Design, synthesis, and biological evaluation of 3-(1H-1,2,3-triazol-1-yl) benzamide derivatives as potent pan Bcr-Abl inhibitors including the threonine315←isoleucine315 mutant

Article abstract of DOI:10.1021/jm301188x

A series of 3-(1H-1,2,3-triazol-1-yl)benzamide derivatives were designed and synthesized as new Bcr-Abl inhibitors by using combinational strategies of bioisosteric replacement, scaffold hopping, and conformational constraint. The compounds displayed significant inhibition against a broad spectrum of Bcr-Abl mutants including the gatekeeper T315I and p-loop mutations, which are associated with disease progression in CML. The most potent compounds 6q and 6qo strongly inhibited the kinase activities of Bcr-Abl^WT and Bcr-Abl^T315I with IC₅₀ values of 0.60, 0.36 and 1.12, 0.98 nM, respectively. They also potently suppressed the proliferation of K562, KU812 human CML cells, and a panel of murine Ba/F3 cells ectopically expressing either Bcr-Abl^WT or any of a panel of other Bcr-Abl mutants that have been shown to contribute to clinical acquired resistance, including Bcr-Abl^T315I, with IC₅₀ values in low nanomolar ranges. These compounds may serve as lead compounds for further development of new Bcr-Abl inhibitors capable of overcoming clinical acquired resistance against imatinib.

Full text of DOI:10.1021/jm301188x

Article
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of 3‑(1H‑1,2,3-Triazol-1-
yl)benzamide Derivatives as Potent Pan Bcr-Abl Inhibitors Including
the Threonine315→Isoleucine315 Mutant
Yupeng Li,†,‡,§ Mengjie Shen,†,‡,§ Zhang Zhang,† Jinfeng Luo,† Xiaofen Pan,† Xiaoyun Lu,†
Huoyou Long,† Donghai Wen,† Fengxiang Zhang,† Fang Leng,† Yingjun Li,†,‡ Zhengchao Tu,†
Xiaomei Ren,† and Ke Ding*,†
†Institute of Chemical Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, #190 Kaiyuan
Avenue, Guangzhou 510530, China
‡Graduate University of Chinese Academy of Sciences, #19 Yuquan Road, Beijing 100049, China
S
* Supporting Information
ABSTRACT: A series of 3-(1H-1,2,3-triazol-1-yl)benzamide derivatives were
designed and synthesized as new Bcr-Abl inhibitors by using combinational
strategies of bioisosteric replacement, scaffold hopping, and conformational
constraint. The compounds displayed significant inhibition against a broad
spectrum of Bcr-Abl mutants including the gatekeeper T315I and p-loop
mutations, which are associated with disease progression in CML. The most
potent compounds 6q and 6qo strongly inhibited the kinase activities of Bcr-
AblWT and Bcr-AblT315I with IC50 values of 0.60, 0.36 and 1.12, 0.98 nM,
respectively. They also potently suppressed the proliferation of K562, KU812
human CML cells, and a panel of murine Ba/F3 cells ectopically expressing
either Bcr-AblWT or any of a panel of other Bcr-Abl mutants that have been
shown to contribute to clinical acquired resistance, including Bcr-AblT315I, with
IC50 values in low nanomolar ranges. These compounds may serve as lead
compounds for further development of new Bcr-Abl inhibitors capable of
overcoming clinical acquired resistance against imatinib.
approved as the second-line drugs to treat adult patients in all
phases of CML with resistance to drug 1 (Figure 1).9
Bafetinib10 and bosutinib11 have also been developed into
late-stage of clinical trials. Despite of the effectiveness of these
second-generation compounds to inhibit most of the Bcr-Abl
mutants, subsets of mutants remain resistant.12 Particularly,
INTRODUCTION
■
Chronic myelogenous leukemia (CML) is a hematological
malignancy representing about 20% of adult leukemia and
characterized by the presence of the Philadelphia (Ph)
chromosome. The chromosome is a truncated version of
chromosome 22 resulting from a reciprocal translocation
between chromosomes 9 and 22. The chimeric Bcr-Abl gene
generated by the translocation encodes a fusion protein having
constitutively active kinase activity.1 This fusion protein Bcr-Abl
kinase has become a well-validated target for development of
small molecular inhibitors to treat CML.2 The first-generation
Bcr-Abl inhibitor imatinib (1, STI571) has achieved great
clinical success and became the first-line drug for the treatment
of CML.3 However, emerging acquired resistance to drug 1 can
be a major challenge,4 with resistance rates of 3−4% reported in
newly diagnosed chronic phase CML patients, up to 40−50%
or over 80%, respectively, in accelerated or blastic phase
disease.5 The point mutations in the kinase domain of Bcr-Abl
are a major mechanism to imatinib resistance, and more than
100 point mutations have been identified.6
none of the second-generation inhibitors inhibits Bcr-AblT315I
,
the “gatekeeper” mutation that accounts for approximately 15−
20% of all clinical acquired mutations.4,13 Bcr-AblT315I -induced
drug resistance remains an unmet clinical challenge for CML
treatment.14
Thr315 residue is critical for the binding of most Bcr-Abl
inhibitors, regardless of whether they target the active or
inactive conformation of the kinase, and in the T315I mutant
loss of the side-chain hydroxyl group abrogates the possibility
of a key hydrogen bond between the inhibitors and Thr315
residue.6b The bulky isoleucine side chain also hinders the
inhibitors from accessing an important adjacent hydrophobic
pocket.7b,15 Several classes of inhibitors have been identified
that potently suppress the function of Bcr-AblT315I mutant.9b,16
To overcome the resistance against drug 1, several classes of
second-generation kinase inhibitors have been developed,
among which nilotinib (2)7 and dasatinib (3)8 have been
Received: August 12, 2012
Published: October 22, 2012
© 2012 American Chemical Society
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Figure 1. Chemical structures of FDA-approved Bcr-Abl inhibitors and newly developed molecules bafetinib, bosutinib, and DCC-2036.
Figure 2. Design of 3-(1H-1,2,3-triazol-1-yl)benzamide derivatives as new Bcr-Abl inhibitors.
PPY-A is a pyrrolopyridine compound binding to the active
conformation of Bcr-Abl and effectively inhibited T315I mutant
with an IC50 value of 20 nM.17 c-Src/Abl dual inhibitor
discontinued due to cardiac toxicity including QTc prolonga-
tion.25
Most recently, some “third-generation” Bcr-Abl inhibitors
were disclosed to inhibit almost the full range of Bcr-Abl kinase
domain mutations as well as the native kinase.26 A novel type II
Bcr-Abl inhibitor GNF-7 (4) was reported to strongly inhibit
Bcr-AblT315I and other mutants with low nanomolar IC50 values.
It also showed promising in vivo efficacy without appreciable
toxicity.27 Ponatinib (5, AP24534)28 and DCC-203629 have
been advanced for clinical investigation. The overall clinical
benefits of these inhibitors are eagerly awaited. Herein, we
report the design, synthesis, and biological evaluation of 3-(1H-
1,2,3-triazol-1-yl)benzamide derivatives as new pan Bcr-Abl
inhibitors that potently inhibit native Bcr-Abl kinase and a
TG100598 displayed an IC50 value of 3.4 nM in an enzymatic
18
assay against Bcr-AblT315I
.
Non-ATP-competitive or allosteric
inhibitors (ON01238019 or GNF-220) were also identified to
potently inhibit Bcr-AblT315I. Several ATP-competitive aurora
kinase inhibitors such as MK-0457 (Tozasertib, VX680),21
PHA-739358,22 AT9283,23 and XL-22824 have been found to
be active against Bcr-AblT315I. Some of the aurora inhibitors
have been advanced into clinical investigation. However, these
molecules have to be formulated for intravenous admin-
istration, and clinical development for MK-0457 has been
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a
Scheme 1. Synthesis of Compounds 6a−6d and 6m−6r
a
Reagents and conditions: (a) 6 N HCl, NaNO2, NaN3, 0 °C to rt, 99%; (b) 4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)aniline, tBuOK,
t
THF, −20 °C to rt, 61%; (c) 11, CuSO4, sodium ascorbate, BuOH/water (1:1), 90 °C, 22−69%; (d) (i) trimethylsilylacetylene, 10 mol % of
Pd(PPh3)2Cl2, 10 mol % of CuI, Et3N, acetonitrile, reflux; (ii) K2CO3, methanol, rt, 68−80% (two steps).
a
wide-range of Bcr-Abl kinase domain mutants, including the
gatekeeper Bcr-AblT315I
Scheme 2. Synthesis of Compounds 6e−6i
.
Molecular Design. Several cocrystal structures of Bcr-Abl
with different type II third-generation inhibitors have been
reported.27,30 Structural analysis of chemical features of the
inhibitors (i.e., 4 and 5) and their interactions with Bcr-Abl
kinase revealed that most of the compounds possessed some
key structural elements which formed crucial interactions with
the protein: (1) a heterocyclic moiety (so-called “head region”)
binding to the adenine pocket and forming a hydrogen bond
network with residues in the hinge region; (2) a methylphenyl
group (“middle part”) occupying the hydrophobic pocket
behind the gatekeeper residue; (3) an additional hydrophobic
group (“tail region”) binding the pocket induced by the DFG-
out conformation; (4) a suitable linker between the head region
and the middle part to avoid the steric clash with the Ile315
residue of Bcr-Abl. On the basis of this observation, a series of
3-(1H-1,2,3-triazol-1-yl)benzamide derivatives were designed as
Bcr-Abl inhibitors in which a 1,2,3-triazol moiety was utilized as
the linker between the head region and the middle part (Figure
2).
Chemistry. The synthesis of compounds 6a−6d and 6m−
6r with alternative hinge-binding templates is illustrated in
Scheme 1. Briefly, methyl 3-azido-4-methylbenzoate (8) was
prepared by diazotization of methyl 3-amino-4-methylbenzoate
(7) and coupled with 4-((4-methylpiperazin-1-yl)methyl)-3-
(trifluoromethyl)aniline to produce the benzamide (9). The
coupling of 9 with 11, which was prepared through a classical
Sonogashira reaction,31 following deprotection afforded final
products 6a−6d and 6m−6r in moderate yields. It is
noteworthy that the coupling reaction worked well for
substrates with unprotected pyrazole, pyrrole, or amine
functionalities, although a slight decrease of isolated yields
was observed.
a
Reagents and conditions: (a) amines, ethanol, 80 °C, 75−98%; (b)
(i) trimethylsilylacetylene, 10 mol % of Pd(PPh3)2Cl2, 10 mol % of
CuI, Et3N, acetonitrile, reflux; (ii) K2CO3, methanol, rt, 64−90% (two
steps); (c) 9, CuSO4, sodium ascorbate, BuOH/water (1:1), 90 °C,
t
14−62%.
Compounds 6qj−6ql were synthesized from 3-bromo-5-
(trifluoromethyl)benzenamine (20). Compounds 21 were
prepared by applying our previously developed protocol.32
The inhibitors with different triazoles or imidazoles (6qj−6ql)
could be synthesized similarly from the procedures outlined in
Scheme 4.
