Synthesis of 4-Hydroxy-7,8-dimethoxyisochroman-3-one and Its Plant Growth-Regulating Properties on Tobacco (Nicotiana tabacum cv. Petit Havana)

Article abstract of DOI:10.1021/jf0351117

The synthesis of 6,7-dimethoxy-4-hydroxyisochroman-3-one 10 from 2,3-dimethoxytoluene (11) via glyoxylate 12 is reported. Compound 10 strongly inhibited vegetative growth of tobacco plants, whereas developmental patterns (protein levels, protein profile, pigments, and chlorophylls) were not affected. Morphological changes were observed in the leaves of the treated plants.

Full text of DOI:10.1021/jf0351117

J. Agric. Food Chem. 2004, 52, 1923−1927 1923
Synthesis of 4-Hydroxy-7,8-dimethoxyisochroman-3-one and Its
Plant Growth-Regulating Properties on Tobacco (Nicotiana
tabacum cv. Petit Havana)
DARIÄO A. BIANCHI,† NICOLAÄ S E. BLANCO,‡ NEÄ STOR CARRILLO,‡ AND
TEODORO S. KAUFMAN*,†
Instituto de Qu´ımica Orga´nica de S´ıntesis (CONICET-UNR) and Instituto de Biolog´ıa Molecular y
Celular de Rosario (CONICET-UNR), Facultad de Ciencias Bioqu´ımicas y Farmace´uticas, Universidad
Nacional de Rosario, Suipacha 531, S2002LRK Rosario, Repu´blica Argentina
The synthesis of 6,7-dimethoxy-4-hydroxyisochroman-3-one 10 from 2,3-dimethoxytoluene (11) via
glyoxylate 12 is reported. Compound 10 strongly inhibited vegetative growth of tobacco plants, whereas
developmental patterns (protein levels, protein profile, pigments, and chlorophylls) were not affected.
Morphological changes were observed in the leaves of the treated plants.
KEYWORDS: Synthesis; plant growth regulation; isochroman-3-one; tobacco (Nicotiana tabacum cv.
Petit Havana)
INTRODUCTION
Several natural and synthetic benzopyran derivatives (iso-
chromanes, isocoumarines, flavonoids, chromones) have dem-
onstrated interesting effects on plant growth. Sclerin (1) and
sclerotinin A (2) promote seed germination and shoot elongation
of castor bean, mung bean, rice, and other plants (1, 2), while
6-hydroxymellein (3) is an inhibitor of pollen development (3)
and several of its derivatives impaired the germination of Setaria
italica and Lepidium satiVum (4).
Cutler et al. (5) reported that isochromane 4 (6) significantly
inhibits etiolated wheat coleoptile growth; they also prepared
derivatives and analogues of 4, many of which were found to
act as auxin polar transport inhibitors (7, 8). More recently,
Michel (9) isolated dihydroisocoumarin 5 and the known
phytotoxin 6 from the culture broth of the fungus causing Dutch
elm disease, a vascular disease that kills the trees; the presence
of phytotoxic compound 5 in infected trees has also been
detected. In addition, it has been shown that hydrangenol (7),
isolated from the flowers of Hydrangea hortensia, has syner-
gistic effects on elongation caused by gibberellin (10), and the
effects of coumarin and substituted coumarins on plant growth
have long been known (11).
On the other hand, Taniguchi and co-workers tested the
activity of 1- and 4-substituted isochroman-3-ones as plant
growth inhibitors (12), after demonstrating that 2-substituted
phenylacetic acid derivatives are capable of inducing antigeo-
tropism and inhibition of radicle and hypocotyl elongation of
seedling plants (13, 14). These authors found that most
isochroman-3-ones without substituent at C-1 had no significant
growth-regulating activity on rice seedlings.
It has also been reported that the flavonoids apigenin (8) and
quercetin (9) are able to perturb auxin transport (15-18). These
natural products have been proposed as endogenous auxin polar
transport regulators. Finally, Tobler et al. (19) have shown that
certain 4-substituted isochroman-3-ones can act as antidotes,
being capable of reducing the damage caused to wheat by the
herbicide clodinafop, thus allowing the selective control of
weeds in useful plant cultivations.
* To whom correspondence should be addressed: fax +54-341-4370477;
e-mail tkaufman@fbioyf.unr.edu.ar.
† Instituto de Qu´ımica Orga´nica de S´ıntesis.
‡ Instituto de Biolog´ıa Molecular y Celular de Rosario.
10.1021/jf0351117 CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/05/2004

1924 J. Agric. Food Chem., Vol. 52, No. 7, 2004
Bianchi et al.
