Synthesis and anti-inflammatory activity of fluorinated isocoumarins and 3,4-dihydroisocoumarins

Article abstract of DOI:10.1016/j.jfluchem.2007.02.021

The synthesis of several fluorinated isocoumarins and 3,4-dihydroisocoumarins are described. The structures of the synthesized compounds were confirmed by spectral and elemental analysis. All synthesized compounds were evaluated for their anti-inflammatory and antioxidant activity. These compounds were found to present antioxidant and anti-inflammatory activities. A few of them were found to be significantly active in vivo against ear edema in mice produced by 12-O-tetradecanoylphorbol-13-acetate (TPA) and also good radical scavengers.

Full text of DOI:10.1016/j.jfluchem.2007.02.021

Journal of Fluorine Chemistry 128 (2007) 641–646
www.elsevier.com/locate/fluor
Synthesis and anti-inflammatory activity of fluorinated isocoumarins and
3,4-dihydroisocoumarins
b
Ghulam Qadeer a, Nasim Hasan Rama a, , M.L. Gardun˜o-Ramırez
*
´
a Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
b
´
´
Centro de Investigaciones Quımicas, Universidad Autonoma del Estado de Morelos,
Avenida Universidad 1001, Col. Chamilpa, C.P. 62210 Cuernavaca,
Morelos, Mexico
Received 22 January 2007; received in revised form 26 February 2007; accepted 28 February 2007
Available online 3 March 2007
Abstract
The synthesis of several fluorinated isocoumarins and 3,4-dihydroisocoumarins are described. The structures of the synthesized
compounds were confirmed by spectral and elemental analysis. All synthesized compounds were evaluated for their anti-inflammatory
and antioxidant activity. These compounds were found to present antioxidant and anti-inflammatory activities. A few of them were found to
be significantly active in vivo against ear edema in mice produced by 12-O-tetradecanoylphorbol-13-acetate (TPA) and also good radical
scavengers.
# 2007 Elsevier B.V. All rights reserved.
Keywords: Synthesis; Fluorinated isocoumarins; 3,4-Dihydroisocoumarins; Anti-inflammatory activity; Antioxidant activity
1. Introduction
inflammatory, anti-allergic and enzyme inhibitory activity
[8]. Naturally occurring isocoumarins containing halogens
have been seldom reported, examples of naturally occurring
isocoumarins containing fluorine are not known yet. However
a few examples of naturally occurring isocoumarins contain-
ing chlorine and bromine have been reported. 4-Chloro-3-[(4-
fluorophenyl) methoxy] isocoumarin has been found [9] to
be quite effective inhibitor for human Q31 granzyme A,
murine and human granzyme A isolated from cytotoxic T
lymphocytes. This isocoumarin derivative has also been
found [10] to be useful in the treatment of emphysema as
serine protease inhibitor. 6-(2-Chloro-4-trifluromethylphe-
noxy)-3,4-dihydroisocoumarin has been used [11] as an
herbicide, which almost totally controlled the growth of
Schinochloa crusgall, Sinapis alba and other weeds. In
continuation of our previous studies [12–14] and important
biological activities associated with fluoro substituted
isocoumarins and 3,4-dihydroisocoumarins prompted us to
synthesize some new 3-difluorophenylisocoumarins and their
conversion to the corresponding 3,4-dihydroisocoumarin in
order to check their anti-inflammatory and antioxidant
activities. General synthetic scheme is shown as follows
(Scheme 1).
Inflammation is a complex phenomenon involving inter-
relationships between humoral or cellular reactions and a
number of inflammatory mediators. It is a usual symptom
covering different pathologies, and there are still many
questions to be answered in order to understand the
inflammatory process as well as a need for better-tolerated
and more efficient non-steroidal anti-inflammatory drugs. In
the pathways of the inflammatory process, the implication of
free radicals is particularly important. It has also been reported
that anti-inflammatory drugs may be effective in the prevention
of free radical-mediated damage [1].
Isocoumarins and 3,4-dihydroisocoumarins have been
reported to have multiple biological activities. The iso-
coumarin nucleus is an abundant structural motif in natural
products [2]. Many constituents of the steadily growing class
of known isocoumarins exhibit valuable biological properties
such as antifungal [3,4], antitumor or cytotoxic [5–7], anti-
* Corresponding author. Tel.: +92 51 2211034.
E-mail address: nasimhrama@yahoo.com (N.H. Rama).
