Identification and development of non-cytotoxic cell death modulators: Impact of sartans and derivatives on PPARγ activation and on growth of imatinib-resistant chronic myelogenous leukemia cells

Article abstract of DOI:10.1016/j.ejmech.2020.112258

4’-((2-Propyl-1H-benzo[d]imidazol-1-yl)methyl)-[1,1′-biphenyl]-2-carboxylic acid derived from telmisartan was identified as lead for the design of cell death modulators. In this study, we evaluated the efficacy of telmisartan itself and other sartans in combination with imatinib against K562-resistant cells. The findings were directly used to further optimize the lead structure. Telmisartan and candesartan cilexetil represented the most effective sartans, thus the influence of carboxyl/methyl carboxylate groups at positions 7 (compounds 6, 7) or 4 (compounds 12–14) at the benzimidazole core was studied. Additionally, according to the results of a former structure-activity study, telmisartan was transformed to the related amide (1). Telmisartan amide 1, as well as the esters 6 and 12 markedly sensitized the resistant CML cells to imatinib treatment. Correlation with their potency to activate PPARγ is not given. Candesartan cilexetil, telmisartan and 1 showed the profile of partial agonists at PPARγ with EC₅₀ values of 4.2, 4.3 and 9.1 μM, respectively, while 6 and 12 caused only marginal intrinsic activation at 10 μM (A_max = 22% and 13%). However, the repression of the STAT5 phosphorylation relates with the possibility to sensitize K562-resistant CML cells to imatinib treatment. It is worth mentioning that all compounds were per se non-cytotoxic at relevant concentrations.

Full text of DOI:10.1016/j.ejmech.2020.112258

European Journal of Medicinal Chemistry 195 (2020) 112258
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Identification and development of non-cytotoxic cell death
modulators: Impact of sartans and derivatives on PPAR activation
g
and on growth of imatinib-resistant chronic myelogenous leukemia
cells
,
,
, *
Anna M. Schoepf a, Stefan Salcher b c, Petra Obexer b d, Ronald Gust a
a Department of Pharmaceutical Chemistry, Institute of Pharmacy, CMBI - Center for Molecular Biosciences Innsbruck, University of Innsbruck, CCB -
Centrum for Chemistry and Biomedicine, Innrain 80-82, 6020, Innsbruck, Austria
b Tyrolean Cancer Research Institute, Innrain 66, 6020, Innsbruck, Austria
c Department of Internal Medicine V, Medical University Innsbruck, Anichstraße 35, 6020, Innsbruck, Austria
d Department of Pediatrics II, Medical University Innsbruck, Innrain 66, 6020, Innsbruck, Austria
a r t i c l e i n f o
a b s t r a c t
4’-((2-Propyl-1H-benzo[d]imidazol-1-yl)methyl)-[1,10-biphenyl]-2-carboxylic acid derived from telmi-
sartan was identified as lead for the design of cell death modulators. In this study, we evaluated the
efficacy of telmisartan itself and other sartans in combination with imatinib against K562-resistant cells.
The findings were directly used to further optimize the lead structure. Telmisartan and candesartan
cilexetil represented the most effective sartans, thus the influence of carboxyl/methyl carboxylate groups
at positions 7 (compounds 6, 7) or 4 (compounds 12e14) at the benzimidazole core was studied.
Additionally, according to the results of a former structure-activity study, telmisartan was transformed to
the related amide (1). Telmisartan amide 1, as well as the esters 6 and 12 markedly sensitized the
Article history:
Received 4 December 2019
Received in revised form
12 February 2020
Accepted 18 March 2020
Available online 26 March 2020
Keywords:
Sartans
resistant CML cells to imatinib treatment. Correlation with their potency to activate PPAR
Candesartan cilexetil, telmisartan and 1 showed the profile of partial agonists at PPAR with EC50 values
of 4.2, 4.3 and 9.1 M, respectively, while 6 and 12 caused only marginal intrinsic activation at 10 mM
g is not given.
Peroxisome proliferator-activated receptor
activity
Structure-activity relationship
Chronic myeloid leukemia
Imatinib resistance
Cell death modulators
g
g
m
(Amax ¼ 22% and 13%). However, the repression of the STAT5 phosphorylation relates with the possibility
to sensitize K562-resistant CML cells to imatinib treatment. It is worth mentioning that all compounds
were per se non-cytotoxic at relevant concentrations.
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
imatinib and other TKIs to completely eradicate leukemic cells
[3,4]. Quiescent hematopoietic stem cells are highly insensitive to
The treatment of cancer was revolutionized since tyrosine ki-
nase inhibitors (TKIs) entered the market [1]. Chronic myeloid
imatinib and remain viable in CML patients [5]. Besides primary
resistance to imatinib, acquired resistance can be observed after
prolonged treatment due to loss of response [2]. Per definition
sustained deep molecular response (MR) is characterized by nearly
undetectable BCR-ABL1 transcripts (PCR analyses). Only some 10%
of patients, who were treated with imatinib and reached deep MR,
are expected to remain in stable treatment-free remission, while
the majority experiences a relapse [6]. Hence, the cure of CML
cannot be achieved and research focuses on the overcome of ima-
tinib resistance as well as the induction of sustained deep MR.
The angiotensin II type 1 receptor blocker (ARB) and partial
leukemia (CML),
a hematopoietic malignancy, was the first
neoplastic disease with rationally designed therapy. In 95% of all
patients the BCR-ABL1 fusion protein is overexpressed, which is
associated with increased tyrosine kinase activity. Imatinib, the
most intensely studied TKI, acts as a competitive inhibitor at the
ATP binding site of the BCR-ABL1 kinase. This leads to death of
target cells along with mild adverse effects [1,2].
A major limitation of the therapy, however, is the failure of
peroxisome proliferator-activated receptor gamma (PPARg) agonist
telmisartan was already studied as anti-cancer agent. It revealed
promising results towards TKI-resistant non-small cell lung cancer
* Corresponding author.
E-mail address: ronald.gust@uibk.ac.at (R. Gust).
https://doi.org/10.1016/j.ejmech.2020.112258
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
A.M. Schoepf et al. / European Journal of Medicinal Chemistry 195 (2020) 112258
Abbreviations
PE
petroleum ether
THF
HRMS
Amax
SD
SEM
ctr
tetrahydrofuran
TKI
CML
MR
ARB
PPAR
STAT5
tyrosine kinase inhibitor
chronic myeloid leukemia
molecular response
angiotensin II type 1 receptor blocker
peroxisome proliferator-activated receptor gamma
signal transducer and activator of transcription 5
phosphorylated signal transducer and activator of
transcription 5
high-resolution mass spectrometry
intrinsic activation
standard deviation
standard error of the mean
control
g
pio.
telmi.
pioglitazone
telmisartan
pSTAT5
cande. cil. candesartan cilexetil
PI
propidium iodide
structure-activity relationship
methanol
cande.
irbe.
lo.
candesartan
irbesartan
losartan
SAR
MeOH
EG
ethylene glycol
olme. med. olmesartan medoxomil
PyBOP
benzotriazol-1-yl-oxytripyrrolidino-phosphonium
hexafluorophosphate
hydroxybenzotriazole
N,N-diisopropylethylamine
ethyl acetate
olme.
