Synthesis, characterization and phytotoxic activity of hydroxylated isobenzofuran-1(3H)-ones

Article abstract of DOI:10.1016/j.molstruc.2013.12.059

Two hydroxylated isobenzofuranones 3 and 4 were synthesized from benzoic acids. The compounds were fully characterized by IR, NMR (¹H and ¹³C), HRMS, and X-ray crystallography. Compounds 3 and 4 crystallized in the space group Pc and

Full text of DOI:10.1016/j.molstruc.2013.12.059

Journal of Molecular Structure 1061 (2014) 61–68
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Synthesis, characterization and phytotoxic activity of hydroxylated
isobenzofuran-1(3H)-ones
a
a
b,
b
c
d
R.R. Teixeira a, , J.L. Pereira , S.F. Da Silva , S. Guilardi , D.A. Paixão , C.P.A. Anconi , W.B. De Almeida ,
⇑
⇑
J. Ellena e, G. Forlani f
a Departmento de Química, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil
b Instituto de Química, Universidade Federal de Uberlândia, Uberlândia, Minas Gerais, Brazil
c Departamento de Química, Universidade Federal de Lavras, Lavras, Minas Gerais, Brazil
d Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
e Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil
f Department of Life Science and Biotechnology, University of Ferrara, Ferrara, Italy
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
ꢀ
ꢀ
ꢀ
ꢀ
Compounds 3 and 4 crystallized in
the space group Pc and P21/n,
respectively.
DFT calculations confirmed
undoubtedly their NMR chemical
shifts.
A good agreement was also obtained
for B3LYP/6-31G(d,p) theoretical and
experimental IR spectra.
Isobenzofuran-1(3H)-ones 3 and 4
interfere with the radicle growth of
monocotyledonous and
O
HO
O
3
O
O
HO
dicotyledonous species.
4
a r t i c l e i n f o
a b s t r a c t
Article history:
Two hydroxylated isobenzofuranones 3 and 4 were synthesized from benzoic acids. The compounds were
fully characterized by IR, NMR (1H and 13C), HRMS, and X-ray crystallography. Compounds 3 and 4
crystallized in the space group Pc and P21/n, respectively. DFT calculations were used to confirm
undoubtedly their NMR chemical shifts. Biological assays showed that these compounds are capable of
interfering with the radicle growth of monocotyledonous and dicotyledonous species, whereas the
photosynthetic electron transport chain was substantially unaffected.
Received 17 September 2013
Received in revised form 17 December 2013
Accepted 18 December 2013
Available online 2 January 2014
Keywords:
Isobenzofuran-1(3H)-ones
Phthalides
Ó 2013 Elsevier B.V. All rights reserved.
X-ray analysis
DFT calculations
Phytotoxicity
1. Introduction
Several natural and synthetic compounds possess a
c-butirolac-
⇑
Corresponding authors. Tel.: +55 31 3899 3209/34 3239 4444; fax: +55 31 3899
3065/34 3239 4208.
tone fused to benzene ring. These substances, known as
a
isobenzofuran-1(3H)-ones (phthalides), have attracted attention
of various research groups due to their wide range of medicinal
E-mail addresses: robsonr.teixeira@ufv.br (R.R. Teixeira), silvana@ufu.br
(S. Guilardi).
0022-2860/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2013.12.059

62
R.R. Teixeira et al. / Journal of Molecular Structure 1061 (2014) 61–68
O
O
O
Pd(OAc)2 (10 mol%)
7
1
8
9
H3CO
6
H3CO
OCH3
H3CO
COOH
K2HPO4 (3 equiv.), 140 oC
CH2Br2, sealed tube
O
O
2
O
5
+
3
4
1b
1
BBr3, CH2Cl2
0 o
r.t.
C
O
7
4
1
8
6
HO
2
O
5
9
3
3
O
O
O
Pd(OAc)2 (10 mol%)
COOH
KHCO3 (3 equiv.), 140 o
CH2Br2, sealed tube
C
O
O
O
+
H3CO
H3CO
H3CO
OCH3
2
2b
BBr3, CH2Cl2
0 o
r.t.
C
O
O
HO
4
Fig. 1. Synthesis of hydroxylated isobenzofuranones 3 and 4.
Table 1
Experimental and theoretical (B3LYP/6-311+G(2d,p) 13C NMR and 1H NMR chemical shifts (in ppm), evaluated with respect to TMS, for 6-hydroxyisobenzofuran-1(3H)-one 3 and
5-hydroxyisobenzofuran-1(3H)-one 4. Methanol was used as solvent.
Compound
Carbon
3
4
Expt.
Theoretical
Deviationa (%)
Expt.
