New Inhibitor Targeting Signal Transducer and Activator of Transcription 5 (STAT5) Signaling in Myeloid Leukemias

Article abstract of DOI:10.1021/acs.jmedchem.7b00369

Signal transducers and activators of transcription 5 (STAT5s) are crucial effectors of tyrosine kinase oncogenes in myeloid leukemias. Inhibition of STAT5 would contribute to reducing the survival of leukemic cells and also tackling their chemoresistance. In a first screening experiment, we identified hit 13 as able to inhibit STAT5 phosphorylation and leukemic cell growth. The synthesis of 18 analogues of 13 allowed us to identify one compound, 17f, as having the most potent antileukemic effect. 17f inhibited the growth of acute and chronic myeloid leukemia cells and the phosphorylation and transcriptional activity of STAT5. Importantly, 17f had minimal effects on bone marrow stromal cells that play vital functions in the microenvironment of hematopoietic and leukemic cells. We also demonstrated that 17f inhibits STAT5 but not STAT3, AKT, or Erk1/2 phosphorylation. These results suggest that 17f might be a new lead molecule targeting STAT5 signaling in myeloid leukemias.

Full text of DOI:10.1021/acs.jmedchem.7b00369

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Article
A New Inhibitor Targeting Signal Transducer and Activator
of Transcription 5 (STAT5) Signaling in Myeloid Leukemias
Ludovic Juen, Marie Brachet-Botineau, Cécile Parmenon, Jérôme Bourgeais,
Olivier Hérault, Fabrice Gouilleux, Marie-Claude Viaud-Massuard, and Gildas Prié
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00369 • Publication Date (Web): 27 Jun 2017
Downloaded from http://pubs.acs.org on June 28, 2017
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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A New Inhibitor Targeting Signal Transducer and
Activator of Transcription 5 (STAT5) Signaling in
Myeloid Leukemias
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†,#
§,‡,#
†
§,‡
Ludovic Juen, Marie Brachet-Botineau,
Cécile Parmenon, Jérôme Bourgeais, Olivier
§
,‡
,‡
†
,†
Hérault, Fabrice Gouilleux,* Marie-Claude Viaud-Massuard and Gildas Prié*
†
Equipe IMT "Innovation Moléculaire et Thérapeutique" - GICC UMR 7292 CNRS - Université
de Tours - Labex SYNORG - Faculté de Pharmacie - 31 avenue Monge - 37200 Tours - France.
‡
Equipe LNOx "Niche leucémique & métabolisme oxydatif" - GICC UMR 7292 CNRS -
Université de Tours - Faculté de Médecine - Bâtiment Dutrochet - 10bis boulevard Tonnellé -
3
7032 Tours - France.
§
CHRU de Tours, Service d’Hématologie Biologique, 2 boulevard Tonnellé - 37044 Tours -
France.
KEYWORDS: STAT5, signal transduction, myeloid leukemias, pharmacological inhibitors, 4,4-
dimethyl-1,2,3,4-tetrahydroquinoline.
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ABSTRACT:
Signal Transducer and Activator of Transcription 5 (STAT5) are crucial effectors of tyrosine
kinase oncogenes in myeloid leukemias. Inhibition of STAT5 would contribute to reducing the
survival of leukemic cells and also tackling their chemoresistance. In a first screening
experiment, we identified hit 13 as able to inhibit STAT5 phosphorylation and leukemic cell
growth. The synthesis of 18 analogs of 13 allowed us to identify one compound, 17f, as having
the most potent antileukemic effect. 17f inhibited the growth of acute and chronic myeloid
leukemia cells and phosphorylation and transcriptional activity of STAT5. Importantly, 17f had
minimal effects on bone marrow stromal cells that play vital functions in the microenvironment
of hematopoietic and leukemic cells. We also demonstrated that 17f inhibits STAT5 but not
STAT3, AKT or Erk1/2 phosphorylation. These results suggest that 17f might be a new lead
molecule targeting STAT5 signaling in myeloid leukemias.
