DNA binding, antiviral activities and cytotoxicity of new furochromone and benzofuran derivatives

Article abstract of DOI:10.1007/s12272-011-1006-2

Bromination of visnagin (1) afforded 9-bromovisnagin (2) which on its alkaline hydrolysis afforded the 3-acetyl benzofuran derivative (3). The condensation of (3) with hydrazine hydrate, phenylhydrazine and/or hydroxylamine hydrochloride afforded the corr

Full text of DOI:10.1007/s12272-011-1006-2

Arch Pharm Res Vol 34, No 10, 1623-1632, 2011
DOI 10.1007/s12272-011-1006-2
DNA Binding, Antiviral Activities and Cytotoxicity of New Furochromone
and Benzofuran Derivatives
Omaima Mohamed Abdelhafez1, Nehad Ahmed Abedelatif1, and Farid A. Badria2
2
1Chemistry of Natural Products, National Research Center, Dokki, Cairo 72427, Egypt and Pharmacognosy, Faculty of
Pharmacy, Mansoura University, Egypt
(Received January 10, 2011/Revised June 7, 2011/Accepted June 23, 2011)
Bromination of visnagin (
afforded the 3-acetyl benzofuran derivative (
hydrate, phenylhydrazine and/or hydroxylamine hydrochloride afforded the corresponding
pyrazole derivatives (4a ) and isoxazole derivative (4c). On the other hand, when compound
was condensed with some aromatic aldehydes, this yielded corresponding -unsaturated
keto derivatives (5a ). Furthermore, when was subjected to chlorosulfonation, the visnagin-
sulfonylchloride derivative was afforded, which on amidation using morpholine, a sulona-
mido derivative ( ) was obtained. Alkaline hydrolysis of the latter compound yielded 7-N-mor-
pholinosulsamidobenzofuran ( ) which was condensed with some aromatic aldehydes to yield
the corresponding chalcone compounds (9a ). Demethylation of visnagin afforded norvisnagin
10). The reaction of 10 with ethylbromoacetate in dry acetone yielded the ester benzopyran
1
) afforded 9-bromovisnagin (
2
) which on its alkaline hydrolysis
3
). The condensation of (
3
) with hydrazine
,
b
3
α, β
-e
1
6
7
8
-e
(
derivative (11) which reacted with hydrazine hydrate to afford the corresponding hydrazide
derivative (12) and this was condensed with 3,4,5-trimethoxybenzaldehyde to give the corre-
sponding hydrazone (13). A thaizolidinone derivative (14) was obtained by condensation of
(13) with thioglycolic acid. Chloromethylation of norvisnagin afforded a 4-chloromethyl deriv-
ative (15) which reacted with different primary and secondary amines to yield the correspond-
ing ethylamino derivative (16a, b). Moreover, mannich bases (16a, b) and (17a-c) were
obtained by reacting norvisnagin with different primary and secondary amines in the pres-
ence of formalin but benzoylation of (16a
,
b
) and (17a
). The prepared compounds were tested for their interaction with DNA; bromovisnagin
showed the highest affinity and compounds 15 8a > 14 > 16b 17a, and 16a showed
also was the most active as
16a, and 18a were found to be
-c) afforded 4-oxybenzoyl derivative
(18a-e
2
6
,
,
,
,
,
moderate activity in decreasing potency. Moreover, compound
15 14
2
antiviral agent toward HS-I virus and compounds
6,
7
,
,
,
moderately active. CD50 of the active compounds were also measured.
Key words: Visnagin, Norvisnagin, Pyrazolines, Isoxazolines, DNA affinity, Antiviral, Cyto-
toxicity
that some benzofuran derivatives show antiviral,
antitumor (Rida et al., 2006; Galal et al., 2009), and
INTRODUCTION
Furochromones of Ammi visnaga such as visnagin DNA binding activities. In addition, some groups as
have been known to exhibit many biological and phy- pyrazole (Park et al., 2005; Bouabdallah et al., 2006;
siological activities (Duarte et al., 1999, 2000; Ragab Tan et al., 2006), isoxazole (Kochetkov and Sokolov,
et al., 2007; Lee et al., 2010). Further, it is well known 1963; Rescifina et al., 2006), chalcones (Dimmock et
al., 1999; El-Subbagh et al., 2000; Kamal et al., 2008),
Mannich bases (Shivarama Holla et al., 2003; Wenzel
et al., 2010), sulfonamides (Scozzafava et al., 2003;
García-Giménez et al., 2009) and thiazolidinones (Abd
Correspondence to: Omaima Mohamed Abdelhafez, Chemistry
of Natural Products, National Research Center, Dokki, Cairo
72427, Egypt
Tel: 202-2-370-4124, Fax: 202-2-3337-1718
E-mail: ommryan@yahoo.com
Elhafez et al., 2003; Rao et al., 2004; Sriram et al.,
˘
2005; Terzioglu et al., 2006; Wu et al., 2006; Balzarini
1623

1624
O. M. Abdelhafez et al.
et al., 2007; Mijatovic et al., 2008; Lv et al., 2010) also water. After cooling, the formed solid product was
exhibited different antiviral, antitumor, and DNA filtered off, air dried and crystallized (Table I).
binding activities.