Theoretical
Deviationa (%)
13C NMR assignments
C-1
C-3
C-4
C-5
C-6
C-7
C-8
C-9
172.5
70.1
176.8
70.2
2.5
0.1
2.0
0.4
2.4
2.1
2.9
4.0
173.7
71.3
178.0
71.3
2.4
0.0
0.6
5.8
3.3
3.0
2.4
13.1
123.3
122.8
158.6
109.7
126.6
138.3
125.8
123.3
162.4
112.1
130.2
143.8
110.9
159.8
123.9
127.7
124.4
139.5
110.2
169.1
119.8
131.5
121.4
157.8
1H NMR assignments
H-3
H-4
H-5
H-6
H-7
5.25
7.18
7.15
–
5.25
7.55
7.25
–
0.0
5.1
1.4
–
5.25
6.9
–
6.95
7.67
5.25
6.97
–
7.17
7.90
0.0
1.0
–
3.2
3.1
7.39
7.44
0.7
a
Percent deviation between experimental and theoretical values (|expt ꢁ theor|/|expt|) ꢂ 100.
properties which include antiproliferative [1], anticonvulsant [2],
antioxidant [3], antiplatelet [4] and anti-HIV [5] activities, and
the ability to inhibit ATP synthesis in the chloroplast [6]. Phtha-
lides are also valuable synthetic intermediates such as in the prep-
aration of phenolic derivatives via Houser annulation [7] and in the
synthesis of 4,8-dihydroxyisochromanones [8].
In addition to the study of their biological properties [9,10], we
are also interested in elucidating the molecular features of iso-
benzofuran-1(3H)-ones [11,12]. In this view, herein we describe
the results of a structural investigation of hydroxylated phthalides
employing a combination of NMR analyses, XRD techniques and
DFT calculations. Their phytotoxic activities were also assessed.

R.R. Teixeira et al. / Journal of Molecular Structure 1061 (2014) 61–68
63
a pad of Celite. The filtrate was concentrated under reduced pres-
sure and the residue was purified by silica gel column chromatog-
raphy (hexane–ethyl acetate 2:1 v/v) to afford the title compound
in 59% yield (681 mg, 4.15 mmol) as a white solid. M.p. 116.9–
118.4 °C. IR (selected bands, cmꢁ1): 3003, 2925, 2837, 1733,
1600, 1585, 1488, 1454, 1431, 1267, 1206, 1028, 973, 869, 749,
690, 545. 1H NMR (300 MHz, MeOH-d4) d: 3.87 (s, 3H, AOCH3),
5.26 (s, 2H, H-3), 7.22–7.38 (m, 3H, H-4, H-5 and H-7); 13C NMR
(75 MHz, MeOH-d4) d: 56.0 (AOCH3), 69.7 (C-3), 107.7 (C-7),
123.1 (C-5), 123.3 (C-4), 127.3 (C-8), 139.1 (C-9), 160.8 (C-6),
171.4 (C-1). HREIMS m/z (M+H+): Calcd for C9H8O3, 165.0552;
found: 165.0608.
2. Experimental
2.1. Synthesis
2.1.1. Materials and methods
Commercially available 3-methoxy benzoic acid, 4-methoxy
benzoic acid, palladium(II) acetate, dibromomethane, potassium
dihydrogen phosphate, potassium bicarbonate, boron tribromide
1.0 mol Lꢁ1 solution in dichloromethane were purchased from Sig-
ma–Aldrich and utilized without further purification. Dichloro-
methane was purified as described in Perrin and Armarego [13].
Infrared spectra were recorded on a Varian 660-IR, equipped with
GladiATR scanning from 4000 to 500 cmꢁ1. HRMS data were re-
corded under ESI conditions on a micrOTOF—QII Bruker spectrom-
eter. Melting points are uncorrected and were obtained with a
MQAPF-301 melting point apparatus (Microquimica, Campinas,
Brazil). Analytical thin layer chromatography was carried out on
TLC plates recovered with 60GF254 silica gel. Column chromatog-
raphy was performed over silica gel (60–230 mesh).
2.1.3. Synthesis of 5-methoxy isobenzofuran-1(3H)-one 2
A tube of 40 mL equipped with a magnetic stir bar was charged
with palladium(II) acetate (67.3 mg, 0.30 mmol), potassium bicar-
bonate (750 mg, 7.50 mmol), 4-methoxybenzoic acid (456 mg,
3.00 mmol) and dibromomethane (12 mL). The tube was sealed
with a Teflon cap and the reaction mixture was stirred at 140 °C
for 18 h. After this time, the mixture was filtered through a pad
of Celite. The filtrate was concentrated under reduced pressure
and the residue was purified by silica gel column chromatography
eluted with hexane–ethyl acetate (2:1 v/v) to afford 5-meth-
oxyisobenzofuran-1(3H)-one 2 in 33% yield (164 mg, 1.00 mmol).