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INTRODUCTION
The Signal Transducer and Activator of Transcription factors 5A and B are two closely related
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STAT family members that play a major role in normal and leukemic haematopoiesis. Persistent
activation of these transcription factors is frequently found in leukemias as a consequence of
deregulated tyrosine kinase activity. Phosphorylation of STAT5 is triggered by tyrosine kinase
oncogenes (TKO) such as Fms-like receptor tyrosine kinase 3 with internal tandem duplications
D816V
V617F
(Flt3-ITD), Kit
, Bcr-Abl and JAK2
which have been characterized in various myeloid
2–6
malignancies. STAT5 is a crucial effector of Bcr-Abl, the major transforming agent in chronic
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,8
myeloid leukemia (CML). The development of Bcr-Abl tyrosine kinase inhibitors (TKI) such
as Imatinib Mesylate (IM) has revolutionized the treatment of CML. Despite this success story,
IM is not curative due to its inability to completely eradicate leukemic stem cells (LSC) that are
responsible for initiation and relapse of CML. Moreover, the occurrence of Bcr-Abl mutations
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during progression of the disease promotes the resistance of CML cells to IM treatment.
Therefore, there is an urgent need for alternative therapeutic strategies to cure CML. In this
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regard, STAT5 fulfils all criteria of a major drug target in CML. High STAT5 expression levels
were shown not only to enhance IM resistance in CML cells but also to trigger Bcr-Abl
mutations by inducing the production of reactive oxygen species (ROS) responsible for DNA
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,11,12
damage.
Moreover, STAT5 was shown to play a key role in the maintenance of CML stem
13
cells that are resistant to chemotherapy. Several approaches have been used to target STAT5 in
leukemias. Among them, cell-based screening with small molecules libraries of already approved
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drugs allowed the identification of pimozide as a potential STAT5 inhibitor in CML cells.
Although pimozide inhibits the transcriptional activity of STAT5, the use of high concentrations
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suggests that effects of this molecule might be indirect. More recently, salicylic acid-based
molecules targeting the SH2 domain required for dimerization and transcriptional activity of
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STAT5 were shown to inhibit the growth of CML cells. One of these inhibitors binds STAT5
protein in a nanomolar range; furthermore it induces apoptosis and inhibits tyrosine
phosphorylation of STAT5 in a micromolar range. A last approach is to target STAT5 activity
through the activation of peroxisome proliferator-activated receptor gamma (PPARγ). Indeed the
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existence of a cross talk between protein PPAR and STAT5 has been discussed.
For
instance, antidiabetic drugs such as glitazones that are PPARγ agonists were shown to have
antileukemic activity. Activation of PPARγ by glitazones decreases expression and/or
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phosphorylation of STAT5 in CML cells.
Importantly, Prost et al. showed that combining
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pioglitazone with IM triggers apoptosis of CML stem cells. The mechanism by which these
leukemic stem cells are killed in response to this drug combination is not yet clear. Moreover,
several reports indicated that antileukemic effects of glitazones might be dependent on “off-
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target” mechanisms.
We previously described the synthesis and pharmacological evaluation of 24 PPARα/γ ligands
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targeting Type 2 diabetes.
All molecules synthesized during the project shared a diversely
substituted 4,4-dimethyl-1,2,3,4-tetrahydroquinoline scaffold, linked to an aryl or an heteroaryl
ring by an ethoxy chain. This series was comprised of PPARα/γ agonist, antagonist and inactive
compounds. Inhibitory effects of some representative molecules of the series were evaluated on
the growth of a Bcr-Abl expressing cell line (KU812 cells) and STAT5 activity.
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Figure 1. Hit compound 13
All molecules showed none to moderate activity on KU812 cell proliferation with a maximum
inhibition of 55% at 10 µM for compound 13 (Figure 1). Interestingly the latter was the only
compound to significantly inhibit STAT5 phosphorylation (supp. info). We also showed that 13
was inactive on PPARα/γ suggesting that inhibition of cell growth and STAT5 phosphorylation
by this molecule occurs through a PPARγ independent pathway. Therefore, pharmacological
exploitation of this “off-target” effect is of interest for the development of new antileukemic
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molecules that are devoid of side effects associated with PPARγ activation.
A collection of 18 derivatives of hit compound 13 was synthesized and a structure/activity
analysis was performed to determine their growth inhibitory effects on various myeloid leukemia
cells. Experiments were conducted in CML cell lines (KU812 and K562) and acute myelogenous
leukemia (AML) cell lines in which STAT5 is constitutively active (KG1a and MV-4-11).
