Effects of Pimozide Derivatives on pSTAT5 in K562 Cells

Article abstract of DOI:10.1002/cmdc.201700234

STAT5 is a transcription factor, a member of the STAT family of signaling proteins. STAT5 is involved in many types of cancer, including chronic myelogenous leukemia (CML), in which this protein is found constitutively activated as a consequence of BCR-ABL expression. The neuroleptic drug pimozide was recently reported to act as an inhibitor of STAT5 phosphorylation and is capable of inducing apoptosis in CML cells in vitro. Our research group has synthesized simple derivatives of pimozide with cytotoxic activity and that are able to decrease the levels of phosphorylated STAT5. In this work we continued the search for novel STAT5 inhibitors, synthesizing compounds in which the benzoimidazolinone ring of pimozide is either maintained or modified, in order to obtain further structure–activity relationship information for this class of STAT5 inhibitors. Two compounds of the series showed potent cytotoxic activity against BCR-ABL-positive and pSTAT5-overexpressing K562 cells and were able to markedly decrease the levels of phosphorylated STAT5.

Full text of DOI:10.1002/cmdc.201700234

Accepted Article
Title: EFFECTS OF PIMOZIDE DERIVATIVES ON pSTAT5 IN K562
CELLS
Authors: Riccardo Rondanin, Daniele Simoni, Martina Maccesi, Romeo
Romagnoli, Stefania Grimaudo, Rosaria Maria Pipitone, Maria
Meli, Antonio Cascio, and Manlio Tolomeo
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10.1002/cmdc.201700234
ChemMedChem
FULL PAPER
EFFECTS OF PIMOZIDE DERIVATIVES ON pSTAT5 IN K562
CELLS
Riccardo Rondanin,*[a] Daniele Simoni,[a] Martina Maccesi,[a] Romeo Romagnoli,[a] Stefania Grimaudo,[c]
Rosaria Maria Pipitone,[c] Maria Meli,[d] Antonio Cascio,[d] and Manlio Tolomeo*[b]
[a]
Dr. R. Rondanin, Prof. D. Simoni, Dr. M. Maccesi, Prof. R. Romagnoli
Dipartimento di Scienze Chimiche e Farmaceutiche
University of Ferrara
via Fossato di Mortara 17, 44121 Ferrara, Italy
E-mail: rnd@unife.it
[b]
Dr. M. Tolomeo
Dipartimento di Scienze per la Promozione della Salute e Materno Infantile
Centro Interdipartimentale di Ricerca in Oncologia Clinica
Università di Palermo
via del Vespro 129, 902127 Palermo, Italy.
E-mail: mtolomeo@hotmail.com
[c]
[d]
Prof. S. Grimaudo, Dr. M.R. Pipitone
Dipartimento Biomedico di Medicina Interna e Specialistica
Università di Palermo
via del Vespro 129, 90127 Palermo, Italy.
Dr. M. Meli, Prof A. Cascio
Dipartimento di Scienze per la Promozione della Salute e Materno Infantile,
Università di Palermo
via del Vespro 129, 90127 Palermo
Abstract: STAT5 is a transcription factor, component of the STAT
family of signalling proteins. STAT-5 is involved in many types of
cancer, including chronic myelogenous leukemia (CML), where this
protein is found constitutively activated as a consequence of the
BCR-ABL expression. Recently, literature has reported that
neuroleptic drug pimozide can act as inhibitor of STAT5
phosphorylation and is capable to induce apoptosis in CML cells in
vitro. Our group has synthesized simple derivatives of pimozide, with
cytotoxic activity and able to reduce the levels of phosphorylated
STAT5. In this work we continue the search for novel STAT5
inhibitors, synthesizing compounds which maintain or modify the
benzoimidazolinone ring of pimozide, in order to obtain further
structure-activity relationship of this class of STAT5 inhibitors. Two
compounds of the series showed a potent cytotoxic activity against
BCR-ABL positive and pSTAT5 overexpressing K562 cells and were
able to decrease markedly the levels of phosphorylated STAT5.
STAT5A and STAT5B.9 Src-homology 2 domain (SH2) is a
crucial part of the STAT5 factor, docked and phosphorylated by
cytokine activated receptors or non receptors kinases. When
phosphorylated, STAT5 is able to homo- or heterodimerize
before nucleus translocation and induction of genes
transcription.10
As a matter of fact, in recent years, literature has reported the
development of some interesting classes of different compounds
that interact with the STAT5 function, with a mechanism related
to inhibition of the SH2 portion, and sometimes discriminating
between A and B isoforms.11-13 There is however an ongoing
interest in finding new structures able to interact with STAT5
signal.
Using a chemical library of compounds known to be safe in
humans, with a cell-based screening, Nelson and coworkers
identified pimozide (1), a neuroleptic drug used to treat Tourette
syndrome, as a STAT5 inhibitor. They reported that pimozide
decreases levels of STAT5 activated by BCR/ABL, being
14-16
effective against both imatinib-sensitive and -resistant cells.
