Synthesis, molecular properties prediction and cytotoxic screening of 3-(2-aryl-2-oxoethyl)isobenzofuran-1(3H)-ones

Article abstract of DOI:10.1016/j.bmcl.2016.04.065

In the present investigation, a collection of nineteen 3-(2-aryl-2-oxoethyl)isobenzofuran-1(3H)-ones was synthesized and screened for their cytotoxic activity against a panel of three leukemia cancer cell lines. The compounds were prepared via ZrOCl₂·8H₂O catalyzed condensation reactions between phthalaldehydic acid and different acetophenones. The reactions were carried out free of solvent and the isobenzofuran-1(3H)-ones were obtained in good yields (80-92%). The identities of the synthesized compounds were confirmed upon IR and NMR (¹H and ¹³C) spectroscopy as well as high resolution mass spectrometry analyses. Structures of compounds 1, 4 and 16 were also investigated by X-ray analysis. The synthesized compounds were submitted to in vitro bioassays against HL-60, K562 and NALM6 cancer cell lines using MTT cytotoxicity assay. After 48 h of treatment, twelve derivatives were able to reduce cell viability and presented IC₅₀ values equal to or below 20 μmol L^-1 against at least one of the evaluated lineages. The most active compound corresponded to 3-(3-methylphenyl-2-oxoethyl)isobenzofuran-1(3H)-one (18) (IC₅₀ values obtained for HL-60, K562 and NALM6 were, respectively, 13.5 μmol L^-1, 8.83 μmol L^-1, and 5.24 μmol L^-1). In addition, compound 18 was capable of triggering apoptosis on NALM6 cells. All isobenzofuranones herein evaluated did not present cytotoxicity on peripheral blood mononuclear cells (PBMC), suggesting selective cytotoxic effect on leukemic cells. A computational study allowed prediction of pharmacokinetics and drug-likeness properties of the synthesized compounds. DFT calculations were performed to obtain the energy values of HOMO, LUMO, and dipole moments of isobenzofuranones.

Full text of DOI:10.1016/j.bmcl.2016.04.065

Accepted Manuscript
Synthesis, Molecular Properties Predicition and Cytotoxic Screening of 3-(2-
aryl-2-oxoethyl) Isobenzofuran-1(3H)-ones
Angélica Faleiros Maia, Raoni Pais Siqueira, Fabrício Marques de Oliveira,
Joana Gasperazzo Ferreira, Silma Francielle da Silva, Clarice Alves Dale
Caiuby, Leandro Licursi de Oliveira, Sérgio Oliveira de Paula, Rafael Aparecido
Carvalho Souza, Silvana Guilardi, Gustavo Costa Bressan, Róbson Ricardo
Teixeira
PII:
S0960-894X(16)30441-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.04.065
BMCL 23827
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 March 2016
21 April 2016
22 April 2016
Please cite this article as: Maia, A.F., Siqueira, R.P., Oliveira, F.M.d., Ferreira, J.G., Silva, S.F.d., Caiuby, C.A.D.,
Oliveira, L.L.d., Paula, S.O.d., Souza, R.A.C., Guilardi, S., Bressan, G.C., Teixeira, R.R., Synthesis, Molecular
Properties Predicition and Cytotoxic Screening of 3-(2-aryl-2-oxoethyl) Isobenzofuran-1(3H)-ones, Bioorganic &
Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.04.065
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Synthesis, Molecular Properties Predicition and
Cytotoxic Screening of 3-(2-aryl-2-oxoethyl) Isobenzofuran-
1(3H)-ones
Angélica Faleiros Maiaa, Raoni Pais Siqueirab, Fabrício Marques de Oliveirac, Joana
Gasperazzo Ferreirab, Silma Francielle da Silvaa, Clarice Alves Dale Caiubya, Leandro
Licursi de Oliveirad, Sérgio Oliveira de Paulad , Rafael Aparecido Carvalho Souzae,
Silvana Guilardie, Gustavo Costa Bressanb*, Róbson Ricardo Teixeiraa*
aDepartamento de Química, Universidade Federal de Viçosa, Av. P.H. Rolfs, S/N,
Viçosa, MG, 36570-900, Brazil
bDepartmento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa,
Av. P.H. Rolfs, S/N, Viçosa, MG, 36570-900, Brazil
cInstituto Federal de Minas Gerais (IFMG), Campus Ouro Branco, Rua Afonso
Sardinha, 90, Ouro Branco, MG, 36420-000, Brazil
dDepartamento de Biologia Celular, Universidade Federal de Viçosa, Av. P.H. Rolfs,
S/N, Viçosa, MG, 36570-900, Brazil
eInstituto de Química, Universidade Federal de Uberlândia, Av. João Naves de Ávila,
2160 - Bloco 1D, Santa Mônica, Uberlândia, MG, 39400-902, Brazil
* Corresponding authors: Tel.: +55 31 3899 3209; fax: +55 31 3899 3065; E-mail
address: robsonr.teixeira@ufv.br (Róbson Ricardo Teixeira)
1

