Targeting ALK2: An Open Science Approach to Developing Therapeutics for the Treatment of Diffuse Intrinsic Pontine Glioma

pubs.acs.org/jmc

Article

Targeting ALK2: An Open Science Approach to Developing

Therapeutics for the Treatment of Diﬀuse Intrinsic Pontine Glioma

́

Deeba Ensan, David Smil, Carlos A. Zepeda-Velazquez, Dimitrios Panagopoulos, Jong Fu Wong,

Eleanor P. Williams, Roslin Adamson, Alex N. Bullock, Taira Kiyota, Ahmed Aman, Owen G. Roberts,

Aled M. Edwards, Je ﬀ A. O ’Meara, Methvin B. Isaac, and Rima Al-awar *

Cite This: https://dx.doi.org/10.1021/acs.jmedchem.0c00395

Read Online

sı

*

Metrics & More

Article Recommendations

Supporting Information

ACCESS

ABSTRACT: Diﬀuse intrinsic pontine glioma is an aggressive

pediatric cancer for which no eﬀective chemotherapeutic drugs

exist. Analysis of the genomic landscape of this disease has led to

the identiﬁcation of the serine/threonine kinase ALK2 as a

potential target for therapeutic intervention. In this work, we

adopted an open science approach to develop a series of potent

type I inhibitors of ALK2 which are orally bio-available and brain-

penetrant. Initial eﬀorts resulted in the discovery of M4K2009, an

analogue of the previously reported ALK2 inhibitor LDN-214117.

Although highly selective for ALK2 over the TGF-βR1 receptor

ALK5, M4K2009 is also moderately active against the hERG

potassium channel. Varying the substituents of the trimethox-

yphenyl moiety gave rise to an equipotent benzamide analogue M4K2149 with reduced oﬀ-target aﬃnity for the ion channel.

Additional modiﬁcations yielded 2-ﬂuoro-6-methoxybenzamide derivatives (26a−c), which possess high inhibitory activity against

ALK2, excellent selectivity, and superior pharmacokinetic proﬁles.

binding to type II BMP receptors is transduced by type I BMP

receptors, which promote the translocation of downstream

eﬀector proteins (SMADs) to the nucleus where they can

regulate the transcription of target genes via chromatin

remodeling.^4,5Aberrant BMP signaling is implicated in a

number of diseases,⁵such as ﬁbrodysplasia ossiﬁcans

progressiva (FOP). Germline mutations (c.617G>A;

p.R206H) in the juxtamembrane glycine−serine (GS)-rich

domain of activin receptor-like kinase-2 (ALK2) confer gain-

of-function activity to the type I BMP receptor and contribute

to the abnormal skeletal phenotype observed in individuals

aﬀected by FOP.^3,6Somatic missense mutations in the ACVR1

gene encoding ALK2 have also been reported in approximately

24% of children with the rare pediatric disease diﬀuse intrinsic

pontine glioma (DIPG),⁶with a higher prevalence of mutation

occurring in the serine/threonine kinase domain of the

receptor.⁷

INTRODUCTION

■

The design and development of brain-penetrant kinase

inhibitors as a therapy for the treatment of primary central

nervous system (CNS) tumors entail numerous challenges.

This is in part due to the remarkably diﬀerent structural

properties that CNS drugs and kinase inhibitors have.

Approved CNS drugs, for instance, have fewer hydrogen

bond donors (HBDs), lower molecular weights, and half the

topological polar surface area (tPSA) of kinase inhibitors on

average.¹Elevated expression levels of eﬄux transporters at the

blood−brain barrier (BBB) constitute an additional obstacle

that drugs must overcome in order to reach therapeutically

relevant concentrations at sites of lesion.¹CNS drug exposure

is further limited by the endothelial tight junctions of the BBB,

which impede paracellular transport.²Despite these diﬃculties,

the recent approval of Lorlatinib by the FDA for the treatment

of metastatic anaplastic lymphoma kinase-positive nonsmall

cell lung cancer demonstrates that the development of BBB

penetrant kinase inhibitors is possible. There are multiple

kinases in addition to the anaplastic lymphoma kinase that play

pivotal roles in oncogenesis. Of interest to us are proteins

involved in the bone morphogenetic protein (BMP) signaling

pathway.

DIPG is a grade IV tumor originating in the glial tissue of

the pons.³Children aﬀected by the disease have a 5-year

Received: March 6, 2020

BMPs are a group of cytokines that modulate a plethora of

physiological processes, including musculoskeletal develop-

ment and neural diﬀerentiation.³The signal elicited by BMP

https://dx.doi.org/10.1021/acs.jmedchem.0c00395

J. Med. Chem. XXXX, XXX, XXX−XXX

A

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 1. Inhibitory and oﬀ-target activity of previously reported ALK2 inhibitors and novel analogues. Reported values were obtained from the

corresponding references.^15,17,18Measured values were determined utilizing a radioactive biochemical kinase assay.

relative survival rate of less than 1%.⁸Treatment options are

limited to focal radiation therapy because of the sensitive area

in which the tumor resides and the failure of currently available

chemotherapeutic drugs to prolong survival.^8,9The mechanism

by which ALK2 contributes to DIPG pathogenesis has not yet

been elucidated.^3,7,10However, a recent study by Carvalho and

coworkers demonstrated that shRNA knockdown of ACVR1

elicits apoptosis in HSJD-DIPG-007 cells, harboring ACVR1

R206H mutations in conjunction with histone H3.3 K27M

mutations.¹¹Their work suggests that DIPG cells are

dependent on enhanced BMP signaling. This was further

recapitulated in their orthotopic patient-derived xenograft

model in which administration of two ALK2 inhibitors

extended survival compared to controls.¹¹Although targeting

the serine/threonine kinase may constitute a viable treatment,

monotherapies are seldom eﬃcacious for DIPG.¹²Targeting

proteins with the potential to restore normal epigenetic

signatures, such as histone deacetylase (HDAC), has gained

momentum in recent years.¹²It is likely that the most

beneﬁcial treatment option for patients will consist of

combinatorial therapies.

Several inhibitors of ALK2 have emerged in the past

decade,¹³including the pyrazolo[1,5-α]pyrimidine compound

LDN-193189,^14,15as well as a relatively new class of 3,5-

diarylpyridine analogues: K02288, LDN-213844, and LDN-

214117.¹⁶⁻¹⁸Triazolamine CP466722 represents another

novel chemotype with moderate binding aﬃnity for ALK2

and unparalleled selectivity over other proteins in the serine/

threonine kinase receptor (STKR) family.¹⁹Structure−activity

relationship (SAR) studies surrounding this new scaﬀold

should also be explored. As LDN-214117 has been reported to

have low cytotoxic activity and excellent kinome-wide

selectivity,¹⁷we sought to explore whether additional

modiﬁcations to the hinge-binding pyridyl core could improve

ALK2 potency and selectivity over the closely related TGF-βRI

receptor ALK5. Cardiotoxicity and gastrointestinal inﬂamma-

tion are adverse eﬀects of ALK5 inhibition.²⁰Therefore, a

major focus of our SAR studies was to synthesize analogues

with reduced oﬀ-target aﬃnity for this receptor. Shifting the

methyl substituent from the C-2 position of the pyridyl core of

LDN-214117 to the C-4 position (M4K2009) maintained

potency and selectivity as determined by the in vitro and cell-

based assays employed throughout our study (Figure 1).²¹

Although M4K2009 has good structural and physicochem-

ical properties, it poses the risk of eliciting torsades de pointes

arrythmia in vivo because of the moderate aﬃnity it has for the

protein product encoded by the human ether-a-go-go related

gene (hERG) (IC₅₀= 8 μM).²¹Additional modiﬁcations made

to the trimethoxyphenyl moiety led to the identiﬁcation of the

benzamide analogue M4K2149, which has a hERG IC₅₀of >50

μM and comparable inhibitory activity against ALK2 (Figure

1). In our pursuit of a potent, selective, orally bioavailable, and

brain-penetrant type I inhibitor of ALK2, M4K2149 was an

excellent starting point from which to expand our SAR studies.

Our initial work with M4K2009 revealed that the inhibitor

was equipotent against both wild-type (WT) and mutant

ALK2 (G328V, R206H, and R258G) in the biochemical kinase

assay.²¹These results are in alignment with the data generated

by Mohedas and coworkers.¹⁷Utilizing a thermal shift kinase

assay, they were able to demonstrate that FOP-causing

mutations in both the GS and serine/threonine kinase domain

of ALK2 had negligible eﬀects on the kinase’s aﬃnity for type I

inhibitors.¹⁷As FOP and DIPG patients harbor very similar

mutations in the ACVR1 gene,³the inhibitory activity of the

compounds in our series was determined primarily against WT

ALK2.

The potency and selectivity of our analogues was assessed

using a radioactive in vitro kinase assay, employing LDN-

193189 as a control. To test the activity of the compounds in

cells, a HEK293 cell-based NanoBRET assay from Promega

was used. In this assay, the competitive displacement of a

ﬂuorescent tracer (PBI-6908) from the binding pocket of

ALK2 by test compounds elicits reductions in BRET ratios,

which are used to generate IC₅₀values. Cell-based potency

against ALK5 was subsequently determined using a dual

luciferase assay (DLA) in HEK293 cells.

The biological evaluation of these compounds was made

possible by the pro bono contributions of Reaction Biology

Corporation. This work, which was initiated by the open

science pharmaceutical company M4K Pharma Inc., was

B

https://dx.doi.org/10.1021/acs.jmedchem.0c00395

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 1. Synthesis of Compounds 7a−b and 8a−c

a

Reagents and conditions: (a) Pd(dppf)Cl₂·DCM, Na₂CO₃, dioxane/H₂O, 85 °C, overnight; (b) B₂pin₂, Pd(dppf)Cl₂·DCM, KOAc, dioxane, 110

°C, 4 h; (c) aryl halide (5a−b), Pd(dppf)Cl₂·DCM, Na₂CO₃, dioxane/H₂O, 100 °C, 2 h; (d) 7 N NH₃in MeOH, 90 °C, 3 d; (e) TFA, DCM, rt,

overnight; (f) aryl halide (5c−e), XPhos Pd G2, K₃PO₄, dioxane/H₂O, 100 °C, 3 h.

performed in collaboration with the not-for-proﬁt organiza-

tions, the Ontario Institute for Cancer Research (OICR) and

the Structural Genomics Consortium (SGC). Adopting an

open science approach enabled us to freely share and discuss

results with experts in the ﬁeld, forging collaborations that

advanced the science without the delays associated with

conﬁdentiality agreements and intellectual property owner-

ship.²²

Suzuki−Miyaura coupling of 15 with the boronate ester 16

aﬀorded the methyl ester intermediate 17, which was

converted to the corresponding primary, secondary, or tertiary

amide via aminolysis or base-catalyzed hydrolysis followed by

EDC-mediated coupling with the desired amine. Deprotection

using TFA or HCl aﬀorded the compounds M4K2149 and

18a−b. A similar synthetic route was used to access analogues

20a−e, although additional transformations beyond the

standard Suzuki−Miyaura coupling and deprotection were

not required, as the boronate esters 19a−e already had the

desired amide substituents installed.

The synthesis of analogues 26a−f (Scheme 3) was initiated

by the nucleophilic aromatic substitution of 4-bromo-2,6-

diﬂuorobenzonitrile (21) with sodium methoxide to yield both

4-bromo-2-ﬂuoro-6-methoxybenzonitrile (22a) and 2,6-dime-

thoxybenzonitrile (22b). Both intermediates were hydrolyzed

to the corresponding amides 5d−e using hydrogen peroxide

and an aqueous solution of sodium hydroxide. Miyaura

borylation of 5d−e followed by Suzuki−Miyaura coupling

with 3-bromo-5-chloro-4-methylpyridine (10) aﬀorded 24a−b,

which were subjected to a second coupling reaction with a

variety of (4-(piperazin-1-yl)phenyl)boronate ester derivatives

(25a−c) to furnish the ﬁnal compounds 26a−f in excellent

yields.

Structure Activity Relationship Studies. It has been

disclosed by Mohedas and coworkers that the pyridyl nitrogen

of LDN-213844 participates in a key hydrogen bond

interaction with the backbone amide of H286 in the hinge

region of ALK2 (Figure 2B).¹⁷Our own crystallographic

eﬀorts led to the generation of a co-crystal structure of

M4K2149 with the kinase in high resolution, which revealed

that the same interaction had been preserved (PDB code

6T6D). Furthermore, the trimethoxyphenyl motif of LDN-

213844 was reported to occupy a hydrophobic pocket of

ALK2, where the meta-methoxy group participates in a water-

mediated hydrogen bond with K235 (PDB code 4BGG).¹⁷

Substitution of the para-methoxy group of LDN-213844 with

RESULTS AND DISCUSSION

■

Synthesis of Analogues. The synthetic route employed to

prepare M4K2149 and related analogues ultimately depended

on the commercial availability of starting materials, ease of

synthesis, and reaction eﬃciency. The compounds were

initially accessed as depicted in Scheme 1. Suzuki−Miyaura

coupling of 3,5-dibromo-4-methylpyridine (2) with 1

generated intermediate 3, which subsequently underwent

Miyaura borylation to yield the boronate ester 4a. Aromatic

methyl esters 5a−b were coupled with 4a to aﬀord

intermediates 6a−b, which were transformed to the corre-

sponding primary amides 7a−b by reﬂuxing in methanolic

ammonia, which was then followed by the removal of the

carbamate protecting groups with triﬂuoroacetic acid (TFA).

The preparation of analogues 8a−c followed a similar synthetic

route in which 4a was coupled with the aromatic amides 5c−e

and then deprotected with TFA. Approximately 50% of

intermediate 3 was converted to the undesired dehalogenated

side product 4b in step b, which contributed to signiﬁcantly

low yields for the ﬁnal products.

To overcome yield constraints, a second synthetic scheme

was devised in which a wide variety of boronate esters were

coupled with the pyridyl derivatives 3, 10, or 15 (Scheme 2).

The synthesis of the carboxylic acid intermediates 13a−b was

accomplished via a two-step, one-pot Suzuki−Miyaura

coupling sequence. HATU-mediated coupling with ammonium

chloride followed by deprotection furnished the ﬁnal amide

regioisomers 14a−b.

C

https://dx.doi.org/10.1021/acs.jmedchem.0c00395

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 2. Synthesis of Compounds 14a−b, M4K2149, 18a−b, and 20a−e

a

Reagents and conditions: (a) Pd(dppf)Cl₂·DCM, Na₂CO₃, dioxane/H₂O, 100 °C, 3 h; (b) XPhos Pd G2, K₃PO₄, dioxane/H₂O, 100 °C, 3 h; (c)

NH₄Cl, HATU, DIPEA, DCM, rt, 3 h; (d) TFA, DCM, rt, 1 h; (e) 7 N NH₃in MeOH, 75 °C, 3 d; (f) methylamine, MeOH, 85 °C, 5 h; (g) KOH,

THF/H₂O, rt, 2 h; (h) dimethylamine, HOBt, EDC, DIPEA, DCM/DMF, 50 °C, overnight; (i) 4 M HCl in dioxane, MeOH, rt, 30 min.

a primary amide results in the establishment of a direct

hydrogen bond between the carbonyl O of the amide and the

well with the results obtained by NanoBRET and DLA (Table

1). Moving the methoxy group from an ortho- to a meta-

position with respect to the amide (14b) improved the

biochemical potency compared to 14a; however, this did not

translate into a signiﬁcant improvement in cell-based potency.

