Synthesis, antiproliferative activities, and computational evaluation of novel isocoumarin and 3,4-dihydroisocoumarin derivatives

Article abstract of DOI:10.1016/j.ejmech.2016.01.051

A series of novel isocoumarin derivatives were synthesized using Castro-Stephens cross-coupling. Moreover, novel 3,4-dihydroisocoumarin derivatives were obtained by catalytic hydrogenation of the corresponding isocoumarin precursors. The antiproliferative

Full text of DOI:10.1016/j.ejmech.2016.01.051

Accepted Manuscript
Synthesis, antiproliferative activities, and computational evaluation of novel
isocoumarin and 3,4-dihydroisocoumarin derivatives
Keller G. Guimarães, Rossimiriam P. de Freitas, Ana L.T.G. Ruiz, Giovanna
F. Fiorito, João E. de Carvalho, Elaine F.F. da Cunha, Teodorico C. Ramalho,
Rosemeire B. Alves
PII:
S0223-5234(16)30059-9
10.1016/j.ejmech.2016.01.051
EJMECH 8341
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 October 2015
Revised Date: 26 January 2016
Accepted Date: 28 January 2016
Please cite this article as: K.G. Guimarães, R.P. de Freitas, A.L.T.G. Ruiz, G.F. Fiorito, J.E. de
Carvalho, E.F.F. da Cunha, T.C. Ramalho, R.B. Alves, Synthesis, antiproliferative activities, and
computational evaluation of novel isocoumarin and 3,4-dihydroisocoumarin derivatives, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.01.051.
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ACCEPTED MANUSCRIPT
1
Synthesis, antiproliferative activities, and computational evaluation of novel isocoumarin and
3
,4-dihydroisocoumarin derivatives
a
a
b
b
b
Keller G. Guimarães, Rossimiriam P. de Freitas, Ana L. T. G. Ruiz, Giovanna F. Fiorito, João E. de Carvalho,
c
c
,a
Elaine F. F. da Cunha, Teodorico C. Ramalho, Rosemeire B. Alves*
a
Laboratório de Síntese Orgânica (Labsinto), Departamento de Química, Universidade Federal de Minas Gerais, Belo Horizonte, MG,
1270-901, Brazil
3
b
Divisão de Farmacologia e Toxicologia, Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas (CPQBA), Universidade
Estadual de Campinas, Campinas, SP, CP 6171, 13083-970, Brazil
c
Laboratório de Química Computacional, Departamento de Química, Universidade Federal de Lavras, CP 3037, 37200-000, Lavras, MG,
Brazil.
*
Corresponding author. Rosemeire B. Alves, rosebrondi@yahoo.com.br; Tel.: +55 31 34095721; fax: + 55 31 34095700.
Abstract
A series of novel isocoumarin derivatives were synthesized using Castro–Stephens cross-coupling. Moreover,
novel 3,4-dihydroisocoumarin derivatives were obtained by catalytic hydrogenation of the corresponding
isocoumarin precursors. The antiproliferative activity of all compounds was evaluated in vitro in different tumor
cells. Furthermore, docking calculations were performed for the kallikrein 5 (KLK5) active site to predict the
possible mechanism of action of this series of compounds. Theoretical findings indicate that the 3,4-
dihydroisocoumarin derivative 10a forms hydrogen bonds with Ser190 and Gln192 residues of KLK5. This
derivative was the most active compound in the series with potent antiproliferative activity and high selectivity
-
1
index (SI > 378.79) against breast cancer cells (MCF-7, GI50 = 0.66 µg mL ). This compound represents a
promising matrix for developing new antiproliferative agents.
Keywords: isocoumarin; 3,4-dihydroisocoumarin; Castro–Stephens reaction; antiproliferative

ACCEPTED MANUSCRIPT
2
1
. Introduction
Cancer is a significant public health problem and a major cause of death in humans [1]. Despite many efforts to
fight cancer, successful treatment of certain types of tumors continues to be challenging because of their
aggressiveness, the complex mechanisms underlying malignant cell metastasis, chemoresistance, and the lack of
selectivity of some drugs [2]. Thus, the development of new, safe, and effective anticancer agents through the
synthesis of simple small molecules is necessary.
Isocoumarins, including their 3,4-dihydro derivatives, are isolated from a variety of natural sources and have
diverse biological activities such as antifungal, antimicrobial [3], antiallergic [4], immunomodulatory [5],
enzyme inhibitory [6-8], antiangiogenic [9], and antioxidant properties [10]. The different biological activities of
the natural compounds belonging to this class are thought to be because of the large structural variety found
among these compounds [11].
Several natural and synthetic coumarins or 3-substituted- 3,4-dihydroisocoumarins have significant cytotoxic
and antitumor activities. For instance, cytogenin 1, a 3-hydroxymethyl coumarin, first isolated in 1990 from
Streptoverticillium eurocidicum, was the first natural isocoumarin that showed anticancer activity against
experimental tumor cells and human cancer cells [12]. NM-3 2, a synthetic analogue of cytogenin in phase I of
clinical tests, potentiates the antineoplastic effects of other chemotherapeutic agents and inhibits angiogenesis
[
13]. 3-Arylisocoumarin 3 has potential anticancer and antimicrobial activity [14]. The compound 185322 4, an
inhibitor of microtubule assembly, induces mitotic arrest and apoptosis of multiple myeloma cells [15]. The
chiral 3-methyl-3,4-ochratoxin A 5, a mycotoxin isolated from Aspergillus ochraceus, shows nephrotoxic,
hepatotoxic, carcinogenic, and teratogenic properties [16]; further, the chiral 3-pentyl-3,4-dihydrocoumarin 6 has
cytotoxic properties [3,7,8].
OH
O
OH
O
OH
O
O
O
O
O
OH
OH
OH
O
O
O
OH
1
2
3
COOH
NH
Ph
OH
O
O
O
O
N
O
O
O
O
O
O
4
Cl
5
6
Figure 1. Representative examples of biologically active isocoumarin and 3,4-dihydroisocoumarin.

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3
Typically, homophthalic acid derivatives are used as starting materials in the synthesis of isocoumarins and 3-
substituted-3,4-dihydrocoumarins [17,18]. The recently developed cross-coupling reactions catalyzed by
transition metals have facilitated the development of new methods for the synthesis of isocoumarins and 3,4-
dihydroisocoumarins [11,19,20]. A cross-coupling reaction known as the Castro–Stephens reaction [21],
catalyzed by Cu (I) has been successfully used to synthesize these compounds. Wang and coworkers modified
the methodology of this reaction, making it a one-pot reaction [22].
On the basis of the previously described biological activities of this class of compounds, we designed and
synthesized a series of 3-substituted isocoumarin derivatives and a series of 3-substituted 3,4-
dihydroisocoumarin derivatives using a modified Castro–Stephens coupling reaction. The antiproliferative
activity of the new compounds was evaluated in vitro against some tumor cell strains, and we performed
theoretical investigations for a potential molecular target.
2
. Results and Discussion
2
.1. Chemistry
To synthesize novel isocoumarin derivatives, we used a one-pot reaction between a terminal alkyne and a
derivative of the 2-halobenzoic acid. The first step was the acquisition of some simple terminal alkynes (Scheme
1
). Most of the alkynes were obtained by modification of both the commercially available propargyl alcohol (7a)
and 4-pentyn-1-ol (7b). The choice of different alkynes governed a structural variability in the final products.
O
3
i
4
7c
OH
n
ii
7
7
a
b
(n=1)
(n=3)
OMs
iii
O
n
n
3
7
f
(n=1)
N
7
7
d
e
(n=1)
(n=3)
7g
(n=3)
o
Scheme 1. Reagents and conditions: (i) 7b (1.0 equiv), NaH (3.0 equiv), 1-iodopentane (3.0 equiv), THF, 0 C-r.t., 16h (7c:
7% yield); (ii) 7a or 7b (1.0 equiv), Et N (1.5 equiv), MsCl (1.7 equiv), CH Cl , -40 °C, 1h (7f: 97% yield, 7g: quant.) ;
8
3
2
2
o
(
iii) 7f or 7g (1.0 equiv), NaH (3.0 equiv), 3-pyridinepropanol (1.5 equiv), THF, 0 C-r.t., 16h (7d: 79% yield, 7e: 83%
yield).
After obtaining the desired alkynes, we synthesized the isocoumarin derivatives in a one-pot reaction catalyzed
by Cu (I) and trans-4-hydroxy-
L-proline as a ligand. The presence of an amino acid ligand is thought to inhibit
the formation of phthalides, which are common byproducts in these kinds of reactions. We did not detect these

