Discovery of Potent, Selective, and Brain-Penetrant 1 H-Pyrazol-5-yl-1 H-pyrrolo[2,3- b]pyridines as Anaplastic Lymphoma Kinase (ALK) Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.8b01630

Anaplastic lymphoma kinase (ALK), a member of the receptor tyrosine kinase family, is predominantly expressed in the brain and implicated in neuronal development and cognition. However, the detailed function of ALK in the central nervous system (CNS) is s

Full text of DOI:10.1021/acs.jmedchem.8b01630

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Article
Discovery of Potent, Selective, and Brain-Penetrant 1#-Pyrazol-5-yl-1#-
pyrrolo[2,3-b]pyridines as Anaplastic Lymphoma Kinase (ALK) Inhibitors
Makoto Fushimi, Ikuo Fujimori, Takeshi Wakabayashi, Tomoaki Hasui, Youichi Kawakita, keisuke imamura,
Tomoko Kato, Morio Murakami, Tsuyoshi Ishii, Yorifumi Kikko, Maki Kasahara, Atsushi Nakatani, Yuto Hiura,
Maki Miyamoto, Kumar Singh Saikatendu, Hua Zou, Scott Weston Lane, J. David Lawson, and Hiroshi Imoto
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01630 • Publication Date (Web): 22 Apr 2019
Downloaded from http://pubs.acs.org on April 22, 2019
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Discovery of Potent, Selective, and Brain-Penetrant
1
H-Pyrazol-5-yl-1H-pyrrolo[2,3-b]pyridines as
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Anaplastic Lymphoma Kinase (ALK) Inhibitors
†
,†
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†
Makoto Fushimi, Ikuo Fujimori,* Takeshi Wakabayashi, Tomoaki Hasui, Youichi Kawakita,
†
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†
†
Keisuke Imamura, Tomoko Kato, Morio Murakami, Tsuyoshi Ishii, Yorifumi Kikko, Maki
†
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‡
‡
Kasahara, Atsushi Nakatani, Yuto Hiura, Maki Miyamoto, Kumar Saikatendu, Hua Zou,
‡
‡
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Scott Weston Lane, J. David Lawson, and Hiroshi Imoto
AUTHOR ADDRESSES
†
Neuroscience Drug Discovery Unit, Research, Takeda Pharmaceutical Company Limited, 26-1,
Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
‡
Takeda California, Inc., 10410 Science Center Drive, San Diego, California 92121, United
States
KEYWORDS. ALK, structure-based drug design (SBDD), tropomyosin receptor kinases (Trks),
brain penetration, phosphorylated-ALK, cognitive improvement
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ABSTRACT
Anaplastic lymphoma kinase (ALK), a member of the receptor tyrosine kinase family, is
predominantly expressed in the brain and implicated in neuronal development and cognition.
However, the detailed function of ALK in the central nervous system (CNS) is still unclear. To
elucidate the role of ALK in the CNS, it was necessary to discover a potent, selective, and
brain-penetrant ALK inhibitor. Scaffold hopping and lead optimization of
N-(2,4-difluorobenzyl)-3-(1H-pyrazol-5-yl)imidazo[1,2-b]pyridazin-6-amine 1 guided by a
cocrystal structure of compound 1 bound to ALK resulted in the identification of
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(
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6-(1-(5-fluoropyridin-2-yl)ethoxy)-1-(5-methyl-1H-pyrazol-3-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)
(2S)-2-methylmorpholin-4-yl)methanone 13 as a highly potent, selective, and brain-penetrable
compound. Intraperitoneal administration of compound 13 significantly decreased the
phosphorylated-ALK (p-ALK) levels in the hippocampus and prefrontal cortex in the mouse
brain. These results suggest that compound 13 could serve as a useful chemical probe to elucidate
the mechanism of ALK-mediated brain functions and the therapeutic potential of ALK inhibition.
INTRODUCTION
Protein tyrosine kinases are enzymes that phosphorylate protein substrates on the tyrosine
residues. They can be classified into receptor tyrosine kinases (RTKs) and non-RTKs. RTKs are
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membrane-spanning cell surface proteins that mediate the transduction of extracellular signals to
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the cytoplasm. They play an important role in the activation of intracellular signaling systems
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that regulate various cellular events, including cell proliferation, migration, metabolism,
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differentiation, and survival. Therefore, when chemically probing the specific roles of an
individual RTK, it is important for the tool compound to be selective for the targeted kinase.
