General chemical synthesis details
All reactions were performed under an atmosphere of nitrogen or argon unless otherwise specified. Anhydrous methanol (MeOH), acetonitrile, and dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) were used as received. Anhydrous tetrahydrofuran (THF) and toluene were obtained from a PureSolv MD 7 solvent purification system (Innovative Technology, Inc., Oldham, UK). Other commercially available chemicals (Sigma-Aldrich) were used as received. Analytical thin-layer chromatography was performed using Merck aluminum-backed silica gel 60 F254 (0.2 mm) plates (Merck, Darmstadt, Germany), which were visualised using shortwave (254 nm) UV fluorescence. Flash chromatography was performed using Merck Kieselgel 60 (230–400 mesh) silica gel, with the eluent mixture reported as the volume/volume ratio (%). Melting point ranges (m.p.) were measured in open capillaries using a Stanford Research Systems (SRS, Sunnyvale, CA, USA) MPA160 melting point apparatus with a ramp rate of 0.5–2.0 °C/min. Nuclear magnetic resonance (NMR) spectra were recorded at 300 K using either a Bruker AVANCE DRX 300 (300 MHz), AVANCE DRX400 (400.1 MHz), or AVANCE III 500 Ascend (500.1 MHz) spectrometer (Bruker, Billerica, MA, USA). The data are reported as chemical shift (δ ppm) relative to the residual protonated solvent resonance, relative integral, multiplicity (s = singlet, br s = broad singlet, d = doublet, t = triplet, quat. = quartet, quin. = quintet, m = multiplet), coupling constants (J Hz), and assignment. Assignment of signals was assisted by correlation spectroscopy (COSY), distortionless enhancement by polarisation transfer (DEPT), heteronuclear single quantum coherence (HSQC), and heteronuclear multiple-bond correlation (HMBC) experiments where necessary. Low-resolution mass spectra (LRMS) were recorded using electrospray ionisation (ESI) on a Finnigan LCQ ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). High-resolution mass spectra (HRMS) were run on a Bruker 7T Apex-Qe Fourier transform (FT) ion cyclotron resonance mass spectrometer equipped with an Apollo II ESI/APCI/MALDI dual source (Bruker) by the Mass Spectrometry Facility of the School of Chemistry at the University of Sydney. Infrared (IR) absorption spectra were recorded on a Bruker ALPHA FT-IR spectrometer (Bruker) as solid or thin film from ethanol, and the data are reported as vibrational frequencies (cm−1).
Preparation of N-pentyl-3-trifluoroacetylindole (19)
Sodium hydride (60% dispersion in mineral oil, 137 mg, 3.42 mmol) was added portion-wise to an ice-cold solution of indole (200 mg, 1.71 mmol) in N,N-dimethylformamide (DMF, 6 mL) and stirred for 10 min. 1-Bromopentane (225 µL, 1.80 mmol) was added, and the mixture was warmed to ambient temperature and stirred for 1 h. The mixture was re-cooled to 0 °C, and trifluoroacetic anhydride (600 µL, 4.28 mmol) was added dropwise. The reaction mixture was warmed to ambient temperature and stirred for 1 h. The reaction mixture was poured onto ice (75 mL) and extracted with CH2Cl2 (3 × 75 mL). The combined organic extracts were washed with H2O (100 mL) and brine (100 mL), dried (MgSO4), and concentrated under reduced pressure, giving 19, following purification by flash chromatography [hexane/ethyl acetate (EtOAc), 94:6 v/v], as a yellow oil (450 mg, 94%).
1H NMR (300 MHz, CDCl3): δ 8.43 (1H, d, J = 8.7 Hz), 7.93 (1H, s), 7.42–7.36 (3H, m), 4.21 (2H, t, J = 6.9 Hz), 1.95 (2H, quin., J = 7.2 Hz), 1.43–1.32 (4H, m), 0.92 (3H, t, J = 6.6 Hz); 13C NMR (75 MHz, CDCl3): δ 174.8 (q, CO, J = 34.5 Hz), 137.5 (d, CH, J = 4.5 Hz), 136.8 (quat.), 127.3 (quat.), 124.6 (CH), 124.0 (CH), 122.8 (CH), 117.3 (q, CF3, J = 289.5 Hz), 110.5 (CH), 109.5 (quat.), 47.8 (CH2), 29.5 (CH2), 29.0 (CH2), 22.3 (CH2), 14.0 (CH3); 19F NMR (282 MHz, CDCl3): δ −72.2 (3F, s); LRMS (+ ESI): m/z 306 ([M + Na]+, 100%); IR (diamond cell, thin film): 3124 (w), 2959 (m), 2933 (m), 2863 (w), 1662 (s), 1527 (s), 1397 (m), 1286 (m), 1181 (s), 1132 (s), 878 (m), 751 (m).
Preparation of 1-N-pentyl-3-indazole methyl carboxylate (22)
Potassium tert-butoxide (350 mg, 3.12 mmol) was added to an ice-cold solution of methyl-3-indazole carboxylate (500 mg, 2.84 mmol) in THF (15 mL), warmed to ambient temperature, and stirred for 1 h. 1-Bromopentane (415 µL, 2.98 mmol) was added and stirred at ambient temperature for 48 h and then heated to reflux for 24 h. The reaction mixture was cooled to ambient temperature, poured into H2O (100 mL), and extracted with diethyl ether (Et2O, 3 × 100 mL). The combined organic extracts were dried (MgSO4) and concentrated under reduced pressure, giving 22, following purification by flash chromatography (hexane/EtOAc, 90:10 v/v), as a colourless oil (585 mg, 84%).
1H NMR (300 MHz, CDCl3): δ 8.24 (1H, d, J = 8.0 Hz), 7.50–7.44 (2H, m), 7.35–7.27 (1H, m), 4.47 (2H, t, J = 7.4 Hz), 4.04 (3H, s), 1.97 (2H, quin., J = 7.0 Hz), 1.32 (4H, m), 0.87 (3H, t, J = 6.6 Hz); 13C NMR (75 MHz, CDCl3): δ 163.3 (CO), 140.6 (quat.), 134.6 (quat.), 126.8 (CH), 123.9 (quat.), 123.1 (CH), 122.3 (CH), 109.7 (CH), 52.1 (CH2), 50.1 (CH3), 29.7 (CH2), 29.0 (CH2), 22.4 (CH2), 14.0 (CH3); LRMS (+ ESI): m/z 269 ([M + Na]+, 60%), 515 ([2M + Na]+, 100%); IR (diamond cell, thin film): 2954 (m), 2932 (m), 2860 (w), 1709 (s), 1477 (s), 1215 (s), 1159 (s), 1117 (s), 751 (s).