Among these molecules, 17f was found to be the most active in blocking STAT5
phosphorylation and leukemic cell growth. We also demonstrated that 17f selectively inhibits
STAT5 phosphorylation but not STAT3, Erk1/2 and Akt signaling which also play a critical role
in myeloid leukemia cell proliferation and survival. Interestingly, 17f was almost inactive in
inhibiting the growth of bone marrow stromal cells that are major components of the
hematopoietic and leukemic microenvironment.
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CHEMISTRY
Hit 13 scaffold was composed of indole and 4,4-dimethyl-1,2,3,4-tetrahydroquinoline rings,
linked by an ethoxy chain attached by carbon C-5 of the indole and nitrogen N-1 of the
tetrahydroquinoline (Scheme 1). This scaffold was substituted by an acetic acid group on the
nitrogen N-1 of the indole and by a cyclohexylcarbonyl group on the carbon C-6 of the
tetrahydroquinoline.
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Scheme 1. Previous synthetic route of hit compound 13
First convergent synthesis of hit 13 followed the synthetic route developed in our previous
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PPAR project. Acylation of intermediate CP25i with cyclohexyl carbonyl chloride in the
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presence of graphite followed by Williamson alkylation with ethyl 2-(5-hydroxy-1H-indol-1-
yl)acetate and further saponification led to 13. This method had some drawbacks including poor
reproducibility of the acylation step and low yield of the alkylation step (<15%). Moreover, this
procedure set the central scaffold substituents from the start, making new pharmacomodulations
on indole and tetrahydroquinoline rings inconvenient. In order to study SAR in our current
STAT5 project, we needed to readily synthetize a series of hit 13 analogs. To do this, we
designed an alternative divergent synthesis. Starting from key intermediates 6a and 6b obtained
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in satisfying yields, the central scaffold was further modified to produce the desired series
(Scheme 2).
a
Scheme 2. Synthesis of key intermediates 6a and 6b
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a
Reagent and conditions: (a) 3,3-dimethyl-acryloyl chloride, dry pyridine, 0 °C to rt, 5 h; (b)
AlCl , dry dichloromethane, 0 °C to rt, 1 h; (c) BH .THF, dry toluene, 0 °C to reflux, 12 h; (d)
NaH, BnBr, dry DMF, 0 °C to rt, 2 h; (e) CuI, 3,4,7,8-tetramethyl-1,10-phenanthroline, Cs CO ,
dry ethylene glycol, 130 °C, 72 h; (f) 1) IBX, DCE, 80 °C, 2 h; 2) Filtration; 3) 4,4-dimethyl-
1,2,3,4-tetrahydroquinoline or amine 3, NaBH(AcO) , rt, 3 h.
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Synthesis of the key intermediates 6a and 6b started with protection of the N-1 position of 5-
bromoindole by a benzyl group. The protected indole 4 was then etherified by Ullmann-type
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coupling. To achieve this, several ligands were tested including 8-hydroxyquinoline and 1,10-
phenanthroline, however best results were obtained with electron rich 3,4,7,8-tetramethyl-1,10-
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phenanthroline to afford 5 with a 61% yield. In order to carry out reductive amination, various
reagents to oxidize alcohol 5 were assessed, including PCC, oxalyl chloride (according to Swern
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oxidation), Dess-Martin periodinane and 2-iodoxybenzoic acid (IBX). As the aldehyde
intermediate was unstable, IBX oxidation was chosen as it allowed an easy purification by
simple filtration (indeed, excess of IBX and reduced by-product 2-iodosobenzoic acid (IBA) are
insoluble at rt in DCE). Moreover, the filtrate was used in the next step without further treatment,
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since DCE is one of the best reaction solvent for reductive aminations. 4,4-Dimethyl-1,2,3,4-
tetrahydroquinoline or 6-bromo-4,4-dimethyl-1,2,3,4-tetrahydroquinoline 3, obtained from
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aniline or 4-bromoaniline, were then introduced in the presence of NaBH(AcO) to form 6a
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and 6b respectively with 77% and 64% yields over two steps (Scheme 2).