Anaplastic lymphoma kinase (ALK) is a member of the insulin receptor (IR) class of RTKs.
The ALK gene is highly expressed in the central nervous system (CNS), and the knock-out (KO)
of the ALK gene in mice leads to various interesting phenotypes: enhancement of cognitive
functions in spatial memory and reduction of anxiety- and depression-like behaviors without any
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apparent morphological deficits. The endogenous ligand of ALK has yet to be established and
the detailed mechanism of the ALK signaling pathway for these phenotypes is still unclear. In the
meantime, fusion proteins of the ALK kinase domain with other genes produced by
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translocations or other chromosomal rearrangements have been discovered in several malignant
cells. Unlike endogenous ALK that is expressed in the brain, the fusion proteins show
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dysregulated expression and activation leading to oncogenesis.
In 2011, a small molecule
inhibitor of ALK, crizotinib (Figure 1), was approved by the U.S. Food and Drug Administration
(
(
FDA) for the treatment of echinoderm microtubule-associated protein-like 4 fused ALK
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EML4–ALK) positive non-small cell lung cancer (NSCLC).
Several ALK inhibitors such as
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ceritinib (Novartis) , brigatinib (Ariad, currently Takeda) , and alectinib (Roche/Chugai) were
also studied for their potential as an anti-cancer drug during our research period. Interestingly,
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Pfizer reported a brain-penetrant ALK inhibitor, lorlatinib, to target brain metastases in 2014.
However, there is no report supporting the development of ALK inhibitors to elucidate the
mechanism of ALK-mediated brain functions. Herein, we report the discovery of a highly
selective and brain-penetrant ALK inhibitor that demonstrated a decrease in phosphorylated-ALK
levels in mouse brain. Furthermore, we describe our structure-based drug design (SBDD) driven
lead generation and optimization efforts resulting in the identification of
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H-pyrazol-5-yl-1H-pyrrolo[2,3-b]pyridine derivatives as a new class of ALK inhibitor.
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Figure 1. Structures of some reported ALK inhibitors.
RESULTS AND DISCUSSION
Lead Generation. To identify a potent, selective, and brain-penetrable ALK inhibitor, we
initiated our efforts with a high-throughput screening of our corporate compound library. An
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N-benzyl-3-(1H-pyrazol-5-yl)imidazo[1,2-b]pyridazin-6-amine derivative was identified as a hit
with moderate ALK inhibitory activity. The initial structure-activity relationship (SAR) studies
around the hit compound led to a highly potent ALK inhibitor 1 in enzymatic and cellular assays
with IC50 values of 2 nM and 6 nM, respectively (Table 1). This is comparable to crizotinib that
showed IC50 values of 1.8 nM and 37 nM in our enzymatic and cellular assays, respectively.
However, compound 1 also exhibited extremely potent TrkA inhibitory activity (IC50 = 0.1 nM)
and moderate kinase selectivity in an enzymatic kinase panel that consisted of 39 different
kinases (see supporting information). Compound 1 is a chiral compound and its absolute
configuration was determined as S-form. An R-isomer of compound 1 showed lower ALK
inhibitory activity with IC50 value of 1000 nM.
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Table 1. In vitro Profiles of Compound 1 Derived from Hit Compound
a
Assays
IC50 (nM)
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ALK enzyme
(1.8–2.2)
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(4.2–10)
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(0.096-0.11)
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ALK cell
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IC50 values and 95% confidence intervals (given in parentheses) were calculated by nonlinear
b
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regression analysis of percent inhibition data (n = 2).
Phospho-ALK levels were measured in cell assay.
ATP concentration is 1 μM.
