Evaluation of toxicity on epithelial and tumor cells of biaryl dipeptide tyrosines

Article abstract of DOI:10.1016/j.ejmech.2016.02.062

We report a method to obtain biaryl dipeptide tyrosine via Suzuki-Miyaura and alkynyl dipeptide tyrosine by Sonogashira cross-coupling reactions. Analysis of the biological action of biaryl dipeptide tyrosine 4d compound showed its ability to impair the metabolism and proliferation of SK-Mel-28 human melanoma lineage cells, independently of mitochondrial membrane depolarization, apoptosis and necrosis. Moreover, 4d compound did not cause toxicity to human umbilical vein endothelial cells (HUVEC), suggesting its toxic specificity to cancer cells.

Full text of DOI:10.1016/j.ejmech.2016.02.062

European Journal of Medicinal Chemistry 114 (2016) 1e7
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Evaluation of toxicity on epithelial and tumor cells of biaryl dipeptide
tyrosines
Stanley N.S. Vasconcelos a, Carine C. Drewes b, Leonard de Vinci Kanda Kupa b,
b
a, *
ꢀ
Sandra H.P. Farsky , Helio A. Stefani
a
ꢀ
^
^
~
~
Departamento de Farmacia, Faculdade de Ciencias Farmaceuticas, Universidade de Sao Paulo, Sao Paulo, SP, Brazil
Departamento de Analises Clínicas e Toxicologicas, Faculdade de Ciencias Farmaceuticas, Universidade de Sao Paulo, Sao Paulo, SP, Brazil
b
ꢀ
ꢀ
^
^
~
~
a r t i c l e i n f o
a b s t r a c t
Article history:
We report a method to obtain biaryl dipeptide tyrosine via SuzukieMiyaura and alkynyl dipeptide
tyrosine by Sonogashira cross-coupling reactions. Analysis of the biological action of biaryl dipeptide
tyrosine 4d compound showed its ability to impair the metabolism and proliferation of SK-Mel-28 hu-
man melanoma lineage cells, independently of mitochondrial membrane depolarization, apoptosis and
necrosis. Moreover, 4d compound did not cause toxicity to human umbilical vein endothelial cells
(HUVEC), suggesting its toxic specificity to cancer cells.
Received 22 December 2015
Received in revised form
23 February 2016
Accepted 24 February 2016
Available online 2 March 2016
© 2016 Elsevier Masson SAS. All rights reserved.
Keywords:
Tyrosine
Alkyne
Dipeptide
Tumor
Melanoma
1. Introduction
in Positron Emission Tomography (PET) to detect neoplasms [4,5].
In this context, tyrosine derivatives have been considered prom-
Structural component of proteins and peptides, amino acids
have been used as building blocks in the synthesis of more complex
molecules and biologically active compounds, this fact is due to the
low cost of amino acids, high availability and low toxicity. In this
sense the tyrosine, a nonessential amino acid, but proteinogenic, is
one of the few aromatic amino acids and being the only carrier of a
phenolic nucleus, ortho director for alkylation and acylation re-
actions it is also used in obtaining of biaryl subunit via cross-
coupling reactions [1].
The functionalization of tyrosine cores is a synthetic strategy
that will achieve unnatural peptides, assigning a higher biological
potential of this class of compounds, as an example of peptides
containing tyrosine residues with known biological activity, the
ustiloxin D, a potent antimitotic agent, isolated from the fungus
Ustaliginoidea virens and recently synthetically exploited by Hutton
and co-workers [2], as well as valorphin, with antiproliferative
properties against tumor cells [3] (Fig. 1).
ising tracers candidates because they take into account the
lengthened half-live of tyrosine in comparison to other amino
acids, such as methionine [4,6,7]. Indeed, the efficacy of fluo-
ralkyltyrosine compounds as tracers has been shown in experi-
mental models and human cancer [8e13].
Melanoma is a malignancy of melanocytes which are pigment-
producing cells found in the skin, iris and rectum. The rate of ma-
lignant melanoma has increased in the last few years and it is
estimated that there will be more than 73,000 new cases in 2015 in
the United States (National Cancer Institute of NIH, 2015). Mela-
noma has a high metastatic index and patients with stage IV mel-
anoma have a poor prognosis, with a mean survival of 8e10 months
[14,15]. Advanced stages of disease are resistant to established
therapeutic approaches, including chemotherapy, surgical excision,
radiotherapy and immunotherapy [15].
