Targeting triple-negative breast cancer cells with 6,7-bis(hydroxymethyl)- 1H,3H-pyrrolo[1,2-c]thiazoles

Article abstract of DOI:10.1016/j.ejmech.2014.04.008

Further studies on 6,7-bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazoles as anticancer agents against breast cancer are reported, allowing to demonstrate the potential of these compounds for the therapy of the triple-negative breast cancer, the most challe

Full text of DOI:10.1016/j.ejmech.2014.04.008

European Journal of Medicinal Chemistry 79 (2014) 273e281
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Targeting triple-negative breast cancer cells with 6,7-
bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazoles
,
b
,
,
b
,
,c
Kathleen Santos a, Mafalda Laranjo a c, Ana Margarida Abrantes a c, Ana F. Brito a
,
Cristina Gonçalves b d, Ana Bela Sarmento Ribeiro b d, M. Filomena Botelho a
,
,
,
,b,c
Maria I.L. Soares e, Andreia S.R. Oliveira e, Teresa M.V.D. Pinho e Melo e
,
*
a Biophysics Unit, Faculty of Medicine of University of Coimbra, Azinhaga de Santa Comba, Celas, 3004-548 Coimbra, Portugal
b CIMAGO e Center of Investigation in Environment, Genetics and Oncobiology, Faculty of Medicine of University of Coimbra, Azinhaga de Santa Comba,
Celas, 3004-548 Coimbra, Portugal
c IBILI e Institute for Biomedical Imaging and Life Science, Faculty of Medicine of University of Coimbra, Azinhaga de Santa Comba, Celas,
3004-548 Coimbra, Portugal
d Applied Molecular Biology, University Clinic of Haematology, Faculty of Medicine of University of Coimbra, Coimbra, Portugal
e Department of Chemistry, University of Coimbra, 3004-535 Coimbra, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Further studies on 6,7-bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazoles as anticancer agents against
breast cancer are reported, allowing to demonstrate the potential of these compounds for the therapy of
the triple-negative breast cancer, the most challenging tumors in clinical practice. These compounds
were assayed for their in vitro cytotoxicity on several human breast cancer cell lines (MCF7, HCC1954 and
HCC1806 cell lines). Particularly interesting were the results obtained for 4-hydroxyphenyl substituted
derivative, which proved to be the most promising compound regarding HCC1806 cell line, a triple-
negative breast cancer. The effects of the two most active compounds on cell survival, viability, cell
cycle, DNA damage and expression of proteins related to cell death pathways were studied. The reported
results consolidate the potential of 6,7-bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazoles for the therapy
of breast cancer, particularly the triple-negative.
Received 1 February 2014
Received in revised form
1 April 2014
Accepted 4 April 2014
Available online 4 April 2014
Keywords:
6,7-Bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-
c]thiazole
Antitumor activity
Ó 2014 Elsevier Masson SAS. All rights reserved.
Breast cancer
Triple-negative breast cancer
1. Introduction
BRCA genes have also been described as having a BRCAness
phenotype and are as sensitive to chemotherapy [3].
Breast cancer is the most common cause of tumor malignancy in
woman [1]. The breast cancer susceptibility genes (BRCA) 1 and 2
are the most associated with breast cancer. BRCA 1 gene is impor-
tant in the normal breast development promoting the expression of
estrogen receptors (ER) and BRCA2 gene is responsible for the
mechanism of DNA repair by homologous recombination (HR).
Mutations in BRCA genes result in a more aggressive and metastatic
phenotype, usually referred as BRCAness phenotypes. However,
these are the types of cancer more susceptible to chemotherapy
due to their compromised DNA repair mechanism [2]. Even triple-
negative breast cancers (TN e which have negative histochemical
confirmation for ER, progesterone receptor, RP, and human
epidermal growth factor receptor 2, HER2), without mutations in
The triple-negative breast cancer is an extremely aggressive
form of breast cancer that occurs most often in young women. It is
difficult to treat successfully because there are no targeted thera-
pies available as it lacks the receptors usually used for clinical
stratification. Triple-negative breast cancers tend to present higher
grades, larger tumors, higher metastasis incidence and a shorter
time of recurrence compared with other breast cancers types. The
lack of adequate therapeutic response for TN breast cancers makes
the search for new anti-cancer agents extremely relevant. Thus, we
were particularly interested in finding compounds with anti-triple-
negative breast cancer properties.
In recent years we have studied chiral hydroxymethyl-1H,3H-
pyrrolo[1,2-c]thiazoles as promising antiproliferating agents
against breast cancer [4,5], leading to the synthesis of chiral 6,7-
bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazoles as the most
promising scaffold for the design of new potent anticancer com-
pounds against breast cancer.
* Corresponding author.
E-mail address: tmelo@ci.uc.pt (T.M.V.D. Pinho e Melo).
http://dx.doi.org/10.1016/j.ejmech.2014.04.008
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

274
K. Santos et al. / European Journal of Medicinal Chemistry 79 (2014) 273e281
Structureeactivity relationship (SAR) studies of several 1H,3H-
pyrrolo[1,2-c]thiazole derivatives (e.g. 1e6) against breast adeno-
carcinoma MCF7 cell line allowed the establishment of structural
features for antitumor activity, Fig. 1 [4,5]. It was demonstrated that
only 1H,3H-pyrrolo[1,2-c]thiazoles bearing hydroxymethyl sub-
stituents show in vitro anticancer activity. On the other hand, the
position of the hydroxymethyl substituent is crucial. In fact, de-
rivative 1 with this group at C-6 is active whereas the C-7
substituted derivative 2 showed low activity. Nevertheless, the
presence of two hydroxymethyl groups leads to a slight improve-
ment in activity against MCF7 breast cancer cell lines as illustrated
Scheme 1. Synthesis of 1H,3H-pyrrolo[1,2-c]thiazoles 9. Reagents and conditions: (i)
Ac2O, DMAD, reflux, 4 h, 8a: 52%, 8b: 42%; (ii) DCM, LiAlH4 in diethyl ether, 0 ꢀC; (iii)
reflux, 1.5 h, 9a: 58%, 9b: 52%.
by 6,7-bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazole
3 with
IC50 value of 1.1 M. Furthermore, the combined presence of a
m
phenyl group at C-3 and a methyl group at C-5 in the 1H,3H-pyrrolo
[1,2-c]thiazole ring system is essential to ensure high cytotoxicity.
Interestingly, pyrrolo[1,2-c]thiazole 6 showed even better perfor-
mance against breast cancer cell lines than the corresponding
enantiomer 3.
