Synthesis and antimicrobial activities of some new 3-(chlorophenylethyl)-, 3-(chlorophenylethenyl)-isocoumarins and their dihydro derivatives

Article abstract of DOI:10.2174/157017809789869528

The bioactive 3-(chlorophenylethyl)- and 3-(chlorophenylethenyl)- isocoumarins were synthesized by the condensation of simple homophthalic acid with 3-chlorophenylpropionoyl chlorides and chlorocinnamoyl chlorides, respectively. Alkaline hydrolysis of the

Full text of DOI:10.2174/157017809789869528

Letters in Organic Chemistry, 2009, 6, 579-584
579
Synthesis and Antimicrobial Activities of Some New 3-(Chlorophenyl-
ethyl)-, 3-(Chlorophenylethenyl)-Isocoumarins and their Dihydro
Derivatives
Gul S. Khana, G. Qadeera, Nasim H. Ramaa and Khalid Mohammed Khan*,b
aDepartment of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan.
bH.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of
Karachi, Karachi-75270, Pakistan
Received January 06, 2009: Revised May 29, 2009: Accepted May 29, 2009
Abstract: The bioactive 3-(chlorophenylethyl)- and 3-(chlorophenylethenyl)-isocoumarins were synthesized by the
condensation of simple homophthalic acid with 3-chlorophenylpropionoyl chlorides and chlorocinnamoyl chlorides,
respectively. Alkaline hydrolysis of these isocoumarins gave the respective keto-acids. (dl)-3,4-Dihydroisocoumarins
were obtained by the reduction of the keto-acids to racemic hydroxyl-acids, followed by cyclodehydration using acetic
anhydride. These compounds were tested for their antimicrobial and cytotoxicity (Brine shrimp lethality) bioassay.
Bioassay indicated that some compounds show good antimicrobial activities and positive lethality.
Keywords: Isocoumarins, dihydroisocoumarins, synthesis, antimicrobial screening.
The isocoumarin nucleus is an abundant structural motif
in natural products [1]. Many constituents of the steadily
growing class of known isocoumarins exhibit valuable
biological properties, such as antifungal [2], antitumor or
cytotoxic [3], antiinflammatory, antiallergic, and enzyme
inhibitory activities [4]. Naturally occurring haloisoco-
umarins and halo-3,4-dihydroisocomuarins are very rare,
however, a few examples of naturally occurring chlorine
containing isocoumarins are known [5]. Naturally occurring
isocoumarins having chlorine are bactabolins A, B, and C
isolated from Pseudomonas yoshitomiensis [6], whereas LLZ
1640-5 was isolated from laderale culture of unidentified
fungus [7]. Bactabolin A has been reported to be active
against bacteria and viruses [8]. Some chloroisocoumarins
are strong inhibitors of numerous serine proteases [9, 10].
of 3-(chlorophenylethyl)- and 3-(chlorophenylethenyl)
isocoumarins 3a-f and conversion of the later into correspon-
ding (dl)-3-(chlorophenylethyl)-3,4-dihydroisocoumarins 6a-
c are achieved by using this method. Antimicrobial,
cytotoxicity activities of the synthesized compounds are also
described. Reaction of commercial homophthalic acid with
chlorophenylpropionyl and chlorophenylcinnamoyl chlorides
2a-f at elevated temperature afforded 3-(chlorophenylethyl)-
and 3-(chlorophenylethenyl)isocoumarins 3a-f in 70-78%
yield . The isocoumarins showed the characteristic 1H singlet
of the isocoumarins moiety (H-4) at 6.48, 6.77, 6.75, 6.40,
1
6.42, and 6.41 ppm, respectively, in H NMR. IR spectra
showed the lactonic carbonyl absorption at 1721, 1719,
1722, 1720, 1727, 1726 cm,-1 respectively. In IR spectrum of
3d-f, double bond appeared at 1611, 1612, and 1616 cm,-1
respectively, while these peaks are absent in the
isocoumarins 3a-c. Alkaline hydrolysis of 3a-c furnished 2-
[4’-(chlorophenyl)-2’-oxobutyl]benzoic acids 4a-c were
accomplished in 81-85% yield. The keto-acids 4a-c showed
2H singlets at ꢀ 3.30, 3.25, and 3.30 ppm (H-4, ArCH2) in
Biological activities associated with chloroisocoumarins
prompted us to synthesize more halogenated 3-substituted
isocoumarins as a continuation of our efforts [11-17] towards
the synthesis of bioactive isocoumarins, herein, we wish to
report the synthesis of a number of 3-(chlorophenylethyl)-,
3-(chlorophenylethenyl)isocoumarins 3a-f and (dl)-3-(chlo-
rophenylethyl)-3,4-dihydroisocoumarins 6a-c. The synthe-
sized compounds were also screened for antimicrobial and
cytotoxicity (Brineshrimp lethality) activities.