In this sense, there is great demand for methods that allow the
synthesis of a variety of functionalized tyrosine dipeptide de-
rivatives. Herein, we report an efficient and general access method
for the synthesis of biaryl dipeptide tyrosine derivatives via the
SuzukieMiyaura reaction and 3-alkynyl dipeptide tyrosine via
Sonogashira coupling. Furthermore, we show that the 4d derivative
had an anti-proliferative effect on the human melanoma cell line
Moreover, amino acid derivatives have been employed as tracers
* Corresponding author.
E-mail address: hstefani@usp.br (H.A. Stefani).
http://dx.doi.org/10.1016/j.ejmech.2016.02.062
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

2
S.N.S. Vasconcelos et al. / European Journal of Medicinal Chemistry 114 (2016) 1e7
Fig. 1. Peptide tyrosine derivatives with known biological activity.
SK-Mel-28, with selectivity over normal human umbilical vein
endothelial cells (HUVEC).
(1.2 eq) 1 (0.5 mmol) TEA (1.2 eq), DCM, 15 h. Yield related to the
isolated products.
2. Results and discussion
2.2. Biological activity
2.1. Chemistry
A MTT assay showed that a solution of 4d impaired the meta-
bolism of SK-Mel-28 cells but not HUVEC. This effect was observed
24 or 48 h after incubation of the cells with two concentrations of
the 4d solutions (Fig. 2). As results from MTT assays may indicate
alterations of mitochondrial membrane potential, we further
evaluated if 4d solutions could affect mitochondrial membrane
depolarization. The results showed equivalent depolarization in
HUVEC or SK-Mel-28 cells treated with PBS, vehicle or 4d solutions
(Fig. 3). Nevertheless, the positive control valinomycin, caused
depolarization in both HUVEC and SK-Mel-28 cells, indicating the
efficacy of the experimental procedure (Fig. 3).
A reduction in SK-Mel-28 cell metabolism may lead to impaired
cell proliferation. Therefore cell proliferation assays were per-
formed to further examine the biological action of solution 4d. As
shown in Fig. 4, incubation of SK-Mel-28 cells with both concen-
trations of 4d led to lower cell numbers than when cells were
incubated with vehicle, and an equivalent number of DMSO-
incubated cells (positive control) (Fig. 4). Moreover, as expected,
the proliferation of 4d-treated HUVEC was similar to that of
vehicle-treated cells and higher than that of DMSO-treated cells
(Fig. 4), indicating that 4d did not affect HUVEC proliferation.
Apoptosis or necrosis impair cell metabolism; therefore, we
investigated if 4d could induce apoptosis or necrosis in SK-Mel-
28 cells. Results showed that 4d did not induce cell death in the
concentrations evaluated, as the percentage of SK-Mel-28 or
HUVEC in apoptosis, late apoptosis or necrosis was similar in
vehicle-treated or 4d-treated cells (Fig. 5).
Together, these results indicate that the biaryl dipeptide tyro-
sine derivative is toxic to melanoma but not to epithelial cells. SK-
Mel-28 lineage was chosen to test the toxicity because it is a ma-
lignant melanoma cell line. Melanoma development is aggressive,
especially in the metastatic form, and no efficient treatments have
been proposed until now [18]. Therefore, novel or complementary
pharmacological approaches are needed for the treatment of mel-
anoma. The results from this study show that further biological
tests should be conducted to determine if biaryl dipeptide tyrosine
derivatives could be used as therapeutic agents. It is important to
mention that 4d selectivity to SK-Mel-28 cells over HUVEC
strengthens our data, as the endothelium consists epithelial cells
covering the luminal surface of all vessels, and therefore, it is the
first barrier to chemical agents during tissue distribution from the
blood circulation [19,20].
Our strategy included the preparation of dipeptides 3a¡c
through the formation of a peptide bond between tyrosine-methyl
ester 1 and N-Boc-tyrosine 2, using hydroxybenzotriazole (HOBt),
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), triethyl-
amine (TEA) and dichloromethane (DCM), in a 15 h reaction at
room temperature. Using the same methodology it was possible to
obtain the peptide 3a in 57% yield with an iodine atom in the
structure, as well as the peptides 3b and 3c containing one iodine
atom in each aromatic ring, which were achieved in 48% and 57%
yield respectively. From peptides core containing iodine we per-
formed SuzukieMiyaura or Sonogashira cross-coupling reactions
[16,17].
