Patent ID: 12195476

EMBODIMENTS

The following examples further explain the invention, however, the examples don't constitute a restriction or limitation to the scope of the invention.

Information and using conditions of the instruments used in the invention are as follows

No.InstrumentModelManufacturerTest procedure1High performanceAgilent 1200,AgilentInstrument: Agilent 1200 DAD HPLCliquidDADSystem or Similar configurationchromatographColumn: Waters XBridge Shield RP18(UPLC)4.6 × 150 mm, 3.5 μmMobile phase: A: 0.05% phosphoricacid aqueous solution;B: Acetonitrile2LC-MSAgilent 1200AgilentInstrument: Agilent 1200 HPLC/6100SQ System Column: Agilent XDB-C18,4.6 mm × 50 mm, 1.8 μmMobile phase: A: 0.05% TFA in water;B: 0.05% TFA in acetonitrile31HNMRAVANCEIII400BRUKERInstrument: BRUKER AVANCE IIIMHz400 MHz Ultrashield-Plus Digital NMRSpectroscopyExperiment: N PROTON 1Hexperiment (default parameters)4X-ray powderD8 AdvanceBRUKERLight source is CuK. X-ray intensity isdiffractometer40 KV/40 mA. Scanning mode is(XRPD)Theta-theta. Scanning angle range is 4-40°. Step length is 0.05°. Scanningspeed is 0.5 seconds/step.5DifferentialQ1000TAWeighing 2-4 mg of sample,scanningtransferring to an unsealed aluminumcalorimeterpan, the equilibrium of the sample is(DSC)reached in nitrogen flow (50 mL/min) at25° C., raising the temperature from25° C. to 300° C. at 10° C./min.6ThermogravimetricQ500TAWeighing 10-20 mg of sample,analyzer (TGA)transferring to a platinum sample plate,raising the temperature from 25° C. to300° C. at 10° C./min in sample nitrogenflow (60 mL/min) and balance nitrogenflow (40 mL/min).7Dynamic vaporAdvantage 1SurfaceWeighing about 10 mg of sample,sorption analyzerMeasurementsetting the temperature at 25° C., drying(DVS)Systemfor 60 minutes at a humidity of 0% RH.and determining the moistureabsorption characteristics of sampleswhen the humidity changes from 0% RHto 95% RH, and dehumidificationcharacteristics of samples when thehumidity changes from 95% RH to0% RH. Humidity change step is 5% RH.When the mass change rate dm/dt is lessthan 0.002%, it is considered as scalebalance. Mass change rate less than0.01%/min within 5 minutes is thebalance criterion in the test and themaximum equilibration time is 2 hours.

Information about materials and reagents used in the invention is as follows:

MaterialPurity/gradeLot No.ManufacturerCompound of formula V≥98.0%KM1009-1804001ZINNOVACompound of formula IV≥98.0%KM1008-1804001ZINNOVAN,N-DiisopropylethylamineARKM261A-1801002Shanghai Qiao Chemical Science(DIPEA)Co., Ltd.Ethanol/160321047BNanjing Chemical Reagent Co., Ltd.MethanolARP1176856GENERAL-REAGENTTetrahydrofuranAR gradeP1167158GENERAL-REAGENTDichloromethaneARP1216848GENERAL-REAGENTEthyl acetate (EA)ARP1080359GENERAL-REAGENTAcetoneARP1160778GENERAL-REAGENTPurified waterMilli-QPrepared on theMilli-Qsame dayMethyl tert-butyl etherARP1135054GENERAL-REAGENTIsopropanolHPLC6553IU13Anhui Fulltime Specialized Solvent& Reagent Co., Ltd.Palladium on carbonARKM416A-1603001Shaanxi Rock New Materials Co.,(Pd/C)Ltd.(R)-lactamideARKM1012-1806001Shanghai Zhongnai BiologicalMedical Technology Co., Ltd.Et3O—BF4ARKM416A-1603001Shaanxi Rock New Materials Co.,Ltd.Hydrochloric acidARGM007-1809001Suzhou Zhoushi Chemical ReagentCo., Ltd.Potassium carbonateIndustrialGM009-1804001Shanghai Weitang Industry Co., Ltd.grade

EXAMPLES

Preparation of a Compound of Formula III

Example 1 Preparation of a Compound of Formula III

Ethanol (4 mL), a compound of formula IV (0.20 g, 1.0 eq), a compound of formula V (0.18 g, 1.0 eq), and DIPEA (0.39 g, 3.0 eq) were added to a 25 mL three-necked flask, then the system was stirred; under nitrogen protection, the system was heated to reflux (70-80° C.), and stirred overnight at the reflux temperature; the system was cooled to room temperature (15-20° C.), solids were precipitated during cooling; water (4 mL) was added to the system dropwise, the system was stirred for 2 hours at room temperature (15-20° C.); then the system was filtered, the filter cake was washed with ethanol solution (2 mL, V/V, 1:1), the filter cake was dried under vacuum at a temperature between 45-50° C. for 16 hours; about 0.21 g of yellow solid was obtained, with a LC-MS purity of 96.4% (214 nm) and a yield of 69%.

MS-ESI: [M+1]+: 303.1

1H NMR (400 MHz, CDCl3): 9.238 (s, 1H), 8.400 (d, 1H), 7.968 (d, 1H), 6.987 (d, 1H), 4.537-4.613 (m, 1H), 4.305-4.350 (m, 1H), 3.661-3.722 (m, 1H), 3.313-3.366 (m, 1H), 2.590-2.699 (m, 2H), 2.407-2.454 (m, 1H), 1.815-2.035 (m, 1H), 1.688-1.806 (m, 2H).

Example 2 Preparation of a Compound of Formula III

Ethanol (120 mL, 20 V), a compound of formula IV (6.0 g, 1.0 eq), a compound of formula V (5.4 g, 1.01 eq) and DIPEA (11.7 g, 3.0 eq) were added to a 250 mL three-necked flask, the system was stirred; under nitrogen protection, the system was heated to 70-80° C. (internal temperature), and the temperature was maintained and the system was stirred for 8 hours; the system was cooled to room temperature (15-20° C.), solids were precipitated during cooling; water (120 mL, 20 V) was added to the system dropwise, the system was stirred for 2 hours at room temperature (10-15° C.); the system was filtered, the filter cake was washed with ethanol solution (30 mL, 1:1); the filter cake was dried under vacuum at 50° C. for 16 hours; about 7.7 g of yellow solid was obtained, with an HPLC purity of 95.5% and a yield of 84.3%.

The MS-ESI and1H NMR data are consistent with example 1.

Example 3 Preparation of a Compound of Formula III

Ethanol (5 mL, 10V), a compound of formula IV (0.50 g, 1.0 eq), a compound of formula V (0.45 g, 1.01 eq), and DIPEA (0.98 g, 3.0 eq) were added to a 25 mL three-necked flask, the system was stirred; under nitrogen protection, the system was heated to 70-80° C., allowed the system to reflux and the system was stirred for 5 hours; the system was cooled to room temperature (15-20° C.), solids were precipitated during cooling; water (5 mL, 10V) was added to the system dropwise, the system was stirred for 2 hours at room temperature (10-15° C.); the system was filtered, the filter cake was washed with ethanol solution (1:1) (1.5 mL, 3V), the filter cake was dried under vacuum at 50° C. for 16 hours; about 0.54 g of brown solid was obtained, with an HPLC purity of 95.4% and a yield of 71%.

The MS-ESI and1H NMR data are consistent with example 1.

Example 4 Preparation of a Compound of Formula III

Ethanol (5 mL, 10V), a compound of formula IV (0.50 g, 1.0 eq), a compound of formula V (0.45 g, 1.01 eq) and DIPEA (0.72 g, 2.2 eq) were added to a 25 mL three-necked flask, the system was stirred; under nitrogen protection, the system was heated to 70-80° C., allowed the system to reflux and the system was stirred for 5 hours; the system was cooled to room temperature (15-20° C.), solids were precipitated during cooling; water (7.5 mL, 15V) was added to the system dropwise, the system was stirred for 1 hour at room temperature (10-15° C.); the system was cooled to 5-10° C., the system was stirred for 2 hours; the system was filtered, the filter cake was washed with ethanol solution (1:1) (1.5 mL, 3V), the filter cake was dried under vacuum at 50° C. for 16 hours; about 0.57 g of brown solid was obtained, with an HPLC purity of 91.4% and a yield of 75%.

The MS-ESI and1H NMR data are consistent with example 1.

Example 5 Preparation of a Compound of Formula III

Ethanol (50 mL, 10V), a compound of formula IV (5.0 g, 1.0 eq), a compound of formula V (4.5 g, 1.01 eq) and DIPEA (7.2 g, 2.2 eq) were added to a 250 mL three-necked flask, the system was stirred; under nitrogen protection, the system was heated to 70-80° C., allowed the system to reflux and the system was stirred for 5 hours; the system was cooled to room temperature (15-20° C.), solids were precipitated during cooling; water (75 mL, 15V) was added to the system dropwise, the system was stirred for 1 hour at room temperature (10-15° C.); the system was cooled to 5-10° C. and was stirred for 2 hours; the system was filtered, the filter cake was washed with ethanol solution (1:1, 15 mL), the filter cake was dried under vacuum at 50° C. for 16 hours; about 6.6 g of yellow solid was obtained, with an HPLC purity of 94.2% and a yield of 86.7%.

The MS-ESI and1H NMR data are consistent with example 1.

