General procedure for fluorination-reductive amination protocol
A 30% solution of either (R)-14 or (S)-14 (30%) catalyst in THF (18 mL) and i-PrOH (1.8 mL) was cooled to -10°C. The aldehyde (1.0 equiv) was added followed by a slow addition of N-fluorobenzenesulfonimide (1.0 equiv, 0.60 mg, 2.0 mmol) in THF over 30 min and the reaction stirred for 12 h at – 10°C and it was then cooled to -78°C, quenched with Et2O (15 mL) and filtered through a pad of silica gel, eluting with Et2O. Dimethyl sulfide (1 mL) was added and the resulting mixture was washed with saturated NaHCO3 (3 x 30 mL), brine (30 mL) and dried over MgSO4, filtered and concentrated. The resulting fluorinated aldehyde was dissolved in DCE (22 mL), followed by addition of the amine (1.1 equiv), and then NaBH(OAc)3 (2.0 equiv, 848 mg, 4.0 mmol) was added and the reaction stirred for 18 h at RT. The reaction was quenched with saturated NaHCO3 (20 mL), and the product extracted into EtOAc (3 x 20 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo.
( S )-4-Amino-3-fluorobutanoic acid (3-F-GABA) (8)
Benzyl (S)-4-(dibenzylamino)-3-fluorobutanoate 13 (1.0 equiv, 65 mg, 0.16 mmol) and Pd(OH)2/C 20 wt.% (2.8%, 20mg, 0.005 mmol) were added to MeOH (5 mL) under argon. The reaction was stirred for 46 h at RT under a H2 atmosphere (1 atm). The catalyst was filtered through a pad of Celite® and the eluant concentrated in vacuo. The residue dissolved in water (5 mL) and washed with Et2O (5 mL) and the aqueous was submitted to reverse phase column chromatography on supported silica (C18) eluting with water. The desired product 8 was obtained as a white solid with (90%, 18 mg, ee 78%). 1H NMR (400 MHz, D2O) δ 5.05–5.30 (m, 1H, CHF), 3.18–3.40 (m, 2H, CH2), 2.47–2.72 (m, 2H, CH2). 13C NMR (101 MHz, D2O) δ 129.7 (CO2), 129.5(CO2), 88.9 (CHF), 87.3 (d, J = 171.5 Hz, CHF), 51.3 (d, J = 20.6 Hz, N-CH2), 47.6 (d, J = 22.4 Hz, CH2-CO2). 19F NMR (376 MHz, D2O) δ -185.6.
( F S,R)-F-Cinacalcet (20)
Prepared with (S)-14 according to the general procedure from 3-(trifluoromethyl)benzenepropanal 19 (1.0 equiv, 340 µL, 2.0 mmol) and (R)-(1-naphthyl)ethylamine (1.1 equiv, 350 µL, 2.2 mmol). The product was purified by chromatography eluting with hexane/EtOAc. Cinacalcet 20 was obtained as a yellow oil (28%, 199 mg, de 98%). [α]20D -47.0 (c 0.54, CHCl3). 1H NMR (400 MHz, MeOD) δ 7.22–8.15 (m, 10H, Ph), 4.69–4.87 (m, 1H, CHF), 4.66 (q, J = 6.7 Hz, 1H, N-CH), 2.62–2.96 (m, 4H, CH2), 1.47 (d, J = 6.7 Hz, 3H, CH3). 13C NMR (101 MHz, MeOD) δ 132.9 (CArom), 129.6 (CArom), 129.0 (CArom), 127.2 (CArom), 126.2 (CArom), 125.2 (CArom), 121.5 (CArom), 89.8 (d, J = 173.0 Hz, CHF), 53.25 (CH-N), 48.9 (d, J = 20.6 Hz, CH2), 37.7 (d, J = 19.2 Hz, CH2), 18.91 (CH3). 19F NMR (377 MHz, MeOD) δ -63.9 (CF3), -186.2 (CHF). ESI-MS calculated for C22H21NF [M + H+] = 376.1688, observed [M + H+] = 376.1678.
