Synthesis, antimicrobial, and antiviral activities of some new 5-sulphonamido- 8-hydroxyquinoline derivatives

Article abstract of DOI:10.1007/s12272-012-0602-0

A series of fused pyranopyrazole and pyranoimidazole, namely 5-(3,6-diamino-4-aryl-5-carbonitrile-pyrano(2,3-c)pyrazol-2-yl) sulphonyl-8-hydroxyquinolines (5a-e), 5-(6-amino-4-aryl-5-carbonitrile-pyrano(2, 3-c)pyrazol-3-yl)sulphonamido-8-hydroxyquinolines (6a-e), 5-(2-thioxo-4-aryl-5- carbonitrile-6-amino-pyrano(2,3-d)imidazol-2-yl)sulphonyl-8-hydroxyquinolines (10a-e), and 5-(2-oxo-4-aryl-5-carbonitrile-6-amino-pyrano(2,3-d)imidazol-2-yl) sulphonyl-8-hydroxyquinolines (11a-e), have been prepared via condensation of some arylidine malononitriles with 5- sulphonamido-8-hydroxyquinoline derivatives 3, 4, 8 and 9. All the synthesized compounds were screened for their antimicrobial activities, and most of the tested compounds showed potent inhibition growth activity towards Escherichia coli, Pseudomonas aeruginosa (Gramnegative bacteria). Furthermore, six selected compounds were tested for their antiviral activity against avian paramyxovirus type1 (APMV-1) and laryngotracheitis virus (LTV), and the results showed that a concentration range of 3-4 μg per mL of compounds 2, 3, and 4 showed marked viral inhibitory activity for APMV-1 of 5000 tissue culture infected dose fifty (TCID ₅₀) and LTV of 500 TCID₅₀ in Vero cell cultures based on their cytopathic effect. Chicken embryo experiments show that compounds 2, 3, and 4 possess high antiviral activity in vitro with an inhibitory concentration fifty (IC₅₀) range of 3-4 μg per egg against avian APMV-1 and LTV and their toxic concentration fifty (CC₅₀) of 200-300 μg per egg.

Full text of DOI:10.1007/s12272-012-0602-0

Arch Pharm Res Vol 35, No 6, 955-964, 2012
DOI 10.1007/s12272-012-0602-0
Synthesis, Antimicrobial, and Antiviral Activities of Some New 5-Sul-
phonamido-8-hydroxyquinoline Derivatives
Emad M. Kassem1, Eslam R. El-Sawy2, Howaida I. Abd-Alla2, Adel H. Mandour2, Dina Abdel-Mogeed1, and
Mounir M. El-Safty3
2
1Department of Therapeutic Chemistry, National Research Centre, Dokki 12622, Giza, Egypt, Department of Natural
3
Compounds Chemistry, National Research Centre, Dokki 12622, Giza, Egypt, and Central Laboratory for Evaluation of
Veterinary Biologics, Abbassia 13181, Cairo, Egypt
(Received September 13, 2011/Revised November 23, 2011/Accepted November 30, 2011)
A series of fused pyranopyrazole and pyranoimidazole, namely 5-(3,6-diamino-4-aryl-5-carbo-
nitrile-pyrano(2,3-c)pyrazol-2-yl)sulphonyl-8-hydroxyquinolines (5a-e), 5-(6-amino-4-aryl-5-car-
bonitrile-pyrano(2,3-c)pyrazol-3-yl)sulphonamido-8-hydroxyquinolines (6a-e), 5-(2-thioxo-4-aryl-
5-carbonitrile-6-amino-pyrano(2,3-d)imidazol-2-yl)sulphonyl-8-hydroxyquinolines (10a-e), and
5-(2-oxo-4-aryl-5-carbonitrile-6-amino-pyrano(2,3-d)imidazol-2-yl) sulphonyl-8-hydroxyquino-
lines (11a-e), have been prepared via condensation of some arylidine malononitriles with 5-
sulphonamido-8-hydroxyquinoline derivatives
were screened for their antimicrobial activities, and most of the tested compounds showed
potent inhibition growth activity towards Escherichia coli Pseudomonas aeruginosa (Gram-
3, 4, 8 and 9. All the synthesized compounds
,
negative bacteria). Furthermore, six selected compounds were tested for their antiviral activ-
ity against avian paramyxovirus type1 (APMV-1) and laryngotracheitis virus (LTV), and the
results showed that a concentration range of 3-4 µg per mL of compounds 2, 3, and 4 showed
marked viral inhibitory activity for APMV-1 of 5000 tissue culture infected dose fifty (TCID50)
and LTV of 500 TCID50 in Vero cell cultures based on their cytopathic effect. Chicken embryo
experiments show that compounds
inhibitory concentration fifty (IC50) range of 3-4
and their toxic concentration fifty (CC50) of 200-300
2
,
3
, and
4
possess high antiviral activity in vitro with an
g per egg against avian APMV-1 and LTV
g per egg.
