Patent Application
20080027035
The following examples are provided so that the invention might be more fully understood. These examples are illustrative only and should not be construed as limiting the invention in any way.
A mixture of 127 mg (1.04 mmol) of benzoic acid, 219 mg (1.14 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 154 mg (1.14 mmol) of 1-hydroxybenzotriazole (HOBt) and 271 μl (1.56 mmol) of diisopropylethylamine (DIEA) in 2 mL of N,N-dimethylformamide is stirred at room temperature for 10 minutes, before a solution of 250 mg (1.24 mmol) of (2S)-2-aminomethyl-pyrrolidine-1-carboxylic acid tert-butyl ester in 2 mL of N,N-dimethylformamide is added and stirring continued overnight. The solution is diluted with 50 mL of ethyl acetate, washed sequentially with 5% citric acid aqueous solution, saturated aqueous sodium bicarbonate solution, and brine, dried over sodium sulphate and the solvent is removed under reduced pressure. Purification of the crude product by flash chromatography (silica gel, eluent: 0% to 10% of ethyl acetate in cyclohexane) gives the title compound.
1H-NMR δ (ppm)=1.40 (s, 9H), 1.75-1.88 (m, 4H), 3.39-3.50 (m, 1H), 3.90-3.98 (m, 1H), 7.41-7.47 (m, 4H), 7.78-7.81 (m, 1H), 8.34-8.39 (m, 1H).
LC/MS (IV) rt 2.79, m/z 368 (M+Na+CH3CN)+.
A solution of 20.0 mg (0.07 mmol) of (2S)-(benzoylamino-methyl)-pyrrolidine-1-carboxylic acid tert-butyl ester (step 1) in 1.0 mL of dichloromethane and 0.5 mL of trifluoroacetic acid is stirred at room temperature for 30 minutes and then evaporated under reduced pressure to give the title compound.
The intermediates in Table 1 are synthesized according to the procedure shown for Example 1.
(2S)-Benzyloxymethyl-pyrrolidine-1-carboxylic acid tert-butyl ester. (For the synthesis see also J. Med. Chem.; 42; 4; 1999; 677-690)
A solution of 500 mg (2.48 mmol) of (2S)-hydroxymethyl-pyrrolidine-1-carboxylic acid tert-butyl ester in 2 mL of tetrahydrofuran is added dropwise to a slurry of 119.2 mg (60% dispersion in oil, 2.98 mmol) of sodium hydride in 2 mL of tetrahydrofuran at 0° C. and the mixture stirred for 5 minutes. 325 μL (119 mg, 2.73 mmol) of benzylbromide is added and the reaction is allowed to warm to room temperature and stirred overnight. Water and 1N hydrochloric acid solution are added and the mixture is extracted with ethyl acetate. The collected organic phases are washed sequentially with a saturated aqueous sodium bicarbonate solution, brine and water, then dried over sodium sulphate and evaporated under reduced pressure. The crude mixture is purified using flash chromatography (silica gel, eluent: 0% to 10% ethyl acetate in cyclohexane) to afford the title compound.
LC/MS (IV) rt 3.41, m/z 233 (M+H-Boc+AcCN)+.
A solution of 300 mg (1.03 mmol) of (2S)-benzyloxymethyl-pyrrolidine-1-carboxylic acid tert-butyl ester (step 1) in 1.5 mL of dichloromethane and 1.5 mL of trifluoroacetic acid is stirred at room temperature for 1 h and then evaporated under reduced pressure. The crude mixture is diluted in 5 mL of dichloromethane and stirred for 1 h with 1.43 g (4.12 mmol) of (polystyrylmethyl)trimethylammonium bicarbonate, then filtered and evaporated under reduced pressure to give the title compound.
1H-NMR δ (ppm)=1.34-1.42 (m, 1H), 1.59-1.81 (m, 3H), 2.76-2.86 (m, 2H), 3.27-3.33 (m, 4H), 4.47 (s, 2H), 7.29-7.24 (m, 5H).
LC/MS (III) rt 2.62, m/z 192 (M+H)+.
A solution of 150 mg (0.75 mmol) of N-Boc-prolinol and 33 mg (0.82 mmol) of sodium hydride in 1 mL of tetrahydrofuran is stirred for 10 minutes at room temperature. 128 mg (0.90 mmol) of methyliodide in 0.5 mL THF are added and the reaction is stirred 1 h. Methanol is added and the solvent is evaporated under reduced pressure. To the crude material 1N hydrochloric acid solution is added and the mixture is extracted with ethyl acetate. The collected organic phases are washed with brine and water, then dried over sodium sulphate and evaporated under reduced pressure. The crude mixture is purified using flash chromatography (silica gel) to afford the title compound.
LC/MS (II) rt 4.46, m/z 201 (M+H-CH3)+.
A solution of 51 mg (0.24 mmol) of the product from step 1 in 1 mL of trifluoroacetic acid and 2 mL of dichloromethane is stirred at room temperature for 1 h and then evaporated under reduced pressure. The product is isolated in the form of a TFA-salt.
1H-NMR δ (ppm)=1.50-1.65 (m, 1H), 1.80-2.10 (m, 3H), 3.05-3.25 (m, 2H), 3.30 (s, 3H), 3.40-3.46 (m, 1H), 3.51-3.56 (m, 1H), 3.60-3.75 (m, 1H), 8.55 (s, 3H), 9.18 (s, 3H).
To a solution of 100 mg (0.50 mmol) of (2S)-hydroxymethyl-pyrrolidine-1-carboxylic acid tert-butyl ester in 500 μL tetrahydrofuran is added sodium hydride (40 mg, 60% dispersion in oil, 0.99 mmol) and the mixture is stirred for 10 minutes. (Bromomethyl)cyclopropane (202 mg, 1.49 mmol) is added and the reaction is heated in the microwave for 10 minutes at 100° C. Ethyl acetate is added and the mixture is washed with brine (3×), then dried over sodium sulphate and evaporated under reduced pressure. The crude mixture is purified using flash chromatography (silica gel, eluent: 10% ethyl acetate in cyclohexane) to afford the title compound.
LC/MS (II) rt 4.55, m/z 256 (M+H-CH3)+.
Obtained from the product of step 1 according to the procedure described for steps 2 in Example 1.
To 1.02 g (1.12 mmol) of polymer bound triphenylphosphine in 5 mL dichloromethane 156 mg (0.89 mmol), diethylazodicarboxylate (DEAD) is added at 0° C. and the mixture is stirred for 5 minutes. To the mixture a solution of 150 mg (0.75 mmol) of Boc-L-prolinol, 70 mg (0.75 mmol) of phenol and 116 μl (1.13 mmol) of triethylamine in 2 mL dichloromethane are added and the reaction is allowed to warm to room temperature and stirred over 60 h. The polymer is filtered off and the solution is evaporated under reduced pressure. The crude mixture is purified using flash chromatography (silica gel, eluent: 0% to 20% ethyl acetate in cyclohexane) to afford the title compound.
LC/MS (III) rt 5.68, m/z 263 (M+H-CH3)+.
Obtained from the product of step 1 according to the procedure described for steps 2 in Example 1.
1H-NMR δ (ppm)=1.65-1.80 (m, 1H), 1.86-2.03 (m, 2H), 2.07-2.18 (m, 1H), 3.05-3.15 (m, 2H), 3.90 (bs, 1H), 4.07 (dd, 1H), 4.23 (dd, 1H), 6.93-6.97 (m, 3H), 7.26-7.32 (m, 2H), 8.72 (bs, 1NH), 9.27 (bs, 1NH).
LC/MS (III) rt 2.77, m/z 178 (M+H)+.
The compounds in Table 2 are synthesized according to the procedure shown for example 13.
1H-NMR δ (ppm) = 1.68-1.82 (m, 1H), 1.86-2.03(m, 2H), 2.08-2.21 (m,1H), 3.16-3.30 (m, 2H),3.87-4.01 (m, 2H), 4.17(dd, 1H), 4.34 (dd, 1H),7.11 (d, 2H), 7.76 (d, 2H),8.70-8.89 (br s, 1NH),9.23-9.43 (br s, 1NH).
1H-NMR δ (ppm) = 1.66-1.81 (m, 1H), 1.82-2.04(m, 2H), 2.06-2.21 (m,1H), 3.16-3.30 (m, 2H),3.85-4.02 (m, 2H), 4.14(dd, 1H), 4.32 (dd, 1H),7.05-7.56 (m, 4H), 8.71-8.87 (br s, 1NH), 9.20-9.39 (br s, 1NH).
1H-NMR δ (ppm) = 1.77-1.97 (m, 2H), 1.99-2.23(m, 2H), 3.19-3.32 (m,2H), 3.94-4.09 (m, 2H),4.29 (dd, 1H), 4.38 (dd,1H), 7.12 (t, 1H), 7.23 (d,1H), 7.60-7.77 (m, 2H),8.75-8.95 (br s, 1NH),9.17-9.33 (br s, 1NH).
1H-NMR δ (ppm) = 1.66-1.81 (m, 1H), 1.84-2.03(m, 2H), 2.06-2.20 (m,1H), 3.22-3.28 (m, 2H),3.77-3.97 (m, 2H), 4.05(dd, 1H), 4.20 (dd, 1H),6.93-7.00 (m, 2H), 7.07-7.16 (m, 2H), 8.64-8.80(br s, 1NH), 9.17-9.32(br s, 1NH).
1H-NMR δ (ppm) = 1.63-1.81 (m, 1H), 1.84-2.03(m, 2H), 2.06-2.18 (m,1H), 3.22-3.31 (m, 2H),3.74-3.98 (m, 2H), 4.08(dd, 1H), 4.25 (dd, 1H),6.74-6.87 (m, 3H), 7.32(q, 1H), 8.70-8.82 (br s,1NH), 9.16-9.35 (br s,1NH).