DMEM
FCS
RPMI
PBS
olmesartan
Dulbecco’s Modified Eagle Medium
fetal calf serum
Roswell Park Memorial Institute
phosphate-buffered saline
HOBt
DIPEA
EA
EtOH
ethanol
[7], adult T-cell leukemia/lymphoma [8], and cholangiocarcinoma
[9]. Prost et al. [10] assessed the activation of PPAR and the sub-
sequent inhibition of STAT5 as main principle to overcome resis-
tance. Therefore, we evaluated 4-substituted 4’-((2-propyl-1H-
benzo[d]imidazol-1-yl)methyl)-[1,10-biphenyl]-2-carboxylic acids,
increased cell death modulating effects (see results). Therefore,
telmisartan has been transformed to an amide (1) following our
previous studies. Additionally, a carboxyl group was introduced at
position 4 or 7 (analogously to candesartan) at the benzimidazole
core of the 4’-((2-propyl-1H-benzo[d]imidazol-1-yl)methyl)-[1,10-
biphenyl]-2-carboxylic acid, which represents our lead structure
(Fig. 1). The compounds were investigated in relation to their
respective methyl ester. STAT5 expression was determined to get an
insight into the mode of action. Cytotoxicity was investigated in the
COS-7 cell line by a modified MTT assay and further in the HS-5 cell
line by PI FACS analyses.
g
initially designed as telmisartan-derived partial PPAR
g agonists
[11], against imatinib-resistant cells. After transformation to their
2-carboxamide derivatives, they represented strong sensitizers to
imatinib therapy in the resistant K562 CML cell line. The respective
carboxylic acids were ineffective [12]. These findings draw our
attention to other ARBs as possible cell death modulators.
Oura et al. [13] already evaluated the sartans telmisartan, irbe-
sartan, losartan, and valsartan regarding their inhibitory and anti-
proliferative impact on different cancer cell lines, such as hepato-
cellular carcinoma cells. As a result, growth inhibition, induction of
2. Results and discussion
2.1. Synthesis
~
cell cycle arrest, and apoptosis were observed. Additionally, Inigo
et al. [14] showed that losartan inhibits cell growth and induces
apoptosis in K562 CML cells. These studies, however, did not pro-
vide an adequate treatment option for cancer therapy as the con-
centrations to achieve an efficacy were relatively high. Besides, the
necessity to successfully circumvent the occurring drug resistance
was not considered yet. Therefore, we evaluated selected sartans
The synthesis of 1 (see Scheme 1) was performed analogously to
Shan et al. [15], reacting telmisartan with the phosphonium salt
based coupling reagent PyBOP, the base DIPEA and NH4Cl.
The syntheses of compounds 6, 7 and 12e14, depicted in
Schemes 2 and 3, are based on a previously described procedure
[12].
2-Amino-3-nitrobenzoic acid (2) was esterified to 3 with H2SO4
in anhydrous MeOH [16]. The formation of the respective butyric
anilide (4) was realized by heating 3 with butyric anhydride and
concentrated HCl. N-Alkylation with methyl 4’-(bromomethyl)-
[1,10-biphenyl]-2-carboxylate in the presence of NaH gave com-
pound 5. In an one-pot reaction the reduction of the nitro group as
well as ring closure were achieved by applying the reducing agent
SnCl2 and subsequent heating in EtOH. Ester cleavage of 6 with 2 N
KOH in THF/EtOH finally yielded 7 [17]. This reaction pathway
exclusively resulted in 7-substituted benzimidazole derivatives
(Scheme 2).
The 4-substituted compounds were synthesized as depicted in
Scheme 3. Compound 2 was directly reduced with SnCl2 to the 1,2-
phenylenediamine derivative 8. Then, 8 was transformed to the bis-
butyric anilide 9 with butyric anhydride. Heating with 4 N HCl
yielded the respective benzimidazole (10), which was esterified
with H2SO4 in anhydrous MeOH. N-Alkylation of 11 with methyl 4’-
(bromomethyl)-[1,10-biphenyl]-2-carboxylate resulted in an
isomeric mixture of 4- and 7-substituted benzimidazole
for PPARg activation and eradication of imatinib-resistant cells. The
sartans should cover a wide range of structural features. Telmi-
sartan and candesartan (Fig. 1) comprise an N-([1,10-biphenyl]-4-
ylmethyl)-1H-benzo[d]imidazole core, while irbesartan, losartan,
and olmesartan consist of an N-([1,10-biphenyl]-4-ylmethyl)-1H-
imidazole substructure (Fig. 1).
Compared to telmisartan, the 2-COOH group at the biphenyl
moiety is bioisosterically replaced by a tetrazole ring in all other
drugs. Furthermore, the heterocyclic cores of olmesartan and can-
desartan are substituted with a carboxylic group, respectively,
which is either attached directly to the imidazole (olmesartan) or to
the benzimidazole (candesartan). Both drugs are clinically applied
as ester-prodrugs, which were also included in the present study.
The ability of this broad spectrum of compounds to activate PPAR
g
in a transactivation assay and to induce cell death in K562-
imatinib-resistant cells by PI (propidium iodide) FACS analyses
was assessed according to previous findings [10,12].
The results of candesartan and its prodrug candesartan cilexetil
revealed the high relevance of modified carboxyl groups for

A.M. Schoepf et al. / European Journal of Medicinal Chemistry 195 (2020) 112258
3
Fig. 1. Structures of the ARBs candesartan cilexetil (prodrug), candesartan, irbesartan, olmesartan medoxomil (prodrug), olmesartan, losartan, telmisartan and the derived lead.
derivatives, from which 12 was separated by column chromatog-
raphy. The 7-substituted isomer was not obtained in sufficient
amount and purity within this synthesis pathway, since the 4-
substituted isomer was preferably formed (ratio of about 4:1).
The subsequent hydrolysis of 12 with 2 N KOH in THF/EtOH yielded
the mono-methyl ester derivative 13. Only upon the use of stronger
alkaline conditions and increased temperature, the bis-carboxylic
acid 14 was formed.
COS-7 cells were transiently transfected with the plasmids
pGal5-TK-pGL3, pGal4-hPPAR DEF, pRenilla-CMV, and subse-
quently stimulated with the sartans or the synthesized compounds,
respectively. Telmisartan and the full PPAR agonist pioglitazone
served as references. The maximum activation of pioglitazone was
set to 100% and its half-maximal effective concentration
g
g
(EC50 ¼ 1.6
m
M) was calculated from the concentration-activation
curve (Fig. 2, concentrations 0.05e20
m
M). The EC50 ¼ 4.3
mM and
Compounds 6, 7 and 12e14 were characterized applying 1H and
13C NMR spectroscopy as well as high-resolution mass spectrom-
etry (HRMS). HPLC was performed to assess purity. The synthesis
protocols and the analytic data of intermediates as well as target
compounds are submitted as supplementary data.