Compounds 6qm−6qs with different R3 substituents were
prepared by using commercially available or self-prepared 3-
amino-4-substituted benzoates (23) as the starting materials,
following procedures similar to that of other substrates
(Scheme 5).
RESULTS AND DISCUSSION
■
Given the strong potency of inhibitor 4 against Bcr-AblWT and
the clinical resistance relevant mutants, we initially selected the
imidazo[1,2-b]pyridazine moiety, which was one of the most
important pharmacophores of 4, as the potential head template
to interact with the hinge region of the kinase. The 4-((4-
methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)benzenamine
tail group was utilized because of its physicochemical properties
to keep ideal hydrophilic−lipophilic balance of the molecules.
This group was also widely found in the chemical structures of
many other kinase inhibitors.15a,33
For the compounds containing 2-amino pyrimidinyl moieties
(6e−6l, Scheme 2), N-substituted 5-bromo-2-amines 13 were
first prepared by SNAr displacement of 5-bromo-2-chloropyr-
imidine (12) with different amines. The final compounds could
be obtained through procedures similar to that in Scheme 1.
The synthesis of 6qa−6qi is outlined in Scheme 3. 4-Methyl-
3-(trifluoromethyl)aniline (15) was protected with a mono-Boc
group to provide intermediate 16. Compound 17 was obtained
by NBS bromination of 16, followed by amine substitution and
deprotection to afford intermediates 18. Compounds 6qa−6qi
were readily synthesized using procedures similar to that in
Scheme 1.
A computational docking study suggested that 6a could bind
to the ATP binding site of the nonphosphorylated (DFG-out)
form of Bcr-AblWT and different Bcr-Abl mutants, including
Bcr-AblT315I, with similar binding modes to that of inhibitor 4
(Figure 3A,B). The imidazo[1,2-b]pyridazine moiety in 6a
formed an essential hydrogen bond with the NH of Met318 in
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a
Scheme 3. Synthesis of 6qa−6qi Containing Different Hydrophilic Groups
a
Reagents and conditions: (a) di-tert-butyl dicarbonate, Na2CO3, dioxane/water (1:1), rt, 99%; (b) NBS, AIBN, CCl4, reflux, 98%; (c) (i)
hydrophilic amine, Et3N, THF, 50 °C; (ii) TFA, DCM, rt, 45−88%; (d) 8, tBuOK, THF, −20 °C to rt, 32−86%; (e) 11q, CuSO4, sodium ascorbate,
tBuOH/water (1:1), 90 °C, 14−43%.
hydrogen bonds with Glu286 and Asp381 and the trifluor-
omethylphenyl group bound deeply into the hydrophobic
pocket. The 1,2,3-triazol linker could form favorable van der
Waals interactions with the gatekeeper Ile315 of Bcr-AblT315I
without causing steric clash.
Scheme 4. Synthesis of Inhibitors 6qj−6ql with Different
a
Triazoles or Imidazoles
Compound 6a inhibited Bcr-AblWT and the Bcr-AblT315I
mutant as assessed using a well-established FRET-based Z′-
Lyte assay.34 Compound 4 was used as the positive control to
validate the screening conditions and for direct comparison. As
shown in Table 1, compound 4 potently inhibited the
enzymatic activity of Bcr-AblWT and Bcr-AblT315I mutant with
mean IC50 values of 1.25 and 0.97 nM, respectively, which is
comparable to the reported data.28 The IC50 value of
compound 6a was 2.67 nM against Bcr-AblWT under the
same screening conditions. The kinase inhibition of 6a was
further validated by investigating its suppression on the growth
of murine Ba/F3 cells ectopically expressing Bcr-AblWT. Further
evaluation indicated that it strongly inhibited the proliferation
of Bcr-AblWT -positive K562 and KU812 CML cells. It also
inhibited the growth of our imatinib-resistant K562R cells,
shown to carry a Q252H mutation in Bcr-Abl, with a mean IC50
value of 1.4 nM. However, its inhibitory activity against Bcr-
AblT315I mutant was significantly lower than that of wild-type
a
Reagents and conditions: (a) triazoles or imidazoles, 1-(5,6,7,8-
tetrahydroquinolin-8-yl)ethanone, CuI, Cs2CO3, DMF, Ar, 130 °C,
50−71%; (b) 6 N HCl, NaNO2, NaN3, 0 °C to rt, 79−95%; (c) 11q,
CuSO4, sodium ascorbate, BuOH/water (1:1), 90 °C, 8−68%.
t
the hinge region of Bcr-Abl. The tail part of the molecule also
participated in hydrogen bonds and hydrophobic interactions
with the DFG motif, among which the amide formed two
a
Scheme 5. Synthesis of Compounds 6qm−6qs with Different R3 Substituents
a
Reagents and conditions: (a) 6 N HCl, NaNO2, NaN3, 0 °C to rt, 47−98%; (b) methyl 3-azido-4-methylbenzoate, tBuOK, THF, −20 °C to rt, 10−
52%; (c) 11q, CuSO4, sodium ascorbate, tBuOH/water (1:1), 90 °C, 13−18%; (d) cyclopropylboronic acid, Pd(dppf)Cl2-DCM, K3PO4, dioxane, Ar,
90 °C; (e) (i) TFA, NH4Cl, THF/water (1:1); (ii) Fe powder, refluxing, 44% (two steps of d and e).
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Figure 3. Binding models of inhibitor 6a and 6q to Bcr-Abl kinase. Hydrogen bonds are indicated by yellow hatched lines to key amino acids. (A)
Binding model of 6a with Bcr-AblWT (PDB: 3cs9). (B) Binding model of 6a with Bcr-AblT315I (PDB: 3ik3). (C) Binding model of 6q with Bcr-AblWT
(PDB: 3cs9). (D) Binding model of 6q with Bcr-AblT315I (PDB: 3ik3).
kinase (mean IC50 92 nM). It also poorly inhibited the growth
of murine Ba/F3 cells stably expressing Bcr-AblT315I (IC50 >10
μM), which might be due to poor solubility.
substituted groups in the 2-amino moiety had a significant
impact on the bioactivities of compounds. With increasing size
of the substituted groups, the potencies decreased markedly.
This might be due to the fact that the substituted groups could
form steric clash to affect their potential hydrogen bond
interaction with the hinge of Bcr-Abl. For instance, N-methyl
analogue 6f displayed activities similar to the N-unsubstituted
inhibitor 6e, but the N-benzyl 6k showed a 46-fold loss in
potency. Not surprisingly, the N,N-disubstituted compound 6l
was almost totally inactive in both enzymatic and cellular
assays.
Inspired by the contribution of the two hydrogen bonds
between the 2-N-methyl amino pyrimidinyl template and the
hinge of Bcr-Abl to the kinase inhibition, a series of compounds
containing either a pyrrolo[2,3-b]pyridinyl or 1H-pyrazolo[3,4-
b]pyridinyl motif were designed by utilizing a conformation-
constraining strategy. As shown in Table 1, 6n, 6q, and 6r
displayed similar potencies against Bcr-AblWT to those of 6e
and 6f. However, their inhibitory activities against Bcr-AblT315I
were significantly increased by factors of 20, 22, and 10,
respectively, compared to that of 6f. The cell growth inhibitory
activities of the compounds were also obviously improved. The
importance of the hydrogen bond network was further
demonstrated by selective removal of the hydrogen bond
donor or receptor moieties. Compound 6m lacking a hydrogen
bond donor group showed mean IC50 values against Bcr-AblWT
and Bcr-AblT315I of 22.6 and 206 nM, respectively, which was
38 and 200 times less potent than 6q. On the other hand, the
removal of a hydrogen bond acceptor (6o and 6p) also caused
dramatic potency loss compared to 6n and 6q.
Other heterocyclic moieties such as 3-imidazo[1,2-a]-
pyrimidinyl (6b), 3-pyrazolo[1,5-a]pyrimidinyl (6c), or 3-lH-
imidazo[1,2-a]pyridinyl (6d) were introduced to maintain the
potential hydrogen bond with the hinge region of Bcr-Abl.
Compounds 6b and 6c displayed activities similar to that of 6a
in biochemical and cellular assays. Interestingly, 6d was 5−6
times more potent than 6a in the enzymatic inhibition assay.
The superiority of 6d became more significant in cell
proliferation inhibition assays.
Our docking study has suggested that the imidazo[1,2-
b]pyridazine moiety in 6a could form a hydrogen bond with the
NH of M318 in the hinge region of Bcr-Abl (Figure 3A). We
hypothesized that the bioactivity might be improved by
introducing an additional hydrogen-donating moiety into the
molecule to provide another hydrogen bond with the hinge
region. A 2-N-substituted amino pyrimidinyl motif has
previously been successfully used as a hinge-binding template
to catch two pairs of hydrogen bonds in the design of receptor
tyrosine kinase inhibitors.35 Therefore, a series of 3-(4-(2-
(substituted amino)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-N-
phenylbenzamides were designed and synthesized as new Bcr-
Abl inhibitors. Computational studies suggested that com-
pound 6f could form two hydrogen bonds with the NH and the
CO motif of M318 in the hinge region of Bcr-Abl
(Supporting Information). Biological evaluation revealed that
compound 6f strongly inhibited the activities of Bcr-AblWT and
Bcr-AblT315I with IC50 values of 0.78 and 10.6 nM, respectively,
which was about 3−8 times more potent than 6a. This
compound was also significantly more active than 6a
suppressing Bcr-Abl-dependent cell proliferation. Further
structure−activity relationship analysis demonstrated that the
We have successfully designed and synthesized 6n, 6q, and
6r as new highly potent inhibitors by applying combinational
strategies of bioisosteric replacement, scaffold hopping, and
conformational constraint. The compounds potently inhibited
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a
Table 1. Bioactivities of New Bcr-Abl Inhibitors
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Table 1. continued
a
b
The data are means of data from at least three independent experiments (IC50, nM). The data are means of results from two independent
c
experiments. The K562R is a self-established cell line which carries a Q252H Bcr-Abl mutation.
the enzymatic functions of Bcr-AblWT and Bcr-AblT315I with
IC50 values in low nanomolar ranges. They also strongly
suppressed the proliferation of K562, K562R, and KU812
human CML cells and murine Ba/F3 cells ectopically
expressing Bcr-AblWT and Bcr-AblT315I. Taking 6q as an
example, the structure−activity relationships were further
explored by investigating the influence of different R1, R2,
and R3 groups.