Replacement of the phenyl ring with ﬁve-membered hetero-

cycles gave rise to the thiophene analogues 7a−b and 8a,

which were proﬁled to further investigate the eﬀect that the

amide geometry had on potency and selectivity. A greater than

40-fold decrease in inhibitory activity against ALK2 was

measured for 7a and 7b, while 8a was found to be completely

inactive against the kinase in both assays. It became evident

that positioning the primary amide on a six-membered

aromatic ring para to the hinge-binding pyridyl core was

critical for maintaining key binding interactions with ALK2.

+

NH₃group of K235 (Figure 2A). Additionally, the phenyl

ring of M4K2149 stacks between G289 and V214,²³while the

+

protonated piperazine NH₂is in close proximity to D293.

This is suggestive of an electrostatic interaction. An intra-

molecular hydrogen bond between the amide NH₂and O of

the ortho-methoxy substituent can also be observed in the co-

crystal structure.

Our initial SAR studies focused on varying the substitution

pattern of the amide in order to determine if the group could

interact with other residues in the pocket, such as D354 of the

DLG motif or E248.²³Inverting the amide and methoxy

substituents (14a) resulted in a complete loss of activity

against ALK2 in the biochemical kinase assay. This correlated

D

https://dx.doi.org/10.1021/acs.jmedchem.0c00395

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 3. Synthesis of Compounds 26a−f

a

Reagents and conditions: (a) NaH, MeOH, dioxane, rt, overnight; (b) H₂O₂, NaOH, EtOH/H₂O, overnight; (c) B₂pin₂, Pd(dppf)Cl₂·DCM,

KOAc, dioxane, 110 °C, 4 h; (d) Pd(dppf)Cl₂·DCM, Na₂CO₃, dioxane/H₂O, 100 °C, 3 h; (e) XPhos Pd G2, K₃PO₄, dioxane/H₂O, 100 °C, 3 h.

Figure 2. (A) Cocrystal structure of M4K2149 (light yellow) with ALK2 (PDB code 6T6D). Hydrogen bonds are established with H286 and

K235. The benzamide moiety of M4K2149 occupies a hydrophobic pocket (green) of ALK2 and is ﬂanked by several hydrogen bond-donating

(blue; K235) and hydrogen bond-accepting residues (red; D354 and E248). The protonated piperazine motif is in close proximity to D293,

indicative of an electrostatic interaction. (B) Cocrystal structure of LDN-213844 (light yellow) with ALK2 (PDB code 4BGG). M4K2149 and

LDN-213844 have similar modes of binding.

The consideration of several physicochemical parameters,

such as lipophilicity (cLogP), tPSA, and number of HBD, is

pertinent in the design of small molecule inhibitors that must

penetrate the BBB to exert their pharmacological eﬀects. The

ideal values that brain-penetrant drugs should have vary

between reviews.²⁴In the case of lipophilicity, the consensus is

that immoderate increases in cLogP should be avoided.

Although increasing the lipophilicity of a drug typically

enhances potency and permeability, concomitant increases in

nonspeciﬁc tissue binding also occur, which would ultimately

decrease the concentration of the free drug at its intended site

of action within the brain.²⁴The number of HBD that a

molecule possesses, in addition to its tPSA, can also inﬂuence

its ability to permeate the BBB. Increasing the value of either

physicochemical parameter also risks recognition by eﬄux

transporters, such as P-glycoprotein (P-gp).^1,2

With these guidelines in mind, we sought to determine if

mono- and di-N-methylation of the primary amide, as well as

its cyclization to form the corresponding isoindolinone

analogue, could be tolerated. The eﬀects of these modiﬁcations

were two-fold. In addition to decreasing the number of HBD,

the tPSA was reduced to below 70 Å²for 18a−b and 20a,²⁵

which is in an optimal range for CNS-penetrant drugs.²⁴These

structural changes had only moderate eﬀects on the

lipophilicity of the three analogues. Unfortunately, these

compounds were discovered to be completely inactive against

ALK2 (Table 2), indicating that the binding aﬃnity of the

benzamides may be sensitive to steric eﬀects. Consequently, we

decided to incorporate only the primary amide motif in the rest

of our analogues.

To determine if the methoxy group of M4K2149 was critical

for maintaining potency, compound 20b was proﬁled. Removal

E

https://dx.doi.org/10.1021/acs.jmedchem.0c00395

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 1. Inhibitory and Oﬀ-Target Activities of 14a−b, 7a−b, and 8a

a

b

Average of duplicate measurements. Average of triplicate measurements.

of the methoxy substituent reduced inhibitory activity against

ALK2 by 28-fold. This result led us to suspect that the

methoxy group oriented the amide into a conformation that

was ideal for ALK2 binding. The incorporation of intra-

molecular hydrogen bonds and electrostatic interactions to

mask HBD is a technique commonly employed to enhance

brain penetration.^1,24,26−28In an attempt to exploit these

interactions and probe for additional ones in the vicinity of the

benzamide ring, we proﬁled compounds 20c and 20d, which

featured a one-carbon homologation of the methoxy group and

a bioisosteric replacement of the methoxy for a ﬂuorine atom,

respectively. Both modiﬁcations, however, failed to improve

biochemical potency against ALK2.

Incorporation of both a ﬂuorine and methoxy substituent

ortho to the amide gave rise to compound 8b, which not only

had a biochemical ALK2 potency comparable to that of

M4K2149 (ALK2 IC₅₀= 24 nM) but also an improved

selectivity proﬁle over ALK5. These results correlated well with

those obtained in the NanoBRET and DLA assays (Table 2).

Exactly how the ﬂuorine substituent contributes to this

enhancement in selectivity is yet to be elucidated, although

two possible explanations exist. We surmised that the electron-

withdrawing nature of the ﬂuorine atom decreases the ability of

the carbonyl O to act as a hydrogen bond acceptor,²⁸thereby

reducing the strength of intermolecular interactions that may

be more critical for ligand binding to ALK5 than ALK2. The

second possible explanation focuses on how the halogen

substituent aﬀects the conformation of the amide with respect

to the phenyl ring. For anisole and benzamide motifs, which

typically adopt planar topologies, ortho substituents can force

the methoxy or amide groups out of the plane of the benzene

ring in order to reduce allylic strain.^28,29We postulated that a

similar phenomenon could be occurring in the case of 8b and

that this change in conformation is better tolerated by ALK2

than ALK5.

We decided to introduce a larger substituent ortho to the

amide in order to conﬁrm whether the latter hypothesis was

correct. Replacing the ﬂuorine atom of 8b with a chlorine atom

(20e) increased the biochemical selectivity over ALK5 to

greater than 200-fold, suggesting that our proposed explan-

ation is a reasonable one. However, NanoBRET ALK2 IC₅₀

values were similar for both 8b and 20e, indicating that the

modiﬁcation has little impact on ALK2 potency. To determine

if electron-donating groups could be tolerated at this position

as well, 8c was prepared. This analogue had the greatest

structural similarity to our lead compound M4K2009.

F

https://dx.doi.org/10.1021/acs.jmedchem.0c00395

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 2. Inhibitory and Oﬀ-Target Activities of 8b−c, 18a−b, 20a−e

a

b

Average of duplicate measurements. Average of triplicate measurements.

Although 8c was the most potent analogue in our series, it

suﬀered from poor selectivity (biochemical selectivity of 9-fold

over ALK5). Additionally, there was a substantial diﬀerence in

its biochemical and cell-based potencies (biochemical ALK2

IC₅₀= 5 nM vs NanoBRET ALK2 IC₅₀= 178 nM).

Permeability, Selectivity, and Pharmacokinetic Stud-

ies. Having identiﬁed several potent analogues, we decided to

focus our eﬀorts on improving the pharmacokinetic (PK)

proﬁles of two: 8b and 8c. In order to assess the permeability

of these compounds, they were tested in a Caco-2 assay, which

revealed that both analogues were poorly permeable and being

recognized by eﬄux transporters (eﬄux ratios for both 8b and

8c were >30) (see Tables 4 and 2 in the Supporting

Information). In an attempt to reduce eﬄux, the terminal

piperazine nitrogens of 8b and 8c were capped with various

alkyl groups to generate 1-methyl-, 1-isopropyl-, and 1,2,6-

trimethylpiperazine analogues (26a−f).³⁰In addition to

reducing the number of HBD, methylation of the terminal

piperazine nitrogen oﬀered the additional advantage of

attenuating pK_a,^25,31which is often associated with a decrease

G

https://dx.doi.org/10.1021/acs.jmedchem.0c00395

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 3. Inhibitory and Oﬀ-Target Activity of 2-Fluoro-6-methoxybenzamide and 2,6-Dimethoxybenzamide Analogues, 26a−f

a

b

c

d

Average of duplicate measurements. Average of triplicate measurements. Average of quadruplicate measurements. Average of quintuplicate

measurements.

in P-gp-mediated eﬄux.²⁴The rationale behind incorporating

methyl groups at positions 1, 2, and 6 of the piperazine groups

was to increase the molecular rigidity of analogues 26c and

26f. This is a strategy that is commonly employed to enhance

brain penetration and oral bioavailability.²⁴As it has been

reported that the piperazine motif of LDN-193189 is a

metabolic liability,³²increasing the steric bulk around this

group was also done to improve the inhibitors’ ADME proﬁle

in vivo.

As anticipated, the permeability (P_{app_AB}) of the 2-ﬂuoro-6-

methoxybenzamide analogues was increased from 0.3 × 10⁻⁶

cm/s (8b) to around 5.0 × 10⁻⁶cm/s for 26a−c. This was

accompanied by a concomitant reduction in the eﬄux ratio

(from >30 for 8b to less than 3.0 for 26a and 26b) (Table 4).

An enhancement in selectivity over ALK5 was also observed

for these compounds. This was more pronounced in the

biochemical kinase assay (Table 3). Unfortunately, similar

results were not obtained for the 2,6-dimethoxybenzamide

analogues (26d−f). Piperazine alkylation appeared to have

little eﬀect on reducing eﬄux (see the Supporting Information,

Table 2). However, 26d−f demonstrated excellent inhibitory

activity against ALK2 in the NanoBRET assay. Although

M4K2149 and 8c diﬀer by only one methoxy group, the latter

analogue had a signiﬁcantly higher eﬄux ratio (8.1 vs >30).²¹

We suspected that the extra electron-donating group was

increasing the hydrogen bond acceptor potential of the amide

carbonyl, which was being recognized by one of the eﬄux

transporters expressed by the Caco-2 cells. Consequently,

these analogues were excluded from further proﬁling.

Prior to assessing the PK proﬁles of the 2-ﬂuoro-6-

methoxybenzamide analogues in vivo, the metabolic stabilities

of 8b and 26a−c were evaluated in mouse and human liver

microsomal (MLM and HLM) stability assays. All four

analogues exhibited moderate to high stability in both in

vitro assays, with compounds 8b and 26b demonstrating the

highest degree of stability after a 60 min incubation period at

37 °C (>85% remaining) (see the Supporting Information,

Table 3). Oral administration of a 10 mg/kg dose of 8b in

female CB17 SCID mice (n = 3) gave rise to suboptimal values

for C_max(97 ng/mL), t_1/2(1.31 h), and AUC_inf(296 ng·h/

mL). Given the poor intrinsic permeability of 8b, these results

were not surprising. A signiﬁcant improvement in PK

properties was observed for analogues 26a and 26b, both of

which yielded a greater than 17-fold increase in C_max, 13-fold

H

https://dx.doi.org/10.1021/acs.jmedchem.0c00395

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 4. In Vitro Permeability and Oral In Vivo PK Studies of 2-Fluoro-6-methoxybenzamide Analogues

Table 5. Inhibitory Activity of Benzamide Analogues against WT and DIPG-Linked Mutant Forms of ALK2

increase in AUC_inf, and a doubling of t_1/2(Table 4). The two

analogues were also assessed for their ability to penetrate the

BBB in the same strain of mice. Oral administration of these

compounds at a 100 mg/kg dose gave rise to average total

brain concentrations of 777 and 1595 ng/g and total brain-to-

plasma ratios (B/P) of 0.178 and 0.132 for 26a and 26b,

respectively. Although these values are moderate, the use of B/

P ratios to assess brain permeability is generally not

encouraged. The extent of brain penetration is typically

evaluated based on the ratio of the unbound brain

concentration to the unbound plasma concentration (K_p,uu).²

Whether these analogues require additional modiﬁcations to

enhance BBB permeability will ultimately depend on the value

of this parameter.

To ensure that a favorable hERG proﬁle had been

maintained for the 2-ﬂuoro-6-methoxybenzamide analogues,

their potencies against the hERG potassium channel were

assessed using a HEK293 cell-based patch-clamp assay. 26a

and 26b had optimal hERG IC₅₀values of >30 μM, while 26c

was slightly more potent against the ion channel (IC₅₀= 19

μM) (see the Supporting Information, Table 4). To further

investigate the oﬀ-target activity of these analogues, we proﬁled

them in a 375-member kinase panel. At a concentration of 1

μM for each of the three compounds, fewer than 5% of the

kinases showed a greater than 50% reduction in enzymatic

activity. Excluding ALK1, 2, 3, and 6, the kinases ARAF,

MAP4K4, MINK, and TNK1 were the most sensitive to

inhibition by 26a−c (see the Supporting Information, Table

5). We were also encouraged by the results obtained in an in

I

https://dx.doi.org/10.1021/acs.jmedchem.0c00395

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

solvents. The yields given refer to chromatographically puriﬁed and

spectroscopically pure compounds. Compounds were puriﬁed using a

Biotage Isolera One system by normal phase chromatography using

vitro CYP inhibition assay, which showed that these analogues

had negligible inhibitory activity (IC₅₀> 50 μM) against 7

CYP isoforms (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and

3A4) (see the Supporting Information, Table 4). Altogether,

these results demonstrate that we were able to meet our

objective of designing selective and orally bioavailable

inhibitors of ALK2. Furthermore, proﬁling 7 analogues against

3 ALK2 mutants (R206H, G328V, and R258G) in a

radioactive in vitro kinase assay revealed that, similar to

M4K2009, the analogues had comparable potencies against

both WT and mutant ALK2 (Table 5). These ﬁndings conﬁrm

that the benzamide inhibitors developed in this series have the

potential to regulate aberrant BMP signaling in patients

harboring these mutations.

̈

Biotage SNAP KP-Sil or Sfar Silica D columns (part no.: FSKO-1107/

FSRD-0445) or by reverse-phase chromatography using Biotage

̈

SNAP KP-C18-HS or Sfar C18 D columns (part no.: FSLO-1118/

FSUD-040). If additional puriﬁcation was required, compounds were

puriﬁed by solid phase extraction (SPE) using Biotage Isolute Flash

SCX-2 cation exchange cartridges (part nos.: 532-0050-C and 456-

0200-D). Products were washed with two cartridge volumes of

MeOH and eluted with a solution of MeOH and NH₄OH (9:1 v/v).

Preparative chromatography was carried out using a Waters 2767

injector with the collector attached to PDA UV/Vis and SQD mass

detectors. An XSelect CSH Prep C18 5 μm OBD 19 mm × 100 mm

(part no.: 186005421) or Xselect CSH Prep C18 5 μm 10 mm × 100

mm (part no.: 186005415) column was used for puriﬁcation. Final

compounds were dried using the Labconco Benchtop FreeZone

Freeze-Dry System (4.5 L Model). ¹H and proton-decoupled ¹⁹F

NMRs were recorded on a Bruker AVANCE-III 500 MHz

spectrometer at ambient temperature. Residual protons of CDCl₃,

DMSO-d₆, and CD₃OD solvents were used as internal references.

Spectral data are reported as follows: chemical shift (δ in ppm),

multiplicity (br = broad, s = singlet, d = doublet, dd = doublet of

doublets, m = multiplet), coupling constants (J in Hz), and proton

integration. Compound purity was determined by UV absorbance at

254 nm during tandem liquid chromatography/mass spectrometry

(LCMS) using a Waters Acquity separation module. All ﬁnal

compounds had a purity of ≥95% as determined using this method.