1
the H NMR spectrum. The ketonic and carboxylic carbonyl
absorptions of these keto-acids in IR spectrum were
observed at 1700-1720, respectively. Sodium borohydride
reduction of the keto-acids 4a-c afforded the corresponding
racemic hydroxyacids 5a-c, which underwent cyclodehy-
dration on refluxing with acetic anhydride to (dl)-3-
(chlorophenyl-ethyl)-3,4-dihydroisocoumarins 6a-c. The
methylene protons (H-4) adjacent to the newly generated
chiral center (C3) in dihydroisocoumarins 6a-c appeared as
multiplets at ꢀ 3.0-3.3, 3.0-3.3, 3.0-3.3 ppm, respectively.
The H-3 protons appeared as a multiplet at ꢀ 4.56, 4.54, and
4.54 ppm, respectively. The ꢀ-lactonic carbonyl absorption
appeared at 1703, 1717, 1703 cm,-1 respectively, in the IR
spectrum. HREI-MS of M+ ions of all the synthesized
compounds were in good agreement with the calculated
RESULTS AND DISCUSSION
Condensation of the acid chloride with homophthalic
acid is useful for the preparation of 3-substituted isoco-
umarins skeleton [18]. Herein, a short and efficient synthesis
*Address correspondence to this author at the H.E.J. Research Institute of
Chemistry, International center for Chemical and Biological Sciences,
University of Karachi, Karachi-75270, Pakistan; Tel: 0092214824910; Fax:
0092214819018; E-mail: khalid.khan@iccs.edu; hassaan2@super.net.pk
1570-1786/09 $55.00+.00
© 2009 Bentham Science Publishers Ltd.

580 Letters in Organic Chemistry, 2009, Vol. 6, No. 7
Khan et al.
O
OH
O
HO
O
O
R
OH
C l
R
SOCl2
30 min
R
5% KOH in water
C2H5OH
O
200 °C, 4h
O
1a-f
2a-f
3a-f
O
HO
O
HO
O
O
R
R
R
Ac2O
Cl
NaBH4
O
OH
6a-c
5a-c
4a-c
Cl
d ) R =
e ) R =
a ) R =
b ) R =
Cl
Cl
Cl
f ) R =
c ) R =
Cl
Scheme 1.
values providing further confirmation for their structures as
shown Scheme 1.
activities of compounds 3b, 4b, and 6b reach to 90% at 200
μg/ml. The other synthesized compounds also show
antifungal activity but not too good. None of the synthesized
compounds show antifungal activity against Candida
albicans and Candida glabrata.
Isocoumarins,
dihydroisocoumarins,
and
related
compounds were tested by agar tube dilution method [19] for
their in vitro fungicidal bioassay. Results were reported as
linear growth inhibition (LGI) against some human
pathogens (e.g. Trichophyton longifusus, Candida albicans,
Aspergillus flavus, Microsporum canis, Fusarium solani,
Candida glabrata). Linear Growth Inhibition results of
compounds are given in Table 1.
All the synthesized compounds were screened for
antibacterial activity against four Gram-negative and two
Gram-positive bacterial strains i.e. Gram negative are
Escherichia
coli, Shigella
flexnari,
Pseudomonas
aeruginosa, and Salmonella typhi and two Gram-positive
bacterial stains are Bacillus subtilis and Staphyloccus
aureus. Using agar well diffusion method [20] as a literature
protocol. The results of antibacterial activity are displayed in
Table 2.
