Discovery of 3-(4-sulfamoylnaphthyl)pyrazolo[1,5-a]pyrimidines as potent and selective ALK2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.09.006

The pyrazolo[1,5-a]pyrimidine LDN-193189 is a potent inhibitor of activin receptor-like kinase 2 (ALK2) but is nonselective for highly homologous ALK3 and shows only modest kinome selectivity. Herein, we describe the discovery of a novel series of potent and selective ALK2 inhibitors by replacing the quinolinyl with a 4-(sulfamoyl)naphthyl, yielding ALK2 inhibitors that exhibit not only excellent discrimination versus ALK3 but also high kinome selectivity. In addition, the optimized compound 23 demonstrates good ADME and in vivo pharmacokinetic properties.

Full text of DOI:10.1016/j.bmcl.2018.09.006

Accepted Manuscript
Discovery of 3-(4-sulfamoylnaphthyl)pyrazolo[1,5-a]pyrimidines as potent and
selective ALK2 inhibitors
Jian-kang Jiang, Xiuli Huang, Khalida Shamim, Paresma R. Patel, Arthur Lee,
Amy Q. Wang, Kimloan Nguyen, Gregory Tawa, Gregory D. Cuny, Paul B. Yu,
Wei Zheng, Xin Xu, Philip Sanderson, Wenwei Huang
PII:
S0960-894X(18)30737-6
https://doi.org/10.1016/j.bmcl.2018.09.006
BMCL 26027
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 May 2018
31 July 2018
4 September 2018
Please cite this article as: Jiang, J-k., Huang, X., Shamim, K., Patel, P.R., Lee, A., Wang, A.Q., Nguyen, K., Tawa,
G., Cuny, G.D., Yu, P.B., Zheng, W., Xu, X., Sanderson, P., Huang, W., Discovery of 3-(4-
sulfamoylnaphthyl)pyrazolo[1,5-a]pyrimidines as potent and selective ALK2 inhibitors, Bioorganic & Medicinal
Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.09.006
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Graphical Abstract
Discovery of 3-(4-sulfamoylnaphthyl)pyrazolo[1,5-a]pyrimidines as potent
and selective ALK2 inhibitors
Jian-kang Jianga,*, Xiuli Huanga, Khalida Shamima, Paresma R. Patela, Arthur Leea, Amy Q.
Wanga, Kimloan Nguyena, Gregory Tawaa, Gregory D. Cunyb, Paul B. Yuc, Wei Zhenga, Xin
Xua, Philip Sandersona and Wenwei Huanga,*
1

Discovery of 3-(4-sulfamoylnaphthyl)pyrazolo[1,5-a]pyrimidines as potent
and selective ALK2 inhibitors
Jian-kang Jianga,*, Xiuli Huanga, Khalida Shamima, Paresma R. Patela, Arthur Leea, Amy Q.
Wanga, Kimloan Nguyena, Gregory Tawaa, Gregory D. Cunyb, Paul B. Yuc, Wei Zhenga, Xin Xua,
Philip Sandersona and Wenwei Huanga,*
a National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive,
Bethesda, Maryland, 20892-3370, USA
b Department of Pharmacological and Pharmaceutical Sciences, University of Houston, 4849 Calhoun Road, Health
Building 2, Room 7036, Houston, Texas 77204, USA
c Brigham and Women’s Hospital and Harvard Medical School, Division of Cardiovascular Medicine, 20 Shattuck
Street, Thorn Biosciences 1219, Boston, Massachusetts 02115, USA
*Corresponding authors: 9800 Medical Center Drive, Rockville MD, USA, 20892; Email address:
jiankangj@mail.nih.gov; Tel.: 1-301-827-7165 (J. K. Jiang) or Email address: huangwe@mail.nih.gov; Tel. 1-301-
827-7164 (W. Huang).
Abbreviations: SAR=structure-activity relationship; Boc=t-butoxycarbonyl; ADME: absorption, distribution, metabolism and excretion;
RLM= rat liver microsomal; PAMPA= parallel artificial membrane permeability assay.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The pyrazolo[1,5-a]pyrimidine LDN-193189 is a potent inhibitor of activin receptor-
like kinase 2 (ALK2) but is nonselective for highly homologous ALK3 and shows only
modest kinome selectivity. Herein, we describe the discovery of a novel series of potent
and selective ALK2 inhibitors by replacing the quinolinyl with a 4-(sulfamoyl)naphthyl,
yielding ALK2 inhibitors that exhibit not only excellent discrimination versus ALK3
but also high kinome selectivity. In addition, the optimized compound 23 demonstrates
good ADME and in vivo pharmacokinetic properties.
Keywords:
LDN-193189
FOP
2018 Elsevier Ltd. All rights reserved.
ALK2
Pyrazolo[1,5-a]pyrimidine
Sulfamoylnaphthyl
Fibrodysplasia ossificans progressiva (FOP) is an extremely rare and devastating disease that
affects one per two million of the population at any time.1-2 Although normal at birth except for
deformed hallux, FOP patients develop heterotopic ossification (HO) of muscles, fascia and
connective tissues induced by postnatal trauma, injury or inflammation, resulting in severe
immobility and shortened life expectancy.3-4 Genetic sequence analysis found >96% of FOP
patients harbor an arginine to histidine mutation near the glycine-serine (GS) rich domain
(c.617G>A; p.R206H) of ACVR1 gene encoding ALK2,5 one of seven bone morphogenetic
protein (BMP) type 1 receptor kinases (ALK1-7). While ALK2 is typically thought to transduce
BMP ligand signaling via phosphorylation of downstream SMAD effectors 1/5/8, recent
investigations found that the ALK2R206H mutant gains responsiveness to activin A, resulting in
dysregulated phosphorylation of SMAD1/5/8 and ultimately, the ectopic formation of
endochondral bone.6-7
2