0022-1139/$ – see front matter # 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.jfluchem.2007.02.021

642
G. Qadeer et al. / Journal of Fluorine Chemistry 128 (2007) 641–646
Scheme 1. Synthesis of fluoro-substituted isocoumarins.
2. Results and discussion
borohydride reduction of keto-acids 4(a–d) afforded the
corresponding racemic hydroxy acids 5(a–d), which were
cyclodehydrated with acetic anhydride to produce 3-difluor-
ophenyl-3,4-dihydroisocoumarin 6(a–c) which exhibited the
2.1. Synthesis
Condensation of the acid chloride with homophthalic acid is
useful for the preparation of 3-substituated isocoumarins
skelton [14]. A short and efficient synthesis of 3-(difluor-
ophenyl)isocoumarins 3(a–d) using this method and conversion
of 3(a–d) into corresponding racemic 3-(difluorophenyl)-3,4-
dihydroisocoumarins 6(a–d) were achieved and anti-inflam-
matory and antioxidant activities of the synthesized compounds
were examined. Difluorobenzoic acids 1(a–d) were converted
into their respective acid chlorides 2(a–d) by reaction with
thionyl chloride. Direct condensation of acid chlorides 2(a–d)
with homophthalic acid at 200 8C afforded 3-difluoropheny-
lisocoumarins 3(a–d) in 77–80% yield, which were purified by
column chromatography. These isocoumarins 3(a–d) exhibited
carbonyl absorptions at 1704, 1707, 1708 and 1703 cmꢀ1
,
respectively. The typical AB pattern for C3–H and ABX pattern
1
for C4–H protons were observed in H NMR spectrum of the
compound 6a. Thus, each of the C4–H showed a doublet of
doublet at d 3.46 and 3.81 ppm and another doublet of doublet
observed at d 5.92 ppm for C3–H.
2.2. Biological activities
2.2.1. Anti-inflammatory activity
The synthesized compounds 3(a–d), 4(a–d), 5(a–d) and
6(a–d) were evaluated by measuring their anti-inflammatory
activity against TPA-induced inflammation in mice, and the
inhibitory activities were compared with that of indomethacin,
a commercially available anti-inflammatory drug. At a dose of
1.0 mg/ear, the compounds 3b, 4d and 5a showed good activity
while the remaining compounds showed lower or nearly equal
activity compared to that of indomethacin (91.35% at a dose of
1.0 mg/ear). For anti-inflammatory evaluation, these com-
pounds did not achieve a structural similar analysis as for the
antioxidant activity, because, in case of anti-inflammatory
activity we have other factors that intervene in the biological
response (solubility, absorption, distribution, etc.). Due to the
lack of exact data related to the solubility, absorption and
distribution of each compound tested, it is difficult to clearly
show the structure–activity relationship concerning the anti-
inflammatory activity. However, we can qualitatively evaluate
the activity by taking account of these factors.
1
characteristic 1H-singlet at d 6.86–6.93 ppm for C4–H in H
NMR. In IR spectra of isocoumarins 3(a–d) lactonic carbonyl
absorptions were observed at 1703–1709 cmꢀ1. The molecular
ion peak in the mass spectrum of isocoumarin 3a was obtained
at m/z 258. Alkaline hydrolysis of isocoumarins 3(a–d)
afforded 2-(difluoro benzoylmethyl)benzoic acids 4(a–d).
Isocoumarins 3(a–d) were obtained on refluxing keto-acids
4(a–d) with acetic anhydride (reversible reaction). Sodium
The in vivo anti-inflammatory activity of these compounds
indicated these compounds as a possible candidate for the
development of new drugs to treat symptoms associated with
inflammatory diseases, such as osteoarthritis and arteriosclero-
sis. Further studies on the assessment of the COX-1/COX-2
selectivity index and inhibitory potency are in progress (Fig. 1).
Fig. 1. Anti-inflammatory activity of the synthesized compounds.

G. Qadeer et al. / Journal of Fluorine Chemistry 128 (2007) 641–646
643
2.2.2. Antioxidant activity
A mixture of homophthalic acid (11.3 mmol) and difluor-
obenzoyl chlorides (62.0 mmol) 2(a–d) was heated at 200 8C
under reflux for 4 h. The reaction mixture was dissolved in ethyl
acetate and aqueous solution of sodium carbonate was added in
order to remove the unreacted homophthalic acid. The organic
layer was separated, concentrated and chromatographed on
silica gel using petroleum ether (40–80 8C) as eluent to afford
isocoumarins 3(a–d) as solids, which were further purified by
recrystallization from methanol.
