Development of 'DFG-out' inhibitors of gatekeeper mutant kinases

Article abstract of DOI:10.1016/j.bmcl.2012.06.036

HG-7-85-01(22) and HG-7-86-01(26) are thiazolo[5,4-b]pyridine containing type II tyrosine kinase inhibitors with potent cellular activity against both wild-type and 'gatekeeper' mutant T315I- Bcr-Abl. Here we report on the 'hybrid design' concept and subs

Full text of DOI:10.1016/j.bmcl.2012.06.036

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5297–5302
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of ‘DFG-out’ inhibitors of gatekeeper mutant kinases
Hwan Geun Choi a,d, Jianming Zhang a,d, Ellen Weisberg b, James D. Griffin b, Taebo Sim a,c,d
,
Nathanael S. Gray a,d,
⇑
a Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA
b Department of Medical Oncology, Dana-Farber Cancer Institute, United States
c Life Sciences Research Division Korea Institute of Science and Technology (KIST), 39-1, Hawolgok-dong, Seongbuk-gu, Seoul 136-791, Republic of Korea
d Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, 250 Longwood Ave, SGM 628, Boston, MA 02115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
HG-7-85-01(22) and HG-7-86-01(26) are thiazolo[5,4-b]pyridine containing type II tyrosine kinase inhib-
itors with potent cellular activity against both wild-type and ‘gatekeeper’ mutant T315I- Bcr-Abl. Here we
report on the ‘hybrid design’ concept and subsequent structure activity guided optimization efforts that
resulted in the development of these inhibitors.
Received 30 April 2012
Accepted 11 June 2012
Available online 23 June 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
T315I Bcr-Abl
Kinase inhibitor
Gatekeeper mutant
Type II inhibitor
Thiazolo[5,4-b]pyridine
Despite the tremendous success of the first generation tyrosine
kinase inhibitor imatinib for the treatment of chronic myelogenous
leukemia (CML), a portion of the patient population eventually
develops resistance.1,2 Three ‘second generation’ inhibitors: niloti-
nib,3 dasatinib4 and bosutinib,5 have been approved to treat pa-
tients with imatinib resistance however these drugs remain
gion adjacent to the ATP-binding site created by the flip of the
‘DFG-motif’ to the portion of a Type I inhibitor that makes contacts
with the hinge region of the kinase.15 We designed the thiazolo-
pyridine tyrosine kinase inhibitors reported here by hybridizing
the hinge interacting thiazole functionality of the Type I inhibitor
dasatinib with the 3-trifluoromethylbenzamide pharmacophore
present in Type II inhibitors such as imatinib, nilotinib and
sorafenib.
The first hybrid compounds that we designed are exemplified
by HG-7-85-01 (22) and HG-7-86-01 (26) and contain a thiazolo-
pyridine core, a 3-trifluoromethylbenzamide Type II ‘tail’ and
various groups appended to the thiazolopyridine (Fig. 1). The
synthesis of the thiazolo[5,4-b]pyridine core commenced with a
Suzuki coupling between commercially available 2-chloro-5-nitro-
pyridine and various phenylboronic acids (Scheme 1). The nitro
group was reduced using 5% Pd/C and the resulting product was
readily brominated using N-bromosuccinimide at low tempera-
ture. One-pot 2-(methylthio)thiazole formation was accomplished
using potassium ethyl xanthogenate and iodomethane to yield
compound 5. The sulfide group was oxidized with oxone to sulfone
compound 6 which could be easily displaced using ammonia in
isopropanol. Saponification of ester compound 8 followed by
amide coupling using HATU and DIEA provided the target
compound 9a.
ineffective against T315I ‘gatekeeper’ mutant form of Bcr-Abl.6
A
‘third generation’ of type II inhibitors that effectively inhibit the
T315I Bcr-Abl mutant has recently been developed. These inhibi-
tors include AP24163,7 AP24534,7 DCC-2036,8 BGG463,9 GNF-7,10
DSA series compounds,11 and a series of alkynyl inhibitors,12 of
which only AP24534 and DCC-2036 are reported to be undergoing
clinical evaluation. Recently, we described the biological character-
ization of two Type II tyrosine kinase inhibitors: HG-7-85-01(22)13
and HG-7-86-01(26)14 which can potently inhibit the proliferation
of cells expressing the major imatinib-resistant gatekeeper
mutants T315I-BCR-ABL, T670I-Kit, T674M/I-PDGFR
a
and
T341M/I-Src and which potently and selectively target mutant
FLT3. Herein, we describe the hybrid design strategy and medicinal
chemistry effort leading to the development of these ATP-
competitive type II inhibitors.
We have previously reported on the use of a rational ‘hybrid-
design’ strategy to convert well known Type I scaffolds into
corresponding Type II inhibitors. This approach consists of
appending the moiety from a Type II inhibitor that occupies the re-
The synthesis of compounds 10–13 was accomplished by acyl-
ation or amidation of the NH2 moiety in 9a. Urea formation to form
14 was accomplished via acylation of 9a with 4-nitrophenyl
chloroformate followed by displacement of the 4-nitrophenyl
⇑
Corresponding author.
E-mail address: nathanael_gray@dfci.harvard.edu (N.S. Gray).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.036