( F R,R)-F-Cinacalcet (21)
Prepared with (R)-14 according to the general procedure from 3-(trifluoromethyl)benzenepropanal 19 (1.0 equiv, 340 µL, 2.0 mmol) and (R)-(1-naphthyl)ethylamine (1.1 equiv, 350 µL, 2.2 mmol). The product was purified by chromatography eluting with hexane/EtOAc. Cinacalcet 21 was obtained as a yellow oil with (50%, 355 mg, de 98%). [α]20D -15.4 (c 0.37, CHCl3). 1H NMR (400 MHz, MeOD) δ 7.82–7.88 (m, 1H, Ph), 7.74 (d, J = 8.2 Hz, 1H, Ph), 7.62 (d, J = 7.2 Hz, 1H, Ph), 7.28–7.53 (m, 8H, Ph), 8.17 (d, J = 8.4 Hz, 1H, Ph), 4.64–4.86 (m, 2H, CHF, N-CH), 2.57–3.01 (m, 4H, CH2), 1.47 (d, J = 6.6 Hz, 3H, CH3). 13C NMR (101 MHz, MeOD) δ 132.9 (CArom), 129.63, 129.0 (CArom), 128.9 (CArom), 127.1 (CArom), 126.2 (CArom), 125.2 (CArom), 123.5 (CArom), 121.7 (CArom), 89.9 (d, J = 176.6 Hz, CHF), 53.31 (CH-N), 49.0 (d, J = 18.1 Hz, CH2), 37.7 (d, J = 18.6 Hz, CH2), 18.58 (CH3). 19F NMR (376 MHz, MeOD) δ -64.1, -185.9. ESI-MS calculated for C22H21NF [M + H+] = 376.1688, observed [M + H+] = 376.1679.
( F S,R)-F-Fendiline (27)
Prepared with (S)-14 according to the general procedure from 3,3-diphenylpropanal 26 (1.0 equiv, 421 mg, 2.0 mmol) and (R)-α-methylbenzylamine (1.1 equiv, 280 µL, 2.2 mmol). The product was purified by chromatography eluting with hexane/EtOAc. Fendiline 27 was obtained as a yellow oil (56%, 373 mg, de 80%). [α]20D +17.2 (c 0.57, CHCl3). 1H NMR (400 MHz, MeOD) δ 7.46–7.35 (m, 5H, Ph), 7.34–7.17 (m, 10H, Ph), 5.68–5.49 (m, 1H, CHF), 4.41 (q, J = 6.9 Hz, 1H, CH), 4.20 (dd, J = 16.9, 8.2 Hz, 1H, N-CH), 3.02–2.92 (m, 2H, CH2), 1.63 (d, J = 6.9 Hz, 3H, CH3). 13C NMR (101 MHz, MeOD) δ 129.3 (CArom), 129.2 (CArom), 128.7 (CArom), 128.2 (CArom), 127.8 (CArom), 127.1 (CArom), 126.8 (CArom), 90.2 (d, J = 183.5 Hz, CHF), 58.1 (CH-N), 53.5 (d, J = 19.5 Hz, CH2), 18.7 (CH3). 19F NMR (376 MHz, MeOD) δ -189.9. ESI-MS calculated for C23H24NF [M + H+] = 334.1971, observed [M + H+] = 334.1956.
( F R,R)-F-Fendiline (28)
Prepared with (R)-14 according to the general procedure from 3,3-diphenylpropanal 26 (1.0 equiv, 421 mg, 2.0 mmol), and (R)-α-methylbenzylamine (1.1 equiv, 280 µL, 2.2 mmol). The product was purified by chromatography eluting with hexane/EtOAc. Fendiline 28 was obtained as a yellow oil (50%, 333 mg, de 90%). [α]20D +20.7 (c 0.45, CHCl3). 1H NMR (400 MHz, MeOD) δ 7.52–7.40 (m, 7H, Ph), 7.38–7.18 (m, 9H, Ph), 5.51 (dt, J = 50.0, 7.9 Hz, 1H, CHF), 4.44 (m, 1H, CH), 4.24 (dd, J = 18.4, 7.8 Hz, 1H, N-CH), 3.26 (dt, J = 13.8, 10.8 Hz, 1H, CH2), 2.92 (ddd, J = 35.8, 13.8, 1.9 Hz, 1H, CH2), 1.67 (t, J = 7.1 Hz, 3H, CH3). 13C NMR (101 MHz, MeOD) δ 129.4 (CArom), 129.2 (CArom), 128.6 (CArom), 128.3 (CArom), 128.3 (CArom), 127.7 (CArom), 127.3 (CArom), 127.1 (CArom), 126.9 (CArom), 90.7(d, J = 179.1 Hz, CHF), 58.8 (CH-N), 53.5 (d, J = 21.1 Hz, CH2), 17.98 (CH3). 19F NMR (377 MHz, MeOD) δ -189.8. ESI-MS calculated for C23H24NF [MH+] = 334.1971, observed [MH+] = 334.1957.