The radical scavenging effects of synthetic compounds
against stable free radical DPPH (2,2-diphenyl-2-picrylhydra-
zyl hydrate) were measured spectrophotometrically. Values for
their IC50 are shown in Table 1. Compounds 4a, 4d, 5b and 5d
were found to be good radical scavengers with IC50 below 100,
97.0, 93.3, 99.5 and 94.5 mM, respectively. Compound 5b was
the most effective DPPH scavenger with an IC50 value of 93.3.
Compounds 3(a–d) and 6(a–d) were considerably inactive with
IC50 values over 1000 mM.
3.1.1. Preparation of 3-(30,50-difluorophenyl) isocoumarin
(3a)
3. Experimental
Yield, 80% white solid; mp, 149–151 8C. IR (KBr, n,
1
The difluorobenzoic acids were purchased from Aldrich.
Melting points were determined in open capillaries using
Gallenkemp melting point apparatus and were uncorrected. The
infrared spectra were recorded on a Hitachi model 270-50
cmꢀ1): 2920, 1704, 1569, 1241, 1142. H NMR (CDCl3) d
6.81 (1H, m, H-40), 6.94 (1H, s, H-4), 7.40 (2H, dd, J = 1.98,
8.30 Hz, H-20, 60), 7.50 (1H, dd, J = 7.88 Hz, H-7), 7.54 (1H,
d, J = 7.40 Hz, H-5), 7.74 (1H, m, H-6), 8.31 (1H, d,
J = 7.92 Hz, H-8); EIMS (70 eV): m/z (%) 258.10 (100%)
(M+). Anal. Calcd. for C15H8F2O2: C, 69.77; H, 3.12; O,
12.39; F, 14.71. Found: C, 69.73; H, 3.10; O, 12.44; F,
14.73%.
1
spectrophotometer as KBr disks or as neat liquids. H NMR
(400 MHz) spectra were recorded on a Bruker AM-400 as
CDCl3 solution using TMS as an internal standard while EIMS
were recorded on a MAT-112-S machine. The petroleum ether
used corresponds to the fraction with a boiling range of 40–
80 8C.
3.1.2. Preparation of 3-(20,30-difluorophenyl) isocoumarin
(3b)
Yield, 77% white solid; mp, 156–158 8C. IR (KBr, n,
3.1. General synthetic procedure for the isocoumarin
derivatives 3(a–d)
1
cmꢀ1): 2935, 1707, 1566, 1242, 1141. H NMR (CDCl3) d
7.19 (1H, s, H-4), 7.36 (1H, m, H-40), 7.44 (1H, m, H-50),
7.54 (1H, dd, J = 8.2 Hz, H-7), 7.68 (1H, d, J = 7.36 Hz, H-
5), 7.74 (1H, m, H-60), 7.92 (1H, m, H-6), 8.31 (1H, d,
J = 7.90 Hz, H-8); EIMS (70 eV): m/z (%) 258.10 (100%)
(M+). Anal. Calcd. for C15H8F2O2: C, 69.77; H, 3.12; O,
12.39; F, 14.71. Found: C, 69.79; H, 3.13; O, 12.31; F,
14.77%.
A mixture of difluorobenzoic acids 1(a–d) (63.3 mmol) and
thionyl chloride (76.0 mmol) was heated for 30 min in the
presence of a drop of DMF under reflux. Completion of the
reaction was determined by the stoppage of SO2 evolution.
Removal of excess of thionyl chloride under reduced pressure
afforded difluorobenzoyl chlorides 2(a–d).
3.1.3. Preparation of 3-(20,40-difluorophenyl) isocoumarin
(3c)
Table 1
Anti-inflammatory and antioxidant activities of the synthesized compounds
Yield, 78% white solid; mp, 131–133 8C. IR (KBr, n,
3(a–d), 4(a–d), 5(a–d) and 6(a–d)
1
cmꢀ1): 2927, 1709, 1562, 1248, 1141. H NMR (CDCl3) d
Entry
Compound
% of inhibition
of inflammation
(1 mg/ear)
Antioxidant
6.93 (1H, m, H-30), 6.99 (1H, m, H-50), 7.12 (1H, s, H-4), 7.50
(1H, dd, J = 3.0, 7.8 Hz, H-7), 7.53 (1H, d, J = 7.56 Hz, H-5),
7.73 (1H, m, H-6), 7.99 (1H, m, H-60), 8.30 (1H, d,
J = 7.89 Hz, H-8); EIMS (70 eV): m/z (%) 258.00 (83.22%)
(M+). Anal. Calcd. for C15H8F2O2: C, 69.77; H, 3.12; O,
12.39; F, 14.71. Found: C, 69.72; H, 3.11; O, 12.48; F,
14.69%.