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Scheme 3. Synthesis of hit compound 13 and its analogs
a
Reagent and conditions: (a) CuCN, DMF, reflux, 6 h; (b) 1) cyclohexylmagnesium chloride,
CuBr, dry THF, reflux, 2 h; 2) H O, H SO 15%; (c) t-BuOK, DMSO, O , rt, 3 h; (d) ethylene
glycol, p-toluenesulfonic acid monohydrate, toluene, reflux, 6 h; (e) HCl 0.1 M, THF, rt, 5 h; (f)
NaH, ethyl bromoacetate or methyl iodide, dry DMF, 0 °C to rt, 2 h; (g) LiOH, THF/H O, 0 °C
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Conversion of aryl bromide 6b to the corresponding nitrile 7, followed by nucleophilic
addition of cyclohexylmagnesium chloride catalyzed by CuBr and subsequent hydrolysis led to
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the ketone 8 (Scheme 3). Attempts of benzyl deprotection of 8 in the presence of potassium
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tert-butoxide under air (1 atm) led to hydroxylation at the α-carbon of the ketone.
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this, several procedures were assessed to protect ketone 8 as the ketal 9,
among which, the
classical method using ethylene glycol and catalytic p-toluenesulfonic acid monohydrate with a
Dean Stark trap gave the best result, nevertheless with a low 37% yield. The long reaction time
required at high temperature led to partial decomposition of the starting ketone. However after
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undergo benzyl deprotection to afford 10 with 83% yield. Deprotection of the latter afforded
ketone 11, whereas N-alkylation with ethyl bromoacetate followed by saponification led to 13.
Deprotection of 6a, conducted with potassium tert-butoxide under air (1 atm) in DMSO afforded
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4 with 78% yield. N-alkylation of 14 by treatment with sodium hydride and methyl iodide led
to analogs 15a. Same reaction using ethyl bromoacetate, followed by saponification led to acid
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5c with 31% yield over two steps.
a
Scheme 4. Synthesis of analogs 16, 17a-i and 18
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Reagent and conditions: (a) t-BuOK, DMSO, O , rt, 3 h; (b) corresponding boronic acid,
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K CO , PdCl (PPh ) , DMF/H O, 90 °C, 1-14 h; (c) bis(pinacolato)diboron, KOAc, PdCl (dppf),
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dry dioxane, 80 °C, 21 h; (d) 2-chloropyridine, K PO , PdCl (dppf), dioxane/H O, 100 °C, 4 h.
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Various aryl and heteroaryl groups were introduced on the tetrahydroquinoline ring of 16 using
Suzuki cross-coupling, affording 17a-i analogs with moderate to good yields (41-83%) (Scheme
4
, Table 1). Boronic acids used for the Suzuki coupling were commercially available except 1-
methyl-4-[[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]sulfonyl]piperazine which was
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synthesized in 2 steps from 4-bromobenzene-1-sulfonyl chloride.
Table 1. Structure of analogs 17a-i
Entry
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Compound
R
Time (h)
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Yield (%)
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Challenging introduction of the 2-pyridinyl group directly from aryl bromide 16 using MIDA
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boronic ester
led to only traces of product and degradation of the starting material. As a
result, we decided to convert aryl bromide 16 to the corresponding boronic acid pinacol ester,
which was directly engaged in a Suzuki coupling with 2-chloropyridine to yield compound 18.
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RESULT AND DISCUSSION
Hit compound 13, an inactive analog of PPAR agonists that have been developed in our lab,
was found to inhibit STAT5 phosphorylation in KU812 cells 72 h after treatment at 10 µM.
However, proliferation and viability assays showed only a moderate cytotoxic efficiency on
KU812 and K562 cells, with EC of 0.2 mM and 8 mM respectively.
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As acid and ketone substituents of hit 13 were chosen in accordance with PPAR activity, we
studied their relevance toward the inhibition of STAT5 activity and cytotoxic efficiency on CML
cell lines, while keeping hit 13 central scaffold. In this purpose and in order to study structure
activity relationships, pharmacomodulations on indole and tetrahydroquinoline rings were
conducted.
Initial screenings were carried out to determine the growth inhibitory property of all newly
synthesized compounds. KU812 and K562 cells were treated with 10 µM of each compound or
DMSO as control and the kinetics of cell growth were determined by MTT and trypan blue dye
exclusion assays (data for compounds with antiproliferative activity lower than 13 are shown in
supp. info).