Current treatments for FOP are limited to corticosteroids and non-steroidal anti-inflammatory
drugs for symptomatic relief and bisphosphonates during acute flares. However, none of these
interventions have been demonstrated to prevent the formation of HO, or modify the natural course
of disease. Several drug candidates for inhibiting the formation of HO by diverse mechanisms of
action are in various stages of clinical development including an activin A neutralizing antibody,8
a selective RAR- agonist (palovarotene)9 and more recently the immunosuppressive agent
rapamycin.10-11 These strategies, while promising, have yet to demonstrate efficacy in human
randomized clinical trials for FOP. They are not known to modify disease progression and have
distinct side effect profiles, which may or may not limit their use in the target population. Thus,
FOP remains a significant unmet medical need. Several chemotypes of ALK2 inhibitors have been
reported12-23 including LDN-193189 and K02288 (Figure 1). However, kinome-wide profiling
showed LDN-193189 has only modest selectivity with no discrimination for ALK3.16 With this in
mind, we initiated medicinal chemistry efforts to identify novel next-generation ALK2 inhibitors
with improved selectivity over ALK3 as compared to published tool compounds.
ALK2 and ALK3 share approximately 63% sequence homology in their kinase domains. The
crystal structure of LDN-193189 bound to ALK2 (PDB: 3Q4U) indicates that the nitrogen of the
quinolinyl ring forms a critical water-mediated hydrogen bond with the carboxyl side-chain of
Glu248, which is conserved in BMP type 1 receptors including ALK3. Therefore, we envisioned
replacing the quinolinyl moiety at the 3-position of the pyrazolo[1,5-a]pyrimidine core with other
aromatics to exploit subtle differences in these regions of ALK2 and ALK3 in order to achieve
better selectivity.
In addition to targeting the quinolinyl moiety, introduction of steric hindrance to the solvent
exposed piperazine ring was pursued in order to reduce potential CYP450-mediated dealkylation
of this group. This metabolic pathway was observed for LDN-193189 (unpublished data) which
results in formation of the corresponding aniline (a potential genotoxin). Therefore, the initial set
of designed derivatives utilized cis-2,6-dimethylpiperazine as the solvent exposed group and
different aromatic moieties at the 3-position of the pyrazolo[1,5-a]pyrimidine core. Compounds 1-
11 were prepared as shown in Scheme 1. Reaction of malonaldehyde 24 and pyrazole-5-amine
(25) gave pyrazolopyrimidine 26 in 81% yield. Bromide 29 was prepared through a Pd2(dba)3/t-
-
Bu3PH+BF4 catalyzed Buchwald-Hartwig amination of 26 followed by treatment with N-
bromosuccinamide (NBS). With 26 in hand, compound 1-11 were synthesized by using Suzuki-
3