1
cm-1; H NMR (δ) 3.62 (br s, 1H, w1/2 ) 8 Hz, OH), 3.87 (s, 3H,
OMe), 3.88 (s, 3H, OMe), 5.10 (br s, 1H, H-4), 5.16 (d, 1H, J ) 14.5
Hz, ArCH2O), 5.64 (d, 1H, J ) 14.5 Hz, ArCH2O), 6.98 (d, 1H, J )
8.4 Hz, H-6), and 7.30 (d, 1H, J ) 8.4 Hz, H-5); 13C NMR (δ) 55.86
(OMe-7), 61.04 (OMe-8), 64.06 (C-1), 67.24 (C-4), 113.01 (C-6),
118.92 (C-7a), 122.90 (C-5), 126.59 (C-4a), 144.40 (C-7), 151.66 (C-
8) and 173.76 (C-3). HRMS for C11H12O5: calcd, 224.06847; found,
224.06823.
Herein, we report the synthesis of 7,8-dimethoxy-4-hydroxy-
isochroman-3-one (10) from commercially available 2,3-
dimethoxytoluene (11) and its growth-regulating properties on
tobacco (Nicotiana tabacum cv. Petit Havana) plants. This is
of importance because although certain isochromans have
demonstrated influence on plant growth, the activity of 4-hy-
droxyisochroman derivatives has not been tested. Interestingly,
the presence of a benzylic oxygen substituent has been linked
to the biological activity of certain natural products bearing an
isochroman moiety such as the pyranonaphthoquinone antibiot-
ics (20).
4-Hydroxy-7,8-dimethoxyisochroman-3-one (10) and 7,8-Dimeth-
oxyisochroman-3,4-diol (14) from Glyoxylate 12. To a stirred solution
of glyoxylate 12 (1000 mg, 3.22 mmol) in anhydrous MeOH (20 mL)
were successively added NaBH4 (243.6 mg, 6.45 mmol), K2CO3 (568
mg, 4.12 mmol), and NaOH (1.5 mL, 1.5 mmol), and the reaction was
stirred overnight at room temperature. Diluted HCl was added until
pH ) 2, and after stirring for 15 min, the reaction was diluted with
brine (20 mL) and extracted with EtOAc (4 × 30 mL). The combined
organic extracts were washed with brine (10 mL), dried (Na2SO4), and
concentrated under reduced pressure. Chromatographic purification of
the residue furnished R-hydroxyketone 10 (268 mg, 37%), whose
spectral data were in agreement with those of the compound obtained
by lactonization of l3 (vide supra). Increasing solvent polarity provided
R-hydroxylactol 14 (175 mg, 24%) as a solid consisting in a 1:1 mixture
of diastereomers; Rf ) 0.35 (hexanes-EtOAc 3:7). IR (KBr, v) 3392,
3262, 2953, 2929, 1495, 1448, 1281, 1230, 1128, 1087, 1020, and 997
cm-1. Major diastereomer: 1H NMR (δ) 2.04 (d, 1H, J ) 9.8 Hz, OH-
3), 3.00 (d, 1H, J ) 4.4 Hz, OH-4), 3.82 (s, 3H, OMe), 3.88 (s, 3H,
OMe), 4.44 (br dd, 1H, J ) 4.4 and 9.8 Hz, H-3), 4.74 (d, 1H, J )
16.1 Hz, H-1), 5.15 (d, 1H, J ) 16.1 Hz, H-1), 5.20 (t, 1H, J ) 4.4
Hz, H-4), 6.90 (d, 1H, J ) 8.5 Hz, H-5), and 7.19 (d, 1H, J ) 8.5 Hz,
H-6); 13C NMR (δ) 55.70 (OMe-7), 60.03 (OMe-8), 62.50 (C-1), 66.41
(C-4), 93.27 (C-3), 111.88 (C-5), 125.37 (C-8a), 127.01 (C-6), 128.16
(C-4a), 143.72 (C-7), and 152.00 (C-8). Minor diastereomer: 1H NMR
(δ) 2.16 (d, 1H, J ) 7.7 Hz, OH-3), 3.48 (d, 1H, J ) 3.0 Hz, OH-4),
3.86 (s, 3H, OMe), 3.98 (s, 3H, OMe), 4.44 (br dd, 1H, J ) 2.0 and
7.7 Hz, H-3), 4.84 (d, 1H, J ) 15.9 Hz, H-1), 4.97 (dd, 1H, J ) 2.0
and 3.0 Hz, H-4), 4.98 (d, 1H, J ) 15.9 Hz, H-1), 6.90 (d, 1H, J ) 8.5
Hz, H-5), and 7.19 (d, 1H, J ) 8.5 Hz, H-6); 13C NMR (δ) 55.70
(OMe-7), 59.58 (C-1), 60.03 (OMe-8), 67.91 (C-4), 95.12 (C-3), 111.79
(C-5), 124.48 (C-8a), 126.27 (C-6), 127.37 (C-4a), 143.64 (C-7), and
151.65 (C-8). HRMS for C11H14O5: calcd, 226.08413; found, 226.08383.