Abstract
In the present investigation, a collection of nineteen 3-(2-aryl-2-oxoethyl)
isobenzofuran-1(3H)-ones was synthesized and screened for their cytotoxic activity
against a panel of three leukemia cancer cell lines. The compounds were prepared via
ZrOCl28H2O catalyzed condensation reactions between phthalaldehydic acid and
different acetophenones. The reactions were carried out free of solvent and the
isobenzofuran-1(3H)-ones were obtained in good yields (80-92%). The identities of the
13
synthesized compounds were confirmed upon IR and NMR (1H and C) spectroscopy
as well as high resolution mass spectrometry analyses. Structures of compounds 1, 4
and 16 were also investigated by X-ray analysis. The synthesized compounds were
submitted to in vitro bioassays against HL-60, K562 and NALM6 cancer cell lines
using MTT cytotoxicity assay. After 48 h of treatment, twelve derivatives were able to
reduce cell viability and presented IC50 values equal to or below 20 µmol L-1 against at
least one of the evaluated lineages. The most active compound corresponded to 3-(3-
methylphenyl-2-oxoethyl) isobenzofuran-1(3H)-one (18) (IC50 values obtained for HL-
60, K562 and NALM6 were, respectively, 13.5 mol L-1, 8.83 mol L-1, and 5.24 mol
L-1). In addition, compound 18 was capable of triggering apoptosis on NALM6 cells.
All isobenzofuranones herein evaluated did not present cytotoxicity on peripheral blood
mononuclear cells (PBMC), suggesting selective cytotoxic effect on leukemic cells. A
computational study allowed prediction of pharmacokinetics and drug-likeness
properties of the synthesized compounds. DFT calculations were performed to obtain
the energy values of HOMO, LUMO, and dipole moments of isobenzofuranones.
Keywords: isobenzofuran-1(3H)-one; phthalides; 3-(2-aryl-2-oxoethyl) isobenzofuran-
1(3H)-ones; cytotoxicity
2

Highlights
 A series of nineteen 3-(2-aryl-2-oxoethyl) isobenzofuran-1(3H)-ones was
prepared
 Isobenzofuranones were screened against HL-60, K562 and NALM6 cancer cell
lines
 The most active compound 18 triggered early events of apoptosis on NALM6
 Compounds did no present cytotoxicity against PBMC cells.
Graphical abstract
compound 18 was capable of triggering early
events of apoptosis on NALM6 cells
3

Cancer is a worldwide disease that affects around fourteen million people every
year. Moreover, at least eight million of deaths caused by this disease are estimated to
occur annually.1 Hematological malignancies (leukemia or lymphoma) are the fourth
most diagnosed cancer in adults (around 9% of the cases), but are responsible for
around 50% of the cases in children younger than 15.2,3 Although several
chemotherapeutic regimes have been successfully approached for fighting cancer
nowadays, the way on how patients respond to treatments is still a puzzle. Furthermore,
most chemotherapies are associated with severe side-effects and cancer resistance,
highlighting the necessity of a constant effort toward the development of novel
therapies.4
Heterocycles play an important role in several branches of chemistry.5 A myriad
of natural products possessing important biological activities are heterocyclic in nature.6
The vast majority of agrochemicals and pharmaceuticals (approximately more than
70%) bears at least one heterocyclic ring.7,8 In addition, several dyes (such as malveine),
polymers, and luminophores have heterocyclic moieties.
The isobenzofuran-1(3H)-ones, also known as phthalides, are a class of
heterocycles presenting a benzene ring (I) fused to a -lactone one (II) (Figure 1).
During the last decades several natural products containing this structural motif have
been isolated and presenting important biological activities.9 In particular, phthalides
functionalized at C-3 position (Figure 1) display useful medicinal properties.10-15 It
stands out the success story of 3-butylisobenzofuran-1(3H)-one (also known as n-
butylphthalide, NBP, Figure 1), a compound which nowadays has been used clinically
as antiplatelet drug for ischemia-cerebralapoplexy.16,17 This example illustrates the
potential utility of isobenzofuranone scaffold for pharmaceutical purposes.
4

