2
R. Grzywa et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
and inhibition of the mitochondrial respiratory chain or NF-
j
B
mainly due to its high and diverse biological activity. The analysis
of several, previously evaluated, structural analogues of SFN indi-
cates that its anticancer potency strongly depends on the presence
2
activity. In addition to the wide range of molecular targets associ-
ated with the antiproliferative activity of ITCs, the NAC and
Cys-conjugates of isothiocyanates show inhibitory activity towards
histone deacetylase (HDAC). In many cancer cells increased HDAC
activity leads to dysregulation of the cell proliferation mechanisms.
Inhibition of HDAC results in an enhanced histone acetylation and
subsequently the activation of genes important for cell cycle con-
trol and apoptosis (e.g., p21, bax). The observed in vitro effects of
HDAC inhibition are cell cycle arrest in G2/M phase and apoptosis,
which coincides with the cellular response to ITCs.10
1
4
of a second, polar group in the structure.
Such structural
requirements may explain a dramatic loss of activity of compound
13—mono-NAC-ITC conjugate bearing the unsubstituted n-butyl
side chain. Its antiproliferative activity was over 64-fold lower than
the one observed for compound 2 in the LoVo cell line (with an IC50
value of 130
lM) and more than 27-fold lower in the LoVo/DX cell
line (IC50 = 134
lM). Moreover, similar activity was observed for
compound 12 and to a lesser extent for compound 14, both
representing simple, aliphatic NAC derivatives of mono-ITCs.
Activity that was similar to the one obtained for compound 2
towards the LoVo cell line, was observed for compound 5 with
Limited bioavailability and structure-dependent activity of nat-
6
ural ITCs are the catalyst for the development of new, synthetic
isothiocyanates, with several examples displaying anticancer
activity at a level similar to the natural products. Amongst the
analysed compounds, the phenethyl isothiocyanate (PEITC)
analogue—3,4-methyelendioxybenzyl isothiocyanate displayed a
an IC50 value of 2.59
lM; however, the inhibition of the viability
in the LoVo/DX cell line was over 1.6-times lower (IC50 = 7.93
lM)
than in the case of compound 2. The significant difference between
n-butyl and the long trioxa moiety connecting thiocarbamoyl-mer-
capturate groups of compounds 2 and 5, respectively, indicate that
there is no direct correlation between the activity of the analysed
NAC derivatives of diisothiocyanates and the length of the alkyl
‘spacer’ connecting thiocarbamoyl groups, especially considering
the decrease in antiproliferative activity observed for analogues
with the intermediate length of the aliphatic linker (compounds
3 and 4). Nevertheless, the IC50 values of all analysed, aliphatic
6
-fold increase in the NF-jB inhibitory activity when compared
1
1
to the parent compound. Similarly, 2,2-diphenylethyl ITC induces
the apoptosis of breast cancer cells to a greater extent than natural
benzyl isothiocyanate (BITC).12 In our previous study we described
a series of new, structurally diverse ITCs bearing dialkoxyphospho-
ryl functionality with antiproliferative activity comparable to
PEITC and BITC. In general, the previous results suggest that there
is no distinct structure–activity relationship of isothiocyanates that
may facilitate anticancer potency; however, the presence of the
second functional group (e.g., polar, aromatic) seems to be impor-
tant for the antiproliferative activity of ITCs.14
1
3
diisothiocyanate mercapturic acids were lower than 5
lM as
evaluated in the LoVo cell line, and below 15
cell line.
