ChemMedChem
10.1002/cmdc.201900566
FULL PAPER
[
50]
Diphenyl disulfide (1): White solid; 25 % yield; m.p. 56 °C, 56-58 °C
;
([CPM in sample with inhibitor – background CPM] ÷ [CPM in sample
without inhibitor – background CPM] x 100).
1
H-NMR (300 MHz, CDCl
3
): δ=7.19-7.32 (m, 6H; Ar-H), 7.49 (d, J=8.64
): δ=127.1 (CH), 127.5 (CH),
37.0 (C); IR (KBr, cm ): 1575, 1475 (C=C); HRMS (ESI): m/z calcd. for
13
Hz, 4H; Ar-H); C-NMR (75 MHz, CDCl
3
-
1
1
C
Cell lines and culture conditions: Tumor cell lines: MCF-7 (ATCC-
HTB-22, breast adenocarcinoma); 786-0 (ATCC-CRL-1932, renal cell
adenocarcinoma); PC-03 (ATCC-CRL-1435, prostate adenocarcinoma);
HEPG2 (ATCC-HB-8065, hepatocellular carcinoma); HT-29 (ATCC-HTB-
38, colorectal adenocarcinoma) and MDA-MB-231 (ATCC HTB-26, triple
negative breast cancer cells) were cultivated in RPMI-1640 (Roswell
Park Memorial Institute Medium). B16-F10 (ATCC CRL-6322, melanoma
cells) and non-tumor cell line NIH/3T3 (ATCC CRL-1658 murine
fibroblast) were cultivated in DMEM (Dulbecco’s Modified Eagle Medium)
+
H S
12 10 2
: 218.0218 [M] ; found: 218.0219.
Bis(4-methylphenyl) disulfide (2): White solid; 57 % yield; m.p. 43 °C,
[
51]
1
4
3-44 °C
J=7.89 Hz, 4H; Ar-H), 7.37 (d, J=8.04 Hz, 4H; Ar-H); C-NMR (75 MHz,
CDCl ): δ=21.0 (CH ), 128.5 (CH), 129.8 (CH), 133.9 (C), 137.4 (C); IR
KBr, cm ): 1568, 1488 (C=C); HRMS (ESI): m/z calcd. for C14
3 3
; H-NMR (300 MHz, CDCl ): δ=2.32 (s, 6H; CH ), 7.10 (d,
13
3
3
-
1
(
2
H
14
S
2
:
+
46.0513 [M] ; found: 246.0553.
-
Sigma Aldrich®. The culture media were supplemented with 10% fetal
bovine serum - Invitrogen® and 1% antibiotics (streptomycin - 100 µg/mL
and penicillin 100 IU/mL - Sigma Aldrich®). All cell lines were incubated
in a humidified chamber at 37 °C in a 5% CO atmosphere.
2
Bis(4-methoxyphenyl) disulfide (3): Yellow solid; 9 % yield; m.p. 42 °C,
[
52] 1
4
2-43 °C
J=8.88 Hz, 4H; Ar-H), 7.38 (d, J=8.88 Hz, 4H); C-NMR (75 MHz,
CDCl ): δ=55.4 (OCH ), 114.6 (CH), 128.4 (C), 132.7 (CH), 159.9 (C); IR
KBr, cm ): 1589, 1492 (C=C); HRMS (ESI): m/z calcd. for C14
3 3
; H-NMR (300 MHz, CDCl ): δ=3.78 (s, 6H; OCH ), 6.81 (d,
13
3
3
-
1
(
14 2 2
H O S :
Cell proliferation assay: The SRB assay was carried out as described
+
[56]
278.0429 [M] ; found: 278.0425.