In addition to their broad spectrum of biological activities, the phthalides are also
valuable synthetic building blocks.18,19
O
7
1
6
2
O
II
I
5
3
4
ISOBENZOFURAN-1(3H)-ONE NUCLEUS
O
O
O
O
N
H
O
CH3
CH3
OH
H
O
O
H3C
Br
O
H3C
O
OH
O
O
ANTIPLATELET ACTIVITY
ANTAGONIST OF CCR5
HUMAN RECEPTOR
GABA ANTAGONIST
O
O
O
OMe
O
R
OR
N
H
MeO
O
O
X =OH, H or X = O
R = H or Me
O
C13H27
X
R = -OCH3 or -SOCH3
CYTOTOXICITY
ANXIOLYTIC EFFECT
CYTOTOXICITY
O
O
NPB
MARKETED DRUG FOR
ISCHEMIA-CEREBRALAPOPLEXY
Figure 1. Structures of some C-3 functionalized isobenzofuran-1(3H)-ones and their
associated biological properties.
We have been involved in the search of bioactive compounds that can be useful
for the treatment of leukemia20 as well as in the synthesis and biological evaluation of
C-3 functionalized isobenzofuran-1(3H)-ones. Our investigations have demonstrated
phytotoxic21 along with cytotoxic effects of phthalides containing aromatic and alicyclic
5

groups attached to C-3 position of the isobenzofuranonone nucleus.22 Concerning their
cytotoxic effects, these phtalides were evaluated against K562 and U937 leukemia cell
lines. It was found that two compounds (Figure 2) displayed superior cytotoxic effects
against K562 cell line compared to etoposide (VP-16) used as positive control in the
biological assays.22
Figure 2. Results from previous isobenzofuranone screening against K562 cell line
compared to etoposide (VP-16) [From reference 22].
These results prompted us to investigate further chemical modifications at C-3
position of isobenzofuranone nucles in order to afford derivatives endowed with
cytotoxic effects. We describe in this paper the synthesis of nineteen 3-(2-aryl-2-
oxoethyl) isobenzofuran-1(3H)-ones. Their cytotoxic activity was evaluated against the
cancer cell lines HL-60 (acute myelogenous leukemia), K562 (chronic myelogenous
leukemia) and NALM6 (B cell acute lymphoblastic leukemia). The effect of the most
active compound, 3-(3-methylphenyl-2-oxoethyl) isobenzofuran-1(3H)-one (18), on
triggering cell death events was also assessed by flow cytometry assays. In silico drug
like properties of the evaluated compounds were determined and the results are also
discussed.
6

The 3-(2-aryl-2-oxoethyl) isobenzofuran-1(3H)-ones 1-19 (Figure 3) were
synthesized via condensation reactions between phthalaldehydic acid and different
acetophenones [18].23 The reactions were carried out free of solvent and catalyzed by
ZrOCl28H2O a versatile catalyst that has been involved in several synthetically useful
transformations.24-27 The compounds were obtained in good yields (80-92%) after
purification by column chromatography followed by recrystallization.28
O
O
O
O
7
4
6
5
OH
CHO
Ar
CH3
.
1
2
O
O
3
ZrOCl2 8H2O
solvent free
140 oC
OH
O
1´
2´
Ar
1-19
Reaction Time Yield
Compound
Ar
(hours)
(%)
Phenyl
3
4
4
4
3.5
3
3
3
3
3
3
3
3
91
80
89
81
88
89
89
90
91
91
89
92
90
80
92
89
90
91
90
1
2
3
4
5
6
7
8
2-hydroxy phenyl
3-hydroxy phenyl
4-hydroxy phenyl
2-nitro phenyl
3-nitro phenyl
4-nitro phenyl
2-bromo phenyl
3-bromo phenyl
4-bromo phenyl
2-fluoro phenyl
3-fluoro phenyl
4-fluoro phenyl
2-methoxy phenyl 3.5
3-methoxy phenyl 3
4-methoxy phenyl 2
9
10
11
12
13
14
15
16
17
18
19
2-methyl phenyl
3-methyl phenyl
4-methyl phenyl
3
3
3
Figure 3. Synthesis of isobenzofuranones 1-19.
7

