Total synthesis and cytotoxic activity of stellatin

Article abstract of DOI:10.1080/10286020.2010.544654

Stellatin (3,4-dihydro-8-hydroxy-7-hydroxymethyl-6-methoxyisocoumarin) (8), an extrolite of fungal genera Emericella and Aspergillus, was synthesized. Thus, Vilsmeier-Haack formylation of methyl ester of 3,5-dimethoxy-4- methylphenylacetic acid (1) to aff

Full text of DOI:10.1080/10286020.2010.544654

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Total synthesis and cytotoxic activity of
stellatin
Aamer Saeed a , Zaman Ashraf a & Hummera Rafique a
a Department of Chemistry, Quaid-I-Azam University, Islamabad,
45320, Pakistan
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cytotoxic activity of stellatin, Journal of Asian Natural Products Research, 13:02, 97-104
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Journal of Asian Natural Products Research
Vol. 13, No. 2, February 2011, 97–104
Total synthesis and cytotoxic activity of stellatin
Aamer Saeed*, Zaman Ashraf and Hummera Rafique
Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan
(Received 7 September 2010; final version received 30 November 2010)
Stellatin (3,4-dihydro-8-hydroxy-7-hydroxymethyl-6-methoxyisocoumarin) (8), an
extrolite of fungal genera Emericella and Aspergillus, was synthesized. Thus,
Vilsmeier–Haack formylation of methyl ester of 3,5-dimethoxy-4-methylphenylacetic
acid (1) to afford the formyl ester (2) followed sulfamic acid–sodium chlorite
oxidation of the aldehydic function to yield the carboxy ester (3). Chemoselective
reduction of ester function in the latter using NaBH4/THF/MeOH furnished the
corresponding hydroxy acid (4) that on cyclodehydration afforded the 3,4-dihydro-6,8-
dimethoxy-7-methylisocoumarin (5). Benzylic bromination of the C-7 methyl in 5
using NBS/benzoyl peroxide to give the 7-bromomethyldihydroisocoumarin
(6) followed the nucleophilic substitution using aqueous acetone to provide
7-hydroxymethyl-dihydroisocoumarin (7). Finally, the regioselective demethylation
of 8-methoxyl group using anhydrous magnesium iodide furnished the stellatin (8). The
dihydroisocoumarins (5–8) were screened for cytotoxic activity against human
keratinocyte cell line and were found to exhibit moderate to good activity.
Keywords: dihydroisocoumarin; stellatin; Aspergillus variecolor; cytotoxic activity
1. Introduction
pathway; hence, most of them possess a
C-3 alkyl/aryl substituent and C-8 oxy-
genation as in phyllodulcin and hydran-
genol, the low-calorie sweetening agents,
or a C-6/C-8 dioxygenation [1,3]. Com-
mon examples of the latter include 6-
methoxymellein, diaporthin, fusamarin,
asperentin derivatives, agrimonolide,
canescins A&B, sclerotinins A&B, feralo-
lide, achlisocoumarin, Ononis natrix
dihydroisocoumarins, scorzocreticin,
hiburipyranone [12–16], and cerocphorins
A-C, antifungal and cytotoxic metabolites
from Cercophora areolata [6]. Stellatin
(Figure 1) is a novel phenolic dihydroiso-
coumarin metabolite first isolated from the
mycelium of Aspergillus variecolor (syn.
Aspergillus stellatus) and its structure was
determined by the chemical and modern
NMR spectroscopic techniques as
Isocoumarins and dihydroisocoumarins are
the extrolites (secondary metabolites) of a
wide variety of fungi, bacteria, plants,
marine organisms, and also among insect
venoms and pheromones, exhibiting a wide
variety of structural diversity and biologi-
cal activities [1–3]. Nearly, 300 isocou-
marins derivatives have been isolated from
various natural sources, exhibiting a wide
spectrum of biological activities such as
antiallergic, antimicrobial [4], immuno-
modulatory [5], cytotoxic [6], antifungal,
anti-inflammatory [7], antiangiogenic [8],
anticalmodulin-sensitive cGMP phospho-
diesterease effects [9], antileukemic [10],
and anti-HIV activities [11].