Example 6 Preparation of a Compound of Formula III

Ethanol (180 mL, 10V), a compound of formula IV (17.8 g, 1.0 eq), a compound of formula V (16.0 g, 1.01 eq), and DIPEA (25.7 g, 2.2 eq) were added to a 500 mL three-necked flask, the system was stirred; under nitrogen protection, the system was heated to 70-80° C., allowed the system to reflux and the system was stirred for 5 hours; the system was cooled to room temperature (15-20° C.), solids were precipitated during cooling; water (270 mL, 15V) was added to the system dropwise, the system was stirred for 1 hour at room temperature (10-15° C.); the system was cooled to 5-10° C. and was stirred for 2 hours; the system was filtered, the filter cake was washed with ethanol solution (ethanol:water=1:1.5, v/v, 40 mL), the filter cake was dried under vacuum at 50° C. for 16 hours; about 23.0 g of brown solid was obtained, with an HPLC purity of 95.3% and a yield of 85.2%.

The MS-ESI and1H NMR data are consistent with example 1.

Example 7 Preparation of a Compound of Formula III

Ethanol (1000 mL, 10V), a compound of formula IV (100 g, 1.0 eq), a compound of formula V (89.9 g, 1.01 eq), and DIPEA (143.2 g, 2.2 eq) were added to a 3000 mL three-necked flask, the system was stirred; under nitrogen protection, the system was heated to 85-90° C. (internal temperature, about 75° C.), allowed the system to reflux and the system was stirred for 10 hours; the system was cooled to room temperature (15-20° C.), solids were precipitated during cooling; water (1500 mL, 15V) was added to the system dropwise, the system was stirred for 1 hour at room temperature (10-15° C.); the system was cooled to 5-10° C. and was stirred for 2 hours; the system was filtered, the filter cake was washed with ethanol solution (1:1.5, v/v, 200 mL), the filter cake was dried with an air blower at 50° C. for 16 hours; about 130 g of reddish brown solid was obtained, with an HPLC purity of 94.2% and a yield of 85.5%.

The MS-ESI and1H NMR data are consistent with example 1.

Example 8 Preparation of a Compound of Formula III

Ethanol (2000 mL, 10V), a compound of formula IV (200 g, 1.0 eq), a compound of formula V (179.7 g, 1.01 eq), and DIPEA (286.4 g, 2.2 eq) were added to a 5000 mL three-necked flask, the system was stirred; under nitrogen protection, the system was heated to 70-80° C. (internal temperature, about 65-70° C.), allowed the system to reflux and the system was stirred for 16 hours; the system was cooled to room temperature (15-20° C.), solids were precipitated during cooling; water (3000 mL, 15V) was added to the system dropwise, the system was stirred for 1 hour at room temperature (10-15° C.); the system was cooled to 5-10° C. and was stirred for 2 hours; the system was filtered, the filter cake was washed with ethanol solution (1:1.5, v/v, 400 mL), the filter cake was dried with an air blower at 50° C. for 16 hours; about 251 g of reddish brown solid was obtained, with an HPLC purity of 93.4% and a yield of 78.1%.

The MS-ESI and1H NMR data are consistent with example 1.

Example 9 Preparation of a Compound of Formula III

Ethanol (5000 mL, 10V), a compound of formula IV (500 g, 1.0 eq), a compound of formula V (450 g, 1.01 eq), and DIPEA (723 g, 2.2 eq) were added to a 20000 mL three-necked flask, the system was stirred; under nitrogen protection, the system was heated to 80-90° C. (internal temperature, about 70-80° C.), allowed the system to reflux and the system was stirred for 16 hours; the system was cooled to room temperature (25-30° C.), solids were precipitated during cooling; water (7500 mL, 15V) was added to the system dropwise, the system was stirred for 1 hour at room temperature (25-30° C.); the system was cooled to 10-15° C. and was stirred for 2 hours; the system was filtered, the filter cake was washed with ethanol solution (1:1.5, v/v, 1000 mL), the filter cake was dried in an at 50-55° C. under vacuum for 24 hours; about 623 g of product was obtained, with an HPLC purity of 93.7%, residual ethanol of 0.5%, a content of 93.1%, and a content yield of 76.2%.

The MS-ESI and1H NMR data are consistent with example 1.

Example 10 Preparation of a Compound of Formula III

Ethanol (100 mL, 10V), a compound of formula IV (10.0 g, 1.0 eq), a compound of formula V (9.0 g, 1.01 eq), and DIPEA (14.3 g, 2.2 eq) were added to a 500 mL three-necked flask, the system was stirred; the system was heated to 70-80° C., allowed the system to reflux and the system was stirred for 16 hours; the system was cooled to room temperature (20-30° C.), solids were precipitated during cooling; water (150 mL, 15V) was added to the system dropwise, the system was stirred for 2 hours at room temperature (20-30° C.); the system was cooled to 5-10° C. and was stirred for 2 hours; the system was filtered, the filter cake was washed with ethanol solution (1:1.5, v/v, 25 mL), the filter cake was dried in an oven at 50-55° C. under vacuum for 16 hours; about 13.7 g of product was obtained, with an HPLC purity of 93.7% and a yield of 90%.

The MS-ESI and1H NMR data are consistent with example 1.

Example 11 Preparation of a Compound of Formula III

Ethanol (17 kg, 10V), a compound of formula IV (2.2 Kg, 1.0 eq), a compound of formula V (1.98 Kg, 1.01 eq), and DIPEA (3.19 Kg, 2.2 eq) were added to a R0462 reactor, the system was stirred; under nitrogen protection, the system was heated to 75-80° C. (internal temperature, about 70-80° C.), the system was stirred for 16 hours; the system was cooled to room temperature (15-25° C.), solids were precipitated during cooling; water (33 Kg, 15V) was added to the system dropwise, the system was stirred for 2 hours at room temperature (10-15° C.); the system was cooled to 5-10° C. and was stirred for 4 hours; the system was filtered, the filter cake was washed with ethanol solution (ethanol:water=1:2, v/v, 6.2 Kg), the filter cake was dried under the vacuum condition of ≤−0.08 MPa in a jacket at 45-55° C. for 16 hours; 2.64 Kg of brown solid was obtained, with an HPLC purity of 94.0%, a content of 93.4%, and a content yield of 79.04%.

The MS-ESI and1H NMR data are consistent with example 1.

Preparation of a Compound of Formula II

Example 12 Preparation of a Compound of Formula II

A compound of formula III (5.0 g), THF (50 mL, 10 V) and palladium on carbon (0.75 g, 10% Pd/C, 50% water wet) were added to a 100 mL stainless steel high pressure reactor successively; the system was purged with nitrogen for 5 times and then hydrogen for 5 times; the pressure of system was increased to 0.50 MPa with hydrogen, and the system was heated to 25-35° C. and the temperature was maintained, the system was stirred for 24 hours; the reaction solution was filtered with diatomite, the filter cake was washed with THF (20 mL), the filtrate was concentrated to dryness, 4.2 g of brown solid was obtained, with an HPLC purity of 94.9% and a yield of 93.3%.

MS-ESI: [M+1]+: 273.1

1H NMR (400 MHz, CDCl3): 7.988 (s, 1H), 7.688 (d, 1H), 6.805 (d, 1H), 4.190-4.338 (m, 3H), 3.584-3.648 (m, 1H), 3.147-3.206 (t, 1H), 2.594-2.651 (d, 2H), 2.318-2.364 (m, 1H), 1.917-1.974 (m, 1H), 1.633-1.738 (m, 1H), 1.456-1.525 (m, 1H).

Example 13 Preparation of a Compound of Formula II

A compound of formula III (120.0 g), THF (2400 mL, 20 V) and palladium on carbon (18 g, 10% Pd/C, 50% water wet) were added to a 5000 mL stainless steel high pressure reactor successively; the system was purged with nitrogen for 5 times and then hydrogen for 5 times; the pressure of system was increased to 0.50 MPa with hydrogen, and the system was heated to 25-35° C. and the temperature was maintained, the system was stirred for 24 hours; the reaction solution was filtered with diatomite, the filter cake was washed with THF (600 mL) (until TLC almost does not show fluorescence), the filtrate was concentrated to obtain 130 g of black semi-oily solid, with an HPLC purity of 91.7% and a yield of 120.26%. an HPLC purity

The MS-ESI and1H NMR data are consistent with example 12.

Example 14 Preparation of a Compound of Formula II

A compound of formula III (100.0 g), THF (2000 mL, 20 V) and palladium on carbon (15.0 g, 10% Pd/C, 50% water wet) were added to a 5 L stainless steel high pressure reactor successively; the system was purged with nitrogen for 5 times and then hydrogen for 5 times; the pressure of system was increased to 0.5-1.0 MPa with hydrogen, the temperature of the jacket was set at 30° C. and the system was stirred for 16 hours with the temperature maintained, the reaction solution was filtered with diatomite, the filter cake was washed with THF (1000 mL), 3877 g of a compound of formula II in THF was obtained. Post-treatment 1: the above filtrate (1820 g, about 40 g of a compound of formula II calculated according to a 100% yield) was concentrated to (2-3 V, 80-120 mL) with rotary evaporator, the system was exchanged with ethanol (150 mL×2) to (2-3 V, 80-120 mL); 78 g of a compound of formula II in ethanol was obtained, with a content of 47.25%, content yield of 92.14%.

Post-treatment 2: the above filtrate (450 g, about 10 g of a compound of formula II calculated according to a 100% yield) was concentrated to dryness with rotary evaporator; 10.5 g of brownish red solid was obtained.

Post-treatment 3: the above filtrate (450 g, about 10 g of a compound of formula II after calculation) was placed in a flask, and was concentrated to about 30-40 mL (3-4 V) with rotary evaporator; the concentrate residue was exchanged with ethanol (50 mL×2) to about 30-40 mL (3-4 V); black oily concentrate residue was obtained, the concentrate residue was directly fed to the next step of reaction.

The MS-ESI and1H NMR data are consistent with example 12.