1H-NMR δ (ppm) = 1.68-1.80 (m, 1H), 1.86-2.03(m, 2H), 2.06-2.20 (m,1H), 3.15-3.26 (m, 2H),3.84-3.99 (m, 2H), 4.33(dd, 1H), 4.50 (dd, 1H),6.83 (d, 1H), 7.00 (dd,1H), 7.70-7.77 (m, 1H),8.14 (dd, 1H), 8.60-8.77(br s, 1NH), 9.07-9.27 (brs, 1NH).
To a solution of 150 mg (0.75 mmol) of (2S)-aminomethyl-pyrrolidine-1-carboxylic acid tert-butyl ester and 117 μL (90.0 mg, 0.90 mmol) of triethylamine in 3 mL of dichloromethane is added 62 μL (85.7 mg, 0.80 mmol) of benzenesulfonylchloride at 0° C. The mixture is stirred for 1.5 h at room temperature and then evaporated under reduced pressure to give a crude mixture containing approx. 70% of the title compound, which is taken directly to the next step.
LC/MS (I) rt 4.34, m/z 241 (M-+H-Boc)+.
A solution of 231 mg (approx. 70% purity, 0.47 mmol) of (2S)-(benzenesulfonylaminomethyl)-pyrrolidine-1-carboxylic acid tert-butyl ester (Step 1) in 1.5 mL of dichloromethane and 0.5 mL of trifluoroacetic acid is stirred at room temperature for 2 h and then evaporated under reduced pressure. The oily product is diluted in 5 mL of dichloromethane and filtered through aluminium oxide (eluent: 0% to 10% methanol in dichloromethane). The collected fractions are concentrated to give the title compound.
1H-NMR δ (ppm)=1.53-1.65 (m, 1H), 1.81-2.05 (m, 3H), 2.91-3.16 (m, 5H), 3.50-3.55 (m, 1H), 7.27-7.29 (m, 1H), 7.58-7.60 (m, 2H), 7.78-7.80 (m, 2H), 7.99 (t, J=7.6 Hz, 1H), 9.15 (bs, 1H).
LC/MS (I) rt 2.11, m/z 241 (M+H)+.
The compounds in Table 3 are synthesized according to the procedure shown for Example 20.
To a solution of 45 mg (0.15 mmol) of 2-(cyclopropanesulfonylamino-methyl)-pyrrolidine-1-carboxylic acid tert-butyl ester (step 1, example 18) in 1 mL of tetrahydrofuran, 7.1 mg (0.30 mmol) of sodium hydride in 0.5 mL THF is added and the reaction is stirred 5 minutes. 14 μl (0.22 mmol) of methyliodide are added slowly and reaction is stirred overnight. The solvent is evaporated under reduced pressure, the crude material is dissolved in ethyl acetate and washed sequentially with 5% aqueous citric acid solution and saturated aqueous sodium bicarbonate solution, and brine, dried over sodium sulphate and the solvent is removed under vacuum. The crude material is used without further purification in the next step.
LC/MS (V) (5-90%, 5 min): rt 2.85, m/z 382 (M+H+Na+AcCN)+.
Obtained from the product of step 1 according to the procedure described for steps 2 in Example 1.
LC/MS (V) (5-90%, 5 min): rt 0.22, m/z 241 (M+H+Na)+.
A solution of 150 mg (0.75 mmol) of (2S)-aminomethyl-pyrrolidine-1-carboxylic acid tert-butyl ester and 86 μL (93.7 mg, 0.79 mmol) of phenyl isocyanate in 3 mL of dioxan is stirred at 90° C. for 5 h. After evaporation of the solvent under reduced pressure, the crude mixture (approx. 50% content of title product) is used in the next step without further purification.
LC/MS (III) rt 4.18, m/z 320 (M+H)+.
A solution of 253 mg (approx. 0.37 mmol) of (2S)-[(3-phenyl-ureido)-methyl]-pyrrolidine-1-carboxylic acid tert-butyl ester (step 1) in 0.5 mL of trifluoroacetic acid and 1.0 mL of dichloromethane is stirred at room temperature for 2 h and then evaporated under reduced pressure. The crude mixture is dissolved in 2 mL of a 1M ammonia solution in methanol, concentrated under reduced pressure and then purified using flash chromatography (aluminium oxide, eluent: 0% to 10% methanol in dichloromethane containing 0.1% of ammonia) to give the title compound.
1H-NMR δ (ppm)=1.35-1.40 (m, 1H), 1.78-1.81 (m, 5H), 2.85-2.84 (m, 2H), 3.02-3.22 (2H), 4.35 (bs, 2H), 6.38 (bs, 1H), 6.83 (t, J=10.0 Hz, 1H), 7.16 (t, J=10.0 Hz, 2H), 7.35 (d, J=10.0 Hz, 2H), 8.65 (bs, 0.1H), 8.77 (bs, 0.9H).
LC/MS (I) rt 1.88, m/z 220 (M+H)+.
To a solution of 100 mg (0.99 mmol) 3-azetidine carboxylic acid in 15 mL THF is added 5 mL of saturated sodium bicarbonate solution and 238 mg (1.09 mmol) di-tert-butyl dicarbonate. The mixture is stirred overnight at room temperature, acidified with 5% aqueous hydrochloric acid and extracted three times with ethyl acetate. The combined organic layers are washed with brine and dried over sodium sulphate. Removal of the solvent in vacuum yields the product that was used without further purification for the next step.
LC/MS (II) rt 2.08, m/z 187 (M+H-CH3)+.
A mixture of 212 mg (0.99 mmol) of the crude material from step 1 and 241 mg (1.49 mmol) CDI in 15 mL dry tetrahydrofurane is stirred for 2 h at room temperature, then cooled to 0° C. and a suspension of 56 mg (1.49 mmol) sodium borohydride in water added quickly. After another 1 h at 0° C. acetone is added, the mixture allowed to warm to room temperature and the solvent removed. The remaining material is dissolved in ethyl acetate and water, the layers separated and the organic layer washed with 5% citric acid, saturated sodium bicarbonate solution and brine. Drying over sodium sulphate and removal of the solvent affords the alcohol.
LC/MS (II) rt 1.81, m/z 173 (M+H-CH3)+.
To a solution of 94 mg (0.5 mmol) Boc protected hydroxymethyl azetidine (step 2) in 5 mL of THF was added 354 mg (0.5 mmol) fluorous triphenyl phosphine and 47 mg (0.5 mmol) phenol. The mixture was cooled down to 0° C. and 405 mg (0.5 mmol) fluorous diethyl azodicarboxylate (DEAD) was added and allowed for warm up to room temperature. The reaction was stirred for 3 days, evaporated to dryness over 1 g of alumina. Alumina containing the reaction product was placed over fluorous silica cartridge and washed with methanol:water 4:1 eluent (4×1 mL). The filtrate was concentrated under reduced pressure and subjected to preparative TLC (silica, hexanes:ethyl acetate 1:1) to afford the title product.
LC/MS (II) rt 1.89, m/z 164 (M+H-Boc)+.
A solution of 34.0 mg (0.13 mmol) of 3-phenoxymethyl-azetidine-1-carboxylic acid tert-butyl ester (step 3) in 300 μL of trifluoroacetic acid and 300 μL of dichloromethane is stirred at room temperature for 30 minutes and then evaporated under reduced pressure to give the title compound.
Obtained from 3-hydroxymethyl-azetidine-1-carboxylic acid tert-butyl ester and pyridin-2-ol according to the procedure described for steps 3 and 4 in Example 28.
LC/MS (II) rt 0.25, m/z 165 (M+H)+.
To 170 mg (0.90 mmol) of 3-hydroxymethyl-azetidine-1-carboxylic acid tert-butyl ester in 10 mL dry dichloromethane 155 μl (1.08 mmol) triethylamine and 75 μl (0.99 mmol) methanesulfonic acid chloride are added at 0° C. After 4 h at 0° C. dichloromethane (50 mL) is added and the organic layer washed twice with brine. The organic layer is dried over sodium sulphate and the solvent removed, yielding crude material that is directly taken to the next step.
LC/MS (II) rt 2.35, m/z 251 (M+H-CH3)+.
A mixture of 3-methanesulfonyloxymethyl-azetidine-1-carboxylic acid tert-butyl ester (266 mg, 0.90 mmol, step 1) and 176 mg (2.70 mmol) sodium azide in 10 mL dry N,N-dimethylformamide is heated to 90° C. for 1 h. For workup 60 mL of ethyl acetate are added and the organic layer is washed thoroughly with brine (3×), dried over sodium sulphate and concentrated under reduced pressure. Purification by flash chromatography on silica gel (cyclohexane to 20% ethylacetate in cyclohexane) yields the azide.
LC/MS (II) rt 2.57, m/z 198 (M+H-CH3)+.
73 mg (0.35 mmol) of 3-azidomethyl-azetidine-1-carboxylic acid tert-butyl ester (step 2) dissolved in 20 mL methanol, 1 mL ammonia (2M in MeOH) and Pd/C (5% with 50% water) added and the mixture stirred at 1 atm H2 for 1 h. Filtration over Celite and evaporation of the solvent affords the crude amine that is taken directly to the next step.
LC/MS (IV) rt 1.75, m/z 172 (M+H-CH3)+.
39 mg (0.21 mmol) of 3-aminomethyl-azetidine-1-carboxylic acid tert-butyl ester (step 3) and 32 μl (0.25 mmol) triethylamine are dissolved in dichloromethane and 17 μl (0.23 mmol) of benzenesulfonylchloride added at 0° C. The reaction mixture is subsequently stirred for 1 h and diluted with dichloromethane. The organic layer is washed with 5% citric acid, saturated sodium bicarbonate solution and brine and dried over sodium sulphate. The crude product is purified by flash chromatography on silica gel (cyclohexane to 20% ethyl acetate in cyclohexane).
