A facile synthesis of new 3,3-disubstituted phthalides of pharmacological interest

Article abstract of DOI:10.1007/s00044-012-0049-3

A series of new phthalides of pharmacological interest were synthesized by a protocol involving condensation of two γ-keto acids, 2-(4-isopropylbenzoyl)benzoic acid (1a) and 2-(4-isopropyl-3-nitrobenzoyl) benzoic acid (1b) with phenolic compounds in the presence of catalytic quantity of concentrated sulphuric acid. The method is simple, efficient, economical and environmentally benign as the reaction is carried out under solvent free condition attempting a green approach. Structural characterization of these newly synthesized compounds was accomplished by IR, UV, ¹H NMR, ¹³C NMR, Mass spectral data, elemental analysis and chemical reactions. Some of the synthesized phthalides were found to exhibit antifungal and antibacterial activity against various human pathogenic bacterial and fungal strains.

Full text of DOI:10.1007/s00044-012-0049-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-012-0049-3
ORIGINAL RESEARCH
A facile synthesis of new 3,3-disubstituted phthalides
of pharmacological interest
•
Tanu Chamoli M. S. M. Rawat
Received: 11 December 2011 / Accepted: 27 March 2012
Ó Springer Science+Business Media, LLC 2012
Abstract A series of new phthalides of pharmacological
interest were synthesized by a protocol involving conden-
sation of two c-keto acids, 2-(4-isopropylbenzoyl)benzoic
acid (1a) and 2-(4-isopropyl-3-nitrobenzoyl)benzoic acid
(1b) with phenolic compounds in the presence of catalytic
quantity of concentrated sulphuric acid. The method is
simple, efficient, economical and environmentally benign
as the reaction is carried out under solvent free condition
attempting a green approach. Structural characterization of
these newly synthesized compounds was accomplished by
IR, UV, 1H NMR, 13C NMR, Mass spectral data, elemental
analysis and chemical reactions. Some of the synthesized
phthalides were found to exhibit antifungal and antibacte-
rial activity against various human pathogenic bacterial and
fungal strains.
ischemia (Lin et al., 2005). The rhizome of Ligusticum
wallichii (family-Umbelliferae) has been used by Chinese
for several thousand years to relieve headache, abdominal
pain, cardiovascular and gynaecological diseases (Ko et al.,
1983). The butylidenephthalide, the most potent antispas-
modic agent isolated from this crude drug was prepared by
a synthetic method and found to be useful as a coronary
dilator and antihypertensive drug (Ko et al., 1994). Chiral
3-substituted phthalides are found to possess significant
pharmacological properties and are also used as versatile
building blocks for large number of medicinally important
compounds (Knepper et al., 2004; Witulski et al., 2002).
Natural products like 3-butylphthalide have been success-
fully synthesized due to their potent biological activities
(Zhang et al., 2010). Flexible racemic synthesis of
phthalide-containing antibiotics that inhibit Helicobacter
pylori have been carried out in a convergent fashion by
Wittig coupling (Brimble et al., 2005). Antimicrobial
activity of celery (Apium graveolens) leaves and roots is
also due to the presence of 3-substituted phthalides in them
(Sipailiene et al., 2005).
Keywords Phthalides ꢀ Phenolic compounds ꢀ
2-(4-Isopropylbenzoyl)benzoic acid ꢀ
2-(4-Isopropyl-3-nitrobenzoyl)benzoic acid ꢀ
Antibacterial and antifungal activities
During recent years, there has been a remarkable
attraction towards the development of convenient and
efficient methodologies for the synthesis of phthalides
(Zhang et al., 2009; Karnik and Kamath, 2008; Lin and
Sun, 2008; Zhou and Jiang, 2007; Patil and Karnik, 2007;
Chang et al., 2007; Pedrosa et al., 2006; Huang et al.,
2006; Kosoka et al., 2005; Tanaka et al., 2004; Witulski
and Zimmerman, 2002; Lei et al., 2002; Everaere et al.,
2001). The above mentioned literature reports related with
the wide spectrum biological activities of phthalides
encouraged us to undertake the synthesis of some new
3-substituted phthalides which are expected to have a
number of pharmacological applications. In these com-
pounds carbon-3 of the phthalide structure is attached with
Introduction
Phthalides constitute an important group of biologically
active compounds which are associated with a wide range
of pharmacological activities including action on central
nervous system, anti-angina, anti-platelet aggregation, anti-
smooth muscle proliferation, anti-thrombosis, cardiac
function modulation and protection against cerebral
T. Chamoli ꢀ M. S. M. Rawat (&)
Department of Chemistry, H.N.B. Garhwal University,
Srinagar (Garhwal) 246174, Uttarakhand, India
e-mail: msmrawat@gmail.com
123

Med Chem Res
cumene and phenolic structural moieties. Cumene contain-
ing essential oils have been found to exhibit antibacterial
activity, and hence, it is used in bioremediation studies.
Bacteria used in the bioremediation of trichloroethylene
exhibited an increased capability to degrade trichloroethyl-
ene in the presence of cumene (Suttinun et al., 2009).
Phenols and their derivatives form an important group of
compounds possessing biological activities. Various plant
flavonoids are reported to have antitumour, antioxidant and
antimicrobial activities due to the presence of a phenolic
moiety in their structure (Seeram et al., 2006). Pathogen-
esis of many chronic diseases, such as cancer, cardiovas-
cular and neurodegenerative diseases caused by reactive
oxidative species has attracted much attention and require
novel antioxidants to combat the reactive oxidative species
(Shahidi, 1997; Thomas, 1997). Some antioxidant phar-
macophores have been identified to facilitate the discovery
of antioxidants, among which catechol has been given the
highest attention (Zhang, 2005, 2000; Zhang et al., 2003a,
b; Foti et al., 2002; Hussain et al., 2003; Chichirau et al.,
2005; Flueraru et al., 2005). Catechol and pyrogallol are
reported as allelochemicals belonging to the group of
phenolic compounds synthesized in plants possessing
antimicrobial activities (Kocac¸alıs¸kan et al., 2006). Res-
orcinol and phenol derivatives from plant sources have
been found to have anticancer and antibacterial activities
(Barbini et al., 2006; Pormord et al., 2006; Mekawey et al.,
2009). Both synthetic and natural phloroglucinol com-
pounds exhibit biological properties such as antispasmodic,
antiviral, antiprotozoal and antimicrobial activities (Singh
et al., 2009).
The molecular manipulation of promising lead compounds
continues to be a widely used approach for the development of
new or betterdrugs. Thisapproachaimsatcombinationoftwo
or more different pharmacophoric units or groups into a single
entity having more potent or entirely new therapeutic prop-
erties. Thus, it is anticipated that addition of hydroxyphenyl
and isopropylbenzene moieties to the phthalide structure, will
enhance the biological activity of these compounds. The
synthesized compounds may be regarded as ‘phenol–phthal-
ide–cumeneconjugates’.Afewofthesynthesizedcompounds
were evaluated for their antibacterial and antifungal activities.
Chemistry
The synthesis of 3,3-disubstituted phthalides (4a–9a and
4b–9b) was accomplished by condensing two c-keto acids,
2-(4-isopropylbenzoyl)benzoic acid (1a) and 2-(4-iso-
propyl-3-nitrobenzoyl)benzoic acid (1b), with various
phenols (phenol, resorcinol, catechol, quinol, phloroglu-
cinol and pyrogallol) in the presence of catalytic quantity
of concentrated sulphuric acid under solvent free reaction
condition attempting to follow a green approach. The
c-keto acids 1a and 1b reacted with phenols (3a–f) through
their lactol tautomeric form 2a and 2b, respectively, to give
the desired phthalides (4a–9a and 4b–9b) as depicted in
Scheme 1. Their physical and analytical data are presented
in Table 1. The occurrence of keto-lactol tautomerism in
c-keto acids and c-formyl acids is thoroughly investigated,
and it is well known that in many chemical reactions these
acids participated through their cyclic lactol form (Valter
and Flitsch, 1985; Zhang et al., 2003a, b). The phenols
were taken in slight excess of molecular proportion than
the c-keto acids, and with excess phenols, the whole acid
From the foregoing account, it is amply evident that
phthalides, phenolic compounds and cumene or isopropyl-
benzene are bestowed with excellent medicinal properties.