Example 15 Preparation of a Compound of Formula II

THF (240 mL, 20 V), a compound of formula III (12.0 g), and palladium on carbon (1.8 g, 5% Pd/C, 50% water wet) were added to a 500 mL three-necked flask successively; the system was purged with nitrogen for 5 times and then hydrogen for 5 times; the system was stirred for 48 hours with the temperature maintained at room temperature (25-30° C.) and a hydrogen pressure (about 0.1 MPa); the reaction solution was filtered, the filter cake was washed with THF (60 mL), the combined filtrate was concentrated to 20-30 mL with a rotary evaporator, then was exchanged with ethanol (60 mL×2) to 20-30 mL; 24 g of a compound of formula II in ethanol was obtained, the solution was used directly for the next step of reaction.

The MS-ESI and1H NMR data are consistent with example 12.

Example 16 Preparation of a Compound of Formula II

THF (1500 mL, 15 V), a compound of formula III (100 g), and palladium on carbon (15 g, 5% Pd/C, 50% water wet) were added to a 5000 mL three-necked flask successively; the system was purged with nitrogen for 5 times and then hydrogen for 5 times; the system was stirred for 48 hours with the temperature maintained at room temperature (20-25° C.) and a hydrogen pressure (about 0.1 MPa); the reaction solution was filtered, the filter cake was washed with THF (200 mL), the combined filtrate was concentrated to 200-300 mL with a rotary evaporator; 185.6 g of a compound of formula II in THF was obtained, with an HPLC purity of 94.2%, a content of 43.2%, and a content yield of 94.0%.

The MS-ESI and1H NMR data are consistent with example 12.

Example 17 Preparation of a Compound of Formula II

THF (12400 mL, 20 V), a compound of formula III (620 g), and palladium on carbon (93 g, 5% Pd/C, 50% water wet) were added to a 20000 mL three-necked flask successively; the system was purged with nitrogen for 5 times and then hydrogen for 5 times; the system was stirred for 48 hours with the temperature maintained at room temperature (30-35° C.) and a hydrogen pressure (about 0.1 MPa); the reaction solution was filtered with diatomite (200 g), the filter cake was washed with THF (1200 mL), the combined filtrate was concentrated to 1200-1800 mL with a rotary evaporator; 1664 g of a compound of formula II in THF was obtained, with an HPLC purity of 93.8%, a content of 34.57%, and a content yield of 110.6%.

The MS-ESI and1H NMR data are consistent with example 12.

Example 18 Preparation of a Compound of Formula II

THF (140 ml, 70 V), a compound of formula III (2.0 g) and palladium on carbon (0.3 g, 5% Pd/C, 50% water wet) were added to a 250 mL high pressure reactor; the high pressure reactor was covered and the nut was screwed tight; the system was purged with nitrogen for 3 times and then hydrogen for 3 times; the high pressure reactor was charged with hydrogen to about a pressure of 0.50±0.05 MPa, the inlet valve was then closed; the stirring apparatus was started at a rotating speed of 500 r/min; hydrogen pressure in the high pressure reactor was maintained at 0.5±0.05 MPa between 25-35° C., the high pressure reactor was stirred and the system was reacted for 96 hours; the reaction solution was filtered with diatomite (10 g), the filter cake was washed with THF (60 mL); the combined filtrate was concentrated with rotary evaporator to dryness to obtain 1.8 g of semi-oily solid, with an HPLC purity of 91.2%, a yield of 99.9%.

The MS-ESI and1H NMR data are consistent with example 12.

Example 19 Preparation of a Compound of Formula II

THF (167 Kg, 70 V), a compound of formula III (2.64 Kg) and palladium on carbon (0.4 Kg, 5% Pd/C, 50% water wet) were added to a 500 L high pressure reactor; the system was purged with nitrogen for 5 times and then hydrogen for 5 times; the high pressure reactor was charged with hydrogen to about a pressure of 0.50±0.05 MPa, the inlet valve was then closed; the stirring apparatus was started; hydrogen pressure in the high pressure reactor was maintained at 0.5±0.05 MPa between 25-35° C., the high pressure reactor was stirred and the system was reacted for 120 hours; the reaction solution was filter-pressed, the filter cake was washed with THF (13 Kg); the combined filtrate was distillated under reduced pressure (to 2V-3V) to obtain 11 Kg of a compound of formula II in THF, with an HPLC purity of 90.7%, a content of 18.5%, and a content yield of 91.9%.

The MS-ESI and1H NMR data are consistent with example 12.

Example 20 Preparation of a Compound of Formula II

THF (60 mL, 12 V), a compound of formula III (5.0 g) and palladium on carbon (0.75 g, 5% Pd/C, 50% water wet) were added to a 100 mL stainless steel high pressure reactor; the system was purged with nitrogen for 5 times and then hydrogen for 5 times; the high pressure reactor was charged with hydrogen to about a pressure of 0.5-1.0 MPa, the jacket temperature was set to 30° C., and the system was stirred for 42 hours with the temperature maintained; after the reaction was completed, the reaction solution was filtered with diatomite, the filter cake was washed with THF (100 mL); 197.8 g of a compound of formula II in THF was obtained; the solution was concentrated with a rotary evaporator to (2-3 V, 10-15 mL); the system was exchanged with ethanol (25 mL×2) to (2-3 V, 10-15 mL); the obtained compound of formula II in ethanol was directly used for the next step of reaction.

The MS-ESI and1H NMR data are consistent with example 12.

Preparation of a Compound of Formula I

Example 21 Preparation of a Compound of Formula I

THF (60 mL, 12 V), (R)-lactamide (6.6 g, 4.0 eq) and Et3O—BF4(13.9 g, 4.0 eq) were added to a 250 mL three-necked flask #1, the system was stirred; the materials in three-necked flask #1 were stirred under nitrogen protection for later use; a compound of formula II (5.0 g, 1.0 eq) and ethanol (80 mL, 16 V) were added to another 250 mL three-necked flask #2; the system was heated to 70±5° C. under nitrogen protection; the materials in three-necked flask #1 were added to three-necked flask #2 with a syringe dropwise within 10-20 minutes; the system was heated to 85±5° C. (internal temperature was in the range of 72-75° C.) under nitrogen protection for reacting for 2 hours; the system was cooled to room temperature; the reaction liquid was concentrated with a rotary evaporator until there was basically no fraction flowing out; 1M HCl (80 mL) was added to the residual concentrated liquid, the pH was about 1 (determined with a pH test paper); the system was extracted four times with DCM (50 mL×4); the pH of the aqueous phase was adjusted to 7-8 with saturated sodium bicarbonate solution; the system was stirred at room temperature for 0.5 hour, then was filtered, the filter cake was washed with water (60 mL) and EA (10 mL), respectively; the filter cake was dried under vacuum at 50° C. for 16 hours; 4.3 g of faint yellow solid was obtained, with a purity of 95.0%; the solid was dissolved with methanol (30 mL); 4.1 g of silicon based metal eliminator and 1.0 g of activated carbon were added, the system was heated to 50° C. and stirred for 1 hour, then was cooled, filtered, washed with methanol (30 mL); the filtrate was concentrated with rotary evaporator until there was basically no fraction flowing out; methanol (10 mL) and MTBE (25 mL) were added to the residue, the system was heated to 50° C., and was stirred for 0.5 hour, then was cooled, the system was cooled to 10±5° C. and stirred for 0.5 hour; filtered, the filter cake was washed with MTBE (25 mL); the filter cake was dried under vacuum at 50° C. for 16 hours, 3.2 g of faint yellow solid was obtained, with a purity of 97.9%.

MS-ESI: [M+1]+: 327.6

1H NMR (400 MHz, CDCl3): 8.988 (s, 1H), 7.922 (d, 1H), 7.175 (d, 1H), 5.200-5.265 (m, 1H), 4.859-4.942 (m, 1H), 4.350-4.406 (t, 1H), 4.020-4.108 (m, 2H), 3.067 (d, 1H), 2.619-2.779 (m, 3H), 2.108-2.269 (m, 2H), 1.790-1.895 (m, 3H).

Example 22 Preparation of a Compound of Formula I

THF (650 mL, 12 V), (R)-lactamide (70.6 g, 4.0 eq) and Et3O—BF4(150.6 g, 4.0 eq) were added to a 1000 mL three-necked flask #1, the system was stirred; the materials in three-necked flask #1 were stirred under nitrogen protection for later use; a compound of formula II (54 g, 1.0 eq) and ethanol (860 mL, 16 V) were added to another 2000 mL three-necked flask #2; the system was heated to 70±5° C. under nitrogen protection; the materials in three-necked flask #1 were slowly added to three-necked flask #2 dropwise within 1 hour; the system was heated to 85±5° C. (internal temperature was in the range of 72-75° C.) under nitrogen protection for reacting for 2 hours; the system was cooled to room temperature; the reaction liquid was concentrated with a rotary evaporator until there was basically no fraction flowing out; 1M HCl (450 mL) was added to the residual concentrated liquid, the pH was about 1 (determined with a pH test paper); the system was extracted four times with DCM (270 mL×4); the pH of the aqueous phase was adjusted to 7-8 with saturated sodium bicarbonate solution; the system was stirred at room temperature for 0.5 hour, then was filtered, the filter cake was washed with water (540 mL); MTBE (270 mL) was added to the filter cake, the system was stirred at room temperature for 0.5 hour, filtered, the filter cake was washed with MTBE (108 mL); the filter cake was dried under vacuum at 50° C. for 16 hours; 49.2 g of light yellow solid was obtained, with an HPLC purity of 94.2%; the solid was dissolved with methanol (380 mL); silicon based metal eliminator (44 g) and activated carbon (5.4 g) were added, the system was heated to 50° C. and stirred for 1 hour, then was cooled, filtered, washed with methanol (430 mL); the filtrate was concentrated with a rotary evaporator to (80-110 mL, 1.5 V-2 V); MTBE (540 mL) was added to the residue, the system was heated to 50° C., and was stirred for 1 hour, then was cooled to 10±5° C. and stirred for 0.5 hour; filtered, the filter cake was washed with MTBE (270 mL); 42.4 g of filter cake was obtained, with an HPLC purity of 96.9%; the filter cake was dried under vacuum at 50° C. for 16 hours, 41.0 g of light yellow solid was obtained, with an HPLC purity of 96.7%, a yield of 63.3%.