LC/MS (IV) rt 2.64, m/z 312 (M+H-CH3)+.
Obtained from the product of step 4 according to the procedure described for step 2 in example 1.
LC/MS (IV) rt 1.73, m/z 227 (M+H)+.
Obtained from 1-Boc-piperidine-3-carboxylic acid according to the procedure described for example 30.
LC/MS (IV) rt 1.87, m/z 255 (M+H)+.
A mixture of 44.8 mg (0.15 mmol) of (3R)-tert-butoxycarbonylamino-4-[2-fluoro-phenyl]-butyric acid, 28.3 mg (0.21 mmol) of 1-hydroxybenzotriazole (HOBt), 39.9 mg (0.21 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 100 μL (98.2 mg, 0.76 mmol) of diisopropylethylamine in 2.5 mL of N,N-dimethylformamide is stirred for 5 minutes. After addition of 50.0 mg (0.17 mmol) of (2S)-benzyloxymethylpyrrolidine (Example 10) in 0.5 mL of N,N-dimethylformamide, the mixture is stirred for further 16 h. The solution is diluted with 5 mL of 1N hydrochloric acid solution and extracted twice with 10 mL of dichloromethane. The collected organic phases are washed with brine and water, dried over sodium sulphate and evaporated under reduced pressure. The residue is purified using flash chromatography (silica gel, eluent: 0% to 10% methanol in dichloromethane) to afford the title compound.
LC/MS (I) rt 5.68, m/z 471 (M+H)+.
A solution of 8.00 mg (0.017 mmol) of {(3R)-[(2S)-benzyloxymethyl-pyrrolidin-1-yl]-1-(2-fluoro-benzyl)-3-oxo-propyl}]-carbamic acid tert-butyl ester (Step 1) in 0.5 mL of trifluoroacetic acid and 1 mL of dichloromethane is stirred at room temperature for 1 h and then evaporated under reduced pressure. The crude mixture is purified using HPLC (eluent: 5% to 95% acetonitrile in water with 0.1% of trifluoroacetic acid) to afford the title compound.
1H-NMR δ (ppm)=1.79-1.87 (m, 3H), 2.85-2.92 (m, 1H), 2.98-3.05 (m, 1H), 3.21-3.32 (m, 5H), 3.43-3.47 (m, 1H), 3.69 (bs), 3.93-3.95 (m, 0.3H), 4.05-4.10 (m, 0.7H), 4.41-4.45 (m, 3H), 7.11-7.18 (m, 2H), 7.21-7.32 (m, 7H), 7.94 (bs, 2H).
LC/MS (I) rt 3.60, m/z 371 (M+H)+.
The compounds in Table 4 are synthesized according to the procedure shown for example 32.
1H-NMR δ (ppm) = 1.68-1.94 (m, 4H), 2.49-2.58(m, 1H), 2.62-3.06 (m,3H), 3.13-3.45 (m, 7H),3.65-3.79 (m, 1H), 3.93-4.08 (m, 1H), 7.10-7.19(m, 2H), 7.26-7.36 (m,2H), 7.90-8.02 (br s, 3H).
1H-NMR δ (ppm) = 1.81-2.06 (m, 4H), 2.52-2.62(m, 1H), 2.71-3.09 (m,3H), 3.30-3.43 (m, 2H),3.67-3.83 (m, 1H), 3.85-4.10 (m, 2H), 4.13-4.27(m, 1H), 7.02-7.12 (m,4H), 7.23-7.33 (m, 2H),7.67-7.78 (m, 2H), 7.90-8.06 (br s, 3H).
1H-NMR δ (ppm) = 1.76-2.06 (m, 4H), 2.50-2.61(m, 1H), 2.69-3.07 (m,3H), 3.20-3.40 (m, 2H),3.65-4.11 (m, 3H), 4.15-4.27 (m, 1H), 6.85-6.98(m, 3H), 7.10-7.42 (m,6H), 7.98 (br s, 3H).
1H-NMR δ (ppm) = 1.83-2.07 (m, 4H), 2.48-2.58(m, 1H), 2.70-3.08 (m,3H), 3.24-3.45 (m, 2H),3.70-3.80 (m, 1H), 3.85-4.08 (m, 2H), 4.15-4.29(m, 1H), 7.08-7.20 (m,2H), 7.22-7.50 (m, 6H),7.89-8.05 (br s, 3H).
1H-NMR δ (ppm) = 1.88-2.07 (m, 4H), 2.48-2.59(m, 1H), 2.68-3.08 (m,3H), 3.28-3.48 (m, 2H),3.67-3.79 (m, 1H), 3.98-4.30 (m, 3H), 6.99-7.38(m, 6H), 7.53-7.72 (m,2H), 7.87-8.00 (br s, 3H).
1H-NMR δ (ppm) = 1.76-2.06 (m, 4H), 2.49-2.57(m, 1H), 2.68-3.08 (m,3H), 3.22-3.42 (m, 2H),3.70-4.00 (m, 3H), 4.11-4.27 (m, 1H), 6.85-6.94(m, 2H), 6.99-7.20 (m,4H), 7.24-7.33 (m, 2H),7.92-8.04 (br s, 3H).
1H-NMR δ (ppm) = 1.85-1.97 (m, 4H), 2.74-2.99(m, 4H), 3.25-3.40 (m,2H), 3.66-3.81 (m, 1H),4.11-4.34 (m, 3H), 6.72-6.82 (dd, 1H), 6.88-7.00(m, 1H), 7.08-7.19 (m,2H), 7.24-7.36 (m, 2H),7.60-7.72 (m, 1H), 7.89-8.01 (br s, 3H), 8.04-8.13(m, 1H).
1H-NMR δ (ppm) = 1.78-2.07 (m, 4H), 2.49-2.58(m, 1H), 2.69-3.08 (m,3H), 3.21-3.43 (m, 2H),3.69-4.05 (m, 3H), 4.12-4.27 (m, 1H), 6.64-6.80(m, 3H), 7.08-7.36 (m,5H), 7.92-8.04 (br s, 3H).
1H-NMR δ (ppm) = 1.79-2.09 (m, 4H), 2.51-2.54(m, 1H), 2.60-3.01 (m,3H), 3.21-3.47 (m, 2H),3.69-3.91 (m, 2H), 4.00-4.05 (m, 1H), 4.17-4.28(m, 1H), 6.64-6.84 (m,3H), 7.13-7.41 (m, 5H),7.90 (bs, 3H.
1H-NMR δ (ppm) = 0.12-0.16 (m, 2H), 0.41-0.47(m, 2H); 0.95 (m, 1H),1.81 (m, 4H), 2.33 (m,2H), 2.72 (m, 2H3.17-3.43 (m, 7H), 4.01 (m,1H), 7.46-7.53 (m, 2H),8.20 (s, 1H).
Using a procedure similar to those outlined for example 32, the following compounds were prepared.
Obtained from (3R)-tert-butoxycarbonylamino-4-(2-fluoro-phenyl)-butyric acid and N-pyrrolidin-(2S)-ylmethyl-benzamide (Example 1) according to the procedure described for step 1 in example 32.
LC/MS (II) rt 2.99, m/z 506 (M+Na)+.
Obtained from the product of step 1 according to the procedure described for step 2 in example 32.
1H-NMR δ (ppm)=1.75-1.95 (m, 5H), 2.78-3.20 (m, 3H), 3.32-3.37 (m, 2H), 3.69-3.80 (m, 0.5H), 3.90-3.97 (m, 0.2H), 4.18-4.20 (m, 0.4H), 7.12-7.19 (m, 2H), 7.28-7.33 (m, 5H), 7.37-7.52 (m, 2H), 7.73-7.76 (m, 3H), 7.92 (bs, 2H), 8.38-8.66 (m, 0.7H), 8.62-8.66 (m, 0.3H).
LC/MS (II) rt 2.02, m/z 384 (M+H)+.
The compounds in Table 5 are synthesized according to the procedure shown for Example 43.
1H-NMR δ (ppm) = 1.74-2.01 (m, 4H), 1H overlapswith the DMSO signal,2.73-3.19 (m, 3H), (m,3H), 3.21-3.43 (m, 4H),3.61-3.77 (m, 1H), 3.87-3.99 (m, 0.3 H), 4.17-4.27(m, 0.5 H), 7.06-7.16 (m,2H), 7.21-7.31 (m, 2H),7.35-7.55 (m, 2H), 7.71-7.95 (m, 4H), 8.37-8.47(m, 0.5H), 8.62-8.72 (m,0.3H).
1H-NMR δ (ppm) = 1.76-2.01 (m, 4H), 1H overlapswith the DMSO signal,2.77-3.18 (m, 3H), 3.24-3.44 (m, 4H), 3.66-3.82(m, 1H), 3.87-4.01 (m, 0.4H), 4.13-4.27 (m, 0.6 H),7.15-7.54 (m, 5H), 7.71-7.96 (m, 6H), 8.38-8.46(m, 0.5H), 8.62-8.70 (m,0.3H).
1H-NMR δ (ppm) = 1.68-2.00 (m, 4H), 1H overlapswith the DMSO signal,2.73-3.09 (m, 3H), 3.22-3.45 (m, 5H), 3.94-4.02(m, 0.4 H), 4.18-4.27 (m,0.6 H), 7.08-7.20 (m, 2H),7.23-7.43 (m, 2H), 7.51-7.64 (m, 1H), 7.85-8.05(m, 5H), 8.54-8.65 (m,1H), 8.74-8.85 (m, 0.5 H),8.95-9.05 (m, 0.3 H).
