Novel bicyclic pyrazoles as potent ALK2 (R206H) inhibitors for the treatment of fibrodysplasia ossificans progressiva

Article abstract of DOI:10.1016/j.bmcl.2021.127858

Mutant activin receptor-like kinase-2 (ALK2) is associated with the pathogenesis of fibrodysplasia ossificans progressiva, making it an attractive target for therapeutic intervention. We synthesized a new series of bicyclic pyrazoles and evaluated their mutant ALK2 enzyme inhibitory activities, leading to the identification of 8 as the most potent inhibitor. This compound showed moderate microsomal metabolic stability and human ether-a-go-go related gene (hERG) safety. In C2C12 cells carrying mutant ALK2 (R206H), 8 efficiently inhibited the bone morphogenetic protein (BMP)-induced alkaline phosphatase activity.

Full text of DOI:10.1016/j.bmcl.2021.127858

Bioorg. Med. Chem. Lett. 38 (2021) 127858
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel bicyclic pyrazoles as potent ALK2 (R206H) inhibitors for the
treatment of fibrodysplasia ossificans progressiva
a
b
c
d
a
,*
Hirofumi Yamamoto , Naoki Sakai , Satoshi Ohte , Tomohiro Sato , Katsuhiko Sekimata
,
b
b
b
b
Takehisa Matsumoto , Kana Nakamura , Hisami Watanabe , Chiemi Mishima-Tsumagari ,
b
e
d
f
Akiko Tanaka , Yoshinobu Hashizume , Teruki Honma , Takenobu Katagiri ,
Kohei Miyazono , Hiroshi Tomoda , Mikako Shirouzu , Hiroo Koyama
g
c
b
a
a
Drug Discovery Chemistry Platform Unit, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
b
c
Drug Discovery Structural Biology Platform Unit, RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan
Department of Microbial Chemistry, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
Drug Discovery Computational Chemistry Platform Unit, RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045,
d
Japan
e
RIKEN Program for Drug Discovery and Medical Technology Platforms, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
f
Division of Biomedical Sciences, Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka-shi, Saitama 350-1241, Japan
Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
g
A R T I C L E I N F O
A B S T R A C T
Keywords:
Mutant activin receptor-like kinase-2 (ALK2) is associated with the pathogenesis of fibrodysplasia ossificans
progressiva, making it an attractive target for therapeutic intervention. We synthesized a new series of bicyclic
pyrazoles and evaluated their mutant ALK2 enzyme inhibitory activities, leading to the identification of 8 as the
most potent inhibitor. This compound showed moderate microsomal metabolic stability and human ether-a-go-
go related gene (hERG) safety. In C2C12 cells carrying mutant ALK2 (R206H), 8 efficiently inhibited the bone
morphogenetic protein (BMP)-induced alkaline phosphatase activity.
Fibrodysplasia ossificans progressiva (FOP)
Activin receptor-like kinase-2 (ALK2)
Bicyclic pyrazole derivatives
Fibrodysplasia ossificans progressiva (FOP) is a rare disorder that
manifests as progressive heterotopic ossification in the muscles, ten-
dons, or ligaments.1 FOP is caused by abnormal activation of bone
morphogenetic protein (BMP) signaling due to highly recurrent muta-
tions, including R206H in ALK2 (encoded by ACVR1), one of the type-I
receptors for BMPs, resulting in an alteration in the intracellular glycine-
serine-rich domain of the enzyme.1,2 Another disease caused by ALK2
mutations, including ALK2 (R206H), is diffuse intrinsic pontine glioma
inhibitors may compensate for the lack of drug candidates and provide a
further proof-of-concept of ALK2 inhibition for these diseases. Previ-
ously, we reported a series of bis-heteroaryl pyrazoles as hit ALK2 in-
hibitors exemplified by RK-59638 (Fig. 1).1
,2
1,12
X-ray crystal structure analysis showed that RK-59638 binds to the
1
1,12
ALK2 (R206H) ATP binding pocket (PDB code 6ACR) (Fig. 1).
The
pharmacophore of RK-59638 was found to be characterized by two
chemical features: an aminopyrimidine hinge binder and 3-pyridyl back
pocket binder. Additionally, the methoxy group of the anisidine moiety
is exposed to the solvent region. The pyrazole moiety is located in the
sugar pocket of the enzyme. In contrast, fused morpholinopyrazole
motifs are expected to affect the activities of these compounds by the
(
DIPG), a fatal cancer that forms in the brainstem of children.2,3 Thus,
the inhibition of the BMP signaling pathway, which acts on ALK2
2
(
R206H), may be an effective treatment for FOP and/or DIPG.