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4
byproducts during our experiments. All the planned isocoumarins were obtained successfully (Scheme 2). The
benzoic acid derivatives 2-iodobenzoic acid (8a) and 2-bromo-5-methoxybenzoic acid (8b) are commercially
available.
O
O
R
1
R1
OH
O
iv
+
7
a-e
R
2
X
n
8
9
8
a
(R
1
1
=H; X=I)
9a (n=1; R
9b (n=1; R
1
=H; R
=OMe; R
=OMe; R
2
=OH)
=OH)
=OH)
=O-n-pentyl)
=O-3-propylpyridine)
=O-3-propylpyridine)
8b
(R
=OMe; X=Br)
1
2
9c
9d
9e
9f
(n=3; R
1
2
(n=3; R
(n=1; R
1
1
=OMe; R
=OMe; R
2
2
(n=3; R
1
=OMe; R
2
Scheme 2 - Reagents and conditions: (iv): 8a or 8b (1.0 equiv), 7 (1.0 equiv), CuI (20 mol%), trans-4-hydroxy-
L
-proline
o
(
20 mol%), K CO (2.0 equiv), DMSO, 70 C, 16h (isolated yields: 9a: 54%; 9b: 51%; 9c: 73%; 9d: 69%; 9e: 65%; 9f:
2
3
7
1%).
The observed yields of the compounds 9a (54%), 9b (51%), 9c (73%), and 9d (69%) suggest that the size of the
alkyne chain influences the performance of the reaction. Compounds 9e and 9f were obtained in moderate to
good yields (65% and 71%, respectively). Compound 9c was subjected to a subsequent mesylation reaction,
which resulted in an intermediate 9i. Subsequently, 9i reacted with both the commercial 1-phenyl-1H-tetrazole-
5
-thiol and 5-phenyl-1H-tetrazole, thus resulting in compounds 9g and 9h (Scheme 3). We did not detect any
isomer for compound 9h.
O
O
O
O
v
O
O
O
S
3
N
N
R
1
ii
O
9g
N
N
9c
OMs
3
Ph
9i
N
vi
N
N
N
3
9h
Ph
Scheme 3. Reagents and conditions: (ii) 9c (1.0 equiv), Et
3
N
(1.5 equiv), MsCl (1.7 equiv), CH
2 2
Cl ,
-
40 °C, 1h (9i: 93% yield); (v) 9i (1.0 equiv), 1-phenyl-1H-tetrazole-5-thiol (1.5 equiv), K CO (3.0 equiv), acetone, reflux,
2
3
2
8
4h (9g: 90% yield); (vi) 9i (1.0 equiv), 5-phenyl-1H-tetrazole (1.5 equiv), K CO (3.0 equiv), acetone, reflux, 24h (9h:
2 3
3% yield).

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5
The obtained isocoumarins were then submitted to Pd/C-catalyzed hydrogenation under pressure, generating
their respective 3,4-dihydroisocoumarins as racemic mixtures (10) in good to high yields (Scheme 4). The
compounds bearing the substituent O-3-propylpyridine, 9e and 9f, did not react under the catalytic
hydrogenation conditions used. The results of the catalytic hydrogenation are shown in Table 1.
O
O
R
1
R1
O
vii
O
R2
R2
n
n
9
10
9
a
b
c
d
e
f
(n=1; R
(n=1; R
1
=H; R
2
=OH)
10a (n=1; R1=H; R2=OH)
10b (n=1; R1=OMe; R2=OH)
10c (n=3; R1=OMe; R2=OH)
10d (n=3; R1=OMe; R2=O-n-pentyl)
10g (n=3; R1=OMe; R2=1-phenyl-1H-tetrazole-5-thiol)
10h (n=3; R1=OMe; R2=5-phenyl-1H-tetrazole)
9
9
9
9
9
9
9
1
=OMe; R
2
=OH)
(n=3; R
(n=3; R
(n=1; R
1
=OMe; R
2
=OH)
1
1
=OMe; R
=OMe; R
2
2
=O-n-pentyl)
=O-3-propylpyridine)
(n=3; R
(n=3; R
(n=3; R
1 2
=OMe; R =O-3-propylpyridine)
g
h
1
1
=OMe; R
=OMe; R
2
=1-phenyl-1H-tetrazole-5-thiol)
=5-phenyl-1H-tetrazole)
2
2
Scheme 4. Reagents and conditions: (vii) 9 (1.0 equiv), Pd/C 10% (10%w/w), H (250-500 psi), methanol or THF, 8-24h.
For yields: see Table 1.
Table 1 – Reduction of the isocoumarins (9) to their respective 3,4-dihydroisocoumarins (10).
2
Starting material Pressure of H (psi) Solvent Reaction time (h) Product Isolated yield (%)
9
9
9
a
b
c
250
250
250
250
500
500
300
300
MeOH
MeOH
MeOH
MeOH
THF
THF
MeOH
MeOH
8
8
8
10a
10b
10c
10d
-
98
Quant.
94
89
-
-
84
81
9
d
8
9
9
e
f
g
24
24
16
16
-
9
9
10g
10h
h
2
.2. Antiproliferative studies
We examined the antiproliferative activities of compounds from series (9a-h, 10a-d, and 10g-h) according to the
methodology described by Developmental Therapeutics Program NCI/NIH (http://http://dtp.nci.nih.gov/; Monks
et al. 1990); we examined the antiproliferative activities against various tumor cells (U251 [glioma], MCF-7
[
breast cancer], NCI-ADR/RES [ovarian phenotype of multidrug resistance], 786-0 [kidney], NCI H460 [lung,
non-small cells], PC-3 [prostate], and HT29 [colon]) and the non-tumor human immortalized keratinocytes
HaCaT). Doxorubicin was used as a standard drug.
(
Here, the antiproliferative activity is expressed as the concentration producing 50% of cell growth inhibition or
-1
cytostatic effect (GI µg mL ) for each cell type (Table 2). Almost all the evaluated compounds showed some
50
inhibition of cell growth. Some compounds showed dose-dependent inhibition of cell proliferation.