µ
µ
Key words: 8-Hydroxyquinoline-5-sulphonyl chloride, Pyrano(2,3-c)pyrazole, Pyrano(2,3-d)
imidazole, Antimicrobial, Antiviral activity
al., 2008), anti-parasitic (Kouznetsov et al., 2007) and
antiviral activities (Chen et al., 2009; Jia et al., 2009).
INTRODUCTION
Heterocyclic systems with quinoline nuclei repre- Due to their broad range of biological activities, quino-
sent privileged moieties in medicinal chemistry and line compounds have been considered good starting
are ubiquitous sub-structures associated with biologi- materials for the search for novel antimicrobial and
cally active natural products. Quinoline derivatives anti-viral agents. Accordingly, the present work is
have been shown to display a wide spectrum of bio- aimed at synthesis of new quinoline derivatives via
logical activities such as antibacterial (Hoemann et cyclization of 8-hydroxyquinoline-5-sulphonyl chloride.
al., 2002; Hussein et al., 2009; Lilienkampf et al., 2009), Moreover, the study includes testing of the target
antifungal (Vargas et al., 2003; Meléndez Gómez et compounds for their expected antimicrobial and anti-
viral activities.
Correspondence to: Howaida I. Abd-Alla, Department of Natu-
ral Compounds Chemistry, National Research Centre, Dokki
12622, Giza, Egypt
Fax: 2-02-3337-0931
E-mail: howaida_nrc@yahoo.com
MATERIALS AND METHODS
Melting points were determined in open capillary
tubes on an Electrothermal 9100 digital melting point
955

956
E. M. Kassem et al.
apparatus (Büchi) and are uncorrected. Elemental
Preparation of:
analyses were performed on a Perkin-Elmer 2400 an- 5-(2-Acetyl-3-amino-5-oxo-1,2-dihydropyrazol-4-yl)
alyzer and were found to be within ± 0.4% of the the- sulphonyl-8-hydroxyquinoline (2)
oretical values (Table I). IR spectra were recorded on A mixture of 8-hydroxyquinoline-5-sulphonyl chloride
a Perkin-Elmer 1600 FTIR in KBr discks. 1H-NMR
(1) (2.4 g, 0.01 mol) and 2'-acetyl-2-cyanoaceto-hydra-
spectra were measured with JEOL 270 MHz (JEOL) zide (1.3 g, 0.01 mol) in dioxane (20 mL) containing tri-
and the analysis carried out at Micro-analytical Unit, ethylamine (1 mL) was refluxed for 3 h. After cooling, the
National Research Center, Cairo, Egypt as well as with formed precipitate was filtered off, washed with water,
Bruker 300 MHz (Bruker) and the analysis carried air dried, and recrystalized from aqueous ethanol.
out at Micro-analytical Center, Faculty of Science,
Cairo University, Egypt. The samples were measured 5-(3-Amino-5-oxo-1,2-dihydropyrazol-1-yl)sulphon-
in DMSO-d6 and chemical shifts were recorded in
relative to TMS as an internal standard. Mass spectra A mixture of 8-hydroxyquinoline-5-sulphonyl chloride
(EI) were run at 70 eV with a Jeol-JMS-AX500 mass spec- ) (2.4 g, 0.01 mol) and 2-cyanoacetic acid hydrazide
δ ppm yl-8-hydroxyquinoline (3)
(1
trometer. The spectral data of the newly synthesized (0.99 g, 0.01 mol) in dioxane (20 mL) containing tri-
compounds are listed in (Table II). 8-Hydroxyquino- ethylamine (1 mL) was refluxed for 2 h. The solid that
line-5-sulphonyl chloride, 2-cyanoacetic acid hydrazide, formed on hot was filtered off, washed with water, air
3-amino-5-pyrazolone, 2'-acetyl-2-cyanoacetohydrazide dried, and recrystalized from absolute ethanol.
and arylidene malononitrile were prepared as reported
(Corson and Stoughton, 1928; Graham et al., 1949; 5-(5-Oxo-1,2-dihydropyrazol-3-yl)sulphonamido-8-
Heibron, 1965; Bankovskis et al., 1979; Callejo et al., hydroxyquinoline (4)
1990).
A mixture of 8-hydroxyquinoline-5-sulphonyl chloride
Table I. Physical and analytical properties of the new prepared compounds
Analysis (%) (calculated/found)
H
Compd.
No.
M. P.
(oC)
Yield
(%)
Formula (M. Wt.)
C
N
2
3
4
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
C14H12N4O5S (348.33)
C12H10N4O4S (306.30)
C12H10N4O4S (306.30)
C22H16N6O4S (460.47)
C22H15ClN6O4S (494.91)
C22H15N7O6S (505.46)
C23H18N6O5S (490.49)
C24H21N7O4S (503.53)
C22H14N6O4S (458.45)
C22H13 ClN6O4S (492.89)
C22H13 N7O6S (503.45)
C23H16 N6O5S (488.48)
C22H19N7O4S (501.52)
C11H10N2O5S (282.27)
C12H9N3O4S2 (323.35)
C12H9N3O5S (307.28)
C22H13N5O4S2 (475.50)
C22H12 ClN5O4S2 (509.99)
C22H12N6O6S2 (520.50)
C23H15N5O5S2 (505.53)
C24H18N6O4S2 (518.08)
C22H13N5O5S (459.43)
C22H12 ClN5O5S (493.88)
C22H12N6O7S (504.43)
C23H15N5O6S (489.46)
C24H18N6O5S (502.50)
222-224
222-224
312-314
218-220
253-255
263-265
310-312
166-168
76-78
289-291
205-207
199-200
164-166
215-217
205-207
290-292
271-273
243-245
221-223
305-307
176-178
207-209
199-201
350 dec.