Our computational investigation suggested that 6q could
tightly bind to the ATP binding site of the nonphosphorylated
(DFG-out) forms of Bcr-AblWT and different Bcr-Abl mutants,
including Bcr-AblT315I (Figure 3B). The 1H-pyrazolo[3,4-
b]pyridinyl moiety formed two hydrogen bonds with the
hinge region of Bcr-Abl. It was also demonstrated that the
trifluoromethylphenyl group in 6q bound deeply into a
hydrophobic pocket formed by DFG-out conformation. It
was further suggested that the methylpiperazine moiety pointed
toward the solvent-accessible domain of an allosteric binding
pocket, and a positively charged terminal anime group might
form favorable hydrogen bond(s) with the carbonyl oxygen
atoms of the residues. The contribution of the terminal anime
group has been clearly demonstrated in cocrystal structures of
Bcr-Abl with inhibitor 1, 4, or others.15a,30,36
Therefore, we further investigated the potential influence of
the tail region by introducing different hydrophilic groups in
the R1 or R2 position. Consistent with our docking analysis, the
1-(4-methyl)piperazinyl group in compound 6q was important
for its strong inhibition against Bcr-Abl kinases. Although the
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compounds with 1-pyrrolidinyl (6qa), 1-piperidinyl (6qd), or
N-morpholino (6qe) moieties displayed similar suppressive
potencies against Bcr-AblWT to that of 6q, their potencies
against Bcr-AblT315I decreased approximately 30−250-fold. For
instance, the IC50 values of compound 6q against Bcr-AblWT
and Bcr-AblT315I were 0.60 and 1.12 nM, while the
corresponding values for compound 6qd were decreased to
2.56 and 272 nM, respectively. Further investigation also
revealed that the potency loss of compound 6qd was able to be
partially restored by introducing a 4-dimethylamino or 4-(1-(4-
methyl)piperazin)yl group at the original piperidinyl moiety
(6qg and 6qh). Compounds 6qb and 6qc with a terminal (S)-
or (R)-dimethylamino group as well as 6qi were also more
potent than the parent compound 6qa. When the 1-(4-
methyl)piperazinyl group was replaced with the 1-(4-methyl)-
1,4-diazepanyl group, the resulting compound 6qf was almost
equally potent to compound 6q. It was noteworthy that
compounds displayed comparable potencies when the 3-
methyl-1H-imidazol (6qj), 1H-1,2,4-triazol (6qk), or 3-
methyl-1H-1,2,4-triazol (6ql) group was introduced at the R2
position.
Several studies have suggested that the “flag methyl” group at
the R3 position was important for drug 1 and other inhibitors to
maintain a favorable conformation for binding with Bcr-Abl
kinase and imparting kinase selectivity.33,37 A brief investigation
was also conducted to evaluate its impact on inhibitory
activities of new inhibitors, and the results are summarized in
Table 1. It was shown that the methyl group in 6q could be
replaced with Cl (6qo) or Br (6qp) atoms without obvious
influence on the kinase inhibition and cell proliferation
inhibition. When the methyl group was replaced by H
(6qm), F (6qn), or an ethyl group (6qq), the resulting
inhibitors displayed inhibition against Bcr-AblWT and anti-
proliferation on K562, K562R, KU812, and Ba/F3 cells
expressing Bcr-AblWT similar to that of 6q. However, their
activities on Bcr-AblT315I kinase and Ba/F3 cells expressing Bcr-
AblT315I were dramatically decreased. The results also clearly
revealed that large hydrophobic groups were detrimental to the
activities both on kinase and cellular levels. For instance,
replacement of the methyl group at the R3 position with a
cyclopropyl (6qr) or isopropyl (6qs) group caused 20−500-
fold potency loss in Bcr-AblWT and Bcr-AblT315I inhibition.
As the two most potent compounds shown in Table 1, the
effects of 6q and 6qo were further investigated on the activation
of Bcr-Abl and its downstream signals in cells harboring
different status of Bcr-Abl kinases. K562 is a human CML cell
line with positive Bcr-AblWT, and Ba/F3 T315I cells are murine
Ba/F3 cells ectopically expressing Bcr-AblT315I. As shown in
Figure 4, 6q and 6qo dose-dependently inhibited the
phosphorylation of Bcr-Abl in both K562 and Ba/F3 cells
expressing Bcr-AblT315I. The phosphorylation level of the
downstream signal proteins such as CrkL and STAT5 were
also obviously decreased, while the total protein levels of Bcr-
Abl, CrkL, and STAT5 and GAPDH remained unchanged.
Further evaluation also suggested that 6q and 6qo could
potently induced apoptosis and G0/G1 phase arrest in both
K562 and Ba/F3 T315I cells (Supporting Information). These
data further supported strong target inhibition of the designed
compounds.
Figure 4. Compounds 6q and 6qo dose-dependently inhibit the
phosphorylation of Abl, CrkL, and STAT5 in both K562 (A,B) and
Ba/F3 cells transformed by Bcr-AblT315I (C,D). (A) 6q suppresses the
activation of Abl and downstream signals in K562 cells; (B) 6qo
suppresses the activation of Abl and downstream signals in K562 cells;
(C) 6q suppresses the activation of Abl and downstream signals in Ba/
F3 T315I cells; (D) 6qo suppresses the activation of Abl and
downstream signals in Ba/F3 T315I cells. The data are representative
of three independent experiments.
inhibitory activities of 6q and 6qo against a panel of other
resistance relevant Bcr-Abl mutants were further investigated by
evaluating their antiproliferation effects on Ba/F3 cells stably
expressing different Bcr-Abl mutants (Table 2). The results
clearly demonstrated that the compounds displayed strong
activity against all of the mutant Bcr-Abl-dependent Ba/F3
cells, which might be due to the fact that the mutated residues
did not form direct interaction with the compound in most of
the mutants. It was noteworthy that the compounds were more
than 30 times less potent on untransformed Ba/F3 cells than
Table 2. Inhibitory Activities of 6q and 6qo against 14 Types
a
of Bcr-Abl Mutants as Well as Bcr-AblWT
transformed Ba/F3 cells
1
2
3
6q
6qo
WT
1834
424
65.2
10.3
21.0
150
4.50
0.83
1.71
6.55
1.29
0.58
0.12
1714
0.31
5.72
1.91
1.94
1.72
0.58
0.51
6589
2.44
0.40
1.43
5.49
1.61
0.37
0.12
13.9
0.28
3.10
2.96
1.44
3.73
0.35
1.67
416.4
2.49
0.45
4.17
7.55
2.97
0.30
0.61
11.9
0.30
2.75
3.41
2.31
5.40
0.52
1.97
372.3
M351T
F486S
Q252H
Y253H
E355G
M244V
T315I
H396P
E255K
E255V
F359V
H396R
Y253F
G250E
parent
2547
3763
17083
362
314
8.38
1.54
775
129
3005
110
6.34
292
2951
8615
960
473
159
2377
1444
1863
18520
87.2
60.7
38.6
40750
In addition to mutations in the gatekeeper region (T315I/
A), a variety of other mutations in the p-loop region (e.g.,
G250E, Q252H, Y253, E255K/V) and hinge region (F317L/V)
conferring imatinib resistance have also been identified.6c The
a
Antiproliferation assays of the compounds are evaluated by using cell
counting kit (CCK-8). Data are the means of three experiments (IC50,
nM).
10040
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Journal of Medicinal Chemistry
Article
2.45 (br, 8H), 2.23 (s, 3H), 2.21 (s, 3H); MS (ESI) m/z 433 [M + H]
.
those expressing Bcr-Abl kinases, supporting the selective
inhibition on Bcr-Abl kinase.
+
5-Ethynyl-1H-pyrazolo[3,4-b]pyridine (11q). A mixture of 5-
bromo-1H-pyrazolo[3,4-b]pyridine (10.0 g, 0.05 mol), Cu (I) iodide
(0.5 g, 0.5 mmol), and Pd(PPh3)2Cl2 (1.8 g, 0.5 mmol) were added to
a 250 mL round-bottom flask with a reflux condenser. The flask was
evacuated and backfilled with argon (3 cycles). Acetonitrile (50.0 mL),
Et3N (21.0 mL, 0.1 mol), and trimethylsilylacetylene (21.0 mL, 0.1
mol) were added by syringe at room temperature. After stirring at 100
°C for 20 h, the reaction mixture was filtered and concentrated. The
residue was dissolved in methanol (50.0 mL) with addition of K2CO3
(14.0 g, 0.1 mol). The reaction mixture was stirred at room
temperature for 0.5 h and then filtered and concentrated for further
purification by flash column chromatography on silica gel to afford the
desired product as gray solid (4.9 g, yield 68%): 1H NMR (400 MHz,
CDCl3) δ 11.27 (s, 1H), 8.69 (d, J = 1.6 Hz, 1H), 8.25 (d, J = 2.0 Hz,
In summary, we have successfully designed and synthesized a
series of 3-(1H-1,2,3-triazol-1-yl)benzamide derivatives as new
Bcr-Abl inhibitors which maintain significant inhibition against
a broad spectrum of Bcr-Abl mutants including the most
refractory gatekeeper T315I mutant associated with disease
progression in CML. The most potent compounds 6q and 6qo
strongly inhibited the kinase activities of Bcr-AblWT and Bcr-
AblT315I with mean IC50 values of 0.60, 0.36 and 1.12, 0.98 nM,
respectively. They also potently inhibited the proliferation of
K562, KU812 human CML cells, and a panel of murine Ba/F3
cells ectopically expressing Bcr-AblWT and a broad spectrum of
other Bcr-Abl mutants contributing to clinical TKI resistance,
including Bcr-AblT315I. Western blot analysis on their effects on
Bcr-Abl signal pathway further supported their strong kinase
inhibition against Bcr-Abl and Bcr-AblT315I. These compounds
may serve as new promising lead compounds for further
development of new Bcr-Abl inhibitors overcoming the clinical
acquired resistance against imatinib.
1
1H), 8.11 (s, 1H), 3.17 (s, 1H); H NMR (400 MHz, DMSO-d) δ
13.89 (br, 1H), 8.59 (d, J = 1.6 Hz, 1H), 8.41 (d, J = 1.6 Hz, 1H), 8.17
(s, 1H), 4.28 (s, 1H).
11a−11d and 11m−11r were prepared by the method similar to
that of 11q.
3-(4-(1H-Pyrazolo[3,4-b]pyridin-5-yl)-1H-1,2,3-triazol-1-yl)-4-
methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)-
phenyl)benzamide (6q). To a solution of 5-ethynyl-1H-pyrazolo[3,4-
b]pyridine 11q (1.5 g, 10 mmol) and 3-azido-4-methyl-N-(4-((4-
methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)benzamide 9
(4.5 g, 10 mmol) in tert-butyl alcohol/water (1:1, 20.0 mL) were
added CuSO4 (0.17 g, 1 mmol) and sodium ascorbate (0.41 g, 2
mmol). Then the mixture was heated at 90 °C overnight. The reaction
mixture was cooled to room temperature and extracted three times
with ethyl acetate. Then the combined organic phases were washed
with brine, dried over anhydrous sodium sulfate, concentrated, and
purified by silica gel column chromatography to give desired product
EXPERIMENTAL SECTION
■
Chemistry. Reagents and solvents were obtained from commercial
suppliers and used without further purification. Flash chromatography
was performed using silica gel (300−400 mesh). All reactions were
monitored by TLC, using silica gel plates with fluorescence F254 and
1
UV light visualization. H and 13C NMR spectra were recorded on a
Bruker AV-400 spectrometer at 400 MHz and Bruker AV-500
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 ESI-MS was recorded on an Agilent 1200
HPLC-MSD mass spectrometer or Applied Biosystems Q-STAR Elite
ESI-LC-MS/MS mass spectrometer, respectively. The purity of
compounds was determined by reverse-phase high-performance liquid
chromatography (HPLC) analysis confirming to be over 95% (>95%).