While some ALK2 inhibitors have been shown to be efficacious in
disease animal models of FOP and DIPG, only BLU-782, saracatinib, and
CH- interaction with Tyr219 on the phosphate-binding loop (P loop)
π
KER-047 are currently under clinical trials for treating FOP.2
,4–10
and by a water-mediated hydrogen bond network with Ser290 and
Asp293 (Fig. 1).
Therefore, the development of novel and specific ALK2 mutant
Abbreviations: ALK2, activin receptor-like kinase-2; ALP, alkaline phosphatase; BMP, bone morphogenetic protein; DIPG, diffuse intrinsic pontine glioma; DMF, N,
N-dimethylformamide; FOP, fibrodysplasia ossificans progressiva; Herg, human ether-a-go-go related gene; MDCK, Madin-Darby canine kidney cells; SAR, structure-
activity relationship.
*
Corresponding author.
E-mail address: katsuhiko.sekimata@riken.jp (K. Sekimata).
https://doi.org/10.1016/j.bmcl.2021.127858
Received 27 November 2020; Received in revised form 1 February 2021; Accepted 5 February 2021
Available online 18 February 2021
0
960-894X/© 2021 Elsevier Ltd. All rights reserved.

H. Yamamoto et al.
Bioorganic & Medicinal Chemistry Letters 38 (2021) 127858
Fig. 1. Schematic 2D representation of the binding mode of RK-59638 in ALK2 (R206H) and our predicted binding mode of fused morpholinopyrazole 1 in
ALK2 (R206H).
In this study, we identified a series of novel bicyclic pyrazole ALK2
R206H) inhibitors by chemical modification of hit RK-59638. We also
of mouse myoblastic C2C12 cells into osteoblasts by measuring the in-
hibition of BMP-induced alkaline phosphatase (ALP) activity, which is a
marker of osteoblast differentiation.13
(
evaluated the effects of representative compounds on the differentiation
Table 1
Inhibition of ALK2 (R206H) enzyme activity, permeability, and efflux profiles.
ꢀ
6
Cpd No.
RK-
R
% inhibition at 0.3
a,b,c
IC50
Permeability Papp A to B (10 cm/
c,e
Efflux ratio B to A/A to B MDR (B to A/A
c,e
a,c,d
μ
M
(μM)
s)
to B)
–
38.8
0.684
0.167
NT
NT
NT
NT
5
9638
1
2
NT
58.0
NT
NT
NT
NT
NT
NT
3
ꢀ 3.9
4
5
73.0
NT
NT
NT
NT
NT
NT
0.0658
6
7
NT
NT
0.0217
0.0247
0.906
4.366
10.2 (53.9)
1.4 (6.1)
8
NT
0.0182
8.686
1.6 (5.8)
a
ALK2(R206H) enzyme assays were conducted by Reaction Biology Corporation using the ‘HotSpot’ assay platform and kinase Assay Protocol. Measured at ATP
1
1
concentration (10 µM); for experimental details, see Ref.
.
b
The percent inhibition values are the averages of two or more tests.
NT, not tested.
c
d
e
IC50 values are the averages of two or more tests.
11
These studies were conducted by Sumika Chemical Analysis Service, Ltd. (SCAS) using in vitro/vivo pharmacokinetic screening.
2

H. Yamamoto et al.
Bioorganic & Medicinal Chemistry Letters 38 (2021) 127858
Table 2
1
1
Inhibition of ALK2 (R206H) enzyme activity.
a
Cpd No.
R
IC50 (nM)
8
18.2
9
276
665
1
1
a
0
1
2890
IC50 values are the averages of two or more tests.
Table 3
1
1
Inhibition of ALK2 (R206H) enzyme activity.
a
Cpd No.
Ra-Rb
IC50 (nM)
8
CH
CH
CH
2
2
2
OCH
CH CH
(CH CH
2
2
CH
2
18.2
28.9
24.9
Fig. 2. (A) Binding mode of 7 in the ATP sites of ALK2 (R206H). Secondary
structure elements of ALK2 (R206H) are represented by ribbon model. Residues
forming ATP-binding pocket are shown as white stick model. Compound 7 is
represented by yellow stick model. The hydrogen bonds involved in the inter-
action of ALK2 (R206H) and 7 are shown in green dashed line. Water molecules
are shown as purple sphere. (B) Superposition of the ALK2 (R206H)—RK-59638
complex (green) and ALK2 (R206H)—7 complex (purple).