The identities of compounds 1-19 were confirmed upon IR, NMR (1H and 13C) as
well as mass spectrometry analyses.28 The IR spectra of 1-19 exhibited strong
diagnostic IR absorptions in the 1747-1785 cm-1 range (for -COO- stretching) and in the
1639-1691 cm-1 range (for -CO- stretching) concordant with carbonyl stretching
frequencies. Diagnostic bands for other functional groups (such as –NO2 and –OH)
were also noticed. The 13C NMR spectrum analyses also confirmed the presence of the
1
carbonyl groups in the structure of phthalides 1-19. In the H NMR spectra, the
diastereotopics –CH2CO- hydrogens were observed as a pair of doublet of doublets. The
molecular formulas of the compounds were confirmed upon high resolution mass
spectrometry analyses.28
We were able to obtain good crystals of compounds 1, 4 and 16 which allowed
their structures to be investigated by X-ray diffraction.29 Concerning compound 16, its
crystallographic data is presented in Table 1. The molecular structure of this compound
with anisotropic displacement ellipsoids (30% probability) and numbering scheme is
shown in Figure 4. Selected bond lengths, bond angles and torsion angles are
summarized in Table 2.
8

Table 1. Crystallographic data and details for compound 16
Compound 16
Empirical formula
Formula weight (g mo )
282.28
Temperature (K)
100(2)
Crystal system
Monoclinic
Space group
P /n
Unit cell dimensions (Å,º)
a
5.0961(1)
b
11.2354(2)
c
23.6337(6)
β
92.543(1)
Volume (ų), Z
1351.86(5), 4
1.387
0.099
Calculated density (g c )
 (m )
F(000)
592
Crystal size (mm)
θ range (º)
Limiting indices
Reflections collected
Independent reflections
Goof
0.461x0.114x0.108
0.41 to 27.1
-6,6; -14,14; 0,30
16676
2330[R(int) = 0.051]
1.119
Data/restraints/parameters
R indices [I > 2σ(I)]
R indices (all data)
2330 / 0 /190
R = 0.0415, wR = 0.1162
R = 0.0581, wR = 0.1441
0.343 and -0.310
Largest diff. peak and hole (e )
;
Figure 4. An ORTEP drawing of 16 with atom-numbering scheme. Displacement
ellipsoids are drawn at the 30% probability level.
9

Table 2. Selected bond lengths (Å), angles (°) and torsion angles (°) for compound 16
Bond length
C(1)―O(1)
C(1) ―O(2)
C(3) ―O(2)
C(1)―C(8)
C(3)―C(9)
C(2’)―O(3)
C(1)―O(1)
1.204(2)
1.366(2)
1.463(2)
1.469(2)
1.511(2)
1.207(6)
1.204(2)
Bond Angles
O(1) ―C(1)―C(8)
O(2) ―C(3)―C(9)
C(9) ―C(3)―C(1’)
C(7) ―C(8)―C(1)
C(4) ―C(9)―C(3)
C(2’) ―C(1’)―C(3)
O(4) ―C(6’)―C(7’)
O(4) ―C(6’)―C(5’)
130.9(1)
103.5(1)
117.9(1)
128.8(1)
131.3(1)
112.0(1)
115.3(1)
124.4(1)
Torsion angles
C(1) ―O(2)―C(3)―C(1’)
O(2) ―C(3) ―C(1’) ―C(2’)
C(4)―C(9)―C(3)―C(1’)
C(9)―C(3)―C(1´)―C(2’)
C(8) ―C(9) ―C(3) ―C(1’)
C(3)―C(1´)―C(2´)―C(3’)
128.1(1)
164.5(1)
60.5(2)
-79.1(2)
-121.8(1)
-175.6(1)
Compound 16 crystallizes in the monoclinic space group P21/n. In the crystal
structure of 16 there is one C-H…O intra molecular interaction involving the O3 atom.
…
Inversion dimmers are linked by pairs of C7-H7 O1 interactions generate (10) ring
motifs,30 which are connected by two C-H…O intermolecular interactions forming
ribbons along b axis. Furthermore, two C-H… interactions link the ribbons into a three-
dimensional supramolecular network (Figure 5).
10

