A fast-track chemistry effort was initiated to evaluate the structure-activity relationship of the 3- and 6-positions of the pyrazolo[1,5-a]pyrimidine scaffold of the known bone morphogenetic protein (BMP) inhibitors. This medicinal chemistry effort led to the identification of a potent and selective compound for ALK2 versus ALK3. The potency contributions of several 3-position substituents were evaluated with subtle structural changes leading to significant changes in potency. From these studies, a novel 5-quinoline molecule was identified and designated an MLPCN probe molecule, ML347, which shows >300-fold selectivity for ALK2 and presents the community with a selective molecular probe for further biological evaluation.

Probe Structure & Characteristics

1. Recommendations for Scientific Use of the Probe

ML347 (CID 44577753) is a potent inhibitor when tested in a cell-based BMP-responsive luciferase reporter assay (BMP4) (IC₅₀ = 152 nM). In order to further explore the selectivity of ML347 against ALK1, ALK2, ALK3 and ALK6 we further evaluated the compound against a panel of kinases and were able to show that ML347 is selective for ALK2 (32 nM) versus ALK3 (10,800 nM) and ALK6 (9830 nM). This selectivity for ALK2 over ALK3 is unprecedented in the primary literature and should make ML347 an invaluable research tool in order to interrogate the biology of BMP signaling at the subtype resolution. ML347 was also evaluated in a Tier 1 DMPK panel and it was shown to be highly cleared in human and mouse liver microsomes and moderate/highly cleared in rat. The compound was shown to have ∼1% free fraction in plasma in all three species tested. Thus, the compound can be used for both in vitro and in vivo studies provided the route of administration is non-oral (IP, for example).

2. Materials and Methods

BMP-responsive luciferase reporter assays. BMP-responsive C2C12BRA cells, stably transformed with the Id1 promoter-firefly luciferase reporter; kind gift of D, Rifkin, NYU Medical Center) were seeded in 96-well plates, and incubated overnight with the compounds and BMP4 (50 ng/mL). The cells were then lysed, and cell extracts were then subjected to the firefly luciferase assay using Steady-Glo luciferase assay kit (Promega). The results were normalized to cell titers, as measured using Cell Titer-Glo luminescence assay (Promega).

Kinase assay. All kinase assays were conducted by Reaction Biology Corp (Malvern, PA), as previously reported. In brief, compounds were tested at 10 concentrations by 3-fold serial dilutions starting at 100 mM. In vitro kinase reactions were carried out in the presence of 10 μM (33P)gATP. Eleven human kinases tested were the BMP type-I receptors ALK-1/ACVRL1, ALK2/ACRV1, ALK3/BMPR1A and ALK6/BMPR1B, the TGFb type-I receptors ALK4/ACVR1B and ALK5/TGFbR1, the BMP type-II receptor (BMPR2), the TGFb type-II receptor (TGFbR2), VEGF type-II receptor (KDR/VEGFR2), AMP-activated protein kinase (AMPK-A1/B1/G1) and the human platelet-derived growth factor receptor-β (PDGFRβ).

DMPK Methods. In vitro: The metabolism of ML347 was investigated in human, rat and mouse hepatic microsomes (BD Biosciences, Billerica, MA) using substrate depletion methodology (% test article remaining). A potassium phosphate-buffered reaction mixture (0.1 M, pH 7.4) of test article (1 μM) and microsomes (0.5 mg/mL) was pre-incubated (5 min) at 37°C prior to the addition of NADPH (1 mM). The incubations, performed in 96-well plates, were continued at 37 °C under ambient oxygenation and aliquots (80 μL) were removed at selected time intervals (0, 3, 7, 15, 25 and 45 min). Protein was precipitated by the addition of chilled acetonitrile (160 μL), containing glyburide as an internal standard (50 ng/mL), and centrifuged at 3000 rpm (4°C) for 10 min. Resulting supernatants were transferred to new 96-well plates in preparation for LC/MS/MS analysis. The in vitro half-life (t_1/2, min, Eq. 1), intrinsic clearance (CL_int, mL/min/kg, Eq. 2) and subsequent predicted hepatic clearance (CL_hep, mL/min/kg, Eq. 3) were determined employing the following equations:

