Bis-Heteroaryl Pyrazoles: Identification of Orally Bioavailable Inhibitors of Activin Receptor-Like Kinase-2 (R206H)

Abstract

Mutant activin receptor-like kinase-2 (ALK2) was reported to be closely associated with the pathogenesis of fibrodysplasia ossificans progressiva (FOP) and diffuse intrinsic pontine glioma (DIPG), and therefore presents an attractive target for therapeutic intervention. Through in silico virtual screenings and structure–activity relationship studies assisted by X-ray crystallographic analyses, a novel series of bis-heteroaryl pyrazole was identified as potent inhibitors of ALK2 (R206H). Derived from in silico hit compound RK-59638 (6a), compound 18p was identified as a potent inhibitor of ALK2 (R206H) with good aqueous solubility, liver microsomal stability, and oral bioavailability.

Introduction

Fibrodysplasia ossificans progressiva (FOP) is a rare disorder that shows progressive heterotopic ossification in the muscles, tendons, or ligaments with an incidence of 1 in 2 million individuals.^1,2) FOP is caused by abnormal activation of bone morphogenetic protein (BMP) signaling due to highly recurrent mutations, including R206H in activin receptor-like kinase-2 (ALK2) (encoded by ACVR1 gene), a subtype of BMP type-I receptors, resulting in an alteration in the intracellular glycine-serine-rich domain of the enzyme. Diffuse intrinsic pontine glioma (DIPG), a fatal brain cancer that forms in the brainstem of children, is another disease caused by the R206H mutation of ALK2.³⁾ Thus, the inhibition of the BMP signaling pathway, which acts on ALK2 (R206H), might offer an effective treatment for FOP and/or DIPG.⁴⁾

While several ALK2 inhibitors were shown to be efficacious in animal models of FOP,⁵⁾ orally efficacious inhibitors of ALK2 (R206H) have yet to be reported. For example, LDN-193189 and LDN-212854 are potent inhibitors of ALK2 (R206H) (IC₅₀ = 20 and 11 nM, respectively), but they are poorly aqueous soluble and highly bound to serum protein (Table 1). We attempted to find orally bioavailable, small-molecule ALK2 (R206H) inhibitors with appropriate biophysical properties.

Table 1. Chemical Profiles of LDN-193189 and Hit Compound RK-59638 (6a)^a)

IC50 ALK2 (R206H) (µM)	0.020	0.684
Aqueous solubility
@ pH 7.4 (µM)	10.6	6.4
Human PPB (%) @ 10 µMb)	97.8	85
HLM/MLMc)
(% remaining after 60 min)	68.4/25.9	40.0/0.0
Caco-2 (pH 6.5/7.4)
Papp A to B (10−6 cm/s)	2.0	52.3
hERG. Automated pach-clamp
(% inhibition @ 10 µM)	81.2	25.4
In vitro toxicity HepG2 cell
(% viability @ 30 µM)	77	78

a) All studies were conducted by Cerep (United States) using in vitro ADME/Tox assays. b) Plasma protein binding. c) HLM = human liver microsomes, MLM = mouse liver microsomes.

In general, computational approaches generate a number of hit compounds obtained from a large compound library via ligand-based two-dimensional and three-dimensional similarity searches, docking simulation with proteins, and a machine-based learning algorithm.⁶⁾ A combination of virtual and biochemical assays can lead to acceleration of the identification of hit compounds. In this study, we describe the results of structural design of ALK2 (R206H) inhibitors supported by X-ray crystallographic studies and structure–activity relationship (SAR) development.

Results and Discussion

Structural Design Supported by X-Ray Crystallographic Analysis

Our in silico and in vitro screening studies identified a series of various ALK2 (R206H) inhibitors.⁷⁾ Among them, the bis-heteroaryl pyrazole series⁸⁾ was investigated to determine their crystal structures, identify pharmacophores, and provide a direction for structural modification. The crystal structures revealed that compound 6a binds to the ALK2 (R206H) ATP-binding pocket in a twisted conformation via hydrogen bonds and hydrophobic interactions (Fig. 1). Compound 6a formed two hydrogen bonds between the aminopyrimidine moiety and the main chain amine and carboxyl oxygen of the His286 residue in the hinge region of the enzyme. Another hydrogen bond is formed between the nitrogen of the 3-pyridyl group of compound 6a and the carboxyl group of the conserved Glu248 side chain from the αC helix via a conserved water molecule. The pyrazole moiety of compound 6a was located in the sugar pocket of the enzyme. The methoxy group of the anisidine moiety was exposed to the solvent region. Introduction of polar groups to the pyrazole and anisidine moieties of compound 6a was expected to result in improved enzyme inhibitory activity, aqueous solubility, and liver microsomal stability. For the SAR development of compound 6a, 3-pyridyl, pyrazole, and anisidine moieties were structurally modified to achieve improved ALK2 (R206H) enzyme inhibitory activity.

Fig. 1. X-Ray Crystal Structure of Compound 6a in Mutated ALK2 (R206H)

Binding mode of compound 6a in ALK2 (R206H). Secondary structural elements of ALK2 (R206H) are represented by ribbon model. Residues forming ATP-binding pockets are shown as white stick models. Compound 6a is represented by the yellow stick model. The hydrogen bonds involved in the interaction of ALK2 (R206H) and compound 6a are shown by green dashed lines. Water molecules are shown as red spheres.

Chemistry

Synthesis of compounds 6a and 6c–e is shown in Chart 1.^9,10) Protection of commercially available pyrazole 1 with a 2-(trimethylsilyl)ethoxymethyl (SEM) group afforded 2 as a 1 : 1.4 mixture of regioisomers. Oxidation of compound 2 with m-chloroperbenzoic acid afforded sulfone 3, which was converted to compound 4 via subsequent nucleophilic displacement of the sulfone moiety with p-anisidine. Palladium-catalyzed coupling of compound 4 with appropriate aryl boronates and subsequent deprotection of the SEM group afforded 6a and 6c–e. Synthesis of compound 6b is shown in Chart 2. Since palladium-catalyzed coupling of compound 4 with 2-pyridyl boronate failed to provide the desired compound 5b, an alternate synthetic route was required for compound 6b.¹⁰⁾ Bromination of commercially available compound 7 with N-bromosuccunimide yielded bromide 8. The pyrazole nitrogen of compound 8 was protected by an SEM group generating a 1 : 1 mixture of regioisomers. Regioisomer 9 was chromatographically separated and converted to boronic acid 10, which was then subjected to the palladium-catalyzed coupling reaction with 2,4-dichloropyrimidine to afford compound 11. Displacement of the chlorine of compound 11 with anisole and subsequent deprotection of the SEM group gave compound 6b.

Chart 1

Reagents and conditions: (a) SEMCl, Cs₂CO₃, DMF, r.t.; (b) mCPBA, THF, 0°C to r.t.; (c) p-anisidine, THF, rlx; (d) heteroaryl-B(OH)₂, Pd(dba₃)₂, K₃PO₄, PCy₃, dioxane, rlx; (e) TBAF, THF, rlx.

Chart 2

Reagents and conditions: (a) NBS, DMF, 0°C; (b) SEMCl, Cs₂CO₃, DMF, r.t.; (c) triisopropyl borate, n-BuLi, THF, −78°C; (d) 2,4-dichloropyrimidine, PdCl₂(PPh₃)₂, Na₂CO₃aq, DME, 80°C; (e) p-anisidine, THF, rlx; (f) TBAF, THF, rlx.

N-Alkylation of pyrazole 6a was carried out according to the general method described in Chart 3.⁹⁾ Under basic reaction conditions, N-alkylation of compound 6a provided regioisomeric mixtures comprising N2-isomer as the major product and N1-regioisomer as the minor product. Chromatographic separation of the mixtures gave 13a–h.

Chart 3

Reagents and conditions: (a) R-X, K₂CO₃, DMF, r.t. to 50°C.

Structural modifications of the p-anisidine moiety of compound 6a were carried out following the synthetic route depicted in Chart 4.^8–10) Commercially available methyl nicotinate 14 was reacted with the lithium enolate of 2-chloro-4-methylpyrimidine to give ketone 15. Treatment of compound 15 with the dimethylformamide dimethylacetal followed by the cyclization reaction with hydrazine yielded pyrazole 16. As described above, N-alkylation of the pyrazole 16 afforded the N2-isomer 17 as the major product and the N1-regioisomer as the minor product. As shown in Chart 4, compound 18a was prepared by cross-coupling using 4-(methoxylbenzoyl) zinc chloride¹¹⁾ and the amino-pyrimidine derivatives 18b–p were prepared using the same method as in the nucleophilic substitution reaction.