Majority of the natural isocoumarins
being of polyketide origin are derived
biogenetically from acetate-polymalonate
*Corresponding author. Email: aamersaeed@yahoo.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
DOI: 10.1080/10286020.2010.544654
http://www.informaworld.com

98
A. Saeed et al.
Alternatively, if it is of pentaketide origin,
MeO
the methyl carbon of the chain-initiating
unit must be lost. Both of these processes
are unprecedented in polyketide biosyn-
thesis. Biosynthetic studies involving the
incorporation of 13C-labeled acetates and
methionine supported the polyketide bio-
synthetic pathway for stellatin, as a bis-C-
methylated tetraketide [24].
O
HOH2C
OH
O
Figure 1. Structure of stellatin (3,4-dihydro-8-
hydroxy-7-hydroxymethyl-6-methoxyisocou-
marin, 8).
Taking into consideration the unique
structure, interesting biosynthesis and for
making it available for bioevaluation, an
efficient synthesis of stellatin was desired.
3,4-dihydro-8-hydroxy-7-hydroxymethyl-
6-methoxyisocoumarin. It is also an
extrolite of Emericella venezuelensis,
Emericella variecolor, and Emericella
heterothallica [17].
2. Results and discussion
Stellatin is unique in being unsubsti-
tuted at both C-3 and C-4 and in having a
carbon substituent at C-7. The only
structural relatives known to date are the
8-hydroxyisocoumarin and 3,4-dihydro-8-
hydroxyisocoumarin found in the defen-
sive secretion of the tenebrionid beetle,
Apsena pubescens [18], and 5-formyl-3,4-
dihydroisocoumarin isolated from a med-
icinal plant Centaurium erythraea [19].
Those having a C-7 carbon substituent
include stoloniferol A from the sea squirt-
derived fungus, Penicillium stoloniferum
[20], and the antimalarial dihydroisocou-
marins from endophytic fungus Geotri-
We envisaged the synthesis of 8 using 3,5-
dimethoxy-4-methylphenylacetic acid (1)
as the key starting material, itself prepared
from the commercial p-toluic acid. Thus,
esterification of p-toluic acid followed the
nuclear bromination using swamping
catalyst effect involving bromine and an
excess of anhydrous AlCl3 to afford the
3,5-dibromo-4-methylbenzoic acid. Cop-
per (I) chloride-catalyzed nucleophilic
substitution of the latter with methoxide
provided 3,5-dimethoxy-4-methylbenzoic
acid [25] which was then converted into
3,5-dimethoxy-4-methylphenylacetic acid
(1) by the standard homologation
sequence. Methyl ester of the acid (1)
was subjected to Vilsmeier–Haack for-
mylation to furnish methyl 2-formyl-3,5-
dimethoxyphenyl acetate (2). The peaks
for CHO at dH 9.75 and dC 179.3 were
observed in 1H NMR and 13C NMR
spectra, respectively. Oxidation of the
aldehydic function was achieved using
sulfamic acid and sodium chlorite at 08C in
79% yield to afford carboxy ester (3). The
carbonyl absorption in the IR spectrum
shifted from 1690 to 1715 cm21 in
addition to broad absorption at
3265 cm21 for carboxylic hydroxyl. In
the 1H NMR spectrum, the singlet for
carboxylic hydrogen at d 8.19 and down-
field shift from d 179.3 to d 197.7 for
carboxylic carbon were noted in the 13C
chum sp. [21].
A closely related
isochroman mycotoxin 3,7-dimethyl-8-
hydroxy-6-methoxyisochroman and its
derivatives have been isolated from
Penicillium corylophilum, which signifi-
cantly inhibit the etiolated wheat coleop-
tile growth and also act as auxin polar
transport inhibitors [22]. A novel chro-
mane derivative related to stellatin was
isolated along with stellatin from
E. heterothallica [23].
The study of biosynthesis of stellatin is
also very interesting. The overall structure
is consistent with a polyketide origin.
However, if stellatin is a tetraketide, then
C-3 must be derived by the introduction of
a methyl group from C1-pool on the
methyl carbon of the chain-initiating unit.

