Novel pyrrolopyrimidine derivatives induce p53-independent apoptosis via the mitochondrial pathway in colon cancer cells

Article abstract of DOI:10.1016/j.cbi.2020.109236

A series of novel pyrrolopyrimidine urea derivatives were synthesized and evaluated for their anticancer activity against colon cancer cell lines. Compounds showed the remarkable cytotoxic activity on HCT-116 wt cell line. The most potent compound 4c (IC₅₀ = 0.14 μM) induced apoptosis in HCT-116 wt and HCT-116 p53?/? cell lines. Otherwise, treatment of HCT-116 BAX?/?BAK?/? cells with compound 4c didn't lead to activation of apoptosis, suggesting that compound 4c induces apoptotic cell death by activating BAX/BAK-dependent pathway. Moreover, while the compound 4c increase the activation of caspase-3 and caspase-9 levels in HCT-116 wt and HCT-116 p53?/? cells, caspase-3 or caspase-9 activation was not observed in HCT-116 BAX?/?BAK?/? cells. In addition, compound 4c induced mitochondrial apoptosis in cells grown as oncospheroids, which better mimic the in vivo milieu of tumors. 4c treatment also activated JNK along with inhibition of prosurvival kinases such as Akt and ERK 1/2 in HCT-116 wt and HCT-116 p53 ?/? cells as well as in HCT-116 BAX?/?BAK?/? cells. Notably, our results indicated that compound 4c induced mitochondrial apoptosis through activation p53-independent apoptotic signaling pathways.

Full text of DOI:10.1016/j.cbi.2020.109236

Chemico-Biological Interactions 330 (2020) 109236
Contents lists available at ScienceDirect
Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
Novel pyrrolopyrimidine derivatives induce p53-independent apoptosis via
the mitochondrial pathway in colon cancer cells
a,
*
b
b
Zühal Kilic-Kurt , Yeliz Aka , Ozgur Kutuk
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Ankara, Turkey
b
Baskent University School of Medicine, Department of Immunology, Adana Dr. Turgut Noyan Medical and Research Center, Adana, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of novel pyrrolopyrimidine urea derivatives were synthesized and evaluated for their anticancer activity
against colon cancer cell lines. Compounds showed the remarkable cytotoxic activity on HCT-116 wt cell line.
Pyrrolopyrimidine
Ureas
The most potent compound 4c (IC50 = 0.14 μM) induced apoptosis in HCT-116 wt and HCT-116 p53ꢀ /ꢀ cell
p53-independent apoptosis
Cancer
lines. Otherwise, treatment of HCT-116 BAXꢀ /ꢀ BAKꢀ /ꢀ cells with compound 4c didn’t lead to activation of
apoptosis, suggesting that compound 4c induces apoptotic cell death by activating BAX/BAK-dependent
pathway. Moreover, while the compound 4c increase the activation of caspase-3 and caspase-9 levels in HCT-
1
16 wt and HCT-116 p53ꢀ /ꢀ cells, caspase-3 or caspase-9 activation was not observed in HCT-116 BAXꢀ /
ꢀ
BAKꢀ /ꢀ cells. In addition, compound 4c induced mitochondrial apoptosis in cells grown as oncospheroids,
which better mimic the in vivo milieu of tumors. 4c treatment also activated JNK along with inhibition of
prosurvival kinases such as Akt and ERK 1/2 in HCT-116 wt and HCT-116 p53 ꢀ /ꢀ cells as well as in HCT-116
BAXꢀ /ꢀ BAKꢀ /ꢀ cells. Notably, our results indicated that compound 4c induced mitochondrial apoptosis
through activation p53-independent apoptotic signaling pathways.
1
. Introduction
Pyrrolopyrimidine is a useful scaffold due to being structural ana-
prepared a series of thienopyrrolopyrimidine derivatives and evaluated
their cytotoxic activity against lung, colon and leukemia cell lines.
Among them, compound I (Fig. 1) showed significant cytotoxic activity
against acute leukemia and colorectal cancer cell lines with IC50 value of
27 and 50 nM, respectively. Brazidec et al. reported 2-substituted pyr-
rolopyrimidine derivatives as dual inhibitors of Aurora A/B kinases [8].
Compound II (Fig. 1) showed strong inhibitory activity against Aurora
A/B kinases as well as anti-proliferative activity toward the HCT116 cell
line with IC50 value of 4 nM. Oguro et al. designed and synthesized a
series of pyrrolo[3,2-d]pyrimidine derivatives to obtain strong inhibi-
tory activity against FGFR kinase [9]. Compound III (Fig. 1) exhibited
potent inhibitor activity against FGFR1 (IC50 = 14 nM) and VEGFR2
(IC50 = 9 nM) kinases as well as both VEGF- (IC50 = 38 nM) and
FGF-stimulated HUVEC proliferation (IC50 = 30 nM) [10].
logues of purines and its wide pharmacological profile such as anti-
cancer, antiviral, antibacterial and anti-inflammatory agents [1]. To
date, several drug molecules based on pyrrolopyrimidine scaffold were
developed as anticancer agents. Ruxolitinib, Tofacitinib [2], Oclaci-
tinib [3] and Baricitinib [4] (Fig. 1) were found as potent inhibitors of
JAK kinases. Ribociclib (Fig. 1), a selective and small-molecule inhib-
itor of cyclin dependent kinases 4/6, is developed for the treatment of
HR-positive, HER2-negative advanced or metastatic breast cancers in
combination with an aromatase inhibitor [5]. NVP-AEW541 (Fig. 1)
was described as highly selective insulin-like growth factor I receptor
(
IGF-IR, IC50 = 0.086
μ
M) inhibitor compared to closely related insulin
receptor (InsR) kinase (IC50 = 2.3
μM) [6]. CCT128930 (Fig. 1) was
It has been known that compounds involving urea moiety have a
significant role due to their potent anticancer activities. Also, some
properties of ureas such as hydrogen bonding capabilities, upgrading
target selectivity and regulating physicochemical properties make them
as pharmacophore in many biologically active molecules in drug design
applications [11]. Some of the FDA approved urea based anti-cancer
drugs such as Sorafenib, Regorafenib and Linifanib are used as
discovered as a potent ATP-competitive Akt inhibitor using fragment-
and structure-based approaches. It also exhibits selectivity against Akt2
with IC50 value of 6 nM and inhibition of phosphorylation of several
downstream Akt biomarkers in U87MG tumor xenografts [7]. After the
finding these promising compounds, plenty of pyrrolopyrimidine-based
compounds have been reported with anticancer activity. Tichy et al.