In order to obtain an alkenyl dipeptide derivative, the peptide 3a
it was reacted with potassium trans-styryltrifluoroborate as sub-
strate, using Pd(OAc)2 in 10 mol%, K2CO3 as base in MeOH, the
dipeptide 4a was achieved in 89% yield. To obtain two different
biarylic fragments, we prepared a SuzukieMiyaura coupling reac-
tion between the dipeptide 3b and the potassium phenyl-
trifluoroborate salt, the reaction led the exclusive formation of
desired cross-coupling product 4b that after purification was iso-
lated in 52% yield. Since iodine was still present in the peptide
fragment, this allowed us to use a new cross-coupling reaction to
get different biarylic subunits in peptides fragments. Therefore,
dipeptide
4b
was
reacted
with
potassium
3-
thiophenetrifluoroborate salt under SuzukieMiyaura conditions
to give the compound 4c in 42% yield.
The reactivity of the dipeptide 3b was observed in the bis
coupling reaction, when we used two equivalent of potassium 4-
methoxyphenyltrifluoroborate salt and one equivalent of 3b un-
der appropriate conditions for SuzukieMiyaura cross-coupling, just
the compound 4d was observed, after flash chromatography, the
product could be obtained in 61% of yield.
The SuzukieMiyaura products were obtained in the peptide
fragments without phenol group protection; but when the Sono-
gashira cross-coupling reaction was performed, it was necessary to
protect the phenol; the reaction without prior protection has led to
the desired product in low yields. In this sense, using Pd(dppf)
Cl.2CH2Cl2 as a catalyst, CuI as a co-catalyst, TEA and THF, dipeptide
3c and ethynyltrimethylsilane were reacted via a Sonogashira re-
action to give the desired product 4e in 89% yield in a single step
(Scheme 1).
The mechanism involved in the selectivity of 4d has not been
identified, but it is possible that cancer cells may uptake the
Peptide bond conditions: 2 (0.5 mmol) HOBt (1.1 eq), EDC

S.N.S. Vasconcelos et al. / European Journal of Medicinal Chemistry 114 (2016) 1e7
3
Peptide bond conditions: 2 (0.5 mmol) HOBt (1.1 eq), EDC (1.2 eq) 1 (0.5 mmol) TEA (1.2 eq),
DCM, 15 h. Yield related to the isolated products.
Scheme 1. Functionalization of tyrosine dipeptides.
compound with more effciency than epithelial cells, as cancer cells
have more amino acids and increased protein metabolism [21,22].
Based in this phenotypic characteristic of cancer cells, tyrosine
derivatives have been employed as tracers, as previously
mentioned. Here we show, for the first time, that the tyrosine de-
rivatives may work as teranostic compounds, with the ability to
detect and induce mechanisms of cancer cell toxicity. These results
pave the way for future studies to investigate the mechanism by
which biaryl dipeptide tyrosine derivatives may be preferentially
captured by cancer cells, leading to more selectivity as cytotoxic
agents.
toxicity of a biaryl dipeptide tyrosine derivative was demonstrated
in a melanoma cell lineage, but not to non-tumoral epithelial cells,
suggesting that these compounds may have selective cytotoxic
potential.
4. Experimental section
4.1. Chemistry
All of the starting materials were commercial grade and used
without further purification. 1H NMR 13C NMR and spectra were
recorded on a Bruker DPX 300 at 300 MHz and 75 MHz, respec-
tively, using CDCl3. Chemical shifts are reported in ppm, referenced
to the solvent signal of CDCl3 or tetramethylsilane (TMS) as the
3. Conclusion
The combination of tyrosine fragments via peptide bond fol-
lowed by functionalization via SuzukieMiyaura and Sonogashira
cross-coupling reactions afforded structurally and biologically
interesting dipeptide compounds. Moreover, for the first time, the
internal reference. Data are reported as follows: chemical shift (d),
multiplicity, coupling constant (J) in Hertz and integrated intensity.
Abbreviations to denote the multiplicity of a particular signal are: s
(singlet), d (doublet), t (triplet), q (quartet), quint (quintet), sex

4
S.N.S. Vasconcelos et al. / European Journal of Medicinal Chemistry 114 (2016) 1e7
Fig. 2. Effect of biaryl dipeptide tyrosine derivative (compound 4d) on human um-
bilical vein endothelial cell (HUVEC) and melanoma cell (SK-Mel-28) metabolism.
