Discovery, synthesis and characterization of a series of 7-aryl-imidazo[1,2-a]pyridine-3-ylquinolines as activin-like kinase (ALK) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.127418

The activin-like kinases are a family of kinases that play important roles in a variety of disease states. Of this class of kinases, ALK2, has been shown by a gain-of-function to be the primary driver of the childhood skeletal disease fibrodysplasia ossificans progressiva (FOP) and more recently the pediatric cancer diffuse intrinsic pontine glioma (DIPG). Herein, we report our efforts to identify a novel imidazo[1,2-a]pyridine scaffold as potent inhibitors of ALK2 with good in vivo pharmacokinetic properties suitable for future animal studies.

Full text of DOI:10.1016/j.bmcl.2020.127418

Journal Pre-proofs
Discovery, synthesis and characterization of a series of 7-aryl-imidazo[1,2-
a]pyridine-3-ylquinolines as activin-like kinase (ALK) inhibitors
Darren W. Engers, Sean R. Bollinger, Andrew S. Felts, Anish K. Vadukoot,
Charles Williams, Anna L. Blobaum, Craig W. Lindsley, Charles C. Hong,
Corey R. Hopkins
PII:
S0960-894X(20)30529-1
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127418
BMCL 127418
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
25 June 2020
13 July 2020
14 July 2020
Please cite this article as: Engers, D.W., Bollinger, S.R., Felts, A.S., Vadukoot, A.K., Williams, C., Blobaum,
A.L., Lindsley, C.W., Hong, C.C., Hopkins, C.R., Discovery, synthesis and characterization of a series of 7-aryl-
imidazo[1,2-a]pyridine-3-ylquinolines as activin-like kinase (ALK) inhibitors, Bioorganic & Medicinal
Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127418
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Design, synthesis and characterization of a
series of 7-aryl-imidazo[1,2-a]pyridin-3-
ylquinolines as activin-like kinase (ALK)
inhibitors
Leave this area blank for abstract info.
Darren W. Engers, Sean R. Bollinger, Andrew S. Felts, Anish K. Vadukoot, Charles Williams, Anna
L. Blobaum, Craig W. Lindsley, Charles C. Hong, and Corey R. Hopkins

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Discovery, synthesis and characterization of a series of 7-aryl-imidazo[1,2-
a]pyridine-3-ylquinolines as activin-like kinase (ALK) inhibitors
a
a
a
b
f
Darren W. Engers , Sean R. Bollinger , Andrew S. Felts , Anish K. Vadukoot , Charles Williams , Anna L.
a
a,c,d,e
f
b,*
Blobaum , Craig W. Lindsley
, Charles C. Hong , and Corey R. Hopkins
a
Warren Center for Neuroscience Drug Discovery, Vanderbilt University, Nashville, TN 37232, USA
Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198-6125, USA
b
c
Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA
d
Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
Department of Biochemistry, Vanderbilt University, Nashville, TN 37232, USA
e
f
Division of Cardiovascular Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The activin-like kinases are a family of kinases that play important roles in a variety of disease
states. Of this class of kinases, ALK2, has been shown by a gain-of-function to be the primary
driver of the childhood skeletal disease fibrodysplasia ossificans progressiva (FOP) and more
recently the pediatric cancer diffuse intrinsic pontine glioma (DIPG). Herein, we report our efforts
to identify a novel imidazo[1,2-a]pyridine scaffold as potent inhibitors of ALK2 with good in vivo
pharmacokinetic properties suitable for future animal studies.
Keywords:
2
009 Elsevier Ltd. All rights reserved.
BMP inhibitors
Activin-like kinase inhibitors
ALK2
ALK3
—
——
*
Corresponding author. Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE
6
8198, USA; Tel.: +1 402 559 9729; Fax: +1 402 559 5643. Email address: corey.hopkins@unmc.edu

