Identification of GZD824 as an orally bioavailable inhibitor that targets phosphorylated and nonphosphorylated breakpoint cluster region-Abelson (Bcr-Abl) kinase and overcomes clinically acquired mutation-induced resistance against imatinib

Article abstract of DOI:10.1021/jm301581y

Bcr-Abl^T315I mutation-induced imatinib resistance remains a major challenge for clinical management of chronic myelogenous leukemia (CML). Herein, we report GZD824 (10a) as a novel orally bioavailable inhibitor against a broad spectrum of Bcr-Abl mutants including T315I. It tightly bound to Bcr-Abl^WT and Bcr-Abl^T315I with K_d values of 0.32 and 0.71 nM, respectively, and strongly inhibited the kinase functions with nanomolar IC₅₀ values. The compound potently suppressed proliferation of Bcr-Abl-positive K562 and Ku812 human CML cells with IC ₅₀ values of 0.2 and 0.13 nM, respectively. It also displayed good oral bioavailability (48.7%), a reasonable half-life (10.6 h), and promising in vivo antitumor efficacy. It induced tumor regression in mouse xenograft tumor models driven by Bcr-Abl^WT or the mutants and significantly improved the survival of mice bearing an allograft leukemia model with Ba/F3 cells harboring Bcr-Abl^T315I. GZD824 represents a promising lead candidate for development of Bcr-Abl inhibitors to overcome acquired imatinib resistance.

Full text of DOI:10.1021/jm301581y

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Article
Identification of GZD824 as an orally bioavailable inhibitor that targets
phosphorylated and non-phosphorylated Bcr-Abl and overcomes
clinically acquired mutation-induced resistance against imatinib
Ke Ding, Xiaomei Ren, zhang zhang, Xiaofeng pan, Zhengchao Tu, Xiaoyun Lu, Yupeng
Li, Wang Deping, Donghai Wen, Huoyou Long, Jinfeng Luo, Yubing Feng, Xiaoxi Zhuang,
Fengxiang Zhang, Jianqi Liu, Fang Leng, Xingfen lang, Yang Bai, Miaoqin She, and Jingxuan Pan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm301581y • Publication Date (Web): 09 Jan 2013
Downloaded from http://pubs.acs.org on January 16, 2013
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Identification of GZD824 as an Orally Bioavailable
Inhibitor that Targets Phosphorylated and Nonꢀ
phosphorylated Breakpoint Cluster RegionꢀAbelson
BcrꢀAbl) Kinase and Overcomes Clinically Acquired
MutationꢀInduced Resistance against Imatinib
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(
Xiaomei Ren, α β Xiaofen Pan, α δ Zhang Zhang, α δ Deping Wang, α Xiaoyun Lu, Yupeng Li,
,
,
δ
,
,
,
ϕ
α
α, ϕ
α
α
α
α
α
α
Donghai Wen, Huoyou Long, Jinfeng Luo, Yubing Feng, Xiaoxi Zhuang, Fengxiang Zhang,
α
α
α
α
α
α
β
Jianqi Liu, Fang Leng, Xingfen Lang, Yang Bai, Miaoqin She, Zhengchao Tu, Jingxuan Pan
and Ke Ding α
*
α
Institute of Chemical Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of
Sciences, #190 Kaiyuan Avenue, Guangzhou 510530, China
β
nd
Zhongshan Medical School, Sun YatꢀSen University, #74 2 Zhongshan Road, Guangzhou 510080,
China
ϕ
Graduate University of Chinese Academy of Sciences, # 19 Yuquan Road, Beijing 100049, China
δ
These authors contributed equally to this work.
*Corresponding author: Tel: +86ꢀ20ꢀ32015276. Fax: +86ꢀ20ꢀ32015299.
EꢀMail: ding_ke@gibh.ac.cn
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RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
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T315I
ABSTRACT. The BcrꢀAbl
mutation induced imatinibꢀresistance remains a major challenge for
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clinical management of chronic myelogenous leukemia (CML). Herein, we report GZD824 (10a) as a
novel orally bioavailable inhibitor against a broad spectrum of BcrꢀAbl mutants including T315I. It
WT
T315I
tightly bound to BcrꢀAbl and BcrꢀAbl
with K values of 0.32 and 0.71 nM, respectively, and
d
strongly inhibited the kinase functions with nM IC50 values. The compound potently suppressed
proliferation of BcrꢀAblꢀpositive K562 and Ku812 human CML cells with IC values of 0.2 and 0.13
5
0
nM, respectively. It also displayed good oral bioavailability (48.7%), a reasonable halfꢀlife (10.6 hrs)
and promising in vivo antiꢀtumor efficacy. It induced tumor regression in mouse xenograft tumor
WT
models driven by BcrꢀAbl or the mutants, and significantly improved the survival of mice bearing an
T315I
allograft leukemia model with Ba/F3 cells harboring BcrꢀAbl
. GZD824 represents a promising lead
candidate for development of BcrꢀAbl inhibitors to overcome acquired imatinibꢀresistance.
KEYWORDS: chronic myeloid leukemia (CML), GZD824, BcrꢀAbl, imatinib resistance, T315I
mutation
INTRODUCTION
The BcrꢀAbl inhibitor imatinib (1) has provided significant clinical benefit and become the firstꢀline
+
drug for conventional treatment of Philadelphia chromosome positive (Ph ) chronic myeloid leukemia
(CML). However, emerging acquired resistance to drug 1 has become a major challenge for clinical
1
ꢀ6
management of CML. For imatinibꢀtreated CML patients, the 2ꢀyear incidence of resistance reaches
0% in the blastic phase, 40ꢀ50% in the accelerated phase, and 8ꢀ10% in the chronic phase. The
occurrence of point mutations in the Abl kinase domain is the primary mechanism underlying imatinibꢀ
8
7
ꢀ9
resistance.
More than 100 resistanceꢀrelated BcrꢀAbl mutants have been identified in the clinic,
among which the “gatekeeper” T315I is most common mutation, as it accounts for approximately
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5~20% of all clinically acquired mutants. Great efforts have been devoted to identifying novel BcrꢀAbl
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inhibitors to overcome imatinib resistance. Nilotinib (2) , dasatinib (3) and bosutinib have been
approved as secondꢀline drugs to treat adult patients in all phases of CML with resistance to drug 1.
12
Many other inhibitors, such as bafetinib, have also been developed in lateꢀstage clinical trials (Figure
1
). However, the secondꢀgeneration inhibitors are not capable of inhibiting the most refractory BcrꢀAbl
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T315I
14ꢀ16
T315I
mutant.
treatment.
T315 is one of the most critical residues for the binding of type I and type II inhibitors in the ATPꢀ
BcrꢀAbl
ꢀinduced drug resistance remains an unmet clinical challenge for CML
1
7, 18
binding pocket of BcrꢀAbl kinase.
The T315I mutation disfavors a key hydrogen bonding
interaction between the classical BcrꢀAbl inhibitors and the kinase, whereas the bulky isoleucine side
chain of I315 prevents the inhibitors from accessing an important hydrophobic pocket at the back of the
ATPꢀbinding domain. Moreover, the T315I mutation is also associated with a conformational change of
the kinase from DFGꢀout to DFGꢀin, which further disrupts the binding of type II inhibitors to the Abl
1
9ꢀ22
kinase domain.
mutations in the pꢀloop region (i.e., G250E, Q252H, Y253H, and E255K/V) and the hinge region
F317L/V) that confer imatinib resistance have also been identified. Notably, the inhibitory activity of 2
In addition to a mutation in the kinase gatekeeper region (T315I/A), various
(
and 3 against the pꢀloop (Y253H and E255K/V) and the hinge of BcrꢀAbl mutants (F317V),
23, 24, 25
respectively, has been reported to be relatively limited.
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CF3
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1 STI-571, imatinib
2 nilotinib
Cl
Cl
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CN
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OH
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dasatinib
bosutinib
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H
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F
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NH
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CF3
8
(ponatinib, AP24534)
9 (DCC-2036)
Figure 1. Chemical structures of FDAꢀapproved and newly developed BcrꢀAbl inhibitors.