On one hand, deletion of the cyclohexyl carbonyl group on the tetrahydroquinoline led to
compound 15c, which lost antiproliferative efficiency after 48 h on K562 cells and had no effect
on KU812 cells. On the other hand, removal of the acetic acid group led to compound 11, which
showed the same activity than 13 on K562 cells but lost it at 72 h. On KU812 cells,
antiproliferative potency of 11 could be suspected at 24 h but was lost after 48 h. Finally, we
removed both terminal substituents to yield 14, which showed a better improvement of cytotoxic
efficiency on K562 and KU812 cell lines. The activity was better than 13 and interestingly,
growth inhibition was observed only after 24 h treatment (Figure 2).
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Figure 2. (A) Growth kinetics of KU812 cells treated with 10 µM of each compound as
indicated or DMSO as control; (B) Growth kinetics of K562 cells treated with each compound or
DMSO as control as in (A) (n = 3 in triplicates, data are mean ± SEM).
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To investigate further the importance of the free indole, intermediates 6a and 15b, respectively
N-alkylated with a benzyl group and an acetate group, were both evaluated and found to be
inactive. To rule out steric hindrance effects and to validate the importance of the free indole as
hydrogen bond donor, 14 was N-alkylated with the minimally hindering methyl group to yield
compound 15a. As the latter showed no activity, the free nitrogen was identified as essential for
the efficiency.
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From this point, the free nitrogen was retained on the indole while the 6-position of the
tetrahydroquinoline was investigated further. To obtain a convergent and facile route for
pharmacomodulations, introduction of a bromine atom on this position yielded 16, found to have
a similar activity than 13 (Figure 3).
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Figure 3. (A) Growth kinetics of KU812 cells treated with 10 µM of each compound as
indicated or DMSO as control; (B) Growth kinetics of K562 cells treated with each compound or
DMSO as control as in (A) (n = 3 in triplicates, data are mean ± SEM).
Starting from 16, a small collection of aryl and heteroaryl substituents were introduced i.e.
phenyl, 4-fluorophenyl, 3-nitrophenyl, 3-thiophenyl, 2-furanyl, 3-pyridinyl, 6-chloropyridin-3-yl,
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-pyrininyl, and 4-((4-methylpiperazin-1-yl)sulfonyl)phenyl which led to compounds 17a-i
Figure 3).
7f was identified as the most antiproliferative molecule in KU812 and K562 cells while 17g
(
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was as efficient as 17f in the growth inhibition of KU812 cells. Compared to hit 13, their
activities were clearly increased. 17c with a 3-nitrophenyl group possessed the same electronic
distribution than 17f, however was inactive. Moreover, 17a with the phenyl group retained the
same volume than 17f but was inactive. We assumed the hydrogen bond acceptor character of
the nitrogen of the pyridine of 17f was required for the activity. By changing the position of the
nitrogen of the pyridine, from 3 to 4, the antiproliferative efficiency of 17h, still potent, was
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slightly reduced compared to 17f and 17g. Changing the position of the nitrogen of the pyridine
from 3 to 2, leading to 18, resulted in a decrease of antiproliferative activity.
Five compounds (14, 16, 17f-h) with similar or better antiproliferative activities than hit 13,
were selected from this initial screening and tested for their capacity to induce apoptosis in
KU812 cells. We observed that 17f, 17g and 17h significantly increased the number of apoptotic
cells as measured by AnnexinV/7AAD staining (Figure 4).
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Figure 4. Representative flow cytometry histogram of KU812 cells cultured for 48 h with
indicated compounds (10 µM) or control DMSO. Cells were stained with FITC-Annexin V and
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-AAD and the percentages of apoptotic cells were then evaluated by flow cytometry (n = 3 in
triplicates, data are mean ± SEM).
EC values for these molecules were also calculated and clearly indicated that 17f was the
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experiments with 17f in K562 cells were also realized by AnnexinV/7AAD staining (supp. info).