1 ꢁ 103 cells/well of HUVEC (A) or SK-Mel-28 cells (B) were incubated with medium
Fig. 3. Effect of biaryl dipeptide tyrosine derivative (compound 4d) on human um-
bilical vein endothelial cell (HUVEC) and melanoma cell (SK-Mel-28) mitochondrial
membrane potential. 1 ꢁ 104 cells/well of SK-Mel-28 (A) or HUVEC (B) were incubated
R10 (control), vehicle (0.1% DMSO), or 4d (0.1 or 1
mM) for 14, 24, or 48 h. Cell
metabolism was assessed using a MTT assay and values are represented as mean SEM
of three independent assays. Data are expressed as % of control group. 10% DMSO was
used as positive control. Significant differences from R10 (control) are *p < 0.05,
**p < 0.01 and ***p < 0.001 assessed by two-way ANOVA followed by the Tukey's test.
with medium R10 (control), vehicle (0.1% DMSO), or 4d (0.1 or 1
m
M) for 48 h and the
g/mL) by flow
M) for 24 h as a positive control.
mitochondrial membrane potential was determined using JC-1 (1.5
cytometry. Cells were treated with valinomycin (200
m
m
Values are represented as mean SEM of two independent assays. Significant differ-
ences from R10 (control) are *p < 0.05, and ***p < 0.001 assessed by one-way ANOVA
followed by the Tukey's test.
(sextet) and m (multiplet). Column chromatography was per-
formed using silica gel (230e400 mesh). Thin Layer Chromatog-
raphy (TLC) was performed using silica gel UV254, 0.20 mm
thickness. High-Resolution Mass Spectra were obtained using a
high-resolution ESI-TOF mass spectrometer Shimadzu LCMS-IT-
TOF.
(t, J ¼ 7.9 Hz, 2H), 6.57 (d, J ¼ 7.7 Hz, 1H), 5.16 (s, 1H), 4.72 (d,
J ¼ 7.0 Hz, 1H), 4.28 (s, 1H), 3.68 (s, 3H), 2.99e2.85 (m, 4H), 1.40 (s,
9H); 13C NMR (75 MHz, CDCl3)
d 171.59, 171.35, 155.67, 155.26,
154.53, 139.13, 130.73, 130.39, 129.32, 115.75, 115.19, 85.01, 53.43,
52.50, 37.54, 36.51, 28.28; HRMS (ESI-TOF) m/z, calcd. for
24H29IN2O7 þ Hþ: 585.1097, found: 585.1094.
4.1.1. General procedure for dipeptide formation
C
Product 3b was obtained as a yellow oil. Yield 170 mg (48%). 1H
A solution of 2 (140 mg, 0.5 mmol) in dry DCM was cooled to
0
ꢀC and 1-hydroxybenzotriazole (1.1 eq) was added, followed by
NMR (300 MHz, CDCl3) d 7.49 (d, 1H, J 1.8 Hz), 7.36 (d, 1H, J 1.8 Hz),
EDC (1.2 eq). After 1 h, the mixture was allowed to warm to room
temperature at which point compound 1 (179 mg, 0.5 mmol) dis-
solved in DMF was added, followed by TEA (1.2 eq). The reaction
mixture was stirred for 15 h. Then the reaction mixture was diluted
with DCM and washed with a saturated solution of NH4Cl
(3 ꢁ 20 mL). The organic phase was collected, dried with MgSO4,
filtered and the solvent was removed under vacuum. The product
was purified by silica flash chromatography and eluted with ethyl
acetate/hexane 5:5.
7.25e7.20 (m, 2H), 7.00 (d, 1H, J 7.9 Hz), 6.86 (dd, 1H, J 1.8, 8.3 Hz),
6.78 (d, 1H, J 8.0 Hz), 6.66 (d, 1H, J 7.7 Hz), 5.19 (d, 1H, J 7.2 Hz), 4.74
(dd, 1H, J 6.0, 13.0 Hz), 4.47 (d, 1H, J 7.8 Hz), 4.30 (s, 1H), 3.69 (s, 3H),
3.02e2.89 (m, 4H), 1.41 (s, 9H); 13C NMR (75 MHz, CDCl3)
d 171.35,
157.55, 155.57, 154.78, 154.67, 139.28, 130.72, 130.62, 129.83, 129.13,
115.31, 115.26, 84.93, 60.55, 53.48, 52.64, 42.34, 28.32, 23.46, 14.19;
HRMS (ESI-TOF) m/z, calcd. for C24H28I2N2O7 þ Hþ: 710.2973,
found: 710.2965.