Plasma Protein Binding. Protein binding of ML347 was determined in human, rat and mouse plasma via equilibrium dialysis employing Single-Use RED Plates with inserts (ThermoFisher Scientific, Rochester, NY). Briefly plasma (220 μL) was added to the 96 well plate containing test article (5 μL) and mixed thoroughly. Subsequently, 200 μL of the plasma-test article mixture was transferred to the cis chamber (red) of the RED plate, with an accompanying 350 μL of phosphate buffer (25 mM, pH 7.4) in the trans chamber. The RED plate was sealed and incubated 4 h at 37 °C with shaking. At completion, 50 μL aliquots from each chamber were diluted 1:1 (50 μL) with either plasma (cis) or buffer (trans) and transferred to a new 96 well plate, at which time ice-cold acetonitrile (2 volumes) was added to extract the matrices. The plate was centrifuged (3000 rpm, 10 min) and supernatants transferred to a new 96 well plate. The sealed plate was stored at −20 °C until LC/MS/MS analysis.

Liquid Chromatography/Mass Spectrometry Analysis. In vitro experiments. ML347 was analyzed via electrospray ionization (ESI) on an AB Sciex API-4000 (Foster City, CA) triple-quadrupole instrument that was coupled with Shimadzu LC-10AD pumps (Columbia, MD) and a Leap Technologies CTC PAL auto-sampler (Carrboro, NC). Analytes were separated by gradient elution using a Fortis C18 2.1 × 50 mm, 3.5 μm column (Fortis Technologies Ltd, Cheshire, UK) thermostated at 40 °C. HPLC mobile phase A was 0.1% NH₄OH (pH unadjusted), mobile phase B was acetonitrile. The gradient started at 30% B after a 0.2 min hold and was linearly increased to 90% B over 0.8 min; held at 90% B for 0.5 min and returned to 30% B in 0.1 min followed by a re-equilibration (0.9 min). The total run time was 2.5 min and the HPLC flow rate was 0.5 mL/min. The source temperature was set at 500°C and mass spectral analyses were performed using multiple reaction monitoring (MRM) utilizing a Turbo-Ionspray® source in positive ionization mode (5.0 kV spray voltage). LC/MS/MS analysis was performed employing a TSQ Quantum^ULTRA that was coupled to a ThermoSurveyor LC system (Thermoelectron Corp., San Jose, CA) and a Leap Technologies CTC PAL auto-sampler (Carrboro, NC). Chromatographic separation of analytes was achieved with an Acquity BEH C18 2.1 × 50 mm, 1.7 μm column (Waters, Taunton, MA).

2.2. Probe Chemical Characterization

Probe compound ML347 (CID 44577753, SID 136349469) was prepared according to the above scheme and had the following characterization. 5-(6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinolone. LCMS: R_T = 0.586 min, >98% @ 215 and 254 nM, m/z = 352.7 [M + H]⁺. ¹H NMR (300 MHz, d-DMSO): δ 9.52 (s, 1H), 8.99-8.94 (m, 2H), 8.57 (s, 1H), 8.41 (d, J = 8.0 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.90-7.80 (m, 4H), 7.53 (dd, J = 8.1, 4.0 Hz, 1H), 7.11 (d, J = 8.5 Hz, 2H), 3.92 (s, 3H). HRMS, calc'd for C₂₂H₁₇N₄O [M + H]⁺, 353.1402; found 353.1403.

Solubility. Solubility for ML347 in PBS was determined to be 4 μM, which is ∼26-fold higher than the cellular IC₅₀ for BMP inhibition.

Stability. Stability was determined for ML337 at 23 °C in PBS (no antioxidants or other protectorants and DMSO concentration below 0.1%). After 48 hours, ∼29% of the initial concentration of ML347 remained in solution. The loss is most likely due to limited solubility in buffer.

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	Percent Remaining (%)
Compound	0 min	15 min	30 min	90 min	24 Hour	48 Hour
ML347, CID 44577753	100 ± 0.0	94.0 ± 1.2	93.3 ± 2.7	91.0 ± 1.7	31.3 ± 1.0	28.6 ± 0.5

Compounds added to the SMR collection (MLS#s): MLS004797119 (ML347, CID 44577753, 25 mg); MLS004797120 (CID 60182382, 8.5 mg); MLS004797121 (CID 60182399, 6.3 mg); MLS004797122 (CID 60182380, 6.05 mg); MLS004797123 (CID 60182372, 6.26 mg); MLS004797124 (CID 60182397, 7.4 mg)

2.3. Probe Preparation

Probe compound ML347 (CID 44577753, SID 136349469) was prepared according to the above scheme and had the following characterization. 5-(6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline.