Then, in
a
recent work,17 we demostrated that simple
Introduction
modifications of pimozide structure allowed to obtain derivatives
(as compound 3c) more potent in decreasing activated STAT5
levels in BCR-ABL expressing cells. Modifications were in part
suggested by our previous work on compound TR120 that
strongly decreased STAT5 expression in K562 cells.18 Moreover,
considering that 4,4-di(p-fluorophenyl)butyl chain of pimozide
plays a role in promoting the CNS effects of the drug, we
changed the piperidine nitrogen substitution from the original
Signal transducers and activators of transcription (STATs) are a
family of proteins involved in signal transduction which are
activated by multiple pathways, including the janus-associated
kinases (JAKs), FLT3, and inflammatory cytokines.1,2 There is
evidence of an important and complex role of JAK2/STAT5
signalling pathway in some prostate and breast cancers
evolution.3 Moreover, STAT5 and many intracellular signalling
cascades are activated by the constitutively active cytoplasmic
tyrosine kinase BCR-ABL, characteristic for chronic myeloid
leukemia (CML) and BCR-ABL-positive acute lymphoblastic
leukemia (ALL).4-6 For this reason, the system JAK2/STAT5 has
been identified as a plausible target to inhibit BCR-ABL positive
CML,7,8 especially in case of resistance to treatment with
tyrosine kinase inhibitors. More recently, it has been reported
that BCR-ABL is able to differently affect the two isoforms
4,4-di(p-fluorophenyl)butyl
diarylbutanoyl groups and obtained derivatives with
chain
into
analogue
4,4-
a
comparable ability to inhibit activated STAT5. In the present
study, a new series of pimozide analogues lacking the original
4,4-di(p-fluorophenyl)butyl chain have been synthesized in order
to find more effective compounds on BCR-ABL expressing
leukemia cell lines. Simple modifications were introduced in
previously reported active compounds 3a,c, in haloacyl
1
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10.1002/cmdc.201700234
ChemMedChem
FULL PAPER
Scheme 1. Synthesis of compounds 8 and 9. Reagents and conditions: a)
(Boc)2O, MeOH; b) DMF, NaH then CH3I; c) TFA, CH2Cl2; d) chloroacetyl
chloride, TEA, CHCl3; e) NaI, acetone.
appendage or in the benzimidazolone ring. Moreover, we tested
the role of piperidine portion in a molecule simplification strategy.
In fact, in the piperidine-containing derivatives, we choose to
maintain the amide function instead of amine for the piperidine
nitrogen and acylating it with different halogenated substituents.
This led to the discovery of two derivatives endowed with a more
potent cytotoxic and STAT5 inhibitory activity where compared
to pimozide and compound 3c.
Benzoxazolinone compounds 13 and 14 were instead obtained
starting from 2-aminophenol, which was reacted with N-benzyl-
4-piperidone in the presence of sodium triacetoxyborohydride to
yield intermediate 10, then transformed into 11 with carbonyl
diimidazole. Piperidine nitrogen of intermediate 11 was
debenzylated and Boc protected with hydrogen atmosphere in
the presence of catalytic amounts of 10% Palladium on carbon
and Boc2O. Boc protection allowed a much easier recovery of
debenzylated intermediate. Then transformation of 12 into 13
and 14 was performed following the same procedures as
described for the preparation of 8 and 9 from 6.
H
N
CH2I
OCH3
O
OCH3
N
O
F
NH
OCH3
N
O
O
H3CO
Pimozide (1)
TR120 (2)
K562 IC50 = 0.12 µM
K562 IC50 = 5 µM
F
O
O
N
OH
O
OH
a
b
Bz
H
N
+
N
N
H
NH2
N
O
Bz
10
N
11
N
3a: X = Cl; K562 IC50 = 7.3 µM
3b: X = Br; K562 IC50 = 2.4 µM
3c: X = I; K562 IC50 = 1.8 µM
Bz
c
N
O
N
O
CH2X
O
O
O
d
13: X = Cl
14: X = I
N
e
Figure 1. Chemical structure of pimozide, TR120 and first derivatives acting
N
12
as p-STAT5 inhibitors.
CH2X
O
N
Boc
Results
Scheme 2. Synthesis of compounds 13 and 14. Reagents and conditions: a)
NaH(AcO)3, CH2Cl2; b) carbonyl diimidazole, CH2Cl2; c) H2, 10% Pd/C, Boc2O,
MeOH; d) TFA, CH2Cl2, then chloroacetyl chloride, TEA, CHCl3; e) NaI,
acetone.
Chemistry.
The synthesis of compounds modified at benzimidazolinone ring
is displayed in Schemes 1 and 2. Starting from commercially
available compound 4, the N-3 methylation was conducted with
methyl iodide after Boc protection at piperidine ring and
subsequent deprotonation with sodium hydride. After Boc
deprotection of intermediate 6 with trifluoroacetic acid, a simple
acylation of piperidine nitrogen with chloroacetyl chloride led to
compound 8, that was in part transformed to compound 9 by
halogen exchange reaction adding sodium iodide in acetone
solution.
In Scheme 3 are reported the syntheses of the derivatives
without the piperidine ring (15 and 16), obtained by direct
acylation
with
chloroacetyl
chloride
of
commercial
benzimidazolinone after sodium hydride deprotonation and the
possible ion exchange as described before for compound 9.
Moreover, in Scheme 3 are also reported the compounds 17-20,
obtained by simple acylation of piperidine nitrogen of starting 4,
with acyl halides or carboxylic acid with condensing
carbodiimide reagent.