Selectivity Improvement over Trks. It is well known that the Trk family is located in nerve cells
and mediates the survival and differentiation of neuronal cells via interactions with various
neurotrophic factors such as nerve growth factor (NGF) and brain-derived neurotrophic factor
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BDNF). Thus, for the tool compounds to accurately probe ALK function in the brain they
should be selective over the Trk family kinases. SBDD is a frequently utilized method for the
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development of selective kinase inhibitors. To support our SBDD efforts, we cocrystallized
compound 1 with the ALK kinase domain and successfully generated a cocrystal structure of 1 in
the ALK active site (Figure 2). The cocrystal structure revealed that the pyrazole ring interacted
with the hinge residues Glu1197 and Met1199. The nitrogen at the 5-position of the
imidazo[1,2-b]pyridazine core allows the pyrazole to adopt a coplanar orientation relative to the
core by alleviating the steric clash with the pyrazole 4-hydrogen that would arise if a carbon were
at the same position. This coplanarity well mimics the adenine structure of ATP, and it may
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explain the high potency of compound 1. Interestingly, the residue of Leu1256 formed three
CH-π interactions with the imidazole ring, the pyridazine ring in the imidazo[1,2-b]pyridazine
core, and the 2,4-difluorophenyl group, leading to robust stabilization of the L-shaped
conformation of the molecule. Next, we analyzed the difference of the amino acid sequence in the
ATP binding pocket between ALK and TrkA to improve selectivity over the Trks. Four amino
acid residues in ALK were identified for consideration: Ala1200, His1124, Leu1196, and
Leu1198. Of these, we focused on Leu1198, an amino acid in the hinge region of ALK which is a
tyrosine in all the Trk family proteins. The side chains of Leu1198 and the corresponding Tyr590
in TrkA extended on the left obliquely upward from the hinge towards the P-loop adjacent to the
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-position of the imidazo[1,2-b]pyridazine core (Figure 2). The bulky Tyr residue led to a
narrower binding space in the Trk family relative to ALK. Thus, we concluded that targeting this
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space should lead to better selectivity over Trks. Conversely, we chose not to target the other
three residues, His1124, Leu1196, and Ala1200: 1) His1124 could not be analyzed due to its
structural disorder, 2) the modification of the methyl group on the pyrazole ring adjacent to
Leu1196 resulted in a decrease of ALK inhibitory activity (data not shown), and 3) Ala1200 and
the corresponding Arg592 in TrkA were considered challenging to study a specific interaction
since the residues were oriented toward the outside of the ligand binding pocket.
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Figure 2. Cocrystal structure of compound 1 bound to the ATP binding site of human ALK
protein (PDB code 6EDL). Different residues in ALK (green) and TrkA (yellow, PDB code 4F0I)
around compound 1 (magenta). Glu1197 and Met1199 are hinge residues. The imidazole portion
of His1124 in ALK is not shown due to poor electron density for the sidechain.
To access the Leu1198 region, the imidazo[1,2-b]pyridazine (which lacks an attachment
point at the 1-position) was converted to 1H-pyrrolo[2,3-b]pyridine 2, which could then be
modified at the corresponding position (Figure 3A). Compound 2 demonstrated moderate ALK
inhibitory activity in both enzymatic and cellular assays with IC50 values of 14 nM and 140 nM,
respectively. We envisioned that the introduction of 3-substituent groups that would clash with
the TrkA Tyr residue would facilitate improving the selectivity of compound 2 over Trks. This
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assumption encouraged us to examine the introduction of sulfonyl and carbonyl moieties attached
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the bulky R group would be directed in such a way to avoid the repulsion with the adjacent
4-position of the 1H-pyrrolo[2,3-b]pyridine core, which would be beneficial to the acquisition of
selectivity. In addition, the sulfonyl and carbonyl groups were predicted to have complementary
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perpendicular or moderately twisted, respectively (Figure 3C).
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Figure 3. (A) Results of scaffold hopping. (B) Design to improve selectivity over Trks. (C)
Comparison of three-dimensional bulkiness between sulfonyl and carbonyl moieties.
We assessed the hypothesis mentioned above with sulfone and amide derivatives at the
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Table 2 summarizes the results. As expected, methyl sulfone 3 showed increased potency for
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ALK and reduced activity for TrkA when compared with those of the compound 2. With respect
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0
increased the cellular potency and the ligand lipophilicity efficiency (LLE) of compound 4.
