Synthesis and bioactivity of novel amino-pyrazolopyridines

Article abstract of DOI:10.1016/j.ejmech.2014.08.008

Here we describe the synthesis and biological activity of novel amino-pyrazolopyridines with anti-NF-κB and pro-apoptotic potential. α-Methylene ketones were used as a starting point for synthesis of amino-pyrazolopyridine 3. The alkylidene malononitriles 1 were obtained by the Knoevenagel reaction of ketones with malononitriles. Vilsmeier-Haack reaction allowed direct access to 2-chloro-3-cyanopyridines 2. Those products, by refluxing with hydrazine hydrate, allowed cyclization to amino-pyrazolopyridines 3a-g, which were not previously described in the literature. Bioactivity results indicated that amino-pyrazolopyridines 3a, 3b and 3g induced apoptotic cell death in K562 cancer cells with an IC₅₀ of 36.5 ± 3.9 μM, 27.6 ± 4.5 μM and 35.0 ± 2.3 μM, respectively, after 72 h. In addition, compounds 3a, 3b and 3g exerted NF-κB inhibition activity with an IC₅₀ of 4.7 ± 1.6 μM, 6.9 ± 1.9 μM and 39.8 ± 3.9 μM, respectively, after 8 h in K562 cells activated with TNFα. Compounds 3b and 3g showed interesting differential toxicity as viability of peripheral blood mononuclear cells (PBMCs) from healthy donors remained largely unaffected by this treatment.

Full text of DOI:10.1016/j.ejmech.2014.08.008

European Journal of Medicinal Chemistry 85 (2014) 450e457
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and bioactivity of novel amino-pyrazolopyridines
,
,
Barbora Orlikova a b, Wafaa Chaouni c d, Marc Schumacher a, Mina Aadil d,
Marc Diederich b *, Gilbert Kirsch d
,
, *
a
ꢀ
^
Laboratoire de Biologie Moleculaire et Cellulaire du Cancer, Fondation de Recherche Cancer et Sang, Hopital Kirchberg, 9 Rue Edward Steichen, 2540
Luxembourg, Luxembourg
b Department of Pharmacy, College of Pharmacy, Seoul National University, Seoul, 151-742, Republic of Korea
c
ꢀ
ꢀ
ꢀ
Laboratoire d'Ingenierie Moleculaire et Biochimie Pharmacologique (LIMBP), Universite de Lorraine, SRSMC, Institut Jean Barriol, 1 Boulevard Arago,
57070, France
d
ꢀ
Laboratoire de Chimie Bioorganique et Analytique (LCBA), Universite Hassan II Mohammedia-Casablanca, FST-Mohammedia, BP 146, 20800
Mohammedia, Morocco
a r t i c l e i n f o
a b s t r a c t
Article history:
Here we describe the synthesis and biological activity of novel amino-pyrazolopyridines with anti-NF-kB
Received 19 July 2014
Received in revised form
1 August 2014
Accepted 4 August 2014
Available online 5 August 2014
and pro-apoptotic potential. a-Methylene ketones were used as a starting point for synthesis of amino-
pyrazolopyridine 3. The alkylidene malononitriles 1 were obtained by the Knoevenagel reaction of ke-
tones with malononitriles. VilsmeiereHaack reaction allowed direct access to 2-chloro-3-cyanopyridines
2. Those products, by refluxing with hydrazine hydrate, allowed cyclization to amino-pyrazolopyridines
3aeg, which were not previously described in the literature.
Bioactivity results indicated that amino-pyrazolopyridines 3a, 3b and 3g induced apoptotic cell death
Keywords:
in K562 cancer cells with an IC50 of 36.5 3.9
72 h. In addition, compounds 3a, 3b and 3g exerted NF-
6.9 1.9 M and 39.8 3.9 M, respectively, after 8 h in K562 cells activated with TNFa
m
M, 27.6 4.5
B inhibition activity with an IC50 of 4.7 1.6
. Compounds 3b
m
M and 35.0 2.3
m
M, respectively, after
VilsmeiereHaack
Amino-pyrazolopyridine
Inflammation
Apoptosis
Cancer
NF-kB pathway
k
m
M,
m
m
and 3g showed interesting differential toxicity as viability of peripheral blood mononuclear cells (PBMCs)
from healthy donors remained largely unaffected by this treatment.