In an effort to produce new structures with better anticancer
activity and that may give additional knowledge on SAR, based on
structure 3, we synthesized and evaluated the effect of replacing
the phenyl substituent at C-3 by a 4-methoxyphenyl group 9a and
by the more hydrophilic 4-hydroxyphenyl group 9b. The 6,7-
bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazole 12, derived from
penicillamine, has also been studied in order to determine whether
the presence of two methyl groups at C-1 would lead to enhance
cytotoxic activity. The new compounds were assayed for their
in vitro cytotoxicity on several human breast cancer cell lines,
including TN breast cancer cell lines.
these reaction conditions the N-acylation of thiazolidines occurs in
situ, followed by 1,3-dipolar cycloaddition via a bicyclic münchnone
intermediate. The chirality at C-4 of the starting thiazolidine is lost
and the chirality at C-2 (C-3 in the product) is retained giving
1H,3H-pyrrolo[1,2-c]thiazole-6,7-dicarboxylates 8 as single enan-
tiomers with R configuration at C-3. The cycloaddition of the
münchnone generated from thiazolidine 7b gave 3-(4-
acethoxyphenyl)-1H,3H-pyrrolo[1,2-c]thiazole 8b, resulting from
the acetylation of the initially formed dimethyl (3R)-3-(4-
hydroxyphenyl)-5-methyl-1H,3H-pyrrolo[1,2-c]thiazole-6,7-
dicarboxylate. Reduction of 1H,3H-pyrrolo[1,2-c]thiazoles 8 with
lithium aluminum hydride afforded the corresponding 6,7-
bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazoles 9 in moderate
yield. In the case of acetylated derivative 8b simultaneous depro-
tection of the hydroxyl group has occurred giving compound 9b.
A similar synthetic strategy was applied in the synthesis of (3S)-
3-phenyl-1,1,5-trimethyl-6,7-bis(hydroxymethyl)-1H,3H-pyrrolo
So far our studies were limited to cytotoxicity of the compounds
synthesized. However, to understand the mechanism of action and
the effects on cancer biology further studies were needed. There-
fore, we evaluated the effects of two promising compounds on cell
survival, viability, cell cycle, DNA damage and expression of pro-
teins related to cell death pathways.
[1,2-c]thiazole
phenylthiazolidine-4-carboxylic acid (10) was prepared as
mixture of 2R,4S- and 2S,4S-diastereoisomers from the reaction of
-penicillamine with benzaldehyde. The 1,3-dipolar cycloaddition
(12)
(Scheme
2).
5,5-Dimethyl-2-
a
D
of the münchnone, generated from thiazolidine 10, gave 1H,3H-
pyrrolo[1,2-c]thiazole-6,7-dicarboxylate 11 as a single enantiomer
with S configuration. 6,7-Bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]
thiazole 12 was obtained in 51% yield by the reduction of com-
pound 11 with lithium aluminum hydride.
2. Results and discussion
2.1. Chemistry
Chiral 6,7-bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazoles 9a
and 9b were prepared as outlined in Scheme 1, following a known
general synthetic procedure [4]. 2-Phenylthiazolidine-4-carboxylic
acids 7 were obtained as a mixture of 2R,4R- and 2S,4R-di-
2.2. Biological evaluation
astereoisomers from the reaction of L-cysteine with the appropriate
aldehyde. Thiazolidines 7 were heated in a solution of acetic an-
hydride in the presence of dimethyl acetylenedicarboxylate. Under
2.2.1. In vitro antiproliferative activities and SAR
The new compounds were evaluated for their in vitro anti-
proliferative activity against human breast cancer representative
of HER2þ (HCC1954), RHþ (MCF7) and TN (HCC1806) tumors
(Table 1). In the HCC1954 cell line none of the compounds affect cell
proliferation at 24 h of incubation. However, when cells were
incubated for 48 h with the studied compounds a significant
decrease in IC50 values was observed (3, 9a and 9b p < 0.001; 6
p ¼ 0.019; 12 p ¼ 0.017). 3 is the most promising anti-proliferative
Scheme 2. Synthesis of 1H,3H-pyrrolo[1,2-c]thiazole 12. Reagents and conditions: (i)
Ac2O, DMAD, reflux, 4 h, 11: 73%; (ii) DCM, LiAlH4 in diethyl ether, 0 ꢀC; (iii) reflux,
1.5 h, 12: 51%.
Fig. 1. Cytotoxicity against MCF7 breast cancer human cell line (72 h incubation time)
[4,5].

K. Santos et al. / European Journal of Medicinal Chemistry 79 (2014) 273e281
275
Table 1
lines was carried out (Table 1). However, the IC50 values obtained
for 12 were rather high in comparison with other studied com-
pounds. Thus, from current and previous results, we can conclude
that substitution at C-1 by methyl groups does not improve anti-
cancer activity of 1H,3H-pyrrolo[1,2-c]thiazole derivatives. More-
over, 12 has the S absolute configuration at C-3, which potentiates
cytotoxic activity against MCF7 cell line, as we already discussed.
These results indicate that the substitution pattern in the pyrrolo
[1,2-c]thiazole ring system plays a more significant role in the anti-
proliferative activity than the absolute configuration at C-3.
IC50 values of the compounds 3, 6, 9 and 12 for 24, 48, 72 and 96 h of incubation in
the MCF7, HCC1954 and HCC1806 cell lines.a
Compd
Incubation time
IC50 (m
M)b
HCC1806
HCC1954
MCF7
3
24 h
48 h
72 h
96 h
24 h
48 h
72 h
96 h
24 h
48 h
72 h
96 h
24 h
48 h
72 h
96 h
24 h
48 h
72 h
96 h
>100
81.4
51.3
13.9
>100
63.1
78.5
28.7
89.0
28.0
19.4
12.3
80.3
26.4
11.9
5.4
98.2
27.0
26.2
24.9
>100
56.8
14.8
11.8
>100
84.4
56.1
19.2
>100
65.6
46.6
27.4
99.5
69.0
55.9
33.5
12.25
1.95
1.05
0.65
6
1.755
0.545
0.545
0.305
>100
46.9
2.2.2. Cell cultures are sensitive to treatment
9a
9b
12
Since 1H,3H-pyrrolo[1,2-c]thiazoles 3 and 9b are promising due
to their anti-proliferative activity against HRþ MCF7 cell line and TN
HCC1806 cell lines, we tested the long-term sensitivity and survival
of a cell culture to the treatment performing the clonogenic assay.
As we can observe in Fig. 2, all treatments reduces significantly
(p < 0.001) the surviving factor of both cell cultures. It is further
noticed that HCC1806 cell line is particularly sensitive to treatment
since surviving factor is below 2%.
These results show that HCC1806 cell line in particular presents
high sensitivity to chemotherapy with these compounds, proving
that this cell line presents a BRCAness phenotype. It is further
noticed that treatment with 3, which is the compound that pre-
sents the higher IC50 value, is the one that most affects HCC1806
survival.