Purification of a Compound of Formula I:

A compound of formula I (41 g) was dissolved with methanol; silica gel (50 g) was added to the solution, the system was concentrated to dryness for later use; silica gel (200 g) was added to the chromatographic column, the column was compacted with an air pump; a compound of formula I mixed with silica gel was added to the chromatographic column, the column was compacted with an air pump; the chromatographic column was eluted with an eluent (VMeOH:VDCM=1:100-1:30); qualified components were collected, concentrated to dryness; the product was dried under vacuum at 50° C. for 16 hours; 36 g of off-white solid was obtained, with an HPLC purity of 98.5%.

The MS-ESI and1H NMR data are consistent with example 21.

Example 23 Preparation of a Compound of Formula I

THF (60 mL, 6 V), (R)-lactamide (13.2 g, 4.0 eq) and Et3O—BF4(27.9 g, 4.0 eq) were added to a 100 mL three-necked flask #1, the system was stirred; the materials in #1 were stirred under nitrogen protection for later use; a compound of formula II (10 g, 1.0 eq) and ethanol (100 mL, 10 V) were added to another 250 mL three-necked flask #2; the system was heated to 70±5° C. under nitrogen protection; the materials in three-necked flask #1 were slowly added to three-necked flask #2 dropwise within 20 minutes; the system was heated to 80±5° C. (internal temperature was in the range of 72-75° C.) under nitrogen protection for reacting for 0.5 hour; the system was cooled to room temperature 20-30° C.; the reaction liquid was concentrated to about 50-80 mL with a rotary evaporator between 30-40° C.; water (100 mL, 10 V) was added to the system, then the system was concentrated with a rotary evaporator between 30-40° C. until there was basically no fraction flowing out; the system was cooled to 20-30° C.; the temperature of the system was controlled at 20-30° C., 12M HCl (5.5 g) was used to adjust the pH of the system to 2-3, the system was extracted with ethyl acetate (50 mL×2, 5V×2); the organic phase was discarded, and the aqueous phase was transferred to a flask; the temperature of the system was controlled at 20-30° C., the pH of the system was adjusted to 8-9 with saturated potassium carbonate solution (23 g); the temperature of the system was controlled at 20-25° C., the system was stirred for 2 hours, then was filtered, the filter cake was washed with water (50 mL) and MTBE (50 mL); the filter cake was dried with an air blower at 50° C. for 24 hours, 18 g of earth yellow solid was obtained, with an HPLC purity of 93.5%.

The MS-ESI and1H NMR data are consistent with example 21.

Example 24 Preparation of a Compound of Formula I

THF (120 mL, 12 V), (R)-lactamide (13.2 g, 4.0 eq) and Et3O—BF4(27.8 g, 4.0 eq) were added to a 250 mL three-necked flask #1, the system was stirred; the materials in #1 were stirred under nitrogen protection for later use; a compound of formula II (10 g, 1.0 eq) and ethanol (140 mL, 14 V) were added to another 500 mL three-necked flask #2; the system was heated to 40-45° C. (internal temperature) under nitrogen protection; the materials in three-necked flask #1 were added to three-necked flask #2 dropwise within 1 hour; the system was maintained at 40-45° C. (internal temperature) under nitrogen protection for reacting for 4.5 hours; the system was cooled to room temperature, and water (20 mL, 2V) was added; the system was concentrated with a rotary evaporator at 30-40° C. until there was basically no fraction flowing out; the system was cooled to 20-30° C.; the temperature of the system was controlled at 20-30° C., 12M HCl (3 mL) was used to adjust the pH of the system to 2-3, the system was extracted with ethyl acetate (50 mL×2, 5V×2); the organic phase was discarded, and the aqueous phase was transferred to a flask; the temperature of the system was controlled at 20-30° C., the pH of the system was adjusted to 8-9 with 50% potassium carbonate solution (15 mL); the temperature of the system was controlled at 20-25° C., the system was stirred for 2 hours, then was filtered, the filter cake was washed with water (50 mL) and acetone (50 mL); the crude product was triturated and stirred with water (50 mL) at 20-25° C. for 1 hour; the system was filtered, the filter cake was washed with water (50 mL) and acetone (50 mL); the filter cake was dried with an air blower at 50° C. for 24 hours, 17.8 g of khaki solid was obtained, with an HPLC purity of 95.3%.

The MS-ESI and1H NMR data are consistent with example 21.

Example 25 Preparation of a Compound of Formula I

THF (60 mL, 12 V), (R)-lactamide (6.6 g, 4.0 eq) and Et3O—BF4(13.9 g, 4.0 eq) were added to a 250 mL three-necked flask #1, the system was stirred; the materials in three-necked flask #1 were stirred under nitrogen protection for later use; a compound of formula II (5 g, 1.0 eq) and ethanol (70 mL, 14 V) were added to another 250 mL three-necked flask #2; the system was heated to 40-45° C. (internal temperature) under nitrogen protection; the materials in three-necked flask #1 were added to three-necked flask #2 dropwise within 20 minutes; the system was maintained at 40-45° C. (internal temperature) under nitrogen protection for reacting for 3 hours; the system was cooled to room temperature and was filtered, the filter cake was washed with THF (10 mL); water (10 mL, 2V) was added to the filtrate; the filtrate was concentrated with a rotary evaporator to 10-20 mL (2V-4V), the concentrated residue was exchanged with ethyl acetate (25 mL×2) and concentrated to 10-20 mL (2V-4V); water (50 mL, 10V) was added to the concentrated residue; the internal temperature was controlled at 20-25° C., 12M HCl (4.1 g) was used to adjust the pH of the system to 1-2; activated carbon (0.5 g) was added to the system, and the system was stirred at room temperature for 2 hours, and was filtered, the filter cake was washed with water (10 mL) and 1M HCl (10 mL); the combined filtrate was extracted with ethyl acetate (25 mL×2), the organic phase was discarded; the internal temperature was controlled at 20-25° C., the pH of the system was adjusted to 9-10 with saturated potassium carbonate solution (15 g); the internal temperature was controlled at 15-20° C., the system was stirred for 1 hour, and was filtered, the filter cake was washed with water (10 mL); the filter cake was triturated with acetone aqueous solution (50 mL, V/V=1:1) for 1 hour; the system was filtered, the filter cake was washed with acetone aqueous solution (10 mL, V/V=1:1); the filter cake was dried with an air blower at 50° C. for 24 hours; 5.0 g of pale gray solid was obtained, with an HPLC purity of 95.6%, and a yield of 83.5%;

Purification of a Compound of Formula I:

5.0 g of the obtained solid and methanol (40 mL) were added to a flask, and were stirred for 10 minutes at room temperature, the materials were basically dissolved and the solution was clear; activated carbon (0.5 g) and silica gel (4.0 g) were added to the system; the system was heated to 50-55° C., the temperature was maintained and the system was stirred for 2 hours, then was filtered with silica gel (5 g), the filter cake was washed with methanol (50 mL); the filtrate was concentrated with a rotary evaporator to 5-10 mL; MTBE (50 mL) was added to the concentrated residue; the system was heated to reflux, and was allowed for reflux for 1 hour; the system was cooled to 5-10° C., the temperature was maintained and the system was stirred for 1 hour and was filtered, the filter cake was washed with MTBE; the filter cake was dried with a drying oven under vacuum at 50° C. for 16 hours; 3.0 g of off-white solid was obtained, with a yield of 60% and a purity of 97.9%; the filtrate was concentrated to dryness to obtain 1.4 g of yellow solid.

The MS-ESI and1H NMR data are consistent with example 21.

Example 26 Preparation of a Compound of Formula I

THF (55 mL, 12 V), (R)-lactamide (5.9 g, 4.0 eq) and Et3O—BF4(12.6 g, 4.0 eq) were added to a 250 mL three-necked flask #1, the system was stirred; the materials in #1 were stirred under nitrogen protection for later use; a compound of formula II (4.5 g, which is 5.0 g of a compound of formula III in example 20 calculated according to a yield of 100%) and ethanol (70 mL, 15.5 V) were added to another 250 mL three-necked flask #2; the system was heated to reflux under nitrogen protection; the materials in three-necked flask #1 were dropwise added to three-necked flask #2 at one time; under nitrogen protection, the system was heated to 85±5° C. (internal temperature 74-76° C.) for reacting for 1 hour; the system was cooled to room temperature and was filtered, the filter cake was washed with THF (10 mL); water (10 mL, 2V) was added to the filtrate; the filtrate was concentrated with a rotary evaporator to 10-20 mL (2V-4V), the concentrated residue was exchanged with ethyl acetate (25 mL×2) and concentrated to 10-20 mL (2V-4V); water (50 mL, 10V) was added to the concentrated residue; the internal temperature was controlled at 20-25° C., 12M HCl (3.6 g) was used to adjust the pH of the system to 1-2; activated carbon (0.5 g) was added to the system, and the system was stirred at room temperature for 2 hours, and was filtered, the filter cake was washed with water (10 mL) and 1M HCl (10 mL); the combined filtrate was extracted with ethyl acetate (25 mL×2), the organic phase was discarded; the internal temperature was controlled at 20-25° C., the pH of the system was adjusted to 9-10 with saturated potassium carbonate solution (15 g); the internal temperature was controlled at 15-20° C., the system was stirred for 1 hour, and was filtered, the filter cake was washed with water (10 mL); the filter cake was triturated with acetone aqueous solution (50 mL, V/V=1:1) for 1 hour; the system was filtered, the filter cake was washed with acetone aqueous solution (10 mL, V/V=1:1); the filter cake was dried with an air blower at 50° C. for 16 hours; 4.9 g of pale gray solid was obtained, with an HPLC purity of 94.5%;

Purification of a Compound of Formula I:

The obtained solid (4.9 g) and ethanol (100 mL) were added to a flask, and were stirred at room temperature for 10 minutes, the materials were basically dissolved and the solution was clear; silica gel (5.0 g, 1×) was added to the system, the system was concentrated with a rotary evaporator to dryness for later use; the crude compound of formula I mixed with silica gel was allowed to pass a silica gel column (40 g, 8×), the column was eluted with a mixed solution of ethyl acetate and ethanol (VEA:VEtOH=100:1-30:1); the components were tested with TLC, components containing the product were collected and concentrated to dryness; 0.5 g of white product was obtained with a purity of 95.0% and 2.9 g of faint yellow product was obtained with a purity of 98.7%; ethyl acetate (30 mL) and ethanol (3 mL) were added to a flask containing 2.9 g of the product; the system was heated to reflux, and allowed the system to reflux for 1 hour; the system was cooled to 5-10° C., the temperature was maintained and the system was stirred for 1 hour, and was filtered, the filter cake was washed with ethyl acetate (5 mL); the filter cake was dried with a drying oven under vacuum at 50° C. for 16 hours; 2.3 g of faint yellow to off-white solid was obtained, with an HPLC purity of 99.6%. There were no impurities >0.1%. The total yield of steps 2 and 3 was 42.7%.