M.p. 113.4–114.7 °C. IR (selected bands, cmꢁ1): 3032, 2922, 2852,
1736, 1601, 1489, 1452, 1361, 1333, 1261, 1146, 1036, 986, 773.
1H NMR (300 MHz, CDCl3) d: 3.89 (s, 3H, AOCH3), 5.25 (s, 2H,
2.1.2. Synthesis of 6-methoxy isobenzofuran-1(3H)-one 1
A tube of 40 mL equipped with a magnetic stir bar was charged
with palladium (II) acetate (156.8 mg, 0.70 mmol), potassium dihy-
drogen phosphate (3654 mg, 21.0 mmol), 3-methoxy benzoic acid
(1064 mg, 7.00 mmol) and dibromomethane (28 mL). The tube
was sealed with a Teflon cap and the reaction mixture was stirred
at 140 °C for 36 h. After this time, the mixture was filtered through
6-hydroxy isobenzofuran: Theoretical IR spectrum
5-hydroxy isobenzofuran: Theoretical IR spectrum
100
100
C=C
50
50
C=C
C=O
0
0
C=O
2400 2300 2200 2100 2000 1900 1800 1700 1600
Frequency / cm -1
2400
2300 2200 2100 2000 1900 1800 1700 1600
Frequency / cm -1
(a)
(b)
6-hydroxy isobenzofuran: Experimental IR spectrum
100
5-hydroxy isobenzofuran: Experimental IR spectrum
90
C=C
C=O
80
C=C
60
C=O
2400 2300 2200 2100 2000 1900 1800 1700 1600
Frequency / cm-1
2400 2300 2200 2100 2000 1900 1800 1700 1600
Frequency / cm-1
(c)
(d)
Fig. 2. B3LYP/6-31G(d,p) theoretical (a and b) and experimental (c and d) IR spectra for compounds 3 and 4.

64
R.R. Teixeira et al. / Journal of Molecular Structure 1061 (2014) 61–68
Fig. 3. ORTEP drawings of compounds 3 and 4 with the atom numbering scheme. Displacement ellipsoids for non-H atoms were drawn at the 30% probability level.
Table 2
Crystallographic data and details of diffraction experiments for the compounds 3 and 4.
Compound
3
4
Empirical formula
Formula weight (g molꢁ1
Temperature (K)
Crystal system
C8H6O3
150.13
100(2)
Monoclinic
Pc
C8H6O3
150.13
293(2)
Monoclinic
P21/n
)
Space group
Unit cell dimensions (Å,°)
a
7.066(6)
7.6506(15)
b
3.880(3)
12.349(4)
c
12.445(11)
7.8409(11)
b
105.85(5)
328.2(5), 2
1.519
0.996
117.453(10)
657.3(2), 4
1.517
0.118
Volume (Å3), Z
Calculated density (g cmꢁ3
)
Absorption coefficient (mmꢁ1
F(000)
)
156
312
Crystal size (mm)
2h range for data collection (°)
Limiting indices
0.28 ꢂ 0.16 ꢂ 0.11
0.24 ꢂ 0.11 ꢂ 0.07
6.51–63.14
3.08–27.47
ꢁ7, 8; ꢁ4.4; ꢁ12, 11
1111/754 [R(int) = 0.0346]
1.171
ꢁ9.9; ꢁ15.15; ꢁ10.9
5950/1461 [R(int) = 0.0338]
1.039
Reflections collected/unique
Goodness-of-fit on F2
Data/restraints/parameters
754/2/100
1461/0/100
Final R indices [I > 2
R indices (all data)
r
(I)]
R = 0.0451, wR = 0.1160
R = 0.0457, wR = 0.1199
0.198 and ꢁ0.200
R = 0.0533, wR = 0.1430
R = 0.0807, wR = 0.1633
0.198 and ꢁ0.193
Largest difference peak and hole (e Aꢁ3
)
1=2
R ¼
R
ðjjFoj ꢁ jFcjÞ=
R
jFoj; wR ¼ ½
R
wðjF2o j ꢁ jF2c jÞ2=
R
wjF2oj ꢃ
.
2
H-3), 6.91 (d, 1H, J = 0.6 Hz, H-4), 7.02 (dd, 1H, J = 8.4, 0.6 Hz, H-6),
7.80 (d, 1H, J = 8.4 Hz, H-7). 13C NMR (75 MHz, CDCl3) d 56.1
(AOCH3), 69.3 (C-3), 106.2 (C-4), 116.7 (C-6), 118.2 (C-8), 127.4
(C-7), 149.6 (C-9), 164.9 (C-5), 171.1 (C-1). HREIMS m/z (M+H+):
Calcd for C9H8O3, 165.0552; found: 165.0606.
