Synthesis and identification of GZD856 as an orally bioavailable Bcr-AblT315I inhibitor overcoming acquired imatinib resistance

Article abstract of DOI:10.1080/14756366.2016.1250757

Bcr-Abl^T315I induced drug resistance remains a major challenge to chronic myelogenous leukemia (CML) treatment. Herein, we reported GZD856 as a novel orally bioavailable Bcr-Abl^T315I inhibitor, which strongly suppressed the kinase activities of both native Bcr-Abl and the T315I mutant with IC₅₀ values of 19.9 and 15.4 nM, and potently inhibited proliferation of corresponding K562, Ba/F3^WT and Ba/F3^T315I cells with IC₅₀ values of 2.2, 0.64 and 10.8 nM. Furthermore, GZD856 potently suppressed tumor growth in mouse bearing xenograft K562 and Ba/F3 cells expressing Bcr-Abl^T315I. Thus, GZD856 may serve as a promising lead for the development of Bcr-Abl inhibitors overcoming acquired imatinib resistance.

Full text of DOI:10.1080/14756366.2016.1250757

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and identification of GZD856 as an orally
bioavailable Bcr-AblT315I inhibitor overcoming
acquired imatinib resistance
Xiaoyun Lu, Zhang Zhang, Xiaomei Ren, Deping Wang & Ke Ding
To cite this article: Xiaoyun Lu, Zhang Zhang, Xiaomei Ren, Deping Wang & Ke Ding (2017)
Synthesis and identification of GZD856 as an orally bioavailable Bcr-AblT315I inhibitor overcoming
acquired imatinib resistance, Journal of Enzyme Inhibition and Medicinal Chemistry, 32:1, 331-336,
DOI: 10.1080/14756366.2016.1250757
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Date: 24 March 2017, At: 00:11

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 32, NO. 1, 331–336
http://dx.doi.org/10.1080/14756366.2016.1250757
RESEARCH ARTICLE
Synthesis and identification of GZD856 as an orally bioavailable Bcr-AblT315I
inhibitor overcoming acquired imatinib resistance
a,b
b
b
a,b
Xiaoyun Lua,b , Zhang Zhang , Xiaomei Ren , Deping Wang and Ke Ding
ꢀ
ꢀ
aSchool of Pharmacy, Jinan University, Guangzhou, China; State Key Laboratory of Respiratory Diseases, Guangzhou Institutes of Biomedicine
b
and Health, Chinese Academy of Sciences, Guangzhou, China
ABSTRACT
ARTICLE HISTORY
Bcr-AblT315I induced drug resistance remains a major challenge to chronic myelogenous leukemia (CML)
treatment. Herein, we reported GZD856 as a novel orally bioavailable Bcr-AblT315I inhibitor, which strongly
suppressed the kinase activities of both native Bcr-Abl and the T315I mutant with IC50 values of 19.9 and
15.4 nM, and potently inhibited proliferation of corresponding K562, Ba/F3WT and Ba/F3T315I cells with IC50
values of 2.2, 0.64 and 10.8 nM. Furthermore, GZD856 potently suppressed tumor growth in mouse bearing
xenograft K562 and Ba/F3 cells expressing Bcr-AblT315I. Thus, GZD856 may serve as a promising lead for
the development of Bcr-Abl inhibitors overcoming acquired imatinib resistance.
Received 18 August 2016
Revised 12 September 2016
Accepted 13 September 2016
KEYWORDS
Bcr-Abl; chronic
myelogenous leukemia;
imatinib clinical resistance;
scaffold hopping; T315I
mutant
Introduction
(2)9,10, dasatinib (3)11,12 and bosutinib (4)13 have been approved
for the treatment of adults in all phases of CML with resistance to
imatinib (Figure 1(A)). However, these second-generation inhibitors
are not capable of inhibiting all the kinase mutants identified in
patients, especially the most notably Bcr-AblT315I gatekeeper
mutant that represents 15–20% of all clinical acquired resistan-
ces14. Thus, CML containing the T315I mutation remains a serious
medical problem in clinic.