The MS-ESI and1H NMR data are consistent with example 21.

Example 27 Preparation of a Compound of Formula I

THF (120 mL, 12 V), (R)-lactamide (13.2 g, 4.0 eq) and Et3O—BF4(27.8 g, 4.0 eq) were added to a 250 mL three-necked flask #1, the system was stirred; the materials in #1 were stirred under nitrogen protection for later use; ethanol (140 mL, 14V) and the concentrated residue (containing about 10 g of a compound of formula II) obtained after post-treatment 3 in example 14 was added to another 500 mL three-necked flask #2; the system was heated to 45±2° C. under nitrogen protection; the materials in three-necked flask #1 were dropwise added to three-necked flask #2 at one time; under nitrogen protection, the system was heated to 45±5° C. (internal temperature 45° C.) for reacting for 6 hours; the system was cooled to room temperature and was filtered, the filter cake was washed with THF (20 mL); water (20 mL, 2V) was added to the filtrate; the filtrate was concentrated with a rotary evaporator to 20-40 mL (2V-4V), the concentrated residue was exchanged with ethyl acetate (50 mL×2) and concentrated to 20-40 mL (2V-4V); water (90 mL, 10V) was added to the concentrated residue; the internal temperature was controlled at 20-25° C., 12M HCl (8.0 g) was used to adjust the pH of the system to 1-2; activated carbon (1.0 g) was added to the system, and the system was stirred at room temperature for 2 hours, and was filtered, the filter cake was washed with water (20 mL) and 1M HCl (20 mL); the combined filtrate was extracted with ethyl acetate (50 mL×2), the organic phase was discarded; the internal temperature was controlled at 20-25° C., the pH of the system was adjusted to 9-10 with saturated potassium carbonate solution (28 g); the internal temperature was controlled at 15-20° C., the system was stirred for 1 hour, and was filtered, the filter cake was washed with water (20 mL); the filter cake was triturated with acetone aqueous solution (100 mL, V/V=1:1) for 1 hour; the system was filtered, the filter cake was washed with acetone aqueous solution (10 mL, V/V=1:1); the filter cake was dried with an air blower at 50° C. for 16 hours; 15.0 g of pale gray solid was obtained, with a purity of 92.4%.

Purification of a Compound of Formula I:

The obtained solid (10 g) and ethanol (100 mL) were added to a flask; the system was heated to 50-60° C., and stirred at 50-60° C. for 30 minutes, the materials were basically dissolved and the solution was clear; silica gel (20.0 g, 2×) was added to the system, the system was concentrated with a rotary evaporator to dryness for later use; the crude compound of formula I mixed with silica gel was allowed to pass a silica gel column (100 g, 10×), the column was eluted with ethyl acetate until there was basically no former impurities (about 1.5 L), then was eluted with a mixed solution of ethyl acetate and ethanol (VEA:VEtOH=100:1-30:1, first 0.5 L of VEA:VEtOH=100:1, then 1.5 L of VEA:VEtOH=30:1); the components were tested with TLC, components containing the product were collected; qualified components were concentrated by rotary evaporation to (50 mL, 5V); the solvent was exchanged with ethyl acetate (50 mL×2) to (50 mL, 5V); the system was cooled to 5-10° C., and was stirred for 2 hours between 5-10° C.; filtered, the filter cake was washed with ethyl acetate (5 mL); the mother liquid was concentrated to dryness to obtain 0.6 g of product; the filter cake was dried with a drying oven under vacuum at 50° C. for 16 hours; 4.6 g of off-white solid was obtained, with a purity of 98.1%. The total yield of steps 2 and 3 was 57.6%.

The MS-ESI and1H NMR data are consistent with example 21.

Example 28 Preparation of a Compound of Formula I

THF (660 mL, 12 V), (R)-lactamide (72 g, 4.0 eq) and Et3O—BF4(153.5 g, 4.0 eq) were added to a 1000 mL three-necked flask #1, the system was stirred; the materials in #1 were stirred under nitrogen protection for later use; the compound of formula II in THF prepared in example 16 (127.3 g, 55 g of a compound of formula II after calculation) was added to a flask, the system was exchanged with EtOH (280 mL×2) and concentrated to 120-180 mL, the concentrated residue was transferred to a 2000 mL three-necked flask #2, and ethanol (770 mL, 14 V) was added, under nitrogen protection, the system was heated to 45-50° C. (internal temperature); the materials in three-necked flask #1 were added to three-necked flask #2 dropwise within about 1 hour; under nitrogen protection, the temperature was maintained at 45-50° C. (basically at about 45° C.) for reacting for 3 hours; the system was cooled to room temperature and was filtered, the filter cake was washed with THF (60 mL); water (110 mL, 2V) was added to the filtrate; the filtrate was concentrated with a rotary evaporator to 110-220 mL (2V-4V), the concentrated residue was exchanged with ethyl acetate (280 mL×2) and concentrated to 110-220 mL (2V-4V); water (550 mL, 10V) was added to the concentrated residue; the internal temperature was controlled at 20-25° C., 12M HCl (35 g) was used to adjust the pH of the system to 1-2; activated carbon (5.5 g) was added to the system, and the system was stirred at room temperature for 2 hours, and was filtered, the filter cake was washed with water (110 mL) and 1M HCl (10 mL); the combined filtrate was extracted with ethyl acetate (280 mL×2), the organic phase was discarded; the internal temperature was controlled at 20-25° C., the pH of the system was adjusted to 9-10 with 50% potassium carbonate solution (180 g); the internal temperature was controlled at 15-20° C., the system was stirred for 2 hours, and was filtered, the filter cake was washed with water (110 mL); the filter cake was triturated with acetone aqueous solution (550 mL, V/V=1:1) for 2 hours; the system was filtered, the filter cake was washed with acetone aqueous solution (110 mL, V/V=1:1); the filter cake was dried with an air blower at 50° C. for 16 hours; 80 g of pale gray solid was obtained, with an HPLC purity of 96.4%, a content of 50.32%, a content yield of 61.1%.

Purification of a Compound of Formula I:

The obtained solid (80 g), silica gel (160 g, 2×) and ethanol (800 mL) were added to the flask; the system was heated to 50-60° C., and stirred at 50-60° C. for 30 minutes, the system was concentrated with a rotary evaporator to dryness for later use; the crude compound of formula I mixed with silica gel was allowed to pass a silica gel column (800 g, 10×), the column was eluted with ethyl acetate until there was basically no former impurities, and the column was eluted with a mixed solution of ethyl acetate and ethanol (VEA:VEtOH=100:1-30:1); the components were tested with TLC, components that showed essentially impurity-free by TLC were collected; qualified components were concentrated by a rotary evaporator to (˜200 mL); the concentrated residue was exchanged with methanol (˜200 mL) twice; the concentrated residue was exchanged with MTBE (˜200 mL) twice; MTBE (˜300 mL) was added to the concentrated residue; the system was heated to reflux and allowed the system to reflux for 1 hour; the system was cooled to 5-10° C., stirred between 5-10° C. for 2 hours; filtered, the filter cake was washed with MTBE (30 mL); the filter cake was dried with a drying oven under vacuum at 50° C. for 16 hours; 39 g of faint yellow solid was obtained, with an HPLC purity of 99.7% and there were no impurities >0.1%.

The MS-ESI and1H NMR data are consistent with example 21.

