Identification of 1H-pyrazolo[3,4-b]pyridine derivatives as potent ALK-L1196M inhibitors

Article abstract of DOI:10.1080/14756366.2019.1639694

Anaplastic lymphoma kinase (ALK) has been recognised as a promising molecular target of targeted therapy for NSCLC. We performed SAR study of pyrazolo[3,4-b]pyridines to override crizotinib resistance caused by ALK-L1196M mutation and identified a novel and potent L1196M inhibitor, 10g. 10g displayed exceptional enzymatic activities (<0.5 nM of IC₅₀) against ALK-L1196M as well as against ALK-wt. In addition, 10g is an extremely potent inhibitor of ROS1 (<0.5 nM of IC₅₀) and displays excellent selectivity over c-Met. Moreover, 10g strongly suppresses proliferation of ALK-L1196M-Ba/F3 and H2228 cells harbouring EML4-ALK via apoptosis and the ALK signalling blockade. The results of molecular docking studies reveal that, in contrast to crizotinib, 10g engages in a favourable interaction with M1196 in the kinase domain of ALK-L1196M and hydrogen bonding with K1150 and E1210. This SAR study has provided a useful insight into the design of novel and potent inhibitors against ALK gatekeeper mutant.

Full text of DOI:10.1080/14756366.2019.1639694

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Identification of 1H-pyrazolo[3,4-b]pyridine
derivatives as potent ALK-L1196M inhibitors
Yunju Nam, Dongkeun Hwang, Namdoo Kim, Hong-Seog Seo, Khalid B. Selim
& Taebo Sim
To cite this article: Yunju Nam, Dongkeun Hwang, Namdoo Kim, Hong-Seog Seo, Khalid B.
Selim & Taebo Sim (2019) Identification of 1H-pyrazolo[3,4-b]pyridine derivatives as potent ALK-
L1196M inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 1426-1438, DOI:
10.1080/14756366.2019.1639694
To link to this article: https://doi.org/10.1080/14756366.2019.1639694
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 1426–1438
https://doi.org/10.1080/14756366.2019.1639694
RESEARCH PAPER
Identification of 1H-pyrazolo[3,4-b]pyridine derivatives as potent
ALK-L1196M inhibitors
a
a
b
a,c
d,e
Yunju Nam , Dongkeun Hwang , Namdoo Kim , Hong-Seog Seo , Khalid B. Selim and Taebo Sima,d
ꢀ
ꢀ
aKU-KIST Graduate School of Converging Science and Technology, Korea University, Seoul, Republic of Korea; NDBio Therapeutics Inc., Incheon,
Republic of Korea; Cardiovascular Center, Korea University Guro Hospital, Seoul, Republic of Korea; Chemical Kinomics Research Center, Korea
Institute of Science and Technology (KIST), Seoul, Republic of Korea; Department of Pharmaceutical Organic Chemistry, Mansoura University,
b
c
d
e
Mansoura, Egypt
ABSTRACT
ARTICLE HISTORY
Received 30 November 2018
Revised 18 June 2019
Accepted 26 June 2019
Anaplastic lymphoma kinase (ALK) has been recognised as a promising molecular target of targeted ther-
apy for NSCLC. We performed SAR study of pyrazolo[3,4-b]pyridines to override crizotinib resistance
caused by ALK-L1196M mutation and identified a novel and potent L1196M inhibitor, 10g. 10g displayed
exceptional enzymatic activities (<0.5 nM of IC50) against ALK-L1196M as well as against ALK-wt. In add-
ition, 10g is an extremely potent inhibitor of ROS1 (<0.5 nM of IC50) and displays excellent selectivity over
c-Met. Moreover, 10g strongly suppresses proliferation of ALK-L1196M-Ba/F3 and H2228 cells harbouring
EML4-ALK via apoptosis and the ALK signalling blockade. The results of molecular docking studies reveal
that, in contrast to crizotinib, 10g engages in a favourable interaction with M1196 in the kinase domain
of ALK-L1196M and hydrogen bonding with K1150 and E1210. This SAR study has provided a useful
insight into the design of novel and potent inhibitors against ALK gatekeeper mutant.
KEYWORDS
Anaplastic lymphoma
kinase; ALK-L1196M
mutant; pyrazolopyridine-
based inhibitor
Introduction
crizotinib18,20,28–30. The ALK gatekeeper mutation L1196M is the
most frequent secondary mutation taking place in NSCLC patients
and, consequently, a significant effort has been made to identify
novel and potent L1196M inhibitors31. These efforts have led to
the development of second-generation ALK inhibitors including
ceritinib32,33, alectinib10, lorlatinib8–9, brigatinib34,35, ensartinib36
and entrectinib11 that circumvent the acquired secondary muta-
tions21,37 (Figure 1).
As part of continuing studies aimed at discovering structurally
distinct and potent ALK-L1196M inhibitors, we carried out SAR
investigation using pyrazolopyridine derivatives bearing 3-fluoro-
phenyl sulfone moiety. The substances utilised in this study were
designed based on the reported 3-amino-5-substituted indazole-
based ALK inhibitors including entrectinib11,38. SAR exploration
using novel pyrazolopyridine derivatives led to identification of
Anaplastic lymphoma kinase (ALK), a member of the insulin recep-
tor tyrosine kinase superfamily, was originally identified as NPM
(nucleophosmin)-ALK fusion protein generated by (2;5)(p23:q35)
chromosomal translocation in anaplastic large cell lymphoma
(ALCL)1,2. ALK gene rearrangements result in various ALK fusion
oncogenes including EML4 (echinoderm microtubule-associated
protein like 4)–ALK3 in lung cancers, TPM3 (tropomyosin 3)-ALK in
ALCL4 and TPM3/4–ALK5 in inflammatory myofibroblastic tumours
(IMT)6. ALK gene aberrations found in several human cancers
include rearrangement, activating point mutations7 such as
F1174L/R1275Q, and gene amplification. ALK has received a great
deal of attention as a promising therapeutic target for targeted
cancer therapy and, as a result, enormous efforts have been
devoted to developing ALK inhibitors. While various scaffolds such
as pyrimidine, aminopyridine8,9, benzo[b]carbazolone10, indazole11
have been exploited to identify new ALK inhibitors, 2,4-diaryla-
mino pyrimidines2,12–21 have been the most common molecular
platform for discovering ALK inhibitors including ceritinib and
brigatinib. Crizotinib22,23 was approved in 2011 as an ALK inhibitor
for treatment of NSCLC patients harbouring EML4-ALK fusion
oncogene. This substance was also approved in 2016 for treat-
ment of ROS1-positive NSCLC. In 2018, crizotinib was given a
breakthrough therapy designation to treat ALK-positive relapsed/
refractory anaplastic large cell lymphoma (ALCL)24–27 patients.
However, acquired secondary mutations (L1196M, G1269A,
10 g as
a novel and potent inhibitor against ALK-L1196M
(IC50 < 0.5 nM) as well as against ALK-wt (IC50 < 0.5 nM). It is worth-
while noting that entrectinib11,38 inhibits ALK-wt with an IC50
value of 12 nM.
Experimental
Chemistry
Unless otherwise described, all commercial reagents and solvents
were purchased from commercial suppliers and used without fur-
ther purification. All reactions were performed under N2 atmos-
F1174L, S1206Y, 1151 T-ins, L1152R, C1156Y and G1202R) occur- phere in flame-dried glassware. Reactions were monitored by TLC
ring in the ALK kinase domain resulted in resistance to with 0.25 mm E. Merck precoated silica gel plates (60 F254).
CONTACT Taebo Sim
tbsim@kist.re.kr
Chemical Kinomics Research Center, Korea Institute of Science and Technology (KIST), 5, Hwarang-ro 14-gil, Seongbuk-
gu, Seoul 136-791, Republic of Korea
ꢀ
These authors contributed equally to this work.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1427
F
Cl
N
Cl
NH2
O
O
HN
N
NH
O
S
O
O
N
CN
F
N
O
Cl
N
N
N
H
N
O
N
CN
N
N
O
N
H2N
N
H
HN
Crizotinib (approved)
Ceritinib (approved)
Alectinib (approved)
Lorlatinib (approved)
O
F
N
O
H
P
F
H
N
Cl
H
N
N
N
O
N
N
N
N
O
N
Cl
F
N
O
Cl
N
NH
N
H
NH
N
O
O
N
N
NH2
Ensartinib (phase lll)
Entrectinib (phase lll)
Brigatinib (approved)
Figure 1. ALK inhibitors on market or under clinical trials.