0.56 mmol). Suitable crystals for XRD analysis were obtained by
slow crystallization from water. M.p. 198.6–201.3 °C. IR (selected
bands, cmꢁ1): 3252 (broad band), 2959, 2922, 2851, 1726, 1623,
1599, 1491, 1447, 1366, 1312, 1052, 990, 771, 542 cmꢁ1 1H NMR
.
(300 MHz, MeOH-d4) d: 5.25 (s, 2H, H-3), 7.15–7.18 (m, 2H, H-5
and H-4), 7.39 (d, 1H, J = 9.0 Hz, H-7). 13C NMR (75 MHz, MeOH-
d4) d: 70.1 (C-3), 109.7 (C-7), 122.8 (C-5), 123.3 (C-4), 126.6
(C-8), 138.3 (C-9), 158.6 (C-6), 172.5 (C-1). HREIMS m/z (M+H+):
Calculated for C8H7O3, 151.0395; found: 151.0370.
2.1.4. Synthesis of 6-hydroxy isobenzofuran-1(3H)-one 3
A 25 mL two necked round bottom flask, under nitrogen atmo-
sphere, was charged with 6-methoxy isobenzofuran-1(3H)-one 1
(100 mg, 0.61 mmol) and anhydrous dichloromethane (5 mL). The
resulting mixture was magnetically stirred and cooled in ice bath
for 10 min. After that, 1.8 mL of 1.0 mol Lꢁ1 BBr3 solution in dichlo-
romethane (1.8 mmol) was added dropwise. Then, the reaction
mixture under continuous magnetic stirring was allowed to warm
up to room temperature. Subsequently, 5 mL of distilled water
were added and the mixture was transferred to a separatory funnel
and extracted with ethyl acetate (3 ꢂ 25 mL). The combined organ-
ic extracts were dried over magnesium sulphate, filtrated and con-
centrated under reduced pressure. The residue was purified by
column chromatography eluted with hexane–ethyl acetate (2:1
v/v) to afford compound 3 as yellow solid in 92% yield (84 mg,
2.1.5. Synthesis of 5-hydroxy isobenzofuran-1(3H)-one 4
Compound 4 was synthesized in 83% yield using a similar pro-
cedure to that described for compound 3. Structure of phtalide 4 is
supported by the data described below. Suitable crystals for XRD
analysis were obtained by slow crystallization from acetone. Pale
yellow solid. M.p. 226.8–227.3 °C. IR (selected bands, cmꢁ1):
3261 (broad band), 2925, 2855, 1714, 1597, 1432, 1340, 1275,
1057, 1007, 937 cmꢁ1 1H NMR (300 MHz, MeOH-d4) d: 5.25 (s,
.
2H; H-3), 6.95–6.90 (m, 2H; H-6 and H-4); 7.67 (d, J = 9.0 Hz, 1H,
H-7). 13C NMR (75 MHz, MeOH-d4) d: 71.3 (C-3), 110.9 (C-4),
123.9 (C-6), 124.4 (C-8), 127.7 (C-7), 139.5 (C-9), 159.8 (C-5),

R.R. Teixeira et al. / Journal of Molecular Structure 1061 (2014) 61–68
65
Table 3
173.7 (C-1). HREIMS m/z (M+H+): Calculated for C8H7O3, 151.0395;
Bond lengths (Å) and angles (°) for compounds 3 and 4.
found: 151.0369.
Compound
3
4
Expt.
Theor.
Expt.
Theor.
2.2. X-ray Crystallography
Bond lengths
O(2)AC(1)
O(2)AC(2)
O(1)AC(1)
C(1)AC(8)
C(8)AC(3)
C(8)AC(7)
C(7)AC(6)
C(6)AC(5)
C(5)AC(4)
C(4)AC(3)
C(3)AC(2)
O(3)AC(6)
O(3)AC(5)
1.350(4)
1.463(5)
1.211(5)
1.464(5)
1.379(5)
1.387(4)
1.387(5)
1.397(6)
1.404(5)
1.394(5)
1.496(5)
1.371(4)
–
1.37
1.44
1.20
1.48
1.39
1.39
1.39
1.40
1.39
1.39
1.50
1.37
1.354(2)
1.453(2)
1.219(2)
1.453(2)
1.380(2)
1.381(3)
1.378(2)
1.393(3)
1.394(3)
1.379(2)
1.493(3)
–
1.38
1.44
1.20
1.47
1.38
1.39
1.38
1.40
1.40
1.39
1.50
A white crystal with approximate dimensions of 0.28 ꢂ 0.16
ꢂ 0.11 mm of the compound 3 was selected for data collection.