Example 29 Preparation of a Compound of Formula I

THF (11 Kg, 12 V), (R)-lactamide (1.64 Kg, 5.0 eq) and Et3O—BF4(3.50 Kg, 5.0 eq) were added to a 100 L reactor #1, the system was stirred; the materials in #1 were stirred under nitrogen protection for later use; the compound of formula II in THF (5.4 Kg) prepared in example 19 was exchanged by ethanol twice (4.2 Kg×2), ethanol (11.2 Kg, 14V) was added to the system and then the system was transferred to 100 L reactor #2; under nitrogen protection, the system was heated to 45-50° C. (internal temperature); the materials in reactor #1 were added to reactor #2 dropwise within about 1 hour; under nitrogen protection, the temperature of the system was maintained at 45-50° C. for reacting for 3 hours; THF (1.0 Kg, 12V), (R)-lactamide (0.16 Kg, 0.50 eq) and Et3O—BF4(0.35 Kg, 0.50 eq) were added to a 5 L flask, stirred and dissolved; the solution was added to reactor #2 dropwise and reacted for 2 hours with the temperature maintained; the system was cooled to room temperature, filtered, the filter cake was washed with THF (1.0 Kg); water (2 Kg, 2 V) was added to the filtrate; the filtrate was concentrated with a rotary evaporator to 3 L (2 V-4 V); the concentrated residue was exchanged with ethyl acetate (4 Kg×2) to 3 L (2 V-4 V); water (10 Kg, 10 V) was added to the concentrated residue; the internal temperature was controlled at 20-25° C., 12M HCl was used to adjust the pH of the system to 1-2; activated carbon (0.1 Kg) was added to the system, and the system was stirred at room temperature for 2 hours, and was filtered, the filter cake was washed with water (2 Kg) and 1M HCl (2 Kg); the combined filtrate was extracted with ethyl acetate (3.90 Kg×2), the organic phase was discarded; the internal temperature was controlled at 20-25° C., the pH of the system was adjusted to 9-10 with 50% potassium carbonate solution; the internal temperature was controlled at 15-20° C., the system was stirred for 2 hours, and was filtered, the filter cake was washed with water (2 Kg); the filter cake was triturated with acetone aqueous solution (8.9 Kg, V/V=1:1) at 25-30° C. for 2 hours; the system was cooled to 5-10° C., then the system was triturated for 2 hours, then was filtered, the filter cake was washed with acetone aqueous solution (1.86 Kg, V/V=1:2); the filter cake was dried under vacuum at 50-55° C., 1.3 Kg of solid was obtained, with a content of 39.9%, an HPLC purity of 97.1%.

Purification of a Compound of Formula I:

The above solid (1.3 Kg) and ethanol (6.50 Kg) were added to a rotary flask; the system was heated to 45-55° C., and was stirred for 30 minutes between 45-55° C., the materials were basically dissolved and the solution was clear; silica gel (2.60 kg) was added to the rotary flask, the flask was placed in a 50-60° C. water bath, concentrated under reduced pressure until there was basically no fraction flowing out; the system was exchanged with ethyl acetate (6.50 Kg) until there was basically no fraction flowing out, then stored for later use; a chromatographic column was prepared and washed, silica gel (13.00 Kg, 200-300 meshes) was added to the silica gel column; silica gel column was compacted with ethyl acetate (26.0 Kg); the prepared samples were added to the silica gel column, the samples were placed evenly; the column was eluted with ethyl acetate (52.0 Kg), then the column was eluted with a mixed solution of ethyl acetate and ethanol (VEA:VEtOH=100:1, 65.6 Kg), a mixed solution of ethyl acetate and ethanol (VEA:VEtOH=80:1, 64.7 Kg), a mixed solution of ethyl acetate and ethanol (VEA:VEtOH=50:1, 123.1 Kg), a mixed solution of ethyl acetate and ethanol (VEA:VEtOH=30:1, 64.8 Kg); components with a purity >99.0% by HPLC were collected; components with a purity >99.0% by HPLC were pumped into a reactor and concentrated to the minimum stirring volume; 4.0 Kg of methanol was pumped into the reactor, concentrated to the minimum stirring volume; 6.50 Kg×5 of MTBE was pumped into the reactor, concentrated to the minimum stirring volume; 6.50 Kg of MTBE was pumped into the reactor; the system was heated to reflux, and allowed the system to reflux for 1 hour; the system was cooled to 5-10° C., and stirred for 2 hours between 5-10° C.; filtered, the filter cake was washed with MTBE (1.3 Kg); 260 g of wet product was obtained, the filter cake was dried with a drying oven under vacuum between 50-55° C. for 16 hours; 0.250 Kg of product was obtained after drying with an HPLC purity of 100.0%.

The MS-ESI and1H NMR data are consistent with example 21.

In all the following examples of preparing crystal form 1 of a compound of formula I, crystal form A of a hydrochloride of a compound of formula I, crystal form B of a sulfate of a compound of formula I, crystal form C of a hydrobromide of a compound of formula I and crystal form D of a phosphate of a compound of formula I, a compound of formula I was used as a starting material.

Preparation of Crystal Form 1 of a Compound of Formula I

Example 30

A compound of formula I (172.6 g) and acetone (2705 g, ˜ 3500 mL) were added to a 5000 mL three-necked flask; the system was heated to 50-60° C., and was stirred for 2 hours between 50-60° C.; the system was cooled to room temperature (25-30° C.); stirred at room temperature (25-30° C.) for 24 hours; the system was distilled under reduced pressure, the volume of the liquid in the flask was distilled to about 0.8-0.9 L; the liquid was cooled to 15-25° C., about 4.3 Kg purified water was added; the system was stirred at room temperature for 2 hours; then cooled to 5-10° C., then was stirred for 2 hours; the system was subjected to suction filtration, the filter cake was washed with water, the filter cake was dried with an air blower between 50-55° C. for 16 hours; 158.8 g of off-white solid was obtained, with a yield of 92.0%. Upon testing, the off-white solid is crystal form 1 of a compound of formula I. The XRPD pattern, DSC thermogram, TGA thermogram and DVS isotherm plot of the crystal form 1 are shown inFIGS.1,2A,3and4respectively.

Example 31

A compound of formula I (100 mg) was dissolved with acetone (2 mL) between 50-55° C. and the solution was clear, the solution was cooled to room temperature and stirred for about 16 hours, 2 mL of water was added, the system was stirred at room temperature for 2 hours, cooled to 10-15° C., then was stirred for 2 hours, filtered, the solid was collected. Upon testing, the solid is crystal form 1 of a compound of formula I. The XRPD pattern of the crystal form is consistent withFIG.1.

Example 32

A compound of formula I (200 mg) was dissolved with 9 mL of methanol/water (9:1) at room temperature and the solution was clear, 5 mg of seed crystal of crystal form 1 of a compound of formula I was added, solids were precipitated, the system was stirred overnight at room temperature, water (18 mL) was added to the system, then the system was stirred at room temperature for 4 hours, cooled to 5-10° C. and stirred for 1 hour, subjected to suction filtration, dried under vacuum (about 50° C.) to obtain 180 mg of off-white solid. Upon testing, the off-white solid is crystal form 1 of a compound of formula I. The XRPD pattern of the crystal form is consistent withFIG.1.

Example 33

The crystallization method that is the same as that in Example 32 was adopted. The crystallization solvent was changed to acetone/water (4:1) to prepare crystal form 1 of a compound of formula I. The XRPD pattern of the crystal form is consistent withFIG.1.

Crystal form 1 of a compound of formula I can also be prepared with the following method:

Slurrying and Crystallization at Room Temperature

Example 34

About 20 mg of a compound of formula I was weighed then placed into a glass bottle, an appropriate amount of THF was added, the bottle was subjected to ultrasonication for 5 minutes to obtain suspension, the sample bottle was wrapped with tinfoil paper to protect the bottle from light and the bottle was placed on a Labquaker rotator and rotated 360 degrees at room temperature; the suspension sample was centrifuged on Day 10, the solid residue on the bottom was collected, the solvent was evaporated to dryness and an off-white solid was obtained. Upon testing, the off-white solid is crystal form 1 of a compound of formula I. The XRPD pattern of the crystal form is consistent withFIG.1.

Example 35-Example 40

The crystallization method that is the same as that in Example 34 was adopted. The crystallization solvent was changed to methyl tert-butyl ether, water, acetone, isopropanol, dichloromethane, and ethanol to prepare crystal form 1 of a compound of formula I. Results are shown in Table 6 below.

TABLE 6ExampleCrystal form of anumberCrystallization solventcompound of formula I35Methyl tert-butyl etherCrystal form 136WaterCrystal form 137AcetoneCrystal form 138IsopropanolCrystal form 139DichloromethaneCrystal form 140EthanolCrystal form 1

The XRPD pattern of crystal form 1 of a compound of formula I prepared in Examples 35-40 is consistent withFIG.1.

Slurrying and Crystallization at 50° C.

Example 41

About 20 mg of a compound of formula I was weighed and placed into a glass bottle, an appropriate amount of THF was added, the bottle was subjected to ultrasonication for 5 minutes to obtain suspension, the sample bottle was wrapped with a tinfoil paper to protect the bottle from light and the bottle was placed on a shaking incubator at 50° C. constant temperature for the suspension to be triturated; the suspension sample was centrifuged on Day 10, the solid residue on the bottom was collected, the solvent was evaporated to dryness and an off-white solid was obtained. Upon testing, the off-white solid is crystal form 1 of a compound of formula I. The XRPD pattern of the crystal form is consistent withFIG.1.

Example 42-Example 45

The crystallization method that is the same as that in Example 41 was adopted. The crystallization solvent was changed to methyl tert-butyl ether, water, acetone and isopropanol to prepare crystal form 1 of a compound of formula I. Results are shown in Table 7 below.

TABLE 7ExampleCrystal form of anumberCrystallization solventcompound of formula I42Methyl tert-butyl etherCrystal form 143WaterCrystal form 144AcetoneCrystal form 145IsopropanolCrystal form 1

The XRPD pattern of crystal form 1 of a compound of formula I prepared in Examples 42-45 is consistent withFIG.1.

Example 46

The crystallization method that is the same as that in Example 41 was adopted. The crystallization solvent was changed to methanol to prepare crystal form 1 of a compound of formula I. The XRPD of the crystal form is consistent withFIG.1and it is crystal form 1 of a compound of formula I. However, the DSC of the crystal form shows an additional peak at about 151° C., as shown inFIG.2B.

Heating of Saturated Solution—Rapid Cooling and Crystallization

Example 47

About 30 mg of a compound of formula I was weighed and placed into a bottle, an appropriate amount of acetone was added to the bottle, the sample bottle was placed on a magnetic heating stirrer, the temperature of a water bath was controlled at 50±2° C., the rotating speed was 200 rpm, sample was heated to promote dissolution, the temperature was maintained for 15 minutes after the sample was dissolved, the supersaturated solution was filtered with a 0.45 μm membrane when hot, the successive filtrate was transferred to a new bottle, then the bottle was immediately placed into a −20° C. refrigerator overnight, the solvent system with solids precipitated was centrifuged and then the solids were collected, the solvent was evaporated to dryness naturally to obtain an off-white solid. Upon testing, the solid is crystal form 1 of a compound of formula I. The XRPD pattern of the crystal form is consistent withFIG.1.