R2
R*
R1
H
R*
R2
R3
R*
R1
O
R3
OH
R4
R5
3
R4
OH
R5
O
O
O
O
O
1
2
4 -9
3d, 7 : R2=R3=R5=H; R1=R4=OH
3e, 8 : R2=R4=H; R1=R3=R5=OH
3f, 9 : R4=R5=H; R1=R2=R3=OH
3a, 4 : R1=R2=R4=R5=H; R3=OH
3b, 5 : R2=R4=R5=H; R1=R3=OH
3c, 6 : R1=R4=R5=H; R2=R3=OH
1a, 2a, 4a-9a (R* = H) and 1b, 2b, 4b-9b (R* = NO2)
Scheme 1 Synthesis of phthalides 4–9
123

Med Chem Res
Table 1 Physical and analytical data of phthalides 4a–9a and 4b–9b
Phthalide
Condensation
Appearance
(microcrystalline) (°C)
m.p.
Yield Mol.
(%) Formula
Analysis (%)
Found (Calcd.)
C
Temp.
(°C)
Duration
(h)
H
N
4a 3-(p-Hydroxyphenyl)-3-(4-
isopropylphenyl)phthalide
130–140 5
110–120 3
Light brown
140–142 89
200–202 56
C23H20O3 80.41 (80.21) 5.78 (5.85)
–
4b 3-(p-Hydroxyphenyl)-3-(4-
isopropyl-3-
nitrophenyl)phthalide
Pinkish brown
C
23H19NO5 70.99 (70.94) 4.96 (4.92) 3.68
(3.60)
5a 3-(2,4-Dihydroxyphenyl)-3-(4- 110–120 0.5
isopropylphenyl)phthalide
Pinkish brown
Purple
162–164 98
180–182 74
C23H20O4 76.41 (76.65) 5.47 (5.59)
–
5b 3-(2,4-Dihydroxyphenyl)-3-
(4-isopropyl-3-
nitrophenyl)phthalide
125–135 0.5
C
23H19NO6 68.21 (68.14) 4.79 (4.72) 3.57
(3.46)
6a 3-(3,4-Dihydroxyphenyl)-3-(4- 105–115 1.5
isopropylphenyl)phthalide
Grey
135–137 76
280–282 50
C
C
23H20O4 76.50 (76.65) 5.38 (5.59)
–
6b 3-(3,4-Dihydroxyphenyl)-3-(4- 120–130 1.5
isopropyl-3-
nitrophenyl)phthalide
Dark brown
23H19NO6 68.22 (68.14) 4.81 (4.72) 3.55
(3.46)
7a 3-(2,5-Dihydroxyphenyl)-3-(4- 140–150 1.5
isopropylphenyl)phthalide
Light brown
Brown
200–202 65
170–172 50
C23H20O4 76.74 (76.65) 5.61 (5.59)
–
7b 3-(2,5-Dihydroxyphenyl)-3-
(4-isopropyl-3-
nitrophenyl)phthalide
130–140 1.5
C
23H19NO6 68.20 (68.14) 4.79 (4.72) 3.57
(3.46)
8a 3-(2,4,6-Trihydroxyphenyl)-3- 170–180 1
(4-isopropylphenyl)phthalide
Brownish orange 230–232 98
Brown [300 64
C23H20O5 73.27 (73.39) 5.28 (5.36)
–
8b 3-(2,4,6-Trihydroxyphenyl)-3- 180–190 0.5
(4-isopropyl-3-
nitrophenyl)phthalide
C
23H19NO7 65.70 (65.55) 4.60 (4.54) 3.46
(3.32)
9a 3-(2,3,4-Trihydroxyphenyl)-3- 135–145 0.5
(4-isopropylphenyl)phthalide
Brownish green 185–187 86
Green [300 56
C
C
23H20O5 73.60 (73.39) 5.36 (5.36)
–
9b 3-(2,3,4-Trihydroxyphenyl)-3- 120–130 0.5
(4-isopropyl-3-
nitrophenyl)phthalide
23H19NO7 65.65 (65.55) 4.65 (4.54) 3.49
(3.32)
taken reacted as lactol. The homogeneity of the synthesized
products was ascertained by TLC. Their structures were
nitrobenzoyl)benzoic acid (1b) reacted with phenols
(3a–f) through their cyclic lactol tautomeric forms 2a and
2b, respectively, as shown in Scheme 1. The phthalide 5a,
b on acetylation and bromination afforded diacetyl (10a, b)
and dibromo (11a, b) derivatives, respectively, as given in
Scheme 2. All the synthesized compounds (4–11) are the
unsymmetrically substituted phthalides in which carbon-3
of phthalide structure is attached to two different phenyl
nuclei, one being phenolic bearing one, two or three
hydroxyl groups, and the other non-phenolic having a
isopropyl group with or without nitro group. During the
present study, in some of the synthesized phthalides, a nitro
group has also been incorporated in the cumene moiety
because in many molecules nitro group has been found to
act as a pharmacophore. Chloramphenicol is a fascinating
example of a naturally occurring medicinally useful nitro
compounds. The antiproliferative effect of some phthalide
derivatives possessing nitro group have shown cytotoxicity
1
elucidated by elemental analysis, IR, H NMR, 13C NMR,
Mass spectral data and chemical reactions such as acety-
lation, bromination and caustic potash fusion. The repre-
sentative phthalides 5a and 5b when subjected to
acetylation and bromination afforded their corresponding
diacetyl (10a and 10b) and dibromo (11a and 11b) deriv-
atives, respectively, while on fusion with caustic potash
each of them was degraded into the starting c-keto acid 1a
or 1b and resorcinol as shown in Scheme 2.
Results and discussion
The synthesis of phthalides (4a–9a and 4b–9b) involves a
condensation reaction in which c-keto acids, 2-(4-isopro-
pylbenzoyl)benzoic acid (1a) and 2-(4-isopropyl-3-
123

Med Chem Res
Br
R*
R*
R*
HO
OH
Br
HO
OH
AcO
OAc
Acetylation
Bromination
O
O
O
O
O
5a,b
O
11a,b
10a,b
KOH Fusion
R*
O
HO
OH
OH
+
O
1a,b
1a, 5a, 10a, 11a (R*= H) and 1b, 5b, 10b, 11b (R*= NO2)
Scheme 2 Acetylation, bromination and KOH fusion of phthalides 5a, b
against human tumour cell lines (Kohn et al., 2006). High
physiological activity in the compounds containing nitro
group (nitrophenols, nitrobenzenes and nitrofurans) is well
known.
basis of their IC50 and MIC values which were determined
by standard procedures. Results of their antibacterial and
antifungal activities are presented in Tables 2 and 3.