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6
Results of the average activity (mean logGI50) indicated that compounds belonging to series 9 were more
promising as cytostatic agents than those belonging to series 10, because unlike one compound belonging to
series 10 (10d), four compounds belonging to series 9 (9c-f) showed the lowest values of mean logGI (Table
5
0
2
). These results indicate that the unsaturation between C-3 and C-4 in the isocoumarin structure contributes to
the cytostatic effect.
Among isocoumarins of series 9, isocoumarin 9d was the most active compound (mean logGI = 1.3), which
5
0
showed selective inhibition of breast (MCF-7), multidrug resistant ovarian (NCI-ADR/RES), and prostate (PC-3)
cancer cell lines (Table 2). Isocoumarins 9c and 9e showed a mean antiproliferative effect (mean logGI50 = 1.4)
similar to that of 9d without exhibiting selective inhibition of any lineage (Table 2). These results suggested that
introduction of a methoxy group on C-8 (compounds 9c-e) contributes to the cytostatic effect especially when an
alkyl ether is introduced on the side chain at C-3 (compound 9d) (Table 2).
Results from the 3,4-dihydroisocoumarins series (10a-d, 10g-h) indicated that the presence of a methoxy group
on C-8 and an alkyl ether on the side chain at C-3 (compound 10d) resulted in an active compound with a mean
inhibition (mean logGI = 1.5) similar to that observed for 9d without exhibiting selective inhibition of any
50
lineage (Table 2) unlike that observed for 10d.
Considering the effect on each tumor cell line, the most active of all the evaluated compounds was the 3,4-
dihydroisocoumarin 10a. Interestingly, this molecule was active only against the breast cancer cell line (MCF-7)
-1
with a very low GI50 (0.66 µg mL ) and a very high selectivity index (SI) >378. Its respective isocoumarin, 9a,
showed much lower activity against this cell line (Table 2).
-
1
Table 2 - GI : Growth Inhibition 50 - concentration required to inhibit 50% of cellular growth (µg mL )
50
#
U251 MCF-7 NCI-ADR/RES 786-0 NCI-H460 PC-3 HT29 Mean logGI50
HaCat
<0.025
222.0
134.4
29.0
5.5
Doxo. <0.025 <0.025
0.14
*
0.025
*
<0.025
*
0.085 0.13
117.8 168.4
-1.2
>2.3
1.9
9
9
a
*
89.3
32.6
25.8
6.0
b
167.3
28.9
19.4
28.5
26.3
43.3
67.4
*
160.4
28.1
7.4
28.0
19.3
*
37.6
26.9
25.5
28.6
28.7
51.1
224.2
*
93.2
29.1
32.0
29.6
25.2
*
49.2
25.9
8.0
80.3
29.1
28.3
25.8
25.0
*
9
c
1.4
9
d
1.3
9
e
26.5
23.8
45.7
75.2
0.66
25.0
26.3
144.5
179.8
*
1.4
26.2
27.0
*
9
f
1.4
9
9
g
>2.2
>2.3
>2.3
h
*
*
*
*
1
0a
*
*
*
*

ACCEPTED MANUSCRIPT
7
1
0b
0c
*
250
80.8
32.3
18.6
28.2
*
*
101.6
31.9
*
*
175.4
*
>2.4
2.0
*
130.3
32.3
*
1
135.0
32.2
*
93.3
42.3
132.1
142.1
135.6
33.7
*
70.7 132.0
1
1
1
0d
0g
0h
29.5
45.1
23.9
43.5
13.7
31.2
1.5
>2.1
>2.1
226.3
*
210.8
*
Tumor human cell lines: U251 = glioma; MCF-7 = breast; NCI-ADR/RES = multidrug resistance ovary; 786-0 = kidney;
NCI-H460 = lung, non-small cells; PC-3 = prostate; HT29 = colon. Non-tumor human cell line: HaCat = human
-1
immortalized keratinocytes; * = >250 ꢀg mL ; # = arithmetical mean value of GI considering only tumor cell lines.
50
Finally, almost all SI values calculated for both series 9 and 10 were below or equal to 1.00 (Table 3).
Doxorubicin, a chemotherapy agent currently used in cancer treatment, also exhibits a low SI value; therefore,
the SI results for the compounds of series 9 and 10 suggest that in vivo toxicological studies should be performed
to confirm the safety of these compounds.
Table 3 - Selective index (SI) for each synthesized compound
Cell lines
Compounds
U251 MCF-7 NCI-ADR/RES 786-0 NCI-H460 PC-3
HT29
9
a
-
2.49
4.12
-
0.84
1.03
0.74
0.94
1.40
-
-
-
1.88
2.73
1.12
0.69
1.05
1.03
>1.73
>1.39
-
1.32
9
b
0.80
1.00
0.28
0.92
1.03
>5.77
>3.71
-
3.57
1.08
0.22
0.92
0.94
>4.89
>1.12
-
1.44
1.00
0.17
0.89
1.07
-
1.67
1.00
0.19
1.02
1.08
-
9
c
1.12
9
d
0.92
9
e
0.99
9
f
1.13
9
g
>5.47
>3.32
>378.79
>1.00
1.12
9
h
-
-
-
1
0a
0b
0c
0d
0g
-
-
-
1
-
-
-
-
>1.43
1.84
1.09
-
1
0.97
1.00
-
1.40
0.76
>1.89
1.28
1.01
-
0.96
0.96
-
0.99
0.74
1
1.00
1
>13.44
>5.54 >18.25

ACCEPTED MANUSCRIPT
8
1
0h
>2.29
>8.87
>1.76
-
>1.19
>10.46 >8.01
Tumor human cell lines: U251 = glioma; MCF-7 = breast; NCI-ADR/RES = multidrug resistance ovary; 786-0 = kidney;
NCI-H460 = lung, non-small cells; PC-3 = prostate; HT29 = colon.
2
.3 Docking Studies
To determine the potential mode of action of all compounds synthesized in this study, we docked the novel
isocoumarins derivatives obtained into the kallikrein 5 (KLK5) active site to simulate a binding mode of this
kind of ligand into that target protein. Human KLK5 is a member of the human tissue kallikrein family, which
comprises 15 kallikrein-like serine peptidases (KLKs) [23]. Recent studies report that isocoumarins are powerful
KLK5 inhibitors and a strong correlation has been observed between the biological activity of isocoumarin,
KLK5 inhibition and the antiproliferative activity against tumor cell lines [24]. Although kallikreins are
attractive targets for the development of novel therapeutics for many pathological conditions, including
neoplastic diseases, an in-depth understanding of the mechanism of action of kallikreins is required, which
requires an interplay of experimental and theoretical techniques. Molecular docking techniques are suitable tools
to adjust ligands at target sites to estimate interaction energy [25]. Currently, those molecular-mechanics
methods are well-established techniques applied on numerous occasions [26]. Although the function of the
KLKs in tissues is not clear, growing evidence indicates that overexpression of KLK5 occurs in endocrine-
related malignancies, including ovarian, breast, and testicular cancer [27]. Hence, a strong correlation between
the biological activity of isocoumarins and the KLK5 inhibition has been observed [27]. Thus, new inhibitors for
KLK5 should be developed as novel anticancer drugs. We calculated the interaction energies between each
isocoumarin and KLK5 residue within a radii of 6 Å around the ligand and the results were then compared with
the predicted GI50. Partial least square (PLS) regression was used to model the relationship between the
independent energy term values and GI50 values. The determination coefficient of 0.81 guarantees that GI50
values are well reproduced by the docking scores and, therefore, this receptor-based approach can be used to
understand the interactions governing their biological activities. The experimental and predicted activity and the
residual values for the compound set, including the MolDock score and hydrogen bond energies are shown in
Table 4. We analyzed the hydrogen bonds formed between the 14 compounds and KLK5, and we observed that:
(
i) all compounds interact through the carbonyl group of the isocoumarin ring hydrogen bonds and Ser190; (ii)
all compounds, except 9g, 10a, and 10g, interact through the oxygen atom at the R2 position with Ser 195; (iii)
compounds 9a and 9b interact with Cys191; (iv) compounds 9g, 9h, 10a, 10c, 10d, and 10g interact with
Gln192; and (v) compound 9h interacts with Ala221. The superimposition of the best conformation of
isocoumarin inside the KLK5 active site and the amino acid residues that interact through hydrogen bonds is
shown in Figure 2. Compound 9d exhibited the lowest Escore (more stable complexes), which indicated that