Miyaura coupling reactions with either aryl boronic acids or aryl pinacol boronic esters followed
by removal of the Boc protecting group with acid.
As depicted in Table 1, introduction of the cis-2,6-dimethylpiperazine in compound 1 retained
potent ALK2 and ALK3 enzyme inhibitory activity (IC50= 9 nM and 7 nM for ALK2 and ALK3,
respectively). However, replacing the quinolinyl moiety with 4-(methylsulfonyl)phenyl
(compound 2) resulted in a significantly weaker ALK2 inhibitor (IC50= ~2 M). Interestingly, for
derivative 3 with a 4-(sulfamoyl)phenyl group in the place of the quinolinyl, the potency for ALK2
inhibition improved ~4-fold (IC50= 526 nM) compared to 2. Adding either a methyl (compound 4)
or an amino (compound 5) at the ortho position of the 4-sulfamoyl improved the ALK2 inhibitory
activity by ~2-fold over 3. Shifting the sulfamoyl group from the 4- to 3-position (compound 6)
improved ALK2 inhibitory activity another 2-fold. To our delight, 7, with a 4-(sulfamoyl)naphthyl
moiety at the 3-position of pyrazolo[1,5-a]pyrimidine core, showed markedly improved ALK2
inhibitory activity (IC50= 19 nM) and excellent selectivity against ALK3. However, mono and di
N–methylation of the S(O)2NH2 group proved detrimental for ALK2 potency as shown by analogs
8 and 9, suggesting that the unsubstituted sulfamoyl might be critical for binding and well-
accommodated in the active site of the enzyme. Shifting the sulfamoyl group from the 4- to 5-
position on the naphthyl ring (compound 10) led to ~3-fold decreased potency compared to 3 and
its selectivity against ALK3 also decreased. Replacing the 4-sulfamoyl with a 4-
methylsulfonamido group also resulted in significantly decreased ALK2 inhibitory activity as
demonstrated by 11.
Table 1. ALK2 and ALK3 inhibitory activity of 1-11, which contain various groups at the 3-
position of the pyrazolo[1,5-a]pyrimidine.
4

ALK2
(IC50, nM) (IC50, nM)
ALK3
Selectivity
(-fold)
Compd
Ar
LDN-193189
17
9
19
7
1.0
0.8
1
2
1971
>10,000
NA
526
246
ND
NA
3
4
16550
67.3
291
109
>10,000
2534
NA
5
6
23.2
5

19
3466
182.4
NA
7
8
326
>10,000
5990
54
64750
444
10.8
8.2
9
10
796
8897
11.2
11
*Selectivity= IC50 (ALK3)/IC50 (ALK2); **NA= not available; ***ND= not determined.
Based on the initial SAR, we identified a new ALK2 inhibitor (7) with a 4-(sulfamoyl)naphthyl
ring attached to the pyrazolo[1,5-a]pyrimidine core that exhibited potent ALK2 potency and
excellent selectivity for ALK2 versus ALK3. We constructed a computational model of the
binding mode of 7 with ALK2 based on the reported co-crystal structure of LDN-193189 and
ALK2 (Figure 2). The model revealed similar interactions and conformations at the hinge and the
solvent-exposed region of ALK2.24 However, the oxygen atoms of the 4-sulfamoyl group forms
two direct hydrogen bonds with the enzyme, one with the amino side-chain of Lys235 and the
other with the backbone amide NH of Asp354. We also generated a homology model of ALK3
using the ALK2 structure as a template. Compound 7 was then modeled into the ALK3 homology
model binding pocket. Although both Lys235 and Asp354 are conserved in ALK2 and ALK3
enzymes, the sulfonamides were generally less potent against ALK3. The potency difference
probably stems from the fact that the hinge residues of ALK3 are different than for ALK2. In
particular, there is a histidine pair in ALK2 (His286 and His284) which in ALK3 is replaced by a
His312 and Asp310 pair. When binding to ALK2 compound 7 forms a hydrogen bond with the
6

backbone nitrogen of His286 and the imidazole of His286 forms an edge on Pi stack interaction
with His284. In the case of ALK3 however, His 312’s histidine forms a hydrogen bond with the
carboxylate of Asp310. This hydrogen bond pulls His312 towards Asp310 causing a puckered
confirmation that pulls the hinge away from the ligand. We speculate that this reduces the ability
of the pyrazolo-pyrimidine core to form a hydrogen bond with His312 in ALK3’s hinge making
compound 7 less potent against ALK3, Figure 3. Also, this computational model seems to explain
the potency difference observed between compounds 7 and 9 against ALK2. As per model, the
potency difference might arise due to the clash that the compound 9 methyl groups has with the
back-end of the ALK2 pocket (Shown in Supporting information, Figure 1B). This steric clash
does not exist for compound 7 which has hydrogen atoms in those positions (Shown in Supporting
information, Figure 2A).
Figure 2. Computational modeling of the interaction of 7 with ALK2.
7