1
1
2
3
2
2
2
)
2
a
IC50 values are the averages of two or more tests.
To study the activity of fused morpholinopyrazole compound, we
performed molecular docking simulations using the ALK2 (R206H) and
RK-59638 (PDB code 6ACR) based on the GLIDE SP mode.14 The docked
model for RK-59638 agreed well with the reported crystal structure
coordinates. The binding mode and score of fused morpholinopyrazole 1
were analyzed in comparison to RK-59638. Compound 1 adopted the
same binding mode as RK-59638. The docking scores of RK-59638 and 1
were ꢀ 8.5123 and ꢀ 8.9590, respectively. As shown by docking analysis,
Table 4
Chemical profiles of RK-59638 and 8.
RK-
8
5
9638
Biochemical
IC50 ALK2 (R206H) (nM)IC50 ALK2
R206H) (nM)
683.7
18.2
1
exhibited a four-fold increase in enzyme inhibitory activity compared
(
to RK-59638, suggesting that the fused ring is important for the inter-
action with ALK2 (R206H) (Table 1). Compound 1 was then further
optimized. In the structure-activity relationship (SAR) of solvent-
exposed anisidine moiety, para-substituted 1 and 4 exhibited substan-
tial levels of activity, whereas meta-substituted 2 and ortho-substituted 3
showed decreased activity (Table 1). Saturated heterocyclic 5 and 6,
containing oxygen or nitrogen at the end of the structure, were found to
be mildly and highly potent, respectively. Piperazine 6 showed low
a
Cellular
Metabolic
stability
hERG
IC50 ALP (nM)
>100
37.3
56.0/
40.5
3.6
HLM/RLM (% remaining after 60
14.8/2.1
b
min)
Automated patch-clamp (% inhibition
25.4
c
at 10 M)
μ
a
Details of ALP assays using C2C12 cells carrying mutant ALK2 (R206H) were
1
5,16
described previously
b
. IC50 values are the averages of two or more tests.
These studies were conducted by Sumika Chemical Analysis Service, Ltd.
ꢀ
6
(SCAS) using in vitro/vivo pharmacokinetic screening.
permeability (A to B Papp = 0.906 × 10 cm/s) and a high efflux ratio
53.9), which was greater than the efflux ratio (46.0) of digoxin, a
c
These studies were conducted by Cerep, Ltd. using in vitro ADME.
(
known substrate of MDR1, in MDR1-MDCKII cell (Table 1). Low efflux
has been shown to be important for achieving sufficient exposure in
inhibitory activity, considerable permeability, and acceptable P-gp-
mediated efflux. To explore the SAR of the 3-pyridyl moiety of 8 sur-
rounded by the hydrophobic back pocket of the enzyme, the pyridine
moiety was replaced with a phenyl or heteroaryl (Table 2). Compounds
1
1
rat. The high efflux ratio of 6 was improved by capping the NH group
of the piperazine moiety of 6 as an N-methyl group (7) and N-methox-
yethyl group (8) (Table 1). Compound 8 showed the strongest enzyme
3

H. Yamamoto et al.
Bioorganic & Medicinal Chemistry Letters 38 (2021) 127858
Scheme 1. Reagents and conditions: (a) SOCl
2
, ethanol, quant; (b) NaOH, tetrahydrofuran, 90%; (c) SOCl2, DMF, 88%; (d) (Diphenylmethylene)hydrazine, pyridine,
CH
DMF, 77%; (j) 2-Chloro-4-(tributylstannyl)pyrimidine, Pd(PPh
-methylpyrimidine, lithium hexamethyldisilazide, 73%; (m) 1-Boc-4-(4-aminophenyl)piperazine, 43%; (n) 20, pyridine, ethanol, 74%; (o) NaH, DMF, 43%; (p)
Trifluoroacetic acid, CH Cl , 80%; (q) 1-bromo-2-methoxyethane, N,N-diisopropylethylamine, acetonitrile, 72%.
2
Cl
2
, 99%; (e) NaH, tetrahydrofuran, 83%; (f) HClaq, 88%; (g) 20, pyidine, ethanol, 59%; (h) Sodium ethoxide, toluene, 74%; (i) N-iodosuccinimide, NaHCO
3
,
3 4
)
, CuI, DMF, 59%; (k) 4-(4-Boc-piperazin-1-yl)aniline, Methanol, tetrahydrofuran, 56%; (l) 2-chloro-
4
2
2
9
–11 showed significant decreases in enzyme inhibitory activity, sug-
were evaluated in ALK2 (R206H) – expressing C2C12 cells to determine
5,16
gesting that the 3-pyridyl group is essential for interacting with the
enzyme. Finally, the fused bicyclic pyrazole moiety of 8 was optimized.