IC50 (mM)
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
3a
3b
3c
3d
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
6c
6d
ꢀ12.5a
103.7
25.3
>1000
>1000
>1000
>1000
97.0
64.8
31.3
85.4
322.4
81.0
463.3
3.1.4. Preparation of 3-(30,40-difluorophenyl) isocoumarin
(3d)
92.2
93.3
93.1
337.2
Yield, 79% white solid; mp, 156–159 8C. IR (KBr, n,
81.8
ꢀ64.9a
99.5
1
cmꢀ1): 2923, 1703, 1565, 1247, 1189. H NMR (CDCl3) d
862
35.1
94.5
6.88 (1H, s, H-4), 7.23 (1H, m), 7.51 (1H, m, H-20), 7.53 (1H,
dd, J = 1.0, 7.8 Hz, H-5), 7.72 (1H, m, H-50), 7.74 (1H, m, H-
6), 7.75 (1H, d, J = 1.24, 7.8, 11.2 Hz, H-60), 8.30 (1H, dd,
J = 8.0 Hz, H-8); EIMS (70 eV): m/z (%) 258.10 (100%)
(M+). Anal. Calcd. for C15H8F2O2: C, 69.77; H, 3.12; O,
12.39; F, 14.71. Found: C, 69.71; H, 3.13; O, 12.41; F,
14.75%.
88.2
>1000
>1000
>1000
>1000
Not determine
29.4
86.1
ꢀ14.8a
ꢀ54.7a
91.4
Indomethacin
Quercetin
Not determine
a
Values with negative sign represent pro-inflammatory activity.

644
G. Qadeer et al. / Journal of Fluorine Chemistry 128 (2007) 641–646
3.2. General synthetic procedure for the keto-acid
derivatives 4(a–d)
3.3. General synthetic procedure for the
dihydroisocoumarin derivatives 6(a–d)
A solution of isocoumarins 3(a–d) in ethanol (50 mL) and
5% potassium hydroxide (100 mL) were refluxed for 4 h.
Ethanol was removed from the reaction mixture by distillation.
Ice-cold water (20 mL) was added and the reaction mixtures
were acidified with hydrochloric acid. The reaction mixture
was then extracted with dichloromethane (3ꢁ 20 mL). The
extracts were dried (Na2SO4) and solvent was rotary evaporated
to yield crude solid benzoic acids 4(a–d), which were
recrystallized from methanol.
To a solution of the keto-acids 4(a–d) (2.07 mmol) dissolved
in 1% potassium hydroxide solution (25 mL), sodium
borohydride (0.25 g) was added and the reaction mixture
was stirred for 1 h at room temperature. After acidification with
hydrochloric acid, the reaction mixture was extracted with ethyl
acetate (2ꢁ 50 mL). The usual workup gave the crude hydroxy-
acids 5(a–d), which were dissolved in acetic anhydride (1 mL)
and heated under reflux for 2 h. The reaction mixture was
cooled, water (25 mL) was added and the reaction mixture was
stirred overnight. The crystals that deposited were collected by
filtration and the filtrate was extracted with dichloromethane
(2ꢁ 20 mL). The solvent was removed under reduced pressure.
The crude dihydroisocoumarin 6(a–d) was purified by column
chromatography on silica gel using petroleum ether as an
eluent.
3.2.1. Preparation of 20-(300,500-difluorobenzoylmethyl)
benzoic acid (4a)
Yield, 80% colorless crystals; mp, 124–126 8C. IR (KBr, n,
1
cmꢀ1): 3300–3250, 2920, 2733, 1708, 1470, 1142. H NMR
(D2O) d 4.61 (1H, s, H-10), 6.98 (1H, dd, J = 8.25, 16.6 Hz, H-
400), 7.23 (1H, d, J = 7.4 Hz, H-3), 7.37 (1H, dd, J = 7.6 Hz, H-
4), 7.51 (2H, m, H-200,600), 7.77 (1H, m, H-4), 8.08 (1H, d,
J = 7.23 Hz, H-6); EIMS (70 eV): m/z (%) 276.20 (M+). Anal.
Calcd. for C15H10F2O3: C, 65.22; H, 3.65; O, 17.38; F, 13.76.
Found: C, 65.24; H, 3.66; O, 17.34; F, 13.76%.