1H-NMR δ (ppm) = 1.72-1.98 (m, 4H), 2.51-2.57(m, 1H), 2.78-3.15 (m,3H), 3.22-3.44 (m, 4H),3.69-3.84 (m, 1H), 3.89-4.02 (m, 0.4 H), 4.14-4.25(m, 0.6 H), 7.34-7.64 (m,6H), 7.65-7.77 (m, 3H),7.81-8.01 (m, 3H), 8.38-8.46 (m, 0.6 H), 8.62-8.72(m, 0.3 H).
1H-NMR δ (ppm) = 1.72-1.97 (m, 4H), 2.52-2.58(m, 1H), 2.70-2.91 (m,1H), 2.97-3.19 (m, 3H),3.23-3.41 (m, 3H), 3.74-3.88 (m, 1H), 3.88-3.95(m, 0.4 H), 4.15-4.22 (m,0.6 H), 7.25-7.31 (m, 2H),7.34-7.54 (m, 5H), 8.01(bs, 3H), 8.38-8.44 (m,0.5H), 8.63-8.71 (m,0.3H).
1H-NMR δ (ppm) = 1.74-1.98 (m, 4H), 1H overlapswith the DMSO signal,2.76-2.88 (m, 2H), 2.93-3.18 (m, 1H), 3.21-3.42(m, 4H), 3.67-3.76 (m,1H), 3.85-3.95 (m, 0.4 H),4.14-4.26 (m, 0.6 H),7.17-7.36 (m, 5H), 7.37-7.53 (m, 3H), 7.73-7.93(m, 5H), 8.37-8.43 (m, 0.7H), 8.60-8.68 (m, 0.3 H).
1H-NMR δ (ppm) = 1.75-2.01 (m, 4H), 1H overlapswith the DMSO signal,2.72-3.16 (m, 3H), 3.26-3.44 (m, 4H), 3.66-3.80(m, 1H), 3.92-3.99 (m, 0.4H), 4.16-4.22 (m, 0.6 H),7.03-7.13 (m, 1H), 7.28-7.54 (m, 5H), 7.71-7.93(m, 5H), 8.37-8.47 (m, 0.7H), 8.62-8.70 (m, 0.3 H).
A mixture of 23 mg (0.08 mmol) of (3R)-tert-butoxycarbonylamino-4-[3-chloro-phenyl]-butyric acid, 34.2 mg (0.09 mmol) O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), and 39.3 μL (0.22 mmol) of diisopropylethylamine in 2.5 mL of N,N-dimethylformamide is stirred for 10 minutes at room temperature. 25.4 mg (0.09 mmol) of 2-methanesulfonyl-N-pyrrolidin-2-ylmethyl-benzamide (example 2) and 19.6 μL (0.11 mmol) of diisopropylethylamine in 1 mL of N,N-dimethylformamide is added to the solution and the mixture is stirred overnight. The solvent is evaporated under reduced pressure. The crude material is dissolved in ethyl acetate and washed sequentially with 5% citric acid aqueous solution and saturated aqueous sodium bicarbonate solution, dried over sodium sulphate and the solvent is removed under reduced pressure. The crude product is used in the next step without further purification.
Obtained from the product of step 1 according to the procedure described for steps 2 in Example 32.
LC/MS (8 min 10-70%) rt 3.13, m/z 478 (M+H)+.
The compounds in Table 6 are synthesized according to the procedure shown for Example 51.
1H-NMR δ (ppm) = 1.77-1.97 (m, 4H), 2.51-2.57(m, 1H), 2.79-3.04 (m,2H), 3.22 (s, 3H), 3.28-3.43 (m, 3H), 3.68-3.82(m, 1H), 3.90-3.98 (m, 0.3H), 4.19-4.26 (m, 0.7 H),7.16-7.38 (m, 4H), 7.67-7.78 (m, 1H), 7.84-7.93(m, 3H), 8.01-8.20 (m,2H), 8.29 (m, 0.7H), 8.35(m, 0.3H), 8.69-8.75 (m,0.7H), 8.93-9.01 (m,0.3H).
1H-NMR δ (ppm) = 0.57-0.76 (m, 4H), 1.45-1.58(m, 1H), 1.73-1.92 (m,4H), 2.70-2.72 (m, 1H),2.79-2.99 (m, 3H), 3.113.39 (m, 4H), 3.66-3.83(m, 1H), 3.94-4.20 (m,1H), 7.16-7.21 (m, 1H),7.27-7.39 (m, 3H), 7.79-7.90 (bs, 3H), 7.95-8.00(m, 0.7H), 8.19-8.28 (m,0.3H).
2-Aminomethyl-pyrrolidine-1-carboxylic acid tert-butyl ester (200 mg, 1.00 mmol) is dissolved in 1 mL methanol. Triethylamine (113 μl, 1.10 mmol) and trifluoroacetic acid anhydride (210 mg, 0.99 mmol) are added sequentially and the reaction is stirred at room temperature overnight. The solvent is evaporated under reduced pressure and the crude material is purified by flash chromatography (silica gel, eluent: 0% to 30% ethyl acetate in cyclohexane) to afford the title compound.
LC/MS (IV) rt 2.81, m/z 282 (M+H-CH3)+.
Obtained from the product of step 1 according to the procedure described for step 2 in example 1.
1H-NMR δ (ppm)=1.62-1.68 (m, 1H), 1.82-2.10 (m, 3H), 3.13-3.35 (m, 2H), 3.43-3.65 (m, 3H), 8.50 (bs, 1NH), 9.11 (bs, 1NH), 9.60 (bs, 1H).
LC/MS (IV) rt 1.15, m/z 197 (M+H)+.
Obtained from the product of step 3 and 3-tert-butoxycarbonylamino-4-(2-fluoro-phenyl)-butyric acid according to the procedure described for step 1 in example 32.
LC/MS (IV) rt 3.05, m/z 498 (M+Na)+.
(1-(2-Fluoro-benzyl)-3-oxo-3-{2-[(2,2,2-trifluoro-acetylamino)-methyl]-pyrrolidin-1-yl}-propyl)-carbamic acid tert-butyl ester (Step 3, 155 mg, 0.33 mmol) is dissolved in 1 mL methanol and 2 mL of a 0.4 N barium hydroxide solution are added. The reaction is stirred overnight at room temperature. The solvent is evaporated under reduced pressure, water is added and the crude material is extracted with dichloromethane. The solvent is evaporated and the crude material is redissolved in a mixture methanol/dichloromethane, dried over sodium sulphate and the solvent is evaporated under reduced pressure. The crude material is used in the next step without further purification.
1H-NMR δ (ppm)=1.28 (m, 9H), 1.70-1.95 (m, 4H), 2.32-2.50 (m, 2H), 2.60-2.90 (m, 3H), 3.10-3.50 (m, 4H), 4.00-4.15 (m, 1H), 6.63 (bs, 1H), 7.03-7.08 (m, 2H), 7.18-7.22 (m, 2H).
LC/MS (IV) rt 2.27, m/z 380 (M+H)+.
Obtained from the product of step 4 and 3-methoxy-benzoyl chloride according to the procedure described for steps 1 in Example 32.
LC/MS (II) rt 2.97, m/z 536 (M+H+Na)+.
Obtained from the product of step 5 according to the procedure described for steps 2 in Example 1.
LC/MS (II) rt 2.09, m/z 414 (M+H)+.
The compounds in Table 7 are synthesized according to the procedure shown for Example 54.
1H-NMR δ (ppm) = 1.75-1.90 (m, 4H), 2.76-3.41(m, 8H), 2H overlap withthe water signal, 7.11-7.32 (m, 6H), 7.43-7.60(m, 2H), 7.95 (bs, 3H),8.24 (bs, 0.7 H), 8.47 (bs,0.3H).
1H-NMR δ (ppm) = 1.68-2.03 (m, 4H), 2.69-3.16(m, 4H), 3.24-3.41 (m,4H), 3.65-3.84 (m, 1H),3.87-3.99 (m, 0.3 H),4.11-4.28 (m, 0.7 H),7.08-7.21 (m, 2H), 7.25-7.52 (m, 3H), 7.69-7.80(m, 2H), 7.84-8.03 (m,3H), 8.65-8.80 (m, 2.5 H),8.95-9.00 (m, 0.3H).
1H-NMR δ (ppm) = 1.71-2.03 (m, 4H), 1H overlapswith the DMSO signal,2.67-3.08 (m, 3H), 3.20-3.40 (m, 4H), 2H) overlapwith the water signal7.09-7.21 (m, 2H), 7.26-7.39 (m, 2H), 7.41-7.54(m, 1H), 7.86-8.02 (m,3H), 8.06-8.20 (m, 1H),8.56-8.64 (m, 0.5H), 8.64-8.72 (m, 1H), 8.80-8.87(m, 0.3H), 8.89-9.01 (m,1H).
1H-NMR δ (ppm) = 1.72-2.02 (m, 4H), 1H overlapswith the DMSO signal,2.68-3.12 (m, 3H), 3.22-3.45 (m, 7H), 3.65-4.26(m, 2H), 7.09-7.21 (m,2H), 7.23-7.34 (m, 2H),7.44-7.58 (m, 1H), 7.61-7.77 (m, 2H), 7.89-8.01(m, 3H), 8.46-8.53 (m, 0.5H), 8.71-8.80 (m, 0.2H).