Chart 4

Reagents and conditions: (a) 2-chloro-4-methylpyrimidine, LiHMDS, THF, −30°C to r.t.; (b) (MeO)₂CHNMe₂, AcOH, toluene, 110°C; (c) NH₂NH₂ H₂O, EtOH, 0°C; (d) EtI, K₂CO₃, DMF, r.t.; (e) 4-MeOC₆H₄CH₂ZnCl, K₂CO₃, Pd(PPh₃)₄ or amine, MeOH, rlx.

SAR Study and Pharmacokinetics

First, to explore the SAR of the 3-pyridyl moiety of compound 6a surrounded by the hydrophobic pocket of the enzyme, the pyridine moiety was replaced with various heteroaryl groups (Table 2). Compounds 6b–e showed significant decreases in enzyme inhibitory activity, suggesting that the 3-pyridyl group is essential for hydrogen bonding with the enzyme. Moreover, the bioactivities of the 6a analogue possessing the 3-pyridyl group have not yet been determined. Second, substitution on N1 or N2 of the pyrazole ring of compound 6a possessing the 3-pyridyl group was investigated (Table 3). Compound 13a exhibited moderate enzyme inhibitory activity, poor cell permeability, and a high efflux ratio in Caco-2 cells, which may have unfavorable effects on oral bioavailability. In contrast, compounds 13b, 13d, and 13f showed improved cell permeability and efflux compared with 13a. The activities of compounds 13e–h clearly indicated that the transposition of the alkyl group from the N2 position to the N1 position leads to a significant decrease in the enzyme inhibitory activity. The most potent compound 13f was then selected for further optimization.

Table 2. Inhibition of ALK2 (R206H) Enzyme Activity^a)

a) IC₅₀ values are the averages of two or more tests. Values marked as greater than (>) indicate that half-maximum inhibition was not achieved at the highest concentration tested.

Table 3. Inhibition of ALK2 (R206H) Enzyme Activity and Caco-2 Assay

a) The percentage inhibition values are the averages of two or more tests. b) Apparent permeability coefficient measured in the Caco-2 assay. The efflux ratio was calculated by comparing P_app B to A with P_app A to B. c) NT, not tested.

As described above, the aminopyrimidine moiety of compound 6a interacted with the His286 residue of the enzyme at two points (Fig. 1). This finding is consistent with the significant loss of inhibitory activity when the amino group of the amino-pyrimidine of compound 6a was replaced by a methylene group in compound 18a (Table 4). Among methoxy-substituted phenyls, 13b and 18b exhibited good activities, whereas ortho-substituted 18c showed decreased activity. Introduction of a difluoromethoxy group to compound 18d led to an improvement in liver microsomal stability. Additionally, saturated heterocyclic compounds 18e–g, 18i–n, 18o, and 18p containing nitrogen or oxygen at the end of the structure were found to be highly potent with good liver microsomal stability. Based on the desirable attributes, including enzyme inhibitory activity and liver microsomal stability, 18g was selected for pharmacokinetic studies in rats (Table 5). The relatively poor exposure of 18g may be explained by low permeability (Caco-2 A to B P_app = 0.26 × 10⁻⁶ cm/s) and high efflux ratio (154) in Caco-2 cells. Detailed efflux ratios were determined using Madin–Darby canine kidney (MDCK) cells, and the multidrug resistance (MDR) profiles of these compounds were determined (Table 4). The results showed that the terminal NH or NH₂ group of piperidine, piperazine, or 4-aminopiperidine in 18f, 18g, and 18i–k, was associated with increased efflux rates. However, we were able to reduce the efflux rates by capping the NH group of the piperazine moiety of compound 18g as an N-methyl group (18o). Similarly, replacement of N-methylpiperazine in compound 18o with piperidine (18h) or morpholine (18p) resulted in a reduced efflux rate. Compounds 18o and 18p showed a 2- to 3-fold decrease in enzyme inhibitory activity, unlike 18g, but 18p showed good aqueous solubility, plasma protein binding (solubility 200.1 µM; human plasma protein binding 91.8%), moderate liver microsomal stability, and low P-gp-mediated efflux. Furthermore, 18p exhibited good oral bioavailability in rats (F = 56%). ALK1–6 are known to transduce signals for BMPs, transforming growth factor-β (TGF-β), and activins. The ALK isoform selectivity of compound 18p was studied (Table 6). Compared with LDN-193189, compound 18p showed less selectivity for the TGF-β type I receptors, ALK4 and ALK5, and higher selectivity for ALK3. In our future study, the efficacy of compounds 18p and 18o will be evaluated in animal models of FOP and/or DIPG.

Table 4. Inhibition of ALK2 (R206H) Enzyme Activity, Permeability, Efflux, and Metabolic Profiles

a) IC₅₀ values are the averages of two or more tests. Values marked as greater than (>) indicate that half-maximum inhibition was not achieved at the highest concentration tested. b) NT, not tested. c) Apparent permeability coefficient measured in the MDCK cell assay. d) The efflux ratio was calculated by comparing P_app B to A with P_app A to B. In parentheses, the MDR efflux ratio using an MDR1/MDCK assay utilizing MDCK cells transfected with the gene that encodes human P-glycoprotein. e) Percentage remaining at 60 min assessed in human, rat, and mouse liver microsoms.

Table 5. Pharmacokinetics in Rats

Cpd. No.	p.o. (5* or 3 mg/kg)			i.v. (1 mg/kg)
	Cmax (µM)	AUC (µM·h)	t1/2 (h)	AUC (µM·h)	Vd (L/kg)	CL (mL/min/kg)	F%
18g*	BLQ	BLQ	BLQ	0.46	13.7	85.7	ND
18p	2.33	2.75	1	1.64	0.56	23.8	56

BLQ, below the lower limit of quantification (<5.0 ng/mL).

Table 6. ALK Selectivity IC₅₀ for LDN19318 and 18p

Cpd. No.	ALK1	ALK2	ALK3	ALK4	ALK5	ALK6
	IC50 (nM)a) and selectivityb)
LDN-193189	7 (0.4)	23.0 (1.2)	5 (0.3)	408 (20.4)	425 (21.3)	13 (0.7)
18p	45.8 (1.8)	112.1 (4.4)	637 (24.9)	43.9 (1.7)	119 (4.6)	25.1 (1.0)

a) IC₅₀ values are the averages of two or more tests. b) In parentheses, the selectivity values are calculated by comparing each ALK with ALK2 (R206H).

In conclusion, we identified a novel series of ALK2 (R206H) inhibitors with enzyme inhibitory activity comparable to that of LDN-193189. Additionally, compound 18p exhibited good attributes including aqueous solubility, plasma protein binding, and good pharmacokinetic profiles.

Experimental

All reagents and solvents were obtained from commercial sources and used as received. ¹H-NMR spectra were recorded with tetramethylsilane as an internal standard using a JEOL JNM-Ex 270 MHz spectrometer. Automated column chromatographic separations were performed on a flash chromatography (Biotage ZIP^®; Biotage, United Kingdom and Amino Inject Column; Yamazen, Osaka, Japan). LC/MS analysis was performed on a Waters Aquity Ultra Performance LC (Waters, United States) with a 2.1 × 50 mm Waters Aquity UPLC BEH C18 1.7 µm column. The column temperature was 40°C, with a run time of 2 min, flow rate of 0.6 mL/min, and a mixture of acetonitrile and water containing 0.1% trifluoroacetic acid with a gradient of 10–90% as an eluting solvent. The mass spectrometry data were acquired on a SQD2 quadrupole mass spectrometer (Waters).