145-147
279-281
60
66
85
20
18
30
22
20
22
20
18
30
34
90
70
60
18
30
22
24
20
33
20
18
25
20
48.27/48.33
47.05/47.22
47.05/47.21
57.38/57.54
53.39/53.21
52.28/52.01
56.32/56.43
57.25/57.44
57.64/57.87
53.60/53.88
52.84/52.91
56.55/56.76
57.48/57.65
46.80/46.91
44.58/44.78
46.90/47.01
55.57/55.50
51.82/52.00
50.77/50.89
54.65/54.77
55.59/55.61
57.51/57.68
53.50/53.77
52.38/52.49
56.44/56.23
57.36/57.43
3.44/3.21
3.26/3.34
3.26/3.45
3.50/3.67
3.05/2.99
2.99/3.03
3.70/3.87
4.20/4.35
3.27/3.47
2.63/2.44
2.58/2.76
3.30/3.55
3.79/3.66
3.54/3.66
2.78/3.00
2.93/3.00
2.76/2.88
2.37/2.55
2.32/2.55
2.99/3.02
3.50/3.52
2.85/3.00
2.45/2.65
2.40/2.65
3.09/2.99
3.61/3.77
16.09/15.99
18.30/18.55
18.30/18.55
18.25/18.44
16.98/17.01
19.40/19.60
17.13/17.33
19.47/19.44
18.34/18.11
17.05/17.21
19.48/19.55
17.20/17.44
19.56/19.77
9.92/10.01
13.00/13.22
13.68/13.77
17.73/17.88
13.73/13.82
16.15/16.32
13.85/14.00
16.21/16.42
15.24/15.28
14.16/14.33
16.66/16.82
14.13/14.22
16.72/16.99
7
8
9
10a
10b
10c
10d
10e
11a
11b
11c
11d
11e

Bioactive New 5-Sulphonamido-8-hydroxyquinolines
957
Table II. Spectral characterization of the new prepared compounds
Compd.
No.
IR
Mass
(m/z, %)
1H-NMR (
δ, ppm)
(ν
max cm−1)
3420 (OH), 3200 & 3106 3163 (NH & 10.45 (s, 1H, OH), 8.01 (s, 1H, NH), 7.20-
NH2), 1702 & 1656 (C=O), 1618 (C=N), 7.88 (m, 5H, Ar-H), 4.66 (s, 2H, NH2), 2.99
348 (M+, 10), 291 (3), 209
(30), 57 (100)
2
1577 (C=C)
(3H, s, COCH3)
10.55 (s, 1H, OH), 9.52 (s, 1H, NH), 8.82 &
3335 (OH), 3209 & 3163 (NH & NH2), 9.11 (2d, 2H, H-2 & H-4 quinoline), 6.90 &
1686 (C=O), 1651 (C=N), 1606 (C=C), 7.81 (2d, 2H, H-6 & H-7 quinoline), 7.51 (m,
306 (M+, 46), 205 (100),
99 (50), 89 (45), 77 (30)
3
1385 & 1188 (SO2N)
1H, H-3 quinoline), 5.62 (s, 2H, NH2), 4.44
ppm (s, 1H, CH-pyrazole)
3325 (OH), 3218 & 3164 (NH & NH2), 10.56 (s, 1H, OH), 9.52 (s, 1H, NH), 7.51-
1687 (C=O), 1651 (C=N), 1601 (C=C), 8.65 (m, 5H, Ar-H quinoline), 5.66 (s, 1H,
4
1368 & 1163 (SO2N)
SO2NH), 4.24 ppm (s, 2H, CH2-pyrazole)
3358 (OH), 3200 & 3103 (NH & NH2), 10.56 (s, 1H, OH), 9.51 (s, 2H, NH2), 8.42 (s,
2230 (CN), 1641 (C=N), 1597 (C=C), 1H, H-pyrane), 7.66-8.12 (m, 10H, Ar-H),
5a
5b
5c
5d
1340 & 1131 (SO2N)
4.24 (s, 2H, NH2)
3421 (OH), 3200 & 3102 (NH & NH2),
2223 (CN), 1599 (C=N), 1565 (C=C),
1370 & 1126 (SO2N)
494 (M+, 10), 496 (M++2,
3), 383 (20), 205 (30), 111
(70), 89 (100), 77 (30)
3400 (OH), 3200 & 3103 (NH & NH2), 9.91 (s, 1H, OH), 8.91 (s, 2H, NH2), 8.47 (s,
2230 (CN), 1641 (C=N), 1597 (C=C), 1H, H-pyrane), 7.67-8.19 (m, 9H, Ar-H),
1346 & 1132 (SO2N)
5.66 (s, 2H, NH2)
3400 (OH), 3218 & 3100 (NH & NH2),
2200 (CN), 1620 (C=N), 1587 (C=C),
1346 & 1132 (SO2N), 1009 (C-O-C)
490 (M+, 1), 33 (40), 355
(100), 205 (50), 87 (46)
11.44 (s, 1H, OH), 8.01 (s, 1H, H-pyrane),
7.88 (s, 2H, NH2), 6.67-7.61 (m, 9H, Ar-
quinoline), 4.14 (s, 2H, NH2), 2.99 (s, 6H,
2CH3)
3445 (OH), 3212 & 3135 (NH & NH2),
2219 (CN), 1601 (C=N), 1529 (C=C),
1369 & 1131 (SO2N)
5e
4200 (OH), 3320 & 3200 (NH & NH2), 10.56 (s, 1H, OH), 8.91 (s, 1H, NH), 8.56 (s,
2215 (CN), 1620 (C=N), 1365 & 1131 2H, NH2), 7.44-8.32 (m, 5H, Ar-H
6a
6b
6c
(SO2N), 1009 (C-O-C)
quinoline), 7.03-7.37 (m, 5H, Ar-H)
3350 (OH), 3318 & 3191 (NH & NH2),
2219 (CN), 1618 (C=N), 1365 & 1136
(SO2N), 1019 (C-O-C), 740 (Cl)
492 (M+, 12), 494 (M++2,