The X-ray diffraction data were collected on a Bruker APEX II dif-
fractometer equipped with a CCD area detector and a graphite
monochromator utilizing Cu
Ka radiation (k = 1.54178 Å) at
100(2) K. Data collection, cell refinement and data reduction were
made using APEX2 software [14]. Final unit cell parameters based
on all reflections were obtained by least squares refinement. The
data were integrated via SAINT [15]. Lorentz and polarization effect
and multi-scan absorption corrections were applied with SADABS
[16]. The integration of the data using a monoclinic unit cell
yielded a total of 1111 reflections to a maximum h angle of
63.14° (0.86 Å resolution), of which 754 were independent (aver-
age redundancy 1.473, completeness = 79.5%, Rint = 3.46%) and
1.357(2)
1.36
Bond angles
C(1)AO(2)AC(2)
O(1)AC(1)AO(2)
O(1)AC(1)AC(8)
O(2)AC(1)AC(8)
C(3)AC(8)AC(7)
C(3)AC(8)AC(1)
C(7)AC(8)AC(1)
C(8)AC(7)AC(6)
C(7)AC(6)AC(5)
C(6)AC(5)AC(4)
C(3)AC(4)AC(5)
C(8)AC(3)AC(4)
C(8)AC(3)AC(2)
C(4)AC(3)AC(2)
O(2)AC(2)AC(3)
O(3)AC(6)AC(7)
O(3)AC(6)AC(5)
O(3)AC(5)AC(6)
O(3)AC(5)AC(4)
111.2(3)
121.3(3)
129.9(3)
108.7(3)
123.7(3)
107.6(3)
128.6(3)
116.6(3)
120.9(4)
121.6(3)
117.3(3)
119.9(3)
109.5(3)
130.6(3)
103.0(3)
116.7(3)
122.4(3)
–
111
123
130
107
123
109
129
117
121
121
118
120
108
132
105
117
122
110.06(14)
119.72(17)
131.21(18)
109.06(14)
121.26(15)
108.27(16)
130.47(15)
118.16(15)
120.72(17)
120.98(15)
117.54(16)
121.34(17)
108.14(15)
130.51(16)
104.44(14)
–
111
122
131
107
121
109
130
118
120
121
118
121
108
131
105
738 (97.88%) were greater than 2r
(F2).
A yellow crystal with approximate dimensions of 0.24 ꢂ 0.11
ꢂ 0.07 mm of the compound 4 was selected for data collection.
Intensity data collections were carried out on an Enraf–Nonius
Kappa-CCD diffractometer using a graphite monochromator with
Mo Ka radiation (0.71073 Å), at room temperature. Data collec-
tions were made using the COLLECT program [17]. The final unit
cell parameters were based on all reflections; integration and scal-
ing of the reflections, correction for Lorentz and polarization effects
were performed with the DENZO-SMN system of programs [18].
The data set consisted a total of 5950 reflections, of which 1461
–
were independent (Rint = 3.38%) and 984 were greater than 2r
(F2).
116.44(17)
122.58(16)
117
122
The structures were solved by direct methods with SHELXS-97
[19]. The models were refined by full-matrix least-squares on F2
with SHELXL-97 [19]. Hydrogen atoms in their calculated positions
were refined using a riding model. The non-hydrogen atoms were
refined anisotropically. Structural representations were drawn
using ORTEP-3 [20] and MERCURY [21]. The program WinGX [22]
was used to prepare materials for publication. Data collections
and experimental details for 3 and 4 are summarized in Table 2.