Chronic myelogenous leukemia (CML) is a hematological stem cell
disorder caused by expansion of lymphocytic or myeloid blasts in
the blood or bone marrow, and resulting from a t (9; 22) recipro-
cal translocation encoding the constitutively active Bcr-Abl tyro-
sine kinase1. Bcr-Abl, a 210-kDa non-receptor protein kinase that
catalyzes the transfer of c-phosphoryl group from ATP to the
hydroxyl group of specific tyrosine residues in proteins, is a vali-
dated target for development of small molecule inhibitors to treat
CML2. The first generation Bcr-Abl inhibitor imatinib (1, STI1571)
has achieved significant clinical benefit and became the first-line
drug for conventional treatment of CML (Figure 1(A))3–5. However,
many patients developed emerging acquired resistance to imatinib
with its widespread used in clinic6. The primary mechanism of
acquired imatinib resistance is the occurrence of point mutations
in the Abl kinase domain, which effects the binding of imatinib in
the ATP-binding site7. To date, more than 100 different point
mutations have been identified in clinic.
To address this unmet need, considerable efforts have been
made to develop the third generation Bcr-Abl inhibitors which can
override the T315I mutation15–18, especially for AP24534 (5)19,20
,
GNF-721 and GZD82422. Among them, only AP24534 (ponatinib,
R
IclusigV) has been approved for the treatment of resistant or
intolerant CML and Phþ ALL patients against imatinib, especially
those harboring Bcr-AblT315I mutation19,20. However, FDA sus-
pended the sale of ponatinib due to the increasing numbers of
blood clots observed in ponatinib-treated patients after 10
months23. Ponatinib was then reauthorized for sale a black-box
warning and revised indication statement for concerns over risks
of its usage24.
To overcome imatinib resistance, several classes of second gen-
eration of Bcr-Abl inhibitors have been developed8. Nilotinib
CONTACT Ke Ding
ding_ke@gibh.ac.cn; Xiaoyun Lu
luxy2016@jnu.edu.cn
School of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou
510632, China
ꢀ
These authors contributed equally.
ß 2017 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, distri-
bution, and reproduction in any medium, provided the original work is properly cited.

332
X. LU ET AL
(A)
(B)
Figure 1. (A) Chemical structures of FDA-approved Bcr-Abl inhibitors; (B) design of GZD856 as new Bcr-Abl inhibitor by scaffold hopping based on ponatinib.
As part of our continuous efforts to identify new molecules followed by the addition of 5 mL of stop solution. Fluorescence sig-
that could target Bcr-AblT315I mutant22,25, here we report the
nal ratio of 445 nm (coumarin)/520 nm (fluorescein) was examined
on EnVision Multilabel Reader (Perkin-Elmer, Inc., Waltham, MA).
The data were analyzed using Graphpad Prism5 (Graphpad
design, synthesis and biological evaluation of GZD856 (6)26 as a
potent Bcr-AblT315I inhibitor by substituting imidazo[1,2-b]pyrida-
Software, Inc., La Jolla, CA). The data were the mean values of
three experiments.
zine (five-fused-six membered ring) of ponatinib with pyrazole[1,5-
a]pyrimidine (six-fused-five membered ring) by scaffold hopping
strategies (Figure 1(B)), which also keep the hydrogen bond with
the residue in kinase hinge region. GZD856 strongly suppressed
the kinase activities of both native Bcr-Abl and the T315I mutant,
and potently inhibited proliferation of corresponding K562, Ba/
F3WT and Ba/F3T315I cells with low nM IC50s. It also displayed
promising in vivo antitumor efficacy in mouse bearing xenograft
Cells and reagents
K562 (Bcr-Abl fusion expression), U-937 and MOLT4 were pur-
chased from ATCC and maintained as recommended by ATCC
(Manassas, VA). Imatinib, dasatinib and nilotinib were purchased
from Biocompounds Pharmaceutical Inc. (Shanghai, China).
Ponatinib was synthesized by ourself. CCK-8 was purchased from
Dojindo Molecular Technologies Inc. (Kumamoto, Japan). Dimethyl
sulfoxide (DMSO) and Cremophor were purchased from Sigma-
Aldrich (North Dorset, UK). Antibodies against Abl, p-Abl, Crkl,
p-Crkl, STAT5 and p-STAT5, respectively, were all purchased from
Cell Signaling Technology, Inc. (Danvers, MA).