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
reactions were used in the synthetic route: first, the Knoevenagel
reaction, applied in our synthetic route, has been well described
Cancer is one of the most deadly diseases worldwide and there-
fore synthesis of novel and potent small molecule anti-cancer agents
is intensifying [1e3]. Chemotherapeutic drugs including cisplatin or
etoposide induce selective cell death in cancer cells [4e6]. In this
[14e17] and the synthetic parameters were optimized to give a good
yield in our laboratory [18]. Second, cyclization reaction was carried
out by a VilsmeiereHaack reaction, as previously described in our
laboratory [18,19]. Treatment of 2-chloro-3-cyanopyridine with
hydrazine monohydrate provided amino-pyrazolopyridine [20].
Based on the reported bioactivity data, we evaluated the cyto-
research field, transcription factor NF-
the development of anti-cancer therapies as the pathological over-
expression of the NF- B pathway is a leading cause of resistance
kB is an important target for
k
toxic and NF-
products in human leukemia cells and demonstrated for the first
time that amino-pyrazolopyridines repress TNF -induced NF-
kB inhibition potential of the novel synthesized
towards chemo- and radiotherapy [7,8]. More specifically, pyr-
azolopyridine derivatives have been reported in the literature to
exert anti-inflammatory [9] and anti-cancer [10,11] properties in
addition to their anti-viral [12] and anti-leishmanial [13] bioactivity.
Considering these potent reported bioactivities, we describe
here a synthesis of amino-pyrazolopyridines based on our previous
synthetic studies with 2-chloro-3-cyanopyridine. Two major key
a
kB
signaling and induce apoptotic cell death in leukemia cells whereas
healthy blood cells remain largely unaffected by this treatment.
2. Material and methods
2.1. Chemistry
* Corresponding authors.
E-mail addresses: marcdiederich@snu.ac.kr (M. Diederich), kirsch@sciences.
univ-metz.fr (G. Kirsch).
The used chemical reagents were analytically pure. All solvents
and liquids were dried and distilled prior to use. Yields were
http://dx.doi.org/10.1016/j.ejmech.2014.08.008
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

B. Orlikova et al. / European Journal of Medicinal Chemistry 85 (2014) 450e457
451
optimized. All melting points were measured in open capillary
tubes. NMR spectra were recoded on a Bruker AC 250 (250 MHZ)
spectrometer in deuteron-chloroform CDCl3 or hexadeuter-
2.1.2.5. Synthesis 7-methyl-6,7,8,9-tetrahydro-3H-pyarazolo[3,4-C]
isoquinolin-1-amine 3e. (Colonn. Chromat.: AcOEt 30%/C6H12),
Aspect Colorless solid, yield 75%, Mp ¼ 243 ꢀC; 1H NMR (DMSO-d6,
odimethylsulfoxide DMSO. Chemical shifts (
d
) are reported in ppm
250 MHz)
d
¼ 1.02e1.04 (d, J ¼ 2.5 Hz, 3H, CH3), 1.33e1.35 (m, 1H,
and coupling constant (J) in Hz. Mass spectrometry has been real-
ized via an Agilent GCeMS system. After chromatographic sepa-
ration by a gas phase on a capillary column, the mass analysis was
performed with a Bruker MICROTOF-Q ESI/QqTOF or a Varion-Ion
spectrometer ESI-FTICR/MS QFT-9 4T. Microanalysis was per-
formed on a Thermo Finnigan EA 1112.
CH), 1.84 (m, 2H, CH2), 2.24e2.34 (m, 1H, CH), 2.76e2.2.83 (m, 1H,
CH), 3.00 (m, 1H, CH), 3.22 (s, 1H, CH), 5.00 (s, 2H, NH2), 7.99 (s, 1H,
Har), 11.77 (s, 1H, NH). 13C NMR (DMSO-d6, 250 MHz)
d
¼ 21.55,
25.05, 28.25, 29.76, 34.02, 104.84, 121.95, 140.00, 147.97, 150.02,
151.65. Anal. Calcl: C, 65.32; H, 6.97; N, 27.70. Found: C, 65.46; H,
6.69; N, 27.07. HRMS (APCI): m/z [C11H14N4 þ Hþ] calcd: 203.12;
Found: 203.13.
2.1.1. Synthesis of 2-chloro-3-cyanopyridine substituted in position
4 and 5 2aeg
These compounds were synthesized according to a known
procedure described by Aadil and et al. [18].