*
Corresponding author.
E-mail address: zkurt@ankara.edu.tr (Z. Kilic-Kurt).
https://doi.org/10.1016/j.cbi.2020.109236
Received 13 April 2020; Received in revised form 10 August 2020; Accepted 24 August 2020
Available online 28 August 2020
0
009-2797/© 2020 Elsevier B.V. All rights reserved.

Z. Kilic-Kurt et al.
Chemico-Biological Interactions 330 (2020) 109236
multi-kinase inhibitors for cancer treatment.
nitrogen atmosphere. Synthesis of the urea derivative 2a was previously
reported [13], which underwent substitution reaction with N,N-dime-
thylethylenediamine to afford compound 4j.
We have previously reported the synthesis and anticancer activities
of a series of 4-(substituted-phenylamino)pyrrolopyrimidines bearing
aryl urea moieties [12,13]. Several compounds showed potent cyto-
toxicity and significant apoptosis inducing effect. In order to compare
the activity and to optimize the scaffold, in this work, several pyrrolo-
pyrimidine derivatives (Fig. 2) containing N-methylpiperazine or N,
N-dimethylaminoethyl moiety instead of hydrophobic aniline group at
2
2
.2. Biological evaluation
.2.1. Anti-proliferative effect toward HCT-116 wild-type cells
To evaluate the effect of the target compounds (4, 4a-j) on the
4
-position of the pyrrolopyrimidine scaffold were designed and syn-
viability of colon cancer cells, parental HCT-116 wild-type (wt) cells
were treated with different concentration of the compounds for 48 h. As
shown in Fig. 3, CellTiter-Glo assay demonstrated that all compounds
(except 4b and 4g) had significant cytotoxic activity with IC50 values of
thesized. Also, effect of the methylene group at the urea side chain to the
activity were evaluated. Anti-proliferative activity and apoptotic
mechanism of the synthesized compounds against colon cancer cell line
were investigated.
ranging from 0.14 to 45.85
μ
M. Chemical structures and IC50 value of
M)
the compounds were shown in Table 1. Compound 4c (IC50 = 0.14
μ
2
. Result and discussion
bearing 4-chloro-3-trifluoromethylphenyl urea moiety exhibited the
best cytotoxic activity against HCT-116 wt cells. Lacking the tri-
fluoromethyl group caused to dramatic decrease in the activity of
2
.1. Chemistry
compound 4b (IC50 = 408.3
with 4-chlorobenzylurea (4h, IC50 = 1.62
improved activity. But, 4-fluorobenzylurea derivative 4g (IC50 = 190.4
M) showed the poor activity, suggesting chloro atom is important for
the activity of the benzylurea derivatives. Replacement of the chloro
atom at 4-position of phenylurea moiety (4c, IC50 = 0.14 M) with
hydrogen (4f, IC50 = 2.7 M) or fluoro atom (4d, IC50 = 8.12 M)
M) containing N,N-
μ
M). Changing the 4-chlorophenylurea (4b)
The synthesis of the target compounds (4a-j) is displayed in Scheme
μM) led to significantly
1
.
Reaction of 2,4-diamino-6-hydroxypyrimidine with chlor-
oacetaldehyde and sodium acetate yielded the 2-amino-3,7-dihydro-4H-
pyrrolo[2,3-d]pyrimidin-4-one 1, which upon reaction with POCl gave
the chloro derivative 2 [14]. N-methylation of compound 2 was per-
formed with NaH/CH in dry dimethylformamide (DMF) [15].
N-methyl piperazine group was introduced to the 4-position of the
compound 3 in the presence of K CO in dry DMF [16]. Target com-
pounds (4a-i) were obtained by the reaction of compound 4 with tri-
phosgene and appropriate amines in the presence of the Et N under
μ
3
μ
3
I
μ
μ
decreased the activity. Compound 4j (IC50 = 17.56
μ
2
3
dimethylamino moiety at the 4-position of pyrrolopyrimidine ring
showed the 2-fold lower cytotoxic activity than N-methylpiperazine
3
derivative 4d (IC50 = 8.12
μM). 2,4-Difluoro derivative 4e had the
Fig. 1. Anticancer compounds based on pyrrolopyrimidine scaffold.
2

Z. Kilic-Kurt et al.
Chemico-Biological Interactions 330 (2020) 109236
signaling is mediated by the pro- and antiapoptotic proteins of the BCL-2
family [17,19]. The dynamic balance between proapoptotic and anti-
apoptotic members of BCL-2 protein family dictate whether cancer cells
respond to cancer therapies or not. In response to various cellular
stresses, BAX and BAK form the oligomers that permeabilize the mito-
chondrial outer membrane, triggering the release of prodeath molecules
into cytosol and activation of caspases. p53 tumor suppressor is mutated
in more than 50% of human cancers. In addition, p53 has been shown to
govern cell death response in response to multiple prodeath signals
including genotoxic stress through regulating the expression of proap-
optotic genes such as Bax, Noxa and Puma [20]. Hence, we took
advantage of HCT-116 cells lacking BAX/BAK or p53 to evaluate the
apoptotic signaling pathways activated by compound 4c, which has best
cytotoxic activity against HCT-116 wt cell lines. Firstly, we explored the
expression of p53, BAX and BAK in HCT-116 wt, HCT-116 p53ꢀ /ꢀ and
HCT-116 BAXꢀ /ꢀ BAKꢀ /ꢀ cells. Immunoblotting results confirmed the
lack of p53 expression in HCT-116 p53ꢀ /ꢀ cells (Fig. 4A). Moreover,
BAX and BAK were undetectable in HCT-116 BAXꢀ /ꢀ BAKꢀ /ꢀ cells.
Next, we tested whether compound 4c induces apoptotic cell death in
HCT-116 cells by activating p53-or BAX/BAK-dependent pathways. As
demonstrated in Figs. 4B and 1.4
μ
M of compound 4c triggered
apoptotic cell death both in HCT-116 wt and HCT-116 p53ꢀ /ꢀ cells.
However, compound 4c failed to induce apoptosis in HCT-116
BAXꢀ /ꢀ BAKꢀ /ꢀ
cells.
Complementing
these
findings,
broad-spectrum caspase inhibitor QVD-OPh significantly inhibited
4
c-induced apoptosis in HCT-116 wt and HCT-116 p53ꢀ /ꢀ cells.