From the biological assay results in Table 1, which
summarize the antifungal activity of the target compounds,
some showed significant activity against the three fungi
Trichphyton Longifusus, Aspergillus Flavus, and Micros-
porum canis. Compounds 4a, 4b, 6a, and 6b exhibited
considerable activities against Trichphyton longifusus,
Aspergillus flavus, and Microsporum canis. Even the
The activity was determined via the growth inhibition of
microorganism, i.e. the zone inhibition, measured in
millimeters. The results indicate that our compounds are
Table 1. Anti-Fungal Activity of Compounds (3b-c, 4a-c, 6a-c) as Linear Growth Inhibition (%) at 200 μg/ml of Media SDA
Compounds
Pathogens
Std. Drugs
Inhibition (%)
3b
3c
4a
4b
4c
6a
6b
6c
Trichphyton longifusus
Candida albicans
Aspergillus flavus
Microsporum canis
Fusarium solani
50
0
30
0
40
0
90
0
0
0
20
0
70
0
50
0
Miconazole
Miconazole
Amphotericin B
Miconazole
Miconazole
Miconazole
100
100
100
100
100
100
0
0
85
50
50
0
90
90
0
85
0
85
85
0
85
90
0
0
90
0
75
0
45
0
15
0
Candida glabrata
0
0
0
0
0
0

Synthesis and Antimicrobial Activities
Letters in Organic Chemistry, 2009, Vol. 6, No. 7
581
Table 2. Antibacterial Activity (In Vitro) of all Synthesized Compounds (3a-f, 4a-c, 6a-c)
Compounds
Imipenem
Bacteria
3a
3b
3c
3d
3e
3f
4a
4b
4c
6a
6b
6c
Escherichia coli
Shigella flexeneri
0
0
0
0
0
0
0
0
12
15
16
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
9
0
0
0
30
27
24
25
33
33
Pseudomonas aeruginosa
Salmonella typhi
12
12
0
20
19
0
0
16
16
0
18
10
0
15
14
0
20
15
0
0
16
0
15
0
Bacillus subtilis
0
Staphyloccus aureus
0
0
12
16
0
0
0
13
10
generally weekly or moderately active and only 3f and 6b
out of twelve compounds tested, demonstrated significant
antibacterial activity. It is evident from Table 2, that
compounds 3f and 6c exhibit significant activity against
Pseudomonas aeruginosa with a zone of inhibition of 20 mm
as compared to the standard drug (imipenem) having zone of
inhibition of 24 mm. Compounds 3b, 3d, and 3e were
completely inactive against all bacterial strains. None of the
compounds were active or weekly active against Escherichia
coli, Shigella flexeneri, and Bacillus subtilis.
capillaries using Gallenkemp melting point apparatus and are
uncorrected. IR spectra were recorded on a Hitachi model
270-50 spectrophotometer as KBr discs, H NMR (400 &
300 MHz) spectra in CDCl3 on a Bruker AM-500 using TMS
as internal standard and HRMS on a MAT-112-S machine at
70ev.
1
General Procedure for the Synthesis of 3-(Chlorophenyl-
ethyl)isocoumarins (3a-c)
Mixtures of homophthalic acid (2.06 g, 11 mmol) and
Compounds 3b, 3c, 3e, 4a, and 6a-c were tested for
cytotoxicity by the brine shrimp lethality bioassay. From the
data collected in Table 3, it is evident that the majority of the
compounds in this study displayed promising cytotoxicity
substituted chlorophenylpropionyl chlorides 2a-c (9.5 g, 47
°
mmol) were heated at 200 C under reflux for four h. The
reaction mixtures were dissolved in ethyl acetate and
aqueous solution of sodium carbonate was added in order to
remove the unreacted homophthalic acid. The organic layers
were separated, concentrated, and chromatographed on silica
(LD
= 1.226 - 7.562 μg/ml). It may be noted that
50
compound 2-[4’-(chlorophenyl)-2’-oxobutyl]benzoic acid 4c
showed highest degree of cytotoxicity (LD50 = 7.562 μg/ml)
than the standard drug and its dihydroisocoumarin derivative
also showed cytotoxicity (2.0978). Interestingly, the 2-[2’-
(chloropheny)-2’-oxobutyl]benzoic acid 4a was completely
inactive but its dihydroisocoumarin showed cytotoxicity
(1.226). We have, however, no information about the mode
of action of these compounds. Further research on the
modification of structure and the mode of action is in
progress.
°
gel using petroleum ether (40-80 C) as eluent to afford 3-
(chlorophenylethyl)isocoumarins 3a-c as solids, which were
further purified by recrystallization from methanol.