Product 3c was obtained as a white solid. Yield 210 mg (57%).
Melting point 132e134 ꢀC. 1H NMR (300 MHz, CDCl3)
d
7.61 (s, 1H),
The product 3a was obtained as a white solid. Yield 166 mg
(57%). Melting point 83e84 ꢀC. 1H NMR (300 MHz, CDCl3)
d
7.34 (s,
7.44 (s, 1H), 7.18e7.10 (m, 1H), 6.96 (dd, J ¼ 8.3, 1.8 Hz, 1H),
1H), 6.97 (d, J ¼ 8.0 Hz, 2H), 6.82 (d, J ¼ 8.3 Hz, 1H), 6.76 (s, 1H), 6.71

S.N.S. Vasconcelos et al. / European Journal of Medicinal Chemistry 114 (2016) 1e7
5
Fig. 5. Effect of biaryl dipeptide tyrosine derivative (compound 4d) on human um-
bilical vein endothelial cell (HUVEC) and melanoma cell (SK-Mel-28) viability.
1 ꢁ 104 cells/well of HUVEC (A) or SK-Mel-28 cells (B) were incubated with medium
Fig. 4. Effect of biaryl dipeptide tyrosine derivative (compound 4d) on human um-
bilical vein endothelial cell (HUVEC) and melanoma cell (SK-Mel-28) proliferation.
1 ꢁ 104 cells/well of HUVEC (A) or SK-Mel-28 cells (B) were incubated with medium
R10 (control), vehicle (0.1% DMSO), or 4d (0.1 or 1
m
M) for 48 h and cell viability was
g/10 L) by flow
SEM of three independent assays. No
R10 (control), vehicle (0.1% DMSO), or 4d (0.1 or 1 mM) for 14, 24, or 48 h and cell
determined using Annexin-V (1:100) and propidium iodide (0.5
m
m
proliferation was determined using a trypan blue assay by optical microscopy. Values
are represented as mean SEM of three independent assays. 10% DMSO was used as
positive control. Significant differences from R10 (control) are *p < 0.05, **p < 0.01 and
***p < 0.001 assessed by one-way ANOVA followed by the Tukey's test.
cytometry. Values are represented as mean
significant differences were found.
(d, J ¼ 7.5 Hz, 2H), 7.38 (d, J ¼ 7.5 Hz, 1H), 7.31 (t, J ¼ 7.6 Hz, 2H), 7.21
(d, J ¼ 7.3 Hz, 1H), 6.99 (d, J ¼ 8.0 Hz, 2H), 6.91 (d, J ¼ 8.3 Hz, 1H),
6.74 (d, J ¼ 8.2 Hz,1H), 6.66 (d, J ¼ 8.1 Hz, 2H), 4.67 (t, J ¼ 6.8 Hz,1H),
4.22 (s, 1H), 3.67 (s, 3H), 3.12e2.65 (m, 4H), 1.33 (s, 9H). 13C NMR
6.76e6.61 (m, 3H), 5.18 (d, J ¼ 8.0 Hz, 1H), 4.75 (q, J ¼ 5.9 Hz, 1H),
4.40e4.27 (m, 1H), 3.82 (s, 6H), 3.69 (s, 3H), 2.97 (q, J ¼ 7.0, 6.4 Hz,
4H), 1.40 (s, 9H); 13C NMR (75 MHz, CDCl3)
d 171.30, 170.82, 157.20,
(75 MHz, MeOH)
d 174.32, 173.26, 157.21, 155.26, 139.58, 131.35,
157.12, 155.28, 140.10, 140.05, 130.75, 130.43, 130.28, 129.86, 111.01,
110.87, 86.07, 85.86, 80.23, 56.39, 56.32, 55.72, 53.32, 52.45, 36.90,
36.54, 28.31; HRMS (ESI-TOF) m/z, calcd. for C26H32I2N2O7 þ Hþ:
739.0377, found: 739.0375.
130.36, 129.62, 129.12, 128.80, 128.46, 128.23, 127.46, 125.77, 124.82,
116.87, 116.21, 80.74, 57.60, 55.27, 52.74, 37.96, 30.42, 28.68. HRMS
(ESI-TOF) m/z, calcd. for C32H36N2O7 þ Hþ: 561.2601, found:
561.2597.