As demonstrated in Fig. 5, treatment of HCT-116 wt and HCT-116
p53ꢀ /ꢀ cells with 1.4
μ
M of compound 4c led to the translocation of
cytochrome c to cytosol. In contrast, we did not observe any alteration of
cytochrome c subcellular localization in HCT-116 BAXꢀ /ꢀ BAKꢀ /ꢀ
cells. Furthermore, we examined the activation of BAX and BAK by using
conformation-specific antibodies. As shown in Fig. 6, treatment with 1.4
Fig. 2. Design of the novel pyrrolopyrimidine derivatives.
moderate activity with IC50 value of 45.88 M. Pyridine urea group at
μ
M of compound 4c resulted in activation of both BAX and BAK in HCT-
μ
1
16 wt and HCT-116 p53ꢀ /ꢀ cells.
the 2-position of pyrrolopyrimidine scaffold (4j, IC50 = 1.46
μM) was
Next, we tested whether compound 4c activates caspase-3 and
well tolerated in cytotoxic activity.
caspase-9 in HCT-116 wt, HCT-116 p53ꢀ /ꢀ and HCT-116 BAXꢀ /
ꢀ
BAKꢀ /ꢀ cells. Compound 4c led to activation of caspase-3 and
2
.2.2. Induction of apoptosis by compound 4c in colon cancer cell lines
Activation of mitochondrial apoptotic pathway has been shown to
caspase-9 in HCT-116 wt and HCT-116 p53ꢀ /ꢀ cells as shown by
fluorometric caspase activation assays (Fig. 7). As expected, we did not
detect caspase-3 or caspase-9 activation in HCT-116 BAXꢀ /ꢀ BAKꢀ /ꢀ
mediate cancer cell death in response to conventional chemotherapy,
radiotherapy and targeted therapies [17,18]. Mitochondrial apoptosis
Scheme 1. Synthesis of the target compounds (4a-j). Reagent and conditions: (a) sodium acetate, rt, 48 h; (b) POCl
3
, reflux, 2 h; (c) NaH/CH
3
I, dry DMF, rt,
◦
◦
overnight; (d) N-methylpiperazine, K
2
CO
3
, dry DMF, 80 C, overnight; (d) i: triphosgen, Et
3
N, 0 C, 3 h; ii: appropriate amines, 1 h, rt.
3

Z. Kilic-Kurt et al.
Chemico-Biological Interactions 330 (2020) 109236
Fig. 3. HCT-116 wt cells were treated with indicated concentrations of the synthesized compounds for 48 h. IC50 values were determined by using CellTiter-Glo assay
mean ± SEM, n = 3 experimental replicates).
(
cells. In support of these findings, staining of HCT-116 wt, HCT-116
We showed that treatment of HCT-116 wt, p53ꢀ /ꢀ and
BAXꢀ /ꢀ BAKꢀ /ꢀ cells with 4c led to decreased phosphorylation of Akt
and ERK1/2 (Fig. 10). 4c treatment also triggered the activation of JNK
in HCT-116 wt, p53ꢀ /ꢀ and BAXꢀ /ꢀ BAKꢀ /ꢀ cells along with
increased phosphorylation. Nonetheless, we did not detect any alter-
ation of p38 phosphorylation (Fig. 10.)
p53ꢀ /ꢀ and HCT-116 BAXꢀ /ꢀ BAKꢀ /ꢀ cells with CellEvent Caspase-
3
/7 Green probe showed robust activation of caspase-3 in HCT-116 wt
and HCT-116 p53ꢀ /ꢀ cells, but not in HCT-116 BAXꢀ /ꢀ BAKꢀ /ꢀ cells
Fig. 8).
Moreover, we evaluated whether 4c would activate cell death
(
signaling in HCT-116 cells grown as oncospheroids. As shown in Fig. 9,
Altogether, these findings revealed that compound 4c activates
mitochondrial apoptotic signaling in colon cancer cells independent of
p53 status. Similar results have been obtained with several anticancer
compounds [23–26]. 5-Fluorouracil induced PKR-mediated apoptosis in
a p53-independent manner in colon cancer cell [27]. Some proteasome
inhibitors such as MG-132, bortezomib, and thiostrepton induced
apoptosis in human cancer cell lines through p53-independent induction
of proapoptotic Noxa but not Puma protein [28]. Mitoguazone was re-
ported as inducer of p53-independent programmed cell death in the
human breast cancer line [29]. Recently, G-quadruplex aptamer has
been identified as a strong antiproliferative agent in p53-deleted colon
cancer cells. Its cytotoxic activity is mediated by the ribosomal protein
uL3 and is associated to the activation of a p53-independent nucleolar
stress pathway [30].
treatment of HCT-116 wt and HCT-116 p53ꢀ /ꢀ , but not HCT-116
BAXꢀ /ꢀ BAKꢀ /ꢀ oncospheroids with 140
μ
M of 4c led to decreased
cell viability (**P < 0.01 vs. untreated oncospheroids). QVD-OPh
inhibited 4c-induced cell death in HCT-116 wt and HCT-116 p53ꢀ /ꢀ
oncospheroids. In line with these findings, we demonstrated that
exposing HCT-116 wt and HCT-116 p53ꢀ /ꢀ oncospheroids to 4c
resulted in increased cleaved PARP, cleaved caspase-3 and caspase-9
(
Fig. 9). Of note, 4c treatment failed to increase cleaved PARP,
caspase-3 or caspase-9 levels in HCT-116 BAXꢀ /ꢀ BAKꢀ /ꢀ
oncospheroids.
Several previous studies have demonstrated the crosstalk between
kinase signaling and cell death signaling pathways [21,22]. Hence, we
explored the effect of 4c treatment on the activation of prosurvival and
prodeath serine-threonine kinases including Akt, ERK1/2, JNK and p38.
4

Z. Kilic-Kurt et al.
Chemico-Biological Interactions 330 (2020) 109236
Table 1
Anti-proliferative activities of the synthesized compounds against HCT-116 wild-type (wt) cells.