Mixture of 4-{5-Iodo-1-[(2-(trimethylsilyl)ethoxy)methyl]-1H-pyrazol-4-yl}-2-(methylthio)pyrimidine and 4-{3-Iodo-1-[(2-(trimethylsilyl)ethoxy)methyl]-1H-pyrazol-4-yl}-2-(methylthio)pyrimidine (2)

To a solution of 4-(5-iodo-1H-pyrazole-4yl)-2-(methylthio)pyrimidine 1 (2.01 g, 6.32 mmol) in N,N-dimethylformamide (DMF) (8 mL) was added Cs₂CO₃ (3.19 g, 9.79 mmol), and the reaction mixture was stirred at room temperature for 1 h. Then, 2-(trimethylsilyl)ethoxymethyl chloride (1.68 mL, 1.60 g, 9.60 mmol) was added slowly, and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with EtOAc, washed with brine, dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by column chromatography over silica gel (n-hexane–EtOAc) to afford the regioisomers 2 (2.47 g, 87%) as clear oil. ¹H-NMR (270 MHz, CDCl₃, major peaks of regioisomers) δ: 8.53 (1H, d, J = 4.9 Hz), 8.15 (1H, s), 7.66 (1H, d, J = 5.4 Hz), 5.46 (2H, s), 3.61–3.67 (2H, m), 2.63 (3H, s), 0.91–0.97 (2H, m), 0.01 (9H, s). LC/MS (electrospray ionization (ESI)): m/z 449 [M + H]⁺.

Mixture of 4-{5-Iodo-1-[(2-(trimethylsilyl)ethoxy)methyl]-1H-pyrazol-4-yl}-2-(methylsulfonyl)pyrimidine and 4-{3-Iodo-1-[(2-(trimethylsilyl)ethoxy)methyl]-1H-pyrazol-4-yl}-2-(methylsulfonyl)pyrimidine (3)

To a solution of regioisomers 2 (2.47 g, 5.51 mmol) in tetrahydrofuran (THF) (40 mL) was added m-chloroperbenzoic acid (mCPBA) (3.17 g, 75%, 13.78 mmol) at 0°C. The reaction mixture was warmed to room temperature, stirred for 17 h, and concentrated in vacuo. The residue was purified twice by column chromatography over silica gel (n-hexane–EtOAc) to afford a mixture of regioisomers 3 (2.63 g, 99%) as a white powder. ¹H-NMR (270 MHz, CDCl₃, major peaks of regioisomers) δ: 8.88 (1H, d, J = 5.1 Hz), 8.33 (1H, s), 8.26 (1H, d, J = 5.4 Hz), 5.47 (2H, s), 3.61–3.67 (2H, t), 3.41 (3H, s), 0.91–0.96 (2H, t), 0.01 (9H, s). LC/MS (ESI): m/z 481 [M + H]⁺.

Mixture of 4-{5-Iodo-1-[(2-(trimethylsilyl)ethoxy)methyl]-1H-pyrazol-4-yl}-N-(4-methoxyphenyl)pyrimidin-2-amine and 4-{3-Iodo-1-[(2-(trimethylsilyl)ethoxy)methyl]-1H-pyrazol-4-yl}-N-(4-methoxyphenyl)pyrimidin-2-amine (4)

A solution of compound 3 (2.62 g, 5.45 mmol) and p-anisidine (2.05 g, 16.65 mmol) in THF (20 mL) was refluxed for 17 h. The reaction mixture was cooled to room temperature, diluted with EtOAc, washed with brine, dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by column chromatography over silica gel (n-hexane–EtOAc) to afford a mixture of regioisomers 4 (1.79 g, 63%) as a beige solid. ¹H-NMR (270 MHz, CDCl₃, major peaks of regioisomers) δ: 8.41 (2H, d, J = 5.1 Hz), 8.05 (1H, s), 7.52 (1H, d, J = 9.2 Hz), 7.38 (1H, d, J = 5.1 Hz), 6.91 (1H, d, J = 8.9 Hz), 5.46 (2H, s), 3.82 (3H, s), 3.64 (2H, t), 0.94 (2H, t), 0.01 (9H, s). LC/MS (ESI): m/z 524 [M + H]⁺.

N-(4-Methoxyphenyl)-4-(5-(pyridin-3-yl)-1-[(2-(trimethylsilyl)ethoxy)methyl]-1H-pyrazol-4-yl)pyrimidin-2-amine and N-(4-Methoxyphenyl)-4-(3-(pyridin-3-yl)-1-[(2-(trimethylsilyl)ethoxy)methyl]-1H-pyrazol-4-yl)pyrimidin-2-amine (5a)

A mixture of compound 4 (104.6 mg, 0.200 mmol), pyridine-3-boronic acid (52.1 mg, 0.424 mmol), Pd₂(dba)₃ (6.7 mg, 0.007 mmol), K₃PO₄ (198.2 mg, 0.934 mmol), H₂O (0.5 mL), dioxane (0.5 mL), and 0.6 M tricyclohexylphosphine (20 µL, 0.012 mmol) was refluxed under an argon atmosphere for 15 h. The reaction mixture was filtered, washed with EtOAc, and concentrated in vacuo. The residue was purified by column chromatography over silica gel (n-hexane–EtOAc) to give a mixture of regioisomers 5a (40.2 mg, 43%) as a yellow oil. ¹H-NMR (270 MHz, CDCl₃, major peaks of regioisomers) δ: 8.86 (1H, d, J = 1.9 Hz), 8.62–8.64 (1H, m), 8.25 (1H, d, J = 5.4 Hz), 8.14 (1H, s), 7.88–7.91 (2H, m), 7.30–7.37 (2H, m), 7.10 (1H, s), 6.79–6.83 (2H, m), 6.56 (1H, d, J = 5.4 Hz), 5.51 (2H, s), 3.80 (3H, s), 3.67–3.80 (2H, s), 0.91–1.01 (2H, m), 0.01 (9H, s). LC/MS (ESI): m/z 475 [M + H]⁺.

N-(4-Methoxyphenyl)-4-(3-(pyridin-3-yl)-1H-pyrazol-4-yl)pyrimidin-2-amine (6a)

To a solution of compound 5a (40 mg, 0.084 mmol) in dry THF (6 mL) was added 1 M TBAF in THF (0.17 µL). The reaction mixture was refluxed for 3.5 h and added to brine, extracted with EtOAc, dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by column chromatography over silica gel (CH₂Cl₂–MeOH) to afford compound 6a (23.3 mg, 81%) as a yellow solid. ¹H-NMR (270 MHz, CD₃OD) δ: 8.70–8.71 (1H, m), 8.49 (1H, dd, J = 1.6, 4.9 Hz), 8.26 (1H, s), 8.24 (1H, s), 7.99–8.03 (1H, m), 7.40–7.44 (1H, m), 7.22 (2H, d, J = 8.9 Hz), 6.82 (2H, d, J = 5.1 Hz), 6.67 (2H, d, J = 8.9 Hz), 3.76 (3H, s). LC/MS (ESI): m/z 345 [M + H]⁺.

Compounds 6c–e were prepared following a procedure similar to that used to prepare compound 6a.

N-(4-Methoxyphenyl)-4-(3-pyridin-4-yl-1H-pyrazol-4-yl)pyrimidin-2-amine (6c)

¹H-NMR (270 MHz, CD₃OD) δ: 8.49–8.52 (2H, m), 8.25–8.28 (2H, m), 7.62–7.64 (2H, m), 7.26 (2H, d, J = 9.2 Hz), 6.83 (1H, d, J = 5.1 Hz), 6.68 (2H, d, J = 9.2 Hz), 3.75 (3H, s). LC/MS (ESI): m/z 345 [M + H]⁺.

N-(4-Methoxyphenyl)-4-(3-pyrimidin-5-yl-1H-pyrazol-4-yl)pyrimidin-2-amine (6d)

¹H-NMR (270 MHz, DMSO-d₆) δ: 13.68 (1H, s), 9.17 (1H, s), 9.07 (1H, s), 8.90 (2H, s), 8.49 (1H, s), 8.35 (1H, d, J = 4.9 Hz), 7.20 (2H, d, J = 8.9 Hz), 6.93 (1H, d, J = 4.9 Hz), 6.58 (2H, d, J = 8.9 Hz), 3.67 (3H, s). LC/MS (ESI): m/z 346 [M + H]⁺.

N-(4-Methoxyphenyl)-4-(3-quinolin-3-yl-1H-pyrazol-4-yl)pyrimidin-2-amine (6e)

¹H-NMR (270 MHz, CD₃OD) δ: 8.92 (1H, d, J = 2.4 Hz), 8.57 (1H, d, J = 1.6 Hz), 8.32 (1H, br  s), 8.29 (1H, d, J = 8.3 Hz), 7.94–8.00 (2H, m), 7.77–7.80 (1H, m), 7.62–7.68 (1H, m), 6.95–6.98 (3H, m), 6.13 (2H, d, J = 8.9 Hz), 3.49 (3H, s). LC/MS (ESI): m/z 395 [M + H]⁺.