MATERIALS AND METHODS
The melting point (uncorrected) was taken on an Ernst Leitz Wetzlar
model 350 hot-stage microscope. Fourier transform infrared (FT-IR)
spectra were determined with a Bruker IFS 25 FT-infrared spectro-
photometer. The 1H and 13C NMR spectra were acquired with a Bruker
AC200-E spectrometer (200.13 MHz for 1H), employing CDCl3 as
solvent; the chemical shifts are expressed in parts per million (ppm)
downfield from the internal standard (TMS). High-resolution mass
spectral data were obtained from the Kent Mass Spectrometry Unit 1
(Kent, U.K.). Reagents and solvents were used as received; dry MeOH
was obtained by distillation from magnesium methoxide. Starting
glyoxylate 12 was prepared in 77% yield (three steps) from com-
mercially available (Aldrich Chemical Co.) 2,3-dimethoxytoluene (11)
by Friedel-Crafts acylation with ethyl oxalyl chloride, followed by
N-bromosuccinimide- (NBS-) mediated benzylic bromination and
subsequent nucleophilic displacement of the bromide with sodium
acetate in hexamethylphosphoramide (HMPA)-toluene (1:3) mixed
solvent (21). Flash column chromatographies were carried out with
silica gel 60 H and eluted with hexanes-EtOAc employing gradient
techniques. All new compounds gave single spots on thin-layer
chromatography (TLC) plates (Merck, art. 5554) run in different
hexanes-ethyl acetate solvent systems. Chromatographic spots were
detected by exposure to UV light (254 nm) followed by spraying with
ethanolic p-anisaldehyde/sulfuric acid reagent and careful heating of
the plates for better selectivity.
Ethyl {2-[(Acetoxy)methyl]-3,4-dimethoxyphenyl}(hydroxy)-
acetate (13). Under a nitrogen atmosphere, a solution of keto ester 12
(21) (1001.4 mg, 3.23 mmol) in absolute ethanol (40 mL) was
successively treated with glacial acetic acid (0.184 mL, 3.23 mmol)
and sodium cyanoborohydride (223.7 mg, 3.55 mmol), and the mixture
was stirred overnight at room temperature. The reaction was quenched
with 1 N NaOH (5 mL), diluted with brine (10 mL), and extracted
with EtOAc (3 × 30 mL). The combined organic extracts were washed
once with brine (5 mL), dried (Na2SO4), concentrated under reduced
pressure, and chromatographed, providing mandelate 13 (918 mg, 91%),
as an oil; Rf ) 0.33 (hexanes-EtOAc 1:1); IR (film, ν) 3460, 2980,
Effect of Compound 10 on the Germination of Tobacco Seeds.
The seeds were sterilized by exposure to sodium hypochlorite (1 mL
of a 1.6% solution) containing Tween 80 (0.05%) during 15 min,
followed by four washings with sterilized distilled water; subsequent
manipulations were carried out under a horizontal laminar flow. The
seeds were germinated in an environment-controlled growth room at
25 °C in Petri dishes (36 seeds/dish) containing 15 mL of MS0 agar
(22) and different concentrations of 10 and a control, employing a 16
h/8 h photoperiod. Fresh weight and percentage of germinated seeds
were measured after 30 days.
1
2850, 1740, 1730, 1600, 1500, 1460, 1280, 1090 and 810 cm-1; H
Effect of Compound 10 on Vegetative Growth of Tobacco Plants.
Two-week-old plants grown in MS0 medium were transferred to
Magenta boxes (4 seedlings/box) and exposed to different concentrations
of 10 (10-3-10-7 M). After 1 month, three plants of each box were
taken for analyses. Shoots and roots were homogenized to a fine powder
by treatment with liquid nitrogen, and the powders were suspended in
1.5 volumes of homogenization solution [50 mM Tris-HCl, pH 7.8, 5
mM MgCl2, 5 mM dithiotreitol, 5 mM ethylenediaminetetraacetic acid
(EDTA), and 1 mM phenylmethanesulfonyl fluoride (PMSF)]. Protein
levels were quantitated according to the spectrophotometric procedure
NMR (δ) 1.22 (s, 3H, J ) 8.6 Hz, OCH2Me), 1.60 (br s, 1H, OH),
2.07 (s, 3H, MeCO2), 3.84 (s, 3H, OMe), 3.86 (s, 3H, OMe), 4.20 (dq,
2H, J ) 1.0 and 8.6 Hz, OCH2Me), 5.35 (s, 2H, ArCH2O), 5.40 (br s,
1H, ArCHOH), 6.92 (d, 1H, J ) 8.6 Hz, ArH) and 7.10 (d, 1H, J )
8.6 Hz, ArH); 13C NMR (δ) 13.88 (MeCH2), 20.86 (MeCO2), 55.56
(OMe-7), 57.60 (ArCH2O), 61.20 (OMe-8), 61.97 (MeCH2O), 69.77
(ArCHOH), 112.71 (C-5), 122.97 (C-6), 128.03 (C-2), 130.88 (C-1),
148.46 (C-4), 152.65 (C-3), 170.73 (CdO), and 173.60 (CdO). HRMS
for C15H20O7: calcd, 312.12087; found, 312.12087.