CH3
H
N
H
N
H
H
N
N
N
b
a
O
a
O
O
O
15: X = Cl
16: X = I
O
N
N
N
b
N
H
N
CH2X
O
N
N
N
5
6
4
H
Boc
Boc
H
N
H
N
c
O
O
c
CH3
CH3
N
N
17: R = CH-(Cl)-CH3
18: R = CH-(Cl)-Ph
19: R = (CH2)3-Cl
20: R = (CH2)2-I
N
N
d
O
O
N
N
N
N
H
7
8: X = Cl
9: X = I
R
O
e
N
N
H
CH2X
O
Scheme 3. Synthesis of compounds 15 - 20. Reagents and conditions: a)
DMF, NaH then chloroacetyl chloride; b) NaI, acetone; c) opportune acyl
chloride, TEA, CHCl3 or, in case of 20, 3-Iodopropanoic acid, EDC
hydrochloride, TEA, CHCl3.
2
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10.1002/cmdc.201700234
ChemMedChem
FULL PAPER
Biological results.
Table 1 and Figure 2a show the cytotoxic effects of new
pimozide derivatives in K562 cells after 48 h of treatment.
Compounds 15, 17, 19 and 20 did not show important cytotoxic
effects on K562 cells (IC50 higher than 30 µM). Compounds 8
and 14 displayed
a cytotoxic activity similar to pimozide.
Compounds 9, 16 and 13 were more active than pimozide, with
the benzoxazolinone derivative 13 that showed a cytotoxic
activity higher than our lead pimozide analogue 3c.18 The most
active compounds (8, 9, 13, 14, 16 and 18) were tested in
K562R, a cell line selected by exposure of K562 cells to
increasing concentrations of imatinib and 10 fold more resistant
to imatinib mesylate than parental cells.18 As shown in Figure 2b,
the cytotoxic effects of our most active compounds on K562R
cells were similar to those observed in parental K562 cells.
a
b
Table 1. IC50 (µM ± SE) and AC50 (µM ± SE) of pimozide and analogues
evaluated in K562 cells after 48 h of treatment.
Compound
IC50 (µM)
5±0.8
AC50 (µM)
10±2.3
5±0.7
7.5±1.1
3.6±0.4
1.8±0.2
11±3
Pimozide
3cc
8
1.8±0.22
4±0.7
9
1.6±0.2
0.75±0.12
5±0.6
13
14
15
16
17
18
19
20
>30
>30
2.5±0.4
>30
5.8±0.7
>20
Figure 2. Cytotoxic activity of new pimozide derivatives used at different
concentrations in K562 cells (a) and K562R cells (b) after 48 h of treatment.
10±2.5
>30
22±5
Then, we examined the influence of the most active compounds
(showing an IC50 ≤ 5 µM) on pSTAT5 expression (Figure 3,
Table 2) and cell cycle distribution in K562 cells (Fig. 4, Table 3).
pSTAT5 expression was determined by flow cytometry after
>30
>30
>30
staining cells with
a fluorochrome-conjugated anti-pSTAT5
[a] Concentration able to inhibit 50% cell growth. [b] Concentration able to
induce apoptosis in 50% of cells. [c] Ref. 18
monoclonal antibody (MoAb); analysis of cell cycle effects was
carried out by flow cytometry after staining cells with propidium
iodide. In Figure 3 the curves expressing the fluorescence of
cells stained with a fluoresceinated anti-pSTAT5 after 24 h
exposure to each compound (thick lines) were compared to
those expressing the fluorescence of untreated cells stained with
an anti-pSTAT5 (dotted lines) and to those stained with an
isotype monoclonal antibody (thin line).
3
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10.1002/cmdc.201700234
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Table 2. Median fluorescence values of K562 cells stained with an anti-
pSTAT5 after 24 h exposure to pimozide analogues (10µM)
Median
Compound
fluorescence
Control (Isotypic MoAb)a
45.7
172.29
117.2
71.3
Control (pSTAT5 MoAb)b
Pimozide
3c
8
133.29
49.87
67.51
62.18
123.19
9
13
14
16
[a] Control (Isotypic MoAb) = K562 cells stained with an isotypic MoAb. [b]
Control (STAT5 MoAb) = K562 cells stained with a STAT5 MoAb.
Cell cycle analysis (Figure 4 and Table 3) revealed that all
compounds that decrease the levels of p-STAT5 caused a
prevalent block in G1. These data are consistent with our
previous observations and with the function of STAT5 to
promote cell cycle progression18.
Figure 3. Intracellular levels of phosphorylated STAT5 in K562 cells evaluated
by flow cytometry after 24 h exposure to each compound. Dotted line: cells
stained with an isotype monoclonal antibody; thin line: cells stained with an
anti-STAT5 monoclonal antibody; thick line: cells stained with an anti-STAT5
monoclonal antibody after 24 h exposure to each compound. (a) 8 (10 µM); (b)
8 (15 µM); (c) 9 (10 µM); (d) 9 (15 µM); (e) 16 (10 µM); (f) 16 (15 µM); (g) 13
(10 µM); (h) 13 (15 µM); (i) 14 (10 µM); (j) 14 (15 µM).