2-(4-Bromo-1H-pyrazol-3-yl)pyridine (8)

A solution of 2-(1H-pyrazole-3-yl) pyridine (3.02 g, 20.80 mmol) and NBS (3.72 g, 20.90 mmol) in DMF (30 mL) was stirred for 2 h in an ice bath. The reaction mixture was poured into ice water, filtered, and dried in vacuo. The residue (4.38 g, 95% yield) as a white solid was reacted without further purification. ¹H-NMR (270 MHz, CDCl₃) δ: 11.46 (1H, br  s), 8.63–8.65 (1H, m), 8.29 (1H, d, J = 8.4 Hz), 7.78–7.85 (1H, m), 7.64 (1H, s), 7.28–7.33 (1H, m). LC/MS (ESI): m/z 223 [M + H]⁺.

2-{4-Bromo-1-[(2-(trimethylsilyl)ethoxy)methyl]-1H-pyrazol-3-yl}pyridine (9)

To a solution of compound 8 (4.38 g, 19.55 mmol) and Cs₂CO₃ (14.01 g, 43.00 mmol) in DMF (35 mL) was added 2-(trimethylsilyl)ethoxymethyl chloride (7.27 mL, 43.01 mmol) at room temperature for 1 h. The reaction mixture was diluted with EtOAc, washed with brine, dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by column chromatography over silica gel (n-hexane–EtOAc) to give compound 9 as a yellow oil (3.23 g, 47%). ¹H-NMR (270 MHz, CDCl₃) δ: 8.73–8.76 (1H, m), 7.98–8.01 (1H, m), 7.73–7.80 (1H, m), 7.30 (1H, s), 7.27–7.30 (1H, m), 5.48 (2H, s), 3.59–3.65 (2H, m), 0.90–1.00 (2H, m), −0.03 (9H, s). LC/MS (ESI): m/z 354 [M + H]⁺.

{3-(Pyridin-2-yl)-1-[(2-(trimethylsilyl)ethoxy)methyl]-1H-pyrazol-4-yl}boronic Acid (10)

To a solution of compound 9 (3.23 g, 9.12 mmol) and triisoppropyl borate (5.47 mL, 23.71 mmol) in THF (50 mL) was added n-BuLi (1.4 M solution in hexane, 8.42 mL, 21.89 mmol) at −78°C. After stirring for 1 h, the reaction mixture was warmed to 0°C for 1 h, quenched with saturated NH₄Cl aqueous solution, and extracted with CH₂Cl₂. The extract was dried over Na₂SO₄ and concentrated in vacuo. The residue was purified by column chromatography over silica gel (hexane–EtOAc) to afford compound 10 (1.92 g, 66% yield) as a white solid. ¹H-NMR (270 MHz, CDCl₃) δ: 8.74 (2H, br  s), 8.52–8.55 (1H, m), 8.32–8.36 (1H, m), 8.01 (1H, m), 7.79–7.86 (1H, m), 7.27–7.32 (1H, m), 5.48 (2H, s), 3.60–3.66 (2H, m), 0.91–0.98 (2H, m), −0.12 (9H, s). LC/MS (ESI): m/z 320 [M + H]⁺.

2-Chloro-4-{3-(pyridin-2-yl)-1-[(2-(trimethylsilyl)ethoxy)methyl]-1H-pyrazol-4-yl}pyrimidine (11)

To a solution of compound 10 (0.51 g, 1.60 mmol), 2,4-dichloropyrimidine (0.29 g, 1.95 mmol), and 2N Na₂CO₃ aq (1.4 mL) in DME–EtOH (3.0 : 6.0 mL) was added Pd(PPh₃)₂Cl₂ (0.08 g, 0.11 mmol). After the replacement of nitrogen, the reaction mixture was heated at 80°C for 4.5 h. The reaction mixture was added to water, extracted with EtOAc, dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by column chromatography over silica gel (n-hexane–EtOAc) to afford compound 11 as a yellow oil (0.60 g, 96% yield). ¹H-NMR (270 MHz, CDCl₃) δ: 8.70–8.73 (1H, m), 8.49 (2H, d, J = 5.7 Hz), 7.83–7.93 (2H, m), 7.76 (1H, d, J = 5.4 Hz), 7.37–7.45 (1H, m), 5.57 (2H, s), 3.70–3.76 (2H, m), 0.99–1.05 (2H, m), 0.52 (9H, s). LC/MS (ESI): m/z 388 [M + H]⁺.

N-(4-Methoxyphenyl)-4-{3-(pyridin-2-yl)-1-[2-(trimethylsilyl)ethoxy)methyl]-1H-pyrazol-4-yl}pyrimidin-2-amine (12)

The title compound 12 was prepared from compound 11 and p-anisidine in a procedure similar to that used to prepare compound 4. ¹H-NMR (270 MHz, CDCl₃) δ: 8.68–8.70 (1H, m), 8.24 (1H, d, J = 5.1 Hz), 8.20 (1H, s), 7.69–7.79 (2H, m), 7.39–7.44 (2H, m), 7.28–7.32 (1H, m), 6.90 (1H, s), 6.81–6.87 (3H, m), 5.53 (2H, s), 3.80 (3H, s), 3.66–3.72 (2H, m), 0.94–1.00 (2H, m), 0.12 (9H, s). LC/MS (ESI): m/z 475 [M + H]⁺.

N-(4-Methoxyphenyl)-4-(3-(pyridin-2-yl-1H-pyrazol-4-yl)pyrimidin-2-amine (6b)

The title compound 6b was prepared from SEM-protected 12 following a procedure similar to that used to prepare compound 6a. ¹H-NMR (270 MHz, CD₃OD) δ: 8.54–8.63 (1H, m), 8.06–8.30 (2H, m), 7.72–7.89 (2H, m), 7.28–7.40 (3H, m), 6.60–6.85 (3H, m), 3.76 (3H, s). LC/MS (ESI): m/z 345 [M + H]⁺.

2-{4-[2-(4-Methoxyanilino)pyrimidin-4-yl]-3-pyridin-3-ylpyrazol-1-yl}ethanol (13a)

To a solution of compound 6a (52.5 mg, 0.152 mmol) and K₂CO₃ (53.2 mg, 0.385 mmol) in DMF (0.4 mL) was added 2-iodoethanol (87.2 g, 0.507 mmol) at 60°C overnight. The reaction mixture was poured into water and extracted with EtOAc. The extract was dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by column chromatography over amino silica gel (CH₂Cl₂–MeOH) to afford the N1-isomer (1.7 mg, 3% yield) and the N2-regioisomer 13a (4.2 mg, 7% yield) as a yellow solid. ¹H-NMR (270 MHz, CDCl₃) δ: 8.83–8.84 (1H, m), 8.60–8.63 (1H, m), 8.23 (1H, d, J = 5.1 Hz), 8.03 (1H, s), 7.88–7.91 (1H, m), 7.34–7.37 (2H, m), 7.31–7.32 (1H, m), 6.93 (1H, br  s), 6.80–6.85 (2H, m), 6.55 (1H, d, J = 5.1 Hz), 4.34–4.37 (2H, m), 4.10–4.13 (2H, m), 3.80 (3H, s). LC/MS (ESI) 389 [M + H]⁺.

Compounds 13b–e were prepared following a procedure similar to that used to prepare compound 13a.

4-[1-(2-Methoxyethyl)-3-pyridin-3-ylpyrazol-4-yl]-N-(4-methoxyphenyl)pyrimidin-2-amine (13b)

¹H-NMR (270 MHz, CDCl₃) δ: 8.85 (1H, d, J = 1.9 Hz), 8.60–8.62 (1H, m), 8.22 (1H, d, J = 5.1 Hz), 8.05 (1H, s), 7.87–7.91 (1H, m), 7.35 (2H, dd, J = 6.8, 2.2 Hz), 7.28–7.31 (1H, m), 6.89 (1H, s), 6.81 (2H, dd, J = 6.8, 2.2 Hz), 6.56 (1H, d, J = 5.1 Hz), 4.38 (2H, t, J = 5.1 Hz), 3.83 (2H, t, J = 5.1 Hz), 3.80 (3H, s), 3.40 (3H, s). LC/MS (ESI): m/z 403 [M + H]⁺.