Low-resolution mass spectrometry was conducted in the positive ion

mode using a Waters Acquity SQD mass spectrometer (electrospray

ionization source) ﬁtted with a PDA detector. Mobile phase A

consisted of 0.1% formic acid in water, while mobile phase B consisted

of 0.1% formic acid in acetonitrile. One of three types of columns

were used: column 1: Acquity UPLC CSH C18 (2.1 × 50 mm, 130 Å,

1.7 μm. part no. 186005296), column 2: Acquity UPLC BEH C8 (2.1

× 50 mm, 130 Å, 1.7 μm. part no. 186002877), or column 3: Acquity

UPLC HSS T3 (2.1 × 50 mm, 100 Å, 1.8 μm. part no. 186003538).

For columns 1 and 2, the gradient went from 90 to 5% mobile phase

A over 1.8 min, maintained at 5% for 0.5 min, then increased to 90%

over 0.2 min for a total run time of 3 min. For column 3, the gradient

went from 98 to 5% mobile phase A over 1.8 min, maintained at 5%

for 0.5 min, then increased to 98% over 0.2 min for a total run time of

3 min, as well. The ﬂow rate was 0.4 mL/min throughout both runs.

All columns were used with the temperature maintained at 25 °C.

High-resolution mass spectrometry was conducted using a Waters

Synapt G2-S quadrupole-time-of-ﬂight (QTOF) hybrid mass

spectrometer system coupled with an Acquity ultra-performance

liquid chromatography (UPLC) system. Chromatographic separations

were carried out on an Acquity UPLC CSH C18 (2.1 × 50 mm, 130

Å, 1.7 μm. part no. 186005296), Acquity UPLC BEH C8 (2.1 × 50

mm, 130 Å, 1.7 μm. part no. 186002877), or Acquity UPLC HSS T3

(2.1 × 50 mm, 100 Å, 1.8 μm. part no. 186003538). The mobile

phases were 0.1% formic acid in water (solvent A) and 0.1% formic

acid in acetonitrile (solvent B). Leucine Enkephalin was used as the

lock mass. MassLynx 4.1 was used for data analysis.

CONCLUSIONS

■

Advances in the development of eﬀective chemotherapeutic

agents for the treatment of DIPG have been limited. This is in

part due to the convoluted genomic signatures of DIPG, which

has made our understanding of its pathogenesis diﬃcult.

Recent identiﬁcation of ALK2 as a target for therapeutic

intervention has prompted the emergence of several classes of

type I kinase inhibitors. In this work, we expanded the SAR of

the 3,5-diarylpyridine inhibitor LDN-214117, which led to the

discovery of a potent benzamide analogue M4K2149 with an

attenuated aﬃnity for the hERG potassium channel. We

determined that we could tailor the selectivity of our analogues

over ALK5 by incorporating halogen substituents at a position

ortho to the amide group of M4K2149. We were also able to

address issues of permeability by capping the NH of the

solvent-exposed piperazine group. The resulting 2-ﬂuoro-6-

methoxybenzamide derivatives 26a−c demonstrated excellent

kinome-wide selectivity and had improved PK properties

compared to their parent compound 8b. Furthermore, the co-

crystal structure of M4K2149 with ALK2 helped us rationalize

potency diﬀerences between analogues in the series and

highlighted structural motifs that were crucial for maintaining

key interactions with the protein. Despite these optimizations,

total brain-to-plasma ratios are inadequate for accurately

assessing the pharmacological activity of 26a−b in the brain.

Measuring the unbound brain concentrations (C_b,u) of these

analogues in vivo is therefore warranted. These data would

ultimately determine whether additional modiﬁcations should

be made to reduce eﬄux/enhance permeability. Nonetheless,

these benzamides represent a new chemotype possessing high

inhibitory activity against both WT and mutant ALK2.

Implementation of an open science model accelerated the

development of these analogues by promoting communication

between the chemists involved in their design and establishing

a pipeline for rapidly generating biological data. Future work

will continue to use open science to develop novel classes of

ALK2 inhibitors. The analogues presented in this study have

the potential to deepen our understanding of the biology of

DIPG and will hopefully pave the way for future chemo-

therapies.

tert-Butyl 4-(4-(5-Bromo-4-methylpyridin-3-yl)phenyl)-

piperazine-1-carboxylate (3). A solution of tert-butyl 4-(4-

(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperazine-1-car-

boxylate (1) (1.549 g, 3.99 mmol), 3,5-dibromo-4-methylpyridine (2)

(0.953 g, 3.80 mmol), [1,12-bis(diphenylphosphino)ferrocene]-

dichloropalladium(II)·DCM complex (0.310 g, 0.38 mmol), and

sodium carbonate monohydrate (1.414 g, 11.40 mmol) in 1,4-dioxane

(16.3 mL) and water (2.7 mL) was heated to 85 °C and stirred

overnight. The reaction mixture was concentrated under reduced

pressure prior to dilution with water (30 mL) and extraction with

EtOAc (3 × 30 mL). The combined extracts were dried over MgSO₄

and concentrated to yield brown oil. The crude material was puriﬁed

by silica gel chromatography (0−50% EtOAc in hexanes) to aﬀord a

white solid (0.770 g, 47% yield). ¹H NMR (500 MHz, CDCl₃): δ 8.61

(s, 1H), 8.31 (s, 1H), 7.20 (d, J = 8.6 Hz, 2H), 6.99 (d, J = 8.8 Hz,

EXPERIMENTAL SECTION

■

Chemistry. All reagents were purchased from commercial vendors

and used without further puriﬁcation. Volatiles were removed under

reduced pressure by rotary evaporation or using the V-10 solvent

evaporator system by Biotage. Very high boiling point (6000 rpm, 0

mbar, 56 °C), mixed volatile (7000 rpm, 30 mbar, 36 °C), and volatile

(6000 rpm, 30 mbar, 36 °C) methods were used to evaporate

J

https://dx.doi.org/10.1021/acs.jmedchem.0c00395

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

2H), 3.65−3.56 (m, 4H), 3.26−3.18 (m, 4H), 2.36 (s, 3H), 1.49 (s,

9H). MS (ESI) m/z: 432.31 [M + H]⁺, 434.38 [M + H]⁺+ 2.

(5-(4-(4-(tert-Butoxycarbonyl)piperazin-1-yl)phenyl)-4-

methylpyridin-3-yl)boronic Acid (4a). A solution of 3 (171 mg,

0.396 mmol), bis(pinacolato)diboron (201 mg, 0.792 mmol), [1,12-

bis(diphenylphosphino)ferrocene]dichloropalladium(II)·DCM com-

plex (32 mg, 0.040 mmol), and potassium acetate (78 mg, 0.792

mmol) in 1,4-dioxane (4 mL) was microwaved at 110 °C for 4 h. The

mixture was transferred to a 15 mL Falcon tube and centrifuged for 1

min at 4000 rpm. The dark brown supernatant was used without

further puriﬁcation in subsequent reactions (190 mg, 57% yield). MS

(ESI) m/z: 397.90 [M + H]⁺.

5-Chloro-4-methoxythiophene-3-carboxamide (5c). The title

compound was prepared using a modiﬁed literature procedure.³³To a

solution of 5-chloro-4-methoxythiophene-3-carboxylic acid (100.0

mg, 0.519 mmol) in DCM (1.5 mL) was added ammonium chloride

(33.3 mg, 0.623 mmol), HATU (237.0 mg, 0.623 mmol), and DIPEA

(271 μL, 1.558 mmol). The reaction mixture was stirred at room

temperature for 3 h. Volatiles were removed under reduced pressure,

and the crude product was puriﬁed by silica gel chromatography (0−

100% EtOAc in hexanes) to aﬀord an oﬀ-white solid (71.1 mg, 72%

yield). ¹H NMR (500 MHz, CDCl₃): δ 7.91 (s, 1H), 7.21 (br s, 1H),

5.72 (br s, 1H), 4.06 (s, 3H). MS (ESI) m/z: 192.27 [M + H]⁺,

194.28 [M + H]⁺+ 2.

4-bromo-3-methoxythiophene-2-carboxylate (5b) (42 mg, 0.167

mmol). The ﬁnal product was a light yellow powder (47 mg, 52%

yield). MS (ESI) m/z: 524.70 [M + H]⁺.

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

pyridin-3-yl)thiophene-2-carboxamide (7a). A 5 mL MW vial

was charged with 6a (20.0 mg, 0.038 mmol). The material was

dissolved in a solution of ammonia in methanol (7 N) (4 mL). The

vial was sealed, and the solution was stirred at 90 °C for 3 days.

Volatiles were removed under reduced pressure, and the crude

material was puriﬁed by silica gel chromatography (0−100% EtOAc in

hexanes). The puriﬁed product was dissolved in DCM (1 mL) and

treated with triﬂuoroacetic acid (88 μL, 1.146 mmol). The solution

was stirred overnight. The product was puriﬁed by SPE. Drying under

high vacuum overnight aﬀorded an oﬀ-white powder (7.8 mg, 47%

1

yield). H NMR (500 MHz, DMSO): δ 8.37 (s, 1H), 8.36 (s, 1H),

7.76 (s, 1H), 7.66 (br s, 1H), 7.31−7.25 (m, 3H), 7.05 (d, J = 8.7 Hz,

2H), 3.51 (s, 3H), 3.22−3.20 (m, 4H), 2.98−2.94 (m, 4H), 2.13 (s,

3H). HRMS (ESI) for C₂₂H₂₄N₄O₂S [M + H]⁺m/z: calcd, 409.1693;

found, 409.1691.

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

pyridin-3-yl)thiophene-2-carboxamide (7b). The title com-

pound was synthesized according to the procedure described for 7a

from 6b (20.0 mg, 0.038 mmol). The ﬁnal product was an oﬀ-white

powder (8.9 mg, 53% yield). ¹H NMR (500 MHz, DMSO): δ 8.38 (s,

1H), 8.36 (s, 1H), 7.76 (s, 1H), 7.66 (br s, 1H), 7.30−7.26 (m, 3H),

7.04 (d, J = 8.7 Hz, 2H), 3.52 (s, 3H), 3.19−3.17 (m, 4H), 2.95−2.91

(m, 4H), 2.13 (s, 3H). HRMS (ESI) for C₂₂H₂₄N₄O₂S [M + H]⁺m/

z: calcd, 409.1693; found, 409.1689.

4-Bromo-2-ﬂuoro-6-methoxybenzamide (5d). The title com-

pound was prepared using modiﬁed literature procedures.^34,35

A

solution of 4-bromo-2-ﬂuoro-6-methoxybenzonitrile (22a) (5.00 g,

21.74 mmol) in EtOH (100.0 mL) was cooled in an ice bath prior to

the addition of an aqueous solution of sodium hydroxide (0.43 M,

65.0 mL). This was followed by the addition of hydrogen peroxide

(30 wt % solution in water) (26.6 mL). The solution was stirred at

room temperature overnight. The reaction mixture was concentrated

under reduced pressure prior to dilution with water (250 mL) and

extraction with EtOAc (3 × 250 mL). The combined organic extracts

were dried over Na₂SO₄, ﬁltered, and concentrated under reduced

pressure to give a white crystalline solid (4.63 g, 80% yield). ¹H NMR

(500 MHz, DMSO): δ 7.84 (br s, 1H), 7.58 (br s, 1H), 7.17 (d, J =

8.5 Hz, 1H), 7.13 (s, 1H), 3.82 (s, 3H). ¹⁹F NMR (471 MHz,

DMSO): δ −115.38. MS (ESI) m/z: 248.20 [M + H]⁺, 250.27 [M +

H]⁺+ 2.

4-Bromo-2,6-dimethoxybenzamide (5e). The title compound

was synthesized according to the procedure described for 5c from 4-

bromo-2,6-dimethoxybenzoic acid (157 mg, 0.600 mmol). The ﬁnal

product was a white solid (100 mg, 64% yield). ¹H NMR (500 MHz,

DMSO): δ 7.51 (br s, 1H), 7.23 (br s, 1H), 6.87 (s, 2H), 3.75 (s,

6H). MS (ESI) m/z: 260.35 [M + H]⁺, 262.29 [M + H]⁺+ 2.

The title compound was alternatively synthesized according to the

procedure described for 5d from 4-bromo-2,6-dimethoxybenzonitrile

(22b) (968 mg, 4.00 mmol), hydrogen peroxide (30 wt % solution in

water) (9.8 mL), and an aqueous solution of sodium hydroxide (2 M,

25.0 mL). The reaction mixture was heated to 110 °C for 8 h. The

solvents were evaporated and the crude material was suspended in

water, ﬁltered, and dried under high vacuum to aﬀord a white

crystalline solid (903 mg, 87% yield).

tert-Butyl 4-(4-(5-(4-Methoxy-5-(methoxycarbonyl)-

thiophen-2-yl)-4-methylpyridin-3-yl)phenyl)piperazine-1-car-

boxylate (6a). A solution of 4a (80 mg, 0.167 mmol), methyl 5-

bromo-3-methoxythiophene-2-carboxylate (42 mg, 0.167 mmol)

(5a), [1,12-bis(diphenylphosphino)ferrocene]dichloropalladium(II)·

DCM complex (14 mg, 0.017 mmol), and sodium carbonate

monohydrate (62 mg, 0.501 mmol) in 1,4-dioxane (2.9 mL) and

water (477 μL) was heated to 100 °C for 2 h. The reaction mixture

was adsorbed onto Celite, and the volatiles were removed under

reduced pressure. The crude product was puriﬁed by silica gel

chromatography (0−100% EtOAc in hexanes) to aﬀord an oﬀ-white

powder (45 mg, 50% yield). MS (ESI) m/z: 524.70 [M + H]⁺.

tert-Butyl 4-(4-(5-(4-Methoxy-5-(methoxycarbonyl)-

thiophen-3-yl)-4-methylpyridin-3-yl)phenyl)piperazine-1-car-

boxylate (6b). The title compound was synthesized according to the

procedure described for 6a from 4a (80 mg, 0.167 mmol) and methyl

4-Methoxy-5-(4-methyl-5-(4-(piperazin-1-yl)phenyl)-

pyridin-3-yl)thiophene-3-carboxamide (8a). A solution of 4a

(60.0 mg, 0.125 mmol), 5c (20.0 mg, 0.104 mmol), XPhos Pd G2

(8.2 mg, 0.010 mmol), and potassium phosphate tribasic (44.3 mg,

0.209 mmol) in 1,4-dioxane (1.8 mL) and water (298 μL) was heated

to 100 °C and stirred for 3 h. The reaction mixture was adsorbed onto

Celite, and volatiles were removed under reduced pressure. The crude

product was puriﬁed by silica gel chromatography (0−100% EtOAc in

hexanes). Further puriﬁcation was carried out by reverse-phase

chromatography [2−95% ACN (0.1% formic acid) in water (0.1%

formic acid)]. The product was dissolved in DCM (1 mL) and treated

with triﬂuoroacetic acid (479 μL, 6.26 mmol). The solution was

stirred for 1 h. The product was puriﬁed by SPE. Freeze-drying for 3

1

days aﬀorded an oﬀ-white powder (6.5 mg, 15% yield). H NMR

(500 MHz, DMSO): δ 8.44 (s, 1H), 8.39 (s, 1H), 8.11 (s, 1H), 7.47

(br s, 1H), 7.44 (br s, 1H), 7.30 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 8.7

Hz, 2H), 3.57 (s, 3H), 3.24−3.21 (m, 4H), 3.01−2.96 (m, 4H), 2.17

(s, 3H). HRMS (ESI) for C₂₂H₂₄N₄O₂S [M + H]⁺m/z: calcd,

409.1693; found, 409.1694.