5298
H. G. Choi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5297–5302
Figure 1. Design rational for the thiazolopyridine scaffold.
O2N
H2N
O
O
O2N
a)
b)
N
O
N
O
N
Cl
R1
R1
1
2a, R1 = H
3a, R1 = H
2b, R1 = Me
2c, R1 = F
3b, R1 = Me
3c, R1 = F
H2N
Br
N
S
O
O
O
S
d)
e)
g)
c)
N
N
O
R1
R1
4a, R1 = H
4b, R1 = Me
4c, R1 = F
5a, R1 = H
5b, R1 = Me
5c, R1 = F
O
S
N
N
S
O
O
O
O
H2N
f)
S
N
O
N
R1
R1
6a, R1 = H
6b, R1 = Me
6c, R1 = F
7a, R1 = H
7b, R1 = Me
7c, R1 = F
N
N
O
O
H2N
H2N
h)
S
S
N
OH
N
N
CF3
H
R1
8a, R1 = H
8b, R1 = Me
8c, R1 = F
9a, R1 = H
Scheme 1. Reagent and conditions: (a) boronic acids[(3-(ethoxycarbonyl)phenylboronic acid(R1 = H), 5-methoxycarbonyl-2-methylphenylboronic acid (R1 = Me), 5-
ethoxycarbonyl-2-fluorophenylboronic acid (R1 = F)], Pd(PPh3)2Cl2, tert-butyl XPhos, 2N Na2CO3(aq), dioxane, 90 °C, 10 h; (b) 5% Pd/C, EtOH, 16 h; (c) NBS, DMF, 0 °C, 5–
10 min; (d) potassium ethyl xanthogenate, AcOH, NMP, 150 °C, 16 h and MeI, 50 °C, 30 min; (e) Oxone, MeOH, THF, Water, rt, 16 h; (f) 2.0 N NH3 in IPA, 90 °C, 24 h; (g)
LiOHÁH2O, THF, MeOH, H2O, rt, 16 h; (h) HATU, DIEA, 3-(trifluoromethyl)aniline, DMF, rt, 16 h.
group with 4-amino-1-Boc-piperidine. The aryl/heteroaryl substi-
coupling reactions between 9a and the appropriate aryl halide
tuted compounds 15–16 were achieved by palladium catalyzed
and protecting group manipulations.