N-(4-Methoxyphenyl)-4-[1-(oxetan-3-yl)-3-pyridin-3-ylpyrazol-4-yl]pyrimidin-2-amine (13c)

¹H-NMR (270 MHz, CDCl₃) δ: 8.86 (1H, d, J = 1.6 Hz), 8.62–8.64 (1H, m), 8.24 (1H, d, J = 5.4 Hz), 8.15 (1H, s), 7.90–7.94 (1H, m), 7.31–7.38 (3H, m), 6.92 (1H, s), 6.81–6.85 (2H, m), 6.55 (1H, d, J = 5.1 Hz), 5.50–5.60 (1H, m), 5.08–5.19 (4H, m), 3.80 (3H, s). LC/MS (ESI): m/z 401 [M + H]⁺.

N-(4-Methoxyphenyl)-4-[3-pyridin-3-yl-1-(2,2,2-trifluoroethyl)pyrazol-4-yl]pyrimidin-2-amine (13d)

¹H-NMR (270 MHz, CDCl₃) δ: 8.83–8.84 (1H, m), 8.64 (1H, d, J = 4.9, 1.6 Hz), 8.26 (1H, d, J = 5.4 Hz), 8.08 (1H, s), 7.88–7.92 (1H, m), 7.31–7.36 (3H, m), 6.91 (1H, s), 6.79–6.85 (2H, m), 6.55 (1H, d, J = 5.1 Hz), 4.81 (2H, m), 3.80 (3H, s). LC/MS (ESI): m/z 427 [M + H]⁺.

4-(1-Ethyl-5-pyridin-3-ylpyrazol-4-yl)-N-(4-methoxyphenyl)pyrimidin-2-amine (13e)

¹H-NMR (270 MHz, CDCl₃) δ: 9.36 (1H, d, J = 5.7 Hz), 8.60–8.73 (3H, s), 8.21 (1H, d, J = 5.1 Hz), 7.92–7.99 (3H, m), 7.07–7.11 (2H, m), 6.73 (1H, d, J = 5.1 Hz), 5.83 (1H, br s), 4.09–4.17 (2H, m), 3.87 (3H, s), 1.40 (3H, t, J = 7.0 Hz). LC/MS (ESI): m/z 373 [M + H]⁺.

4-(1-Ethyl-3-pyridin-3-ylpyrazol-4-yl)-N-(4-methoxyphenyl)pyrimidin-2-amine (13f)

¹H-NMR (270 MHz, CDCl₃) δ: 8.84–8.85 (1H, m), 8.61 (1H, d, J = 5.1, 1.6 Hz), 8.21 (1H, d, J = 5.4 Hz), 7.98 (1H, s), 7.87–7.92 (1H, m), 7.33–7.39 (2H, m), 7.29–7.32 (1H, m), 6.93 (1H, s), 6.79–6.85 (2H, m), 6.53 (1H, d, J = 4.9 Hz), 4.27 (2H, q, J = 7.3 Hz), 3.80 (3H, s), 1.59 (3H, t, J = 7.3 Hz). LC/MS (ESI): m/z 373 [M + H]⁺.

N-(4-Methoxyphenyl)-4-(1-propan-2-yl-5-pyridin-3-ylpyrazol-4-yl)pyrimidin-2-amine (13g)

¹H-NMR (270 MHz, CDCl₃) δ: 8.74 (1H, dd, J = 4.9, 1.6 Hz), 8.66 (1H, d, J = 2.1 Hz), 8.16 (1H, s), 8.14 (1H, d, J = 5.4 Hz), 7.67–7.71 (1H, m), 7.40–7.44 (1H, m), 7.28–7.31 (2H, m), 6.91 (1H, br s), 6.80–6.83 (2H, m), 6.33 (1H, d, J = 5.4 Hz), 4.24–4.34 (1H, m), 3.79 (3H, s), 1.49 (3H, s), 1.46 (3H, s). LC/MS (ESI): m/z 387 [M + H]⁺.

N-(4-Methoxyphenyl)-4-(1-propan-2-yl-3-pyridin-3-ylpyrazol-4-yl)pyrimidin-2-amine (13h)

¹H-NMR (270 MHz, CDCl₃) δ: 8.85 (1H, d, J = 1.4 Hz), 8.61 (1H, d, J = 3.2 Hz), 8.21 (1H, d, J = 5.4 Hz), 8.00 (1H, s), 7.88–7.92 (1H, m), 7.33–7.39 (2H, d, J = 8.9 Hz), 7.29–7.32 (1H, m), 7.02 (1H, br s), 6.82 (2H, d, J = 8.9 Hz), 6.54 (1H, d, J = 5.1 Hz), 4.54–4.64 (1H, m), 3.80 (3H, s), 1.61 (3H, s), 1.59 (3H, s). LC/MS (ESI): m/z 387 [M + H]⁺.

2-(2-Chloropyrimidin-4-yl)-1-(pyridin-3-yl)ethanone (15)

To a solution of methyl nicotinate 14 (26.6 g, 0.194 mol), 2-chloro-4-methylpyridine (25.0 g, 0.194 mol) in THF (250 mL) at −30°C was added LiHMDS (0.389 mol) dropwise. The mixture was stirred for 40 min at −30°C and then for 2 h at room temperature. The solution was diluted with saturated NH₄Cl aqueous solution, and the aqueous phase was extracted with EtOAc. The extracts were dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by chromatography over silica gel (EtOAc). The resulting solid was washed with MTBE to afford compound 15 (30.6 g, 68% yield) as an orange solid. LC/MS (ESI): m/z 234 [M + H]⁺.

2-Chloro-4-(3-(pyridin-3-yl)-1H-pyrazol-4-yl)pyrimidine (16)

A solution of compound 15 (30.6 g, 0.131 mol), N,N-dimethylformamide dimethyl acetal (18.7 g, 0.157 mol), and AcOH (9.44 g, 0.157 mol) in toluene (459 mL) was heated at 110°C for 1.5 h. N,N-dimethylformamide dimethyl acetal (3.74 g, 0.031 mol) was added to the mixture, and it was stirred at 110°C for 1 h. After cooling to 45°C, the mixture was concentrated in vacuo. To a solution of the residue in EtOH (245 mL), AcOH (9.44 g) and hydrazine monohydrate (6.56 g, 0.131 mol) were added and stirred in an ice bath for 75 min. Then ice water was added, filtered, and concentrated in vacuo to afford 16 (19.4 g, 58% yield). ¹H-NMR (270 MHz, CDCl₃) δ: 8.82 (1H, d, J = 2.3 Hz), 8.72 (1H, dd, J = 4.6, 1.6 Hz), 8.42 (1H, d, J = 5.3 Hz), 8.33 (1H, s), 7.90–7.98 (1H, m), 7.39–7.48 (1H, m), 7.07 (1H, d, J = 5.3 Hz). LC/MS (ESI): m/z 258 [M + H]⁺

2-Chloro-4-(1-ethyl-3-(pyridin-3-yl)-1H-pyrazol-4-yl)pyrimidine (17)

To a solution of compound 16 (9.17 g, 35.6 mmol) and K₂CO₃ (7.38 g, 53.4 mmol) in DMF (73 mL) was added ethyl iodide (6.10 g, 39.1 mmol) at room temperature for 6 h. The mixture was poured into water, and the aqueous phase was extracted with EtOAc. The extracts were dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by column chromatography over silica gel (CH₃Cl–MeOH to CH₃Cl–MeOH). The resulting solid was washed with MTBE to afford compound 17 (4.30 g, 42% yield) as a yellow solid. ¹H-NMR (270 MHz, CDCl₃) δ: 8.79 (1H, dd, J = 2.1, 0.8 Hz), 8.68 (1H, dd, J = 4.9, 1.6 Hz), 8.36 (1H, d, J = 5.3 Hz), 8.22 (1H, s), 7.89 (1H, dt, J = 7.7, 2.1 Hz), 7.26–7.41 (1H, ddd, J = 7.8, 4.9, 0.8 Hz), 6.96 (1H, d, J = 5.2 Hz), 4.28 (2H, q, J = 7.3 Hz), 1.58–1.62 (overlapped by a water signal, 5H, m). LC/MS (ESI): m/z 286 [M + H]⁺.