General procedure A: 1-N-alkylation of azaindoles
The appropriate azaindole (1.00 g, 8.46 mmol) in DMF (15 mL) was slowly added to an ice-cold solution of sodium hydride (60% dispersion in mineral oil, 675 mg, 16.9 mmol) in DMF (5 mL) and stirred for 10 min. 1-Bromopentane (1.15 mL, 9.31 mmol) was added, and the mixture was warmed to ambient temperature and stirred for 1 h. The reaction mixture was slowly added to H2O (75 mL) and extracted with EtOAc (3 × 75 mL). The combined organic extracts were dried (MgSO4) and concentrated under reduced pressure.
Preparation of 1-N-pentyl-4-azaindole (28)
Treating 4-azaindole (1.00 g, 8.46 mmol) according to general procedure A gave 28, following purification by flash chromatography (hexane/EtOAc, 70:30 v/v), as an orange oil (1.28 g, 80%).
1H NMR (300 MHz, CDCl3): δ 8.57 (1H, d, J = 5.4 Hz), 8.18 (1H, d, J = 8.4 Hz), 7.67 (1H, d, J = 3.3 Hz), 7.46 (1H, dd, J = 8.4, 5.7 Hz), 7.00 (1H, d, J = 3 Hz), 4.26 (2H, t, J = 7.2 Hz), 1.86 (2H, quin., J = 7.5 Hz), 1.29 (4H, m), 0.88 (3H, t, J = 7.0 Hz); 13C NMR (75 MHz, CDCl3): δ 138.3 (quat.), 136.8 (CH), 135.3 (CH), 132.1 (quat.), 123.9 (CH), 116.1 (CH), 98.3 (CH), 47.5 (CH2), 30.1 (CH2), 28.9 (CH2), 22.2 (CH2), 13.9 (CH3); LRMS (+ ESI): 189 ([M + H]+, 100%); IR (diamond cell, thin film): 2956 (m), 2929 (m), 2860 (w), 1604 (m), 1417 (s), 1322 (m), 1290 (m), 771 (s), 723 (m).
Preparation of 1-N-pentyl-5-azaindole (29)
Treating 5-azaindole (250 mg, 2.12 mmol) according to general procedure A gave 29, following purification by flash chromatography (EtOAc), as a red oil (285 mg, 71%).
1H NMR (300 MHz, CDCl3): δ 8.89 (1H, s), 8.29 (1H, d, J = 5.7 Hz), 7.22 (1H, d, J = 5.7 Hz), 7.10 (1H, d, J = 2.4 Hz), 6.57 (1H, d, J = 1.2 Hz), 4.12 (2H, t, J = 6.9 Hz), 1.82 (2H, quint., J = 7.2 Hz), 1.30 (4H, m), 0..89 (3H, t, J = 7.1 Hz); 13C NMR (75 MHz, CDCl3): δ 144.1 (CH), 140.6 (CH), 139.6 (quat.), 128.8 (CH), 125.5 (quat.), 104.9 (CH), 100.9 (CH), 46.4 (CH2), 30.1 (CH2), 29.1 (CH2), 22.4 (CH2), 14.3 (CH3); LRMS (+ ESI): m/z 189 ([M + H]+, 100%); IR (diamond cell, thin film): 2956 (m), 2929 (m), 2859 (w), 1602 (m), 1476 (m), 1453 (m), 1319 (m), 1295 (m), 890 (m), 801 (m), 724 (s).
Preparation of 1-N-pentyl-6-azaindole (30)
Treating 6-azaindole (1.00 g, 8.46 mmol) according to general procedure A gave 30, following purification by flash chromatography (EtOAc), as a red oil (1.35 g, 85%).
1H NMR (300 MHz, CDCl3): δ 8.77 (1H, s), 8.22 (1H, d, J = 5.4 Hz), 7.50 (1H, d, J = 5.4 Hz), 7.49 (1H, m), 6.47 (1H, m), 4.19 (2H, t, J = 7.2 Hz), 1.87 (2H, quint., J = 7.2 Hz), 1.33 (4H, m), 0.89 (3H, t, J = 6.8 Hz); 13C NMR (75 MHz, CDCl3): δ 138.5 (CH), 133.3 (quat.), 133.2 (quat.), 133.0 (CH), 131.5 (CH), 115.4 (CH), 100.5 (CH), 46.9 (CH2), 30.3 (CH2), 29.2 (CH2), 22.4 (CH2), 14.0 (CH3); LRMS (+ ESI): m/z 189 ([M + H]+, 100%); IR (diamond cell, thin film): 3091 (w), 2956 (s), 2930 (s), 2870 (m), 1682 (m), 1500 (s), 1470 (s), 1320 (s), 1030 (m), 814 (s), 733 (s).
Preparation of 1-N-pentyl-7-azaindole (31)
Treating 7-azaindole (250 mg, 2.12 mmol) according to general procedure A gave 31, following purification by flash chromatography (hexane/EtOAc, 65:35 v/v), as a red oil (217 mg, 68%).
1H NMR (300 MHz, CDCl3): δ 8.32 (1H, d, J = 3.3 Hz), 7.89 (1H, d, J = 7.8 Hz), 7.22 (1H, m), 7.04 (1H, t, J = 4.8 Hz), 6.44 (1H, m), 4.29 (2H, t, J = 7.2 Hz), 1.87 (2H, quint., J = 6.9 Hz), 1.26 (4H, m), 0.88 (3H, t, J = 6.6 Hz); 13C NMR (75 MHz, CDCl3): δ 147.6 (quat.), 142.8 (CH), 128.8 (CH), 128.1 (CH), 120.7 (quat.), 115.6 (CH), 99.3 (CH), 44.7 (CH2), 30.3 (CH2), 29.2 (CH2), 22.5 (CH2), 14.1 (CH3); LRMS (+ ESI): m/z 189 ([M + H]+, 100%); IR (diamond cell, thin film): 3051 (w), 2956 (s), 2926 (s), 2858 (m), 1509 (s), 1425 (s), 1272 (s), 772 (s), 715 (s).
General procedure B: 3-trifluoroacylation of azaindoles
A suspension of aluminium chloride (4.42 g, 33.2 mmol) and the appropriate 1-N-pentyl azaindole (1.25 g, 6.64 mmol) in DMF (25 mL) was stirred at ambient temperature for 1 h. The reaction was cooled to 0 °C and trifluoroacetic anhydride (1.41 mL, 9.96 mmol) was added dropwise, warmed to ambient temperature, and stirred for 5 h. The reaction mixture was slowly added to H2O (75 mL) and extracted with CH2Cl2 (3 × 75 mL). The combined organic extracts were dried (MgSO4) and concentrated under reduced pressure.