Journal of Asian Natural Products Research
99
NMR spectrum. Chemoselective reduction
of ester function in 3 leaving acid group
intact was achieved using NaBH4/
THF/MeOH to give the hydroxy acid (4)
which on cyclodehydration in refluxing
acetic anhydride afforded 3,4-dihydro-6,8-
dimethoxy-7-methylisocoumarin (5). In
the IR spectrum of the dihydroisocou-
marin, the lactonic carbonyl absorption
was observed at 1725 cm21. The protons
of C-4 methylene group were shown as a
triplet at d 2.56 (J ¼ 3.6 Hz) and that for
C-3 methylene were shown slightly down-
field at d 4.25 (J ¼ 3.6 Hz) due to vicinity
of oxygen. Benzylic bromination of the 7-
methyl group using NBS/benzoyl peroxide
was carried out using dry CCl4 to yield the
bromide (6). In the 1H NMR spectrum, the
singlet for 7-methyl protons shifted down-
field from d 2.66 in 5 to d 4.85 in 6. Due to
the presence of the base-sensitive lactone
ring, a mild method for nucleophilic
substitution of 7-bromomethyl group to
7-hydroxymethyl was required. It was
achieved by refluxing the bromide (6) in
aqueous acetone (1:2) for 2 h to provide 7.
The IR spectrum showed a broad band at
3467 cm21 for hydroxyl group, while in
cal data were reported in the original paper
nor was a specimen available for compari-
son purposes.
The Neutralrot-Test was carried out for
cytotoxic activity of compounds 5–8
according to the protocol of the National
Institutes of Health. In the vital dye,
neutral red is taken up by living cells and
then protonated. The cell acts as an ion
trap, allowing the dye not to diffuse out of
it. By destroying the cells, the neutral is
released again and can be determined
photometrically. The absorption is a
measure of cell viability, and a lower
absorption indicates the less living cells.
For the tests, the immortalized human
keratinocyte cell line was used. Incubation
with the test substances was carried out for
3 days and was conducted in two
independent experiments with a number
of parallels. The stock solutions of
substances were prepared in DMSO.
Each of the tested concentration range
was between 1.56 and 100 mM. The
concentrations of the solvent DMSO
were all tested in concentrations of 0.1%.
Etoposide as positive control was carried
in a concentration of 10 mM. Etoposide
showed an IC50 value of 0.8 mM (Figure 2).
The results showed that the cytotoxic
activity increases with an increase in the
concentration of compounds. Stellatin
exhibits higher activity than its precursors.
While compounds 5 and 6 showed moder-
ate to low cytotoxic activity at the highest
concentration (100 mM). However, when
7-methyl group in compound 5 is converted
into 7-hydroxymethyl group in 7, the
cytotoxic activity is increased with a
decrease in viability of up to 45%. Selective
demethylation of the 8-methoxy group in
stellatin 8 resulted in further increase in
cytotoxic activity. Thus, the presence of the
7-hydroxymethyl group and the free 8-
hydroxy group play a vital role in cytotoxic
activity of stellatin.
1
the H NMR spectrum a singlet appeared
at d 2.36.
Regioselective demethylation of 7 was
finally accomplished using Mg/I2 refluxed
in THF/benzene to afford 8-hydroxy-7-
(hydroxymethyl)-6-methoxy-3,4-dihydro-
1H-isochromen-1-one (stellatin) (8,
1
Scheme 1). In the H NMR spectrum, a
singlet at d 10.90 appeared for 8-hydroxy,
while that for 7-hydroxy proton at d 2.29.
The presence of phenolic hydroxyl was
confirmed by a purple ferric chloride test
and solubility in dilute aqueous sodium
hydroxide. In the IR spectrum, the lactonic
carbonyl absorption was lowered from
1714 to 1695 cm21 due to chelation with
8-hydroxyl, which appeared at 3559 cm21
.