Product 4b was obtained as a colorless oil. Yield 171 mg (52%).
4.1.2. General procedure for SuzukieMiyaura coupling
1H NMR (300 MHz, CDCl3)
d 7.37e7.35 (m, 5H), 6.88 (d, 1H, J 7.5 Hz),
K2CO3 (207 mg, 1.5 mmol) was added to a two-necked 25 mL
round-bottom flask under a nitrogen atmosphere containing
Pd(OAc)2 (11.23 mg, 10 mol%), potassium trifluoroborate salt
(0.6 mmol), dipeptide 3a or 3b (0.5 mmol) and MeOH (3 mL). The
reaction mixture was stirred and heated to 60 ꢀC. TLC was used to
monitor the 10-h reaction. The reaction mixture was then diluted
with ethyl acetate and washed with a saturated solution of NH4Cl
(3 ꢁ 20 mL). The organic phase was collected, dried with MgSO4,
filtered and the solvent was removed under vacuum. The product
was purified by silica flash chromatography and eluted with ethyl
acetate/hexane 5:5.
6.82 (s, 1H), 6.77e6.76 (m, 2H), 6.68 (d, 1H, J 8.2 Hz), 6.35 (d, 1H, J
7.5 Hz), 5.78 (s, 1H), 4.95 (s, 1H), 4.73e4.67 (m, 1H), 4.17 (s, 1H), 3.61
(s, 3H), 2.96e2.93 (m, 2H), 2.81e2.79 (m, 2H), 1.32 (s, 9H); 13C NMR
(75 MHz, CDCl3)
d 171.52, 170.83, 155.35, 154.33, 151.93, 139.10,
137.12, 131.08, 130.82, 130.19, 129.68, 129.06, 128.38, 127.75, 127.53,
116.21, 115.17, 85.30, 52.46, 42.38, 37.19, 37.01, 28.27, 23.45; HRMS
(ESI-TOF) m/z, calcd. for C30H33IN2O7 þ Hþ: 661.1411, found:
661.1407.
Product 4c was obtained as a colorless oil. Yield 64 mg (42%). 1H
NMR (300 MHz, CDCl3) d 7.36e7.28 (m, 7H), 7.22e7.19 (m, 1H), 7.07
(s, 1H), 6.86e6.79 (m, 2H), 6.76 to ꢂ6.67 (m, 3H), 6.30 (d, 1H, J
The product 4a was obtained as a white solid. Yield 243 mg
7.5 Hz), 6.11 (s, 1H), 5.75 (s, 1H), 4.96 (s, 1H), 4.69e4.65 (m, 1H), 4.22
(87%). Melting point 133e134 ꢀC. 1H NMR (300 MHz, MeOH)
d 7.51

6
S.N.S. Vasconcelos et al. / European Journal of Medicinal Chemistry 114 (2016) 1e7
(s, 1H), 3.55 (s, 3H), 2.95e2.75 (m, 4H), 1.30 (s, 9H); 13C NMR
(75 MHz, CDCl3) 171.52, 171.07, 152.12, 151.90, 137.51, 137.13,
37 ꢀC and 5% CO2. Subsequently, cells were washed and incubated
with 100 L of MTT solution (0.5 mg/mL in RPMI media) for 3 h in
the dark at 37 ꢀC and 5% CO2. After incubation, the MTT solution was
removed and cells were washed with PBS buffer (300 L). 200
DMSO was then added to each well and. the plate was mixed for
5 min at 500 rpm. The optical density was read by spectropho-
tometry at 570 nm. 10% DMSO was employed as positive control.
The percentage of viable cells was calculated by comparing the
absorbance of treated and untreated cells (incubated with only
RPMI, corresponding to 100% viability).
d
m
131.02, 130.59, 129.68, 129.29, 129.05, 129.03, 128.36, 128.09, 127.71,
127.56, 126.05, 123.05, 116.41, 116.17, 60.44, 53.44, 52.30, 42.34,
37.18, 28.25, 23.45, 14.19; HRMS (ESI-TOF) m/z, calcd. for
m
mL
C
34H36N2O7S þ Hþ: 617.2321, found: 617.2318.
Product 4d was obtained as a colorless oil. Yield 102 mg (61%).