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Table 2. EC values of selected compounds for K562 and KU812 cells
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Cells were treated with concentrations ranging from 100 nM to 50 µM for 48 h. Cell viability
was then determined by MTT assays and EC values were calculated using Origin® software (n
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Interestingly, this inhibitory effect was not blocked by GW9662, an antagonist of PPARγ
indicating that PPARγ activation was not involved in 17f-mediated inhibition of cell growth
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(supp. info). We then addressed whether the biological effect of this molecule correlated with
the suppression of STAT5 activity in KU812 and K562 cells. We first evaluated impact of 17f on
STAT5A and STAT5B gene expression in KU812 cells by qRT-PCR experiments. Results showed
that 17f had no effect on STAT5A and STAT5B mRNA levels in KU812 cells treated for 15 h
with this molecule (Figure 5A). We then analyzed the phosphorylation and expression of STAT5
in KU812 and K562 cells exposed to 17f (10 µM) for 12 h. We observed that 17f inhibited
STAT5 phosphorylation in both leukemia cell lines (Figure 5B).
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Figure 5. (A) qRT-PCR analysis of STAT5A and STAT5B transcripts in KU812 cells treated or
not with 17f (10 μM) or DMSO as control for 15 h. Results are presented as the fold changes in
gene expression in 17f-treated cells normalized to internal control genes (GAPDH and ACTB)
and relative to control cells (normalized to 1) (n = 3 in triplicates, data are mean ± SEM). (B)
Protein extracts from KU812 and K562 cells treated with 17f (+) or DMSO (-) for 12 h were
analyzed by Western blotting to detect P-Y-STAT5 and STAT5 protein expression (n = 2).
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We then addressed whether 17f inhibited STAT5 transcriptional activity in KU812 cells. qRT-
PCR experiments were conducted to determine effects of 17f on STAT5-dependent expression of
target genes such as PIM1, CYCLIN D1, C-MYC and MCL-1. We found that 17f was able to
downregulate PIM1 and C-MYC gene expression but had weak effects on CYCLIN D1 and MCL-
1 gene expression (Figure 6A). We also compared expression of Pim1, Cyclin D1 and Mcl-1
proteins by Western blot analysis and found that expression of Pim1 was strongly inhibited by
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7f in KU812 cells while Cyclin D1 and Mcl-1 protein levels were less affected by this
compound (figure 6B). Cyclin D1 and Mcl-1 expressions are also known to be regulated by
distinct signaling pathways in Bcr-Abl-expressing cells through transcriptional and post-
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transcriptional mechanisms.
We then decided to determine whether 17f might directly
interfere with the transcriptional activation of a reporter gene driven by a STAT5-specific
promoter. KU812 cells were transfected with a construct containing six tandem copies of the
STAT5 response element in front of the minimal TK promoter fused to the luciferase reporter
gene (6x(STAT5)-TK-luc). As control, cells were also transfected with a TK-luciferase vector
without STAT5 response elements (TK-luc). Luciferase activity was then determined 48 hours
post-transfection in cells treated or not with 17f. As expected, constitutive STAT5 activity
induced by Bcr-Abl increased luciferase activity in cells transfected with the STAT5-dependent
promoter construct compared to cells transfected with the control TK-luc vector (figure 6C). This
enhanced luciferase activity was selectively reduced after 17f treatment. Collectively, these data
strongly suggested that 17f inhibits DNA binding and transcriptional activity of STAT5 in CML
cells.
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Figure 6. (A) qRT-PCR analysis of PIM1, CYCLIN D1, C-MYC and MCL-1 expression in
KU812 cells treated with 17f (10 μM) or DMSO (control) for 15 h. Results are presented as the
fold changes in PIM1, CYCLIN D1, C-MYC and MCL-1 gene expression in 17f cells normalized
to internal control genes (GAPDH and ACTB) and relative to untreated cells (normalized to 1) (n
= 3 in triplicates, data are mean ± SEM, *p < 0.05, ***p < 0.001). (B) Expression of Pim1,
Cyclin D1 and Mcl-1 proteins in KU812 cells treated with 10 µM 17f (+) or DMSO (-) for 24
hours. (C) KU812 cells transfected with a 6x(STAT5)-TK-luciferase reporter construct or a
control TK-luciferase vector were treated with 17f (10 µM) or DMSO (control) for 48 hours.
Luciferase activities were next determined as described in material and methods. Luciferase
activity (arbitrary units) in the histogram represents the relative luminescence units (rlu)
values/mg of proteins (n = 3 in triplicates, data are mean ± SEM, ***p < 0.001).