ACCEPTED MANUSCRIPT
9
further studies are required on compounds with alkyl functional group at the R2 position. However, 9d is a very
toxic compound with a very low SI (Table 3). Compound 10a, which was the most active against breast cancer
-1
cells (0.66 µg mL ), was the most selective (SI > 378.79) compound of the studied series. Despite recent
developments in chemotherapy, breast cancer continues to be one the most serious cancers in humans with a
great impact on society and is especially shocking and challenging for young women. Our findings indicate that
1
0a forms hydrogen bonds with Ser190 and Gln192. The differences in the morphology, histological type, tumor
grade, and lymph node status among the different cancer cell lines can lead to differences in the
pharmacokinetics of the studied compounds [28].
Table 4 - Experimental (GI50Exp) and predicted (GI50Pred) activity and residual (GI50Exp-GI50Pred) values for the
-1
compound set. MolDock score and hydrogen bond energies (kcal mol ) from docking studies.
Compounds GI50Exp GI50Pred Residue Energy score Hydrogen bond energy
9
a
3.93
4.31
4.59
5.10
4.60
4.58
3.84
3.75
3.60
3.76
4.15
4.53
4.35
4.62
3.86
4.16
4.55
5.12
4.75
4.49
3.76
3.98
3.77
3.90
3.99
4.14
4.52
4.71
0.07
0.15
0.04
-0.03
-0.15
0.09
0.08
-0.24
-0.16
-0.14
0.16
0.39
-0.18
-0.09
-81.2
-90.55
1.18
-2.02
-2.60
-6.11
-2.18
-2.96
-0.50
-8.40
-4.00
0.58
9
b
9
c
-120.25
-143.79
-118.33
-130.82
-118.39
-115.94
-133.162
-91.53
9
d
9
e
9
f
9
g
9
h
1
0a
0b
0c
0d
0g
0h
1
1
-109.28
-122.98
-138.81
-137.20
-0.95
-3.78
-14.49
-0.15
1
1
1

ACCEPTED MANUSCRIPT
1
0
Figure 2 - Structure of isocoumarin analogs docked into the KLK5 active site. The residues shown are involved in
hydrogen bonding with the compounds.
Figure 3 - Structure of compound 10a docked into the KLK5 active site.
3
. Conclusions
We synthesized a range of new isocoumarin and 3,4-dihydroisocoumarin derivatives. Some of these compounds
exhibited antiproliferative activity in vitro; compound 10a, in particular, showed an excellent activity against the
breast cancer cell line MCF-7 with an excellent SI value.
Results from our docking studies reinforce the observation that the binding orientation of the inhibitors in the
active site of KLK5 is governed by the interaction between the carbonyl group of the isocoumarin ring and
Ser190 and Gln192. However, it is important to mention that some compounds also interact with Cys191 or
Ala221.

ACCEPTED MANUSCRIPT
1
1
Our theoretical and experimental results indicate that compound 10a (Figure 3) is a promising compound and has
selective activity against breast cancer. Furthermore, our findings indicate that structural modifications aiming to
maximize interaction with Ser190 and Gln192 can lead to a better biological activity against cancer cells. In fact,
the determination coefficient of 0.81 between the interaction energy data and experimental results guarantees that
GI50 values are well reproduced by the docking scores.
4
. Materials and Methods
4
.1. Reagents and equipment
Commercially available reagents were purchased from Sigma–Aldrich and used without further purification. The
solvents were purified by distillation. The melting points were determined using a Büchi apparatus and were
uncorrected. Column chromatography was performed on Silica Gel 60 (70–230 mesh, Merck). The progress of
1
13
the reactions was monitored by thin-layer chromatography using Merck silica plates (GF254). H and C NMR
spectra were recorded using a Bruker AVANCE DRX 200 MHz spectrometer by using tetramethylsilane as an
internal standard. The results are presented as the chemical shift
δ in ppm, number of protons, multiplicity, J
values in Hertz, proton position, and carbon position. Multiplicities are abbreviated as follows: s, singlet; brs,
broad signal; d, doublet; dd, double doublet; t, triplet; m, multiplet; qn, quintet; and td, triple doublet. Infrared
spectra were recorded on a Perkin–Elmer Spectrum One SP-IR Spectrometer. High-resolution mass spectra were
recorded using an LC-MS Bruker Daltonics Micro electrospray ionization-time-of-flight mass spectrometer
(
solvent: MeOH/H O, 1:1). All the compounds were purified by instrumental flash chromatography using a
2
Biotage Isolera™ Dalton system. Chromatography was performed in SNAP Ultra 10 g columns using gradient
elution with hexane and ethyl acetate. The elemental analyses were performed on a Perkin-Elmer CHNS model
2
400. A mechanically stirred stainless steel Parr 4560 bomb coupled with a 4282 control module with a PID
temperature controller and tachometer was used as the reaction vessel in catalytic hydrogenation reactions.
4
4
.2. Synthesis
.2.1 General procedure 1
CuI (0.2 equivalent), trans-4-hydroxy-
L-proline (0.2 equivalent), K CO (2.0 equivalents) and the derivative of
2 3
2
-halogenated benzoic acid (1.0 equivalent) were added to a 50 mL round-bottom flask. The flask was sealed
with a rubber septum and purged with nitrogen. Then DMSO enough to obtain a 0.2 mol/L solution of the
benzoic acid derivative was added. Lastly the alkyne (1.0 equivalent) was added. The solution was stirred under
nitrogen atmosphere at 70 °C for 16 hours. The reaction was monitored by TLC. After 16 hours, the reaction
flask was cooled and its contents transferred to a separatory funnel where EtOAc (20 mL) was added. The

ACCEPTED MANUSCRIPT
1
2
mixture was partitioned with water, collecting the organic phase, which was washed with HCl 2 mol/L, saturated
NaHCO and brine. The organic phase was dried over anhydrous sodium sulfate and removed by performing
3
distillation. The remaining residue was purified by column chromatography on silica gel.
4
.2.2 General procedure 2
NaH (3.0 equivalents), prewashed with hexane, was added to a 50 mL round-bottom flask. The flask was sealed
with a rubber septum and attached to a bubbler containing mineral oil. The system was cooled in an ice bath.
Then, a solution 0.2 mol/L of the alcohol (1.0 mmol in 5 mL of dry THF) was added dropwise. The mixture was
stirred until gas release stopped. Lastly the electrophile (3.0 equivalents) was added dropwise. The ice bath was
removed after an hour and the reaction was stirred for 16 hours at room temperature. The reaction was monitored
by TLC. After 16 hours, the reaction was quenched by slow addition of ice water, followed by extraction with
DCM (3 × 25 mL). The organic phase was washed with HCl 2 mol/L, saturated NaHCO and brine, dried over
3
anhydrous sodium sulfate and removed by performing distillation. The remaining residue was purified by
column chromatography on silica gel to produce pure compound.
4
.2.3 General procedure 3
The alcohol (1.0 equivalent) and triethylamine (1.5 equivalent) were added to a 50 mL round-bottom flask
containing dry DCM enough to a 0.3 mol/L concentration. The flask was sealed with a rubber septum and
purged with nitrogen. The mixture was cooled to -40 °C, and methanesulfonyl chloride (1.7 equivalent) was
added to the mixture while being stirred vigorously. After one hour, the mixture was washed with HCl 2 mol/L,
saturated NaHCO and brine, dried over anhydrous sodium sulfate and removed by performing distillation,
3
obtaining the pure mesylate product.
4
.2.4 General procedure 4
Tetrazolium derivative (1.5 mmol in 10 mL of acetone) and potassium carbonate (3.0 equivalents) were added to
a 50 mL round-bottom flask. The reaction mixture was stirred under nitrogen flow for 10 minutes. The
mesylated isocoumarin (1.0 mmol in 10 mL of acetone) was added dropwise. The reaction mixture was stirred
under reflux for 24 hours. After 24 hours, the acetone was removed by performing distillation and water was
added to the residue, followed by extraction with DCM (3 × 25 mL). The organic phase was washed with HCl 2
mol/L, saturated NaHCO and brine, dried over anhydrous sodium sulfate and removed by performing
3
distillation. The remaining residue was purified by column chromatography on silica gel.