4-Hydroxy-7,8-dimethoxyisochroman-3-one (10). A mixture of
mandelate 13 (250 mg, 0.80 mmol) and camphorsulfonic acid (93 mg,
0.4 mmol) in anhydrous MeOH (8 mL) was warmed to 40 °C and
stirred for 6 h. After cooling, the reaction mixture was diluted with
brine (10 mL) and extracted with EtOAc (3 × 10 mL); the organic
extracts were washed with brine (10 mL) and dried over Na2SO4.
Concentration of the solvent in a Vacuum afforded a residue, which
was purified by column chromatography to give R-hydroxylactone 10
(160 mg, 89%), as a white solid, mp 126-128 °C (hexanes-EtOAc);
Rf ) 0.38 (hexanes-EtOAc 1:1). IR (KBr, ν) 3466, 2980, 2870, 1734,
1496, 1456, 1380, 1276, 1258, 1206, 1142, 1086, 1024, 818, and 806
of Bradford against a standard of bovine serum albumin (E1%,1cm
)
6.67 at 279 nm) (23), while the protein profile was determined (25 µg
of protein/lane) by sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (SDS-PAGE) (12% polyacrylamide), followed by staining
with Coomassie Brilliant Blue (24). Proteins were denatured by
incubation during 10 min at 85 °C with a solution containing 60 mM
Tris-HCl, pH 6.8, 1.25% (v/v) 2-mercaptoethanol, 10% (v/v) glycerin,
2% (w/v) sodium dodecyl sulfate, and 0.15 mg/mL Bromophenol Blue.
Pigment levels (carotenes, chlorophyll a, and chlorophyll b) were
determined in ethanolic extracts according to Lichtenthaler (25).

Isochromanone with Plant Growth-Regulating Properties
J. Agric. Food Chem., Vol. 52, No. 7, 2004 1925
Scheme 1a
Scheme 2a
a Reagents and conditions: (a) NaCNBH , AcOH , EtOH, RT, overnight
(91%); (b) camphorsulfonic acid (5 mol %), MeOH, 40 °C, 6 h (89%); (c)
camphorsulfonic acid (5 mol %), MeOH, 40 °C, 6 h (100%).
a Reagents and conditions: (a) (1) EtO CCOCl, AlCl3, CH Cl2, 0°C (94%);
3
gl
2
2
(2) NBS, CCl4, reflux, 90 min (91%); (3) NaOAc, Ac2O, HMPA−PhMe (1:3),
RT, overnight (90%), see ref 21; (b) (1) NaBH , MeOH, K CO , NaOH; (2) dil
4
2
3
HCl, pH ) 2 (14, 24%; 10, 37%).
Table 1. Effect of 10 on the Germination of Tobacco Seeds
Optical Microscopic Observation of the Leaves. The anatomical
study was performed on leaves that were fixed in formaldehyde-acetic
acid-50% ethanol (1:1:18 v/v/v) solution, dehydrated through a graded
n-butanol series (50%, 70%, 85%, 95%, 100%), and embedded in
paraffin wax, according to standard methods. Transversal sections, 6-8
µm thick, of the leaf were then cut, stained with Cresyl Violet, and
mounted in Canada balsam (26, 27). The photomicrographs were taken
with the aid of a Zeiss Axiolab microscope fitted with an MC 80
camera.
concn of 10a (M)
fresh wtb (mg)
% germinationc
control
81.1 ± 5.8
20.6 ± 8.3
25.0 ± 17.0
83 ± 5
82 ± 10
76 ± 11
1 × 10-5
1 × 10-3
a Tobacco seeds were germinated in Murashige−Skoog (MS0) agar (22)
supplemented or not with the indicated concentrations of 10. Experiments were
carried out in duplicate. b Seedlings (shoots and roots) were withdrawn at 4 weeks
after the seeds were planted, extensively rinsed to remove agar debris, and weighed.