Figure 5. Packing diagram of 16, viewed down the c axis. Dashed lines indicate C-
H…O intermolecular interactions.
The geometric parameters for intermolecular interactions in compound 16 are
listed in Table 3.
Table 3. Geometric parameters for intermolecular interactions in compound 16
Donor─H...Acceptor
d(D─H) d(H...A)
d(D...A)
3.083(2)
<(DHA)
126.6
C4─H4...O3
0.93
0.93
0.93
0.93
0.97
0.96
2.44
2.59
2.56
2.39
2.78
2.73
C7─H7...O1i
3.457(2)
3.352(2)
3.300(2)
4.365(2)
3.746(2)
154.5
143.1
165.6
136
C5'─H5'...O1ii
C8'─H8'...O3iii
C1’─H1’2...Cg1 iv
C9’─H9’2...Cg1v
136
Symmetry codes: (i) 4-x, 1-y, 2-z; (ii) 5/2-x, -1/2+y, 3/2-z; (iii) 5/2-x, 1/2+y,
3/2-z; (iv) 1+x,y,z; (v) -1+x,y,z. Cg1 is phenyl ring.
The geometric parameters (Table 2) are within the expected values for this class
of compounds.31 In the isobenzofuran-1(3H)-one ring system the C1-O1 bond distance
is equal to 1.204(2) Å and correspond to formal double bond. The C1-O2 bond length
[1.366(2) Å] indicates significant double bond character and C3-O2 [1.463(2) Å] is
consistent with the expected value for a C-O single bond. The C3-C9 bond is larger than
C1-C8. Similar behavior is observed for phthalide,32 hydroxylated and methoxylated
11

isobenzofuran-1(3H)-ones33-35 and 3-(4-hexyloxyphenyl) isobenzofuran-1(3H)-one.36 In
the 2-aryl-2-oxoethyl moiety, all bond lengths and angles are normal and comparable
with those observed in related compounds.37-39 The double bond length of C2’-O3
corresponds to 1.207(6) Å.
The isobenzofuran-1(3H)-one ring in compound 16 is planar with r.m.s. deviation
of 0.0189 Å from the least-squares plane defined by the ten constituent atoms. The 2-
aryl-2-oxoethyl system is almost planar with r.m.s. deviation of 0.0149 A˚. In
compound 16 the dihedral angles between these planes is 54.12 (3)°.
Crystallography information concerning compounds 1 and 4 can be found in the
supplementary material.
Isobenzofuran-1(3H)-ones 1–19 were biologically screened against HL-60 (acute
myelogenous leukemia), K562 (chronic myelogenous leukemia) and NALM6 (B cell
lymphoblastic leukemia) cell lines in comparison to DMSO (0.4% v/v) using the MTT
cytotoxicity assay.40,41 Results are summarized in Table 4. Twelve out of nineteen
compounds were able to significantly reduce cell viability with IC50 equal to or below
20 µmol L-1 after 48 h against at least one of the evaluated leukemic cells, whereas
compounds 4, 7 and 19 were substantially ineffective. As a general trend, the active
compounds were more effective against HL-60. The occurrence of a remarkable
variability among the compounds suggest that the presence of various substituents in the
aryl group in aliphatic moiety, and their position as well, may strongly interfere with
cell viability. In particular, phtalide 18 containing the 3-methyl phenyl group
corresponded to the most active derivative. Also, the presence of a methoxy phenyl
group (compound 15) gave a derivative which presented IC50 below 10 µmol L-1 against
HL-60. It should be mentioned that no plain correlation could be found between IC50
values and the substitution pattern of the phenyl ring.
12