Thus, we utilized the 5-fluoro-2-pyridyl group instead of the 2,4-fluorophenyl group for further
1
optimization. An increase in the size of the R group in sulfonyl derivatives was detrimental to
ALK inhibitory activity based on the potencies of ethyl, isopropyl, and oxetan-3-yl compounds
(
5−7). However, these bulkier modifications improved the selectivity for ALK over TrkA to a
greater extent. Among the sulfone derivatives tested, isopropyl compound 6 achieved the
highest selectivity with 94-fold higher activity for ALK than for TrkA, though the ALK cellular
inhibitory activity was only moderate (IC50 = 270 nM). Interestingly, the amide derivatives
produced better results. Dimethyl amide 8 demonstrated equipotent ALK inhibition and
decreased TrkA inhibition relative to compound 4, leading to 67-fold ALK selectivity over
TrkA. The cellular activity of compound 8 was also maintained with an IC50 value of 16 nM.
Morpholylamide 9 exhibited substantial improvement in the selectivity (480-fold) while
maintaining cellular potency (IC50 = 23 nM), whereas N-methylpiperazine 10 resulted in a drop
in selectivity. However, dihydropyran 11 without sulfone or amide linkers did not improve the
selectivity over TrkA. These results suggested that the sulfonyl/amide linker was the key to
selectivity over TrkA.
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7
8
9
Table 2. In vitro Profiles of Sulfone and Amide Derivatives at 3-Position of
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1H-pyrrolo[2,3-b]pyridine.
ALK
ALK cell
TrkA
1
2
b
Compd
R
R
LLE
a
a
a
IC50 (nM)
IC50 (nM)
IC50 (nM)
1
4
140
(120–160)
27
2
3
4
5
H
2,4-diF-Ph
2,4-diF-Ph
5-F-2-Py
5-F-2-Py
<10
3.4
5.6
6.8
5.8
(9.8–19)
.2
(0.92–1.5)
.3
(2.5–4.4)
.6
(7.3–13)
1
15
(12–19)
45
(22–33)
13
3
(10–17)
37
(24–84)
110
9
(20–66)
(63–210)
700
(1300–
200)
1
1
8
270
6
7
8
9
5-F-2-Py
5-F-2-Py
5-F-2-Py
5-F-2-Py
5.2
5.7
7.1
6.8
(
(
13–25)
(240–320)
2
2
0
60
730
17–23)
(32–110)
(600–890)
1
.8
16
120
(
(
1.5–2.1)
(14–18)
(87–170)
1200
(1000–
1500)
2
.5
23
2.0–3.2)
(19–27)
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
.1
37
250
10
11
5-F-2-Py
5-F-2-Py
6.0
5.9
(
(
4.9–7.7)
(22–62)
(190–320)
1.9
9.4
2.7
1.5–2.4)
(7.6–12)
(2.4–3.2)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
IC50 values and 95% confidence intervals (given in parentheses) were calculated by nonlinear
b
regression analysis of percent inhibition data (n = 2). LLE = pIC50 – cLogP.
As described above, our efforts succeeded in the identification of sulfone and amide
derivatives with high ALK selectivity over TrkA. To build a rationale for this finding,
cocrystallization was attempted for several of these compounds and two cocrystal structures of
ALK were successfully obtained. The structures of amide 9 (PDB code 6EBW) and sulfone 7
(PDB code 6E0R) aligned with TrkA (PDB code 4F0I) are shown in Figures 4A and 4B,
respectively. The binding mode of amide 9 is consistent with our design hypothesis, where the
amide substituent pointed toward Leu1198 via twisting of the amide plane. The morpholine ring
oriented obliquely to the pyrrolo[2,3-b]pyridine core due to intramolecular strain preventing a
co-planar conformation. In the TrkA active site, compound 9 cannot accommodate this
conformation because of the repulsion between the morpholine ring and Tyr590 thus dramatically
reducing the TrkA inhibitory activity of compound 9. Interestingly, as Figure 4B shows, the
1
binding mode of compound 7 is inconsistent with our expectation that R would orient towards
Leu1198. The sulfonyl group of compound 7 appeared to have more freedom of rotation than the
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8
9
amide group in compound 9. Consequently, the cocrystal structure with the ALK showed the
(
oxetan-3-yl)sulfonyl moiety of compound 7 in the projecting away from the hinge towards the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
vacant solvent-exposed region and created an unfavorable contact between the sulfonyl oxygen
and hinge backbone carbonyl oxygen of Met1199. This likely explains the lower ALK inhibitory
activity of compound 7 relative to compound 9. In the case of the similar compound 4, the
methylsulfonyl group is expected to adopt several conformations, some of which avoid the
repulsion between the sulfonyl oxygen and the Met1199 carbonyl oxygen, thus resulting in
1
tighter binding. As the R substituent becomes bulkier (5−7), it becomes harder to access
conformations that do not create a repulsive interaction. With either greater strain or greater
electrostatic repulsion, with the carbonyl of Met1199, the overall binding energy becomes less
favorable. Regarding the binding to TrkA, we speculate that the narrower binding space does not
allow orientation of the sulfonyl group without forcing one of the sulfonyl oxygens into a
1
repulsive interaction even when the R substitution is as small as a methyl group. While these
analyses provide the rationale behind the improvement of the selectivity over TrkA in the
sulfonyl derivatives, complete understanding at the molecular level requires additional structural
information. Toward this end, a cocrystal with TrkA is currently under investigation.