2.1.2.6. Synthesis
of
6,7,8,9-tetrahydro-3H-pyrazolo[3,4-C]iso-
quinolin-1-amine 3f. Aspect Colorless solid, yield 88%, Mp ¼ 230 ꢀC;
1H NMR (DMSO-d6, 250 MHz)
d
¼ 1.69e1.75 (m, 4H, CH2CH2),
3.06e3.09 (t, J ¼ 7.5 Hz, 2H, CH2), 3.30e3.33 (t, 2H, J ¼ 7.5 Hz, CH2),
5.00 (s, 2H, NH2), 7.99 (s,1H, Har),11.77 (s,1H, NH). 13C NMR (DMSO-
2.1.2. General procedure of synthesis of amino-pyrazolopyridine
3aeg
d6, 250 MHz)
d
¼ 21.67, 22.29, 25.10, 25.57, 104.93, 122.16, 140.39,
147.95, 150.09, 151.59. Anal. Calcl: C, 63.81; H, 6.425; N, 29.765.
Found: C, 62.61; H, 6.26; N, 30.27. HRMS (APCI): m/z
[C10H12N4 þ Naþ] calcd: 211.11; Found: 211.09.
Compound 3 (1.5 mmol) was dissolved in hydrazine hydrate
(3.1 g), the reaction mixture was heated for 2 h at 150 ꢀC. After
cooling, the mixture is poured into water and ice. The obtained
solid was filtered, washed with water twice and crystallized from
cyclohexane.
2.1.2.7. Synthesis of 5-methyl-4-phenyl-1H-pyrazolo[3,4-b]pyridin-
3amine 3g. Aspect Colorless solid, yield 76%, Mp ¼ 217 ꢀC; 1H NMR
(CDCl3, 250 MHz)
d
¼ 2.208 (s, 3H, CH3), 3.66 (s, 2H, NH2), 7.35e7.39
2.1.2.1. Synthesis7-methyl- 6-hydro-3H-benzo[f]pyrazolo[3,4-C]iso-
quinolin-1-amine 3a. Aspect Colorless solid, yield 92%,
(m, 2H, Har), 7.48e7.57 (m, 3H, Har), 8.41 (s, 1H, Har). 13C NMR
(CDCl3, 250 Hz)
d
¼ 15.97, 30.92, 104.93, 122.11, 128.50, 128.54,
Mp ¼ 209 ꢀC; 1H NMR (DMSO-d6, 250 MHz)
d
¼ 1.69e1.19 (m, 3H,
128.61, 128.73, 135.93, 143.54, 147.31, 151.39, 152.11. Anal. Calcl: C,
69.624; H, 5.393; N, 24.982. Found: C, 69.23; H, 5.81; N, 24.46.
HRMS (APCI): m/z [C13H12N4 þ Hþ] calcd: 225.11; Found: 225.11.
CH3), 2.70e2.78 (m, 1H, CH), 3.02e3.18 (m, 2H, CH2), 4.24 (s, 2H,
NH2), 7.33e7.47 (m, 3H, 3Har), 8.12e8.15 (m, 1H, Har), 8.43 (s, 1H,
Har), 10.66 (s, 1H, NH). 13C NMR (CDCl3, 250 Hz)
d
¼ 19.08, 30.39,
36.89, 127.00, 128.86, 129.13, 129.54, 129.81, 131.01, 137.77, 138.26,
147.02, 148.39, 153.83, 174.32. Anal. Calcl: C, 71.97; H, 5.63; N, 23.38.
Found: C, 71.63; H, 5.02; N, 23.12. m/z [C15H14N4 þ Hþ] calcd:
251.12; Found: 251.11.
2.2. Biological assays
2.2.1. Cell culture
TNFa was purchased from Sigma (Bornem, Belgium) and dis-
solved to a concentration of 10 mg/mL in phosphate buffered saline
(PBS) supplemented with 0.5% (w/v) bovine serum albumin (BSA;
Sigma) according to the manufacturer's instructions. K562 (human
chronic myelogenous leukemia) cells (Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, DSMZ, Braunschweig,
Germany) were cultured in RPMI medium (Lonza, Verviers,
Belgium) supplemented with 10% (v/v) fetal calf serum (Lonza) and
1% (v/v) antibiotic-antimycotic (Bio-Whittaker, Verviers, Belgium)
at 37 ꢀC and 5% of CO2. The cells were harvested every 3 days. After
stock defrosting, cells were kept in normal culture conditions for 10
days before experimental application.