The physical and analytical data of the synthesized
phthalides are given in Table 1. The structures of all the
phthalides were established by their analytical and spectral
data (IR, UV, 1H NMR, 13C NMR, Mass). IR spectra of the
synthesized phthalides 4a–9a, 4b–9b and 11a, b exhibited
a strong and broad band in the region between 3,250 and
3,422 cm-1 due to OH stretching vibrations. The broad
Taking into account the various useful pharmacological
properties reported for phenols, cumene and phthalides, the
synthesized compounds are expected to be potentially
useful therapeutic agents. In order to detect the possibility
of antimicrobial activity in these compounds, a few of them
were evaluated for in vitro antimicrobial activity on the
Table 2 Antifungal and antibacterial activity of some of the synthesized phthalides (IC50 lg mL-1
)
Compounds
IC50 (lg mL-1
)
C.
albicans
C.
glabrata
C.
krusei
A.
fumigatus
C.
neoformans
S.
aureus
MRS E. coli
P.
aeruginosa
M.
intracellulare
6a
6b
9a
9b
172.41
[200
65.02
98.94
0.20
45.81
76.28
17.5
15.46
[200
53.23
137.3
[200
[200
[200
0.32
4.96
26.36
18.45
42.3
–
6.81 [200
60.97
[200
116.4
162.8
–
[200
[200
[200
[200
–
[200
27.79 [200
25.48 [200
88.67 [200
18.8
169.8
21.32
0.28
45.7 [200
Amphotericin
B
0.56
0.67
–
–
Ciprofloxacin
–
–
–
–
–
0.11
0.10
0.005
0.11
0.32
123

Med Chem Res
Table 3 Antifungal and antibacterial activity of selected of the synthesized novel phthalides (MIC lg mL-1
)
Compounds
MIC (lg mL-1
)
C.
albicans
C.
glabrata
C.
krusei
A.
fumigatus
C.
neoformans
S.
aureus
MRS E. coli
P.
aeruginosa
M.
intracellulare
6a
6b
9a
9b
[200
[100
[200
[200
0.52
200
[100
[200
83.33
0.63
[200
[100
[200
[200
[200
[100
[200
[200
1.25
[200
[100
[200
[200
0.63
18.75
50.00
25.00
100
12.50 [200
50.00 [200
25.00 [200
133.33 [200
[200
[200
[200
[200
–
[200
[200
[200
[200
–
Amphotericin
B
1.25
–
–
–
Ciprofloxacin
–
–
–
–
–
0.33
0.42
0.013
0.83
0.50
envelope and low frequency of this band indicated that OH
groups are either involved in strong hydrogen bonding or
tautomeric shifts. This broad band between 3,250 and
3,422 cm-1 was found to be absent in 10a, b, instead these
compounds displayed a strong band at 1,755 or 1,760 cm-1
assignable to carbonyl stretching in a phenolic acetate
moiety. All the phthalides, 4–11 showed a sharp and strong
band near 1,740–1,760 cm-1 which could be attributed to
the lactonic carbonyl group present in all the structures.
The stretching vibrations of C–O–C of the lactonic struc-
tures gave a peak at 1,000–1,020 cm-1 and C–O bond
stretching of the phenolic group gave absorption peaks near
1,108–1,120 and 1,250–1,260 cm-1. The presence of two
bands at 690–700 cm-1 and 750–760 cm-1 in all the
compounds is in accordance with the o-disubstituted
phthalide ring of their structures. The nitro group attached
to phenyl group in the structures of 4b–11b gave three
IR absorptions near 1,530–1,550; 1,330–1,350 and
840-850 cm-1 that can be assigned to asymmetric N–O
stretching, symmetric N–O stretching and C–N stretching
vibrations, respectively. The UV spectra (in methanol) of
all the compounds revealed the same pattern of absorption
in Fig. 1. Spectral data and their assignments are given in
experimental section.
The representative compounds 5a and 5b were subjected
to mass spectral analysis. The compound 5a gave molec-
ular ion peak at m/z 359 (M-H)- together with a base peak
at m/z 317 and other peaks at m/z 342, 299, 273 and 239.
The compound 5b gave a peak at m/z 404 (M-H)- and a
base peak at m/z 91 with other peaks at m/z 297, 208, 117.
Mass spectrum of the compound 5a is shown in Fig. 2
while the proposed fragmentation pattern is depicted in
Scheme 3.
Experimental
All the melting points were determined in open capillary
tubes and are uncorrected. UV spectra were taken in
methanol on a Perkin-Elmer Lambda 15 UV/Vis spectro-
photometer. IR spectra were recorded in KBr on a Perkin-
1
Elmer FT-IR Spectrum RX-I spectrometer. The H NMR
spectra were recorded in DMSO-d6 solutions on a Bruker
400 NMR spectrometer using TMS as internal standard.
13C NMR spectra were measured at 100 MHz in DMSO-
d6. Mass spectra were recorded on a GC–MS spectrometer
(Agilent 6890N GC), equipped with 5973 inert mass
selective detector, Agilent Technologies, USA. The sam-
ples were dissolved in dichloromethane and they were
1
at 208–225, 245–290 and 285–340 nm. In the H NMR
spectra (400 MHz, DMSO-d6) of the synthesized phtha-
lides 4–11, the aromatic protons displayed a complex
multiplet in the region d 6.20–8.15. The hydroxyl protons
in 4a–9a, 4b–9b and 11a, b appeared as a singlet from d
5.70 to 9.50. The phthalides 10a, b did not give any signal
for hydroxyl protons, instead, these displayed a sharp sin-
glet at d 1.20 or 1.30 due to the presence of acetoxyl
protons. 1H NMR spectra of all the phthalides (4–11)
showed a doublet at d 1.10–1.30 and septet at d 2.50–3.20
which are given by methyl protons and methine proton,
respectively, present in the isopropyl group. 13C NMR
spectral data of all the compounds are also in good
agreement with the structures assigned to them. For the
purpose of assignment of 13C NMR spectral data, the
carbon atoms of the synthesized phthalides, 4–9
(Scheme 1) are numbered according to the pattern shown
R*
9
3'
6'
8
3"
10
4"
4'
5'
2"
1"
2'
1'
(OH)n
5"
6"
4
3
1
3a
7a
5
O2
6
7
O
[R*=H or NO2, n=1 or 2 or 3]
Fig. 1 Numbering of carbon atoms for 13C NMR spectral assignment
123

Med Chem Res
Fig. 2 Mass spectrum of 5a
OH
HO
O
O
C23H20O4
Mol. Wt. 360
injected into GC–MS at an injection volume of 1 lL. The
MS was operated at a voltage of 70 eV. Ionization was
effected by electro spray ionization technique. The chem-
icals and solvents were procured from E. Merck (Germany)
and Qualigens (India) and used as received without further
purification. The progress of the reactions and homogeneity
of the products was ascertained by TLC. For TLC, plates
coated with alumina–silica gel G (1:1) layers were run in
ethyl acetate–methanol–5 N ammonia (40:40:20).
concentrated hydrochloric acid. The precipitated 1a was
crystallized from the ethanol as yellowish white crystalline
solid (35 g, 86 %), m.p. 136–137 °C [Ref. (Underwood
and Walsh, 1935) m.p. 133–134 °C].
2-(4-Isopropyl-3-nitrobenzoyl)benzoic acid (1b)
2-(4-Isopropylbenzoyl)benzoic acid (26.8 g, 0. 1 mol) was
dissolved in concentrated nitric acid (d, 1.41, 100 ml), the
resulting solution was refluxed for 1 h and then cooled in
an ice bath. The separated solid was filtered and washed
with water and dried. Crystallization of the product from
ethanol gave 1b as yellowish-white crystals (18.23 g,
68.1 %); m.p. 160–162 °C. IR (KBr, m, cm-1): 3400, 2972,
2800, 2700, 1780 (weak), 1690, 1675, 1600, 1580, 1530,
1490, 1450, 1360, 850. Anal. calcd. for C17H15NO5: C,
65.17; H, 4.83; N, 4.47. Found C, 65.90; H, 4.32; N,
4.65 %.
The starting c-keto acid, 2-(4-isopropylbenzoyl)benzoic
acid (1a) was synthesized by Friedel–Crafts phthaloylation
of cumene by modifying the reported procedure (Under-
wood and Walsh, 1935), to get better yield, and to avoid
the formation of sticky and impure product. The acid 1a
was subjected to nitration to get 2-(4-isopropyl-3-
nitrobenzoyl)benzoic acid (1b).
