Aminopyridyl/pyrazinyl spiro[indoline-3,4′-piperidine]-2-ones as highly selective and efficacious c-met/ALK inhibitors

Article abstract of DOI:10.1021/ml400203d

A series of novel aminopyridyl/pyrazinyl-substituted spiro[indoline-3, 4′-piperidine]-2-ones were designed, synthesized, and tested in various in vitro/in vivo pharmacological and antitumor assays. 6-[6-Amino-5-[(1R)-1-(2,6- dichloro-3-fluorophenyl)ethoxy]-3-pyridyl]-1′-methylspiro[indoline-3, 4′-piperidine]-2-one (compound 5b or SMU-B) was identified as a potent, highly selective, well-tolerated, and orally efficacious c-Met/ALK dual inhibitor, which showed pharmacodynamics effect by inhibiting c-Met phosphorylation in vivo and significant tumor growth inhibitions (>50%) in GTL-16 human gastric carcinoma xenograft models.

Full text of DOI:10.1021/ml400203d

Letter
pubs.acs.org/acsmedchemlett
Aminopyridyl/Pyrazinyl Spiro[indoline-3,4′-piperidine]-2-ones As
Highly Selective and Efficacious c‑Met/ALK Inhibitors
Jingrong Li,†,§ Nan Wu,‡,§ Yuanxin Tian,† Jiajie Zhang,*,† and Shuguang Wu*,†
†School of Pharmaceutical Sciences, Southern Medical University, 1838 North Guangzhou Boulevard, Guangzhou 510515, China
‡Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038, China
S
* Supporting Information
ABSTRACT: A series of novel aminopyridyl/pyrazinyl-
substituted spiro[indoline-3,4′-piperidine]-2-ones were de-
signed, synthesized, and tested in various in vitro/in vivo
pharmacological and antitumor assays. 6-[6-Amino-5-[(1R)-1-
(2,6-dichloro-3-fluorophenyl)ethoxy]-3-pyridyl]-1′-
methylspiro[indoline-3,4′-piperidine]-2-one (compound 5b or
SMU-B) was identified as a potent, highly selective, well-
tolerated, and orally efficacious c-Met/ALK dual inhibitor,
which showed pharmacodynamics effect by inhibiting c-Met
phosphorylation in vivo and significant tumor growth
inhibitions (>50%) in GTL-16 human gastric carcinoma xenograft models.
KEYWORDS: Hepatocyte growth factor receptor (HGFR or c-Met), anaplastic lymphoma kinase (ALK), inhibitor, cancer,
aminopyridine, aminopyrazine, spiro[indoline-3,4′-piperidine]-2-one, small molecule
c-Met is a receptor tyrosine kinase known as hepatocyte growth
factor receptor (HGFR). c-Met and its natural ligand
hepatocyte growth factor (HGF), also known as the scatter
factor, are often aberrantly expressed in a wide range of human
cancers, including hereditary papillary renal cell carcinoma,1
lung cancers,2,3 head and neck cancers,4 and gastric cancer.5
Because of the role of dysregulated c-Met/HGF signaling in
human oncogenesis, invasion, and metastasis, targeting the c-
Met/HGF pathway becomes a powerful strategy for anticancer
therapy. Currently half a dozen humanized monoclonal
antibody and more than two dozens of small molecule c-
Met/HGF inhibitors are being evaluated in preclinical and
clinical studies as cancer treatments.6−10 Two small molecule
inhibitors, i.e., Pfizer’s c-Met/ALK dual inhibitor Crizotinib
(PF-02341066) and Exelixis’ VEGFR2/c-Met multikinase
inhibitor Cabozantinib (Figure 1), have been approved by the
FDA, for treatments of late-stage non-small cell lung cancer11
and progressive metastatic medullary thyroid cancer,12
respectively.
number of human cancers, such as neuroblastoma,14 pulmonary
neuroendocrine carcinoma,15 esophageal cancer,16 diffuse large
cell lymphoma,17 and certain types of non-small cell lung
cancer (NSCLC).18 Targeting oncogenic c-Met and ALK
simultaneously has proven to be beneficial in glioblastoma19
and Ewing’s sarcomas.20 Although dual inhibition of c-Met and
ALK sounds like attractive strategy for cancer therapy, there has
not been a lot of known c-Met/ALK dual inhibitors in the
literature. Crizotinib is the first and currently only approved c-
Met/ALK dual inhibitor for treating EML4-ALK fusion
protein-positive NSCLC patients. Other c-Met/ALK dual
inhibitors include Xcovery’s aminopyridazine-based X-376 and
X-396.21 In continuation of our interest in pursuing therapeuti-
cally useful c-Met inhibitors (Figure 2), we report herein a class
of novel aminopyridyl/pyrazinyl-substituted spiro[indoline-
3,4′-piperidine]-2-ones as highly selective and orally efficacious
c-Met/ALK dual inhibitors.