The mechanism of several known antitumor agents
involves the interaction with DNA. Examples include
alkylating agents (e.g. chlorabucil, cyclophosphamide,
4-Methoxy-6-hydroxy-5-(3-methylisoxazol-5-
yl)-7-bromo benzofuran (4c)
A mixture of 9-bromovisnagin (0.3 g, 1 mmol) in 20
Mephalan, streptozocin), antitumor antibiotics (e.g. bleo- mL anhydrous pyridine and hydroxylamine hydro-
mycin, dexorubacin, mithramycin), and various other chloride (0.4 g, 6 mmol) was refluxed for 4 h. The
substances (e.g. cisplatin, chloroquine) (El-Sherbeny reaction mixture was cooled and then acidified with
et al., 1995; El-Bendary and Badria, 2000; El-Subbagh dilute hydrochloric acid (10 mL) and the formed solid
et al., 2000). Based on the interaction of small mole- was filtered off, washed well with water, air dried and
cular weight compounds with DNA, some short term crystallized (Table I).
procedures have been devised that are applicable for
the discovery and evaluation of naturally occurring
Chalcone derivatives (5a-e)
and synthetic compounds that function by this mecha-
A solution of 7-bromovisnaginone (0.5 g, 1.8 mmol)
nism. All the previous findings prompted us to synthe- in 10 mL absolute ethanol, one gram sodium hydroxide
size new derivatives of visnagin with the aforemen- dissolved in 3 mL water and the appropriate aromatic
tioned functional groups: pyrazoles, isoxazoles, chalcones, aldehydes (1.8 mmol) namely, benzaldehyde, 4-chloro-
Mannich bases, sulfonamides, and thiazolidinones to benzaldehyde, 4-methoxybenzaldehyde, thiophenalde-
study their antiviral against herpes simplex type I hyde, and buteraldehyde, was stirred for 1 h at room
virus as well as their cytotoxic and DNA binding temperature and left overnight. After acidification of
activities.
the reaction mixture with dilute hydrochloric acid, the
formed solid was filtered off, washed with water, air
dried and crystallized (Table I).
MATERIALS AND METHODS
Melting points were determined with a Electrother-
mal IA 9000 apparatus and were uncorrected. Elem-
ental microanalysis was performed on an Elementar,
4-Methoxy-7-methyl-5-oxo-5H-7-methylfuro (3,2-
g) (1) benzopyran-9-sulfonyl chloride (6)
Visnagin (1) (2.3 g, 10 mmol) was added portion-wise
Vario EL, at the Microanalytical Center, National to monochlorosulfonic acid (3.4 mL, 5 mmol) within a
Research Center. The infrared spectra were recorded period of 1 h while stirring at room temperature. After
on a Nexus 670 FT-IR FT-Raman spectrometer using complete addition of visnagin, the reaction mixture
potassium bromide discs, at National Research Center. was stirred 1 more h and then poured on ice-water.
The proton nuclear magnetic resonance spectra were The solid formed was filtered off, washed with water,
determined on a Varian mercury 300 MHz spectro- air dried and crystallized to give the title compound
meter, using tetramethylsilane (TMS) as an internal (El-Gamal et al., 1987) (Table I).
standard, at Faculty of Science, Cairo University and
National Research Center. 13Carbon nuclear magnetic
4-Methoxy-7-methyl-5-oxo-5H-furo (3,2-g)-9-N-
morpholino-sulfonamido (1) benzopyran (7)
resonance spectra were determined using 1D-13C NMR
Bruker Jeol-EX 125 MHz spectrometer using TMS as
A mixture of visnagin-9-sulfonyl chloride (6) (3.28 g,
an internal standard. The mass spectra were perform- 10 mmol) in 15 mL dioxin and morpholine (0.87 mL,
ed on Jeol JMS-AX500 mass spectrometer at National 10 mmol) and a few drops triethylamine was refluxed
Research Center. The reactions were followed by thin for 2 h. The deposited product after cooling was filter-
layer chromatography (silica gel, aluminum sheets 60 ed off, air dried to give the title compound (Mandour
F254, Merck) using benzene-ethyl acetate (8:2 v/v) as et al., 1994) (Table I).
eluent and sprayed with iodine-potassium iodide re-
agent.
4-Methoxy-5-acetyl-6-hydroxy-7-N-7-methyl-5-
oxo-5H-furo (3,2-g)-9-N-morpholinosulfonamido
benzofuran (8)
4-Methoxy-6-hydroxy-5-(3-methylpyrazol-5-yl)-
7-bromobenzofuran derivatives (4a, b)
A solution of visnagin-9-morphilinosulfonamide (7)
To a solution of 9-bromovisnagin (2) (0.3 g, 1 mmol) (0.38 g, 1 mmol) in 15% aqueous potassium hydroxide
in 15 mL absolute ethanol, hydrazine hydrate or phenyl was refluxed for 1 h. The reaction mixture was cooled
hydrazine (3 mmol) was added. The reaction mixture and acidified with dilute hydrochloric acid. The form-
was refluxed for 2 h and then diluted with 15 mL of ed product was filtered, washed with water, air dried

New Antiviral Furochromones and Benzofurans
1625
Table I. Physical and analytical data of the prepared compounds
M.P. (oC)
(Solvent)
yield
%
Mol. For
(M.wt)
analysis calc/found %
Comp. No.