The compounds 1-19 were also screened against peripheral blood mononuclear
cells (PBMC), but no significant effect was observed within the evaluated concentration
range (Table 4). This observation provides evidences for the fact that these phthalides
are selective for the analyzed cell lines analyzed.
Table 4. IC50 values (µmol L-1) a obtained when K562, HL-60, NALM6 and PBMC were
treated with compounds 1–19
IC50 (mol L-1)
Compound
K562
HL60 NALM6
PBMC
93.0
31.8
55.3
> 200
100.7
48.0
> 200
57.4
24.3
12.3
23.4
32.9
30.9
46.3
48.2
87.9
38.2
8.83
> 200
15.7
45.7
28.2
> 200
105.9
49.7
> 200
12.0
21.3
62.7
16.0
15.1
12.1
20.1
5.01
58.0
40.2
13.5
> 200
27.9
>200
19.2
113.6
166.3
20.3
163.00
26.3
14.5
7.78
13.3
14.3
24.1
30.0
13.2
61.1
51.2
5.24
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
>200
>200
>200
> 200
>200
>200
> 200
>200
>200
> 200
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
aIC50: compound concentration required to inhibit 50% of viable cells, determined after
48h of continuous treatment.
In following assays, we evaluated if the most active compound on the three
investigated lineages could affect cellular apoptosis program by approaching double
staining by propidium iodide and Annexin V/FITC (Figure 6). After 12 h of treatment
with compound 18, an increase of Annexin V/FITC positive NALM6 was observed in
comparison to treatment with the vehicle DMSO (0.4% v/v). The percentage of cells in
early or late events of apoptosis practically doubled, changing from 6.86% to 11.90%
13

and 0.31% to 0.68% respectively. The absence of necrotic cells in these assays is of
particular interest and represents an interesting finding in terms of application as
anticancer agent. Despite these observations, further in deep biological studies are
certainly needed to determine the exact mechanism of action of derivative 18 in cancer
cells.
Figure 6. Compound 18 induces early apoptosis events on NALM6 cells.To assess cell
death, NALM6 cells were treated with 10 µmol L-1 of compound 18 for 12 h. Cells
treated with the vehicle DMSO (0.4% v/v) were used as controls. Subsequently, the cell
death was evaluated using Annexin V/FITC and propidium iodide label. One
representative experiment of three independent ones is shown.
The synthesized compounds were subjected to a computational study to predict
their physicochemical properties since these are related with absorption, distribution,
metabolism, excretion and toxicity (ADMET) properties.42 A screening was carried out
for compliance to the Lipinski rule of five43 and other related criteria added by Veber44
and Ertl.45 The molecular attributes analyzed included the calculated octan-1-ol/water
partition coefficient (cLogP), number of hydrogen bond acceptors (HBA), number of
hydrogen-bond donors (HBD), molecular weight (MW) and number of rotatable bonds
(nRotb). Another recognized parameter for the membrane permeation and prerequisite
for the bioavailability, the topological polar surface area (TPSA),45 was also considered
(Table 5). Physicochemical parameters (Tables 5 and 6) were calculated using Osiris
Property Explorer46 and Molinspiration (http://www.molinspiration.com/cgi-
14

bin/properties), integral part of some pharmaceutical companies’ in-house substance
registration46 and used by several research groups.22,47-51
The calculated LogP values for compounds 1-19 (2.455–3.743) predict an
appropriate lipophilicity that would facilitate their penetration across biological
membranes, since drugs interacting with enzyme inside the human body have LogP
values between 2 and 5.52
Aqueous solubility of a compound significantly affects its absorption and
distribution characteristics. A parameter used to evaluate aqueous solubility is LogS. All
compounds present solubility values within the desired range (-6.5–0.5).53
Taking into consideration the parameters described above, it is expected that
compounds 1-19 exhibit high bioavailability. This fact is corroborated by the
determined TPSA values (43.376 – 89.2 Å2) and calculated percentages of absorption
(%ABS). In general, the determined TPSA values for compounds 1-19 can be
considered appropriated ones since compounds with TPSA ≥ 140 Å2 are thought to have
poor oral bioavailability and TPSA ≤ 61 Å2 are regarded to have good bioavailability.45
Moreover, compounds presenting TPSA values smaller than 60 Å2 are good ones to
penetrate the blood–brain barrier.47 As can be seen in Table 5, the TPSA values
predicted for phthalides 1 and 8-19 are less than the 60 A2. The percentages of
absorption (%ABS) values are greater than 78% for all compounds, and ten phtalides
out of nineteen exhibit the expected rate of absorption of 94%. In addition, the
compounds 1-19 possess moderate number of rotatable bonds (3–4) and therefore,
exhibit moderate conformational flexibility (Table 5).
Osiris Property Explorer software was used to predict the risk of toxicity of the
synthesized compounds on the following criteria: mutagenic, tumorigenic, irritant and
reproductive effective. Toxicity risk alerts are an indication that the compound may be
15