2-(4-Isopropylbenzoyl)benzoic acid (1a)
A mixture of finely powdered phthalic anhydride (22.2 g,
0.15 mol), cumene (18 g, 0.15 mol) and carbon disulphide
(200 ml) was vigorously agitated for 1 h. Then anhydrous
aluminium chloride (40.2 g, 0.3 mol) was added gradually
with stirring and maintaining the reaction temperature
below 40 °C. The mixture was then gently refluxed on a
steam bath for 3 h and allowed to stand overnight. It was
decomposed with ice and concentrated hydrochloric acid,
and carbon disulphide was distilled off. The semi solid
mass left behind was cooled in an ice-bath and filtered. The
residue was thoroughly washed with cold water and
extracted with 5 % (w/v) warm aqueous solution of
anhydrous sodium carbonate. The extract was boiled with
animal charcoal, filtered, cooled and acidified with
General procedure for the synthesis of phthalides (4–9)
An intimate mixture of c- keto acid 1a or 1b (0.01 mol)
and phenolic compound 3a–f (0.015 mol) was heated in an
oil bath to afford a homogeneous molten mass. To this,
concentrated sulphuric acid (3–5 drops) was added cau-
tiously with stirring by a glass rod, and the heating was
continued for 0.5–5 h (monitored by TLC) to obtain a hard
and brittle mass on cooling. The condensed mass so
obtained was crushed and thoroughly washed with water to
remove the excess phenolic compounds. In case of syn-
thesis of 4a, b, the unreacted phenol was removed by steam
distillation. Then the product was extracted with 2 %
aqueous NaOH and the extract was filtered. Acidification
123

Med Chem Res
OH
HO
O
HO
O
O
5a
m/z 273
-e
- CO2
OH
HO
OH
HO
OH
HO
-H
- 43
O
O
O
(C3H7 )
O
O
O
M+ m/z 360
m/z 359
m/z 317
-H2O
-m/z 120
-m/z 121
-H2O
OH
OH
OH
- 43
(C3H7 )
O
O
O
O
O
m/z 299
O
m/z 342
m/z 239
Scheme 3 Possible mass fragmentation pattern for 5a
of the filtrate by gradual addition of dil. HCl precipitated
the phthalides (4–9). They were purified by repeated
crystallization with aqueous ethanol, dried in an oven at
100 °C and finally over phosphorus pentoxide in a vacuum
desiccator. Preparatory conditions and physical data of the
synthesized phthalides are presented in Table 1. The results
of their spectral studies are given below.
d, ppm): 170.2 (C-1), 156.8 (C-40), 155.5 (C-3a), 147.1
(C-400), 140.2 (C-100), 134.5 (C-5), 132.3 (C-10), 131.7
(C-200/C-600), 130.1 (C-20/C-60), 129.9 (C-7), 128.2 (C-4),
126.9 (C-7a), 126.1 (C-6), 125.6 (C-300/C-500), 115.4 (C-30/
C-50), 80.9 (C-3), 36.0 (C-8), 24.1 (C-9/C-10).
3-(p-Hydroxyphenyl)-3-(4-isopropyl-3-
nitrophenyl)phthalide (4b)
3-(p-Hydroxyphenyl)-3-(4-isopropylphenyl)phthalide
(4a)
IR (KBr, m, cm-1): 3409 (OH), 2920 (aliphatic C–H), 1750
(lactonic[C=O), 1600, 1590, 1500, 1450 (Ar C=C), 1550
(asym. N–O), 1350 (sym. N–O), 1255, 1108 (C–O), 1020
(C–O–C), 840 (C–N), 755, 690 (o-disubstituted phthalide
IR (KBr, m, cm-1): 3393 (OH), 2962 (aliphatic C–H), 1744
(lactonic[C=O), 1610, 1590, 1513, 1466 (Ar C=C), 1255,
1120 (C–O), 1018 (C–O–C), 760, 693 (o-disubstituted
phthalide ring); UV (methanol): kmax 210, 265, 310 nm; 1H
NMR (400 MHz, DMSO-d6, d, ppm): 1.1 (d, 6H, 29 CH3,
J = 6.5 Hz), 2.9 (sept, 1H, –CH\), 6.7 (s, 1H, OH),
6.8–8.0 (m, 12H, Ar–H); 13C NMR (100 MHz, DMSO-d6,
1
ring); UV (methanol): kmax 218, 275, 325 nm; H NMR
(400 MHz, DMSO-d6, d, ppm): 1.2 (d, 6H, 29 CH3,
J = 6.5 Hz), 2.7 (sept, 1H, –CH\), 6.7–8.0 (m, 11H, Ar–
H), 9.7 (s, 1H, OH); 13C NMR (100 MHz, DMSO-d6,
d, ppm): 169.1 (C-1), 158.5 (C-40), 152.9 (C-3a), 146.9
123

Med Chem Res
(C-3000), 142.1 (C-100), 141.5 (C-400), 134.9 (C-5), 132.0
(C-1 ), 130.5 (C-20/C-60), 130.0 (C-6), 129.3 (C-600), 128.2
(C-200), 127.6 (C-500), 127.1 (C-7a), 126.2 (C-7), 125.1 (C-4),
116.0 (C-30/C-50), 90.1 (C-3), 28.19 (C-8), 22.2 (C-9/C-10).
30), 145.1 (C-40), 142.0 (C-100), 138.0 (C-10), 133.9 (C-
5),130.9 (C-200/C-600), 129.7 (C-7), 129.0 (C-60), 128.0 (C-
4), 126.3 (C-7a), 125.9 (C-6), 125.6 (C-300/C-500), 118.2 (C-
50), 115.5 (C-20), 82.5 (C-3), 36.3 (C-8), 24.2 (C-9/C-10).
3-(2,4-Dihydroxyphenyl)-3-(4-
3-(3,4-Dihydroxyphenyl)-3-(4-isopropyl-3-
isopropylphenyl)phthalide (5a)
nitrophenyl)phthalide (6b)
IR (KBr, m, cm-1): 3368 (OH), 2962 (aliphatic C–H), 1740
(lactonic[C=O), 1610, 1590, 1508, 1465 (Ar C=C), 1255,
1113 (C–O), 1018 (C–O–C), 750, 694 (o-disubstituted
phthalide ring); UV (methanol): kmax 208, 285, 320 nm; 1H
NMR (400 MHz, DMSO-d6, d, ppm): 1.1 (d, 6H, 29 CH3,
J = 6.5 Hz), 2.9 (sept, 1H, –CH\), 6.1 and 6.2 (each s, 1H,
29 OH), 7.1–7.85 (m, 11H, Ar–H); 13C NMR (100 MHz,
DMSO-d6, d, ppm): 171.0 (C-1), 157.5 (C-40), 156.7 (C-20),
156.2 (C-3a), 148.2 (C-400), 141.1 (C-100), 135.1 (C-5),
131.0 (C-200/C-600), 130.1 (C-7), 128.2 (C-4), 128.0 (C-60),
126.9 (C-7a), 126.5 (C-6), 125.9 (C-300/C-500), 113.6 (C-10),
110.0 (C-50), 103.9 (C-30), 81.1 (C-3), 35.9 (C-8), 24.5 (C-
9/C-10); MS (70 eV) m/z (%): 359 (M-1, 10), 342 (2), 317
(100), 299 (6), 273 (5), 239 (5).
