6
R. Rondanin et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Table 3
Although it might trace back to a general alkylating property,
the evidenced inhibition of pSTAT5 suggests that a more specific
mechanisms seem to be involved in the cytotoxic activity. On the
other hand, the introduction of 4,4-diaryl butyryl appendage,
apparently much more similar to parent pimozide, gave substan-
tially controversial results. Compounds 20–22 show less cytotoxic
and apoptotic potency than pimozide, whereas 23 is not active.
Moreover, only 21 retain a good inhibition of pSTAT5, instead of
the slightly more cytotoxic non fluorinated analogues 20 and 22.
It is still not known how pimozide might lead to the observed high
inhibition of STAT5 phosphorylation. Reported results are just
excluding a direct inhibition of different STAT5 activator kinases,
such as FLT3 ITD transformed in AML, mutated BCR/ABL in CML,
and various JAK subtypes.9–11 The particular structure–activity
Cell cycle distribution (%) of K562 cells after 24 h exposure to pimozide and pimozide
derivatives
Compound
G1 (%)
S (%)
G2-M (%)
Control
Pimozide (1)
7
8
9
14
20
21
22
39.05
51.28
33.67
47.55
46.96
31.72
34.1
45.94
38.41
60.23
43.23
39.06
62.21
58.57
46.64
31.57
15.01
10.31
6.1
9.22
13.98
6.07
7.33
6.16
20.41
47.2
48.02
relationship of compounds 20–23 suggests
a finely tuned
an anti-pSTAT5 (dotted lines) and to those stained with an isotype
monoclonal antibody. The concentrations of each compound used
for pSTAT5 analysis were scarcely or not cytotoxic on K562 at 24 h.
As shown in Figure 3, pimozide was able to decrease the expres-
sion of pSTAT5 in a dose dependent manner. However, compounds
8 and 9 were more effective than pimozide to reduce the level of
interaction with the biological counterpart(s). Finally, simple
alkyl, N-methyl (4) or -benzyl (5), appendages were found of no
interest.
In summary, a series of pimozide analogues have been synthe-
sized in order to find new growth inhibitors of BCR/ABL expressing
leukemia cell lines without the neuroleptic side effects of parent
compound. Simple modifications regarding the 4,4-di(p-fluoro-
phenyl)butyl portion led to the discovery of two haloacetyl deriv-
atives more potent of pimozide, both as cytotoxic and pSTAT5
inhibitors. Other compounds, more similar to pimozide, bearing
4,4-diarylbut-3-enoyl or 4,4-diarylbutanoyl groups, appeared less
active, but only one demonstrated a concomitant pSTAT5 inhibi-
tory activity. The presented results demonstrated for the first time
that a series of simple derivatives of pimozide maintain a similar or
better behavior toward BCR/ABL expressing leukemia cell lines and
can be seen as a starting point for the development of a novel class
of cancer cells growth inhibitors.
pSTAT5 when used at concentrations of 10 lM and 15 lM (Fig. 3,
panels a, b, e, f, g, h, and Table 2). Compounds 7, 14, 20, 22, were
ineffective causing, in contrast to 8 and 9, a slight increase in
pSTAT5 levels (Fig. 4, Table 2).
Of interest, compound 21 showed an inhibitory activity on
pSTAT5 similar to that of pimozide (Fig. 3 panel c and Fig. 4 panel
e, Table 2).
Cell cycle analysis (Fig. 5 and Table 3) revealed that compounds
able to induce a decrease in pSTAT5 expression, such as pimozide,
8, 9 and 21 caused a prevalent block in G1; in contrast, compounds
inactive on pSTAT5 induced an arrest of cells in S phase, with the
exception of compound 22 that blocked cells in G1 without effects
on pSTAT5. These data are in line with our previous observations17
and with the function of STAT5 to promote cell cycle progression.
In our study, pimozide was effective in reducing intracellular
pSTAT5 expression showing the maximum effect at the concentra-
Acknowledgment
This work was financially supported in part by Ministero
dell’Università e della Ricerca Scientifica e Tecnologica (PRIN 2009).
tion of 30 lM. Moreover pimozide was endowed with cytotoxic
activity on K562 cells. However, our compounds 8 and 9 were
more active than pimozide either as cytotoxic agents or pSTAT5
inhibitors. In fact, 9 was 2.7, 2.0, and 1.49 times more potent than
pimozide as cytotoxic, apoptotic and pSTAT5 inhibitor agent
respectively. Compound 8 was slightly less potent than 9. Of inter-
est, compound 21 was less cytotoxic than pimozide but shows a
pSTAT5 inhibitory activity slightly higher than pimozide. All these
compounds, included pimozide, caused a block in G1 phase of cell
cycle.
Supplementary data
Supplementary data (details for the synthesis and spectroscopic
characterization of target compounds; evaluation of cytotoxicity,
apoptosis, clonal assay for CFU-GM and of intracellular proteins)
associated with this article can be found, in the online version, at
References and notes
Anticonvulsive and antipsychotic drugs can induce blood
dyscrasias.22 Toxic suppression of hematopoietic precursors by
prolonged administration has been observed after the use of phe-
nothiazines. Also new atypical neuroleptic drugs can induce toxic
suppression of hematopoietic precursors.23 Pimozide is an antipsy-
chotic drug of the diphenylbutylpiperidine class. It has not been
shown to have important adverse effects on normal hematopoietic
cells. In fact, while pimozide can decrease colony formation of
bone marrow progenitor cells derived from patients with CML, it
has almost no effect on hematopoietic progenitors derived from
healthy donors.9
Our most active pimozide derivatives 7, 8 and 9 showed on
normal hemopoietic cells effects similar to those displayed by
pimozide.
The most active derivatives seem to underline the importance
of a structural characteristic evidenced in previous results regard-
ing TR120. Indeed, the haloacetamide group represents the most
promising appendage with a clear trend of activity I > Br > Cl, both
as cytotoxic and pSTAT inhibitors.
Rondanin, Riccardo