–
2.4. Radicle elongation assay on filter paper
Bioassays were carried out using Petri dishes (90 mm diameter)
with one sheet of Whatman No. 1 filter paper as substrate. Germi-
nation and growth were conducted in aqueous solutions at con-
trolled pH by using 0.02 mol Lꢁ1 [N-morpholino]ethanesulfonic
acid (MES) buffer, brought to pH 6.0 with NaOH 1.0 mol Lꢁ1. Com-
pounds to be assayed were dissolved in dimethyl sulfoxide (DMSO)
at different concentrations and these solutions were diluted with
buffer (0.5% v/v of DMSO) so that test concentrations for the com-
pound (10ꢁ3 and 10ꢁ5 mol Lꢁ1) were reached. This procedure facil-
itated the solubility of the compounds. Twenty seeds of each target
species (Allium cepa and Cucumis sativus) were placed in each Petri
dish. Treatments and negative controls (buffered aqueous solu-
tions with DMSO and without any tested compound) were added
(5 mL) to each Petri dish. Petri dishes were sealed with Parafilm
to ensure closed-system models. Seeds were further incubated at
25 °C in a controlled-environment growth chamber, in the absence
2.3. DFT calculations
Density Functional Theory (DFT) [23] calculations using the
B3LYP functional [24,25] and the 6-31G(d,p) and 6-311+G(2d,p)
basis sets [26,27] (named B3LYP/6-31G(d,p) and B3LYP/6-
311+G(2d,p) levels) were carried out for geometry optimization
and also harmonic frequency calculations which yield directly
the infrared (IR) spectrum. The crystallographic structures (X-ray
coordinates) were used as starting point for B3LYP geometry opti-
mization procedures. The gauge-independent atomic orbital
(GIAO) method implemented by Wolinski, Hilton and Pulay [28]
was used for DFT calculations of 1H and 13C magnetic shielding
constants (
tive to the TMS, taken as reference. The solvent effect (methanol:
= 32.7) was accounted for using the polarizable continuum model
r), with chemical shifts (d), obtained on a d-scale rela-
Table 4
e
Hydrogen bonds for compounds 3 and 4.
(PCM) by the integral equation formalism (IEFPCM) [29], in single
point NMR calculations on the fully optimized geometries in the
vacuum. Such theoretical procedure that enables undoubtedly
the identification of NMR chemical shifts of the isobenzofuranones
3 and 4 was previously used successfully in conformational analy-
sis [30,31]. In addition to NMR spectra, the calculated theoretical IR
spectra were used for comparison with experimental spectroscopic
data at the B3LYP/6-31G(d) level of theory. All calculations were
carried out with the Gaussian-03 program [32] and the theoretical
spectra have been obtained according to Dos Santos and co-work-
ers’ procedure [33].
DonorAHꢄ ꢄ ꢄAcceptor
d(DAH)
d(Hꢄ ꢄ ꢄA)
d(Dꢄ ꢄ ꢄA)
<(DHA)
Compound 3
O(3)AHꢄ ꢄ ꢄO(1)i
0.84
0.95
0.99
0.99
1.91
2.53
2.53
2.56
2.699(4)
3.450(5)
3.430(5)
3.255(5)
155.8
163.7
151.7
127.2
C(5)AH(5)ꢄ ꢄ ꢄO(2)i
C(2)AH(2B)ꢄ ꢄ ꢄO(3)ii
C(2)AH(2A)ꢄ ꢄ ꢄO(3)iii
Compound 4
O(3)AHꢄ ꢄ ꢄO(1)iv
0.82
1.93
2.735(2)
167.1
Symmetry codes: (i) x + 1, y + 1, z; (ii) x ꢁ 1, ꢁy + 2, z ꢁ 1/2; (iii) x ꢁ 1, ꢁy + 1,
z ꢁ 1/2; (iv) x ꢁ 1, y, z ꢁ 1.

66
R.R. Teixeira et al. / Journal of Molecular Structure 1061 (2014) 61–68
Fig. 4. A portion of the crystal packing of compound 3 viewed approximately down the b axis. Dotted lines denote OAHꢄ ꢄ ꢄO and CAHꢄ ꢄ ꢄO interactions. Centroids of benzene
rings are drawn as red balls and lactone rings as pink balls. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article.)
Fig. 5. A packing diagram of compound 4. The OAHꢄ ꢄ ꢄO and
p–p interactions are shown as dotted lines. Centroids of benzene rings are drawn as red balls. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web version of this article.)
of light. Bioassays took 7 days. After growth, plants were frozen at
ꢁ10 °C for 24 h to avoid subsequent growth during the measure-
ment process. This facilitated the handling of the plants and
allowed a more accurate measurement of radicle elongation. The
radicle length was measured to the nearest millimeter. All treat-
ments were replicated four times in a completely randomized
design. The percentage of radicle growth inhibition or stimulation
was calculated in relation to the radicle length of the control. A.
cepa and C. sativus seeds (TOPSEED brand) were purchased from
Agristar do Brazil, Petrópolis, Rio de Janeiro State, Brazil.
Table 5
Effect of isobenzofuran-1(3H)-ones 1–4 on radicle growth of C. sativus and A. cepa
seedlings.
Compound
A. cepa
C. sativus
10ꢁ3 mol Lꢁ1
10ꢁ5 mol Lꢁ1
10ꢁ3 mol Lꢁ1
10ꢁ5 mol Lꢁ1
Inhibition or stimulation (%)
Inhibition or stimulation (%)
1
2
3
4
ꢁ36.3
ꢁ63.1
ꢁ23.8
ꢁ29.1
+2.64
ꢁ40.0
+1.30
ꢁ10.6
ꢁ57.0
ꢁ37.7
ꢁ38.9
ꢁ14.4
ꢁ11.7
ꢁ5.60
ꢁ3.20
+4.62
(ꢁ) Means inhibitory effect; (+) means stimulatory effect.