32.7
6.6
>100
86.2
53.9
29.5
>100
41.7
26.9
21.3
>100
>100
>100
>100
a
Cells were incubated with DMSO solutions of the selected compounds, washed
and then cell proliferation was evaluated by MTT test.
b
Concentration needed to inhibit cell proliferation by 50% as determined from
doseeresponse curves by exponential decay fitting (r2 > 0.9).
2.2.3. Cell mass and total protein production is reduced
compound against HCC1954 cells and the less promising is 9a. In
the MCF7 cell line, 3 and 6 are the most promising compounds
The total protein content was analyzed after treatment of MCF7
and HCC1806 cell lines with 1H,3H-pyrrolo[1,2-c]thiazoles 3 and 9b
(Fig. 3). HCC1806 cell line reduces the total protein content to about
50% when treated with any of the compounds. In the MCF7 cell line,
treatment with 3 produces similar results to those of HCC1806 cell
line, but treatment with 9b reduces total protein content to
15.4 ꢁ 9.4%. The reduction of protein content showed statistical
significance in all conditions of treatment in both cell lines
(p < 0.001). SRB assay allows us to conclude that cell mass is
significantly reduced with treatment, which indicates that cell
viability is also significantly reduced.
concerning anti-proliferative activity with IC50 values of 0.6
0.3 M, respectively, for 96 h incubation time. On the other hand,12
showed no anti-proliferative activity against MCF7 cell line, in the
concentrations tested, up to 100 M. Despite the fact that 9a and 9b
mM and
m
m
showed moderate anti-proliferative activity in MCF7 cell line, the
decreasing IC50 value over time has statistical significance
(p < 0.05). In the HCC1806 cell line, 9b is the most promising
compound with significant decrease of IC50 value over time
(p < 0.05), with IC50 value of 5.4 mM for 96 h incubation time. All the
other compounds showed a significant decrease in the IC50 value
going from 24 h to 48 h incubation time (p < 0.001), being 3 and 6
the less active compounds in this cell line.
2.2.4. PT induce cell death preferentially by late apoptosis and
necrosis
The design of new compounds has been guided considering 3 as
the lead compound [5] and the structural modifications were
thought not only to obtain derivatives with higher anti-
proliferation activity, but also to gather data regarding SAR. We
showed that the presence of the phenyl group at C-3 seemed to be
important to the activity of the compounds [5]. However, in pre-
vious studies, the possibility of replacing the phenyl group by other
aryl groups such as 4-methoxyphenyl and 4-hydroxyphenyl sub-
stituents was not explored. In this context, we synthesized 3-(4-
methoxyphenyl)-1H,3H-pyrrolo[1,2-c]thiazole 9a and 3-(4-
hydroxyphenyl)-1H,3H-pyrrolo[1,2-c]thiazole 9b. Regarding MCF7
cell line the new derivatives show lower activity than the lead
compound 3. However, these compounds presented better anti-
cancer activity than 1H,3H-pyrrolo[1,2-c]thiazole 3, against TN
cell line, HCC1806, which is more challenging to treat and lack a
targeted therapy. Particularly interesting were the results obtained
for 4-hydroxyphenyl substituted derivative 9b, which prove to be
the most promising compound regarding HCC1806 cell line, a TN-
breast cancer.
In view of the high cytotoxicity exhibited against MCF7 and
HCC1806 by 1H,3H-pyrrolo[1,2-c]thiazoles 3 and 9b and SRB assay
results which indicates a reduced viable cell mass, flow cytometry
studies were performed with IC50 values of these compounds at
48 h to determine cell viability and the induced mechanism of cell
death (necrosis or apoptosis). The graphs showed in Fig. 4 presents
the percentage and standard deviation for populations of four
different cell states: living cells (negative for An-V and PI), apoptotic
cells (negative for PI and positive for An-V), late apoptotic or
necrotic cells (positive for An-V and PI) and necrotic cells (negative
for An-V and positive for PI).
For the MCF7 cell line it is observed that 3 induces a decreasing
in the viable cells population (p ¼ 0.015) and an increasing in the
late apoptotic/necrotic (p ¼ 0.048) and in the necrotic populations
(p ¼ 0.001). 9b effects in MCF7 cell line decreased viable cells
population to 21.4 ꢁ 4.2% (p < 0.001), and increased apoptotic
(p ¼ 0.001), late apoptotic/necrotic (p < 0.001) and necrotic
(p ¼ 0.006) populations, comparing to the control.
In the TN cell line, HCC1806, both 3 and 9b decreases viable cells
population (3: p < 0.001; 9b: p ¼ 0.022) and increases late
apoptosis/necrosis (3: p < 0.001; 9b: p ¼ 0.011) and necrosis (3:
p < 0.001; 9b: p ¼ 0.019). HCC1806 cell line is characterized by a
2bp insertion after the nucleotide 256 in the 4th exon in 17p13.1
So far, all the compounds studied in our group were unsub-
stituted at C-1. Thus, 1,1-dimethyl-1H,3H-pyrrolo[1,2-c]thiazole 12
was synthesized and its biological evaluation as anticancer activity
against HCC1806, HCC1954 and MCF7 breast cancer human cell

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K. Santos et al. / European Journal of Medicinal Chemistry 79 (2014) 273e281
Fig. 2. Survival factor of MCF7 and HCC1806 cell lines incubated with 3 and 9b during 48 h. Results are presented as mean ꢁ SD (n ꢂ 8). The * represent significant differences
between the respective control population. Statistical significance: ***p < 0.001.
Fig. 3. Protein content of MCF7 and HCC1806 cell lines incubated with 3 and 9b during 48 h. Results are presented as mean ꢁ SD (n ꢂ 8). The * represent significant differences
between the respective control population. Statistical significance: ***p < 0.001.
Fig. 4. Cell viability of MCF7 and HCC1806 cell lines incubated with 3 and 9b during 48 h. Cell viability results represent the percentage of viable cells, cells in apoptosis, cells in late
apoptosis/necrosis and cells in necrosis and are presented as mean ꢁ SD (n ꢂ 5). The * represent significant differences between the respective control population. Statistical
significance: *p < 0.05; **p < 0.01; ***p < 0.001.
chromosome [6], which encodes p53, causing a frame shift which
results in a shorter protein with only 122 aminoacids. Therefore
HCC1806 lacks p53 expression7 due to the degradation of a non-
functional p53 protein and, as consequence, cellular death by
apoptosis is diminished. That is the reason why p53 loss is corre-
lated with multidrug resistance and also with defective cell pro-
liferation control, inefficient DNA repair and genetic instability and
that contributes to the aggressive phenotype due to cumulative
genetic alteration [7].
increased, although with time cells are able to recover as observed
with the clonogenic assay, which means that, initially the majority
of cells are affected and die but the remaining cells are able to
recover after treatment. On the other hand HCC1806 is more
resistant to cell death and clearly cell death through apoptosis is
compromised since cell death is mainly necrotic with both treat-
ments but its long-term survival is significantly reduced to
1.4 ꢁ 0.3%.