A mixture of 70.0 mg (0.21 mmol) of (3R)-tert-butoxycarbonylamino-4-[2-fluoro-phenyl]-butyric acid, 31.3 mg (0.23 mmol) of 1-hydroxybenzotriazole, 45.0 mg (0.23 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 56 μL (0.31 mmol) of diisopropylethylamine in 1 mL of dichloromethane is stirred for 30 minutes at 0° C. After addition of 87.0 mg (0.26 mmol) of cyclopropanecarboxylic acid (pyrrolidin-2-ylmethyl)-amide (Example 4) in 1 mL of dichloromethane and another 56 μL (0.31 mmol) of diisopropylethylamine, the mixture is stirred overnight at room temperature. The solution is diluted with dichloromethane, washed sequentially with 5% citric acid aqueous solution, saturated aqueous sodium bicarbonate solution, and brine, dried over sodium sulphate and the solvent is removed under vacuum. Purification of the crude product by flash chromatography (silica gel, eluent: 5% to 10% of ethyl acetate in cyclohexane) gives the title compound.
A solution of the product from step 1 in 30% trifluoroacetic acid in dichloromethane is stirred at 0° C. for 1 h and then 1 mL methanol is added. The solvent is evaporated under reduced pressure. The crude mixture is dissolved in dichloromethane and the solvent is removed under reduced pressure. This procedure is repeated 3-4 times. The crude material is purified using HPLC (eluent: 5% to 95% acetonitrile in water with 0.1% of trifluoroacetic acid) to afford the title compound.
1H-NMR δ (ppm)=0.60-0.66 (m, 4H), 1.45-1.54 (m, 1H), 1.70-1.90 (m, 4H), 2.75 (m, 1H), 2.81-3.00 (m, 2H), 3.12-3.21 (m, 2H), 3.45-3.49 (m, 3H), 3.68-3.81 (m 1.5H), 3.98 (m, 0.7H), 7.43-7.62 (m, 2H), 8.10 (bs, 0.6H), 8.14 (s, 0.7H), 8.22 (s, 0.2H), 8.38 (bs, 0.4H)
LC/MS (10 min, 1-30%) rt 6.81, m/z 384 (M+H)+.
The compounds in Table 8 are synthesized according to the procedure shown for example 63.
1H-NMR δ (ppm) = 1.15-1.30 (m, 4H), 1.64-1.88(m, 4H), 2.42 (m, 1H),2.61-2.68 (m, 1H), 2.82(m, 2H), 2.90-3.00 (m,1H), 3.12-3.38 (m, 3H),3.52-3.58 (m, 1.3H), 3.86(m, 0.5H), 4.10 (m, 0.8H),7.43-7.55 (m, 2H), 7.95(bs, 0.6H), 8.10 (bs,0.4H), 8.22 (s, 1H).
1H-NMR δ (ppm) = 0.61-0.76 (m, 4H), 1.66-2.10(m, 5H), 2.54 (m, 2H),2.75-2.85 (m, 4H), 3.13(m, 3H), 3.52 (m, 3H),4.20 (m, 1H), 7.44-7.55(m, 2H), 8.17 (s, 0.7H).
Obtained from (3R)-tert-butoxycarbonylamino-4-(2-fluoro-phenyl)-butyric acid and phenyl-pyrrolidin-(2S)-ylmethyl-amine according to the procedure described for step 1 in example 32.
LC/MS (III) rt 4.16, m/z 455 (M+H)+.
Obtained from the product of step 1 according to the procedure described for step 2 in example 32.
1H-NMR δ (ppm)=1.76-1.90 (m, 6H), 2.77-3.35 (m, 10H), 3.70-3.79 and 4.10-4.15 (2m, 1H), 4.90 (bs), 6.42-6.47 (2m, 1H), 6.54-6.61 (m, 2H), 6.95-7.03 (m, 1H), 7.13-7.20 (m, 2H), 7.30-7.35 (m, 2H), 7.96 (bs, 2H).
LC/MS (III) rt 2.84, m/z 354 (M+H)+.
The compounds in Table 9 are synthesized according to the procedure shown for Example 66.
1H-NMR δ (ppm) = 1.752.09 (m, 8H), 2.52-2.67(m, 2H), 2.62-3.16 (m,6H), 3.29-3.43 (m, 2H),2.52-3.83 (m, 3H), 4.17-4.30 (m, 1H), 7.17-7.22(m, 1H), 7.27-7.41 (m,3H), 7.95 (bs, 3H), 8.30(bs, 1H).
20 mg (0.05 mmol) [3-(2-aminomethyl-pyrrolidin-1-yl)-1-(2-fluoro-benzyl)-3-oxo-propyl]-carbamic acid tert-butyl ester (step 4, example 50) and 25 μl (0.16 mmol) diisopropylamine are dissolved in 1 mL NMP. 21 mg (0.16 mmol) of 6-chloronicotinonitrile are added at room temperature. The reaction mixture is stirred for 3 hours at room temperature and for 3 hours at 80° C. After cooling to room temperature the solvent is evaporated under reduced pressure and the crude product is purified by flash chromatography on silica gel (2% methanol in dichloromethane).
LC/MS (II) rt 2.85, m/z 482 (M+H)+.
The product of step 1 is dissolved in 1 mL of 4N hydrochloric acid in dioxane. The solution is stirred for 1 hour at room temperature and the solvent is evaporated under reduced pressure. The crude material is redissolved in methanol and the solvent is evaporated under reduced pressure to give the title compound.
LC/MS (II) rt 2.09, m/z 382 (M+H)+.
The compounds in Table 10 are synthesized according to the procedure shown for Example 68.
1H-NMR δ (ppm) = 1.79-2.01 (m, 4H), 2.34-2.43(m, 2H), 2.80-2.88 (m,1H), 2.92-3.02 (m, 2H),3.20-3.38 (m, 2H), 3.46-3.53 (m, 1H), 3.63-3.76(m, 1H), 3.95-4.05 (m,1H), 7.12-7.18 (m, 2H),7.25-7.34 (m, 2H), 7.68-7.75 (m, 1H), 7.95-8.02(m, 4H), 8.06-8.10 (m,1H), 8.20-8.32 (bs, 2H).
Obtained from (3R)-tert-butoxycarbonylamino-4-(2-fluoro-phenyl)-butyric acid and N-pyrrolidin-(2S)-ylmethyl-benzensulfonamide (Example 20) according to the procedure described for step 1 in example 32.
LC/MS (III) rt 4.98, m/z 542 (M+Na)+.
Obtained from the product of step 1 according to the procedure described for step 2 in example 32.
1H-NMR δ (ppm)=1.75-1.85 (m, 4H), 2.48-2.49 (m, 1H), 2.62-2.72 (m, 1H), 2.72-3.04 (m, 3H), 3.22-3.28 (m, 2H), 3.64-3.75 (m, 1H), 3.90-3.94 (m, 0.7H), 4.70 (bs), 7.09-7.15 (m, 2H), 7.21-7.31 (m, 2H), 7.50-7.66 (m, 4H), 7.70-7.77 (m, 2H), 7.89 (bs, 1H).
LC/MS (III) rt 3.16, m/z 442 (M+Na)+.
The compounds in Table 11 are synthesized according to the procedure shown for Example 70.
1H-NMR δ 1.70-1.91 (m, 4H), 2.64-3.05(m, 3H), 3.42-3.55 (m,2H), 3.63-3.71 (m, 3H),3.86-4.11 (m, 1.5 H),4.50-4.61 (m, 1H), 7.12-7.36 (m, 4H), 7.47-7.62(m, 3H), 7.64-7.79 (m,2H), 7.91-8.30 (bs, 2H).
1H-NMR δ (ppm) = 0.90-0.96 (m, 4H), 1.76-1.94(m, 5H), 2.40 (m, 1H),2.74-3.80 (m, 2H), 2.82-2.98 (m, 1H), 3.17-3.36(m, 5H), 3.87-4.00 (m,1H), 7.08 (bs, 0.7H),7.39-7.84 (m, 2H), 8.22(bs, 1H).
1H-NMR δ (ppm) = 1.76-1.92 (m, 4H), 2.38 (m,1H), 2.74 (m, 2H), 2.91(m, 3H), 3.10-3.50 (m,4H), 3.87-3.97 (m, 3H),7.05 (bs, 0.6H), 7.38-7.47(m, 2H), 8.24 (bs, 0.7H).
1H-NMR δ (ppm) = 1.82-1.87 (m, 4H), 2.46-2.51(m, 1H), 2.82-3.10 (m,3H), 3.22-3.50 (m, 4H),3.64-3.74 (m, 1H), 3.88-3.99 (m, 1H), 7.49-7.59(m, 2H), 8.16 (s, 0.4H).
1H-NMR δ (ppm) = 1.82-1.88 (m, 4H), 2.42 (m,1H), 2.82 (m, 1H), 3.00(m, 1H), 3.12-3.58 (m,6H), 3.86-3.99 (m, 1H),4.32-4.45 (m, 2H), 7.42-7.59 (m, 2H), 8.18 (s,0.4H).
15 mg (0.04 mmol) [3-(2-aminomethyl-pyrrolidin-1-yl)-1-(2-fluoro-benzyl)-3-oxo-propyl]-carbamic acid tert-butyl ester (step 4, example 51) and 8 μL (0.06 mmol) triethylamine are dissolved in 1 μL dichloromethane. 11 mg (0.05 mmol) of 3,4-dimethoxy-benzenesulfonyl chloride are added at room temperature. The reaction mixture is stirred overnight at room temperature and the solvent is evaporated under reduced pressure and the crude product is used in the next step without further purification.
Obtained from the product of step 1 according to the procedure described for step 2 in Example 32.
LC/MS (IV) rt 2.21, m/z 480 (M+H)+.
The compounds in Table 12 are synthesized according to the procedure shown for example 78.