Reaction progress was monitored by TLC analysis using a UV 13.01 (bs, 1H), 8.32 (d, J ¼ 2.6 Hz, 1H), 8.13 (d, J ¼ 2.6 Hz, 1H),
lamp, ninhydrin, or p-anisaldehyde stain for detection purposes. 7.39–7.33 (m, 1H), 7.10–7.01 (m, 3H). LCMS (ESI) m/z:
247.22 [M þ H]þ.
All solvents were purified by standard techniques. Purification of
reaction products was carried out by silica gel column chromatog-
raphy using Kieselgel 60 Art. 9385 (230 ꢁ 400 mesh). The purities
2-Chloro-5-((3-fluorophenyl)thio)nicotinonitrile (4)
of all compounds were shown to be over 95% by using Waters
LCMS system (Waters 2998 photodiode array detector, a Waters
3100 mass detector, a Waters SFO system fluidics organiser, a
Water 2545 binary gradient module, a Waters reagent manager
and a Waters 2767 sample manager) using a SunFireTM C18 col-
umn (4.6 mm ꢂ 50 mm, 5 mm particle size): solvent gradient ¼ 60%
(or 95%) A at 0 min, 1% A at 5 min. Solvent A ¼ 0.035% TFA in
H2O; solvent B ¼ 0.035% TFA in MeOH; flow rate 3.0 (or 2.5) mL/
min. 1H and 13 C NMR spectra were obtained by using a Bruker
400 MHz FT-NMR (400 MHz for 1H, and 100 MHz for 13 C) spec-
trometer. Standard abbreviations are used for denoting the signal
multiplicities.
To a solution of compound 3 (620 mg, 2.52 mmol) in POCl3
(3.9 mL, 0.04 mmol) was added PCl5 (1.47 g, 7 mmol) under nitro-
gen atmosphere at 0 ꢃC. The reaction mixture was stirred at
100 ꢃC for 5 h, cooled to room temperature, diluted with water,
and extracted with DCM. The organic layers were dried over
Na2SO4, filtered, and concentrated. The resulting residue was sub-
jected to silica gel column chromatography (50% EtOAc/hexane)
to afford 4 (620 mg, 93%) as a brown solid. 1H NMR (400 MHz,
DMSO-d6) d 8.64 (d, J ¼ 2.4 Hz, 1H), 8.54 (d, J ¼ 2.4 Hz, 1H),
7.49–7.43 (m, 1H), 7.34–7.30 (m, 1H), 7.27 (d, J ¼ 7.8 Hz, 1H),
7.24–7.19 (m, 1H). LCMS (ESI) m/z: 265.27 [M þ H]þ.
2-Chloro-5-((3-fluorophenyl)sulfonyl)nicotinonitrile (5)
5-bromo-2-oxo-1,2-dihydropyridine-3-carbonitrile (2)
To a solution of compound 4 (600 mg, 2.27 mmol) in dry DCM
(5 mL, 0.45 M) was added m-CPBA (1.37 g, 3.5 mmol) under nitro-
gen atmosphere at 0 ꢃC. The reaction mixture was stirred at room
temperature for 5 h, diluted with Na2SO3 saturated aqueous solu-
tion, and extracted with DCM. The organic layer was washed with
brine, dried over Na2SO4, filtered and concentrated. The resulting
residue was subjected to silica gel column chromatography
(30–50% EtOAc/hexane) to afford 5 (530 mg, 79%) as a yellow
solid. 1H NMR (400 MHz, DMSO-d6) d 9.27 (d, J ¼ 2.4 Hz, 1H), 9.17
(d, J ¼ 2.4 Hz, 1H), 7.99 (dd, J ¼ 8.3, 1.6 Hz, 1H), 7.94 (d, J ¼ 7.8 Hz,
1H), 7.74 (td, J ¼ 8.0, 5.6 Hz, 1H), 7.67–7.62 (m, 1H). LCMS (ESI) m/z:
297.22 [M þ H]þ.
To a solution of 2-oxo-1,2-dihydropyridine-3-carbonitrile (15 g,
124.9 mmol) in acetic acid (93 mL, 0.5 M) was added Br2 (9.6 mL,
374.7 mmol) slowly under nitrogen atmosphere. The reaction mix-
ture was stirred for 5 h at room temperature, diluted with Na2S2O3
aqueous solution and extracted with EtOAc. The organic layer was
dried over Na2SO4, filtered, and concentrated to afford 2 (21.1 g,
85%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) d 12.86 (bs,
1H), 8.39 (d, J ¼ 2.8 Hz, 1H), 8.09 (d, J ¼ 2.8 Hz, 1H). LCMS (ESI) m/z:
200.22 [M þ H]þ.
5-((3-Fluorophenyl)thio)-2-oxo-1,2-dihydropyridine-3-carboni-
trile (3)
A mixture of Compound 2 (450 mg, 2.26 mmol), 3-fluorobenzene-
thiol (0.25 mL, 2.34 mmol), K2CO3 (500 mg, 3.62 mmol), and copper
iodide (900 mg, 4.73 mmol) in dry DMF (4.5 mL) was stirred at
120 ꢃC for 15 h. The reaction mixture was cooled to room tem-
perature, filtered, quenched with water, and extracted by DCM.
The organic layer was dried over Na2SO4, filtered and concen-
trated. The resulting crude product was subjected to flash column
2–(5-((3-Fluorophenyl)sulfonyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)i-
soindoline-1,3-dione (6)
A mixture of compound 5 (520 mg, 1.75 mmol), and hydrazine
monohydrate (0.22 mL, 4.54 mmol) in EtOH (3 mL, 0.6 M) was
stirred at 80 ꢃC for 7 h, then cooled to room temperature, diluted
with water, and extracted with DCM. The organic layer was dried
chromatography on silica gel (10–50% EtOAc/hexane) to afford 3 over Na2SO4, filtered, and concentrated. The resulting crude prod-
(500 mg, 90%) as yellow solid. 1H NMR (400 MHz, DMSO-d6) d uct was diluted with 1,4-dioxane (7 mL) and treated with phthalic

1428
Y. NAM ET AL.
anhydride (360 mg, 2.43 mmol). The mixture was stirred at 100 ꢃC was stirred at room temperature for 1 h. The mixture was concen-
trated and the generated residue was subjected to silica gel col-
for 15 h, filtered and concentrated. The residue was subjected to
umn chromatography (50–80% EtOAc/hexane) to afford 11
(150 mg, 88%) as a pale yellow solid. H NMR (400 MHz, DMSO-d6)
flash column chromatography on silica gel (30–70% EtOAc/hex-
ane) to afford 6 (530 mg, 72%) as a white solid. H NMR (400 MHz,
1
1
d 10.86 (bs, 1H), 8.93 (d, J ¼ 2.4 Hz, 1H), 8.81 (d, J ¼ 2.4 Hz, 1H),
7.93–7.90 (m, 1H), 7.87–7.82 (m, 3H), 7.69–7.64 (m, 1H), 7.58–7.53
(m, 1H), 7.27–7.18 (m, 15H), 6.59 (d, J ¼ 8.8 Hz, 2H), 5.87 (bs, 2H).
13 C NMR (100 MHz, DMSO-d6) d 165.87, 163.47, 160.99, 153.10,
152.46, 146.95, 143.37, 143.30, 142.57, 141.92, 134.88, 132.62,
132.55, 130.53, 129.68, 129.54, 127.85, 127.23, 124.10, 124.07,
121.62, 121.41, 119.24, 115.11, 114.87, 112.77, 110.85, 78.36. LCMS
(ESI) m/z: 654.22 [M þ H]þ.
DMSO-d6) d 14.70 (bs, 1H), 9.20 (d, J ¼ 2.4 Hz, 1H), 9.10 (d,
J ¼ 2.0 Hz, 1H), 8.07–8.04 (m, 2H), 8.00–7.97 (m, 2H), 7.89–7.86 (m,
1H), 7.85 (d, J ¼ 8.0 Hz, 1H), 7.2–7.66 (m, 1H), 7.59–7.57 (m, 1H).
LCMS (ESI) m/z: 423.22 [M þ H]þ.