On the other hand, MCF7 cell line expresses the wild-type p53
[7], which could explain the duality of response to treatment in the
clonogenic assay and viability and types of cell death. That is, cell
viability is reduced to about 55/60% when both cell lines are treated
with 3 and also late apoptotic/necrotic and necrotic populations are
significantly increased which means that the response to treatment
is probably p53 independent. However, when cells are treated with
9b their response is very distinct. MCF7 viability is significantly
reduced to 21.4 ꢁ 4.2% and the apoptotic population is significantly
2.2.5. p53 expression is influenced by 1H,3H-pyrrolo[1,2-c]thiazole
3
Although the p53 profile of the two cell lines is known, we
confirmed that the HCC1806 cell line lacks its expression. Never-
theless we evaluated p53 expression by western blot to access this
protein in the MCF7 cell line in response to treatment.
As we can observe in Fig. 5, p53 is increased with treatment in
the MCF7 cell line, with a significant effect with 9b treatment
(p < 0.009). This result corroborates the increasing of the apoptotic

K. Santos et al. / European Journal of Medicinal Chemistry 79 (2014) 273e281
277
For instance, poly(ADP-ribose)polymerase-1 (PARP-1) promotes
cell death by apoptosis when DNA damage is moderate and cell
death by necrosis in the presence of extensive DNA damage, but
also the balance between apoptosis and necrosis is dependent of
the cell type [10]. With that in mind 1H,3H-pyrrolo[1,2-c]thiazole 3
could cause severe DNA damage and activate PARP-1 causing
necrotic cell death, and also HCC1806 cell line could be more sen-
sitive to the activation of this protein since both treatments cause
necrotic cell death.
2.2.7. 1H,3H-pyrrolo[1,2-c]thiazole blocks cell-cycle progression
preferentially at S phase
The growing evidence that these compounds could induce DNA
damage led to the analysis the progress/blockade of cell cycle of
MCF7 and HCC1806 cell lines after treatment with 3 and 9b. In the
MCF7 cell line, both 3 and 9b, as represented in Fig. 7, decreased
population of cells in the G0/G1 (3 and 9b: p < 0.001) and increased
cell population in the S phase (3 and 9b: p < 0.001). A blockade in
the G2/M phases is induced by compound 3 (p ¼ 0.001) but not by
9b (p > 0.05). Both intra-S [11] and the G2/M [12] checkpoints are
mostly related to stalled replication forks that can derived from
DNA inter-strand crosslinks. When this particular type of DNA
damage is detected, ataxia telangiectasia and Rad3 related (ATR) is
activated and promotes mainly checkpoint protein kinase 1 (Chk1)
phosphorylation [12], a protein responsible for the intra-S phase
arrest in order to promote DNA repair [11]. Also, Chk1-P is able to
activate p53 protein, upregulating p21WAF1 which inhibit the
cyclin-dependent kinase 1-cyclin B complex (Cdk1-cyclin B) and
cause cell cycle G2/M phase arrest [12].
As can be observed in Fig. 7, 3 induces a blockade in the G0/G1
phase in HCC1806 cell line (p ¼ 0.001), but has no effect in the other
phases of the cell cycle, indicating a state of quiescence where cells
stop dividing [13]. Similarly to treatment in the MCF7 cell line, 9b
produced a decreased population of HCC1806 cells in the G0/G1
(p < 0.001) and an accentuated accumulation of cells in the S phase
(p < 0.001) and in the pre-G0 phase (p ¼ 0.011). These results
indicate DNA degradation and intra-S phase arrest with the
objective to promote DNA repair [11] as previously explained.
Fig. 5. p53 Protein expression. The graphic represents ratio of p53 expression versus
b-
actin and the image refers to a representative immunoblot of p53 and -actin. Results
b
are presented as mean ꢁ SD (n ꢂ 3). The * represent significant differences between
the respective control population. Statistical significance: **p < 0.01.
population after 9b treatment, and the slight non significant in-
crease of p53 expression compared to control, after 3 treatment,
explains why cell death is not related to apoptosis.
2.2.6. 1H,3H-pyrrolo[1,2-c]thiazole increases Bax/Bcl-2 ratio
We also assessed the ratio of the expression of Bax, a pro-
apoptotic protein, and Bcl-2, an anti-apoptotic protein, two pro-
teins involved in the apoptotic cell death (Fig. 6). Briefly, this
pathway is mainly activated in response to DNA damage, which can
activate p53 resulting in the expression of Bax and repression of
BCl-2 proteins. Later, Bax leads to the cytochrome c release from
mitochondria and later caspase activation, ultimately causing cell
death by apoptosis [7,8].
Comparing to the control, the ratio Bax/Bcl-2 is increased in all
treatments, however only treatment with 9b increase this ratio
with statistical significance (MCF7: p ¼ 0.003; HCC1806: p < 0.001).
As we can observe Bax/Bcl-2 ratio in the MCF7 cell line is consistent
with results of cell death and p53 expression, where only treatment
with 9b was able to induce a significant increase in the apoptotic
cell population.
2.2.8. Comet assay in the MCF7 and HCC1806 cell lines
Because of the chemical structure of the compounds it was
proposed that they could be able to act by mono and bis-alkylation
of DNA. The alkaline comet assay (pH > 13) allows to detect DNA
damage in individual eukaryotic cells, namely, single strand breaks,
double strand breaks, alkaline labile sites, and transient repair sites
[14]. As can be observed in Fig. 8, cells treated with IC50 and with
three times more this concentration do not show evidence of DNA
breaks, in contrast with the positive controls with hydrogen
Despite the fact that HCC1806 cell line does not express p53, the
Bax/Bcl-2 ratio is significantly increased with 9b treatment. This
might have to do with the default Bax activation when anti-
apoptotic proteins such as Bcl-2 are neutralized or their expres-
sion is diminished [9]. However cell death is mainly necrotic, which
means that other mechanisms of cell death might be predominant.
Fig. 6. Ratio of the mean fluorescence intensity of Bax and the mean fluorescence intensity of Bcl2 in MCF7 and HCC1806 cell lines incubated with 3 and 9b during 48 h. Results are
presented as mean ꢁ SD (n ꢂ 5). The * represent significant differences between the respective control population. Statistical significance: **p < 0.01; ***p < 0.001.