Amax ¼ 60.5% of telmisartan are in agreement with our previous
findings [12].
Candesartan cilexetil showed the same profile as telmisartan
(Fig. 2, A). It achieved an Amax of 59.8% at 10
mM and an EC50 value of
4.2 M (Table 1). At the highest concentration used, the maximum
m
effect was attained in both cases, which is lower than that of pio-
glitazone. Interestingly, candesartan itself did not cause receptor
activation (Fig. 2, A) [21].
2.2. Biological activity
Irbesartan activated PPARg to only 8.1% at 10 mM. Its potency
Referring to previous findings [10,18e20], the ability of the
cannot be calculated, as the concentration-activation curve did not
reach the maximum (Fig. 2, B). Losartan, olmesartan medoxomil,
and olmesartan (Fig. 2, C) were even completely inactive.
compounds to activate PPAR
imatinib resistance was studied.
g and their potency to circumvent
The structure-activity relationship (SAR) clearly indicated that a
high PPARg binding requires a benzimidazole core as realized in
2.2.1. Activation of PPARg in a transactivation assay
Telmisartan, candesartan cilexetil, candesartan, irbesartan, los-
artan, olmesartan medoxomil, and olmesartan as well as the syn-
thesized compounds 1, 6, 7, and 12e14 were evaluated in a dual-
luciferase reporter assay to assess their potency (EC50 values) and
telmisartan. Regarding the benzimidazole derivative candesartan,
only the ester-prodrug candesartan cilexetil yielded good receptor
binding, while hydrolysis of the ester to candesartan strongly
decreased the affinity. To further evaluate this effect, a COOH group
was introduced in position 7 (6, 7) or 4 (12e14) at the benzimid-
azole of the lead and the results were compared with that of the
intrinsic activity (Amax) towards PPAR
Table 1 refer to a concentration of 10
g
m
. The results (Amax) listed in
M, according to the highest
one used in the cytotoxicity assay with K562-resistant cells.

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A.M. Schoepf et al. / European Journal of Medicinal Chemistry 195 (2020) 112258
Fig. 2. Concentration-dependent activation of PPARg. COS-7 cells were transiently transfected with the plasmids pGal5-TK-pGL3, pGal4-hPPARgDEF, pRenilla-CMV, and subse-
quently stimulated with the two references pioglitazone ( ) and telmisartan ( ), as well as the ARBs, candesartan cilexetil ( ), candesartan ( ), irbesartan ( ), losartan ( ),
olmesartan medoxomil ( ), olmesartan ( ), and the compounds 1 ( ), 6 ( ), 12 ( ), 13 ( ), 7 ( ), 14 ( ), respectively. The data represent the mean SD of ꢀ3 independent
experiments with threefold determination.

A.M. Schoepf et al. / European Journal of Medicinal Chemistry 195 (2020) 112258
5
Scheme 1. Derivatization of telmisartan to the amide 1. Reagents and conditions: PyBOP, NH4Cl, DIPEA, anhydrous DMF, 0 ꢁC to rt.
Scheme 2. Synthesis of compounds 6 and 7. Reagents and conditions: (a) H2SO4, anhydrous MeOH, 65 ꢁC; (b) butyric anhydride, concentrated HCl, 120 ꢁC; (c) methyl 4’-(bro-
momethyl)-[1,1’-biphenyl]-2-carboxylate, NaH, anhydrous DMF, rt; (d) SnCl2 ꢂ 2 H2O, EtOH, 80 ꢁC; (e) 2 N KOH, THF, EtOH, 80 ꢁC.
Scheme 3. Synthesis of compounds 12, 13, and 14. Reagents and conditions: (a) SnCl2 ꢂ 2 H2O, HCl, EtOH, rt; (b) butyric anhydride, concentrated HCl, 120 ꢁC; (c) 4 N HCl, 100 ꢁC; (d)
H2SO4, anhydrous MeOH, 65 ꢁC; (e) methyl 4’-(bromomethyl)-[1,1’-biphenyl]-2-carboxylate, NaH, anhydrous DMF, rt; (f) 2 N KOH, THF, EtOH, 80 ꢁC; (g) 6 N KOH, EG, 160 ꢁC.
respective methyl esters.
benzimidazole, was inactive too. Esterification of the 2-COOH as
well marginally increased the efficiency (2-CO2CH3 (13) 4.5% < 2/4-
CO2CH3 (12) 13%).
Derivatization of telmisartan to the amide 1 also lowers the
receptor activation and potency (Fig. 2, D). From the curve in Fig. 2,
In relation to the lead, a strong decrease of intrinsic activity was
observed for 6, 7 and 12e14 (Fig. 2, E-F). The 7-CO2CH3 group at the
benzimidazole together with the 2-CO2CH3 group reduced Amax at
10
mM from 58.5% (lead) to 22% (6) and even to 0% after hydrolysis
of the esters (7). Compound 14, which bears a 4-COOH at the
D an Amax of 41.9% and an EC50 value of 9.1 mM were calculated.

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A.M. Schoepf et al. / European Journal of Medicinal Chemistry 195 (2020) 112258
Table 1
combination therapy of imatinib with the full PPAR
glitazone. Following this approach, we investigated the cell death
modulating effects of the partial PPAR agonists telmisartan, tel-
misartan amide 1, and candesartan cilexetil as well as the less/non-
agonistic ARBs candesartan, irbesartan, losartan, olmesartan
medoxomil, olmesartan and the carboxyl/methyl carboxylate
g agonist pio-
Intrinsic activity (Amax) and potency (EC50) determined in a transactivation assay
with PPAR
g.
g
compound
Amax at 10
m
M [%]a,c
EC50 [m
M]b,c
candesartan cilexetil
candesartan
irbesartan
losartan
olmesartan medoxomil
olmesartan
1
6
7
12
13
14
59.8 6.5
0.9 0.1
8.1 0.5
1.6 0.3
0.7 0.1
0.5 0.1
41.9 2.3
22.0 2.2
0
13.0 0.3
4.5 1.2
0
100
60.5 4.1
4.2 0.7
n.d.
n.d.
n.d.
n.d.
n.d.
9.1 0.8
n.d.
n.d.
n.d.
bearing
4‘-((2-propyl-1H-benzo[d]imidazol-1-yl)methyl)-[1,1’-
biphenyl]-2-carboxylic acid derivatives 6, 7, 12e14 in K562-
resistant CML cells.
To prove imatinib resistance, the K562 and K562-resistant cell
lines were comparatively examined upon treatment with
increasing concentrations of imatinib [12]. In the K562 cell line
1 mM of imatinib induced 46% cell death, whereas in the K562-
n.d.
n.d.