Compound
R
F
1
R
2
R
3
X
Y
HCT116 wt IC50
13.22
3.34
(μM)
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
H
H
H
H
H
F
–
CH
CH
CH
CH
CH
CH
CH
CH
N
Cl
Cl
F
H
–
408.3
0.14
CF
CF
H
3
3
–
–
8.12
F
–
45.88
2.7
H
F
CF
H
3
H
H
H
H
–
–
g
h
i
CH
CH
–
2
190.4
1.62
Cl
H
–
H
2
H
1.46
j
–
–
–
17.56
Fig. 4. A) Expression of p53, BAX and BAK was
determined in HCT-116 wt, HCT-116 p53ꢀ /ꢀ and
HCT-116 BAXꢀ /ꢀ BAKꢀ /ꢀ cells by means of immu-
noblot analysis. Actin was probed as loading control.
B) HCT-116 wt, HCT-116 p53ꢀ /ꢀ and HCT-116
BAXꢀ /ꢀ BAKꢀ /ꢀ cells were treated with 1.4
μ
M of
compound 4c with or without 10
μ
M broad-spectrum
caspase inhibitor QVD-OPh. Apoptotic cell death was
determined by Annexin V-FITC/PI staining. Data
were shown as mean ± SEM, **P < 0.01.
3
. Conclusion
In this work, a series of pyrrolopyrimidine urea derivatives were
synthesized and evaluated for their cytotoxic activity against HCT-116
wt cell line. Compound 4c exhibited the strongest activity with IC50
value of 0.14
μ
M and induced mitochondrial apoptotic pathway in HCT-
1
16 colon cancer cells. This effect was dependent on the presence of BAX
and BAK, which are multidomain proapoptotic BCL-2 proteins that form
oligomers to facilitate mitochondrial outer membrane permeabilization.
As expected, compound 4c failed to activate mitochondrial apoptosis in
HCT-116 BAXꢀ /ꢀ BAKꢀ /ꢀ cells. Our findings also showed that acti-
vation of BAX and BAK was complemented by the release of cytochrome
c into cytosol as well as the activation of caspase-3 and -9 in HCT-116 wt
and HCT-116 p53ꢀ /ꢀ cells. p53 tumor suppressor is frequently deac-
tivated through mutations in the DNA-binding domain in cancer cells
Fig. 5. HCT-116 wt, HCT-116 p53ꢀ /ꢀ and HCT-116 BAXꢀ /ꢀ BAKꢀ /ꢀ cells
were treated with 1.4 M 4c for 48 h. Cytosolic and mitochondrial fractions
μ
were blotted for cytochrome c. CoxIV was probed as loading control for mito-
chondrial fractions.
[
20]. Several studies have shown that p53 transcriptionally regulates
proapoptotic BCL-2 proteins PUMA, NOXA, BAX and Apaf-1, which are
5

Z. Kilic-Kurt et al.
Chemico-Biological Interactions 330 (2020) 109236
Fig. 6. HCT-116 wt and HCT-116 p53ꢀ /ꢀ cells were treated with 1.4
μ
M of compound 4c for 16 h. Cells were stained with active conformation-specific BAX (6A7)
or BAK (Ab-1) antibodies followed by incubation with FITC-conjugated secondary antibody. Active BAK-related immunofluorescence was analyzed by flow
cytometry. Dark gray, control cells; light gray, treated cells.
critical components of mitochondrial apoptosis signaling [31–34]. In
particular, PUMA and NOXA contribute to DNA damage-induced
apoptosis [35,36]. Notably, our data showed that compound 4c acti-
vated mitochondrial apoptosis independent of p53. Importantly, 4c
treatment activated JNK along with inhibition of prosurvival kinases Akt
and ERK 1/2 in HCT-116 wt and HCT-116 p53 ꢀ /ꢀ cells as well as in
HCT-116 BAXꢀ /ꢀ BAKꢀ /ꢀ cells, in which it failed to trigger apoptosis.
Thus, 4c modulates various kinases upstream of mitochondrial apoptosis
signaling independent of its ability to activate cell death. Correspond-
ingly, 4c induced mitochondrial apoptosis in cells grown as oncosphe-
roids, which better mimic the in vivo milieu of tumors. Nonetheless,
pharmacodynamic and pharmacokinetic studies are warranted before
studying the effect of 4c in in vivo models of colon cancer including
patient-derived xenograft models. In summary, these biochemical and
functional data provided a solid rationale to target colon cancer cells by
compound 4c.
6

Z. Kilic-Kurt et al.
Chemico-Biological Interactions 330 (2020) 109236
Fig. 7. HCT-116 wt, HCT-116 p53ꢀ /ꢀ and HCT-116 BAXꢀ /ꢀ BAKꢀ /ꢀ cells were treated with 1.4
μ
M of compound 4c for 36 h. Activation of caspase-3 and caspase-
9
was evaluated by fluorometric caspase activation assays. Columns, mean relative fluorescence units (RFU) from three experimental repeats; bars, SEM; **P < 0.01.
Fig. 8. HCT-116 wt, HCT-116 p53ꢀ /ꢀ and HCT-116 BAXꢀ /ꢀ BAKꢀ /ꢀ cells were treated with 1.4
μ
M of compound 4c for 36 h. Cells were stained with CellEvent
◦
Caspase-3/7 Green Detection Reagent at 37 C for 30 min, counterstained with Hoechst 33342 to visualize nuclei and imaged by using Evos FLOID digital microscopy
system. Scale bars, 100
. Experimental section
.1. Chemistry
μ
m. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
4
plates. The ESI spectra were recorded on a Waters ZQ Micromass LC-MS
spectrometer (Waters Corporation, Milford, MA, USA) with ESI source as
ionization. Elemental analysis was performed using a Leco-932 CHNS–O
analyzer (Leco, St. Joseph, MI, USA) within ±0.4% of the theoretical
values.
4
4
.1.1. General
Commercial reagents were used without further purification. The
NMR spectra (400 MHz) were recorded on Varian Mercury 400 NMR
spectrometer (Varian Inc., Palo Alto, CA, USA) using tetramethylsilane
as internal standard. TLC was performed on F254 (Merck) silica gel
4.1.2. Synthesis of 2-amino-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-
one (1)
2,4-Diamino-6-hydroxypyrimidine (1 eq), chloroacetaldehyde (1 eq)
7

Z. Kilic-Kurt et al.
Chemico-Biological Interactions 330 (2020) 109236
Fig. 9. HCT-116 wt, HCT-116 p53ꢀ /ꢀ and HCT-116 BAXꢀ /ꢀ BAKꢀ /ꢀ oncospheroids were grown in 24-well Algimatrix culture plates. Growth of oncospheroids
were evaluated by using Evos FLOID microscopy system. Scale bars, 100 m. Cell viability was evaluated in oncospheroids following treatment with 4c (140 M) with
or without 1 mM broad-spectrum caspase inhibitor QVD-OPh for 48 h by using alamarBlue assay. Results are mean values from three independent experiments
mean ± SEM, **P < 0.01 by two-tailed t-test). Activation of intrinsic apoptotic signaling in oncospheroids following treatment with 4c (140 M) for 48 h was
evaluated by detecting cleaved PARP, caspase-3 and caspase-9 by using immunoblotting. Actin was immunoblotted as loading control.