H. G. Choi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5297–5302
5299
Table 1
Table 2
Cellular antiproliferative activity of compounds 9a–16
SAR of non-bonded sulfur and oxygen(nitrogen) interaction derivatives
N
N
S
O
O
N
HN
R2
HN
R3
N
S
N
N
H
CF3
N
N
H
CF3
Compd R2
Cellular antiproliferative activity (EC50
M)
,
Compd R3
Cellular antiproliferative activity (EC50
,
l
l
M)
Bcr-Abl
(wt)a
Bcr-Abl
Ba/F3c
Bcr-Abl (wt)
Bcr-Abl (T315I)
BaF3
(T315I)b
20
21
22
23
H
Acetyl
>10
0.139
>10
>10
4.16
3.085
3.532
9a
10
H
>10
>10
>10
>10
0.193
0.156
0.276
Cyclopropanecarbonyl 3.085
2-Pyridinyl 0.146
5.0
3.77
O
11
12
0.42
>10
0.96
>10
0.91
>10
O
HN
O
O
13
14
5.21
7.6
>10
>10
>10
N
N
O
HN
O
7.18
HN
HN
N
N
15
16
1.90
6.21
2.41
>10
6.74
>10
N
HN
N
S
O
O
a
b
c
Cellular antiproliferative activity (EC50
Cellular antiproliferative activity (EC50
Cytotoxicity (EC50, lM) on wt-Ba/F3.
,
,
l
l
M) on wt Bcr-Abl-Ba/F3.
M) on mutant Bcr-Abl-T315I-Ba/F3.
To evaluate the ability of the compounds to inhibit Bcr-Abl in a
cellular context we used the murine pre-B cell line Ba/F3 trans-
formed with the Bcr-Abl oncogene. Wild-type Ba/F3 cells prolifer-
ate only in the presence of interleukin-3 (IL-3) while Ba/F3 cells
transformed with oncogenic kinases such as Bcr-Abl become capa-
ble of growing in the absence of IL-3. This provides a robust and
commonly used assay for selective kinase inhibition.16 The small-
est compound 9a did not display antiproliferative activity against
wild-type Bcr-Abl or T315I-Bcr-Abl and the larger compounds
12–16 possessed only weak anti-proliferative activity effect (Table
1). Interestingly, only one compound containing a small cyclopro-
pyl amide displayed submicromolar antiproliferative activity
against Bcr-Abl and T315I-Bcr-Abl. Compound 11 exhibited EC50’s
of 420 nM on wild-type Bcr-Abl and 960 nM onT315I-Bcr-Abl;
however 11 also showed a similar antiproliferative effect against
parental Ba/F3 cells suggesting that targets other than Bcr-Abl
may contribute to the antiproliferative activity of this compound.
To explore this chemical series further we next prepared com-
pounds 20-23 which possess an extended Type II tail containing
an (4-ethyl-piperazin-1-yl)methyl moiety appended to the 4-posi-
tion of the trifluoromethylbenzamide (Table 2). This chemical
maneuver resulted in an approximate 6-fold improvement in cellu-
lar antiproliferative activity relative to 11 against T315I-Bcr-Abl
and a 3-fold decrease in cytotoxicity against parental Ba/F3 cells
(Table 2, compound 22).
Figure 2. Compound 22 (turquois sticks) forms five hydrogen-bonding interactions
with Src; (A) Two hydrogen bonds are made with the hinge region: between the
thiazole N and backbone NH of M341 and between the cyclopropyl amide NH and
the backbone carbonyl of M341. (B) Hydrogen bonding interactions exist between
the benzamide carbonyl of 22 and the backbone NH of D404 of the DFG-motif,
between the benzamide NH and side-chain carboxylate of E310 from the aC-helix,
and from the presumably protonated distal piperazine nitrogen and the backbone
carbonyls of V383 and H384.
compounds 21–22 contributes to their conformation and biological
activity.17 This is supported by the similar cellular potencies of 21–
22 with the 2-pyridine derivative 23. Compound 23 is predicted to
exhibit a similar bioactive conformation to 21–22 as a result of a
lone pair of electrons from the pyridine N interacting with the un-
filled orbitals of the thiazole sulfur as shown by previous computa-
tional analysis.18 Finally we established the bioactive conformation
of this series of compounds by elucidation of a co-crystal structure
We hypothesized that an intramolecular interaction between
the thiazole sulfur and the oxygen atom of the adjacent amide in

5300
H. G. Choi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5297–5302
Table 3
Cellular antiproliferation activity of analogs 24–33
Compd
Structure
Cellular antiproliferative activity (EC50, lM)
Bcr-Abl (wt)
Bcr-Abl (T315I)
BaF3
2.30
NH
N
N
S
O
O
24
2.31
2.30
HN
O
N
N
N
H
CF3
N
N
N
S
25
HN
O
>10
>10
>10
N
H
CF3
N
S
O
O
HN
O
26
27
28
0.38
0.001
0.03
0.19
0.06
0.56
1.99
1.23
4.67
N
N
N
H
N
N
N
S
N
HN
O
N
N
H
CF3
CF3
N
S
O
O
HN
O
N
N
N
H
N
N
N
S
N
HN
O
N
29
30
0.02
0.02
0.30
0.59
>10
>10
N
H
CF3
F
F
CF3
N
S
O
HN
O
N
N
H
N
N
N
S
N
H
N
HN
O
N
31
32
0.2
0.78
>10
N
N
N
CF3
O
O
O
N
S
H
N
H
N
HN
O
CF3
0.018
0.013
0.69
N
N
N
S
N
H
N
H
N
HN
O
N
33
1.24
0.55
>10
CF3
between HG-7-85-01(22) and Src (Fig. 2, PDB code: 4AGW). The
structure revealed that the distance between the sulfur and oxygen
is 2.7 Å which is shorter than the sum of the normal oxygen and
sulfur van der Waals radii of 3.3 Å. Consistent with the ‘hybrid-de-
sign’ strategy, compound 22 was confirmed to bind to the DFG-out
’inactive’ conformation of Src.11
We next further explored substitution on the 3-trifluorophenyl
moiety which occupies the pocket created by the ‘DFG-out’ flip of
the activation loop (Table 3 and 24–26). Compound 24, which
eliminates the methylene spacer between the aryl group and
piperazine in 22, lost approximately 10-fold cellular activity rela-
tive to 22. Compound 25 lacking the CF3 group was completely
inactive up to the highest concentration tested (10 lM). Com-
pound 26, which possesses the ‘tail’ moiety of nilotinib exhibited
slightly diminished potency relative to compound 22 but substan-
tially improved the overall kinase selectivity as demonstrated be-
low. We next explored the effects of introducing a flag-methyl or
2-fluoro onto the central phenyl ring (Table 3and 27–30). The com-
pounds exhibited enhanced activity on Bcr-Abl but not on T315I-
Bcr-Abl. For example, compound 27 is an exceptionally potent as