Means and standard deviations are expressed per Petri dish. c With reference to
36 ± 5 seeds per Petri dish.
RESULTS AND DISCUSSION
Numerous synthetic strategies have been put in practice for
the elaboration of molecules carrying the isochroman skeleton
(28); however, few of them allow the direct synthesis of
isochroman-3-ones (29, 30) and only scattered procedures yield
4-hydroxyisochroman derivatives (31-35). Furthermore, oxida-
tion of the C-4 position of the isochroman skeleton is a difficult
task due to the ease with which C-1 undergoes oxidation (36,
37).
Table 2. Number of Leaves, Fresh Weight, and Total Foliar Area of
Tobacco Plants Grown in MS0 Medium Containing Different
Concentrations of 10a
concn of 10b (M)
no. of leaves
fresh wtc (g)
total foliar area (cm )
2
control
9
9
9
8
0.83 ± 0.04
0.74 ± 0.00
0.44 ± 0.18
0.24 ± 0.10
111.3
102.4
45.8
1 × 10-7
1 × 10-5
1 × 10-3
23.7
We decided to develop a strategy in which the 4-hydroxy
substituent is introduced early in the synthesis and the hetero-
cyclic ring containing the lactone moiety is accessed by acid-
catalyzed lactonization of an appropriate precursor. This pro-
vided an extremely simple and straightforward synthesis of the
desired compound 10. In a first approach depicted in Scheme
1, submission of the easily accessible glyoxylate 12 (21) to
reduction with sodium borohydride in dry methanol, to which
1.3 equiv of potassium carbonate and 0.45 equiv of NaOH had
been added, directly furnished the desired R-hydroxylactone 10,
presumably through the intermediacy of mandelate 13. However,
the isolated yield was only 37% and the expected R-hydroxyl-
actone required separation from more polar overreduction
products, such as the R-hydroxylactol 14, produced in 24%
yield.
Therefore, a two-step synthesis was devised. Initially, and in
order to avoid overreduction, glyoxylate 12 was selectively
converted into mandelate 13 in 91% yield with sodium
cyanoborohydride in absolute EtOH to which acetic acid was
added, as shown in Scheme 2; then, compound 13 was submitted
to a camphorsulfonic acid-promoted lactonization in dry MeOH,
smoothly affording R-hydroxylactone 10 as the sole product in
89% isolated yield.
a Two-week-old plants were treated during 1 month. b Experiments were run in
duplicate. c Fresh weight corresponds to the whole plant.
acid promotion resulted exclusively in transesterification of the
starting material to the related methyl ester 15, which was unable
to cyclize under the acidic conditions tested. On the other hand,
basic hydrolysis of both ester groups of 12 followed by acidic
workup also failed to give the expected cyclized product.
Therefore, the route shown in Scheme 2 can be considered as
the simplest and most efficient one to access 10.
To study the activity of compound 10, its effect on the
germination of tobacco seeds was next evaluated. Analysis of
the results collected in Table 1 evidenced that although 10 had
no statistically significant effect on the percentage of germinat-
ing seeds (76% ( 11% in the presence of 10-3 M 10 vs 83%
( 5% in the control lot), a clear growth inhibition effect was
observed after germination. At 30 days, the fresh weight of the
plants exposed to 10-5 or 10-3 M 10 was 70-75% lower than
that of the corresponding controls.
In a second series of experiments, vegetative development
was studied in 2-week-old plants, grown in MS0 agar devoid
of 10. After exposure of the plants to the R-hydroxylactone at
concentrations ranging from 10-3 to 10-7 M during 1 month,
analysis of morphologic variables indicated important differ-
ences in fresh weight and total foliar area, as shown in Table 2.
It was observed that reduction of starting glyoxylate 12 to
mandelate 13 must be carried out before the cyclization step,
since attempts to lactonize 12 in MeOH under camphorsulfonic

1926 J. Agric. Food Chem., Vol. 52, No. 7, 2004
Bianchi et al.
Figure 1. Morphological changes in leaves of tobacco plants (top) exposed to different concentrations of compound 10 and detailed views of the primary
veins of the above samples (bottom). Left, control specimen; center, leaf of a plant exposed to 10 at a concentration of 10-5 M; right, leaf of a plant
exposed to compound 10 at a concentration of 10-3 M.
intercellular spaces and the abaxial epidermis also presents
stomata; in addition, the primary vein, with a mean diameter
of 100-110 µm, is well-developed in the abaxial face, having
mature vessels of 10-12 µm in diameter.