μ
μ
(
μ
and sodium acetate (1 eq) was dissolved in the mixture of DMF:H
2
O
1H NMR (400 MHz, DMSO‑d
H-5), 6.60 (s, 2H, NH
), 7.09 (d, J = 3.2 Hz, H-6) [15].
6
): δ = 3.57 (s, 3H, CH ), 6.25 (d, J = 4 Hz,
3
(
100:16) and stirred for 48 h at room temperature. Then, the solvent was
2
evaporated under reduced pressure. Water (50 ml) was added and
brown precipitate was filtered. The filtrate was evaporated and ethanol
was added. The white precipitate was filtered to obtain the compound 1
in 86% yield [14].
4.1.5. Synthesis of 7-methyl-4-(4-methylpiperazin-1-yl)-7H-pyrrolo[2,3-d]
pyrimidin-2-amine (4)
The mixture of 4-chloro-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-
amine (3, 0,677 g; 3.72 mmol), N-methylpiperazine (4.1 ml; 37.19
4
.1.3. Synthesis of 4-chloro-7H-pyrrolo[2,3-d]pyrimidin-2-amine (2)
mmol) and K
2
CO
3
(1.5 g; 11.16 mmol) in dry DMF (10 ml) were stirred
◦
2
-Amino-3H-pirolo[2,3-d]pirimidin-4(7H)-on (1, 2.95 g; 0.02 mol)
at 80 C overnight. After that, the mixture was quenched with water and
◦
was suspended in 25 ml of POCl
cooling, excess of POCl
crude product was poured on ice-water mixture and neutralized to pH =
with aqueous ammonia. Brown precipitate (1.53 g) was filtered and
purified by column chromatography using dichlorometane:methanol
10:01) mixture to obtain pure compound 2 (0.37 g). Also, the filtrate
was extracted with EtOAc (3 × 50 ml), then organic phase was dried
over anhydrous Na SO and evaporated under reduced pressure to
afford the pure product (0.3 g). Finally, total yield is 20% [14].
3
and refluxed for 2 h at 110 C. After
extracted with EtOAc (3 × 50 ml). Organic phase was dried over Na
2 4
SO
3
was evaporated under reduced pressure. The
and evaporated under reduced pressure. The crude product was purified
by column chromatography using hexane:ethylacetate (8:2) mixture to
◦
7
obtain pure compound 4 (0.45 g, 49%). Mp: 64 C. MS (ESI, 70eV) m/z:
1
247.3 (M + H). H NMR (400 MHz, DMSO‑d
6
): δ = 2.19 (s, 3H, CH
3
),
),
(
2.36 (t, 4H, H-b), 3.51 (s, 3H, CH
3
), 3.75 (t, 4H, H-a), 5.64 (s, 2H, NH
2
1
3
6.35 (d, 1H, J = 4.0 Hz, H-5), 6.77 (d, 1H, J = 3.6 Hz, H-6). C NMR
(100 MHz, DMSO‑d
2
4
6
): δ = 31.07, 45.28, 46.27, 55.07, 96.29, 100.94,
122.12, 154.31, 157.64, 159.38.
4
.1.4. Synthesis of 4-chloro-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-amine
4.1.6. General synthesis method of the compounds (4a-i)
(
3)
To the solution of 7-methyl-4-(4-methylpiperazin-1-yl)-7H-pyrrolo
[2,3-d]pyrimidin-2-amine (1 eq) in dry dioxane, triphosgen (1 eq) and
To the solution of 4-chloro-7H-pyrrolo[2,3-d]pyrimidin-2-amine (2,
.248 g; 1.47 mmol) in dry DMF (10 ml), NaH (35 mg; 1.47 mmol) was
◦
0
triethylamine (2.5 eq) was added at 0 C and the mixture was stirred for
◦
added at 0 C and stirred for 1 h. Then, CH
3
I (0.1 ml; 1.62 mmol) was
3 h at room temperature under nitrogen atmosphere. After that,
appropriate amine derivatives (2 eq) was added and stirred for 1 h.
Then, the solvent was evaporated and aceton was added. The precipitate
was filtered and washed with cold water and crystallized from ethanol or
ethylacetate to give the final products in moderate yield.
added and stirred at room temperature overnight. After that, the mixture
was quenched with water and extracted with EtOAc (3 × 30 ml).
2 4
Organic phase was dried over Na SO and evaporated under reduced
pressure. The crude product was purified by column chromatography
using hexane:ethylacetate (9:1) mixture to obtain pure compound 3
(
0.15 g, 38%). MS (ESI, 70eV) m/z: 183.17 (M + H), 185.14 (M + H+2).
8

Z. Kilic-Kurt et al.
Chemico-Biological Interactions 330 (2020) 109236
Fig. 10. HCT-116 wt, HCT-116 p53ꢀ /ꢀ and HCT-116 BAXꢀ /ꢀ BAKꢀ /ꢀ were treated with 4c (140
μ
M) for 16 h. Phosphorylation of Akt, ERK 1/2, JNK and p38 was
evaluated by using cell-based ELISA assays. Data were shown as normalized fluorescence units (RFU) representing mean ± SEM of four independent experiments
(
**P < 0.01 by two-tailed t-test, vs. untreated cells).
4
.1.6.1. 1-(4-Fluorophenyl)-3-(7-methyl-4-(4-methylpiperazin-1-yl)-7H-
3.77 (t, 4H, H-a), 6.67 (d, 1H, J = 3.6 Hz, H-5), 7.19 (d, 1H, J = 3.6 Hz,
◦
′
pyrrolo[2,3-d]pyrimidine-2-yl)urea (4a). Yield 26%; mp: 223–225 C.