HPLC instrument: Dionex Summit HPLC (column: Diamonsil C18,
5.0 μm, 4.6 × 250 mm (Dikma Technologies); detector: PDA-100
photodiode array; injector: ASI-100 autoinjector; pump: p-680A).
Elution, MeOH in water; flow rate = 1.0 mL/min.
1
as white solid (1.31 g, yield 22%): H NMR (400 MHz, DMSO-d) δ
13.81 (s, 1H), 10.60 (s, 1H), 9.15 (d, J = 9.6 Hz, 2H), 8.77 (s, 1H),
8.26 (s, 1H), 8.19 (d, J = 5.2 Hz, 2H), 8.14 (d, J = 6.4 Hz, 1H), 8.06
(d, J = 6.8 Hz, 1H), 7.72 (dd, J = 6.4 Hz, 2.8 Hz, 2H), 3.56 (s, 2H),
2.36 (br, 10H), 2.15 (s, 3H); 13C NMR (125 MHz, DMSO-d) δ
164.04, 146.89, 144.77, 137.95, 137.15, 136.09, 133.65, 133.11, 132.26,
131.81, 131.24, 129.10, 127.38 (J = 30 Hz), 126.15, 125.36, 125.07,
123.53, 123.18, 123.01, 119.70, 117.27, 114.36, 57.40, 54.68, 52.66,
45.68, 17.70; HR MS (EI) calcd for C29H28F3N9O 576.2442 [M +
H]+; found 576.2434, purity 96.4% (tR 6.67 min).
Compounds 6a−6d and 6m−6r were prepared from compound 9
and the corresponding reactant 11 by a method similar to that of 6q.
5-Bromo-N-cyclohexylpyrimidin-2-amine (13j). A solution of 5-
bromo-2-chloropyrimidine 12 (1.93 g, 10.0 mmol) and cyclohexyl-
amine (2.29 mL, 20 mmol) in ethanol (15.0 mL) was heated at 80 °C
with stirring in a sealed tube overnight. The reaction mixture was
cooled to room temperature. After evaporation of the solvent, the off-
white solid was disssolved in ethyl acetate. Then the organic phase was
washed with water, dried over anhydrous sodium sulfate, and
concentrated to give desired product as off-white solid (2.4 g, yield
Methyl 3-Azido-4-methylbenzoate (8). Methyl 3-amino-4-methyl-
benzoate 7 (8.4 g, 0.05 mol) was dissolved in HCl (6.0 mol/L, 40.0
mL, 0.24 mol) at room temperature, cooled to 0 °C, treated with a
solution of NaNO2 (3.57 g in 8.4 mL of H2O, 0.05 mol) and stirred for
10 min at −5 to ∼0 °C. Sodium azide (3.97 g in 12.6 mL of H2O, 0.06
mol) was added slowly, and the mixture was stirred at room
temperature for 2 h. The mixture was diluted with ethyl acetate, and
the organic layer was washed with brine, dried over anhydrous sodium
sulfate, and concentrated to give the crude product as yellow oil which
was pure enough for subsequent reaction (9.7 g, yield 99%): 1H NMR
(400 MHz, CDCl3) δ 7.70 (d, J = 1.2 Hz, 1H), 7.63 (dd, J = 7.6 Hz,
1.2 Hz, 1H), 7.15 (d, J = 8.0 Hz, 1H), 3.85 (s, 3H), 2.18 (s, 3H).
3-Azido-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-
(trifluoromethyl)phenyl)benzamide (9). To a mixture of 8 (9.6 g, 0.5
mol) and 4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)-
aniline (13.7 g, 0.5 mol) in anhydrous THF (50.0 mL) was slowly
added potassium tert-butoxide (16.8 g, 0.15 mol) in anhydrous THF
(50.0 mL) at −20 °C. Then the reaction mixture was stirred for 1 h,
then slowly warmed to room temperature and stirred for another 8 h.
After evaporation of the solvent, the residue was disssolved in ethyl
acetate, washed with brine, dried over anhydrous sodium sulfate, and
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography to afford the desired compound as
1
94%): H NMR (400 MHz, CDCl3) δ 8.24 (s, 2H), 5.06 (d, J = 5.6
Hz, 1H), 3.78−3.69 (m, 1H), 2.03−1.99 (m, 2H), 1.77−1.71 (m, 2H),
1.66−1.62 (m, 1H), 1.46−1.35 (m, 2H), 1.27−1.16 (m, 3H).
N-Cyclohexyl-5-ethynylpyrimidin-2-amine (14j). A mixture of 5-
bromo-N-cyclohexylpyrimidin-2-amine 13j (1.28 g, 5.0 mmol), Cu(I)
iodide (0.048 g, 0.25 mmol), and Pd(PPh3)2Cl2 (0.18 g, 0.25 mmol)
were added to a sealed tube. The tube was evacuated and backfilled
with argon (3 cycles). Acetonitrile (15.0 mL), Et3N (2.1 mL, 15.0
mmol), and trimethylsilylacetylene (2.1 mL, 15.0 mmol) were added
by syringe at room temperature. After stirring at 100 °C for 24 h, the
reaction mixture was filtered and concentrated. The residue was
dissolved in methanol (20.0 mL) and treated with K2CO3 (1.4 g, 10.0
mmol). The reaction mixture was stirred at room temperature for 0.5
h and then filtered and concentrated for further purification by flash
column chromatography on silica gel to afford the desired product as
1
red solid (13.5 g, yield 61%): H NMR (400 MHz, CDCl3) δ 7.81−
1
7.77 (m, 3H), 7.73 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 1.4 Hz, 1H), 7.39
(dd, J = 7.8 Hz, 1.4 Hz, 1H), 7.21 (d, J = 8.3 Hz, 1H), 3.57 (s, 3H),
yellow solid (0.9 g, yield 89%): H NMR (400 MHz, CDCl3) δ 8.36
(s, 2H), 5.24 (d, J = 6.4 Hz, 1H), 3.87−3.78 (m, 1H), 3.16 (s, 1H),
10041
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2.04−2.00 (m, 2H), 1.77−1.72 (m, 2H), 1.66−1.61 (m, 1H), 1.47−
1.36 (m, 2H), 1.27−1.18 (m, 3H).
3-Azido-4-methyl-N-(4-((4-methyl-1,4-diazepan-1-yl)methyl)-3-
(trifluoromethyl)phenyl)benzamide (19f). To a solution of methyl 3-
azido-4-methylbenzoate 8 (1.03 g, 5.4 mmol) and 4-((4-methyl-1,4-
diazepan-1-yl)methyl)-3-(trifluoromethyl)aniline 18f (1.3 g, 4.5
mmol) in anhydrous THF (5.0 mL) was added potassium tert-
butoxide (1.5 g, 12.6 mmol) in anhydrous THF (15.0 mL) slowly at
−20 °C. Then the reaction mixture was stirred for 1 h then slowly at
room temperature and stirred overnight. After evaporation of the
solvent, the residue was diluted with ethyl acetate. The organic layer
was washed with brine, dried over anhydrous sodium sulfate, and
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography to give the desired compound as
Other N-substituted 5-ethynylpyrimidin-2-amines 14e−14l were
synthesized by using the procedure similar to that of 14j.
3-(4-(2-(Cyclohexylamino)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-4-
methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)-
phenyl)benzamide (6j). To a solution of N-cyclohexyl-5-ethynyl-
pyrimidin-2-amine 14j (0.20 g, 1.0 mmol) and 3-azido-4-methyl-N-(4-
((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)-
benzamide 9 (0.43 g, 1.0 mmol) in tert-butyl alcohol/water (1:1, 4.0
mL) were added CuSO4 (16 mg, 0.1 mmol) and sodium ascorbate (40
mg, 0.2 mmol). Then the mixture was heated at 90 °C overnight. The
reaction mixture was cooled to room temperature and extracted three
times with ethyl acetate. The combined organic phases were washed
with brine, dried over anhydrous sodium sulfate, concentrated, and
purified by silica gel column chromatography to give desired product
as white solid (0.31 g, yield 50%): 1H NMR (400 MHz, CDCl3) δ 8.71
(s, 2H), 8.41 (s, 1H), 7.95−7.88 (m, 5H), 7.78 (d, J = 8.8 Hz, 1H),
7.51 (d, J = 7.6 Hz, 1H), 5.21 (d, J = 7.6 Hz, 1H), 3.88−3.86 (m, 1H),
3.63 (s, 2H), 2.50 (br, 8H), 2.35 (s, 3H), 2.30 (s, 3H), 2.07−2.04 (m,
2H), 1.75 (m, 2H), 1.67−1.64 (m, 1H), 1.48−1.39 (m, 2H), 1.29−
1.24 (m, 3H); 13C NMR (125 MHz, CDCl3) δ 164.30, 161.45, 155.66,
143.58, 137.99, 136.47, 136.37, 134.12, 133.53, 132.24, 131.40, 129.27
(J = 32.4 Hz), 128.57, 125.08, 124.71, 123.36, 122.90, 119.75, 117.69,
113.39, 57.77, 55.23, 53.14, 49.83, 46.05, 33.13, 25.68, 24.80, 18.22;
HR MS (EI) calcd for C33H38F3N9O 634.3224 [M + H]+; found
634.3214, purity 99.5% (tR 13.24 min).
Compounds 6e−6l were prepared from compound 9 and the
corresponding reactant 14 by a method similar to that of 6j.
tert-Butyl 4-Methyl-3-(trifluoromethyl)phenylcarbamate (16). To
4-methyl-3-(trifluoromethyl)aniline 15 (25.0 g, 0.14 mol) in dioxane
(50.0 mL) was added sodium carbonate (22.3 g, 0.21 mol) in water
(50.0 mL) followed by di-tert-butyl dicarbonate (39.0 mL, 0.17 mol).
After stirring at room temperature overnight, the reaction was diluted
with EtOAc, washed with brine, dried over anhydrous Na2SO4, and
evaporated under reduced pressure to give the desire product as red
solid which was used without further purification (39.0 g, yield 99%):
1H NMR (400 MHz, CDCl3) δ 7.61 (d, J = 2.0 Hz, 1H), 7.42 (d, J =
1
yellow oil (1.5 g, yield 74%): H NMR (400 MHz, CDCl3) δ 8.65 (s,
1H), 7.95 (s, 1H), 7.92 (d, J = 8.8 Hz, 1H), 7.73 (d, J = 1.2 Hz, 1H),
7.67 (d, J = 8.4 Hz, 1H), 7.53 (dd, J = 8.0 Hz, 1.2 Hz, 1H), 7.26−7.25
(m, 1H), 3.76 (s, 2H), 3.16−3.14 (m, 2H), 3.03−3.01 (m, 2H), 2.81−
2.78 (m, 2H), 2.62 (s, 3H), 2.06−2.00 (m, 4H).