2–(5-((3-Fluorophenyl)sulfonyl)-1-trityl-1H-pyrazolo[3,4-b]pyridin-3-
yl)isoindoline-1,3-dione (7)
To
a solution of 6 (600 mg, 1.42 mmol) and TEA (0.4 mL,
2.87 mmol) in dry DCM (22 mL) was added triphenylmethyl chlor-
ide (520 mg, 1.87 mmol) slowly at 0 ꢃC. The mixture was stirred for
0.5 h, quenched with water, and extracted by DCM. The organic
layers were dried over Na2SO4, filtered, and concentrated. The
resulting residue was subjected to silica gel column chromatog-
raphy (25–50% EtOAc/hexane) to afford 7 (700 mg, 75%) as a yel-
low solid. 1H NMR (400 MHz, DMSO-d6) d 9.04 (d, J ¼ 2.4 Hz, 1H),
8.95 (d, J ¼ 2.4 Hz, 1H), 8.04–8.01 (m, 2H), 7.98–7.95 (m, 2H),
7.92–7.82 (m, 2H), 7.71–7.65 (m, 1H), 7.60–7.55 (m, 1H), 7.30–7.06
(m, 15H). LCMS (ESI) m/z: 665.27 [M þ H]þ.
General procedure A for the synthesis of compounds 10a–10h
To a solution of 8 (1 equiv) in pyridine (0.1 M) was added various
acid chlorides (1.05 equiv) and DMAP (0.6 equiv) at 0 ꢃC under
nitrogen atmosphere. The mixture was stirred at room tempera-
ture for 0.5 h, then diluted with water, and extracted with DCM.
The organic layer was dried over Na2SO4, filtered, and concen-
trated. The residue was diluted with dry DCM (0.5 M), and added
slowly with TFA (2 equiv) at 0 ꢃC. The mixture was stirred at room
temperature for 0.5 h, diluted with water, and extracted with
DCM. The organic phase was washed with brine, dried over
Na2SO4, filtered and concentrated. The resulting crude product
was subjected to flash column chromatography on silica gel.
5-((3-Fluorophenyl)sulfonyl)-1-trityl-1H-pyrazolo[3,4-b]pyridin-3-
amine (8)
A suspension of compound 7 (1 g, 1.5 mmol) and hydrazine mono-
hydrate (0.22 mL, 4.5 mmol) in THF (15 mL) and EtOH (15 mL) was
stirred at room temperature for 1 h. The mixture was concentrated
and the generated residue was subjected to silica gel column
chromatography (20–50% EtOAc/hexane) to afford 8 (680 mg,
85%) as a pale yellow solid. 1H NMR (400 MHz, DMSO-d6) d 8.23
(d, J ¼ 2.4 Hz, 1H), 8.68 (d, J ¼ 2 Hz, 1H), 7.84–7.79 (m, 2H),
7.71–7.65 (m, 1H), 7.58–7.54 (m, 1H), 7.31–7.14 (m, 15H), 6.27 (bs,
2H). 13 C NMR (100 MHz, DMSO-d6) d 163.59, 161.10, 153.83,
149.59, 146.87, 143.96, 143.89, 143.16, 132.77, 132.69, 131.61,
129.47, 128.66, 127.90, 127.74, 126.95, 123.89, 123.86, 121.52,
121.31, 120.97, 114.7, 114.63, 109.67, 77.55. LCMS (ESI) m/z:
535.27 [M þ H]þ.
4-Cyano-N-(5-((3-fluorophenyl)sulfonyl)-1H-pyrazolo[3,4-b]pyridin-
3-yl)benzamide (10a)
General procedure A was used to transform 8 (50 mg, 0.09 mmol)
to the target compound. The resulting residue was subjected to
flash column chromatography on silica gel (50–100% THF/hexane)
to afford 10a (14.2 mg, 37%) as a yellow solid. 1H NMR (400 MHz,
DMSO-d6) d 14.09 (bs, 1H), 11.67 (bs, 1H), 9.15 (s, 1H), 9.10 (s, 1H),
8.24 (d, J ¼ 8.0 Hz, 2H), 8.06 (d, J ¼ 8.0 Hz, 2H), 7.93 (d, J ¼ 8.4 Hz,
1H), 7.89 (d, J ¼ 7.6 Hz, 1H), 7.72–7.66 (m, 1H), 7.59–7.55 (m, 1H).
13 C NMR (100 MHz, DMSO-d6) d 164.79, 163.67, 161.19, 152.70,
148.62, 143.96, 143.89, 141.84, 137.79, 135.33, 132.96, 132.82,
132.74, 129.51, 129.44, 124.03, 124.00, 121.62, 121.41, 118.72,
115.00, 114.87, 114.76, 107.71. LCMS (ESI) m/z: 422.27 [M þ H]þ.
4–(1,3-Dioxoisoindolin-2-yl)-N-(5-((3-fluorophenyl)sulfonyl)-1-trityl-
1H-pyrazolo[3,4-b]pyridin-3-yl)benzamide (9i)
N-(5-((3-Fluorophenyl)sulfonyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)-3-
(trifluoromethyl)benzamide (10b)
To a solution of 8 (500 mg, 0.94 mmol) and DMAP (50 mg,
0.41 mmol) in pyridine (10 mL) was added 4–(1,3-dioxoisoindolin-
2-yl)benzoyl chloride (400 mg, 1.4 mmol) dropwise at 0 ꢃC under
nitrogen atmosphere. The mixture was stirred at room tempera-
ture for 1 h, then diluted with water, and extracted with DCM. The
organic layer was dried over Na2SO4, filtered and concentrated.
The resulting crude was subjected to silica gel column chromatog-
raphy (25–80% EtOAc/hexane) to afford 9i (680 mg, 92%) as a yel-
low solid. 1H NMR (400 MHz, DMSO-d6) d 11.53 (bs, 1H), 9.03 (s,
1H), 8.85 (s, 1H), 8.21 (d, J ¼ 8.4 Hz, 2H), 8.02–8.00 (m, 2H),
7.95–7.93 (m, 2H), 7.88 (d, J ¼ 8.0 Hz, 1H), 7.71–7.63 (m, 3H),
7.59–7.55 (m, 1H), 7.31–7.21 (m, 15H). LCMS (ESI) m/z:
784.27 [M þ H]þ.
General procedure A was used to transform 8 (50 mg, 0.09 mmol)
to the target compound. The resulting residue was subjected to
flash column chromatography on silica gel (40–90% THF/hexane)
to afford 10 b (21.5 mg, 51%) as a white solid. 1H NMR (400 MHz,
DMSO-d6) d 14.08 (bs, 1H), 11.69 (bs, 1H), 9.16 (d, J ¼ 2.0 Hz, 1H),
9.10 (d, J ¼ 2.4 Hz, 1H), 8.46 (bs, 1H), 8.39 (d, J ¼ 7.6 Hz, 1H), 8.02
(d, J ¼ 8 Hz, 1H), 7.94–7.91 (m, 1H), 7.89 (d, J ¼ 8.0 Hz, 1H), 7.82 (t,
J ¼ 7.6 Hz, 1H), 7.72–7.66 (m, 1H), 7.59–7.54 (m, 1H). 13 C NMR
(100 MHz, DMSO-d6) d 165.11, 163.51, 161.02, 152.54, 148.37,
143.83, 143.76, 142.47, 142.20, 135.28, 132.65, 132.57, 130.35,
130.08, 129.08, 127.81, 123.85, 121.43, 121.22, 117.99, 114.82,
114.58, 112.87, 107.60. LCMS (ESI) m/z: 465.22 [M þ H]þ.