IR (KBr, m, cm-1): 3497 (OH), 2950 (aliphatic C–H), 1749
(lactonic[C=O), 1600, 1580, 1500, 1450 (Ar C=C), 1550
(asym. N–O), 1350 (sym. N–O), 1255, 1116 (C–O), 1020
(C–O–C), 850 (C–N), 753, 700 (o-disubstituted phthalide
1
ring); UV (methanol): kmax 225, 280, 330 nm; H NMR
(400 MHz, DMSO-d6, d, ppm): 1.2 (d, 6H, 29 CH3,
J = 6.5 Hz), 2.7 (sept, 1H, –CH\), 6.6–8.05 (m, 10H, Ar–
H), 8.85 and 9.0 (each s, 1H, 29 OH); 13C NMR
(100 MHz, DMSO-d6, d, ppm): 169.2 (C-1), 152.4 (C-3a),
148.2 (C-3), 147.0 (C-300), 145.8 (C-40), 142.3 (C-100),
141.2 (C-400), 137.5 (C-10), 134.2 (C-5), 130.2 (C-6), 129.5
(C-600), 129.0 (C-60), 128.1 (C-200), 127.8 (C-500), 127.1 (C-
7a), 126.6 (C-7), 125.2 (C-4), 117.5 (C-50), 115.1 (C-20),
90.2 (C-3), 28.5 (C-8), 22.4 (C-9/C-10).
3-(2,4-Dihydroxyphenyl)-3- (4-isopropyl-3-
3-(2,5-Dihydroxyphenyl)-3-(4-
nitrophenyl)phthalide (5b)
isopropylphenyl)phthalide (7a)
IR (KBr, m, cm-1): 3402 (OH), 2950 (aliphatic C–H), 1740
(lactonic[C=O), 1609, 1590, 1500, 1450 (Ar C=C), 1530
(asym. N–O), 1350 (sym. N–O), 1250, 1133 (C–O), 1020
(C–O–C), 850 (C–N), 757, 690 (o-disubstituted phthalide
IR (KBr, m, cm-1): 3404 (OH), 2961 (aliphatic C–H), 1750
(lactonic[C=O), 1604, 1580, 1500, 1466 (Ar C=C), 1253,
1117 (C–O), 1000 (C–O–C), 764, 690 (o-disubstituted
phthalide ring); UV (methanol): kmax 210, 250, 300 nm; 1H
NMR (400 MHz, DMSO-d6, d, ppm): 1.1 (d, 6H, 29 CH3,
J = 6.5 Hz), 2.9 (sept, 1H, –CH\), 6.5 and 6.6 (each s, 1H,
29 OH), 7.0–8.1 (m, 11H, Ar–H); 13C NMR (100 MHz,
DMSO-d6, d, ppm): 170.1 (C-1), 152.0 (C-3a), 151.7 (C-
50), 148.3 (C-20), 146.5 (C-400), 141.2 (C-100), 133.8 (C-5),
130.8 (C-200/C-600), 129.8 (C-7), 128.1 (C-4), 126.7 (C-7a),
126.1 (C-300/C-500), 125.7 (C-6), 122.5 (C-10), 118.2 (C-30),
116.0 (C-60), 114.3 (C-40), 82.6 (C-3), 36.0 (C-8), 24.0
(C-9/C-10).
1
ring); UV (methanol): kmax 215, 290, 330 nm; H NMR
(400 MHz, DMSO-d6, d, ppm): 1.2 (d, 6H, 29 CH3,
J = 6.5 Hz), 2.8 (sept, 1H, –CH\), 6.15 and 6.25 (each s,
1H, 29 OH), 6.7–8.0 (m, 10H, Ar–H); 13C NMR
(100 MHz, DMSO-d6, d, ppm): 169.2 (C-1), 158.0 (C-40),
156.8 (C-20), 152.1 (C-3a), 147.0 (C-300), 142.5 (C-100),
141.4 (C-400), 134.2 (C-5), 133.5 (C-60),130.2 (C-6), 129.5
(C-600), 128.2 (C-200), 127.8 (C-500), 127.0 (C-7a), 126.5(C-
7), 125.3 (C-4), 113.1 (C-10), 109.5 (C-50), 104.5 (C-30),
90.0 (C-3), 28.2 (C-8), 22.4 (C-9/C-10); MS (70 eV) m/z
(%): 404 (M-1, 9), 297 (9), 208 (14), 117 (66), 91 (100).
3-(2,5-Dihydroxyphenyl)-3- (4-isopropyl-3-
nitrophenyl)phthalide (7b)
3-(3,4-Dihydroxyphenyl)-3-(4-
isopropylphenyl)phthalide (6a)
IR (KBr, m, cm-1): 3384 (OH), 2950 (aliphatic C–H), 1749
(lactonic[C=O), 1600, 1580, 1500, 1450 (Ar C=C), 1550
(asym. N–O), 1350 (sym. N–O), 1250, 1113 (C–O), 1015
(C–O-C), 850 (C–N), 753, 700 (o-disubstituted phthalide
IR (KBr, m, cm-1): 3398 (OH), 2962 (aliphatic C–H), 1740
(lactonic[C=O), 1604, 1580, 1500, 1450 (Ar C=C), 1250,
1114 (C–O), 1020 (C–O-C), 745, 600 (o-disubstituted
phthalide ring); UV (methanol): kmax 215, 260, 295 nm; 1H
NMR (400 MHz, DMSO-d6, d, ppm): 1.3 (d, 6H, 29 CH3,
J = 6.5 Hz), 3.0 (sept, 1H, –CH\), 7.2–8.1 (m, 11H, Ar–
H), 9.1 (s, 2H, 29 OH); 13C NMR (100 MHz, DMSO-d6,
d, ppm): 170.5 (C-1), 151.3 (C-3a), 148.1 (C-400), 147.8 (C-
1
ring); UV (methanol): kmax 220, 270, 320 nm; H NMR
(400 MHz, DMSO-d6, d, ppm): 1.2 (d, 6H, 29 CH3,
J = 6.5 Hz), 2.7 (sept, 1H, –CH\), 6.5–8.0 (m, 10H, Ar–
H), 9.2 (s, 2H, 29 OH); 13C NMR (100 MHz, DMSO-d6,
d, ppm): 169.5 (C-1), 152.5 (C-3a), 150.0 (C-50), 148.2
(C-20), 147.1 (C-300), 142.7 (C-100), 141.2 (C-400), 135.0
123

Med Chem Res
(C-5), 130.2 (C-6), 129.6 (C-600), 128.1 (C-200), 127.6
(C-500), 127.1 (C-7a), 126.7 (C-7), 125.0 (C-4), 123.1
(C-10), 117.5 (C-30), 117.0 (C-60), 115.3 (C-40), 90.0 (C-3),
28.4 (C-8), 22.3 (C-9/C-10).
120.0 (C-60), 113.8 (C-10), 110.5 (C-50), 84.9 (C-3), 36.2
(C-8), 24.3 (C-9/C-10).
3-(2,3,4-Trihydroxyphenyl)-3-(4-isopropyl-3-
nitrophenyl)phthalide (9b)
3-(2,4,6-Trihydroxyphenyl)-3-
IR (KBr, m, cm-1): 3421 (OH), 2950 (aliphatic C–H), 1750
(lactonic[C=O), 1600, 1580, 1500, 1450 (Ar C=C), 1550
(asym. N–O), 1330 (sym. N–O), 1250, 1108 (C–O), 1020
(C–O–C), 850 (C–N), 752, 690 (o-disubstituted phthalide
(4-isopropylphenyl)phthalide (8a)
IR (KBr, m, cm-1): 3392 (OH), 2960 (aliphatic C–H), 1751
(lactonic[C=O), 1609, 1580, 1500, 1465 (Ar C=C), 1250,
1158 (C–O), 1020 (C–O–C), 760, 690 (o-disubstituted
phthalide ring); UV (methanol): kmax 220, 260, 325 nm; 1H
NMR (400 MHz, DMSO-d6, d, ppm): 1.0 (d, 6H, 29 CH3,
J = 6.5 Hz), 2.8 (sept, 1H, –CH\), 5.7 (broad s, 3H, 39
OH), 7.0–8.0 (m, 10H, Ar–H); 13C NMR (100 MHz,
DMSO-d6, d, ppm): 170.4 (C-1), 158.9 (C-40), 158.7 (C-20/
C-60), 152.1 (C-3a), 146.9 (C-400), 141.5 (C-100), 135.0
(C-5), 130.5 (C-200/C-600), 129.6 (C-7), 128.5 (C-4), 126.5
(C-7a), 126.3 (C-6), 126.0 (C-300/C-500), 110.5 (C-10), 96.1
(C-30/C-50), 82.5 (C-3), 36.1 (C-8), 24.3 (C-9/C-10).
