Synthesis of the calcilytic ligand NPS 2143

Summary (R)-3 (NPS 2143) is a negative allosteric modulator of the human calcium-sensing receptor (CaSR) and as such represents an important pharmacological tool compound for studying the CaSR. Herein, we disclose for the first time a complete experimental description, detailed characterisation and assessment of enantiomeric purity for (R)-3. An efficient, reproducible and scalable synthesis of (R)-3 that requires a minimum of chromatographic purification steps is presented. (R)-3 was obtained in excellent optical purity (er > 99:1) as demonstrated by chiral HPLC and the pharmacological profile for (R)-3 is in full accordance with that reported in the literature.


Experimental section General information
All anhydrous reactions were carried out in oven or flame-dried glassware, under nitrogen. Solvents were of chromatography grade and dried using an SG Water solvent purification system (CH 2 Cl 2 , THF) or with 3 Å molecular sieves (Et 2 O, MeCN, EtOH, DMSO, acetone and toluene). Commercially acquired chemicals were used without further purification. Aqueous sulfate buffer (pH ≈ 2) was prepared by dissolving 1.5 mol Na 2 SO 4 in 0.5 mol H 2 SO 4 and adding H 2 O to a total volume of 2000 mL. Flash column chromatography and dry column vacuum chromatography (DCVC) [1] were carried out according to standard procedures using silica gel 60 (40-63 µm and 15-40 µm mesh, respectively).

Thin-layer chromatography (TLC)
TLC was carried out on Merck precoated silica gel 60 F 254 plates and visualised using UV (254 nm), I 2 /SiO 2 , KMnO 4 or ninhydrin stain. Retention factor, R f , values were rounded to the nearest 0.05.

Melting point (mp)
Melting points were recorded on a Stanford Research System (SRS) OptiMelt capillary melting-point apparatus and are uncorrected.

Fourier transform infrared spectroscopy (FTIR)
FT-IR was recorded neat on a Perkin-Elmer Spectrum One IR spectrometer with a universal ATR accessory, and the signals are reported in wavenumbers S3 (cm −1 ). Solid samples were either loaded directly or dissolved in CH 2 Cl 2 and loaded, then allowing the solvent to evaporate before recording the spectrum.

Optical rotation ( )
Optical rotation values were measured at 27 °C on a Perkin-Elmer 241 polarimeter using a sodium vapor lamp (589 nm) and are reported in units of 10 −1 × deg × dm 2 × g −1 . Anhydrous HPLC grade MeOH or CHCl 3 were used as solvent, and the concentration is reported as c (gram/100 mL).

Nuclear magnetic resonance spectroscopy (NMR)
NMR spectra were recorded on a 400 MHz Bruker Avance instrument.

Low-resolution mass spectrometry (LRMS)
LRMS were recorded on a Bruker Esquire 3000 plus instrument connected to an Agilent 1200 HPLC system, using an electrospray ionization (ESI) mass detector.

High-resolution mass spectrometry (HRMS)
High-resolution mass spectra were recorded on a Micromass Q-TOF 1.5, UB137.

yl)amino)propoxy)benzonitrile hydrochloride ((R)-3)
According to the procedure by Marquis et al. [2] epoxide (R)-16 (0.24 g, 1.13 mmol) and amine 6 (0.23 g, 1.13 mmol) were dissolved in anhydrous EtOH (6.5 mL) and stirred under reflux for 20 h. The mixture was allowed to cool to ambient temperature and then concentrated in vacuo to afford the crude product as a colorless foam. Purification by column chromatography
All analytical data for rac-3 were identical to those reported above for (R)-3.
The slurry was cooled in an ice/water bath and 4 M aqueous HCl (25 mL) was added dropwise. After 15 min the cooling bath was removed and the mixture was stirred vigorously overnight. The reaction mixture was transferred to a 1 L beaker with ethanol (70 mL) and cooled in an ice-water bath. Saturated aqueous NaHCO 3 was added slowly to pH 8 (≈100 mL) and the cooling bath was removed. The slurry was filtered on filter paper and washed with ethanol

IP-One Assay
For the pharmacological studies a HEK293 cell line stably transfected with rat CaSR (HEK293-CaSR) was used, which had previously been characterised [5].