1.6 0.3
4.3 0.5
resistant cells only 17% dead cells were detected by PI FACS ana-
lyses. Thus, imatinib resistance in the latter cell line was confirmed
(for detailed information see SI, Fig. S19).
pioglitazone
telmisartan
a
All tested compounds alone (10
ble to the vehicle (DMSO) treated control (Fig. 4, A: ctr.; cell death
rate: 7%) or the imatinib control (1 M; Fig. 4, B: ctr.; cell death rate:
mM) revealed effects compara-
Amax indicates the response of the compounds related to the maximum acti-
M).
EC50 values were calculated by means of the concentration-activation curves
(Fig. 2).
vation of pioglitazone (100%, 10
m
b
m
17%) in K562-resistant cells. The same is true, if imatinib, cande-
sartan, olmesartan medoxomil, olmesartan, losartan, 7, or 14 were
combined with imatinib. The compounds could not sensitize the
cells to imatinib treatment. In contrast, imatinib administered
together with irbesartan and candesartan cilexetil caused 31% and
42% cell death, together with telmisartan or its amide 1 even 63%
and 89%, respectively (Fig. 4, B). Also 6 and 12 were highly active
sensitizers. This effect is nearly independent on the position of the
carboxylic ester at the benzimidazole core (cell death rate: 83%
each). However, ester cleavage reduced the effects. Compounds 7
and 14 were completely inactive (cell death rate: 12%). The mono
ester 13 was of medium activity (cell death rate: 56%).
c
Data represent the mean
SD of ꢀ3 independent experiments (n.d., not
determinable).
2.2.2. Modified MTT assay
Pioglitazone, the selected sartans, and all synthesized target
compounds were evaluated in a modified MTTassay regarding their
cytotoxic potential in COS-7 cells. The assay is based on the for-
mation of purple colored formazan derivatives indicating cell
viability. This process is driven by NAD(P)H-dependent cellular
dehydrogenases. Their enzyme activity correlates with functional
mitochondria, which reflects the number of viable cells [22]. The
metabolic activity of actively growing cells markedly decreases
upon exposure to cytotoxic molecules.
The potency of telmisartan, 1, 6, 12, and 13 as cell death mod-
ulators for imatinib was further concentration-dependently proven
in K562-resistant CML cells. The cells were incubated for 72 h with
Neither pioglitazone and the sartans, nor the compounds 1, 6, 7,
12e14 affected the metabolic activity of COS-7 cells at a concen-
tration of 10
1
m
M of imatinib in combination with increasing concentrations
(1e10 M) of the selected compounds (Fig. 5).
1, 6, and 12 showed nearly equal concentration-activity curves.
At 2.5 M they mediated a cell death rate of about 50e60% for
imatinib. Compound 13 showed the same activity profile at reduced
concentrations. Only at 5 and 10 M, the effects were somewhat
lower (54% and 56% dead cells, respectively). In contrast, telmi-
sartan was less active up to a concentration of 2.5 M and reached
m
M (used concentration in the cell death assays). At a
m
concentration of 20
m
M, which was applied within the trans-
activation assay, only 12 caused a slightly stronger decrease in
metabolic activity of about 20% (Fig. 3). Nevertheless, all com-
pounds can be stated as non-cytotoxic.
m
m
2.2.3. Induction of cell death
m
Prost et al. [10] described to overcome imatinib resistance by the
Fig. 3. Metabolic activity of COS-7 cells treated with 20
mM of either vehicle (ctr., DMSO), pioglitazone (pio.), telmisartan (telmi.), candesartan cilexetil (cande. cil.), candesartan
(cande.), irbesartan (irbe.), losartan (lo.), olmesartan medoxomil (olme. med.), olmesartan (olme.), 1, 6, 7, or 12e14. The mean values þSD of ꢀ3 independent experiments are shown.

A.M. Schoepf et al. / European Journal of Medicinal Chemistry 195 (2020) 112258
7
Fig. 4. Induction of cell death in K562-resistant cells, treated with telmisartan (telmi.), candesartan cilexetil (cande. cil.), candesartan (cande.), irbesartan (irbe.), losartan (lo.),
olmesartan medoxomil (olme. med.), olmesartan (olme.), and the compounds 1, 6, 7, 12e14 either without (A) or in combination with 1 M of imatinib (B). Ctr. represents the
m
vehicle treated control without (A) or with imatinib (1
m
M, B). Dead cells were detected by PI FACS analyses. Data represent the mean þSEM of ꢀ4 independent experiments.
the efficiency of 13 at 5 and 10
mM.
None of the compounds repressed the STAT5 expression in
K562-resistant cells treated with imatinib (Fig. 7; see also Fig. S20).
However, imatinib combined with the compounds 1, 6, or 12 and to
2.2.4. Cytotoxicity assay
a small extent also telmisartan or 13 repressed the phosphorylation
status of STAT5 (Fig. 7).
2.2.4.1. PI FACS analyses. The human non-malignant cell line HS-5
was used to exclude a general cytotoxicity of all compounds. PI
FACS analyses were applied to detect cell death. None of the com-
pounds showed cytotoxic properties up to a concentration of
In case of 7 and 14, neither an influence on STAT5 nor on pSTAT5
regulation was observed. These results are in good agreement with
those of the PI FACS analyses. It is very likely that regulation of the
STAT5 expression is part of the mode of action regarding the
investigated cell death modulators. The inhibition of STAT5, which
is a guardian protein of quiescence and stemness of leukemia cells,
by the investigated compounds might lead to eradication of the
stem cell pool. However, participation of other targets or pathways
cannot be excluded yet.
10
mM. Due to control-like effects, they can be stated as non-
cytotoxic (Fig. 6).
2.2.5. Immunoblot analyses
As a possible mode of action repression of the STAT5 activity is
discussed. Inhibition of STAT5 was reported to be essential for TKI-
induced cell death in resistant CML cells [23]. It is strongly involved
in the development of imatinib resistance and the maintenance of
insensitive stem cells [10,24]. Therefore, telmisartan as well as all
synthesized compounds were investigated to inhibit the expression
or the phosphorylation status of STAT5 at tyrosine 694/699, which
is a hallmark for activated STAT5, by immunoblot analyses.
2.3. Discussion
Prost et al. [10] stated to overcome imatinib resistance by co-
administration with the full PPAR
g
agonist pioglitazone.

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A.M. Schoepf et al. / European Journal of Medicinal Chemistry 195 (2020) 112258
Fig. 5. PI FACS analyses of K562-resistant cells treated with 1
mM of imatinib in combination with increasing concentrations (1e10 mM) of telmisartan (telmi.), 1, 6, 12, or 13,
respectively. The mean values þSEM of ꢀ4 independent experiments are shown.
Fig. 6. Induction of cell death in HS-5 cells, treated with DMSO (ctr.), telmisartan (telmi.), candesartan cilexetil (cande. cil.), candesartan (cande.), irbesartan (irbe.), losartan (lo.),
olmesartan medoxomil (olme. med.), olmesartan (olme.), 1, 6, 7, and 12e14, respectively. The mean values þSEM of ꢀ3 independent experiments are shown.