278
K. Santos et al. / European Journal of Medicinal Chemistry 79 (2014) 273e281
Fig. 7. Cell cycle analysis of MCF7 and HCC1806 cell lines incubated with 3 and 9b during 48 h. Cell cycle results show the percentage of cell populations in the sub-Go, G0/G1, S and
G2/M phases. Results are presented as mean ꢁ SD (n ꢂ 5). The * represent significant differences between the respective control population. Statistical significance: *p < 0.05;
**p < 0.01; ***p < 0.001.
peroxide. In fact, cells from treatment have the same appearance as
the negative controls. However, we cannot exclude the possibility
of intrastranded or interstranded crosslinking formation. Inter-
stranded crosslinking is also a cytotoxic form of DNA damage once
DNA strands separation is prevented and DNA replication is
blocked. Concerning comet assay the presence of crosslinks results
in less DNA migration [15].
against breast cancer, we introduce the potential of these com-
pounds also for the triple-negative breast cancer, the most chal-
lenging tumors in clinical practice.
The study allowed us to redraw some conclusions regarding
structureeactivity relationships. Thus, the results of the in vitro
activity have demonstrated that the presence of methyl groups at
C-1 in the 1H,3H-pyrrolo[1,2-c]thiazole ring system is prejudicial
for their activity, in all cell lines studied. The presence of a
hydroxyphenyl group at C-3 seems to improve the anti-cancer ac-
tivity for the TN cell line. These results, together with our previous
ones, indicate that there is a differential anti-cancer activity con-
cerning the biochemical profile of the cell lines, therefore future
modifications must be envisioned taking this into account.
Furthermore, it is the first time that cellular effects beyond
cytotoxicity are explored adding a new and valuable role in the
screening of our compounds, giving a first insight into their
mechanism of action. Both cell lines are sensitive to 3 and 9b being
the TN cell line HCC1806 survival the most affected/reduced.
Viability is also decreased and cell death related proteins p53, Bax
and Bcl-2 are significantly altered, occurring cell cycle blockage
mainly in S-phase and cell death mostly by late apoptosis and ne-
crosis. The reported results indicate that the studied compounds
may induce DNA damage.
3. Conclusion
Herein, in the continuation of our series of studies on the
hydroxymethyl-1H,3H-pyrrolo[1,2-c]thiazoles as anticancer agents
Our studies in this family of compounds consolidate the po-
tential of hydroxymethyl-1H,3H-pyrrolo[1,2-c]thiazoles for the
therapy of breast cancer, particularly the triple-negative, an asset to
continue with pre-clinic studies.
4. Experimental section
4.1. Synthetic procedures
4.1.1. General methods
1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were
recorded on a Bruker Avance III 400 MHz spectrometer. Chemical
shifts (d) are expressed in parts per million (ppm) related to in-
ternal TMS, and coupling constants (J) are in hertz. The following
abbreviations are used in the assignment of NMR signals: s
(singlet), d (doublet), m (multiplet), bs (broad singlet). IR spectra
were recorded on a Nicolet 6700 FTIR spectrometer. HRMS spectra
were obtained on a VG Autospect M spectrometer (TOF MS EIþ or
ESI). Melting points were determined in open glass capillary with
an Electrothermal melting point apparatus and are uncorrected.
Optical rotations were measured on an Optical Activity AA-5
electrical polarimeter. Flash column chromatography was per-
formed with silica gel 60 as the stationary phase. TLC analyses
were carried out on Merck Silica gel 60 F254 plates. 1H,3H-Pyrrolo
[1,2-c]thiazoles 3 [4] and 6 [5] were prepared as described in the
literature.
Fig. 8. Representative images of the Comet Assay in the MCF7 and HCC1806 cell lines
after 48 h of incubation with 3 and 9b in the concentrations equal to the IC50 and three
times this value. A and H: Control, untreated cell cultures, 100ꢃ; B and I: positive
control, 100ꢃ; C and J: positive control, 400ꢃ; D and K: 3 IC50 treated cultures, 100ꢃ; E
and L: 3 3ꢃ IC50 treated cultures, 100ꢃ; F and M: 9b IC50 treated cultures, 100ꢃ; G and
N: 9b 3ꢃ IC50 treated cultures, 100ꢃ.

K. Santos et al. / European Journal of Medicinal Chemistry 79 (2014) 273e281
279
4.1.2. General procedure for the synthesis of 1,3-thiazolidine-4-
carboxylic acids 7
A solution of the appropriate aldehyde (40.0 mmol) in ethanol
(30 mL) was added to a solution of L-cysteine (5.0 g, 40.0 mmol) in
water (40 mL). After stirring overnight at room temperature the
product was filtered and washed with diethyl ether.
chromatography [hexane-ethyl acetate (3:1), hexane-ethyl acetate
(2:1), then hexane-ethyl acetate (1:1)] gave 8b as a white solid
(2.0 g, 42%); mp 90e92 ꢀC (from ethyl acetate/hexane); IR (KBr):
nmax ¼ 1766, 1734, 1709, 1437, 1367, 1296, 1215, 1095 cmꢄ1; 1H NMR
(400 MHz, CDCl3):
d
¼ 2.03 (3H, s), 2.26 (3H, s), 3.81 (6H, m), 4.29
(1H, d, J ¼ 14.9 Hz), 4.44 (1H, d, J ¼ 14.9 Hz), 6.32 (1H, s), 7.07 (4H, s,
ArH); 13C NMR (100 MHz, CDCl3):
d
¼ 11.37, 20.97, 29.88, 51.29,
4.1.2.1. 2-(4-Methoxyphenyl)-1,3-thiazolidine-4-carboxylic acid (7a).
Yield: 89% (8.5 g), white solid, mp 166e168 ꢀC (lit. [16] 156e158 ꢀC).
51.45, 64.23, 106.83, 117.36, 122.34, 126.84, 130.62, 137.59, 140.34,
150.90, 163.84, 165.10, 168.94; HRMS (EI-TOF) m/z 389.0935 (Mþ,
1
The H NMR spectrum obtained at 40 ꢀC showed the presence of
C19H19NO6S requires 389.0933).
the two diastereoisomers (2R,4R) and (2S,4R) (ratio 48:52); 1H NMR
(400 MHz, (CD3)2SO):
d
¼ 3.05e3.10 and 3.14e3.18 (1H, 2ꢃ m, ABX),
4.1.4.3. Dimethyl (3S)-1,1,5-trimethyl-3-phenyl-1H,3H-pyrrolo[1,2-c]
thiazole-6,7-dicarboxylate (11). Purification by flash chromatog-
raphy [hexane-ethyl acetate (3:1), then hexane-ethyl acetate (2:1)]
gave 7 as a yellowish oil (3.1 g, 73%); IR (KBr): nmax ¼ 1709, 1531,
3.29e3.32 and 3.35e3.39 (1H, 2ꢃ m, ABX), 3.75 and 3.76 (3H, 2ꢃ s),
3.85e3.89 and 4.23e4.26 (1H, 2ꢃ m, ABX), 5.46 and 5.62 (1H, 2ꢃ s,
CHAr), 6.89 and 6.93 (2H, 2ꢃ d, J ¼ 8.6 Hz, ArH), 7.37 and 7.44 (2H,
2ꢃ d, J ¼ 8.5 Hz, ArH).