N-(5-((3-Fluorophenyl)sulfonyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)-4-
(trifluoromethyl)benzamide (10c)
General procedure A was used to transform 8 (50 mg, 0.09 mmol)
to the target compound. The resulting residue was subjected to
4-Amino-N-(5-((3-fluorophenyl)sulfonyl)-1-trityl-1H-pyrazolo[3,4-
b]pyridin-3-yl)benzamide (11)
A mixture of compound 9i (200 mg, 0.26 mmol) and hydrazine
monohydrate (0.06 mL, 1.3 mmol) in THF (4 mL) and EtOH (4 mL) flash column chromatography on silica gel (40–90% THF/hexane)

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1429
to afford 10c (18.7 mg, 45%) as a white solid. 1H NMR (400 MHz, DMSO-d6) d 13.94 (bs, 1H), 11.07 (bs, 1H), 9.12 (d, J ¼ 2.4 Hz, 1H),
9.07 (d, J ¼ 2.4 Hz, 1H), 8.00 (d, J ¼ 8.8 Hz, 2H), 7.93–7.88 (m, 2H),
7.71–7.66 (m, 1H), 7.58–7.53 (m, 1H), 7.03 (d, J ¼ 8.8 Hz, 2H),
3.33–3.31 (m, 4H), 2.46–2.43 (m, 4H), 2.23 (s, 3H). 13 C NMR
(100 MHz, DMSO-d6) d 164.94, 163.16, 160.68, 153.35, 152.21,
147.94, 143.54, 143.48, 142.21, 135.08, 132.28, 132.21, 129.69,
128.58, 123.49, 121.48, 121.05, 120.83, 114.46, 114.21, 113.29,
107.30, 79.12, 64.85, 54.29, 46.70, 45.68, 15.11. LCMS (ESI) m/z:
495.27 [M þ H]þ.
DMSO-d6) d 14.07 (bs, 1H), 11.65 (bs, 1H), 9.16 (d, J ¼ 2.0 Hz, 1H),
9.10 (d, J ¼ 1.6 Hz, 1H), 8.29 (d, J ¼ 8.0 Hz, 2H), 7.94 (d, J ¼ 8.4 Hz,
2H), 7.92–7.88 (m, 2H), 7.72–7.66 (m, 1H), 7.59–7.56 (m, 1H). 13 C
NMR (100 MHz, DMSO-d6) d 164.92, 163.61, 161.13, 152.65, 148.55,
143.91, 143.85, 141.86, 137.50, 135.27, 132.75, 132.67, 132.43,
132.11, 129.56, 129.36, 125.85, 125.81, 125.78, 125.64, 123.96,
123.93, 122.93, 121.55, 121.34, 114.93, 114.69, 107.65. LCMS (ESI)
m/z: 465.22 [M þ H]þ.
N-(5-((3-Fluorophenyl)sulfonyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)-
3–(4-methylpiperazin-1-yl)benzamide (10h)
N-(5-((3-Fluorophenyl)sulfonyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)-4-
methoxybenzamide (10d)
General procedure A was used to transform 8 (50 mg, 0.09 mmol)
to the target compound. The resulting residue was subjected to
flash column chromatography on silica gel (50–100% THF/hexane)
to afford 10 h (9 mg, 20%) as a white solid. 1H NMR (400 MHz,
DMSO-d6) d 11.35 (bs, 1H), 9.13 (d, J ¼ 2.0 Hz, 1H), 9.08 (d,
J ¼ 2.4 Hz, 1H), 7.95–7.91 (m, 1H), 7.89 (d, J ¼ 8.0 Hz, 1H), 7.71–7.66
(m, 1H), 7.64 (bs, 1H), 7.59–7.54 (m, 1H), 7.5 (d, J ¼ 7.6 Hz, 1H), 7.38
(t, J ¼ 8.4 Hz, 1H), 7.21–7.18 (m, 1H), 3.26–3.24 (m, 4H), 2.48–2.47
(m, 4H), 2.24 (s, 3H). LCMS (ESI) m/z: 495.27 [M þ H]þ.
General procedure A was used to transform 8 (50 mg, 0.09 mmol)
to the target compound. The resulting residue was subjected to
flash column chromatography on silica gel (30–70% THF/hexane)
to afford 10d (13.3 mg, 35%) as a white solid.1H NMR (400 MHz,
DMSO-d6) d 13.96 (bs, 1H), 11.24 (bs, 1H), 9.10 (d, J ¼ 17.6 Hz, 2H),
8.11 (d, J ¼ 7.6 Hz, 2H), 7.93–7.88 (m, 2H), 7.69–7.68 (m, 2H),
7.58–7.56 (m, 1H), 7.09 (d, J ¼ 8 Hz, 2H), 3.86 (s, 3H). 13 C NMR
(100 MHz, DMSO-d6) d 165.24, 163.52, 162.73, 152.57, 148.35,
143.86, 143.80, 135.32, 132.66, 132.58, 130.56, 129.05, 125.47,
123.84, 121.44, 121.22, 114.84, 114.59, 114.04, 107.65, 55.81. LCMS
(ESI) m/z: 426.27 [M þ H]þ.
4-Amino-N-(5-((3-fluorophenyl)sulfonyl)-1H-pyrazolo[3,4-b]pyridin-
3-yl)benzamide (12)
To a solution of 11 (50 mg, 0.076 mmol) in DCM (1 mL) was added
TFA (0.1 mL, 0.15 mmol) dropwise. The mixture was stirred at
room temperature for 0.5 h, diluted with water, and extracted
with DCM. The organic phase was washed with brine, dried over
Na2SO4, filtered and concentrated. The resulting crude product
was subjected to flash column chromatography on silica gel
(40–80% THF/hexane) to afford 12 (16 mg, 51%) as a pale yellow
4-(Dimethylamino)-N-(5-((3-fluorophenyl)sulfonyl)-1H-pyrazolo[3,4-
b]pyridin-3-yl)benzamide (10e)
General procedure A was used to transform 8 (50 mg, 0.09 mmol)
to the target compound. The resulting residue was subjected to
flash column chromatography on silica gel (50–90% THF/hexane)
to afford 10e (10.1 mg, 26%) as a yellow solid. 1H NMR (400 MHz,
DMSO-d6) d 13.91 (bs, 1H), 10.99 (bs, 1H), 9.12 (d, J ¼ 2.0 Hz, 1H),
9.06 (d, J ¼ 2.0 Hz, 1H), 8.01 (d, J ¼ 8.8 Hz, 2H), 7.92 (d, J ¼ 8.4 Hz,
1H), 7.89 (d, J ¼ 8.0 Hz, 1H), 7.71–7.65 (m, 1H), 7.58–7.53 (m, 1H),
6.77 (d, J ¼ 8.8 Hz, 2H), 3.02 (s, 6H). 13 C NMR (100 MHz, DMSO-d6)
d 165.64, 163.69, 161.21, 153.26, 152.75, 148.45, 144.08, 144.02,
142.91, 135.69, 132.82, 132.75, 130.28, 129.06, 124.00, 121.58,
121.37, 119.55, 114.98, 114.74, 111.25, 107.85, 67.50, 49.08, 34.86,
30.64, 25.60. LCMS (ESI) m/z: 440.27 [M þ H]þ.
1
solid. H NMR (400 MHz, DMSO-d6) d 13.89 (bs, 1H), 10.87 (bs, 1H),
9.06 (d, J ¼ 6.0 Hz, 2H), 7.93–7.84 (m, 4H), 7.71–7.65 (m, 1H),
7.58–7.54 (m, 1H), 6.61 (d, J ¼ 8.4 Hz, 2H), 5.87 (bs, 2H). 13 C NMR
(100 MHz, DMSO-d6) d 165.70, 163.68, 161.19, 153.22, 152.73,
148.41, 144.09, 144.02, 142.94, 135.63, 132.80, 132.72, 130.52,
129.00, 124.00, 123.97, 121.55, 121.34, 119.58, 114.96, 114.72,
113.04, 107.85, 31.15. LCMS (ESI) m/z: 412.27 [M þ H]þ.
General procedure B for the synthesis of compounds 13a–13c
To a solution of 11 (1 equiv) in pyridine (0.1 M) was added various
acid chlorides (1.05 equiv) and DMAP (0.6 equiv) at 0 ꢃC under
nitrogen atmosphere. The mixture was stirred at room tempera-
ture for 0.5 h, then diluted with water, and extracted with DCM.
The organic layer was dried over Na2SO4, filtered, and concen-
trated. The residue was diluted with dry DCM (0.5 M) and treated
slowly with TFA (2 equiv) at 0 ꢃC. The mixture was stirred at room
temperature for 0.5 h, diluted with water, and extracted with
DCM. The organic phase was washed with brine, dried over
Na2SO4, filtered and concentrated. The resulting crude product
was subjected to flash column chromatography on silica gel.