harmful concerning the risk category specified. Isobenzofuranones 1-19 present low risk
of mutagenic, reproductive effective and tumorigenic, but high risk of being irritant.
This is an important information but not decisive for a drug. In fact, among the 3343
commercially available drugs used to develop Osiris Property Explorer software, 4%
had high risk of being irritant and 4% medium risk according to this software package
(http://www.organic-chemistry.org/prog/peo/tox.html).
Table 5. Predicted physicochemical parameters of 3-(2-aryl-2-oxoethyl) isobenzofuran-
1(3H)-ones (1-19)
Comp
%ABS
MW
cLogP LogS
TPSA
.
Rulea
1
-
≤ 500
-
-
≤ 140
94.0
87.1
87.1
87.1
78.2
78.2
78.2
94.0
94.0
94.0
94.0
94.0
94.0
90.8
90.8
90.8
94.0
94.0
94.0
252.269 2.958
268.268 2.899
268.268 2.455
268.268 2.479
297.266 2.869
297.266 2.893
297.266 2.917
331.165 3.719
331.165 3.743
331.165 3.767
270.259 3.074
270.259 3.098
270.259 3.122
282.295 2.967
282.295 2.991
282.295 3.015
266.296 3.359
266.296 3.383
266.296 3.407
-3.65
-3.36
-3.36
-3.36
-4.11
-4.11
-4.11
-4.49
-4.49
-4.49
-3.96
-3.96
-3.96
-3.67
-3.67
-3.67
-4.00
-4.00
-4.00
43.376
63.604
63.604
63.604
89.2
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
89.2
89.2
43.376
43.376
43.376
43.376
43.376
43.376
52.610
52.610
52.610
43.376
43.376
43.376
16

Table 5. continued
Comp. HBA HBD
nViol nRotB
Volume
Rulea
1
≤ 10
3
4
4
4
6
6
6
3
3
3
3
3
3
4
4
4
≤ 5
0
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
< 2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
< 10
3
3
3
3
4
4
4
3
3
3
3
3
3
4
4
4
225.192
233.209
233.209
233.209
248.526
248.526
248.526
243.077
230.123
230.123
230.123
230.123
230.123
250.737
250.737
250.737
241.752
241.752
241.752
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
3
3
3
3
3
3
%ABS, percentage of absorption [%ABS = 109 – (0.345 x TPSA)]; cLogP = calculated
lipophilicity; TPSA, topological polar surface area; HBA, number of H-bond acceptors;
HBD, number of H-bond donors; nViol, number of violations; nRotB, number of
rotatable bonds. arepresent the ideal values according to Lipinski,43 Veber44 and Ertl45.
Drug score values can be used to judge the compound overall potential to qualify
it for a drug. The values are the combination of drug likeness, toxicity risk, and some
physicochemical parameters, such as cLogP, solubility, and molecular weight. The
negative values of the drug-likeness calculations, between -13.4 and -0.08 (Table 6)
indicate that compounds of the series 1-19 do not contain fragments that are frequently
present in commercial drugs.54,55 Drug-score values are low probably due to the little
similarity to commercially available drugs and the high risk for the irritant category
(Table 6).
17