by Skehan and coworkers. Cell suspensions (3500-7500 cells per well)
were seeded in 96 wells plates, incubated and allowed to stabilize for 24
h. The cells were treated with different sample concentrations (0.25; 2.5;
25 and 250 µg/mL) prepared in DMSO and then diluted in culture
medium. DMSO (0.25%) alone did not affect cell viability in comparison
to the untreated controls. Doxorubicin (DOX) was taken as a positive
control only for validation experiments, and used at a ten times lower
concentration (Fauldoxo®/LIBBS). The initial optical density (OD) for the
SRB assay was measured at the same moment as compounds were
added to the samples (Time 0) and after a 48 h treatment, (exceptionally
for line cell 786-0, it was performed independent experiments at time
periods of 12, 24 and 48h. The OD of each well was determined by
measuring at 540 nm using a microplate reader (SpectraMax 190
microplate reader - Molecular Devices), and the absorbance values were
used to determine the percentage of cell growth, calculated using the
S-Phenyl benzenethiosulfonate (4): White solid; 71 % yield; m.p. 38 °C,
[
26]
1
3
6-38 °C
;
H-NMR (300 MHz, CDCl
3
): δ=7.28-7.48 (m, 7H; Ar-H),
): δ=127.5 (CH), 127.8
13
7.53-7.58 (m, 3H; Ar-H); C-NMR (75 MHz, CDCl
3
(
CH), 128.8 (CH), 129.4 (CH), 131.4 (C), 133.6 (CH), 136.5 (CH), 142.9
-
1
(C); IR (KBr, cm ): 1575, 1475 (C=C), 1326, 1146 (S=O); HRMS (ESI):
+
+
+
m/z calcd. for
2
12 10 2 2 12 10 2 2
C H O S +H : 251.0194 [M+H] ; C H O S +Na =
+
73.0008 [M+Na] ; found: 251.0210, 273.0001.
S-(4-methylphenyl) 4-methylbenzenesulfonothioate (5): White solid;
[
53] 1
1
(
0 % yield; m.p. 72 °C, 72-74 °C
s, 3H; CH ), 2.40 (s, 3H; CH ), 7.12 (d, J=8.1 Hz, 2H; Ar-H), 7.19 (d,
J=8.5 Hz, 2H; Ar-H), 7.22 (d, J=8.4 Hz, 2H; Ar-H), 7.44 (d, J=8.2 Hz, 2H;
3
; H-NMR (300 MHz, CDCl ): δ=2.36
3
3
13
Ar-H); C-NMR (75 MHz, CDCl
3
): δ=21.4 (CH
3 3
), 21.6 (CH ), 124.6 (C),
[
57]
software Soft Max Pro 6.3 as described by Monks and coworkers. The
results were expressed as a curve of cell growth versus compound
concentration. GI50 values (50% cell growth inhibition, TGI (total cell
growth inhibition) and LC50 (50% cell death) were determined by non-
linear regression analysis using data graphic software (Origin version
1
1
27.6 (CH), 129.3 (CH), 130.2 (CH), 136.4 (CH), 140.4 (C), 142.0 (C),
-
1
44.5 (C); IR (KBr, cm ): 1590, 1488 (C=C), 1323, 1139 (S=O); HRMS
+
+
+
(
ESI): m/z calcd. for C14
H
14
O
2
S
2
+H : 279.0507 [M+H] ; C14
H
14
O
2
S
2
+Na :
+
+
+
301.0321 [M+Na] ; C14
H
14
O
2
S
2
+K : 317.0061 [M+K] ; found: 279.0496,
301.0322, 317.0068.
6.0). This assay was performed in three independent experiments, and
the results are expressed in molarity. The compounds were classified as
S-(4-methoxyphenyl)-4-methoxybenzenesulfonothioate (6): White
either active (GI50<10 μM), moderately active (GI50 between 10 and 100
[
26]
1
solid; 22 % yield; m.p. 85 °C, 84 °C
δ=3.81 (s, 3H; OCH ), 3.85 (s, 3H; OCH
.85 (d, J=8.6 Hz, 2H; Ar-H), 7.25 (d, J=8.4 Hz, 2H; Ar-H), 7.48 (d, J=8.9
;
H-NMR (300 MHz, CDCl
3
):
[58]
μM), or inactive (GI50>100 μM).