The spectroscopic data (1H and 13C NMR,
mass spectrum) of the synthetic compound
was identical with that of the natural
product [17]; however, no physico-chemi-
In summary, the first total synthesis
of stellatin, a natural bioactive dihydro-

100
A. Saeed et al.
MeO
H3C
CH2COOH
MeO
CH2COOMe
i) MeOH/H+
NH2SO3H/NaClO2
ii) POCl3/DMF
H3C
CHO
OMe
(1)
(2)
OMe
OH
MeO
CH2COOMe
MeO
H3C
NaBH4/THF/MeOH/
4h reflux
Ac2O
H3C
COOH
COOH
OMe
OMe
(3)
(4)
MeO
H3C
MeO
NBS/BPO/
dry CCl4
Aqueous acetone
(1:1) 3h reflux
O
O
BrH2C
O
OMe
O
OMe
(5)
(6)
MeO
MeO
Mg/I2/dry THF:PhH
O
O
HOH2C
HOH2C
OMe
O
O
OH
(7)
(8)
Scheme 1. Synthesis route to stellatin (8).
isocoumarin and its cytotoxic activity has
been reported.
spectrophotometer. 1H NMR and 13C
NMR spectra were determined in CDCl3
solutions at 300 MHz on a Bruker AM-
300 spectrophotometer. Mass spectra
3. Experimental
(EI, 70 eV) were performed on a
GCMS instrument, and elemental ana-
lyses with a LECO-183 CHNS analyzer.
All the compounds were purified by thin
layer chromatography using silica gel
HF-254 from Merck (Darmstadt,
Germany).
3.1 General experimental procedures
The solvents were purified and dried
according to the standard procedures
before using. The dried solvents were
stored under molecular sieves (4A).
Standard procedures were employed for
the purification and drying of solvents.
Melting points were recorded using a
digital Gallenkamp (SANYO) model
MPD BM 3.5 apparatus and are
uncorrected. FT-IR spectra were
recorded using an FTS 3000 MX
3.2 Methyl (3,5-dimethoxy-4-methyl
phenyl) acetate
A stirred solution of 3,5-dimethoxy-4-
methylphenylacetic acid (1) (5.0 g,

Journal of Asian Natural Products Research
101
1008C for 2 h and stirred overnight at room
temperature. Then, the reaction mixture
was poured into the aqueous solution of
sodium acetate (10%, 10 ml) and shaked
vigorously. Methyl (2-formyl-3,5-
dimethoxy-4-methylphenyl) acetate (2)
was precipitated out as yellowish precipi-
tates (1.9 g, yield 84%), R*f : 0.55, mp 51–
538C, IR (KBr): nmax 3029 (CZH), 1722
120
100
80
60
40
20
0
5
6
7
8
10
25
50
100
1000
Concentration (µM)
(CvO), 1690 (CHO), 1545 (CvC) cm21
;
1H NMR (CDCl3, d ppm): d 9.75 (1H, s
CHO), 7.96 (1H, s, H-6), 3.42 (3H, s, 3-
OCH3), 3.25 (3H, s, 5-OCH3), 3.11 (3H, s,
CO2CH3), 2.92 (2H, s, ArZCH2), 2.80
(3H, s, ArZCH3); 13C NMR (CDCl3, d
ppm): d 179.3 (aldehyde CvO), 162.4
(ester CvO), 136.7 (C3, C5), 131.9 (C2),
126.2 (C6), 121.3 (C4), 117.5 (C1), 61.6
(ester OCH3), 57.3 (ArZOCH3), 39.1
(ArZCH2), 32.0 (ArZCH3); MS (70 eV):
m/z (%) 252 [M]þz (25), 251 (65), 224 (49),
223 (34), 165 (100), 29 (31); Elemental
analysis: Found: C, 61.67%, H, 6.16%;
calcd for C13H16O5: C, 61.90%, H, 6.34%.
Figure 2. Cytotoxic activities of compounds
5–8.
23.8 mmol) in dry methanol (30 ml) was
treated dropwise with conc. H2SO4 (5 ml).