1H-NMR δ (ppm) = 1.66-1.90 (m, 4H), 2.58-3.15(m, 5H), 3.15-3.35 (m,2H), 3.62-3.76 (m, 2H),1H overlaps with thewater signal, 7.03-7.13(m, 2H), 7.17-7.35 (m,2H), 7.42-7.65 (m, 4H),7.82-7.89 (m, 0.7H), 8.05-8.22 (bs, 3H).
1H-NMR δ (ppm) = 1.73-1.90 (m, 4H), 2.72-2.91(m, 3H), 2.95-3.13 (m,3H), 3.14-3.31 (m, 1H),3.62-3.77 (m, 2H), 1Hoverlaps with the watersignal, 7.03-7.16 (m, 2H),7.18-7.34 (m, 2H), 7.50-7.73 (m, 3H), 7.80-7.97(m, 3H), 8.07-8.12 (bs,3H).
1H-NMR δ (ppm) = 1.68-1.90 (m, 4H), 1H overlapswith the water signal,2.59-2.76 (m, 1H), 2.82-3.09 (m, 3H), 3.16-3.40(m, 2H), 3.61-3.80 (m,2H), 3.86-3.95 (m, 1H),7.09-7.18 (m, 2H), 7.21-7.52 (m, 5H), 7.66-8.01(m, 5H).
1H-NMR δ (ppm) = 1.68-1.90 (m, 4H), 1H overlapswith the water signal,2.72-2.89 (m, 2H), 2.92-3.09 (m, 2H), 3.16-3.33(m, 2H), 3.65-3.82 (m,2H), 3.85-3.97 (m, 1H),7.03-7.17 (m, 2H), 7.20-7.47 (m, 5H), 7.58-8.02(m, 5H).
Obtained from (3R)-tert-butoxycarbonylamino-4-(2-fluoro-phenyl)-butyric acid and 1-phenyl-3-pyrrolidin-(2S)-ylmethyl urea (Example 27) according to the procedure described for step 1 in Example 32.
LC/MS (III) rt 4.79, m/z 521 (M+Na)+.
Obtained from the product of step 1 according to the procedure described for step 2 in Example 32.
1H-NMR δ (ppm)=1.79-1.83 (m, 4H), 2.27-2.31 (m, 1H), 2.67-2.69 (m, 2H), 3.29-3.37 (m, 5H), 3.87 and 3.97 (2m, 1H), 6.20 (m, 0.6H), 6.40 (m, 0.2H), 6.81-6.86 (m, 1H), 7.06-7.39 (m, 8H), 8.41 (bs, 0.5H), 8.52 (bs, 0.2H).
LC/MS (III) rt 3.12, m/z 421 (M+Na)+.
Obtained from (3R)-tert-butoxycarbonylamino-4-(2-fluoro-phenyl)-butyric acid and N-piperidin-(2S)-ylmethyl-benzamide (example 7) according to the procedure described for Example 32.
1H-NMR δ (ppm)=1.25-1.75 (m, 6H), 2.60-3.05 (m, 4H), 3.25-3.55 (m, 4H partially hidden by water signal), 3.65-3.75 (m, 0.5H), 3.95-4.05 (m, 0.5H), 4.28-4.37 (m, 0.3 H), 4.76-4.84 (m, 0.7H), 7.09-7.52 (m, 7H), 7.64 (m, 1H), 7.76-7.94 (m, 4H), 8.25-8.35 (m, 0.7H), 8.55-8.62 (m, 0.3H).
LC/MS (IV) rt 2.16, m/z 398 (M+H)+.
Obtained from (3R)-tert-butoxycarbonylamino-4-(2-fluoro-phenyl)-butyric acid and N-piperidin-(2S)-ylmethyl-benzenesulfonamide (example 25) according to the procedure described for Example 32.
LC/MS (II) rt 2.26, m/z 434 (M+H)+.
Obtained from (3R)-tert-butoxycarbonylamino-4-(2-fluoro-phenyl)-butyric acid and N-piperidin-3-ylmethyl-benzenesulfonamide (Example 31) according the procedure described for example 32.
1H-NMR δ (ppm)=1.05-1.35 (m, 2H), 1.40-1.75 (m, 3H), 2.24-2.05 (m, 1H), 2.52-2.75 (m, 4H), 2.80-3.20 (m, 3H), 3.50-3.75 (m, 2H), 4.00-4.12 (m, 0.6H), 4.22-4.35 (m, 0.4H), 7.10-7.20 (m, 2H), 7.26-7.35 (m, 2H), 7.50-7.70 (m, 3H), 7.72-7.74 (m, 2H), 7.85 (bs, 3H).
LC/MS (II) rt 2.13, m/z 434 (M+H)+.
Obtained from (3R)-tert-butoxycarbonylamino-4-(2-fluoro-phenyl)-butyric acid and N-azetidin-3-yl-methyl-benzylamide (example 8) according the procedure described for example 32.
1H-NMR δ (ppm)=1.28 (m, 2H), 2.61-2.92 (m, 2H), 2.95-3.02 (m, 1H), 3.43-3.49 (m, 2H), 3.57-3.70 (m, 2H), 3.74-3.91 (m, 2H), 4.01-4.09 (m, 1H), 7.13-7.19 (m, 2H), 7.29-7.32 (m, 2H), 7.38-7.49 (m, 3H), 7.76-7.80 (m, 2H), 7.92 (bs, 3H), 8.51 (m, 1H).
LC/MS (II) rt 1.92, m/z 370 (M+H)+.
Obtained from (3R)-tert-butoxycarbonylamino-4-(2-fluoro-phenyl)-butyric acid N-Azetidin-3-ylmethyl-benzenesulfonamide (Example 30) according to the procedure described for example 32.
1H-NMR δ (ppm)=1.20-2.29 (m, 2H), 2.52-2.64 (m, 1H), 2.82-3.03 (m, 4H), 3.39-3.49 (m, 1H), 3.56-3.80 (m, 3H), 3.91-4.00 (m, 1H), 7.08-7.18 (m, 2H), 7.26-7.33 (m, 2H), 7.52-7.64 (m, 3H), 7.74-7.77 (m, 3H), 8.04 (bs, 3H).
LC/MS (II) rt 1.99, m/z 406 (M+H)+.
A mixture of 33.0 mg (0.11 mmol) of (3R)-tert-butoxycarbonylamino-4-[2-fluoro-phenyl]-butyric acid, 16.0 mg (0.21 mmol) of 1-hydroxybenzotriazole, 23.0 mg (0.12 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 114 μL (0.65 mmol) of diisopropylethylamine in 2.5 mL of DCM is stirred for 5 minutes. After addition of 48.0 mg (0.11 mmol) of 3-phenoxymethyl-azetidine (Example 28), the mixture is stirred overnight The solution is diluted with dichloromethane, washed with a saturated aqueous bicarbonate solution and brine, dried over sodium sulphate and evaporated under reduced pressure. The residue is purified using flash chromatography (silica gel, eluent: 25% cyclohexane in ethyl acetate) to afford the title compound.
LC/MS (115) rt 3.21, m/z 443 (M+H)+.
A solution of 20.0 mg (0.045 mmol) of 1-(2-fluoro-benzyl)-3-oxo-3-(3-phenoxymethyl-azetidin-1-yl)-propyl]-carbamic acid tert-butyl ester in 300 μL of trifluoroacetic acid and 700 μL of dichloromethane is stirred at room temperature for 1 h and then evaporated under reduced pressure to give the title compound.
1H-NMR δ (ppm)=2.32 (d, 2H), 2.86-3.06 (m, 3H), 3.60-3.70 (m, 3H), 3.80-4.00 (m, 2H), 4.01-4.17 (m, 2H), 6.91 (t, 3H), 7.15 (t, 2H), 7.23-7.34 (m, 4H), 8.01 (bs, 3H).
LC/MS (II) rt 2.35, m/z 343 (M+H)+.
The compounds in Table 13 are synthesized according to the procedure shown for Example 92.
1H-NMR δ (ppm) = 2.31(d, 2H), 2.85-3.04 (m,3H), 3.50-4.05 (m, 4H),4.12 (q, 1H), 4.37-4.39(m, 2H), 6.77 (t, 3H),6.94-6.98 (m, 1H), 7.13-7.18 (m, 2H), 7.31-7.34(m, 2H), 7.64-7.71 (m,1H), 7.96 (bs, 3H), 8.09-8.11 (m, 1H).
Obtained from (3R)-tert-butoxycarbonylamino-4-(2-fluoro-phenyl)-butyric acid and N-piperidin-3-ylmethyl-benzylamide (example 9) according to the procedure described for example 32.
Prepared as a mixture of diastereomers.
LC/MS (II) rt 2.14, m/z 398 (M+H)+.
To 10.31 g (0.10 mol) of benzonitrile dissolved in 40 mL methanol is added 20.73 g (0.15 mole) of finely powdered potassium carbonate. To this is added, in small portions with stirring, 13.89 g (0.20 mol) of hydroxylamine hydrochloride dissolved in 120 mL of methanol. The mixture is then refluxed for 5 hours and, after cooling to room temperature, the solvent is removed under reduced pressure. The residue is taken up in 50 mL of water and 200 mL of chloroform. The organic layer is separated, washed twice with 30 mL of water, and dried over magnesium sulphate. The mixture is then filtered and evaporated under reduced pressure. The residue is crystallised with diethyl ether to afford the title compound.
M.P.: 77-79° C.
To 40.05 g (129.7 mmol; 23.7 mL) of trichloroacetic anhydride in a 250 mL round bottom flask protected with a calcium chloride drying tube is added portionwise, with stirring, at room temperature over 20 minutes, 8.82 g (64.80 mmol) of the product from step 1. When addition is complete, the mixture is heated to 90-120° C. for 75 minutes, and the hot mixture is then poured into a stirred ice-water solution. The resulting solid is crystallised with hexane or diethylether to give the title compound.