2.1.2.2. Synthesis
6,7-dihydro-3H-benzo[f]pyrazolo[3,4-C]iso-
quinolin-1-amine 3b. Aspect: colorless solid; yield: 85%;
Mp ¼ 204 ꢀC; 1H NMR: (DMSO-d6, 250 MHz)
d
¼ 2.80 (m, 4H,
CH2CH2), 4.24 (s, 2H, NH2), 7.37e7.48 (m, 3H, Har), 8.12e8.15 (d,
J ¼ 7.5 Hz, 1H, Har), 8.40 (s, 1H, Har), 10.30 (s, 1H, NH). 13C NMR
(DMSO-d6, 250 MHz)
d
¼ 25.02, 28.75, 100.90, 124.21, 126.75,
127.86, 129.00, 129.32, 131.29, 137.07, 139.30, 146.66, 148.33, 153.51.
Anal. Calcl: C, 71.169; H, 5.11; N, 23.712. Found: C, 71.15; H, 5.42; N,
23.22. HRMS (APCI): m/z [C14H12N4 þ Hþ] calcd: 273.11; Found:
273.11.
Cells
were
pre-treated
with
synthesized
amino-
2.1.2.3. Synthesis of 3,6,7,8-tetrahydrocyclopenta[d]pyrazolo[3,4-b]
pyrazolopyridines at indicated concentrations and time periods.
Amino-pyrazolopyridines were dissolved in 100% dimethyl sulf-
oxide (DMSO) (SigmaeAldrich, Bornem, Belgium) and the amount
of DMSO in cell culture did not exceed 0.4%.
Healthy blood samples from donors were kindly provided as
buffy coats by the Red Cross (Luxembourg). A 1-1 dilution of blood
and RPMI Medium was added to a Ficoll (Paque PRENIUM, GE
Healthcare, Diegem, Belgium) layer and then subjected to a
centrifugation (400 g for 20 min). Then, after 2e3 washes of the
pellet with RPMI medium, the isolated peripheral blood mono-
nuclear cells (PBMCs) were incubated at 37 ꢀC and 5% CO2 in a
humidified atmosphere for 24 h prior to treatment with the syn-
thesized compounds.
pyridin-1-amine 3c. Aspect Colorless solid; yield 99%, Mp ¼ 240 ꢀC;
1H NMR: (DMSO-d6, 250 MHz)
d
¼ 2.02e2.11 (m, 2H, CH2),
2.84e2.90 (t, J ¼ 7.5 Hz, 2H, t, CH2), 3.15e3.21 (t, J ¼ 7.5 Hz, 2H, t,
CH2), 5.15 (s, 2H, NH2), 8.18 (s, 1H, Har), 11.79 (s, 1H, NH). 13C NMR
(DMSO-d6, 250 MHz)
d
¼ 24.77, 29.07, 30.75, 103.86, 129.50, 144.66,
146.83, 147.39, 152.31. Anal. Calcl: C, 62.053; H, 5.786; N, 32.161.
Found: C, 62.01; H, 5.26; N, 32.13. HRMS (APCI): m/z [C9H10N4 þ Hþ]
calcd: 175.09; Found: 175.09.
2.1.2.4. Synthesis of 4-ethyl-5-methyl-1H-pyrazolo[3,4-b]pyridin-3-
amine 3d. Aspect Colorless solid, yield 62%, Mp ¼ 206 ꢀC; 1H
NMR (DMSO-d6, 250 MHz)
d
¼ 1.12e1.18 (m, 3H, CH3), 2.26 (s, 3H,
CH3), 2.87e2.96 (m, 2H, CH2), 5.02 (s, 2H, NH2), 8.08 (s, 1H, Har). 13
NMR (DMSO-d6, 250 Hz)
C
d
¼ 14.38, 14.51, 105.04, 120.48, 145.67,
2.2.2. Cell viability assessment
147.29, 150.44, 152.41. Anal. Calcl: C, 61.343; H, 6.86; N, 31.794.
Found: C, 61.15; H, 6.81; N, 31.86. HRMS (APCI): m/z [C9H12N4 þ Hþ]
calcd: 177.11; Found: 177.11.
Percentages of cell survival were evaluated using Promega's
CellTiter-Glo™ Luminescent Cell Viability Assay (Promega, Leiden,
Netherlands) kit, according to the manufacturer's instructions.

452
B. Orlikova et al. / European Journal of Medicinal Chemistry 85 (2014) 450e457
Alternatively, Trypan Blue staining was used to determine cell
integrity (Supplementary data). Data were normalized to the con-
trol and reported as percentage of viable cells. Apoptosis estimation
was performed as previously described by analyzing nuclear
morphology upon staining with Hoechst 33342 (1
mg/mL) [21].