Preparation of 1-N-pentyl-3-trifluoroacyl-4-azaindole (32)
Treating 28 (1.55 g, 7.17 mmol) according to general procedure B gave 32, following purification by flash chromatography (hexane/EtOAc, 70:30 v/v), as an off-white solid (1.48 g, 78%).
m.p. 101.5–103.0 °C; 1H NMR (300 MHz, CDCl3): δ 8.75 (1H, d, J = 4.5 Hz), 8.10 (1H, s), 7.76 (1H, d, J = 8.4 Hz), 7.29 (1H, t, J = 6.6 Hz), 4.23 (2H, t, J = 7.2 Hz), 1.92 (2H, quin., J = 6.9 Hz), 1.41–1.32 (4H, m), 0.90 (3H, t, J = 6.9 Hz); 13C NMR (75 MHz, CDCl3): δ 173.8 (q, CO, J = 35.3 Hz), 147.1 (CH), 144.8 (quat.), 139.0 (q, CH, J = 4.5 Hz), 130.0 (quat.), 118.8 (CH), 118.1 (CH), 116.9 (q, CF3, J = 288.8 Hz), 109.4 (quat.), 48.1 (CH2), 29.5 (CH2), 28.9 (CH2), 22.2 (CH2), 13.9 (CH3); 19F NMR (282 MHz, CDCl3): δ −72.69 (3F, s); LRMS (+ ESI): m/z 591 ([2 M + Na]+, 100%); IR (diamond cell, thin film): 3027 (w), 2956 (m), 2932 (m), 2871 (w), 1674 (s), 1525 (s), 1382 (s), 1282 (s), 1176 (s), 1135 (s), 880 (s), 785 (s), 725 (s).
Preparation of 1-N-pentyl-3-trifluoroacyl-5-azaindole (33)
Treating 29 (1.25 g, 6.64 mmol) according to general procedure B gave 33, following purification by flash chromatography (hexane/EtOAc, 45:55 v/v), as a red oil (1.18 g, 63%).
1H NMR (300 MHz, CDCl3): δ 9.63 (1H, s), 8.55 (1H, d, J = 4.5 Hz), 7.94 (1H, m), 7.34 (1H, d, J = 5.7 Hz), 4.21 (2H, t, J = 7.2 Hz), 1.92 (2H, quin., J = 7.2 Hz), 1.42–1.31 (4H, m), 0.92 (3H, t, J = 7.1 Hz); 13C NMR (75 MHz, CDCl3): δ 174.8 (q, CO, J = 36.3 Hz), 145.8 (CH), 143.9 (CH), 141.0 (quat.), 137.9 (q, CH, J = 4.6 Hz), 123.4 (quat.), 116.9 (q, quat., J = 290.8 Hz), 109.8 (quat.), 105.6 (CH), 47.7 (CH2), 29.6 (CH2), 28.9 (CH2), 22.3 (CH2), 13.9 (CH3); 19F NMR (282 MHz, CDCl3): δ −72.7 (3F, s); LRMS (+ ESI): m/z 307 ([M + Na]+, 100%); IR (diamond cell, thin film): 2959 (m), 2933 (m), 2864 (w), 1675 (s), 1527 (s), 1394 (m), 1295 (m), 1184 (s), 1141 (s), 1112 (s), 881 (s).
Preparation of 1-N-pentyl-3-trifluoroacyl-6-azaindole (34)
Treating 30 (1.25 g, 6.64 mmol) according to general procedure B gave 34, following purification by flash chromatography (hexane/EtOAc, 55:45 v/v), as a red oil (217 mg, 95%).
m.p. 80.5–82.5 °C; 1H NMR (300 MHz, CDCl3): δ 8.89 (1H, s), 8.53 (1H, d, J = 5.1 Hz), 8.25 (1H, d, J = 5.4 Hz), 8.01 (1H, s), 4.31 (2H, t, J = 7.2 Hz), 1.97 (2H, quin., J = 6.9 Hz), 1.42-1.30 (4H, m), 0.92 (3H, t, J = 6.9 Hz); 13C NMR (75 MHz, CDCl3): δ 174.8 (q, CO, J = 35.3 Hz), 143.1 (CH), 139.3 (q, CH, J = 4.5 Hz), 133.7 (CH), 133.7 (quat.), 132.6 (quat.), 116.8 (q, CF3, J = 288.8 Hz), 116.7 (CH), 109.1 (quat.), 48.3 (CH2), 29.7 (CH2), 28.9 (CH2), 22.2 (CH2), 13.8 (CH3); 19F NMR (282 MHz, CDCl3): δ −72.9 (3F, s); LRMS (+ ESI): m/z 307 ([M + Na]+, 100%); IR (diamond cell, thin film): 3118 (w), 2960 (m), 2938 (m), 2867 (m), 1681 (s), 1606 (s), 1525 (s), 1294 (s), 1188 (s), 1147 (s), 995 (m), 829 (m).
Preparation of 1-N-pentyl-3-trifluoroacyl-7-azaindole (35)
Treating 31 (200 mg, 1.06 mmol) according to general procedure B gave 35, following purification by flash chromatography (hexane/EtOAc, 80:20 v/v), as a red oil (235 mg, 78%).
1H NMR (300 MHz, CDCl3): δ 8.63 (1H, d, J = 7.8 Hz), 8.46 (1H, d, J = 1.5 Hz), 8.07 (1H, s), 7.32 (1H, t, J = 4.5 Hz), 4.37 (2H, t, J = 7.2 Hz), 1.95 (2H, quint., J = 6.6 Hz), 1.37 (4H, m), 0.91 (3H, t, J = 6.3 Hz); 13C NMR (75 MHz, CDCl3): δ 148.3 (quat,), 145.7 (CH), 137.3 (q, CH, J = 4.4 Hz), 131.1 (CH), 119.8 (CH), 119.6 (quat.), 117.0 (q, quat., J = 290.7 Hz), 108.1 (quat.), 46.1 (CH2), 29.8 (CH2), 29.0 (CH2), 22.3 (CH2), 14.0 (CH3), unresolved CO due to F splitting; 19F NMR (282 MHz, CDCl3): δ −72.5 (3F, s); LRMS (+ ESI): m/z 307 ([M + Na]+, 100%); IR (diamond cell, thin film): 3119 (w), 2958 (w), 2933 (w), 2863 (w), 1671 (s), 1527 (s), 1385 (s), 1128 (s), 1104 (s), 882 (s).
General procedure C: hydrolysis of methyl esters, trifluoroacetyl and benzyl sulfonyl groups
Aqueous NaOH (4 M, 3.87 mL, 15.5 mmol) was added dropwise to a stirred solution of the appropriately substituted methyl ester, trifluoroacetyl compound, or benzyl sulfonyl-protected indole (2.58 mmol) in MeOH (20 mL), and allowed to stir at ambient temperature (indazole derivatives) or reflux (indole/azaindole derivatives) for 18 h. The reaction mixture was added to H2O (75 mL) and washed with Et2O (75 mL). The aqueous layer was acidified to pH ~2 using 1 M aq. HCl and extracted with Et2O (3 × 75 mL). The combined organic extracts were dried (MgSO4) and concentrated under reduced pressure.