1441, 1396, 1294, 1213, 1172 cmꢄ1 1H NMR (400 MHz, CDCl3):
;
d
¼ 1.84 (3H, s), 1.90 (3H, s), 1.95 (3H, s), 3.80 (3H, s), 3.84 (3H, s),
6.34 (1H, s, CHPh), 7.07e7.09 (2H, m, ArH), 7.31e7.36 (3H, m, ArH);
13C NMR (100 MHz, CDCl3):
4.1.2.2. 2-(4-Hydroxyphenyl)-1,3-thiazolidine-4-carboxylic acid (7b).
Yield: 99% (8.9 g), white solid, mp 165e167 ꢀC (lit. [16] 167e169 ꢀC).
The 1H NMR spectrum showed the presence of the two di-
astereoisomers (2R,4R) and (2S,4R) (ratio 50:50); 1H NMR
d
¼ 11.26, 30.78, 31.92, 51.55, 51.58,
52.86, 64.17, 106.28, 117.67, 125.80, 128.79, 129.20, 129.21, 140.38,
145.73, 164.98, 165.50; HRMS (EI-TOF) m/z 359.1195 (Mþ,
(400 MHz, (CD3)2SO):
d
¼ 3.02e3.07 and 3.13e3.17 (1H, 2ꢃ m, ABX),
C19H21NO4S requires 359.1191).
3.25e3.28 and 3.34e3.37 (1H, 2ꢃ m, ABX), 3.82e3.86 and 4.24e
4.26 (1H, 2ꢃ m, ABX), 5.40 and 5.54 (1H, 2ꢃ s, CHAr), 6.70 and 6.74
(2H, 2ꢃ d, J ¼ 8.4 Hz, ArH), 7.25 and 7.31 (2H, 2ꢃ d, J ¼ 8.1 Hz, ArH).
4.1.5. General procedure for the synthesis of 6,7-
bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazole carboxylates 9
and 12
4.1.3. 5,5-Dimethyl-2-phenyl-1,3-thiazolidine-4-carboxylic acid
(10)
A solution of benzaldehyde (0.54 g, 5.1 mmol) in methanol
(7 mL) was slowly added to a solution of D-penicillamine (0.69 g,
A solution of the appropriate 5-methyl-1H,3H-pyrrolo[1,2-c]
thiazole-carboxylate 8 or 11 (2.9 mmol) in dry dichloromethane
(30 mL) was added dropwise to a suspension of lithium aluminum
hydride (2.2 eq., 0.24 g, 6.4 mmol) in anhydrous diethyl ether
(40 mL) at 0 ꢀC. The solution was refluxed for 1.5 h after the addition
was completed and then cooled on an ice bath. The excess of hy-
dride was carefully decomposed by addition of ethyl acetate fol-
lowed by slow addition of water (0.3 mL), NaOH 15% (0.3 mL) and
water (0.9 mL). The mixture was filtered through celite and the
inorganic residue was washed with several portions of hot
dichloromethane. The filtrate was dried (Na2SO4) and the solvent
evaporated off. The crude product was purified by flash chroma-
tography [hexane-ethyl acetate] or recrystallization.
4.6 mmol) in methanol (90 mL). After stirring overnight at room
temperature the mixture was concentrated under reduced pressure
and the residue was washed with diethyl ether to give a white solid
(1.02 g, 93%). mp 160e162 ꢀC. The 1H NMR spectrum showed the
presence of the two diastereoisomers (2R,4S) and (2S,4S) (ratio
38:62); Minor isomer: 1H NMR (400 MHz, CD3OD):
d
¼ 1.49 (3H, s),
1.71 (3H, s), 3.95 (1H, s), 5.92 (1H, CHPh), 7.54e7.55 (5H, m, ArH);
Major isomer: 1H NMR (400 MHz, CD3OD):
d
¼ 1.46 (3H, s), 1.74 (3H,
s), 3.82 (1H, s), 5.68 (1H, CHPh), 7.37e7.41 (5H, m).
For the synthesis of compound 8b 3.3 equivalents of lithium
aluminum hydride (0.36 g, 9.6 mmol) were used. In this case the
excess of hydride was eliminated by the addition of ethyl acetate
followed by slow addition of water (0.4 mL), NaOH 15% (0.4 mL) and
water (1.2 mL).
4.1.4. General procedure for the synthesis of 1H,3H-pyrrolo[1,2-c]
thiazole carboxylates 8 and 11
A solution of the appropriate thiazolidine-4-carboxylic acid
(12.0 mmol), dimethyl acetylenedicarboxylate (1.5 equiv.,
18.0 mmol) and Ac2O (40 mL) was heated at 110 ꢀC during the 4 h.
The reaction was cooled to room temperature and was diluted with
CH2Cl2 (100 mL). The organic phase was washed with saturated
aqueous solution of NaHCO3 and with water, dried (Na2SO4) and
the solvent evaporated off. The crude product was purified by flash
chromatography [hexane-ethyl acetate].
4.1.5.1. (3R)-6,7-Bis(hydroxymethyl)-3-(4-methoxyphenyl)-5-
methyl-1H,3H-pyrrolo[1,2-c]thiazole (9a). Recrystallization with
diethyl ether gave 9a as a white solid (0.52 g, 58%); mp 119e121 ꢀC;
IR (KBr): nmax ¼ 3392, 1614, 1514, 1435, 1338, 1286, 1255, 1172,
1009 cmꢄ1
;
1H NMR (400 MHz, CDCl3):
d
¼ 1.84 (3H, s), 2.38 (1H,
bs), 2.53 (1H, bs), 3.79 (3H, s), 4.08 (1H, d, J ¼ 12.8 Hz), 4.28 (1H, d,
J ¼ 12.8 Hz), 4.48 (1H, d, J ¼ 12.0 Hz), 4.53 (1H, d, J ¼ 12.0 Hz), 4.59
(2H, s), 6.21 (1H, s, CHAr), 6.84 (2H, d, J ¼ 8.4 Hz, ArH), 7.02 (2H, d,
4.1.4.1. Dimethyl
(3R)-3-(4-methoxyphenyl)-5-methyl-1H,3H-pyr-
rolo[1,2-c]thiazole-6,7-dicarboxylate (8a). Purification by flash
chromatography [hexane-ethyl acetate (2:1)] gave 8a as a white
solid (2.3 g, 52%); mp 80e82 ꢀC (from diethyl ether); IR (KBr):
J ¼ 8.4 Hz, ArH); 13C NMR (100 MHz, CDCl3):
¼ 9.96, 27.62, 55.33,
d
56.48, 56.79, 64.26, 113.30, 114.34, 123.09, 123.46, 127.25, 131.49,
nmax ¼ 1736, 1697, 1516, 1444, 1288, 1209, 1088 cmꢄ1
;
1H NMR
133.38, 159.73; HRMS (EI-TOF) m/z 305.1085 (Mþ, C16H19NO3S re-
20
(400 MHz, CDCl3):
d
¼ 2.00 (3H, s), 3.80 (3H, s), 3.83 (6H, s), 4.30
quires 305.1086); [
a
]
¼ þ 230 (c 1, CH2Cl2).