Briefly, cells were incubated at 37 ꢀC for 15 min with the DNA-
specific dye and analyzed by fluorescence microscope.
2.2.3. Transient transfection and luciferase reporter gene assay
K562 cells were transiently transfected as described previously
[22]. For each electroporation, we used 5
gene construct containing five repeats of a consensus NF-
(pGL4.32[luc2P/NF- B-RE/Hygro], Promega, Leiden, Netherlands)
and 5 g of a Renilla luciferase plasmid (phRG-TK, Promega, Leiden,
mg of a luciferase reporter
k
B site
k
m
Netherlands). After electroporation cells were re-suspended in
normal culture medium (RPMI, 10% FCS) and cultured at 37 ꢀC and
5% CO2 for 24 h. Then, cells were harvested and re-suspended in
fresh growth medium (RPMI, 0.1% FCS) to a final concentration of
1 ꢁ 106 cells/mL and pre-treated for 2 h with the indicated con-
centrations of amino-pyrazolopyridines. After 2 h of pre-treatment,
cell were activated by 20 ng/mL of TNF
incubation period of 6 h. After 8 h of total treatment time, 75
Dual-Glo™ luciferase reagent (Promega, Leiden, Netherlands) were
added to 75 L of the cellular suspension for 10 min incubation at
22 ꢀC before luciferase activity measurement. Afterwards, 75
L of
a
, followed by an additional
mL of
m
m
Dual-Glo™ Stop&Glo Reagent (Promega, Leiden, Netherlands) were
added for 10 min at 22 ꢀC to the cell suspension to assay Renilla
activity. An Orion microplate luminometer (Berthold, Pforzheim,
Germany) was used to measure luciferase and Renilla activity. The
results are expressed as a ratio of arbitrary units of firefly luciferase
activity normalized to Renilla luciferase activity.
Fig. 1. Synthesis and chemical structures of amino-pyrazolopyridines. A) Synthesis of
2-chloro-3-cyanopyridines substituted in position 4 and 5 2aeg through Knoevenagel
reaction followed by a VilsmeiereHaack reaction and Condensation of 2-chloro-3-
cyanopyridines with hydrazine hydrate leading to the formation of amino-pyrazolo-
pyridines 3aeg. Reagent and conditions: (i) malononitrile, ammonium acetate, acetic
acid, toluene, reflux, 24 h. (ii) phosphorus oxychloride, dimethylformamide, NP/KF,
70e80 ꢀC, 3 h. (iii) hydrazine hydrate, reflux, 2e3 h. B) Chemical structures of syn-
thesized amino-pyrazolopyridines 3aeg.
2.2.4. Human CXCL8/IL-8 immunoassay
IL-8 concentrations in culture supernatants of activated K562
cells were measured by sandwich ELISA (R&D Systems, Abingdon,
United Kingdom). According to the manufacturer's guide, 50
cell supernatants were added with 100 L of assay diluent to anti-
IL-8 pre-coated wells followed by 2 h incubation at RT. After
washing, a polyclonal peroxidase-conjugated anti-IL-8 antibody
was added for another 60 min at RT. Colorimetric visualization and
protein dosage were developed by addition of the H2O2 þ TMB
(tetramethylbenzidine) containing substrate. After a 30 min reac-
tion at room temperature (RT) in the dark, the enzymatic reaction
was stopped by addition of H2SO4 and optical densities were
measured at a wavelength of 450 nm.
mL of
m
3. Results
The goal of this article was the synthesis of previously unknown
amino-pyrazolopyridine 3 via cyclization of intermediary 2-chloro-
3-cyanopyridines 2, prepared from a-methylene ketones through a
Knoevenagel reaction and VilsmeiereHaack reaction [14e19]. A
cyclization of the 2-chloro-3-cyanopyridines 2 with hydrazine hy-
drate led to the formation of amino-pyrazolopyridines 3. In a sec-
ond part, a bioactivity study of the synthesized products was
carried out. In detail, this study evaluated differential cytotoxic
activity of novel amino-pyrazolopyridines and determined inhibi-
2.2.5. Caspase-glo® 3/7 assay
Measurement of caspase 3/7 activities was assessed by homo-
geneous Caspase-Glo®3/7 luminescent assay (Promega) following
manufacturer's guide. Briefly, 3 ꢁ 105 cells/mL were pre-treated or
not for 1 h with 50 mM of Caspase Inhibitor I (Z-VAD (OMe)-FMK,
tory potential of novel amino-pyrazolopyridines on TNF
a-induced
Calbiochem), following treatments with amino-pyrazolopyridines
3a, 3b and 3g at 40 M for 8 h, 24 h and 48 h. After treatment
period, 75 L of cell culture were mixed with 75 L of Caspase-
NF- B pathway in human leukemia cells.
k
m
m
m
Glo®3/7 reagent and incubated at room temperature for 1 h.