The bone morphogenetic proteins (BMPs) play critical roles in
embryonic pattern formation.1-3 There have been more than a
dozen BMP proteins identified in mammals and these can be
further subclassified depending on their structures.3 The BMP-1
receptors activate intracellular proteins, such as Smad, and have
seven different types of receptors, termed activin-like kinase
1
(
ALK1-7). Of these, three receptors have been shown to bind
BMP ligands during mammalian skeletal development (ALK2,
4
ALK3 and ALK6) , in addition to a variety of other disease states
5, 6
such as iron-refractory iron-deficient anemia (IRIDA) , non-
7
alcoholic steatohepatitis (NASH) , Duchenne muscular dystrophy
8
, 9
10-12
13
(
DMD) , cancer
and liver fibrosis. More recent, genetic
studies have shown that two of these receptors play critical roles
in two devastating childhood diseases: fibrodysplasia ossificans
1
4, 15
progressiva (FOP)
and diffuse intrinsic pontine glioma
Genetic studies have found that FOP patients and
20% of DIPG patients experience a gain-of-function mutation in
1
6, 17
(DIPG).
~
1
8, 19
the ACVR1 gene which encodes ALK2.
FOP is an autosomal
dominant disorder characterized by heterotopic ossification and is
caused by the ACVR1 mutation; however, the role in DIPG is
under study. There are no effective treatments for FOP, although
a clinical trial with a monoclonal antibody that blocks Activin A
2
0
is underway , and a trial for an RAR- agonist (palovarotene) was
recently halted due to safety concerns.21 Thus, identification of
new, small molecules is still a major unmet medical need for this
devastating disease.
Our laboratory has been interested in developing a selective
ALK2 inhibitor for some time and we have recently published our
efforts in the area. In addition, other laboratories have published
work on the same pyrazolo[1,5-a]pyrimidine scaffold, with the
most recent publication possessing the sulfonamide moiety.22 The
2
3
first compounds discovered in this area were dorsomorphin (1)
2
4
25
followed by LDN-193189 (2) , followed by DMH1 (3) and
these compounds were shown to be active in in vivo models of
FOP. These compounds were followed by ML347 (4) and LDN-
2
6
Figure 1. Previously reported pyrazolo[1,5-a]pyrimidine scaffold as ALK
2
7
2
12854 (5) , two compounds that were ALK2 preferring
inhibitors.
inhibitors (compared to ALK3, 337-fold and 66-fold selectivity,
respectively). More recently, compound 6 was reported as a
Our efforts were concentrated on the development of a new,
core scaffold for this series of compounds. After several attempts,
we identified the imidazo[1,2-a]pyridine core as a suitable
replacement system as potent ALK inhibitors. The synthesis of
the imidazo[1,2-a]pyridine compounds is shown in Scheme 1. 4-
bromo-2-aminopyridine, 7, was reacted with the boronic ester, 8,
2
2
selective ALK2 inhibitor (715-fold selectivity). In addition to
the pyrazolo[1,5-a]pyrimidine scaffold, compounds based off the
K02288, 7, trimethoxyaryl moiety, leading to 8, have been recently
2
8, 29
reported.
Although these compounds represent breakthroughs
in the area, they still suffer from some pharmacokinetic (PK)
liabilities and thus, new compounds are needed in order to further
investigate therapies for these diseases. Herein, we report our
efforts toward an imidazo[1,2-a]pyridine scaffold as potent ALK
inhibitors.
30
under palladium cross-coupling conditions to yield 9. Next, the
2
-aminopyidine was cyclized to the imidazo[1,2-a]pyridine, 10,
using chloroacetaldehyde under basic conditions. The final targets
were realized by reacting the appropriate aryl chloride or aryl
bromide under palladium catalysis to directly access the coupled
products, 11a-k.