1
ring); UV (methanol): kmax 215, 260, 310 nm; H NMR
(400 MHz, DMSO-d6, d, ppm): 1.2 (d, 6H, 29 CH3,
J = 6.5 Hz), 2.8 (sept, 1H, –CH\), 6.2-8.5 (m, 9H, Ar–H),
9.5 (broad s, 3H, 39 OH); 13C NMR (100 MHz, DMSO-
d6, d, ppm): 169.8 (C-1), 152.3 (C-3a), 150.4 (C-20), 150.2
(C-40), 146.8 (C-300), 143.0 (C-100), 141.3 (C-400), 140.1
(C-30), 134.5 (C-5), 134.0 (C-60), 131.7 (C-600), 130.0
(C-6), 128.2 (C-200), 127.6 (C-500), 127.0 (C-7a), 126.1
(C-7), 125.4 (C-4), 115.1 (C-10), 108.2 (C-50), 88.5 (C-3),
28.6 (C-8), 22.5 (C-9/C-10).
Acetylation of phthalide 5a and 5b: Synthesis of 3-(2,4-
diacetoxyphenyl)-3-(4-isopropylphenyl)phthalide (10a)
and 3-(2,4-diacetoxyphenyl)-3- (4-isopropyl-3-
nitrophenyl)phthalide (10b)
3-(2,4,6-Trihydroxyphenyl)-3-(4-isopropyl-3-
nitrophenyl)phthalide (8b)
IR (KBr, m, cm-1): 3397 (OH), 2950 (aliphatic C–H), 1718
(lactonic[C=O), 1610, 1500, 1450 (Ar C=C), 1550 (asym.
N–O), 1350 (sym. N–O), 1250, 1115 (C–O), 1020 (C–O–C),
840 (C–N), 754, 700 (o-disubstituted phthalide ring); UV
A mixture of the phthalide, 5a or 5b (1 g), acetic anhydride
(20 mL) and fused sodium acetate (3.0 g) was refluxed at
130–140 °C to get a diacetyl derivative 10a as a pale
yellow solid (0.8 g) and 10b as a light brown solid (0.9 g).
These were purified by recrystallization from acetone. The
analytical and spectral data of the acetyl derivatives are
given below.
10a: m.p. 152–153 °C; % yield: 65; IR (KBr, m, cm-1):
IR (KBr, m, cm-1): 2960 (aliphatic C–H), 1750, 1760
(lactone and acetoxyl[C=O), 1600, 1585, 1500, 1460 (Ar
C=C), 1250, 1115 (C–O), 1025 (C–O–C), 760, 700
(o-disubstituted phthalide ring); UV (methanol): kmax 210,
270, 285 nm; 1H NMR (400 MHz, DMSO-d6, d, ppm): 1.0
(d, 6H, 29 CH3, J = 6.5 Hz), 2.4 (s, 6H, acetoxyl), 2.9
(sept, 1H, –CH\), 6.9–8.1 (m, 11H, Ar–H); 13C NMR
(100 MHz, DMSO-d6, d, ppm): 170.3 (C-1), 169.2 (CO,
acetoxyl), 153.8 (C-3a), 150.6 (C-20), 150.1 (C-40), 146.2
(C-600), 140.8 (C-100), 135.2 (C-5), 131.2 (C-200/C-600), 130.5
(C-7), 128.3 (C-4), 128.1 (C-60), 126.8 (C-7a), 126.1 (C-300/
C-500), 125.9 (C-6), 123.5 (C-10), 119.0 (C-50), 115.8 (C-30),
84.3 (C-3), 36.2 (C-8), 24.4 (C-8/C-9), 20.8 (–CH3, acet-
oxyl); Anal. calcd. for C27H24O6: C.72.96; H, 5.44. Found:
C, 73.02; H, 5.58 %.
1
(methanol): kmax 210, 270, 340 nm; H NMR (400 MHz,
DMSO-d6, d, ppm): 1.1 (d, 6H, 29 CH3, J = 6.5 Hz), 3.1
(sept, 1H, –CH\), 5.50, 5.55, 5.60 (each s, 1H, 39 OH),
7.0–8.1(m, 9H, Ar–H); 13C NMR (100 MHz, DMSO-d6, d,
ppm): 169.5 (C-1), 158.9 (C-40), 158.2 (C-20/C-60), 152.1
(C-3a), 147.1 (C-300), 142.8 (C-100), 141.3 (C-400), 134.6 (C-
5), 130.0 (C-6), 129.2 (C-600), 128.3 (C-200), 127.9 (C-500),
127.2 (C-7a), 126.8 (C-7), 125.0 (C-4), 110.3 (C-10), 97.1
(C-30/C-50), 90.1 (C-3), 28.6 (C-8), 22.3 (C-9/C-10).
3-(2,3,4-Trihydroxyphenyl)-3-
(4-isopropylphenyl)phthalide (9a)
IR (KBr, m, cm-1): 3422 (OH), 2962 (aliphatic C–H), 1751
(lactonic[C=O), 1600, 1575, 1500, 1466 (Ar C=C), 1250,
1112 (C–O), 1020 (C–O–C), 764, 690 (o-disubstituted
phthalide ring); UV (methanol): kmax 210, 245, 290 nm; 1H
NMR (400 MHz, DMSO-d6, d, ppm): 1.0 (d, 6H, 29 CH3,
J = 6.5 Hz), 2.8 (sept, 1H, –CH\), 6.1, 6.2, 6.3 (each s,
1H, 39 OH), 6.8–8.1 (m, 10H, Ar–H); 13C NMR
(100 MHz, DMSO-d6, d, ppm): 170.0 (C-1), 153.2 (C-3a),
148.9 (C-40), 148.7 (C-20), 146.8 (C-400), 145.0 (C-30),
140.9 (C-100), 135.1 (C-5), 130.7 (C-200/C-600), 130.2 (C-7),
128.5 (C-4), 126.9 (C-7a), 126.3 (C-6), 126.2 (C-300/C-500),
10b: m.p. 120–122 °C; % yield: 75; IR (KBr, m, cm-1):
2960 (aliphatic C–H), 1750, 1755 (lactone and acetoxyl
[C=O), 1605, 1585, 1510, 1460 (Ar C=C), 1535 (asym.
N–O), 1350 (sym. N–O), 1260, 1115 (C–O), 1020 (C–O–C),
123

Med Chem Res
840 (C–N), 760, 700 (o-disubstituted phthalide ring); UV
1
170.0 (C-1), 153.2 (C-3a), 153.1 (C-20), 151.8 (C-40), 146.9
(C-500), 142.1 (C-100), 141.5 (C-400), 135.2 (C-5), 132.2
(C-600), 130.0 (C-6), 129.4 (C-200), 128.1 (C-600), 127.2
(C-300), 127.1 (C-7a), 126.4 (C-7), 125.7 (C-4), 118.0 (C-10),
110.5 (C-50), 109.2 (C-30), 86.0 (C-3), 28.6 (C-8), 22.3 (C-9/
C-10); Anal. calcd. for C23H17Br2NO6: C, 49.05; H, 3.04; Br,
28.3; N, 2.49. Found C, 49.11; H, 3.08; Br, 28.7; N, 2.52 %.