In addition to c-Met, Crizotinib also inhibits anaplastic
lymphoma kinase (ALK). ALK is a member of the insulin
receptor superfamily of tyrosine kinases.13 It plays a role in a
Figure 2. Compounds 322 and 423 as c-Met inhibitors.
Received: May 24, 2013
Accepted: July 12, 2013
Figure 1. Crizotinib (1) and Cabozantinib (2).
© XXXX American Chemical Society
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It was found previously that the 1′-methyl-2-oxo-spiro-
[indoline-3,4′-piperidine] moiety when attached to the triazolo-
[4,5-b]pyrazine core was well tolerated in c-Met kinase.22 We
decided to introduce this group to the typical kinase hinge
binding group aminopyridyl, which presented in the chemical
structure of Crizotinib (1). Therefore, a series of novel 1′-
methyl-2-oxo-spiro[indoline-3,4′-piperidine]-substituted ami-
nopyridines and aminopyrazines24 were designed, computa-
tionally modeled, synthesized, and tested in in vitro and in vivo
assays. Compounds 5a−b, 6a−b, and 7a−b, shown in Figure 3
were selected examples to be discussed in this letter.
molecules including the 2,6-dichloro-3-fluorobenzyloxyl and 2-
aminopyridyl overplayed perfectly except for the piperidinyl-
pyrazolyl group in Crizotinib and the 1′-methyl-2-oxo-spiro-
[indoline-3,4′-piperidine] moiety in compound 5b. The
piperidinyl of Crizotinib and the spiro portion of 5b were
solvent exposed and therefore should not have a significant
impact on potency even though they adopted a different
conformations. Figure 5 showed the detailed interaction of 5b
Figure 5. Interactions of compound 5b with the amino acid residues in
the ATP binding pocket of c-Met kinase.
with the c-Met kinase. The 2-aminopyridyl group formed a pair
of bidentate hydrogen bonds with hinge residues Pro1158
(yellow) and Met1160 (red). The hydroxyl oxygen of Tyr1159
(blue) formed a hydrogen bond with the indolin-2-one NH. In
the lipophilic back pocket, where the 2,6-dichloro-3-fluoro
phenyl ring sat, the fluoro group formed a hydrogen bond with
Asp1222 NH (magenta). The phenyl ring formed a π−π
stacking interaction with the phenyl ring of Tyr1230 (green).
To test the modeling results, aminopyridyl compound 5b and
its regioisomer 5a as well as their close analogues of
aminopyrazines 6a−b were synthesized. Achiral analogues
7a−b were also prepared for comparison purpose.
Figure 3. Novel aminopyridyl/pyrazinyl-substituted spiro compounds
(5a−b, 6a−b, and 7a−b).
Docking studies revealed that compounds of 5a−b and 6a−b
types fit very well in the crystal structure of c-Met kinase
domain (PDB code, 2WGJ), Figure 4 showed the docking
model of compound 5b in the cocrystal structure of Crizotinib
bound to the c-Met kinase. It was found that most parts of the
Scheme 1 showed the synthetic methods for compounds 5a−
b, 6a−b, and 7a−b. Compounds 5a−b were prepared by
Suzuki coupling of bromide 10a25 and boronates 11m or 11n.22
Compounds 6a−b were synthesized in a similar fashion except
that bromide 10b was used. The required intermediate 10b was
obtained by reaction of commercially available 3,5-dibromopyr-
azin-2-amine (8) and (1R)-1-(2,6-dichloro-3-fluorophenyl)-
ethanol (9) under basic condition. Compounds 7a−b were
generated by Suzuki coupling of intermediate 13 with 11m or
11n. Compound 13 was made in 4 steps from 2,6-dichloro-3-
fluorobenzoic acid (12) by reduction of acid into alcohol,
Mitsunobu reaction of the alcohol with 3-hydroxyl-2-nitro-
pyridine, reduction of the nitro into amino group, and
bromination of the pyridine ring with NBS.