2-Fluoro-6-methoxy-4-(4-methyl-5-(4-(piperazin-1-yl)-

phenyl)pyridin-3-yl)benzamide (8b). The title compound was

synthesized according to the procedure described for 8a from 4a (77

mg, 0.161 mmol) and 5d (40 mg, 0.161 mmol). The ﬁnal product was

an oﬀ-white powder (20 mg, 29% yield). ¹H NMR (500 MHz,

DMSO): δ 8.38 (s, 1H), 8.34 (s, 1H), 7.89 (br s, 1H), 7.58 (br s,

1H), 7.30 (d, J = 8.6 Hz, 2H), 7.04 (d, J = 8.6 Hz, 2H), 6.98−6.95

(m, 2H), 3.85 (s, 3H), 3.19−3.15 (m, 4H), 2.94−2.89 (m, 4H), 2.19

(s, 3H). ¹⁹F NMR (471 MHz, DMSO): δ −116.73. HRMS (ESI) for

C₂₄H₂₅FN₄O₂[M + H]⁺m/z: calcd, 421.2034; found, 421.2040.

2,6-Dimethoxy-4-(4-methyl-5-(4-(piperazin-1-yl)phenyl)-

pyridin-3-yl)benzamide (8c). The title compound was synthesized

according to the procedure described for 8a from 4a (190 mg, 0.396

mmol) and 5e (82 mg, 0.317 mmol). The ﬁnal product was an oﬀ-

1

white powder (45 mg, 32% yield). H NMR (500 MHz, DMSO): δ

8.36 (s, 1H), 8.35 (s, 1H), 7.57 (br s, 1H), 7.31 (d, J = 8.6 Hz, 2H),

7.23 (br s, 1H), 7.04 (d, J = 8.7 Hz, 2H), 6.72 (s, 2H), 3.78 (s, 6H),

3.19−3.15 (m, 4H), 2.93−2.89 (m, 4H), 2.20 (s, 3H). HRMS (ESI)

for C₂₅H₂₈N₄O₃[M + H]⁺m/z: calcd, 433.2234; found, 433.2228.

5-(5-Chloro-4-methylpyridin-3-yl)-2-methoxybenzoic Acid

(11a). The title compound was synthesized according to the

procedure described for 6a from 3-bromo-5-chloro-4-methylpyridine

(10) (41 mg, 0.200 mmol) and 5-borono-2-methoxybenzoic acid (9a)

K

https://dx.doi.org/10.1021/acs.jmedchem.0c00395

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(39 mg, 0.200 mmol). DMF (1.6 mL) and water (428 μL) were used

as the solvents.

The crude material was used without puriﬁcation in the subsequent

cross-coupling reaction (56 mg, 87% yield). MS (ESI) m/z: 278.30

[M + H]⁺, 280.30 [M + H]⁺+ 2.

3-(5-Chloro-4-methylpyridin-3-yl)-5-methoxybenzoic Acid

(11b). The title compound was synthesized according to the

procedure described for 6a from 3-bromo-5-chloro-4-methylpyridine

(10) (41 mg, 0.200 mmol) and 3-carboxy-5-methoxyphenylboronic

acid (9b) (39 mg, 0.200 mmol). DMF (1.6 mL) and water (428 μL)

were used as the solvents.

The crude material was used without puriﬁcation in the subsequent

cross-coupling reaction (56 mg, 90% yield). MS (ESI) m/z: 278.23

[M + H]⁺, 280.30 [M + H]⁺+ 2.

pressure to aﬀord a light beige solid (160 mg, 99% yield), which was

used without further puriﬁcation in the subsequent reaction. MS

(ESI) m/z: 518.57 [M + H]⁺.

tert-Butyl 4-(4-(5-(4-Carbamoyl-3-methoxyphenyl)-4-meth-

ylpyridin-3-yl)phenyl)piperazine-1-carboxylate (M4K2149).

To a solution of 17 (0.160 g, 0.309 mmol) in MeOH (3.0 mL) at

room temperature was added a solution of ammonia in MeOH (7 N)

(4.4 mL). The resulting mixture was heated to 75 °C for 3 days prior

to cooling back down to room temperature, removing all solvents

under reduced pressure, and triturating the residue from EtOAc with

hexanes. The beige precipitate was collected by ﬁltration and washed

with hexanes. The product was subsequently dissolved in MeOH (5.0

mL) and treated with HCl (4.0 M in dioxane, 1.0 mL). The solution

was stirred for 30 min prior to the removal of solvents under reduced

pressure. The product was puriﬁed by SPE. The ﬁnal compound was

dried under vacuum overnight to give an oﬀ-white solid (75 mg, 60%

5-(5-(4-(4-(tert-Butoxycarbonyl)piperazin-1-yl)phenyl)-4-

methylpyridin-3-yl)-2-methoxybenzoic Acid (13a). The title

compound was synthesized according to the procedure described for

8a from 11a (56 mg, 0.200 mmol) and 1 (140 mg, 0.360 mmol). The

crude product was puriﬁed by reverse-phase chromatography [2−95%

ACN (0.1% formic acid) in water (0.1% formic acid)]. The puriﬁed

intermediate was used immediately in the subsequent reaction.

3-(5-(4-(4-(tert-Butoxycarbonyl)piperazin-1-yl)phenyl)-4-

methylpyridin-3-yl)-5-methoxybenzoic Acid (13b). The title

compound was synthesized according to the procedure described for

8a from 11b (56 mg, 0.200 mmol) and 1 (140 mg, 0.360 mmol). The

crude product was puriﬁed by reverse-phase chromatography [2−95%

ACN (0.1% formic acid) in water (0.1% formic acid)]. The puriﬁed

intermediate was used immediately in the subsequent reaction.

2-Methoxy-5-(4-methyl-5-(4-(piperazin-1-yl)phenyl)-

pyridin-3-yl)benzamide (14a). The title compound was synthe-

sized according to the procedure described for 5c from 13a.

Deprotection with triﬂuoroacetic acid (459 μL, 6.00 mmol),

puriﬁcation by SPE, and freeze-drying for 2 days aﬀorded the ﬁnal

1

yield). H NMR (500 MHz, MeOD): δ 8.35 (s, 1H), 8.34 (s, 1H),

8.10 (d, J = 7.9 Hz, 1H), 7.33 (d, J = 8.6 Hz, 2H), 7.20 (s, 1H), 7.12

(d, J = 8.3 Hz, 3H), 4.04 (s, 3H), 3.32−3.26 (m, 4H), 3.13−3.07 (m,

4H), 2.24 (s, 3H). HRMS (ESI) for C₂₄H₂₆N₄O₂[M + H]⁺m/z:

calcd, 403.2129; found, 403.2128.

tert-Butyl 4-(4-(5-(3-Methoxy-4-(methylcarbamoyl)phenyl)-

4-methylpyridin-3-yl)phenyl)piperazine-1-carboxylate (18a).

To a solution of 17 (42 mg, 0.081 mmol) in MeOH (811 μL) was

added methylamine, 33 wt % in EtOH (1.0 mL). The solution was

stirred at 85 °C for 5h. The solvents were removed under reduced

pressure prior to the crude material being triturated from a minimum

amount of EtOAc and hexanes. The product was ﬁltered and dried

under air, then dissolved in DCM (213 μL), treated with

triﬂuoroacetic acid (100 μL, 1.310 mmol), and stirred for 1 h. The

solution was concentrated under reduced pressure prior to

puriﬁcation by reverse-phase chromatography [2−95% ACN (0.1%

formic acid) in water (0.1% formic acid)]. The product was puriﬁed

by SPE. Freeze-drying for 2 days aﬀorded a white powder (7.68 mg,

1

product as a yellow powder (5.96 mg, 7% yield over 4 steps). H

1

NMR (500 MHz, DMSO): δ 8.32 (s, 1H), 8.29 (s, 1H), 7.81 (d, J =

2.1 Hz, 1H), 7.71 (br s, 1H), 7.59 (br s, 1H), 7.56 (dd, J = 8.5, 2.2

Hz, 1H), 7.31 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.6 Hz, 1H), 7.04 (d, J

= 8.6 Hz, 2H), 3.95 (s, 3H), 3.21−3.18 (m, 4H), 2.97−2.93 (m, 4H),

2.14 (s, 3H). HRMS (ESI) for C₂₄H₂₆N₄O₂[M + H]⁺m/z: calcd,

403.2129; found, 403.2126.

20% yield). H NMR (500 MHz, MeOD): δ 8.32 (s, 1H), 8.30 (s,

1H), 8.01 (d, J = 7.9 Hz, 1H), 7.30 (d, J = 8.6 Hz, 2H), 7.15 (s, 1H),

7.12−7.06 (m, 3H), 4.00 (s, 3H), 3.30−3.27 (m, 4H), 3.12−3.08 (m,

4H), 2.98 (s, 3H), 2.21 (s, 3H). HRMS (ESI⁺) for C₂₅H₂₈N₄O₂[M +

H]⁺m/z: calcd, 417.2285; found, 417.2288.

4-(5-(4-(4-(tert-Butoxycarbonyl)piperazin-1-yl)phenyl)-4-

methylpyridin-3-yl)-2-methoxybenzoic Acid. To a suspension of

17 (54 mg, 0.104 mmol) in THF (695 μL) and water (695 μL) was

added potassium hydroxide pellets (12 mg, 0.209 mmol). The

suspension was stirred at room temperature for 2 h. The reaction

mixture was diluted with water (35 mL) and extracted with Et₂O (1 ×

20 mL). The aqueous layer was carefully acidiﬁed to a pH of 5 and

extracted with DCM (3 × 20 mL). The Et₂O and DCM layers were

combined, dried over MgSO₄, ﬁltered, and concentrated under

reduced pressure to aﬀord an oﬀ-white solid (50 mg, 93% yield). MS

(ESI) m/z: 504.60 [M + H]⁺.

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

pyridin-3-yl)benzamide (14b). The title compound was synthe-

sized according to the procedure described for 5c from 13b.

Deprotection with triﬂuoroacetic acid (0.459 mL, 6.00 mmol),

puriﬁcation by reverse-phase chromatography and SPE and freeze

drying for 2 days aﬀorded the ﬁnal product as an oﬀ-white powder

1

(4.52 mg, 6% yield over 4 steps). H NMR (500 MHz, DMSO): δ

8.36 (s, 1H), 8.35 (s, 1H), 8.02 (br s, 1H), 7.52−7.49 (m, 1H), 7.49−

7.47 (m, 1H), 7.44 (br s, 1H), 7.31 (d, J = 8.6 Hz, 2H), 7.17−7.15

(m, 1H), 7.05 (d, J = 8.7 Hz, 2H), 3.85 (s, 3H), 3.23−3.20 (m, 4H),

2.99−2.95 (m, 4H), 2.15 (s, 3H). HRMS (ESI) for C₂₄H₂₆N₄O₂[M

+ H]⁺m/z: calcd, 403.2129; found, 403.2137.

2-Methoxy-N,N-dimethyl-4-(4-methyl-5-(4-(piperazin-1-yl)-

phenyl)pyridin-3-yl)benzamide (18b). To a solution of 4-(5-(4-

(4-(tert-butoxycarbonyl)piperazin-1-yl)phenyl)-4-methylpyridin-3-

yl)-2-methoxybenzoic acid (50 mg, 0.099 mmol), HOBt (16 mg,

0.119 mmol), and EDC (18 mg, 0.119 mmol) in DCM (894 μL) and

DMF (99 μL) was added DIPEA (43 μL, 0.248 mmol) and

dimethylamine, 2.0 M in THF (50 μL, 0.099 mmol). The solution

was stirred at 50 °C overnight. The reaction mixture was diluted with

water (5 mL) and DCM (5 mL). The organic layer was separated,

dried over MgSO₄, ﬁltered, and concentrated under reduced pressure

to aﬀord a sticky yellow solid. The solid was dissolved in DCM (886

μL) and treated with triﬂuoroacetic acid (339 μL, 4.430 mmol). The

solution was stirred for 45 min prior to puriﬁcation by reverse-phase

chromatography [2−95% ACN (0.1% formic acid) in water (0.1%

formic acid)]. The product was puriﬁed by SPE. Freeze-drying for 2

tert-Butyl 4-(4-(5-Chloro-4-methylpyridin-3-yl)phenyl)-

piperazine-1-carboxylate (15). The title compound was synthe-

sized according to the procedure described for 6a from 10 (320 mg,

1.550 mmol) and 1 (722 mg, 1.860 mmol). The ﬁnal product was an

1

oﬀ-white crystalline solid (510 mg, 85% yield). H NMR (500 MHz,

CDCl₃): δ 8.48 (s, 1H), 8.30 (s, 1H), 7.21 (d, J = 8.7 Hz, 2H), 6.99

(d, J = 8.7 Hz, 2H), 3.63−3.59 (m, 4H), 3.24−3.19 (m, 4H), 2.33 (s,

3H), 1.49 (s, 9H). MS (ESI) m/z: 388.56 [M + H]⁺, 390.57 [M +

H]⁺+ 2.

tert-Butyl 4-(4-(5-(3-Methoxy-4-(methoxycarbonyl)phenyl)-

4-methylpyridin-3-yl)phenyl)piperazine-1-carboxylate (17).

The title compound was synthesized according to the procedure

described for 8a from 15 (120 mg, 0.309 mmol) and 3-methoxy-4-

methoxycarbonylphenylboronic acid, pinacol ester (16) (90 mg,

0.309 mmol). The solvents used were butan-1-ol (2 mL) and water

(476 μL). The reaction mixture was diluted with water (20 mL) and

extracted with EtOAc (3 × 20 mL). The combined organic fractions

were dried over Na₂SO₄, ﬁltered, and concentrated under reduced

1

days aﬀorded a white powder (13 mg, 23% yield). H NMR (500

MHz, MeOD): δ 8.33−8.30 (m, 2H), 7.35−7.28 (m, 3H), 7.12−7.04

(m, 4H), 3.90 (s, 3H), 3.28−3.24 (m, 4H), 3.12 (s, 3H), 3.08−3.04

(m, 4H), 2.94 (s, 3H), 2.22 (s, 3H). HRMS (ESI) for C₂₅H₂₈N₄O₂

[M + H]⁺m/z: calcd, 431.2442; found, 431.2439.

L

https://dx.doi.org/10.1021/acs.jmedchem.0c00395

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

4-Bromo-2-(hydroxymethyl)benzonitrile. To a solution of 4-

bromo-2-formylbenzonitrile (630 mg, 3.00 mmol) in MeOH (7.5

mL) cooled in an ice bath was added sodium borohydride (125 mg,

3.30 mmol). The reaction mixture was stirred for an hour at 0 °C

prior to quenching with water (20 mL). Volatiles were removed under

reduced pressure, and the aqueous layer was extracted with EtOAc (3

× 50 mL). The combined organic fractions were washed with brine,

dried over NaSO₄, ﬁltered, and concentrated under reduced pressure

to aﬀord a yellow-brown solid, which was used without further

puriﬁcation in the subsequent reaction (637 mg, 85% yield). MS

(ESI) m/z: 212.28 [M + H]⁺, 214.22 [M + H]⁺+ 2.

4-Bromo-2-(methoxymethyl)benzonitrile. To a solution of 4-

bromo-2-(hydroxymethyl)benzonitrile (400 mg, 1.89 mmol) in THF

(6.3 mL) cooled in an ice bath was added sodium hydride, 60% in

mineral oil (181 mg, 7.54 mmol). The solution was stirred for 30 min

prior to the addition of iodomethane (1.4 mL, 22.63 mmol). The

reaction mixture was stirred for an additional 2 h, then quenched with

water (50 mL) and extracted with EtOAc (3 × 50 mL). The

combined organic fractions were dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure prior to puriﬁcation by silica gel

chromatography (0−80% EtOAc in hexanes) to aﬀord the ﬁnal

product (89 mg, 20% yield).