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2
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. X-ray crystal structures of the amide compound 9 (A) and the sulfone compound 7 (B)
bound to the ATP binding site of human ALK protein (gray, PDB 6EBW and 6E0R, respectively).
The relative position of Tyr590, based on the PDB 4F0I crystal structure, is shown in yellow.
Avoidance of Multidrug Resistance Protein 1 (MDR1) Efflux. The blood-brain barrier
tightly regulates the passage of molecules from the blood into the brain and highly expresses
various efflux transporters, represented by P-glycoprotein (P-gp) which is referred to by its gene
2
2
name MDR1. Brain penetrant molecules need to have good membrane permeability and avoid
being an MDR1 substrate. As Table 2 shows, the selectivity over TrkA was dramatically
improved by the introduction of the morpholylamide group at the 3-position of the
1
H-pyrrolo[2,3-b]pyridine core. However it was revealed that compound 9 demonstrated high
P-gp efflux (MDR1 BA/AB ratio = 18), presumably because of its increased polarity.
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9
One of the practical approaches to avoid being an MDR1 substrate is removing or
2
3
weakening hydrogen-bond donors (HBDs) and hydrogen-bond acceptors (HBAs). Using the
cocrystal structures for guidance, we explored removing HBAs and HBDs that did not form
essential interactions with the ALK active site. The cocrystal structure of compound 9 with ALK
revealed that the benzylamine NH at the 6-position of 1H-pyrrolo[2,3-b]pyridine did not form
any interactions with either structural waters or amino acid residues in the ALK protein.
Therefore, we replaced the benzylamine group with a benzyl ether group to remove one of the
HBDs.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. In vitro Profiles of Benzyl ether Derivatives at 6-Position of 1H-pyrrolo[2,3-b]pyridine.
ALK
ALK cell
TrkA
MDR
3
Compd
R
a
a
a
b
IC50 (nM) IC50 (nM)
IC50 (nM)
ratio
6
.2
69
1400
c
1
2
3.1
1.0
O
N
(5.2–7.4)
(37–130)
(1000–1800)
Me
(S)
2
.9
27
2600
d
13
O
N
(2.5–3.4)
(19–39)
(1600–4300)
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Me
(R)
4.6
42
3400
d
1
4
1.3
O
N
(3.5–6.1)
(29–61)
(2200–5100)
a
IC50 values and 95% confidence intervals (given in parentheses) were calculated by nonlinear
b
regression analysis of percent inhibition data (n = 2). Multidrug resistance protein 1 (MDR1)
c
d
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
efflux ratio in P-glycoprotein (P-gp)-overexpressing cells. Racemic compound. Both of 13 and
1
4 are enantiomerically pure compounds.
Table 3 summarizes the results of this effort. As expected, benzyl ether 12 significantly
improved the MDR1 efflux ratio while maintaining potent enzymatic and cellular ALK inhibitory
activities. The shielding of the oxygen atom on the morpholine ring by a methyl group further
improved the MDR1 efflux ratio (13 and 14). The (S)-isomer compound 13 demonstrated better
enzymatic and cellular ALK potency than (R)-isomer compound 14.
Determination of Eutomer and Distomer by Methoxyether Derivatives. For the determination
of the eutomer and the distomer of the ether-linker derivatives, the chiral compound 15 and 16
were prepared. The compounds 13 and 14 were assumed to be eutomers because they were
derivatized from the common intermediate 52a with the eutomer 15 in Table 4. According to the
benzylamine derivatives in Table 2, the absolute configuration of the eutomer of the benzyl ether
derivatives was also estimated as S-form. The absolute configuration of the methyl group at the
benzyl position could not be determined because the single crystallization was not achieved
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9
despite a number of single-crystallization trials. The endeavor to determine the absolute
configuration of 13 will be continued.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. Determination of Eutomer and Distomer by Methoxyether Derivatives.