The compounds were first screened in MKN45 cell
mechanistic assay by measuring the inhibition of phosphor-
ylation of c-Met signals. Crizotinib was used as the reference
compound. The results are listed in Table 1.
It was found that compounds 5a−b and 6a−b were very
potent in cells with IC50 values in the same range with
Figure 4. Overlay of Crizotinib (magenta) and compound 5b (gray) in
c-Met kinase domain (PDB code: 2WGJ).
B
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ACS Medicinal Chemistry Letters
Letter
a
Scheme 1. Synthesis of Compounds 5a−b, 6a−b, and 7a−b
Figure 6. Kinase dendrogram of compound 5b (SMU-B) at [drug] =
100 nM in DiscoveRx’s scanEDGESM against 97 wild-type and mutant
kinases.
nistic assay against c-Met, ALK, and AXL. The results are
summarized in Table 2.
a
Reagents and conditions: (a) NaH, THF, reflux, 20 h, 78%. (b)
Pd(PPh3)4, DME−H2O (4:1), K2CO3, 80 °C. Yields: 86% for 5a; 82%
for 5b; 54% for 6a; 67% for 6b; 85% for 7a; 78% for 7b. (c) BH3,
THF, reflux, 24 h, 75%. (d) 3-Hydroxyl-2-nitropyridine, DIAD, PPh3,
THF, 0 °C, 16 h, 90%. (e) Fe, HCl (aq, 20 mol %), EtOH, 80 °C, 30
min, 95%. (f) NBS, CH3CN, 0 °C, 30 min, 60%.
Table 2. Biochemical and Cell Mechanistic IC50s of
Compound 5b (SMU-B)
a
b
biochemical IC50 (nM)
cell mechanistic IC50 (nM)
c
target
[ATP] (μM)
5b
cell line
5b
Crizotinib
c-Met
ALK
AXL
10
1.87
MKN45
karpas 299
MEF
22
20
50
<0.5
28.9
39
110
Table 1. c-Met Cell Mechanistic IC50s of Compounds 5a−b,
6a−b, and 7a−b in MKN-45 Cells and Modeling Scores
100
300
a
b
Data generated by Reaction Biology Corp. Data generated by
c
a
c-Met cell mechanistic IC50 (μM) in MKN-
modeling
b
ProQinase GmbH. [ATP] = Km.
compd
45 cells
score
Crizotinib
0.020
0.020
0.019
0.023
0.012
>10.0
8.67
8.68
8.67
9.11
8.95
6.70
6.91
Compound 5b potently inhibited c-Met, ALK, and AXL
kinases with IC50s of 1.87, <0.5, and 28.9 nM, respectively, in
biochemical assays, and 22, 39, and 300 nM, respectively, in
cellular kinase assays. It was equipotent against c-Met (22 vs 20
nM) and 2.8-fold more potent than Crizotinib against ALK (39
vs 110 nM) in cells. The c-Met cell IC50 data of compound 5b
generated by ProQinase in MKN45 cells matched well with our
in house data (22 vs 19 nM, Tables 1 and 2). Encouraged by
the high biochemical and cell mechanistic potencies against c-
Met, we determined the antiproliferative activities of compound
5b in four cell lines with different c-Met status (Table 3).
Compound 5b had 20 nM IC50 in inhibiting the growth of
human gastric carcinoma cell line GTL-16 with Met gene
amplification but was less potent in non-small cell lung cancer
(NSCLC) lines H1993 and H441 with IC50s of 1.58 and 2.82
μM, respectively. Compound 5b was not potent in human
glioblastoma cell line U87MG with HGF/Met autocrine
formation.
The excellent in vitro profile of compound 5b warranted
further studies of this compound in vivo including pharmaco-
kinetics (PK), pharmacodynamics (PD), and tumor growth
inhibition (TGI) in xenograft models. The PK studies were
carried out in BALB/c mice following i.v. and p.o. routes of
administration, and PD studies conducted in GTL-16 nu/nu
mouse xenografts following a single oral dose at 20, 40, and 80
mg/kg, respectively. The results were summarized in Table 4. It
was found that compound 5b was orally bioavailable with 48%
bioavailability in mice. In PD studies, compound 5b
significantly inhibited phosphorylation of c-Met kinase at 20
mg/kg, and completely stopped the phospho-c-Met signals in
5a
5b (SMU-B)
6a
6b
7a
7b
9.0
a
b
Data generated in house. Data generated using Surflex-Dock
program.