3-(2-Chlorophenethyl)-1H-isochromen-1-one (3a)
°
Yield 77%; m.p 70 C; IR ꢁmax (KBr) 2930, 1720, 1072
1
cm-1, H NMR (acetone-d6) ꢂ 2.57 (2H, t, J = 7.52, 8.1 Hz,
H-1ꢃ), 2.98 (2H, t, J = 8.0 Hz, H-2ꢃ), 6.40 (1H, s, H-4), 7.15-
7.23 (2H, m, H-4ꢄ,6ꢄ), 7.3-7.34 (2H, m, H-3ꢄ,5ꢄ), 7.50 (1H,
dd, J = 1.9, 8.2 Hz, H-7), 7.60 (1H, d, J = 7.5 Hz, H-5), 7.71
(1H, ddd, J = 7.3, 0.9 Hz, H-6), 8.12 (1H, dd, J = 7.3, 0.9
Hz, H-8) ppm; EIMS (70ev): m/z (%) 284 (M+, 37.5), 286
(M+ +2, 11.85), 159 (60.4), 125 (100), 103 (32.3); HRMS:
m/z Found; 284.0602 (Cl35) (Calcd for C17H13O2Cl35;
284.0604) & Found; 286.0572 (Cl37), (Calcd for C-
17H13O2Cl37; 286.0575).
The findings of this research suggest that isocoumarins
and related compounds may be used as potent bioactive
antibacterial and antifungal agents. Furthermore, the
methods used in this research to synthesize isocoumarins and
related compounds may be useful to enhance the fungicidal
potential for agricultural as well as pharmaceutical purposes.
These studies may serve as a basis for the further chemical
modification on the isocoumarin moiety, which may be the
incorporation of an NH2 or SH group or an amidic
functionality into the isocoumarin residue. These
functionalities may help to enhance the activity of
isocoumarins and dihydroisocoumarins and may lead to the
development of a potential bioactive derivative of clinical
interest without having severe effects attributed to ꢀ-lactam
ring.
3-(3-Chlorophenethyl)-1H-isochromen-1-one (3b)
°
Yield 75%; m.p 85 C; IR ꢁmax (KBr) 2935, 1727, 1072
cm-1; 1H NMR (acetone- d6) ꢂ 2.83 (2H, t, J = 7.2,8.1 Hz, H-
1ꢃ), 2.99 (2H, t, J = 7.2, 8.1 Hz, H-2ꢃ), 6.42 (1H, s, H-4),
7.14-7.18 (2H, m, H-4ꢄ, 5ꢄ), 7.19 (1H, dd, J = 7.6, 1.4 Hz,
H-6ꢄ), 7.25 (1H, bs, H-2ꢄ), 7.44 (1H, dd, J = 7.0, 7.8 Hz, H-
7), 7.5 (1H, ddd, J = 7.5, 1.2 Hz, H-6), 8.11 (1H, dd, J = 8.6,
1.2 Hz, H-8) ppm; EIMS (70ev): m/z (%) 284 (M+, 37.5),
286 (M+ +2, 11.8), 159 (3.4), 125 (100), 103 (11.8); HRMS:
m/z Found; 284.0609 (Cl35) (Calcd for C17H13O2Cl35;
EXPERIMENTAL SECTION
&
Found; 286.0576 (Cl37) (Calcd for C-
General Experimental Procedures
284.0604)
17H13O2Cl37; 286.0575).
Chlorocinnamic acids were purchased from Aldrich.
Melting points of the compounds were determined in open

582 Letters in Organic Chemistry, 2009, Vol. 6, No. 7
Khan et al.
Table 3. Brine Shrimp (Artemia salina) Lethality Bioassay
Code No
3b
Dose (μg/ml)
No of Shrimps
No. of Survivors
LD50 (μg/ml)
STD. DRUG
LD50 (μg/ml)
Remarks
100
10
1
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
24
30
30
1
--------
Etoposide
7.4625
No Cytotoxicity
100
10
1
Positive
Lethality
3c
3e
4a
4c
6a
6b
6c
6
1.6963
3.9484
-------
Etoposide
Etoposide
Etoposide
Etoposide
Etoposide
Etoposide
Etoposide
7.4625
7.4625
7.4625
7.4625
7.4625
7.4625
7.4625
18
3
100
10
1
Positive
Lethality
11
21
24
30
30
7
100
10
1
No Cytotoxicity
100
10
1
Positive
Lethality
17
19
0
7.562
100
10
1
Positive
Lethality
1.226
2
17
10
30
30
0
100
10
1
--------
2.0978
No Cytotoxicity
100
10
1
Postive
Lethality
9
18
3-(4-Chlorophenethyl)-1H-isochromen-1-one (3c)
3-(chlorophenylethenyl)]isocoumarins 3d-f as solids, which
were further purified by recrystallization from methanol.