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3
4
.2.5 General procedure 5
The isocoumarin (50 mg), Pd/C (5 mg) and 25 mL of the solvent were added to the reaction vessel. The reactor
was closed and three cycles of vacuum/argon were made. The vessel was pressurized with hydrogen up to the
reported pressure. The reaction mixture was stirred for 8 to 24 hours. The reaction was monitored by TLC every
®
8
hours. At the end, the mixture was filtered in Celite and the solvent was removed by performing distillation.
The remaining residue was purified by column chromatography on silica gel.
Synthesis of 5-(pentyloxy)pent-1-yne (7c)
1
4
6
8
10
2
O
11
3
5
7
9
4
-pentyn-1-ol (1.0 mmol) and 1-iodopentane (3 mmol) were used according to general procedure 2 to produce
the compound 7c.
1
Colorless oil; yield 87%; H NMR (200 MHz, CDCl
3
): δ 0.90 (3H, t, J = 6.4 Hz, H-11), 1.27-1.36 (4H, m, H-9
and H-10), 1.57 (2H, qn, J = 6.2 Hz, H-8), 1.77 (2H, qn, J = 6.6 Hz, H-4), 1.94 (1H, t, J = 2.4 Hz, H-1), 2.29
1
3
(
2H, td, J = 6.6 and 2.4 Hz, H-3), 3.40 (2H, t, J = 6.6 Hz, H-5), 3.49 (2H, t, J = 6.2 Hz, H-7); C NMR (50
MHz, CDCl ): 14.1 (C-11), 15.4 (C-3), 22.7 (C-10), 28.5, 28.8 and 29.6 (C-4, C-8 and C-9), 68.5 (C-1), 69.1
C-7), 71.1 (C-5), 84.0 (C-2).
δ
3
(
Synthesis of 3-(3-(prop-2-yn-1-yloxy)propyl)pyridine (7d)
3
5
7
13
N
8
12
O
4
2
6
1
9
11
10
Prop-2-yn-1-ol (2 mmol) was used according to general procedure 3 to produce the mesylate intermediate 7f in
7% yield. 3-pyridinepropanol (1.5 mmol) and 7f (1.0 mmol) were used according to general procedure 2 to
produce compound 7d.
9
1
Pale brown oil; yield: 79%; H NMR (200 MHz, CDCl
3
): δ 1.60-1.90 (2H, m, H-6), 2.35 (1H, s, H-1), 2.57 (2H,
t, J = 7.5 Hz, H-7), 3.38 (2H, t, J = 6.0 Hz, H-5), 4.00 (2H, s, H-3), 7.00-7.10 (1H, m, H-12), 7.37 (1H, d, J = 6.3
13
Hz, H-13), 8.32 (2H, br s, H-9 and H-11); C NMR (50 MHz, CDCl ):
δ
29.3 and 30.7 (C-6 and C-7), 58.0 (C-
3
3
), 68.6 (C-5), 74.4 (C-1), 79.7 (C-2), 123.2 (C-12), 135.8 (C-13), 136.9 (C-8), 147.2 (C-11), 149.9 (C-9).

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4
Synthesis of 3-(3-(pent-4-yn-1-yloxy)propyl)pyridine (7e)
3
5
7
9
15
N
2
10
1
4
O
6
1
4
8
1
1
13
1
2
Pent-4-yn-1-ol (2 mmol) was used according to general procedure 3 to produce the mesylate intermediate 7g in
7% yield. 3-pyridinepropanol (1.5 mmol) and 7g (1.0 mmol) were used according to general procedure 2 to
produce compound 7e.
9
1
Pale brown oil; yield: 83%; H NMR (200 MHz, CDCl ):
δ
1.72 (2H, qn, J = 7.2 Hz, H-8), 1.81 (2H, dt, J = 7.0
and 6.2 Hz, H-4), 1.90 (1H, t, J = 2.7 Hz, H-1), 2.23 (2H, td, J = 7.0 and 2.7 Hz, H-3), 2.63 (2H, t, J = 7.2 Hz, H-
), 3.35 (2H, t, J = 6.2 Hz, H-5 or H-7), 3.43 (2H, t, J = 6.2 Hz, H-5 or H-7), 7.13 (1H, dd, J = 7.8 and 4.7 Hz,
H-14), 7.44 (1H, dt, J = 7.8 and 1.8 Hz, H-15) 8.36 (1H, dd, J = 4.7 and 2.0 Hz, H-13), 8.39 (1H, d, J = 2.0 Hz,
3
9
13
H-11); C NMR (50 MHz, CDCl ):
δ
15.3 (C-3), 28.7 and 29.6 (C-4 and C-8), 31.1 (C-9), 68.6 (C-1), 69.1 and
3
6
9.6 (C-5 and C-7), 84.0 (C-2), 123.4 (C-14), 136.1 (C-15), 137.3 (C-10), 147.3 (C-13), 150.0 (C-11).
Synthesis of 3-(hydroxymethyl)-1H-isochromen-1-one (9a)
11
O
6
1
2
5
4
9
10
O
13
8
OH
3
7
12
2
-Iodobenzoic acid (1 mmol) and prop-2-yn-1-ol (1 mmol) were used according to general procedure 1 to
produce compound 9a.
o
1
Yellow solid; 54% yield; m.p. (hexane) 92.2-93.6 C; H NMR (200 MHz, CDCl ):
δ
3.75 (1H, br s, OH), 4.45
3
(
2H, s, H-12), 6.52 (1H, s, H-7), 7.32 (1H, d, J = 8.0 Hz, H-3), 7.40 (1H, t, J = 8.0 Hz, H-1), 7.62 (1H, t, J = 8.0
13
Hz, H-2), 8.13 (1H, d, J = 8.0 Hz, H-6); C NMR (50 MHz, CDCl
3
): δ 61.3 (C-12), 103.2 (C-7), 120.4 (C-5),
1
25.8 (C-1), 128.4 (C-6), 129.6 (C-3), 135.1 (C-2), 137.0 (C-4), 156.0 (C-8), 162.9 (C-10); HRMS (ESI) m/z:
+
[
M+H] calcd for C H O , 177.0552; found, 177.0558; elemental analysis calcd for C H O : C 68.177, H
10 8 3 10 8 3
4
.577; found: C 68.701, H 4.817.

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1
5
Synthesis of 3-(hydroxymethyl)-7-methoxy-1H-isochromen-1-one (9b)
11
1
2
O
6
3
1
O
5
9
1
0
O
1
3
15
8
OH
2
4
7
14
2
-Bromo-5-methoxybenzoic acid (1 mmol) and prop-2-yn-1-ol (1 mmol) were used according to general
procedure 1 to produce compound 9b.
o
1
Yellow solid; 51% yield; m.p. (hexane) 128.4-129.0 C; H NMR (200 MHz, CDCl ):
δ 2.66 (1H, br s, OH),
3
13
3
.77 (3H, s, H-13), 4.36 (2H, s, H-14), 6.39 (1H, s, H-7), 7.10-7.30 (2H, m, H-2 and H-3), 7.52 (1H, s, H-6); C
NMR (50 MHz, CDCl
3
):
δ
55.9 (C-13), 61.6 (C-14), 103.3 (C-7), 110.3 (C-6), 121.8 (C-5), 124.8 (C-2), 127.5
+
(
C-3), 130.7 (C-4), 153.6 (C-8), 159.8 (C-1), 162.9 (C-11); HRMS (ESI) m/z: [M+H] calcd for C H O ,
11
10
4
2
4
07.0657; found, 207.0237; elemental analysis calcd for C H O : C 64.074, H 4.888; found: C 63.799, H
11 10 4
.821.
Synthesis of 3-(3-hydroxypropyl)-7-methoxy-1H-isochromen-1-one (9c)
11
12
O
6
1
O
5
9
10
O
1
3
15
17
8
OH
2
4
3
7
14
16
2
-Bromo-5-methoxybenzoic acid (1 mmol) and pent-4-yn-1-ol (1 mmol) were used according to general
procedure 1 to produce compound 9c.
1
Yellow oil; 73% yield; H NMR (200 MHz, CDCl ):
δ 1.70-2.10 (2H, m, H-15), 2.58 (2H, t, J = 8.0 Hz, H-14),
3
13
3
.66 (2H, t, J = 6.0 Hz, H-16), 3.81 (3H, s, H-13), 7.10-7.30 (2H, m, H-2 and H-3), 7.55 (1H, s, H-6); C NMR
(
(
50 MHz, CDCl
3
):
δ
29.8 and 30.0 (C-14 and C-15), 55.8 (C-13), 61.5 (C-16), 103.1 (C-7), 109.9 (C-6), 121.1
+
C-5), 124.6 (C-2), 126.8 (C-3), 131.3 (C-4), 155.4 (C-8), 159.2 (C-1), 163.4 (C-10); HRMS (ESI) m/z: [M+H]
calcd for C H O , 235.0970; found, 235.0888; elemental analysis calcd for C H O : C 66.656, H 6.024;
13
14
4
13 14
4
found: C 66.484, H 5.917.