Table 3. Determination of Pigment Levels of Plants Exposed to
Different Concentrations of 10
chlorophyll a
(µg/mg of
fresh weight)
chlorophyll b
(µg/mg of
fresh weight)
carotenes
(µg/mg
of fresh weight)
proteins
(µg/mg
of fresh weight)
concn of
10 (M)
When leaves of plants exposed to 10-5 M compound 10 were
examined (center), images of the same zone revealed distinct
cell deterioration and abnormal features, including disorganiza-
tion of the parenchymatous structure and distortion of the cell
wall. In addition, the primary vein size showed a slightly reduced
size, with a mean diameter of 80-85 µm, and presented scanty
vessels, transversed by a narrow ray of parenchyma. The mature
vessel size was also reduced to a mean diameter of 7-8 µm.
control
10-7
10-5
10-3
1.05 ± 0.25
1.23 ± 0.01
1.21 ± 0.11
0.84 ± 0.13
0.34 ± 0.11
0.37 ± 0.02
0.39 ± 0.02
0.26 ± 0.06
0.21 ± 0.04
0.25 ± 0.01
0.25 ± 0.04
0.20 ± 0.04
2.44 ± 0.25
2.78 ± 0.00
2.72 ± 0.20
2.22 ± 0.82
Values of 0.44 ( 0.18 and 0.24 ( 0.10 g were recorded for the
fresh weight of plants exposed to concentrations of the test
compound of 10-5 and 10-3 M, respectively, compared to 0.83
( 0.04 g in the control plants, while the foliar area measured
Furthermore, when leaves from plants exposed to 10-3
M
10 were evaluated, it was observed that the cross-sections of
the leaves showed a large parenchymatous mesophyll, with
chloroplasts firmly pressed against the plasmalemma and the
wall by a notoriously large central vacuole. A slight decline in
the number and pigmentation of the chloroplasts was also
detected. In addition, it was observed that the primary vein was
composed of minute groups of vessels of small size, having a
mean diameter of 50 µm, with the mature vessel size of only
5-6 µm in diameter.
It has been reported that certain isochroman derivatives
behave as auxin transport inhibitors (5-8, 12-14), producing
similar effects on different model systems; therefore, the
observed growth changes may be a result of the auxin transport
inhibiting activity of R-hydroxylactone 10, which, despite
lacking a C-1 substituent, was active at lower doses than
1-substituted isochroman-3-ones (12).
In conclusion, a short and efficient synthesis of R-hydroxyl-
actone 10 from the commercially available 2,3-dimethoxytoluene
was described. Compound 10 inhibited the vegetative growth
of tobacco seedlings, leading to lower fresh weight of the treated
plants but without affecting protein and pigment levels. Changes
observed are compatible with inhibition of cellular division,
resulting in severe limitations in the enlargement of the
meristematic tissue. The collected results suggest that compound
10 may act as an auxin transport inhibitor.
for plants grown in media containing 10-5 and 10-3
M
R-hydroxylactone 10 were 45.8 and 23.7 cm2, respectively; this
represents 41% and 21.3% of the foliar area of the control plants.
On the other hand, the protein profiles, as well as the levels
of total proteins (23), carotenes, and chlorophylls (a and b) (24)
were also determined (Table 3), and no statistically significant
differences were observed between the seedlings grown in media
containing compound 10, at a concentration of 10-3 M, and
the corresponding controls.
Then, it can be concluded that treatments did not affect
important metabolic pathways such as the synthesis of the major
proteins and pigments, even at the highest concentration of the
compound. The lack of effect on germination (Table 1) or in
the number of leaves per plant (Table 2) also suggest that the
developmental pattern was not affected, with the toxicity being
limited to a significant inhibition of vegetative growth (cell
division and/or expansion) that prevented plants from entering
the flowering stage.
When the leaves were microscopically examined, morpho-
logical changes were clearly distinguished, as shown in the
Figure 1. In the control specimen (left), the cross-sections of
the leaves displayed their normal structure; dorsiventrally
compressed, with a unistratified adaxial epidermis with well-
developed cells. They also showed a smooth cuticle with
stomata, the mesophyll was dorsiventral, and only one layer of
palisade parenchyma was observable, with cells rich in chlo-
roplasts, firmly pressed against the plasmalemma and the wall
by a central vacuole. It was also detected that the spongy
mesophyll contains laxly arranged cells widely separated by
ACKNOWLEDGMENT
We are also indebted to Dr. Martha A. Gattuso for the
microscopy work.

Isochromanone with Plant Growth-Regulating Properties
J. Agric. Food Chem., Vol. 52, No. 7, 2004 1927
LITERATURE CITED
(20) de Koning, C. B.; Green, I. R.; Michael, J. P.; Oliveira, J. R. A
novel synthesis of substituted 4-hydroxybenzo[c]pyran quinones.