1H NMR (300 MHz, CDCl3)
d 7.35 (d, 4H, J 8.4 Hz), 7.02e6.96 (m, 6H),
6.87e6.79 (m, 4H), 6.42 (d, 1H, J 7.6 Hz), 5.70 (s, 1H), 5.04 (s, 1H),
4.80e4.74 (m, 1H), 4.32 (s, 1H), 3.84 (s, 6H), 3.65 (s, 3H), 2.99e2.88
(m, 4H), 1.40 (s, 9H); 13C NMR (75 MHz, CDCl3)
d 171.53, 171.05,
159.22, 159.20, 151.93, 131.17, 130.99, 130.21, 129.31, 128.31, 128.06,
128.02, 127.54, 116.08, 116.01, 114.53, 114.48, 55.34, 53.44, 52.28,
42.32, 28.25, 23.46; HRMS (ESI-TOF) m/z, calcd. for
4.4. Cell death
A flow cytometry-based method using Annexin V (Becton and
Dickinson, San Jose, CA, USA) and Propidium Iodide dye (PI; Sig-
maeAldrich, St Louis, MO, USA) was employed to determine the
ability of a solution of 4d to induce apoptosis or necrosis, respec-
tively. Annexin-V binds to phosphatidylserine exposed on
apoptotic cell membranes and PI stains necrotic cells, which lose
plasma membrane integrity. Briefly, 1 ꢁ 104 cells/well were plated
and incubated for 48 h. Thereafter, cells were washed with PBS,
trypsinized, centrifuged (600 ꢁ g, 10 min) and incubated with FITC-
conjugated Annexin V diluted in binding buffer (1:100) for 30 min.
C
38H32N2O9 þ Hþ: 671.2969, found: 671.2960.
4.1.3. General procedure for Sonogashira cross-coupling
Dipeptide 3c (74 mg, 0.1 mmol), Pd(dppf)Cl.2CH2Cl2 (16.2 mg,
0.02 mmol), and CuI (38 mg, 0.2 mmol) were added to a dried flask
under nitrogen atmosphere. Freshly distilled THF (4 mL) was added
using a syringe, then Na2CO3 (21 mg, 0.2 mmol) was added and the
resulting solution was stirred for 10 min. Ethynyltrimethylsilane
(0.6 mmol) was added slowly. The resulting solution was stirred at
60 ꢀC. The reaction was monitored by TLC. The mixture was poured
into 10 mL saturated NH4Cl, and then extracted with ethyl acetate
(15 mL) three times. The organic layer was combined, dried with
MgSO4, filtered and the solvent was evaporated. The resulting
residue was purified by silica gel chromatography and eluted with
ethyl acetate/hexane.
After this period, 0.5 mg/10 mL PI in binding buffer was added and
10,000 events were acquired in a flow cytometer (FACS Canto,
Becton and Dickinson, San Jose, CA, USA). The percentage of
apoptotic (FITC positive and PI negative), necrotic (FITC negative
and PI positive), late apoptotic (FITC and PI positive) and living cells
(FITC and PI negative) was quantified.
The product 4e was obtained as a yellow oil. Yield 60.3 mg (89%).
1H NMR (300 MHz, CDCl3)
d
7.53 (qd, J ¼ 7.6, 4.9, 3.2 Hz, 2H),
4.5. Cell proliferation
7.30e7.23 (m, 3H), 7.13e7.09 (m, 1H), 6.92 (dd, J ¼ 8.5, 2.3 Hz, 1H),
6.75 (d, J ¼ 8.7 Hz, 1H), 4.93 (s, 1H), 4.75 (q, J ¼ 6.4 Hz, 1H), 4.26 (d,
J ¼ 7.4 Hz, 1H), 3.86 (s, 6H), 3.70 (s, 3H), 3.06e2.86 (m, 4H), 1.43 (s,
After cell plating (1 ꢁ 104 cells/well) and adhesion, Trypan Blue
dye (SigmaeAldrich, St Louis, MO, USA) was used to quantify cell
proliferation of SK-Mel-28 or HUVEC after incubation with a solu-
tion of 4d or vehicle for 14, 24 or 48 h. A haemocytometer was used
to count the number of cells. Results are expressed as absolute
number of counted cells.
9H), 0.26 (s, 18H). 13C NMR (75 MHz, CDCl3)
d 171.24, 170.64, 159.42,
159.36, 135.16, 134.78, 134.66, 130.77, 130.62, 128.25, 127.42, 112.38,
111.00,110.80,100.97, 98.64, 60.30, 55.85, 55.79, 53.26, 52.23, 36.87,
28.19, 0.00. HRMS calcd. for C36H50N2O7Si2 þ Hþ: 679.3350. Found:
679.3355.
