Broad spectrum alkynyl inhibitors of T315I Bcr-Abl

Article abstract of DOI:10.1016/j.bmcl.2010.05.043

A series of alkyne-containing type II inhibitors with potent inhibitory activity of T315I Bcr-Abl has been identified. The most active compound 4 exhibits an EC₅₀ of less than 1 nM against wild-type Bcr-Abl and an EC₅₀ of 10 nM again

Full text of DOI:10.1016/j.bmcl.2010.05.043

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4196–4200
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Broad spectrum alkynyl inhibitors of T315I Bcr-Abl
Xianming Deng a, Sang Min Lim a,b, Jianming Zhang a, Nathanael S. Gray a,
*
a Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, and Department of Cancer Biology, Dana-Farber Cancer Institute,
250 Longwood Ave, SGM 628, Boston, MA 02115, United States
b Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of alkyne-containing type II inhibitors with potent inhibitory activity of T315I Bcr-Abl has
been identified. The most active compound 4 exhibits an EC50 of less than 1 nM against wild-type
Bcr-Abl and an EC50 of 10 nM against T315I mutant but is broadly active against a number of other
kinases.
Received 21 April 2010
Accepted 12 May 2010
Available online 19 May 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Kinase inhibitors
T315I Bcr-Abl
The success of Bcr-Abl inhibitor imatinib (GleevecÒ, Novartis
Pharma AG) for the treatment of Chronic Myelogenous Leuke-
mia (CML) has provided the paradigm for targeting dominant
oncogenes with small molecules.1,2 Imatinib resistance is rare
in chronic phase patients, however for patients with blast crisis
phase CML or Philadelphia chromosome-positive CML, resistance
is common after an initial response in the first year.3,4 To ad-
dress these relapses, two more potent ATP-site directed agents:
nilotinib (AMN107)5 and dasatinib (BMS-354825)6 have been
approved as second-line therapies. Although both compounds
inhibit most of the protein mutants that induce resistance to
imatinib, neither compound is capable of inhibiting the so-
called ‘gatekeeper’ T315I mutant.7 Because of the clinical impor-
tance of this mutation, there has been intense interest in the
synthesis of novel inhibitors that are able to circumvent this
mutation.
gatekeeper mutants, BCR-ABL-T315I, Kit-T670I, PDGFRa-T674M/
I, as well as Src-T341M/I.13 HG-7-85-01 was designed as a hybrid
between the type I inhibitor dasatinib and the type II inhibitor,
nilotinib. Specifically, a superposition of the Abl-bound conforma-
tion of dasatinib (PDB code: 2GQG)15 and nilotinib (PDB code:
3CS9)5 guided the choice of how to connect the aminothiazole
hinge-interacting motif of dasatinib with the N-(3-(trifluoro-
methyl)phenyl)-benzamide substructure of nilotinib, which is
known to be responsible for inducing the ‘DFG-out’ flip that is
characteristic of type II kinase inhibitors. Our results demonstrate
that it is possible to design a type II inhibitor that can circumvent
F3C
N
N
Recently, several compounds from the Type-II class8 that rec-
ognize the ‘DFG-out’ conformation have been reported to inhibit
T315I. These include cyclic urea compound 14,9 BGG463,10
AP24163,11 DSA series compounds,12 HG-7-85-0113 and AP245
34.14 A co-crystal structure of T315I with AP24534, an imi-
dazo[1,2b]-pyridazine-based multitargeted inhibitor demon-
strates how this compound can circumvent a larger residue at
the gatekeeper site.14
O
N
N
H
HN
N
S
O
HG-7-85-01
H
N
In our efforts to identify new molecular scaffolds that could
target T315I mutant of Bcr-Abl, we recently reported the discov-
ery of HG-7-85-01, a small molecule type II inhibitor that inhibits
the proliferation of cells expressing the major imatinib-resistant
N
CF3
N
O
N
Hinge head Linker
type II tail
* Corresponding author. Tel.: +1 617 582 8590.
E-mail address: nathanael_gray@dfci.harvard.edu (N.S. Gray).
Figure 1. Scaffold design strategy.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.043