ACCEPTED MANUSCRIPT
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6
Synthesis of 7-methoxy-3-(3-(pentyloxy)propyl)-1H-isochromen-1-one (9d)
11
1
2
O
6
3
1
O
5
9
10
O
17
13
15
19
21
8
O
2
4
2
2
7
14
16
18
20
2
-Bromo-5-methoxybenzoic acid (1 mmol) and 7c (1 mmol) were used according to general procedure 1 to
produce compound 9d.
1
Colorless oil; 69% yield; H NMR (200 MHz, CDCl ):
δ 0.92 (3H, t, J = 6.0 Hz, H-22), 1.25-1.40 (4H, m, H-20
3
and H-21), 1.40-1.70 (2H, m, H-19), 1.80-2.15 (2H, m, H-15), 2.64 (2H, t, J = 8.0 Hz, H-14), 3.46 (4H, t, J = 6.0
Hz, H-16 and H-18), 3.90 (3H, s, H-13), 6.26 (1H, s, H-7), 7.10-7.30 (2H, m, H-2 and H-3), 7.66 (1H, s, H-6);
1
3
3
C NMR (50 MHz, CDCl ): δ 14.1 (C-22), 22.6 (C-21), 27.2 and 28.4 (C-19 and C-20), 29.5 and 30.2 (C-14 and
C-15), 55.7 (C-13), 69.4 and 71.1 (C-16 and C-18), 102.9 (C-7), 109.9 (C-6), 121.2 (C-5), 124.5 (C-2), 126.7 (C-
+
3
3
7
), 131.3 (C-4), 155.6 (C-8), 159.1 (C-1), 163.2 (C-10); HRMS (ESI) m/z: [M+H] calcd for C H O ,
18
24
4
05.1747; found, 305.1744; elemental analysis calcd for C H O : C 71.027, H 7.947; found: C 70.899, H
18
24
4
.971.
Synthesis of 7-methoxy-3-((3-(pyridin-3-yl)propoxy)methyl)-1H-isochromen-1-one (9e)
11
1
2
O
6
3
22
19
1
O
5
4
9
23
18
21
1
0
O
24
13
16
8
O
N 20
2
7
14
15
17
2
-Bromo-5-methoxybenzoic acid (1 mmol) and 7d (1 mmol) were used according to general procedure 1 to
produce compound 9e.
1
Pale brown oil; 65% yield; H NMR (200 MHz, CDCl ):
δ
1.70-1.90 (2H, m, H-16), 2.61 (2H, t, J = 6.0 Hz, H-
3
1
7), 3.43 (2H, t, J = 7.0 Hz, H-15), 3.75 (3H, s, H-13), 4.15 (2H, s, H-14), 6.33 (1H, s, H-7), 7.00-7.30 (3H, m,
13
H-2, H-3 and H-22), 7.38 (1H, d, J = 8.0 Hz, H-23), 7.54 (1H, d, J = 2.0 Hz, H-6); C NMR (50 MHz, CDCl
3
):
δ
1
1
3
4
29.5 (C-16), 31.0 (C-17), 55.9 (C-13), 69.2 and 70.1 (C-14 and C-15), 104.2 (C-7), 110.3 (C-6), 122.0 (C-5),
23.5 (C-22), 124.6 (C-2), 127.4 (C-3), 130.5 (C-4), 136.1 (C-23), 137.1 (C-18), 147.5 (C-21), 150.0 (C-19),
+
51.6 (C-8), 159.8 (C-1), 162.6 (C-10); HRMS (ESI) m/z: [M+H] calcd for C H NO , 326.1392; found,
19
19
4
4
26.1716; elemental analysis calcd for C19H19NO : C 70.139, H 5.886, N 4.305; found: C 69.932, H 6.214; N
.160.

ACCEPTED MANUSCRIPT
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7
Synthesis of 7-methoxy-3-(3-(3-(pyridin-3-yl)propoxy)propyl)-1H-isochromen-1-one (9f)
11
1
2
O
6
3
25
22
1
O
5
9
26
21
24
1
0
O
17
13
19
15
8
O
N 23
2
4
7
14
16
18
20
2
-Bromo-5-methoxybenzoic acid (1 mmol) and 7e (1 mmol) were used according to general procedure 1 to
produce compound 9f.
1
Pale brown oil; 71% yield; H NMR (200 MHz, CDCl ):
δ
1.60-1.95 (4H, m, H-15 and H-19), 2.50-2.70 (4H, m,
3
H-14 and H-20), 3.20-3.45 (4H, m, H-16 and H-18), 3.76 (3H, s, H-13), 6.14 (1H, s, H-7), 6.70-7.10 (3H, m, H-
2
2
6
4
, H-25 and H-26), 7.19 (1H, d, J = 7.8 Hz, H-3), 7.34 (1H, d, J = 2.4 Hz, H-6), 8.00-8.20 (2H, m, H-22 and H-
13
4); C NMR (50 MHz, CDCl ):
δ 26.8 and 29.2 (C-15 and C-19), 29.0 and 30.6 (C-14 and C-20), 55.3 (C-13),
3
9.2 (C-16 and C-18), 102.5 (C-7), 109.5 (C-6), 120.8 (C-5), 123.0 (C-25), 124.2 (C-2), 126.3 (C-3), 130.9 (C-
), 135.6 (C-26), 136.9 (C-21), 146.9 (C-24), 149.7 (C-22), 155.0 (C-8), 158.7 (C-1), 162.8 (C-10); HRMS (ESI)
+
m/z: [M+H] calcd for C21
H
23NO
4
, 354.1705; found, 354.1948; elemental analysis calcd for C21
H
23NO
4
: C
7
1.369, H 6.560, N 3.963; found: C 71.805, H 6.638; N 4.044.
Synthesis of 7-methoxy-3-(3-((1-phenyl-1H-tetrazol-5-yl)thio)propyl)-1H-isochromen-1-one (9g)
11
1
2
O
6
3
1
O
5
9
1
0
O
17
13
15
22
8
S
18
N
2
4
N 21
1
6
7
14
19
2
4
N
23
N
25
20
24
2
6
25
9
c (3 mmol) was submitted to general procedure 3 to produce compound 9i in 93% yield. 9i (1 mmol) and 1-
phenyl-1H-tetrazole-5-thiol (1.5 equiv) were used according to general procedure 4 to produce compound 9g.
o
1
Colorless solid; 90% yield; m.p. (hexane/DCM 9:1) 109.1-110.7 C; H NMR (200 MHz, CDCl
3
):
δ 2.00-2.30
(
2H, m, H-15), 2.61 (2H, t, J = 7.0 Hz, H-14), 3.37 (2H, t, J = 7.0 Hz, H-16), 3.78 (3H, s, H-13), 6.23 (1H, s, H-
1
3
7
2
), 7.10-7.30 (2H, m, H-2, H-3), 7.40-7.60 (4H, m, H-6, H-24, H-25 and H26); C NMR (50 MHz, CDCl ): δ
3
6.4 (C-15), 32.0 (C-14), 32.2 (C-16), 55.7 (C-13), 103.5 (C-7), 109.9 (C-6), 121.1 (C-5), 123.7 (C-24), 124.5
(
C-2), 126.8 (C-3), 129.8 and 130.2 (C-25 and C-26), 130.9 (C-4), 133.5 (C-23), 153.8 (C-18), 154.0 (C-8),