Example 48

The crystallization method that is the same as that in Example 47 was adopted. The crystallization solvent as changed to THF to prepare crystal form 1 of a compound of formula I. The XRPD spectrum of crystal form 1 of 1 compound of formula I prepared is consistent withFIG.1.

Heating of Saturated Solution—Slow Cooling and Crystallization

Example 49

About 30 mg of a compound of formula I was weighed and placed into a bottle, an appropriate amount of acetone was added to the bottle, the sample bottle was placed on a magnetic heating stirrer, the temperature of a water bath was controlled at 50±2° C., the rotating speed was 200 rpm, the sample was heated to promote dissolution, the temperature was maintained for 15 minutes after the sample was dissolved, the supersaturated solution was filtered with a 0.45 μm membrane when hot, the successive filtrate was transferred to a new bottle, then was slowly cooled to room temperature at a rate of 6° C./h, stored in a refrigerator the next day for about 24 hours (2-8° C.), the solvent system with solids precipitated was centrifuged and then the solids were collected, the solvent was evaporated to dryness naturally to obtain an off-white solid. Upon testing, the off-white solid is crystal form 1 of a compound of formula I. The XRPD pattern of the crystal form is consistent withFIG.1.

Example 50

The crystallization method that is the same as that in Example 49 was adopted. The crystallization solvent was changed to dichloromethane to prepare crystal form 1 of a compound of formula I. The XRPD pattern of the crystal form of a compound of formula I prepared is consistent withFIG.1.

Anti-Solvent Crystallization

Example 51-Example 62

About 30 mg of a compound of formula I was weighed and placed into a bottle, a certain volume of good solvent was added, the bottle was subjected to ultrasonication at room temperature to disperse the sample to evenly. If the solution was clear, more solid sample was added and ultrasonicating was continued to promote dissolution to obtain a supersaturated solution of the sample in the good solvent. The solution was filtered with a 0.45 μm membrane, the initial filtrate was discarded, the successive filtrate was transferred to a new bottle, an anti-solvent with a volume that is 10 times that of the good solvent was added to the bottle during stirring and the stirring was maintained at 50 rpm, the solvent system with solids precipitated was centrifuged and an off-white solid was obtained. Upon testing, the off-white solid prepared in Examples 51-62 is crystal form 1 of a compound of formula I. The XRPD pattern of the crystal form is consistent withFIG.1. Results are shown in Table 8 below.

TABLE 8ExampleCrystallization solventCrystal form of aNumber(good solvent - anti-solvent)compound of formula I51Methanol - water (1:10)Crystal form 152Ethanol - water (1:10)Crystal form 153THF-methyl tert-butyl etherCrystal form 1(1:10)54Ethanol-methyl tert-butylCrystal form 1ether (1:10)55Methanol-methyl tert-butylCrystal form 1ether (1:10)56Acetone-methyl tert-butylCrystal form 1ether (1:10)57Isopropanol-methyl tert-butylCrystal form 1ether (1:10)58THF - dichloromethane (1:10)Crystal form 159Ethanol-dichloromethane (1:10)Crystal form 160Methanol-dichloromethane (1:10)Crystal form 161Acetone-dichloromethane (1:10)Crystal form 162Isopropanol - dichloromethaneCrystal form 1(1:10)

Preparation of Crystal Form a of a Hydrochloride of a Compound of Formula I

Example 63

About 200 mg of a compound of formula I was weighed and placed into a bottle, 4 mL of ethanol was added, the bottle was subjected to ultrasonication and heating until the sample was completely dissolved, the sample bottle was placed on a magnetic heating stirrer, magnetic stirring was conducted and during stirring, 1.32 ml of hydrochloric acid in ethanol (the concentration of the hydrochloric acid in ethanol was 50 mg/mL) was slowly added dropwise, a white precipitate was produced, the bottle was covered with a cap tightly at room temperature and was stirred for 1 day, the suspending reaction liquid was then centrifuged, the collected solid was dried under vacuum at 40° C. overnight, a hydrochloride solid of a compound of formula I was obtained. Upon testing, the solid is crystal form A of a hydrochloride of a compound of formula I. The XRPD pattern and the DSC thermogram of the crystal form were shown inFIGS.5and6.

Example 64

About 50 mg of a compound of formula I was weighed and placed into a bottle, 1 mL of ethanol was added, the bottle was subjected to ultrasonication and heating until the sample was completely dissolved, the sample bottle was placed on a magnetic heating stirrer, magnetic stirring was conducted and during stirring, 0.33 ml hydrochloric acid in ethanol (the concentration of the hydrochloric acid in ethanol was 50 mg/mL) was slowly added dropwise, a white precipitate was produced, the bottled was covered with a cap tightly at room temperature and was stirred for 1 day, the suspending reaction liquid was then centrifuged, the collected solid was dried under vacuum at 40° C. overnight, a hydrochloride solid of a compound of formula I was obtained. Upon testing, the solid is crystal form A of a hydrochloride of a compound of formula I. The XRPD pattern of the crystal form is consistent withFIG.5.

Preparation of Crystal Form B of a Sulfate of a Compound of Formula I

Example 65

About 200 mg of a compound of formula I was weighed and placed into a bottle, 4 mL of ethanol was added, the bottle was subjected to ultrasonication and heating until the sample was completely dissolved, the sample bottle was placed on a magnetic heating stirrer, magnetic stirring was conducted and during stirring, 1.4 ml of sulfuric acid in ethanol (the concentration of the sulfuric acid in ethanol was 50 mg/mL) was slowly added dropwise, a white precipitate was produced, the bottle was covered with a cap tightly at room temperature and was stirred for 1 day, the suspending reaction liquid was centrifuged, the collected solid was dried under vacuum at 40° C. overnight, a sulfate solid of a compound of formula I was obtained. Upon testing, the solid is crystal form B of a sulfate of a compound of formula I. The XRPD pattern, DSC thermogram, TGA thermogram and DVS isotherm plot were shown inFIGS.7-10.

Example 66

About 50 mg of a compound of formula I was weighed and placed into a bottle, 1 mL of ethanol was added, the bottle was subjected to ultrasonication and heating until the sample was completely dissolved, the sample bottle was placed on a magnetic heating stirrer, magnetic stirring was conducted and during stirring, 0.35 ml of sulfuric acid in ethanol (the concentration of the sulfuric acid in ethanol was 50 mg/mL) was slowly added dropwise, a white precipitate was produced, the bottle was covered with a cap tightly at room temperature and was stirred for 1 day, the suspending reaction liquid was centrifuged, the collected solid was dried under vacuum at 40° C. overnight, a sulfate solid of a compound of formula I was obtained. Upon testing, the solid is crystal form B of a sulfate of a compound of formula I. The XRPD pattern is consistent withFIG.7.

Preparation of Crystal Form C of a Hydrobromide of a Compound of Formula I

Example 67

About 200 mg of a compound of formula I was weighed and placed into a bottle, 4 mL of ethanol was added, the bottled was subjected to ultrasonication and heating until the sample was completely dissolved, the sample bottle was placed on a magnetic heating stirrer, magnetic stirring was conducted and during stirring, 2.4 ml of hydrobromic acid in ethanol (the concentration of the hydrobromic acid in ethanol was 50 mg/mL) was slowly added dropwise, a white precipitate was produced, the bottle was covered with a cap tightly at room temperature and was stirred for 1 day, the suspending reaction liquid was centrifuged, the collected solid was dried under vacuum at 40° C. overnight, a hydrobromide solid of a compound of formula I was obtained. Upon testing, the solid is crystal form C of a hydrobromide of a compound of formula I. The XRPD pattern and DSC thermogram were shown inFIGS.11and12.

Example 68

About 50 mg of a compound of formula I was weighed and placed into a bottle, 1 mL of ethanol was added, the bottle was subjected to ultrasonication and heating until the sample was completely dissolved, the sample bottle was placed on a magnetic heating stirrer, magnetic stirring was conducted and during stirring, 0.60 ml of hydrobromic acid in ethanol solution (the concentration of the hydrobromic acid in ethanol was 50 mg/mL) was slowly added dropwise, a white precipitate was produced, the bottle was covered with a cap tightly at room temperature and was stirred for 1 day, the suspending reaction liquid was centrifuged, the collected solid was dried under vacuum at 40° C. overnight, a hydrobromide solid of a compound of formula I was obtained. Upon testing, the solid is crystal form C of a hydrobromide of a compound of formula I. The XRPD pattern of the crystal form is consistent withFIG.11.

Preparation of Crystal Form D of a Phosphate of a Compound of Formula I

Example 69

About 200 mg of a compound of formula I was weighed and placed into a bottle, 4 mL of ethanol was added, the bottled was subjected to ultrasonication and heating until the sample was completely dissolved, the sample bottle was placed on a magnetic heating stirrer, magnetic stirring was conducted and during stirring, 1.56 ml of phosphoric acid in ethanol (the concentration of the phosphoric acid in ethanol was 50 mg/mL) was slowly added dropwise, a white precipitate was produced, the bottle was covered with a cap tightly at room temperature and was stirred for 1 day, the suspending reaction liquid was centrifuged, the collected solid was transferred to another bottle, 3-4 ml of a mixed solvent: acetone/water (9/1, V/V) was added, magnetic stirring was conducted at room temperature overnight, the bottle was centrifuged, the solid was collected, and dried under vacuum at 40° C. overnight, a phosphate solid of a compound of formula I was obtained. Upon testing, the solid is crystal form D of a phosphate of a compound of formula I. The XRPD pattern, DSC thermogram, TGA thermogram and DVS isotherm plot are shown inFIGS.13-16.