X. Deng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4196–4200
4197
Table 1
Table 2
SAR of mono-heterocyclic head alkynyl analogs
SAR of bi-heterocyclic head alkynyl analogs
R1
H3C
H
N
R2
CF3
Ar2
Ar1
O
II
12-16, 18, R1 = Me, 17,19, R1 = H
N
N
CF3
CF3
CF3
12-17, R2
=
18-19, R2
=
20, R2
=
I
N
N
N
O
N
HN
HN
N
N
NH
O
O
Compds Ar1
Cellular antiproliferative activity (EC50
M)
,
Compds Ar1
Cellular antiproliferative activity (EC50
, lM)
l
Bcr-Abla (wt) Bcr-Ablb (T315I) BaF3c
Bcr-Abla (wt)
Bcr-Ablb (T315I)
BaF3c
3
<0.001
0.092
1.59
2.05
12
<0.001
0.080
0.56
1.00
N
N
N
H
O
4
5
<0.001
<0.001
0.010
0.013
Me
13
0.026
1.00
N
H
N
N
H
N
N
O
1.80
3.98
14
<0.001
0.037
0.24
0.014
5.52
8.37
0.060
3.32
6.76
N
N
N
N
H
H
N
H
N
15
16
6
0.041
2.58
N
N
N
O
N
7
8
<0.001
0.006
0.99
3.03
2.19
2.49
O
N
N
17
18
19
20
0.011
0.037
0.015
0.070
0.22
0.47
0.30
>10.0
N
N
N
N
N
H
N
N
N
>10.0
9
<0.001
0.165
0.86
N
H
H2N
a
b
c
Cellular antiproliferative activity (EC50
Cellular antiproliferative activity (EC50
Cytotoxicity (EC50, lM) on wt-Ba/F3.
,
,
l
l
M) on wtBcr-Abl-Ba/F3.
M) on mutant Bcr-Abl-T315I-Ba/F3.
2.83
N
H
2.63
7.86
N
H
the T315I Bcr-Abl ‘gatekeeper’ mutation by bridging the ATP and
allosteric binding sites using a linker segment that can accommo-
date a larger gatekeeper residue. Here we report on our efforts in
applying this strategy towards the synthesis of type II inhibitors
using an alkyne as a linear linkage segment that can traverse a
larger gatekeeper residue. A number of compounds from this ser-
ies exhibit highly potent activities against both the wild-type and
T315I mutant of Bcr-Abl.
a
b
c
Cellular antiproliferative activity (EC50
Cellular antiproliferative activity (EC50
Cytotoxicity (EC50, lM) on wt-Ba/F3.
,
,
l
M) on wtBcr-Abl-Ba/F3.
lM) on mutant Bcr-Abl-T315I-Ba/F3.
were synthesized analogously using different heteroaromatic io-
dides or bromides in the final coupling step.
Molecular modeling suggested that the triple-bond linkage
should be used to connect the toluene moiety of imatinib/niloti-
nib with a variety of heterocycles that would be capable of
forming hydrogen bonding interactions with the kinase hinge re-
gion (Fig. 1). This scaffold is exemplified by structures I and II
(Tables 1 and 2). Concise synthetic routes were developed to
prepare I and II (Schemes 1 and 2). A Sonogashira coupling16
was used as the key reaction in both synthetic routes. Scheme
1 depicts the synthesis of compound 3, starting with the amide
condensation of freshly prepared 3-iodo-4-methylbenzoyl chlo-
ride with 4-((4-ethyl-piperazin-1-yl)methyl)-3-(trifluoromethyl)-
benzenamine to afford the iodo-intermediate 1. Alkyne interme-
diate 2 was obtained using a Sonogashira coupling of intermedi-
ate 1 with ethynyltrimethylsilane followed by deprotection of
the TMS group. The final product 3 was obtained using another
Sonogashira coupling of 2 with 3-iodopyridine. Compounds 4–9
Synthesis of 12 was accomplished by introduction of an ethynyl
group to 5-bromo-1H-pyrrolo[2,3-b]pyridine followed by coupling
with the iodo-intermediate 1 (Scheme 2). Compounds 13–20 were
obtained following this synthetic route.
To assess the cellular activity of the compounds, we tested them
against parental, wild-type and T315I Bcr-Abl transformed Ba/F3
cells. Wild-type Ba/F3 cells proliferate only in the presence of
interleukin-3 (IL-3) while Ba/F3 cells transformed with oncogenic
kinases such as Bcr-Abl become capable of growing in the absence
of IL-3 and provides a robust and commonly used assay for selec-
tive kinase inhibition.17
The first compound 3 we synthesized exhibited an EC50 of
less than 1 nM on wild-type Bcr-Abl and an EC50 of 92 nM on
T315I. The EC50 against parental Ba/F3 cells was 1.59 lM, dem-
onstrating that the antiproliferative activity was derived from
on-target inhibition of Bcr-Abl. Encouraged by this result, we