1H-NMR (300 MHz, CDCl3) δ=7.60-7.45 (m, 3H), 8.15 (m, 2H).
Prepared following the procedure outlined for Example 95 according to Scheme F.
Obtained from pyridine-2-carbonitrile and hydroxylamine hydrochloride according to Step 1 in Example 95.
M.P.: 115-117° C.
Obtained from N-hydroxy-pyridine-2-carboxamide (Example 96, Step 1) according to Step 2 in Example 95.
1H-NMR (300 MHz, DMSO-d6) δ=7.68 (2×dd, 1H), 8.07 (ddd, 1H), 8.14 (dd, 1H), 8.81 (m, 1H).
Prepared following the procedure outlined for Example 95 according to Scheme F.
Obtained from 3-chlorobenzonitrile and hydroxylamine hydrochloride according to Step 1 in Example 95.
M.P.: 115-118° C.
Obtained from 3-chloro-N-hydroxy-benzamidine (Example 97, Step 1) according to Step 2 in Example 95.
1H-NMR (300 MHz, CDCl3) δ=7.44 (dd, 1H), 7.55 (ddd, 1H), 8.04 (ddd, 1H), 8.12 (dd, 1H).
Prepared following the procedure outlined for Example 95 according to Scheme F.
Obtained from 3-fluorobenzonitrile and hydroxylamine hydrochloride according to Step 1 in Example 95.
M.P.: 74-76° C.
Obtained from 3-fluoro-N-hydroxy-benzamidine (Example 98, Step 1) according to Step 2 in Example 95.
1H-NMR (300 MHz, CDCl3) δ=7.26 (m, 1H), 7.51 (m, 1H), 7.75 (m, 1H), 7.93 (m, 1H).
Prepared following the procedure outlined for Example 95 according to Scheme F.
Obtained from 4-methanesulphonyl-benzonitrile and hydroxylamine hydrochloride according to Step 1 in Example 95.
M.P.: 115-118° C.
Obtained from N-hydroxy-4-methanesulphonyl-benzamidine (Example 99, Step 1) according to Step 2 in Example 95.
1H-NMR (300 MHz, CDCl3) δ=3.13 (s, 3H), 8.12 and 8.38 (m, 4H).
Following examples are prepared according to Schemes G and H.
164.40 mg (0.62 mmol) of 3-phenyl-5-trichloromethyl-[1,2,4]oxadiazole (Example 95) and 150.0 mg (0.75 mmol) of (2S)-aminomethyl-pyrrolidine-1-carboxylic acid tert-butyl ester is stirred in 5 mL of dry N,N-dimethylformamide at 60° C. for 12 hours. The progression of the reaction is monitored by TLC (Kieselgel Merck 5554 sheets, eluent: hexane-ethylacetate 2:1). The mixture is evaporated to dryness under reduced pressure and the residue is purified by preparative thin layer-chromatography using the same solvent system to afford the title compound.
1H-NMR (300 MHz, CDCl3) δ=1.45 (s, 9H), 1.62-2.16 (m, 4H), 3.28-3.70 (m, 4H), 4.17 (m, 1H), 7.30 (m, 1H), 7.41 (m, 3H), 7.90 (m, 2H).
The product from Step 1, (2S)-[3-phenyl-[1,2,4]oxadiazol-5-ylamino)-methyl]pyrrolidine-1-carboxylic acid tert-butyl ester, is dissolved in 4 mL of dichloromethane then 8 mL of saturated HCl/dioxane solution is added. After the mixture is stirred for 2 hours the solvent is evaporated under reduced pressure to yield the title compound, which is used directly in the next step without further purification and characterisation.
In a 25 mL round-bottomed flask is stirred for 2 hours under nitrogen a mixture of 74.90 mg (0.38 mmol) of (3R)-tert-butoxycarbonylamino-4-(2-fluoro-phenyl)-butyric and 64.70 mg (0.40 mmol; 1.05 eq.) of 1,1′-carbonyldiimidazole in 5 mL of dry 1,2-dichloroethane. Separately, 106.70 mg, (0.38 mmol) of 3-phenyl-[1,2,4]oxadiazol-5-yl)pyrrolidin-(2S)-ylmethylamine hydrochloride (Example 100, Step 2) and 107.90 mg, (0.83 mmol; 145.0 μL; 2.2 eq.) of N,N-diisopropylethylamine is stirred in 4 mL of dry 1,2-dichloroethane for 15 minutes and this solution is poured into the butyric acid and 1,1′-carbonyldiimidazole reaction mixture prepared above. Stirring is continued overnight at room temperature, then the mixture is boiled for 5 hours. The solution is cooled to room temperature, washed successively with a 5% citric acid solution, saturated sodium hydrogen carbonate solution, water and brine, dried over magnesium sulphate, filtered and the solvent is removed under reduced pressure. The residue is subjected to preparative thin layer chromatography on silica gel (eluent: dichloroethane/ethanol 5:1) to afford the title compound which is taken directly into the next step without further characterisation.
(1R)-(2-fluorobenzyl)-3-oxo-3-{(2S)-[(3-phenyl-[1,2,4]oxadiazol-5-ylamino-methyl]-pyrrolidin-1-yl}-propyl)carbamic acid tert-butyl ester, the product from Step 3, is dissolved in 4 mL of dichloromethane then 10 mL of saturated HCl/dioxane solution is added. The mixture is stirred for 2 hours then is the solvent removed under reduced pressure to yield the title compound. If the residue is a solid it is taken up in diethyl ether and hexane and filtered. Otherwise, if the residue is an oil, this is taken up in 10 mL of dioxane and the solvent is evaporated to dryness. This procedure is repeated two times to afford the title compound.
1H-NMR (300 MHz, CDCl3) δ=1.82-2.00 (m, 4H), 2.66-2.70 (m, 1H), 2.84 (bs, 1H), 3.21 (bs, 1H), 3.33-3.53 (m, 5H), 4.01 (bs, 1H), 4.39 (bs, 1H), 6.92-7.03 (m, 2H), 7.13-7.21 (m, 1H), 7.32-7.47 (m, 3H), 7.58 (bs, 1H), 7.88-7.90 (m, 2H), 7.94-8.06 (m, 1H), 8.66 (m, 3H).
LC/MS (Method VII) m/z 424 [M+H]+.
Prepared according to the procedure above outlined for Example 100 Steps 1 to 4, according to Schemes G and H.
Obtained from 2-(5-trichloromethyl-[1,2,4]oxadiazol-3-yl)-pyridine (Example 96) and (2S)-aminomethyl-pyrrolidine-1-carboxylic acid tert-butyl ester, synthesised according to the procedure for Example 100, Step 1.
1H-NMR (300 MHz, CDCl3) δ=1.45 (s, 9H), 1.60-2.20 (m, 4H), 3.20-3.45 (m, 4H), 4.15 (m, 1H), 7.38 (m, 1H), 7.50 (m, 1H), 7.79 (dd, 1H), 8.08 (dd, 1H), 8.78 (dd, 1H).
Obtained from (2S)-[(3-Pyridin-2-yl-[1,2,4]oxadiazol-5-ylamino)-methyl]-pyrrolidine-1-carboxylic acid tert-butyl ester (Example 101, Step 1), and synthesised according to the procedure for Example 100, Step 2.
Obtained from (3-pyridin-2-yl-[1,2,4]oxadiazol-5-yl)-pyrrolidin-(2S)-ylmethylamine dihydrochloride (Example 101, Step 2) and (3R)-tert-butoxycarbonylamino-4-(2-fluorophenyl)-butyric acid, and synthesised according to Example 100, Step 3.
Obtained from (1R)-(2-fluorobenzyl)-3-oxo-3-{(2S)-[(3-pyridin-2-yl-[1,2,4]oxadiazol-5-ylamino)-methyl]-pyrrolidin-1-yl}-propyl)-carbamic acid tert-butyl ester (Example 101, Step 3), and synthesised according to Example 100, Step 4.
1H-NMR (300 MHz, CDCl3+DMSO-d6) δ=1.88-1.99 (m, 4H), 2.50-2.58 (m, 1H), 2.62-2.68 (m, 1H), 2.97-3.03 (m, 1H), 3.12-3.17 (m, 1H), 3.33-3.40 (m, 3H), 3.50-3.54 (m, 1H), 3.70-3.74 (m, 1H), 4.25-4.27 (m, 1H), 7.04-7.16 (m, 2H), 7.24-7.35 (m, 2H), 7.53-7.58 (m, 1H), 7.95-8.06 (m, 2H), 8.19-8.24 (bs, 3H), 8.43-8.46 (m, 1H), 8.70-8.76 (m, 1H).
LC/MS (Method VII) m/z 425 [M+H]+
Prepared according to the procedure above outlined for Example 100, steps 1-4 according to Schemes G and H.
Obtained from 3-(3-Chlorophenyl)-5-trichloromethyl-[1,2,4]oxadiazole (Example 97) and (2S)-aminomethyl-pyrrolidine-1-carboxylic acid tert-butyl ester, synthesised according to the procedure for Example 100, Step 1.
Obtained from (2S)-{[3-(3-chlorophenyl)-[1,2,4]oxadiazol-5-ylamino]-methyl}-pyrrolidine-1-carboxylic acid tert-butyl ester (Example 102, Step 1), and synthesised according to the procedure for Example 100, Step 2.
1H-NMR (300 MHz, CDCl3+d6-DMSO) δ=1.75-2.20 (m, 4H), 3.20-3.30 (m, 2H), 3.71 (m, 2H), 3.81 (m, 1H), 7.40-7.50 (m, 2H), 7.85 (ddd, 1H), 7.88 (dd, 1H), 8.57 (m, 1H), 9.00 and 9.42 (bm, 2H).