T315I
Several small molecules capable of inhibiting the BcrꢀAbl
mutant in biochemical and cellular
2
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assays have been reported. Examples include the type I inhibitor PPYꢀA, cꢀSrc/Abl dual inhibitor
TG100598 and nonꢀATPꢀcompetitive or allosteric inhibitors (ON012380 or GNFꢀ2, respectively).
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Several ATPꢀcompetitive Aurora kinase inhibitors, such as MKꢀ0457 (4) , PHAꢀ739358 (5)
,
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T315I
AT9283 (6) and XLꢀ228 (7), have been reported to inhibit BcrꢀAbl
. However, most of these
compounds appear to lack oral bioavailability, as they were formulated for intravenous administration.
Furthermore, the clinical development of compound 4 has been discontinued because of its toxicity.
T315I
Although 6 potently inhibits the activity of the BcrꢀAbl
mutant and BcrꢀAbl variants with
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mutations in the pꢀloop and hinge regions, the mechanism underlying this broad spectrum of inhibition
and the relative selectivity of this compound for BcrꢀAbl over other offꢀtarget kinases require further
investigation. As imatinibꢀresistant mutants are increasingly observed in the clinic, nextꢀgeneration
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“panꢀBcrꢀAbl inhibitors” are required to not only overcome T315I resistance but also display broad
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inhibitory activity towards multiple BcrꢀAbl mutants.
Recently, several “thirdꢀgeneration” BcrꢀAbl
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inhibitors capable of inhibiting nearly the full range of BcrꢀAbl kinase domain mutants in addition to
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the native enzyme were disclosed, among which AP24534 (8) and DCC2036 (9) are under clinical
investigation.
Herein, we report the identification of GZD824 (10a) as a new orally bioavailable pan BcrꢀAbl
inhibitor with potency against a wide range of BcrꢀAbl mutants, including the highly refractory
T315I
G250E
Q252H
E255K
gatekeeper mutant BcrꢀAbl
; pꢀloop region mutants BcrꢀAbl
, BcrꢀAbl
and BcrꢀAbl
;
F317L
and the ATPꢀbinding domain hinge region mutant BcrꢀAbl
, in addition to the native enzyme.
Furthermore, compound 10a induced complete tumor regression in xenografted or allografted mice,
WT
including those receiving K562, Ku812 human CML cells, or Ba/F3 cells harboring BcrꢀAbl
or a
panel of resistanceꢀrelated mutants. Compound 10a also extended survival in an allograft mice model
T315I
utilizing Ba/F3ꢀBcrꢀAbl
cells.
MOLECULAR DESIGN AND COMPUTATIONAL STUDIES
Compound 8 is a recently disclosed pan BcrꢀAbl inhibitor that demonstrated promising efficacy in
3
4
imatinibꢀresistant CML patients in a phase II clinical investigation. Xꢀray crystallographic analyses
WT
revealed that compound 8 bound the DFGꢀout modes of BcrꢀAbl
and the BcrꢀAbl mutants, and that
the imidazo[1,2ꢀb]pyridazine core acted as an essential hydrogen bond acceptor for the NH of Met318
WT
in the hinge region of the kinase. The binding mode of compound 8 for BcrꢀAbl
critically differed
from that of compound 1 in that a hydrogen bond was not formed with the hydroxyl group of Thr315.
T315I
Compound 8 therefore did not suffer the loss of binding affinity for the BcrꢀAbl
mutant that was
observed for compound 1. The ethynyl linkage of 8 could provide a favorable van der Waals interaction
with the I315 mutated residue. Based on the structural information and interaction mode of 8 with the
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kinases, a series of 1Hꢀpyrazolo[3,4ꢀb]pyridine derivatives was designed and synthesized as novel Bcrꢀ
Abl inhibitors. We sought to increase the panꢀBcrꢀAbl binding affinity relative to 8 by introducing an
additional hydrogen bond, as this scaffold was predicted to be capable of acting as both a hydrogen
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bond donor and a hydrogen bond acceptor for the hinge region of the kinase (Figure 2).
NH
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R1 = H, methyl, methoxyl,
cyclo-propyl, or phenyl
N
N
R1
N
N
R2 = H, methyl, ethyl, chloro,
or cyclo-propyl,
N
N
H
H
N
R2
N
N
N
O
O
CF3
CF
3
AP24534 (8)
10
Figure 2. Novel BcrꢀAbl inhibitors designed by introducing a 1Hꢀpyrazolo[3,4ꢀb]pyridine core.
Computational studies were performed to investigate the potential binding mode of the initially
designed compound 10a (R = H; R = Me) with the wildꢀtype and mutant forms of BcrꢀAbl. The results
1
2
suggested that 10a could tightly bind to the ATPꢀbinding site of nonꢀphosphorylated (DFGꢀout) BcrꢀAbl
WT
T315I
and different BcrꢀAbl mutants, including the clinicalꢀresistanceꢀrelated BcrꢀAbl
, using modes
similar to that of 8. The 1Hꢀpyrazolo[3,4ꢀb] pyridine core occupied the adenine pocket of BcrꢀAbl
kinase to form a hydrogen bond donorꢀacceptor network with the backbone of M318 in the hinge
region. The amide moiety formed two additional hydrogen bonds with E286 and D381, and the
trifluoromethylphenyl group bound deeply into the hydrophobic pocket. The alkyne moiety of 10a could
T315I
make favorable van der Waals interactions with the gatekeeper I315 of BcrꢀAbl
without causing
steric clash. The methylpiperazine group may form hydrogen bond interactions with I360 or H361
Figure 3A and 3B).
(
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Figure 3. The predicted binding modes of 10a with the wildꢀtype and mutant forms of BcrꢀAbl.
Hydrogen bonds to key amino acids are indicated by yellow hatched lines. The following binding modes
T315I
are shown: A) 10a with the nonꢀphosphorylated BcrꢀAbl
mutant (GScore = ꢀ14.82 kcal/mol); B)
1
0a with nonꢀphosphorylated native BcrꢀAbl (GScore = ꢀ13.94 kcal/mol); C) 10a with the
T315I
phosphorylated BcrꢀAbl
mutant (GScore = ꢀ8.63 kcal/mol); and D) 10a with phosphorylated native
BcrꢀAbl (GScore = ꢀ9.76 kcal/mol). The crystal structures of type I conformations of wildꢀtype and Bcrꢀ
T315I
Abl
mutant were taken from PDB (ID 3QRI and 3IK3). The PDB codes of the crystal structures of
T315I
type II conformations of wildꢀtype and BcrꢀAbl
mutant were 2GQG and 2V7A, respectitively.
Interestingly, further computational analyses indicated that 10a could also bind to the ATPꢀbinding
site of phosphorylated BcrꢀAbl with a DFGꢀin conformation. Because of the significant differences of
activation loop between the DFGꢀin and DFGꢀout conformation of BcrꢀAbl, the predicted binding mode
of 10a is greatly different from the mode proposed for nonꢀphosphorylated kinases. As shown in Figure
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C and 3D, a large part of 10a extended out of the ATPꢀbinding pocket and protruded toward solventꢀ
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T315I
exposed section of the protein. In the predicted binding pose of 10a with activated BcrꢀAbl
, a
hydrogen bond donorꢀacceptor network could form between the 1Hꢀpyrazolo[3,4ꢀb] pyridine core and
the carboxyl group of E329. The methylpiperazine group extended to the pocket form by the Pꢀloop, the
DFG motif and the catalytic loop, and may form a hydrogen bond interaction with the backbone of
G250. For the DFGꢀin conformation of wideꢀtype BcrꢀAbl (PDB ID 2GQG), Y253 of the Pꢀloop
pointed to the activation loop and seemed to engage in a hydrogen bond with the backbone carbonyl of
R367, and this motif couldn’t be occupied by the ligand. In the docking pose of phosphorylated native
BcrꢀAbl (Figure 3D), 10a extended out further toward solventꢀexposed protein, and the
methylpiperazine group could form hydrogen bond interactions with D325. The 1Hꢀpyrazolo[3,4ꢀb]
pyridine moiety pointed to the α Cꢀhelix and probably formed a hydrogen bond interaction with E286.