6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidine (3). Equally divided amongst two 20 mL μwave vials, to a solution of 1H-pyrazol-3-amine, 2, (1.17 g, 14.0 mmol, 1.0 eq) in 5% AcOH/EtOH (12 mL/ vial) was added 2-(4-methoxyphenyl)malonaldehyde, 1, (2.5 g, 14 mmol, 1.0 eq). The reaction was heated to 170 °C for 20 min under microwave irradiation. Upon cooling, a precipitate formed. Filtration of the reaction provided an off white solid (2.9 g, 92% yield). LCMS: R_T = 0.620 min, >98% @ 215 and 254 nM, m/z = 226.1 [M + H]⁺.

3-iodo-6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidine (4). To a solution of 6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidine (2.90 g, 12.9 mmol, 1.0 eq) in DMF (30 mL) at rt was added N-iodosuccinimide (3.05 g, 13.5 mmol, 1.05 eq). After 16 h, the brown slurry was diluted with H₂O (100 mL) and Brine (15 mL). The mixture was extracted with EtOAc (100 mL). The aqueous layer was re-extracted with EtOAc (100 mL) and the collected organic layers were washed with H₂O (2 × 30 mL), 10% sodium thiosulfate (20 mL), Brine (30 mL) and dried (MgSO₄). After filtration, the solution was concentrated to afford 3-iodo-6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidine, 4, as an off-white solid. The material was taken through without further purification. LCMS: R_T = 0.781 min, >98% @ 215 and 254 nM, m/z = 352.0 [M + H]⁺.

5-(6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline (ML347). In a μwave vial, 3-iodo-6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidine, 4, (1.0 g, 2.9 mmol, 1.0 eq), quinolin-5-ylboronic acid (0.54 g, 3.1 mmol, 1.1 eq), and Pd(dppf)Cl₂•CH₂Cl₂ (116 mg, 0.143 mmol, 0.05 eq) were added. The solid mixture was evacuated under vacuo and purged with Argon (3x). To the mixture was added 1,4-dioxane (13 mL), followed by a solution of K₃PO₄ (1.2 g, 5.7 mmol, 2.0 eq) in H₂O (5 mL). The reaction was heated to 120 °C for 30 min under microwave irradiation. The reaction was added to EtOAc:H₂O (1:1, 200 mL). The organic layer was separated, washed with H₂O (3 × 25 mL), Brine (25 mL), dried (MgSO4), filtered and concentrated. The material was purified by reverse-phase HPLC (20-55% acetonitrile: H₂O w/0.1% TFA) to provide 5-(6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline (ML347) (0.47 g, 47% yield). LCMS: R_T = 0.586 min, >98% @ 215 and 254 nM, m/z = 352.7 [M + H]⁺. ¹H NMR (300 MHz, DMSO-d₆): δ 9.52 (s, 1H), 8.99-8.94 (m, 2H), 8.57 (s, 1H), 8.41 (d, J = 8.0 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.90-7.80 (m, 4H), 7.53 (dd, J = 8.1, 4.0 Hz, 1H), 7.11 (d, J = 8.5 Hz, 2H), 3.92 (s, 3H). HRMS, calc'd for C₂₂H₁₇N₄O [M + H]⁺, 353.1402; found 353.1403.

3. Results

3.1. Dose Response Curves for Probe

Figure 3In vitro molecular pharmacology characterization of ML347

Concentration-response curves of for ML347 in ALK2 kinase assay (A) and ALK3 kinase assay (B).

ML347 is an extraordinarily selective compound which exhibits >3,000 fold selectivity for ALK2 over ALK4, ALK5, BMPR2, TGFBR2, AMPK and KDR/VERGR2, and over 300 fold selectivity for ALK2 over ALK2 and ALK6.

4. Discussion

5. References

1.

Chaikuad A, Alfano I, Shrestha B, Muniz JRC, Petrie K, Fedorov O, Phillips C, Bishop S, Mahajan P, Pike ACW, von Delft F, Arrowsmith CH, Edwards AM, Weigelt J, Bountra C, Knapp S, Bullock A. Structure of the bone morphogenetic protein receptor ALK2 and implications for fibrodysplasia ossificans progressiva. J. Biol. Chem. 2012;287:36990–36998. [PMC free article: PMC3481300] [PubMed: 22977237]