Structure-activity relationship of 3,5-diaryl-2-aminopyridine ALK2 inhibitors reveals unaltered binding affinity for fibrodysplasia ossificans progressiva causing mutants

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Open Access on 08/07/2015

Structure−Activity Relationship of 3,5-Diaryl-2-aminopyridine ALK2

Inhibitors Reveals Unaltered Binding Aﬃnity for Fibrodysplasia

Ossiﬁcans Progressiva Causing Mutants

Agustin H. Mohedas,^†,¶You Wang,^‡,¶Caroline E. Sanvitale,^§Peter Canning,^§Sungwoon Choi,^‡

Xuechao Xing,^‡Alex N. Bullock,^§Gregory D. Cuny,*^,‡,∥and Paul B. Yu*^,⊥

^†Harvard−MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, 77 Massachusetts Avenue,

Cambridge, Massachusetts 02139, United States

^‡Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Brigham and Women’s Hospital and

Harvard Medical School, 65 Landsdowne Street, Cambridge, Massachusetts 02139, United States

^§Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, United Kingdom

^∥Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Science and Research Building 2, Room 549A,

Houston, Texas 77204, United States

^⊥Department of Medicine, Cardiovascular Division, Brigham and Women’s Hospital, 75 Francis Street, Boston, Massachusetts 02115,

United States

S

* Supporting Information

ABSTRACT: There are currently no eﬀective therapies for

ﬁbrodysplasia ossiﬁcans progressiva (FOP), a debilitating and

progressive heterotopic ossiﬁcation disease caused by activat-

ing mutations of ACVR1 encoding the BMP type I receptor

kinase ALK2. Recently, a subset of these same mutations of

ACVR1 have been identiﬁed in diﬀuse intrinsic pontine glioma

(DIPG) tumors. Here we describe the structure−activity

relationship for a series of novel ALK2 inhibitors based on the

2-aminopyridine compound K02288. Several modiﬁcations

increased potency in kinase, thermal shift, or cell-based assays

of BMP signaling and transcription, as well as selectivity for

ALK2 versus closely related BMP and TGF-β type I receptor

kinases. Compounds in this series exhibited a wide range of in vitro cytotoxicity that was not correlated with potency or

selectivity, suggesting mechanisms independent of BMP or TGF-β inhibition. The study also highlights a potent 2-

methylpyridine derivative 10 (LDN-214117) with a high degree of selectivity for ALK2 and low cytotoxicity that could provide a

template for preclinical development. Contrary to the notion that activating mutations of ALK2 might alter inhibitor eﬃcacy due

to potential conformational changes in the ATP-binding site, the compounds demonstrated consistent binding to a panel of

mutant and wild-type ALK2 proteins. Thus, BMP inhibitors identiﬁed via activity against wild-type ALK2 signaling are likely to

be of clinical relevance for the diverse ALK2 mutant proteins associated with FOP and DIPG.

inhibitor of diﬀerentiation (Id) gene family. Functional and

anatomic speciﬁcity of BMP signaling is regulated by the

spatiotemporal expression of ligands and their cognate

receptors as well as the expression of endogenous BMP

antagonists such as noggin.^5,6

Inappropriate BMP signaling has been shown to contribute

to the pathophysiology of various disease processes.⁷One of

the most striking examples of BMP signaling-related disease is

seen in ﬁbrodysplasia ossiﬁcans progressiva (FOP), a rare and

disabling genetic disease aﬀecting approximately 2500 people

worldwide.⁸While individuals with the classical form of FOP

INTRODUCTION

■

Bone morphogenetic proteins (BMPs) are members of the

transforming growth factor-beta (TGF-β) signaling family,

which includes over 30 diﬀerent ligands.¹BMP signaling is

essential for numerous processes, including cell fate determi-

nation, embryonic patterning, and iron homeostasis.^2,3The

BMP signaling cascade parallels that of TGF-β signaling. BMP

ligand dimers bind to transmembrane receptor complexes

consisting of two constitutively active type II receptor kinases

(BMPRII, ACTRIIA, or ACTRIIB), which transphosphorylate

and activate two type I receptor kinases (ALK1, ALK2, ALK3,

or ALK6).⁴Activated type I receptors phosphorylate eﬀector

proteins (SMAD1/5/8) that complex with SMAD4, translocate

to the nucleus, and activate BMP responsive genes such as the

Received: November 24, 2013

A

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are nearly normal at birth except for cervical and hallux joint

deformities, during early life they develop progressive formation

of endochondral bone in muscles, fascia, and ligaments, leading

to severe immobility, pain, and premature mortality. A highly

conserved gain-of-function mutation in the glycine−serine

(GS) rich domain of the BMP type-I receptor ALK2

(c.617G>A; p.R206H) accounts for more than 98% of cases

of classic FOP.^9,10Several other FOP-causing gain-of-function

mutations in both the GS and kinase domains of ALK2 have

also been described in nonclassic or variant forms of FOP.¹⁰⁻¹⁴

Recently, several of the mutations identiﬁed in classic and

nonclassic forms of FOP have been observed to arise in a

proportion of tumors in diﬀuse intrinsic pontine glioma, a

deadly childhood tumor also without eﬀective therapies.¹⁵⁻¹⁸

The consistency of this ﬁnding across diverse patient cohorts by

several independent groups suggests an important role of

somatic activating mutations of ACVR1 in this disease,

however, the pathogenetic role of these mutant proteins is

currently under investigation.

BMP and TGF-β signaling inhibition in biochemical and

cellular assays, selectivity, and cytotoxicity. These studies were

pursued as part of an eﬀort to elucidate the BMP type I

receptor inhibitor pharmacophore, while producing a set of

compounds with greater utility as physiologic probes. This SAR

provides unique insights into features of 2-aminopyridine

derivatives that are required for potent and selective inhibition

of ALK2 versus closely related BMP and TGF-β receptors. We

found that substitution of the 3-phenol with 4-phenylpiperazine

greatly increased potency in cells, yielding a series of

compounds more likely to be useful as probes of ALK2

function. These included a 2-methylpyridine derivative that

exhibited potent and relatively selective inhibition of ALK2

activity in cell-based and in vitro kinase assays, high selectivity

across the kinome, and low cytotoxicity. Additionally, we used

this novel set of derivatives to demonstrate for the ﬁrst time

that FOP-causing mutations do not aﬀect inhibitor binding

aﬃnity as compared to wild-type ALK2. This ﬁnding strongly

suggests that ATP-competitive kinase inhibitors identiﬁed on

the basis of their activity against endogenous BMP signaling,

such as dorsomorphin and its derivatives, or by their aﬃnity for

wild-type ALK2, as in the case of K02288, will inhibit with

equal potency the mutant ALK2^R206Hfound in classical FOP as

well as the other GS- and kinase-domain mutants of ALK2 that

have been described in nonclassical or variant FOP or DIPG.

These results describe a novel series of speciﬁc and potent

probe compounds for the interrogation of BMP signaling that

may have therapeutic potential for FOP and other diseases of

maladaptive or inappropriate BMP signaling.

We and others have previously reported the discovery and

development of small molecule inhibitors of BMP type-I

receptors such as dorsomorphin, LDN-193189, LDN-212854,

and DMH1, all of which are based on the pyrazolo[1,5-

a]pyrimidine scaﬀold (Figure 1).¹⁹⁻²¹These compounds have

RESULTS AND DISCUSSION

■

Chemistry. A series of 2-amino-3-(3,4,5-trimethoxyphenyl)-

pyridine derivatives were synthesized according to the

procedures outlined in Scheme 1. Commercially available 2-

Scheme 1. General Procedure for the Synthesis of 2-Amino-

a

3-(3,4,5-trimethoxyphenyl)pyridine Derivatives

a

Reagents and conditions: (a) 3,4,5-trimethoxyphenylboronic acid,

MeCN/DMF, Na₂CO₃(aqueous, 1 M), 10 mol % Pd(PPh₃)₄, 90 °C,

8 h, 80%; (b) arylboronic acid, DME, Na₂CO₃(aqueous, 1 M), 10 mol

%, Pd(PPh₃)₄, 90 °C, 8 h, 40−85%.

Figure 1. Previously described BMP inhibitors.

proven to be useful chemical reagents for the study of in vitro

phenomenon, and several have demonstrated in vivo eﬃcacy in

a mouse model of FOP.^21,22More recently we described a

structurally distinct BMP type-I receptor inhibitor, K02288,

which is based on a 2-aminopyridine scaﬀold and demonstrated

greater kinome-wide selectivity than LDN-193189.²³The 2-

aminopyridine scaﬀold is also found in crizotinib, which was

recently approved by the FDA for the treatment of nonsmall

cell lung cancer in patients with activating mutations in the

anaplastic lymphoma kinase.²⁴Despite the high aﬃnity and

selectivity of K02288 for BMP receptors in thermal shift and in

vitro kinase assays, it has comparatively weak potency in cell-

based assays.²¹

amino-5-bromo-3-iodopyridine (1) was coupled with 3,4,5-

trimethoxyphenylboronic acid under Suzuki reaction conditions

to give 2 in 80% yield.²⁵⁻²⁷This intermediate was subjected to

a second Suzuki reaction with a range of boronic acids to

furnish the target compounds 3 in 40−85% yield.

A variety of 2-substituted 3-aryl-5-(piperazinylphenyl)-

pyridine derivatives were synthesized according to the method

outlined in Scheme 2. An aryl group was ﬁrst introduced at the

3-position of pyridine 1 or 4 via a Suzuki coupling to provide 5

in 65−85% yield. This was followed by a second Suzuki

reaction using [(N-Boc)piperazin-1-yl]phenylboronic acid

pinacol esters to generate 6, which was deprotected using

triﬂuoroacetic acid (TFA) in dichloromethane at room

temperature to give amines 7. Starting material 4 (R =

NHMe or NMe₂) was prepared by reductive amination of 1 in

In this article, we describe a structure−activity relationship

(SAR) study of K02288 with respect to ALK2 binding aﬃnity,

B

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of the 3-phenol (Figure 2a,b), chosen either to mimic hydrogen

bonding of the phenol with Asp293 or to introduce an

electropositive charge (e.g., a protonated amine) to mediate an

ionic interaction with Asp293, thus maintaining a potentially

important interaction. To gain insight into the potency and

selectivity for BMP vs TGF-β signaling, derivatives were tested

for their ability to bind BMP type I receptor ALK2 and TGF-β

type I receptor ALK5, using an in vitro thermal shift kinase

assay (Figure 2c). This type of assay has been previously shown

by us and others to be highly predictive of biochemical kinase

inhibition activity,³¹which was also measured in a selected

subset of the derivatives (Figures 2−5). Tm shift data were

found to correlate highly (r²≥ 0.8) with biochemical inhibition

data (Supporting Information, Figure 2). To assess the potency

and selectivity of these compounds in cells, inhibition of BMP6-

induced transcriptional activity (BRE-Luciferase) and TGF-β1-

induced transcriptional activity (CAGA-Luciferase) was meas-

ured for each of the compounds (Figure 2c), using cell lines

(C2C12 for BMP6 and HEK293T for TGF-β1) previously

shown to express a complement of BMP or TGF-β receptors

required for ligand-mediated signaling.³²In general, the

magnitude of ΔT_mfor ALK2 and ALK5 correlated inversely

with the log IC₅₀for inhibition of BMP and TGF-β-induced

transcriptional activity (Figure 1d) but with some minor

exceptions. Notably, K02288 exhibited a large thermal shift for

ALK2 kinase protein (ΔT_m= 13.2 °C), consistent with potent

inhibition of ALK2 activity by biochemical assay (IC₅₀= 34

nM) but was substantially weaker in the cell-based assay of

BMP6 activity (IC₅₀= 421 nM, Figure 2c). Of the variants at

the 3-phenol position, compound 13 exhibited the best in vitro

inhibition of ALK2, whereas compound 15 demonstrated the

best cell-based activity. The occasional discordance between

biochemical (ΔT_mand enzymatic) and functional assays

(ligand-induced transcription) highlighted the need for multiple

assays in an SAR aimed at identifying physiologic probes with

useful potency and selectivity.

Scheme 2. General Procedure for the Synthesis of Various 2-

Substituted 3-Aryl-5-(piperazinylphenyl)pyridine

Derivatives

a

Reagents and conditions: (a) arylboronic acid, MeCN/DMF,

Na₂CO₃(aqueous, 1 M), 10 mol % Pd(PPh₃)₄, 90 °C, 8 h, 65−

85%; (b) [(N-Boc)piperazin-1-yl]phenylboronic acid pinacol ester,

DME, Na₂CO₃(aqueous, 1 M), 10 mol % Pd(PPh₃)₄, 90 °C, 8 h, 70−

75%; (c) TFA, DCM, rt, 12 h, 100%.

dichloromethane using various amounts of paraformaldehyde in

the presence of NaBH(OAc)₃.