A
B
Figure 3. Compound 7 modeled into the ALK2 (pink) and ALK3 (orange) binding pockets. A.
The hinge backbones for ALK2 and ALK3 are shown as a lines. Hinge residues His 284, 286
(ALK2) and Asp310, His312 (ALK3) are explicitly shown. B. ALK2-ALK3 hinge region
sequence alignment
Although 7 demonstrated promising potency and selectivity for ALK2, early in-house ADME
profiling showed this compound had poor permeability, as measured in PAMPA (5.5x10-6 cm/s)
and aqueous solubility (1.8 g/ml at pH 7.4). To further improve both ALK2 potency and ADME
properties, more comprehensive SAR efforts were carried out focusing on changes to the 6-
position of the pyrazolo[1,5-a]pyrimidine core via compounds 12-23. Each new analog was
assessed not only for its inhibition of ALK2 and ALK3, but also for its early ADME parameters,
including RLM stability, PAMPA permeability, and aqueous solubility at pH 7.4.
8

As shown in Table 2, replacing the phenyl ring with a pyridinyl linker resulted in a >20-fold
decreased ALK2 inhibitory activity as exemplified by analogs 12, 13 and 14, although 14 showed
significant improvement in PAMPA permeability with the more lipophilic 2-(piperidinyl)ethoxy
moiety. Analog 15 with a phenylpiperidinyl ring showed modest improvement in PAMPA
permeability, but was only ~5-fold less potent (IC50= 93 nM) than 7, suggesting that the phenyl
linkage might reinstate potency for ALK2. Analogs with a 2-(piperidinyl)ethoxy chain such as 16
and 17 exhibited IC50 values < 100 nM for ALK2 with significantly improved PAMPA
permeability, although 17 showed shorter half-life in RLM compared to 16, which might be
derived from favorable cleavage of the tertiary C-O bond. Analog 18 with a five-membered
pyrrolidinyl ring showed ~10-fold improvement of aqueous solubility compared to 16 and retained
good RLM stability and PAMPA permeability. Despite its optimized ADME properties, 18 was
still ~3-fold less potent than LDN-193189 for ALK2 thus more optimization at the 6-position of
the core was pursued. Gratifyingly, benzylic amine analogs such as 19-21 showed improved
inhibitory potency for ALK2 compared to 18, although their PAMPA permeability decreased
dramatically. This might be attributed to the polar primary amine functionality. Incorporation of a
pyrrolidinyl ring to give analog 22 not only maintained similar ALK2 inhibitory activity as 19, but
regained PAMPA permeability (467x10-6 cm/s). Finally, adding a methyl group at the benzylic
position culminated in the discovery of compound 23 that was not only ~2-fold more potent than
7, but also showed >700-fold selectivity against ALK3. It also exhibited amenable ADME
properties as indicated by its excellent PAMPA permeability, acceptable RLM stability (t1/2= 22.8
min) and aqueous solubility at pH 7.4 (7 g/ml or 14 M), which is ~4-fold increase compared to
7 (1.8 g/mL at pH 7.4).
Table 2. ALK2 and ALK3 inhibitory activity, RLM stability, PAMPA permeability and aqueous
solubility of 7-23, which represent changes to the 6-position of the pyrazolo[1,5-a]pyrimidine.
ALK2
(IC50, nM) (IC50, nM)
ALK3
Selectivity
(-fold)
Aq. sol.
PAMPA
(x10-6 cm/s) (g/mL)
RLM
t1/2 (min)
Compd
R
9

19
3466
182.4
>30
5.5
1.8
7
422
1026
738
>10,000
>10,000
>10,000
NA
NA
NA
>30
9.4
8.3
8.8
1.4
<1
<1
12
13
14
20.9
364.7
93
>10,000
NA
>30
78.6
<1
15
63
47
>10,000
>10,000
NA
NA
>30
6.1
481.5
3.2
<1
16
17
1920.5
48
25
>10,000
12830
NA
>30
ND
317
33
18
19
513.2
14.2
7.6
29
>10,000
NA
>30
9.4
10.6
20
16
27
8195
512.2
NA
ND
ND
467
<1
8
21
22
>10,000
21.9
9
6439
715.4
22.8
1116.0
7.0
23
To better understand the enzyme selectivity of 23, it was profiled against other BMP type 1
receptor kinases (ALK1, ALK4, ALK5 and ALK6) and four upstream BMP type 2 receptor
10