Tetrahedron Lett. 1997, 38, 5055-5056.
(21) Kaufman, T. S. Sulfonamidoacetal cyclization-based synthesis
of a tetrahydrooxazaphenalene lactone related to the ABC-ring
system of the stephaoxocanes. Heterocycles 2001, 55, 323-
330.
(22) Murashige, T.; Skoog, F. A revisited medium for rapid growth
and bioassays with tobacco tissue cultures. Physiol. Plant. 1962,
15, 473-497.
(23) Bradford, M. M. A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal. Biochem. 1976, 72, 248-254.
(24) Ausubel, F. M.; Brent, R.; Kingston, R. E.; Moore, D. D.;
Seidman, J. G.; Smith, J. A.; Struhl, K. Current Protocols in
Molecular Biology; John Wiley & Sons: New York, 1987.
(25) Lichtenthaler, H. K. Chlorophylls and carotenoids: pigments of
photosynthetic biomembranes. Methods Enzymol. 1987, 148,
350-382.
(26) O’Brien, P. T.; McCully, M. E. The Study of Plant Structure.
Principles and Selected Methods; Termacarphi, Pty. Ltd., Mel-
bourne, Australia, 1981; p 199.
(27) Ruzin, S. Plant Microtechnique and Microscopy; Oxford Uni-
versity Press: New York, 1999; p 187.
(1) Satomura, Y.; Sato, A. Isolation and physiological activity of
sclerin, a metabolite of Sclerotinia fungus. Agric. Biol. Chem.
1965, 29, 337-344.
(2) Barber, J. A.; Garson, M. J.; Staunton, J. The biosynthesis of
fungal metabolites: sclerin, a plant growth hormone from
Sclerotinia sclerotiorum. J. Chem. Soc., Perkin Trans. 1 1981,
2584-2593.
(3) Shimada, A.; Kusano, M.; Takeuchi, S.; Fujioka, S.; Inokuchi,
T.; Kimura, S. Aspterric acid and 6-hydroxymellein, inhibitors
of pollen development in Arabidopsis thaliana, produced by
Aspergillus terreus. Z. Naturforsch. 2002, 57c, 459-464.
(4) Stadler, M.; Anke, H.; Sterner, O. Metabolites with nematicidal
and antimicrobial activities from the ascomycete Lachnum
papyraceum (Karst.) Karst. III. Production of novel isocoumarin
derivatives, isolation, and biological activities. J. Antibiot. 1995,
48, 261-266.
(5) Cutler, H. G.; Arrendale, R. F.; Cole, P. D.; Davis, E. E.; Cox,
R. H. 3,7-Dimethyl-8-hydroxy-6-methoxyisochroman from Peni-
cillium corylophilium: plant growth regulatory activity. Agric.
Biol. Chem. 1989, 53, 1975-1978.
(6) Cox, R. H.; Hernandez, O.; Dorner, J. W.; Cole, R. J.; Fennel,
D. I. A new isochroman micotoxin isolated from Penicillium
steckii. J. Agric. Food Chem. 1979, 27, 999-1001.
(7) Cutler, H. G.; Majetich, G.; Tian, X.; Spearing, P. Synthesis of
3,7-dimethyl-8-hydroxy-6-methoxyisochroman, the 3,7-dimethyl-
6-hydroxy-8-methoxy isomer, and their ester and ether deriva-
tives: plant growth regulatory activity. J. Agric. Food Chem.
1997, 45, 1422-1429.
(8) Cutler, H. G.; Majetich, G.; Tian, X.; Spearing, P. Synthesis of
isochromans and their derivatives. U.S. Patent 5,922,889; Chem.
Abstr. 1998, 131, 73559x.
(9) Michel, A. Me´tabolites secondaires d’Ophiostoma noVo-ulmi et
de Ceratocystis fimbriata sp. platani, pathoge`nes de l’orme et
du platane. Thesis, Universite´ de Neuchaˆtel, 2001; available at
http://www.unige.ch/cyberdocuments/unine/theses2001/MichelA/
these_front.html.
(28) Markaryan, E. A.; Samodurova, A. G. Advances in the chemistry
of isochroman. Russ. Chem. ReV. 1989, 58, 479-493.
(29) Kraiss, G.; Povarny, M.; Scheiber, P.; Nador, K. Selective
reduction of diethyl homophthalate with diisobutylaluminum
hydride. Tetrahedron Lett. 1973, 2359-2360.
(30) Yoneda, E.; Kaneko, T.; Zhang, S.-W.; Takahashi, S. Selective
synthesis of 3-isochromanones by rhodium-catalyzed carbonyl-
ation of 2-alkynylbenzyl alcohols under water-gas shift reaction
conditions. Tetrahedron Lett. 1998, 39, 5061-5064.