3.3.1. Preparation of 2-[20-hydroxy-20-(300,500-
difluorophenyl)ethyl] benzoic acid (5a)
Yield, 69% colorless crystals; mp, 132–133 8C. IR (KBr, n,
1
cmꢀ1): 3300–3250, 2920, 2733, 1708, 1470, 1142. H NMR
(D2O) d 2.33 (1H, dd, J = 8.22, 15.6 Hz, H-10b), 2.67 (1H, dd,
J = 6.24, 15.6 Hz, H-10a), 4.49 (1H, dd, J = 7.12, 14.20 Hz, H-
20), 6.91 (3H, m, H-200,400,600), 7.23 (1H, d, J = 7.4 Hz, H-3),
7.37 (1H, dd, J = 7.6 Hz, H-5), 7.77 (1H, m, H-4), 8.08 (1H, d,
J = 7.23 Hz, H-6); EIMS (70 eV): m/z (%) 278.00 (M+). Anal.
Calcd. for C15H10F2O3: C, 64.75; H, 4.35; O, 17.25; F, 13.66.
Found: C, 64.24; H, 4.66; O, 17.34; F, 13.76%.
3.2.2. Preparation of 20-(200,300-difluorobenzoylmethyl)
benzoic acid (4b)
Yield, 70% colorless crystals; mp, 151–154 8C. IR (KBr, n,
1
cmꢀ1): 3300–3250, 2920, 2735, 1704, 1471, 1145. H NMR
(D2O) d 4.65 (1H, s, H-10), 6.98 (1H, dd, J = 8.02, 16.6 Hz, H-
400,500), 7.27 (1H, d, J = 7.14 Hz, H-3), 7.31 (1H, dd, J = 7.6 Hz,
H-5), 7.51 (2H, m, H-600), 7.73 (1H, m, H-4), 8.05 (1H, d,
J = 7.21 Hz, H-6); EIMS (70 eV): m/z (%) 276.20 (M+). Anal.
Calcd. for C15H10F2O3: C, 65.22; H, 3.65; O, 17.38; F, 13.76.
Found: C, 65.29; H, 3.62; O, 17.38; F, 13.71%.
3.3.2. Preparation of 2-[20-hydroxy-20-(200,300-
difluorophenyl)ethyl] benzoic acid (5b)
Yield, 75% colorless crystals; mp, 144–146 8C. IR (KBr, n,
1
cmꢀ1): 3300–3250, 2922, 2737, 1701, 1475, 1141. H NMR
3.2.3. Preparation of 20-(200,400-difluorobenzoylmethyl)
benzoic acid (4c)
(D2O) d 2.53 (1H, dd, J = 8.20, 15.16 Hz, H-10b), 3.01 (1H, dd,
J = 6.14, 15.06 Hz, H-10a), 4.62 (1H, dd, J = 7.10, 14.29 Hz,
H-20), 6.69 (1H, m, H–H-400), 6.99 (2H, m, H-500,600), 7.43 (1H,
d, J = 7.4 Hz, H-3), 7.39 (1H, dd, J = 7.6 Hz, H-5), 7.67 (m, H-
4), 8.03 (1H, d, J = 7.21 Hz, H-6); EIMS (70 eV): m/z (%)
278.00 (M+). Anal. Calcd. for C15H10F2O3: C, 64.75; H, 4.35;
O, 17.25; F, 13.66. Found: C, 64.77; H, 4.46; O, 17.07; F,
13.70%.
Yield, 77% colorless crystals; mp, 174–177 8C. IR (KBr, n,
1
cmꢀ1): 3300–3250, 2924, 2731, 1708, 1477, 1140. H NMR
(D2O) d 4.62 (1H, s, H-10), 6.79 (1H, m, H-300), 6.97 (1H, dd,
J = 8.23, 16.6 Hz, H-500), 7.23 (1H, d, J = 7.4 Hz, H-3), 7.35
(1H, dd, J = 7.6 Hz, H-5), 7.51 (2H, m, H-600), 7.71 (1H, m, H-
4), 8.10 (1H, d, J = 7.25 Hz, H-6); EIMS (70 eV): m/z (%)
276.20 (M+). Anal. Calcd. for C15H10F2O3: C, 65.22; H, 3.65;
O, 17.38; F, 13.76. Found: C, 65.19; H, 3.67; O, 17.39; F,
13.75%.