H. G. Choi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5297–5302
5301
N
A. General synthetic route for Table 1
O
HN
O
S
R3
N
N
H
CF3
a)
10 - 13
14
N
O
N
S
HN
O
H2N
S
b)
c)
HN
N
N
CF3
N
N
H
CF3
H
O
9a
HN
N
S
O
HN
R4
N
N
H
CF3
15 - 16
B. General synthetic route for reverse amide and urea compounds
N
N
H
HN
O
H
N
H2N
b)
N
a)
S
S
N
Boc
R4
N
Boc
17
18
N
S
N
HN
O
H
c)
HN
O
NH2
N
N
S
N
19
31-33
Scheme 2. Reagent and conditions; (A) (a) acyl chloride, pyridine, DCM, 0 °C, 4 h or acid, EDCI, HOBT, DMAP, DMF, rt, 8 h, (b) i. 4-nitrophenyl chloroformate, pyridine, DCM, rt,
1 h, (ii) tert-butyl 4-aminopiperidine-1-carboxylate, TEA, THF, rt, 4 h, (iii) TFA, DCM, 4 h, (c) (i) tert-butyl 4-(6-chloro-2-methylpyrimidin-4-yl)piperazine-1-carboxylate,
Pd2(dba)3, Xanthphos, K2CO3, 2-BuOH, 90 °C, 4 h, (ii) TFA, DCM, 4 h; (B) (a) Cyclopropanecarbonyl chloride, pyridine, DCM, 0 °C, 4 h, (b) TFA, DCM, 4 h, (c) (i) benzoic acid,
HATU, DIEA, DMF, 16 h. or tert-butyl 4-aminopiperidine-1-carboxylate, TEA, THF, rt, 4 h.
Figure 3. Kinase Selectivity of HG-7-85-01(22) and HG-7-86-01(26).
inhibitor of wild-type Bcr-Abl dependent Ba/F3 cell proliferation.
We also investigated the effect of reversing the amide between
the central phenyl and CF3-substituted ring and replacing the
amide with a urea moiety. The synthesis of these compounds is de-
picted in Scheme 2B. Reversing the amide bond orientation, com-
pound 33 compared to 22, resulted in approximately a fourfold
reduction in cellular activity against T315I-Bcr-Abl. Interestingly,
introduction of a urea linkage (32) resulted in 5- to 10- fold

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H. G. Choi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5297–5302
Table 4
Pharmacokinetic results following intravenous 1 mg/kg and oral 2 mg/kg dosing
Compd
Route of administration
t1/2 (h)
Tmax
Cmax (ng/ml)
AUC (min⁄ng/ml)
AUC (%extrapolated)
Cl (ml/min/kg)
MRT (h)
VSS (L/kg)
%F
22
PO
IV
PO
IV
4.7
5.8
3.7
2.4
2.7
0.08
1.1
53.1
291.7
32.0
27733
73800
8160
35
35.3
21
74.4
13.7
303.3
9.5
7.7
8.1
5.3
3.1
18.8
6.5
1.7
26
3.7
0.14
671.7
111167
8.7
improved activity on both wild-type and T315I Bcr-Abl, but in-
creased cytotoxicity towards parental Ba/F3 cells by approximately
fivefold.
References and notes
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M.19 The kinases which interacted with 22 and 26 with
an Ambit score less than 10% of the DMSO control are highlighted
with a red circle in a spot tree (Fig. 3, Supplementary data contains
full screening data for 22 and 26). To provide a measure of overall ki-
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ratio of the number of kinases inhibited below a score of ten versus
total number of kinases tested at a specified concentration. This
analysis revealed that compound 26 possess an S(10) of 0.36 and
compound 22 possesses an S(10) of 0.10 at a concentration of 10 lM.
We assessed the pharmacological properties of compound 22
and 26 in rats following oral and intravenous delivery (Table 4)
prior to performing the murine tumor models which we have pre-
viously reported.13 HG-7-85-01(22) has limited oral bioavailability
(%F = 18.8), a moderate half-life (T1/2 = 5.8 h), a relatively low max-
imal serum concentration (Cmax = 292 ng/mL at 2 mg/kg) and a rel-
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Acknowledgment
We thank Nam Doo Kim for assistance with molecular modeling
and Sara Buhrlage for critical reading of the manuscript. We
acknowledge research funding from Novartis and NIH (R01
CA130876-02).
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
06.036.