All compounds tested decreased the expression of pSTAT5 in a
dose dependent manner (Figure 3, Table 2). Compound 9 was
the most potent pSTAT5 inhibitor of the series. In fact, as shown
in Table 2, the median fluorescence value of K562 cells stained
with anti-pSTAT5 MoAb after 24h exposure to compound 9 at
10 µM was similar to that observed in the control stained with an
isotypic MoAb, indicating the ability of this compound to inhibit
completely the phosphorylation of STAT5. Previously, we
observed that compound 3c was markedly more potent than
pimozide in reducing the levels of pSTA5 in K562 cells. However,
compound 9 revealed an inhibitory activity on pSTAT5 higher
than that observed with our lead compound 3c. Also compounds
13 and 14 used at the concentration of 10µM showed a potent
inhibitory effect on phosphorylation of STAT5. Compounds 16
and 8 were slightly less active on pSTAT5 than 9, 13 and 14.
Figure 4. Effects of pimozide derivatives on DNA content per cell following
exposure of K562 cells for 24 h. (a) control; (b) 8 (6 µM); (c) 9 (2 µM); (d) 16 (3
µM); (e) 13 (8 µM); (f) 14 (2 µM).
4
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10.1002/cmdc.201700234
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information about the relative importance of the piperidine
portion. Despite we had poor expectations for this strategy,
results were somehow surprising. As a matter of fact, compound
15 was found not active, while 16 demonstrated a better activity
than pimozide. The anti-STAT5 monoclonal antibody test of 16,
moreover, displayed a moderate activity at 10 µM that rose to
very good at higher concentration (15 µM). This is not a trivial
finding since the piperidine ring has been linked to the
determinism of QT prolongation by antipsychotic agents making
a piperidine-less structure a desirable feature for a STAT5
inhibitor compound.19
Table 3. Cell cycle distribution (%) of K562 cells after 24 h exposure to
pimozide derivatives.
Compound
G1 (%)
34.24
44.37
50.67
48.04
49.01
45.42
S (%)
48.04
50.52
44.92
47.84
46.03
50.57
G2/M (%)
17.72
5.11
Control
8
9
4.42
13
14
16
4.12
4.96
4.01
Conclusions
In the search of new molecules able to inhibit the uncontrolled
proliferation of BCR-ABL expressing cell lines, pimozide was
identified as a promising, although unexpected, structure able to
interfere with the pSTAT-5 constitutive overexpression. In our
previous and present studies, we discovered that important
modifications of pimozide structure lead to compounds still or
even more active than pimozide itself. Apart from di(p-
fluorophenyl)-butyl side chain, in the present study we found that
also the benzoimidazolinone ring or the piperidine portion can be
chemically modified resulting in compounds highly active as
growth inhibitors and apoptosis inducers and able to lower the
phosphorylated STAT-5 content.
The presence of a haloacetyl group gives the idea of an
important possible contribute of covalent bonds formations in the
mechanism of these derivatives, but in the case of the most
active compound 13, a chloroacetyl bearing molecule that
overcomes the corresponding iodoacetyl 14 activity, there is the
idea that other specific mechanisms can be preferably involved.
We have no specific evidence that our compounds alkylate
intracellular proteins, thereby interfering with STAT activation,
and we feel this possibility is too speculative to discuss at this
time. As reported in the previous literature of Nelson and
coworkers, pimozide action toward pSTAT5 levels is not yet
supported by a known mechanism, and direct interaction with
the STAT5 protein or its SH2 domain is still not demonstrated. At
the same time, also the mechanism by which the present new
molecules modulates STAT5 expression remains to be
elucidated and it is an intriguing problem currently under
investigation.
Discussion
On the basis of previous promising results, we explored further
modifications of compound 3c, by maintaining the amide group
on the piperidine nitrogen and acylating it with different
halogenated substituents in order to study the structure-activity
relationship of the STAT5 inhibitors. Compounds 17-20
demonstrated in our tests that the substitution of haloacetyl
portion with other haloacyl groups is generally detrimental for the
activity. Only phenylchloroacetic derivative 18 retained an
appreciable IC50 (10 µM) and AC50 toward K562 cells. More
interesting results were obtained investigating the role of the
nitrogen N3 of the benzoimidazolinone core in the activity of the
compounds.
Two derivatives 8 and 9 have been synthesized with methylated
N3 and can be directly compared with 3a and 3c. In this case,
the growth inhibition and apoptotic potencies are in the same
range of concentrations. In the immunofluorescence test, both
compounds 8 and 9 are able to inhibit pSTAT5 signal when
added at 15 µM but only 9 is effective also at 10 µM
concentration, showing a median fluorescence value close to the
one obtained with Isotypic MoAb control.
Replacement of the nitrogen at
3
position of the
benzoimidazolinone core with an oxygen atom (compounds 13
and 14) allow to maintain a high growth inhibition and pro-
apoptotic activity. Indeed, the cytotoxic activity of compound 14
was similar to pimozide while, of note, compound 13 showed a
marked increase of cytotoxic activity (IC50 0.75 µM). Interestingly,
on one hand, chloro-substituted compound 13 resulted more
potent than iodo-substitued analog 14, and on the other hand
compounds 3c and 9 (iodo-substituted) were found more potent
than respective chloro substituted analogs 3a and 8. Therefore,
the peculiar relative potency of compounds demonstrated in the
13 vs 14 system is in contrast with a trend that could be
envisaged in the other cases, for example, considering a role of
different alkylating power of iodoacetyl vs chloroacetyl groups.