The methylphenyl group occupied the ATPꢀbinding site to make favorable hydrophobic interactions
with the side chains of F317 and M318. However, the results clearly demonstrated that the predicted
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T315I
WT
GScores of 10a with DFGꢀout conformations of BcrꢀAbl
mutant and BcrꢀAbl
(ꢀ14.82 kcal/mol
and ꢀ13.94 kcal/mol, respectively) were significantly lower than that with DFGꢀin conformations (ꢀ8.63
kcal/mol and ꢀ9.76 kcal/mol, respectively), suggesting that 10a prefers to bind with DFGꢀout
conformation of BcrꢀAbl. A recent study on DCC2036 (9) also implied that 10a might bind with
the phosphorylated BcrꢀAbl and the mutants, which exist predominately in the type I conformation, by
3
5a
inducing them to adopt a DFGꢀoutꢀlike conformation.
CHEMISTRY
The designed inhibitors were readily prepared using palladiumꢀcatalyzed Sonogashira coupling
3
7
reactions as the key steps (Scheme 1 and 2). Briefly, commercially available or selfꢀprepared methyl
3ꢀiodoꢀbenzoates (11) were treated with 4ꢀ((4ꢀmethylpiperazinꢀ1ꢀyl) methyl)ꢀ3ꢀ(trifluoromethyl)
benzenamine (12) under basic conditions to yield the amides (13). The intermediates 13 were reacted
with ethynyltrimethylsilane under palladium catalysis to afford the Sonogashira coupling products,
which were further deprotected to produce the terminal alkynes 14. The coupling of compounds 14 with
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aromatic bromides 18 under Sonogashira conditions, followed by deprotection, afforded the designed
inhibitor 10. Alternately, compound 10b was obtained by a palladiumꢀcatalyzed Sonogashira coupling
reaction of 13 with the readily prepared aromatic ethyne 19. Compounds 18a-18c and 19 were readily
prepared using 5ꢀbromoꢀ1Hꢀpyrazolo[3,4ꢀb]pyridine (15) as the starting material, whereas 20 was
synthesized from methyl 5ꢀbromoꢀ2ꢀchloronicotinate (21) in three steps. The methanesulfonic acid salt
of 10a (GZD824) was prepared after the free base was reacted with methanesulfonic acid in a
quantitative yield. Compounds 24a, 24b and 25 were prepared from the key intermediate 14 via
Sonogashira coupling with 5ꢀbromoꢀ1Hꢀindazole (26a), 5ꢀbromoꢀ1ꢀmethylꢀ1Hꢀpyrazolo[3,4ꢀb]pyridine
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(26b) or 6ꢀbromoꢀ[1,2,4]triazolo[1,5ꢀa]pyrimidine (27), respectively (Scheme 2).
Scheme 1. Chemical syntheses of designed inhibitors 10a – 10i.
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
N
N
N
I
N
R2
a
O
a). R2 = Me,
b). R2 = H,
+
I
COOCH3
F3C
N
H
c). R2 = Et,
F3C
NH2
d). R2 = cyclo-Pr,
e). R2 = Cl,
R2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
11
HN
12
13
N
R1
c
N
N
N
N
b
d
N
O
HN
O
R2
F3C
N
CF3
H
a). R = H, R = Me
b). R = H, R = H
c). R = H, R = Et
d). R = H, R = cyclo-Pr
e). R = H, R = Cl
f). R = Me, R = Me
g). R = cyclo-Pr, R = Me
h). R = Ph, R = Me
i). R =MeO, R = Me
1
2
10
1
2
14
1
2
H
1
2
N
N
1 2
N
1
2
1
2
1
2
1
9
1 2
c
SEM
SEM
H
H
N
N
N
N
N
N
N
e
N
N
N
g
f
N
N
Br
Br
Br
Br
I
I
R1
a). R = Me,
15
16
17
18
1
b). R = cyclo-Pr,
1
c). R = Ph,
1
MeO
OMe
H
N
N
N
N
Cl
N
N
N
N
h
i
j
N
N
CO Me
Br
2
Br
Br
Br
OH
OH
OMe
2
1
22
23
20
N
N
HN
N
HN
N
N
N
N
k
N
HN
HN
2 CH SO H
3
3
CF3
O
CF3
O
10a
GZD824
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o
Reagents and conditions: a) tꢀBuOK, THF, ꢀ20 C to r.t., argon, 40ꢀ91%; b) Pd(dppf) Cl , CuI,
2
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
o
compound 19, DIPEA, DMF, 80 C, 40ꢀ75%; c) (i) Pd(dppf) Cl , CuI, ethynyltrimethylsilane, DIPEA,
CH CN, 60 C; (ii) K CO , MeOH. 79%; d) (i) Pd(dppf) Cl , CuI, compounds 18a-c, DIPEA, DMF, 80
C; (ii) tꢀBu NF, THF, reflux, 5 hrs. 29ꢀ45 %; e) NIS, 1,2ꢀdichloroethane, 66%; f) SEMꢀCl, NaH, DMF,
63%; g) R B(OH) Pd(dppf)Cl , K PO , dioxane, 37ꢀ57%; h) NH NH ꢀH O, EtOH, 80 C, overnight,
2
2
o
3
2
3
2
2
o
4
o
1
2,
2
3
4
2
2
2
9
4
3%; i) NaOH, DMSO, pꢀmethoxylbenzyl chloride, rt, 1 hr, 62%; j) NaH, MeI, DMF, rt, overnight,
7%; k) methanesulfonic acid, EtOH, 97%.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 2. Chemical syntheses of designed inhibitors 24a, 24b, and 25.
N
N
N
Y N
X
N
Y
N
O
N
X
a
N
HN
F3C
N
+
H
Br
CF3
O
14
26
24
a) X = CH, Y = H
b) X = N, Y = Me
a) X = CH, Y = H
b) X = N, Y = Me
N
N
N
N
N
N
N
N
N
N
a
O
+
N
N
HN
F3C
N
Br
H
CF3
O
14
27
25
o
Reagents and conditions: a) Pd(dppf) Cl , CuI, DIPEA, DMF, 80 C, 50ꢀ55%.
2
2
RESULTS AND DISCUSSION
The kinase inhibitory activity of designed compounds 10a ꢀ 10i, 24 and 25 against different types of
3
8
Abl kinases were evaluated by using a wellꢀestablished FRETꢀbased Z’ꢀLyte assay. Three FDAꢀ
approved BcrꢀAbl inhibitors [imatinib (1), nilotinib (2) and dasatinib (3)] were used as positive controls
to validate the screening conditions. Inhibitor 8 was also included for a direct comparison. As shown in
WT
Table 1, under the experimental conditions, 1 potently inhibited the enzymatic activity of Abl
with
35a
an IC50 value of 98.2 nM, which is highly comparable to the reported data.
However, 1 was
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significantly less potent for kinases harboring mutations. Although 2 and 3 were potently inhibited Abl
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
WT
and several of the mutants, they were inactive against the resistanceꢀrelated T315I mutant. As
observed in the reported data, compound 8 suppressed the enzymatic activity of native Abl kinase and
34
all of the 7 resistanceꢀrelated mutants tested, including the T315I mutation, with low nM IC values.