4a
C
H
N
S
Br
Cl
208-10
(ethanol)
235-37
(ethanol)
189-90
(ethanol)
158-60
(ethanol)
190-192
(methanol)
175-177
(ethanol)
147-149
(ethanol)
379-381
(methanol)
187-89
57
61
50
90
62
70
95
50
30
60
65
58
60
43
85
80
87
53
70
95
65
61
85
65
66
75
82
81
75
62
C13H11BrN2O3
(323.14)
C19H15BrN2O3
(399.24)
C13H10BrNO4
(324.13)
C18H13BrO4
(373.2)
C18H12BrClO4
(407.64)
C19H15BrO5
(403.22)
C16H11BrO4S
(379.23)
C15H15BrO4
(339.18)
C13H9ClO6S
(328.72)
C17H17NO7S
379.38
C15H17NO7S
(355.36)
C22H20ClNO7S
(477.91)
48.30
48.56
57.14
57.02
48.15
48.37
57.91
57.96
53.01
52.91
56.58
56.28
50.66
50.83
53.10
52.90
3.40
3.63
3.75
3.62
3.09
3.28
3.4
3.69
2.95
2.89
3.72
3.69
2.90
2.83
4.43
4.39
8.67
8.69
7.01
6.92
4.32
4.31
24.73
24.69
20.01
20.09
24.65
24.92
21.41
20.99
19.60
19.54
19.82
19.76
21.07
21.12
23.56
23.44
4b
4c
5a
5b
8.70
8.69
5c
5d
8.46
8.54
5e
6
(methylen chloride)
258-260
(ethanol)
170-171
(ethanol)
123-125
(methanol)
179-80
7
8
9a
55.29
55.11
59.26
59.23
59.26
59.23
4.19
4.03
5.35
5.28
5.35
5.28
2.93
2.90
5.76
5.71
5.76
5.71
6.71
6.77
6.77
6.87
6.59
6.76
7.42
7.64
9b
C23H23O8S
(473.5)
(methanol)
203-5
(ethanol)
156-8
9c
C24H26N2O7S
(486.54)
10
C12H8O4
(glacial acetic acid)
138-41
(216.19)
11
C16H14O
63.57
63.49
58.33
58.09
61.80
61.69
57.77
57.94
4.64
4.72
4.16
4.51
4.72
4.43
4.44
4.66
(ethanol)
247-9
(ethanol)
237-40
(ethanol)
171-3
(ethanol)
278-80
(chloroform)
262-5
(ethanol)
286-8
(ethanol)
165-7
(ethanol)
184-6
(ethanol)
157-9
(ethanol)
188-90
(ethanol)
312-14
(ethanol)
318-20
(ethanol)
320-22
(ethanol)
290-2
(ethanol)
(302.28)
12
C14H12N2O5
(288.26)
9.70
9.66
6.01
6.23
5.18
5.49
13
C24H22N2O8
(466.44)
C26H24N2O9S
(540.54)
C14H11ClO4
(278.69)
C14H13NO4
(259.26)
C18H14N2O4
(322.31)
C18H19NO4
(313.35)
C17H17NO5
(315.32)
C17H19NO4
301.34
C21H17NO5
(363.36)
C25H18N2O5
(426.42)
C25H23NO5
(417.45)
C24H21NO6
(419.43)
14
5.93
5.87
15
16a
16b
17a
17b
17c
18a
18b
18c
18d
18e
64.86
64.89
67.08
67.09
5.02
5.08
4.35
4.33
5.41
5.36
8.70
8.69
69.42
69.39
70.42
70.38
71.94
71.89
68.74
68.69
71.11
71.09
4.68
4.66
4.22
4.13
5.52
5.53
5.01
5.03
5.68
5.63
3.86
3.89
6.57
6.49
3.36
3.39
3.34
3.29
3.46
3.39
C24H23NO5
(405.44)

1626
O. M. Abdelhafez et al.
to give the title compound (Mandour et al., 1994) absolute ethanol containing a few drops of acetic acid
(Table I).
was refluxed for 5-7 h. The reaction mixture was
evaporated to 1/3 of its volume and the formed solid
was filtered off, air dried to obtain the title compound
Chalcones derivatives (9a-c)
To a solution of visnaginone-7-N-morpholinosulfona- (Table I).
mido ( ) (0.71 g, 2 mmol) in 5 mL ethanol, 0.5 g sodium
hydroxide in 3 mL water and (2 mmol) of the chosen
8
4-Oxyacetamido-N-[3(3-thiazolidinyl-4-oxo-2(3,
4,5-trimethoxyphenyl]-7-methyl-5-oxo-5H-furo-
(3,2-g) benzopyran (14)
aldehydes, namely,
benzaldehyde, and
was added. The reaction mixture was stirred for 1 h at
p
-methoxybenzaldehyde,
p-chloro-
p-N,Ndimethylaminobenzaldehyde
A mixture of the hydrazone (13) (0.5 g, 1 mmol) and
room temperature and left overnight, then acidified mercaptoacetic acid (0.2 mL, 2 mmol) in 15 mL dry
with dilute hydrochloric acid. The precipitate formed benzene was refluxed for 13 h and the reaction pro-
was filtered off, air dried and crystallized (Table I).
gress was checked by TLC. The reaction mixture was
triturated with boiling water followed by diethyl ether
(Table I).