Obtained from [3-(3-chlorophenyl)-[1,2,4]oxadiazol-5-yl]-pyrrolidin-(2S)-ylmethylamine hydrochloride (Example 102, Step 2) and (3R)-tert-butoxycarbonylamino-4-(2-fluorophenyl)-butyric acid, and synthesised according to Example 100, Step 3.
1H-NMR (300 MHz, CDCl3) δ=1.38 (s, 9H), 1.80-2.10 (m, 4H), 2.50 and 2.90 (m, 2×2H), 3.20-3.70 (m, 4H), 4.23 (m, 1H), 4.38 (m, 1H), 5.38 (m, 1H), 7.00-7.25 (m, 4H), 7.32 (bm), 7.35 (m, 1H), 7.42 (m, 1H), 7.87 (ddd, 1H), 7.97 (dd, 1H).
Obtained from [3-(2S)-{[3-(3-chlorophenyl)-[1,2,4]oxadiazol-5-ylamino]-methyl}-pyrrolidin-1-yl)-(1R)-(2-fluorobenzyl)-3-oxo-propyl]-carbamic acid tert-butyl ester (Example 102, Step 3), and synthesised according to Example 100, Step 4.
1H-NMR (300 MHz, CDCl3) δ=1.82-1.99 (m, 4H), 2.67-2.73 (m, 1H), 2.83-2.89 (m, 1H), 3.21-3.25 (m, 1H), 3.39-3.53 (m, 5H), 3.99-4.03 (m, 1H), 4.40-4.42 (m, 1H), 6.93-6.97 (m, 1H), 7.00-7.04 (m, 1H), 7.14-7.19 (m, 1H), 7.21-7.40 (m 3H), 7.59 (bs, 1H), 7.76-7.78 (m, 1H), 7.87 (s, 1H), 8.66 (bs, 3H).
LC/MS (Method VII) m/z 458 [M+H]+
Prepared according to the procedure above outlined for Example 100, steps 1-4, according to Schemes G and H.
Obtained from 3-(3-fluorophenyl)-5-trichloromethyl-[1,2,4]oxadiazole (Example 98) and (2S)-aminomethyl-pyrrolidine-1-carboxylic acid tert-butyl ester, synthesised according to the procedure for Example 100, Step 1.
Obtained from (2S)-{[3-(3-Fluorophenyl)-[1,2,4]oxadiazol-5-ylamino]-methyl}-pyrrolidine-1-carboxylic acid tert-butyl ester (Example 103, Step 1), and synthesised according to the procedure for Example 100, Step 2.
1H-NMR (300 MHz, CDCl3+d6DMSO) δ=1.75-2.20 (m, 4H), 3.10-3.38 (m, 2H), 3.69 (m, 2H), 3.81 (m, 1H), 7.25 (m, 1H), 7.48 (m, 1H), 7.66 (ddd, 1H), 7.78 (dd, 1H), 8.62 (1H, m, NH), 8.90 and 9.42 (bm, 2H).
Obtained from [3-(3-fluorophenyl)-[1,2,4]oxadiazol-5-yl]-pyrrolidin-(2S)-ylmethylamine hydrochloride (Example 103, Step 2) and (3R)-tert-butoxycarbonylamino-4-(2-fluorophenyl)-butyric acid, and synthesised according to Example 100, Step 3.
Obtained from [(1R)-(2-fluorobenzyl)-3-((2S)-{[3-(3-fluorophenyl)-[1,2,4]oxadiazol-5-ylamino]-methyl}pyrrolidin-1-yl)-3-oxo-propyl]-carbamic acid tert-butyl ester (Example 103, Step 3), and synthesised according to Example 100, Step 4.
1H-NMR (300 MHz, CDCl3) δ=1.79-2.06 (m, 4H), 2.73-2.83 (m, 2H), 3.21-3.27 (m, 1H), 3.38-3.54 (m, 3H), 3.58-3.72 (m, 2H), 3.97-4.05 (m, 1H), 4.42-4.49 (m, 2H), 6.93-6.97 (m, 1H), 7.01-7.04 (m, 1H), 7.12-7.17 (m, 2H), 7.30-7.41 (m, 2H), 7.62-7.65 (m, 1H), 7.74-7.76 (d,1H), 8.32 (bs, 1H), 8.62 (bs, 3H).
LC/MS (Method VII) m/z 442 [M+H]+
Prepared according to the procedure above outlined for Example 100, steps 1-4, according to Schemes G and H.
Obtained from 3-(4-methanesulphonyl-phenyl)-5-trichloromethyl-[1,2,4]oxadiazole (Example 99) and (2S)-aminomethyl-pyrrolidine-1-carboxylic acid tert-butyl, synthesised according to the procedure for Example 100, Step 1.
1H-NMR (300 MHz, CDCl3) δ=1.45 (s, 9H), 1.60-2.18 (m, 4H), 3.05 (s, 3H), 3.25-3.70 (m, 4H), 4.17 (m, 1H), 7.46 (m, 1H), 8.00 (m, 2H), 8.20 (m, 2H).
Obtained from (2S)-{[3-(4-methanesulphonylphenyl)-[1,2,4]oxadiazol-5-ylamino]-methyl}pyrrolidine-1-carboxylic acid tert-butyl ester (Example 104, Step 1), and synthesised according to the procedure for Example 100, Step 2.
Obtained from [3-(4-methanesulphonylphenyl)-[1,2,4]oxadiazol-5-yl]-pyrrolidin-(2S)-ylmethylamine hydrochloride (Example 104, Step 2) and (3R)-tert-butoxycarbonylamino-4-(2-fluoro-phenyl)-butyric acid, and synthesised according to Example 100, Step 3.
Obtained from [(1R)-(2-Fluorobenzyl)-3-((2S)-{[3-(4-methanesulphonylphenyl)-[1,2,4]oxadiazol-5-ylamino]-methyl}pyrrolidin-1-yl)-3-oxo-propyl]-carbamic acid tert-butyl ester (Example 104, Step 3), and synthesised according to Example 100, Step 4.
1H-NMR (300 MHz, CDCl3) δ=1.80-2.03 (m, 4H), 2.75-2.81 (m, 2H), 3.07 (s, 3H), 3.21-3.27 (m, 1H), 3.34-3.55 (m, 4H), 3.60-3.68 (m, 1H), 3.96-4.06 (m, 1H), 4.43-4.50 (m, 1H), 6.94-6.98 (m, 1H), 7.02-7.05 (m, 1H), 7.16-7.21 (m, 1H), 7.30-7.34 (m, 1H), 7.98 (d, 2H), 8.14 (d, 2H), 8.35 (bs, 1H), 8.64 (bs, 3H).
LC/MS (Method VII) m/z 502 [M+H]+.
The following compound (example 105) was prepared according to the following experimental procedure:
To 200 μL of a 0.6M N,N-dimethylformamide solution of (3R)-tert-butoxycarbonylamino-4-[2-fluoro-phenyl]-butyric acid including 1.5 eq of triethylamine in a well of a 96-microtiterplate (96-MTP) 200 μL of a 0.6M N,N-dimethylformamide solution of O-(benzotrialzol-1-yl)-N-N-N,N-tetramethyluronium hexafluorophosphate (HBTU) are added. After 15 min. at room temperature, 200 μL of a 0.5M N,N-dimethylformamide solution of the corresponding amine and 11 μL (0.15 mmol) of N-methylmorpholine are added and the reaction mixture is stirred overnight at 50° C. Solvents are removed under reduced pressure, 500 μL of a solution of trifluoroacetic acid in dichloromethane (33% v/v) is added and the reaction mixture is stirred for 2 h at room temperature. After removal of the solvents under reduced pressure, 500 μL of methanol are added and the crude material is purified using preparative HPLC with a 10 min. linear gradient from 5% to 95% acetonitrile in water (0.1% TFA) to afford the title compounds.
LC/MS (VI) (1-30%, 8 min) rt 5.28, m/z 295 (M+H)+.
Further examples from this series are exemplified below:
Inhibition of DPP-IV peptidase activity was monitored with a continuous fluorimetric assay. This assay is based on the cleavage of the substrate Gly-Pro-AMC (Bachem) by DPP-IV, releasing free AMC. The assay is carried out in 96-well microtiterplates. In a total volume of 100 μL, compounds are preincubated with 50 DPP-IV employing a buffer containing 10 mM Hepes, 150 mM NaCl, 0.005% Tween 20 (pH 7.4). The reaction is started by the addition of 16 μM substrate and the fluorescence of liberated AMC is detected for 10 minutes at 25° C. with a fluorescence reader (BMG-Fluostar; BMG-Technologies) using an excitation wavelength of 370 nm and an emission wavelength of 450 nm. The final concentration of DMSO is 1%. The inhibitory potential of the compounds were determined. DPP-IV activity assays were carried out with human and porcine DPP-IV (see below); both enzymes showed comparable activities-include.
Soluble human DPP-IV lacking the transmembrane anchor (Gly31-Pro766) was expressed in a recombinant YEAST-strain as Pre-Pro-alpha-mating fusion. The secreted product (rhuDPP-IV-Gly3l-Pro766) was purified from fermentation broth (>90% purity) and used for inhouse screening.
In the table are listed the IC50 values for inhibition of DPP-IV peptidase activity determined in assays as described above. The IC50 values were grouped in 3 classes: a≦100 nM; b≧101 nM and ≦1001 nM; c≧1001 nM≦2000 nM.
| Number | Date | Country | Kind |
|---|---|---|---|
| 03028211.5 | Dec 2003 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP04/14040 | 12/9/2004 | WO | 00 | 6/15/2007 |