The results showed that the IC50 value of alicyclic type 12 and 13 were
lower than that of 8 (Table 3). X-ray crystallographic analyses were
conducted to examine the SAR in a structural context. It was expected
that soaking 8 would be difficult because the N-methoxyethyl group of 8
caused a steric clash with a neighboring protomer in the crystal. Thus, 7
possessing the N-methyl group was selected for crystal structure anal-
ysis. A previous study revealed that 7 directly binds to the ALK2
their ability to inhibit BMP4-induced ALP activity.1
biochemical marker of osteoblastic differentiation of C2C12 cells.
ALP is a useful
1
3
Compound 8 showed higher potency than RK59638, which was
consistent with the biochemical ALK2 (R206H) assay (Table 4). More-
over, 8 showed improvements in human and rat microsomal stability
and reduced the human ether-a-go-go related gene (hERG) inhibition
compared with RK-59638 (Table 4).
The synthesis of 8 was achieved via two divergent routes: route A
included the Stille cross-coupling reaction and route B included a
nucleophilic substitution reaction using lithium hexamethyldisilazide
(Scheme 1).
(
R206H) ATP-binding pocket in the same manner as RK-59638 (PDB
code 6ACR) (Fig. 2A).
Compound 7 formed two hydrogen bonds between the amino-
pyrimidine moiety and the main chain amine and carbonyl of the His286
in the hinge region of the enzyme. Three hydrogen bonds were formed
between the nitrogen of the 3-pyridyl group of 7, the carboxyl group of
First, key intermediate 20 was synthesized. Commercially obtained
p-dioxanone 14 was treated with thionyl chloride in ethanol to give the
corresponding ethyl ester 15.17 Carboxylic acid 16 was formed by base
hydrolysis of 15 and then treated with thionyl chloride in N,N-dime-
1
7
Glu248 from the
αC helix, and the amino group of Lys235 from the β3
thylformamide (DMF) to give the corresponding acid chloride 17.
strand via conserved water molecules. A hydrogen bond was formed
between the nitrogen of the pyrazole ring of 7 and the amino group of
Lys235 via a conserved water molecule. Another weak hydrogen bond
might have formed between the oxygen atom of the morpholinopyrazole
moiety of 7 and Asp293 and Ser290 via a conserved water molecule. The
oxygen atom and the water molecule were 3.4 Å apart. Moreover, the
morpholine moiety was located near Tyr219. The methylene carbon
atoms at the morpholine moiety and the aromatic ring of Tyr219 were
Compound 17 was coupled using benzophenone hydrazine with pyri-
dine to give the amide product 18.18 Compound 19 was obtained by
intramolecular dehydrohalogenation of 18 with sodium hydride in
tetrahydrofuran, followed by deprotection using aqueous hydrochloric
acid to give the intermediate 20.18 Next, as shown in Scheme 1, for route
A, commercially obtained 3-oxo-3-(pyridin-3-yl)propionic acid ethyl
ester 21 was condensed with the hydrazide 20 in pyridine to give the
corresponding acyl hydrazone 22.19 Compound 22 was cyclized and
hydrolyzed using sodium ethoxide to give carboxylic acid 23. Acid 23
4
.0 Å apart, and they formed a CH-
π
interaction. Structural super-
◦
position of RK-59638 and 7 showed that the fused morpholinopyrazole
motif of 7 induced a significant positional change in Tyr219 on the P
was treated with N-iodosuccinimide in DMF at 25 C to give the corre-
sponding iodide 24.18 The Stille reaction of iodide 24 with 2-Chloro-4-
loop (Fig. 2B), suggesting that the motif is important for CH-
π
interac-
(tributylstannyl)pyrimidine produced chloride 25. Displacement of the
1
1
tion with Tyr219 in ALK2 (R206H).
chloride 25 with 1-Boc-4-(4-aminophenyl)piperazine gave 26. As
In addition to the biochemical ALK2 (R206H) assay, RK-59638 and 8
shown in Scheme 1, for route B, commercially available methyl
4

H. Yamamoto et al.
Bioorganic & Medicinal Chemistry Letters 38 (2021) 127858
nicotinate 27 was reacted with the lithium enolate of 2-chloro-4-meth-
ylpyrimidine to give ketone 28.11 Displacement of the chloride 28
with 1-Boc-4-(4-aminophenyl)piperazine and subsequent condensation
with hydrazide 20 in pyridine gave the corresponding acyl hydrazone
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.127858.