Preparation of N-pentyl-3-indole carboxylic acid (20)
Treating 19 (450 mg, 1.59 mmol) according to general procedure C gave 20 as a white solid (323 mg, 88%).
m.p. 106.5–108.0 °C; 1H NMR (300 MHz, MeOD-d4): δ 8.09 (1H, dd, J = 6.5, 1.9 Hz), 7.93 (1H, s), 7.42–7.28 (3H, m), 4.17 (2H, t, J = 7.2 Hz), 1.90 (2H, quin., J = 7.0 Hz), 1.40–1.32 (4H, m), 0.90 (3H, t, J = 6.6 Hz), COOH signal not observed; 13C NMR (75 MHz, MeOD-d4): δ 168.8 (CO2H), 138.1 (quat.), 136.3 (CH), 128.3 (quat.), 123.6 (CH), 122.6 (CH), 122.4 (CH), 111.3 (CH), 107.7 (quat.), 47.7 (CH2), 30.7 (CH2), 30.0 (CH2), 23.3 (CH2), 14.2 (CH3); LRMS (– ESI): m/z 230 ([M–H]−, 100%); IR (diamond cell, thin film): 3106 (w), 2925 (m), 2856 (w), 2525 (bs), 1649 (s), 1526 (s), 1461 (m), 1273 (m), 1204 (s), 1117 (m), 940 (m), 731 (s).
Preparation of 1-N-pentyl-3-indazole carboxylic acid (23)
Treating 22 (600 mg, 2.58 mmol) according to general procedure C gave 23 as a white solid (510 mg, 85%).
m.p. 76.5–78.0 °C; 1H NMR (300 MHz, MeOD-d4): δ 8.26 (1H, d, J = 8.1 Hz), 7.52–7.44 (2H, m), 7.35 (1H, t, J = 7.8 Hz), 4.48 (2H, t, J = 7.2 Hz), 1.99 (2H, quin., J = 7.2 Hz), 1.34 (4H, m), 0.89 (3H, t, J = 6.0 Hz), COOH signal not observed; 13C NMR (75 MHz, MeOD-d4): δ 165.4 (CO2H), 142.0 (quat.), 135.9 (quat.), 127.9 (CH), 124.5 (quat.), 124.0 (CH), 122.9 (CH), 111.0 (CH), 49.7 (CH2), 30.4 (CH2), 29.8 (CH2), 23.1 (CH2), 14.1 (CH3); LRMS (– ESI): m/z 231 ([M–H]−, 100%); IR (diamond cell, thin film): 3053 (bs), 2956 (m), 2931 (m), 2860 (w), 1687 (s), 1503 (s), 1218 (s), 1176 (s), 1121 (s), 752 (s).
Preparation of 1-N-pentyl-4-azaindole-3-carboxylic acid (36)
Treating 32 (1.48 g, 5.21 mmol) according to general procedure C gave 36 as an off-white solid (1.18 g, 97%).
m.p. 112.0–113.5 °C; 1H NMR (300 MHz, MeOD-d4): δ 8.48 (1H, d, J = 4.5 Hz), 8.06 (1H, s), 7.78 (1H, d, J = 8.3 Hz), 7.26 (1H, dd, J = 8.6, 4.8 Hz), 4.18 (2H, t, J = 7.1 Hz), 1.87 (2H, quin., J = 7.2 Hz), 1.38–1.24 (4H, m), 0.87 (3H, t, J = 7.1 Hz), COOH signal not observed; 13C NMR (75 MHz, MeOD-d4): δ 164.4 (CO2H), 144.4 (quat.), 143.7 (CH), 136.1 (CH), 129.2 (quat.), 118.9 (CH), 117.9 (CH), 106.6 (quat.), 47.8 (CH2), 29.8 (CH2), 28.9 (CH2), 22.3 (CH2), 13.9 (CH3); LRMS (+ ESI): m/z 255 ([M + Na]+, 100%); IR (diamond cell, thin film): 3358 (bs), 3120 (w), 2948 (m), 2928 (m), 2864 (m), 1666 (m), 1538 (m), 1373 (m), 1143 (m), 1032 (m), 962 (m), 782 (s), 723 (s), 491 (s).
Preparation of 1-N-pentyl-5-azaindole-3-carboxylic acid (37)
33 (600 mg, 2.11 mmol) was treated according to general procedure C. Once the reaction was complete, the reaction mixture was concentrated under reduced pressure and immediately subjected to flash chromatography (hexane/EtOAc/acetic acid, 91:8:1 v/v), giving 37 as an off-white solid (425 mg, 87%).
m.p. 185.0–188.0 °C; 1H NMR (300 MHz, MeOD-d4): δ 9.36 (1H, s), 8.36 (1H, d, J = 6.9 Hz), 8.18 (1H, s), 7.84 (1H, d, J = 6.3 Hz), 4.34 (2H, t, J = 7.2 Hz), 1.89 (2H, quint., J = 7.4 Hz), 1.39-1.28 (4H, m), 0.89 (3H, t, J = 7.1 Hz), COOH signal not observed; 13C NMR (75 MHz, MeOD-d4): δ 168.0 (CO2H), 143.3 (quat.), 141.2 (CH), 139.0 (CH), 136.8 (CH), 125.0 (quat.), 113.1 (quat.), 108.5 (CH), 48.1 (CH2), 30.8 (CH2), 29.9 (CH2), 23.3 (CH2), 14.2 (CH3); LRMS (+ ESI): m/z 233 ([M + H]+, 100%); IR (diamond cell, thin film): 3105 (w), 2956 (m), 2859 (w), 2391 (bs), 1677 (w), 1538 (m), 1475 (m), 1186 (s), 1007 (s), 805 (s).
Preparation of 1-N-pentyl-6-azaindole-3-carboxylic acid (38)
34 (900 mg, 3.17 mmol) was treated according to general procedure C. Once the reaction was complete, the reaction mixture was concentrated under reduced pressure and immediately subjected to flash chromatography (CH2Cl2/MeOH, 94:6 v/v), giving 38 as an off-white solid (385 mg, 52%).
m.p. 159.0–160.5 °C; 1H NMR (300 MHz, MeOD-d4): δ 8.90 (1H, s), 8.26 (1H, d, J = 5.6 Hz), 8.22 (1H, s), 8.13 (1H, d, J = 5.7 Hz), 4.37 (2H, t, J = 7.1 Hz), 1.92 (2H, quin., J = 7.1 Hz), 1.41–1.26 (4H, m), 0.89 (3H, t, J = 7.1 Hz), COOH signal not observed; 13C NMR (75 MHz, MeOD-d4): δ 167.9 (CO2H), 140.6 (CH), 139.3 (CH), 135.1 (quat.), 134.5 (CH), 133.5 (CH), 117.5 (quat.), 109.4 (quat.), 48.3 (CH2), 31.0 (CH2), 29.9 (CH2), 23.2 (CH2), 14.2 (CH3); LRMS (+ ESI): m/z 233 ([M + H]+, 100%); IR (diamond cell, thin film): 2930 (w), 2861 (w), 1694 (s), 1479 (s), 1183 (m), 1131 (m), 1029 (s), 822 (m).