°
Yield 74%; m.p 109 C; IR ꢀmax (KBr) 2940, 1726, 1080
cm-1; 1H NMR (acetone- d6) ꢁ 2.83 (2H, t, J = 7.8 Hz, H-1ꢂ),
3.00 (2H, t, J = 7.9 Hz, H-2ꢂ), 6.40 (1H, s, H-4), 7.2-7.3 (4H,
m, H-2ꢃ, 3ꢃ, 5ꢃ, 6ꢃ), 7.44 (1H, d, J = 7.8 Hz, H-7), 7.5 (1H,
dd, J = 7.6 Hz, H-5), 7.72 (1H, ddd, J = 7.5, 1.2 Hz, H-6),
8.12 (1H, dd, J = 7.8, 1.2 Hz, H-8) ppm; EIMS (70ev): m/z
(%) 284 (M+, 11.4), 286 (M+ +2, 3.56), 159 (3.4), 125 (100),
103 (11.8); HRMS: m/z Found; 284.0601 (Cl35) (Calcd for
C17H13O2Cl35; 284.0604) & Found; 286.0571 (Cl37) (Calcd
for C17H13O2Cl37; 286.0575).
3-[-2-(2-Chlorophenyl)ethenyl]-1H-isochromen-1-one (3d)
Yield 73%; m.p 162 °C; IR ꢀmax (KBr) 3030, 1721, 1611,
1
1059 cm-1; H NMR (acetone-d6) ꢁ 6.48 (1H, s, H -4), 6.67
(1H, d, J = 15.9 Hz, H-2ꢂ), 7.39 (1H, d, J = 15.4 Hz, H-1ꢂ),
7.19-7.27 (2H, m, H-3ꢃ, 5ꢃ), 7.37-7.42 (2H, m, H-4ꢃ, 6ꢃ),
7.46 (1H, dd, J = 7.5 Hz, H-7), 7.59 (1H, dd, J = 5.9, 1.6 Hz,
H-5), 7.67 (1H, ddd, J = 7.0, 1.2 Hz, H-6), 8.27 (1H, dd, J =
7.8, 0.9 Hz, H-8) ppm; EIMS (70ev): m/z (%) 282 (M+,
29.4), 247 (21.2), 191 (61.4), 165 (6.6), 89 (100); HRMS:
m/z Found; 282.0442 (Cl35) (Calcd for C17H11O2Cl35;
282.0448)
&
Found; 284.0420 (Cl37) (Calcd for
General Procedure for the Synthesis of 3-(Chlorophenyl-
ethenyl)isocoumarins (3d-f)
C17H11O2Cl37; 284.0418).
3-[-2-(3-Chlorophenyl)ethenyl]-1H-isochromen-1-one (3e)
Yield 70%; m.p 178 °C; IR ꢀmax (KBr) 3026, 1721, 1612,
Mixtures of homophthalic acid (2.27 g, 12 mmol) and
substituted chlorocinnamoyl chlorides 2d-f (10.4 g, 51
°
1
1061 cm-1; H NMR (acetone-d6) ꢁ 6.77 (1H, s, H-4), 7.05
mmol) were heated at 200 C under reflux for four h. The
mixtures were dissolved in ethyl acetate and aqueous
solution of sodium carbonate was added in order to remove
the unreacted homophthalic acid. The organic layers were
separated, concentrated, and chromatographed on silica gel
using petroleum ether (40-80 °C fractions) as eluent to afford
(1H, d, J = 16.0 Hz, H-2ꢂ), 7.29 (1H, d, J = 16.0 Hz, H-1ꢂ),
7.32 (1H, m, H-3ꢃ), 7.54 (1H, dd, J = 7.78, 7.9 Hz, H-4ꢃ),
7.52-7.55 (2H, m, H-7, 5ꢃ), 7.58 (1H, dd, J = 8.1, 3.2 Hz, H-
5), 7.67 (1H, bs, H-2ꢃ), 7.77 (1H, ddd, J = 7.0, 1.2 Hz, H-6),
8.17 (1H, d, J = 7.8, 0.9 Hz, H-8) ppm; EIMS (70ev): m/z

Synthesis and Antimicrobial Activities
Letters in Organic Chemistry, 2009, Vol. 6, No. 7
583
(%) 282 (M+, 84.8), 284 (M+ +2, 25.1), 219 (100), 191
(77.7), 165 (12.3); HRMS: m/z Found; 282.0431 (Cl35)
(Calcd for C17H11O2Cl35; 282.0448) & Found; 284.0424
(Cl37) (Calcd for C17H11O2Cl37; 284.0418).