K562 and Ba/F3 cells expressing Bcr-AblT315I
.
Methodology
Computational study
The structure of Bcr-AblT315I protein was retrieved from the
Protein Data Bank (PDB code: 3IK3), which was published by
O'Hare et al.20 The protein was processed using protein prepar-
ation wizard, which assigns bond orders and adds hydrogen and
missing atoms. GZD856 was built by in LigPrep (LigPrep, version
Stably transformed Ba/F3 cells
The Ba/F3 cell lines stably Bcr-AblWT and Bcr-AblT315I mutant were
self-established by following procedures similar to those described
by von Bubnoff27. Briefly, wild-type Bcr-Abl p210 was cloned into
pcDNA3.1(þ) (Invitrogen, Carlsbad, CA). Point mutations were intro-
duced to pcDNA3.1(þ) Bcr-Abl using the QuickChange XL Site-
Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Ba/F3 cells were
transfected with the constructs using Amaxa Cell Line Nucleofector
Kit V (Lonza, Cologne, Germany) by electroporation. Stable lines
were selected using Transfected Cells Cloning Kit (Stem Cell
Technologies, Vancouver, Canada) with G418 (Merck, Whitehouse
Station, NJ) and withdrawal of interleukin-3 (IL-3, R&D). Ba/F3 stable
cell lines were verified by monitoring both DNA sequences through
DNA sequencing and protein expression levels of the correspond-
ing Bcr-Abl mutants through Western blotting analysis. Their
responses to the imatinib, nilotinib and dasatinib were also hired
for selecting the right clones. Parent Ba/F3 cells were cultured in
€
2.5, Schrodinger, LLC, New York, NY, 2011) module using OPLS-
2005 force field. Molecular docking was performed in Glide mod-
€
ule (Glide, version 5.7, Schrodinger, LLC, New York, NY, 2011) with
standard precision scoring function.
FRET-based Z0-lyte assay detecting peptide substrate
phosphorylation
The effects of GZD856 on the kinase activity of Bcr-Abl and its
mutants were assessed in 384-well plates using the FRET-based
Z0-Lyte assay system according to manufacturer’s instructions
(Invitrogen, Carlsbad, CA). Briefly, 10 mL per well reactions con-
tained ATP concentration at 10 mM (for Bcr-Abl wildtype) or 5 mM
(for T315I mutant), 2 mM Tyr2 peptide substrate in 50 mM HEPES
(pH 7.5), 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, 0.0247 mg/mL
Bcr-Abl, and inhibitors as appropriate. The reaction was performed RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and
IL-3 (10 ng/mL), while all Bcr-Abl-transformed Ba/F3 stable cell lines
were cultured in the similar medium except without IL-3.
at room temperature for 2.0 h, and then 5 mL of development
reagent was added for a further 2 h room temperature incubation

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
333
Stably K562R (Q252H) cells
Synthesis of GZD856
Imatinib-resistant K562 cells which expressed Bcr-Abl Q252H were Reagents and solvents were obtained from commercial suppliers
self-established. Briefly, K562 cells were treated with a range of
concentrations of imatinib (from 0.1 mM to 5 mM) over a 3 month
period. Single clones were then selected and identified through
DNA sequencing, and their response to imatinib, nilotinib and
dasatinib were monitored as an internal reference.
and used without further purification. Flash chromatography was
performed using silica gel (300–400 mesh). All reactions were
monitored by TLC, silica gel plates with fluorescence F254 were
used and visualized with UV light. 1H and 13C NMR spectra were
recorded on a Bruker AV-400 spectrometer at 400 MHz and Bruker
AV-500 spectrometer at 125 MHz, respectively (Bruker, Billerica,
MA). Coupling constants (J) are expressed in hertz (Hz). Chemical
shifts (d) of NMR are reported in parts per million (ppm) units rela-
tive to internal control (TMS). The low or high resolution of ESI-MS
was recorded on an Agilent 1200 HPLC-MSD mass spectrometer
(Santa Clara, CA) or Applied Biosystems Q-STAR Elite ESI-LC-MS/MS
mass spectrometer (Foster City, CA), respectively.