2), 396 (100), 330 (30),
206 (45), 111 (40), 87 (50)
3419 (OH), 3354 & 3255 (NH & NH2),
2223 (CN), 1664 (C=N), 1584 (C=C),
1348 & 1178 (SO2N), 1040 (C-O-C)
11.24 (s, 1H, OH), 9.91 (s, 1H, NH), 8.87 (s,
2H, NH2), 7.11-8.37 (m, 9H, Ar-H )
(1) (2.4 g, 0.01 mol) and 3-amino-5-pyrazolone (0.99 g, ed off, washed with water, air dried, and recrystalized
0.01 mol) in dioxane (20 mL) containing triethylamine from absolute ethanol.
(1 mL) was refluxed for 3 h. After cooling, the formed
precipitate was filtered off, washed with water, air dried, 5-(6-Amino-4-aryl-5-carbonitrile-pyrano(2,3-c)pyra-
and recrystalized from aqueous ethanol.
zol-3-yl)sulphonamido-8-hydroxyquinolines (6a-
e)
5-(3,6-Diamino-4-aryl-5-carbonitrile-pyrano(2,3-c) A solution of the appropriate arylidene malononitriles
pyrazol-2-yl)sulphonyl-8-hydroxyquinolines (5a- (0.01 mol) and compound (3.06 g, 0.01 mol) in dioxane
e) containing triethylamine (1 mL) was refluxed for 3-6
A solution of the appropriate arylidene malononitriles h. After cooling, the formed precipitate was filtered
(0.01 mol) and compound (3.06 g, 0.01 mol) in diox- off, washed with water, air dried, and recrystalized
4
3
ane containing triethylamine (1 mL) was refluxed for from absolute ethanol.
3-6 h. After cooling, the formed precipitate was filter-

958
E. M. Kassem et al.
2-(2-(8-Hydroxyquinolin-5-yl)sulphonamido)acetic pounds was determined in vitro using the disc diffusion
acid (7) method (Barry and Thornsberry, 1980) against a variety
A suspension of 8-hydroxyquinoline-5-sulphonyl chloride of pathogenic microorganisms: Escherichia coli Pseu-
) (0.24 g, 0.001 mol) and glycine (0.07 g, 0.001 mol) domonas aeruginosa (Gram-negative bacteria), Staph-
,
(1
in a saturated solution of potassium carbonate (5 mL, ylococcus aureus, Bacillus cereus (Gram-positive bac-
1.1 mol/L) was heated at 50oC for 10 min and then at teria), and one strain of fungi (Candida albicans). They
100oC for 30 min. After cooling, the reaction mixture were isolated from clinical samples and identified to
was neutralized with diluted hydrochloric acid (1:1). the species level according to different API 20E systems
The precipitate that formed was filtered off and recry- (Analytab Products) (bioMerieux). The antimicrobial
stallized from dioxane.
activities of the tested compounds were estimated by
placing presterilized filter paper discs (6 mm in diam-
5-(2-Thioximidazolidinon-1-yl)sulphonyl-8-hydroxy eter) impregnated with different doses of the tested
quinoline (8)
A suspension of compound
compounds (100, 50 and 25
7 (3.38 g, 0.012 mol), acetic MacConky agar media for bacteria and on Sabouraud
µg per disc) on Nutrient and
anhydride (6.3 g, 0.067 mol), anhydrous pyridine (15 dextrose agar for the fungi. Dimethyl formamide (DMF)
mL) and ammonium thiocyanate (1.2 g, 0.015 mol) was was used as a solvent for impregnation. The inhibition
heated at 110oC for 1 h. The volatiles were removed in zones (IZ) of the tested compounds were measured
vacuo, and the residue was suspended in water (100 after 24-48 h incubation at 37oC for bacteria and after
mL) and stirred for 1 h. The solid formed was filtered 5 days incubation at 28oC for fungi. Cefotaxime (CTX)
off, air dried, and recrystallized from benzene-petroleum [a standardized 30
ether (60-80oC).