At least in this system, a more complex and specific mechanism
seems to be important for the biological potency. In the anti-
STAT5 monoclonal antibody test, both 13 and 14 showed the
ability to act at 10 µM concentration, resulting almost equal or
slightly more active than 3c.
Experimental Section
Chemistry
General procedures
Reagents were purchased from commercial suppliers and used without
further purification. Nuclear magnetic resonance spectra (1H-NMR e 13C-
NMR) were determined in d6-DMSO or CDCl3 solutions and recorded with
a Bruker AC-200 or a Varian Mercury Plus 400 spectrometers. Peak
positions are given in parts per million downfield from tetramethylsilane
as the internal standard; J values are expressed in hertz. Mass analyses
were performed on Finnigan MAT 95 instrument with BE geometry using
Finally, two compounds, 15 and 16, were synthetized without the
piperidine ring but directly functionalized with halogenated acetyl
substituents at N1 of benzimidazolone ring in order to obtain
double
focusing
electrospray
(ESI)
technique.
Thin
layer
chromatographies (TLCs) were performed on silica gel F-254 to evaluate
reaction courses and mixtures. Flash chromatography was performed on
5
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10.1002/cmdc.201700234
ChemMedChem
FULL PAPER
Merck 60 silica gel (0.063-0.200mm). The anhydrification of solvents was
performed following standard techniques. All non-aqueous reactions
were run in argon atmosphere. Solutions containing the final products
were dried over Na2SO4, then filtered and concentrated in vacuo using
rotatory evaporator.
To a solution of 8 (100 mg, 0.324 mmol) in acetone, NaI was added (97
mg, 2 eq.). The solution was kept under stirring for 24 h. After a filtration
the solution was concentrated in vacuo. Residue was dissolved in ethyl
acetate and washed with H2O and brine. After the removal of the solvent
under reduced pressure and chromatographic purification, the desired
product 9 is obtained (90 mg). Yield 70%.
Tertbutyl-4(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidin-1-
carboxylate (5)
1 H NMR (CDCl3, 200 MHz): δ 1.80-1.95 (m, 2H), 2.22-2.60 (m, 2H), 2.70-
2.90 (m, 1H), 3.20-3.40 (m, 1H), 3.40 (s, 3H), 3.75-3.88 (m, 2H), 3.90-
4.10 (m, 1H), 4.50-4.70 (m, 1H), 4.70-4.90 (m,1H), 6.97-7.13 (m, 4H);
To a solution of 4 (436 mg, 2 mmol) in methanol (10 mL) was added di
tertbutyl dicarbonate (480 mg, 2.2 mmol) and the solution was kept
overnight under stirring. The solvent was then evaporated, the residue
solubilized in ethyl acetate, and washed with aqueous citric acid 0.5 M,
water, saturated solution of NaHCO3 and brine. After drying and solvent
evaporation the product 5 was obtained in 85% yield.
13 C NMR (CDCl3, 400 MHz): δ 3.98, 27.15, 28.74, 28.92, 42.07, 46.96,
50.48, 107.77, 108.95, 121.17, 127.77, 130.17, 153.78, 166.39;
MS(ESI): 399.71 [M+1].
1 H NMR (CDCl3, 200 MHz): δ 1.45 (s, 9H), 1.75-1.90 (m, 2H), 2.20-2.40
(m, 2H), 2.75-2.95 (m, 2H), 4.25-4.4 (m, 2H), 4.35-4.6 (m, 1H), 6.9-7.15
(m, 4H), 9.25 (s, 1H).
N-(1-Benzylpiperidin-4-yl)-2-aminophenol (10)
To a solution of 2-aminophenol (436 mg, 4 mmol) in dry CH2Cl2, N-
benzyl-4-piperidone (757 mg, 4 mmol) and sodium triacetoxyborohydride
(1.27 gr, 6 mmol) were added, obtaining a yellow solution. The solution
was kept under stirring at room temperature for 14 h. The mixture is
washed with saturated NaHCO3 and brine. After removal of the organic
solvent in vacuo and purification on column chromatography, the desired
product 10 was obtained as beige crystalline solid, 850 mg. Yield 75 %.
Tertbutyl-4(3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-
yl)piperidin-1-carboxylate (6)
To a dispersion of NaH 60% (94 mg, 2.2 mmol) in dry DMF (3 mL) was
added dropwise a solution of 5 (637 mg, 2 mmol) in dry DMF, and stirring
was kept for 30 min. Then, CH3I (101 µL, 2 mmol) was added and the
solution was stirred for 6h. The reaction, was quenched with water and
the solvent evaporated. H2O and AcOEt were then added and the
organic solution was separeted. Three extractions of the aqueous phase
were performed with AcOEt, while the combined organic phases were
washed with, brine, then dried over Na2SO4 and evaporated.
Chromatographic purification gave the product 6, 577 mg. Yield 87%.
1 H NMR (CDCl3, 200 MHz): δ 1.40-1.65 (m, 2H), 2.03-2.20 (m, 4H), 2.93
(d, J = 18, 2H), 3.20-3.38 (m, 1H), 3.63 (s, 2H), 6.56-6.80 (m, 4H), 7.25-
7.40 (m, 5H).