3-(4-(1H-Pyrazolo[3,4-b]pyridin-5-yl)-1H-1,2,3-triazol-1-yl)-4-
methyl-N-(4-((4-methyl-1,4-diazepan-1-yl)methyl)-3-
(trifluoromethyl)phenyl)benzamide (6qf). To a solution of 5-ethynyl-
1H-pyrazolo[3,4-b]pyridine 11q (0.30 g, 2.1 mmol) and 3-azido-4-
methyl-N-(4-((4-methyl-1,4-diazepan-1-yl)methyl)-3-(trifluoro-
methyl)phenyl)benzamide 19f (0.3 g, 0.7 mmol) in tert-butyl alcohol/
water (1:1, 5.0 mL) were added CuSO4 (11 mg, 0.07 mmol) and
sodium ascorbate (28 mg, 0.14 mmol). Then the mixture was heated
at 90 °C overnight. The reaction mixture was cooled to room
temperature and was extracted three times with ethyl acetate. Then the
combined organic phases were washed with brine, dried over
anhydrous sodium sulfate, concentrated, and purified by silica gel
column chromatography to give the desired product as yellow solid
1
(0.24 g, yield 59%): H NMR (400 MHz, DMSO-d) δ 13.84 (s, 1H),
10.60 (s, 1H), 9.16 (s, 1H), 9.14 (d, J = 1.2 Hz, 1H), 8.77 (d, J = 1.2
Hz, 1H), 8.26 (s, 1H), 8.20 (s, 1H), 8.17 (s, 1H), 8.14 (d, J = 6.4 Hz,
1H), 8.06 (d, J = 6.4 Hz, 1H), 7.76 (d, J = 6.8 Hz, 1H), 7.71 (d, J = 6.4
Hz, 1H), 3.69 (s, 2H), 2.64−2.60 (m, 4H), 2.58−2.55 (m, 2H), 2.52
(m, 2H), 2.35 (s, 3H), 2.24 (s, 3H), 1.72−1.70 (m, 2H); 13C NMR
(100 MHz, DMSO-d) δ 163.94, 151.46, 146.78, 144.67, 137.70,
136.98, 136.00, 133.39, 133.09, 131.61, 130.96, 128.88, 127.14 (J =
31.0 Hz), 126.04, 125.55, 124.93, 123.45, 122.80, 119.62, 117.26,
114.24, 57.25, 57.15, 55.92, 54.19, 53.81, 46.14, 26.79, 17.46; MS
(ESI) m/z 590 [M + H]+; HR MS (EI) calcd for C30H30F3N9O
590.2598 [M + H]+; found 590.2590, purity 97.0% (tR 18.19 min).
Compounds 6qa−6qi were prepared from compound 11q and the
corresponding compound 19 by a method similar to that of 6qf.
3-(4-Methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)aniline (21j). A
mixture of CuI (190 mg, 1 mmol), 4-methyl-1H-imidazole (1.64g, 20
mmol), Cs2CO3 (3.25g, 10 mmol), 3-bromo-5-(trifluoromethyl)-
aniline 20 (2.40g, 10 mmol), 1-(5,6,7,8-tetrahydroquinolin-8-yl)-
ethanone (350 mg, 2 mmol), and DMF (30 mL) was added to a
round-bottom flask. The flask was evacuated and backfilled with argon
(this procedure was repeated three times), and then the mixture was
heated at 130 °C for 24 h under argon. After cooling to room
temperature, the solvent was removed under vacuum and the residue
was purified by column chromatography on silica gel to afford the
crude product. The crude product was recrystallized from toluene to
afford 21j as a white solid (1.7 g, 71%): 1H NMR (400 MHz, DMSO-
d) δ 8.06 (s, 1H), 7.35 (s, 1 H), 6.97 (s, 1 H), 6.93 (s, 1 H), 6.81 (s, 1
H), 5.87 (br, 2H), 2.15 (s, 3H); MS (ESI) m/z 242 [M + H]+.
3-Azido-4-methyl-N-(3-(4-methyl-1H-imidazol-1-yl)-5-
(trifluoromethyl)phenyl)benzamide (22j). To a solution of methyl 3-
azido-4-methylbenzoate 8 (0.19 g, 1.0 mmol) and 3-(4-methyl-1H-
imidazol-1-yl)-5-(trifluoromethyl)aniline 21j (0.24 g, 1.0 mmol) in
anhydrous THF (2.0 mL) was added potassium tert-butoxide (0.34 g,
3.0 mmol) in anhydrous THF (5.0 mL) slowly at −20 °C and 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 diluted with ethyl acetate. The organic layer
was washed with brine, dried over anhydrous sodium sulfate, and
concentrated for purification by silica gel column chromatography to
7.6 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 6.53 (br, 1H), 2.41 (d, J = 1.6
Hz, 3H), 1.52 (s, 9H).
tert-Butyl 4-(Bromomethyl)-3-(trifluoromethyl)phenylcarbamate
(17). To a preheated solution of CCl4 (50.0 mL) were added tert-butyl
4-methyl-3-(trifluoromethyl)phenylcarbamate 16 (18.3 g, 0.066 mol),
NBS (23.6 g, 0.133 mol), and AIBN (1.1 g, 6.6 mmol) in one portion.
After heating under reflux overnight, the mixture was filtered. The
filtrate was washed with brine, dried over anhydrous Na2SO4, and
concentrated to red oil which was used without further purification
1
(23.0 g, yield 98%): H NMR (400 MHz, CDCl3) δ 7.68 (d, J = 1.6
Hz, 1H), 7.54 (d, J = 8.8 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 6.80 (br,
1H), 4.59 (s, 2H), 2.76 (s, 9H).
4-((4-Methyl-1,4-diazepan-1-yl)methyl)-3-(trifluoromethyl)-
aniline (18f). tert-Butyl 4-(bromomethyl)-3-(trifluoromethyl)phenyl-
carbamate 17 (1.8 g, 5.0 mmol) was added to anhydrous THF (10.0
mL) followed by N-methylhomopiperazine (1.5 mL, 12.0 mmol) and
Et3N (2.1 mL, 15.0 mmol). The stirred mixture was heated at 50 °C
overnight. After evaporation of the solvent, the residue was diluted
with ethyl acetate. The organic layer was washed with brine, dried over
anhydrous NaSO4, and concentrated for purification by silica gel
column chromatography (DCM/CH3OH = 20:1, 0.5% NH3·H2O) to
1
give desired product as yellow oil (1.75 g, yield 88%): H NMR (400
MHz, CDCl3) δ 7.71 (d, J = 8.4 Hz, 1H), 7.63 (d, J = 2.0 Hz, 1H),
7.49 (d, J = 8.0 Hz, 1H), 6.75 (s, 1H), 3.70 (s, 2H), 2.71−2.66 (m,
6H), 2.63−2.60 (m, 2H), 2.37 (s, 3H), 1.86−1.80 (m, 2H), 1.51 (s,
9H). Then the product (1.75 g, 4.5 mmol) was added to a solution of
DCM (5.0 mL) and TFA (5.0 mL). After stirring for 1 h at room
temperature, the solvent was evaporated off under reduced pressure.
The residue was neutralized with saturated NaHCO3 and extracted
with ethyl acetate. The combined organic layers were dried and
concentrated to yellow oil which was used directly in the next step (1.3
g, quantitative).
1
afford the desired compound as white solid (0.37 g, yield 92%): H
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NMR (400 MHz, DMSO-d) δ 10.71 (s, 1H), 8.28 (s, 1 H), 8.21 (s, 1
H), 8.12 (s, 1 H), 7.81 (s, 1 H), 7.75−7.74 (m, 2H), 7.49 (s, 1 H),
7.44 (d, J = 8.0 Hz, 1H), 2.24 (s, 3H), 2.18 (s, 3H); MS (ESI) m/z
401 [M + H]+.
4H), 2.15 (s, 3H), 1.82−1.75 (m, 1H), 1.01−0.99 (m, 2H), 0.85−0.84
(m, 2H); 13C NMR (125 MHz, DMSO-d) δ 163.97, 151.82, 146.74,
144.84, 143.22, 138.10, 136.42, 133.50, 132.24, 132.17, 131.23, 129.60,
127.43 (J = 30.0 Hz), 126.09, 125.71, 125.29, 124.29 (J = 272.5 Hz),
123.56, 123.52, 119.53, 117.30, 114.38, 57.42, 55.99, 54.69, 52.66,
45.68, 18.51, 11.07, 9.74; HR MS (EI) calcd for C31H30F3N9O
602.2598 [M + H]+; found 602.2590, purity 95.5% (tR 7.63 min).
Compounds 6qm−6qs were prepared from compound 11q and the
corresponding compound 25 by a method similar to that of 6qr.
Computational Study. The structures of Bcr-AblWT protein and
Bcr-AblT315I protein were retrieved from the Protein Data Bank (PDB
code: 3CS9.pdb and 3IK3.pdb). The proteins were processed using
protein preparation wizard, which assigns bond orders and adds
hydrogens and missing atoms. Inhibitors were built by in LigPrep
3-(4-(1H-Pyrazolo[3,4-b]pyridin-5-yl)-1H-1,2,3-triazol-1-yl)-4-
methyl-N-(3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)-
phenyl)benzamide (6qj). 5-Ethynyl-1H-pyrazolo[3,4-b]pyridine 11q
(0.24 g, 1.7 mmol) and 3-azido-4-methyl-N-(3-(4-methyl-1H-
imidazol-1-yl)-5-(trifluoromethyl)phenyl)benzamide 22j (0.22 g, 0.55
mmol) was added to a solution of THF/water (1:1, 5.0 mL) followed
by CuSO4 (8.8 mg, 0.55 mmol) and sodium ascorbate (22 mg, 1.1
mmol). Then the mixture was heated at 90 °C overnight. The reaction
mixture was cooled to room temperature and extracted three times
with ethyl acetate. The combined organic phases were washed with
brine, dried over anhydrous sodium sulfate, concentrated, and purified
by silica gel column chromatography to give desired product as yellow
(LigPrep, version 2.5, Schrodinger, LLC, New York, NY, 2011)
̈
module using OPLS-2005 force field. Molecular docking was
1
solid (0.21 g, yield 70%): H NMR (400 MHz, DMSO-d) δ 13.81 (s,
performed in Glide module (Glide, version 5.7, Schrodinger, LLC,
̈
1H), 10.84 (s, 1H), 9.18 (s, 1H), 9.14 (s, 1H), 8.77 (s, 1H), 8.50 (s,
1H), 8.36 (s, 1H), 8.26 (s, 1H), 8.24 (s, 1H), 8.17 (s, 2H), 7.79 (s,
1H), 7.76 (d, J = 7.6 Hz, 1H), 7.60 (s, 1H), 2.37 (s, 3H), 2.21 (s, 3H);
13C NMR (125 MHz, DMSO-d) δ 164.82, 151.90, 147.32, 145.27,
141.52, 138.01, 137.76, 136.61, 135.30, 134.19, 133.15, 132.43, 131.32
(J = 27.5 Hz), 129.64, 126.63, 125.59, 123.96 (J = 271.2 Hz), 123.48,
120.13, 115.87, 115.29, 114.84, 112.76, 18.18, 9.02; HR MS (EI) calcd
for C27H20F3N9O 544.1816 [M + H]+; found 544.1805, purity 95.0%
(tR 5.20 min).
Compounds 6qk and 6ql were prepared from compound 11q and
the corresponding compound 22 by a method similar to that of 6qj.