Subsequently, compound 6 was
3
3
), 6.82 (d, J=8.5 Hz, 2H; Ar-H),
evaluated by the SRB cytotoxicity assay against the cell line 786-0
ATCC-CRL-1932) in treatment periods of 12, 24 and 48 h.
6
(
13
Hz, 2H; Ar-H); C-NMR (75 MHz, CDCl
3 3 3
): δ=55.4 (OCH ), 55.7 (OCH ),
1
1
13.8 (CH), 114.9 (CH), 118.9 (C), 129.9 (CH), 134.9 (C), 138.3 (CH),
-
1
Morphological analysis: The AO/EB staining assay permits the
identification of viable, apoptotic, and necrotic cells. It was used to
evaluate death induction in 786-0 cells (ATCC-CRL-1932) after treatment
for periods of 24 and 48 h with compound 6 at the same concentrations
used for cytotoxicity tests. In order to carry out this assay, the cells were
collected and resuspended in phosphate-buffered saline (PBS). The
slides were prepared with 10 µL of the cell suspension and 1 µL of the
stain mixture containing ethidium bromide (100 μg/mL) and acridine
orange (100 μg/mL). Three experiments were performed, and 100
treated cells were analyzed by fluorescence, 400x magnification
62.2 (C), 163.5 (C); IR (KBr, cm ): 1591, 1495 (C=C), 1323, 1139
+
(S=O), 1261, 1020 (C-O); HRMS (ESI): m/z calcd. for C14
H
H
14
O
4
S
2
+H :
+
+
+
+
311.0406 [M+H] ; C14
H
14
O
4
S
2
+Na : 333.0220 [M+Na] ; C14
14
O
4
S
2
+K :
+
348.9959 [M+K] ; found: 311.0400, 333.0219, 348.9958.
Biological assays
Tubulin assays: Tubulin assembly and inhibition of colchicine binding
to tubulin were performed as described before. In the assembly assay,
the tubulin concentration was 10 µM, and the parameter measured was
the extent of assembly after 30 min at 30 °C. In the colchicine binding
[
54]
[
55]
(Olympus BX41). For the image acquisition we used excitation wave of
420-490 nm and barrier filter of 520 nm. Morphological alterations were
3
assay, the tubulin concentration was 1.0 µM, the [ H]colchicine
also evaluated by optical microscopy after treatment with compound 6 at
concentration was 5.0 µM, and the inhibitor concentration was 5 or 50
µM, as indicated. Incubation was for 10 min at 37 °C, a time point chosen
because the control reaction is about 40-60% complete. After the 10 min
incubation, samples were diluted with 2 mL of ice cold water, placed on
ice, and then filtered through a stack of two DEAE-cellulose filters. In
each experiment there were samples without tubulin for determination of
background radiolabel retained by the filters and with tubulin but no
inhibitor for determination of control binding of colchicine. Typically, the
background filters retained about 5% as much radiolabel as the filters
with tubulin only. The background radiolabel was substracted from all
samples to detrmine the % inhibition caused by each potential inhibitor
concentrations near the values of the GI50, TGI and LC50, for 24 or 48 h.
Quantification of caspase-3 by flow cytometry: The assay was
performed using the antibody PE Rabbit Anti-Active Caspase-3 (BD
Pharmingen). The cells of the line 786-0 (ATCC-CRL-1932) were seeded
in 6 well plates (4x105/well), and they were allowed to stabilize for 24 h.
The cells were treated with compound 6 at concentrations near the GI50
,
TGI and, LC50 values for 24 or 48 h. After each incubation period, the
cells were collected, washed twice with cold PBS, fixed, and
permeabilized with BD Cytofix/Cytoperm for 20 min. Then, the cells were
8
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