1
1b
55.8
1
1
1c
1
1d
10.4
1
1e
148
14
1
1f
1
1
1g
1h
15
135
Scheme 1. Synthesis of the imidazo[1,2-a]pyridine scaffold.
Reagents and conditions: (a) Pd(dppf)Cl
2
, 1,4-dioxane, K
3
PO
4
,
,
1
1
1i
1j
38.1
o
H O, W, 120 C, 30 min; (b) chloroacetaldehyde, NaHCO
2
3
EtOH, reflux; (c) R-Cl or R-Br, KOAc, Pd(OAc)
2
, DMA, W, 200
o
C, 30 min.
17.3
Our initial efforts centered around the southern portion of the
molecule as represented in Table 1. Starting with the 4-quinoline,
1
1a, which has been used in the previous scaffold, was shown to
1
1k
17.8
be active against ALK2 (IC50 = 24 nM). As this particular moiety
has been shown to have significant metabolic liabilities (oxidation
of the 2-position), we wanted to move away from the unsubstituted
aALK2 data were obtained at Reaction Biology
Malvern, PA)
(
4
-quinoline and thus investigated alternative heterocyclic
replacements. The 3-thieno[3,2-d]pyrimidine, 11b, was potent
against ALK2 (IC50 = 56 nM) and moving the nitrogen one spot to
the 3-thieno[2,3-b]pyrazine, 11c, led to a significant improvement
in ALK2 potency (IC50 = 1 nM). Rearranging the attachment from
the 3-position of the 6,5-systems to the 7-position led to
compounds 11d-f. The 7-benzo[d]thiazole, 11d, and 7-thieno[3,2-
b]pyridine, 11f, were active against ALK2 (IC50 = 10 and 14 nM,
respectively), and, interestingly, the 6-methyl-[1,2,4]triazolo[1,5-
a]pyridine, 11e, was much less active against ALK2 (IC50 = 148
nM). Reinvestigation of the 4-quinoline moiety led to the
identification of potent compounds, starting with the 6-fluoro-4-
quinoline, 11g (IC50 = 15 nM). Incorporation of a substituent in
the 2-position in order to alleviate the metabolic liability (2-
methyl), led to compounds that all retained activity (11i-k), with
the exception of the 2-methyl-6-trifluoromethyl (11h), which was
much less potent.
Having identified ALK2 potent compounds, we next evaluated
these compounds for hepatic microsomal stability in an established
intrinsic clearance assay (Table 2). All of the compounds tested
showed high instability in both human and mouse liver
microsomes. In order to better understand the reason for the
instability, we performed a soft-spot analysis and it was revealed
that N-demethylation of the piperazine ring was the primary source
of instability.
Table 2.
Hepatic microsomal stability of selected compounds.
Human liver
microsomes
(mL/min/kg)
CLINT
100
192
47.7
Mouse liver microsomes
(mL/min/kg)
a
a
Cmpd
CLHEP
17.4
18.9
14.6
17.8
17.9
CLINT
2481
1263
201
1492
1078
CLHEP
86.8
84.0
62.1
84.9
83.1
1
1
1
1
1b
1d
1e
1j
Table 1.
SAR around the southern portion of the imidazo[1,2-
a]scaffold.
118
120
1
a
1k
Data obtained at Warren Center for Neuroscience Drug
Discovery, Vanderbilt University, Franklin, TN
In order to identify compounds with better microsomal
stability, we investigated alternative substituents on the piperazine
system including removal of the N-substituent as well as adding
steric bulk in order to minimize the demethylation. In addition, we
introduced carbon-linked analogs as the N-linked analogs could,
potentially, release an aniline upon metabolism. These analogs
were synthesized as described in Scheme 2. Starting material 12,
was reacted with the boronic acid, 13, under normal coupling
a
ALK2 (IC50
nM)
,
Cmpd
1a
Structure
1
24

conditions (Cs
2
CO
3
, Pd(dppf)Cl
2
) to yield 14.30 Next, the
piperazine and 2-morpholine were also productive changes
bromoquinoline, 15, was coupled directly with the imidazo[1,2-
leading to the promising analogs (19n-s, IC50 = < 5 nM).
o
a]pyridine, 14 (Pd(OAc) , KOAc, W, 130 C), to yield the
2
Table 3.
precursor material to the final cyclization. The aldehyde, 16, was
subjected to the initiation of the SnAP chemistry (tin(Sn) Amine
Protocol) pioneered by Bode et al. by condensation with the
respective amino tin reagents, 17, under anhydrous conditions (4
SAR around the northern portion of the molecule.
3
1-33
Å MS, CH
intermediate was subjected to the copper-mediated cyclization
Cu(OTf) , HFIP, 2,6-lutidine, CH Cl , 12 h, rt) to yield the
2
Cl
2
, rt) to yield the imine intermediate, 18.
This
(
2
2
2
cyclized material, which was then subjected to acid mediated Boc
removal to obtain the desired compounds, 19.
ALK2a
R
Cmpd
9a
R1 R2 (IC , nM)
50
1
H
F
F
H
H
F
8.0
1
9b
9c
10.9
32.2
1
1
9d
F
F
<5
1
9e
F
F
H
F
216
1
9f
31.7
1
1
9g
9h
H
F
H
H
H
H
H
46.3
14.7
6.3
1
1
9i
9j
H
H
H
15.6
148
Scheme 2. Synthesis of carbon-linked analogs. Reagents and
conditions: (a) Cs
2
CO
C, 15 min., 67%; (b) Pd(OAc)
min., 68%; (c) 4 Å MS, CH Cl , 12 h, rt; (d) Cu(OTf)
lutidine, CH Cl , 12 h, rt, 30-50%; (e) when X = NHBoc: TFA,
CH Cl , rt, 12 h, 45-65%.
3
, Pd(dppf)Cl
, KOAc, DMF, W, 130 C, 10
, HFIP, 2,6-
2 2
, 1,4-dioxane/H O, W, 120
o
o
2
2
2
2
1
9k
2
2
2
2
1
9l
H
F
H
H
37
Having identified three southern quinoline moieties that had
ALK2 potency, we utilized these in our next round of SAR as they
have also been shown to impart stability to the quinoline ring
system (Table 3). We utilized the 2-methyl fluorinated quinolines
as the southern moiety for this SAR exploration as these were all
shown to be potent. Thus, incorporation of the NH-piperazine
19m
9n
>10,000
1
H
F
H
H
F
<5
<5
<5
<5
<5
(
19a-c) was well tolerated (IC50 = 8 – 32 nM). Addition of the
larger isopropyl group produced an active compound (19d, IC50
5 nM), and introduction of methyl groups flanking the N-Me
=
19o
19p
<
piperazine were moderately active (19e) to active (19f) (IC50 = 216
and 32 nM, respectively) as was the bulky morpholine, 19g (IC50
F
1
9q
9r
H
F
H
H
=
46 nM). In addition to the phenyl spacer, we examined the
pyridine replacement with good results (19g-19l) (IC50 = 6.3 – 148
nM). The ether linked compound, however, was inactive (19m,
IC50 = 10,000 nM). Lastly, introduction of the carbon-linked 2-
1