Methyl 4-Cyclopropyl-3-nitrobenzoate (27). To a solution of
methyl 4-bromo-3-nitrobenzoate 26 (1.0 g, 3.9 mmol) and
cyclopropylboronic acid (0.34 g, 3.9 mmol) in dioxane (10.0 mL)
were added Pd(dppf)Cl2-DCM (64 mg, 0.078 mmol) and K3PO4
(2.08 g, 7.8 mmol). The reaction mixture was evacuated and backfilled
with argon (3 cycles). After stirring at 90 °C overnight, the reaction
mixture was filtered and concentrated. The residue was diluted with
EtOAc and washed with brine. Then the organic layer was dried over
anhydrous sodium sulfate and concentrated to afford the desired
product which was used directly in the next step due to difficulty in
purification by column chromatography.
New York, NY, 2011) with standard precision scoring function.
FRET-Based Z′-Lyte Assay Detecting Peptide Substrate
Phosphorylation. The effects of compounds on the kinase activity
of Bcr-Abl and its mutants were assessed in 384-well plates using the
FRET-based Z′-Lyte assay system according to the manufacturer’s
instructions (Invitrogen, Carlsbad, CA, USA). Briefly, 10 μL per well
reactions contained ATP concentration at 10 μM (for Bcr-Abl wild-
type) or 5 μM (for T315I mutant), 2 μM Tyr2 peptide substrate in 50
mM HEPES (PH 7.5), 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA,
0.0247 μg/mL Bcr-Abl, and inhibitors as appropriate. The reaction was
performed at room temperature for 2.0 h, and then 5 μL of
development reagent was added for a further 2 h room temperature
incubation followed by the addition of 5 μL of stop solution.
Fluorescence signal ratio of 445 nm (coumarin)/520 nm (fluorescein)
was examined on EnVision Multilabel Reader (Perkin-Elmer, Inc.).
The data were analyzed using Graphpad Prism5 (Graphpad Software,
Inc.). The data were the mean values of three experiments.
Cells and Reagents. The leukemia cell lines (K562, KU812) were
purchased from ATCC and maintained as recommended by ATCC
(Manassas, VA, USA). Imatinib, dasatinb, and nilotinib were
purchased from Biocompounds Pharmaceutical Inc. (Shanghai,
China). CCK-8 was purchased from Dojindo Molecular Technologies
Inc. (Kumamoto, Japan). Dimethyl sulfoxide (DMSO) and
Cremophor were purchased from Sigma-Aldrich (Dorset, USA).
Antibodies against Abl, p-Abl, Crkl, p-Crkl, STAT5, p-STAT5 were all
purchase from Cell Signaling Technology Inc. (Danvers, USA).
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
μL/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% = (As − Ac)/(Ab − Ac) × 100%, and the data
were further analyzed using Graphpad Prism5 (Graphpad Software,
Inc.) (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-Amino-4-cyclopropylbenzoate (23r). To a solution of
methyl 4-cyclopropyl-3-nitrobenzoate 27 (20 g, 91 mmol) in THF−
water (1:1) (200 mL) were added CF3COOH (4.38 g, 73 mmol) and
NH4Cl (0.48 g, 9.1 mmol). After heating under reflux, Fe powder
(20.5 g, 364 mmol) was added in portion. The reaction was stirred for
a further 3 h. The reaction mixture was filtered and concentrated. The
residue was diluted with EtOAc and washed with brine. Then the
organic layer was dried over anhydrous sodium sulfate, concentrated,
and purified by column chromatography to afford the desired product
1
as yellow solid (7.64 g, yield 44% of two steps): H NMR (400 MHz,
DMSO-d) δ 7.25 (d, J = 2.0 Hz, 1H), 7.06 (dd, J = 8.0 Hz, 2.0 Hz,
1H), 6.87 (d, J = 8.0 Hz, 1H), 5.27 (s, 2H), 3.78 (s, 3H), 1.75−1.71
(m, 1H), 0.92−0.88 (m, 2H), 0.56−0.52 (m, 2H).
Methyl 3-Azido-4-cyclopropylbenzoate (24r). This compound was
synthesized through an analogous procedure to that used for the
synthesis of 8 (yield 95%): 1H NMR (400 MHz, CDCl3) δ 7.78 (d, J =
2.0 Hz, 1H), 7.70 (dd, J = 8.0 Hz, 1.6 Hz, 1H), 6.86 (d, J = 8.0 Hz,
1H), 3.92 (s, 3H), 2.16−2.11 (m, 1H), 1.06−1.02 (m, 2H), 0.75−0.71
(m, 2H).
3-Azido-4-cyclopropyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-
(trifluoromethyl)phenyl)benzamide (25r). This compound was
synthesized through an analogous procedure to that used for the
synthesis of 9 (yield 15%): m/z 459 [M + H]+.
3-(4-(1H-Pyrazolo[3,4-b]pyridin-5-yl)-1H-1,2,3-triazol-1-yl)-4-cy-
clopropyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoro-
methyl)phenyl)benzamide (6qr). This compound was synthesized
through an analogous procedure to that used for the synthesis of 6q
(yield 25%): 1H NMR (400 MHz, DMSO-d) δ 9.16 (s, 1H), 9.13 (d, J
= 1.6 Hz, 1H), 8.76 (d, J = 1.6 Hz, 1H), 8.25 (s, 1H), 8.18 (s, 2H),
8.14 (d, J = 8.0 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.70 (d, J = 8.4 Hz,
1H), 7.37 (d, J = 8.4 Hz, 1H), 3.56 (s, 2H), 2.38 (br, 4H), 2.33 (br,
Stably Transformed Ba/F3 Cells. The Ba/F3 cell lines stably
expressing native Bcr-Abl or various Bcr-Abl mutants were established
by following a procedure similar to that described by von Bubnoff.38
Briefly, wild-type Bcr-Abl p210 was cloned into pCDNA3.1(+)
(Invitrogen, Carlsbad, CA, USA). Point mutations were introduced
to pCDNA3.1(+) Bcr-Abl using the QuickChange XL site-directed
mutagenesis kit (Stratagene, La Jolla, CA, USA). Ba/F3 cells were
transfected with the constructs using transfected cells cloning kit
(Stem Cell Technologies, Vancouver, Canada) by electroporation.
Stable lines were selected in Amaxa cell line nucleofector kit V (Lonza,
Cologne, Germany) with G418 (Merck, Whitehouse Station, USA)
and withdrawal of interleukin-3 (IL-3, R&D). All of the Ba/F3 stable
cell lines were verified by monitoring both DNA sequences through
DNA sequencing and protein expression levels of the corresponding
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Journal of Medicinal Chemistry
Article
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 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. Imatininib-
resistant K562 cells was self-established. Briefly, K562 cells were
treated with graded imatinib (from 0.1 to 5 μM) during a 3 month
period. Single clones were then selected and identified through DNA
sequencing, and their responds toward imatinib, nilotinib, and dasatinb
were also monitored as references.
Western Blot Analysis. The Western blot analysis was carried out
by following the protocol described before.39 Briefly, after the
indicated treatment, cell lysates were collected dissolving cells in 1×
SDS sample lysis buffer. After being sonicated and boiled, the
supernatant of 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 1× TBS containing 0.1%
Tween-20 and 5% nonfat milk, and 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, USA).
Ser, serine; R, Arg, arginine; TKI, tyrosine-kinase inhibitor;
ATP, adenosine triphosphate
REFERENCES
■
(1) (a) Sawyers, C. L. Chronic myeloid leukemia. N. Engl. J. Med.
1999, 340 (17), 1330−1340. (b) Faderl, S.; Talpaz, M.; Estrov, Z.;
O’Brien, S.; Kurzrock, R.; Kantarjian, H. M. The biology of chronic
myeloid leukemia. N. Engl. J. Med. 1999, 341 (3), 164−1672.
(c) Deininger, M. W. N.; Goldman, J. M.; Melo, J. V. The molecular
biology of chronic myeloid leukemia. Blood 2000, 96 (10), 3343−
3356. (d) Sattler, M.; Griffin, J. D. Molecular mechanisms of
transformation by the Bcr-Abl oncogene. Semin. Hematol. 2003, 40
(2 Suppl 2), 4−10.
(2) (a) Goldman, J. M.; Melo, J. V. Chronic myeloid leukemia
Advances in biology and new approaches to treatment. N. Engl. J. Med.
2003, 349 (15), 1451−1464. (b) Druker, B. J. Imatinib as a Paradigm
of Targeted Therapies. In Advances in Cancer Research; Academic
Press: New York, 2004; Vol. 91, pp 1−30.
(3) (a) Capdeville, R.; Buchdunger, E.; Zimmermann, J.; Matter, A.
Glivec (STI571, imatinib), a rationally developed, targeted anticancer
drug. Nat. Rev. Drug Discov. 2002, 1 (7), 493−502. (b) Druker, B. J.
Inhibition of the Bcr-Abl tyrosine kinase as a therapeutic strategy for
CML. Oncogene 2002, 21 (56), 8541−8546. (c) Deininger, M.;
Buchdunger, E.; Druker, B. J. The development of imatinib as a
therapeutic agent for chronic myeloid leukemia. Blood 2005, 105 (7),
2640−2653. (d) Wong, S.; Witte, O. N. The BCR-ABL story: Bench
to bedside and back. Annu. Rev. Immun. 2004, 22, 247−306.
(4) (a) Daub, H.; Specht, K.; Ullrich, A. Strategies to overcome
resistance to targeted protein kinase inhibitors. Nat. Rev. Drug Discov.
2004, 3 (12), 1001−1010. (b) Hochhaus, A.; La Rosee, P. Imatinib
therapy in chronic myelogenous leukemia: Strategies to avoid and
overcome resistance. Leukemia 2004, 18 (8), 1321−1331.
ASSOCIATED CONTENT
* Supporting Information
■
S
More chemical information of the final compounds, the
preliminary pharmacokinetics analysis on compounds 6q and
6qo and other biological data as well as computational docking
results can be obtained in Supporting Information. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(5) Druker, B. J.; Guilhot, F.; O’Brien, S. G.; Gathmann, I.;
Kantarjian, H.; Gattermann, N.; Deininger, M. W.; Silver, R. T.;
Goldman, J. M.; Stone, R. M.; Cervantes, F.; Hochhaus, A.; Powell, B.
L.; Gabrilove, J. L.; Rousselot, P.; Reiffers, J.; Cornelissen, J. J.;
Hughes, T.; Agis, H.; Fischer, T.; Verhoef, G.; Shepherd, J.; Saglio, G.;
Gratwohl, A.; Nielsen, J. L.; Radich, J. P.; Simonsson, B.; Taylor, K.;
Baccarani, M.; So, C.; Letvak, L.; Larson, R. A. Five-year follow-up of
patients receiving imatinib for chronic myeloid leukemia. N. Engl. J.
Med. 2006, 355 (23), 2408−2417.
AUTHOR INFORMATION
Corresponding Author
*Tel: +86-20-32015276. Fax: +86-20-32015299. E-mail: ding_
ke@gibh.ac.cn.
■
Author Contributions
§These authors contributed equally to this work.