D
(1H, d, J ¼ 14.9 Hz), 4.47 (1H, d, J ¼ 14.9 Hz), 6.27 (1H, s), 6.86 (2H, d,
J ¼ 8.6 Hz), 7.02 (2H, d, J ¼ 8.6 Hz); 13C NMR (100 MHz, CDCl3):
4.1.5.2. (3R)-6,7-Bis(hydroxymethyl)-3-(4-hydroxyphenyl)-5-
methyl-1H,3H-pyrrolo[1,2-c]thiazole (9b). Recrystallization with
diethyl ether gave 9b as a pale brown solid (0.43 g, 52%);
mp > 220 ꢀC (with decomposition); IR (KBr): nmax ¼ 3263, 1612,
1518, 1427, 1363, 1277, 1240, 982 cmꢄ1; 1H NMR (400 MHz, CD3OD):
d
¼ 11.42, 29.98, 51.38, 51.56, 55.36, 64.87, 106.71, 114.59, 117.38,
127.23, 130.73, 131.93, 140.43, 160.08, 164.08, 165.36; HRMS (EI-
TOF) m/z 361.0988 (Mþ, C18H19NO5S requires 361.0984).
4.1.4.2. Dimethyl (3R)-3-(4-acethoxyphenyl)-5-methyl-1H,3H-pyr-
rolo[1,2-c]thiazole-6,7-dicarboxylate (8b). Purification by flash
d
¼ 1.84 (3H, s), 4.07 (1H, d, J ¼ 13.1 Hz), 4.29 (1H, d, J ¼ 13.1 Hz),
4.43 (2H, s), 4.52 (1H, d, J ¼ 12.1 Hz), 4.56 (1H, d, J ¼ 12.1 Hz), 6.30

280
K. Santos et al. / European Journal of Medicinal Chemistry 79 (2014) 273e281
(1H, s, CHAr), 6.71 (2H, d, J ¼ 8.4 Hz, ArH), 6.92 (2H, d, J ¼ 8.4 Hz,
ArH); 13C NMR (100 MHz, CD3OD):
(final DMSO concentration 1%). SRB assay was performed as
described [19]. Briefly, cells were washed with ultra-pure water and
twice with PBS. Cells were then incubated with 1% acetic acid in
methanol for 30 min prior to incubation with 0.4% SRB (Sigma
AldrichÒ, EUA) dissolved in 1% acetic acid (Sigma AldrichÒ, EUA) in
methanol (Sigma AldrichÒ, EUA) for 1 h in the dark. After incuba-
tion SRB crystals were dissolved in 10 mM TriseNaOH, pH 10.
d
¼ 9.94, 28.12, 56.16, 56.62,
65.24, 114.13, 116.45, 124.06, 124.32, 128.47, 132.93, 134.34, 158.72;
HRMS (ESI-TOF) m/z 314.0829 ([M þ Na]þ, C15H17NO3SNa requires
20
314.0821); [
a]
¼ þ245 (c 1, MeOH).
D
4.1.5.3. (3S)-6,7-Bis(hydroxymethyl)-1,1,5-trimethyl-3-phenyl-
1H,3H-pyrrolo[1,2-c]thiazole (12). Purification by flash chroma-
tography [hexane-ethyl acetate (1:2), then hexane-ethyl acetate
(1:4)] gave 12as a colorless oil (0.45 g, 51%); IR (film): nmax ¼ 3375,
4.2.5. Cell viability and types of cell death
For analysis of cell viability and types of cell death, 2 ꢃ 106 cells
were incubated overnight to allow attachment and then were
treated with IC50 of 3 or 9b for 48 h (final DMSO concentration
0.1%). Then, cells were collected, centrifuged and washed with PBS
1537, 1456, 1433, 1402, 1363, 1120, 989 cmꢄ1 1H NMR (400 MHz,
;
CDCl3):
d
¼ 1.78 (3H, s), 1.83 (3H, s), 1.85 (3H, s), 4.49 (1H, d,
J ¼ 12.1 Hz, CH2), 4.53 (1H, d, J ¼ 12.1 Hz, CH2), 4.65 (2H, s, CH2),
6.30 (1H, s, CHPh), 7.06e7.09 (2H, m, ArH), 7.29e7.32 (3H, m, ArH);
prior to incubation with binding buffer, 2.5
propidium iodide (kit Immunotech) for 15 min, at 4 ꢀC in the dark.
400 L PBS were added and samples were analyzed in a FACSCa-
mL An-VeFITC and 1 mL
13C NMR (100 MHz, CDCl3):
d
¼ 9.87, 32.64, 33.82, 52.00, 55.28,
56.27, 64.04, 111.90, 121.86, 123.90, 125.91, 128.36, 128.96, 140.00,
m
141.82; HRMS (EI-TOF) m/z 303.1295 (Mþ, C17H21NO2S requires
libur cytometer (BD Biosciences, EUA). Excitation was set at
488 nm, and the emission filters were set at for An-VeFITC and
propidium iodide, respectively.
303.1293); ½a 2D0
¼ ꢄ205ðc 1; CH2Cl2Þ:
ꢅ
4.2. Biological evaluation
4.2.6. P53 expression
4.2.1. Cell culture
Western Blot was used to evaluate the expression of p53 protein.
Cells were treated with the IC50 of 3 or 9b during 48 h. Total protein
extracts were prepared on ice using a solution of radioimmuno
precipitation assay (RIPA) buffer supplemented with cOmplete
Mini (Roche). After sonication and centrifugation at 14,000 g, the
samples were kept at ꢄ80 ꢀC prior use. Protein content was
determined by bicinchoninic (BCA) method. Sodium dodecyl sul-
fate polyacrylamide gel electrophoresis (SDS-PAGE) was held using
a 10% acrylamide gel for 20 min at 80 V followed by 160 V till 1 h
10 min. Subsequently proteins were electrotransferred to nitro-
cellulose (PVDF) membranes at 100 V during 1 h. Blocking of PVDF
membranes was performed with 4% bovine serum albumin (BSA) in
tris-buffered saline tween-20 (TBS-T) for 1 h at room temperature
with stirring. Incubation with primary antibodies, Actin (Sigmae
Aldrich) and p53 (mouse anti-human p53-DO7, Santa Cruz
Biotechnology, Inc.) was performed overnight at 4 ꢀC. After several
washes with TBS-T, membranes were incubated with secondary
antibody (anti-mouse antibody) for 1 h at room temperature. New
washes were performed and ultimately the blots were stained with
fluorescent reagent elemental chlorine free (ECF) and reading was
performed in 9000 Typhoon FLA equipment. Quantification of
fluorescence was performed using the ImageQuant Software (GE
Healthcare).