3-Aryl-5-(4-piperazinylphenyl)pyridine derivatives containing

a methyl substituent in the 2-position of the pyridine were

prepared using the method outlined in Scheme 3. The 2-

chloropyridine 8 was treated with trimethylboroxine in the

presence of a palladium catalyst to generate 2-methylpyridine 9

in 90% yield.²⁸The carbamate protecting group was again

removed with TFA to generate 10.

On the basis of several physiochemical properties, including

calculated octanol−water partition coeﬃcient (cLogP), dis-

tribution coeﬃcient (cLogD_7.4), topological polar surface area

(tPSA in Å²), and number of hydrogen bond donors, acceptors

(HBD and HBA) and rotatable bonds (Supporting Informa-

tion, Table 1), the prepared compounds are predicted to be cell

permeable and orally absorbed.^29,30

SAR of Solvent Exposed Position. We previously demon-

strated that K02288 exhibits similar potency to LDN-193189 in

biochemical kinase assays for inhibiting ALK2 and related BMP

type I receptor kinases but was surprisingly less active in cell-

based reporter assays using constitutively active BMP type I

receptors.^21,23We speculated the relatively weaker activity of

K02288 in cells might be due to poor solubility or impaired

interactions with solvent water molecules that might be

addressed via modiﬁcations in the solvent-exposed domain.

We created six derivatives of K02288 by modifying the 3-

phenol substituent, which in the co-crystal structure of ALK2

occupied the solvent-exposed hydrophobic channel at the

entrance of the ATP pocket (Supporting Information, Figure

1). Here, several functional groups were used as replacements

In addition to altering potency, modiﬁcations to the solvent-

exposed 3-phenol showed signiﬁcant alterations in selectivity.

Replacing 3-phenol with 4-phenol (11) increased potency

against BMP6 signaling by ∼20-fold compared to K02288

while retaining a modest degree of selectivity for BMP6 versus

TGF-β1 signaling (28-fold, Figure 2c). Adding a 3-methoxy

group to the 4-phenol (12) reduced BMP6 inhibition modestly,

with similar selectivity. Replacing the 3-phenol with a

bioisosteric methylsulfonamide (13) improved BMP6 inhib-

ition compared to K02288 but decreased selectivity. The

largest increase in potency occurred with the replacement of

the 3-phenol with 3- or 4-phenylpiperazine, as previously done

with LDN-193189, likely due to the increased polarity of this

a

Scheme 3. Synthesis of a 2-Methyl 3-Aryl-5-(4-piperazinylphenyl)pyridine Derivatives

a

Reagents and conditions: (a) trimethylboroxine, 1,4-dioxane, K₂CO₃(2 equiv), 20 mol % Pd(PPh₃)₄, 110 °C, 8 h, 90%; (b) TFA, DCM, rt, 12 h,

100%.

C

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Figure 2. Potency and selectivity of K02288 derivatives based on thermal shift, biochemical kinase activity, and ligand induced transcriptional activity

assays. (a) The 2-aminopyridine scaﬀold of K02288. (b) Modiﬁcations to the solvent exposed domain (R₁) of K02288. (c) Thermal shift (ΔT_m),

biochemical enzymatic inhibition (IC₅₀) for ALK2 and ALK5 kinase proteins, and inhibition of cell-based BMP6 and TGF-β1-induced

transcriptional activity (IC₅₀) by K02288 derivative compounds (nd = not determined). (d) Correlation of thermal shift and cell-based BMP/TGF-β

inhibition assays.

substituent resulting in both improved inhibitor aqueous

solubility and increased enthalpic interactions with solvent

water molecules.¹⁹Compounds 14 and 15 demonstrated 70−

100-fold increases in BMP6 inhibition (IC₅₀= 6 nM, and 4

nM) compared to K02288, with modest improvements in

selectivity. Compound 15 is structurally similar to previously

disclosed aminopyridine inhibitors of interleukin-2-inducible T-

cell kinase (ITK) and pyridine benzamide inhibitors of protein

kinase D (PKD).^33,34To further investigate the type I receptor

selectivity of 15, cells were transfected with adenoviruses

expressing constitutively active BMP type I receptors (caALK1,

caALK2, and caALK3) and constitutively active activin or TGF-

β type I receptors (caALK4 and caALK5) in low serum

conditions and in the absence of exogenous ligand (Supporting

Information, Figure 3). Derivative 15 was most potent against

BMP receptors caALK2 and caALK3 with IC₅₀measurements

of ∼3.5 nM, whereas the activin/TGF-β type I receptors and

caALK1 were inhibited with with an IC₅₀measurements of ∼20

nM, with nearly complete extinction of BMP and TGF-β type I

receptor signaling at approximately 250 nM. Taken together,

these data demonstrate that replacing the 3-phenol in the

solvent exposed region of K02288 with 4-phenylpiperazine

dramatically improved its potency in cells but with relatively

poor selectivity for BMP versus TGF-β signaling. These results

prompted us to explore structural variants at other positions

that might reﬁne selectivity while retaining gains in potency

aﬀorded by modiﬁcation of the solvent-exposed 3-phenol with

4-phenylpiperazine in 15.

SAR of Hydrophobic Pocket Position. Further modiﬁcations

of potent compound 15 were performed to develop an SAR for

the 3,4,5-trimethoxyphenyl group (R₂) (Figure 3a,b) to identify

the role of each methoxy group on potency and selectivity. The

trimethoxyphenyl group has previously been shown to interact

with the hydrophobic back pocket in ALK2 where it forms

water-mediated hydrogen bonds with the catalytic lysine

residue (K235) (Supporting Information, Figure 1). Com-

pounds were again proﬁled in thermal shift, biochemical

enzyme inhibition, and cell-based luciferase reporter assays

(Figure 3b,c).

Removal of any of the methoxy groups resulted in a

signiﬁcant decrease in BMP inhibition. However, particular

methoxy groups were more crucial than others. For example,

removing one of the 3-methoxy groups (16) resulted in a 35-

fold loss in potency compared to 15, although selectivity for

BMP6 inhibition versus TGF-β increased. Removing the 4-

methoxy group (17) decreased activity 10-fold but did not

improve selectivity. Combining these changes (18) demon-

strated that the 4-methoxy group contributed less signiﬁcantly

to BMP6 inhibition as compared to either meta-methoxy group.

As expected, removal of both meta-methoxy groups, while

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Figure 3. Potency and selectivity of compound 15 derivatives based on thermal shift, biochemical kinase activity, and ligand induced transcriptional

activity assays. (a) The 2-aminopyridine scaﬀold of 15. (b) Modiﬁcations to the ATP-binding pocket hydrophobic domain (R₂) of compound 15. (c)

Thermal shift (ΔT_m), biochemical enzymatic inhibition (IC₅₀) for ALK2 and ALK5 kinase proteins, and inhibition of cell-based BMP6 and TGF-β1-

induced transcriptional activity (IC₅₀) by compound 15 derivatives (nd = not determined). (d) Correlation of thermal shift and cell-based BMP/

TGF-β inhibition assays.

retaining the para-methoxy group (20) drastically reduced

potency by almost 500-fold. Incorporating the 3,4-dimethoxy

groups into a benzo-1,4-dioxane (19) resulted in decreased

activity compared to 16, perhaps reﬂecting disruption of the

hydrogen bond with ALK2 residue K235. In addition,

increasing the steric bulk of the 3-alkoxy group (21) or

replacing the 4-methoxy with a chlorine (22) or a methyl (23)

were also not productive. In conclusion, of the compounds

studied the 3,4,5-trimethoxphenyl group resulted in the best

balance between BMP6 inhibition and selectivity over TGF-β.

This is likely due to its greater molecular volume for occupying

the hydrophobic pocket in ALK2 while retaining hydrogen

bond acceptors in the meta-positions of the phenyl ring. Future

studies will seek to optimize binding in the hydrophobic pocket

by replacing the 3,4,5-trimethoxyphenyl entirely with a diverse

set of aryl and heteroaryl moieties.

aminopyridine (R₃, Figure 4a,b), a region that was previously

shown to interact with the hinge region of the ALK2 kinase

domain. Here, the amine was within hydrogen bonding

distance to the backbone carbonyl of H284, as well as the

gatekeeper residue T283 of ALK2 (Supporting Information,

Figure 1). Both residues are changed in ALK5 (D281 and S280,

respectively). Secondary and tertiary amines such as 24 and 25,

respectively, exhibited reduced potency in both the thermal

shift and cell-based assays. Similarly, 28, in which the primary

amine is replaced with a methoxy substituent, exhibited

decreased potency, suggesting that bulky substituents are not

well tolerated at this position. Notably, these compounds

exhibited negligible thermal shift despite detectable albeit low

activity in cell-based assays (Figure 4c). Despite the high degree

of correlation between the thermal shift and cell-based assays

(Supporting Information, Figure 4), compounds that exhibit

very low ΔT_mmay exhibit measurable inhibition in cells at

moderately high concentrations, potentially due to cytotoxicity

SAR of Hinge Binding Position. To further explore the SAR

of 15, we modiﬁed the primary amine residue of the 2-

E

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Figure 4. Potency and selectivity of K02288 derivatives based on thermal shift, biochemical kinase activity, and ligand induced transcriptional activity

assays. (a) The 2-aminopyridine scaﬀold of 15. (b) Modiﬁcations to the primary amine kinase hinge binding domain (R₃) of compound 15. (c)

Thermal shift (ΔT_m), biochemical enzymatic inhibition (IC₅₀) for ALK2 and ALK5 kinase proteins, and inhibition of cell-based BMP6 and TGF-β1-

induced transcriptional activity (IC₅₀) by compound 15 derivatives (nd = not determined). (d) Correlation of thermal shift and cell-based BMP/

TGF-β inhibition assays.

at high concentrations (>50 μM, Supporting Information,

Figure 5). Replacing the primary amine with hydrogen (26)

resulted in only a modest decline in potency and signiﬁcantly

increased selectivity for BMP6 versus TGF-β signaling,

suggesting that the primary amine hydrogen bond to the

hinge backbone is more critical for binding to ALK5 (D281)

than ALK2 (H284). Finally, we used two other small

substituents, e.g., chlorine (27) and methyl (10) groups to

explore the possibility that ALK2 is less sensitive than ALK5 to

substituents at this position. Although both compounds lost

potency relative to 26, there was a signiﬁcant increase in

selectivity for BMP over TGF-β signaling, with both showing

greater than 150-fold selectivity in cell-based assays. In

particular, 10 remained relatively potent with a biochemical

IC₅₀of 24 nM for ALK2, a cell-based IC₅₀for BMP6 of

approximately 100 nM, and 164-fold selectivity for BMP6

versus TGF-β1. The activities of compounds 15, 26, and 10 in

the thermal shift kinase assay as well as two diﬀerent cell-based

assays (ligand induced transcription and constitutively active

type I receptor transcriptional activity) are summarized in

Supporting Information, Table 2. In each of the various assays,

compound 10 (LDN-214117) exhibited improved selectivity

for ALK2 versus ALK5 signaling, consistent with a high degree

of selectivity for BMP versus TGF-β signaling. In fact, when the

activity of 10 was measured against closely related BMP type I

receptors ALK1−3 via kinase assays, it appeared to inhibit

ALK2 more potently than ALK3 by more than 40-fold (Figure

6a), a degree of selectivity which rivals previously reported

compound LDN-212854.²¹We tested whether or not

compounds with improved receptor selectivity such as 10 or

26 might also exhibit more selective inhibition of BMP ligands.

We found that both of these compounds impacted the

transcriptional activity of a BMP-responsive luciferase reporter

(BRE-Luc) in response to BMP6, a known ALK2 ligand, more

potently than that of BMP2 or BMP4, classic ligands of ALK3

(Figure 6b−d).³⁵In fact, compound 10 inhibited BMP6

preferentially to BMP2 and BMP4 by approximately 10-fold,

whereas K02288 and compound 15 inhibited BMP2, -4, and -6

with equal potency. These data support the concept that

increased selectivity of 10 and 26 for ALK2 translates into

increased selectivity for the activity of ligands which signal

primarily through ALK2, a class which include BMP6 and

BMP7.³⁶These results further highlight that the 2-position of

the pyridine in the K02288 series can be exploited to achieve

reasonably potent and highly selective BMP inhibitors,

presumably via optimization of hinge domain interactions.