Notes
(6) (a) O’Hare, T.; Walters, D. K.; Stoffregen, E. P.; Jia, T.; Manley,
P. W.; Mestan, J.; Cowan-Jacob, S. W.; Lee, F. Y.; Heinrich, M. C.;
Deininger, M. W.; Druker, B. J. In vitro activity of Bcr-Abl inhibitors
AMN107 and BMS-354825 against clinically relevant imatinib-
resistant Abl kinase domain mutants. Cancer Res. 2005, 65 (11),
4500−4505. (b) O’Hare, T.; Eide, C. A.; Deininger, M. W. N. Bcr-Abl
kinase domain mutations, drug resistance, and the road to a cure for
chronic myeloid leukemia. Blood 2007, 110 (7), 2242−2249.
(c) Jabbour, E.; Kantarjian, H.; Jones, D.; Talpaz, M.; Bekele, N.;
O’Brien, S.; Zhou, X.; Luthra, R.; Garcia-Manero, G.; Giles, F.; Rios,
M. B.; Verstovsek, S.; Cortes, J. Frequency and clinical significance of
BCR-ABL mutations in patients with chronic myeloid leukemia treated
with imatinib mesylate. Leukemia 2006, 20 (10), 1767−1773.
(d) Melo, J. V.; Chuah, C. Resistance to imatinib mesylate in chronic
myeloid leukaemia. Cancer Lett. 2007, 249 (2), 121−132.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank National Basic Research Program of China (No.
2010CB529706), Major Projects in National Science and
Technology “Creation of major new drugs” (No.
2013ZX0910204), National Natural Science Foundation
(Nos. 21072192, 21102146), key Project on Innovative Drug
of Guangdong province (No. 2011A080501013), Key Project
on Innovative Drug of Guangzhou City (Grant Nos. 2009Z1-
E911, 2010J-E551, 12C34061592), and the grant from Chinese
Academy of Sciences (CAS) for their financial support.
ABBREVIATIONS USED
(7) (a) O’Hare, T.; Walters, D. K.; Deininger, M. W.; Druker, B. J.
AMN107: tightening the grip of imatinib. Cancer Cell 2005, 7 (2),
■
Bcr, breakpoint cluster region; Abl, abelson murine leukemia
viral oncogene; T, Thr, threonine; I, Ile, isoleucine; CML,
chronic myelogenous leukemia; WT, wild-type; DFG, Asp-Phe-
Gly; Boc, tert-butoxycarbonyl; THF, tetrahydrofuran; rt, room
temperature; NBS, N-bromosuccinimide; AIBN, azodiisobutyr-
onitrile; TFA, trifluoroacetic acid; DCM, dichloromethane;
DMF, dimethylformamide; E, Glu, glutamic acid; Q, Gln,
glutamine; H, His, histidine; M, Met, methionine; A, Ala,
alanine; G, Gly, glycine; Y, Try, tyrosine; K, Lys, lysine; V, Val,
valine; F, Phe, phenylalanine; L, Leu, leucine; P, Pro, proline; S,
117−119. (b) Weisberg, E.; Manley, P. W.; Breitenstein, W.; Bruggen,
̈
J.; Cowan-Jacob, S. W.; Ray, A.; Huntly, B.; Fabbro, D.; Fendrich, G.;
Hall-Meyers, E.; Kung, A. L.; Mestan, J.; Daley, G. Q.; Callahan, L.;
Catley, L.; Cavazza, C.; Mohammed, A.; Neuberg, D.; Wright, R. D.;
Gilliland, D. G.; Griffin, J. D. Characterization of AMN107, a selective
inhibitor of native and mutant Bcr-Abl. Cancer Cell 2005, 7 (2), 129−
141.
(8) Shah, N. P.; Tran, C.; Lee, F. Y.; Chen, P.; Norris, D.; Sawyers, C.
L. Overriding imatinib resistance with a novel ABL kinase inhibitor.
Science 2004, 305 (5682), 399−401.
10044
dx.doi.org/10.1021/jm301188x | J. Med. Chem. 2012, 55, 10033−10046

Journal of Medicinal Chemistry
Article
(9) (a) Quintas-Cardama, A.; Kantarjian, H.; Cortes, J. Flying under
the radar: the new wave of BCR-ABL inhibitors. Nat. Rev. Drug Discov.
2007, 6 (10), 834−848. (b) Weisberg, E.; Manley, P. W.; Cowan-
Jacob, S. W.; Hochhaus, A.; Griffin, J. D. Second generation inhibitors
of Bcr-Abl for the treatment of imatinib-resistant chronic myeloid
leukaemia. Nat. Rev. Cancer 2007, 7 (5), 345−356.
(10) Kimura, S.; Naito, H.; Segawa, H.; Kuroda, J.; Yuasa, T.; Sato,
K.; Yokota, A.; Kamitsuji, Y.; Kawata, E.; Ashihara, E.; Nakaya, Y.;
Naruoka, H.; Wakayama, T.; Nasu, K.; Asaki, T.; Niwa, T.;
Hirabayashi, K.; Maekawa, T. NS-187, a potent and selective dual
Bcr-Abl/Lyn tyrosine kinase inhibitor, is a novel agent for imatinib-
resistant leukemia. Blood 2005, 106 (12), 3948−3954.
(11) Puttini, M.; Coluccia, A. M.; Boschelli, F.; Cleris, L.; Marchesi,
E.; Donella-Deana, A.; Ahmed, S.; Redaelli, S.; Piazza, R.; Magistroni,
V.; Andreoni, F.; Scapozza, L.; Formelli, F.; Gambacorti-Passerini, C.
In vitro and in vivo activity of SKI-606, a novel Src-Abl inhibitor,
against imatinib-resistant Bcr-Abl+ neoplastic cells. Cancer Res. 2006,
66 (23), 11314−11322.
(12) (a) Kantarjian, H.; Giles, F.; Wunderle, L.; Bhalla, K.; O’Brien,
S.; Wassmann, B.; Tanaka, C.; Manley, P.; Rae, P.; Mietlowski, W.;
Bochinski, K.; Hochhaus, A.; Griffin, J. D.; Hoelzer, D.; Albitar, M.;
Dugan, M.; Cortes, J.; Alland, L.; Ottmann, O. G. Nilotinib in
imatinib-resistant CML and Philadelphia chromosome-positive ALL.
N. Engl. J. Med. 2006, 354 (24), 2542−2551. (b) Talpaz, M.; Shah, N.
P.; Kantarjian, H.; Donato, N.; Nicoll, J.; Paquette, R.; Cortes, J.;
O’Brien, S.; Nicaise, C.; Bleickardt, E.; Blackwood-Chirchir, M. A.;
Iyer, V.; Chen, T. T.; Huang, F.; Decillis, A. P.; Sawyers, C. L.
Dasatinib in imatinib-resistant Philadelphia chromosome-positive
leukemias. N. Engl. J. Med. 2006, 354 (24), 2531−2541.
(13) O’Hare, T.; Eide, C. A.; Deininger, M. W. New Bcr-Abl
inhibitors in chronic myeloid leukemia: keeping resistance in check.
Expert Opin. Invest. Drugs 2008, 17 (6), 865−878.
(14) Bradeen, H. A.; Eide, C. A.; O’Hare, T.; Johnson, K. J.; Willis, S.
G.; Lee, F. Y.; Druker, B. J.; Deininger, M. W. Comparison of imatinib
mesylate, dasatinib (BMS-354825), and nilotinib (AMN107) in an N-
ethyl-N-nitrosourea (ENU)-based mutagenesis screen: high efficacy of
drug combinations. Blood 2006, 108 (7), 2332−2338.
Jahnke, W.; Cowan-Jacob, S. W.; Li, A. G.; Iacob, R. E.; Sim, T.;
Powers, J.; Dierks, C.; Sun, F.; Guo, G. R.; Ding, Q.; Okram, B.; Choi,
Y.; Wojciechowski, A.; Deng, X.; Liu, G.; Fendrich, G.; Strauss, A.;
Vajpai, N.; Grzesiek, S.; Tuntland, T.; Liu, Y.; Bursulaya, B.; Azam, M.;
Manley, P. W.; Engen, J. R.; Daley, G. Q.; Warmuth, M.; Gray, N. S.
Targeting Bcr-Abl by combining allosteric with ATP-binding-site
inhibitors. Nature 2010, 463 (7280), 501−506.
(21) Giles, F. J.; Cortes, J.; Jones, D.; Bergstrom, D.; Kantarjian, H.;
Freedman, S. J. MK-0457, a novel kinase inhibitor, is active in patients
with chronic myeloid leukemia or acute lymphocytic leukemia with the
T315I BCR-ABL mutation. Blood 2007, 109 (2), 500−502.
(22) (a) Fancelli, D.; Moll, J.; Varasi, M.; Bravo, R.; Artico, R.; Berta,
D.; Bindi, S.; Cameron, A.; Candiani, I.; Cappella, P.; Carpinelli, P.;
Croci, W.; Forte, B.; Giorgini, M. L.; Klapwijk, J.; Marsiglio, A.;
Pesenti, E.; Rocchetti, M.; Roletto, F.; Severino, D.; Soncini, C.;
Storici, P.; Tonani, R.; Zugnoni, P.; Vianello, P. 1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazoles: identification of a potent Aurora
kinase inhibitor with a favorable antitumor kinase inhibition profile. J.
Med. Chem. 2006, 49 (24), 7247−7251. (b) Modugno, M.; Casale, E.;
Soncini, C.; Rosettani, P.; Colombo, R.; Lupi, R.; Rusconi, L.; Fancelli,
D.; Carpinelli, P.; Cameron, A. D.; Isacchi, A.; Moll, J. Crystal
structure of the T315I Abl mutant in complex with the aurora kinases
inhibitor PHA-739358. Cancer. Res. 2007, 67 (17), 7987−3990.
(c) Gontarewicz, A.; Balabanov, S.; Keller, G.; Colombo, R.; Graziano,
A.; Pesenti, E.; Benten, D.; Bokemeyer, C.; Fiedler, W.; Moll, J.;
Brummendorf, T. H. Simultaneous targeting of Aurora kinases and
Bcr-Abl kinase by the small molecule inhibitor PHA-739358 is
effective against imatinib-resistant BCR-ABL mutations including
T315I. Blood 2008, 111 (8), 4355−4364.
(23) Howard, S.; Berdini, V.; Boulstridge, J. A.; Carr, M. G.; Cross, D.
M.; Curry, J.; Devine, L. A.; Early, T. R.; Fazal, L.; Gill, A. L.;
Heathcote, M.; Maman, S.; Matthews, J. E.; McMenamin, R. L.;
Navarro, E. F.; O’Brien, M. A.; O’Reilly, M.; Rees, D. C.; Reule, M.;
Tisi, D.; Williams, G.; Vinkovic, M.; Wyatt, P. G. Fragment-based
discovery of the pyrazol-4-yl urea (AT9283), a multitargeted kinase
inhibitor with potent aurora kinase activity. J. Med. Chem. 2009, 52
(2), 379−388.
(15) (a) Schindler, T.; Bornmann, W.; Pellicena, P.; Miller, W. T.;
Clarkson, B.; Kuriyan, J. Structural mechanism for STI-571 inhibition
of abelson tyrosine kinase. Science 2000, 289 (5486), 1938−1942.