The mixture was refluxed for 8–9 h. The
reaction was monitored by TLC. After the
completion of the reaction, the mixture
was concentrated to 55 ml and extracted
with ethyl acetate (3£ 50 ml). The extract
was washed with saturated brine, dried,
and concentrated to give crude oil that was
distilled to afford methyl ester (4.7 g,
yield 88.18%), R*f : 0.7, mp 38–408C, IR
(KBr): nmax 3023 (CZH), 1734 (CvO),
1
1573 (CvC) cm21; H NMR (CDCl3, d
3.4 2,4-Dimethoxy-6-(2-methoxy-2-
oxoethyl)-3-methylbenzoic acid (3)
ppm): d 7.45 (2H, s, H-2, H-6), 3.96 (6H,
s, 2£ OCH3), 3.54 (2H, s, ArZCH2), 3.47
(3H, s, COOCH3), 2.55 (3H, s, ArZCH3);
13C NMR (CDCl3, d ppm): 168.2 (CvO),
132.5 (C3, C5), 128.3 (C2, C6), 119.4
(C4), 112.2 (C1), 68.5 (ester OCH3), 55.3
(ArZOCH3), 36.9 (CH2), 28.6 (ArZCH3);
MS (70 eV): m/z (%) 224[M]þz (46), 193
(43), 165 (100), 59 (12); Elemental
analysis: Found: C, 64.28%, H, 7.14%;
calcd for C12H16O4: C, 64.02%, H,
6.96%.
Formyl ester (2) (6.3 g, 25.0 mmol) and
sulfamic acid (8.3 g, 86.0 mmol) in 150 ml
H2O:THF:DMSO (20:0:1) at 08C were
treated with NaClO2 (7.24 g, 80.0 mmol)
in 20 ml H2O. The reaction mixture was
stirred for 20 min at 08C and then diluted
with ethyl acetate (100 ml), washed with
saturated aqueous ammonium chloride
(2£ 13 ml), and saturated aqueous sodium
chloride (130 ml). Organic layer was dried
over anhydrous sodium sulfate and evap-
orated to afford the keto acid (3) (6.6 g,
yield 79%), R*f : 0.4, mp 164–1668C, IR
(KBr): nmax 3265 (OZH), 3037 (CZH),
1734 (CvO), 1715 (COOH), 1562 (CvC)
3.3 Methyl (2-formyl-3,5-dimethoxy-4-
methyl phenyl) acetate (2)
1
Phosphorous
oxychloride
(1.61 g,
cm21; H NMR (CDCl3, d ppm): d 8.19
10.0 mmol) was added dropwise into a
stirred solution of methyl (3,5-dimethoxy-
4-methyl phenyl) acetate (2.0 g, 8.9 mmol)
in freshly distilled DMF (10 ml) at 558C.
Reaction mixture was heated at about
(1H, s, COOH), 7.66 (1H, s, H-6), 3.82
(3H, s, 3-OCH3), 3.67 (3H, s, 5-OCH3),
3.63 (3H, s, CO2CH3), 2.54 (2H, s,
ArZCH2), 2.25 (3H, s, ArZCH3); 13C
NMR (CDCl3, d ppm): d 197.8 (carboxylic

102
A. Saeed et al.
CvO), 168.5 (ester CvO), 139.3 (C2,
C4), 134.3 (C6), 127.1 (C5), 120.6 (C3),
114.1 (C1), 66.0 (ester OCH3), 55.4
(ArZOCH3), 35.0 (ArZCH2), 29.8
(ArZCH3); MS (70 eV): m/z (%) 268
[M]þz (32), 251 (51), 224 (65), 165 (100),
45 (25); Elemental analysis: Found: C,
58.04%, H, 5.76%; calcd for C13H16O6: C,
58.20%, H, 5.97%.
refluxed for 1 h. Then, the reaction mixture
was poured into ice-cold water and
extracted with ethyl acetate (3£ 20 ml).