Cellular antiproliferation assay using cell counting kit (CCK-8)
Cells in the logarithmic phase were plated in 96-well culture
dishes (ꢁ3000 cells/well). Twenty-four hours later, cells were
treated with the corresponding compounds or vehicle control at
the indicated concentration for 72 h. CCK-8 was added into the
96-well plates (10 mL/well) and incubated with the cells for 3 h.
OD450 and OD650 were determined by a microplate reader.
Absorbance rate (A) for each well was calculated as
OD450–OD650. The cell viability rate for each well was calculated
as V% ¼ (As ꢂ Ac)/(Ab ꢂ Ac) ꢃ 100%, and the data were further
analyzed using Graphpad Prism5 (Graphpad Software, Inc., La
Jolla, CA) (As, absorbance rate of the test compound well; Ac,
absorbance rate of the well without either cell or test compound;
Ab, absorbance rate of the well with cell and vehicle control).
Methyl 3-ethynyl-4-methylbenzoate (9)
To a solution of methyl 3-iodo-4-methylbenzoate (7) (2.76 g,
10 mmol) and trimethylsilyl acetylene (0.98 g, 10 mmol) in aceto-
nitrile (20 mL) were added Pd(dppf)Cl2 (0.07 g, 0.1 mmol), CuI
(0.19 g, 1 mmol) and triethylamine (10 mL). The mixture was stirred
at 80 ꢄC for 2 h under argon atmosphere. The reaction mixture
was filtered through a pad of Celite. The filtrate was concentrated,
and the resulting residue was solved in CH3OH (25 mL) and
treated with K2CO3 (9.8 g, 60 mmol) solution, and the mixture was
stirred at room temperature for 3 h. The reaction solution was con-
centrated, and EtOAc and H2O were added to the residue. The
organic layer was separated, and the aqueous layer was exacted
with EtOAc (3 ꢃ 50 mL). The combined layers were dried over
Na2SO4 and concentrated. The resulting crude was further purified
by flash chromatography to give the product as a white solid
(1.56 g, 90.0%). 1H NMR (400 MHz, DMSO-d6) d 7.93 (d, J ¼ 1.2 Hz,
1H), 7.85 (dd, J ¼ 8.0 Hz, 1.6 Hz, 1H), 7.44 (d, J ¼ 8.4 Hz, 1H), 4.49 (s,
1H), 3.84 (s, 3H), 2.44 (s, 3H). LC–MS (ESI): m/z 175 [M þ H]þ; 173
[M ꢂ H]ꢂ.
Western blot analysis
The Western blot analysis was carried out by following the proto-
col described previously22. Briefly, after the indicated treatment,
cell lysates were collected, dissolving cells in 1ꢃ SDS sample lysis
buffer. After being sonicated and boiled, the supernatant of the
cell lysate was used for Western blot analysis. Cell lysates were
loaded to 8–12% SDS-PAGE and separated by electrophoresis.
Separated proteins were then electrically transferred to a PVDF
film. After being blocked with 1ꢃ TBS containing 0.1% Tween-20,
and 5% nonfat milk, the film was incubated with corresponding
primary antibody followed by HRP-conjugated secondary antibody.
The protein lanes were visualized using ECL Western Blotting
Detection Kit (GE Healthcare, Piscataway, NJ).
Methyl 4-methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)-
benzoate (11)
Animal studies
To a solution of 9 (385 mg, 2.2 mmol) in DMF (10 mL), 6-bromopyr-
azolo[1,5-a]pyrimidine (396 mg, 2 mmol), N,N-diisopropylethyl-
amine (193 mg, 1.5 mmol), Pd(PPh3) Cl2 (7 mg, 0.01 mmol) and CuI
(19 mg, 0.1 mmol) were placed in a vial with rubber septum. The
mixture underwent three cycles of vacuum/filling with Ar. The mix-
ture was stirred at 80 ꢄC overnight and then quenched with H2O.