kinases (ACVR2A, ACVR2B, BMPR1 and TGFR2)25 (Table 3). The results indicated 23 was
highly selective among both BMP type 1 and 2 receptor kinases with the exception of ALK1 (IC50=
51 nM), which was not surprising considering the high sequence homology in the ATP binding
pockets of ALK1 and ALK2.26 In addition, LDN-193189, 7 and 23 were evaluated for their cellular
activity through measuring their inhibition of BMP6 (an ALK2 ligand) induced transcriptional
activity (BRE-Luciferase) in C2C12 cells as previously reported.27 While compound 23 (EC50 = 8
nM) had similar cellular activity as LDN-193189 (EC50 = 14 nM), compound 23 was about 11-
fold more potent than compound 7 (EC50 = 91 nM). It should be noted that 23’s high selectivity
against ALK5 (also called TGFR1) and TGFR2 might be advantageous since it is well known
that chronic inhibition of TGF-signaling is associated with immunosuppression, physeal
hypertrophy and cardiac valvulopathy.28-29 Also kinome-wide profiling of 21, which is structurally
similar to 23, at 1 M found only five non-BMP receptor kinases (ARK5, DDR1, MAP4K5,
RIPK2 and TNIK) out of 252 kinases with >80% inhibition (for the detailed results, see
Supplementary Material), further showcasing the high selectivity of this class of ALK2 inhibitors
versus other kinases.
Table 3. Enzymatic activities of analog 23 for BMP type 1 and type 2 receptor kinases (IC50, nM).
Enzyme ALK1 ALK4 ALK5 ALK6
ACVR2A ACVR2B BMPR2
TGFR2
Compd
51
18330 18650 13140
2370
3130
>10,000
698
23
Pharmacokinetic parameters of 23 were determined in C57BL/6 mice after single
intraveneous (IV) and oral (PO) administration of 3 mg/kg for each route (Table 4). This analog
showed a relative low plasma clearance (CLp) of 16 mL/min/kg and a moderate half-life of 3-4
hours. It appears that the compound distributed extensively to the tissues based on its large steady-
state volume of distribution (Vdss = 4.6 L/kg). The oral bioavailability (%F) was 24%.
Table 4. In vivo pharmacokinetic parameters of 23 in mice.
Pharmacokinetic Parameters of 23 in C57BL/6 Mice (3 mg/kg)
Route
IV
3220
3.5
PO
782
4.3
67
AUC0→∞ (hr●ng/mL)
t1/2 (h)
Cmax (ng/mL)
CL (mL/min/kg)
Vdss (L/kg)
%F
16
4.6
24
In summary, we discovered a novel class of selective ALK2 inhibitors with 4-
(sulfamoyl)naphthyl as the pendant substituent at the 3-position of the pyrazolo[1,5-a]pyrimidine
core. Compound 23 not only showed excellent ALK2 inhibitory activity, which was comparable
to LDN-193189, but excellent selectivity against other kinases including BMP type 1 and 2
11

receptor kinases. Compound 23 also showed acceptable in vivo pharmacokinetics therefore
positioning this class of ALK2 inhibitors for further development towards the treatment of FOP.
Acknowledgments
This work was supported by the Intramural Research Programs of the National Center for
Advancing Translational Sciences. The authors thank members of analytical and compound
management groups of National Center for Advancing Translational Sciences of NIH. G.D.C
acknowledges support from the American Heart Association (15GRNT22970025). P.B.Y
acknowledges support from NIH/NIAMS (AR057374). The authors also thank Dr. Catherine A.
Evans of Keros Therapeutics for critical review and valuable suggestions.
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Supplementary Material
Supplementary material includes the experimental procedure for the measurement of IC50
values for ALK1-6, the method of computer modeling of interaction of ALK2 and ALK3
binding with analog 7, the synthetic procedures and spectral characterization of all analogs (1-
23) and the profiling of the inhibition of analog 21 against 252 kinases at 1 M concentration.
Graphical Abstract
Discovery of 3-(4-sulfamoylnaphthyl)pyrazolo[1,5-a]pyrimidines as potent
and selective ALK2 inhibitors
Jian-kang Jianga,*, Xiuli Huanga, Khalida Shamima, Paresma R. Patela, Arthur Leea, Amy Q.
Wanga, Kimloan Nguyena, Gregory Tawaa, Gregory D. Cunyb, Paul B. Yuc, Wei Zhenga, Xin
Xua, Philip Sandersona and Wenwei Huanga,*
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 Identification of a novel class of 4-sulfamoylnaphthyl series ALK2 inhibitor
 Excellent selectivity for ALK2 over ALK3 with additional overall kinome selectivity
 Key distinctive features identified in the ALK3 homology model to explain ALK2 vs
ALK3 selectivity
15