The combined ethyl acetate extract was
washed with 1% NaHCO3 and then with
water. Ethyl acetate was evaporated under
reduced pressure to afford 6,8-dimethoxy-
7-methyl-3,4-dihydro-1H-isochromen-1-
one (5). Yield 78%, R*f : 0.6, mp 145–
1478C, IR (KBr): nmax 3010 (CZH), 1702
(CvO), 1591 (CvC) cm21 1H NMR
;
3.5 2,4-Dimethoxy-6-(2-hydroxyethyl)-
3-methylbenzoic acid (4)
(CDCl3, d ppm): d 7.48 (1H, s, H-5), 4.25
(2H, t, J ¼ 3.6 Hz, H-3), 3.90 (6H, s, 6-
OCH3, 8-OCH3), 2.66 (3H, s, ArZCH3),
2.56 (2H, t, J ¼ 3.6 Hz, H-4); 13C NMR
(CDCl3, d ppm) d 163.9 (C1), 152.3 (C6,
C8), 140.8 (C4a), 134.6 (C8a), 108.9 (C7),
103.7 (C5), 65.9 (C3), 56.4 (6-OCH3, 8-
OCH3), 27.4 (C4), 26.2 (ArZCH3); MS
(70 eV): m/z (%) 222 [M]þz (52), 194 (45),
192 (100), 164 (31), 30 (21); Elemental
analysis: Found: C, 64.75%, H, 6.19%;
calcd for C12H14O4: C, 64.86%, H, 6.30%.
Keto acid (3) (0.5 g, 1.86 mmol) and
sodium borohydride (0.84 g, 22.32 mmol)
were suspended in freshly distilled THF
(10 ml). The reaction mixture was stirred
for 15 min at 658C and then methanol
(10 ml) was added dropwise for 30 min.
The mixture was refluxed for 4 h, then
cooled to room temperature, and treated
with saturated ammonium chloride sol-
ution (10 ml). Stirring was continued for
1 h, then acidified with dilute hydrochloric
acid, and extracted with ethyl acetate
(3£ 20 ml). The extract was dried, evap-
orated to afford hydroxyl acid (4) (0.35 g,
yield 78%), R*f : 0.3, mp 72–748C, IR
(KBr): nmax 3481 (OZH), 3009 (CZH),
3.7 6,8-Dimethoxy-7-(bromomethyl)-
3,4-dihydro-1H-isochromen-1-one (6)
To a stirred solution of dihydroisocou-
marin (5) (0.5 g, 2.25 mmol) in dry carbon
tetrachloride (10 ml), N-bromosuccini-
mide (0.6 g, 3.37 mmol) and benzoyl
peroxide (7.5 mg) were added. The reac-
tion mixture was refluxed for 6 h, then
cooled, filtered, and washed with a little
carbon tetrachloride. The solvent was
rotary evaporated to leave 7-bromo dihy-
droisocoumarin (6). Yield 81%, R*f : 0.6,
mp 91–938C, IR (KBr): nmax 3013 (CZH),
2926 (ArZCZH), 1718 (CvO), 1582
(CvC) cm21; 1H NMR (CDCl3, d ppm): d
7.50 (1H, s, H-5), 4.85 (2H, s, CH2ZBr),
4.24 (2H, t, J ¼ 3.6 Hz, H-3), 3.90 (6H, s, 6-
OCH3, 8-OCH3), 2.58 (2H, t, J ¼ 3.6 Hz,
H-4); 13C NMR (CDCl3, d ppm): d 169.2
(C1), 155.4 (C6, C8), 144.1 (C4a), 110.7
(C7), 106.2 (C8a), 104.6 (C5), 67.4 (C3),
53.7 (6-OCH3, 8-OCH3), 39.5 (CH2ZBr),
26.4 (C4); MS (70 eV): m/z (%) 200 [M]þz
(24), 302 [M þ 2] (24), 272 (39), 221 (19),
1710 (CvO), 1574 (CvC) cm21 1H
;
NMR (CDCl3, d ppm): d 8.22 (1H, s,
COOH), 7.48 (1H, s, H-5), 4.21 (2H, t,
J ¼ 3.8 Hz, H-10), 3.90 (3H, s, 2-OCH3),
3.75 (3H, s, 4-OCH3), 2.65 (2H, t,
J ¼ 3.8 Hz, H-20), 2.51 (3H, s, ArZCH3);
13C NMR (CDCl3, d ppm): d 190.5
(COOH), 166.2 (C2), 162.4 (C4), 141.3
(C6), 108.1 (C3), 105.6 (C1), 101.2 (C5),
63.2 (C20), 56.4 (2-OCH3, 4-OCH3), 32.3
(C10), 28.8 (ArZCH3); MS (70 eV): m/z
(%) 240 [M]þz (32), 223 (24), 196 (43),
165 (100), 45 (19), 31 (37); Elemental
analysis: Found: C, 59.87%, H, 6.57%;
calcd for C12H16O5: C, 60.00%, H, 6.66%.