ALK2
R206H)
ALK3/
3
.8
5.6
<5
8.8
<5
<5
<5
<5
<5
(
1
9s
F
F
5.6
<
5
BMPR1A
aALK2 data were obtained at Reaction Biology
ALK4
ALK5
ALK6
464
1,260
18.4
568.7
1,267
29.4
596.1
1,285
40.9
192
310
54.5
390
717
188
(Malvern, PA)
BMPR2
TGFR2
AMPK
KDR
>10,000
1,250
>10,000
5,930
1,250
>50,000
587.2
13,590
13,410
7,450
>50,000
170.7
18,600
20,560
>10,000
>50,000
4,590
20,000
9,350
2,820
>50,000
498
11,500
36,900
6,903
Having identified the next generation of compounds, we tested
these compounds for hepatic microsomal stability (Table 4).
Removing the N-alkyl group improved the stability (19a-c) in
human and mouse liver microsomes, with 19b,c being stable in
human. Introducing steric bulk to the piperidine ring system
provided some improvement (19d-f); however, the compounds
were not stable enough to progress. Introducing the pyridine ring
system as a replacement for the central phenyl ring led to
significant instability (19g,k). Moving to the carbon-linked series,
the piperazine containing compounds showed improved stability
over the morpholine based compounds (19n-o vs. 19q-s) in both
human and mouse liver microsomes.
PDGFR
a
Selectivity data were obtained at Reaction Biology (Malvern, PA)
Next, we further evaluated the three top compounds based on
stability and selectivity in additional assays including plasma
protein binding, CYP inhibition and in vivo pharmacokinetic (PK)
studies in animals to assess their potential as drug candidates
Table 6). The compounds had modest free fraction in both human
and mouse plasma (%f = 1.0 – 2.2), with 19c having the best
(
u
overall binding profile. In addition, the compounds did not have
any significant CYP inhibition in the five CYP enzymes tested
(1A2, 3A4, 2D6, 2C9, 2C19). Next, we performed an IV/PO
pharmacokinetic cross-over study in rats. All three compounds
showed modest clearance in rats (CLp = 13 – 17 mL/min/kg) and
excellent half-life (t1/2 = 5 hr) and showed good oral bioavailability
(F = 30 – 45%). Lastly, the compounds were evaluated in a
plasma:brain tissue distribution study in order to evaluate their
ability to cross the blood-brain barrier (BBB). The compounds
were dosed (10 mg/kg) and then measured for brain penetration at
1 h. 19b showed excellent brain penetration (Brain:Plasma (B:P)
1.6) while 19a and 19c displayed modest brain penetration.
Overall, these compounds display in vivo PK attributes that would
be amenable to a once-a-day dosing regimen. Recently, 19b, was
shown to be active in an animal model of iron-refractory iron-
deficient anemia (IRIDA), further bolstering this scaffold as a
potential translatable scaffold.
Table 4.
Hepatic microsomal stability of selected compounds
Human liver
microsomes
mL/min/kg)
Mouse liver microsomes
(mL/min/kg)
(
Cmpd
CLINT
28.9
12.3
0.82
33.3
92.4
113
CLHEP
12.2
7.7
CLINT
87.7
207
131
27.3
130
186
265
352
<49.5
<49.5
<49.5
198
100
97.1
CLHEP
44.4
62.7
53.4
21.0
53.2
60.6
67.2
71.7
<32.9
<32.9
<32.9
97.8
47.4
47.7
a
1
1
1
1
1
1
1
1
1
1
1
1
1
1
9a
9b
9c
a
a
0.79
12.9
17.1
17.7
17.5
17.3
<10.7
12.4
14.0
17.5
17.4
17.5
a
9d
a
9e
9f
a
a
a
b
b
b
b
9g
9k
9n
9o
9p
9q
106
98.1
<23.3
32.8
46.7
139
Table 6.
a
b
Further profiling of the lead compounds .
9r
9s
134
139
b
19a
19b
19c
a
Plasma Protein Binding (%Fu)
Data obtained at Warren Center for Neuroscience Drug
Discovery, Vanderbilt University, Franklin, TN. Data obtained at