lM for the LoVo/DX
The main goal of the present study was to investigate the
antiproliferative activity of a series of structurally diverse diisoth-
iocyanate mercapturic acids towards cancer cells. Additionally, we
have analysed the structure–activity relationship for obtained
compounds in comparison with the selected mono-isothiocyanate
mercapturic acids as well as non-conjugated mono- and diisothio-
cyanates, including naturally occurring isothiocyanates—BITC and
PEITC. The antiproliferative activity was evaluated on the human
colon adenocarcinoma cell line (LoVo) and the doxorubicin-resis-
tant human colon adenocarcinoma cell line (LoVo/DX) by means
of the sulforhodamine B (SRB) assay. The impact of the compounds
under study on cells viability is presented as a 50% growth inhibi-
tory concentration (IC50) with doxorubicin used as reference. As a
result of the fact that histone deacetylases are an emerging target
for new chemotherapeutics, and isothiocyanate metabolites
More significant differences in antiproliferative activity were
observed for cyclic representatives of NAC-conjugates of diisothio-
cyanates with the most potent being trans-1,4-cyclohexanedithio-
carbamoyl-mercapturate (7). The IC50 value for inhibition of LoVo
cells viability was 5.21
active NAC conjugate towards the LoVo/DX cell line with an IC50
value of 3.45 M. The shift in the thiocarbamoyl-mercapturate
lM, nevertheless compound 7 was the most
l
groups substitution in the cyclohexane ring from the 1,4-position
in 7 to the 1,2-position in 6 resulted in an almost complete loss
of activity. Interestingly, the close analogue of compound 7 with
a methylsulfoxide substituent in the position occupied by the sec-
ond thiocarbamoyl-mercapturate group was described by Posner
and co-workers as an NAD(P)H quinone oxidoreductase 1 inducer
with only a 2-fold decrease in activity in comparison to SFN,
whereas unsubstituted cyclohexane-ITC showed a 280-times lower
1
9
(
mainly Cys- and NAC-conjugates of ITC) have shown inhibitory
activity than that of SFN. This example again indicates that the
second, polar group incorporated into the isothiocyanate structure
enables an increase in the anticancer activity of ITCs. However,
compound 6 demonstrated that unsuitable positioning of such a
substituent may significantly diminish its biological activity. The
aromatic representatives of analysed NAC-conjugates of diisothio-
cyanates (compounds 9–11) showed a moderate antiproliferative
activity towards HDAC, the present study includes an analysis of
the reported diisothiocyanate mercapturic acids potential to inhi-
bit HDAC activity. The measurements were performed in the
LoVo/DX cell lysate, used as a source of crude enzyme, by the indi-
rect spectrofluorometric assay.1
5,16
The synthetic protocols were
1
7
based on known methods of synthesis of isothiocyanates and
1
8
mercapturic acids and were described in detail in the Supple-
mentary material along with the description of the biological
experiments and the full characterisation of resultant compounds.
Since the relationship between the structure of ITCs and their
biological activity is not straightforward, the designed series of
activity with the IC50 values being above 20 lM.
The introduction of an aromatic ring in compound 9 instead of a
cyclohexane ring (7) resulted in the considerable loss of activity:
4.5-fold for the LoVo and 5.6-fold for LoVo/DX cell line. Moreover,
in contrast to aliphatic diisothiocyanate mercapturic acids, NAC
derivatives of the diisothiocyanate bearing aromatic group showed
a similar or lower impact on cancer cells viability than aromatic
mono-NAC–ITC conjugates. The IC50 values for BITC (15) and PEITC
1
1 NAC-conjugates of diisothiocyanates (Table 1, compounds
1–11) included structures bearing aliphatic, cyclic as well as
aromatic groups. The antiproliferative activity has been observed
in the micromolar range for both tested cell lines. The most
promising group among obtained compounds were aliphatic ana-
logues of diisothiocyanates with 1,4-butanedithiocarbamoyl-mer-
capturate (2) being the most active compound towards the LoVo
(16) NAC-conjugates were, respectively, 11.7
the LoVo and 16.8 M and 7.7 M in the LoVo/DX cell lines. How-
ever, p-methoxybenzyl-thiocarbamoyl-marcapturate (17) was
even more active than natural ITCs with IC50 values of 3.7
(LoVo) and 4.2 M (LoVo/DX). An observed improvement in
lM and 12.7 lM in
l
l
l
M
cell line (IC50 = 2.02
LoVo/DX (IC50 = 4.84
l
l
M) and one of the most potent towards
M). Interestingly, the n-butyl chain of
l
antiproliferative activity, in comparison with NAC-BITC, was
attributed to the presence of a polar substituent in the para
position of the aromatic ring.
compound 2 corresponds to the length of the alkyl chain in
sulforaphane—probably the most intensively studied natural ITC,