4-(1-Ethyl-3-(pyridin-3-yl)-1H-pyrazol-4-yl)-2-(4-methoxybenzyl)pyrimidine (18a)

To a solution of compound 17 (150 mg, 0.525 mmol) and Pd(PPh₃)₄ (30 mg, 0.026 mmol) in THF (5 mL) was added 4-(methoxybenzoyl) zinc chloride (2.0 mL) dropwise at room temperature. The mixture was stirred for 8 h at 60°C. The solution was poured into saturated NH₄Cl aqueous solution and Na₂ ethylenediaminetetraacetic acid (EDTA), and the aqueous phase was extracted with EtOAc. The extract was dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by chromatography over silica gel (CH₂Cl₂–MeOH) to afford compound 18a (74.25 mg, 38% yield) as a clear oil. ¹H-NMR (270 MHz, CDCl₃) δ: 8.81–8.82 (1H, m), 8.62–8.65 (1H, m), 8.45 (1H, d, J = 5.4 Hz), 8.06 (1H, s), 7.85–7.89 (1H, m), 7.27–7.34 (2H, m), 7.23–7.25 (2H, m), 6.81–6.91 (3H, m), 4.28 (2H, m), 4.16 (2H, s), 3.78 (3H, s), 1.58 (3H, t), 1.57–1.60 (overlapped by a water signal, 5.8H, m). LC/MS (ESI): m/z 372 [M + H]⁺.

4-(1-Ethyl-3-pyridin-3-ylpyrazol-4-yl)-N-(3-methoxyphenyl)pyrimidin-2-amine (18b)

A solution of compound 17 (50 mg, 0.175 mmol) and m-anisidine (19.7 µL, 21.6 mg, 0.175 mmol) in MeOH (0.5 mL) was heated for 22 h in a sealed tube. After cooling to room temperature, the mixture was concentrated in vacuo. The residue was purified by column chromatography over amino silica gel (CH₂Cl₂–MeOH) to afford compound 18b (35 mg, 53% yield) as a beige solid. ¹H-NMR (270 MHz, CDCl₃) δ: 8.83–8.84 (1H, m), 8.61–8.63 (1H, m), 8.23 (1H, d, J = 5.1 Hz), 8.00 (1H, s), 7.87–7.92 (2H, m), 7.29–7.34 (1H, m), 7.14–7.21 (2H, m), 6.97–7.03 (1H, m), 6.77 (1H, s), 6.55 (1H, d, J = 5.1 Hz), 4.23–4.32 (2H, m), 2.32 (3H, s), 1.59 (3H, t). LC/MS (ESI): m/z 373 [M + H]⁺.

4-(1-Ethyl-3-pyridin-3-ylpyrazol-4-yl)-N-(2-methoxyphenyl)pyrimidin-2-amine (18c)

The title compound 18c was prepared from compound 17 and o-anisidine following a procedure similar to that used to prepare compound 18b. ¹H-NMR (270 MHz, CDCl₃) δ: 8.85–8.86 (1H, m), 8.62 (1H, d, J = 1.6, 4.6 Hz), 8.23–8.28 (2H, m), 8.04 (1H, s), 7.88–7.92 (1H, m), 7.71 (1H, s), 7.30–7.35 (1H, m), 6.87–6.94 (3H, m), 6.56 (1H, d, J = 5.4 Hz), 4.25–4.33 (2H, m), 1.61 (3H, t, J = 7.3 Hz). LC/MS (ESI): m/z 373 [M + H]⁺.

N-[4-(Difluoromethoxy)phenyl]-4-(1-ethyl-3-pyridin-3-ylpyrazol-4-yl)pyrimidin-2-amine (18d)

The title compound 18d was prepared from compound 17 and 4-(difluoromethoxy)aniline following a procedure similar to that used to prepare compound 18b. ¹H-NMR (270 MHz, CDCl₃) δ: 8.83 (1H, d, J = 1.1 Hz), 8.61–8.62 (1H, m), 8.26 (1H, d, J = 2.7 Hz), 7.99 (1H, s), 7.90 (1H, d, J = 4.3 Hz), 7.46 (2H, d, J = 4.6 Hz), 7.29–7.34 (1H, m), 7.01–7.23 (3H, m), 6.31–6.62 (2H, m), 4.27–4.31 (2H, m), 1.57–1.63 (overlapped by a water signal, 15H, t). LC/MS (ESI): m/z 409 [M + H]⁺.

4-(1-Ethyl-3-pyridin-3-ylpyrazol-4-yl)-N-(4-piperidin-4-yloxyphenyl)pyrimidin-2-amine (18e)

Boc-protected 18e, tert-butyl 4-{4-[(4-(1-ethyl-3-(pyridin-3-yl)-1H-pyrazol-4-yl)pyrimidin-2-yl)amino]phenoxy}piperidine-1-carboxylate, was prepared from compound 17 and tert-butyl 4-(4-aminophenoxy)piperidine-1-carboxylate following a procedure similar to that used to prepare compound 18b. To a solution of boc-protected 18e (170 mg, 0.314 mmol) in CH₂Cl₂ was added trifluoroacetic acid (607 µL, 904.4 mg, 7.932 mmol) in an ice bath. The mixture was warmed to room temperature and stirred for 16 h. The solution was added to saturated NaHCO₃ aqueous solution, and the aqueous phase was extracted with CH₂Cl₂. The extract was washed with brine, dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by column chromatography over amino silica gel (CH₂Cl₂–MeOH) to afford compound 18e (104.8 mg, 75% yield) as a yellow solid. ¹H-NMR (270 MHz, CDCl₃) δ: 8.84–8.85 (1H, m), 8.60–8.62 (1H, m), 8.22 (1H, d, J = 5.1 Hz), 7.98 (1H, s), 7.87–7.92 (1H, m), 7.35 (2H, d, J = 8.6 Hz), 7.29–7.32 (1H, m), 7.07 (1H, s), 6.83 (2H, d, J = 8.9 Hz), 6.54 (1H, d, J = 5.1 Hz), 4.23–4.36 (3H, m), 3.12–3.19 (2H, m), 2.71–2.80 (2H, m), 2.20 (1H, br s), 1.99–2.06 (2H, m), 1.66–1.72 (2H, m), 1.59 (3H, t). LC/MS (ESI): m/z 442 [M + H]⁺.

4-(1-Ethyl-3-pyridin-3-ylpyrazol-4-yl)-N-(4-piperidin-4-ylphenyl)pyrimidin-2-amine (18f)

The title compound 18f was prepared from compound 17 and tert-butyl 4-(4-aminophenyl)-1-piperidinecarboxylate following a procedure similar to that used to prepare compound 18e. ¹H-NMR (270 MHz, CDCl₃) δ: 8.77–8.88 (1H, m), 8.54 (1H, dd, J = 4.6, 1.6 Hz), 8.17 (1H, d, J = 4.9 Hz), 7.93 (1H, s), 7.80–7.85 (1H, m), 7.34 (2H, d, J = 8.4 Hz), 7.23–7.27 (1H, m), 7.05 (2H, d, J = 8.6 Hz), 6.97 (1H, s), 6.49 (1H, d, J = 5.4 Hz), 4.21 (2H, q, J = 7.3 Hz), 3.14–3.18 (2H, m), 2.66–2.74 (2H, m), 2.49–2.56 (1H, m), 1.58–1.81 (4H, m), 1.53 (3H, t, J = 7.3 Hz). LC/MS (ESI): m/z 426 [M + H]⁺.

4-(1-Ethyl-3-pyridin-3-ylpyrazol-4-yl)-N-(4-piperazin-1-ylphenyl)pyrimidin-2-amine (18g)

The title compound 18g was prepared from compound 17 and tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate following a procedure similar to that used to prepare compound 18e. ¹H-NMR (270 MHz, CDCl₃) δ: 8.55–8.86 (1H, m), 8.61–8.62 (1H, m), 8.21 (1H, d, J = 5.1 Hz), 7.98 (1H, s), 7.87–7.91 (1H, m), 7.35 (2H, d, J = 8.6 Hz), 7.30–7.31 (1H, m), 6.93 (1H, s), 6.86 (2H, d, J = 8.9 Hz), 6.51 (1H, d, J = 5.4 Hz), 4.23–4.31 (2H, m), 3.05–3.12 (8H, m), 1.58–1.63 (3H, m). LC/MS (ESI): m/z 427 [M + H]⁺.