(b) Tokarski, J. S.; Newitt, J. A.; Chang, C. Y.; Cheng, J. D.; Wittekind,
M.; Kiefer, S. E.; Kish, K.; Lee, F. Y.; Borzillerri, R.; Lombardo, L. J.;
Xie, D.; Zhang, Y.; Klei, H. E. The structure of Dasatinib (BMS-
354825) bound to activated ABL kinase domain elucidates its
inhibitory activity against imatinib-resistant ABL mutants. Cancer
Res. 2006, 66 (11), 5790−5797.
(16) (a) Schenone, S.; Bruno, O.; Radi, M.; Botta, M. New insights
into small-molecule inhibitors of Bcr-Abl. Med. Res. Rev. 2011, 31 (1),
1−41. (b) Choi, H. G.; Zhang, J.; Weisberg, E.; Griffin, J. D.; Sim, T.;
Gray, N. S. Development of ’DFG-out’ inhibitors of gatekeeper mutant
kinases. Bioorg. Med. Chem. Lett. 2012, 22 (16), 5297−5302.
(17) Zhou, T.; Parillon, L.; Li, F.; Wang, Y.; Keats, J.; Lamore, S.; Xu,
Q.; Shakespeare, W.; Dalgarno, D.; Zhu, X. Crystal structure of the
T315I mutant of Abl kinase. Chem. Biol. Drug Des. 2007, 70 (3), 171−
181.
(18) Cao, J. G.; Fine, R.; Gritzen, C.; Hood, J.; Kang, X. S.;
Klebansky, B.; Lohse, D.; Mak, C. C.; McPherson, A.; Noronha, G.;
Palankl, M. S. S.; Pathak, V. P.; Renick, J.; Solla, R.; Zenga, B. Q.; Zhu,
H. The design and preliminary structure−activity relationship studies
of benzotriazines as potent inhibitors of Abl and Abl-T3151 enzymes.
Bioorg. Med. Chem. Lett. 2007, 17 (21), 5812−5818.
(19) Gumireddy, K.; Baker, S. J.; Cosenza, S. C.; John, P.; Kang, A.
D.; Robell, K. A.; Reddy, M. V.; Reddy, E. P. A non-ATP-competitive
inhibitor of BCR-ABL overrides imatinib resistance. Proc. Natl. Acad.
Sci. U.S.A. 2005, 102 (6), 1992−1997.
(24) Shah, N.; Kasap, C.; Paquette, R.; Cortes, J.; Pinilla, J.; Talpaz,
M.; Bui, L.; Clary, D. In Targeting drug-resistant CML and Ph+-ALL
with the spectrum selective protein kinase inhibitor XL228. Blood
2007, 474.
(25) Green, M. R.; Woolery, J. E.; Mahadevan, D. Update on Aurora
kinase targeted therapeutics in oncology. Expert Opin. Drug Discovery
2011, 6 (3), 291−307.
(26) (a) Lu, X. Y.; Cai, Q.; Ding, K. Recent developments in the third
generation inhibitors of Bcr-Abl for overriding T315I mutation. Curr.
Med. Chem. 2011, 18 (14), 2146−2157. (b) Quintas-Cardama, A.;
Kantarjian, H.; Cortes, J. Third-generation tyrosine kinase inhibitors
and beyond. Semin. Hematol. 2010, 47 (4), 371−380.
(27) Choi, H. G.; Ren, P.; Adrian, F.; Sun, F.; Lee, H. S.; Wang, X.;
Ding, Q.; Zhang, G.; Xie, Y.; Zhang, J.; Liu, Y.; Tuntland, T.;
Warmuth, M.; Manley, P. W.; Mestan, J.; Gray, N. S.; Sim, T. A type-II
kinase inhibitor capable of inhibiting the T315I “gatekeeper” mutant of
Bcr-Abl. J. Med. Chem. 2010, 53 (15), 5439−5448.
(28) O’Hare, T.; Shakespeare, W. C.; Zhu, X. T.; Eide, C. A.; Rivera,
V. M.; Wang, F.; Adrian, L. T.; Zhou, T. J.; Huang, W. S.; Xu, Q. H.;
Metcalf, C. A.; Tyner, J. W.; Loriaux, M. M.; Corbin, A. S.; Wardwell,
S.; Ning, Y. Y.; Keats, J. A.; Wang, Y. H.; Sundaramoorthi, R.; Thomas,
M.; Zhou, D.; Snodgrass, J.; Commodore, L.; Sawyer, T. K.; Dalgarno,
D. C.; Deininger, M. W. N.; Druker, B. J.; Clackson, T. AP24534, a
pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently
inhibits the T315I mutant and overcomes mutation-based resistance.
Cancer Cell 2009, 16 (5), 401−412.
(29) Eide, C. A.; Adrian, L. T.; Tyner, J. W.; Mac Partlin, M.;
Anderson, D. J.; Wise, S. C.; Smith, B. D.; Petillo, P. A.; Flynn, D. L.;
Deininger, M. W.; O’Hare, T.; Druker, B. J. The ABL switch control
inhibitor DCC-2036 is active against the chronic myeloid leukemia
mutant BCR-ABLT315I and exhibits a narrow resistance profile.
Cancer Res. 2011, 71 (9), 3189−3195.
(20) (a) Adrian, F. J.; Ding, Q.; Sim, T.; Velentza, A.; Sloan, C.; Liu,
Y.; Zhang, G.; Hur, W.; Ding, S.; Manley, P.; Mestan, J.; Fabbro, D.;
Gray, N. S. Allosteric inhibitors of Bcr-abl-dependent cell proliferation.
Nat. Chem. Biol. 2006, 2 (2), 95−102. (b) Zhang, J.; Adrian, F. J.;
10045
dx.doi.org/10.1021/jm301188x | J. Med. Chem. 2012, 55, 10033−10046

Journal of Medicinal Chemistry
Article
(30) Zhou, T.; Commodore, L.; Huang, W. S.; Wang, Y.; Thomas,
M.; Keats, J.; Xu, Q.; Rivera, V. M.; Shakespeare, W. C.; Clackson, T.;
Dalgarno, D. C.; Zhu, X. Structural mechanism of the Pan-BCR-ABL
inhibitor ponatinib (AP24534): Lessons for overcoming kinase
inhibitor resistance. Chem. Biol. Drug Des. 2011, 77 (1), 1−11.
(31) Chinchilla, R.; Najera, C. The Sonogashira reaction: A booming
methodology in synthetic organic chemistry. Chem. Rev. 2007, 107 (3),
874−922.
(32) Wang, D.; Zhang, Z.; Lu, X.; Feng, Y.; Luo, K.; Gan, J.; Yingxue,
L.; Wan, J.; Li, X.; Zhang, F.; Tu, Z.; Cai, Q.; Ren, X.; Ding, K. Hybrid
compounds as new Bcr/Abl inhibitors. Bioorg. Med. Chem. Lett. 2011,
21 (7), 1965−1968.
(33) Huang, W. S.; Metcalf, C. A.; Sundaramoorthi, R.; Wang, Y.;
Zou, D.; Thomas, R. M.; Zhu, X.; Cai, L.; Wen, D.; Liu, S.; Romero, J.;
Qi, J.; Chen, I.; Banda, G.; Lentini, S. P.; Das, S.; Xu, Q.; Keats, J.;
Wang, F.; Wardwell, S.; Ning, Y.; Snodgrass, J. T.; Broudy, M. I.;
Russian, K.; Zhou, T.; Commodore, L.; Narasimhan, N. I.;
Mohemmad, Q. K.; Iuliucci, J.; Rivera, V. M.; Dalgarno, D. C.;
Sawyer, T. K.; Clackson, T.; Shakespeare, W. C. Discovery of 3-[2-
(imidazo[1,2-b]pyridazin-3-yl)ethynyl]-4-methyl-N-{4-[(4-methylpi-
perazin-1-y l)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 (12), 4701−4719.
(34) Chang, S.; Zhang, L.; Xu, S.; Luo, J.; Lu, X.; Zhang, Z.; Xu, T.;
Liu, Y.; Tu, Z.; Xu, Y.; Ren, X.; Geng, M.; Ding, J.; Pei, D.; Ding, K.
Design, synthesis, and biological evaluation of novel conformationally
constrained inhibitors targeting epidermal growth factor receptor
threonine790 → methionine790 mutant. J. Med. Chem. 2012, 55 (6),
2711−2723.
(35) Thomas, M.; Huang, W. S.; Wen, D.; Zhu, X.; Wang, Y.;
Metcalf, C. A.; Liu, S.; Chen, I.; Romero, J.; Zou, D.; Sundaramoorthi,
R.; Li, F.; Qi, J.; Cai, L.; Zhou, T.; Commodore, L.; Xu, Q.; Keats, J.;
Wang, F.; Wardwell, S.; Ning, Y.; Snodgrass, J. T.; Broudy, M. I.;
Russian, K.; Iuliucci, J.; Rivera, V. M.; Sawyer, T. K.; Dalgarno, D. C.;
Clackson, T.; Shakespeare, W. C. Discovery of 5-(arenethynyl) hetero-
monocyclic derivatives as potent inhibitors of BCR-ABL including the
T315I gatekeeper mutant. Bioorg. Med. Chem. Lett. 2011, 21 (12),
3743−3748.
(36) (a) Nagar, B.; Bornmann, W. G.; Pellicena, P.; Schindler, T.;
Veach, D. R.; Miller, W. T.; Clarkson, B.; Kuriyan, J. Crystal structures
of the kinase domain of c-Abl in complex with the small molecule
inhibitors PD173955 and imatinib (STI-571). Cancer Res. 2002, 62
(15), 4236−4243. (b) Horio, T.; Hamasaki, T.; Inoue, T.; Wakayama,
T.; Itou, S.; Naito, H.; Asaki, T.; Hayase, H.; Niwa, T. Structural
factors contributing to the Abl/Lyn dual inhibitory activity of 3-
substituted benzamide derivatives. Bioorg. Med. Chem. Lett. 2007, 17
(10), 2712−2717.
(37) Zimmermann, J.; Buchdunger, E.; Mett, H.; Meyer, T.; Lydon,
N. B. Potent and selective inhibitors of the Abl-kinase: Phenylamino-
pyrimidine (PAP) derivatives. Bioorg. Med. Chem. Lett. 1997, 7 (2),
187−192.
(38) von Bubnoff, N.; Veach, D. R.; van der Kuip, H.; Aulitzky, W. E.;
Sanger, J.; Seipel, P.; Bornmann, W. G.; Peschel, C.; Clarkson, B.;
Duyster, J. A cell-based screen for resistance of Bcr-Abl-positive
leukemia identifies the mutation pattern for PD166326, an alternative
Abl kinase inhibitor. Blood 2005, 105 (4), 1652−1659.
(39) Ren, X. M.; Duan, L.; He, Q. A.; Zhang, Z.; Zhou, Y.; Wu, D.
H.; Pan, J. X.; Pei, D. Q.; Ding, K. Identification of niclosamide as a
new small-molecule inhibitor of the STAT3 signaling pathway. ACS
Med. Chem. Lett. 2010, 1 (9), 454−459.
10046
dx.doi.org/10.1021/jm301188x | J. Med. Chem. 2012, 55, 10033−10046