N-(5-((3-Fluorophenyl)sulfonyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)-4-
morpholinobenzamide (10f)
General procedure A was used to transform 8 (50 mg, 0.09 mmol)
to the target compound. The resulting residue was subjected to
flash column chromatography on silica gel (50–90% THF/hexane)
to afford 10f (11.5 mg, 27%) as a white solid. 1H NMR (400 MHz,
DMSO-d6) d 13.95 (bs, 1H), 11.10 (bs, 1H), 9.12 (s, 1H), 9.07 (s, 1H),
8.03 (d, J ¼ 8.0 Hz, 2H), 7.93–7.88 (m, 2H), 7.69–7.68 (m, 1H),
7.58–7.56 (m, 1H), 7.04 (d, J ¼ 8.0 Hz, 2H), 3.75 (bs, 4H), 3.29 (m,
4H). 13 C NMR (100 MHz, DMSO-d6) d 165.44, 16.68, 161.20, 154.03,
152.73, 148.47, 144.05, 143.99, 142.70, 135.59, 132.80, 132.72,
130.18, 129.11, 123.98, 122.54, 121.57, 121.36, 114.98, 114.74,
113.69, 107.81, 66.37, 47.57. LCMS (ESI) m/z: 482.27 [M þ H]þ.
4–(3,3-Dimethylureido)-N-(5-((3-fluorophenyl)sulfonyl)-1H-pyra-
zolo[3,4-b]pyridin-3-yl)benzamide (13a)
N-(5-((3-Fluorophenyl)sulfonyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)-
4–(4-methylpiperazin-1-yl)benzamide (10g)
General procedure
B was used to transform 11 (50 mg,
0.076 mmol) to the target compound. The resulting residue was
General procedure A was used to transform 8 (50 mg, 0.09 mmol)
to the target compound. The resulting residue was subjected to
flash column chromatography on silica gel (50–100% THF/hexane)
subjected to flash column chromatography on silica gel (50–100%
THF/hexane) to afford 13a (16 mg, 43%) as a white solid. H NMR
1
(400 MHz, DMSO-d6) d 13.96 (bs, 1H), 11.18 (bs, 1H), 9.13 (d,
to afford 10 g (16 mg, 36%) as a white solid. 1H NMR (400 MHz, J ¼ 2.4 Hz, 1H), 9.08 (d, J ¼ 2.0 Hz, 1H), 8.64 (bs, 1H), 8.02 (d,

1430
Y. NAM ET AL.
J ¼ 8.8 Hz, 2H), 7.94–7.88 (m, 2H), 7.71–7.65 (m, 3H), 7.59–7.56 (m, Anti-proliferation assay
1H), 2.96 (bs, 6H). 13 C NMR (100 MHz, DMSO-d6) d 165.54, 163.67,
Cells (ALK wt-TEL, ALK L1196M-TEL and Parental Ba/F3: 1 ꢂ 104,
161.19, 155.78, 152.72, 148.49, 145.12, 144.04, 143.97, 142.54,
135.51, 132.81, 132.73, 129.34, 129.18, 125.94, 124.01, 123.99,
121.58, 121.37, 118.70, 114.98, 114.73, 107.80, 36.75. LCMS (ESI) m/
z: 483.27 [M þ H]þ.
H2228: 5 ꢂ 103) were plated in 96 well tissue culture plates. Each
compound was added to each well at 10 dose points of three-fold
serial dilution in DMSO. After treatment with each compound for
72 h, CTG assay solution (Promega, # G7572) was added to each
well. Cell proliferation was assessed by measuring the lumines-
cence using a 96 well plate reader (EnVision 2013).
N-(5-((3-Fluorophenyl)sulfonyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)-4-
propionamidobenzamide (13b)
Western blot
General procedure
B was used to transform 11 (50 mg,
0.076 mmol) to the target compound. The resulting residue was
Cells were harvested and lysed using IP buffer containing 50 mM
HEPES (pH 7.4), 1% Triton X-100, 2 mM EDTA, 150 mM NaCl,
2.5 mM NaF, 5 mM Na3VO4, protease inhibitor cocktail tablet
(Roche, # 11–878-580–001). The protein concentration was deter-
subjected to flash column chromatography on silica gel (50–100%
1
THF/hexane) to afford 13b (18 mg, 50%) as a white solid. H NMR
(400 MHz, DMSO-d6) d 13.99 (bs, 1H), 11.26 (bs, 1H), 10.19 (bs, 1H),
9.13 (d, J ¼ 2.4 Hz, 1H), 9.08 (d, J ¼ 2.0 Hz, 1H), 8.08 (d, J ¼ 8.8 Hz, mined by Bradford assay. Proteins were separated using SDS-PAGE
and transferred onto NC membrane. The membranes were
blocked using 5% skim milk in TBS-T buffer. The rabbit polyclonal
antibody against phospho-ALK (Tyr1604, # 3341), phospho-ERK1/2
2H), 7.94–7.91 (m, 1H), 7.89 (d, J ¼ 8 Hz, 1H), 7.76 (d, J ¼ 8.4 Hz,
2H), 7.71–7.66 (m, 1H), 7.58–7.56 (m, 1H), 2.38 (q, J ¼ 7.6 Hz, 2H),
1.10 (tr, J ¼ 7.6 Hz, 3H). 13 C NMR (100 MHz, DMSO-d6) d 172.91,
(Thr202/Tyr204, # 8544), phospho- PLC-gamma (Tyr783, # 14008),
phospho-STAT3 (Tyr705, # 4074) and cleaved caspase3 (# 9661)
were purchased from Cell Signalling Technologies, and PARP1/2
(H-250, # sc-7150) and anti-b-actin (# sc-47778) antibody was
obtained from Santa Cruz Biotechnology. All primary antibodies
were diluted in TBS-T at 1:1000. Each primary antibody was incu-
bated overnight at 4 ꢃC, followed by the secondary antibody treat-
ment for 1 h at room temperature. Secondary antibodies were
purchased from genDEPOT. Proteins were detected using ECL sub-
strate, and then exposed to X-ray film.
165.30, 163.57, 161.09, 152.61, 148.41, 143.92, 143.85, 143.28,
142.34, 135.38, 132.69, 132.61, 129.60, 129.12, 127.42, 123.91,
123.88, 121.47, 121.26, 118.49, 114.88, 114.64, 107.69, 30.00, 9.85.
LCMS (ESI) m/z: 468.27 [M þ H]þ.
N-(5-((3-Fluorophenyl)sulfonyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)-4-
(methylsulfonamido)benzamide (13c)
General procedure
B was used to transform 11 (50 mg,
0.076 mmol) to the target compound. The resulting residue was
subjected to flash column chromatography on silica gel (50–100%
1
THF/hexane) to afford 13c (12 mg, 32%) as a pale yellow solid. H
Apoptosis analysis and cell cycle arrest
NMR (400 MHz, DMSO-d6) d 14.01 (bs, 1H), 11.30 (bs, 1H), 9.12 (d,
J ¼ 2.0 Hz, 1H), 9.08 (d, J ¼ 2.0 Hz, 1H), 8.10 (d, J ¼ 8.4 Hz, 2H),
7.93–7.88 (m, 2H), 7.71–7.66 (m, 1H), 7.59–7.54 (m, 1H), 7.32 (d,
J ¼ 8.4 Hz, 2H), 3.12 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) d
164.69, 161.19, 152.72, 148.61, 143.98, 143.91, 141.98, 135.40,
134.63, 132.82, 132.74, 130.28, 129.87, 129.55, 129.40, 129.21,
125.42, 125.37, 124.02, 121.61, 121.41, 114.99, 114.74, 107.74.
LCMS (ESI) m/z: 490.27 [M þ H]þ.
After 24–48 h following compound treatment, cells were harvested
and stained with annexin V (1:500 diluted in annexin V buffer) for
30 min and PI (1:200) solution for 30 min. 1 ꢂ 106 cells were ana-
lysed using flow cytometric analysis. For the cell cycle arrest ana-
lysis, cells were harvested by trypsinisation and fixed with 70%
ethanol overnight at ꢁ20 ꢃC. The next day, cells were harvested
and washed with cold DPBS at 500 ꢂ g. The cells were suspended
in RNase/PI solution (Cell Signalling Technologies, # 4087).
Permeability assessment for 10g using Caco-2 cells
Cell culture and reagent
Stock solutions (10 mM) of reference compounds and 10g were
diluted to a concentration of 10 mM with the transport buffer
(HBSS þ 1%BSA) and the test compounds were applied to the
apical or basolateral side of the cell monolayer. Permeability of
the test compounds from A to B direction or B to A direction was
assessed in duplicate over a 120 min incubation at 37 ꢃC and 5%
CO2 with a relative humidity of 95%. In addition, the efflux ratio
of each compound was also determined. Reference compounds
and 10g were quantified by LC-MS/MS analysis based on the peak
area ratio of analyte/the internal standard.