3.3.3. Preparation of 2-[20-hydroxy-20-(200,400-
difluorophenyl)ethyl] benzoic acid (5c)
3.2.4. Preparation of 20-(300,400-difluorobenzoylmethyl)
benzoic acid (4d)
Yield, 79% colorless crystals; mp, 129–130 8C. IR (KBr, n,
1
cmꢀ1): 3300–3250, 2923, 2730, 1705, 1477, 1145. H NMR
Yield, 69% colorless crystals; mp, 132–135 8C. IR (KBr, n,
(D2O) d 2.57 (1H, dd, J = 8.20, 15.56 Hz, H-10b), 2.99 (1H, dd,
J = 6.21, 15.56 Hz, H-10a), 4.75 (1H, dd, J = 7.10, 14.25 Hz, H-
20), 6.55 (2H, m, H-300,500), 7.01 (1H, dd, J = 8.25, 16.6 Hz,
H-600), 7.23 (1H, d, J = 7.4 Hz, H-3), 7.45 (1H, dd, J = 7.6 Hz,
H-5), 7.69 (1H, m, H-4), 8.10 (1H, d, J = 7.23 Hz, H-6); EIMS
(70 eV): m/z (%) 278.00 (M+). Anal. Calcd. for C15H10F2O3: C,
64.75; H, 4.35; O, 17.25; F, 13.66. Found: C, 64.67; H, 4.26; O,
17.40; F, 13.67%.
1
cmꢀ1): 3300–3250, 2927, 2737, 1710, 1473, 1141. H NMR
(D2O) d 4.62 (1H, s, H-10), 6.89 (1H, dd, J = 8.22, 16.16 Hz, H-
500), 7.24 (1H, d, J = 7.14 Hz, H-3), 7.33 (1H, dd, J = 7.16 Hz,
H-5), 7.55 (2H, m, H-200,600), 7.73 (1H, m, H-4), 8.03 (1H, d,
J = 7.21 Hz, H-6); EIMS (70 eV): m/z (%) 276.20 (M+). Anal.
Calcd. for C15H10F2O3: C, 65.22; H, 3.65; O, 17.38; F, 13.76.
Found: C, 65.22; H, 3.69; O, 17.30; F, 13.79%.

G. Qadeer et al. / Journal of Fluorine Chemistry 128 (2007) 641–646
645
3.3.4. Preparation of 2-[20-hydroxy-20-(300,400-
difluorophenyl) ethyl] benzoic acid (5d)
7.21 (1H, m, H-60), 7.41 (1H, m, H-7), 7.55 (1H, dd, J = 1.22,
7.48 Hz, H-5), 7.88 (1H, m, J = 1.8, 6.90 Hz, H-6), 8.13 (1H, d,
J = 7.79 Hz, H-8); EIMS (70 eV): m/z (%) 260.00 (4.5%)
(M + 1). Anal. Calcd. for C15H8F2O2: C, 69.23; H, 3.87; O,
12.30; F, 14.60. Found: C, 69.23; H, 3.03; O 13.07; F, 14.62%.
Yield, 75% colorless crystals; mp, 121–123 8C. IR (KBr, n,
cmꢀ1): 3300–3250, 2933, 2733, 1703, 1467, 1143. H NMR
1
(D2O) d 2.55 (1H, dd, J = 8.02, 15.36 Hz, H-10b), 2.89 (1H, dd,
J = 6.12, 15.6 Hz, H-10a), 4.85 (1H, dd, J = 7.01, 14.22 Hz,
H-20), 6.71 (2H, m, H-200,500), 6.87 (1H, dd, J = 8.20, 16.6 Hz,
H-600), 7.43 (1H, d, J = 7.4 Hz, H-3), 7.55 (1H, dd, J = 7.6 Hz,
H-5), 7.61 (1H, m, J = 1.6, 7.90 Hz, H-4), 8.09 (1H, d,
J = 7.23 Hz, H-6); EIMS (70 eV): m/z (%) 278.00 (M+). Anal.
Calcd. for C15H10F2O3: C, 64.75; H, 4.35; O, 17.25; F, 13.66.
Found: C, 64.60; H, 4.28; O, 17.49; F, 13.63%.
4. Biological assay
4.1. Anti-inflammatory activity
4.1.1. Experimental animals
Adult male Wistar CD-1 mice with a body weight ranging
from 20 to 25 g were used. All animals had free access to food
and water and were kept on a 12/12 h light–dark cycle.