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Our computational studies suggest that the 1Hꢀpyrazolo[3,4ꢀb]pyridine core in 10a could form a
hydrogen bond donorꢀacceptor network with the hinge region of BcrꢀAbl to achieve better protein
WT
binding than 8 (Figure 2). The novel inhibitor 10a indeed displayed strong inhibition of Abl
and a
panel of mutants with IC values that were approximately 2ꢀ3 folds lower than the values obtained for
5
0
T315I
E255K
compound 8. For instance, the IC values of 8 were 1.39, 1.08 and 1.43 nM against Abl
, Abl
50
G250E
and Abl
, respectively, whereas the corresponding values for 10a were 0.68, 0.27 and 0.71 nM,
respectively (Table 1). To confirm the key contribution of the dual hydrogen bond network formed by
the 1Hꢀpyrazolo[3,4ꢀb]pyridine core with the hinge region of BcrꢀAbl to the activity of 10a, compounds
2
3a, 23b and 24 were designed and synthesized. Compound 23a lacks a hydrogen bond acceptor in the
hingeꢀbinding region. The IC50 values of this compound were 13.9, 82.2, 34.8, 48.6, 29.1, 12.7, 20.1
WT
T315I
E255K
G250E
Q252H
H396P
M351T
Y253F
and 52.6 nM against Abl , Abl
, Abl
, Abl
, Abl
, Abl
, Abl
and Abl
,
respectively; thus, this compound was 40ꢀ120ꢀfold less potent than 10a. The removal of a hydrogen
bond donor in the hingeꢀbinding region (23b and 24) also induced an obvious loss of potency.
A minor structural modification was also introduced to investigate the potential impact of R and R
1
2
on Abl kinase inhibition (Table 1). Although many studies have demonstrated that the “flagꢀmethyl”
group at the R position is critical for compound 1 and many other BcrꢀAbl inhibitors to maintain their
2
kinase inhibition, compound 10b, in which the methyl group was removed, displayed potency
comparable to that of 10a. However, when the methyl group in the R position was replaced by slightly
2
larger substituents, such as ethyl (10c), cyclopropyl (10d) or Cl (10e), the kinase inhibitory activity was
T315I
E255K
G250E
decreased. For instance, the IC values of compound 10d against Abl
, Abl
and Abl
were
5
0
53.73, 11.38, and 28.71 nM, respectively; thus, this compound was 40ꢀ100ꢀfold less potent than 10a.
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The results also clearly revealed that substitution at the R position greatly impacts the inhibitory
1
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
potency of compounds 10f – 10i. Although compound 10f with a methyl group in the R position
1
displayed a potency similar to that of inhibitor 10a, compounds 10g (R = cyclic propyl), 10h (R =
1
1
phenyl), and 10i (R = methoxyl) were obviously less potent than 10a. For example, the IC values for
1
50
WT
T315I
1
0i were approximately 25 times higher than those for 10a against Abl
or the Abl
mutant. The
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
detrimental effect of R substitution may be related to increased steric hindrance that makes it
1
unfavorable for the 1Hꢀpyrazolo[3,4ꢀb]pyridine core to form hydrogen bonds with the hinge region of
BcrꢀAbl kinase.
WT
a
Table 1. Inhibitory activity of the designed compounds against Abl
and different mutants.
N
R1
HN
N
N
N
HN
R2
CF3
O
10
cpds
R1
Abl1 inhibition (IC , nM)
50
Nativ
E255 G250 Q252 H396 M351 Y253
R2
Me
H
T315I
e
K
E
H
P
T
F
0
0
.34± 0.68± 0.27± 0.71± 0.15± 0.35± 0.29± 0.35±
.07 0.11 0.03 0.05 0.01 0.11 0.02 0.05
10a
H
H
0
0
.58± 0.76± 0.99± 0.81± 0.39± 0.46± 0.36± 0.17±
.10 0.17 0.11 0.09 0.10 0.03 0.01 0.01
10b
1
.92± 2.36± 4.93± 4.08± 1.14± 9.39± 0.94± 1.28±
10c
H
Et
0.07 0.15 0.34 1.17 0.13 0.55 0.09 0.07
Cyclo 7.1±0. 53.73 11.38 28.71 3.27± 2.16± 2.93± 4.44±
Pr ±1.20 ±2.37 ±5.20 0.20 0.18 0.23 1.11
10d
H
ꢀ
7
0
0
.83± 1.09± 2.16± 3.47± 0.58± 0.65± 0.57± 0.77±
.13 0.05 0.07 0.35 0.15 0.11 0.05 0.03
10e
H
Cl
0
0
.75± 0.75± 1.30± 1.51± 0.61± 0.53± 0.63± 0.54±
.15 0.09 0.28 0.05 0.07 0.12 0.06 0.05
10f
Me
Me
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Cyclo
Pr
0.84± 2.07± 2.90± 4.08± 0.92± 0.43± 0.93± 1.15±
1
2
3
4
5
6
7
8
9
10g
Me
Me
Me
ꢀ
0.01
0.35
0.05
0.39
0.27
0.12
0.35
0.07
4.8±1. 3.38± 10.58 9.02± 3.47± 3.79± 2.06± 2.57±
10h
Ph
0
5
0.09 ±0.55 0.83 0.03 0.08 0.06 0.56
7.9±0. 15.81 16.53 36.56 5.02± 2.72± 4.28± 5.15±
39 ±0.58 ±3.09 ±1.27 2.07 0.09 0.12 0.09
1
0i
MeO
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13.9± 82.2± 34.8± 48.6± 29.1± 12.7± 20.1± 52.6±
.58 0.79 5.87 7.27 0.59 0.00 1.35 2.48
2
4a
0
1.49± 3.20± 5.40± 5.04± 1.08± 0.71± 0.76± 1.06±
24b
0.05 0.09 0.21 0.09 0.10 0.11 0.11 0.07
3.20± 6.10± 9..52± 18.30 2.03± 1.38± 1.33± 3.26±
25
1
0.83
0.63
1.07
±5.29
0.77
0.29
0.07
0.01
3
59.9
9
2
8.2±
.65
485.8
±55.7
115±2 173.9 114.3 749.6
.3 ±15.8 ±5.80 ±66.7
5155
±123.
5
43.5± 702.4 27.8± 215.3 24.2± 15.8± 12.8± 29.9±
2
3
5.8
±37.7
5.7
±16.8
0.9
1.8
5.7
3.2
0
0
.26±
.03
0.21± 0.25± 0.24± 0.30± 0.22± 0.17±
0.11 0.05 0.13 0.05 0.03 0.01
1450
0
0
.33±
.03
0.32± 0.43± 0.41± 0.50±
0.01 0.15 0.22 0.18
1.39± 1.08± 1.43±
0.07
0.09
0.13
8
(
0.37)
b
(0.44) (0.34) (0.30) (0.30)
b
(2.0)
b
b
b
b
a
The kinase inhibitory activities were determined using a FRETꢀbased Z’ꢀLyte assay. The data
b
34
represent the mean values of 3 independent experiments. Reported data.
The direct binding affinity of compound 10a with the native Abl kinase and the Abl mutants was
determined by using a Kinome screening assay based on activeꢀsiteꢀdependent competition (conducted
by Ambit Bioscience, San Diego, USA). Compound 10a tightly bound to the ATPꢀbinding sites of the
kinases of native Abl and retained the same affinity towards six other frequently detected mutants,
including the most refractory gatekeeper mutant T315I as well as those with point mutations in the pꢀ
loop (Q252H, E255K), the ATPꢀbinding hinge (F317L/I), the SH2ꢀcontact region (M351T) and the
activation loop (H396P) (Table 2). Notably, 10a displayed similar binding affinities for nonꢀ
phosphorylated and phosphorylated forms of Abl kinase. For instance, the binding constant values (Kd)
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were 0.32 and 0.34 nM for 10a to bind with nonphosphorylated and phosphorylated Abl, respectively.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
T315I
Compound 10a also tightly bound with nonphosphorylated and phosphorylated Abl
mutants with
K values of 0.71 and 3.2 nM, respectively. The selectivity profile of 10a against a panel of 442 kinases
d
was also investigated at a concentration of 10 nM which was about 33 times of its K values on native
d
Abl (Supporting Information). The results clearly revealed that compound 10a also exhibited strong
binding with bꢀRAF, DDR1, FGFR, Flt3, Kit, PDGFRα, PDGFRβ, RET, Src, Tie1 and Tie2 and
several other kinases, indicating that this compound might be used as a lead compound for treatment of
many other types of cancer. However, the potential safety issue of 10a related to its relative kinase
selectivity requires further investigation.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Binding affinity of 10a to different mutants of Abl as determined by an activeꢀsiteꢀdependent
a
competitive binding assay (Kinomescan Screening).