Therefore, we focused our attention on the potency of the partial
PPAR agonist telmisartan and related sartans (candesartan cilex-
etil and candesartan, irbesartan, losartan, olmesartan medoxomil
as well as olmesartan). The purpose of this study was on the one
acid derivatives 7 and 14 were completely inactive.
g
All compounds were non-cytotoxic against COS-7 and HS-5 cells
as well as K562 imatinib-resistant CML cells. When the compounds
were tested together with imatinib on the K562-resistant cell line,
telmisartan, candesartan cilexetil, irbesartan, 1, 6, 12, and 13 abro-
hand the investigation of their PPARg activating potential and on
the other hand the determination of their cell death modulating
effects. This work continues a recently published SAR study with 4-
substituted derivatives of the lead 4’-((2-propyl-1H-benzo[d]imi-
dazol-1-yl)methyl)-[1,10-biphenyl]-2-carboxylic acid with the
gated the resistance. Especially in presence of 1, 6, and 12 (10 mM)
imatinib caused maximum cytotoxic effects, even higher than in
sensitive K562 cells (89% vs 46%).
These findings clearly demonstrate that CO2CH3 or CONH2
groups (especially a derivatization of the 2-COOH group) are posi-
tive structural modifications, whereas COOH groups either at po-
sition 4 or 7 of the benzimidazole reduce the cell death modulating
objective to establish a correlation between PPAR
circumvention of imatinib resistance in K562-resistant cells.
Telmisartan and candesartan cilexetil were partial PPAR
g activation and
g
ago-
nists, all other sartans were more or less inactive. Also candesartan
did not activate the receptor. This led to the question if a carboxyl
group at the benzimidazole (either at position 4 or 7) generally
decreases the binding. Accordingly, the lead was modified (com-
pounds 7 and 14) and the related methyl esters (6, 12, 13) were as
well included in our investigations. Additionally, telmisartan was
derivatized to an amide (1) following the positive results described
effects. A correlation with the PPARg activation does not exist.
To get an insight into the mode of action, the influence on the
STAT5/pSTAT5 expression in K562-resistant cells in the presence of
imatinib was studied. The ability of telmisartan, 1, 6, 12, and 13 to
repress the STAT5 phosphorylation correlates with their potency to
circumvent imatinib resistance in these cells.
In conclusion, no clear connection of overcoming imatinib
in Ref. [12]. While 1 represented a partial PPAR
g
agonist, 6, 12, and
resistance and activating PPAR
g was observed. Moreover, the
13 only marginally stimulated the gene expression. The carboxylic
compounds themselves did not exhibit cytotoxic effects.

A.M. Schoepf et al. / European Journal of Medicinal Chemistry 195 (2020) 112258
9
Fig. 7. STAT5 and pSTAT5 expression in K562-resistant cells treated with 1
mM of imatinib alone (ctr.) or together with 10 mM of telmisartan (telmi.), 1, 6, 12, 13, 7, and 14 for 6 h (A).
GAPDH served as loading control. Densitometric analyses (B) were performed with the ImageJ 1.48 software. Control cells were set as 100%.
Nevertheless, the proven repression of the STAT5 phosphorylation
upon combination of imatinib with telmisartan, 1, 6, 12, or 13 is
related to the mode of action. This is in agreement with the theory
of Rousselot et al. [18] and Schafranek et al. [23], stating the erad-
ication of the CML stem cell pool by silencing its trigger STAT5.
Consequently, an increased deep MR to imatinib is assumed.
4. Experimental section
4.1. Chemistry
4.1.1. General materials and methods
Compound 2, all reagents as well as other chemicals were pur-
chased from Sigma-Aldrich, TCI Chemicals, or Alfa Aesar and used
without further purification. Telmisartan, irbesartan, losartan,
candesartan cilexetil, and olmesartan medoxomil were isolated
from tablets that were received from the pharmacy. The ester-
prodrugs candesartan cilexetil and olmesartan medoxomil were
hydrolyzed by established procedures and their analytical data was
in accordance with the literature. The solvents dichloromethane
(DCM), ethanol (EtOH), ethyl acetate (EA), and methanol (MeOH)
were distilled before use, whereas petroleum ether (PE) was used
directly. Ethylene glycol (EG), N,N-dimethylformamide (DMF), N,N-
diisopropylethylamine (DIPEA), and tetrahydrofuran (THF) were
purchased in appropriate quality. Column chromatography was
performed applying classic standard procedures and/or medium
pressure liquid chromatography. For the latter an Isolera One 3.0
Flash purification system (Biotage) was employed. Silica gel 60
3. Conclusions
The sartans are a very interesting class of drugs regarding their
potential to overcome imatinib resistance. The representatives
telmisartan and the prodrug candesartan cilexetil, but not cande-
sartan itself, were efficient sensitizers to imatinib treatment in
K562-resistant CML cells, whereas irbesartan, losartan, olmesartan
medoxomil, and olmesartan were inactive.
The design of 6, 12, and 13 bearing substructures of each tel-
misartan and candesartan yielded highly active imatinib sensi-
tizers. Their potency to treat imatinib-resistant CML cells was
completely abrogated upon hydrolysis of the methyl esters to the
carboxylic acids (7 and 14). However, the derivatization of telmi-
sartan to its carboxamide derivative 1 resulted in a very effective
compound.
(particle size 40e63
mm, 230e240 mesh) served as stationary
phase. Thin layer chromatography (TLC) was carried out using
Polygram SIL G/UV254 polyester foils covered with a 0.2 mm layer of
silica gel and a fluorescence indicator (Macherey-Nagel). TLC plates
were visualized with UV-light at 254 nm or 366 nm. Nuclear
magnetic resonance spectra (NMR) were recorded using a 200 MHz
Gemini (now Agilent), a 400 MHz Avance 4 Neo (Bruker), or a
600 MHz Avance II (Bruker) spectrometer. Deuterated dimethyl
sulfoxide (DMSO‑d6) or acetone (acetone-d6) were used as solvents
Compound 1 showed moderate intrinsic activity towards PPARg,
while 6, 7, 12e14, candesartan, irbesartan, losartan, olmesartan
medoxomil, or olmesartan were inactive. Although 1, telmisartan,
and candesartan cilexetil acted by partial agonism at PPARg, the
mechanism of action is presumably dependent on the phosphory-
lation status of STAT5. Inhibition of this pro-survival transcription
factor was proven for telmisartan and the developed imatinib
sensitizers. Notably, none of the tested compounds exhibited
cytotoxic properties.
Summing up, this study demonstrated that derivatives of tel-
misartan or candesartan are capable cell death modulators to
overcome imatinib resistance. Regarding the optimization of the
lead structure, a benzimidazole scaffold definitely is favored over
an imidazole core and compounds bearing more than one car-
boxylic acid reveal no sensitizing effect.