ALK
IC50 (nM)
3.2
TrkA
Compd
Chirality
(S) or (R)
(R) or (S)
a
a
IC50 (nM)
1
1
5
6
<10
(
2.2–4.8)
2800
1700–4700)
1500
(
(1200–1800)
a
IC50 values and 95% confidence intervals (given in parentheses) were calculated by nonlinear
regression analysis of percent inhibition data (n = 2).
Selectivity and Pharmacokinetic Profiles of Compound 13. With the promising in vitro
profiles, such as the high enzymatic and cellular ALK inhibitory activity, the excellent selectivity
over TrkA, and the lower MDR1 efflux ratio, compound 13 was additionally characterized in
kinase selectivity assays (Table 5). Compound 13 displayed good selectivity in the enzymatic
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
kinase panel with almost negligible inhibition for all kinases except for focal adhesion kinase
(
FAK) at 100 nM. A follow-up enzymatic assay for FAK under 0.5 μM ATP concentration
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
determined the IC50 value to be 25 nM, indicating 10-fold ALK selectivity over FAK. The more
broad-spectrum kinase selectivity has also been examined by Kinome Scan (Supporting
Information, Table S2). It was revealed that compound 13 showed good selectivity over TrkB and
TrkC that play an important role in CNS. The CNS penetration of compound 13 was also
evaluated in mice. As Table 6 shows, after a single intraperitoneal administration at 100mg/kg
dose, brain and plasma samples were collected at 0.5 h, 1.0 h, and 2.0 h. The highest brain
concentration and plasma partition coefficient (Kp) values were observed at 1.0 h after the
administration. The Kp value properly reflected the MDR1 efflux ratio of compound 13. In
addition, the plasma protein binding and brain tissue binding of compound 13 depicted as an
unbound fraction (fu) was measured as 0.08 and 0.017, respectively.
Table 5. Enzymatic Kinase Selectivity Profile of Compound 13.
a
a
a
kinase
FAK
% inhibition
kinase
MAPKAPK2
IRK
% inhibition
kinase
PI3K
SRC
% inhibition
83
21
12
9
3
2
−2
−2
-4
Aurora B
PKA
PKC
2
p38
TrkA
VEGFR2
ASK1
2
cMET
ROCK1
−5
−5
CK1
6
−1
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7
8
9
CDK2
PLK1
5
5
3
3
EGFR
−1
−1
−2
−2
IRAK4
JAK1
−7
−8
MEK1
CDC7
EphA5
ERK1
GSK3
CHEK1
AKT1
−9
−11
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
% inhibition at 100 nM.
a
Table 6. Mouse Pharmacokinetic Properties of 13 in Plasma and Brain .
b
Concentration (μg/mL)
b,c
p
d
Time (h)
0.5
K
K
puu
Plasma
26.2
Brain
33.0
33.4
1.26
1.28
0.27
0
0
.27
.11
1
2
.0
.0
26.2
11.6
10.3
0.515
a
b
c
1
00 mg/kg intraperitoneal administration in mice. Average of three mice. Brain-to-plasma
d
ratio. calculated with fu in plasma and brain of 0.08 and 0.017, respectively.
Effect of Compound 13 on Phosphorylation Level of ALK in Mouse’s Brain. After determining
that compound 13 had acceptable kinase selectivity and brain penetration, it was then evaluated
for in vivo efficacy. The pharmacodynamic effect of compound 13 was investigated by measuring
the level of p-ALK in the mouse’s brain via western blot at 1 h after the intraperitoneal
administration. As Figure 5 shows, compound 13 decreased p-ALK levels in the hippocampus in
a dose-dependent manner with a statistical significance at 30 mg/kg and higher. In addition, this
compound significantly decreased the p-ALK level in the prefrontal cortex (PFC) at 100 mg/kg.