4-Hydroxy-7-methyl-5-oxo-5H-furo (3,2-g) (1) ben-
zopyran (norvisnagin) (10)
To a solution of visnagin (1) (1 g, 4.3 mmol) in 15
mL water, 15 mL concentrated hydrochloric acid was
added. The reaction mixture was refluxed for 1 h where
4-Methoxy-5-oxo-7-methyl-9-chloromethylfuro
(3,2-g) benzopyran (15)
Norvisnagin (10) (2 g, 9 mmol) was dissolved in 15
chromone dissolved and after 20 min, a precipitate mL glacial acetic acid by gentle heating. To this solu-
began to form and the amount increased gradually. tion, 25 mL concentrated hydrochloric acid and 10 mL
After cooling, the precipitate was filtered off and air of 40% formalin were added. The mixture was allowed
dried to give the title compound (Schönberg and Aziz, to stand for 30 min at room temperature. The formed
1953) (Table I).
product was filtered off, washed with water and air
dried (Hishmat et al., 1971) (Table I).
4-Ethyl-oxyacetate-7-methyl-5-oxo-5H-furo (3,2-
g) benzopyran (11)
To a solution of norvisnagin (10) (0.2 g, 1 mmol) and
anhydrous potassium carbonate (0.28 g, 2 mmol) in 20
mL dry acetone, ethylbromoacetate (0.12 mL, 1.1 mmol)
4-Hydroxy-5-oxo-7-methyl-9-methylaminomethyl
(16a) and 9-methylamino (2-pyridyl) (16b) furo
(3,2-g) benzopyrans
To a solution of (15) (0.26 g, 1 mmol) in 15 mL dry
was added. The reaction mixture was refluxed while benzene, 1 mmol of the appropriate amines namely,
stirring for 8 h. The reaction mixture was filtered ethylamine or 2-aminopyridine, was added. The reac-
while hot and the residue was washed with 20 mL hot tion mixture was refluxed for 2 h. The reaction mix-
dry acetone. The filtrate was evaporated till dryness ture was concentrated to half its volume. The formed
and the residue was triturated with 20 mL diethyl- product was filtered off and air dried (Table I).
ether. The obtained solid was filtered off to give 11
(Table I).
Mannich bases (16a, b and 17a-c)
To a solution of norvisnagin (10) (0.2 g, 1 mmol) in
15 mL dry ethanol, 1 mmol of the appropriate amines
namely, ethylamine, 2-aminopyridine, diethylamine,
4-Oxyacetic acid hydrazide-7-methyl-5-oxo-5-H-
furo-(3,2-g) benzopyran (12)
To a solution of 98% hydrazine (0.1 mL, 2 mmol) in piperidine or morpholine and 0.2 mL of 40% formalin
20 mL absolute ethanol, a solution of the ester (11 were added. The reaction mixture was refluxed for 1-2
)
(0.3 g, 1 mmol in 20 mL ethanol) was added dropwise h. The reaction mixture was concentrated to half its
while stirring. The reaction mixture was warmed in a volume and left to cool. The formed product was filter-
water bath (60-70oC) for 6 h. The reaction mixture ed off, air dried and crystallized to obtain the Mannich
was evaporated in vacuo to 1/3 of its volume and the bases 16a, b and 17a-c (Abdel-Alim and Aboulezz,
formed precipitate was filtered off, air dried and 1986) (Table I).
recrystallized (Table I).
4-Benzoyloxo-5-oxo-7-methyl-9-methylfuro (3,2-
4-Oxyacetic acid (3,4,5-trimethoxyphenylhydra- g) benzopyran derivatives (18a-e)
To a solution of 1 mmol of compounds 16a
,
b and/or
zono)-7-methyl-5-oxo-furo (3,2-g) benzopyran (13)
A mixture of hydrazide (12) (0.23 g, 1 mmol) and 17a-c in 10 mL benzene, benzoyl chloride (0.23 mL, 2
3,4,5-trimethoxybenzaldehyde (1 mmol) in 20 mL mmol) and 0.5 g anhydrous potassium carbonate were

New Antiviral Furochromones and Benzofurans
1627
Table II. Activity of compounds in methyl green/DNA
displacement assay*
added. The reaction mixture was refluxed for 4-5 h,
filtered while hot and the filtrate was evaporated until
dry. Two drops of water and ethanol were added to
remove any inorganic matter, the formed product was
filtered off and air dried (Table I).