Preparation of 1-N-pentyl-7-azaindole-3-carboxylic acid (39)
Treating 35 (200 mg, 0.70 mmol) according to general procedure C gave 39 as an off-white solid (135 mg, 83%).
m.p. 143.0–147.5 °C; 1H NMR (300 MHz, MeOD-d4): δ 8.43 (1H, d, J = 7.2 Hz), 8.31 (1H, m), 8.23 (1H, m), 7.25 (1H, s), 4.33 (2H, t, J = 6.0 Hz), 1.88 (2H, quin., J = 6.0 Hz), 1.41–1.15 (4H, m), 0.88 (3H, m), COOH signal not observed; 13C NMR (75 MHz, MeOD-d4): δ 167.9 (CO2H), 148.8 (quat.), 144.5 (CH), 136.5 (CH), 131.3 (CH), 121.0 (quat.), 118.9 (CH), 106.8 (quat.), 46.2 (CH2), 30.9 (CH2), 29.9 (CH2), 23.3 (CH2), 14.3 (CH3); LRMS (–ESI): m/z 231 ([M–H)−, 100%); IR (diamond cell, thin film): 2930 (w), 2856 (w), 1685 (s), 1533 (s), 1257 (s), 1138 (s), 798 (s), 755 (s).
General procedure D: amidation of 1-N-pentyl-3-indole/indazole/azaindole carboxylic acids with adamantylamine hydrochloride via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) hydrochloride coupling
N,N-Diisopropylethylamine (340 µL, 1.95 mmol) was added dropwise to a stirred solution of the appropriate 1-N-pentyl-3-indole/indazole/azaindole carboxylic acid (0.39 mmol), adamantylamine hydrochloride (0.41 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 150 mg, 0.78 mmol), and 1-hydroxybenzotriazole (HOBt, 119 mg, 0.78 mmol) in DMSO (5 mL), and was stirred at ambient temperature for 18 h. The reaction mixture was added to sat. aq. NaHCO3 (75 mL) and extracted with EtOAc (3 × 75 mL). The combined organic extracts were dried (MgSO4) and concentrated under reduced pressure.
Preparation of N-(adamantan-1-yl)-1-pentyl-1H-indole-3-carboxamide (APICA, 4)
Treating 20 (576 mg, 2.50 mmol) according to general procedure D gave 4, following recrystallisation from isopropanol/H2O, as a white solid (787 mg, 86%).
m.p. 140.0–141.0 °C; 1H NMR (500 MHz, CDCl3): δ 7.87 (1H, d, J = 7.5 Hz), 7.65 (1H, s), 7.36 (1H, d, J = 8.0 Hz), 7.28–7.22 (2H, m), 5.71 (1H, br s, NH), 4.11 (2H, t, J = 7.2 Hz), 2.19 (6H, br s), 2.14 (3H, br s), 1.84 (2H, quin., J = 7.3 Hz), 1.76 (6H, m) 1.39–1.24 (4H, m), 0.88 (3H, t, J = 7.0 Hz); 13C NMR (125 MHz, CDCl3): δ 164.0 (CO), 136.7 (quat.), 131.5 (CH), 125.3 (quat.), 122.3 (CH), 121.2 (CH), 120.0 (CH), 112.3 (quat.), 110.4 (CH), 52.2 (quat.), 46.9 (CH2), 42.4 (CH2), 36.6 (CH2), 29.8 (CH), 29.7 (CH2), 29.1 (CH2), 22.4 (CH2), 14.0 (CH3).
Preparation of N-(adamantan-1-yl)-1-pentyl-1H-indazole-3-carboxamide (APINACA, 7)
Treating 23 (100 mg, 0.43 mmol) according to general procedure D gave 7, following purification by flash chromatography (hexane/EtOAc, 95:5 v/v), as a white solid (128 mg, 81%).
m.p. 66.1–68.8 °C; 1H NMR (300 MHz, CDCl3): δ 8.38 (1H, d, J = 8.1 Hz), 7.38–7.34 (2H, m), 7.27–7.22 (1H, m), 6.80 (1H, s), 4.34 (2H, t, 6.9 Hz), 2.23–2.18 (6H, m), 2.17–2.10 (3H, m), 1.93 (2H, quin., 6.9 Hz), 1.79–1.69 (6H, m), 1.40–1.30 (4H, m), 0.89 (3H, t, J = 6.3 Hz); 13C NMR (75 MHz, CDCl3): δ 162.2 (CO), 141.0 (quat.), 138.2 (quat.), 126.6 (CH), 123.3 (CH), 123.0 (quat.), 122.4 (CH), 109.2 (CH), 52.0 (CH2), 49.5 (quat.), 42.1 (CH2), 36.6 (CH2), 29.7 (CH), 29.6 (CH2), 29.1 (CH2), 22.4 (CH2), 12.0 (CH3); LRMS (+ ESI): m/z 388 ([M + Na]+, 100%); HRMS (+ ESI): m/z calculated [M + Na]+ 388.2359, found 388.2360; IR (diamond cell, thin film): 3303 (w), 2908 (s), 2846 (m), 1647 (s), 1529 (s), 1491 (m), 1451 (m), 1357 (m), 1341 (m), 1289 (m), 1218 (m), 1139 (m), 1005 (m), 859 (m), 772 (m), 748 (s).
Preparation of N-(adamantan-1-yl)-1-pentyl-1H-pyrrolo[3,2-b]pyridine-3-carboxamide (AP4AICA, 8)
Treating 36 (200 mg, 0.86 mmol) according to general procedure D gave 8, following purification by flash chromatography (hexane/EtOAc, 70:30 v/v), as a white solid (210 mg, 67%).
m.p. 212.0–214.0 °C; 1H NMR (300 MHz, CDCl3): δ 8.69 (1H, br s), 8.47 (1H, s), 7.97 (1H, s), 7.65 (1H, d, J = 8.1 Hz), 7.16 (1H, t, J = 3.9 Hz), 4.12 (2H, t, J = 6.3 Hz), 2.13 (6H, m), 2.04 (3H, m), 1.83–1.69 (8H, m), 1.35–1.21 (4H, m), 0.88 (3H, t, J = 6.9 Hz); 13C NMR (75 MHz, CDCl3): δ 163.4 (CO), 143.5 (CH), 134.8 (CH), 129.8 (quat.), 127.1 (quat.), 117.6 (CH), 116.9 (CH), 112.0 (quat.), 51.8 (CH2), 47.2 (quat.), 42.2 (CH2), 36.8 (CH2), 29.9 (CH2), 29.8 (CH2), 29.0 (CH2), 22.4 (CH2), 14.0 (CH3); LRMS (+ ESI): m/z 388 ([M + Na]+, 100%); HRMS (+ ESI): m/z calculated [M + Na]+ 388.2359, found 388.2362; IR (diamond cell, thin film): 3257 (w), 3065 (w), 2902 (s), 2849 (m), 1635 (s), 1562 (s), 1432 (m), 1276 (m), 1255 (m), 782 (s).