ed content of 30 g per disc, was used in the disc diffu-
sion test, Hoechst-Roussel Pharmaceuticals)] and Pi-
5-(2,4-Dioxo-imidazolidin-1-yl)sulphonyl-8-hydroxy pracillin (Pipracillin (PIP) 100 g per disc manufac-
µg CTX disc (BBL; Lot 104026, assay-
µ
µ
quinoline (9)
tured by Bristol-Myers Squibb) were used as reference
A suspension of compound
8
(1.77 g, 0.0055 mol), chlo- drugs for the bacteria, whereas Nystatin (30 unit per
roacetic acid (10 g, 0.1 mol) and water (3 mL) was disc) manufactured by Bristol-Myers Squibb, Giza,
heated at 120oC for 12 h in a sand bath. The reaction Egypt, [European unit = 0.04
g per disc] was used as
albicans).
µ
mixture was then diluted with water (50 mL) and set the reference drug for the fungi (
C.
aside in a refrigerator at 5oC. The solid formed was
filtered off, air dried, and recrystallized from benzene-
petroleum ether (60-80oC).
Antiviral evaluation
Viruses
Live Avian paramyxovirus type1 (APMV-1) and
5-(2-Thioxo-4-aryl-5-carbonitrile-6-amino-pyrano Laryngotracheitis virus (LTV) were obtained from the
(2,3-d)imidazol-2-yl)sulphonyl)-8-hydroxyquino- Strains Bank of Central Laboratory for Evaluation of
lines (10a-e)
Veterinary Biologics (CLVB), Cairo, Egypt.
A solution of the appropriate arylidene malononitriles
(0.01 mol) and compound
8
(3.23 g, 0.01 mol) in diox-
Cell line
ane containing triethylamine (1 mL) was refluxed for
Vero (Normal, African green monkey kidney) cell
3-6 h. After cooling, the formed precipitate was filtered culture was kindly obtained from Veterinary Vaccines
off, air dried, and recrystalized from absolute ethanol. and Serum Research Institute, Cairo, Egypt. Cells
were cultured in sterile growth medium RPMI-1640
5-(2-Oxo-4-aryl-5-carbonitrile-6-amino-pyrano(2,3-
d)imidazol-2-yl) sulphonyl-8-hydroxyquinolines activated new born calf serum (Sigma-Aldrich, USA
(11a-e) origin) and antibiotics (1000 I.U. per mL Penicillin,
A solution of the appropriate arylidene malononitriles 100 g per mL Streptomycin and 25 g per mL Am-
(0.01 mol) and compound
(Sigma-Aldrich) and supplemented with 10% heat
µ
µ
9
(3.07 g, 0.01 mol) in diox- photericin B, Gibco). The cells were maintained at
ane containing triethylamine (1 mL) was refluxed for 37oC in a humidified atmosphere with 5% CO2, and
3-6 h. After cooling, the formed precipitate was filtered they were subcultured twice a week. The virus was
off, air dried, and recrystalized from absolute ethanol. propagated in Vero cells, and the infective titer of the
stock solution was 10−7TCID50 per mL (50% tissue
culture infective dose). Viruses were adapted on Vero
cells throughout seven successive passages by which
Biological assay
Antimicrobial evaluation
The antimicrobial activity of the synthesized com- the virus showed distant cytopathic effects (degener-

Bioactive New 5-Sulphonamido-8-hydroxyquinolines
959
ation and floatation of the infected cells) on the 3rd day
after infection.
In Vero cell cultures
These assays were performed in nine tissue culture
plates of 24-wells, according to Cox et al. (Cox et al.,
1996). Confluent monolayers of Vero cells were infected
Specific pathogen free egg
Specific pathogen free (SPF) embryonated chicken with 5000 tissue culture infected dose fifty (TCID50)
eggs were obtained from Nile SPF Farm, Koam Oshiem, per 0.2 mL per well of APMV-1 or 500 TCID50 of ILTV
Fayoum, Egypt.
and incubated for 2 h (for virus adsorption); then
inoculum was decanted, followed by the addition of
different ten-fold concentrations to each test sample
In vitro cytotoxicity screening
Cytotoxicity of the tested compounds was determin- separately (from 3-5 µg per mL per well of each con-
ed using the MTT [3-(4,5-dimethylthiazoyl-2-yl)2,5-di- centration). The virus infectivity control and each test
phenyltetrazolium bromide] assay (Mosmann, 1983). compound cytotoxicity control were prepared separately.