These results clearly demonstrated that compound 13 inhibited ALK autophosphorylation not
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
only in the in vitro cellular system but also in vivo in the mouse brain, suggesting that it could be
useful as a chemical probe to elucidate the brain functions mediated by ALK. The hippocampus
is a brain area that is responsible for various functions including spatial memory, contextual
recognition, and emotional control, while the prefrontal cortex is implicated in executive,
cognitive, emotional, and motivational functions. Interestingly, some of these functions were
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
-6
observed to be augmented in the ALK knock-out mice as reported. It is expected that
behavioral tests with compound 13 in various animal models would contribute to understanding
the role of ALK in the brain functions; hence, further investigation will be carried out.
Figure 5. Decrease of phosphorylated ALK levels in mouse’s (A) hippocampus and (B)
prefrontal cortex after 1 h of the intraperitoneal administration of compound 13 at 10, 30, and 100
mg/mL/kg to the ICR mice. Data are presented as the mean + SEM (n = 3); p-values < 0.025 vs.
vehicle by one-tailed Williams’ test.
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9
CHEMISTRY
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme
1
shows
the
synthesis
NIS
of
1.
N-Benzyl-3-(1H-pyrazol-5-yl)imidazo[1,2-b]pyridazin-6-amine
6-Chloroimidazo[1,2-b]pyridazine
17
was
iodized
by
to
afford
6
-chloro-3-iodoimidazo[1,2-b]pyridazine 18. The methylpyrazole moiety was introduced by
Suzuki-coupling reaction with the THP-protected pyrazole boronic ester 22 to give the
intermediate 19. chiral 1-(2,4-difluorophenyl)ethanamine was substituted by
A
Buchwald-Hartwig reaction followed by the deprotection of the THP group to give the target
compound 1. The pyrazole boronic ester 22 was prepared in a two-step reaction; THP protection
and regioselective borylation from the commercially available material 20.
a
Scheme 1. Synthesis of N-Benzyl-3-(1H-pyrazol-5-yl)imidazo[1,2-b]pyridazin-6-amine 1
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and conditions: (a) NIS, DMF, 90 °C; (b) 22, Pd(PPh
3
)
4
, Na
2
CO
3
, DME-H
2
O, reflux;
(c) Pd (dba) , (±)-BINAP, NaOt-Bu, 1-(2,4-difluorophenyl)ethanamine, toluene, reflux, then 4 N
2
3
HCl/EtOAc, EtOAc-MeOH, 0 °C; (d) dihydropyrane, TFA, toluene, reflux; (e) n-BuLi,
triisoprolyl borate, THF, -78 °C, then pinacol, acetic acid, rt.
Scheme 2 shows the syntheses of 1H-pyrrolo[2,3-b]pyridine derivatives 2 and 11.
1
H-pyrrolo[2,3-b]pyridine templates 23a–b were oxidized by m-CPBA to afford N-oxides 24a–b
and the subsequent substitution by each chiral benzylamine 28–29 gave the intermediates 25a–b.
The methylpyrazole moiety was introduced by Ullmann coupling reaction with the
THP-protected iodopyrazole 27 to give the intermediates 26a–26b. The deprotection of the THP
group under an acidic condition gave the target compound 2. Preparation of the 3-substituted
compound 11 was accomplished by Suzuki-reaction with boronic acid pinacol ester followed by
the deprotection of the THP group. The iodopyrazole 27 was prepared by regioselective
iodination from the THP protected pyrazole 21.
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a
Scheme 2. Synthesis of 1H-pyrrolo[2,3-b]pyridine Derivatives
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and conditions: (a) m-CPBA, Et
0 °C; (c) 27, CuI, (±)-trans-1,2-diaminocyclohexane, Cs
HCl/MeOH, MeOH, 0 °C; (e) boronic acid pinacol ester, PdCl
2
O, 0 °C; (b) m-CBA, Me
CO , dioxane, reflux; (d) 2 N
(AmPhos) , K CO , toluene-H O,
, THF, -78 °C to rt.