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STAT5 is also constitutively phosphorylated in AML cells and particularly in myeloid
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leukemia cells expressing the FLT3-ITD oncogene. To exclude the possibility that 17f-mediated
inhibition of STAT5 and cell growth is a peculiarity of cells expressing Bcr-Abl, we evaluated
effects of 17f on STAT5 phosphorylation in two AML cell lines. MV-4-11 cells expressing
FLT3-ITD and KG1a, an immature AML cell line, were exposed to 17f for 12 h and
phosphorylation of STAT5 was then analyzed by Western blot. Results showed that 17f strongly
reduced STAT5 phosphorylation in both cell lines (Figure 7A). We then explored whether 17f
also inhibited the growth of these leukemic cells. MV-4-11 and KG1a cells were treated with 17f
(10 µM) and growth kinetics were determined by MTT and trypan blue dye exclusion assays.
We found that 17f suppressed the growth and viability of these AML cells (Figure 7B).
Importantly, EC values (KG1a: 2.64 µM; MV-4-11: 3.55 µM) showed that 17f was even more
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active in AML cells than in CML cells (Table 3). Interestingly enough, the EC value obtained
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µM), a STAT5-SH2 inhibitor that selectively binds to STAT5 in a nanomolar range. In
addition, we also found that 17f induces apoptosis of KG1a and MV-4-11 cells indicating that
17f inhibits STAT5-dependent cell survival (figure 7C).
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Figure 7. (A) Protein extracts from MV-4-11 and KG1a cells treated with 17f (+) or DMSO (-)
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for 12 h were analyzed by Western blotting to detect P-Y
-STAT5 and STAT5 protein
expression. (B) Growth kinetics of KG1a (left) and MV-4-11 (right) cells cultured in absence
DMSO) or presence of 17f (10 µM) were determined by MTT assays (n = 3 in triplicates, data
(
are mean ± SEM). (C) Representative flow cytometry histogram of KG1a and MV-4-11 cells
cultured for 48 h with 17f (10µM) or DMSO (control). Cells were stained with FITC-Annexin V
and 7-AAD and the percentages of apoptotic cells were then evaluated by flow cytometry (n = 3
in triplicates, data are mean ± SEM, *p < 0.05).
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was then determined by MTT assays and EC values were calculated using Origin® software (n
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To determine the selectivity of 17f on STAT5 phosphorylation, we analyzed effects of this
molecule on STAT3, Akt and Erk1/2 signaling. KG1a, MV-4-11 and KU812 cells were treated
with 17f (10 µM) for 24 h and phosphorylation levels of STAT3, Akt and Erk1/2 proteins were
analyzed by Western blot analysis (Figure 8). Phosphorylation levels were also determined by
band intensity quantification (supp. info). While phosphorylation of STAT5 was clearly inhibited
by 17f in all cell lines, effects on STAT3, Akt and Erk1/2 phosphorylation after 24 h treatment
were almost negligible. Collectively, our findings suggest that the growth-suppressive effect of
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Figure 8. Protein extracts from KG1a, MV-4-11 and KU812 cells treated with 17f (+) or
DMSO (-) for 24 h were analyzed by Western blotting to detect the phosphorylated forms of
STAT5, STAT3, Akt and Erk1/2 and the total forms of these proteins (n = 2). Actin expression
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Bone marrow stromal and mesenchymal stem cells (MSC) are major cellular components of
the leukemic and hematopoietic microenvironment and play a vital role in the survival and
chemoresistance of leukemic cells. We then analyzed effects of 17f on freshly isolated MSC
from human bone marrow. We also included in these experiments HS-27a cells, a human bone
marrow stromal cell line that shares similar biological properties with MSC. HS-27a cells
constitutively express phosphorylated STAT3 but not phosphorylated STAT5 proteins while
phosphorylation of STAT5 and STAT3 are not detectable in primary MSC (data not shown).
Results showed that 17f does not significantly or weakly inhibits HS-27a and MSC cell growth
with EC values 10 to 20 fold higher than those required to inhibit leukemic cell growth (Figure
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A, Table 4). Interestingly, we also found that 17f does not affect the phosphorylation of STAT3
in HS-27a cells (Figure 9B). These findings indicated that 17f selectively inhibits myeloid
leukemia cell growth while sparing normal bone marrow stromal cells of the leukemic niche.