(methanol): kmax 210, 280, 310 nm; H NMR (400 MHz,
DMSO-d6, d, ppm): 1.0 (d, 6H, 29 CH3, J = 6.5 Hz), 2.2
(s, 6H, acetoxyl), 3.2 (sept, 1H, –CH\), 7.5–8.15 (m, 10H,
Ar–H); 13C NMR (100 MHz, DMSO-d6, d, ppm): 170.0
(C-1), 168.0 (CO, acetoxyl), 153.0 (C-3a), 150.1 (C-20),
149.5 (C-40), 146.8 (C-300), 142.1 (C-100), 141.5 (C-400),
134.6 (C-5), 129.8 (C-6), 129.4 (C-60), 129.2 (C-600), 128.1
(C-200), 127.8 (C-500), 127.1 (C-7a), 126.2 (C-7), 125.4
(C-4), 123.0 (C-10), 118.5 (C-50), 115.5 (C-30), 88.5 (C-3),
28.5 (C-8), 22.5 (C-9/C-10), 20.5 (–CH3, acetoxyl); Anal.
calcd. for C27H23NO8: C, 66.25; H, 4.74; N, 2.86 Found: C,
66.55; H, 4.76; N, 2.89 %.
Caustic potash fusion of 5a and 5b
The phthalide 5a or 5b (1 g) was mixed with a paste of
KOH pellets (10 g) in water and the mixture was strongly
heated for 1.5 h. The fused mass was cooled, dissolved in
water and filtered. The filtrate was acidified with dil. HCl,
when a solid (A) was obtained. It was filtered and the
filtrate was shaken with ether. Evaporation of the ether
produced another solid (B). The solid A was identified as
2-(4-isopropylbenzoyl)benzoic acid (1a) or 2-(4-isopropyl-
3-nitrobenzoyl)benzoic acid (1b), and the solid B was
identified as resorcinol by direct comparison (m.m.p.,
co-TLC and superimposable IR spectra) with their
authentic samples.
Bromination of phthalide 5a and 5b: Synthesis of 3-
(3,5-dibromo-2,4-dihydroxyphenyl)-3-(4-
isopropylphenyl)phthalide (11a) and 3-(3,5-dibromo-
2,4-dihydroxyphenyl)-3-(4-isopropyl-3-
nitrophenyl)phthalide (11b)
The phthalide 5a or 5b (1.0 g) was dissolved in ethanol
(10 mL) and to the resulting solution, an excess of bromine
(2 mL) was added gradually with constant shaking. The
contents were left overnight. The deposited solid mass was
thoroughly washed with water and crystallized from acetic
acid to get dibromo compounds 11a as a brick-red powder
(1.2 g) and 11b as a brownish orange powder (1.0 g). The
analytical and spectral data of the synthesized dibromo
derivatives are given below.
Biological activity
The synthesized phthalides (4a–11a and 4b–11b) are expec-
ted to exhibit a large array of biological properties. During the
present study we have evaluated a few phthalides for their
antibacterial and antifungal activities. The phthalides 3-(3,4-
dihydroxyphenyl)-3-(4-isopropylphenyl)phthalide (6a),
3-(3,4-dihydroxyphenyl)-3-(4-isopropyl-3-nitrophenyl)phthal-
ide (6b), 3-(2,3,4-trihydroxyphenyl)-3-(4-isopropylphenyl)
phthalide (9a) and 3-(2,3,4-trihydroxyphenyl)-3-(4-isopropyl-
3-nitrophenyl)phthalide (9b) were tested for their in vitro
antibacterial activity against Staphylococcus aureus ATCC
29213 (Sa), methicillin-resistant S. aureus ATCC 33591
(MRS), Escherichia coli ATCC 35218 (Ec), Pseudomonas
aeruginosa ATCC 27853 (Pa) and Mycobacterium intracellu-
lare ATCC 23068 (Mi). The in vitro antifungal activity of the
phthalides 6a, 6b, 9a and 9b was determined against Candida
albicans ATCC 90028 (Ca), Candida glabrata ATCC 90030
(Cg), Candida krusei ATCC 6258 (Ck), Cryptococcus neo-
formans ATCC 90113 (Cn) and Aspergillus fumigatus ATCC
204305 (Af). The compounds selected for the antimicrobial
screening contain catechol and pyrogallol structural units in
their structures. The rationale to select these compounds is the
fact that catechol and pyrogallol are allelo chemicals belonging
to phenolic compounds synthesized in plants (Kocac¸alıs¸kan
et al., 2006), and significant antibacterial activity is associated
withthem (Zhang, 2005,2000; Zhang et al., 2003a,b;Fotiet al.,
2002; Hussain et al., 2003; Chichirau et al., 2005; Flueraru
et al., 2005).
11a: m.p. 280–282 °C; % yield: 86; IR (KBr, m, cm-1):
3250 (OH), 2960 (aliphatic C–H), 1760 (lactonic [C=O),
1605, 1590, 1500, 1460 (Ar C=C), 1260, 1120 (C–O), 1010
(C–O–C), 755, 695 (o-disubstituted phthalide ring); UV
1
(methanol): kmax 215, 260, 335 nm; H NMR (400 MHz,
DMSO-d6, d, ppm): 1.1 (d, 6H, 29 CH3, J = 6.5 Hz), 3.0
(sept, 1H, –CH\), 6.9–7.9 (m, 12H, Ar–H), 9.5 (broad s,
2H, 29 OH); 13C NMR (100 MHz, DMSO-d6, d, ppm):
170.9 (C-1), 153.4 (C-3a), 153.0 (C-20), 152.1 (C-40), 146.3
(C-600), 141.6 (C-100), 135.6 (C-5), 131.4 (C-200/C-600), 131.0
(C-60), 130.0 (C-7), 128.1 (C-4), 126.7 (C-300/C-500), 126.3
(C-7a), 126.2 (C-6), 117.5 (C-10), 109.8 (C-50), 109.0
(C-30) 84.9 (C-3), 36.1 (C-8), 23.5 (C-9/C-10); Anal. calcd.
for C23H18O4Br2: C, 53.31; H, 3.50; Br, 30.84. Found C,
53.52; H, 3.48; Br, 30.69 %.
11b: m.p. 288–290 °C; % yield: 72; IR (KBr, m, cm-1):
3300 (OH), 2955 (aliphatic C–H), 1760 (lactonic [C=O),
1610, 1590, 1500, 1460 (Ar C=C), 1530 (asym. N–O),
1340 (sym. N–O), 1260, 1110 (C–O), 1020 (C–O–C), 845
(C–N), 758, 695 (o-disubstituted phthalide ring); UV
1
(methanol): kmax 220, 290, 340 nm; H NMR (400 MHz,
DMSO-d6, d, ppm): 1.1 (d, 6H, 29 CH3, J = 6.5 Hz), 2.5
(sept, 1H, –CH\), 6.1(broad s, 1H, 29 OH), 7.0–8.1 (m,
8H, Ar–H); 13C NMR (100 MHz, DMSO-d6, d, ppm):
123

Med Chem Res
R*
R*
O
(OH)n
(OH)n
HS
OH
NH2
Cysteine
OH
S
NH2
O
OH
O
O
O
Phthalide (4-11)
Scheme 4 Possible formation of phthalide–cysteine adduct
Phthalide - cysteine adduct
All organisms were tested using modified versions of the
CLSI (formerly NCCLS) methods. For all organisms
excluding M. intracellulare and A. fumigatus, optical
density was used to monitor the growth (NCCLS, 2002a, b;
NCCLS, 2006). Media supplemented with 5 % Alamar
BlueTM (BioSource International, Camarillo, CA) was uti-
lized for growth detection of M. intracellulare (NCCLS,
2003; Franzblau et al., 1998) and A. fumigatus (NCCLS,
2002a, b). Samples (dissolved in DMSO) were serially-
diluted in 20 % DMSO/saline and transferred (10 lL) in
duplicate to 96 well flat bottom microplates. Inocula were
prepared by correcting the OD630 of microbe suspensions
in incubation broth [RPMI 1640/0.2 % dextrose/0.03 %
glutamine/MOPS at pH 6.0 (Cellgro) for Candida spp.,
Sabouraud Dextrose for C. neoformans, cation-adjusted
Mueller–Hinton (Difco) at pH 7.3 for Staphylococcus spp.,
E. coli, and P. aeruginosa, 5 % Alamar BlueTM(BioSource
International, Camarillo, CA) in Middlebrook 7H9 broth
with OADC enrichment, pH 7.0 for M. intracellulare, and
5 % Alamar BlueTM/RPMI 1640 broth (0.2 % dextrose,
0.03 % glutamine, buffered with 0.165 M MOPS at
pH 7.0) for A. fumigatus to afford an assay volume of
200 lL and final target inocula of Candida spp. and
C. neoformans: 1.5 9 103; M. intracellulare: 2.0 9 106;
Staphylococcus spp., E. coli, P. aeruginosa: 5.0 9
M. intracellulare at 37 °C and 10 % CO2 for 70–74 h. IC50
(concentrations that afford 50 % inhibition relative to
controls) were calculated using XLfit 4.2 software (IDBS,
Alameda, CA) using fit model 201. The MIC was defined
as the lowest test concentration that allows no detectable
growth (for M. intracellulare and A. fumigatus, no colour
change from blue to pink). Results of antimicrobial
screening of compounds are given in Tables 2 and 3.