3.3.5. Preparation of (DL)-3-(30,50-difluorophenyl)-3,4-
dihydroisocoumarin (6a)
Yield, 69% white solid; mp, 121–122 8C. IR (KBr, n, cmꢀ1):
2920, 1704, 1244, 1142. H NMR (CDCl3) d 3.46 (1H, dd,
4.1.2. TPA-induced mouse ear edema
1
Mouse ear edema was evaluated following the protocol
previously described [15], using groups of three male CD-1
mice. Edema was induced by topical application of 2.5 mg per
ear of TPA dissolved in EtOH. Solutions of the compounds
(1 mg/ear) and the standard drug indomethacin (1 mg/ear) as
reference, dissolved in different solvents according to their
solubility, respectively, were applied to both sides of the right
ear (10 mL each side) simultaneously with TPA. The ear
swelling was measured before TPA application and 4 h after,
and the edema was expressed as the increase in thickness. The
results are shown in Table 1.
J = 2.92, 16.37 Hz, H-4b), 3.81 (1H, dd, J = 4.09, 13.04 Hz, H-
4a), 5.92 (1H, dd, J = 2.95, 13.10 Hz, H-3), 6.90 (1H, m, H-40),
7.12 (2H, m, H-20,60), 7.28 (1H, m, H-7), 7.51 (1H, dd, J = 2.15,
7.68 Hz, H-5), 7.67 (1H, dd, J = 3.37, 5.67 Hz, H-6), 8.14 (1H,
d, J = 7.58 Hz, H-8); EIMS (70 eV): m/z (%) 261.20 (4%)
(M + 1). Anal. Calcd. for C15H8F2O2: C, 69.23; H, 3.87; O,
12.30; F, 14.60. Found: C, 69.23; H, 3.03; O 13.07; F, 14.62%.
3.3.6. Preparation of (DL)-3-(20,30-difluorophenyl)-3,4-
dihydroisocoumarin (6b)
Yield, 80% white solid; mp, 118–121 8C. IR (KBr, n, cmꢀ1):
2935, 1707, 1244, 1141. H NMR (CDCl3) d 3.09 (1H, dd,
1
4.2. Antioxidant activity
J = 2.92, 16.37 Hz, H-4b), 3.31 (1H, dd, J = 4.09, 16.31 Hz, H-
4a), 5.76 (1H, dd, J = 2.94, 12.16 Hz, H-3), 6.61 (1H, m, H-40),
6.71 (m, H-60), 7.46 (1H, m, H-50), 7.54 (1H, dd, J = 2.24,
7.90 Hz, H-7), 7.62 (1H, dd, J = 2.5, 7.57 Hz, H-5), 7.68 (1H,
dd, J = 3.20, 5.72 Hz, H-6), 8.13 (1H, d, J = 7.27 Hz, H-8);
EIMS (70 eV): m/z (%) 261.20 (51%) (M + 1). Anal. Calcd. for
C15H8F2O2: C, 69.23; H, 3.87; O, 12.30; F, 14.60. Found: C,
69.23; H, 3.03; O 13.07; F, 14.62%.
4.2.1. DPPH radical scavenging assay
Radical scavenging activity of compounds against stable
DPPH (2,2-diphenyl-2-picrylhydrazyl hydrate, Sigma–Aldrich
Chemie, Steinheim, Germany) was determined spectrophoto-
metrically. When DPPH reacts with an antioxidant compound,
which can donate hydrogen, it is reduced. The changes in
colour (from deep-violet to light-yellow) were measured at
515 nm on a UV–vis light spectrophotometer (Spectronic
Genesys 8, Rochester, USA).
3.3.7. Preparation of (DL)-3-(20,40-difluorophenyl)-3,4-
dihydroisocoumarin (6c)
Radical scavenging activity of compounds was measured by
slightly modified method of Brand-Williams [16], as described
below. Compound solutions were prepared by dissolving an
appropriate amount of each compound in ethanol. The solution
of DPPH in ethanol was prepared just before UV measure-
ments. 1.5 mL of this solution was mixed with 0.5 mL of
compound solutions in 1 dm path length disposable micro-
cuvettes (final mass ratio of compound with DPPH was
approximately 3:1). The samples (at 1, 10, 100, 500 and
1000 mM) were kept in the dark for 30 min at room temperature
and then the decrease in absorption was measured. For the
control absorption of blank sample containing the same amount
of ethanol and DPPH solution was measured. The experiment
was carried out in triplicate. Radical scavenging activity was
calculated by the following formula:
Yield, 80% white solid; mp, 121–122 8C. IR (KBr, n, cmꢀ1):
2935, 1707, 1244, 1141. H NMR (CDCl3) d 3.09 (1H, dd,
1
J = 2.92, 16.37 Hz, H-4b), 3.31 (1H, dd, J = 4.09, 16.31 Hz, H-
4a), 5.76 (1H, dd, J = 2.94, 12.16 Hz, H-3), 6.61 (1H, m, H-40),
6.71 (m, H-60), 7.46 (1H, m, H-50), 7.54 (1H, dd, J = 2.24,
7.90 Hz, H-7), 7.62 (1H, dd, J = 2.5, 7.57 Hz, H-5), 7.68 (1H,
dd, J = 3.20, 5.72 Hz, H-6), 8.13 (1H, d, J = 7.27 Hz, H-8);
EIMS (70 eV): m/z (%) 261.20 (4%) (M + 1). Anal. Calcd. for
C15H8F2O2: C, 69.23; H, 3.87; O, 12.30; F, 14.60. Found: C,
69.23; H, 3.03; O 13.07; F, 14.62%.