ALK wt-TEL, ALK L1196M-TEL transformed Ba/F3 and H2228 cell
lines were cultured in RPMI1640 (Welgene, # LM011-01). The cul-
ture media was supplemented with 10% foetal bovine serum
(Hyclone), Antibiotic-Antimycotic solution (Welgene, # LS203-01)
containing 10,000 U/mL penicillin, 10 mg/mL streptomycin and
25 mg/mL amphotericin B in 0.85% NaCl. Parental Ba/F3 cells were
cultured in RPMI1640 media supplemented with 10% foetal
bovine serum and 1% penicillin/streptomycin solution in the pres-
ence of IL-3. The cells were maintained in a humidified atmos-
phere containing 5% CO2 at 37 ꢃC.
Molecular docking study
Biochemical kinase assay
X-ray co-crystal structures of ALK kinase domain complexed with
crizotinib (PDB code: 2XP2) and ALK kinase domain in complex
with entrectinib (PDB code: 5FTO) were retrieved from Protein
Data Bank. Molecular docking studies were carried out by meth-
ods previously reported40.
Biochemical kinase assay was performed by methods previously
reported39. Kinase reactions of all test compounds except 10g
were carried out at 10 mM ATP. Kinase reaction of 10g was per-
formed at three different concentrations of ATP (10/50/100 mM).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1431
Statistics
Rationale for the design of pyrazolo[3,4-b]pyridines and
SAR study
Statistical analysis was performed using GraphPad Prism (Ver
6.01). All values are expressed as the mean standard deviation.
The goal of the investigation described below was to identify
unique and potent inhibitors against both ALK-wt and ALK-
L1196M. Based on the observation that entrectinib11 strongly
inhibits ALK-L1196M41, we selected the indazole platform design
strategy because substances in this family have not been widely
explored in efforts focusing on the discovery of ALK inhibitors
while they have been investigated11 in our other kinase programs.
Additional considerations led us to choose the closely related pyr-
azolo[3,4-b]pyridine group, which is superior to indazole in terms
of cLogP, as the core structure. It should be noted that ALK inhib-
ition properties of this type of substances have not been eluci-
dated previously. It was reported42 that a fluorine group at the C3
position of the phenyl group in crizotinib contributes to potency
and binding efficiency, and that entrectinib contains a related flu-
orophenyl moiety. These findings prompted us to incorporate a 3-
fluorophenyl group in the newly designed pyrazolo[3,4-b]pyridine
based ALK inhibitors. Moreover, we envisioned that the methylene
linker between indazole core and difluorophenyl ring of entrecti-
nib could be optimised to promote additional interaction with
ALK. In addition, the results of docking studies suggested that the
methylene linker in entrectinib could be replaced by a sulfone
group. These considerations led us to design a new family poten-
tial ALK inhibitors that contain a common 5-((3-fluorophenyl)sul-
fonyl)-1H-pyrazolo[3,4-b]pyridine moiety and various head groups
that should reside in solvent exposed region.
Results and discussion
Chemistry
As shown in Scheme 1, synthesis of the pyrazolo[3,4-b]pyridine
derivatives commenced with bromination of commercially avail-
able 2-oxo-1,2-dihydropyridine-3-carbonitrile 1 using bromine in
AcOH to give bromide 2, which was substituted with 3-fluoroben-
zenethiol using CuI and K2CO3 to afford 3 in 90% yield. Sulphide
3 was treated with PCl5/POCl3 to give the chloride 4 (93%), which
was oxidised using m-CPBA to furnish the sulfone 5. Subjection of
5 to aminopyrazole ring formation conditions using hydrazine fol-
lowed by protection of the resulting amine group with phthalic
anhydride yielded 6 in 72% yield. The pyrazole moiety of 6 was
protected with a trityl group to give 7 and phthalimide protecting
group of 7 was removed using hydrazine to afford the aminopyra-
zole 8. Amide coupling reactions of 8 with various benzoyl chlor-
ides were performed in the presence of DMAP to produce the
amides 9a-i. The trityl groups in 9a-h and 11 were removed using
TFA to yield the target the pyrazolo[3,4-b]pyridine derivatives
10a-h and 12, respectively. The aniline 11 was transformed to the
corresponding urea, amide and sulfonamide followed by removal
of trityl group to give the target derivatives 13a-c.
H
N
H
N
H
N
Cl
N
O
C
O
C
O
C
a
b
c
C
S
F
F
Br
S
F
N
N
N
N
O
1
2
3
4
7
H
Trt
N
N
N
N
N
O
S
Cl
N
N
N
O
S
O
F
d
e, f
g
O
S
N
O
O
N
C
F
O
N
O
O
5
8
6
H
N
Trt
Trt
N
N
N
N
R1
N
O
S
O
h
i
j
R1
N
O
S
O
N
O
S
O
F
F
F
NH
H2N
NH
O
O
9a-i
9d/10d, R1 = 4-methoxy
10a-h
9g/10g, R1 = 4-methylpiperazin-1-yl
9a/10a, R1 = 4-cyano
9b/10b, R1 = 3-trifluoromethyl 9e/10e, R1 = 4-dimethylamino 9h/10h, R1 = 3-methylpiperazin-1-yl
9c/10c, R1 = 4-trifluoromethyl 9f/10f, R1 = 4-morpholino
Trt
H
N
O
H
N
N
H2N
N
Trt
R2
N
h
k, j
N
N
N
O
S
N
O
S
O
N
O
N
O
S
O
F
F
NH
NH
F
O
NH
O
O
9i
11
13a-c
O
j
H
N
H2N
N
N
O
13a, R2 = 3,3-dimethylureido
13b, R2 = propionyl
13c, R2 = methanesulfonyl
S
F
NH
O
12
O
Scheme 1. Synthesis of derivatives 10a-h, 12, 13a-ca.
aReagents and conditions: (a) Br2, Acetic acid, rt (85%); (b) 3-fluorobenzenethiol, copper iodide, K2CO3, DMF, 120ꢃC (90%); (c) PCl5, POCl3, 100 ꢃC (93%); (d) m-CPBA, Na2SO3, DCM, 0 ꢃC (79%);
(e) hydrazine monohydrate, EtOH, 80 ꢃC; (f) phthalic anhydride, 1,4-dioxane, 100ꢃC (72% over 2 steps); (g) triphenylmethyl chloride, TEA, DCM, 0 ꢃC (75%); (h) hydrazine monohydrate, THF,
EtOH, rt (85%); (i) various benzoyl chloride, DMAP, pyridine, 0 ꢃC (70–85%); (j) TFA, DCM, rt (35–45% over 2 steps); (k) for 13a, dimethylcarbamic chloride, DMAP, pyridine, 0 ꢃC (43%); for 13b,
propionyl chloride, DMAP, pyridine, 0 ꢃC (50%); for 13c, methanesulfonyl chloride, DMAP, pyridine, 0 ꢃC (32%).

1432
Y. NAM ET AL.
Table 1. Kinase-inhibitory activities of 1H-pyrazolo[3,4-b]pyridine derivatives against ALK-wt and ALK-L1196M.
H
N
N
N
O
S
O
F
R1
NH
O
Enzymatic activity (IC50, nM)a,b
Entry
R1
–
ALK WT
25 c
ALK L1196M
980 c
crizotinib
10a
NC
453
4400
Inactive
69
10b
10c
10d
F3C
F3C
O
N
19
10e
10f
7.3
0.6
0.7
O
N
1.4
N
N
10g
< 0.5
< 0.5
10h
12
19
24
13
34
N
N
H2N
H
N
N
13a
143
O
O
H
N
13b
60
39
15
39
H
O
S
N
13c
O
aRadiometric kinase assay.
b‘Inactive’ means that kinase activity is inhibited by less than 50% even at 10 lM concentration of compound.
cActivity value from the reference15
.