3-(1-Benzylpiperidin-4-yl)benzo[d]oxazole-2(3H)-one (11)
To a solution of 10 (841 mg, 2.97 mmol) dissolved in dry CH2Cl2, (15 mL),
carbonyldiimidazole (528 mg, 1.1 eq) was added. The obtained solution
was yellow-orange and it was kept under stirring for 16 h. After dilution
with more CH2Cl2 and washing with H2O and brine, the organic phase
was concentrated in vacuo. The chromatography column furnished
product 11 as white crystal solid (810 mg). Yield 88%.
1 H NMR (CDCl3, 200 MHz): δ 1.45 (s, 9H), 1.75-1.90 (m, 3H), 2.20-2.45
(m, 2H), 2.75-2.95 (m, 2H), 3.41 (s, 3H), 4.27-4.34 (m, 2H), 4.35-4.57 (m,
1H), 6.90-7.17 (m, 4H).
1-[1-(2-Chloroacetyl)piperidin-4-yl]3-metil-1H-benzo[d]imidazol-
2(3H)-one (8)
1 H NMR (CDCl3, 200 MHz): δ 1.82-1.87 (m, 2H), 2.10-2.25 (m, 2H), 2.25-
2.57 (m, 2H), 2.98-3.17 (m, 2H), 3.57 (s, 2H), 4.10-2.35 (m, 1H), 7.05-
7.35 (m, 9H).
To a solution of compound 6 (331 mg, 1 mmol) dissolved 3 mL of CH2Cl2,
trifluoroacetic acid (3 mL) was added. The solution was kept under
stirring at room temperature for 30 minutes. After removal of the solvent
under reduced pressure,
5 mL of CHCl3 were added and then
Tertbutyl-4-(2-oxobenzo[d]oxazole-3-(2H)-yl)piperidin-1-carboxylate
(12)
triethylamine (0.35 µL, 2.5 mmol). A solution of chloroacetyl chloride (135
mg, 1.2 mmol) in CHCl3 (3 mL) was finally added dropwise under stirring
at 0° C. The solution was kept under stirring at room temperature
overnight. The mixture was washed with HCl 5%, H2O, saturated solution
of NaHCO3 and brine. After removal of the solvent under reduced
pressure, the solution was concentrated using AcOEt. The
chromatography purification furnished the desired product 8, 121 mg.
Yield 40%.
To
a solution of 11 (440 mg, 1.42 mmol) in EtOH, di-tert-butyl
dicarbonate (370 mg, 1.2 eq) and catalytic amount of 10% Palladium on
carbon were added. The mixture was kept under stirring in a hydrogen
atmosphere for 2 h. Catalyzer was then filtered out and the solution was
concentrated in vacuo obtaining
a yellow oil. The chromatography
column furnished white crystals of 12, 380 mg. Yield 84%.
1 H NMR (CDCl3, 200 MHz): δ 1.945 (m, 2H), 2.23-2.60 (m, 2H), 2.70-
2.90 (m, 1H), 3.20-3.40 (m, 1H), 3.413 (s, 3H), 3.98-4.10 (m, 1H), 4.12-
4.15 (m, 2H), 4.30-4.50 (m, 1H), 4.75-4.90 (m, 1H), 6.95-7.10 (m, 4H);
1 H NMR (CDCl3, 200 MHz): δ 1.49 (s, 9H), 1.81-1.96 (m, 2H), 2.17-2.40
(m, 2H), 2.75-2.95 (9mm, 2H), 4.20-4.40 (m, 3H), 7.07-7.23 (m, 4H).
3-(1-(2-Chloroacetyl-piperidin-4-yl)benzo[d]oxazol-2-(3H)-one (13)
13 C NMR (CDCl3, 400 MHz): δ 27.22, 28.92, 29.68, 41.14, 42.19, 46.14,
50.58, 107.84, 180.97, 121.26, 121.26, 127.85, 130.22, 153.77, 165.15;
The procedure was similar as described for 8, starting from 12 (380 mg,
1.18 mmol) Chromatography column furnished product 13 as white solid,
225 mg. Yield 65%.
MS[ESI]: 308.32 [M+1].
1-[1-(2-Iodoacetyl)piperidin-4-yl]3-methyl-1H-benzo[d]imidazol-
2(3H)-one (9)
1 H NMR (CDCl3, 200MHz): δ 1.90-2.12 (m, 2H), 2.17-2.52 (m, 2H), 2.66-
2.85 (m, 1H), 3.20-3.60 (m, 1H), 4.06-4.24 (m, 3H), 4.35-4.50 (m, 1H),
4.78-4.85 (m, 1H), 7.04-7.24 (m, 4H).
6
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10.1002/cmdc.201700234
ChemMedChem
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13 C NMR (CDCl3, 400 MHz): δ 28.53, 29.26, 41.05, 41.85, 45.81, 51.99,
pressure, the chromatography purification furnished 17 as a white solid,
109.47, 110.52, 122.60, 123.92, 129.60, 142.72, 153.87, 165.19;
205 mg. Yield 66%.
MS(ESI): 295.44 [M+1].