Binding affinity (K nM)
d
Abl1
Nonꢀphosphorylated
form
Phosphorylated form
Wild type
T315I
0.32
0.71
0.46
NA
0.34
3.20
NA
Q252H
E255K
F317I
0.28
NA
1.8
H396P
M351T
0.18
NA
NA
0.23
a
TM
Binding constant values (K ) were determined from Ambit KINOMEscan . The data represent the
d
mean values of 2 independent experiments.
The BcrꢀAblꢀinhibitory activity of 10a was further validated at the cellular level after its
antiproliferative activity was evaluated in stably transformed Ba/F3 cells whose growth was driven by
native BcrꢀAbl or BcrꢀAbl mutants, which are mostly responsible for imatinib resistance observed in the
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3
9 ꢀ 41
clinic.
The mutants included T315I, L248V, G250E, Q252H, Y253H/F, E255K/V, F317L/V,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
M351T, E355G, F359V, H396R and F486S, some of which have been frequently reported to be
moderately resistant or resistant to compounds 2 or 3. As shown in Table 3, 10a potently inhibited the
growth of Ba/F3 cells expressing native BcrꢀAbl with an IC value of 1.0 nM, which was equivalent to
5
0
the potency of compound 3 (1.9 nM) and much higher than the potency of compounds 1 (460 nM) and 2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(22 nM).
Highly consistent with its potent biochemical kinase inhibition and tight protein binding affinity, 10a
also strongly inhibited the proliferation of Ba/F3 cells that stably expressed the most refractory BcrꢀAbl
T315I
mutant, with an IC value of 7.1 nM. All of the 3 FDAꢀapproved drugs (1 ꢀ 3) did not inhibit the
5
0
proliferation of these cells. Furthermore, 10a displayed a similar potency of inhibition against Ba/F3
cells expressing 14 other resistanceꢀrelevant BcrꢀAbl mutants. Notably, 10a was > 50 times less active
against parental Ba/F3 cells than Ba/F3 cells expressing BcrꢀAbl kinases, which supports its selective
inhibition of BcrꢀAbl kinase.
Table 3. Inhibition of cellular proliferation as determined using the CCKꢀ8 assay (IC values, ꢀM) for
5
0
a
1
0a and 3 clinically approved BcrꢀAbl inhibitors in Ba/F3 cells expressing a panel of BcrꢀAbl mutants.
Growth inhibition (IC , ꢀM)
5
0
Ba/F3
Cells
Mutated
region
1
2
3
10a
Parental 13.5±2.1
WT 0.5±0.1
L248V 12.2±0.8
15.1±0.7
0.022±0.005
3.0±0.5
2.5±0.3
0.170±0.05
0.001±0.0003
0.0073±0.0031
0.006±0.0007
0.006±0.001
0.011±0.005
0.003±0.001
0.0081±0.0012
Wildꢀtype
Pꢀloop
0.0019±0.0004
0.029±0.011
0.0051±0.0012
0.008±0.002
0.017±0.011
0.0023±0.0012
0.023±0.009
G250E
Q252H
Y253F
5.2±1.3
3.6±0.9
9.2±2.3
0.15±0.12
0.12±0.05
1.4±0.7
Y253H 12.5±2.5
E255V >10
1.0±0.3
1.2±0.1
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E255K 5.6±0.87
0.17±0.07
16.0±1.9
0.0032±0.0006 0.0035±0.0011
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Gate
keeper
T315I
12.2±2.1
3.6±1.7
0.0071±0.0013
Hinge
region
F317L
F317V
3.3±0.1
9.5±3.7
0.16±0.03
2.9±0.5
0.019±0.013
0.025±0.006
0.0027±0.0010
0.0040±0.0010
SH2ꢀ
contact
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
M351T 0.24±0.06 0.003±0.001 0.0002±0.00003 0.0002±0.00007
region
E355G
1.6±0.1
0.13±0.03
0.015±0.005
0.0026±0.0007
Substrateꢀ
binding
region
F359V 0.59±0.13
H396R 11.1±2.7
0.12±0.07
21.1±3.9
0.0013±0.0003 0.0023±0.0005
3.8±2.4 0.0055±0.0010
Aꢀloop
Cꢀ
terminal
lobe
F486S
1.0±0.3 0.0095±0.0015 0.0009±0.0001 0.0010±0.0003
a
IC50 values are determined by using the cell counting kit (CCKꢀ8) assay. The data represent the
mean values of 3 independent experiments.
The antiproliferative activity of 10a was also examined against a panel of leukemia cells with
differing BcrꢀAbl status. Taxol was utilized as a positive control to validate the screening conditions.
Table 4 shows that 10a strongly inhibited the growth of leukemia cells positively expressing BcrꢀAbl,
including K562 and Ku812 human CML cells as well as SUPꢀB15 human acute lymphocyte leukemia
(ALL) cells, with IC values of 0.21, 0.13 and 2.5 nM, respectively. As a further confirmation of 10a’s
5
0
BcrꢀAblꢀselective effect, 10a was significantly less potent against the proliferation of MOLT4 and HLꢀ
0 leukemia cells, which are negative for BcrꢀAbl expression. Not surprisingly, the cytotoxic agent
6
Taxol, which was included in our experiment as a control, inhibited the growth of all tested cells with
similar IC50 values. K562R is a selfꢀestablished imatinibꢀresistant K562 cell line that has been
demonstrated to carry a Q252H mutation in BcrꢀAbl. 10a also potently inhibited the proliferation of this
cell line with an IC value of 4.5 nM. Collectively, these results strongly indicate that 10a selectively
5
0
inhibits the proliferation of BcrꢀAbl ꢀpositive tumor cells.
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Journal of Medicinal Chemistry
Table 4. Compound 10a selectively and potently inhibits the proliferation of BcrꢀAbl positive leukemia
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
cells.
IC values (nM, (X±SD)
5
0
Cell lines
K562
10a
TAXOL
5.1±0.5
7.0±0.5
5.3±3.2
8.0±3.4
2.3±0.2
2.7±0.9
5.8±1.5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.2±0.1
K562R(Q252H)
Ku812
4.5±0.7
0.13±0.02
2.5±1.0
SUPꢀB15
Uꢀ937
390.2±153.4
26.3±10.2
348.9±158.2
MOLT4
HLꢀ60
a
IC values were determined by using the cell counting kit (CCKꢀ8) assay. The data represent the
5
0
mean values of 3 experiments.
To further validate the cellular kinase inhibitory activity of 10a, we examined its effects on the
activation of BcrꢀAbl and its downstream signals in BcrꢀAblꢀpositive K562 CML cells and stably
T315I
transformed Ba/F3 cells expressing native BcrꢀAbl or BcrꢀAbl
(Figure 4) by Western blot analyses.
10a efficiently suppressed the activation of BcrꢀAbl as well as downstream Crkl and STAT5 in a
concentrationꢀdependent manner in K562 CML cells. Similar inhibition was also observed in Ba/F3
T315I
cells expressing native BcrꢀAbl or BcrꢀAbl
. Not surprisingly, none of the 3 FDAꢀapproved drugs
T315I
demonstrated an obvious suppression of BcrꢀAbl
activation.
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
)
M
n
1
0a (nM)
0
5
(
0
1
2
5 10 20
0 0.1 0.3 1 3 10 30 100
0 0.1 0.3 1 3 10 30 100
pBcr-Abl
Bcr-Abl
pStat5
Stat5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
pCrkl
Crkl
Beta-actin
K562
Ba/F3-Bcr-Abl-WT
Ba/F3-BCR-ABL-T315I
Figure 4. Compound 10a inhibits BcrꢀAbl signaling in K562 and Ba/F3 stable cell lines expressing
T315I
native BcrꢀAbl or BcrꢀAbl
. Cells were treated with 10a at the indicated concentrations for 4.0 hours
and whole cell lysates were then subjected to western blot analyses. The results represent 3 independent
experiments (1, imatinib; 2, nilotinib; 3, dasatinib).