EtOAc and more H2O were added for extraction. The combined
organic layer was dried over Na2SO4, filtered, and concentrated,
and the resulting residue was purified by chromatography, giving
the title compound as a white solid (465 mg, 80.0%). 1H NMR
(400 MHz, DMSO-d6) d 9.56 (s, 1H), 8.70 (s, 1H), 8.32 (s, 1H), 8.08 (s,
1H), 7.89 (d, J ¼ 7.6 Hz, 1H), 7.50 (d, J ¼ 8.0 Hz, 1H), 6.82 (s, 1H),
3.86 (s, 3H), 2.56 (s, 3H). 13C NMR (125 MHz, DMSO-d6) d 165.4,
150.9, 146.5, 146.3, 145.3, 138.2, 132.2, 130.3, 129.5, 127.6, 122.0,
104.6, 97.3, 90.2, 87.8, 52.2, 20.5. LC–MS: m/z 292 [M þ H]þ.
Male BALB/c or SCID nude mice were purchased from Vital River
Laboratory Animal Technology Inc. (Beijing, China). All animal
studies were approved by the Institutional Animal Use and Care
Committee of Guangzhou Institute of Biomedicine and Health,
Chinese Academy of Science.
Mice xenograft models
K562 and Bcr-AblT315I cells were re-suspended in normal saline
(NS) solution (2.5 ꢃ 107 and 1 ꢃ 107 cell/mL respectively). A 0.2 mL
amount of cell suspension was injected subcutaneously into the
right flank of each mouse. Mice were randomly grouped based on
the tumor volume when the mean tumor volume reached
100–200 mm3. GZD856 and imatinib were dissolved in a vehicle
containing 1% DMSO, 22.5% Cremophor, 7.5% ethanol and 69%
NS. Mice were treated for the 16 consecutive days once daily by
oral gavage with GZD856 (10 mg/kg), imatinib (50 mg/kg) and
vehicle, respectively. Tumor volume and body weight were moni-
tored once every 2 days. Tumor volume was calculated as the
4-Methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-
(trifluoromethyl)phenyl)-3-(pyrazolo[1,5-a]pyrimidin-6-
ylethynyl)benzamide (6, GZD856)
LꢃW2/2 (L and W are the length and width of the tumor, respect- A solution of methyl 4-methyl-3-(pyrazolo[1,5-a]pyrimidin-6-yle-
ively). Tumor volume data were analyzed with the one-way
ANOVA method using software SPSS 17.0 (SPSS Inc., Chicago, IL).
thynyl)-benzoate (145 mg, 0.5 mmol) and 4-((4-methylpiperazin-1-
yl)methyl)-3-(trifluoromethyl)aniline
(137 mg,
0.5 mmol)
in

334
X. LU ET AL
anhydrous THF (10 mL) was added potassium tert-butoxide suggested that GZD856 can be a potent Bcr-Abl inhibitor over-
(336 mg, 3 mmol) in anhydrous THF (10 mL) slowly at ꢂ20 ꢄC and
coming acquired imatinib resistance. However, imatinib and niloti-
nib were both significantly less potent for Bcr-AblT315I mutation.
stirred for 1 h. Then the reaction mixture was slowly warmed to
room temperature and stirred for another 8 h. After evaporation of
the solvent, the residue was re-dissolved in ethyl acetate. Then the
organic phase was washed with brine, dried over anhydrous
Molecular docking studies
sodium sulfate, concentrated under reduced pressure. The residue
was purified through silica gel column chromatography to the
desired compound as yellow solid (200 mg, 75%). 1H NMR
(400 MHz, DMSO-d6), 2.17 (s, 3H), 2.37 (m, 8H), 2.60 (s, 3H), 3.57 (s,
2H), 6.85 (d, J ¼ 2.0 Hz, 1H), 7.54 (d, J ¼ 8.0 Hz, 1H), 7.71 (d,
J ¼ 8.4 Hz, 1H), 7.96 (dd, J ¼ 8.0, 3.29 Hz, 1H), 8.06 (d, J ¼ 2.00 Hz,
1H), 8.21 (dd, J ¼ 4.2, 2.0 Hz, 2H), 8.34 (d, J ¼ 2.0 Hz, 1H), 8.72 (d,
J ¼ 2.0 Hz, 1H), 9.58 (d, J ¼ 2.00 Hz, 1H), 10.56 (s, 1H). 13C NMR
(125 MHz, DMSO-d6) d 165.5, 151.8, 147.4, 147.2, 144.8, 139.1,
139.0, 133.1, 132.9, 132.0, 131.7, 130.8, 129.4, 126.3, 124.4, 122.5,
118.2, 105.6, 98.2, 91.6, 88.4, 58.3, 55.5, 53.5, 46.5, 21.3. MS (ESI),
m/z: 533 (M þ H)þ. HRMS (EI): calcd for C29H27F3N6O: 533.2198
[M þ H]þ; found: 533.2266.