( F S,R)-F-Tecalcet (24)
Prepared with (S)-14 according to the general procedure from 3-(2-chlorophenyl)propanal 23 (1.0 equiv, 337 mg, 2.0 mmol) and (R)-3-methoxy-α-methylbenzylamine (1.1 equiv, 365 µL, 2.2 mmol). The product was purified by chromatography eluting with hexane/EtOAc and was obtained as a yellow (54%, 347 mg, de 86%). [α]20D +22.6 (c 0.67, CHCl3). 1H NMR (400 MHz, MeOD) δ 7.44–7.34 (m, 2H, Ph), 7.33–7.24 (m, 3H, Ph), 7.06–6.97 (m, 3H, Ph), 5.23–4.99 (m, 1H, CHF), 4.35 (q, J = 6.5 Hz, 1H, CH-N), 3.85 (s, 3H, O-CH3), 3.21–2.93 (m, 4H, CH2), 1.65 (dd, J = 6.8, 2.7 Hz, 3H, CH3). 13C NMR (101 MHz, MeOD) δ 131.7 (CArom), 130.2 (CArom), 129.3 (CArom), 128.7 (CArom), 126.9 (CArom), 119.1 (CArom), 114.3 (CArom), 112.8 (CArom), 88.8 (d, J = 174.7 Hz, CHF), 58.31 (CH-N), 54.44 (CH3-O), 48.8 (d, J = 17.8 Hz, CH2), 36.0 (d, J = 19.9 Hz, CH2), 19.06 (CH3). 19F NMR (377 MHz, MeOD) δ -186.8. ESI-MS calculated for C18H21ONClF [MH+] = 322.1374, observed [MH+] = 322.1365.
( F R,R)-F-Tecalcet (25)
Prepared with (R)-14 according to the general procedure from 3-(2-chlorophenyl)propanal 23 (1.0 equiv, 337 mg, 2.0 mmol) and (R)-3-methoxy-α-methylbenzylamine (1.1 equiv, 365 µL, 2.2 mmol). The product was purified by chromatography eluting with hexane/EtOAc and was obtained as a yellow oil (56%, 360 mg, de 86%). [α]20D -27.4 (c 0.50, CHCl3). 1H NMR (400 MHz, MeOD) δ 7.44–7.34 (m, 3H), 7.34–7.15 (m, 6H), 7.07–7.00 (m, 3H), 5.07–4.87 (m, 1H, CHF), 3.84 (s, 3H, O-CH3), 3.61 (t, J = 6.5 Hz, 1H, CH-N), 3.23–2.95 (m, 2H, CH2), 2.90–2.78 (m, 2H, CH2), 1.70 (d, J = 6.8 Hz, 3H, CH3). 13C NMR (101 MHz, MeOD) δ 131.7 (CArom), 130.4 (CArom), 129.3 (CArom), 129.0 (CArom), 128.8 (CArom), 127.4 (CArom), 127.2 (CArom), 126.9 (CArom), 126.7 (CArom), 119.2 (CArom), 114.7 (CArom), 113.1 (CArom), 88.8 (d, J = 176.9 Hz, CHF), 58.96 (N-CH), 54.48 (O-CH3), 48.7 (d, J = 12.2 Hz, CH2), 35.8 (d, J = 16.9 Hz, CH2), 32.3, 29.5, 17.9 (CH3). 19F NMR (377 MHz, MeOD) δ -186.4. ESI-MS calculated for C18H21ONClF [M + H+] = 322.1374, observed [M + H+] = 322.1366.