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Figure 9. (A) Growth kinetics of HS-27a cells (left) and MSC (right) cultured in absence
(DMSO) or presence of 17f (10 µM) were determined by MTT assays (n = 3 in triplicates, data
are mean ± SEM). (B) Extracts from HS-27a cells treated with 17f (+) or DMSO (-) were
analyzed by Western blotting to detect the phosphorylation and expression of STAT3.
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Table 4. EC values of 17f for HS-27a and MSC cells (3 donors)
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Cells were treated with concentrations ranging from 1 µM to 100 µM for 48 h. Cell viability
was then determined by MTT assays and EC values were calculated using Origin® software (n
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3 in triplicates, data are mean ± SEM) (supp. info).
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Journal of Medicinal Chemistry
Page 28 of 85
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In summary, among the 18 analogs of hit 13 that have been synthesized, compound 17f was
found to be the most potent inhibitor of AML and CML cell growth with encouraging EC values.
Interestingly, the antileukemic effect of 17f was even more pronounced in AML cells than in
CML cells. Key structural features for 17f-mediated antiproliferative effect are the free nitrogen
of the indole ring along with the C-6 tetrahydroquinoline 3-pyridinyl substitution.
We also demonstrated that 17f inhibits STAT5 phosphorylation in AML and CML and
STAT5-dependent transcriptional activity. Importantly, we found that 17f has no or weak impact
on major cellular components of the leukemic niche and does not affect STAT3, Akt or Erk1/2
signaling in leukemia cells suggesting that 17f selectively targets myeloid leukemia cells
addicted to STAT5 signaling. These data also indicate that 17f or derivatives might be of
potential interest to analyze their impact on leukemia cells in contact with their stromal
microenvironment.
In the aim of improving cell growth and STAT5 phosphorylation inhibition, in AML and CML
cells, alternative modifications on the central scaffold of 17f are being investigated. For instance,
modification of the linker and substitution of indole by its isosteres indazole and 7-azaindole are
currently undertaken.
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Page 29 of 85
Journal of Medicinal Chemistry
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EXPERIMENTAL SECTION
CHEMISTRY
Material and methods
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All commercial materials were used without further purification. For anhydrous and inert
reactions, the glassware was heated with a heat gun while several vacuum-dry argon cycles were
performed. The thin layer chromatography (TLC) studies were performed using commercial pre-
coated aluminium sheets silica gel (60 Å, F ) marketed by Merck and revealed under UV 254
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and 365 nm lighting. The purifications by chromatography on silica gel columns were carried out
on an ISCO purification unit, Combi Flash RF 75 PSI, with Redisep flash silica gel columns (60
Å, 230-400 mesh, grade 9385). NMR spectra were measured on a Bruker Ultrashield 300
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spectrometer, 300 MHz ( H), 282 MHz ( F) and 75 MHz ( C) in deuterated chloroform
(
(
(
CDCl ) or dimethylsulfoxide (DMSO-d ). Chemical shifts are given in parts per million (ppm)
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δ relative to residual solvent peak for H and C). The notations used are: δ: chemical shift
ppm); s: singlet; d: doublet; dd: doublet of doublet; t: triplet; m: multiplet; br: broad signal; J:
coupling constant (Hz). The HRMS was performed by the mass spectrometry service on a Q-
Exactive spectrometer from Thermo Scientific using the electrospray ionization (ESI) technique.
Melting points (mp) were taken in open capillaries on a Büchi melting point apparatus Model B-
5
40. If necessary, the purity was determined by high performance liquid chromatography
(HPLC). Purity of all final compounds was 95% or higher. HPLC analyses were carried out with
a LaChrom Elite system [Hitachi L-2130 (pump) and L-2400 (UV-detector)] using 254 nm UV
for detection. The column was a Phenomenex Synergi™ C-12, 4 µm particle size (100 mm × 4.6
mm), supported by Phenomenex Security Guard cartridge kit C12 (4.0 mm × 3.0 mm); elution
was performed with 0.2% (by volume) of TFA in water (solvent A), and acetonitrile (solvent B);
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