DNA active compounds DNA/methyl green IC50, µg/mL
2
6
15
7
17a
14
16a
16b
18a
28 ± 3
40 ± 1
41 ± 3
47 ± 1
72 ± 2
68 ± 2
70 ± 1
84 ± 2
51 ± 3
< 85
DNA affinity
DNA binding assay (DNA/compound using RP-
TLC)
TLC plates (RP-18 F254; 0.25 mm; Merck) were
redeveloped with MeOH-H2O (8:2). Test compounds
were then applied (5 mg/mL in MeOH) at the origin,
followed by addition of DNA (1 mg/mL in H2O and
MeOH solution) at the same position on the origin.
The plates were then developed with the same solvent
system and the position of DNA was determined by
spraying with anisaldehyde reagent which yields a
blue color on reaction with DNA and the intensity of
the color is proportional to the quantity of DNA added
Remaining compounds
*Values represent the concentration (means ± S.D., n = 3
to 5 separate determinations) required to decrease the
initial absorbance of the DNA/methyl green solution by
50%.
Herpes simplex type I (HS-I) was the gift of Dr. R.
to the plate. Ethididium bromide was used as a posi- G. Hughes, Roswell Park Memorial Institute, Buffalo,
tive control.
NY. Virus stocks were prepared as aliquots in culture
medium from Vero cells infected at multiplicity of 1
Colorimetric assay for compounds that bind to virion per 10 cells and cultured for 3 days. They were
DNA stored at
−
80oC. Working stocks were prepared by
DNA/methyl green (20 ng Sigma) was suspended in titering virus by serial dilution in culture medium and
100 mL of 0.05 M Tris-HCl buffer, pH 7.5 containing assayed in triplicate on Vero monolayers in the wells
7.5 mM MgHSO4 and stirred at 37oC with a magnetic of microtiter plates. Viral suspensions that gave about
stirrer for 24 h. Unless otherwise indicated, samples 30 plaques per well were stored at 4oC until use. Vero
to be tested were dissolved in EtOH in Eppendorff African green monkey kidney cells were purchased
tubes. Solvent was removed under vacuum and 200 from Viromed Laboratories, Minnetonka, MN and grown
µL of DNA-methyl green solution was added to each in Dulbecco’s modified Eagle’s medium supplemented
tube. The absorption maxima for DNA/methyl green with 10% (v/v) calf serum (Hyclone Laboratories), 60
complex is 642.5-645 nm. Samples were incubated in mg/mL Penicillin G and 100 mg/mL streptomycin
the dark at ambient temperature. After 24 h, the final sulfate maintained at 37oC in a humidified atmos-
absorbance of samples was determined. Readings were phere containing about 15% (v/v) CO2 in air. All media
corrected for initial absorbance and normalized as a components were obtained from Sigma Chemical Co.,
percentage of the untreated DNA-methyl green com- St. Louis, MO, unless otherwise indicated. Vero stocks
plex absorbance value. IC50 values were determined were maintained at 34oC in cultured flasks filled with
for each compound as shown in Table II.
medium supplemented with 1% (v/v) calf serum. Sub-
cultures for virus titration or antiviral screening were
grown in microtiter plates (Falcon Microtest III 96-
wells trays, Becton Dickinson Labware) by suspend-
Antiviral and cytotoxic assays
Sample preparation
Samples were prepared for assay by dissolving in 50 ing Vero cells in medium following trypsin-EDTA treat-
mL of DMSO and diluting the aliquots into sterile ment, diluting in medium containing 10% calf serum
culture medium at 0.4 mg/mL. These solutions were to 2 × 104 cells per in 200 mL culture, aliquoting into
diluted again to 0.02 mg/mL in sterile medium and each well and cultured until confluency.
the two solutions were used as stocks to test samples
at 100, 50, 20, 10, 5, 2, and 1 mg/mL in microtiter plates.
Procedures
Microtiter trays with confluent monolayer cultures
with Vero cells were inverted, the medium was shaken
Virus assay and culture
The compounds were tested for antiviral activity out and replaced with serial dilutions of sterile ex-
against Herpes simplex type I (HS-I) grown in Vero tracts in triplicate in 100 L medium followed by
African green monkey Kidney cells. titered virus in 100 L medium containing 10% (v/v)
µ
µ

1628
O. M. Abdelhafez et al.
calf serum. In each tray, the last row of wells was re- of Variance “ANOVA” followed by Tukey-Kramer mul-
served for controls that were not treated with com- tiple comparison test. Statistical analysis was carried
pounds or virus. The trays were cultured for 66 h. The out using InStat 2 Computer Program (Graphpad
trays were inverted onto a pad of paper towels; the re- software INC V2.04).
maining cells were rinsed carefully with medium and
fixed with 3.7% (v/v) formaldehyde in saline for at
least 20 min. The fixed cells were rinsed with water and
examined visually. Antiviral activity was identified as
confluent, relatively unaltered monolayers of stained
RESULTS AND DISCUSSION
Chemistry
We described here a convenient approach for the
Vero cells treated with HS-I. Cytotoxicity was esti- preparation of bromovisnagin (
mated as the concentration that caused approximately by bromination of visnagin (
) using bromine in acetic
50% loss of the monolayer around the plaques caused acid (Starkowsky, 1959). Condensation of
with hydra-
by HS-I.