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X. Deng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4196–4200
O
O
N
I
I
N
c
a, b
OH
N
CF3
H
1
O
N
TMS
O
N
N
N
CF3
N
d
N
CF3
H
H
2
N
e
O
N
N
N
CF3
H
3
Scheme 1. Synthetic route of 3. Reagents and conditions: (a) SOCl2, reflux, 1 h; (b) 4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)-benzenamine, DIEA, CH2Cl2,
0 °C to rt, 56% over two steps; (c) ethynyltrimethylsilane, Pd(PPh3)4, CuI, DIEA, DMF, rt, 62%; (d) TBAF, THF, rt, 72%; (e) 3-iodopyridine, Pd(PPh3)4, CuI, DIEA, DMF, 50 °C,
72%.
H
N
H
H
of imatinib, nilotinib and AP24534 suggests that the methyl
group favors the twisted conformation required for high affinity
binding.5,14,18 The orientation of the amide found in nilotinib
(12) is favored over the reverse amide orientation found in imati-
nib (20).
The selectivity of this scaffold was assessed using
KINOMEscan™ (Ambit Biosciences, San Diego, CA),19 a high-
throughput method for screening kinase inhibitors against a pa-
nel of 442 kinases. Compounds 3 and 12, were screened at a
N
N
N
N
N
a
b
Br
TMS
11
10
H
N
N
c
O
N
N
N
CF3
H
concentration of 10 lM. This analysis revealed that the com-
12
pounds possessed an extremely broad selectivity profile with
compounds 3 and 12 inhibiting 44% and 46% of kinases on the
panel respectively, with a score of less than 10% of the DMSO
control. The kinase hits for 12 with ambit score less 0.1% of
the DMSO control were highlighted in a spot tree20 (Fig. 2,
please see Supplementary data for full screening data of 12
and 3). The potently targeted kinases were mainly from the
TK, TKL, STE, and CMGC groups. The selectivity scores19 for 3
(S10 = 0.427) and 12 (S10 = 0.449) indicate that these compounds
are considerably less specific relative to compounds such as
HG-7-85-01 (S10 = 0.056).13 As compounds 3 and 12 are highly
rigid structures they are most likely only to bind with high affin-
ity to the ‘DFG-out’ conformation as confirmed by the recent co-
structure of AP24534 with T315I Bcr-Abl.14 These kinase profil-
ing results demonstrate that a very large number of kinases
can be potently targeted in this conformation and inhibitors 3
and 12 represent starting points for the design of multitargeted
inhibitors with the potential to target diverse combinations of
kinase targets.
In summary, we have used a structure-based design approach
to design a new type II scaffold using an alkyne as a linker seg-
ment between a heterocyclic hinge-interacting motif and a triflu-
oromethylphenylamide motif that binds to the pocket created by
the ‘DFG-out’ conformation. The compounds exhibit very potent
cellular activity against both the wild-type and T315I Bcr-Abl. De-
spite being extremely promiscuous kinase inhibitors, compounds
such as 3 and 12 are not general cytotoxic agents and exhibit up
to 1000-fold selectivity for Bcr-Abl dependent cellular growth.
Further medicinal chemistry efforts are in progress to develop
analogs from this compound series whose multitargeted inhibi-
tion profile is tailored for optimal activity against particular can-
cer genotypes.
Scheme 2. Synthetic route of 12. Reagents and conditions: (a) ethynyltrimethylsi-
lane, Pd(PPh3)4, CuI, DIEA, DMF, 50 °C, 55%; (b) TBAF, THF, rt, 77%; (c) 1, Pd(PPh3)4,
CuI, DIEA, DMF, rt, 82%.
next prepared a small set of compounds to investigate the SAR
and validate our design strategy (Table 1). Introduction of a
substituted amino-group to the pyridine 6-position (4 and 5)
resulted in an approximate eight-fold improvement relative to
3 against T315I mutant Bcr-Abl. This might be attributed to
the introduction of an additional hydrogen bond to the kinase
hinge from the amino-group. Incorporation of a carbonyl group,
as is present in Sorafenib at the 2-position, afforded compound
6 which was less potent against both wild-type and T315I Bcr-
Abl relative to 3. Replacing the pyridine head with pyrimidine
and pyrazine resulted in approximately equipotent compounds
7 and 8 against wild-type but decreased potency on T315I mu-
tant. The identification of highly potent compounds 3–5 clearly
validates our design strategy. The results also demonstrate that
T315I Bcr-Abl is less potently inhibited relative to wild-type by
this inhibitor series.
We next investigated the effects of using 6-5 and 6-6 fused
heterocyclic rings such as 7-aza-indole, imidazopyridine, pyrido-
pyrazine, and benzofuran as hinge-interacting motifs (Table 2).
Most of the resulting compounds exhibited EC50 values below
100 nM against wild-type Bcr-Abl, but only compounds 12 and
14 exhibited EC50 values below 100 nM against T315I. A compar-
ison of potencies of compounds 12 and 17 demonstrate that the
presence of the toluene methyl group is an important structural
element for achieving potent inhibition against both wild-type
and T315I Bcr-abl. An analysis of the Abl-bound conformations

X. Deng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4196–4200
4199
Figure 2. KINOMEscan™ profiling of 12. Compounds were screened at a concentration10
circles and include: ABL, ARG, BLK, PITSLRE, CRIK, FMS/CSFR, DDR1, EphA8, FGFR4, HPK1, JNK2, KIT, LOK, LYN, GCK, MUSK, MYO3B, p38
SRC, TAO1, TAO2, TAO3, and TIE2. The kinase dendrogram was adapted and reproduced with permission from Cell Signaling Technology, Inc.
l
M against 442 kinases and the most potently bound kinases are indicated by red
a
, p38b, PDGFR , PDGFRb, RET, RIPK1,
a
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This work was supported by the NIH, R01 CA130876-02. We
thank Ambit Biosciences for technical support in the compound
selectivity profiling. The kinase dendrogram was adapted and
reproduced with permission from Cell Signaling Technology, Inc.
(http://www.cellsignal.com).
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Supplementary data
Supplementary data associated with this article can be found, in
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