Mode of action of phthalides on biological systems
Despite of the extensive studies on biological activity of
phthalides, very little attention has been paid towards their
mode of action. However, it has been suggested that the
presence of five membered lactone ring in phthalides is
responsible for their bioactivity (Beck and Chou, 2007).
The amino acid cysteine, and certain enzymes (containing
mercapto groups) play an important role in growth and
normal activity of bacteria and other microorganisms. On
the basis of an experiment involving the action of buty-
lidenephthalide on hairless mouse, Sekia et al. (2000)
showed the formation of butylidenephthalide–cysteine
adduct as a urinary metabolite. This work suggested that
occurrence of this reaction in bacteria inhibits their growth
and activity. Examination of the structures of the various
phthalides (4–11) synthesized in the present study indicates
their ability to react with cysteine to form adducts
(Scheme 4) and show antimicrobial activity.
105 CFU mL-1 and A. fumigatus: 2.7 9 104 CFU mL-1
.
Final sample test concentrations are 1/100th the DMSO
stock concentration.
Drug controls [Ciprofloxacin (ICN Biomedicals, Ohio)
for bacteria and Amphotericin B (ICN Biomedicals, Ohio)
for fungi] are included in each assay. All organisms are
read at either 530 nm using the Biotek Powerwave XS
plate reader (Bio-Tek Instruments, Vermont) or 544
ex/590 em, (M. intracellulare, A. fumigatus) using the
Polarstar Galaxy Plate Reader (BMG LabTechnologies,
Germany) prior to and after incubation Candida spp.
at 35 °C for 46–50 h; Staphylococcus spp., E. coli and
P. aeruginosa at 35 °C for 16–20 h; C. neoformans at
35 °C for 70–74 h; A. fumigatus at 35 °C for 46–50 h and
Conclusion
New 3-substituted phthalides of biological interest are
synthesized in 50–98.6 % yield by the condensation of
2-(4- isopropylbenzoyl)benzoic acid and 2-(4-isopropyl-3-
nitrobenzoyl)benzoic acid with phenols without the use of
a solvent. The procedure is simple, convenient, efficient,
environmentally benign and economic as it involves the
use of easily available cheap chemicals.
123

Med Chem Res
Everaere K, Scheffler JL, Mortreuxand A, Carpentier JF (2001)
Stereoselective synthesis of 3-substituted phtalides via asym-
metric transfer hydrogenation using well-defined ruthenium
catalysts under neutral conditions. Tetrahedron Lett 42:1899–
1901
Flueraru M, Chichirau A, Chepelev LL, Willmore WG, Durst T,
Charron M, Barclay LRC, Wright JS (2005) Cytotoxicity and
cytoprotective activity in naphthalene diols depends on their
tendency to form naphthoquinones. Free Radic Biol Med 39:
1368–1377
Foti MC, Johnson ER, Vinqvist MR, Wright JS, Barclay LRC, Ingold
KU (2002) Naphthalene diols: a new class of antioxidants
intramolecular hydrogen bonding in catechols, naphthalene
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Franzblau SG, Witzig RS, McLaughlin JC, Torres P, Madico G,
Hernandez A, Degnan MT, Cook MB, Quenzer VK, Ferguson
RM, Gilman RH (1998) Rapid low-technology MIC determina-
tion with clinical mycobacterium tuberculosis isolates by using
the microplate alamar blue assay. J Clin Microbiol 36:362–366
Huang LL, Xu MH, Lin GQ (2006) Catalytic enantioselective
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5624–5625
All the tested compounds (6a, 6b, 9a and 9b) have
shown promising antibacterial activity especially against S.
aureus ATCC 29213 and methicillin-resistant S. aureus
ATCC 33591 (as can be seen from Tables 2, 3). Compound
9b also showed promising antifungal activity against
C. glabrata ATCC 90030 (Table 3). Although, the phtha-
lides taken for in vitro antimicrobial screening were found
to be much less active than the Ciprofloxacin and
Amphotericin B, which were used as control drugs. But in
view of growing incidences of drug resistance in micro-
organisms, and remarkable bioenhancing property exhib-
ited by some less or non-active compounds, further
appropriate investigations of anticipated biological prop-
erties (including in vivo antimicrobial screening) of these
phthalides are warranted to explore their significant thera-
peutic potential. It is well known that in vivo assessment is
necessary even for drugs with little or no activity under
best in vitro conditions (Emmons et al., 1977).
The anticancer and antioxidant properties of these
compounds are under investigation and results of this
ongoing work will be communicated in near future. The
phthalides reported in this paper can be evaluated for other
possible biological properties as well as used as lead
molecules for the development of new drugs.
Hussain HH, Babic G, Durst T, Wright JS, Flueraru M, Chichirau A,
Chepelev LL (2003) Development of novel antioxidants: design,
synthesis, and reactivity. J Org Chem 68:7023–7032
Karnik AV, Kamath SS (2008) Cascade enantioselective synthesis of
3-arylphthalides using chiral auxiliary route. Synthesis 12:
1832–1834
¨
Knepper K, Ziegert RE, Brase S (2004) Solid-phase synthesis of
isoindolinones and naturally-occurring benzobutyrolactones
(phthalides) using a cyclative-cleavage approach. Tetrahedron
60:8591–8603
Acknowledgments The authors are highly thankful to Dr.
M. Vairamani, Head, Analytical Chemistry Division, National Centre
for Mass Spectrometry, Indian Institute of Chemical Technology,
Hyderabad, India for providing mass spectral data. Authors also thank
Prof. Alice M. Clark and Dr. Melissa Jacob, National Center for
Natural Products Research, School of Pharmacy, University of Mis-
sissippi, Mississippi (USA), for providing generous help for the
antimicrobial studies.
Ko WC, Lin LC, Lin SH, Hwang PY, Hsu CY, Wang GY, Chang CW
(1983) Comparison of some alkylidenephthalides on their
antispasmodic effects. J Taiwan Pharm Assoc 35:155–161
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effects of butylidenephthalide. Phytother Res 8:321–326
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catechol and pyrogallol as allelochemicals.
61c:639–642
Z Naturforsch
Kohn LK, Pavam CH, Veronese D, Coelho F, De Carvalho JE,
Almeida WP (2006) Antiproliferative effect of Baylis–Hillman
adducts and a new phthalide derivative on human tumor cell
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