2.5. Measurement of the photosynthetic electron transport
The chloroplastic electron transport chain was measured by fol-
lowing the light-dependent reduction of ferricyanide by isolated
thylakoid membranes, as previously described [10,34]. Briefly,
deveined plant material was homogenized in 100 mL of ice-cold
20 mM Tricine-NaOH buffer (pH 8.0) containing 10 mM NaCl,
5 mM MgCl2 and 0.4 M sucrose. The homogenate was filtered
through surgical gauze and the filtrate was centrifuged at 4 °C for
1 min at 500g; the supernatant was further centrifuged for

R.R. Teixeira et al. / Journal of Molecular Structure 1061 (2014) 61–68
67
10 min at 1500g. Pelleted chloroplasts were osmotically swollen by
resuspension in sucrose-lacking buffer, immediately diluted 1:1
with sucrose-containing buffer, and kept on ice in the dark until
1600 cmꢁ1 (C@C stretching) for compound 4 is correctly repro-
duced by the theoretical spectrum, as well as the absorption band
around 1700 cmꢁ1 that corresponds to C@O stretching. The main
characteristic of compound 3 is the drastic reduction of intensity
of the C@C stretching mode which is precisely reproduced by the
theoretical calculations. It should be mentioned that the B3LYP
calculated harmonic frequencies were scaled by a factor of 0.93
in order to obtain an agreement with the experimentally observed
vibrational frequencies which are not true harmonic oscillators.
This procedure was well justified and successfully used in our
previous combined experimental and theoretical work [30]. The
similarities of the compounds 3 and 4 and the correct identification
of the spectral profile reinforce the accuracy of the theoretical pre-
diction at B3LYP level of theory, an aspect that can be explored in
the area of organic synthesis mainly in synthetic routes for which
more than one product is expected.
used. Aliquots of membrane preparations corresponding to 15 lg
chlorophyll were incubated at 24 °C in 1-mL cuvettes containing
20 mM Tricine–NaOH buffer (pH 8.0), 10 mM NaCl, 5 mM MgCl2,
0.2 M sucrose and 1 mM K3[Fe(CN)6]. The assay was initiated by
exposure to saturating light, and the rate of ferricyanide reduction
was measured at 1-min intervals for 15 min. Isobenzofuranones
were dissolved and diluted in DMSO. Their effect upon the photo-
synthetic electron transport was evaluated in parallel assays in
which 2.5% (v/v) of a suitable dilution was added to the reaction
mixture so as to obtain the desired final concentration. Results
were expressed as percentage of controls treated with DMSO alone.
Reported values are mean SE over 4 replicates.
3. Results and discussion
3.3. XRD analysis
3.1. Synthesis of hydroxylated isobenzofuranones
The atom numbering scheme of compounds 3 and 4 are shown
in Fig. 3.
To synthesize isobenzofuran-1(3H)-ones 3 and 4 we started
with 3-methoxy benzoic acid and 4-methoxy benzoic acid
(Fig. 1). Thus, the palladium(II) catalyzed ortho alkylation of these
benzoic acids [35] afforded substances 1 and 2 in, respectively, 59%
and 33% yields after purification by column chromatography. The
diesters 1b and 2b, which are formed as a consequence of the
nucleophilic substitution between the substrate and dibromo-
methane, were also isolated. Dimethylation of isobenzofuranones
1 and 2 with BBr3 [36] gave hydroxylated derivatives 3 (92% yield)
and 4 (83% yield).
Crystallographic data and details of diffraction experiments are
provided in Table 2. Bond distances and angles are given in Table 3.
Theoretical structural data are also given in Table 3 showing a good
agreement with experiment and so corroborating structure deter-
mination. Compound 3 crystallizes in the non-centrosymmetric
space group Pc. In the absence of significant anomalous scatters
in the molecule, attempts to confirm the absolute structure by
refinement of the Flack parameter in the presence of 327 sets of
Friedel equivalents led to an inconclusive value of ꢁ0.2(4).
In both structures the geometric parameters are within the val-
ues expected for this class of compounds [37] and are consistent
with the data reported for similar structures [11,12,37–40]. The
C2AO2 bond lengths [1.463(2) Å (compound 3) and 1.453(2) Å
(compound 4)] are consistent with the expected value for a CAO
single bond and the C1AO2 bond lengths [1.350(2) Å and
1.354(3) Å, respectively, for compounds 3 and 4] have double bond
character. The C2AC3 bond is larger than C1AC8. Similar behavior
is observed for phtalide [41] and methoxylated compounds (1) and
(2) [11,12].