zine hydrate, phenylhydrazine and/or hydroxylamine
The prepared compounds were tested at the Uni- hydrochloride afforded the corresponding pyrazole
versity of Minnesota for their possible antiviral acti- derivatives and isoxazole derivatives 4a
4b, and 4c
vity against herpes simplex type I (HS-I) grown on respectively. On the other hand, 4-methoxy-5-acetyl-6-
Vero African green monkey kidney cells. An improved hydroxy-7-bromobenzofuran
(Starkowsky, 1959)
plaque-reduction assay was used to test the compounds which was prepared by alkaline hydrolysis of
was
2) which was obtained
1
2
,
,
3
2
using aphidicolin as the positive control (Table II). condensed with some aromatic aldehydes namely,
Plaque reduction assays typically use a monolayer of benzaldehyde, 4-chlorobenzaldehyde, 4-methoxyben-
cultured host cells which are allowed to bind virus, zaldehyde, thiophealdehyde, and buteraldehyde to yield
then overlapped with a layer of a medium thickened the corresponding
with agar or another thicker which makes plaque for-
5a ) (Scheme 1). Chlorosulfonation of visnagin
mation possible by preventing mixing due to currents using chlorosulfonic acid at room temperature with-
in the medium. Samples to be tested for antiviral acti- out solvent yielded visnagin-9-sulfonylchloride (
) which
α, β-unsaturated keto derivatives
(
-e
1
6
vity are either incorporated into thickened agar or
absorbed on a paper disc laid on the thickened layer.
The thickened layer can cause several types of techni-
cal problems including toxicity of the thickener to
virus or host cells and absorption or other types of in-
terference with unknown antiviral agents being tested.
Cytotoxicity was estimated as the loss of cell mono-
layers in which the plaques are normally formed (Table
III).
Statistical analysis
The data are expressed as means ± S.E.M. Statisti-
cal analysis was carried out using a One-way Analysis
Table III. Antiviral and cytotoxicity for the tested com-
pounds
% Reduction Minimal antiviral Cytotoxicity
Compound
in plaque
conc. (mg/mL) CD50 (mg/mL)
Aphidicolin
100
100
073
067
069
none
059
none
55
00.005
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.20
0.08
0.10
0.19
0.17
0.15
0.19
0.20
0.20
0.20
2
6
15
7
17a
14
16a
16b
18a
64
Scheme 1

New Antiviral Furochromones and Benzofurans
1629
Scheme 3
Scheme 2
amine and 2-aminopyridine on 15 or by the effect of
on amidation using morpholine in dioxane yielded the primary amines in the presence of formaldehyde on
9-sulfonamido derivative ( ). Alkaline hydrolysis of the norvisnagin (10). The Mannich bases (17a-c) were ob-
7
later compound by 3% aqueous potassium hydroxide tained by the treatment of 10 with the secondary
yielded 4-methoxy-5-acetyl-6-hydroxy-7-N-morpholino- amines, diethylamine, piperidine, and morpholine in
sulfonamido benzofuran (
some aromatic aldehydes namely, 4-chlorobenzaldehyde, bases (16a
4-methoxybenzaldehyde, and dimethyl aminobenzal- zene afforded the corresponding benzoyloxy derivati-
dehyde in alkaline medium to yield the corresponding ves (18a ) (Table IV).
-unsaturated keto derivatives (9a-c) (Scheme 2).
Demethylation of ( ) using hydrochloric acid afforded
8) which was condensed with the presence of formaldehyde. Benzoylation of Mannich
,
b) and (17a-c) by benzoyl chloride in ben-
-e
α, β
1
Pharmacological screening
4-demethyl-visnagin (norvisnagin) (10) which reacted
DNA binding affinity
with ethylbromoacetate in dry acetone in the presence
The compounds were tested for DNA affinity and it
of anhydrous potassium carbonate to yield 4-ethoxy-5- was found that bromovisnagin (
2) showed high affin-
oxo-2- methylfuro[3,2-g] benzopyran (11). The later ity to. Compounds 15 18a 14
6
,
7
,
,
,
,
16b 17a, and 16a
,
compound reacted with hydrazine hydrate to give the showed moderate activity in decreasing order. The
corresponding 4-oxyacetohydrazide derivative (12). The remaining compounds showed weak activity (Table
later compound was further condensed with 3,4,5- II).
trimethoxybenzaldehyde to give the hydrazone deriva-
tive (13) which on its condensation with mecaptoacetic
acid yielded thiazolidinone derivative (14) (Scheme 3).
Antiviral effect and cytotoxicity
The prepared compounds were tested for their pos-
Chloromethylation of demethylvisnagin (10) afforded sible antiviral activity against herpes simplex type I
9-chloromethylnorvisnagin (15). 16a were obtained (HS-I) grown on Vero African green monkey kidney
either by the effect of primary amines such as methyl- cells. An improved plaque-reduction assay was used to
,
b

1630
O. M. Abdelhafez et al.
1
Table IV. IR, H-NMR and MS spectra of the new compounds
Comp.