7.5 (1H, dd, J = 7.1, 1.5 Hz, H-5), 7.7 (1H, ddd, J = 7.9, 1.3
Hz, H-6), 8.02 (1H, dd, J = 8.6, 0.9 Hz, H-8), 11.2 (COOH,
s, exchangeable with D2O) ppm; EIMS (70ev): m/z (%) 302
(M+, 4.4), 248 (3.8), 184 (6.7), 167 (11), 169 (3.1), 139
(22.8), 141 (7.8), 127 (26.9), 118 (100); HRMS: m/z Found;
302.0701 (Calcd for C17H15O3Cl; 302.0710).
3-[-2-(4-Chlorophenyl)ethenyl]-1H-isochromen-1-one (3f)
Yield 78%; m.p 210 °C; IR ꢀmax (KBr) 3063, 1721, 1616,
1
1068 cm-1; H NMR (acetone-d6) ꢁ 6.75 (1H, s, H-4), 6.98
General Procedure for the Synthesis of (dl)-3-
(Chlorophenylethyl)-3,4-dihydroisocoumarin (6a-c)
(1H, d, J = 16.0 Hz, H-2ꢂ), 7.30 (1H, d, J = 16.0 Hz, H-1ꢂ),
7.39 (2H, d, J = 9.2 Hz, H-2ꢃ, 6ꢃ), 7.54 (1H, d, J = 7.2 Hz,
H-7), 7.57 (1H, dd, J = 7.8, 3.4 Hz, H-5), 7.63 (1H, dd, J =
8.5 Hz, H-3ꢃ,5ꢃ), 7.76 (1H, ddd, J = 6.6, 1.5 Hz, H-6), 8.16
(1H, dd, J = 7.9, 0.9 Hz, H-8) ppm;. EIMS (70ev): m/z (%)
282 (M+, 95.5), 284 (M+ +2, 30.28), 254 (20), 219 (56), 191
(100), 165 (10.2), 89 (75); HRMS: m/z Found; 282.0430
(Cl35) (Calcd for C17H11O2Cl35; 282.0448) & Found;
284.0403 (Cl37) (Calcd for C17H11O2Cl37; 284.0418).
Keto-acids 4a-c (1.2 g, 4 mmol) were stirred over night
at room temperature with sodium borohydride (0.26 g, 6.87
mmol) in sodium hydroxide (1% in water, 40 ml). The
reaction mixtures were chilled and acidified with dilute
hydrochloric acid to yield hydroxy-acids 5a-c, which were
cyclodehydrated on refluxing with acetic anhydride for 1 h.
The reaction mixtures were diluted with chilled water (30
ml) and extracted with dichloromethane (100 ml). The
solvent was evaporated to afford (dl)-3- (chlorophenylethyl)-
3,4-dihydroisocoumarins 6a-c.
General Procedure for the Synthesis of 2-[4ꢀ-
(Chlorophenyl)-2ꢀ-oxobutyl]benzoic acid (4a-c)
(dl)-3-(2-Chlorophenethyl)-3,4-dihydro-1H-isochromen-1-
one (6a)
Solutions of 3-(chlorophenylethyl)isocoumarins 3a-c (2.2
g, 8 mmol) in ethanol (29 ml) and potassium hydroxide (5%
in water, 34 ml ) were refluxed for 5 h. After cooling, the
reaction mixtures were evaporated under reduced pressure to
remove ethanol. Cold water (20 ml) was then added and the
reaction mixtures were acidified with dilute hydrochloric
acid and extracted with dichloromethane (2 ꢀ 25 ml). The
solvent was rotary evaporated to afford crude solids, which
were recrystallized from ethyl acetate to give keto-acids 4a-
c.