4-Bromo-2-(methoxymethyl)benzamide. The title compound

was synthesized according to the procedure described for 5d from 4-

bromo-2-(methoxymethyl)benzonitrile (80 mg, 0.354 mmol). The

reaction mixture was stirred at 90 °C for 2 h and then at room

temperature overnight. The crude mixture was diluted with water (10

mL) and extracted with EtOAc (3 × 10 mL). The organic layers were

combined and dried over Mg₂SO₄to aﬀord an oﬀ-white solid (71 mg,

81% yield). ¹H NMR (500 MHz, DMSO): δ 7.84 (br s, 1H), 7.64 (d,

J = 1.9 Hz, 1H), 7.55 (dd, J = 8.2, 2.0 Hz, 1H), 7.46 (br s, 1H), 7.42

(d, J = 8.2 Hz, 1H), 4.58 (s, 2H).

2-(Methoxymethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-

lan-2-yl)benzamide (19c). The title compound was synthesized

according to the procedure described for 4a from 4-bromo-2-

(methoxymethyl)benzamide (50 mg, 0.205 mmol). The dark brown

supernatant was used without puriﬁcation in the subsequent reaction

(60 mg, 78% yield). MS (ESI) m/z: 292.53 [M + H]⁺.

mg, 0.889 mmol). The solution was stirred at 90 °C for 6 h. The

crude was diluted with water (50 mL) and extracted with EtOAc (3 ×

50 mL). The organic layers were combined, dried over Na₂SO₄,

ﬁltered, and concentrated under reduced pressure to give the ﬁnal

product (219 mg, 77% yield). MS (ESI) m/z: 264.25 [M + H]⁺,

1

266.26 [M + H]⁺+ 2, 268.20 [M + H]⁺+ 4. H NMR (500 MHz,

CDCl₃): δ 7.20 (d, J = 1.4 Hz, 1H), 6.99 (d, J = 1.3 Hz, 1H), 5.93 (br

s, 1H), 5.72 (br s, 1H), 3.85 (s, 3H).

2-Chloro-6-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxabor-

olan-2-yl)benzamide (19e). The title compound was synthesized

according to the procedure described for 4a from 4-bromo-2-chloro-

6-methoxybenzamide (100 mg, 0.378 mmol). The dark supernatant

was used without puriﬁcation in the subsequent reaction (113 mg,

36% yield). MS (ESI) m/z: 312.46 [M + H]⁺, 314.41 [M + H]⁺+ 2.

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

isoindolin-1-one (20a). The title compound was synthesized

according to the procedure described for 8a from 15 (75 mg, 0.193

mmol) and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoindolin-

1-one (19a) (50 mg, 0.193 mmol). The ﬁnal product was a white

powder (33 mg, 44% yield). ¹H NMR (500 MHz, DMSO): δ 8.62 (br

s, 1H), 8.37 (s, 1H), 8.34 (s, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.65 (s,

1H), 7.54 (d, J = 7.7 Hz, 1H), 7.31 (d, J = 8.7 Hz, 2H), 7.04 (d, J =

8.8 Hz, 2H), 4.44 (s, 2H), 3.19−3.16 (m, 4H), 2.94−2.90 (m, 4H),

2.15 (s, 3H). HRMS (ESI) for C₂₄H₂₄N₄O [M + H]⁺m/z: calcd,

385.2023; found, 385.2018.

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

benzamide (20b). The title compound was synthesized according to

the procedure described for 8a from 15 (19 mg, 0.048 mmol) and 4-

aminocarbonylphenylboronic acid (19b) (8 mg, 0.048 mmol). The

1

ﬁnal product was a light pink-orange powder (8 mg, 45% yield). H

NMR (500 MHz, DMSO): δ 8.36 (s, 1H), 8.33 (s, 1H), 8.06 (br s,

1H), 7.98 (d, J = 8.3 Hz, 2H), 7.53 (d, J = 8.2 Hz, 2H), 7.42 (br s,

1H), 7.31 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 8.7 Hz, 2H), 3.23−3.19

(m, 4H), 3.02−2.92 (m, 4H), 2.15 (s, 3H). HRMS (ESI) for

C₂₃H₂₄N₄O [M + H]⁺m/z: calcd, 373.2023; found, 373.2014.

2-(Methoxymethyl)-4-(4-methyl-5-(4-(piperazin-1-yl)-

phenyl)pyridin-3-yl)benzamide (20c). The title compound was

synthesized according to the procedure described for 8a from 15 (73

mg, 0.188 mmol) and 19c (60 mg, 0.205 mmol). The ﬁnal compound

4-Bromo-2-chloro-6-ﬂuorobenzonitrile. The title compound

was prepared using a modiﬁed literature procedure.³⁶To a solution of

4-bromo-2-chloro-6-ﬂuoroaniline (2.00 g, 8.91 mmol) in DCM (17.8

mL) was added nitrosonium tetraﬂuoroborate (1.14 g, 9.80 mmol).

The solution was stirred for 1 h at room temperature and then cooled

in an ice bath. Potassium cyanide (1.16 g, 17.82 mmol) was added. A

solution of copper (II) sulfate pentahydrate (4.45 g, 17.82 mmol) in

water (35.0 mL) was then added gradually. The suspension was

stirred for 1 h on ice and then at room temperature for an additional

hour. The reaction mixture was diluted with DCM and a saturated

sodium bicarbonate solution, and then it ﬁltered through Celite. The

organic layer was washed with brine, separated, dried over Na₂SO₄,

ﬁltered, and concentrated under reduced pressure prior to puriﬁcation

by silica gel chromatography (0−50% EtOAc in hexanes) to give the

ﬁnal product (0.398 g, 10% yield).

1

was an oﬀ-white powder (23 mg, 29% yield). H NMR (500 MHz,

CDCl₃): δ 8.45 (s, 1H), 8.37 (s, 1H), 7.93 (d, J = 7.9 Hz, 1H), 7.44

(dd, J = 7.9, 1.7 Hz, 1H), 7.40 (d, J = 1.4 Hz, 1H), 7.28−7.26 (m,

2H), 7.01 (d, J = 8.7 Hz, 2H), 5.74 (br s, 1H), 4.65 (s, 2H), 3.46 (s,

3H), 3.26−3.22 (m, 4H), 3.10−3.06 (m, 4H), 2.17 (s, 3H). HRMS

(ESI) for C₂₅H₂₈N₄O₂[M + H]⁺m/z: calcd, 417.2285; found,

417.2283.

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

yl)benzamide (20d). The title compound was synthesized according

to the procedure described for 8a from 15 (100 mg, 0.258 mmol) and

4-carbamoyl-3-ﬂuorophenylboronic acid (19d) (47 mg, 0.258 mmol).

1

The ﬁnal compound was a white powder (50 mg, 50% yield). H

NMR (500 MHz, MeOD): δ 8.37 (s, 1H), 8.32 (s, 1H), 7.96 (t, J =

8.0 Hz, 1H), 7.38−7.30 (m, 4H), 7.11 (d, J = 8.7 Hz, 2H), 3.29−3.23

(m, 4H), 3.08−3.02 (m, 4H), 2.24 (s, 3H). ¹⁹F NMR (471 MHz,

MeOD): δ −114.60. HRMS (ESI) for C₂₃H₂₃FN₄O [M + H]⁺m/z:

calcd, 391.1929; found, 391.1926.

4-Bromo-2-chloro-6-methoxybenzonitrile. The title com-

pound was prepared using a modiﬁed literature procedure.³⁷To a

solution of 4-bromo-2-chloro-6-ﬂuorobenzonitrile (0.398 g, 1.697

mmol) in 1,4-dioxane (4.6 mL) was added MeOH (178 μL, 4.412

mmol). Sodium hydride, 60% in mineral oil (106 mg, 4.412 mmol)

was added gradually over 1 h. The reaction mixture was stirred for 1 h

at room temperature. The solvents were removed under reduced

pressure, and the crude material was suspended in water and ﬁltered.

The ﬁlter cake was dissolved in DCM, concentrated, and puriﬁed by

silica gel chromatography (0−100% DCM in hexanes) to give the

ﬁnal product (223 mg, 49% yield). MS (ESI) m/z: 246.26 [M + H]⁺,

2-Chloro-6-methoxy-4-(4-methyl-5-(4-(piperazin-1-yl)-

phenyl)pyridin-3-yl)benzamide (20e). The title compound was

synthesized according to the procedure described for 6a from 3 (66

mg, 0.153 mmol) and 19e (59 mg, 0.189 mmol). The material was

deprotected with triﬂuoroacetic acid (350 μL, 4.579 mmol) and

puriﬁed by SPE. Freeze-drying for a day and a half aﬀorded an oﬀ-

1

white powder (24 mg, 36% yield). H NMR (500 MHz, CDCl₃): δ

8.45 (s, 1H), 8.31 (s, 1H), 7.26−7.24 (m, 2H), 7.03 (d, J = 1.1 Hz,

1H), 7.01 (d, J = 8.7 Hz, 2H), 6.81 (d, J = 1.0 Hz, 1H), 5.94−5.87 (br

m, 2H), 3.89 (s, 3H), 3.26−3.22 (m, 4H), 3.10−3.06 (m, 4H), 2.18

(s, 3H). HRMS (ESI) for C₂₄H₂₅ClN₄O₂[M + H]⁺m/z: calcd,

437.1739; found, 437.1740.

1

248.26 [M + H]⁺+ 2, 250.21 [M + H]⁺+ 4. H NMR (500 MHz,

CDCl₃): δ 7.27 (d, J = 1.5 Hz, 1H), 7.04 (d, J = 1.4 Hz, 1H), 3.95 (s,

3H).

4-Bromo-2-chloro-6-methoxybenzamide. 4-bromo-2-chloro-

6-methoxybenzamide was synthesized according to the procedure

described for 5d from 4-bromo-2-chloro-6-methoxybenzonitrile (219

4-Bromo-2-ﬂuoro-6-methoxybenzonitrile (22a) and 4-

Bromo-2,6-dimethoxybenzonitrile (22b). The title compounds

M

https://dx.doi.org/10.1021/acs.jmedchem.0c00395

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

were prepared according to the procedure described for 4-bromo-2-

chloro-6-methoxybenzonitrile from 4-bromo-2,6-diﬂuorobenzonitrile

(21) (2.00 g, 9.17 mmol), MeOH (744 μL, 18.34 mmol) and sodium

hydride, 60% in mineral oil (0.733 g, 18.34 mmol). 4-Bromo-2-ﬂuoro-

6-methoxybenzonitrile was a white crystalline solid (965 mg, 46%

yield). 4-Bromo-2,6-dimethoxybenzonitrile was also a white crystal-

line solid (754 mg, 34% yield). 4-Bromo-2-ﬂuoro-6-methoxybenzoni-

MeOH in EtOAc). Freeze-drying for 1 day aﬀorded white powder

(183 mg, 83% yield). H NMR (500 MHz, DMSO): δ 8.37 (s, 1H),

1

8.34 (s, 1H), 7.89 (br s, 1H), 7.58 (br s, 1H), 7.30 (d, J = 8.7 Hz,

2H), 7.05 (d, J = 8.8 Hz, 2H), 6.98−6.94 (m, 2H), 3.85 (s, 3H),

3.23−3.19 (m, 4H), 2.48−2.45 (m, 4H), 2.23 (s, 3H), 2.19 (s, 3H).

¹⁹F NMR (471 MHz, DMSO): δ −116.74. HRMS (ESI) for

C₂₅H₂₇FN₄O₂[M + H]⁺m/z: calcd, 435.2191; found, 435.2191.

2-Fluoro-4-(5-(4-(4-isopropylpiperazin-1-yl)phenyl)-4-

methylpyridin-3-yl)-6-methoxybenzamide (26b). The title

compound was synthesized according to the procedure described

for 26a from 24a (150 mg, 0.509 mmol) and 4-(4-

isopropylpiperazinyl)phenylboronic acid, pinacol ester (25b) (202

mg, 0.611 mmol). The ﬁnal compound was white powder (142 mg,

1

trile: H NMR (500 MHz, CDCl₃): δ 7.00 (d, J = 8.0 Hz, 1H), 6.94

(s, 1H), 3.95 (s, 3H). ¹⁹F NMR (471 MHz, CDCl₃): δ −103.78 (s).

MS (ESI) m/z: 230.21 [M + H]⁺, 232.21 [M + H]⁺+ 2. 4-Bromo-

1

2,6-dimethoxybenzonitrile: H NMR (500 MHz, CDCl₃): δ 6.73 (s,

2H), 3.91 (s, 6H). MS (ESI) m/z: 242.25 [M + H]⁺, 244.19 [M +

H]⁺+ 2.

1

60% yield). H NMR (500 MHz, DMSO): δ 8.38 (s, 1H), 8.34 (s,

(4-Carbamoyl-3-ﬂuoro-5-methoxyphenyl)boronic Acid

(23a). The title compound was synthesized according to the

procedure described for 4a from 5d (2.48 g, 10.0 mmol). The dark

brown supernatant was used without further puriﬁcation in

subsequent reactions (2.46 g, 84% yield). MS (ESI) m/z: 214.34

[M + H]⁺.

2,6-Dimethoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-

2-yl)benzamide (23b). The title compound was synthesized

according to the procedure described for 4a from 5e (800 mg, 3.08

mmol). The dark brown supernatant was used without further

puriﬁcation in subsequent reactions (945 mg, 70% yield). MS (ESI)

m/z: 308.26 [M + H]⁺.

1H), 7.89 (br s, 1H), 7.58 (br s, 1H), 7.30 (d, J = 8.7 Hz, 2H), 7.04

(d, J = 8.8 Hz, 2H), 6.98−6.95 (m, 2H), 3.85 (s, 3H), 3.23−3.17 (m,

4H), 2.72−2.66 (m, 1H), 2.63−2.57 (m, 4H), 2.19 (s, 3H), 1.02 (d, J

= 6.5 Hz, 6H). ¹⁹F NMR (471 MHz, DMSO): δ −116.74. HRMS

(ESI) for C₂₇H₃₁FN₄O₂[M + H]⁺m/z: calcd, 463.2504; found,

463.2506.

2-Fluoro-6-methoxy-4-(4-methyl-5-(4-((3R,5S)-3,4,5-trime-

thylpiperazin-1-yl)phenyl)pyridin-3-yl)benzamide (26c). The

title compound was synthesized according to the procedure described

for 26a from 24a (156 mg, 0.531 mmol) and (2R,6S)-1,2,6-trimethyl-

4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperazine

(25c) (58.5 mg, 0.177 mmol). The ﬁnal compound was light yellow

powder (36 mg, 43% yield). ¹H NMR (500 MHz, CDCl₃): δ 8.43 (s,

1H), 8.30 (s, 1H), 7.24 (d, J = 8.6 Hz, 2H), 6.99 (d, J = 8.6 Hz, 2H),

6.77 (dd, J = 9.7, 0.5 Hz, 1H), 6.71 (s, 1H), 6.32 (br s, 1H), 6.09 (br

s, 1H), 3.91 (s, 3H), 3.57 (d, J = 11.5 Hz, 2H), 2.86−2.67 (m, 2H),

2.60−2.45 (m, 2H), 2.40 (s, 3H), 2.16 (s, 3H), 1.26 (d, J = 5.8 Hz,

6H). ¹⁹F NMR (471 MHz, MeOD): δ −117.13. HRMS (ESI) for

C₂₇H₃₁FN₄O₂[M + H]⁺m/z: calcd, 463.2504; found, 463.2510.