Q2 Solutions, Indianapolis, IN.
b
hPPB (%f
mPPB (%f )
u
u
)
1.4
1.8
1.0
1.0
1.4
2.2
Rat in vivo PK
IV dosing (0.5 mg/kg, 5% DMSO/5% solutol:EtOH (1:1,
v:v)/90% saline)
Having identified more stable compounds, we next evaluated
the compounds in a selectivity panel against the other ALK
receptors as well as the important ALK2 mutation (R206H) (Table
CLp (mL/min/kg)
17.1
5.9
133.2
7.3
12.9
5.9
144
5.5
16.7
5.4
83.6
6.8
t
1/2 (h)
max (ng/mL)
dss (L/kg)
AUC0-∞ (hr*ng/mL)
5
). The lead compounds were all selective against ALK4, ALK5,
C
BMPR2, TGFR2, KDR and PDGFR. Each of the compounds,
however, did retain activity against ALK1, ALK3 and ALK6.
Importantly, the compounds were potent inhibitors of the R206H
mutation in ALK2, an important mutation found in fibrodysplasia
ossificans progressiva (FOP), a rare inherited connective tissue
disorder. Compounds 19a-c were also profiled against a 369
kinase panel (Reaction Biology, Malvern, PA) in order to assess
their selectivity against a wider range of receptors. The compounds
were inactive against the panel with the exceptions of ABL2,
TNIK, DDR1, SIK1, SIK2 and LIMK1.
V
489.6
701
503.9
PO dosing (3 mg/kg, suspension, 0.1% Tween80 and 0.5%
methyl cellulose)
t
1/2 (h)
4.7
127.7
4
3.9
163
4
3.8
95.7
4
C
max (ng/mL)
tmax (hr)
AUC0-∞ (hr*ng/mL)
%F
1289
45.3
1683
40.0
920.5
29.8
Plasma:Brain Study (10 mg/kg, PO, 1 h)
Plasma (ng/mL)
Brain (ng/g)
B:P
240
84
0.35
101
163
1.61
145
41
0.3
Table 5.
Off-target selectivity of lead compounds 19a-c,n,p
Receptor
ALK1/
ACVRL1
ALK2/
19a
19b
19c
19n
19p
a
In vivo PK studies performed at Jubilent Biosys.
3
9.1
166.3
308.8
<5
89.2
8
.0
10.9
32.2
<5
<5
In summary, we have identified a novel imidazo[1,2-a]pyridine
scaffold as a new entry into ALK inhibitors. The lead optimization
ACVR1

campaign identified three lead compounds (19a-c) that contain a
northern piperazine moiety with 2-methyl-fluorinated quinoline as
the southern portion. These compounds are potent ALK2
inhibitors, including the important R206H mutation, and display
selectivity against other related receptors. Further profiling
showed that these compounds have balanced in vivo PK properties
Acknowledgments
The authors would like to thank La Jolla Pharmaceuticals for
their support of the program to develop ALK inhibitors. The
authors: D.W.E., C.W.L., C.C.H., and C.R.H. have received
funding and royalties from La Jolla Pharmaceuticals.
(low clearance, oral bioavailability, t1/2) amenable to further
advancement into IND-enabling studies. Further progression of
these lead molecules will be reported in due course.
1
9. Pacifici, M., Shore, E. M. Cytokine Growth Factor Rev.
016;27:93.
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Declaration of interests
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1
☐ The authors declare that they have no known
competing financial interests or personal
relationships that could have appeared to influence
the work reported in this paper.
☒
The authors declare the following financial
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interests/personal relationships which may be
considered as potential competing interests:
1
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The authors: D.W.E., C.W.L., C.C.H., and C.R.H. have received funding and royalties from La Jolla Pharmaceuticals.
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