Molecular docking study was performed to investigate the binding
mode of compound GZD856 with Bcr-AblT315I (PDB code: 3IK3)
€
using Glide module (Glide, version 5.7 Schrodinger, LLC, New York,
NY) with standard precision scoring function. GZD856 bound to
the ATP binding site of the DFG-out conformation of Bcr-AblT315I
(Figure 2), which was similar to that of ponatinib19,20. The pyra-
zolo[1,5-a]pyrimidine of GZD856 formed an essential hydrogen
bond with the NH of Met318 in the hinge region of Bcr-AblT315I
.
The amide formed two hydrogen bonds with Glu286 and Asp381,
and the trifluoromethylphenyl group bound deeply into the
hydrophobic pocket. The alkynyl linker made favorable van der
Waals interactions with the gatekeeper Ile315 avoiding steric clash.
Cellular antiproliferation assay
Results and discussion
The antiproliferative activity of GZD856 was also examined against
a panel of leukemia cells with differing Bcr-Abl status (Table 2).
Imatinib, nilotinib, ponatinib and taxol were utilized as positive
controls to validate the screening conditions. As shown in Table 2,
imatinib and nilotinib were both significantly less potent for
K562R (Q252H) and Ba/F3T315I cells. GZD856 strongly suppressed
the proliferation of K562, K562R (Q252H) and murine Ba/F3 cells
ectopically expressing Bcr-AblWT and Bcr-AblT315I, with IC50 values
of 2.2, 67.0, 0.64 and 10.8 nM, respectively, which were equivalent
to the potency of ponatinib and much higher than the potency of
Synthetic route
GZD856 was readily prepared using palladium catalyzed
Sonogashira reaction as the key step, which is illustrated in
Scheme 1. Briefly, the commercial material methyl 3-iodo-4-meth-
ylbenzoate (7) was reacted with ethynyltrimethylsilane (8) under
palladium catalysis to afford the Sonogashira coupling products,
which was further deprotected to produce the alkyne 9. Then,
coupling of alkyne 9 with 6-bromopyrazolo[1,5-a]pyrimidine (10)
under the Sonogashira coupling conditions afforded the key inter-
mediate 11. Ammonolysis of 11 with 4-((4-methylpiperazin-1-yl) imatinib and nilotinib (Table 2). As a confirmation of GZD856’s
methyl)-3-(trifluoromethyl) benzenamine (12) under basic condi- Bcr-Abl-selective effect, GZD856 was significantly less potent
tion yielded the desired GZD856 (6).
against the proliferation of MOLT4 and U937 leukemia cells
Table 1. Inhibitory activities of GZD856 against Bcr-AblWT and
Bcr-AblT315I mutant.
Kinase activities evaluation
The kinase inhibitory activities of GZD856 against Bcr-AblWT and
Bcr-AblT315I were evaluated by using the well established FRET-
based Z’-Lyte assay22,28. Imatinib and nilotinib were used as posi-
tive controls to validate the screening conditions. Under the
screening conditions, imatinib and nolitinib potently inhibited the
enzymatic activity of Bcr-AblWT with IC50 values of 98.2 and
43.5 nM, which were highly consistent to the reported data9,29. As
shown in Table 1, the designed GZD856 potently inhibited Bcr-
AblWT and Bcr-AblT315I with IC50 values of 19.9 and 15.4 nM,
respectively, which displayed similar activity to ponatinib (19),
Kinase inhibition (IC50 nM)
Compounds
Bcr-AblWT
Bcr-AblT315I
GZD856
Imatinib
Nilotinib
Ponatinib
ꢀ
19.9
98.2
43.5
8.6
15.4
5155
702.4
40
ꢀ
19
Reported data
.
Scheme 1. Synthetic route of compound GZD856. Reagents and conditions: (a) i.