2,6-Dimethoxy-4-(4-methyl-5-(4-(4-methylpiperazin-1-yl)-

phenyl)pyridin-3-yl)benzamide (26d). The title compound was

synthesized according to the procedure described for 26a from 24b

(150 mg, 0.489 mmol) and 25a (129 mg, 0.587 mmol). The ﬁnal

4-(5-Chloro-4-methylpyridin-3-yl)-2-ﬂuoro-6-methoxyben-

zamide (24a). The title compound was synthesized according to the

procedure described for 6a from 10 (2.06 g, 10.0 mmol) and 23a

(2.95 g, 10.0 mmol). The ﬁnal compound was an oﬀ-white crystalline

1

solid (1.67 g, 56% yield). H NMR (500 MHz, DMSO): δ 8.63 (s,

1H), 8.38 (s, 1H), 7.90 (br s, 1H), 7.60 (br s, 1H), 6.97−6.94 (m,

2H), 3.83 (s, 3H), 2.33 (s, 3H). ¹⁹F NMR (471 MHz, DMSO): δ

−116.46. MS (ESI) m/z: 294.97 [M + H]⁺, 297.10 [M + H]⁺+ 2.

4-(5-Chloro-4-methylpyridin-3-yl)-2,6-dimethoxybenza-

mide (24b). The title compound was synthesized according to the

procedure described for 6a from 10 (954 mg, 4.62 mmol) and 23b

(946 mg, 3.08 mmol). The ﬁnal compound was an oﬀ-white

crystalline solid (747 mg, 79% yield). ¹H NMR (500 MHz,

DMSO): δ 8.61 (s, 1H), 8.38 (s, 1H), 7.58 (br s, 1H), 7.26 (br s,

1H), 6.70 (s, 2H), 3.77 (s, 6H), 2.34 (s, 3H). MS (ESI) m/z: 307.26

[M + H]⁺.

1

compound was white powder (189 mg, 87% yield). H NMR (500

MHz, DMSO): δ 8.35 (s, 1H), 8.35 (s, 1H), 7.57 (br s, 1H), 7.30 (d,

J = 8.6 Hz, 2H), 7.23 (br s, 1H), 7.05 (d, J = 8.7 Hz, 2H), 6.72 (s,

2H), 3.78 (s, 6H), 3.23−3.19 (m, 4H), 2.48−2.46 (m, 4H), 2.24 (s,

3H), 2.20 (s, 3H). HRMS (ESI) for C₂₆H₃₀N₄O₃[M + H]⁺m/z:

calcd, 477.2391; found, 447.2394.

(2R,6S)-4-(4-Bromophenyl)-1,2,6-trimethylpiperazine. The

title compound was prepared using modiﬁed literature proce-

dures.^38,39A solution of (2R,6S)-1,2,6-trimethylpiperazine (100 mg,

0.780 mmol), 1-bromo-4-iodobenzene (221 mg, 0.780 mmol),

bis(dibenzylideneacetone)palladium(0) (22 mg, 0.039 mmol),

xantphos (68 mg, 0.117 mmol), and lithium tert-butoxide 1.0 M in

THF (2.34 mL, 2.340 mmol) in 1,4-dioxane (3.12 mL) was heated to

110 °C and stirred overnight. Volatiles were removed under reduced

pressure, and the crude material was puriﬁed by silica gel

4-(5-(4-(4-Isopropylpiperazin-1-yl)phenyl)-4-methylpyridin-

3-yl)-2,6-dimethoxybenzamide (26e). The title compound was

synthesized according to the procedure described for 26a from 24b

(150 mg, 0.489 mmol) and 25b (194 mg, 0.587 mmol). The ﬁnal

1

compound was an oﬀ-white powder (179 mg, 75% yield). H NMR

(500 MHz, MeOD): δ 8.31 (s, 1H), 8.30 (s, 1H), 7.33 (d, J = 8.6 Hz,

2H), 7.13 (d, J = 8.7 Hz, 2H), 6.69 (s, 2H), 3.86 (s, 6H), 3.47−3.36

(m, 4H), 3.15−3.01 (m, 5H), 2.22 (s, 3H), 1.27 (d, J = 6.6 Hz, 6H).

HRMS (ESI) for C₂₈H₃₄N₄O₃[M + H]⁺m/z: calcd, 475.2704; found,

475.2705.

2,6-Dimethoxy-4-(4-methyl-5-(4-((3R,5S)-3,4,5-trimethylpi-

perazin-1-yl)phenyl)pyridin-3-yl)benzamide (26f). The title

compound was synthesized according to the procedure described

for 26a from 24b (109 mg, 0.354 mmol) and 25c (58 mg, 0.177

mmol). The ﬁnal compound was white powder (22 mg, 26% yield).

¹H NMR (500 MHz, MeOD): δ 8.31 (s, 1H), 8.29 (s, 1H), 7.30 (d, J

= 8.4 Hz, 2H), 7.09 (d, J = 8.5 Hz, 2H), 6.69 (s, 2H), 3.86 (s, 6H),

3.65 (d, J = 11.8 Hz, 2H), 2.62−2.55 (m, 2H), 2.51−2.44 (m, 2H),

2.37 (s, 3H), 2.23 (s, 3H), 2.16 (s, 2H), 1.23 (d, J = 6.2 Hz, 6H).

HRMS (ESI) for C₂₈H₃₄N₄O₃[M + H]⁺m/z: calcd, 475.2704; found,

475.2699.

Kinase Assay. The biochemical potencies of all compounds were

measured by Reaction Biology Corporation (RBC) (Malvern,

Pennsylvania, United States). Compounds were tested against

ALK2/ACVR1 and ALK5/TGFβ-R1 in a 10-dose IC₅₀mode with a

2-fold serial dilution starting at 1 or 5 μM. Reactions were conducted

at an ATP concentration of 10 μM and Casein concentration of 1

1

chromatography (0−10% MeOH in EtOAc). H NMR (500 MHz,

MeOD): δ 7.32 (d, J = 9.0 Hz, 2H), 6.87 (d, J = 9.0 Hz, 2H), 3.55−

3.49 (m, J = 11.5 Hz, 2H), 2.52−2.46 (m, J = 11.4 Hz, 2H), 2.44−

2.38 (m, 2H), 2.33 (s, 3H), 1.19 (d, J = 6.1 Hz, 6H). MS (ESI) m/z:

283.37 [M + H]⁺, 285.32 [M + H]⁺+ 2.

(2R,6S)-1,2,6-Trimethyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-diox-

aborolan-2-yl)phenyl)piperazine (25c). The title compound was

prepared according to the procedure described for 4a from (2R,6S)-4-

(4-bromophenyl)-1,2,6-trimethylpiperazine (100 mg, 0.353 mmol).

The dark brown reaction mixture was used without puriﬁcation in

subsequent reactions (117 mg, 91% yield). MS (ESI) m/z: 331.46 [M

+ H]⁺.

2-Fluoro-6-methoxy-4-(4-methyl-5-(4-(4-methylpiperazin-

1-yl)phenyl)pyridin-3-yl)benzamide (26a). The title compound

was synthesized according to the procedure described for 8a from 24a

(150 mg, 0.509 mmol) and 4-(4-methylpiperazin-1-yl)phenylboronic

acid (25a) (134 mg, 0.611 mmol). XPhos Pd G3 (21.54 mg, 0.025

mmol) was used as the catalyst. The reaction mixture was adsorbed

onto Celite, and the solvents were removed under reduced pressure.

The crude material was puriﬁed by silica gel chromatography (0−15%

N

https://dx.doi.org/10.1021/acs.jmedchem.0c00395

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

mg/mL. LDN-193189 was tested as a control in a 10-dose IC₅₀mode

with a threefold serial dilution starting at 10 μM. Reductions in

enzymatic activity were determined relative to DMSO controls.

Cell Culture and Transfection. HEK-293 cells were maintained

in Dulbecco’s modiﬁed Eagle medium (DMEM, Gibco) supple-

mented with 10% fetal bovine serum (FBS) (Thermo Fisher) and

penicillin/streptomycin (Thermo Fisher). HEK-293 cells were

transfected with the protein expression or reporter constructs using

FuGENE HD (Promega) according to the manufacturer’s instruc-

tions. Brieﬂy, DNA was diluted into phenol red-free Opti-MEM

(Gibco) at a concentration of 10 μg/mL. Without coming in contact

with the sides of the container, 3 μL of FuGENE HD was added for

each μg of DNA used. After thorough mixing by inversion, FuGENE

HD/DNA complexes were allowed to form by incubation at room

temperature for 20 min. Transfection mixture (1 part) was added to

20 parts of HEK-293 cell suspension with a density of 200,000 cells

per mL (volume/volume). HEK-293 cells were incubated in a

humidiﬁed, 37 °C incubator with 5% carbon dioxide for 24 h before

they are used in the NanoBRET target engagement assay or dual

luciferase reporter assay.

NanoBRET Target Engagement Assay. ALK2-C-terminal

nanoluciferase fusion with the GSSG linker was encoded by the

pFC32K vector (Promega). ALK2-nanoluciferase fusion construct (1

part) was mixed with 9 parts of Transfection Carrier DNA (mass/

mass) (Promega). Transfected cells were trypsinized and resuspended

in Opti-MEM at a density of 200,000 cells per mL. Cells (17 μL) were

dispensed into each well of 384-well ﬂat-bottom polypropylene plate

(Greiner). Working solution (20×) of target engagement tracer PBI-

6908 (Promega) was prepared by diluting DMSO stock in tracer

dilution buﬀer (12.5 mM HEPES pH 7.5, 31.25% PEG-400). Stocks

(1000×) of test compounds in DMSO (Cell Signaling Technology)

were diluted further in Opti-MEM for 10× working solutions. After

the addition of 1 μL of 20× target engagement tracer and 2 μL of 10×

working solutions, contents of the wells were thoroughly mixed by

agitating the plate at 500 rpm for 1 min. Cells were incubated in a

humidiﬁed, 37 °C incubator with 5% carbon dioxide for 2 h prior to

BRET measurement. For bioluminiscence resonance energy transfer

(BRET) measurement, the NanoBRET NanoGlo Substrate and

Extracellular NanoLuc Inhibitor (Promega) were diluted 166× and

500×, respectively, in Opti-MEM to produce 3× working stock. A

PHERAstar FSX microplate reader (BMG Labtech) with the LUM

610-LP 460-80 optical module was used to measure the intensity of

dual emission. A measurement interval of 1 s and gain settings of 3600

and 1879 for 610 and 460 nm, respectively, were used. Milli-BRET

units (mBU) were calculated by dividing the signal measured at 610

nm with the signal measured at 460 nm and multiplying by 1000. The

apparent EC₅₀values of test compounds were estimated using the

[Inhibitor] versus response (three parameters) nonlinear regression

curve ﬁtting function of GraphPad Prism 7.

Dual Luciferase Reporter Assay. CAGA-Luc and Renilla-

luciferase constructs (a gift of Dr Petra Knaus, Free University of

Berlin) were used as the reporter for ALK5 signaling and loading

control, respectively. CAGA-Luc construct (4 parts) was mixed with 1

part of Renilla-luciferase construct (mass/mass). Ten thousand

transfected cells were seeded into each well of 96-well plate

(Corning). Twenty four hours after transfection, the cells were

incubated with 10 ng/mL TGF-β1 (Peprotech, 100-21-10) and test

compounds simultaneously at the concentrations indicated in a

humidiﬁed, 37 °C incubator with 5% carbon dioxide. Twenty-four

hours later, the cells were harvested, lysed, and processed for the

measurement of luciferase activity using the Dual-Luciferase Reporter

Assay System (Promega) according to the manufacturer’s instruc-

tions. Brieﬂy, the culture medium was aspirated completely, and cells

were lysed in 50 μL of 1× PLB with 300 rpm agitation for 30 min.

Cell lysate (10 μL) was dispensed into each well of a 384-well ﬂat-

bottom polypropylene plate (Greiner). The luminescent signal of

ﬁreﬂy and Renilla luciferase activity were measured sequentially using

a PHERAstar FS microplate reader (BMG Labtech) after the addition

of 25 μL of LARII and Stop & Glo, respectively. A measurement

interval of 2 s and gain setting of 3600 were used. The ﬁreﬂy luciferase

signal was normalized to the cell number by division with Renilla

luciferase signal. The relative luciferase unit (RLU) was obtained by

further division with the signal from cells without TGF-β stimulation.

The apparent EC50 values of test compounds were estimated using

the [Inhibitor] versus response (three parameters) nonlinear

regression curve ﬁtting function of GraphPad Prism 7.

Caco-2 Permeability Assay. Caco-2 cells (C2BBe1) were

purchased from American Type Culture Collection, ATCC. Caco-2

cell cultures were routinely maintained in T-75 tissue culture ﬂasks in

DMEM containing 20% FBS, 0.1 mg/mL normocin, and 0.05 mg/mL

gentamicin. These cells were seeded at a density of 40,000 cells/well

on the 24-well polyethylene terephthalate (PET) membrane (1.0 μm

pore size, 0.31 cm²surface area) insert plates. Cell monolayers were

grown for 21 or 22 days at 37 °C with 5% CO₂in a humidiﬁed

incubator. The cell culture medium was replaced twice weekly during

the cell growth period. Prior to beginning the permeability assay, cell

monolayers were rinsed with Hank’s balanced salt solution (HBSS)

twice to remove the residual cell culture medium.

The assay buﬀer comprised HBSS containing 10 mM HEPES and

15 mM glucose at a pH of 7.4. The dosing buﬀer contained 5 μM

metoprolol (positive control), 5 μM atenolol (negative control), and

100 μM Lucifer yellow in the assay buﬀer. The receiving buﬀer

contained 1% bovine serum albumin (BSA) in the assay buﬀer. The

concentration of the test compound was 5 μM in the dosing buﬀer

(ﬁnal DMSO concentration was 0.1%). Digoxin at 10 μM was utilized

as a Pgp substrate control.

For apical to basolateral (A to B) permeability experiment, 0.25 mL

of the dosing buﬀer was added to the apical chambers, and 1.0 mL of

the receiving buﬀer was added to the basolateral chambers of the

assay plate. For the basolateral to apical (B to A) permeability

experiment, 0.25 mL of the receiving buﬀer was added to the apical

chambers, and 1.0 mL of dosing buﬀer was added to the basolateral

chambers of the assay plates. The assay plates were then incubated at

37 °C for 120 min on an orbital shaker at 65 rpm. Sample solutions

were taken from the donor chambers (10 μL) and receiver chambers

(100 μL) after the incubation period. For each sample, there were two

technical replicates. The sample solutions from donor chambers were

diluted ten times with the receiving buﬀer. In order to extract test

compounds and precipitate BSA from sample solutions, three volumes

of acetonitrile (containing 0.5% formic acid and an internal standard)

were added, and the plate was vigorously mixed. Sample solutions

were then centrifuged at 4000 rpm for 10 min to remove debris and

precipitated BSA. Approximately, 150 μL of the supernatant was

subsequently transferred to a new 96-well microplate for LC/MS

analysis. Narrow-window mass extraction LC/MS analysis was

performed for all samples from this study using a Waters Xevo

quadrupole time-of-ﬂight (QTof) mass spectrometer to determine

relative peak areas of parent compounds. The co-dosed positive and

negative controls were also measured for each well to monitor

integrity of cell monolayers and well-to-well variability. The apparent

permeability coeﬃcient (P_app) and post-assay recovery are calculated

using the following equations

P

= V × (dC/dt) × 1/(A × C₀)

app

r

Percent recovery = 100 × ((V × C_r^final) + (V × C_d^final))

r

d

/(V × C₀)

d

where dC/dt is the slope of cumulative concentration in the receiver

compartment versus time, V_ris the volume of the receiver

compartment, V_dis the volume of the donor compartment, A is the

membrane surface area, C₀is the compound initial concentration in

the donor chamber, C^f_r^inalis the cumulative receiver concentration at

the end of the incubation period, and C^f_d^inalis the concentration of the

donor at the end of the incubation period.