2
SO , DIPEA, 28–29, MeCN,
4
8
2
3
2
2
2
3
2
o
1
10 C, then 4 N HCl/EOAc, EtOAc; (f) n-BuLi, I
2
Scheme 3 shows the synthesis of 3-methylsulfonyl-6-amino-1H-pyrrolo[2,3-b]pyridine
derivative 3. The BOC group protected the indole NH group of a commercially available
3
-iodo-1H-pyrrolo[2,3-b]pyridine 30 to enhance the reactivity of the iodine group. The
2
-thiopropionate group was readily introduced at the 3-position of 1H-pyrrolo[2,3-b]pyridine by
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Buchwald-Hartwig coupling reaction. The thiol group with the propionate moiety was
deprotected under a basic condition and, subsequently, substituted by iodomethane. The chiral
benzylamine 28 and methylpyrazole group were introduced by the same method, as shown in
Scheme 2, and the following deprotection reaction of the THP group under an acid condition
afforded 3-sulfonyl derivative 3.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 3. Synthesis of 3-methylsulfonyl-6-amino-1H-pyrrolo[2,3-b]pyridine derivative 3
a
Reagents and conditions: (a) Boc
(dba) , Xantphos, DIPEA, toluene, 70 °C; (c) MeI, KOt-Bu, THF, 0 °C to rt, then 2 N
HCl/MeOH, MeOH, 80 C; (d) m-CPBA, Et
2
O, KOt-Bu, THF, rt; (b) 2-ethylhexyl 3-mercaptopropanoate,
Pd
2
3
o
2
O, rt; (e) m-CBA, Me
2
SO , 28, DIPEA, MeCN,
4
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0 °C; (f) 27, CuI, (±)-trans-1,2-diaminocyclohexane, Cs
2
CO , DME, 90 °C; (g) 4 N HCl/EtOAc,
3
EtOAc, 0 °C.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 4 shows the syntheses of 3-sulfonyl-6-amino-1H-pyrrolo[2,3-b]pyridine
derivatives 4–7. A commercially available 3-iodo-1H-pyrrolo[2,3-b]pyridine 30 was oxidized
into N-oxide 36 to enhance the reactivity of the iodine group. The 2-thiopropionate group was
readily introduced at the 3-position of 1H-pyrrolo[2,3-b]pyridine by Buchwald-Hartwig coupling
reaction. The chiral benzylamines 29 and methylpyrazole group were introduced by the same
method, as shown in Scheme 2. The 2-thiopropionate intermediate 39 was subjected to the
retro-Michael reaction by potassium tert-butoxide to furnish the free thiol group in situ which
was immediately substituted with a series of alkyl halides. The subsequent oxidation reaction into
a sulfonyl group by m-CPBA and the deprotection reaction of the THP group by acid afforded
3
-sulfonyl derivatives 4–7.
a
Scheme 4. Synthesis of 3-alkylsulfonyl-6-amino-1H-pyrrolo[2,3-b]pyridine Derivatives 4–7
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and conditions: (a) m-CPBA, Et
2
O, rt; (b) 2-ethylhexyl 3-mercaptopropanoate,
Pd
2
(dba)
3
, Xantphos, DIPEA, toluene, 80 °C; (c) m-CBA, Me
2
SO
4
, 29, DIPEA, MeCN, 80 °C;
1
(d) 27, CuI, (±)-trans-1,2-diaminocyclohexane, Cs
2
CO
3
, DME, 90 °C; (e) R I, KOt-Bu, THF,
0
°C to rt,; (f) m-CPBA, EtOAc, 0 °C; (g) 4 N HCl/EtOAc, EtOAc, 0 °C.
Scheme 5 shows the syntheses of 3-aminocarbonyl-6-amino-1H-pyrrolo[2,3-b]pyridine
derivatives 8–10. An aldehyde group was introduced at the 3-position of
H-pyrrolo[2,3-b]pyridine 23a utilizing hexamethylenetetramine as a carbonyl source. The
1
subsequent reduction to alcohol which was followed by protection with the TIPS group and
oxidation by m-CPBA provided the intermediate 41. As Scheme 2 shows, the introduction of
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(
R)-5-fluoro-2-pyridylethanamine moiety and 2-methylpyrazole group were conducted by the
same method. An aldehyde group at the 3-position was regenerated via the deprotection of the
TIPS group by TBAF and the oxidation of alcohol by manganese dioxide. The aldehyde 44 was
converted to the carboxylic acid by Pinnick oxidation. The subsequent amidation with the
corresponding amine reagents and the deprotection of the THP group gave the amide derivatives
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
–10.
Scheme 5. Synthesis of 3-Aminocarobonyl-6-amino-1H-pyrrolo[2,3-b]pyridine Derivatives 8–
a
1
0
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