4-(1-Ethyl-3-pyridin-3-ylpyrazol-4-yl)-N-(4-piperidin-1-ylphenyl)pyrimidin-2-amine (18h)

The title compound 18h was prepared from compound 17 and 4-(1-piperidino)aniline following a procedure similar to that used to prepare compound 18b. ¹H-NMR (270 MHz, CDCl₃) δ: 8.85 (1H, dd, J = 2.2, 1.1 Hz), 8.60–8.62 (1H, m), 8.20 (1H, d, J = 5.4 Hz), 7.98 (1H, s), 7.87–7.91 (1H, m), 7.28–7.35 (3H, m), 6.86–6.91 (3H, m), 6.50 (1H, d, J = 5.1 Hz), 4.23–4.31 (2H, m), 3.07–3.11 (4H, m), 1.68–1.77 (overlapped by a water signal, 12H, m), 1.52–1.62 (5H, m). LC/MS (ESI): m/z 426 [M + H]⁺.

N-[4-(4-Aminopiperidin-1-yl)phenyl]-4-(1-ethyl-3-pyridin-3-ylpyrazol-4-yl)pyrimidin-2-amine (18i)

The title compound 18i was synthesized from compound 17 and tert-butyl (1-(4-aminophenyl)piperidin-4-yl)carbamate following a procedure similar to that used to prepare compound 18e. ¹H-NMR (270 MHz, CDCl₃) δ: 8.83–8.87 (1H, m), 8.60 (1H, dd, J = 4.8, 1.8 Hz), 8.21 (1H, d, J = 5.3 Hz), 7.98 (1H, s), 7.89 (1H, dt, J = 7.9, 2.0 Hz), 7.28–7.45 (3H, m), 6.79–7.03 (3H, m), 6.51 (1H, d, J = 4.9 Hz), 4.27 (2H, q, J = 7.4 Hz), 3.50–3.62 (2H, m), 2.61–2.94 (3H, m), 1.87–1.98 (2H, m), 1.59 (3H, t, J = 7.4 Hz), 1.47–1.55 (2H, m). LC/MS (ESI): m/z 441 [M + H]⁺.

4-(1-Ethyl-3-pyridin-3-ylpyrazol-4-yl)-N-(3-piperazin-1-ylphenyl)pyrimidin-2-amine (18j)

The title compound 18j was synthesized from compound 17 and tert-butyl 4-(3-aminophenyl)piperidine-1-carboxylate following a procedure similar to that used to prepare compound 18e. ¹H-NMR (270 MHz, CDCl₃) δ: 8.85–8.86 (1H, m), 8.60–8.63 (1H, m), 8.23 (1H, d, J = 5.4 Hz), 8.03 (1H, s), 7.88–7.92 (1H, m), 7.27–7.35 (3H, m), 7.16–7.20 (1H, m), 7.01–7.03 (1H, m), 6.54–6.63 (2H, m), 4.24–4.31 (2H, m), 3.14–3.18 (4H, m), 3.01–3.06 (4H, m), 1.57–1.62 (3H, m). LC/MS (ESI): m/z 427 [M + H]⁺.

4-(1-Ethyl-3-pyridin-3-ylpyrazol-4-yl)-N-(6-piperazin-1-ylpyridin-3-yl)pyrimidin-2-amine (18k)

The title compound 18k was synthesized from compound 17 and tert-butyl 4-(5-aminopyridin-2-yl)piperazine-1-carboxylate following a procedure similar to that used to prepare compound 18e. ¹H-NMR (270 MHz, CDCl₃) δ: 8.83–8.84 (1H, m), 8.60–8.61 (1H, m), 8.26 (1H, d, J = 2.7 Hz), 8.20 (1H, d, J = 5.1 Hz), 7.98 (1H, s), 7.85–7.90 (1H, m), 7.71–7.74 (1H, m), 7.28–7.33 (1H, m), 6.87 (1H, s), 6.55–6.60 (2H, m), 4.27 (2H, q, J = 7.3 Hz), 3.65–3.69 (4H, m), 3.17–3.21 (4H, m), 1.57–1.62 (3H, m). LC/MS (ESI): m/z 428 [M + H]⁺.

4-(1-Ethyl-3-pyridin-3-ylpyrazol-4-yl)-N-(5-piperazin-1-ylpyridin-2-yl)pyrimidin-2-amine (18l)

The title compound 18l was prepared from compound 17 and tert-butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate following a procedure similar to that used to prepare compound 18e. ¹H-NMR (270 MHz, CDCl₃) δ: 8.86–8.87 (1H, m), 8.59–8.61 (1H, m), 8.30 (1H, d, J = 5.1 Hz), 7.99 (1H, s), 7.84–7.89 (3H, m), 7.65 (1H, br s), 7.29–7.33 (1H, m), 7.11–7.15 (1H, m), 6.64 (1H, d, J = 5.4 Hz), 4.25–4.33 (2H, m), 3.04–3.11 (8H, m), 1.58–1.64 (overlapped by a water signal, 10H, m). LC/MS (ESI): m/z 428 [M + H]⁺.

N-(3-Chloro-4-piperazin-1-ylphenyl)-4-(1-ethyl-3-pyridin-3-ylpyrazol-4-yl)pyrimidin-2-amine (18m)

The title compound 18m was prepared from compound 17 and tert-butyl 4-(4-amino-2-chlorophenyl)piperazine-1-carboxylate following a procedure similar to that used to prepare compound 18e. ¹H-NMR (270 MHz, CDCl₃) δ: 8.83–8.84 (1H, m), 8.61–8.63 (1H, m), 8.23 (1H, d, J = 5.1 Hz), 8.05 (1H, s), 7.88–7.92 (1H, m), 7.79 (1H, d, J = 2.4 Hz), 7.31–7.36 (1H, m), 7.21–7.31 (1H, m), 6.96 (2H, d, J = 8.6 Hz), 6.56 (1H, d, J = 4.9 Hz), 4.25–4.33 (2H, m), 2.98–3.08 (8H, m), 1.58–1.64 (3H, t). LC/MS (ESI): m/z 461 [M + H]⁺.

4-(1-Ethyl-3-pyridin-3-ylpyrazol-4-yl)-N-(3-methyl-4-piperazin-1-ylphenyl)pyrimidin-2-amine (18n)

The title compound 18n was prepared from compound 17 and tert-butyl 4-(4-amino-2-methylphenyl)piperazine-1-carboxylate following a procedure similar to that used to prepare compound 18e. ¹H-NMR (270 MHz, CDCl₃) δ: 8.84–8.85 (1H, m), 8.60–8.62 (1H, m), 8.22 (1H, d, J = 5.4 Hz), 8.01 (1H, s), 7.88–7.92 (1H, m), 7.29–7.36 (3H, m), 6.82 (1H, d, J = 8.4 Hz), 6.88 (1H, s), 6.52 (1H, d, J = 4.9 Hz), 4.24–4.32 (2H, m), 3.01–3.04 (4H, m), 2.83–2.87 (4H, m), 2.29 (3H, s), 1.57–1.63 (3H, m). LC/MS (ESI): m/z 441 [M + H]⁺.

4-(1-Ethyl-3-pyridin-3-ylpyrazol-4-yl)-N-[4-(4-methylpiperazin-1-yl)phenyl]pyrimidin-2-amine (18o)

The title compound 18o was prepared from compound 17 and 4-(4-methyl-piperazinyl)aniline following a procedure similar to that used to prepare compound 18b. ¹H-NMR (270 MHz, CDCl₃) δ: 8.85–8.86 (1H, m), 8.61 (1H, dd, J = 4.6, 1.6 Hz), 8.21 (1H, d, J = 5.1 Hz), 7.98 (1H, s), 7.91 (1H, m), 7.35 (2H, d, J = 9.2 Hz), 7.30–7.32 (1H, m), 6.85–6.89 (3H, m), 6.52 (1H, d, J = 5.4 Hz), 4.24–4.32 (2H, m), 3.16–3.19 (4H, m), 2.58–2.63 (4H, m), 2.37 (3H, s) 1.56–1.62 (3H, m). LC/MS (ESI): m/z 441 [M + H]⁺.