Given its highly promising in vitro kinase inhibition and antiproliferative activity, we further
investigated the preliminary ADME (absorption, distribution, metabolism and excretion) profiles of
compound 10a, and it displayed good metabolic stability as determined by an in vitro rat microsomal
42
stability assay. The halfꢀlife (T ) of 10a was approximately 56 minutes, and its intrinsic clearance
1
/2
4
3
(CL ) was approximately 36.4 ꢀL/min/mg. Furthermore, a cytochrome P450 inhibitory assay
int
revealed that 10a did not obviously inhibit CYP1A2, CYP2D6, CYP3A4 and CYP2E1 at 30 ꢀM.
However, 10a potently inhibited CYP2C9 and CYP2C19 with IC50 values of 0.11 and 0.99 ꢀM
respectively, which indicates that it may be subject to potential drugꢀdrug interactions with other
medicinal products, which are metabolized primarily by CYP2C9 and CYP2C19. The transcellular
diffusional permeability and transport directionality of 10a were also determined by using the wellꢀ
44
established model of Cacoꢀ2 cells, which express efflux transporters, such as Pꢀglycoprotein. The
apicalꢀtoꢀbasolateral and basolateralꢀtoꢀapical permeabilities of 10a were 3.2 and 2.8 nm/s, respectively.
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Journal of Medicinal Chemistry
Thus, 10a exhibits good bidirectional permeability in Cacoꢀ2 cells. The permeability directional ratio
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(efflux ratio) for 10a was 0.90, and 10a thus did not appear to be a substrate for Pꢀglycoprotein efflux
4
5
transporters.
An in vivo pharmacokinetic (PK) evaluation of the bismesylate salt of 10a (GZD824) was also
conducted in rats. Plasma levels of 10a were monitored after a single oral dose of 25 mg/kg. The Cmax
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(390.5 ꢀg/L = 733 nM) occurred at 4.0 hours postꢀdose and was > 500 times higher than the in vitro
IC50 values in BcrꢀAblꢀpositive CML cells and Ba/F3 cells expressing different BcrꢀAbl mutants,
T315I
including BcrꢀAbl
. The halfꢀlife of 10a was 10.6 hours. The plasma levels of 10a were
approximately 270 and 169 nM at 12 and 24 hours postꢀdose, respectively (Table 4). Good oral
bioavailability (48.7%) and an ideal halfꢀlife suggest that 10a may have good in vivo efficacy when
orally administrated.
Table 4. The bismesylate salt of compound 10a (GZD824) displays a good pharmacokinetic profile in
rats.
Pharmacokinetic parameters for 10a
Routes
Cmax
T max
AUC0ꢀt
AUC0ꢀ∞
T1/2
MRT
(hr)
CL
Vss
F
(%)
(ꢀg/L)
(hr)
(ꢀg/L* (ꢀg/L*h) (hr)
(L/h/kg) (L/kg)
h)
I.V.
5mg/kg)
1375.6
390.5
0.067
4.0
2737.3
6699.0
2922.4
7108.2
5.6
6.7
1.7
3.6
13.7
53.2
(
Oral
10.6
17.9
48.7%
(25mg/kg)
The human etherꢀaꢀgoꢀgoꢀrelated gene (hERG) encodes an inward rectifying voltageꢀgated potassium
channel in the heart (IKr) that is involved in cardiac repolarization. Inhibition of the hERG can cause
QTc interval prolongation, which is associated with potential lethal arrhythmias known as torsadesde
4
8
pointes. The potential cardiotoxicity of 10a was also initially examined by using a nonradioactive
+
49
rubidium (Rb ) efflux assay. Compound 10a did not show appreciable activity against the hERG
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potassium channel at concentrations of ≤ 30 ꢀM, which suggests that it may have little or no cardiotoxic
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
effects (Supporting Information).
The in vivo antiꢀtumor effect of 10a was first evaluated in subcutaneous K562 and Ku812 xenograft
models of human CML. The clinical drug 1 was used as a positive control to validate the animal
models. As shown in Figure 5, 10a doseꢀdependently inhibited the growth of the K562 tumor xenograft
when administrated once daily for 14 consecutive days via oral gavage. A significant reduction in tumor
size was observed after only 2 days of treatment at doses of 2, 5 and 10 mg/kg/day (p = 0.03, 0.002 and
0.002, respectively). Compound 10a almost completely eradicated the tumor at doses of 5 and 10
mg/kg/day after only 6 days of treatment, whereas the reference drug 1 only induced tumor stasis at a
dose of 50 mg/kg/day. Even at a dose as low as 1 mg/kg/day, 10a still significantly suppressed tumor
growth (68.3% inhibition after 14 days of dosing, p < 0.01). Notably, after the cessation of treatment (14
days of dosing), there was no sign of tumor recurrence in the following 20 days for the groups that
received 5 and 10 mg/kg/day, and recurrence in the 2 mg/kg/day group was also found to a minor extent
(data not shown). All doses of 10a were well tolerated, with no mortality or significant body loss (< 5%
relative to the vehicleꢀmatched controls) observed during treatment. Similar results were also obtained
in the Ku812 xenograft model, where even 1.0 mg/kg/day of 10a induced complete tumor regression
after a 14ꢀday treatment period (Supporting Information).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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Journal of Medicinal Chemistry
1
2
3
4
5
6
Figure 5. Compound 10a potently suppresses tumor growth in mice bearing xenografted K562 cells as
a model of human CML. Mice bearing K562 xenografts were dosed orally once a day with the indicated
compounds at the indicated dosages for 14 consecutive days (n=10 per group, error bars represent the
standard error). The data are representative of 2 independent experiments.
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The antiꢀtumor activity of 10a was also evaluated in allograft models using Ba/F3 cells expressing
WT
T315I
E255K
G250E
F317L
BcrꢀAbl , BcrꢀAbl
, BcrꢀAbl
, BcrꢀAbl
and BcrꢀAbl
. As described above, BcrꢀAbl
T315I
E255K
G250E
is the most refractory mutant, whereas the pꢀloop mutants BcrꢀAbl
and BcrꢀAbl
and the
F317L
hinge region mutant BcrꢀAbl
are frequently detected mutants that drive clinical resistance to many
E255K
BcrꢀAbl inhibitors. For instance, the BcrꢀAbl
mutant has been reported to be insensitive to the
three classical inhibitors (1, 2 and 3) and the two most recently reported BcrꢀAbl inhibitors, 8 and 9,
F317L
whereas BcrꢀAbl
was also relatively insensitive to 1, 3 and 9. The Pꢀloop mutants were also
46, 47
reported to be associated with poor prognoses in the clinic.
Following an experimental design similar to that of the K562 and Ku812 xenograft studies, animals
were administrated a range of doses of 10a via oral gavage once daily for 14 days. Compound 1 was
again used as a reference compound and administered orally at doses of 50 or 100 mg/kg/day for 14
days. As shown in Figure 6A, 10a doseꢀdependently inhibited tumor growth in the allograft model using
WT
Ba/F3 cells expressing BcrꢀAbl
. A dose of 1.0 mg/kg/day of 10a exhibited in vivo efficacy
comparable to that of 50 mg/kg of the reference drug 1. When the mice received a dose of 5.0
mg/kg/day, 10a induced complete tumor regression. Additionally, 10a displayed promising efficacy in
T315I
the allograft model bearing BcrꢀAbl
ꢀdriven tumors (Figure 6B). Although 10a did not show
T315I
obvious inhibition of tumor growth at doses of 5 or 10 mg/kg/day in the BcrꢀAbl
model, it induced
almost complete tumor regression at a dose of 20 mg/kg/day after a 14ꢀday consecutive treatment. In
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contrast, 100 mg/kg/day of 1 completely failed to exhibit any inhibition of tumor growth in the BcrꢀAbl
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
T315I
model. Moreover, 10a also significantly reduced the tumor burden by alleviating
hepatosplenomegaly, which appears at late stages of this leukemiaꢀlike model and is induced by the
T315I
BcrꢀAbl
ꢀdriven cells (data not shown). Similar efficacy of 10a was also observed in the BcrꢀAbl
G250E
E255K
F317L
and BcrꢀAbl
pꢀloop mutants as well as BcrꢀAbl
hingeꢀregionꢀmutantꢀdriven tumor
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
allografts (Supporting Information).