H-6), 7.65 (d, 1H, J = 9.2 Hz, H-5 ), 7.75 (dd, 1H, J
o
= 8.8 Hz, J = 2.0
m
1
′
′
MS (ESI, 70eV) m/z: 384.6 (M + H). H NMR (400 MHz, DMSO‑d
.25 (s, 3H, CH ), 2.47 (bs, 4H, H-b), 3.70 (s, 3H, CH ), 3.86 (bs, 4H, H-
a), 6.56 (d, 1H, J = 4.0 Hz, H-5), 7.07 (d, 1H, J = 4.0 Hz, H-6), 7.14 (t,
6
): δ =
Hz, H-6 ), 8.25 (d, 1H, J = 2.8 Hz, H-2 ), 9.63 (s, 1H, NH), 12.0 (s, 1H,
2
3
3
NH). 13C NMR spectra of the compound couldn’t be taken due to poor
solubility of the compound. Anal. calcd. for C20
H21ClF
3
N
7 2
O⋅2.3H O (%):
′
′
′
2
H, H-3 ,5 ), 7.55–7.59 (m, 2H, H-2 ,6’), 9.27 (s, 1H, NH), 11.78 (s, 1H,
C, 47.16; H, 5.06; N, 19.25. Found (%): C, 46.86; H, 4.60; N, 19.40.
1
3
NH). C NMR (100 MHz, DMSO‑d
6
): δ = 31.14, 44.68, 45.28, 54.15,
8.03, 100.96, 115.34, 115.56, 120.51, 124.29, 135.30, 150.44, 152.15,
56.44, 158.83. Anal. calcd. for C19 22FN O (%): C, 58.69; H,
9
1
5
4.1.6.4. 1-(4-Fluoro-3-(trifluoromethyl)phenyl)-3-(7-methyl-4-(4-methyl-
H
7
O⋅0.3H
2
piperazin-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-2-yl)urea (4d). Yield 30%;
◦
1
.85; N, 25.21. Found (%): C, 58.35; H, 6.02; N, 25.26.
mp: 203 C. MS (ESI, 70eV) m/z: 452.3 (M + H). H NMR (400 MHz,
DMSO‑d ), 2.41 (t, 4H, H-b), 3.72 (s, 3H, CH ),
): δ = 2.21 (s, 3H, CH
6
3
3
4
.1.6.2. 1-(4-Chlorophenyl)-3-(7-methyl-4-(4-methylpiperazin-1-yl)-7H-
3.85 (t, 4H, H-a), 6.57 (d, 1H, J = 3.6 Hz, H-5), 7.09 (d, 1H, J = 3.2 Hz,
◦
′
′
pyrrolo[2,3-d]pyrimidine-2-yl)urea (4b). Yield 29%; mp: 290 C. MS
H-6), 7.46 (t, 1H, H-5 ), 7.70–7.74 (m, 1H, H-6 ), 8.13 (dd, 1H, J = 2.0
1
13
(
ESI, 70eV) m/z: 400.3 (M + H). H NMR (400 MHz, DMSO‑d
6
): δ = 2.20
Hz, 2.8 Hz, H-2’), 9.50 (s, 1H, NH), 12.20 (s, 1H, NH). C NMR (100
(
s, 3H, CH ), 2.40 (t, 4H, H-b), 3.70 (s, 3H, CH ), 3.85 (t, 4H, H-a), 6.56
3
3
MHz, DMSO‑d
6
): δ = 31.14, 45.0, 45.69, 54.43, 98.14, 101.02, 116.63,
(
d, 1H, J = 3.6 Hz, H-5), 7.07 (d, 1H, J = 3.6 Hz, H-6), 7.35 (d, 2H, J =
117.66, 117.88, 124.36, 124.73, 124.83, 135.80, 150.36, 152.05,
152.18, 152.74, 156.44. Anal. calcd. for C20 O (%): C, 53.21; H,
′
′
′
8
.4 Hz, H-2 ,6 ), 7.59 (d, 2H, J = 8.8 Hz, H-3 ,5’), 9.31 (s, 1H, NH), 11.88
): δ = 31.11, 44.96, 45.61,
4.37, 98.03, 100.97, 120.34, 124.21, 126.01, 128.73, 137.89, 150.38,
51.93, 152.13, 156.42. Anal. calcd. for C19 22ClN O (%): C,
21 4 7
H F N
(
s, 1H, NH). 13C NMR (100 MHz, DMSO‑d
6
4.68; N, 21.71. Found (%): C, 52.92; H, 4.74; N, 21.69.
5
1
5
H
7
O⋅0.05H
2
4.1.6.5. 1-(2,4-Difluorophenyl)-3-(7-methyl-4-(4-methylpiperazin-1-yl)-
6.94; H, 5.55; N, 24.46. Found (%): C, 56.56; H, 5.65; N, 24.48.
7H-pyrrolo[2,3-d]pyrimidine-2-yl)urea
(4e). Yield
25%;
mp:
1
2
25–227 ◦C. MS (ESI, 70eV) m/z: 402.3 (M + H). H NMR (400 MHz,
4
.1.6.3. 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(7-methyl-4-(4-methyl-
DMSO‑d
6
): δ = 2.20 (s, 3H, CH
3 3
), 2.39 (t, 4H, H-b), 3.69 (s, 3H, CH ),
piperazin-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-2-yl)urea (4c). Yield 25%;
3.86 (t, 4H, H-a), 6.58 (d, 1H, J = 3.6 Hz, H-5), 7.04–7.08 (m, 2H),
◦
1
mp: 280 C. MS (ESI, 70eV) m/z: 468.3 (M + H). H NMR (400 MHz,
DMSO‑d ), 2.77 (s, 4H, H-b), 3.29 (s, 3H, CH ),
): δ = 2.49 (s, 3H, CH
7.32–7.38 (m, 1H), 8.29–8.35 (m, 1H), 9.48 (s, 1H, NH), 11.89 (s, 1H,
NH). 13C NMR (100 MHz, DMSO‑d
): δ = 30.85, 44.88, 45.69, 54.48,
6
3
3
6
9

Z. Kilic-Kurt et al.
Chemico-Biological Interactions 330 (2020) 109236
9
1
8.12, 101.10, 103.45, 103.73, 103.96, 111.03, 111.20, 121.63, 124.00,
24.24, 150.15, 151.91, 152.20, 156.52. Anal. calcd. for
4.2. Biological tests
4.2.1. Cell lines
C
19
H
21
F
2
N
7
O⋅0.08H
2
O (%): C, 56.64; H, 5.29; N, 24.33. Found (%): C,
5
6.25; H, 4.63; N, 24.27.