The inhibitory activities of the pyrazolopyridine derivatives achieved with the 4-morpholino derivative 10f, which is also
extremely active against ALK-L1196M (IC50 ¼ 1.4 nM). The SAR
against both ALK-wt and ALK-L1196M were assessed by using an
study led us to identify 10g containing a 4-methylpiperazin-1-yl
group as the most potent inhibitor against both ALK-wt
(IC50 < 0.5 nM) and ALK-1196M (IC50 < 0.5 nM).
in vitro biochemical assay. As shown in Table 1, the kinase-inhibi-
tory activities of the derivatives were highly dependent on the R1
group. For example, 10a containing a 4-cyano group displayed a
reasonable inhibitory activity (IC50 ¼ 453 nM) on ALK-wt.
Introduction of a trifluoromethyl group as R1 (10b and 10c)
resulted in little to no activity against ALK-wt. In contrast, the
4-methoxy containing derivative 10d has an enhanced activity
against ALK-wt (IC50 ¼ 69 nM) and it possesses a high activity
(IC50 ¼ 19 nM) against ALK-L1196M gatekeeper mutation, a value
that is 50-fold higher than that (IC50 ¼ 980 nM) of crizotinib.
Moreover, replacement of the 4-methoxy group by a 4-dimethyla-
mino group led to 10e, which was found to exhibit picomolar
activity against ALK-L1196M. It is worthwhile to note that 10e is
Antiproliferative activities of selected pyrazolo[3,4-b]pyridines
Based on the results arising from studies of the kinase-inhibitory
activities of the pyrazolo[3,4-b]pyridine derivatives against ALK-wt
and ALK-L1196M gatekeeper mutant, we selected the most potent
inhibitors and measured their antiproliferative activities on Ba/F3
cells transformed with ALK-wt/ALK-L1196M and on H2228 non-
small cell lung cancer cells harbouring EML4-ALK. Ba/F3 cell lines
transformed with ALK-wt and ALK-L1196M mutant were employed
more potent against ALK-L1196M (IC50 ¼ 0.7 nM) than against ALK- to assess the ALK inhibition capability of the derivatives in a cellu-
wt (IC50 ¼ 7.3 nM). Picomolar inhibitory activity against ALK-wt was lar context and parental Ba/F3 cells were utilised as controls to

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1433
Table 2. Antiproliferative activities of 1H-pyrazolo[3,4-b]pyridine derivatives against Ba/F3 transformed with ALK and H2228 NSCLC cancer cell.
GI50 (lM)a,b
Ba/F3 cell lines
Entry
H2228 (EML4-ALK)
Parental
ALK wt-TEL
ALKL1196M-TEL
crizotinib
10d
10e
10f
10g
10h
12
13c
0.249 0.06
Inactive
8.538 0.78
1.693 0.40
0.219 0.05
Inactive
1.654 0.13
Inactive
Inactive
0.141 0.08
Inactive
0.726 0.21
Inactive
1.767 0.69
0.916 0.50
0.205 0.06
3.352 0.24
3.869 1.54
4.708 3.02
4.549 0.72
2.527 1.50
0.129 0.02
2.276 0.59
1.980 0.28
5.625 2.34
Inactive
3.495 1.13
15.18 0.52
Inactive
4.033 1.81
9.215 1.92
inactive
aGI50 represents the concentration at which a compound causes half-maximal growth inhibition. GI50 value for parental, Ba/F3 transformed with ALK and H2228 cell
lines were shown as the means standard deviation (SD) of three independent experiments.
b‘Inactive’ means that the proliferation was suppressed by less than 10% even at 50 lM concentration of compound.
Table 3. Cell permeability assessment of 10g using Caco-2 cells.
Mean Pappa (10–6 cm/s)
Mean recovery %
Rank
A to B
B to A
Efflux ratio
A to B
B to A
Papp
Low
High
Low
Moderate
P-gp
Atenolol
Propranolol
Digoxin
10g
1.69
16.05
0.43
2.14
8.42
8.60
4.74
1.27
0.52
20.22
1.85
92.60
84.81
96.82
74.17
91.61
91.66
95.09
92.30
Substrate
2.56
aPapp (A to B) < 2.5: Low permeability, 2.5< Papp (A to B) < 10: Moderate permeability, Papp (A to B) > 10: High permeability.
determine differential cytotoxicities. The antiproliferative activities
of the selected pyrazolo[3,4-b]pyridines were further elucidated
using the H2228 NSCLC cell line, which is an EML4-ALK positive
cancer cell line. As the data in Table 2 show, the overall cellular
activities of the selected pyrazolo[3,4-b]pyridines are relatively
moderate compared with their enzymatic activities. In particular, it
is difficult to understand why 10d is potent against ALK enzyme
Table 4. IC50 values of 10g at various ATP concentrations.
IC50 (nM)a
ALK-wt
[ATP](10 lM)
<0.5
[ATP](50 lM)
[ATP](100 lM)
10g
17.3
24.9
The IC50 values of 10g against ALK-wt were measured depending on ATP con-
but inactive on H2228 and ALK-driven Ba/F3 cells. Among the centrations ranging from 10 to 100 lM.
aRadiometric biochemical kinase assay.
compounds tested, 10g most strongly suppressed proliferation of
both
H2228
(GI50 ¼ 0.219 mM)
and
ALK-driven
Ba/F3
cells (GI50 < 0.205 mM).
high antiproliferative activities on H2228 and ALK-driven Ba/F3
cells. Like 10g, with exception of 10d, all of the other pyra-
zolo[3,4-b]pyridines tested including 10e, 10f, 10h, 12, and 13c,
exhibit higher differential cytotoxicities against ALK-L1196M Ba/F3
cells vs. parental Ba/F3 cells than does crizotinib. Based on the
results of the antiproliferative activity studies, 10g was selected
for further cellular investigations.
In order to understand the discrepancy between enzymatic
and cellular activities, we first assessed the cell permeability of
10g using the human colon carcinoma cell line Caco-2. It was
found that 10g has moderate permeability and is not a substrate
of P-glycoprotein (P-gp) as evidenced by the fact that the efflux
ratio of 10g is 1.85 (Table 3). This finding indicates that the cell
permeability of 10g is not the reason for the discrepancy. We
next measured the kinase-inhibitory activities of 10g against ALK-
wt at three different ATP concentrations because the IC50 value
derived from biochemical kinase assay depends on both Ki and
Km, which are defined by ATP concentration43,44. As described in
Table 4, it was observed that a 10-fold increase in ATP concentra-
tion resulted in a 50-fold decrease in IC50 (IC50 ¼ 24 nM at 100 mM
ATP). It should be noted that the physiological ATP concentration
is around 1 mM and the IC50 value of 10g should be much higher
than 24 nM at 1 mM ATP concentration, which may explain the
discrepancy.
Furthermore, it should be emphasised that 10g more strongly
inhibits (GI50 ¼ 0.129 mM) proliferation of Ba/F3 cells transformed
with ALK-L1196M than crizotinib (GI50 ¼ 0.726 mM). In addition,
10g is 27-fold more potent against ALK-L1196M Ba/F3 cells than
parental Ba/F3 cells while crizotinib is just 2-fold more potent
against ALK-L1196M Ba/F3 cells than parental Ba/F3 cells. These
findings indicate that 10g possesses much more favourable differ-
ential cytotoxicity than does crizotinib. Pyrazolo[3,4-b]pyridine 10f,
Kinase profile of 10g
The inhibitory activities of 10g against ROS1, c-Met, IRK (insulin
receptor kinase), c-Src, and Lyn were assessed by estimating IC50
values derived from biochemical kinase assay (Figure 2). As is
expected by considering its structural similarity to entrectinib, 10g
inhibits ROS1 (IC50 < 1 nM) as well as ALK-wt (IC50 < 0.5 nM). As a
matter of fact, 10g is the most extremely potent inhibitor of ROS1
(IC50 < 1 nM). The activity of 10g against c-Met is low
(IC50 ¼ 3775 nM) and it is over 7000-fold more potent against ALK-
wt (IC50 < 0.5 nM) than c-Met. This observation indicates that 10g
possesses extremely high selectivity over c-Met. It is notable that
crizotinb inhibits c-Met as well as it does ALK with IC50 values less
than 1 nM23. Therefore, 10g is superior to crizotinib in terms of its
selectivity over c-Met. IRK is much less inhibited (IC50 ¼ 404 nM) by
10g than is ALK, which could be significantly advantageous in
terms of toxicity because the alteration of the insulin receptor kin-
the second most potent inhibitor of the ALK enzymes, displays ase activity could causes insulin resistance45. It is of interest to