ACCEPTED MANUSCRIPT
1
8
+
1
59.2 (C-1), 162.9 (C-10); HRMS (ESI) m/z: [M+Na] calcd for C20
H
18
N
4
O
3
S, 417.0997; found, 417.1012;
elemental analysis calcd for C H N O S: C 60.899, H 4.600, N 14.204; found: C 60.637, H 4.490; N 13.953.
20
18
4
3
Synthesis of 7-methoxy-3-(3-(5-phenyl-1H-tetrazol-1-yl)propyl)-1H-isochromen-1-one (9h)
11
1
2
O
6
3
20
1
21
O
5
9
N
10
O
N
1
3
15
1
7
8
N
19
N
1
2
4
8
22
7
14
16
23
23
24
2
4
25
9
i (1 mmol) and 5-phenyl-1H-tetrazole (1.5 equiv) were used according to general procedure 4 to produce
compound 9h.
o
1
Colorless solid; 83% yield; m.p. (hexane/DCM 9:1)105.1-106.4 C; H NMR (200 MHz, CDCl ):
δ
2.30-2.70
3
(
4H, m, H-14 and H-15), 3.86 (3H, s, H-13), 4.69 (2H, t, J = 6.0 Hz, H-16), 6.19 (1H, s, H-7), 7.20-7.30 (2H, m,
13
3
H-2, H-3), 7.30-7.50 (2H, m, H-24 and H-25), 7.90-8.10 (1H, m, H-23); C NMR (50 MHz, CDCl ): δ 26.5 (C-
1
2
1
5), 30.4 (C-14), 52.1 (C-16), 55.8 (C-13), 104.0 (C-7), 110.0 (C-6), 121.3 (C-5), 124.6 (C-25), 126.8 (C-3 and
4), 127.3 (C-22), 128.9 (C-23), 130.4 (C-25), 130.8 (C-4), 153.1 (C-8), 159.4 (C-1), 162.9 (C-18), 165.3 (C-
+
0); HRMS (ESI) m/z: [M+H] calcd for C H N O , 363.1457; found, 363.1543; elemental analysis calcd for
20
18
4
3
C H N O : C 66.288, H 5.007, N 15.461; found: C 66.007, H 4.983; N 15.239.
20
18
4
3
Synthesis of 3-(hydroxymethyl)isochroman-1-one (10a)
11
O
6
5
4
9
1
2
1
0
O
13
8
OH
3
7
12
9
a (0.5 mmol) was used according to general procedure 5 to produce compound 10a.
1
Colorless oil; 98% yield; H NMR (200 MHz, CDCl ):
δ
δ
3.00-3.25 (3H, m, H-12 and OH), 3.70-3.90 (2H, m, H-
3
7
), 4.40-4.70 (1H, m, H-8), 6.90-7.20 (1H, m, H-3), 7.10-7.40 (1H, m, H-1), 7.44 (1H, t, J = 8.0 Hz, H-2), 7.96
1
3
(
(
1H, d, J = 8.0 Hz, H-6); C NMR (50 MHz, CDCl ):
29.3 (C-7), 64.2 (C-12), 79.3 (C-8), 124.7 (C-5), 127.8
3
+
C-1 and C-3), 130.4 (C-6), 134.1 (C-2), 139.1 (C-4), 165.6 (C-10); HRMS (ESI) m/z: [M+H] calcd for

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1
9
C
10
H
10
O
3 10 3
, 179.0708; found, 179.0417; elemental analysis calcd for C10H O : C 67.406, H 5.657; found: C
6
7.876, H 5.671.
Synthesis of 3-(hydroxymethyl)-7-methoxyisochroman-1-one (10b)
11
1
2
O
6
3
1
O
5
9
1
0
O
1
3
15
8
OH
2
4
7
14
9
b (0.5 mmol) was used according to general procedure 5 to produce compound 10b.
o
1
Colorless solid; quantitative; m.p. (hexane)99.8-100.1 C; H NMR (200 MHz, CDCl
3
):
δ 2.60-3.20 (1H, m, H-
7
2
1
), 3.34 (1H, s, OH), 3.70-3.90 (2H, m, H-14), 3.77 (3H, s, H-13), 4.40-4.60 (1H, m, H-8), 6.90-7.20 (2H, m, H-
13
and H-3), 7.48 (1H, s, H-6); C NMR (50 MHz, CDCl ):
δ 28.4 (C-7), 55.7 (C-13), 64.1 (C-14), 79.6 (C-8),
3
13.0 (C-6), 121.9 (C-5), 125.5 (C-2), 128.9 (C-3), 131.2 (C-4), 159.0 (C-1), 165.6 (C-10); HRMS (ESI) m/z:
+
[
M+H] calcd for C11
H
12
O
4
, 209.0814; found, 209.1013; elemental analysis calcd for C11
H
12
O
4
: C 63.454, H
5
.809; found: C 63.050, H 5.931.
Synthesis of 3-(3-hydroxypropyl)-7-methoxyisochroman-1-one (10c)
11
12
O
6
1
O
5
4
9
10
O
13
15
17
8
OH
2
3
7
14
16
9
c (0.5 mmol) was used according to general procedure 5 to produce compound 10c.
1
Colorless oil; 94% yield; H NMR (200 MHz, CDCl ):
δ 1.60-1.90 (4H, m, H-14 and H-15), 2.38 (1H, s, H-17),
3
2
.80-2.90 (1H, m, H-7), 3.66 (2H, t, J = 6.0 Hz, H-16), 3.78 (3H, s, H-13), 4.40-4.60 (1H, m, H-8), 6.90-7.20
13
(
1H, m, H-2 and H-3), 7.50 (1H, d, J = 2.0 Hz, H-6); C NMR (50 MHz, CDCl
3
):
δ 28.1 (C-15), 31.5 (C-14),
3
1
2.5 (C-7), 55.7 (C-13), 62.3 (C-16), 79.1 (C-8), 113.0 (C-6), 121.7 (C-5), 125.8 (C-2), 128.7 (C-3), 131.6 (C-4),
+
59.0 (C-1), 166.0 (C-10); HRMS (ESI) m/z: [M+H] calcd for C H O , 237.1127; found, 237.0953; elemental
13
16
4
analysis calcd for C H O : C 66.087, H 6.826; found: C 66.118, H 6.879.
13
16
4

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0
Synthesis of 7-methoxy-3-(3-(pentyloxy)propyl)isochroman-1-one (10d)
11
1
2
O
6
3
1
O
5
9
10
O
17
13
15
19
21
8
O
2
4
2
2
7
14
16
18
20
9
d (0.5 mmol) was used according to general procedure 5 to produce compound 10d.
1
Colorless oil; 89% yield; H NMR (200 MHz, CDCl
3
):
δ
0.80 (3H, t, J = 6.6 Hz, H-22), 1.10-1.35 (4H, m, H-19
and H-20), 1.30-1.60 (2H, m, H-19), 1.70-2.00 (2H, m, H-15), 2.52 (2H, t, J = 7.2 Hz, H-14), 2.70-2.95 (2H, m,
H-7), 3.20-3.50 (4H, m, H-6 and H-18), 3.78 (3H, s, H-13), 4.40-4.60 (1H, m, H-8), 6.90-7.20 (2H, m, H-2 and
13
H-3), 7.49 (1H, d, J = 2.4 Hz, H-6); C NMR (50 MHz, CDCl ):
δ
14.0 (C-22), 22.5 (C-21), 27.1, 28.3, 29.4 and
3
3
6
0.1 (C-14, C-15, C-19 and C-20), 32.5 (C-7), 55.6 (C-13), 69.3 and 70.9 (C-16 and C-18), 79.0 (C-8), 112.9 (C-
+
), 121.6 (C-3), 125.7 (C-5), 128.6 (C-2), 131.5 (C-4), 158.9 (C-1), 165.9 (C-10); HRMS (ESI) m/z: [M+H]
calcd for C H O , 307,1909; found, 307,2127; found, 237.0953; elemental analysis calcd for C H O : C
18
26
4
18 26
4
7
0.560, H 8.553; found: C 70.168, H 8.651.
Synthesis of 7-methoxy-3-(3-((1-phenyl-1H-tetrazol-5-yl)thio)propyl)isochroman-1-one (10g)
11
1
2
O
6
3
1
O
5
9
1
0
O
17
13
15
22
8
S
18
N
2
4
N 21
1
6
7
14
19
2
4
N
23
N
25
20
24
2
6
25
9
g (0.5 mmol) was used according to general procedure 5 to produce compound 10g.
o
1
Colorless solid; 84% yield; m.p. (hexane/DCM 9:1) 98.3-100.7 C; H NMR (200 MHz, CDCl
3
):
δ 1.70-2.20
(
4H, m, H-15 and H-16), 2.60-2.90 (2H, m, H-14), 3.37 (2H, dd, J = 8.0 and 6.0 Hz, H-7), 3.73 (3H, s, H-13),
1
3
4
.30-4.60 (1H, m, H-8), 6.90-7.20 (2H, m, H-2 and H-3), 7.40-7.60 (4H, m, H-6, H-24, H-25 and H-26); C
24.9 (C-15), 32.3 (C-14), 32.8 (C-7), 33.7 (C-16), 55.6 (C-13), 78.3 (C-8), 112.9 (C-
), 121.6 (C-5), 123.8 (C-24), 125.7 (C-2), 128.6 (C-3), 129.8 (C-25), 130.2 (C-26), 131.2 (C-4), 133.6 (C-23),
NMR (50 MHz, CDCl ):
δ
3
6
1
3
1
+
54.7 (C-18), 158.9 (C-1), 165.4 (C-10); HRMS (ESI) m/z: [M+H] calcd for C H N O S, 397.1334; found,
2
0
20
4
3
97.0883; elemental analysis calcd for C H N O S: C 60.589, H 5.085, N 14.132; found: C 60.503, H 5.185, N
2
0
20
4
3
4.132.