3.3.8. Preparation of (DL)-3-(30,40-difluorophenyl)-3,4-
dihydroisocoumarin (6d)
Yield, 69% white solid; mp, 121–122 8C. IR (KBr, n, cmꢀ1):
2923, 1703, 1243, 1189. H NMR (CDCl3) d 3.08 (1H, dd,
1
ꢀ
ꢁ
AB ꢀ AA
J = 3.10, 16.39 Hz, H-4b), 3.29 (1H, dd, J = 4.4, 16.38 Hz, H-
4a), 5.46 (1H, dd, J = 3.09, 11.91 Hz, H-3), 7.15 (2H, m, H-20,50),
%inhibition ¼
ꢁ 100
AB

646
G. Qadeer et al. / Journal of Fluorine Chemistry 128 (2007) 641–646
[7] R. Rossi, A. Carpita, F. Bellina, P. Stabile, L. Mannina, Tetrahedron 59
(2003) 2067–2081.
where AB is the absorption of blank sample (t = 0 min); AA is
the absorption of tested extract solution (t = 15 min).
[8] J.C. Powers, J.L. Asgian, D. Ekici, K.E. James, Chem. Rev. 102 (2002)
4639–4750.
Acknowledgement
[9] S. Odake, C.M. Kam, L. Narasimhan, M. Poe, J.T. Blake, O. Krahenbuhl,
J. Tschopp, J.C. Powers, Biochemistry 30 (1991) 2217–2225.
[10] D. Hudig, N.J. Allison, C.M. Kam, J.C. Powers, Mol. Immunol. 26 (1989)
793–799 (C.A. 111: 192909h).
The authors gratefully acknowledge funds from the Higher
Education Commission, Islamabad, Pakistan.
[11] F. Fratev, V. Enchev, P. Nikolov, O.E. Polansky, Z. Naturforsch. A: Phys.
Chem. Komophys. 39A (11) (1984) 1143–1151.
References
[12] H.B. Ahmad, N.H. Rama, M. Hussain, M.T. Hussain, M. Qasim, S.
Hameed, M.A. Malana, A. Malik, Indian J. Chem. 42B (2003) 611–
615.
[1] P.L. Hiller, K.O. Hodd, R.L. Wilson, Chem. Biol. Interact. 47 (1983) 293–
305.
[13] Z. Shafiq, M. Arfan, N.H. Rama, S. Hameed, G. Abbas, M.T. Hussain,
Turk. J. Chem. 29 (2005) 321–324.
[2] R.D. Barry, Chem. Rev. 64 (1964) 229–260.
[3] K. Meepagala, G. Sturtz, D. Wedge, J. Agric. Food Chem. 50 (2002)
6989–6992.
[14] F. Zhijin, G. Qadeer, N.H. Rama, Chinese Patent, Application No. CN
1012-2353 200,510,122,353. Priority: CAN 145:188619 AN 2006:593756
CAPLUS p. 24.
[4] O.A. Krupke, A.J. Castle, D.L. Rinker, Mycol. Res. 107 (2003) 1467–1475.
[5] A.C. Whyte, J.B. Gloer, J.A. Scott, D. Malloch, J. Nat. Prod. 59 (1996)
765–769.
[15] B. Williams, M.E. Cuvelier, C. Berset, Lebensmittel Wissenschaft Tech-
nol. 28 (1995) 25–30.
´
[16] Y. Herrera-Salgado, M.L. Gardun˜o-Ramırez, M.Y. Rios, L. Alvarez, J.
[6] Y. Cai, H. Kleiner, D. Johnston, A. Dubowski, S. Botic, W. Ivie, J. Di
Giovanni, Carcinogenesis 18 (1997) 1521–1527.
Nat. Prod. 68 (2005) 1031–1036.