1434
Y. NAM ET AL.
point out that 10g significantly inhibits both c-Src (IC50 ¼ 7 nM) Inhibition by 10g of ALK signalling in ALK-driven cell lines
and Lyn (IC50 ¼ 33 nM), a Src family kinase, because it was
In order to elucidate whether 10g is capable of decreasing the
reported that Lyn46 regulates activation of EGFR in lung cancer
level of ALK phosphorylation and deactivating downstream signal-
cells and c-Src47 is a potential therapeutic target in alectinib-resist-
ling molecules in a cellular context, western blot analysis was car-
ant patients. Therefore, it is anticipated that the remarkable activ-
ried out using H2228 and Ba/F3 cells transformed with ALK-wt/
ities of 10g against both c-Src and Lyn contribute to its potential
ALK-L1196M. In agreement with the results of biochemical kinase
as lead for lung cancer chemotherapy. We next measured the kin-
assays and antiproliferative activity assays, 10g (1 and 10 mM)
ase-inhibitory activities of 10g against other clinically relevant ALK
effectively attenuates ALK autophosphorylation in ALK wt-TEL Ba/
mutants including DFG motif mutant F1174L, aC helix mutant
F3, ALK L1196M-TEL Ba/F3 and H2228 cell lines (Figure 3). Also,
C1156Y, T1151-L1152insT. As shown in Table 5, it was observed
we found that the phosphorylation levels of STAT3, ERK and PLC-
that 10g strongly inhibits all of these mutants with single-digit
gamma, which are ALK downstream signalling molecules, are mod-
nanomolar IC50 values.
erately suppressed by 10g in a dose-dependent manner. Consistent
with the results of biochemical kinase assays and antiproliferative
activity assays, 10g is more effective than crizotinib in inhibiting
ALK autophosphorylation of ALK L1196M-TEL Ba/F3 cells. These
results indicate that 10g effectively inhibits the gatekeeper mutant
ROS1
c-Src
Lyn
< 1 nM
7 nM
ALK-L1196M as well as ALK-wt in a cellular context.
Effects of 10g on apoptosis and cell cycle arrest in ALK-driven
cell lines
33 nM
IRK
404 nM
Apoptosis plays a pivotal role in anticancer therapy and several
ALK inhibitors are able to induce cancer cell apoptosis. Therefore,
we determined whether 10g is capable of inducing apoptosis in
Ba/F3 cells transformed with ALK-TEL. Annexin V-FITC and PI stain-
ing was performed to estimate the rate of formation of apoptotic
cells after 10g treatment in Ba/F3 cells transformed with ALK wt-
TEL and ALK L1196M-TEL. FACS analysis showed that treatment
with 10g for 24 h increases the number of apoptotic cells in a
dose-dependent manner (Figure 4(a,b,d,e)). Furthermore, 10g
induces apoptosis of ALK L1196M-TEL Ba/F3 cells more effectively
than does crizotinib (Figure 4(d,e)). In order to confirm that 10g
induces apoptosis of Ba/F3 cells transformed with ALK wt-TEL and
ALK L1196M-TEL, the presence of apoptosis related protein
markers was assessed using western blotting. Treatment with 10g
results in increases in the levels of cleaved PARP and cleaved cas-
pase-3 (Figure 4(c,f)). The results demonstrate that the antiprolifer-
ative activity of 10g against ALK-driven Ba/F3 cells is associated
with its apoptosis induction capability. The effects of 10g on
apoptosis and cell cycle arrest in H2228 NSCLC cells harbouring
EML4-ALK were also determined. FACS analysis revealed that treat-
ment with 10g for 48 h induces apoptosis in H2228 cells in a dose
3775 nM
c-MET
0
20
40
400
500
4000
5000
IC (nM)
50
Figure 2. Kinase-inhibitory activities of the 10g against ROS1, c-Src, Lyn, IRK
(insulin receptor kinase) and c-Met.
Table 5. Kinase-inhibitory activities of 10g against clinically relevant
ALK mutants.
IC50 (nM)a
Enzyme
10g
ALK-wt
<0.5
<0.5
1.81
8.17
6.19
ALK-L1196M
ALK-C1156Y
ALK-F1174L
ALK-T1151-L1152insT
aRadiometric biochemical kinase assay.
Figure 3. Autophosphorylation of ALK and its downstream signalling are inhibited by 10g. (a) effects of 10g in ALK wt-TEL Ba/F3 (b) ALK L1196M-TEL Ba/F3 (c) H2228
cell line. Ba/F3 cells transformed with ALK wt-TEL, ALK L1196M-TEL and H2228 cells were treated for 2 h with increasing concentration of 10g.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1435
Figure 4. 10g induced apoptosis in Ba/F3 cells transformed with ALK-TEL. These cell lines were incubated with 24 h and 48h, respectively. (a, b) ALK wt-TEL Ba/F3
(d, e) ALK L1196M-TEL Ba/F3 cells were harvested and incubated in a binding buffer containing propidium iodide and annexin V-FITC and then analysed by FACS. t-
ꢀꢀ
ꢀꢀꢀ
p < .005. Treatment with 10g for 36h increased cleaved PARP1/2 and cleaved caspase-3 in Ba/F3 cell transformed with (c) ALK wt-TEL and (f) ALK
test p < .05,
L1196M-TEL Ba/F3 cells.
dependent fashion (Figure 5(a,b)). Also, treatment with 1 mM 10g co-crystal structures of the kinase domain of ALK complexed with
for 24 h leads to a significant enhancement of G1-S arrest in
H2228 cells (Figure 5(c)), suggesting that 10g inhibits cell prolifer-
ation via apoptosis and cell cycle arrest.
crizotinib (PDB code: 2XP2) and entrectinib (PDB code: 5FTO)
using Glide (Figures 6 and 7). Analysis of the results reveals that
10g forms three hydrogen bonds with the backbone carbonyl
oxygen of E1197 and backbone NH-carbonyl group of M1199 in
the hinge region. It is worthwhile to note that 10g is capable of
engaging in a favourable interaction with M1196 in the kinase
domain of ALK-L1196M (Figure 6(b)). It is important to note that
Molecular docking studies of 10g with ALK-wt and ALK-L1196M
To better understand the high kinase-inhibitory activity of 10g on
ALK, molecular docking studies were carried out using the X-ray M1196 sterically clashes with crizotinib and creates unfavourable

1436
Y. NAM ET AL.
Figure 5. Effect on the cell apoptosis and cell cycle arrest in H2228 cells. (a, b) Percent of apoptotic cells detected by FACS analysis in H2228 cells. cells were har-
ꢀꢀꢀ
vested and incubated in a binding buffer containing propidium iodide and annexin V-FITC. t-test
tion of 10 g, fixed and stained with propidium iodide and analysed by using FACS.
p < .005. (c) H2228 cells were incubated with indicated concentra-
Figure 6. Docking study of 10g on ALK kinase domain (a) WT and (b) L1196M based on an X-ray crystal structure (PDB code: 2XP2).
Figure 7. The predicted binding mode (a) of 10g (carbon in green) based on an X-ray crystal structure (PDB code: 5FTO) is superimposed (b) to the X-ray binding
mode of entrectinib (carbon in yellow).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1437
interactions with amino group and methyl substituents of crizoti-
nib28. Moreover, the sulfone group of 10g participates in H-bond-
ing with K1150 in the kinase domain of ALK-L1196M, which might
be the reason for the improved binding affinity of 10g with both
wt and L1196M. In contrast to crizotinib, 10g is involved in a H-
bonding interaction with E1210 in solvent exposed region, which
might also contribute to extremely high potency of 10g on ALK
enzyme. In addition, the predicted binding mode of 10g was
superimposed to the X-ray binding mode of entrectinib (Figure 7).
This superimposition reveals that the sulfone linker in 10g makes
a water molecule-mediated hydrogen bond with K1150 and
D1270 while the methylene linker of entrectinib is not capable of
forming this H-bond.
Funding
This study was financially supported by Korea Institute of Science
and Technology (KIST), the KU-KIST Graduate School of
Converging Science and Technology Program, and Candidate
Development Program (NRF-2016M3A9B5940991) of the National
Research Foundation of Korea funded by the Ministry of Science
and ICT.
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Disclosure statement
No potential conflict of interest was reported by the authors.

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