Eﬄux ratio (ER) is deﬁned as P_app(B-to-A)/P_app(A-to-B).

Liver Microsomal Metabolic Stability Assay. For this assay,

stock solutions of test compounds in DMSO (1 mM) were initially

diluted to a concentration of 40.0 μM using 0.1 M potassium

phosphate buﬀer (pH 7.4). Test compounds were then added to

O

https://dx.doi.org/10.1021/acs.jmedchem.0c00395

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

reaction wells at a ﬁnal concentration of 1 μM which was assumed to

be well below K_mvalues to ensure linear reaction conditions (i.e.,

avoid saturation). The ﬁnal DMSO concentration was kept constant

at 0.1%. Each compound was tested in duplicate for both time points

(0 and 60 min). CD-1 mouse (male) or pooled human liver

microsomes (Corning Gentest) were added to the reaction wells at a

ﬁnal concentration of 0.5 mg/mL (protein). The ﬁnal volume for each

reaction was 100 μL, which included the NADPH-regeneration

solution (NRS) mix (Corning Gentest). This NRS mix comprised

glucose 6-phosphate dehydrogenase, NADP⁺, MgCl₂, and glucose 6-

phosphate. Reactions were carried out at 37 °C in an orbital shaker at

175 rpm. Upon completion of the 60 min time point, reactions were

terminated by the addition of two volumes (200 μL) of ice-cold

acetonitrile containing 0.5% formic acid and an internal standard.

Samples were then centrifuged at 4000 rpm for 10 min to remove

debris and precipitated proteins. Approximately, 150 μL of super-

natant was subsequently transferred to a new 96-well microplate for

LC/MS analysis.

current was measured using a pulse pattern with ﬁxed amplitudes

(conditioning pre-pulse: −80 mV for 25 ms; test pulse: +40 mV for

80 ms) from a holding potential of 0 mV (“zero holding” procedure).

The hERG current was measured as a diﬀerence between the peak

current at 1 ms after the test step to +40 mV and the steady-state

current at the end of the step to +40 mV.

CYP Inhibition Assay. CYP IC₅₀values were generated by

Pharmaron. Their protocol is described below:

Multiple concentrations (1 μL) of the test compound or positive

control compound (CYP1A2: furafylline, CYP2B6: ketoconazole,

CYP2C8: quercetin, CYP2C9: sulfaphenazole, CYP2C19: N-3-

benzylnirvanol, CYP2D6: quinidine, and CYP3A4: ketoconazole)

were transferred to the “Compound Plate”. The concentrations of test

compounds and positive control compounds were 0, 0.2, 1, 2, 10, 50,

200, 2000, and 10,000 μM. The master solution was prepared with

MgCl₂solution (20 μL of 50 mM solution), phosphate buﬀer (100 μL

of 200 mM solution), ultrapure water (56 μL), human liver

microsomes [2 μL of 20 mg/mL stock concentration (Corning

UltraPool HLM 150, Mixed Gender Cat. no.: 452117)], and 1 μL of

substrate [CYP1A2: phenacetin (8 mM stock concentration),

CYP2B6: bupropion (10 mM stock concentration), CYP2C8:

paclitaxel (1 mM stock concentration), CYP2C9: tolbutamide (40

mM stock concentration), CYP2C19: mephenytoin (10 mM stock

concentration), CYP2D6: dextromethorphan (2 mM stock concen-

tration) and CYP3A4: midazolam (1 mM stock concentration), and

testosterone (10 mM stock concentration)]. The master solution was

prewarmed in a water bath at 37 °C for 5 min. The incubated master

solution (179 μL) was transferred to the compound plate. In the

mixed system, the ﬁnal concentrations of the test compounds and

positive control compounds were 0, 0.001, 0.005, 0.01, 0.05, 0.25, 1,

10, and 50 μM. All experiments were performed in duplicate. The

reaction was started with the addition of 20 μL of 10 mM NADPH

solution at the ﬁnal concentration of 1 mM. The reaction was stopped

by the addition of 1.5 volumes of methanol with IS (100 nM

alprazolam, 200 nM imipramine, 200 nM labetalol, and 2 μM

ketoprofen) to the “Incubation Plate” at the designated time points

(20 min for CYP1A2, 2B6, 2C9, 2C19, and 2D6, 5 min for

midazolam-mediated 3A4, and 10 min for testosterone-mediated

3A4). The “Incubation Plate” was centrifuged at 3220 g for 40 min to

precipitate the protein. An aliquot of 100 μL of the supernatant was

diluted using 100 μL ultrapure H₂O, and the mixture was used for

LC/MS/MS analysis. The formation of metabolites was analyzed

using LC/MS/MS. A decrease in the formation of the metabolites in

the peak area to vehicle control was used to calculate an IC₅₀value

(test compound concentration which produces 50% inhibition) using

Excel XLﬁt.

Narrow-window mass extraction LC/MS analysis was performed

for all samples in this study using a Waters Xevo quadrupole time-of-

ﬂight (QTof) mass spectrometer to determine relative peak areas of

test compounds. The percentage remaining values were calculated

using the following equations

A

A₀

% remaining =

× 100

where A is area response after incubation, A₀is area response at initial

time point.

hERG Inhibition Assay. hERG IC₅₀values were generated by

Charles River Laboratories (Cleveland, Ohio, United States). Their

protocol is described below:

Compounds were tested against cloned hERG potassium channels

expressed in HEK293 cells. Chemicals used in solution preparation

were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise

noted and were of ACS reagent-grade purity or higher. Stock

solutions of test articles and the positive control were prepared in

dimethyl sulfoxide (DMSO) and stored frozen. Reference compound

concentrations were prepared fresh daily by diluting stock solutions

into a Charles River proprietary HEPES-buﬀered physiological saline

(HB-PS) solution which was prepared weekly and refrigerated until

use. Because previous results have shown that ≤0.3% DMSO did not

aﬀect channels currents, all test and control solutions contained 0.3%

DMSO. Each test article formulation was sonicated (model 2510/

5510, Branson Ultrasonics, Danbury, CT) at ambient room

temperature for 20 min to facilitate dissolution. Cells were cultured

in DMEM/nutrient mixture F-12 (D-MEM/F-12) supplemented with

10% FBS, 100 U/mL penicillin G sodium, 100 μg/mL streptomycin

sulfate, and 500 μg/mL G418. Before testing, cells in culture dishes

were washed twice with HBSS and detached with accutase.

Immediately before use in the IonWorks Barracuda system, the cells

were washed twice in HB-PS to remove the accutase and resuspended

in 5 mL of HB-PS. The test article eﬀects were evaluated using

IonWorks Barracuda systems (Molecular Devices Corporation, Union

City, CA). HEPES-buﬀered intracellular solution (Charles River

proprietary) for whole cell recordings was loaded into the intracellular

compartment of the Population Patch Clamp (PPC) planar electrode.

Extracellular buﬀer (HB-PS) was loaded into PPC planar electrode

plate wells (11 μL per well). The cell suspension was pipetted into the

wells of the PPC planar electrode (9 μL per well). After establishment

of a whole-cell conﬁguration (the perforated patch), membrane

currents were recorded using a patch clamp ampliﬁer in the IonWorks

Barracuda system. The current recordings were performed one (1)

time before test article application to the cells (baseline) and one (1)

time after application of the test article. Test article concentrations

In Vivo Pharmacokinetic Studies. The pharmacokinetic proﬁles

of 8b, 26a, and 26b were assessed by Pharmaron. Their protocol is

described below:

Test compounds were dissolved ﬁrst in DMSO, then mixed with

47.5% PEG400 and 47.5% DI water with 10% Tween80. The

solutions were thoroughly vortexed after each step and stored at room

temperature. Solutions were freshly prepared on the day of dosing.

Female CB17 SCID mice (n = 3) (6−8 weeks old, 17−20 g weight)

were orally administered a 10 mg/kg dose (10 mL/kg dose volume, 1

mg/mL concentration) of the test compound. Blood samples were

taken via the dorsal metatarsal vein at 0.25, 0.5, 1, 2, 4, 8, and 24 h

post-dosage. Blood samples were transferred into plastic micro-

centrifuge tubes containing the anticoagulant Heparin-Na and

centrifuged at 4000 g for 5 min at 4 °C to obtain plasma. The

samples were stored in a freezer at −75 15 °C prior to analysis.

To determine brain concentrations, female CB17 SCID mice (n =

3) (6−8 weeks old, 17−20 g weight) were orally administered a 100

mg/kg dose (10 mL/kg dose volume, 10 mg/mL concentration) of

the test compound. 4 h post-dose, the animals were terminally

anaesthetized by an increasing concentration of CO₂. Their chest

cavities were opened to expose the heart and an incision at the right

auricle using surgical scissors was done. A syringe full of gentle saline

was pushed into the heart slowly via the left ventricle (saline volume:

∼10 mL). The animal was placed head down at a 45° angle to

̈

were applied to naive cells (n = 4, where n = replicates/

concentration). Each application consisted of addition of 20 μL of

2× concentrated test article solution to the total 40 μL of ﬁnal volume

of the extracellular well of the PPC plate. The duration of exposure to

each compound concentration was ﬁve (5) minutes. The hERG

P

https://dx.doi.org/10.1021/acs.jmedchem.0c00395

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

facilitate blood removal. Brain samples were collected and kept frozen

at −75 15 °C. All brain samples were weighed and homogenized

with phosphate buﬀered saline by brain weight (g) to buﬀer volume

(mL) ratio 1:3 before analysis. The actual concentrations were the

detected value multiplied using the dilution factor.

Concentrations of the test compound in the plasma samples were

analyzed using a LC/MS/MS method. WinNonlin (Phoenix, version

8.0) or other similar software was used for pharmacokinetic

calculations. The following pharmacokinetic parameters were

calculated, whenever possible from the plasma concentration versus

time data:

AUTHOR INFORMATION

Corresponding Author

■

Rima Al-awar − Drug Discovery Program, Ontario Institute for

Cancer Research, Toronto, Ontario M5G 0A3, Canada;

Department of Pharmacology and Toxicology, University of

Toronto, Toronto, Ontario M5S 1A8, Canada; orcid.org/

0000-0002-4185-055X; Phone: +1 (416) 673-8577;

Email: Rima.Alawar@oicr.on.ca

Authors

Deeba Ensan − Department of Pharmacology and Toxicology,

University of Toronto, Toronto, Ontario M5S 1A8, Canada;

Drug Discovery Program, Ontario Institute for Cancer Research,

Toronto, Ontario M5G 0A3, Canada; orcid.org/0000-

0002-3251-1521

David Smil − Drug Discovery Program, Ontario Institute for

Cancer Research, Toronto, Ontario M5G 0A3, Canada;

orcid.org/0000-0002-6232-6087

PO administration: T_1/2, C_max, T_max, AUC_last, AUC_inf, and F.

All animal procedures were in accordance with the regulations of

Institutional Animal Care and Use Committee (IACUC) at

Pharmaron Inc.

Cocrystallization of ALK2 with M4K2149. Protein Expression

and Puriﬁcation. Constructs were prepared by ligation-independent

cloning. The kinase domain of ALK2 (residues 201−499; Uniprot ID,

Q04771) was cloned into pFB-LIC-Bse for the baculoviral expression.

The construct was veriﬁed by sequencing. ALK2 was expressed in Sf9

insect cells grown at 27 °C. Some 72 h postinfection, cells were

harvested and lysed using ultrasonication. ALK2 was initially puriﬁed

by nickel aﬃnity chromatography before subsequent puriﬁcation by

size exclusion chromatography (Superdex 200 16/600). The eluted

protein was stored in 50 mM HEPES, pH 7.5, 300 mM NaCl, 10 mM

DTT. The hexahistidine tag of ALK2 was cleaved using tobacco etch

virus protease after initial nickel puriﬁcation.

Crystallization. Crystallization was achieved at 4 °C using the

sitting-drop vapor diﬀusion method. ALK2 was preincubated with 1

mM M4K2149 at a protein concentration of 11 mg/mL and

crystallized using a precipitant containing 0.1 M citrate pH 4.9, 1 M

ammonium sulfate, and 0.2 M sodium/potassium tartrate. Viable

crystals were obtained when the protein solution was mixed with the

reservoir solution at 2:1 volume ratio. Crystals were cryoprotected

with mother liquor plus 25% ethylene glycol, prior to vitriﬁcation in

liquid nitrogen.

́

Carlos A. Zepeda-Velazquez − Drug Discovery Program,

Ontario Institute for Cancer Research, Toronto, Ontario M5G

0A3, Canada; orcid.org/0000-0002-8130-2232

Dimitrios Panagopoulos − Drug Discovery Program, Ontario

Institute for Cancer Research, Toronto, Ontario M5G 0A3,

Canada; Structural Genomics Consortium, University of

Toronto, Toronto, Ontario M5G 1L7, Canada; Department of

Chemistry, Simon Fraser University, Burnaby, British Columbia

V5A 1S6, Canada

Jong Fu Wong − Structural Genomics Consortium, University of

Oxford, Oxford OX3 7DQ, U.K.

Eleanor P. Williams − Structural Genomics Consortium,

University of Oxford, Oxford OX3 7DQ, U.K.

Roslin Adamson − Structural Genomics Consortium, University

of Oxford, Oxford OX3 7DQ, U.K.

Data Collection. Diﬀraction data were collected at the Diamond

Light Source, station I03 using monochromatic radiation at a

wavelength 0.9763 Å.

Alex N. Bullock − Structural Genomics Consortium, University

of Oxford, Oxford OX3 7DQ, U.K.

Taira Kiyota − Drug Discovery Program, Ontario Institute for

Cancer Research, Toronto, Ontario M5G 0A3, Canada

Ahmed Aman − Drug Discovery Program, Ontario Institute for

Cancer Research, Toronto, Ontario M5G 0A3, Canada; Leslie

Dan Faculty of Pharmacy, University of Toronto, Toronto,

Ontario M5S 3M2, Canada

Phasing, Model Building, Reﬁnement, and Validation. Data were

processed with Xia2 and subsequently scaled using the program

AIMLESS from the CCP4 suite.^40,41Initial phases were obtained by

molecular replacement using the program PHASER and the structure

of ALK2 (Protein Data Bank code 6SRH) as a search model.⁴²The

resulting structure solution was reﬁned using Phenix Reﬁne and

manually rebuilt with COOT.^43,44The complete structure was

veriﬁed for geometric correctness with MolProbity.⁴⁵Data collection

and reﬁnement statistics can be found in Supporting Information

Table 1.

Owen G. Roberts − M4K Pharma Inc., Toronto, Ontario M5G

1L7, Canada

Aled M. Edwards − M4K Pharma Inc., Toronto, Ontario M5G

1L7, Canada; Structural Genomics Consortium, University of

Toronto, Toronto, Ontario M5G 1L7, Canada

Jeﬀ A. O’Meara − Drug Discovery Program, Ontario Institute

for Cancer Research, Toronto, Ontario M5G 0A3, Canada

Methvin B. Isaac − Drug Discovery Program, Ontario Institute

for Cancer Research, Toronto, Ontario M5G 0A3, Canada

Cocrystal images in the article were processed using Molsoft

MolBrowser 3.8.

ASSOCIATED CONTENT

■

sı

* Supporting Information

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c00395

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00395.