SAR of K02288 and LDN-193189 Hybrid Molecules. We

previously described a highly selective pyrazolo[1,5-a]-

pyrimidine based BMP type I receptor kinase inhibitor LDN-

212854 that exhibited biased activity for ALK2. This selectivity

was achieved by a 5-quinoline moiety that interacts with the

same hydrophobic pocket as the 3,4,5-trimethoxy group of

K02288. With this in mind, we synthesized several derivatives

of 15 that replaced the 3,4,5-trimethoxyphenyl with 4- or 5-

F

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Figure 5. Potency and selectivity of K02288 derivatives based on thermal shift, biochemical kinase activity, and ligand induced transcriptional activity

assays. (a) Structure of hybrid derivative molecules. (b) Thermal shift (ΔT_m), biochemical enzymatic inhibition (IC₅₀) for ALK2 and ALK5 kinase

proteins, and inhibition of cell-based BMP6 and TGF-β1-induced transcriptional activity (IC₅₀) by hybrid molecules (nd = not determined). (c)

Correlation of thermal shift and cell-based BMP/TGF-β inhibition assays.

quinolines (Figure 5a). As expected, the 5-quinoline (31)

demonstrated substantially increased selectivity for BMP versus

TGF-β inhibition (Figure 5b). However, all of these

compounds were substantially less potent than 15. Modeling

of these 5-quinoline substituted compounds in the ATP-

binding pocket suggested that binding to the kinase hinge by

the 2-aminopyridine scaﬀold may constrain the quinoline

moiety to a suboptimal position as compared to the

pyrazolo[1,5a]pyrimidine scaﬀolds (Supporting Information,

Figure 6). Conversely, replacing the 5-quinoline of LDN-

212854 with the 3,4,5-trimethoxyphenyl of K02288 yielded 32

that demonstrated potent BMP6 inhibitory activity but with

less selectivity. Finally, hypothesizing that two individual

changes yielding improvements in selectivity might synergize,

we combined the substitutions of the 2-amino group with

hydrogen and the 3,5-dimethoxy group found in 26 and 17 to

yield 33. Although this molecule demonstrated improved

selectivity it had considerably less potency.

Kinome-Wide Selectivity. We previously reported the

kinome-wide selectivity for both K02288 and LDN-193189,

showing that K02288 has a more selective proﬁle with fewer

oﬀ-target kinases inhibited at low (0.1 μM) and high (1.0 μM)

concentrations.^21,23We sought to determine the kinome-wide

selectivity of K02288 derivative compounds 10 (LDN-214117)

and 15, via enzymatic kinase proﬁling of approximately 200

kinases, summarized in the kinome dendrogram shown in

Figure 7. The kinase most highly inhibited by compound 10

(LDN-214117) was ALK2, followed by TNIK, RIPK2, and

ABL1 (detailed results of kinome proﬁling provided in

Supporting Information, Table 3). Although less potent than

15, compound 10 (LDN-214117) demonstrated signiﬁcant

improvement in selectivity across the kinome. At 100 nM and 1

μM, compound 10 inhibited only 0.5% and 3.6% of kinases

proﬁled by more than 50%, whereas compound 15 inhibited

2.1% and 14.4% of kinases proﬁled, respectively. Compound 10

thus exhibited improved kinome selectivity than that previously

reported for LDN-193189, LDN-212854, or K02288.^21,23

FOP Mutations and Inhibitor Binding. The majority of

individuals with FOP harbor the R206H germline mutation

aﬀecting the glycine−serine (GS-) rich regulatory domain of

ALK2.^9,37−39While several of the other known FOP-causing

mutations also involve residues of the GS-domain (i.e., L196P,

R202I, and Q207E), several aﬀect important regulatory sites

within the kinase domain (i.e., G328E/R/W, R258S, G356D,

and R375P).^{11−14,23,40}A subset of both GS-domain and kinase

domain mutations associated with FOP have also been found to

arise somatically in 20−30% of DIPG tumor tissues, frequently

in combination with mutations aﬀecting the loci encoding

histone H3.1¹⁵⁻¹⁸

We and others have shown that much of the enhanced

cellular activity of various FOP-causing ALK2 mutants is

attributable to diﬀerential regulation of the signaling pathway,

i.e., impaired interactions with kinase regulatory protein

FKBP12, and diﬀerential basal versus ligand-induced signaling

activity.^40,41However, there is the possibility that ALK2

mutants have intrinsic diﬀerences in their enzymatic function,

G

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Figure 6. LDN-214117 exhibits selectivity for ALK2-mediated BMP signaling. (a) 10 (LDN-214117) demonstrates selective inhibition of ALK2 and

ALK1 in preference to ALK3 kinase activity. (b) In a cell-based assay measuring BMP-mediated transcription (BRE-Luciferase), K02288 (b) and 15

(c) exhibit relatively limited selectivity for diverse BMP ligands, whereas 26 (d) and 10 (e) exhibit relatively selective inhibition of BMP6 versus

BMP2 or BMP4, consistent with selective inhibition of ALK2- versus ALK3-mediated signaling, respectively.

which could manifest as diﬀerences in aﬃnity for ATP and

altered K_m, with implications for their cellular activity and

susceptibility to inhibitors. We tested this directly by measuring

the K_mfor ATP of wild-type ALK2 and four FOP-causing

ALK2 mutants (L196P, Q207E, G328E, and R258S). The K_m

values for wild type and mutant ALK2 were between 16 and 48

μM (Supporting Information, Table 4). Importantly, none of

the FOP-causing mutants exhibited enhanced aﬃnity for ATP

as compared with wild-type ALK2. Because ATP concen-

trations within cells vary from 1−10 mM,⁴²far in excess of the

calculated K_mvalues, these slight diﬀerences in K_mwould likely

be inconsequential in cells.

A related, long-standing, and clinically relevant question in

the FOP ﬁeld has been whether mutant ALK2 proteins might

exhibit diﬀerential inhibition by, or distinct aﬃnity for,

particular kinase inhibitors, and if so, whether highly speciﬁc

inhibitors could be engineered to target selectively the activity

of these activated mutant proteins. We sought to answer this

question by probing a panel of seven representative mutant

ALK2 proteins with the library of K02288 derivatives

displaying varying potency against wild-type ALK2 using a

thermal shift kinase assay. We found a highly linear correlation

(r²= 0.94−0.99) between the thermal shift induced by these

derivatives with wild-type vs mutant ALK2 proteins (Figure 8).

H

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Figure 7. Compound 10 exhibits increased kinome selectivity. Kinome dendrogram plot for compound 15 (LDN-212838) (a) and compound 10

(LDN-214117) (b) showing an improved selectivity proﬁle for 10 for BMP type I receptor kinases.

selectively target mutant proteins. The pathogenicity of

activating mutations of ACVR1 has been well-established for

FOP, and thus the development of highly selective inhibitors is

an important step in validating ALK2 as a feasible clinical target.

While the presence of ACVR1 mutations appears to be

associated with increased downstream BMP signaling and

transcriptional activity in DIPG tumors,^16,18further work will

need to be performed to determine whether or not these

mutations contribute to the initiation or progression of tumors

in DIPG. The identiﬁcation of selective inhibitors of ALK2 may

help to elucidate the mechanisms of DIPG tumorigenicity and

could potentially be therapeutic if a contribution of BMP

signaling is conﬁrmed.

Cytotoxicity of Kinase Inhibitors. We next sought to

determine the cytotoxicity of these derivatives and to compare

them to many of the current FDA approved kinase inhibitors

(Figure 9). Because hepatotoxicity is one of the most common

reasons for withdrawal of approved drugs, we used HEPG2

cells for evaluation of cytotoxicity.⁴³Compounds were tested in

a large concentration range (1−100 μM) for 4 and 24 h. Upon

the basis of residual cell viability after treatment, compounds

were categorized as having low (>75%), medium (25−75%), or

high (<25%) cytotoxicity. Of the 12 approved kinase inhibitors

tested, only one exhibited high cytotoxicity at 100 μM after 4 h

of incubation, whereas six of the 28 derivatives in our K02288

library exhibited high cytotoxicity after 4 h. Over a 24 h period

binding. (a) Strong correlation of thermal shift data for ATP

Figure 8. FOP-causing ALK2 mutations do not aﬀect inhibitor

four of 12 approved kinase inhibitors showed high cytotoxicity

at 100 μM, whereas 23 of the 28 K02288 derivatives showed

high toxicity. However, 10, which demonstrated good potency

and high BMP selectivity, exhibited very low cytotoxicity. In

fact, cytotoxicity was not correlated with BMP signaling

inhibition, TGF-β inhibition, nor selectivity for BMP versus

TGF-β signaling (Supporting Information, Figure 5). For

example, the highly potent BMP inhibitor 11 was also

noncytotoxic, suggesting that the mechanisms of cytotoxicity

within this series of compounds do not result from eﬀects on

BMP or TGF-β signaling.

competitive kinase inhibitors binding to wild-type ALK2 versus

known FOP causing GS-domain mutations of ALK2 and (b) known

FOP causing kinase domain mutations suggests the potency of ATP

competitive inhibitors are not aﬀected by these disease causing

mutations. m = slope, R²= correlation coeﬃcient.

These results suggest that inhibitors engineered or identiﬁed

against wild-type or mutant ALK2 proteins will have

interchangeable activity against diverse mutant proteins found

in FOP or DIPG. Conversely, these results would preclude the

development of ATP-competitive ALK2 inhibitors which

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Structural Basis of Inhibitor Binding. A number of the most

promising derivatives were tested for co-crystallization with

ALK2 to further understand the binding mode and SAR.

Diﬀraction quality crystals were obtained in the presence of 26,

and the structure of the complex was solved at 2.6 Å resolution

(Figure 9; see Supporting Information, Table 5 for data

collection and reﬁnement statistics).

In the co-crystal structure, 26 was bound to the kinase hinge

region as shown previously for the parent molecule K02288

(Figure 10).²³Both molecules established an ATP-mimetic

hydrogen bond between the pyridine nitrogen and the amide of

H286. Replacement of the 3-phenol and primary amine with 4-

phenylpiperazine and hydrogen, respectively, did not alter the

overall position of 26 but resulted in the loss of the hinge

hydrogen bond interaction between the primary amine and the

carbonyl of H284. The conserved 3,4,5-trimethoxyphenyl

provided hydrophobic interaction as well as a water-mediated

hydrogen bond to the catalytic lysine K235. Docking of 10

produced a similar binding mode, with no signiﬁcant change

resulting from the introduction of the methyl group. Overall,

the ATP pocket occupied by these 3,5-diarylpyridines was

similar to the pyrazolo[1,5-a]pyrimidine scaﬀold of LDN-

193189. However, the two series diﬀered slightly in their hinge

binding orientation resulting in shifts in the position of their

respective hydrophobic pocket groups as well as the shared 4-

phenylpiperazine (Supporting Information, Figure 6).

The selectivity of these molecules for ALK2 over ALK5 likely

results from dynamic conformational diﬀerences between these

kinases as well as the modest number of sequence changes in

the ATP pocket. Perhaps as a result of its smaller serine

gatekeeper residue, the ATP pocket in many ALK5 co-crystal

structures shows a more open conformation than those of

ALK2 with a noticeable movement of the N-lobe away from the

C-lobe (Supporting Information, Figure 1). Such conforma-

tional diﬀerences are expected to change the shape, volume,

and dynamics of the ATP pocket to impact inhibitor binding.

CONCLUSION

■

We have developed a library of BMP receptor kinase inhibitors

based on the 2-aminopyridine scaﬀold of K02288. This library

allowed us to explore the SAR of various functional groups and

resulted in the creation of several potent derivatives. Several of

these compounds demonstrated improved activity, selectivity,

or both, measured using a thermal shift assay, an enzymatic

assay, and cellular assays of BMP/TGF-β-induced transcription,

Figure 9. Cytotoxicity of FDA-approved kinase inhibitor compounds

as compared with BMP inhibitor compounds. Cultured HepG2 cells

were exposed to 1, 10, and 100 μM concentrations of compounds for 4

and 24 h. The average cell viability of three experiments is shown

(green indicating >75%, orange indicating 25−75%, and red <25%

viability).