°
Yield 77%; m.p 89-90 C; IR ꢀmax (KBr) 2926, 1703,
1
1090 cm-1; H NMR (acetone-d6) ꢁ 2.59 (2H, t, J = 7.4, 8.0
Hz, H-2ꢂ), 2.9 (2H, m, H-1ꢂ), 3.00 (1H, m, H-4a), 3.3 (1H, m,
H-4b), 4.56 (1H, m, H-3), 7.15-7.24 (2H, m, H-4ꢃ, 6ꢃ), 7.3-
7.37 (2H, m, H-3ꢃ, 5ꢃ), 7.56 (1H, dd, J = 8.1 Hz, H-7), 7.6
(1H, d, J = 7.3 Hz, H-5), 7.75 (1H, ddd, J = 7.5, 1.2 Hz, H-
6), 7.99 (1H, dd, J = 7.6, 0.9 Hz, H-8) ppm; EIMS (70ev):
m/z (%) 286 (M+, 78.7), 288 (M+ +2,28.8), 251 (12.6), 233
(11.6), 174 (2.4), 167 (7.4), 151 (100), 139 (8.7), 125 (57),
103 (15.6); HRMS: m/z Found; 286.0770 (Cl35) (Calcd for
C17H13O2Cl35; 286.0761) & Found; 288.0723 (Cl 37) (Calcd
for C17H13O2Cl37; 288.0731).
2-[4-(2-Chlorophenyl)-2-oxobutyl]benzoic acid (4a)
°
Yield 81%; m.p. 80 C; IR ꢀmax (KBr) 3300, 2923, 1700,
1
1082 cm-1; H NMR (acetone-d6) ꢁ 2.60 (2H, t, J = 7.4, 8.0
Hz, H-3), 3.02 (2H, t, J = 8.0 Hz, H-4), 3.30 (2H, s, CH2),
7.16-7.26 (2H, m, H-4ꢃ, 6ꢃ), 7.32-7.37 (2H, m, H-3ꢃ, 5ꢃ),
7.48 (1H, dd, J = 8.0 Hz, H-7), 7.51 (1H, dd, J = 7.8 Hz, H-
5), 7.82 (1H, ddd, J = 8.0, 1.2 Hz, H-6), 8.03 (1H, dd, J =
8.8, 1.2 Hz, H-8), 11.2 (1H, s, exchangeable with D2O) ppm;
EIMS (70ev): m/z (%) 284 (M+ -H2O, 10.7), 138 (13.8), 125
(100), 103 (40.5); HRMS: m/z Found; 284.0715 (M+-H2O)
(Calcd for C17H13O2Cl; 284.0710).
(dl)-3-(3-Chlorophenethyl)-3,4-dihydro-1H-isochromen-1-
one (6b)
°
Yield 71%; m.p 71-73 C; IR ꢀmax (KBr) 2927, 1717,
1
1080 cm-1; H NMR (acetone-d6) ꢁ 2.6 (2H, m, H-1ꢂ), 2.86
(2H, J = 7.5 Hz, H-2ꢂ), 3.0 (1H, m, H-4a), 3.3 (1H, m, H-4b),
4.54 (1H, m, H-3), 7.16-7.20 (1H, m, H-4ꢃ, 5ꢃ), 7.23-7.28
(1H, m, H-2ꢃ, 6ꢃ), 7.57 (1H, dd, J = 8.7 Hz, H-7) 7.60
(1H,dd, J = 7.5, 1.4 Hz, H-5), 7.62 (1H, ddd, J = 7.6, 1.2 Hz,
H-6), 8.00 (1H, dd, J = 7.7, 0.9 Hz, H-8) ppm; EIMS (70ev):
m/z (%) 286 (M+, 100), 288 (M+ +2, 36.5), 139 (9.4), 125
(12.5), 119 (45.9), 103 (12.4); HRMS: m/z Found; 286.0770
(Cl35) (Calcd for C17H13O2Cl35; 286.0761) & Found;
288.0741 (Cl 37) (Calcd for C17H13O2Cl37; 288.0731).
2-[4-(3-Chlorophenyl)-2-oxobutyl]benzoic acid (4b)
°
Yield 85%; m.p. 58 C; IR ꢀmax (KBr) 3300, 2920, 1699,
1
1080 cm-1; H NMR (acetone-d6) ꢁ 2.58 (2H, t, J = 7.5 Hz.,
H-3’), 2.88 (2H, t, J = 7.6 Hz, H-4’), 3.25 (2H, s, CH2), 7.16
(1H, dd, J = 7.7 Hz, H-5ꢃ), 7.23 (IH, s, H-2ꢃ), 7.26 (1H, d, J
= 7.1 Hz, H-4ꢃ), 7.28 (1H, d, J = 7.5 Hz, H-6ꢃ), 7.35 (1H,
dd, J = 8.0 Hz, H-7), 7.5 (1H, dd, J = 7.1, 1.5 Hz, H-5), 7.7
(1H, ddd, J = 7.9, 1.3 Hz, H-6), 8.01 (1H, dd, J = 8.9, 1.1
Hz, H-8), 11.2 (COOH, s, exchangeable with D2O) ppm;
EIMS (70ev): m/z (%) 284 (M+ -H2O, 32.4), 286 (15.8), 184
(95), 167 (42), 139 (74.9), 125 (100); HRMS: m/z Found;
284.0711 (M+-H2O) (Calcd for C17H13O2Cl; 284.0710).