Table 6. Drug-likeness (DL) and drug-score (DS) parameters for 3-(2-aryl-2-oxoethyl)
isobenzofuran-1(3H)-ones series (1-19)
Comp DL
DS
Comp. DL
DS
-4.71 0.24
-7.35 0.24
-0.08 0.36
-2.77 0.26
-4.46 0.25
-1.22 0.31
-3.57 0.27
-4.80 0.24
-2.28 0.26
1
2
3
4
5
6
7
8
9
10
-4.30 0.26 11
-3.09 0.27 12
-4.31 0.26 13
-0.99 0.33 14
-10.5 0.24 15
-9.71 0.24 16
-13.4 0.24 17
-7.08 0.21 18
-7.92 0.21 19
-4.14 0.22
Quantum chemical calculations were performed using Spartan 1028 and the
Gaussian 0956 molecular modeling softwares. The frontier orbitals and the dipole
moment may be related to biological activity.57-58 The Highest Occupied Molecular
Orbital (HOMO) and the Lowest Unoccupied Molecular Orbital (LUMO) determine the
way that a molecule interacts with other species. The frontier orbital gap helps
characterize the chemical reactivity of the substance and the dipole moment is an
important molecular descriptor that gives the measure of bond polarity throughout the
molecule. These parameters were determined as previously published by our research
group taking into consideration the solvent effect.22 The calculated values are listed in
Table 7.
18

Table 7. Quantum-chemical descriptors for 3-(2-aryl-2-oxoethyl) isobenzofuran-1(3H)-
ones series 1-19
Compound HOMO LUMO GAPa Dipole Moment
1
-7.26
-6.55
-6.54
-6.55
-7.27
-7.27
-7.27
-7.05
-7.03
-7.07
-7.21
-7.15
-7.22
-6.52
-6.45
-6.54
-6.97
-6.94
-7.01
-2.03
-2.10
-2.10
-2.10
-3.18
-3.13
-3.37
-1.86
-2.19
-2.17
-2.11
-2.15
-2.01
-1.94
-2.12
-1.84
-1.94
-2.00
-1.95
5.23
4.44
4.43
4.44
4.09
4.14
3.90
5.18
4.83
4.90
5.10
5.00
5.20
4.58
4.33
4.70
5.04
4.93
5.06
7.34
5.71
6.60
5.71
8.73
10.67
11.06
7.80
5.41
5.45
8.89
7.37
5.77
8.52
6.60
9.40
7.58
7.49
8.15
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
aGAP, Difference between the HOMO and LUMO. Values calculated
with Gaussian 09 using the B3LYP method with the 6-311++G(2d,p)
basis set.
The highest and lowest energy difference between the HOMO and LUMO (gap
energies) were observed for compounds 1 (5.23 eV) and 7 (3.90 eV), respectively, in
case of using water as a solvent. Compound 7, which has the highest value for the
dipole moment (11.06), proved to be inactive for all cell lines used in the biological
assays. Although there is a considerable variation between the frontier orbital and dipole
moment values for the compounds under investigation, it was not possible to establish a
clear correlation between the energy values of HOMO, LUMO, GAP and dipole
moment and biological activity data.
In summary, we have demonstrated that twelve out of nineteen 3-(2-aryl-2-oxo)-
isobenzofuran-1(3H)-one were capable of significantly inhibiting the viability of the
cancer cells HL-60, K562 and NALM6. This effect seemed to be selective as no
significant activity against PBMCs was observed. The most active derivative 18 was
observed to induce apoptosis which paves the way toward further in deep biological
19

studies of mechanism of action. Considering the obtained biological data as whole, the
substitution pattern of the aryl ring in the aliphatic portion seems to be important in
terms of biological activity.
Acknowledgements
The authors are grateful to the Brazilian agencies Fundação de Amparo à
Pesquisa de Minas Gerais (FAPEMIG - CEX APQ 01287/14) and Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq) for financial support. We are
also grateful to Coordination for the Improvement of Higher Education Personnel -
CAPES/Brazil (CAPES) and Fundação Arthur Bernardes (FUNARBE) for research
fellowships to AFM and GCB, respectively. This work was supported by the National
Program for Academic Cooperation (PROCAD) of the Coordination for the
Improvement of Higher Education Personnel-CAPES/Brazil. The authors also
acknowledge to the Núcleo de Microscopia e Microanálise of Universidade Federal de
Viçosa for the available facilities and technical support with the flow citometry
analyses. The authors also thank Professor Dr Javier Ellena of the IFSC, USP, Brazil,
for the X-ray data collection.
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