4.6. Mitochondrial membrane potential assay
4.2. Cell culture and treatment
5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazolocarbocya-
nine iodide (denoted JC-1) is a cationic lipophilic dye used as an
indicator for monitoring membrane potential [23]. This molecule
enters mitochondria in monomeric form. At an excitation wave-
length of 490 nm, the molecule has an emission wavelength of
527 nm. However, depending on the membrane potential, JC-1
forms aggregates are associated with a large shift in emission
(590 nm). Thus, the color of the dye changes reversibly from green
(read in FITC channel) to greenish orange (read in PE channel) as
the mitochondrial membrane becomes more polarized [24].
1 ꢁ 104 cells/well SK-Mel-28 or HUVEC were plated and after
adhesion were incubated with a solution of 4d or vehicle for 48 h.
After this period, cells were washed with PBS and incubated with
Human skin malignant melanoma cells (SK-Mel-28) and human
umbilical vein endothelial cells (HUVEC) were maintained in RPMI
1540 medium containing 10% fetal bovine serum (Vitrocell
ꢀ
~
Embrolife, Campinas, Sao Paulo, Brazil) at 37 C with 5% CO2. When
cells reached confluency, they were trypsinized (Vitrocell embro-
~
life, Campinas, Sao Paulo Brazil), centrifuged (600 ꢁ g for 10 min),
counted in a haemocytometer, plated, and incubated with culture
medium for 24 h. After adhesion, the culture medium was replaced
with a solution of 4d (0.1 or 1 mM) or vehicle (0.1% DMSO). 4d so-
lution was prepared from a DMSO stock solution (10 mM) and
stored at room temperature in dark conditions. Dilutions were
made in complete culture medium just before the experiments.
JC-1 (1.5 mg/mL, Life Technologies, Walthan, MA, USA) for 30 min at
4.3. Cell metabolism
37 ꢀC and 5% CO2. Thereafter, cells were washed again, trypsinized,
centrifuged (600 ꢁ g, 10 min) and transferred to flow cytometry
tubes. 10,000 events were acquired in a flow Cytometer (FACS
Canto, Becton and Dickinson, San Jose, CA, USA) at 488 nm (exci-
tation) and 529 and 590 (emission for monomeric form and
aggregate form, respectively). For the control, cells were treated
The MTT assay is a colorimetric assay used to quantify cell
metabolism.
MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; SigmaeAldrich; St Louis, MO, USA)
is a yellow compound that is soluble in water and converted to
formazan crystal, a violet insoluble product in water, by the action
of mitochondrial reductases in active cells. 1 ꢁ 103 cells/well SK-
Mel-28 or HUVEC were prepared and treated as described above
and incubated with a solution of 4d or vehicle for 14, 24 or 48 h, at
with medium RPMI 1540 or 200 mM Valinomycin (positive control;
SigmaeAldrich, St Louis, MO, USA) for 30 min before incubation
with JC-1 dye. Data are expressed as the ratio of mean of green
fluorescence intensity to red fluorescence intensity.

S.N.S. Vasconcelos et al. / European Journal of Medicinal Chemistry 114 (2016) 1e7
Amino Acids 47 (2015) 335e344.
7
4.7. Statistics analysis
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H.H. Coenen, K.J. Langen, N. Galldiks, The usefulness of dynamic O-(2-18F-
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The use of dynamic O-(2-18F-fluoroethyl)-L-tyrosine PET in the diagnosis of
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All data are expressed as mean standard error of the mean
(SEM). Comparisons were performed using student's t test when
two groups were compared. ANOVA test followed by Tukey's test
was performed when three or more groups were compared. Sig-
nificant difference was considered with p < 0.05.
Acknowledgments
[12] P. Lohmann, H. Herzog, E.R. Kops, G. Stoffels, N. Judov, C. Filss, N. Galldiks,
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The authors gratefully acknowledge financial support from the
Sao Paulo Research Foundation (FAPESP - grant 2012/00424-2 and
fellowships 2013/17960-7 to S.N.S.V. and 2010/19802-1 to C.C.D)
and The National Council for Scientific and Technological Devel-
opment for a fellowship (308.320/2010e7 to HAS and L.V.K.K).
~
Appendix A. Supplementary data
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Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.02.062.
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