Figure 10. Binding mode of 26. (a) The inhibitor (yellow) forms a single hydrogen bond to the hinge amide of H286 as well as a water-mediated

bond to the catalytic lysine K235. (b) Plot of the interactions of the inhibitor (purple) in the binding pocket of ALK2. The plot was generated by

LigPlot+.⁵⁴

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analyses using an instrument equipped with a quaternary pump and a

SB-C8 column (30 mm × 4.6 mm, 3.5 μm). UV absorption was

monitored at λ = 254 nm. The injection volume was 5 μL. HPLC

gradient went from 5% acetonitrile/95% water to 95% acetonitrile/5%

water (both solvents contain 0.1% triﬂuoroacetic acid) over 1.9 min

with a total run time of 3.0 min and a ﬂow rate of 3.0 mL/min.

Synthesis of 2-Amino-5-bromo-3-(3,4,5-trimethoxyphenyl)-

pyridine (2). A mixture of 5-bromo-3-iodopyridin-2-amine (386 mg,

1.30 mmol), 3,4,5-trimethoxyphenylboronic acid (275 mg, 1.30

mmol), and Pd(PPh₃)₄(180 mg, 0.156 mmol) were added to a

sealed tube. The tube was evacuated and backﬁlled with argon (3

cycles). Acetonitrile (6.0 mL) and DMF (2.5 mL) were added by

syringe at room temperature, followed by (1 M) aqueous Na₂CO₃(2.6

mL, 2.60 mmol). After being stirred at 90 °C for about 8 h, the

reaction mixture was ﬁltered and concentrated. The residue was

puriﬁed by ﬂash column chromatography to give 2 as white solid (235

thus overcoming the limited potency of the parent compound

in cells.

We determined that the solvent-exposed 3-phenol sub-

stituent of K02288 was responsible for its unexpectedly low

activity in cells as compared to kinase assay IC₅₀. By replacing

this group with either a 4-phenol or 4-phenylpiperazine, we

were able to improve potency in cellular assays compared to

K02288 by 20- and 100-fold, respectively. We previously

reported that the 3,4,5-trimethoxyphenyl occupies the rear

hydrophobic pocket to provide excellent shape complementar-

ity and forms water-mediated hydrogen bonds to the catalytic

lysine residue (K235).²³Here we found that the 4-methoxy

group was largely dispensable, while the 3- or 5-methoxy

groups were more critical for maintaining potency. The balance

of selectivity and potency found in the 3,5-dimethoxy derivative

17 suggests further medicinal chemistry optimization is possible

and could yield further insights into the determinants of activity

in the hydrophobic pocket. Within the 2-aminopyridine core,

we found that the primary amine was more critical for TGF-β

than BMP binding aﬃnity and could be replaced with a

nonpolar methyl group to generate a highly BMP selective

compound 10 (LDN-214117), which is signiﬁcantly biased

toward ALK2 and its cognate ligands including BMP6 and also

demonstrates a high degree of kinome selectivity and low

cytotoxicity. Finally, we concluded that replacing the 3,4,5-

trimethoxyphenyl with quinolines as previously described for

pyrazolopyrimidine compounds (LDN-193189 and LDN-

212854) was not an eﬀective strategy and resulted in a

substantial loss of potency.

We used this structurally diverse compound series with

varying degrees of potency to explore the eﬀect of FOP-causing

mutations on inhibitor binding aﬃnity. These compounds

exhibited nearly identical binding aﬃnity for wild-type ALK2

and each of the FOP-causing mutants tested, demonstrating

that ATP-competitive inhibitors active against wild-type protein

will eﬀectively target diverse FOP mutants. While this result

would also suggest that ATP-competitive inhibitors cannot

speciﬁcally target mutant versus wild-type ALK2, one could

envision molecules targeting allosteric sites unique to mutant

proteins to potentially achieve speciﬁcity.

1

mg, 80%). H NMR (500 MHz, CDCl₃) δ 8.11 (d, J = 2.5 Hz, 1H),

7.48 (d, J = 2.5 Hz, 1H), 6.62 (s, 2H), 3.90 (s, 3H), 3.88 (s, 6H). MS

(ESI): 339.0 [M]⁺.

General Synthesis of 2-Amino-5-aryl-3-(3,4,5-

trimethoxyphenyl)pyridines (3). To a solution of 2 (1.0 equiv),

an aryl boronic acid (1.1 equiv), and Pd(PPh₃)₄(0.12 equiv) in DME,

(1 M) aqueous Na₂CO₃(2.0 equiv) was added. The reaction mixture

was stirred under an argon atmosphere at 90 °C for 8 h. The reaction

mixture was ﬁltered and then concentrated. The residue was puriﬁed

by ﬂash column chromatography, eluting with a mixture of

cyclohexane and EtOAc to give products 3.

3-(6-Amino-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl)phenol

(K02288). Yield: 40%. ¹H NMR (500 MHz, CDCl₃) δ 8.48 (d, J = 2.0

Hz, 1H), 7.69 (d, J = 2.0 Hz, 1H), 7.34 (t, J = 7.5 Hz, 1H), 7.20 (d, J =

2.0 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 6.90 (dd, J = 2.0, 7.0 Hz, 1H),

6.68 (s, 2H), 4.81 (br, 2H), 3.91 (s, 3H), 3.89 (s, 6H). HRMS (ESI)

calcd for C₂₀H₂₁N₂O₄353.1501 [M + H]⁺; found 353.1462; purity

95.6% (t_R1.35 min).

4-(6-Amino-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl)phenol

1

(11). Yield: 42%. H NMR (500 MHz, CDCl₃) δ 8.27 (d, J = 2.5 Hz,

1H), 7.57 (d, J = 2.5 Hz, 1H), 7.43−7.41 (m, 2H), 6.92−6.90 (m,

2H), 6.90 (dd, J = 2.0, 7.0 Hz, 1H), 6.69 (s, 2H), 4.64 (br, 2H), 3.91

(s, 3H), 3.89 (s, 6H). HRMS (ESI) calcd for C₂₀H₂₁N₂O₄353.1501

[M + H]⁺; found 353.1490; purity 100.0% (t_R1.32 min).

4-(6-Amino-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl)-2-me-

1

thoxyphenol (12). Yield: 70%. H NMR (500 MHz, CDCl₃) δ 8.27

(d, J = 2.5 Hz, 1H), 7.56 (d, J = 2.5 Hz, 1H), 7.06−6.98 (m, 3H), 6.70

(s, 2H), 4.65 (br, 2H), 3.95 (s, 3H), 3.91 (s, 3H), 3.89 (s, 6H). HRMS

(ESI) calcd for C₂₁H₂₃N₂O₅383.1607 [M + H]⁺; found 383.1603;

purity 98.3% (t_R1.34 min).

N-(3-(6-Amino-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl)-

phenyl)methanesulfonamide (13). Yield: 85%. ¹H NMR (500

MHz, CDCl₃) δ 8.89 (br, 1H), 8.39 (d, J = 2.5 Hz, 1H), 7.61 (d, J =

1.5 Hz, 1H), 7.47−7.40 (m, 3H), 7.34 (dt, J = 1.5, 7.5 Hz, 1H), 6.68

(s, 2H), 5.19 (br, 2H), 3.91 (s, 3H), 3.90 (s, 6H) 3.06 (s, 3H). HRMS

(ESI) calcd for C₂₁H₂₄N₃O₅S 430.1437 [M + H]⁺; found 430.1412;

purity 99.3% (t_R1.34 min).

General Synthesis of 3-Aryl-5-bromopyridines (5). A mixture

of 5-bromo-3-iodopyridin-2-amine (1.0 equiv), arylboronic acid (1.0

equiv), and Pd(PPh₃)₄(0.12 equiv) was added to a sealed tube. The

tube was evacuated and backﬁlled with argon (3 cycles). Acetonitrile

and DMF (3:1 mL) were added by syringe at room temperature,

followed by (1 M) aqueous Na₂CO₃(2.0 equiv). After being stirred at

90 °C for about 8 h, the reaction mixture was ﬁltered and

concentrated. The residue was puriﬁed by ﬂash column chromatog-

raphy to give 5.

General Synthesis of 3-Aryl-5-((N-Boc)-piperazinylphenyl)-

pyridines (6). To a solution of 5 (1.0 equiv), [(N-Boc)piperazin-1-

yl]phenylboronic acid pinacol ester (1.1 equiv), and Pd(PPh₃)₄(0.12

equiv) in DME, (1 M) aqueous Na₂CO₃(2.0 equiv) was added. The

reaction mixture was stirred under argon atmosphere at 90 °C for 8 h.

The reaction mixture was ﬁltered and concentrated. The residue was

puriﬁed by ﬂash column chromatography, eluting with a mixture

cyclohexane/EtOAc to give 6.

The novel series of compounds reported here constitutes an

alternative pharmacophore with discrete properties, including

distinct kinome selectivity, as compared to the pyrazolopyr-

imidine class of BMP inhibitors.²³Several of these compounds,

including 10 (LDN-214117), may be useful as highly selective

probes of BMP-mediated cellular physiology that may provide a

useful complement to the dorsomorphin class of compounds.

Furthermore, this class of BMP inhibitors oﬀers a structurally

distinct template for the development of therapeutics for the

treatment of BMP signaling-mediated diseases such as FOP.

EXPERIMENTAL SECTION

■

Chemistry Material and Methods. Unless otherwise noted, all

reagents and solvents were purchased from commercial sources and

used without further puriﬁcation. The NMR spectra were obtained

using a 300 or 500 MHz spectrometer. All ¹H NMR spectra are

reported in δ units (ppm) and were recorded in CDCl₃and referenced

to the peak for tetramethylsilane (TMS) or in DMSO. Coupling

constants (J) are reported in hertz. Column chromatography was

performed utilizing a CombiFlash Sg 100c separation system with

RediSep disposable silica gel columns. High-resolution mass spectra

were obtained by using AccuTOF with a DART source. All test

compounds reported in this manuscript had a purity ≥95% as

determined by high-performance liquid chromatography (HPLC)

K

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General Synthesis of 3-Aryl-5-(piperazinylphenyl)pyridines

(7). To a stirring solution of the 6 (0.01 mmol) in dry CH₂Cl₂(2 mL)

at 0 °C, triﬂuoroacetic acid (0.2 mL) was slowly added and the

reaction mixture was stirred overnight at room temperature. The

mixture was concentrated under vacuum. The residue was suspected in

ethyl acetate (10 mL), and then a saturated NaHCO₃solution was

added to adjust the pH to 7 at 0 °C. The mixture was extracted with

ethyl acetate (3 × 10 mL). The combined organic layer was dried over

anhydrous Na₂SO₄, ﬁltered, and concentrated in vacuo. The remaining

residue was subjected to column chromatography to furnish 7 as a

white to light-yellow foam.

3-(4-Chloro-3-methoxyphenyl)-5-(4-(piperazin-1-yl)phenyl)-

1

pyridin-2-amine (22). Yield: 82%. H NMR (300 MHz, CDCl₃) δ

8.27 (d, J = 2.1 Hz, 1H), 7.57 (d, J = 2.4 Hz, 1H), 7.48−7.44 (m, 3H),

7.04−6.98 (m, 4H), 4.83 (br, 2H), 3.94 (s, 3H), 3.32−3.29 (m, 3.6H)

and 3.26−3.22 (m, 0.4H) due to rotamer, 3.18−3.15 (m, 3.6H) and

2.74−2.69 (m, 0.4H) due to rotamer. HRMS (ESI) calcd for

C₂₂H₂₄ClN₄O 395.1639 [M + H]⁺; found 395.1647; purity 96.6%

(t_R1.18 min).

3-(3-Methoxy-4-methylphenyl)-5-(4-(piperazin-1-yl)phenyl)-

1

pyridin-2-amine (23). Yield: 84%. H NMR (300 MHz, CDCl₃) δ

8.28 (d, J = 2.4 Hz, 1H), 7.58 (d, J = 2.4 Hz, 1H), 7.47−7.44 (m, 2H),

7.24−7.21 (m, 1H), 7.00−6.97 (m, 3H), 6.93 (d, J = 1.5 Hz, 1H), 4.65

(br, 2H), 3.85 (s, 3H), 3.20−3.16 (m, 4H), 3.07−3.03 (m, 4H), 2.67

(s, 3H). HRMS (ESI) calcd for C₂₃H₂₇N₄O 375.2185 [M + H]⁺;

found 375.2189; purity 100.0% (t_R1.16 min).