Crizotinib. The high potencies were consistent with the
docking results. To our surprise, however, the achiral
compounds 7a−b were much less potent with IC50 values ≥
9 μM although their chemical structures were so similar to
compounds 5a−b and the only difference was with or without
the α-methyl group at the benzylic position. To understand
what factor had contributed to the differences in potency,
modeling scores were generated using the Surflex-Dock
program’s empirical scoring function (Table 1).26,27 The results
showed that compounds 7a−b received lower scores of <7.0
than all other compounds, which had scores >8.6. The α-
methyl group at the benzylic position must have been required
to help maintain the bioactive U-shaped binding mode.
Compound 5b, i.e., SMU-B, was further studied in various in
vitro and in vivo assays. In DiscoveRx’s scanEDGESM panel of
97 kinases,28,29 compound 5b showed high selectivity and only
hit three kinases, i.e., c-Met, ALK, and AXL, with percent
control <10% at 100 nM drug concentration (Figure 6). To
verify this result, compound 5b was tested in Reaction Biology
Corp’s nanoliter kinase assay30 and ProQinase’s cell mecha-
C
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ACS Medicinal Chemistry Letters
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Table 3. Antiproliferative IC50s of Compound 5b (SMU-B)
in Four Different Cell Lines
Figure 7. Tumor growth inhibition (TGI) by compound 5b (SMU-B)
in GTL-16 xenograft models.
c-Met status
cell line antiproliferative IC50 (μM), 5b (SMU-B)
showed pharmacodynamics effect and significant tumor growth
inhibitions in GTL-16 human gastric carcinoma xenograft
models. Compound 5b (SMU-B) represented one of the very
few selective and highly efficacious c-Met/ALK dual inhibitors
with clear mechanism of action (MoA) known to date. The
superior antitumor properties of compound 5b (SMU-B)
justified further studies.
Met amplification
Met amplification
Met overexpression
HGF/Met autocrine
GTL-16
H1993
H441
0.020
1.58
2.82
>10
U87MG
Table 4. Pharmacokinetics and Pharmacodynamics Results
of Compound 5b (SMU-B); Inhibition of Phospho-c-Met in
GTL-16 Xenografts after 4 h Following a Single Oral Dose
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedures and analytical data for compounds
reported in this letter and procedures for in vitro and in vivo
assays. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +86-20-62789415. Fax: +86-20-61648548. E-mail:
zhangjj@smu.edu.cn (J.Z.); shuguang@smu.edu.cn (S.W.).
■
pharmacokinetics in BALB/c mice
dose (mg/kg) dosing route Cmax (μM) AUC0‑∞ (ng.h/mL)
F %
Author Contributions
5
i.v.
1.36
1.45
1229.1
2348.5
100
48
§(J.L. and N.W.) These authors contributed equally to this
work.
20
p.o.
Funding
This study was supported by grants from International Science
and Technology Cooperation Base of Guangdong Provincial
Department of Science and Technology (No.
2009B050900006).
40 and 80 mg/kg dose groups. These results demonstrated that
the in vitro c-Met potencies could be successfully translated
into in vivo activities.
To assess the antitumor potential of compound 5b in vivo,
efficacy studies were carried out in GTL-16 xenograft models.
Tumor-bearing nu/nu mice were dosed orally at 20 and 40 mg/
kg, qd, for 14 days. Tumor sizes and body weights were
measured every other day. The results were shown in Figure 7.
Significant antitumor efficacies of compound 5b (SMU-B)
were observed in this model, i.e., 52% and 87% TGI’s for the 20
and 40 mg/kg dose groups, respectively. There was no
significant body weight loss in all experiment groups (data
not shown), which suggested low toxicity of this compound.
In summary, a series of novel aminopyridyl/pyrazinyl-
substituted spiro[indoline-3,4′-piperidine]-2-ones were de-
signed, synthesized, and tested in various in vitro and in vivo
assays. Selected compounds, especially compound 5b (SMU-
B), showed single-digit nM biochemical and less than 100 nM
cellular potencies against c-Met and ALK kinases. Compound
5b (SMU-B) was highly selective and only hit very few targets
in the 97-kinase panel screening. It was orally bioavailable and
Notes
The authors declare no competing financial interest.
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