(both from Eurisotop or Alfa Aesar). Chemical shifts (d) are given in
parts per million (ppm) and were referenced to the solvent peak or
to tetramethylsilane (TMS) as an internal standard. Coupling con-
stants (J) are reported in Hertz (Hz). Signals are described as fol-
lows: s ¼ singlet, br s ¼ broad singlet, d ¼ doublet, dd ¼ doublet of
doublets, t ¼ triplet, m ¼ multiplet. HRMS was conducted with an
Orbitrap Elite system (Thermo Fisher Scientific). For the determi-
nation of purity, high performance liquid chromatography (HPLC)
was applied, using a Shimadzu Nexera-i LC 2040C 3D with the
autosampler SIL 20A HT, the column oven CTO-10AS VP, the
degasser DGU-20A, the detector SPD-M20A, and the pumps LC-
Based on the established application of telmisartan and cande-
sartan in clinical use, our findings might be beneficial to attain deep
MR in CML patients.

10
A.M. Schoepf et al. / European Journal of Medicinal Chemistry 195 (2020) 112258
20AD. As stationary phase an RP-18 column (dimension
m particle size, Knauer) was used and the chro-
matograms were analyzed with the program Lab-Solutions 5.86
(Shimadzu). Purity of ꢀ95% was assured for all compounds.
mixture was stirred for 30 min. Then, methyl 4’-(bromomethyl)-
[1,10-biphenyl]-2-carboxylate (0.15 g, 0.5 mmol) was added and
stirring was continued for 12 h at room temperature. The reaction
mixture was treated with ice water to double the volume and it was
neutralized using 1 N HCl. After extracting with EA (3 ꢂ), the
organic layers were combined, washed with brine, dried over
anhydrous Na2SO4 and filtered. The solvent was removed under
reduced pressure. Purification was performed by column chroma-
tography (PE/EA, 7:3). Colorless solid, yield: 56%. 1H NMR
125 ꢂ 4 mm, 5
m
4.1.2. Synthesis of 4’-((1,70-dimethyl-20-propyl-1H,30H-[2,50-
bibenzo[d]imidazol]-30-yl)methyl)-[1,10-biphenyl]-2-carboxamide
(1)
Telmisartan (0.15 g, 0.3 mmol) was dissolved in anhydrous DMF
(1 ml) and cooled in an ice bath. PyBOP (0.17 g, 0.3 mmol) dissolved
in anhydrous DMF (1 ml) was added and the reaction mixture was
stirred at 0 ꢁC for 5 min. DIPEA (0.15 g, 1.2 mmol) was added, fol-
lowed by an NH4Cl (0.02 g, 0.3 mmol) suspension in anhydrous
DMF (1 ml). It was stirred for another 30 min at 0 ꢁC. The mixture
was allowed to attain room temperature and stirring was continued
for 16 h. Then, the solution was diluted with water to double the
volume and it was basified to a pH of 9 with 1 N NaOH solution. The
mixture was extracted with EA (3x) to remove HOBt and unreacted
carboxylic acid. The organic phases were combined, washed with
brine, dried over anhydrous Na2SO4 and filtered. EA was removed
under reduced pressure. The resulting product was purified by
column chromatography (DCM/MeOH, 95:5) and flash column
chromatography with gradient elution (PE/EA, 7:3 to 0:1). Colorless
(400 MHz, acetone-d6): d7.81e7.74 (m, 2H, H3, H5’’), 7.67 (dd, 1H,
3J ¼8.1 Hz, 4J ¼1.1 Hz, H7’’), 7.62e7.56 (m,1H, H5), 7.50e7.44 (m, 1H,
H4), 7.40 (dd, 1H, 3J ¼7.7 Hz, 4J ¼1.3 Hz, H6), 7.31e7.23 (m, 3H, H2’,
H6’, H6’’), 7.17 (d, 2H, 3J ¼8.1 Hz, H3’, H5’), 5.64 (s, 2H, NCH2), 3.93 (s,
3H, 4’’-CO2CH3), 3.56 (s, 3H, 2-CO2CH3), 2.95 (t, 2H, 3J ¼7.6 Hz,
CH2CH2CH3), 1.92e1.82 (m, 2H, CH2CH2CH3), 1.03 (t, 3H, 3J ¼7.4 Hz,
CH2CH2CH3). 13C NMR (101 MHz, acetone-d6): 169.3, 167.6, 157.9,
142.9, 142.3, 141.6, 137.8, 136.9, 132.3, 132.2, 131.4, 130.4, 129.7,
128.3, 127.1, 124.8, 122.2, 121.8, 115.1, 52.1, 51.9, 47.2, 21.9, 14.3.
HRMS: m/zcalculated for C27H26N2O4 [MþH]þ: 443.1965, found:
443.1977.
4.1.5. General procedure for the hydrolysis of the bis-methyl esters
The respective bis-methyl ester was dissolved in THF and EtOH
(each ~1e2 ml/mmol). 2 N KOH (~3e4 ml/mmol) was added and
the reaction mixture was heated to 70 ꢁC for 16 h. The solvents were
removed under reduced pressure and the residue was suspended in
water. 6 N HCl was used to obtain a pH of 1. The mixture was
extracted with DCM (3 ꢂ), the organic layers were combined,
washed with brine, dried over anhydrous Na2SO4 and filtered. The
solvent was removed under reduced pressure. Purification was
performed by column chromatography (DCM/MeOH, 9:1).
solid, yield: 17%. 1H NMR (400 MHz, DMSO‑d6):
d7.77e7.73 (m, 1H,
AreH), 7.66e7.56 (m, 3H, AreH, NH), 7.50e7.34 (m, 6H, AreH, NH),
7.32 (dd, 1H, 3J ¼7.6 Hz, 4J ¼1.3 Hz, AreH), 7.30e7.19 (m, 3H, AreH),
7.17 (d, 2H, 3J ¼8.3 Hz, AreH), 5.61 (s, 2H, NCH2), 3.82 (s, 3H, NCH3),
2.93 (t, 2H, 3J¼ 7.6 Hz, CH2CH2CH3), 2.63 (s, 3H, CH3), 1.89e1.77 (m,
2H, CH2CH2CH3), 1.01 (t, 3H, 3J¼ 7.4 Hz, CH2CH2CH3). 13C NMR
(101 MHz, DMSO‑d6): d171.0, 156.2, 154.0, 142.7, 142.5, 139.7, 138.3,
137.4, 136.7, 135.95, 134.7, 129.8, 129.2, 128.7, 128.2, 127.5, 127.1,
126.4, 123.3, 123.2, 122.0, 121.8, 118.7, 110.4, 109.3, 46.0, 31.8, 28.7,
20.7, 16.5, 13.9. HRMS: m/zcalculated for C33H31N5O [MþH]þ:
514.2601, found: 514.2653.