( F S,R )-F-NPS R-467 (30)
Prepared with (S)-14 according to the general procedure from 3-phenylpropanal 29 (1.0 equiv, 266 µL, 2.0 mmol), and (R)-3-methoxy-α-methylbenzylamine (1.1 equiv, 365 µL, 2.2 mmol). The product was purified by chromatography eluting with hexane/EtOAc and was obtained as a yellow oil (47%, 270 mg, de 70%). [α]20D +28.9 (c 0.23, CHCl3). 1H NMR (400 MHz, MeOD) δ 7.39 (t, J = 7.9 Hz, 1H, Ph), 7.34–7.16 (m, 4H, Ph), 7.09–6.97 (m, 3H, Ph), 5.08 (d, J = 50.9 Hz, 1H, CHF), 4.49–4.34 (m, 1H, N-CH), 3.84 (d, J = 1.7 Hz, 3H, O-CH3), 3.28–2.87 (m, 4H, CH2), 1.76–1.60 (m, 3H, CH3). 13C NMR (101 MHz, MeOD) δ 130.4 (CArom), 129.0 (CArom), 128.3 (CArom), 126.8 (CArom), 119.1 (CArom), 114.7 (CArom), 112.9 (CArom), 89.7 (d, J = 168.5 Hz, CHF), 58.5 (N-CH), 54.5 (O-CH3), 48.5 (d, J = 21.2 Hz, CH2), 38.1 (d, J = 17.6 Hz, CH2), 18.4 (CH3). 19F NMR (377 MHz, MeOD) δ -186.3. ESI-MS calculated for C18H22ONF [M + H+] = 288.1764, observed [M + H+] = 288.1757.
( F R,R )-F-NPS R-467 (31)
Prepared with (R)-14 according to the general procedure from 3-phenylpropanal 29 (1.0 equiv, 266 µL, 2.0 mmol) and (R)-3-methoxy-α-methylbenzylamine (1.1 equiv, 365 µL, 2.2 mmol). The product was purified by chromatography eluting with hexane/EtOAc and was obtained as a yellow oil (62%, 356 mg, de 68%). [α]20D +42.6 (c 0.96, CHCl3). 1H NMR (400 MHz, MeOD) δ 7.40–7.34 (m, 1H, Ph), 7.33–7.18 (m, 6H, Ph), 7.04–6.94 (m, 3H, Ph), 4.87–4.77 (m, 1H, CHF), 4.27 (q, J = 6.8 Hz, 1H, CH-N), 3.83 (d, J = 2.2 Hz, 3H, O-CH3), 3.15 (td, J = 13.3, 9.7 Hz, 1H, CH2), 3.04–2.85 (m, 3H, CH2), 1.62 (d, J = 6.8 Hz, 3H, CH3). 13C NMR (101 MHz, MeOD) δ 130.1 (CArom), 129.0 1 (CArom), 128.2 1 (CArom), 126.7 1 (CArom), 119.1 1 (CArom), 114.2 1 (CArom), 112.8 1 (CArom), 90.8 (d, J = 174.5 Hz, CHF), 58.74 (CH-N), 54.42 (O-CH3), 49.0 (d, J = 20.0 Hz, CH2), 38.4 (d, J = 20.0 Hz, CH2), 18.85 (CH3). 19F NMR (377 MHz, MeOD) δ -185.9. ESI-MS calculated for C18H22ONF [M + H+] = 288.1764, observed [M + H+] = 288.1756.
Generation of DNA and FlpIn HEK TREx cells which are stable in expressing c-myc-tagged wild type CaSR in pcDNA5/frt/TO has been described previously [46]. FlpIn HEK TREx CaSR cells were maintained in DMEM cell culture medium, containing 5% FBS, 200µg/mL hygromycin B and 5 µg/mL blasticidin S HCl.
FlpIn HEK293 TRex-expressing CaR cells were seeded in clear 96-well plates coated with poly-D-lysine (50 µg mL− 1, 80,000 cells/well) and incubated overnight in the presence of tetracycline (100 ng mL− 1) to induce CaR expression. The following day, cells were washed with the assay buffer (150 mM NaCl, 2.6 mM KCl, 1.18 mM MgCl2, 10 mM D-Glucose, 10 mM HEPES, 0.1 mM CaCl2, 0.5% BSA and 4 mM probenecid at pH 7.4) and loaded with Fluo-8 AM (1 µM in assay buffer) for 1 hr at 37 oC. Cells were washed once with assay buffer and fresh buffer was added to wells. Extracellular calcium (Ca2 + o) and the calcimimetic compounds were co-added to wells and measurements of Ca2 + i elevations were performed at 37°C using a Flexstation (Molecular Devices). Fluorescence was detected for 60 sec at 490 nm excitation and 520 nm emission and peak fluorescence was used as a readout of the agonist response. Relative peak fluorescence units were normalised to the fluorescence stimulated by 1 µM ionomycin to account for differences in cell number and loading efficiency.