1 H NMR (CDCl3, 200MHz): δ 1.723 (d, J = 6.2, 3H), 1.959 (s, 2H), 2.26-
2.60 (m, 1H), 2.65-2.90 (m, 2H), 3.19-3.40 (m, 2H), 4.134 (m, 1H), 4.07-
4.25 (m, 1H), 4.67 (d, J = 6.4, 1H), 4.79-4.94 (m, 1H), 7.073 (s, 1H),
8.843 (s, 1H);
3-(1-(2-Iodoacetyl-piperidin-4-yl)benzo[d]oxazole-2-(3H)-one (14)
The procedure was similar as described for 9, starting from compound 13
(50 mg, 0.17 mmol). Chromatographic purification gave the desired
product 14 as white solid, 45 mg. Yield 68%.
13 C NMR (CDCl3, 400 MHz): δ 21.05, 28.38, 28.79, 28.97, 42.42, 49.63,
50.18, 109.64, 109.99, 121.46, 121.61, 128.03, 128.67, 154.98, 167.33;
1 H NMR (CDCl3, 200MHz): δ 1.85- 2.05 (m, 2H), 2.21-2.38 (m, 1H), 2.38-
2.63 (m, 1H), 2.67-2.85 (m, 1H), 3.15-3.37 (m, 1H), 3.75-3.85 (m, 2H),
3.86- 4.10 (m, 1H), 4.37-4.57 (m, 1H), 4.75-2.95 (m, 1H), 7.08-7.22 (m,
4H);
MS (ESI): 308.544 [M+1].
(R,
S)
1-[1-(2-Chloro-2-phenylacetyl)-piperidin-4-yl]-1H-
benzo[d]imidazol-2(3H)-one (18)
13 C NMR (CDCl3, 400 MHz): δ 28.42, 28.56, 41.79, 46.69, 51.96, 109.53,
110.53, 122.59, 123.92, 129.56, 142.72, 153.90, 166.51;
(R, S)-Mandelic acid (304 mg, 2 mmol) was dissolved in thionyl chloride
(6 mL). The solution was kept under stirring at room temperature for 24 h
and then it was concentrated in vacuo to obtain the phenylchloroacetic
acid chloride, as a colorless oil, used with no further purification. To a
solution of compound 4 (414 mg, 2 mmol) dissolved in chloroform (7 mL)
and cooled in ice bath, triethylamine (280 µL, 2 mmol) was added.
Phenylchloroacetic acid chloride dissolved in chloroform was then added
dropwise and the solution was kept under stirring at room temperature for
24 h. The mixture was washed with HCl 5%, H2O, saturated solution of
NaHCO3 and brine. After removal of the solvent under reduced pressure,
the chromatography purification gave the desired product 18 as a white
solid, 104 mg. Yield 14%.
MS(ESI): 387.305 [M+1]
1-(2-Chloroacetyl)-1H-benzo[d]-imidazol-2(3H)one (15)
To a mixture of NaH (60%, 80 mg, 2 mmol) in dry DMF (4 mL), 2(1H)-
Benzimidazolone (268 mg, 2 mmol) was added, giving a milky white
solution. The solution was kept under stirring for 30 min. After cooling
with ice bath, chloroacetyl chloride (0.18 mL, 2.2 mmol) was then
cautiously added. The solution was kept under stirring for 24h. The
mixture was then poured into crushed ice, acidified with 5% HCl and
extracted three times with small amounts of ethyl acetate. The combined
organic phases were washed with water and brine, dried and
concentrated in vacuo. The solid was then purified in chromatography
column to obtain product 15 as white solid (135 mg). Yield 32%.
1 H NMR (CDCl3, 200 MHz): δ 2.7-2.98 (m, 2H), 2.05-2.40 (m-2H), 2.63-
2.87 (m, 1H), 3.02-3.23 (m, 1H), 2.9-4.10 (m, 1H), 2.40-2.55 (m, 1H),
5.80-5.95 (m, 1H), 5.78-5.81 (d, J=7.4, 1H), 6.90-7.15 (m, 4H), 7.30-7.60
(m, 5H);
13 C NMR (CDCl3, 400 MHz): δ 28.62, 28.69, 29.03, 42.76, 45.63, 49.76,
109.36, 109.87, 121.04, 121.49, 121.68, 121.76, 127.90, 128.40, 128.61,
128.85, 129.02, 129.25, 154.76, 165.52;
1 H NMR (DMSO, 200MHz): δ 5.05 (s, 2H), 7.03-7.22 (m, 3H), 7.97-8.01
(m, 1H), 11.51 (s, 1H);
13 C NMR (DMSO, 400 MHz): δ 45.45, 109.29, 114.61, 121.79, 124.73,
126.64, 129.29, 151.98, 166.24.
MS (ESI): 370.349 [M+1].
1-[1-(4-Chlorobutyryl)piperidin-4-yl]-1H-benzo[d]imidazol-2(3H)-one
(19)
MS (ESI): 177.15 [M -Cl +1].
1-(2-Iodo acetyl)-1H-benzo[d]-imidazol-2(3H)one (16)
The procedure was similar as described for 17, from compound 4 (217
mg, 1 mmol) and 4-chlorobutyryl chloride (170 µL, 1.5 mmol). After
chromatography purification 19 was obtained as a white solid, 118 mg.