4.1.5.1. 1-((20-Carboxy-[1,10-biphenyl]-4-yl)methyl)-2-propyl-1H-
benzo[d]imidazole-7-carboxylic acid (7). From 6 (0.12 g, 0.3 mmol),
in THF and EtOH (each 0.5 ml) with 2 N KOH (1 ml). Colorless solid,
4.1.3. Synthesis of methyl 1-((2’-(methoxycarbonyl)-[1,10-biphenyl]-
4-yl)methyl)-2-propyl-1H-benzo[d]imidazole-7-carboxylate (6)
After dissolving 5 (2.56 g, 5.2 mmol) in EtOH (70 ml), SnCl2ꢂ 2
H2O (5.89 g, 26.1 mmol) was added and the mixture was heated to
80 ꢁC for 14 h. Water was added to double the volume and a 1 N
NaOH solution was used to obtain a pH of 9. The formed precipitate
was sucked off and washed with EtOH. The filtrate was evaporated,
the residue was suspended in water and extracted with DCM (3 ꢂ).
The organic layers were combined, washed with brine, dried over
anhydrous Na2SO4 and filtered. The solvent was removed under
reduced pressure. Purification was performed by column chroma-
tography (PE/EA, 1:1). Colorless solid, yield: 29%. 1H NMR
yield: 36%. 1H NMR (400 MHz, DMSO‑d6):
d 12.94 (br s, 2H,
2 ꢂ COOH), 7.86 (dd, 1H, 3J ¼8.0 Hz, 4J ¼1.2 Hz, H4’’), 7.69 (dd, 1H,
3J ¼7.6 Hz, 4J ¼1.4 Hz, H3), 7.64 (dd, 1H, 3J ¼7.6 Hz, 4J ¼1.2 Hz, H6’’),
7.56e7.49 (m, 1H, H5), 7.45e7.39 (m, 1H, H4), 7.33e7.19 (m, 4H, H6,
H2’, H6’, H5’’), 6.88 (d, 2H, 3J ¼8.2 Hz, H3’, H5’), 5.90 (s, 2H, NCH2),
2.87 (t, 2H, 3J ¼7.5 Hz, CH2CH2CH3), 1.88e1.75 (m, 2H, CH2CH2CH3),
0.97 (t, 3H, 3J ¼7.4 Hz, CH2CH2CH3). 13C NMR (101 MHz, DMSO‑d6):
169.5,167.7,157.4,140.4,139.7,136.5,132.4,132.2,130.8,130.5,129.1,
128.6, 127.3, 125.7, 124.9, 122.8, 121.0, 117.9, 54.9, 47.6, 28.9, 20.1,
13.8. HRMS: m/z calculated for C25H22N2O4 [MþH]þ: 415.1652,
found: 415.1714.
(400 MHz, DMSO‑d6):
d
7.87 (dd, 1H, 3J ¼8.0 Hz, 4J ¼1.2 Hz, H4’’),
4.1.5.2. 1-((2’-(Methoxycarbonyl)-[1,10-biphenyl]-4-yl)methyl)-2-
propyl-1H-benzo[d]imidazole-4-carboxylic acid (13). From 12
(0.07 g, 0.2 mmol), in THF and EtOH (each 0.2 ml) with 2 N KOH
(0.6 ml). Colorless solid, yield: 75%. 1H NMR (400 MHz, DMSO‑d6):
7.70 (dd, 1H, 3J ¼7.7 Hz, 4J ¼1.5 Hz, H3), 7.62e7.55 (m, 1H, H5), 7.52
(dd, 1H, 3J ¼7.6 Hz, 4J ¼1.2 Hz, H6’’), 7.50e7.42 (m, 1H, H4), 7.36 (dd,
1H, 3J ¼7.8 Hz, 4J ¼1.3 Hz, H6), 7.29e7.22 (m, 1H, H5’’), 7.18 (d, 2H,
3J ¼8.2 Hz, H2’, H6’), 6.85 (d, 2H, 3J ¼8.0 Hz, H3’, H5’), 5.72 (s, 2H,
NCH2), 3.69 (s, 3H, 7’’-CO2CH3), 3.51 (s, 3H, 2-CO2CH3), 2.90 (t, 2H,
3J ¼7.5 Hz, CH2CH2CH3), 1.90e1.75 (m, 2H, CH2CH2CH3), 0.99 (t, 3H,
3J ¼7.4 Hz, CH2CH2CH3). 13C NMR (101 MHz, DMSO‑d6): 168.5,166.4,
157.7, 144.2, 140.7, 139.4, 136.3, 132.1, 131.5, 130.8, 130.4, 129.3,
128.3, 127.5, 125.7, 124.2, 123.1, 120.9, 116.7, 52.3, 51.7, 47.7, 28.9,
20.2,13.8. HRMS: m/z calculated for C27H26N2O4 [MþH]þ: 443.1965,
found: 443.2003.
d
7.88 (d, 1H, 3J ¼7.4 Hz, H5’’), 7.70 (d, 1H, 3J ¼7.4 Hz, H3), 7.59e7.52
(m, 2H, H5, H7’’), 7.49e7.42 (m, 1H, H4), 7.34 (d, 1H, 3J ¼7.6 Hz, H6),
7.26e7.17 (m, 3H, H2’, H6’, H6’’), 7.07 (d, 2H, 3J ¼7.9 Hz, H3’, H5’),
5.59 (s, 2H, NCH2), 3.45 (s, 3H, CO2CH3), 3.15 (t, 2H, 3J ¼7.8 Hz,
CH2CH2CH3), 1.15e0.94 (m, 2H, CH2CH2CH3), 0.43 (t, 3H, 3J ¼7.1 Hz,
CH2CH2CH3). 13C NMR (101 MHz, DMSO‑d6):
d168.3, 167.2, 157.4,
140.7, 139.9, 135.9, 134.8, 131.5, 130.7, 130.4, 129.3, 128.5, 128.4,
127.5, 126.2, 124.6, 112.1, 51.7, 46.0, 13.3. HRMS: m/z calculated for
C
26H24N2O4 [MþH]þ: 429.1809, found: 429.1832.
4.1.4. N-Alkylation of compound 11 to methyl 1-((2’-
(methoxycarbonyl)-[1,10-biphenyl]-4-yl)methyl)-2-propyl-1H-
benzo[d]imidazole-4-carboxylate (12)
11 (0.10 g, 0.4 mmol) was dissolved in anhydrous DMF (0.5 ml).
NaH (0.01 g, 0.5 mmol) was added in small portions and the
4.1.6. Hydrolysis of compound 13 to 1-((20-carboxy-[1,10-biphenyl]-
4-yl)methyl)-2-propyl-1H-benzo[d]imidazole-4-carboxylic acid
(14)
13 (0.04 g, 0.1 mmol) was suspended in EG (0.2 ml) and 6 N KOH

A.M. Schoepf et al. / European Journal of Medicinal Chemistry 195 (2020) 112258
11
(2 ml) was added before heating to 160 ꢁC for 30 h. The reaction
mixture was diluted with water to double the volume. 6 N HCl was
used to reach a pH of 1. The mixture was extracted with DCM (3 ꢂ),
the organic layers were combined, washed with brine, dried over
anhydrous Na2SO4 and filtered. The solvent was removed under
reduced pressure. Purification was performed by column chroma-
tography (DCM/MeOH, 9:1). Colorless solid, yield: 44%. 1H NMR
or 20 mM), respectively, before further incubation for 72 h. A
modified MTT assay (EZ4U kit, Biomedica) was employed to analyze
the metabolic activity according to the manufacturer’s protocol.
