing 1 % FBS per well. The over-layered
cultures were incubated at 37 °C in 5 % CO2 for 3–4 days of plaque formation
monitoring. Upon plaque observation, plates were fixed with 3.7 % formalin in
phosphate-buffered saline for 2 h, washed, and stained with 0.1 % crystal violet
dye. Plaques were counted manually from triplicate wells for each treatment under
examination based on plaque number but not plaque size. Viral counts and
percentage of virus reduction were calculated according to Hayden et al. [26].
The effective dose ED50 (50 % reduction of viral count) for each compound was
calculated from the equation derived from the plot of its standard curve from:
percentage of plaque reduction versus sample concentration. The therapeutic index
was calculated by dividing TC50 by ED50.
Acknowledgments The authors extend their appreciation to the Deanship of Scientific Research at
King Saud University for funding the work through the research group project No. RGP-VPP-0172.
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