N-Methyl-5-(4-(piperazin-1-yl)phenyl)-3-(3,4,5-

trimethoxyphenyl)pyridin-2-amine (24). Yield: 92%. ¹H NMR

(500 MHz, CDCl₃) δ 8.39 (d, J = 2.0 Hz, 1H), 7.50 (d, J = 2.5 Hz,

1H), 7.46−7.45 (m, 2H), 7.00−6.98 (m, 2H), 6.63 (s, 2H), 4.67 (q, J

= 5.0 Hz, 1H), 3.91 (s, 3H), 3.88 (s, 6H), 3.25−3.22 (m, 0.6H) and

3.20−3.18 (m, 3.4H) due to rotamer, 3.08−3.06 (m, 3.6H) and 2.72−

2.70 (m, 0.4H) due to rotamer, 3.01 (d, J = 5.0 Hz, 3H). HRMS (ESI)

calcd for C₂₅H₃₁N₄O₃435.2396 [M + H]⁺; found 435.2396; purity

98.9% (t_R1.10 min).

N,N-Dimethyl-5-(4-(piperazin-1-yl)phenyl)-3-(3,4,5-

trimethoxyphenyl)pyridin-2-amine (25). Yield: 90%. ¹H NMR

(500 MHz, CDCl₃) δ 8.40 (d, J = 2.0 Hz, 1H), 7.62 (d, J = 2.5 Hz,

1H), 7.48−7.46 (m, 2H), 7.00−6.99 (m, 2H), 6.76 (s, 2H), 3.90 (s,

3H), 3.88 (s, 6H), 3.21−3.19 (m, 4H), 3.07−3.05 (m, 4H), 2.78 (s,

6H). HRMS (ESI) calcd for C₂₆H₃₃N₄O₃449.2553 [M + H]⁺; found

449.2575; purity 97.8% (t_R1.14 min).

1-(4-(5-(3,4,5-Trimethoxyphenyl)pyridin-3-yl)phenyl)-

piperazine (26). Yield: 95%. ¹H NMR (500 MHz, CDCl₃) δ 8.78 (d,

J = 2.0 Hz, 1H), 8.71 (d, J = 2.5 Hz, 1H), 7.95 (t, J = 2.5 Hz, 1H),

7.58−7.56 (m, 2H), 7.06−7.04 (m, 2H), 6.80 (s, 2H), 3.95 (s, 6H),

3.91 (s, 3H), 3.25−3.23 (m, 4H), 3.08−3.06 (m, 4H). HRMS (ESI)

calcd for C₂₄H₂₈N₃O₃406.2131 [M + H]⁺; found 406.2142; purity

100.0% (t_R1.20 min).

1-(4-(6-Chloro-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl)-

phenyl)piperazine (27). Yield: 94%. ¹H NMR (300 MHz, CDCl₃) δ

8.57 (d, J = 2.4 Hz, 1H), 7.83 (d, J = 2.7 Hz, 1H), 7.53−7.50 (m, 2H),

7.03−7.00 (m, 2H), 6.70 (s, 2H), 3.92 (s, 3H), 3.90 (s, 6H), 3.32−

3.28 (m, 0.5H) and 3.27−3.24 (m, 3.5H) due to rotamer, 3.10−3.07

(m, 3.5H) and 2.75−2.69 (m, 0.5H) due to rotamer. HRMS (ESI)

calcd for C₂₄H₂₇ClN₃O₃440.1741 [M + H]⁺; found 440.1723; purity

95.6% (t_R1.42 min).

1-(4-(6-Methoxy-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl)-

phenyl)piperazine (28). Yield: 79%. ¹H NMR (500 MHz, CDCl₃) δ

8.34 (d, J = 2.0 Hz, 1H), 7.78 (d, J = 2.5 Hz, 1H), 7.50−7.48 (m, 2H),

7.03−7.01 (m, 2H), 6.81 (s, 2H), 4.02 (s, 3H), 3.90 (s, 9H), 3.28−

3.26 (m, 0.3H) and 3.23−3.21 (m, 3.7H) due to rotamer, 3.08−3.07

(m, 3.7H) and 2.73−2.71 (m, 0.3H) due to rotamer. HRMS (ESI)

calcd for C₂₅H₂₉N₃O₄436.2236 [M + H]⁺; found 436.2265; purity

100.0% (t_R1.38 min).

5-(3-(Piperazin-1-yl)phenyl)-3-(3,4,5-trimethoxyphenyl)-

1

pyridin-2-amine (14). Yield: 82%. H NMR (500 MHz, CDCl₃) δ

8.31 (d, J = 2.5 Hz, 1H), 7.61 (d, J = 2.5 Hz, 1H), 7.35 (d, J = 8.0 Hz,

1H), 7.07 (t, J = 2.0 Hz, 1H), 7.04−7.03 (m, 1H), 6.92−6.90 (m, 1H),

6.70 (s, 2H), 4.68 (br, 2H), 3.91 (s, 3H), 3.89 (s, 6H), 3.21−3.19 (m,

4H), 3.06−3.04 (m, 4H). HRMS (ESI) calcd for C₂₄H₂₈N₄O₃

421.2240 [M + H]⁺; found 421.2215; purity 98.7% (t_R1.12 min).

5-(4-(Piperazin-1-yl)phenyl)-3-(3,4,5-trimethoxyphenyl)-

1

pyridin-2-amine (15). Yield: 77%. H NMR (500 MHz, CDCl₃) δ

8.29 (d, J = 2.0 Hz, 1H), 7.58 (d, J = 2.5 Hz, 1H), 7.47−7.45 (m, 2H),

7.00−6.98 (m, 2H), 6.70 (s, 2H), 4.61 (br, 2H), 3.91 (s, 3H), 3.89 (s,

6H), 3.26−3.24 (m, 0.6H) and 3.20−3.18 (m, 3.4H) due to rotamer,

3.07−3.05 (m, 3.4H) and 2.72−2.70 (m, 0.6H) due to rotamer.

HRMS (ESI) calcd for C₂₄H₂₈N₄O₃421.2240 [M + H]⁺; found

421.2259; purity 98.6% (t_R1.05 min).

3-(3,4-Dimethoxyphenyl)-5-(4-(piperazin-1-yl)phenyl)-

1

pyridin-2-amine (16). Yield: 80%. H NMR (500 MHz, CDCl₃) δ

8.28 (d, J = 2.5 Hz, 1H), 7.57 (d, J = 2.0 Hz, 1H), 7.47−7.45 (m, 2H),

7.06−7.04 (m, 1H), 7.01−6.97 (m, 4H), 4.58 (br, 2 H), 3.94 (s, 3 H),

3.91 (s, 3 H), 3.26−3.24 (m, 0.3H) and 3.20−3.18 (m, 3.7H) due to

rotamer, 3.07−3.05 (m, 3.7H) and 2.72−2.70 (m, 0.3H) due to

rotamer. HRMS (ESI) calcd for C₂₃H₂₇N₄O₂391.2134 [M + H]⁺;

found 391.2142; purity 97.9% (t_R1.08 min).

3-(3,5-Dimethoxyphenyl)-5-(4-(piperazin-1-yl)phenyl)-

1

pyridin-2-amine (17). Yield: 85%. H NMR (500 MHz, CDCl₃) δ

8.27 (d, J = 2.5 Hz, 1H), 7.59 (d, J = 2.5 Hz, 1H), 7.46−7.44 (m, 2H),

7.00−6.98 (m, 2H), 6.63 (d, J = 2.0 Hz, 2H), 6.50 (t, J = 2.5 Hz, 1H),

4.76 (br, 2H), 3.83 (s, 6H), 3.21−3.19 (m, 4H), 3.08−3.06 (m, 4H).

HRMS (ESI) calcd for C₂₃H₂₇N₄O₂391.2134 [M + H]⁺; found

391.2159; purity 97.7% (t_R1.16 min).

3-(3-Methoxyphenyl)-5-(4-(piperazin-1-yl)phenyl)pyridin-2-

1

amine (18). Yield: 82%. H NMR (500 MHz, CDCl₃) δ 8.28 (d, J =

2.0 Hz, 1H), 7.59 (d, J = 2.0 Hz, 1H), 7.46−7.44 (m, 2H), 7.41 (t, J =

8.0 Hz, 1H), 7.09−7.07 (m, 1H), 7.03 (t, J = 2.0 Hz, 1H), 7.00−6.98

(m, 2H), 6.95 (dd, J = 2.0, 8.5 Hz, 1H), 4.69 (br, 2 H), 3.85 (s, 3H),

3.26−3.24 (m, 0.7H) and 3.22−3.20 (m, 3.3H) due to rotamer, 3.09−

3.07 (m, 3.3H) and 2.72−2.70 (m, 0.7H) due to rotamer. HRMS

(ESI) calcd for C₂₂H₂₅N₄O 361.2028 [M + H]⁺; found 361.2043;

purity 97.5% (t_R1.16 min).

3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(4-(piperazin-1-

1

yl)phenyl)pyridin-2-amine (19). Yield: 80%. H NMR (500 MHz,

CDCl₃) δ 8.25 (d, J = 2.5 Hz, 1H), 7.54 (d, J = 2.0 Hz, 1H), 7.45−7.43

(m, 2H), 7.02−7.01 (m, 1H), 6.99−6.96 (m, 4H), 4.63 (br, 2H), 4.31

(s, 4 H), 3.25−3.23 (m, 0.8H) and 3.20−3.18 (m, 3.2H) due to

rotamer, 3.07−3.05 (m, 3.2H) and 2.72−2.70 (m, 0.8H) due to

rotamer. HRMS (ESI) calcd for C₂₃H₂₅N₄O₂389.1978 [M + H]⁺;

found 389.2003; purity 97.0% (t_R1.16 min).

5-(4-(Piperazin-1-yl)phenyl)-3-(quinolin-4-yl)pyridin-2-

1

amine (29). Yield: 79%. H NMR (500 MHz, CDCl₃) δ 9.02 (d, J =

4.5 Hz, 1H), 8.46 (d, J = 2.5 Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 7.79−

7.75 (m, 2H), 7.64 (d, J = 2.5 Hz, 1H), 7.57−7.53 (m, 1H), 7.48−7.43

(m, 3H), 7.01−6.98 (m, 2H), 4.34 (br, 2H), 3.25−3.23 (m, 0.6H) and

3.20−3.18 (m, 3.4H) due to rotamer, 3.07−3.05 (m, 3.4H) and 2.72−

2.70 (m, 0.6H) due to rotamer. HRMS (ESI) calcd for C₂₄H₂₄N₅

382.2032 [M + H]⁺; found 382.1993; purity 97.7% (t_R1.04 min).

5-(3-(Piperazin-1-yl)phenyl)-3-(quinolin-4-yl)pyridin-2-

3-(4-Methoxyphenyl)-5-(4-(piperazin-1-yl)phenyl)pyridin-2-

1

amine (20). Yield: 78%. H NMR (500 MHz, CDCl₃) δ 8.27 (d, J =

2.5 Hz, 1H), 7.55 (d, J = 2.0 Hz, 1H), 7.46−7.42 (m, 4H), 7.02−6.98

(m, 4H), 4.55 (br, 2H), 3.87 (s, 3H), 3.19−3.18 (m, 4H), 3.07−3.05

(m, 4H). HRMS (ESI) calcd for C₂₂H₂₅N₄O 361.2028 [M + H]⁺;

found 361.2055; purity 97.7% (t_R1.20 min).

1

amine (30). Yield: 85%. H NMR (500 MHz, CDCl₃) δ 9.03 (d, J =

4.0 Hz, 1H), 8.48 (s, 1 H), 8.22 (d, J = 8.0 Hz, 1H), 7.80−7.74 (m,

2H), 7.66 (s, 1 H), 7.57 (d, J = 8.0 Hz, 1H), 7.47−7.44 (m, 1H), 7.35

(t, J = 8.0 Hz, 1H), 7.07−7.03 (m, 2H), 6.92 (d, J = 8.5 Hz, 1H), 4.38

(br, 2 H), 3.26−3.22 (m, 1H) and 3.21−3.19 (m, 3H) due to rotamer,

3.05−3.04 (m, 3H) and 2.71−2.66 (m, 1H) due to rotamer. HRMS

(ESI) calcd for C₂₄H₂₄N₅382.2032 [M + H]⁺; found 382.2024; purity

95.1% (t_R1.09 min).

3-(3-Isopropoxyphenyl)-5-(4-(piperazin-1-yl)phenyl)pyridin-

2-amine (21). Yield: 80%. ¹H NMR (300 MHz, CDCl₃) δ 8.29 (d, J =

2.4 Hz, 1H), 7.58 (d, J = 2.4 Hz, 1H), 7.47−7.44 (m, 2H), 7.39 (d, J =

8.4 Hz, 1H), 7.06−6.97 (m, 4H), 6.93−6.89 (m, 1H), 4.63−4.55 (m,

3H), 3.20−3.17 (m, 4H), 3.07−3.04 (m, 4H), 1.37 (d, J = 6.0 Hz,

6H). HRMS (ESI) calcd for C₂₄H₂₉N₄O 389.2341 [M + H]⁺; found

389.2362; purity 100.0% (t_R1.16 min).