1
9
3
0. Compound 30 was cyclized using sodium hydride to give 26.
Finally, Boc-deprotection of 26 using trifluoroacetic acid gave 6 and
subsequent reaction of 6 with 1-bromo-2-methoxyethane and N,N-dii-
sopropylethylamine in DMF gave 8.
References
1
2
3
4
5
6
7
8
Pignolo RJ, Shore EM, Kaplan FS. Pediatr Endocrinol Rev. 2013;10:437–448.
Sekimata K, Sato T, Sakai N. Chem Pharm Bull. 2020;68:194–200.
In summary, we have identified a highly potent ALK2 (R206H) in-
hibitor 8 showing good membrane permeability, strong ALP inhibitory
activity in C2C12 (R206H) cells, moderate microsomal metabolic sta-
bility, and moderate hERG safety. In our future studies, the compound 8
and its derivatives will be evaluated for their pharmacokinetics profile
using in vitro ADME/Tox assays, and their in vivo efficacies in animal
models of FOP and /or DIPG.
Taylor KR, Mackay A, Truffaux N, et al. Nat Genet. 2014;46:457–461.
Yu PB, Deng DY, Lai CS, et al. Nat Med. 2008;14:1363–1369.
Mohedas AH, Xing X, Armstrong KA, et al. ACS Chem Biol. 2013;8:1291–1302.
Hoeman CM, Cordero FJ, Hu G, et al. Nat Commun. 2019;10:1023.
Carvalho D, Taylor KR, Olaciregui NG, et al. Commun Biol. 2019;2:156.
A Phase 1, randomized, double-blind, placebo-controlled, single and multiple
ascending dose and food effect study to evaluate the safety, tolerability, and
pharmacokinetics of BLU-782 when administered orally to healthy adult subjects.
Available at: <https://clinicaltrials.gov/ct2/show/NCT03858075?term=BLU-78
2
&draw=2&rank=1> [Last accessed Jan 24, 2021].
Declaration of Competing Interest
9
Saracatinib trial TO prevent FOP. Available at: <https://clinicaltrials.gov/ct2/sho
w/NCT04307953?term=AZD0530&draw=2&rank=2> [Last accessed Jan 24,
2
021].
The authors declare the following financial interests/personal re-
lationships which may be considered as potential competing interests:
Takenobu Katagiri receives research support from Daiichi-Sankyo, Co.
Ltd. The other authors declare no competing interest in the present
study.
1
0
Randomized, double-blind, placebo-controlled, single and multiple ascending dose
study to evaluate the safety, tolerability and pharmacokinetics of KER-047
administered to healthy male volunteers and postmenopausal female volunteers.
Available at: <https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?
id=376969> [Last accessed Jan 24, 2021].
11 Sekimata K, Sato T, Sakai N, et al. Chem Pharm Bull. 2019;67:224–235.
1
1
1
1
1
1
1
2
3
4
5
6
7
8
Sato T, Sekimata K, Sakai N, et al. ASC Omega. 2020;5:11411–11423.
Katagiri T, Yamaguchi A, Komaki M, et al. J Cell Biol. 1994;127:1755–1766.
GLIDE. Schr o¨ dinger Inc., New York, NY, USA, http://www.schrodinger.com/.
Fukuda T, Kohda M, Kanomata K, et al. J Biol Chem. 2009;284:7149–7156.
Ohte S, Shokawa T, Koyama N, et al. J Antibiot. 2020;73:554–558.
Eickmeier C, Fuchs K, Peters S, et al. WO 2006/103038; 2006.
Acknowledgements
We thank Dr. H. Kojima (Drug Discovery Initiative, The University of
Tokyo) for providing the chemical library. This work was partially
supported by the Platform Project for Supporting in Drug Discovery and
Life Science Research from Japan Agency for Medical Research and
Development (AMED). This work was supported by RIKEN Program for
Drug Discovery and Medical Technology Platforms.
Sawyer JS, Beight DW, Ciapetti P, et al. WO 2002/094833; 2002.
19 Li HY, Wang Y, McMillen WT, et al. Tetrahedron. 2007;63:11763–11770.
5