¹H and ¹⁹F NMR spectra of select compounds,

crystallization methods, Caco-2 and HLM and MLM

data, CYP and hERG inhibition data, and kinase

selectivity panel (PDF)

Author Contributions

Analogues were synthesized by D.E., D.S., and D.P. The

compounds were designed and their syntheses devised by D.S.,

M.B.I., C.A.Z.-V., and D.E. All NanoBRET data was generated

by J.F.W. and A.H. Crystallography experiments were

performed by E.P.W. and R.A. Caco-2 and microsomal

stability studies were conducted by T.K. HRMS were also

generated by T.K. A.N.B., A.A., J.A.O., M.B.I., and R.A.-a. were

involved in the experimental design, the interpretation of data,

and they monitored project progress. O.G.R. and A.M.E.

Molecular formula strings (CSV)

Accession Codes

PBD ID codes: ALK2-M4K2149, 6T6D; ALK2-LDN-213844,

4BGG.

Q

https://dx.doi.org/10.1021/acs.jmedchem.0c00395

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(4) Schmierer, B.; Hill, C. S. TGF β -SMAD signal transduction:

molecular specificity and functional flexibility. Nat. Rev. Mol. Cell Biol.

2007, 8, 970−982.

initiated the project and edited the paper. The manuscript was

written by D.E. and revised by D.S., C.A.Z.-V., M.B.I., and

R.A.-a.

́

(5) Massague, J.; Blain, S. W.; Lo, R. S. TGF β signaling in growth

Notes

control, cancer, and heritable disorders. Cell 2000, 103, 295−309.

(6) Taylor, K. R.; Vinci, M.; Bullock, A. N.; Jones, C. ACVR1

mutations in DIPG: lessons learned from FOP. Cancer Res. 2014, 74,

4565−4570.

The authors declare no competing ﬁnancial interest.

Authors will release the atomic coordinates and experimental

data upon article publication.

(7) 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. Nat. Genet. 2014, 46,

457−461.

ACKNOWLEDGMENTS

■

This work was funded by the Cancer Therapeutics Innovation

Pipeline program at the Ontario Institute for Cancer Research

(OICR). The OICR is funded by the Government of Ontario.

Funding from The Brain Tumour Charity was used to support

the cellular and crystallographic research conducted at the

University of Oxford. The SGC is a registered charity (number

1097737) that receives funds from AbbVie, Bayer Pharma AG,

Boehringer Ingelheim, Canada Foundation for Innovation,

Eshelman Institute for Innovation, Genome Canada through

Ontario Genomics Institute [OGI-055], Innovative Medicines

Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766],

Janssen, Merck KGaA, Darmstadt, Germany, MSD, Novartis

(8) Mathew, R. K.; Rutka, J. T. Diffuse intrinsic pontine glioma:

clinical features, molecular genetics, and novel targeted therapeutics. J.

Korean Neurosurg. Soc. 2018, 61, 343−351.

(9) Warren, K. E. Diffuse intrinsic pontine glioma: poised for

progress. Front. Oncol. 2012, 2, 205.

(10) 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.; Zhang, J.; Baker, S. J. The genomic landscape of diffuse

intrinsic pontine glioma and pediatric non-brainstem high-grade

glioma. Nat. Genet. 2014, 46, 444−450.

(11) Carvalho, D.; Taylor, K. R.; Olaciregui, N. G.; Molinari, V.;

Clarke, M.; Mackay, A.; Ruddle, R.; Henley, A.; Valenti, M.; Hayes,

A.; Brandon, A. D. H.; Eccles, S. A.; Raynaud, F.; Boudhar, A.; Monje,

M.; Popov, S.; Moore, A. S.; Mora, J.; Cruz, O.; Vinci, M.; Brennan, P.

E.; Bullock, A. N.; Carcaboso, A. M.; Jones, C. ALK2 inhibitors

display beneficial effects in preclinical models of ACVR1 mutant

diffuse intrinsic pontine glioma. Commun. Biol. 2019, 2, 156.

(12) Anastas, J. N.; Zee, B. M.; Kalin, J. H.; Kim, M.; Guo, R.;

Alexandrescu, S.; Blanco, M. A.; Giera, S.; Gillespie, S. M.; Das, J.;

Wu, M.; Nocco, S.; Bonal, D. M.; Nguyen, Q.-D.; Suva, M. L.;

Bernstein, B. E.; Alani, R.; Golub, T. R.; Cole, P. A.; Filbin, M. G.; Shi,

Y. Re-programing chromatin with a bifunctional LSD1/HDAC

inhibitor induces therapeutic differentiation in DIPG. Cancer Cell

2019, 36, 528−544.

Pharma AG, Pﬁzer, Sao Paulo Research Foundation-FAPESP,

̃

Takeda, and Wellcome [106169/ZZ14/Z]. We thank Reaction

Biology Corporation for their pro bono contributions in testing

the biological activity of our compounds. D.P. is grateful to

NSERC for the Collaborative Research and Training

Experience 432008-2013 grant. We would like to thank Dr.

Gennady Poda for generating the Oracle database where the

compound data were stored and for providing his expertise on

molecular modeling. We would also like to thank Mehakpreet

Saini for contributing to the writing and editing of ADME

protocols.

ABBREVIATIONS

■

ALK2, activin receptor-like kinase-2; ALK5, activin receptor-

like kinase-5; ATP, adenosine triphosphate; AUC_inf, area under

the curve (extrapolated to inﬁnity); BBB, blood−brain barrier;

BMP, bone morphogenetic protein; B/P, total brain-to-plasma

ratio; C_b,u, unbound brain concentration; cLogP, calculated

lipophilicity; C_max, maximum concentration; CNS, central

nervous system; DIPG, diﬀuse intrinsic pontine glioma;

EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; FOP,

ﬁbrodysplasia ossiﬁcans progressiva; GS, glycine−serine-rich

domain; HATU, hexaﬂuorophosphate azabenzotriazole tetra-

methyl uronium; HBD, hydrogen bond donor; HDAC, histone

deacetylase; hERG, human ether a-go-go related gene; HLM,

human liver microsome; MLM, mouse liver microsome; PK,

pharmacokinetic; P-gp, P-glycoprotein; SAR, structure−

activity relationship; STKR, serine/threonine kinase receptor;

TFA, triﬂuoroacetic acid; TGFβ-R1, transforming growth

factor beta receptor 1; tPSA, topological polar surface area;

WT, wild-type.

(13) Hopkins, C. R. Inhibitors of the bone morphogenetic protein

(BMP) signaling pathway: a patent review (2008-2015). Expert Opin.

Ther. Pat. 2016, 26, 1115−1128.

(14) 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.

(15) Yu, P. B.; Cuny, G. D.; Mohedas, A. H.; Bloch, K. D.; Peterson,

R. T. BMP Inhibitors and Methods of Use Thereof.

WO2014138088A1, 2014.

(16) 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, No. e62721.

(17) Mohedas, A. H.; Wang, Y.; Sanvitale, C. E.; Canning, P.; Choi,

S.; Xing, X.; Bullock, A. N.; Cuny, G. D.; Yu, P. B. Structure-activity

relationship of 3,5-diaryl-2-aminopyridine ALK2 inhibitors reveals

unaltered binding affinity for fibrodysplasia ossificans progressiva

causing mutants. J. Med. Chem. 2014, 57, 7900−7915.

REFERENCES

■

(1) Heffron, T. P. Small molecule kinase inhibitors for the treatment

of brain cancer. J. Med. Chem. 2016, 59, 10030−10066.

(2) Di, L.; Rong, H.; Feng, B. Demystifying brain penetration in

central nervous system drug discovery. J. Med. Chem. 2013, 56, 2−12.

(3) Pacifici, M.; Shore, E. M. Common mutations in ALK2/ACVR1,

a multi-faceted receptor, have roles in distinct pediatric muscu-

loskeletal and neural orphan disorders. Cytokine Growth Factor Rev.

2016, 27, 93−104.

(18) Yu, P. B.; Cuny, G. D.; Mohedas, A. H. Compositions and

Methods for Inhibiting BMP. WO2015148654A1, 2015.

(19) Reinecke, M.; Ruprecht, B.; Poser, S.; Wiechmann, S.; Wilhelm,

́

M.; Heinzlmeir, S.; Kuster, B.; Medard, G. Chemoproteomic

R

https://dx.doi.org/10.1021/acs.jmedchem.0c00395

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

selectivity profiling of PIKK and PI3K kinase inhibitors. ACS Chem.

Biol. 2019, 14, 655−664.

Derivatives Useful as Soluble Guanylate Cyclase Stimulators.

WO2017200825A1, 2017.

(37) Balkovec, J. M.; Bensen, D. C.; Borchardt, A.; Brady, T. P.;

Chen, Z.; Lam, T.; Tari, L. W. Small Molecule Inhibitors of

Dihydrofolate Reductase. WO2016201219A1, 2016.

(38) Pouzet, P.; Hoenke, C.; Nickolaus, P.; Goeggel, R.; Fox, T.;

Fiegen, D.; Klinder, K. Novel Piperazino-dihydrothienopyrimidine

Derivatives. WO2009050236A1, 2009.

(20) Lahn, M.; Herbertz, S.; Sawyer, J. S.; Stauber, A. J.;

Gueorguieva, I.; Driscoll, K. E.; Estrem, S. T.; Cleverly, A. L.;

Desaiah, D.; Guba, S. C.; Benhadji, K. A.; Slapak, C. A. Clinical

development of galunisertib (LY2157299 monohydrate), a small

molecule inhibitor of transforming growth factor-beta signaling

pathway. Drug Des., Dev. Ther. 2015, 9, 4479−4499.

(39) Guerin, D. J.; Bair, K. W.; Caravella, J. A.; Ioannidis, S.; Lancia,

D. R., Jr; Li, H.; Mischke, S.; Ng, P. Y.; Richard, D.; Schiller, S. E. R.;

Shelekhin, T.; Wang, Z. Thienopyridine Carboxamides as Ubiquitin-

speciﬁc Protease Inhibitors. WO2017139778A1, 2017.

(40) Winter, G. xia2: an expert system for macromolecular

crystallography data reduction. J. Appl. Crystallogr. 2010, 43, 186−

190.

(21) Unpublished results. Data can be viewed in presentations

archived at https://m4kpharma.com/blog/ (accessed Apr. 1, 2020).

(22) Morgan, M. R.; Roberts, O. G.; Edwards, A. M. Ideation and

implementation of an open science drug discovery business model -

M4K Pharma. Wellcome Open Res. 2018, 3, 154.

(23) 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 bone morphogenetic protein

receptor ALK2 and implications for fibrodysplasia ossificans

progressiva. J. Biol. Chem. 2012, 287, 36990−36998.

(24) Rankovic, Z. CNS drug design: balancing physicochemical

properties for optimal brain exposure. J. Med. Chem. 2015, 58, 2584−

2608.

(41) Winn, M. D.; Ballard, C. C.; Cowtan, K. D.; Dodson, E. J.;

Emsley, P.; Evans, P. R.; Keegan, R. M.; Krissinel, E. B.; Leslie, A. G.

W.; McCoy, A.; McNicholas, S. J.; Murshudov, G. N.; Pannu, N. S.;

Potterton, E. A.; Powell, H. R.; Read, R. J.; Vagin, A.; Wilson, K. S.

Overview of the CCP4 suite and current developments. Acta

Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 235−242.

(42) 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.

(25) tPSA, pK_aand cLogP values were calculated using ChemDraw

Professional 19.0.

(26) Johnson, T. W.; Richardson, P. F.; Bailey, S.; Brooun, A.; Burke,

B. J.; Collins, M. R.; Cui, J. J.; Deal, J. G.; Deng, Y.-L.; Dinh, D.;

Engstrom, L. D.; He, M.; Hoffman, J.; Hoffman, R. L.; Huang, Q.;

Kania, R. S.; Kath, J. C.; Lam, H.; Lam, J. L.; Le, P. T.; Lingardo, L.;

Liu, W.; McTigue, M.; Palmer, C. L.; Sach, N. W.; Smeal, T.; Smith,

G. L.; Stewart, A. E.; Timofeevski, S.; Zhu, H.; Zhu, J.; Zou, H. Y.;

Edwards, M. P. Discovery of (10R)-7-amino-12-fluoro-2,10,16-

trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno)pyrazolo-

[4,3-h][2,5,11]-benzoxadiazacyclotetradecine-3-carbonitrile (PF-

06463922), a macrocyclic inhibitor of anaplastic lymphoma kinase

(ALK) and c-ros oncogene 1 (ROS1) with preclinical brain exposure

and broad-spectrum potency against ALK-resistant mutations. J. Med.

Chem. 2014, 57, 4720−4744.

(27) Kuhn, B.; Mohr, P.; Stahl, M. Intramolecular hydrogen bonding

in medicinal chemistry. J. Med. Chem. 2010, 53, 2601−2611.

(28) Gillis, E. P.; Eastman, K. J.; Hill, M. D.; Donnelly, D. J.;

Meanwell, N. A. Applications of fluorine in medicinal chemistry. J.

Med. Chem. 2015, 58, 8315−8359.

(29) Johnson, T. W.; Gallego, R. A.; Edwards, M. P. Lipophilic

efficiency as an important metric in drug design. J. Med. Chem. 2018,

61, 6401−6420.

́

(43) 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 macro-

molecular structure solution. Acta Crystallogr., Sect. D: Biol. Crystallogr.

2010, 66, 213−221.

(44) Emsley, P.; Cowtan, K. Coot: model-building tools for

molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004,

60, 2126−2132.

(45) 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−W383.

(30) Sekimata, K.; Sato, T.; Sakai, N.; Watanabe, H.; Mishima-

Tsumagari, C.; Taguri, T.; Matsumoto, T.; Fujii, Y.; Handa, N.;

Honma, T.; Tanaka, A.; Shirouzu, M.; Yokoyama, S.; Miyazono, K.;

Hashizume, Y.; Koyama, H. Bis-heteroaryl pyrazoles: identification of

orally bioavailable inhibitors of activin receptor-like kinase-2

(R206H). Chem. Pharm. Bull. 2019, 67, 224−235.

(31) Khalili, F.; Henni, A.; East, A. L. L. pK_avalues of some

piperazines at (298, 303, 313, and 323) K. J. Chem. Eng. Data 2009,

54, 2914−2917.

(32) 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.

Bioorg. Med. Chem. Lett. 2018, 28, 3356−3362.

(33) Chen, J.; Ding, C. Z.; Dragovich, P.; Fauber, B.; Gao, Z.;

Labadie, S.; Lai, K. W.; Purkey, H. E.; Robarge, K.; Wei, B.; Zhou, A.

Piperidine-dione Derivatives. WO2015140133A1, 2015.

(34) Mascitti, V.; McClure, K. F.; Munchhof, M. J.; Robinson, R. P.,

Jr. 4-(5-Cyano-pyrazol-1-yl)-piperidine Derivatives as GPR 119

Modulators. WO2012069948A1, 2012.

(35) Zhan, W.; Ji, L.; Ge, Z.-m.; Wang, X.; Li, R.-t. A continuous-

flow synthesis of primary amides from hydrolysis of nitriles using

hydrogen peroxide as oxidant. Tetrahedron 2018, 74, 1527−1532.

(36) Whitehead, A.; Ornoski, O.; Raghavan, S.; Berger, R.;

Garfunkle, J.; Yang, Z.; Ji, G.; Jiang, F.; Fu, J. Fused Pyrazine

S

https://dx.doi.org/10.1021/acs.jmedchem.0c00395

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Targeting ALK2: An Open Science Approach to Developing Therapeutics for the Treatment of Diffuse Intrinsic Pontine Glioma

DOI: 10.1021/acs.jmedchem.0c00395

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acs.jmedchem.0c00395

Full text of DOI:10.1021/acs.jmedchem.0c00395

Products guided by the article

R&D Labs maybe for 1400503-15-2

Relevant to this article

Hot Product