HCT-116, HCT-116 p53ꢀ /ꢀ and HCT-116 BAXꢀ /ꢀ BAKꢀ /ꢀ cells
were grown in McCoy’s 5A (Modified) medium (ThermoFisher Scienti-
fic, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal
bovine serum (Sigma, St Louis, MO, USA), 100 IU/ml penicillin, and
4
.1.6.6. 1-(7-Methyl-4-(4-methylpiperazin-1-yl)-7H-pyrrolo[2,3-d]pyrim-
idine-2-yl)-3-(3-(trifluoromethyl)phenyl)urea (4f). Yield 41%; mp:
◦
1
1
98 C. MS (ESI, 70eV) m/z: 434.2 (M + H). H NMR (400 MHz,
DMSO‑d ), 2.43 (t, 4H, H-b), 3.74 (s, 3H, CH ),
): δ = 2.23 (s, 3H, CH
.88 (t, 4H, H-a), 6.59 (d, 1H, J = 3.2 Hz, H-5), 7.10 (d, 1H, J = 3.6 Hz,
100 g/ml streptomycin (ThermoFisher Scientific, Carlsbad, CA, USA) at
μ
◦
37 C and 5% CO .
2
6
3
3
3
′
′
H-6), 7.37 (d, 1H, J = 7.6 Hz, H-4 ), 7.57 (t, 1H, H-5 ), 7.66 (d, 1H, J =
4.2.2. Cell death assays
′
13
8
.8 Hz, H-6 ), 8.15 (s, 1H, H-2’), 9.45 (s, 1H, NH), 12.09 (s, 1H, NH).
C
Cell viability was assessed by using CellTiter-Glo One Solution Assay
according to the manufacturer’s protocol (Promega, Madison, WI, USA).
In brief, 2 × 104 cells were grown in Corning 96-well solid white flat-
bottom microplates. Following incubation with test compounds for 48
h, one volume of CellTiter-Glo One solution equal to the volume of cell
culture medium present in each well was added. Luminescence was
measured by using Spectramax Gemini XPS microplate fluorometer.
Apoptotic cell death was determined by Annexin V-FITC/PI staining kit
(BD Biosciences, San Diego, CA, USA) according to the manufacturer’s
protocols. InSolution QVD-OPh was purchased from Millipore. Briefly,
NMR (100 MHz, DMSO‑d
6
): δ = 31.01, 44.94, 45.53, 54.31, 98.09,
1
1
00.94, 114.79, 118.74, 122.36, 124.28, 125.40, 129.43, 129.74,
30.01, 139.69, 150.39, 152.01, 156.40. Anal. calcd. for
C
20
H
22
F
3
N
7
O⋅0.6H
2
O (%): C, 54.07; H, 5.26; N, 22.07. Found (%): C,
5
3.81; H, 5.10; N, 22.17.
4
.1.6.7. 1-(4-Fluorobenzyl)-3-(7-methyl-4-(4-methylpiperazin-1-yl)-7H-
◦
pyrrolo[2,3-d]pyrimidine-2-yl)urea (4g). Yield 35%; mp: 80 C. MS (ESI,
1
7
3
2
0eV) m/z: 398.63 (M + H). H NMR (400 MHz, DMSO‑d
H, CH ), 2.75 (s, 4H, H-b), 3.30 (s, 3H, CH ), 3.53 (s, 4H, H-a), 4.45 (d,
H, J = 5.6 Hz, CH ), 6.61 (d, 1H, J = 3.6 Hz, H-5), 7.10 (d, 1H, J = 3.2
6
): δ = 2.50 (s,
1
06 cells were grown in 6-well plates and treated with indicated treat-
3
3
ments. Apoptosis was quantified by flow cytometry on FACSCanto (BD
Biosciences, San Diego, CA, USA), followed by analysis using FlowJo v9
software. Results are expressed as mean ± SEM of three independent
experiments. HCT-116 colon cancer cell oncospheroids were grown in
AlgiMatrix 24-well plates (ThermoFisher Scientific) as recommended by
the manufacturer. Algimatrix dissolving buffer (ThermoFisher Scienti-
fic) was used to isolate oncospheroids from 3D matrix for protein
isolation. Cell viability in oncospheroids was evaluated by means of
alamarBlue assay (ThermoFisher Scientific) as described by the manu-
facturer and results were expressed as percentage of cell viability
compared to untreated samples.
2
Hz, H-6), 7.17 (t, 1H), 7.24 (t, 1H), 7.39–7.43 (m, 1H), 7.53–7.56 (m,
H), 9.0 (s, 1H, NH), 9.51 (t, 1H, NH). 13C NMR (100 MHz, DMSO‑d
): δ
31.31, 41.91, 42.70, 42.79, 52.27, 98.55, 100.99, 115.53, 115.74,
15.92, 125.17, 129.70, 129.79, 131.73, 131.81, 136.45, 153.13,
54.89, 156.60. Anal. calcd. for C20 24FN O (%): C,
1
6
=
1
1
5
H
7
O⋅0.7C
4
H
8
O
2
⋅0.2H
2
9.18; H, 6.53; N, 21.18. Found (%): C, 58.78; H, 6.29; N, 21.05.
4
.1.6.8. 1-(4-Chlorobenzyl)-3-(7-methyl-4-(4-methylpiperazin-1-yl)-7H-
◦
pyrrolo[2,3-d]pyrimidine-2-yl)urea (4h). Yield 46%; mp: 136–138 C.