Preparation of N-(adamantan-1-yl)-1-pentyl-1H-pyrrolo[3,2-c]pyridine-3-carboxamide (AP5AICA, 9)
Treating 37 (70 mg, 0.30 mmol) according to general procedure D gave 9, following purification by flash chromatography (EtOAc), as a white solid (68 mg, 62%).
m.p. 155.5–156.5 °C; 1H NMR (300 MHz, CDCl3): δ 9.24 (1H, s), 8.38 (1H, d, J = 5.7 Hz), 7.63 (1H, s), 7.26 (1H, d, J = 6.0 Hz), 5.70 (1H, br s), 4.10 (2H, t, J = 7.2 Hz), 2.18 (6H, m), 2.14 (3H, m), 1.84 (2H, quin., J = 7.2 Hz), 1.75 (6H, m), 1.38–1.27 (4H, m), 0.88 (3H, t, J = 7.1 Hz); 13C NMR (75 MHz, CDCl3): δ 163.4 (CO), 143.4 (CH), 141.6 (CH), 140.5 (quat.), 131.7 (CH), 122.4 (quat.), 113.0 (quat.), 105.4 (CH), 52.2 (CH2), 46.8 (quat.), 42.2 (CH2), 36.6 (CH), 29.9 (CH2), 29.7 (CH3), 29.0 (CH2), 22.3 (CH2), 14.0 (CH3); LRMS (+ ESI): m/z 366 ([M + H]+, 100%); HRMS (+ ESI): m/z calculated [M + Na]+ 388.2359, found 388.2362; IR (diamond cell, thin film): 3343 (bs), 2905 (m), 2851 (m), 1619 (s), 1537 (s), 1516 (s), 1357 (m), 1196 (m), 1141 (m), 799 (m).
Preparation of N-(adamantan-1-yl)-1-pentyl-1H-pyrrolo[2,3-c]pyridine-3-carboxamide (AP6AICA, 10)
Treating 38 (70 mg, 0.30 mmol) according to general procedure D gave 10, following purification by flash chromatography (hexane/EtOAc, 80:20 v/v), as a white solid (88 mg, 80%).
m.p. 154.0–155.5 °C; 1H NMR (500 MHz, CDCl3): δ 8.79 (1H, d, J = 0.9 Hz), 8.35 (1H, d, J = 5.6 Hz), 7.75-7.73 (2H, m), 5.67 (1H, br s), 4.19 (2H, t, J = 7.2 Hz), 2.17 (9H, m), 1.88 (2H, quin., J = 7.3 Hz), 1.77–1.70 (6H, m), 1.36–1.27 (4H, m), 0.87 (3H, t, J = 7.3 Hz); 13C NMR (125 MHz, CDCl3): δ 163.6 (CO), 140.5 (CH), 134.1 (CH), 133.8 (CH), 133.7 (quat.), 130.2 (quat.), 114.5 (CH), 112.2 (quat.), 52.4 (CH2), 47.4 (quat.), 42.3 (CH2), 36.6 (CH2), 30.1 (CH2), 29.7 (CH), 29.1 (CH2), 22.4 (CH2), 14.0 (CH3); LRMS (+ ESI): m/z 366 ([M + H]+, 100%); HRMS (+ ESI): m/z calculated [M + Na]+ 388.2359, found 388.2362; IR (diamond cell, thin film): 3333 (bs), 2904 (s), 2851 (m), 1618 (s), 1537 (s), 1514 (s), 2180 (m), 1245 (m), 821 (m).
Preparation of N-(adamantan-1-yl)-1-pentyl-1H-pyrrolo[2,3-b]pyridine-3-carboxamide (AP7AICA, 11)
Treating 39 (75 mg, 0.32 mmol) according to general procedure D gave 11, following purification by flash chromatography (hexane/EtOAc, 80:20 v/v), as a white solid (88 mg, 75%).
m.p. 149.0–149.5 °C; 1H NMR (300 MHz, CDCl3): δ 9.24 (1H, m), 8.26 (1H, d, J = 8.1 Hz), 7.69 (1H, s), 7.17 (1H, t, J = 4.5 Hz), 5.55 (1H, br s), 4.29 (2H, t, J = 6.6 Hz), 2.17 (9H, m), 1.88 (2H, quin., J = 6.8 Hz), 1.74 (6H, m), 1.33 (4H, m), 0.88 (3H, t, J = 6.7 Hz); 13C NMR (75 MHz, CDCl3): δ 164.0 (CO), 147.8 (quat.), 143.7 (CH), 130.2 (CH), 128.8 (CH), 118.8 (quat.), 117.3 (CH), 110.6 (quat.), 52.3 (CH2), 45.1 (quat.), 42.3 (CH2), 36.6 (CH), 30.1 (CH2), 29.7 (CH), 29.1 (CH2), 22.4 (CH2), 14.1 (CH3); LRMS (+ ESI): m/z 388 ([M + Na]+, 100%); HRMS (+ ESI): m/z calculated [M + Na]+ 388.2359, found 388.2362; IR (diamond cell, thin film): 3322 (bs), 3097 (s), 2905 (s), 2850 (m), 1622 (s), 1538 (s), 1451 (m), 1187 (m).
General procedure E: esterification of 1-N-pentyl-3-indole/indazole/azaindole carboxylic acids with 1-bromoadamantane
A suspension of the appropriate 1-N-pentyl-indole, indazole, or azaindole-3-carboxylic acid (0.43 mmol), 1-bromoadamantane (101 mg, 0.47 mmol), and silver carbonate (178 mg, 0.65 mmol) in N,N-dimethylacetamide (4 mL) was stirred at reflux for 24 h. The reaction was cooled to ambient temperature and concentrated under reduced pressure. The residue was added to H2O (50 mL) and extracted with EtOAc (3 × 50 mL), and the combined organic extracts were dried (MgSO4) and concentrated under reduced pressure.