Pd(dppf)2Cl2, CuI, Et3N, DMF, 80 ꢄC; ii. K2CO3, MeOH, rt, 90%; (b) Pd(PPh3) Cl2, CuI,
DIPEA, DMF, 80%; (c) t-BuOK, THF, ꢂ20 ꢄC, 75%.
Figure 2. The predicted binding mode of GZD856 with Bcr-AblT315I. Hydrogen
bonds are indicated by yellow hatched lines to key amino acids.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
335
Table 2. GZD856 selectively and potently inhibited the proliferation of Bcr-Abl
positive leukemia cells.
IC50 values (nM)
Cell lines
GZD856
Imatinib
Nilotinib
Ponatinib
Taxol
K562
2.2
0.64
10.8
67.0
499.4
2001.0
189
500
10 160
6050
48 500
16 000
6.5
22
1461
350
20 400
9520
0.5
0.16
6.5
ND
7.8
5.1
ND
ND
7.0
2.7
2.3
Ba/F3WT
Ba/F3T315I
K562R (Q252H)
MOLT-4
U937
1.0
ND: not detected.
Figure 4. GZD856 potently suppressed tumor growth in K562 xenograft model of
human CML. Mice bearing K562 xenografts were dosed orally once a day with
GZD856 at 10 mg/kg dosages for 16 consecutive days. The data are representative
of three independent experiments.
Figure 3. GZD856 inhibits Bcr-Abl signaling in K562 and Ba/F3 stable cell lines
expressing Bcr-AblWT and Bcr-AblT315I. Cells were treated with GZD856 at the indi-
cated concentrations for 4.0 h, and whole cell lysates were then subjected to
Western blot analyses. The results represent three independent experiments.
(negative for Bcr-Abl expression), which are much selective than
ponatinib. Further, GZD856 also displayed less potent antiprolifera-
tive activity against non-cancer cells HFL-1 (human embryonic
lung fibroblasts) with IC50 value of 6.78 mM26. The studies sug-
gested that GZD856 could be
a selective anti-cancer drug.
However, the cytotoxic agent taxol inhibited the growth of all
tested cells with similar IC50 values.
Figure 5. GZD856 suppressed tumor growth in xenograft models of Ba/F3 cells
expressing Bcr-AblT315I. Mice bearing xenograft Ba/F3 Bcr-AblT315I cells were orally
dosed with GZD856 and imatinib at the indicated doses. Tumor sizes were moni-
tored every 2 days (n ¼ 10 for each group; error bar represents SE).
Western blot study
To further validate the cellular kinase inhibitory activity of
GZD856, we examined its effects on the activation of Bcr-Abl and
its downstream signals in Bcr-Abl positive K562 CML cells and sta-
bly transformed Ba/F3 cells expressing Bcr-AblWT and Bcr-AblT315I
by Western blot analysis (Figure 3). GZD856 efficiently suppressed
the activation of both Bcr-Abl and downstream Crkl and STAT5 in
dose-dependent manners in K562 CML cells. Similar inhibition was
also observed in the Ba/F3 cells expressing Bcr-AblWT and Bcr-
AblT315I. Not surprisingly, none of the three FDA approved drugs
(imatinib, nilotinib, dasatinib) showed obviously suppression on
well tolerated with no mortality or significant body loss (<5% rela-
tive to vehicle-matched controls) during the treatment.
Similar to that of the K562 xenograft studies, the antitumor effi-
cacy of GZD856 was further evaluated in a xenograft model using
Ba/F3 cell expressing Bcr-AblT315I. The animals were administered
GZD856 at doses of 20 and 50 mg/kg via oral gavage once daily
for 16 days. Imatinib was again used as a reference compound
and administered orally at dose of 100 mg/kg/day for 16 days. As
shown in Figure 5, GZD856 dose-dependently inhibited tumor
growth in the xenograft model bearing Bcr-AblT315I. Although
GZD856 did not show obvious inhibition of tumor growth at dose
of 20 mg/kg/day, it induced almost about 90% tumor regression at
a dose of 50 mg/kg/day after a 16 days consecutive treatment. In
contrast, 100 mg/kg/day of imatinib failed to exhibit inhibition of
tumor growth in the Bcr-AblT315I model.
the activation of Bcr-AblT315I 22
.