Addition of Caspase-Glo®3/7 reagent results in cell lysis, followed
by caspase cleavage of the substrate and generation of a “glow-
type” luminescent signal, produced by luciferase. Luminescence is
proportional to the amount of caspase activity present. The results
are expressed as fold change compared to the control.
3.1. Chemistry
The alkylidene-malonodinitrile 1 prepared by Knoevenagel re-
action from -methylene ketones, which has a methylene active in
position, can serve as intermediates for the synthesis of desired
a
a
heterocycles 3aeg (Fig. 1) [14e18].
The treatment of alkylidene malononitriles 1aeg by Vils-
meiereHaack reagent at 70e80 ꢀC leads to the formation of 2-
chloro-3-cyanopyridines 2aeg substituted in position 4 and 5
(Fig. 1) [18,19]. Reaction of the reactive methylene group of the
alkylidene malononitrile 1 with the VilsmeiereHaack reagent gave
2.2.6. Statistical analysis
Data are expressed as mean S.D. and their signification de-
grees were analyzed by Student's T-tests. P-values below 0.01 were
considered as statistically significant.

B. Orlikova et al. / European Journal of Medicinal Chemistry 85 (2014) 450e457
453
Fig. 2. Effects of amino-pyrazolopyridines on K562 cell viability. A) Effect of amino-pyrazolopyridine analogues on metabolic activity of K562 cells after 24 and 72 h at 40 mM.
Control (Co) corresponds to untreated cells. Etoposide (Eto) served as a positive control. Each value is a mean SD of three determinations. The asterisk indicates a significant
difference compared to control analyzed by t-test (*p < 0.01). B) Dose-dependent inhibition of K562 cell viability by compound 3a, C) by compound 3b and D) by compound 3g is
shown.
an intermediate iminium salt. The latter cyclizes spontaneously
with loss of dimethylamine to the -chloro -cyano pyridines 2aeg.
(Fig. 3BeD, IC50s are indicated). Altogether, these results indicate a
selective cytotoxic effect of compounds 3b and 3g.
a
b
However, this reaction has been accomplished, in general with low
yields from 10 to 15%. The use of natural phosphate (NP) alone or
natural phosphate modified by potassium fluoride (KF/NP) as new
efficient solid catalysts in the VilsmeiereHaack reaction can in-
crease the yield to 60%.
Aminopyrazolopyridine 3aeg substituted in position 4 and 5
with either an aliphatic or cyclic system, however, were not pre-
viously known. We describe here their preparation from 2-
chlorocyanopyridines and hydrazine hydrate as shown in Fig. 1A.
The aromatic nucleophilic substitution of the chlorine by hydrazine
followed by the attack of the cyano group led to the amino pyr-
azoles [20].
3.2.2. Amino-pyrazolopyridines induce apoptotic cell death
To further assess cell death mechanisms, morphology of nuclei
of K562 cells treated with 40 mM of amino-pyrazolopyridines 3a, 3b
and 3g for 72 h was assessed. Results showed condensed nuclei
typical of apoptosis (Fig. 4). In order to strengthen our results, we
evaluated activation of executioner caspases 3/7. Amino-
pyrazolopyridines increased caspase 3/7 activity in K562 cells
reaching a maximum after 24 h (Fig. 5A). In order to generalize our
results, we tested the effects of amino-pyrazolopyridines in two
additional leukemia cell lines (Jurkat and U937). Our results
showed that all three amino-pyrazolopyridines significantly
Hence, the described synthetic route led to the synthesis of
novel amino-pyrazolopyridines substituted in position 4 and 5
previously not described in literature (Fig. 1B).
induced caspase 3/7 activity in Jurkat and U937 cells at 40
mM
(Fig. 5B and C). Compounds 3a and 3b reached the activation peak
after 8 h in both cell lines, while compound 3g induced caspase 3/7
activity after 48 h and 24 h in Jurkat and U937 cells, respectively. In
order to ascertain involvement of caspase-dependent apoptotic
mechanisms, we pre-treated cells with 50 mM of Z-VAD caspase
3.2. Biological activity
inhibitor for 1 h. Under these conditions, induction of caspase 3/7
activity was completely abrogated (Fig. 5AeC). Caspase 3/7 activ-
ities after amino-pyrazolopyridines treatments are summarized in
Table 1.