L

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Article

5-(4-(Piperazin-1-yl)phenyl)-3-(quinolin-5-yl)pyridin-2-

amine (31). Yield: 84%. ¹H NMR (500 MHz, CDCl₃) δ 8.98 (dd, J =

2.0, 4.5 Hz, 1H), 8.44 (d, J = 2.5 Hz, 1H), 8.21 (d, J = 9.0 Hz, 1H),

8.06−8.04 (m, 1H), 7.84−7.81 (m, 1H), 7.63 (d, J = 2.5 Hz, 1H),

7.59−7.58 (m, 1H), 7.48−7.46 (m, 2H), 7.41−7.39 (m, 1H), 7.00−

6.97 (m, 2H), 4.29 (br, 2 H), 3.25−3.23 (m, 0.4H) and 3.20−3.18 (m,

3.6H) due to rotamer, 3.06−3.04 (m, 3.6H) and 2.71−2.69 (m, 0.4H)

due to rotamer. HRMS (ESI) calcd for C₂₄H₂₄N₅382.2032 [M + H]⁺;

found 382.2039; purity 95.2% (t_R0.97 min).

1-(4-(5-(3,5-Dimethoxyphenyl)pyridin-3-yl)phenyl)-

piperazine (33). Yield: 98%. ¹H NMR (300 MHz, CDCl₃) δ 8.79 (d,

J = 2.4 Hz, 1H), 8.73 (d, J = 2.4 Hz, 1H), 7.99 (t, J = 2.1 Hz, 1H),

7.57−7.54 (m, 2H), 7.04−7.02 (m, 2H), 6.76 (d, J = 1.8 Hz, 2H), 6.53

(t, J = 2.1 Hz, 1H), 3.86 (s, 6H), 3.23−3.21 (m, 4H), 3.06−3.04 (m,

4H). HRMS (ESI) calcd for C₂₃H₂₆N₃O₂376.2025 [M + H]⁺; found

376.2023; purity 100.0% (t_R1.26 min).

of ALK2 in complex with 26 grew in a 150 nL drop, mixing the

protein, preincubated with 1 mM compound, with a reservoir solution

containing 0.2 M ammonium citrate and 20% PEG 3350 at 2:1 volume

ratio. Crystals were transferred into a cryoprotective solution prepared

from the mother liquor supplemented with 25% ethylene glycol.

Diﬀraction data were collected at Diamond Light Source, beamline

I04-I, and were processed and scaled with MOSFLM and AIMLESS

from the CCP4 suite.⁴⁵The crystals formed in space group I121 and

contained four protein molecules in the asymmetric unit. The

structure was solved by molecular replacement using PHASER⁴⁶and

the structure of the ALK2-LDN-193189 complex (PDB 3Q4U) as a

search model. Subsequent manual model building was performed

using COOT⁴⁷alternated with reﬁnement in REFMAC.⁴⁸TLS-

restrained reﬁnement was applied in the latter cycles using the input

thermal motion parameters determined by the TLSMD server.⁴⁹The

ﬁnal model was veriﬁed for geometry correctness with PHENIX

validation tools and MOLPROBITY.^50,51Data collection and

reﬁnement statistics are summarized in Supporting Information,

Table 5.

Kinase Assay. Puriﬁed recombinant ALK2 and ALK5 kinase

proteins (Invitrogen), ATP (Sigma), ATP [γ-³²P] (PerkinElmer), and

dephosphorylated casein (Sigma) at ﬁnal concentrations of 2.5 nM, 6

μM, 0.05 μCi μL⁻¹, and 0.5 mg mL⁻¹, respectively, were aliquoted in

kinase buﬀer (Cell Signaling) containing 0.2% bovine serum albumin

supplemented with 10 mM MnCl₂into 96-microwell plates, in

combination with inhibitor compounds diluted at varying concen-

trations in kinase buﬀer (0.01 nM to 100 μM) in triplicate. In other

experiments, puriﬁed recombinant ALK2 mutant kinase proteins³⁴

were incubated with γ-³²P and substrate under similar conditions, but

in the presence of varying concentrations of unlabeled ATP, for the

determination of K_mand V_maxfor each ALK2 mutant. Positive control

samples lacking inhibitor compounds, and negative controls lacking

recombinant kinase, were also measured in triplicate. The mixture was

reacted at RT for 45 min, quenched with a ﬁnal concentration of 2%

phosphoric acid. The reaction mixture was transferred to 96-well P81

phosphocellulose ﬁlter plates (Millipore) and bound for 5 min. The

plates were washed 20 times with 150 μL of 1% phosphoric acid

solution per well by vacuum manifold. Plates were dried at RT for 1 h,

sealed, and assayed with Microscint 20 scintillation ﬂuid (PerkinElm-

er) using a Spectramax L luminometer (Molecular Devices) using the

photon counting setting with an integration time of one second per

well. Data was normalized to positive controls at 100% enzyme

activity, with negative controls being subtracted as background.

GraphPad (Prism software) was used for graphing and regression

analysis by sigmoidal dose−response with variable Hill coeﬃcient, or

by Michaelis−Menten analysis for the determination of K_m.

Luciferase Reporter Assay. C2C12 myoﬁbroblasts cells stably

transfected with BMP responsive element from the Id1 promoter

fused to luciferase reporter gene (BRE-Luc) were generously provided

by Dr. Peter ten Dijke (Leiden University Medical Center, NL).⁵²

Human embryonic kidney 293T cells stably transfected with the TGF-

β responsive element from the PAI-1 promoter fused to luciferase

reporter gene (CAGA-Luc) were generously provided by Dr. Howard

Weiner (Brigham and Women’s Hospital, Boston, MA).⁵³C2C12 Bre-

Luc and 293T CAGA-Luc cells were seeded in DMEM supplemented

with 2% FBS at 20000 cells per well in tissue culture treated 96-well

plates (Costar 3610; Corning). The cells were incubated for 1 h (37

°C and 10% CO₂) and allowed to settle and attach. Compounds of

interest or DMSO were diluted in DMEM and added at ﬁnal

compound concentrations of 1 nM to 10 μM. Cells were then

incubated for 30 min. Adenovirus expressing constitutively active BMP

and TGF-β type I receptors (Ad.caALK1−5), generously provided by

Dr. Akiko Hata (University of California at San Francisco), were

added to achieve a multiplicity of infection (MOI) of 100. Plates were

incubated overnight at 37 °C. Cell viability was assayed with an 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colori-

metric assay (Promega) per the manufacturer’s instructions. Media

was discarded, and ﬁreﬂy luciferase activity was measured (Promega)

according to manufacturer’s protocol. Light output was measured

using a Spectramax L luminometer (Molecular Devices) with an

Synthesis of 1-(4-(6-Methyl-5-(3,4,5-trimethoxyphenyl)-

pyridin-3-yl)phenyl)piperazine (10). A mixture of N-Boc-4-(4-(6-

chloro-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl)phenyl)piperazine (43

mg, 0.080 mmol), trimethylboroxine (46 μL, 0.32 mmol), Pd(PPh₃)₄

(19 mg, 0.016 mmol), and K₂CO₃(22 mg, 0.16 mmol) were added to

a sealed tube. The tube was evacuated and backﬁlled with argon (3

cycles). 1,4-Dioxane (1.0 mL) was added by syringe at room

temperature. After being stirred at 110 °C for 8 h, the reaction

mixture was ﬁltered and concentrated. The residue puriﬁed by ﬂash

column chromatography to give 9 (40 mg, 96%). ¹H NMR (300 MHz,

CDCl₃) δ 8.70 (d, J = 2.4 Hz, 1H), 7.69 (d, J = 2.4 Hz, 1H), 7.54−7.51

(m, 2H), 7.02−6.99 (m, 2H), 6.55 (s, 2H), 3.91 (s, 3H), 3.88 (s, 6H),

3.61−3.58 (m, 4H), 3.21−3.18 (m, 4H), 2.55 (s, 3H), 1.48 (s, 9H).

MS (ESI): 519.5 [M]⁺. The carbamate protecting group of 9 (40 mg)

was removed using the general method previously described using

1

TFA to furnish 10 as a white foam (30 mg, 93%). H NMR (300

MHz, CDCl₃) δ 8.71 (d, J = 2.1 Hz, 1H), 7.70 (d, J = 2.4 Hz, 1H),

7.55−7.52 (m, 2H), 7.03−7.00 (m, 2H), 6.56 (s, 2H), 3.92 (s, 3H),

3.89 (s, 6H), 3.24−3.20 (m, 4H), 3.08−3.05 (m, 4H), 2.55 (s, 3H).

HRMS (ESI) calcd for C₂₅H₃₀N₃O₃420.2287 [M + H]⁺; found

420.2295; purity 95.5% (t_R1.13 min).

Synthesis of 3-(3,4,5-Trimethoxyphenyl)-6-[4-(1-

piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidine (32). This com-

pound was prepared using a reported methodology.⁹¹H NMR (500

MHz, DMSO) δ 9.38 (d, J = 2.0 Hz, 1H), 9.04 (d, J = 2.0 Hz, 1H),

8.80 (s, 1H), 7.75 (d, J = 9.0 Hz, 2H), 7.51 (s, 2H), 7.07 (d, J = 9.0

Hz, 1H), 3.88 (s, 6H), 3.70 (s, 3H), 3.25−3.18 (m, 4H), 2.92−2.90

(m, 4H). HRMS (ESI) calcd for C₂₅H₂₇N₅O₃446.2192 [M + H]+;

found 446.2186; purity 100% (t_R1.43 min).

Thermal Shift Kinase Assay. Thermal melting experiments were

performed using a Real Time PCR machine Mx3005p (Stratagene)

with a protein concentration of 1−2 μM and 10 μM inhibitor as

described previously.⁴⁴Recombinant human kinases for DSF screening

were prepared by SGC using published methods.²³

Protein Expression and Puriﬁcation. The human ALK2 kinase

domain, residues 201−499 including the activating mutation Q207D,

was subcloned into the vector pFB-LIC-Bse. Baculoviral expression

was performed in Sf9 insect cells at 27 °C, shaking at 110 rpm. Cells

were harvested at 72 h postinfection and resuspended in 50 mM

HEPES pH 7.5, 500 mM NaCl, 5 mM imidazole, 5% glycerol, 0.1 mM

TCEP, supplemented with protease inhibitor set V (Calbiochem).

Cells were lysed using a C5 high pressure homogenizer (Emulsiﬂex)

and the insoluble material excluded by centrifugation at 21000 rpm.

Nucleic acids were removed on a DEAE-cellulose column before

puriﬁcation of the N-terminally His-tagged ALK2 protein by Ni-

aﬃnity chromatography. The eluted protein was cleaved with TEV

protease and further puriﬁed by size exclusion chromatography using a

S200 HiLoad 16/60 Superdex column. A ﬁnal clean up step was

performed by reverse puriﬁcation on a Ni-sepharose column and the

puriﬁed protein stored at −80 °C.

X-ray Crystallography. Protein was concentrated to 10 mg/mL

buﬀered in 50 mM HEPES, pH 7.5, 300 mM NaCl, 10 mM DTT, 50

mM ^L-arginine, and 50 mM L-glutamate. Crystallization was performed

using the sitting drop vapor diﬀusion method at 4 °C. Viable crystals

M

dx.doi.org/10.1021/jm501177w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

integration time of one second per well. Data was normalized to 100%

of incremental BRE-Luc activity due to adenoviruses specifying

caALK1, -2, or -3, or the incremental CAGA-Luc activity due to

adenoviruses specifying caALK4 or -5. Graphing and regression

analysis by sigmoidal dose−response with variable Hill coeﬃcient was

performed using GraphPad Prism software.

Cell Viability Assay. HepG2 hepatocarcinoma cells were seeded in

DMEM supplemented with 10% FBS at 25000 cells per well in tissue

culture treated 96-well plates (Costar 3610; Corning). The cells were

incubated for 2 h (37 °C and 5% CO₂) and allowed to settle and

attach. Compounds of interest or DMSO were diluted in DMEM and

added at ﬁnal compound concentrations of 1, 10, and 100 μM. Cells

were incubated for 4 and 24 h, after which the media was discarded.

Cells were lysed by adding 30 μL of passive lysis buﬀer (Promega) and

shaken at RT for 15 min. Cell viability was determined by quantifying

the ATP present in each well by adding 10 μL of Cell Titer Glo

(Promega) per well and measuring the light output Spectramax L

luminometer (Molecular Devices) with an integration time of one

second per well. Data was normalized to 100% viability for cells

receiving only DMSO without any concurrent compound.