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1
Synthesis of 7-methoxy-3-(3-(5-phenyl-1H-tetrazol-1-yl)propyl)isochroman-1-one (10h)
11
1
2
O
6
3
20
1
21
O
5
4
9
N
N
7
10
O
1
3
15
1
8
N
19
N
1
2
8
22
7
14
16
23
23
24
2
4
25
9
h (0.5 mmol) was used according to general procedure 5 to produce compound 10h.
o
1
Colorless solid; 81% yield; m.p. (hexane/DCM 9:1) 92.5-94.2 C; H NMR (200 MHz, CDCl ):
δ
1.55-2.20 (2H,
3
m, H-15), 2.10-2.60 (2H, m, H-14), 2.60-3.00 (2H, m, H-7), 3.77 (3H, s, H-13), 4.30-4.65 (1H, m, H-8), 4.70
(
(
2H, t, J = 6.0 Hz, H-16), 6.90-7.20 (2H, m, H-2 and H-3), 7.30-7.60 (3H, m, H-6, H-24 and H-25), 7.90-8.20
1
3
1H, m, H-23); C NMR (50 MHz, CDCl ):
δ
25.1 (C-15), 31.8 (C-14), 32.5 (C-7), 52.8 (C-16), 55.7 (C-13),
3
7
8.0 (C-8), 113.0 (C-6), 121.8 (C-2), 125.7 (C-5), 126.9 (C-23), 127.4 (C-22), 128.6 (C-3), 129.0 (C-24), 130.4
+
(
C-25), 131.1 (C-4), 159.1 (C-1), 165.4 (C-10); HRMS (ESI) m/z: [M+H] calcd for C H N O , 365.1608;
20 20 4 3
20 4 3
found, 365.2039; elemental analysis calcd for C20H N O : C 65.921, H 5.532, N 15.375; found: C 65.558, H
5
4
4
.752, N 15.065.
.3. Antiproliferative evaluation
.3.1. Cell lines
Human tumor cell lines U251 (glioma, CNS), MCF-7 (human breast adenocarcinoma), NCI-ADR/RES (ovarian
expressing phenotype multiple drug resistance), 786-0 (kidney), NCI-H460 (lung, non-small cells), PC-3
(
prostate), HT-29 (human colon adenocarcinoma) were kindly provided by the National Cancer Institute (NCI-
USA). The immortalized human keratinocyte (HaCaT) cell line was kindly was provided by prof. Dr. Ricardo
Della Coletta (University of Campinas, UNICAMP).
4
.3.2. Antiproliferative activity assay
The in vitro antiproliferative activity assay was performed as described by Monks et al. (1991). All cell lines
were maintained in RPMI 1640 (Gibco, USA) supplemented with 5% (v/v) fetal bovine serum (FBS, Gibco) and
1
% (v/v) penicillin:streptomycin (Nutricell, 1000 U/mL:1000 g/mL) in a humidified atmosphere with 5% CO2,
at 37°C. For the experiments, cell lines were used between passages 5 to 12. Cells in 96 well plates (100
ꢀ
L
4
-1
cells/well, 3 to 5 x 10 cells mL ) were exposed to sample concentrations in DMSO/RPMI (0.25, 2.5, 25 and

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2
2
2
50
ꢀ
g/ml) at 37 °C, in a humidified atmosphere with 5% CO2, for 48 h. Doxorubicin (DOX; 0.025 – 25 µg/mL)
0.25%) did not affect cell viability. Before (T0)
was used as positive control. The final DMSO concentration (
≤
and after sample addition [compound-free (C) and tested (T) cells], cells were fixed with 50% trichloroacetic
acid, and cell proliferation was determined by the spectrophotometric quantification (540 nm) of cellular protein
content using sulforhodamine B assay. Cell proliferation was determined according to the equation 100 x [(T-
T )/C-T ], for T < T
≤ C, and 100 x [(T-T )/T ], for T ≤ T and a concentration-response curve for each cell line
0 0 0
0
0
0
was plotted using software ORIGIN 7.5 (OriginLab Corporation).
4
.3.3. Data analysis
Using the concentration-response curve for each cell line, GI50 (concentration that inhibits cell growth by 50%)
was determined through non-linear regression analysis using the software ORIGIN 8.0 (OriginLab Corporation)
(
(
Shoemaker, 2006). The average activity (mean of log GI ) (Shoemaker, 2006) and the selectivity index (SI)
50
relation GI50 HaCat/GI50 tumor cell line) were calculated for each compound using MS Excel software.
4
.4. Docking studies
In order to obtain a molecular interaction profile of isocoumarin derivatives as antitumor agents, the crystal
structure of kallikrein 5 (KLK5) was obtained [29] from the Protein Data Bank (PDB code: 2PSX) and used for
docking and alignment of the structures synthetized (Table 4). The calculation of the docking energies of the
ligands inside the hK5 active site was performed using the software Molegro Virtual Docker® (MVD®) [30].
This program is able to predict the most likely conformation of how a ligand will bind to a macromolecule [31,
3
2]. In summary, MolDock scoring function is used to automatically superimpose a flexible molecule onto a
template molecule. The docking search algorithm used in MVD is based on an evolutionary algorithm, where
interactive optimization techniques inspired by Darwinian evolution theory and a new hybrid search algorithm
called guided differential evolution are employed [33, 34]. The guided differential evolution algorithm combines
the differential evolution optimization technique with a cavity prediction algorithm during the search process,
which allows for a fast and accurate identification of potential binding modes (poses). The active site exploited
in docking studies was defined, with a subset region of 8.0 Å from the center of the ligand. The interaction
modes of the ligand with the enzyme active site were determined as the highest energy scored protein-ligand
complex used during docking [35].

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3
Appendix A. Supplementary data
1
13
Supplementary data (S1: images of the antiproliferative activity assay, S2: H and C NMR spectra, and S3:
high resolution mass spectra related to this article can be found at http://...
Acknowledgements
This research was supported by grants from CNPq (Conselho Nacional de Desenvolvimento Científico e
Tecnológico), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), FAPEMIG (Fundação
de Amparo à Pesquisa do estado de Minas Gerais) and PRPq (Pró-Reitoria de Pesquisa da UFMG). We thank
UFMG for contributing funding for this study.
The authors have declared no conflict of interest.
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