The sub-confluent cell cultures were trypsinized and Test plates were incubated at 37oC and 5% CO2 for 3
collected. The cells at a concentration of 3 × 103 cells days. Cytotoxicity concentration fifty (CC50) of each
per ml in 100 µL RPM1-1640 culture medium were test compound was determined as the concentration of
incubated for 3 h at 37oC and 5% CO2. The seed cells compounds that induced any deviation of the morph-
were incubated in 96-well microplates (3 × 103 cells ology than the normal control cells in 50% of the VERO
per well) at 37oC and a 5% CO2 incubator for 24 h. cells’ monolayers. Antiviral inhibitory concentration
After 24 h, when the cells became confluent, the super- fifty (IC50) of test compounds was assayed as the con-
natant was flicked off and previously diluted media of centration of compounds that fully inhibited the virus-
100 µL of different concentrations of test compounds cytopathic effect (100 TCID) in 50% of monolayers.
was added to the microplates and kept for incubation Also, the therapeutic index (TI) of samples was ex-
at 37oC in a 5% CO2 incubator for 72 h. The cells were pressed as CC50/IC50 (Reed and Muench, 1938).
periodically checked for granularity, shrinkage, and
swelling. After 72 h, the sample solution in the wells
was flicked off and 100 L of MTT (0.5 mg/mL) was
In embryonated chicken eggs
Groups of 9-11 days old specific pathogen free (SPF)
µ
added to each well. The plates were gently shaken and embryonated chicken eggs (ECEs) were inoculated with
incubated for 4 h at 37oC in a 5% CO2 incubator. The 500 embryo infective dose fifty (EID50) per 0.2 mL per
purple crystals that developed were dissolved in 100 egg of APMV-1 or 50 EID50 of ILTV, immediately follow-
µ
L DMSO, and absorbance was measured using an ed by injection of different concentration of each com-
ELISA Microplate Reader (Bio
-
Rad Laboratories) at a pound (2-500 mg per 0.2 mL per egg) separately. The
wavelength of 570 nm.
virus infectivity control and test sample toxicity con-
trols were inoculated via the chorioallantoic cavity.
In vitro antiviral assay
Test eggs were incubated for 3-4 days at 37oC and 80%
Different nontoxic concentrations of test compounds, humidity. The CC50, IC50, and TI values were determined
i.e. lower than CTC50, were checked for antiviral pro- as mentioned above. APMV-1 infectivity in ECE was
perty by a cytopathic effect (CPE) assay against a detected by haemagglutinating activity of the allantoic
challenge dose of 10 TCID50. Cells were seeded in 96- fluids of the inoculated eggs as measured by the micro-
well microtitre plates with populations of 10,000 cells technique of the haemagglutination (HA) test (Takatsy,
per well and incubated at 37oC in a 5% CO2 atmos- 1956), while ILTV infectivity was determined by the
phere for a period of 48 h. The plates were washed criterion of distension of the abdominal region, mottled
with fresh RPMI-1640 medium and changed into necrotic or hemorrhagic liver, and mortality scores in
maintenance medium containing virus (10 TCID50) embryos. The CC50 and IC50 were calculated by the
and incubated at 37oC for 90 min for adsorption of the method of Reed and Muench (1938).
virus. Then, the cultures were treated with different
dilutions of the test compounds in fresh maintenance
medium and incubated at 37oC for five days. Every 24
h, observations were made and cytopathic effects were
RESULTS AND DISCUSSION
The reaction routes for the synthesis of the title com-
recorded. Anti-APMV-1and anti-ILTV activity was pounds are described in Schemes 1 and 2. A series of 5-
determined by the inhibition of the cytopathic effect substituted sulphonyl-8-hydroxyquinoline derivatives
compared with the control, i.e. the protection offered were prepared. Condensation of 8-hydroxyquinoline-5-
by the test samples to the cells scored (Meyyanathan sulphonyl chloride (1) with 2'-acetyl-2-cyanoacetohy-
et al., 2006). drazide in refluxing dioxane in the presence of tri-

960
E. M. Kassem et al.
ethylamine led to the formation of 5-(2-acetyl-2-amino- signals at 10.55 (s, 1H, OH), 9.52 (s, 1H, NH), 9.11 and
5-oxo-1,2-dihydropyrazol-4-yl)sulphonyl-8-hydroxyqu- 8.82 (2d, 2H, H-2 and H-4 quinoline), 7.81 and 6.90
inoline ( ) (Scheme 1). The reaction may be preceded (2d, 2H, H-6 and H-7 quinoline), 7.51 (m, 1H, H-3
via reaction of a chlorine atom of with an active quinoline), 5.62 (s, 2H, NH2) and 4.44 ppm (s, 1H, CH-
methylene group of followed by intramolecular cycli- pyrazole) (Table I)
zation to give revealed signals at 10.45 The cyclocondensation reactions of compounds
. 1H-NMR of
(s, 1H, OH), 8.01 (s, 1H, NH), 7.20-7.88 (m, 5H, Ar-H), with some arylidenemalononitriles, namely benzyli-
4.66 (s, 2H, NH2), and 2.99 ppm (3H, s, COCH3) denemalononitrile, -chlorobenzylidenemalononitrile,
-nitrobenzylidenemalononitrile, -methoxybenzyli-
with the denemalononitrile, and -(N,N-dimethylamino) benzyli-
2
1
2
3
2
3 and
4
p
(Table II).