(A.N.B.), and the Structural Genomics Consortium (A.N.B.).

The SGC is a registered charity (number 1097737) that

receives funds from AbbVie, Bayer PHARMA AG, Boehringer

Ingelheim, the Canada Foundation for Innovation, the

Canadian Institutes for Health Research, Genome Canada,

GlaxoSmithKline, Janssen, Lilly Canada, the Novartis Research

Foundation, the Ontario Ministry of Economic Development

and Innovation, Pﬁzer, Takeda, and the Wellcome Trust

[092809/Z/10/Z].

ABBREVIATIONS USED

■

BMP, bone morphogenetic protein; TGF-β, transforming

growth factor β; FOP, ﬁbrodysplasia ossiﬁcans progressiva

REFERENCES

■

(1) Kitisin, K.; Saha, T.; Blake, T.; Golestaneh, N.; Deng, M.; Kim,

C.; Tang, Y.; Shetty, K.; Mishra, B.; Mishra, L. TGF-Beta Signaling in

Development. Sci. STKE 2007, 2007, cm1.

(2) Waite, K. A.; Eng, C. From Developmental Disorder to Heritable

Cancer: It’s All in the BMP/TGF-Beta Family. Nature Rev. Genet

2003, 4, 763−773.

ASSOCIATED CONTENT

* Supporting Information

Additional data describing the cell-based activity of 15, the

correlation of IC₅₀and cytotoxicity, the docking model of 10,

diﬀraction data and reﬁnement statistics, kinome selectivity

data, and calculated physiochemical properties. This material is

available free of charge via the Internet at http://pubs.acs.org.

■

S

(3) Andriopoulos, B., Jr.; Corradini, E.; Xia, Y.; Faasse, S. A.; Chen,

S.; Grgurevic, L.; Knutson, M. D.; Pietrangelo, A.; Vukicevic, S.; Lin,

H. Y.; Babitt, J. L. BMP6 Is a Key Endogenous Regulator of Hepcidin

Expression and Iron Metabolism. Nature Genet. 2009, 41, 482−487.

(4) Shi, Y.; Massague, J. Mechanisms of TGF-Beta Signaling from

Cell Membrane to the Nucleus. Cell 2003, 113, 685−700.

(5) Schmierer, B.; Hill, C. S. TGFbeta−SMAD Signal Transduction:

Molecular Specificity and Functional Flexibility. Nature Rev. Mol. Cell

Biol. 2007, 8, 970−982.

Accession Codes

PDB ID code: 4BGG.

AUTHOR INFORMATION

Corresponding Authors

*For P.B.Y.: phone, 857-307-0390; E-mail, pbyu@partners.org.

*For G.D.C.: phone, 713-743-1274; E-mail, gdcuny@central.

uh.edu.

(6) Chen, Y. G.; Hata, A.; Lo, R. S.; Wotton, D.; Shi, Y.; Pavletich,

N.; Massague, J. Determinants of Specificity in TGF-Beta Signal

Transduction. Genes Dev. 1998, 12, 2144−2152.

(7) Hong, C. C.; Yu, P. B. Applications of Small Molecule BMP

Inhibitors in Physiology and Disease. Cytokine Growth Factor Rev.

2009, 20, 409−418.

(8) Kaplan, F. S.; Le Merrer, M.; Glaser, D. L.; Pignolo, R. J.;

Goldsby, R. E.; Kitterman, J. A.; Groppe, J.; Shore, E. M.

Fibrodysplasia Ossificans Progressiva. Best Pract Res. Clin. Rheumatol.

2008, 22, 191−205.

■

Author Contributions

^¶A.H.M. and Y.W. contributed equally. Compounds were

conceived by A.H.M., Y.W., S.C., G.DC., and P.B.Y.

Compounds were synthesized by Y.W., S.C., and X.X.

Biological experiments were conceived and designed by

A.H.M, C.E.S., A.N.B., G.DC., and P.B.Y. These experiments

were conducted by A.H.M. Crystallography experiments were

conducted by C.E.S. and P.C. The manuscript was written and

revised by A.H.M., Y.W., C.E.S, A.N.B., G.D.C., and P.B.Y.

(9) Shore, E. M.; Xu, M.; Feldman, G. J.; Fenstermacher, D. A.;

Brown, M. A.; Kaplan, F. S. A Recurrent Mutation in the BMP Type I

Receptor Acvr1 Causes Inherited and Sporadic Fibrodysplasia

Ossificans Progressiva. Nature Genet. 2006, 38, 525−527.

(10) Kaplan, F. S.; Xu, M.; Seemann, P.; Connor, J. M.; Glaser, D. L.;

Carroll, L.; Delai, P.; Fastnacht-Urban, E.; Forman, S. J.; Gillessen-

Kaesbach, G.; Hoover-Fong, J.; Koster, B.; Pauli, R. M.; Reardon, W.;

Zaidi, S. A.; Zasloff, M.; Morhart, R.; Mundlos, S.; Groppe, J.; Shore,

E. M. Classic and Atypical Fibrodysplasia Ossificans Progressiva

(FOP) Phenotypes Are Caused by Mutations in the Bone

Morphogenetic Protein (BMP) Type I Receptor Acvr1. Hum. Mutat.

2009, 30, 379−390.

(11) Petrie, K. A.; Lee, W. H.; Bullock, A. N.; Pointon, J. J.; Smith, R.;

Russell, R. G.; Brown, M. A.; Wordsworth, B. P.; Triffitt, J. T. Novel

Mutations in Acvr1 Result in Atypical Features in Two Fibrodysplasia

Ossificans Progressiva Patients. PLoS One 2009, 4, e5005.

(12) Ohte, S.; Shin, M.; Sasanuma, H.; Yoneyama, K.; Akita, M.;

Ikebuchi, K.; Jimi, E.; Maruki, Y.; Matsuoka, M.; Namba, A.; Tomoda,

H.; Okazaki, Y.; Ohtake, A.; Oda, H.; Owan, I.; Yoda, T.; Furuya, H.;

Kamizono, J.; Kitoh, H.; Nakashima, Y.; Susami, T.; Haga, N.; Komori,

T.; Katagiri, T. A Novel Mutation of ALK2, L196p, Found in the Most

Benign Case of Fibrodysplasia Ossificans Progressiva Activates BMP-

Specific Intracellular Signaling Equivalent to a Typical Mutation,

R206h. Biochem. Biophys. Res. Commun. 2011, 407, 213−218.

(13) Gregson, C. L.; Hollingworth, P.; Williams, M.; Petrie, K. A.;

Bullock, A. N.; Brown, M. A.; Tobias, J. H.; Triffitt, J. T. A Novel

Acvr1 Mutation in the Glycine/Serine-Rich Domain Found in the

Notes

Brigham and Women's Hospital has applied for patents related

to the use of these compounds for the treatment of FOP, DIPG

and other disorders, and inventors (A.H.M., G.D.C., P.B.Y.)

may be entitled to royalties. LDN-214117 will be available from

Sigma-Aldrich (product SML1119) under a non-exclusive

license for research use only.

ACKNOWLEDGMENTS

■

We thank the staﬀ at the Diamond Light Source beamline I04-1

for help with diﬀraction data collection. This work was

supported by grants from the U.S. National Institutes of

Health DK082971-02S1 (A.H.M.), AR057374-03S1 (A.H.M.),

HL079943, and AR057374 (P.B.Y.) and from the Pulmonary

Hypertension Association (P.B.Y.), a Leducq Foundation

Transatlantic Network of Excellence Award (P.B.Y.), a Howard

Hughes Medical Institute Early Career Physician-Scientist

Award (P.B.Y.), the Harvard NeuroDiscovery Center (Y.W.,

S.C., X.X., G.D.C.), FOP action UK (C.E.S.), FOP France

N

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Journal of Medicinal Chemistry

Article

Most Benign Case of a Fibrodysplasia Ossificans Progressiva Variant

Reported to Date. Bone 2011, 48, 654−658.

T.; Bloch, K. D. BMP Type I Receptor Inhibition Reduces

Heterotopic Ossification. Nature Med. 2008, 14, 1363−1369.

(23) Sanvitale, C. E.; Kerr, G.; Chaikuad, A.; Ramel, M. C.; Mohedas,

A. H.; Reichert, S.; Wang, Y.; Triffitt, J. T.; Cuny, G. D.; Yu, P. B.; Hill,

C. S.; Bullock, A. N. A New Class of Small Molecule Inhibitor of BMP

Signaling. PLoS One 2013, 8, e62721.

(24) Cui, J. J.; Tran-Dube, M.; Shen, H.; Nambu, M.; Kung, P. P.;

Pairish, M.; Jia, L.; Meng, J.; Funk, L.; Botrous, I.; McTigue, M.;

Grodsky, N.; Ryan, K.; Padrique, E.; Alton, G.; Timofeevski, S.;

Yamazaki, S.; Li, Q.; Zou, H.; Christensen, J.; Mroczkowski, B.;

Bender, S.; Kania, R. S.; Edwards, M. P. Structure Based Drug Design

of Crizotinib (Pf-02341066), a Potent and Selective Dual Inhibitor of

Mesenchymal−Epithelial Transition Factor (C-Met) Kinase and

Anaplastic Lymphoma Kinase (ALK). J. Med. Chem. 2011, 54,

6342−6363.

(25) Miyaura, N.; Yanagi, T.; Suzuki, A. The Palladium-Catalyzed

Cross-Coupling Reaction of Phenylboronic Acid with Haloarenes in

the Presence of Bases. Synth. Commun. 1981, 11, 513−519.

(26) Alo, B. I.; Kandil, A.; Patil, P. A.; Sharp, M. J.; Siddiqui, M. A.;

Snieckus, V.; Josephy, P. D. Sequential Directed Ortho Metalation−

Boronic Acid Cross-Coupling Reactions. A General Regiospecific

Route to Oxygenated Dibenzo[b,d]pyran-6-ones Related to Ellagic

Acid. J. Org. Chem. 1991, 56, 3763−3768.

(27) Hilton, S.; Naud, S.; Caldwell, J. J.; Boxall, K.; Burns, S.;

Anderson, V. E.; Antoni, L.; Allen, C. E.; Pearl, L. H.; Oliver, A. W.;

Aherne, G. W.; Garrett, M. D.; Collins, I. Identification and

Characterisation of 2-Aminopyridine Inhibitors of Checkpoint Kinase

2 (Vol 18, Pg 707, 2010). Bioorg. Med. Chem. 2010, 18, 4591−4591.

(28) Gray, M.; Andrews, I. P.; Hook, D. F.; Kitteringham, J.; Voyle,

M. Practical Methylation of Aryl Halides by Suzuki−Miyaura

Coupling. Tetrahedron Lett. 2000, 41, 6237−6240.

(14) Fukuda, T.; Kanomata, K.; Nojima, J.; Kokabu, S.; Akita, M.;

Ikebuchi, K.; Jimi, E.; Komori, T.; Maruki, Y.; Matsuoka, M.;

Miyazono, K.; Nakayama, K.; Nanba, A.; Tomoda, H.; Okazaki, Y.;

Ohtake, A.; Oda, H.; Owan, I.; Yoda, T.; Haga, N.; Furuya, H.;

Katagiri, T. A Unique Mutation of ALK2, G356d, Found in a Patient

with Fibrodysplasia Ossificans Progressiva Is a Moderately Activated

BMP Type I Receptor. Biochem. Biophys. Res. Commun. 2008, 377,

905−909.

(15) Wu, G.; Diaz, A. K.; Paugh, B. S.; Rankin, S. L.; Ju, B.; Li, Y.;

Zhu, X.; Qu, C.; Chen, X.; Zhang, J.; Easton, J.; Edmonson, M.; Ma,

X.; Lu, C.; Nagahawatte, P.; Hedlund, E.; Rusch, M.; Pounds, S.; Lin,

T.; Onar-Thomas, A.; Huether, R.; Kriwacki, R.; Parker, M.; Gupta, P.;

Becksfort, J.; Wei, L.; Mulder, H. L.; Boggs, K.; Vadodaria, B.; Yergeau,

D.; Russell, J. C.; Ochoa, K.; Fulton, R. S.; Fulton, L. L.; Jones, C.;

Boop, F. A.; Broniscer, A.; Wetmore, C.; Gajjar, A.; Ding, L.; Mardis,

E. R.; Wilson, R. K.; Taylor, M. R.; Downing, J. R.; Ellison, D. W.;

Baker, S. J. The Genomic Landscape of Diffuse Intrinsic Pontine

Glioma and Pediatric Non-Brainstem High-Grade Glioma. Nature

Genet. 2014, 46, 444−450.