Human breast carcinoma cell lines HCC1954 and HCC1806 were
cultured in RPMI-1640 medium (Sigma AldrichÒ, EUA) supple-
mented with 10% and 5% of fetal bovine serum (FBS) (Sigma
AldrichÒ, EUA), respectively, 2.5 g Lꢄ1 of
D-(þ)-glucose (Sigma
AldrichÒ, EUA), 400 mM of sodium pyruvate (GibcoÒ, UK),
100 U mLꢄ1 of penicillin and 100 U mLꢄ1 of streptomycin (GibcoÒ,
UK), under an atmosphere containing 5% CO2 at 37 ꢀC. Human
breast adenocarcinoma cell line MCF7 was cultured in DMEM
(Sigma AldrichÒ, EUA) supplemented with 10% of FBS, 100 mM of
sodium pyruvate, 100 U mLꢄ1 of penicillin and 100 U mLꢄ1 of
streptomycin, under an atmosphere containing 5% CO2 at 37 ꢀC.
4.2.2. Cytotoxicity
The cells were plated in 96-well culture plates at a density of
2 ꢃ 104 cells per well and incubated overnight to allow cell
attachment. All compounds were dissolved in dimethylsulphoxide
(DMSO, Sigma AldrichÒ, EUA) at various concentrations and diluted
in culture media. Cells were then incubated with the compounds
for 24, 48, 72 and 96 h. The metabolic activity was determined by
MTT assay [17,18] (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide; MTT, Sigma AldrichÒ, EUA). Briefly, cell cul-
ture were washed with phosphate saline buffer (PBS: 137 mM NaCL,
2.7 mM KCl, 10 mM Na2HPO4.2H2O, 2.0 mM KH2PO4; pH ¼ 7.4) and
incubated with 0.5 mg mLꢄ1 MTT (Sigma AldrichÒ, EUA), pH ¼ 7.4,
at 37 ꢀC for 4 h. Then, formazan crystals were dissolved in acid
isopropanol (0.04 M 37% hydrochloric acid in isopropanol; Sigma
AldrichÒ, EUA). The results allowed to establish doseeresponse
curves and to calculate IC50 values, the concentration required to
inhibit cell proliferation by 50%.
4.2.7. Bax/Bcl-2 analysis
Bax and Bcl-2 expression was also analyzed by flow cytometry
using monoclonal antibodies conjugated with phycoerythrin (PE)
and FITC respectively. Briefly, 2 ꢃ 106 cells were incubated over-
night to allow cell attachment and were treated with IC50 of 3 or 9b
for 48 h (DMSO 0.1%). Cells were collected, centrifuged and washed
with PBS prior to incubation with permeabilizing solution (Intracell
4.2.3. Clonogenic assay
Kit, Immunostep), 3
mL Bax-PE (Santa Cruz Biotechnology, Inc.) and
For the clonogenic assay cells were plated in 6-well culture
plates at a density of 5 ꢃ 105 cells per well and incubated to allow
cell attachment. Incubation with the IC50 of 3 or 9b was made for
48 h. Cells were harvested, counted and 1000, 3000 or 5000 cells
were plated. After 12 days, cells were fixed with methanol, stained
with crystal violet and individual colonies were counted.
3 m
L Bcl-2-FITC (Santa Cruz Biotechnology, Inc.), for 15 min in the
dark. After washing with PBS, analysis was performed in the
FACSCalibur cytometer with excitation at 488 nm, and emission
filter at 530/30 and 585/42 for BCl-2 and Bax, respectively.
4.2.8. Cell cycle analysis
For analysis of DNA content, 2 ꢃ 106 cells were incubated
overnight to allow cell attachment and then were treated with IC50
of 3 or 9b for 48 h (DMSO 0.1%). Cells were collected, centrifuged,
and fixed with ice-cold ethanol (70%) for 30 min in the dark. Then,
cells were washed with PBS and incubated with PI/RNase solution
(Immunostep) for 15 min RT. Cells were analyzed in the
4.2.4. Sulphorhodamine B assay
For total protein production evaluation, cells were plated in 24-
well culture plates at a density of 4 ꢃ 104 cells per well and incu-
bated overnight to allow cell attachment. Cell cultures were incu-
bated with IC50 concentrations of the compounds 3 and 9b for 48 h

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Damage in cell DNA was analyzed with alkaline single-cell gel
electrophoresis, comet assay [20]. Briefly, 5 ꢃ 105 cells were treated
with IC50 of 3 or 9b and with the triple of the first value (DMSO 0.1%
for both cases) for 48 h. Positive control was prepared from nega-
tive control with administration of 20 nM of hydroxide peroxide
(Sigma AldrichÒ, EUA) for 15 min at ꢄ4 ꢀC. Controls and treated
cells were than collected and counted in order to prepare a sus-
pension with 5 ꢃ 104 g mLꢄ1. Cells suspensions were diluted 1:1 in
1% low melting point agarose and applied in Starfrost slides pre-
viously overlaid with 1% normal melting point agarose. The sides
were submerged in alkaline lysis solution (2.5 M NaCl, 100 mM
EDTA, 10 mM Tris, 10% DMSO and 1% Triton x-100, Sigma AldrichÒ,
EUA) overnight. Slides were equilibrated in alkaline electrophoresis
buffer (300 mM NaOH and 1 mM EDTA, pH > 13) and then were
submitted to a potential difference of 25 V and current of 1 A, for
15 min. After electrophoresis, slides were incubated in neutralizing
buffer (0.4 M Tris, pH 7.4) 3 times, 5 min each, and stained with
25 m
g mLꢄ1 ethidium bromide for 20 min. Slides were washed in
distilled water and visualized in a fluorescent inverted microscope
Motic with excitation at 546 nm with a 100 W mercury lamp with
emission at 580/10. Image acquisition was performed in Motic
Images 2.0 (Microscope world, EUA).
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Acknowledgments
[14] P. Moller, The alkaline comet assay: towards validation in biomonitoring of
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Thanks are due to FCT (Project PEst-OE/QUI/UI0313/2014, SFRH/
BD/44957/2008 PhD Grant received by M. Laranjo, SFRH/BD/61378/
2009 PhD Grant received by A. F. Brito), for financial support. We
acknowledge the Nuclear Magnetic Resonance Laboratory of the
Coimbra Chemistry Center (www.nmrccc.uc.pt), University of
Coimbra for obtaining the NMR data.
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Appendix A. Supplementary data
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Supplementary data related to this article can be found at http://
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