Preparation of adamantan-1-yl 1-pentyl-1H-indole-3-carboxylate (APIC, 12)
Treating 20 (100 mg, 0.43 mmol) according to general procedure E gave 12, following purification by flash chromatography (hexane/EtOAc, 93:7 v/v), as an off-white solid (68 mg, 41%).
m.p. 102.0–103.0 °C; 1H NMR (300 MHz, CDCl3): δ 8.21–8.11 (1H, m), 7.75 (1H, s), 7.38–7.19 (3H, m), 4.12 (2H, t, J = 7.1 Hz), 2.40–2.31 (6H, m), 2.28–2.19 (3H, m), 1.86 (2H, quin., J = 7.3 Hz), 1.80–1.65 (6H, m), 1.44–1.25 (4H, m), 0.89 (3H, t, J = 6.5 Hz); 13C NMR (75 MHz, CDCl3): 164.7 (CO), 136.7 (quat.), 134.2 (CH), 126.9 (quat.), 122.5 (CH), 122.0 (CH), 121.6 (CH), 110.0 (CH), 108.9 (quat.), 80.1 (quat.), 47.1 (CH2), 42.1 (CH2), 36.5 (CH), 31.1 (CH2), 29.8 (CH2), 29.1 (CH2), 22.4 (CH2), 14.0 (CH3); LRMS: m/z 388 ([M + Na]+, 100%); HRMS (+ ESI): m/z calculated [M + Na]+ 388.2247, found 388.2245; IR (diamond cell, thin film): 3112 (w), 2904 (s), 2847 (m), 1680 (s), 1530 (s), 1265 (s), 1102 (s), 1058 (s), 734 (s).
Preparation of adamantan-1-yl 1-pentyl-1H-indazole-3-carboxylate (APINAC, 13)
Treating 20 (500 mg, 1.54 mmol) according to general procedure E gave 13, following purification by flash chromatography (hexane/EtOAc, 92:8 v/v), as an off-white solid (215 mg, 38%).
m.p. 109.0–110.0 °C; 1H NMR (300 MHz, CDCl3): δ 8.15 (1H, d, J = 8.1 Hz), 7.46–7.37 (2H, m), 7.26 (1H, t, J = 6.9 Hz), 4.44 (2H, t, J = 7.5 Hz), 2.39–2.37 (6H, m), 2.26–2.23 (3H, m), 1.96 (2H, quin., J = 7.2 Hz), 1.80–1.69 (6H, m), 1.39–1.28 (4H, m), 0.88 (3H, t, J = 6.9 Hz); 13C NMR (300 MHz, CDCl3): δ 161.8 (CO), 140.7 (quat.), 136.3 (quat.), 126.5 (CH), 123.5 (quat.), 122.7(0) (CH), 122.6(9) (CH), 109.7 (CH), 81.9 (quat.), 49.9 (CH2), 41.8 (CH2), 36.4 (CH), 31.1 (CH2), 29.6 (CH2), 29.1 (CH2), 22.4 (CH2), 14.0 (CH3); LRMS (+ ESI): m/z 389 ([M + Na]+, 100%), 755 ([2 M + Na]+, 95%); HRMS (+ ESI): m/z calculated [M + Na]+ 389.2199, found 389.2196; IR (diamond cell, thin film): 2905 (s), 2849 (m), 1690 (m), 1644 (s), 1361 (m), 1305 (s), 1169 (m), 1124 (s), 866 (m), 756 (s), 544 (w).
Preparation of adamantan-1-yl 1-pentyl-1H-pyrrolo[3,2-b]pyridine-3-carboxylate (AP4AIC, 14)
Treating 36 (100 mg, 0.43 mmol) according to general procedure E gave 14, following purification by flash chromatography (hexane/EtOAc, 70:30 v/v), as a white solid (55 mg, 35%).
m.p. 83.5–85.0 °C; 1H NMR (300 MHz, CDCl3): δ 8.64 (1H, d, J = 4.5 Hz), 7.86 (1H, s), 7.62 (1H, d, J = 8.4 Hz), 7.15 (1H, dd, J = 7.8, 4.8 Hz), 4.11 (2H, t, J = 6.9 Hz), 2.34–2.31 (6H, m), 2.22–2.19 (3H, m), 1.85 (2H, quin., J = 7.2 Hz), 1.74–1.67 (6H, m), 1.38–1.25 (4H, m), 0.89 (3H, t, J = 6.3 Hz); 13C NMR (75 MHz, CDCl3): δ 162.6 (CO), 145.5 (CH), 144.7 (quat.), 136.1 (CH), 129.7 (quat.), 117.2 (CH), 117.1 (CH), 109.1 (quat.), 80.2 (quat.), 47.2 (CH2), 41.8 (CH2), 36.5 (CH2), 31.1 (CH), 29.8 (CH2), 29.0 (CH2), 22.3 (CH2), 13.9 (CH3); LRMS (+ ESI): m/z 389 ([M + Na]+, 100%); HRMS (+ ESI): m/z calculated [M + Na]+ 389.2199, found 389.2200; IR (diamond cell, thin film): 3107 (w), 2911 (m), 2856 (w), 1661 (s), 1640 (s), 1517 (s), 1329 (s), 1182 (m), 885 (m), 799 (m), 549 (m).
Preparation of adamantan-1-yl 1-pentyl-1H-pyrrolo[3,2-c]pyridine-3-carboxylate (AP5AIC, 15)
Treating 37 (100 mg, 0.43 mmol) according to general procedure E gave 15, following purification by flash chromatography (hexane/EtOAc, 60:40 v/v), as an off-white solid (79 mg, 50%).
m.p. 101.5–102.5 °C; 1H NMR (300 MHz, CDCl3): δ 9.56 (1H, d, J = 0.9 Hz), 8.52 (1H, d, J = 5.8 Hz), 7.72 (1H, s), 7.33 (1H, dd, J = 5.9, 0.8 Hz), 4.19 (2H, t, J = 7.1 Hz), 2.31–2.28 (6H, m), 2.11–2.06 (3H, m), 1.90 (2H, quin., J = 7.3 Hz), 1.72–1.61 (6H, m), 1.39–1.28 (4H, m), 0.90 (3H, t, J = 7.0 Hz); 13C NMR (75 MHz, CDCl3): δ 162.5 (CO) 145.3 (CH), 143.3 (CH), 141.6 (quat.), 137.8 (CH), 123.0 (quat.), 116.2 (quat.), 105.7 (CH), 74.2 (quat.), 47.5 (CH2), 40.1 (CH2), 36.4 (CH2), 30.1 (CH), 29.5 (CH2), 29.0 (CH2), 22.3 (CH2), 14.0 (CH3); LRMS (+ ESI): m/z 367 ([M + H]+, 100%); HRMS (+ ESI): m/z calculated [M + Na]+ 389.2199, found 389.2199; IR (diamond cell, thin film): 3109 (w), 2909 (m), 2852 (w), 1683 (s), 1533 (m), 1229 (m), 1132 (s), 1055 (s), 757 (m), 421 (m).