Yield 36%.
The procedure was similar as described for 9, starting from compound 15
(125 mg, 0.6 mmol). After chromatographic purification, 16 was obtained
as a white solid (70 mg). Yield 40%.
1 H NMR (CDCl3, 200 MHz): δ 1.80-2.00 (m, 2H), 2.09-2.24 (m, 2H), 2.28-
2.37 (m, 2H), 2.50-2.65 (m, 2H), 2.65-2.80 (m, 1H), 3.10-3.30 (m, 1H),
3.60-3.75 (m, 2H), 4.05-4.18 (m, 1H), 4.41-4.61 (m, 1H), 4.80-4.97 (m,
1H) 7.03-7.22 (m, 4H), 9.43 (s, 1H);
1 H NMR (DMSO, 200 MHz): δ 4.57 (s, 2H), 7.035-7.22 (m, 3H), 7.94-
7.98 (m, 1H), 11.54 (s, 1H);
13 C NMR (DMSO, 400 MHz): δ 109.27, 114.86, 121.81, 124.83, 126.89,
128.89, 151.60, 168.28.
13 C NMR (CDCl3, 400 MHz): δ 27.84, 28.98, 29.77, 31.09, 41.49, 44.94,
45.05, 50.71, 109.24, 109.94, 121.32, 121.58, 127.88, 128.78, 154.94,
170.17;
MS (ESI): 177.15 [M -I +1].
(R, S) 1-[1-(2-Chloropropanoyl)-piperidin-4-yl]-1H-benzo[d]imidazol-
MS (ESI): 322.420 [M+1].
2(3H)-one (17)
1-[1-(3-Iodopropanoyl)piperidin-4-yl]-1H-benzo[d]imidazol-2(3H)-one
(20)
To a solution of compound 4 (217 mg, 1 mmol) in CH2Cl2 (4 mL) was
added triethylamine (0.140 µL, 1 mmol) and, after cooling in ice bath, 2-
chloropropanoyl chloride (145 µL, 1.5 mmol) was added dropwise. The
solution was kept under stirring at room temperature for 24 h. Afterwards,
the mixture was diluted with CH2Cl2 and washed with 5% HCl, saturated
NaHCO3 solution and brine. After removal of the solvent under reduced
To a solution of compound 4 (217 mg, 1 mmol) dissolved in CHCl3 (7 mL),
3-iodopropionic acid (200 mg, 1 mmol), EDC·HCl and HOBT (17 mg, 1.1
mmol) were added. The solution was kept under stirring at room
temperature for 24 h. The mixture was then washed with HCl 1M, H2O,
7
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10.1002/cmdc.201700234
ChemMedChem
FULL PAPER
saturated solution of NaHCO3 and brine. After removal of the solvent
under reduced pressure, the chromatography purification (AcOEt)
furnished 55.5 mg of the desired product 20. Yield 15%.
About 1x106 cells were washed twice with PBS (Sigma) and
resuspended in 100 µL cytofix/cytoperm solution (Becton-Dickinson) at
4˚C. After 20 min the cells were washed twice with BD Perm/Wash™
buffer solution (Becton-Dickinson) and incubated with 20 µL of the
specific fluorochrome(PE)-conjugated monoclonal antibody anti-p-STAT5
(Becton-Dickinson) at 4˚C. After 30 minutes the cells were washed twice
and analysed by flow cytometry. Alternatively, the cells were incubated
with 2 µL PE-conjugated rat anti-mouse IgG1 monoclonal antibody
(Becton-Dickinson) at 4˚C. After 30 minutes the cells were washed twice
and analysed by a FACScan flow cytometer (Becton–Dickinson).
1 H NMR (CDCl3, 200 MHz): δ 1.9 (m, 2H), 2.20-2.35 (m, 2H), 2.60-2.80
(m, 2H), 3.0-3.13 (m, 2H), 3.16-3.37 (m, 2H), 3.97-4.10 (m, 1H), 4.30-
4.63 (m, 1H), 7.05-7.13 (m, 4H), 9.54 (s, 1H);
MS (ESI): 400.556 [M+1].
.
Cell lines and culture
K562 human myeloid cell line was used in this study. K562 express the
anti-apoptotic oncogene BCR-ABL and high levels of phosphorylated
STAT5. Cell lines were grown in RPMI 1640 (Gibco Grand Island, NY,
USA) containing 10% FCS (Gibco), 100 U/mL penicillin (Gibco), 100
µg/mL streptomycin (Gibco), and 2 mM l-glutamine (Sigma Chemical Co.,
St. Louis, MO) in a 5% CO2 atmosphere at 37 °C.
Keywords: STAT5 inhibitors • Pimozide • BCR/ABL expressing
leukemia • Apoptosis • Antiproliferation
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8
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Entry for the Table of Contents
Insert graphic for Table of Contents here.
New derivatives of pimozide were obtained with potent growth inhibitor and apoptotic activity in K562 BCR-ABL expressing leukemia
cell lines. Moreover, detection of overexpressed phosphorylated STAT5, with fluorochrome-conjugated monoclonal antibody anti-p-
STAT5 resulted in strong inhibition when derivatives are used in 10-15 µM concentration, instead of pimozide, active at 30 µM.
9
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