In vivo antitumor studies
Giving its highly promising antiproliferative activity in vitro and
pharmacokinetic (PK) properties in vivo in rats26, GZD856 may pos-
sess good in vivo efficacy when orally administered. Thus, the
in vivo antitumor effect of GZD856 was first evaluated in subcuta-
neous K562 xenograft model of human CML. Imatinib was used as
a positive control at dose of 50 mg/kg/day to validate the animal
models. The animals were dosed orally with GZD856 at
10 mg/kg/day. As shown in Figure 4, GZD856 almost completely
Conclusions
In summary, we have successfully discovered GZD856 as a new
Bcr-Abl inhibitor which maintains significant inhibition against Bcr-
eradicated the tumor at doses of 10 mg/kg/day after 8 days of Abl gatekeeper T315I mutant. GZD856 strongly suppressed both
treatment, whereas imatinib only induced tumor stasis at a dose native Bcr-Abl and the T315I mutant with IC50 values of 19.9 and
of 50 mg/kg/day. Notably, after the cessation of treatment 15.4 nM, and potently inhibited proliferation of corresponding
(16 days of dosing), there was no sign of tumor recurrence in the K562 and Ba/F3T315I cells with IC50 values of 2.2 and 10.8 nM.
following 7 days. Also, at the dose of 10 mg/kg/day, GZD856 was Western blot analysis further supported its kinase inhibition

336
X. LU ET AL
against Bcr-Abl and Bcr-AblT315I. In vivo efficacy studies in mouse 13. Puttini M, Coluccia AM, Boschelli F, et al. In vitro and in vivo
xenograft models of human leukemia demonstrated that GZD856
potently suppresses growth of tumors driven by native Bcr-Abl
and Bcr-AblT315I, which indicated that GZD856 is a promising anti-
cancer lead for further development.
activity of SKI-606, a novel Src-Abl inhibitor, against imati-
nib-resistant cells. Cancer Res
Bcr-Abl þ neoplastic
2006;66:11314–22.
14. Modugno M. New resistance mechanisms for small molecule
kinase inhibitors of Abl kinase. Drug Discov Today Technol
2014;11:5–10.
15. Tanaka R, Kimura S. Abl tyrosine kinase inhibitors for overrid-
ing Bcr-Abl/T315I: from the second to third generation.
Expert Rev Anticancer Ther 2008;8:1387–98.
Disclosure statement
The authors report no declarations of interest.
16. Noronha G, Cao J, Chow CP, et al. Inhibitors of ABL and the
ABL-T315I mutation. Curr Top Med Chem 2008;8:905–21.
17. Schenone S, Brullo C, Botta M. New opportunities to treat
the T315I-Bcr-Abl mutant in chronic myeloid leukaemia: tyro-
sine kinase inhibitors and molecules that act by alternative
mechanisms. Curr Med Chem 2010;17:1220–45.
18. Lu XY, Cai Q, Ding K. Recent developments in the third gen-
eration inhibitors of Bcr-Abl for overriding T315I mutation.
Curr Med Chem 2011;18:2146–57.
Funding
We thank National High Technology Research and Development
(863) for Young Scientists program (No. 2015AA020906),
Guangdong Natural Science Funds for Distinguished Young
Scholar (2015A030306042), the Pearl River S&T Nova Program of
Guangzhou (201506010086), and Guangdong Special Branch
Program Technology Young Talents (2014TQ01R341) for the finan-
cial support.
19. Huang WS, Metcalf CA, Sundaramoorthi R, et al. Discovery
of 3-[2-(imidazo[1,2-b]pyridazin-3-yl)ethynyl]-4-methyl-N-{4-
[(4-methyl-piperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}-
benzamide (AP24534), a potent, orally active pan-inhibitor of
breakpoint cluster region-abelson (BCR-ABL) kinase including
the T315I gatekeeper mutant. J Med Chem 2010;53:4701–19.
20. O'Hare T, Shakespeare WC, Zhu X, et al. AP24534, a pan-
BCR-ABL inhibitor for chronic myeloid leukemia, potently
inhibits the T315I mutant and overcomes mutation-based
resistance. Cancer Cell 2009;16:401–12.
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