p
p
Furthermore, the reaction of compound
1
p
amino group of 2-cyanoacetic acid hydrazide and its denemalononitrile, in refluxing dioxane in the presence
cyclic form 3-amino-5-pyrazolone in refluxing dioxane of triethylamine as a catalyst led to the formation of
in the presence of triethylamine gave the correspond- fused systems 5-(3,6-diamino-4-aryl-5-carbonitrile-pyr-
ing 5-(3-amino-5-oxo-1,2-dihydropyrazol-1-yl)sulphonyl- ano(2,3-c)pyrazol-2-yl)sulphonyl-8-hydroxylquinolines
8-hydroxy-quinoline (
zol-3-yl)sulphonamido-8-hydroxyquinoline (
ively (Scheme 1). The characteristic features of
the absence of the absorption bands for the Cl atom in
3
) and 5-(5-oxo-1,2-dihydropyra-
(5a-e) and 5-(6-amino-4-aryl-5-carbonitrile-pyrano(2,3-
4), respect- c)pyrazol-3-yl)sulphonamido-8-hydroxyquinolines (6a-
3
showed
e), respectively (Scheme 1).
Moreover, the reaction of 8-hydroxyquinoline-5-sul-
the IR spectrum and showed absorption bands at phonyl chloride (
3209, 3163, 1686, 1385 and 1188 cm−1 for NH2, NH, urated potassium carbonate solution led to the forma-
C=O and SO2, respectively. The 1H-NMR of
revealed tion of 2-(2-(8-hydroxyquinolin-5-yl)sulphonamido)acetic
1) with glycine in the presence of sat-
3
Scheme 1. Synthesis of 5-(3,6-diamino-4-aryl-5-carbonitrile-pyrano(2,3-c)pyrazol-2-yl)sulphonyl-8-hydroxylquinolines and
5-(6-amino-4-aryl-5-carbonitrile-pyrano (2,3-c)pyrazol-3-yl)sulphonamido-8-hydroxyquinolines.

Bioactive New 5-Sulphonamido-8-hydroxyquinolines
961
acid (7) (Scheme 2). Heterocyclization of the latter (Table II).
compound via its reaction with ammonium thiocyan-
Condensation of compounds
8
and
9
with some
ate in acetic anhydride in the presence of anhydrous arylidene malononitriles, namely benzylidenemalono-
pyridine gave 5-(2-thioximidazolidinon-1-yl)sulphonyl- nitrile, -chlorobenzylidene-malononitrile, -nitrobenz-
8-hydroxyquinoline ( ) (Scheme 2). The IR spectrum ylidenemalononitrile, -methoxybenzylidene- malono-
of
p
p
8
p
8
showed absorption bands at 1240 cm−1 for C=S nitrile, and
p-(N,N-dimethylamino)benzylidenemalo-
besides the sulphonamido group at 1371 and 1136 cm−1. nonitrile, under reflux in dioxane in the presence of
In addition, its H NMR spectrum revealed singlet triethylamine as a catalyst led to the formation of the
1
signals at 8.76 ppm for NH and at 4.24 ppm for CH2 of fused systems of pyrano(2,3-d)imidazole derivatives
1
the imidazole moieties besides the other signals which 10a
were located at their positions (Table II). NMR of compounds 10a
Acid hydrolysis of compound using aqueous mono- sence of CH2 protons of imidazole and revealed new
chloroacetic acid yielded the corresponding imidazo- singlet signals at 8.99, 9.15, 6.76, 8.87, 8.81, and 8.57
lidine-2,4-dione derivatives (Scheme 2). IR spectrum ppm, respectively, for NH2 (Table II).
-e
and 11a
-
e
, respectively (Scheme 2). The H-
,c,e and 11a,c,e lack the pre-
8
9
of
9 showed no absorption bands for C=S and showed
absorption bands at 1705 & 1715 cm−1 for (C=O) groups
Antimicrobial activity
All the newly synthesized compounds were tested
for their antimicrobial activity against a variety of
pathogenic microorganisms, E. coli
negative bacteria) and S. aureus
positive bacteria), and one strain of fungi (C. albicans
at different doses of the tested compounds (100, 50
and 25 g per disc) (Table III). The results showed
that compounds 5c and were the most highly
,
P. aeruginosa (Gram-
,
B. cereus (Gram-
)
µ
3,
4,
,
8
9
active of all tested compounds with growth inhibition
zones 28, 27, 22, 22 and 20 mm, respectively, at 100
µ
g per disc against E. coli, compared to the reference
drug cefatoxime (32 mm) at 30 g per disc. Also, in-
hibition zones 18, 18, 16, 14 and 14 mm showed growth
at 50 g per disc against E. coli, compared to the
reference drug cefatoxime (22 mm) at 30 g per disc.
On the other hand, compounds and were found
µ
µ
µ
9
3,
4
,
8
to be the most active of all the tested compounds with
growth inhibition zones 19, 20, 18 and 18mm at 100
µ
g per disc against P. aeruginosa, compared to the
reference drugs cefatoxime (22 mm) at 30 g per disc
and piperacillin (20 mm) at 100 g per disc. The rest
µ
µ
of the tested compounds were non-active against all
microorganisms tested.