(dl)-3-(4-Chlorophenethyl)-3,4-dihydro-1H-isochromen-1-
one (6c)
°
Yield 66%; m.p 95-97 C; IR ꢀmax (KBr) 2930, 1703,
1
1089 cm-1; H NMR (acetone-d6) ꢁ 2.58 (2H, m, H-1ꢂ), 2.88
(2H, t, J = 7.7 Hz, H-2ꢂ), 3.0 (1H, m, H-4a), 3.30 (1H, m, H-
4b), 4.54 (1H, m, H-3), 7.20-7.24 (1H, m, H-4ꢃ, 6ꢃ), 7.27-
7.31 (1H, m, H-3ꢃ, 5ꢃ), 7.58 (1H, J = 7.8 Hz, H-7), 7.6 (1H,
dd, J = 7.6 Hz, H-5), 7.7 (1H, ddd, J = 7.5,1.2 Hz, H-6), 7.99
(1H, dd, J = 7.6, 0.9Hz, H-8) ppm; EIMS (70ev): m/z (%)
286 (M+, 36.3), 288 (M+ +2, 12.5), 167 (33.9), 125 (100),
103 (12.9); HRMS: m/z Found; 286.0761 (Cl35) (Calcd for
2-[4-(4-Chlorophenyl)-2-oxobutyl]benzoic acid (4c)
Yield 81%; m.p 102 °C; IR ꢀmax (KBr) 3300, 2930, 1695,
1
1092 cm-1; H NMR (acetone-d6) ꢁ 2.58 (2H, t, J = 7.6 Hz,
H-3’), 2.88 (2H, t, J = 7.5 Hz, H-4’), 3.30 (2H, s, CH2), 7.2-
7.3 (4H, m, H-2ꢃ, 3ꢃ, 5ꢃ, 6ꢃ), 7.35 (1H, dd, J = 8.0 Hz, H-7),

584 Letters in Organic Chemistry, 2009, Vol. 6, No. 7
Khan et al.
C17H13O2Cl35; 286.0761) & Found; 288.0733 (Cl37) (Calcd
for C17H13O2Cl37; 288.0731).
2 ml of methanol only. The solvent was allowed to evaporate
overnight. After two days, when Shrimp larvae were ready 1
ml of seawater and 10 shrimp were added to each vial (30
shrimps/dilution) and the volume was adjusted with seawater
to 5 ml per vial. After 24 h, the number of survivors was
counted and data was analyzed by Finney computer program
to determine LD50 [21-23] Table 3.
Biological Studies
Antifungal Studies
Isocoumarins,
dihydroisocoumarins,
and
related
compounds were tested by agar tube dilution method [19] for
their in vitro fungicidal bioassay. Results were reported as
linear growth inhibition (LGI) against some human
pathogens (e.g Trichophyton longifusus, Candida albicans,
Aspergillus flavus, Microsporum canis, Fusarium solani, and
Candida glaberata). Linear Growth Inhibition results of
compounds are given in Table 1.
ACKNOWLEDGEMENT
The authors at the Department of Chemistry, Quaid-i-
Azam University, Islamabad, Pakistan, highly acknowledged
the financial grant from the Higher Education Commission
and URF Project, Quaid-i-Azam University, Islamabad,
Pakistan.
Antibacterial Studies
Antibacterial activities of all compounds were
determined in vitro by agar well diffusion method [20, 21].
The well were duged in the media with the help of a sterile
metallic borer with centers at least 24 mm a part with two to
eight hours old bacterial in culums contaminating
approximately 104-106 colony forming units (CFU)/ml
spread on the surface of nutrient agar with the help of a
sterile cotton swab rotating firmly against the upper inside
well of the lube to express excess fluid. Entire agar surface
of the plate was streaked with the swab three times turning
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