Preparation of adamantan-1-yl 1-pentyl-1H-pyrrolo[2,3-c]pyridine-3-carboxylate (AP6AIC, 16)
Treating 38 (100 mg, 0.43 mmol) according to general procedure E gave 16, following purification by flash chromatography (hexane/EtOAc, 50:50 v/v), as a white solid (68 mg, 43%).
m.p. 108.5–110.0 °C; 1H NMR (300 MHz, CDCl3): δ 8.78 (1H, s), 8.37 (1H, d, J = 5.1 Hz), 7.97 (1H, d, J = 5.4 Hz), 7.83 (1H, s), 4.21 (2H, t, J = 7.2 Hz), 2.35–2.30 (6H, m), 2.26–2.21 (3H, m), 1.90 (2H, quin., J = 6.9 Hz), 1.78–1.67 (6H, m), 1.37–1.30 (4H, m), 0.89 (3H, t, J = 6.3 Hz); 13C NMR (75 MHz, CDCl3): δ 163.8 (CO), 140.9 (CH), 136.8 (CH), 133.8 (quat.), 133.4 (CH), 131.9 (CH), 116.2 (quat.), 109.0 (quat.), 80.8 (quat.), 47.5 (CH2), 42.0 (CH2), 36.5 (CH2), 31.1 (CH), 30.0 (CH2), 29.0 (CH2), 22.3 (CH2), 14.0 (CH3); LRMS (+ ESI): m/z 367 ([M + H]+, 100%); HRMS (+ ESI): m/z calculated [M + Na]+ 389.2199, found 389.2197; IR (diamond cell, thin film): 3109 (w), 2909 (s), 2848 (m), 1682 (s), 1603 (m), 1526 (s), 1443 (m), 1249 (s), 1194 (m), 1131 (s), 1055 (s), 820 (s), 626 (m), 424 (m).
Preparation of adamantan-1-yl 1-pentyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylate (AP7AIC, 17)
Treating 39 (100 mg, 0.43 mmol) according to general procedure E gave 17, following purification by flash chromatography (hexane/EtOAc 70:30 v/v), as a yellow solid (60 mg, 38%).
m.p. 99.5–101.0 °C; 1H NMR (300 MHz, CDCl3): δ 8.38-8.34 (2H, m), 7.87 (1H, s), 7.17 (1H, dd, J = 8.1, 4.8 Hz), 4.29 (2H, t, J = 7.3 Hz), 2.34–2.29 (6H, m), 2.26–2.20 (3H, m), 1.89 (2H, quin., J = 7.2 Hz), 1.78–1.68 (6H, m), 1.39–1.28 (4H, m), 0.88 (3H, t, J = 7.0 Hz); 13C NMR (75 MHz, CDCl3): δ 164.0 (CO), 147.9 (quat.), 143.8 (CH), 133.9 (CH), 130.1 (CH), 119.3 (quat.), 117.8 (CH), 107.4 (quat.), 80.5 (quat.), 45.2 (CH2), 42.0 (CH2), 36.5 (CH2), 31.1 (CH), 30.0 (CH2), 29.0 (CH2), 22.4 (CH2), 14.0 (CH3); LRMS (+ ESI): m/z 389 ([M + Na]+, 100%); HRMS (+ ESI): m/z calculated [M + Na]+ 389.2199, found 389.2198; IR (diamond cell, thin film): 3105 (w), 2915 (m), 2852 (w), 1686 (s), 1528 (s), 1403 (m), 1256 (s), 1135 (s), 1117 (s), 1058 (s), 773 (m), 758 (s).
In vitro pharmacological assessment
Mouse AtT-20 neuroblastoma cells stably transfected with human CB
1 or human CB
2 have been described previously and were cultured in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum (FBS), 100 U penicillin/streptomycin, and 80 μg/mL hygromycin [
10]. Cells were passaged at 80% confluence as required. Cells for assays were grown in 75 cm
2 flasks and used at 90% confluence. The day before, the assay cells were detached from the flask with trypsin/ethylenediaminetetraacetic acid (Sigma-Aldrich) and resuspended in 10 mL of Leibovitz’s L-15 media supplemented with 1% FBS, 100 U penicillin/streptomycin, and 15 mM glucose for the below membrane potential and Ca5 calcium assays. The cells were plated in a volume of 90 μL in black-walled, clear-bottomed 96-well microplates (Corning Inc., Corning, NY, USA). Cells were incubated overnight at 37 °C in ambient CO
2.
Membrane potential was measured using a fluorometric imaging plate reader (FLIPR) membrane potential assay kit (blue) from Molecular Devices (San Jose, CA, USA), as described previously [
28]. The dye was reconstituted with assay buffer of the following composition (mM): NaCl 145, HEPES 22, Na
2HPO
4 0.338, NaHCO
3 4.17, KH
2PO
4 0.441, MgSO
4 0.407, MgCl
2 0.493, CaCl
2 1.26, glucose 5.56 and bovine serum albumin (BSA) (pH 7.4 and osmolarity 315 ± 5). Prior to the assay, 90 μL/well of the dye solution was added, giving an initial assay volume of 180 μL/well.
Drugs were dissolved at 30 mM in DMSO and aliquoted for storage at −30 °C. On the day of experiment, an aliquot was unfrozen, and a portion diluted to 10 mM in DMSO. Serial dilutions were performed in HEPES-buffered saline (HBS) containing 0.1% BSA. The first 1:10 dilution was made into HBS plus BSA and 1% DMSO. Drugs were added to the cells at 10 times the final concentration, to give a final concentration of DMSO of 0.1% in all wells.
Plates were incubated at 37 °C at ambient CO2 for 60 min. Fluorescence was measured using a FlexStation 3 (Molecular Devices) microplate reader, with cells excited at a wavelength of 530 nm and emission measured at 565 nm. Baseline readings were taken every 2 s for at least 2 min, at which time either drug or vehicle was added in a volume of 20 μL. The background fluorescence of cells without dye or dye without cells was negligible. Changes in fluorescence were expressed as a percentage of predrug fluorescence after subtraction of the changes produced by vehicle addition.
Drugs were initially tested over a concentration range of 1 nM to 30 µM. Full concentration response curves were constructed for drugs which produced an effect greater than 50% of that of CP 55,940 (1 µM; Cayman Chemical, Ann Arbor, MI, USA) when tested at 10 µM. If drugs produced a response less than 50% of CP 55,940 (1 µM) when tested at 10 µM, experiments were repeated at 10 µM only.
Data were analysed with PRISM (GraphPad Software Inc., San Diego, CA, USA), using four-parameter nonlinear regression to fit concentration-response curves. In all plates, a maximum effective concentration of CP 55,940 (1 µM) was added to allow for a ready comparison between experiments.