In compounds 3 and 4, the molecule is essentially planar with
root mean-square deviation to the fitted non-hydrogen atoms of
0.018(2) Å and 0.014(2) Å, respectively. The compound without
substituents attached to the aromatic ring (phtalide) has a devia-
tion of 0.014 Å [41].
The molecular packing in both structures is mainly determined
by intermolecular O3AHꢄ ꢄ ꢄO1 hydrogen bond between the
hydroxyl and carbonyl groups (Table 4). In the crystal of compound
3.2. DFT calculations
NMR chemical shifts (1H and 13C) were undoubtedly identified
through the aid of B3LYP/6-311+G(2d,p) theoretical data for com-
pounds 3 and 4. Calculated and measured chemical shift data are
given in Table 1, along with the deviation between experimental
and theoretical NMR results. A very good agreement between the-
oretical and experimental values was observed for compound 3,
being this compound precisely characterized by the DFT NMR cal-
culations. Data for compout 4 are also very good regarding the 1H
NMR spectra, with an average deviation below 5% observed. The
13C NMR chemical shift shows a sizeable deviation only for C-9 car-
bon atom (13%) with a good agreement found for the other chem-
ical shift values (less than 5% deviation). A similar behavior has
also been found in previous work for epoxide quebrachitol deriva-
tives [30], which do not invalidate the use of theoretical 13C NMR
data for structural determination.
A good agreement was also obtained for B3LYP/6-31G(d,p) the-
oretical and experimental IR spectra in the 2400–1500 cmꢁ1 region
(Fig. 2). These analyses can be very helpful since the IR spectral
profile is also affected by changes in the molecular structure and
immediate local chemical environment. It can be seen from Fig. 2
that the theoretical IR spectrum for both compounds 3 and 4
reproduces well the experimental spectral profile in the 2000–
1500 cmꢁ1 finger print region. The noticeable peak around
3, this hydrogen bond and
assemble the molecules in two-dimensional network along [110]
a
weak C5AH5ꢄ ꢄ ꢄO2 interaction
direction. There are also two CAHꢄ ꢄ ꢄO intermolecular interactions,
where the O3 atom acts as a bifurcated acceptor, and p–p stacking
between adjacent molecules involving benzene rings [Cg1–Cg1 =
3.88 Å; symmetry code: x, ꢁ1 + y, z] and lactone rings [Cg2–
Cg2 = 3.88 Å; symmetry code: (x, 1 + y, z)] forms a three dimen-
sional network (Fig. 4).
Table 6
Effect of isobenzofuranones on light-driven ferricyanide reduction by isolated spinach chloroplasts.
Concentration
10ꢁ5 mol Lꢁ1
3 ꢂ 10ꢁ5 mol Lꢁ1
10ꢁ4 mol L1
3 ꢂ 10ꢁ4 mol L-1
10ꢁ3 mol Lꢁ1
Compound
1
2
3
4
+5.4 ( 2.5)
+2.0 ( 0.6)
+3.6 ( 1.1)
+5.2 ( 1.0)
+2.6 ( 1.7)
+2.3 ( 2.8)
+1.2 ( 1.5)
+7.8 ( 2.9)
+3.5 ( 1.4)
ꢁ0.3 ( 0.6)
+1.3 ( 2.5)
+16.1 ( 6.3)
ꢁ6.9 ( 4.5)
ꢁ13.3 ( 1.9)
ꢁ10.8 ( 3.1)
+20.2 ( 0.8)
ꢁ11.4 ( 2.4)
ꢁ16.4 ( 0.2)
ꢁ23.4 ( 1.1)
+17.6 ( 2.4)
(ꢁ) Means inhibitory effect; (+) means stimulatory effect.

68
R.R. Teixeira et al. / Journal of Molecular Structure 1061 (2014) 61–68
In the crystal packing of compound 4, the hydrogen bond
Estudos Avançados de Materiais/FAMa (projeto FAPESP No. 2009/
54035-4).
O3AHꢄ ꢄ ꢄO1 links the molecules in a two-dimensional network
along [101] direction. The molecules are further connected by
p–p
interactions between benzene rings of molecules related by
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Supplementary material
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We are grateful to FAPEMIG for financial support. This work is a
collaboration research project of Rede Mineira de Química (RQ-
MG) also supported by FAPEMIG. We are also grateful to CNPq
for research fellowship (to J. Ellena) and Laboratório multiusuário
for X-ray diffraction data collection in ApexII Bruker:Facility para