4-(1-Ethyl-3-pyridin-3-ylpyrazol-4-yl)-N-(4-morpholin-4-ylphenyl)pyrimidin-2-amine (18p)

The title compound 18p was prepared from compound 17 and 4-(4-morpholinyl)aniline following a procedure similar to that used to prepare compound 18b. ¹H-NMR (270 MHz, CDCl₃) δ: 8.85–8.86 (1H, m), 8.61–8.62 (1H, m), 8.22 (1H, d, J = 4.9 Hz), 7.98 (1H, s), 7.86–7.91 (1H, m), 7.35–7.40 (2H, m), 7.28–7.33 (1H, m), 6.91 (1H, s), 6.83–6.87 (2H, m), 6.53 (1H, d, J = 5.1 Hz), 4.24–4.32 (2H, m), 3.85–3.89 (4H, m), 3.09–3.13 (4H, m), 1.57–1.62 (3H, m). LC/MS (ESI): m/z 428 [M + H]⁺

Expression and Purification of ALK2 (R206H)

The ALK2 kinase domain including residues 201 to 499 with the R206H mutation was expressed with a recombinant baculovirus expression system. Sf9 cells were inoculated at 27°C and harvested 48 h after infection. The cells were resuspended in 20 mM Tris–HCl pH 8.0, 500 mM NaCl, 10% glycerol, and 20 mM imidazole and disrupted by sonication. Cell debris and insoluble components were clarified with centrifugation. The supernatant was applied to a HisTrap HP 5 mL column (GE Healthcare, Sweden) and eluted with linear gradient using 20 mM Tris–HCl pH 8.0, and 500 mM NaCl, 10% glycerol, 500 mM imidazole. The His-tag was cleaved by TEV protease and removed on the HisTrap HP 5 mL column. The buffer was exchanged to 20 mM Tris–HCl pH 8.0, 500 mM NaCl, 10% glycerol, and 2 mM dithiothreitol (DTT) for further purification. The protein was passed through a HiTrap Q-XL 5 mL column (GE Healthcare) and then purified on a 16/60 HiLoad Superdex 75 prep grade column (GE Healthcare) equilibrated with 50 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.5, 300 mM NaCl, 50 mM L-arginine, 50 mM L-glutamate, and 10 mM DTT.

Crystallization, Data Collection and Structural Analysis

The best crystals of ALK2 (R206H) were grown in 100 mM HEPES, pH 7.6–8.0, and 1.5–1.6 M ammonium sulfate using the sitting-drop vapor diffusion method at 20°C. Each drop contained 500 nL of protein solution at a concentration of 10 mg/mL and an equal volume of crystallization reagent. Crystals were grown spontaneously, although streak seeding helped to obtain relatively bigger crystals. The crystals were soaked in mother liquors supplemented with 5 mM compound 6a for 1 h. Then the crystals were soaked in solution containing 25% ethylene glycol and 75% of mother liquor for cryoptotection and flash-frozen under a nitrogen stream. Diffraction data were collected at the beamline BL17A of the Photon Factory (Tsukuba, Japan) at 100 K and wavelength of 0.9800 Å and processed with XDS.¹²⁾ For structure determination, the software contained in the CCP4 suite¹³⁾ was used. The structure of ALK2 (R206H) was determined by molecular replacement using MOLREP.¹⁴⁾ The coordinate of the ALK2 (Q207D) mutant in complex with an inhibitor (PDBID: 3MTF) was used as the search model. Structural refinement was carried out using PHENIX,¹⁵⁾ with iterative manual model inspection using COOT.¹⁶⁾ Table 7 shows a summary of the results of data collection and structural refinement. The atomic coordinate and structure factor of ALK2 (R206H) in complex with compound 6a were deposited in the Protein Data Bank with the accession code 6ACR.

Table 7. Crystallographic Data and Refinement Statistics

	ALK2 (R206H)_RK-59638 (6a)
PDB ID	6ACR
Data collection
Beamline	BL-17A, Photon Factory KEK
Wavelength (Å)	0.9800
Space group	P21212
Unit cell parameters (Å, °)	a = 85.1, b = 138.4, c = 59.4, α = β = γ = 90.0
Resolution (Å)*	50.0–2.01 (2.13–2.01)
Reflections*	314633 (49976)
Unique reflections*	90544 (14636)
Redundancy*	3.47 (3.41)
Completeness (%)*	99.6 (99.3)
†Rmerge*	0.125 (0.946)
‡Rr.i.m*	0.147 (1.123)
I/σ(I)*	8.59 (1.24)
Refinement
Resolution (Å)	45.04–2.01
Reflections (total)	90502
Reflections (test)	4490
Rcryst	0.2153
Rfree	0.2568
No. of atoms
Protein	4724
Ligand/ion	82
Waters	97
Average B-factors (protein/ligand) (Å2)	33.83/41.52
R.m.s. deviations
Bond lengths (Å)	0.008
Bond angles (°)	0.863

* Values in parentheses are for the outer shell. ^† R_merge = Σ_hklΣ_i|I_i(hkl) − ⟨I(hkl)⟩|/Σ_hklΣ_iI_i(hkl). ^‡ R_r.i.m = R_meas = Σ_hkl(N/N − 1)^1/2Σ_i|I_i(hkl) − ⟨I(hkl)⟩|Σ_hklΣ_iI_i(hkl).

In Vitro Enzyme Assay

ALK enzyme assays were conducted by Reaction Biology (United States) using the ‘HotSpot’ assay platform and kinase assay protocol. This was a 10-point assay, starting from 100 µM to 0.5 nM, performed in duplicate using the ALK inhibitor LDN-193189 as a control. In a single dose screening, the compound was tested with single dose duplicates at a concentration of 0.3 µM. The reaction was carried out at 10 µM ATP concentration. The mixture was incubated for 2 h.

P-Glycoprotein Substrate Assay Using MDR1-MDCK II Cells

In vitro efflux studies were performed at Sumika Chemical Analysis Service (Osaka, Japan), with 10 µM of compound added to either the apical (A) or basolateral (B) side of monolayers of MDCK II cells and MDCK II cells transfected with human MDR1. Apparent permeability (P_app) in the A to B and B to A directions was determined by the quantity of the compound on the opposite side of the membrane using LC/MS/MS and a fluorescence plate reader. The efflux ratio was used as an index of efflux, whereby the efflux ratio = P_app B to A/P_app A to B.

Microsomal Stability Studies

Microsomal stability studies were performed at Sumika Chemical Analysis Service (Osaka, Japan). One micromolar of compounds with 0.5 mg/mL of liver microsomes were incubated at 37°C for 30 or 60 min in a final volume of 50 µL of 125 mM phosphate buffer solution containing 3.5 µM β-NADPH. Aliquots of incubation samples were protein precipitated with cold methanol containing labetalol (internal standard) and centrifuged, and supernatants were analyzed using LC/MS/MS. All incubations were performed in duplicate, and the percentage of parent compounds remaining at the end of incubation was determined by the LC/MS/MS peak area ratio.

Pharmacokinetics

The pharmacokinetic studies were performed at Nemoto Science (Joso, Japan). The pharmacokinetics of the tested compounds were evaluated in male Sprague–Dawley rats (n = 3 or 2). The compounds were dosed intravenously at 1 mg/kg and orally by gavage at 5 mg/kg or 3 mg/kg. For the intravenous administration study, the compounds were formulated in a mixture of 10% (w/v) DMSO, 40% (w/v) PEG, and 50% (w/v) saline. For the oral administration study, the compounds were formulated as a solution in 0.5% (w/v) methyl cellulose. Blood samples were collected at eight time points (0.1, 0.25, 0.5, 1, 2, 4, 6, 24 h). Plasma proteins were precipitated with acetonitrile, and compounds concentrations were determined using LC/MS/MS. Data were analyzed using program of a moment (Excel) analysis¹⁷⁾ and peak plasma concentration (C_max), oral bioavailability (F), exposure (AUC), half-life (t_1/2), volume of distribution (V_d), and clearance (CL) were calculated. The value of F was calculated from the ratio of the dose normalized AUC, with AUC_0–t for animals dosed intravenously and AUC_0–∞ for animals dosed orally.

Acknowledgments

We thank Dr. H. Kojima (Drug Discovery Initiative, the University of Tokyo) for providing the chemical library. This research was partially supported by the Platform Project for Supporting Drug Discovery and Life Science Research from Japan Agency for Medical Research and Development (AMED) under Grant Number JP17am0101086. This research was also supported by the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics and Structural Life Science) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

Conflict of Interest

Katsuhiko Sekimata, Tomohiro Sato, Tomonori Taguri, Akiko Tanaka, Yoshinobu Hashizume, Kohei Miyazono, and Hiroo Koyama are holders of the following patent: WO 2018/124001 (A1). Katsuhiko Sekimata, Tomohiro Sato, Naoki Sakai, Hisami Watanabe, Chiemi Mishima-Tsumagari, Takehisa Matsumoto, Teruki Honma, Akiko Tanaka, Mikako Shirouzu, Yoshinobu Hashizume, and Hiroo Koyama are employees of RIKEN. Kohei Miyazono is an employee of the University of Tokyo.

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