Antiviral activity
In Vero cell cultures
Six selected compounds were tested for their anti-
viral activity against avian paramyxovirus type1
(APMV-1) and laryngotracheitis virus (LTV) using a
virus cytotoxicity effect inhibitory assay. The results
revealed that compounds
5000 TCID50 of APMV-1 and 500 TCID50 of ILTV infec-
tivity in the range of 3, 4, 3 g per mL, respectively
2, 3, and 4 completely inhibit
µ
(Table IV). Substantial therapeutic indices of 66, 75,
and 66 were recorded. The cytotoxicity assay indicated
Scheme 2. Synthesis of 5-(2-thioxo-4-aryl-5-carbonitrile-6-
amino-pyrano(2,3-d)imidazol-2-yl)sulphonyl-8-hydroxyquino-
lines and 5-(2-oxo-4-aryl-5-carbonitrile-6-amino-pyrano(2,3-
d)imidazol-2-yl)sulphonyl-8-hydroxyquinolines.
that CC50 of
2, 3 and 4 were greater than 200, 300 and
200 mg per mL, respectively (Table IV). These results

962
E. M. Kassem et al.
Table III. Antimicrobial activity of the newly synthesized compounds
Inhibition zone (mm)
S. aureus
Compounds ( g per disc)
E. coli
P. aeruginosa
B. cereus
C. albicans
Compd.
No.
µ
100
50
25
100
50
25
100
50
25
100
50
25
100
50
25
2
3
4
5a
5b
5c
5d
5e
6a
6b
6c
17
28
27
19
19
22
17
17
17
17
17
17
17
22
20
18
17
17
17
17
12
12
12
12
12
14
18
18
14
12
16
12
10
9
8
8
8
8
14
14
12
10
10
10
10
9
9
9
9
9
-
12
13
-
-
-
-
-
-
-
-
-
-
9
9
9
8
8
-
18
19
20
16
16
16
14
14
12
12
12
12
12
18
18
16
16
16
14
14
10
10
10
10
10
14
14
14
10
10
12
9
8
8
8
8
-
10
10
-
-
-
-
-
-
-
-
-
-
9
9
-
-
-
14
17
17
12
12
12
12
10
10
12
12
12
12
14
14
14
14
14
12
12
12
12
12
12
12
9
10
12
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
12
14
12
12
10
10
10
10
9
10
12
12
12
14
14
14
14
14
14
14
12
12
12
12
12
-
9
-
-
-
-
-
-
-
-
-
-
-
8
8
8
8
8
8
8
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
12
12
12
10
10
10
10
10
10
10
10
10
10
12
12
9
9
9
9
9
9
9
9
9
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
6d
6e
8
8
8
12
12
10
10
10
-
-
-
-
-
8
9
8
8
8
8
8
8
8
8
8
8
9
10a
10b
10c
10d
10e
11a
11b
11c
11d
11e
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
9
Cefatoxime
32
-
22
-
17
-
22
20
-
18
15
-
12
10
-
31
27
-
26
18
17
10
-
26
20
-
20
15
-
14
10
-
-
-
-
-
-
-
(30
Piperacillin
(100 g per disc)
µg per disc)
µ
Nystatin
(30 unit per disc)
-
-
-
-
40
E. coli
cereus
:
;
Escherichia coli
;
P. aeruginosa
:
Pseudomonas aeruginosa
;
S. aureus
:
Staphylococcus aureus
;
B. cereus
:
Bacillus
C albicans Candida albicans.
:
proved that the three compounds possessed antiviral 200, and 200
µg per egg, respectively, showed 100% of
activity in Vero cells with absence of apparent cytotoxicity. the inoculated eggs without death on the fifth day
after inoculation. Thus, the recorded therapeutic indices
In chicken embryos
The activity of the six selected compounds 2, 3, 4, 7,
of the three compounds were 75, 66, and 50, respect-
ively, in the case of APMV-1 and 66, 50, and 50, re-
8
, and
in allantoic fluids and ILTV infectivity criterion in embryo experiments show that compounds
embryos, showed that 4, 3 and 4 g per 0.2 mL per egg had high antiviral activity in vitro with inhibitory
of compounds , and , respectively, fully reduced concentration fifty (IC50) ranges of 3-4 g per egg
the infectivity of 500 EID50 of APMV-1 and 50 EID50 of against avian APMV-1 and LTV and a toxic concentra-
ILTV (Table V). The toxicity assays of compounds tion fifty (CC50) of 200-300 g per egg.
and in chicken embryos with concentrations of 300, The results showed that the concentration range of
9
, as determined by haemagglutinating activity spectively, in the case of ILTV. In conclusion, chicken
3,
4, and
2
µ
3,
4
2
µ
3,
4,
µ
2

Bioactive New 5-Sulphonamido-8-hydroxyquinolines
963
Table IV. Cytotoxic effect of test compounds on normal
Vero cell lines
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Z7
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Z19
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out the antimicrobial activity screening. Also, the
authors are grateful to the Micro analytical Unit, Na-
tional Research Centre, Cairo, Egypt, for carrying out
elemental analyses, H-NMR and mass spectra and
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