3.2.1. Differential toxicity of amino-pyrazolopyridines
We first evaluated the effect of amino-pyrazolopyridines on
viability of chronic myeloid leukemia K562 cells. Cells were treated
with amino-pyrazolopyridines at 40
mM for 24 and 72 h. Com-
pounds 3a, 3b and 3g decreased more than 50% of K562 cell
viability after 72 h and were selected for further studies (Fig. 2A).
Compound 3a, 3b and 3g dose-dependently down-regulated K562
metabolic activity after 24 and 72 h. (Fig. 2BeD, IC50s are indicated).
Results were confirmed by trypan blue assays (Fig. S1). To assess for
differential toxicity, PBMCs from healthy donors were treated un-
der the same conditions with compounds 3a, 3b and 3g. Results
show no strong effect on PBMC viability for 3b and 3g whereas
compound 3a induced levels of toxicity comparable to K562 cells
3.2.3. TNF
pyrazolopyridines
Our results showed that amino-pyrazolopyridines 3a, 3b and 3g
inhibited TNF -induced NF- B transactivation at 40 M (Fig. 6A).
These three compounds dose-dependently inhibited NF- B acti-
vation in K562 cells (Fig. 6BeD, IC50s are indicated). Importantly,
compounds 3a, 3b and 3g inhibit NF- B at sub-cytotoxic concen-
trations. This includes compound 3g as the IC50 value for NF-
inhibition (39.8 3.9 M) was evaluated after 8 h of treatment
a-induced NF-kB pathway is repressed by amino-
a
k
m
k
k
kB
m

454
B. Orlikova et al. / European Journal of Medicinal Chemistry 85 (2014) 450e457
Fig. 3. Effects of amino-pyrazolopyridines on PMBC viability. A) Effects of amino-pyrazolopyridine analogues on metabolic activity of healthy PBMCs after 24 and 72 h at 40 mM. Control
(Co) corresponds to untreated cells. Etoposide (Eto) served as a positive control. Each value is a mean SD of three determinations. The asterisk indicates a significant difference
compared to control analyzed by t-test (*p < 0.01). B) Dose-dependent inhibition of PBMCs viability by compound 3a, C) by compound 3b and D) by compound 3g is displayed.
Fig. 4. Induction of apoptotic morphology by amino-pyrazolopyridines. K562 cells were stained with Hoechst in order to detect nuclear morphology, non-treated (control) vs. cells
treated with 40 mM of amino-pyrazolopyridines 3a, 3b or 3g for 72 h. 10 mM of etoposide served as a positive control.
Fig. 5. Induction of caspase 3/7 activity in leukemia cell lines. K562 (A), Jurkat (B) and U937 cells (C) were pre-treated or not with Z-VAD at 50
mM for 1 h, followed by amino-
pyrazolopyridines treatment at 40 M for 8, 24 and 48 h. Control corresponds to untreated cells. Asterisk indicates a significant difference compared to control analyzed by t-test (*p < 0.05).
m

B. Orlikova et al. / European Journal of Medicinal Chemistry 85 (2014) 450e457
455
Table 1
showed AT1 receptor affinity [23], anti-inflammatory [9] and
cytotoxic effects [11,24], inhibition of Aurora A kinase [24,25], PI3-
kinase [26], protein kinase B/Akt [27], VEGFR kinase [28] and Polo-
like kinase [29]. Considering these results, it was not surprising that
viability assays confirmed the cell-death inducing properties of
amino-pyrazolopyridines derivatives 3a, 3b and 3g in the leukemia
cell line K562. Hoechst staining showed condensed nuclei wit-
nessing apoptosis induction in K562 cells further validated by
caspase assays. Viability assays on PBMCs from healthy donors
showed that compounds 3b and 3g present interesting differential
toxicity in line with previous reports [10,24].
Activation of caspase 3/7 activity by amino-pyrazolopyridines in leukemia cancer
cells. Results are expressed as fold-change increase compared to untreated cells.