(31) Giles, R. G. F.; Joll, C. A. Complementary diastereoselectivity
in the intermolecular addition of titanium and magnesium
naphtholates to asymmetric lactaldehydes. J. Chem. Soc., Perkin
Trans. 1 1999, 3029-3038, and references therein.
(32) Giles, R. G. F.; Green, I. R.; Oosthuizen, F. J.; Taylor, C. P.
Synthesis of asymmetric 2-benzopyrans. The influence of
aromatic halogen substituents on the intramolecular cyclisation
of enantiopure tethered phenolic lactaldehydes. ArkiVoc 2002,
x, 99-116.
(33) Echarri, R.; Matheu, M. I.; Castillo´n, S. Isochroman “versus”
O-glycoside synthesis. A study of the reaction of 2-alkoxy-2-
fluoro-glycosyl fluorides with alcohols. Tetrahedron 1994, 50,
9125-9134.
(34) Bianchi, D. A.; Ru´a, F.; Kaufman, T. S. Studies on the
intramolecular oxa-Pictet-Spengler rearrangement of 5-aryl-1,3-
dioxolanes to 4-hydroxy-isochromans. Tetrahedron Lett. 2004,
45, 411-415.
(35) Bianchi, D. A.; Cipulli, M. A.; Kaufman, T. S. Model studies
towards stephaoxocanes: Construction of the 2-oxa-4-azaphen-
alene core of stephaoxocanidine and eletefine. Eur. J. Org. Chem.
2003, 4731-4736.
(36) Xu, Y.-C.; Lebeau, E.; Gillard, J. W.; Attardo, G. Regio- and
stereoselective, DDQ-induced oxidative coupling of isochromans
and isothiochroman with alcohols. Tetrahedron Lett. 1993, 34,
3841-3844.
(37) Xu, Y.-C.; Kohlman, D. T.; Liang, S. X.; Erikkson, C. Stereo-
selective, oxidative C-C bond coupling of naphthopyran induced
by DDQ: stereocontrolled total synthesis of deoxyfrenolicin.
Org. Lett. 1999, 1, 1599-1602.
(10) Ases, S.; Cathey, H. M.; Stuart, N. W. Enhancement of
gibberellin growth promoting activity by hydrangenol isolated
from leaves of Hydrangea macrophylla. Plant Physiol. 1960,
35, 816-819.
(11) Cathey, H. M. Physiology of growth retarding chemicals. Annu.
ReV. Plant Physiol. 1964, 15, 271-302.
(12) Watanabe, K.; Taniguchi, E. Systematic syntheses and structure-
activity relationships of substituted and nonsubstituted isochro-
man-3-ones as plant growth regulators. J. Pestic. Sci. 1994, 19,
209-219.
(13) Watanabe, K.; Taniguchi, E. Syntheses and plant growth
regulating activity of 2-substituted phenylacetic acid derivatives.
Agric. Biol. Chem. 1986, 50, 2047-2052.
(14) Watanabe, K.; Taniguchi, E. Syntheses of aryl isoxazolylphen-
ylacetic acid and aryl isoquinolinones and their plant growth-
regulating activities. Agric. Biol. Chem. 1986, 50, 2591-
2595.
(15) Jacobs, M.; Rubery, P. H. Naturally occurring auxin transport
regulators. Science 1988, 241, 346-349.
(16) Winkel-Shirley, B. Biosynthesis of flavonoids and effects of
stress. Curr. Opin. Plant Biol. 2002, 5, 218-223.
(17) Faulkner, I. J.; Rubery, P. H. Flavonoids and flavonoid sulphates
as probes of auxin-transport regulation in Cucurbita pepo
hypocotyl segments and vesicles. Planta 1992, 186, 618-625.
(18) Brown, D. E.; Rashotte, A. M.; Murphy, A. S.; Normanly, J.;
Tague, B. W.; Peer, W. A.; Taiz, L.; Muday, G. K. Flavonoids
act as negative regulators of auxin transport in ViVo in Arabi-
dopsis thaliana. Plant Physiol. 2001, 126, 524-535.
(19) Tobler, H.; Szczepanski, H.; Fory, W. Preparation of heterocyclic
compounds as new antidotes in herbicidal compositions. Euro-
pean Patent WO 97-EP5252; Chem. Abstr. 1998, 128, 257333p.
Received for review September 30, 2003. Revised manuscript received
January 30, 2004. Accepted February 2, 2004. The authors gratefully
acknowledge CONICET, ANPCyT, SECyT-UNR, and Fundacio´n An-
torchas for financial support. D.A.B. and N.E.B. thank CONICET for
their fellowships.
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