No.
IR spectra
m/z
(rel. int. %)
1H-NMR spectra (DMSO-d6
, δ ppm)
(KBr, n cm-1)
3351 (OH), 3134 (NH), 2.25 (3H, s, CH3 pyrazole), 4.0 (3H, s, OCH3), 6.8 (1H, s, H-4× pyrazole), 7.35
4a
322/324 (87%)
399/401 (85%)
323/325 (98%)
372/374 (84%)
1626 (C=N)
(1H, d, H-3), 8.0 (1H, d, H-2), 13.15 (1H, s, NH) and 13.35 (1H-s, OH)
2.45 (3H, s, CH3 pyrazole), 4.1 (3H, s, OCH3), 6.9 (1H, s, H-4× pyrazole), 7.1-
7.3 (5H, m, Ar-H), 7.32 (1H, d, H-3), 7.99 (1H, d, H-2), and 13.25 (1H-s, OH)
4b 3367 (OH), 1624 (C=N)
4c 3415 (OH), 1622 (C=N)
5a 3413 (OH), 1666 (C=0)
5b 3413 (OH), 1668 (C=0)
5c 3413 (OH), 1668 (C=0)
5d 3423 (OH), 1671 (C=0)
2.25 (3H, s, CH3 pyrazole), 4.0 (3H, s, OCH3), 6.5 (1H, s, H-4×), 7.35 (1H, d,
H-3), 8.0 (1H, d, H-2), and 10.25 (1H-s, OH)
4.1 (3H, s, OCH3), 6.2, 6.5 (2H, dd, CH=CH×), 7.4 (5H, m, phenyl), 7.7 (1H,
d, H-3 furan), 8.0 (1H-d, H-2 furan) and 11.2 (1H, s, OH)
4.1 (3H, s, OCH3), 6.2, 6.5 (2H, dd, CH=CH×), 7.2-7.4 (4H, m, phenyl), 7.6
(1H, d, H-3 furan), 8.0 (1H-d, H-2 furan) and 11.1 (1H, s, OH)
407.5/409.5
(70%)
3.99 (6 H, s, OCH3), 6.5, 6.7 (2H, dd, CH=CH), 6.98-7.26 (4H, m, phenyl), 7.2
(1H, d, H-3 furan), 7.82 (1H-d, H-2 furan) and 11.4 (1H, s, OH)
402/404 (41%)
379/381 (50%)
4.15 (3H, s, OCH3), 7.1 (2H, dd, CH=CH), 7.4 (1H, d, H-3), 7.6 (3H, m,
thiophen), 8.0 (1H, d, H-2) and 11.4 (1H, s, OH)
1.1 (3H, t, CH3), 1.41 (2H, m, CH2), 1.99 (2H, t, CH2), 4.14 (3H, s, OCH3), 6.9,
5e 3421 (OH), 1628 (C=0) 7.1 (2H, dd, CH=CH), 7.3 (1H, d, H-3 furan), 7.9 (1H, d, H-2 furan) and 11.4 339/341 (55%)
(1H, s, OH)
1H-NMR (DMSO-d6
3375 (OH), 1668 (C=0), (4H, t, 2CH2O system of morpholine), 4.11 (3H, s, OCH3), 6.5, 6.6 (2H, dd,
, δ, ppm); 3.1 (4H, t, 2CH2N system of morpholine), 3.67
477/479
(4.25:1.53%)
9a
9b
1111, 1333 (SO2N)
CH=CH×), 7.2-7.4 (4H, m, phenyl), 7.89 (1H, d, H-3 furan), 8.00 (1H-d, H-2
furan) and 10.8 (1H, s, OH)
3.2 (4H, t, 2CH2N system of morpholine), 3.67 (4H, t, 2CH2O system of
morpholine), 4.21 (6H, s, OCH3), 6.5, 6.6 (2H, dd, CH=CH×), 7.2-7.4 (4H, m,
phenyl), 7.99 (1H, d, H-3 furan), 8.00 (1H-d, H-2 furan) and 10.5 (1H, s, OH)
3375 (OH), 1668 (C=0),
1111, 1333 (SO2N)
473 (2.02%)
486 (0.07%)
3.2 (4H, t, 2CH2N system of morpholine), 3.4 (6H, s, N(CH3)2), 3.7 (4H, t,
3428 (OH), 1658 (C=0), 2CH2O system of morpholine), 4.25 (3H, s, OCH3), 6.4, 6.5 (2H, dd,
9c
11
1111, 1385 (SO2N)
CH=CH×), 7.3 (4H, m, phenyl), 8.0 (1H, d, H-3), 8.25 (1H-d, H-2) and 10.2
(1H, s, OH)
1755, 1663 (C=0), 1619 ppm1.2 (3H, t, CH3), 2.3 (3H, s, CH3), 4.2 (2H, q, CH2), 4.85 (2H, s, CH2), 6.1
303 (89.5%)
288 (15.8%)
(C=C)
(1H, s, H-6), 7.1 (1H, s, H-9), 7.6 (1H, d, H-3), 8.1 (1H-d, H-2)
3122 (NH), 3227, 3334
12 (NH2), 1676, 1604
2.3 (3H, s, CH3), 4.4 (2H, s, NH2), 5.0 (2H, s, CH2), 6.2 (1H, s, H-6), 7.4 (1H,