3a (40
m
M)
3b (40
m
M)
3g (40 mM)
8 h
24 h
48 h
8 h
24 h
48 h
8 h
24 h
48 h
K562
Jurkat
U937
1.44
8.89
16.51
2.04
5.38
6.27
1.55
2.11
1.48
1.37
2.86
11.68
2.50
2.73
5.84
2.23
2.05
1.74
1.26
1.27
1.73
3.62
1.31
5.87
2.90
1.44
4.35
whereas IC50 value for cell viability (35.0 2.3
after 72 h. Moreover, after 24 h of treatment with compound 3g at
40 M, we did not observe 50% inhibition of cell viability, indicating
mM) was determined
Interestingly, to our best knowledge, no NF-
property of pyrazolopyridines has been reported in literature so far.
Our results, shown by the NF- B assays, indicate that amino-
pyrazolopyridine derivatives 3a, 3b and 3g inhibited TNF
induced NF- B activity in K562 cells with an IC50 of 4.7 1.6 M,
6.9 1.9 M and 39.8 3.9 M, respectively. This new reported
kB inhibition
m
k
that this concentration is sub-toxic up to 24 h.
a
-
To further confirm the inhibition potential of amino-
k
m
pyrazolopyridines on TNF
gated the effect of amino-pyrazolopyridines on interleukin 8 (IL-8),
a gene under the control of NF- B. Results show that compound 3a
dose-dependently inhibited IL-8 release in K562 cells (Fig. 7).
Importantly, K562 cell viability was not affected after 24 h at con-
centrations corresponding to the IC50 of the inhibition of IL-8
a-induced NF-kB activation, we investi-
m
m
activity of amino-pyrazolopyridine derivatives is not surprising as
previously synthesized pyrazolopyridines were reported to affect
signaling pathways tightly associated to the NF-kB transcription
factor. The later is linked to anti-inflammatory activity [9], Aurora A
kinase [25,30], AT1 receptor [23,31], protein kinase B/Akt [27],
VEGFR kinase [28,32] Polo-like kinase [29]. These cell signaling
pathways were all reported to be targets of various pyrazolopyr-
idine derivatives.
k
release (8.2 0.9 mM) (Figs. 3B and S1).
4. Discussion
We hypothesize that inhibition of NF-kB pathway by amino-
pyrazolopyridines may be responsible for the induction of
apoptotic cell death in K562 cells, as concentrations necessary for
The goal of this bioactivity study was the determination of
cytotoxicity and NF- B inhibition potential of newly synthesized
k
NF-
points compared to cytotoxic concentrations. These observations
indicate that inhibition of NF- B activity occurs prior to induction
kB inhibition were lower and/or appeared at the shorter time
amino-pyrazolopyridines.
Over the last years, pyrazolopyridines were published to impact
a large variety of cancer signaling pathways: pyrazolopyridines
k
Fig. 6. Inhibitory effects of amino-pyrazolopyridines on TNF
RG-tk Renilla plasmid for 24 h. After transfection, K562 cells were treated with amino-pyrazolopyridines at 40
Results are expressed as a ratio of the measured luminescence of the firefly luciferase vector and the luminescence of Renilla plasmid. Negative control corresponds to untreated
cells, positive control refers to TNF -treated cells. Curcumin (Cur) has been used as positive inhibitory control. Results are presented as a mean S.D. of three independent ex-
a-induced NF-
k
B activity. A) K562 cells were transiently transfected with firefly luciferase vector (NF-
kB pGL4) and ph-
m
M for 2 h followed by TNF stimulation (20 ng/ml) during 6 h.
a
a
periments. The asterisk indicates a significant difference compared to the positive control analyzed by t-test (*p < 0.01). B) Dose-dependent inhibition of TNF
a-induced NF-kB
activity in K562 cells by compound 3a, C) by compound 3b and D) by compound 3g is shown.

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B. Orlikova et al. / European Journal of Medicinal Chemistry 85 (2014) 450e457
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Acknowledgments
ꢀ ꢀ
M.S. was supported by a Televie grant. Research at the Labo-
ratoire de Biologie Moleculaire et Cellulaire du Cancer (LBMCC) is
financially supported by “Recherche Cancer et Sang” foundation, by
«Recherches Scientifiques Luxembourg» asbl, by «Een Haerz fir
Kriibskrank Kanner» association, the Action Lions “Vaincre le
Cancer” Luxembourg, Televie Luxembourg and the Foundation for
Scientific Cooperation between Germany and Luxemburg for
additional support. BO and MD are supported by the National
Research Foundation (NRF) by the MEST of Korea for Tumor
Microenvironment Global Core Research Center (GCRC) grant,
[grant number 2012-0001184]: MD is also supported by the Seoul
National University Research grant and by the Research Settlement
Fund for the new faculty of SNU.
ꢀ
€
ꢀ ꢀ
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.08.008.

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