Unspecific staining was excluded by subtracting the optical density
of the FCS-containing medium and the substrate. Metabolic activity
in the absence of the compounds (ctr., DMSO) was set to 100% and
served as internal reference [12].
(600 MHz, DMSO‑d6):
d
8.09 (d, 2H, 3J ¼8.4 Hz, H5’’, H7’’), 7.74 (dd,
1H, 3J ¼7.7 Hz, 4J ¼1.4 Hz, H3), 7.69e7.60 (m,1H, H6’’), 7.60e7.54 (m,
1H, H5), 7.49e7.44 (m, 1H, H4), 7.37e7.26 (m, 5H, H6, H2’, H3’, H5’,
H6’), 5.88 (s, 2H, NCH2), 1.76e1.63 (m, 2H, CH2CH2CH3), 0.97 (t, 3H,
3J ¼7.3 Hz, CH2CH2CH3). 13C NMR (151 MHz, DMSO‑d6): 206.5,169.4,
165.6, 156.9, 140.8, 140.3, 132.1, 131.0, 130.5, 129.3, 128.9, 127.5,
126.7, 117.8, 56.0, 30.7, 21.2, 18.6, 13.6. HRMS: m/zcalculated for
4.2.4. Determination of cell death by flow cytometry
Cell death was measured according to the previously described
procedure [27]. K562 or K562-resistant cells (2 ꢂ 105 cells per well)
and HS-5 cells (0.5 ꢂ 105 cells per well) were seeded in 24-well
plates. The ARBs telmisartan, candesartan cilexetil, candesartan,
irbesartan, olmesartan medoxomil, olmesartan, losartan, the com-
pounds 1, 6, 7, 12e14, or vehicle (ctr., DMSO) were added in the
respective concentrations, followed by an incubation for 72 h. After
harvesting of the cells, they were stained with PI/Triton-X100 for
2 h at 4 ꢁC. Forward/sideward scatter analyses were conducted
using a CytomicsFC-500 Beckman Coulter. Stained nuclei in the
sub-G1 marker window were considered to represent dead cells.
The tested compounds were proven to be soluble in the applied
concentrations by microscopy.
C
25H22N2O4[MþH]þ: 415.1652, found: 415.1681.
4.2. Biology
4.2.1. General cell culture methods
The monkey kidney-derived cell line COS-7 (ATCC) was cultured
as monolayer culture in Dulbecco’s Modified Eagle Medium
(DMEM) with 4.5 g/l glucose and 584 mg/l L-glutamine (GE
Healthcare), without phenol red and sodium pyruvate. It was
supplemented with fetal calf serum (FCS 10%, Sigma-Aldrich). The
human bone marrow/stroma cell line HS-5 (ATCC) was maintained
applying the above mentioned medium with 100 U/ml penicillin
4.2.5. Western blot analyses
The protein extracts were prepared as previously described [27].
Immunoblots were incubated with antibodies specific to STAT5
(#94205, Cell Signaling) or pSTAT5 (#05e886R, Merck) and GAPDH
(#2118, Cell Signaling). Prior to the development of the blots by
enhanced chemiluminescence (GE Healthcare) and the measure-
ment with the AutoChemi detection system (BioRad Laboratories),
incubation with anti-rabbit horseradish-peroxidase-conjugated
secondary antibody (GE Healthcare) was performed. Densitometric
analyses were conducted with the ImageJ 1.48 software and control
cells were set as 100%.
and 100 mg/ml streptomycin (both Lonza).
The myelogenous leukemia cell lines K562 and K562-resistant
were received from Ernesto Yague. K562-resistant cells were orig-
inally described as doxorubicin-resistant subclone of K562 cells
(termed KD225 by Hui et al. [25]). These cell lines were cultivated in
Roswell Park Memorial Institute (RPMI) 1640 medium (Lonza)
supplemented with 10% FCS, 2 mM
cillin, and 100 g/ml streptomycin.
L-glutamine, 100 U/ml peni-
m
Incubation of the cells was performed in a humidified atmo-
sphere (5% CO2/95% air) at 37 ꢁC and all cell lines were passaged
twice a week. The final concentration of DMSO never exceeded 0.1%
in cell based assays and vehicle treated controls (ctr., DMSO) were
always included.
Author contributions
The manuscript was written through contributions of all au-
thors. All authors have given approval to the final version of the
manuscript.
4.2.2. PPARg transactivation assay
A dual-luciferase reporter assay (Promega) was employed ac-
cording to the manufacturer’s protocol. COS-7 cells were seeded in
96-well plates (104 cells per well) in triplicates and incubated. After
24 h, the cells were transiently transfected with TransIT-LT1
(MoBiTec) and the three plasmids pGal5-TK-pGL3 (90 ng), pGal4-
hPPARgDEF (9 ng), as well as pRenilla-CMV (3 ng) in phosphate-
buffered saline (PBS), followed by further incubation for 7 h. Sub-
sequently, pioglitazone, the ARBs telmisartan, candesartan cilexetil,
candesartan, irbesartan, olmesartan medoxomil, olmesartan, los-
artan, the compounds 1, 6, 7, 12e14, or vehicle (DMSO), respec-
tively, were added at indicated concentrations. The samples were
incubated for 39 h. After washing with PBS, lysis was induced by
freezing (ꢃ80 ꢁC) of the cells overnight. The appropriate buffers
were added and it was shaken for 30 min to complete lysis. Firefly-
luminescence was measured with the multimode plate reader
EnSpire (PerkinElmer) and normalized with the activity of the co-
transfected pRenilla-CMV [26].
Declarations of competing interest
The authors declare no conflict of interest.
Acknowledgment
This work was supported by the Austrian Research Promotion
Agency FFG [West Austrian BioNMR 858017], the “Kinderkrebshilfe
Südtirol-Regenbogen”, and the “Südtiroler Krebshilfe”. We thank
Christoph Kreutz (Institute of Organic Chemistry, University of
Innsbruck) for his assistance in recording 13C NMR spectra and
Peter Enoh (Department of Pharmaceutical Chemistry, University of
Innsbruck) for technical support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112258.
4.2.3. Determination of metabolic activity
COS-7 cells were seeded in 96-well plates (2 ꢂ 103 cells per well)
in triplicates and incubated for 24 h. Pioglitazone, the ARBs telmi-
sartan, candesartan cilexetil, candesartan, irbesartan, olmesartan
medoxomil, olmesartan, losartan, the compounds 1, 6, 7, 12e14, or
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