The antitumor efficacy of 10a was further evaluated in a mouse allograft leukemia model, in which
T315I
Ba/F3 cells expressing BcrꢀAbl
were further stably transfected with a luciferaseꢀexpressing vector
to facilitate nonꢀinvasive bioluminescent monitoring of tumor cell proliferation in vivo. Ba/F3 cells coꢀ
T315I
expressing BcrꢀAbl
and luciferase were designated as Ba/F3/T315IꢀLuc cells and injected
intravenously into immunoꢀdeficient mice. These cells proliferated in the blood, spleen and bone
marrow, eventually leading to the morbidity and death of the recipients. The in vivo antiꢀtumor efficacy
of 10a was therefore evaluated in this model through the measurement of in vivo luminescence signals
and survival in the recipient mice. Generally, at 7ꢀ10 days after inoculation of the Ba/F3ꢀT315ꢀLuc
cells, the in vivo luminescence signal became detectable in the vehicleꢀtreated control group, which
indicates the success of the engraftment. Treatment with the test compounds was then initiated through
oral gavage once daily for 10 consecutive days.
A
B
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Journal of Medicinal Chemistry
Figure 6. Compound 10a suppresses tumor growth in mice bearing allografted Ba/F3 cells expressing
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
WT
T315I
BcrꢀAbl
(A) and BcrꢀAbl
(B). Mice bearing allografted Ba/F3 cells expressing the indicated
BcrꢀAbl mutants were orally dosed with 10a and 1 at the indicated doses. The tumor sizes were
monitored over time (n=10 for each group; error bars represent the SE). The data are representative of 2
or 3 independent experiments.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
As shown in Figure 7A, luminescence signals representing the populations of Ba/F3ꢀT315IꢀLuc cells
in vivo were detected at days 0 and 7 after initiation of treatment. At day 0, it was clear that the average
population size of Ba/F3ꢀT315IꢀLuc cells was almost identical among different groups before the
treatment was initiated. At day 7, in the vehicleꢀonly group, luminescence signals were observed
throughout the entire bodies of the mice, which suggested that the leukemiaꢀlike Ba/F3ꢀT315Iꢀluc cells
has disseminated in vivo. After treatment with 10a at 5, 10 or 20 mg/kg/day, the luminescence signals
were obviously decreased when compared with the untreated vehicleꢀonly group. Notably, treatment
with 20 mg/kg/day of 10a nearly eliminated the luminescence signals, which indicates complete
suppression of the proliferation of the Ba/F3ꢀT315IꢀLuc cells. Quantification of the luminescence signal
clearly revealed that 10a potently and doseꢀdependently decreased the signal intensity (Figure 7B).
T315I
Although compound 10a demonstrated significant efficacy in this BcrꢀAbl
leukemia model,
reference drug 1 was totally inactive at a dose of 100 mg/kg/day. Highly consistent with its strong
inhibition of the proliferation of the BaF3/T315Iꢀluc cells, treatment with 10a significantly prolonged
the survival of the mice in a doseꢀdependent manner in this allograft model, whereas 100 mg/kg of 1 did
not show any survival benefit (Figure 7C). Indeed, following treatment with 10a for 10 days at doses of
5
, 10 and 20 mg/kg/day, the median survival of the mice was significantly increased to 23.7, 24.6 and
1.9 days, respectively, as compared with 18.9 days for the vehicleꢀtreated group. Additionally,
3
hepatosplenomegaly was commonly observed in vehicleꢀtreated mice in the late stages of the survival
experiment but was barely detected in the 10aꢀtreated mice, which indicates that 10a could efficiently
alleviate the tumor burden in late stage leukemia (data not shown).
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A
B
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Vehicle
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100 mg/kg
0a 1 mg/kg
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10a 2 mg/kg
10a 5 mg/kg
C
Vehicle
1
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100mg/kg
0a 1mg/kg
0a 2mg/kg
0a 5mg/kg
0a 10mg/kg
0a 20mg/kg
1
0a 10 mg/kg
1
0a 20 mg/kg
Time
Day 0
Day 7
Figure 7. Compound 10a efficiently prolongs animal survival in an allograft leukemia tumor model
T315I
using Ba/F3 cells coꢀexpressing BcrꢀAbl
and luciferase. A) Compound 10a suppresses the
T315I
proliferation of leukemiaꢀlike Ba/F3 cells coꢀexpressing BcrꢀAbl
and luciferase; B) Quantification
of the images in B; C) Compound 10a prolongs the survival of leukemic mice.
In summary, we have successfully designed and synthesized a series of novel inhibitors that display
significant inhibition against a broad spectrum of BcrꢀAbl mutants, including those carrying the
gatekeeper T315I and the pꢀloop mutations, which are associated with poor prognosis in CML. One of
the most promising compounds, 10a, tightly binds the phosphorylated and nonꢀphosphorylated forms of
BcrꢀAbl kinase and inhibits the proliferation of BcrꢀAblꢀpositive human CML cells. Furthermore,
compound 10a potently inhibits the proliferation of a panel of murine BaF3 cells ectopically expressing
WT
BcrꢀAbl
and a broad spectrum of other BcrꢀAbl mutants that contribute to clinical TKI resistance,
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Journal of Medicinal Chemistry
T315I
including the most refractory gatekeeper mutant, BcrꢀAbl
. In vivo efficacy studies in mouse
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xenograft or allograft models of human leukemia demonstrated that 10a successfully suppressed the
T315I
G250E,
Q252H,
E255K
growth of tumors driven by native BcrꢀAbl, BcrꢀAbl
, BcrꢀAbl
BcrꢀAbl
BcrꢀAbl
F317L
and BcrꢀAbl
. Compound 10a also provided a significant survival benefit leukemia model mice
T315I
allografted with luciferaseꢀexpressing Ba/F3 cells driven by the mutant BcrꢀAbl
kinase. The
0
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0
potential for compound 10a to overcome all of the BcrꢀAbl mutations that result in clinically acquired
resistance to imatinib indicates that this novel BcrꢀAbl inhibitor is a promising lead candidate for
further development.
EXPERIMENTAL
Chemistry. Reagents and solvents were obtained from commercial suppliers and used without further
purification. Flash chromatography was performed using silica gel (300ꢀ400 mesh). All reactions were
1
monitored by TLC, silica gel plates with fluorescence F254 were used and visualized with UV light. H
1
3
and C NMR spectra were recorded on a Bruker AVꢀ400 spectrometer at 400 MHz and Bruker AVꢀ500
spectrometer at 125 MHz, respectively. Coupling constants (J) are expressed in hertz (Hz). Chemical
shifts (δ) of NMR are reported in parts per million (ppm) units relative to internal control (TMS). The
low or high resolution of ESIꢀMS was recorded on an Agilent 1200 HPLCꢀMSD mass spectrometer or
Applied Biosystems QꢀSTAR Elite ESIꢀLCꢀMS/MS mass spectrometer, respectively. The purity of
compounds was determined to be over 95% (>95%) by reverseꢀphase high performance liquid
chromatography (HPLC) analysis. HPLC instrument: DIONEX SUMMIT HPLC (Column: Diamonsil
C18, 5.0 µm, 4.6 x 250 mm(Dikma Technologies); Detector: PDAꢀ100 Photodiode Array; Injector:
ASIꢀ100 Autoinjector; Pump: pꢀ680A.). Elution: MeOH in water; Flow rate: 1.0 mL/min.