(16) Fontebasso, A. M.; Papillon-Cavanagh, S.; Schwartzentruber, J.;

Nikbakht, H.; Gerges, N.; Fiset, P. O.; Bechet, D.; Faury, D.; De Jay,

N.; Ramkissoon, L. A.; Corcoran, A.; Jones, D. T.; Sturm, D.; Johann,

P.; Tomita, T.; Goldman, S.; Nagib, M.; Bendel, A.; Goumnerova, L.;

Bowers, D. C.; Leonard, J. R.; Rubin, J. B.; Alden, T.; Browd, S.; Geyer,

J. R.; Leary, S.; Jallo, G.; Cohen, K.; Gupta, N.; Prados, M. D.; Carret,

A. S.; Ellezam, B.; Crevier, L.; Klekner, A.; Bognar, L.; Hauser, P.;

Garami, M.; Myseros, J.; Dong, Z.; Siegel, P. M.; Malkin, H.; Ligon, A.

H.; Albrecht, S.; Pfister, S. M.; Ligon, K. L.; Majewski, J.; Jabado, N.;

Kieran, M. W. Recurrent Somatic Mutations in Acvr1 in Pediatric

Midline High-Grade Astrocytoma. Nature Genet. 2014, 46, 462−466.

(17) Taylor, K. R.; Mackay, A.; Truffaux, N.; Butterfield, Y. S.;

Morozova, O.; Philippe, C.; Castel, D.; Grasso, C. S.; Vinci, M.;

Carvalho, D.; Carcaboso, A. M.; de Torres, C.; Cruz, O.; Mora, J.;

Entz-Werle, N.; Ingram, W. J.; Monje, M.; Hargrave, D.; Bullock, A.

N.; Puget, S.; Yip, S.; Jones, C.; Grill, J. Recurrent Activating Acvr1

Mutations in Diffuse Intrinsic Pontine Glioma. Nature Genet. 2014, 46,

457−461.

(29) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.

Experimental and Computational Approaches to Estimate Solubility

and Permeability in Drug Discovery and Development Settings. Adv.

Drug Delivery Rev. 2001, 46, 3−26.

(30) Veber, D. F.; Johnson, S. R.; Cheng, H. Y.; Smith, B. R.; Ward,

K. W.; Kopple, K. D. Molecular Properties That Influence the Oral

Bioavailability of Drug Candidates. J. Med. Chem. 2002, 45, 2615−

2623.

(31) Fedorov, O.; Marsden, B.; Pogacic, V.; Rellos, P.; Muller, S.;

Bullock, A. N.; Schwaller, J.; Sundstrom, M.; Knapp, S. A Systematic

Interaction Map of Validated Kinase Inhibitors with Ser/Thr Kinases.

Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 20523−20528.

(32) Luo, J.; Tang, M.; Huang, J.; He, B. C.; Gao, J. L.; Chen, L.;

Zuo, G. W.; Zhang, W.; Luo, Q.; Shi, Q.; Zhang, B. Q.; Bi, Y.; Luo, X.;

Jiang, W.; Su, Y.; Shen, J.; Kim, S. H.; Huang, E.; Gao, Y.; Zhou, J. Z.;

Yang, K.; Luu, H. H.; Pan, X.; Haydon, R. C.; Deng, Z. L.; He, T. C.

TGFbeta/BMP Type I Receptors ALK1 and ALK2 Are Essential for

BMP9-Induced Osteogenic Signaling in Mesenchymal Stem Cells. J.

Biol. Chem. 2010, 285, 29588−29598.

(33) Ramsden, N.; Wilson, F. Aminopyridine Derivates as Kinase

Inhibitors. Patent WO2008025820 A1, 2008.

(34) Blackaby, W. P.; Charles, M. D.; Ekwuru, C. T.; Foxton, C. H.;

Hammonds, T. R.; Pave, G. A.; Raynham, T. M.; Stevens, A. P.

Pyridine Benzamides and Pyrazine Benzamides Used as Pkd Inhibitors.

Patent WO2008025820 A1, 2008.

(35) Yu, P. B.; Beppu, H.; Kawai, N.; Li, E.; Bloch, K. D. Bone

Morphogenetic Protein (BMP) Type II Receptor Deletion Reveals

BMP Ligand-Specific Gain of Signaling in Pulmonary Artery Smooth

Muscle Cells. J. Biol. Chem. 2005, 280, 24443−24450.

(36) Aoki, H.; Fujii, M.; Imamura, T.; Yagi, K.; Takehara, K.; Kato,

M.; Miyazono, K. Synergistic Effects of Different Bone Morphogenetic

Protein Type I Receptors on Alkaline Phosphatase Induction. J. Cell

Sci. 2001, 114, 1483−1489.

(18) Buczkowicz, P.; Hoeman, C.; Rakopoulos, P.; Pajovic, S.;

Letourneau, L.; Dzamba, M.; Morrison, A.; Lewis, P.; Bouffet, E.;

Bartels, U.; Zuccaro, J.; Agnihotri, S.; Ryall, S.; Barszczyk, M.;

Chornenkyy, Y.; Bourgey, M.; Bourque, G.; Montpetit, A.; Cordero,

F.; Castelo-Branco, P.; Mangerel, J.; Tabori, U.; Ho, K. C.; Huang, A.;

Taylor, K. R.; Mackay, A.; Bendel, A. E.; Nazarian, J.; Fangusaro, J. R.;

Karajannis, M. A.; Zagzag, D.; Foreman, N. K.; Donson, A.; Hegert, J.

V.; Smith, A.; Chan, J.; Lafay-Cousin, L.; Dunn, S.; Hukin, J.; Dunham,

C.; Scheinemann, K.; Michaud, J.; Zelcer, S.; Ramsay, D.; Cain, J.;

Brennan, C.; Souweidane, M. M.; Jones, C.; Allis, C. D.; Brudno, M.;

Becher, O.; Hawkins, C. Genomic Analysis of Diffuse Intrinsic Pontine

Gliomas Identifies Three Molecular Subgroups and Recurrent

Activating Acvr1 Mutations. Nature Genet. 2014, 46, 451−456.

(19) Cuny, G. D.; Yu, P. B.; Laha, J. K.; Xing, X.; Liu, J. F.; Lai, C. S.;

Deng, D. Y.; Sachidanandan, C.; Bloch, K. D.; Peterson, R. T.

Structure−Activity Relationship Study of Bone Morphogenetic Protein

(BMP) Signaling Inhibitors. Bioorg. Med. Chem. Lett. 2008, 18, 4388−

4392.

(20) Hao, J.; Ho, J. N.; Lewis, J. A.; Karim, K. A.; Daniels, R. N.;

Gentry, P. R.; Hopkins, C. R.; Lindsley, C. W.; Hong, C. C. In Vivo

Structure−Activity Relationship Study of Dorsomorphin Analogues

Identifies Selective Vegf and BMP Inhibitors. ACS Chem. Biol. 2009, 5,

245−253.

(21) Mohedas, A. H.; Xing, X.; Armstrong, K. A.; Bullock, A. N.;

Cuny, G. D.; Yu, P. B. Development of an ALK2-Biased BMP Type I

Receptor Kinase Inhibitor. ACS Chem. Biol. 2013, 8, 1291−1302.

(22) Yu, P. B.; Deng, D. Y.; Lai, C. S.; Hong, C. C.; Cuny, G. D.;

Bouxsein, M. L.; Hong, D. W.; McManus, P. M.; Katagiri, T.;

Sachidanandan, C.; Kamiya, N.; Fukuda, T.; Mishina, Y.; Peterson, R.

(37) Nakajima, M.; Haga, N.; Takikawa, K.; Manabe, N.; Nishimura,

G.; Ikegawa, S. The Acvr1 617g>a Mutation Is Also Recurrent in

Three Japanese Patients with Fibrodysplasia Ossificans Progressiva. J.

Hum. Genet. 2007, 52, 473−475.

O

dx.doi.org/10.1021/jm501177w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(38) Bocciardi, R.; Bordo, D.; Di Duca, M.; Di Rocco, M.; Ravazzolo,

R. Mutational Analysis of the Acvr1 Gene in Italian Patients Affected

with Fibrodysplasia Ossificans Progressiva: Confirmations and

Advancements. Eur. J. Hum. Genet. 2009, 17, 311−318.

(39) Lee, D. Y.; Cho, T. J.; Lee, H. R.; Park, M. S.; Yoo, W. J.;

Chung, C. Y.; Choi, I. H. Acvr1 Gene Mutation in Sporadic Korean

Patients with Fibrodysplasia Ossificans Progressiva. J. Korean Med. Sci.

2009, 24, 433−437.

(40) Chaikuad, A.; Alfano, I.; Kerr, G.; Sanvitale, C. E.; Boergermann,

J. H.; Triffitt, J. T.; von Delft, F.; Knapp, S.; Knaus, P.; Bullock, A. N.

Structure of the BMP Receptor ALK2 and Implications for

Fibrodysplasia Ossificans Progressiva. J. Biol. Chem. 2012, 287,

36990−36998.

(41) Bagarova, J.; Vonner, A. J.; Armstrong, K. A.; Borgermann, J.;

Lai, C. S.; Deng, D. Y.; Beppu, H.; Alfano, I.; Filippakopoulos, P.;

Morrell, N. W.; Bullock, A. N.; Knaus, P.; Mishina, Y.; Yu, P. B.

Constitutively Active ALK2 Receptor Mutants Require Type II

Receptor Cooperation. Mol. Cell. Biol. 2013, 33, 2413−2424.

(42) Miller, D. S.; Horowitz, S. B. Intracellular Compartmentalization

of Adenosine Triphosphate. J. Biol. Chem. 1986, 261, 13911−13915.

(43) Issa, A. M.; Phillips, K. A.; Van Bebber, S.; Nidamarthy, H. G.;

Lasser, K. E.; Haas, J. S.; Alldredge, B. K.; Wachter, R. M.; Bates, D. W.

Drug Withdrawals in the United States: A Systematic Review of the

Evidence and Analysis of Trends. Curr. Drug Saf. 2007, 2, 177−185.

(44) Niesen, F. H.; Berglund, H.; Vedadi, M. The Use of Differential

Scanning Fluorimetry to Detect Ligand Interactions That Promote

Protein Stability. Nature Protoc. 2007, 2, 2212−2221.

(45) Leslie, A. G. The Integration of Macromolecular Diffraction

Data. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2006, 62, 48−57.

(46) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M.

D.; Storoni, L. C.; Read, R. J. Phaser Crystallographic Software. J. Appl.

Crystallogr. 2007, 40, 658−674.

(47) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and

Development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010,

66, 486−501.

(48) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement of

Macromolecular Structures by the Maximum-Likelihood Method. Acta

Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53, 240−255.

(49) Painter, J.; Merritt, E. A. Optimal Description of a Protein

Structure in Terms of Multiple Groups Undergoing Tls Motion. Acta

Crystallogr., Sect. D: Biol. Crystallogr. 2006, 62, 439−450.

(50) Davis, I. W.; Leaver-Fay, A.; Chen, V. B.; Block, J. N.; Kapral, G.

J.; Wang, X.; Murray, L. W.; Arendall, W. B., III; Snoeyink, J.;

Richardson, J. S.; Richardson, D. C. Molprobity: All-Atom Contacts

and Structure Validation for Proteins and Nucleic Acids. Nucleic Acids

Res. 2007, 35, W375−383.

(51) Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis,

I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse-

Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R.

J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H.

Phenix: A Comprehensive Python-Based System for Macromolecular

Structure Solution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66,

213−221.

(52) Zilberberg, L.; ten Dijke, P.; Sakai, L. Y.; Rifkin, D. B. A Rapid

and Sensitive Bioassay to Measure Bone Morphogenetic Protein

Activity. BMC Cell Biol. 2007, 8, 41−50.

(53) Oida, T.; Weiner, H. L. Murine Cd4 T Cells Produce a New

Form of TGF-Beta as Measured by a Newly Developed TGF-Beta

Bioassay. PloS One 2011, 6, e18365.

(54) Laskowski, R. A.; Swindells, M. B. Ligplot+: Multiple Ligand−

Protein Interaction Diagrams for Drug Discovery. J. Chem. Inf Model.

2011, 51, 2778−2786.

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Structure-activity relationship of 3,5-diaryl-2-aminopyridine ALK2 inhibitors reveals unaltered binding affinity for fibrodysplasia ossificans progressiva causing mutants

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