3.6 6,8-Dimethoxy-7-methyl-3,4-
dihydro-1H-isochromen-1-one (5)
Hydroxy acid (4) (1.0 g, 4.16 mmol) was
dissolved in acetic anhydride (5 ml) and

Journal of Asian Natural Products Research
103
191 (100), 30 (28); Elemental analysis:
Found: C, 47.72%, H, 4.23%; calcd for
C12H13O4Br: C, 47.84%, H%, 4.31%.
Yield 47%, Rf: 0.5, mp 127–1288C, IR
(KBr): nmax 3559 (OZH), 3001 (CZH),
2920 (ArZCZH), 1695 (CvO), 1593
(CvC) cm21; 1H NMR (CDCl3, d ppm): d
10.90 (1H, s, 8-OH), 7.06 (1H, s, H-5),
4.54 (2H, s, CH2OH), 4.12 (2H, t,
J ¼ 3.7 Hz, H-3), 3.90 (3H, s, 6-OCH3),
2.86 (2H, t, J ¼ 3.7 Hz, H-4), 2.29 (1H, s,
CH2OH); 13C NMR (CDCl3, d ppm): d
166.5 (C1), 158.7 (C8), 151.4 (C6), 143.1
(C4a), 116.9 (C7), 105.2 (C8a), 102.6
(C5), 63.9 (C3), 56.9 (6-OCH3), 47.1
(CH2ZOH), 28.4 (C4); MS (70 eV): m/z
(%) 224 [M]þz (59), 222 (21), 207 (42),
194 (100), 30 (19); Elemental analysis:
Found: C, 59.37%, H, 4.39%; calcd for
C11H10O5: C, 59.45%, H, 4.50%.
3.8 7-(Hydroxymethyl)-6,8-dimethoxy-
3,4-dihydro-1H-isochromen-1-one (7)
6,8-Dimethoxy-7-(bromomethyl)-3,4-
dihydro-1H-isochromen-1-one (6) (1.0 g,
3.32 mmol) was dissolved in a mixture of
water and acetone (10 ml, 1:1). The
reaction mixture was refluxed for 1 h, then
most of the acetone was rotary evaporated,
and the residue was poured into ice-cold
water and the solid was filtered, washed
with water, and dried to afford 7-hydro-
xymethyl dihydroisocoumarin (7). Yield
77%, Rf: 0.4, mp 136–1388C, IR (KBr):
nmax 3467 (OZH), 3010 (CZH), 2919
(ArZCZH), 1714 (CvO), 1587 (CZC)
R*f : (petroleum ether: ethyl acetate,
4:1).
1
cm21; H NMR (CDCl3, d ppm): d 7.62
Acknowledgement
(1H, s, H-5), 4.88 (2H, s, CH2ZOH), 4.20
(2H, t, J ¼ 3.7 Hz, H-3), 3.90 (6H, s, 6-
OCH3, 8-OCH3), 2.56 (2H, t, J ¼ 3.7 Hz,
H-4), 2.36 (1H, s, OZH); 13C NMR
(CDCl3, d ppm): d 168.6 (C1), 157.3 (C6,
C8), 142.1 (C4a), 115.2 (C7), 105.2 (C8a),
102.6 (C5), 64.5 (C3), 55.7 (6-OCH3, 8-
OCH3), 48.6 (CH2ZOH), 27.4 (C4); MS
(70 eV): m/z (%) 238 [M]þz (49), 221 (31),
210 (55), 208 (100), 30 (19); Elemental
analysis: Found: C, 60.38%, H, 5.76%;
calcd for C12H14O5: C, 60.50%, H, 5.88%.
The authors are grateful to the Pakistan Science
Foundation for a research grant under Project
No. PSF/Res/C-QU/Chem(395).
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