3
-Iodo-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)benzamide (13a).
solution of 11a (8.6 g, 31.16 mmol) and 4ꢀ((4ꢀmethylpiperazinꢀ1ꢀyl)methyl)ꢀ3ꢀ
A
(trifluoromethyl)aniline (12) (8.48 g, 31.16 mmol) in anhydrous THF (150.0 mL) was added potassium
o
tertꢀbutoxide (20.9 g, 186.96 mmol) in anhydrous THF (150.0 mL) at ꢀ 20 C for 1.0 hr. Then the
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reaction mixture was slowly warmed to room temperature and stirred for another 2.0 hrs. After the
reaction was finished by TLC, the mixture was poured into 1.0 L water with stirring and then left to
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
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2
2
2
2
2
2
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3
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6
stand overnight. The precipitated brown solid was filtered and dried under reduced pressure to afford
1
the desired product. (14.7 g, yield: 91.4%) H NMR (400 MHz, CDCl ), δ 8.28 (d, J = 1.8 Hz, 1 H),
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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8
9
0
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9
0
7.91 (s, 1 H), 7.86 (dd, J = 1.9, 8.4 Hz, 1 H), 7.83 (s, 1 H), 7.78 (d, J = 8.4 Hz, 1 H), 7.74 (dd, J = 1.8,
8
.0 Hz, 1 H), 7.33 (d, J = 8.0 Hz, 1 H), 3.63 (s, 2 H), 2.50ꢀ2.46 (m, 11 H), 2.29 (s, 3 H).
3
-ethynyl-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)benzamide
(
14). A mixture of 13a (13.7 g, 26.5 mmol), Cu (I) iodide (0.05 g, 0.26 mmol), Pd(PPh ) Cl (0.2 g,
3 2
2
0
.26 mmol) were added to a 250 mL roundꢀbottom flask. The flask was evacuated and backfilled with
argon (3 cycles). Acetonitrile (100.0 mL), DIPEA (18.1 mL, 106 mmol) and trimethylsilylacetylene
o
(
7.48 mL, 53.0 mmol) were added by syringe at room temperature. After stirring at 60 C overnight, the
reaction mixture was filtered and concentrated. The residue was dissolved in methanol (50.0 mL) with
addition of K CO (7.31 g, 53 mmol). The reaction mixture was stirred at room temperature for 10~15
2
3
minutes, and then filtered and concentrated for further purification through flash column
1
chromatography on silica gel to afford the desired product as pale solid. (8.7 g, yield: 79%) H NMR
(
400 MHz, dꢀDMSO), δ 10.49 (s, 1 H), 8.19 (d, J = 2.0 Hz, 1 H), 8.08 (d, J = 1.6 Hz, 1 H), 8.03 (dd, J =
1.6, 8.4 Hz, 1 H), 7.90 (dd, J = 8.0 Hz, 1.6 Hz, 1 H), 7.70 (d, J = 8.4 Hz, 1 H), 7.47 (d, J = 8.0 Hz, 1 H),
.51 (s, 1 H), 3.56 (s, 2 H), 2.46 (s, 3 H), 2.33 (br, 8 H), 2.15 (s, 3 H).
4
3-((1H-Pyrazolo[3,4-b]pyridin-5-yl)ethynyl)-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-
(trifluoromethyl)phenyl)benzamide (10a). A mixture of 13a (2.60 g, 5.0 mol), 19 (0.72 g, 5.0 mmol),
Cu (I) iodide (0.5 g, 0.5 mmol), Pd(PPh ) Cl (1.8 g, 0.5 mmol) were added to a sealed tube. The tube
3
2
2
was evacuated and backfilled with argon (3 cycles). Acetonitrile (50.0 mL), Et N (21.0 mL, 0.1 mol)
3
o
were added by syringe at room temperature. After stirring at 100 C for about 20 hrs, the reaction
mixture was filtered and concentrated. The residue purified by flash column chromatography on silica
1
gel to afford the desired product as yellow solid. Yield: 76%. H NMR (400 MHz, dꢀDMSO), δ 13.94 (s,
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1
H), 10.53 (s, 1 H), 8.74 (d, J = 2.0 Hz, 1 H), 8.53 (d, J = 2.0 Hz, 1 H), 8.22ꢀ8.19 (m, 3 H), 8.07 (dd, J
1.6, 8.4 Hz, 1 H), 7.92 (dd, J = 2.0, 8.0 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1 H), 7.53 (d, J = 8.0 Hz, 1 H),
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
=
1
3
3.56 (s, 2 H), 2.59 (s, 3 H), 2.39 (br, 4 H), 2.33 (br, 4 H), 2.16 (s, 3 H); C NMR (125 MHz, dꢀDMSO),
δ 165.11, 151.44, 151.01, 144.12, 138.59, 134.12, 133.42, 132.61, 132.51, 131.65, 130.95, 130.36,
1
28.56, 127.82 (d, J = 30.0 Hz), 124.76 (d, J = 272.5 Hz), 123.91, 122.60, 117.68, 114.44, 112.21,
0
1
2
3
4
5
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8
9
0
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0
+
92.33, 88.71, 57.88, 55.14, 53.11, 46.13, 20.85; MS (EI): 533 [M+H] ; Purity: 99.77% (t 11.33 min).
R
3-((1H-Pyrazolo[3,4-b]pyridin-5-yl)ethynyl)-N-(4-((4-methylpiperazin-1-yl)methyl)-3-
(trifluoromethyl)phenyl)benzamide (10b). This compound was synthesized by reacting 13b with 19
1
under similar procedure to that of 10a. Yield: 51%. HNMR (400 MHz, dꢀDMSO), δ 13.94 (s, 1 H),
1
0.62 (s, 1 H), 8.73 (d, J = 2.0 Hz, 1 H), 8.52 (d, J = 1.6 Hz, 1 H), 8.23ꢀ8.21 (m, 3H), 8.07 (d, J = 8.4
Hz, 1 H), 8.01 (d, J = 7.6 Hz, 1 H), 7.82 (d, J = 7.6 Hz, 1 H), 7.72 (d, J = 8.4 Hz, 1 H), 7.63 (t, J = 7.6
1
3
Hz, 1 H), 3.57 (s, 2 H), 2.39 (br, 4 H), 2.33 (br, 4 H), 2.15 (s, 3 H); C NMR (125 MHz, dꢀDMSO), δ
1
1
65.25, 151.52, 151.00, 138.51, 135.39, 134.84, 134.14, 133.54, 132.65, 131.68, 130.71, 129.58,
28.62, 127.84, 124.74, 123.94, 122.82, 117.70, 114.41, 111.99, 89.83, 88.55, 57.87, 55.14, 53.12,
+
46.13; MS (EI): 519 [M+H] ; Purity: 99.71% (t 9.73 min).
R
3-((1H-Pyrazolo[3,4-b]pyridin-5-yl)ethynyl)-4-ethyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-
(trifluoromethyl)phenyl)benzamide (10c). This compound was synthesized by reacting 13c with 19
1
under similar procedure to that of 10a. Yield: 40.7%. H NMR (400 MHz, dꢀDMSO), δ 13.95 (s, 1 H),
1
0.56 (s, 1 H), 8.72 (d, J = 2.0 Hz, 1 H), 8.51 (d, J = 2.0 Hz, 1 H), 8.22 (s, 2 H), 8.19 (d, J = 1.6 Hz, 1
H), 8.07 (dd, J = 1.6, 8.4 Hz, 1 H), 7.96 (dd, J = 2.0, 8.4 Hz, 1 H), 7.70 (d, J = 8.4 Hz, 1 H), 7.53 (d, J
= 8.0 Hz, 1 H), 3.57 (s, 2 H), 2.95 (q, J = 7.6 Hz, 2 H), 2.40 (br, 8 H), 2.20 (s, 3 H), 1.30 (t, J = 7.6 Hz,
1
3
3
1
H); C NMR (125 MHz, dꢀDMSO), δ 164.71, 150.90, 150.55, 149.64, 138.18, 133.67, 132.93,
32.27, 131.95, 131.20, 130.83, 18.48, 128.38, 127.38, 124.30, 123.44, 121.45, 117.22, 113.99, 111.76,
+
91.35, 87.98, 57.35, 55.54, 52.39, 45.57, 45.38, 27.13, 14.49; MS (EI): 547 [M+H] ; Purity: 96.31% (t
R
1
3.20 min).
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