s, H-9), 7.55 (1H, d, H-3), 8.1 (1H, d, H-2), 10.4 (1H, s, NH)
(C=0), 1541 (C=C)
3202 (NH), 1697, 1650 2.1 (3H, s, CH3), 3.2 (2H, s, OCH2), 3.8 (3H, s, OCH3), 4.15 (3H, s, 2OCH3),
13 (C=0), 1604 (C=N), 1537 7.1 (1H, s, H-6), 7.4 (1H, s, H-9), 7.6 (1H, d, H-3), 8.1 (1H-d, H-2), 8.45 (1H,
467 (7.9%)
541 (7.3%)
(C=C)
s, CH=N) and 11.25 (1H, s, NH)
2.0 (3H, s, CH3), 3.8 (2H, s, OCH2), 3.8 (9H, s, OCH3), 4.0, 4.3 (2H, dd, CH2
3186 (NH), 1729, 1689, thiazolidinone), 4.70 (1H, s, CH2 thaizolidinone), 6.3 (2H, s, phenyl), 7.2 (1H,
1651 (C=0), 1604 (C=C) s, H-6), 7.4 (1H, s, H-9), 7.6 (1H, d, H-3), 8.1 (1H-d, H-2), and 11.25 (1H, s,
NH)
14
3418 (OH), 3133 (NH), 1.7 (3H, s, CH3), 2.47 (3H, S, N-CH3), 2.9 (2H, s, CH2), 6.13 (1H, s, H-6), 7.6
16a
16b
259 (11%)
322 (1.8%)
1621 (C=0)
(1H, d, H-3), 8.1 (1H-d, H-2), 11.3 (1H, s, NH), 13.15 (1H, s, OH)
3424 (OH), 3133 (NH), 1.7 (3H, s, CH3), 2.7 (2H, s, CH2), 7.2-8.0 (7H, m, aromatic protons), 11.3 (1H,
1676 (C=0)
s, NH), 13.15 (1H, s, OH)
1.51 (6H, m, CH2 piperdine), 1.7 (3H, s, CH3), 2.75 (4H, t, 2CH2N system of
piperdine), 3.70 (2H, s, CH2), 6.13 (1H, s, H-6), 7.3-8.14 (5H, m, phenyl), 8.0
(1H, d, H-3), 8.25 (1H-d, H-2)
1721, 1662 (C=0), 1631
(C=C)
18a
18b
417 (22%)
419 (20%)
1.7 (3H, s, CH3), 3.0 (4H, t, 2CH2N system of morpholine), 3.67 (4H, t,
2CH2O system of morpholine), 3.70 (2H, s, CH2), 6.13 (1H, s, H-6), 7.3-8.14
(5H, m, phenyl), 8.0 (1H, d, H-3), 8.25 (1H-d, H-2)
1721, 1662 (C=0), 1631
(C=C)

New Antiviral Furochromones and Benzofurans
1631
Table 4. Continued
Comp.
No.
IR spectra
m/z
(rel. int. %)
1H-NMR spectra (DMSO-d6
, δ ppm)
(KBr, n cm-1)
1723, 1662 (C=0), 1630 1.1 (6H, t, 2CH3), 1.7 (3H, s, CH3), 2.4 (4H, q, 2CH2N), 3.70 (2H, s, CH2), 6.13
18c
18d
405 (18%)
(C=C)
(1H, s, H-6), 7.3-8.14 (5H, m, phenyl), 8.01 (1H, d, H-3), 8.23 (1H-d, H-2)
3233 (NH), 1710, 1621 1.7 (3H, s, CH3), 2.4 (3H, s, N-CH3), 3.80 (2H, s, CH2), 6.13 (1H, s, H-6), 7.3-
363 (20%)
427 (32%)
(C=0), 1598 (C=C)
8.14 (5H, m, phenyl), 8.01 (1H, d, H-3), 8.23 (1H-d, H-2), 11.2 (1H, s, NH)
1.7 (3H, s, CH3), 4.00 (2H, s, CH2), 6.13 (1H, s, H-6), 6.65-8.11 (4H, m,
pyridyl), 7.3-8.14 (5H, m, phenyl), 8.01 (1H, d, H-3), 8.23 (1H-d, H-2), 11.2
(1H, s, NH)
3136 (NH), 1725, 1695
(C=0), 1598 (C=C)
18e
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test the compounds by using aphidicolin as a positive
control (Table II). Compound was highly active at a
concentration of 0.001 mg/mL compared with the
standard (0.005 mg/mL) whereas compounds 15
14 16a, and 18a were moderately active towards HS-
I. The remaining compounds were inactive towards
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towards the virus were also measured (Table III).
2
6,
7,
,
,
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