1
MS (ESI, 70eV) m/z: 414.5 (M + H), 416.5 (M + H+2). H NMR (400
MHz, DMSO‑d
CH
6
): δ = 2.16 (s, 3H, CH
3
), 2.29 (s, 4H, H-b), 3.51 (s, 3H,
4
.2.3. Protein isolation and immunoblotting
3
), 3.69 (s, 4H, H-a), 4.42 (d, 2H, J = 5.6 Hz, CH
2
), 6.49 (d, 1H, J =
H, NH), 9.62 (t, 1H, NH). 1 C NMR (100 MHz, DMSO‑d
): δ = 30.77,
Whole cell lysates were prepared in 1% CHAPS buffer [5 mM MgCl2,
3
1
4
1
.6 Hz, H-5), 7.0 (d, 1H, J = 4.0 Hz, H-6), 7.35–7.40 (m, 4H), 8.91 (s,
3
140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (w/v) CHAPS, 20 mM Tris-
HCl, pH 7.5 and protease inhibitors (cOmplete ULTRA, Roche)]. Pro-
teins were separated on 10% SDS-PAGE gels, transferred onto PVDF
membranes (Millipore) and then blocked with 5% non-fat dried milk in
PBS-Tween20. Membranes were incubated with primary and secondary
antibodies (GE Healthcare) in a buffer containing 10% milk diluent-
blocking concentrate (KPL), detected with Luminata Crescendo West-
ern HRP substrate (Millipore). Immunoblotting images were processed
by using C-DiGit Blot Scanner (LI-COR Biosciences, Bad Homburg,
Germany) on chemiluminescence mode. Antibodies used for immuno-
blotting were as follows: Cytochrome c (#4272, Cell Signaling), CoxIV
6
2.38, 44.95, 45.62, 54.32, 97.87, 100.87, 123.96, 128.36, 129.26,
31.47, 138.73, 150.98, 152.65, 154.54, 156.37. Anal. calcd. for
C
20
H
7
24ClN O⋅0.08C
4
H
8
O
2
⋅0.01H
2
O (%): C, 57.95; H, 5.90; N, 23.28.
Found (%): C, 57.61; H, 6.09; N, 22.82.
4
.1.6.9. 1-(7-Methyl-4-(4-methylpiperazin-1-yl)-7H-pyrrolo[2,3-d]pyrim-
◦
idine-2-yl)-3-(pyridine-2-yl)urea (4i). Yield 40%; mp: 260 C. MS (ESI,
1
7
3
1
7
9
3
1
0eV) m/z: 367.5 (M + H). H NMR (400 MHz, DMSO‑d
6
): δ = 2.45 (s,
H, CH ), 2.76 (s, 4H, H-b), 3.33 (s, 3H, CH ), 3.72 (s, 4H, H-a), 6.67 (d,
3
3
H, J = 3.6 Hz, H-5), 7.02–7.05 (m, 1H), 7.17 (1H, J = 3.6 Hz, H-6),
(
#4844, Cell Signaling), BAX (#2774, Cell Signaling), BAK (#3814, Cell
.74–7.78 (m, 1H), 8.05 (d, 1H, J = 8.0 Hz), 8.29 (d, 1H, J = 3.2 Hz),
.63 (t, 1H, NH), 12.35 (s, 1H, NH). 1 C NMR (100 MHz, DMSO‑d
3
Signaling), Actin (#8457, Cell Signaling) and p53 (#2527, Cell
Signaling), Cleaved PARP (Asp214) (#5625, Cell Signaling), Cleaved
Caspase-3 (Asp175) (#9661, Cell Signaling) and Cleaved Caspase-9
6
): δ =
1.01, 42.09, 42.57, 51.90, 98.35, 100.77, 112.49, 118.63, 125.15,
38.27, 148.34, 151.09, 151.67, 151.98, 152.21, 155.86. Anal. calcd.
(
Asp315) (#9505, Cell Signaling).
for C18
H
22
N
8
O⋅4.5H
2
O (%): C, 48.31; H, 6.98; N, 25.04. Found (%): C,
4
8.01; H, 6.51; N, 24.83.
4
.2.4. Caspase activation assays
The activity of caspase-3 and caspase-9 was determined by ApoAlert
4
.1.6.10. 1-(4-((2-(Dimethylamino)ethyl)amino)-7-methyl-7H-pyrrolo
Caspase-3 and caspase-9/6 assay kits (Clontech, Takara) as described by
the manufacturer. The release of fluorochrome AFC was analyzed at 400
nm excitation and 505 nm emission for caspase-3 and the release of
fluorochrome AMC was analyzed at 380 nm excitation and 460 nm
emission for caspase-9/6 using Spectramax Gemini XPS microplate
fluorometer. Data shown are mean ± SEM of three independent exper-
iments and expressed in arbitrary fluorescence units per mg of protein.
CellEvent Caspase-3/7 Green ReadyProbes Reagent (Thermo Scientific)
was used to evaluate caspase-3/7 activation. Cells were grown in 6-well
plates, treated with indicated compounds for 36 h and CellEvent
Caspase-3/7 Green ReadyProbes Reagent was added to medium per
[
2,3-d]pyrimidine-2-yl)-3-(4-fluoro-3-(trifluoromethyl)phenyl)urea (4j).
◦
1
Yield: 25%; mp: 195 C. MS (ESI, 70eV) m/z: 440.61 (M + H). H NMR
400 MHz, DMSO‑d ), 3.30 (s, 3H, CH ),
), 6.50 (d, 1H, J = 3.6 Hz, H-5),
(
6
): δ = 2.16 (s, 6H, N-(CH
.55–3.59 (m, 2H, CH ), 3.63 (t, 2H, CH
.95 (1H, J = 3.2 Hz, H-6), 7.45 (t, 1H, H-5 ), 7.66 (s, 1H, H-2 ), 7.80 (t,
3
)
2
3
3
6
1
2
2
′
′
1
3
H), 8.12 (d, 1H, J = 6.8 Hz), 9.37 (t, 1H, NH), 12.20 (s, 1H, NH).
C
NMR spectra of the compound couldn’t be taken due to poor solubility of
the compound. Anal. calcd. for C19 (%): C, 52.12;
O⋅0.4C
H, 5.13; N, 20.65. Found (%): C, 51.71; H, 4.54; N, 20.07.
H
F
21 4
N
7
4 8 2
H O
1
0

Z. Kilic-Kurt et al.
Chemico-Biological Interactions 330 (2020) 109236
supplier’s instructions (2 drops/mL). Nuclei were counterstained with
Hoechst 33342 and cells were imaged by using EVOS FLoid Cell Imaging
Station (ThermoFisher Scientific, Carlsbad, CA, USA). Images were
processed by CoLocalizer Pro for Mac software (Version 3.0.2).
for NMR and elemental analyses.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.cbi.2020.109236.
4
.2.5. Subcellular fractionation
Subcellular fractionation was performed as described before [37].
After indicated treatment with compounds, cells were harvested and
washed in ice-cold PBS and then resuspended in an isotonic buffer [250
mmol/L sucrose, 20 mmol/L HEPES (pH 7.5), 10 mM KCl, 1.5 mM
MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, and protease inhibitors
complete ULTRA, Roche] on ice for 20 min. After incubation, cells were
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[
8] M. Tichý, S. Smolen
́
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