Example 70

About 50 mg of a compound of formula I was weighed and placed into a bottle, 1 mL of ethanol was added, the bottled was subjected to ultrasonication and heating until the sample was completely dissolved, the sample bottle was placed on a magnetic heating stirrer, magnetic stirring was conducted and during stirring, 0.39 ml of phosphoric acid in ethanol solution (the concentration of phosphoric acid in ethanol was 50 mg/mL) was slowly added dropwise, a white precipitate was produced, the bottle was covered with a cap tightly at room temperature and was stirred for 1 day, the suspending reaction liquid was centrifuged, the collected solid was transferred to another bottle, 0.75-1 ml of a mixed solvent: acetone/water (9/1, V/V) was added, magnetic stirring was conducted at room temperature overnight, the bottle was centrifuged, the solid was collected, and dried under vacuum at 40° C. overnight, a phosphate solid of a compound of formula I was obtained. Upon testing, the solid is crystal form D of a phosphate of a compound of formula I. The XRPD pattern of the crystal form is consistent withFIG.13.

Experimental Section

Experimental Example 1 Solid Characterization

DSC, TGA and DVS tests were conducted for crystal form A of a hydrochloride of a compound of formula I, crystal form B of a sulfate of a compound of formula I, crystal form C of a hydrobromide of a compound of formula I and crystal form D of a phosphate of a compound of formula I.

The DSC test results indicated that crystal form B of a sulfate of a compound of formula I (FIG.8) and crystal form D of a phosphate of a compound of formula I (FIG.14) had single and higher melting points, indicating that the thermostabilities of the crystal forms were relatively good. Thermodynamic behaviors of crystal form A of a hydrochloride of a compound of formula I (FIG.6) and crystal form C of a hydrobromide of a compound of formula I (FIG.12) were complex and their crystallinities were lower. Therefore, further DVS and TGA characterizations were conducted for crystal form B of a sulfate of a compound of formula I and crystal form D of a phosphate of a compound of formula I only.

The DVS test results indicated that crystal form B of a sulfate of a compound of formula I (FIG.10) and crystal form D of a phosphate of a compound of formula I (FIG.16) have certain hygroscopicities. Moisture weight gains were 9.4% and 4.7% from 0% RH-95% RH, respectively. There were no significant changes in the crystal forms of the two salts before and after the DVS test.

The TGA test results indicated that crystal form D of a phosphate of a compound of formula I (FIG.15) had a weight loss of 2.862% from 44° C. to 120° C.

On the basis of the characterization results of the crystal forms of the above four salts, their comparison with crystal form 1 of a compound of formula I is shown in Table 9 below.

TABLE 9Crystal form ACrystal form BCrystal form CCrystal form DCrystal form 1of a hydrochlorideof a sulfateof a hydrobromideof a phosphateof a compoundof a compoundof a compoundof a compoundof a compoundof formula Iof formula Iof formula Iof formula Iof formula ICrystallinityExcellentGoodExcellentGoodExcellentInitial melting161° C.No obvious199° C.145° C.207° C.pointmelting pointTGA~5.4%———~2.9%(25-133° C.)(44-120° C.)Hygroscopicity5.2%—9.4%—4.7%(95% RHhygroscopic)“—” means not tested

In conclusion, crystal form A of a hydrochloride of a compound of formula I and crystal form C of a hydrobromide of a compound of formula I have wide melting ranges and the melting points are not obvious; the hygroscopicity of crystal form B of a sulfate of a compound of formula I is stronger than that crystal form D of a phosphate of a compound of formula I. Crystal form 1 of a compound of formula I and crystal form D of a phosphate of a compound of formula I have better crystallinities, single melting points, and relatively lower hygroscopicities.

Experimental Example 2 Solubility Test

4 samples, each of crystal form 1 of a compound of formula I and crystal form D of a phosphate of a compound of formula I each with appropriate amount were weighed and placed into a 4 mL transparent glass bottle respectively, 1 mL of water, simulated gastric fluid (SGF), fasted-state simulated intestinal fluid (FaSSIF) and fed-state simulated intestinal fluid (FeSSIF) were added respectively, sample suspension was obtained and quickly placed on a shaker (37° C., 200 rpm) and the shaker was shaked, the samples were observed 5 minutes later, a quantity of samples or medium were supplemented to obtain mild suspension, samples were taken at 30 minutes, 2 hours, 4 hours and 24 hours, respectively, centrifuged for 10 minutes at 12000 rpm, the supernatants were collected, diluted appropriately and then were tested by a higher performance liquid chromatography. The chromatographic conditions were shown in Table 10.

TABLE 10High performance liquid chromatographicconditions of the solubility testInstrumentAgilent 1200 DAD HPLC systemChromatographicWaters XBridge Shield RP18 4.6 ×column150 mm, 3.5 μmMobile phaseA: 0.05% phosphoric acid aqueous solutionB: AcetonitrileColumn temperature30° C.DetectorDADDetection wavelength232 nmInjection volume10 μLColumn flow rate1.0 mL/minRun time10 minCollection time10 minElution procedureTime (min)A (%)B (%)0.09556.070307.05957.195510955

The sample concentrations were calculated with an external standard method. The test results are shown in Table 11.

TABLE 11Solubility (based on acompound of formula I, mg/mL)SampleMedium30 min2 h4 h24 hCrytstal formWater0.160.180.160.181 of a compoundSimulated gastric16.5216.7116.8816.42of formula Ifluid (SGF)Fasted-state0.170.190.160.17simulated intestinalfluid (FaSSIF)Fed-state simulated0.260.230.220.24intestinal fluid(FeSSIF)Crystal formWater1.851.781.731.80D of a phosphateSimulated gastric30.2229.7329.4730.18of a compound offluid (SGF)formula IFasted-state0.170.170.170.16simulated intestinalfluid (FaSSIF)Fed-state simulated0.360.220.190.21intestinal fluid(FeSSIF)

The results showed that there was no obvious difference in the equilibrium solubilities (24 h) of crystal form 1 of a compound of formula I and crystal form D of a phosphate of a compound of formula I in FaSSIF and FeSSIF. In water and SGF, equilibrium solubilities (24 h) of crystal form D of a phosphate of a compound of formula I were 1.8 mg/mL and 30.18 mg/mL, respectively, that is, 10 times and 2 times that of the crystal form 1 of a compound of formula I, respectively. Solubilities of crystal form 1 of a compound of formula I and crystal form D of a phosphate of a compound of formula I of the invention meet medicinal requirements.

Experimental Examples 3 Stability Test

About 1 mg of crystal form 1 of a compound of formula I and 1 mg of crystal form D of a phosphate of a compound of formula I samples were weighed and each was placed into a 40 mL transparent glass bottle, the samples were placed in a stability chamber in accelerated conditions (40° C./75% RH, open) and at a high temperature (60° C., sealed) respectively. For the open samples, the bottle cap was removed and the bottle neck was covered with aluminium-foil paper stabbed with pinholes to avoid cross contamination; for the closed samples, each bottle was covered with a cap and sealed tightly. Samples were taken at week 1 and week 2, respectively, after diluted with diluent (acetonitrile/water (1/1) (v/v), the liquid phases were injected according to the chromatographic conditions in Table 12 to determine sample purities.

TABLE 12InstrumentAgilent 1200 DAD HPLC systemChromatographicAgilent ZORBAX SB-Phenyl 150*4.6 mmcolumn3.5 umMobile phaseA: 20 mM ammonium acetate aqueous solutionB: MethanolColumn temperature40° C.DetectorDADDetection wavelength220 nmInjection volume5.0 μLColumn flow rate1.0 mL/minRun time42 minCollection time42 minElution procedureTime (min)A (%)B (%)070301446.553.530257535257535.017030427030

The sample purities were calculated with an area normalization method. The test results are shown in Table 13.

TABLE 13Stability study (purity, Area %)60° C.40° C./75% RHCrystalCrystalSample0 day1 week2 weeksform1 week2 weeksformCrystal form 1 of a98.7498.7398.74Unchanged98.7198.77Unchangedcompound of formula ICrystal form D of a99.1399.1199.11Unchanged99.1199.10Unchangedphosphate of acompound of formula I

The results indicated that the appearances of crystal form 1 of a compound of formula I and crystal form D of a phosphate of a compound of formula I samples didn't change within 2 weeks and they were off-white powder. There was no significant difference in their purities and the total related substances didn't increase. The XRPD and DSC tests (FIGS.17-20) indicated that there was no significant difference in the crystal forms and the initial melting points of the two samples compared with those on Day 0, showing that the 2-week physical and chemical stabilities of crystal form 1 of a compound of formula I and crystal form D of a phosphate of a compound of formula I were good at a high temperature (60° C.) and in accelerated conditions (40° C./75% RH).

Experimental Example 4 TYK2 Biochemical Test

An appropriate amount of a compound of formula I was weighed for TYK2 biochemical test.

The test was conducted by Reaction Biology Corp, Malvern, PA (Anastassiadis et al. Nat Biotechnol. 2011; 29(11):1039-45). The step is briefly described as follows.

Reagents:

Basic reaction buffer: 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT and 1% DMSO. The required cofactor was added to each kinase reaction respectively.

Reaction Steps:

1. Preparing the designated substrate in the newly prepared basic reaction buffer;2. Transferring the required cofactor to the above matrix solution;3. Transferring the designated kinase to the substrate solution and mixing well slightly;4. Transferring a compound of formula I in DMSO to the kinase reaction mixture with Acoustic technique (Echo550; nanoliter range), culturing for 20 minutes at room temperature;5. Introducing33P-ATP (specific activity: 10 μCi/μl) to the reaction mixture to trigger a reaction;6. Culturing at room temperature and conducting a kinase reaction for 2 hours;7. Plotting the reaction on P81 ion exchange paper;8. Testing the kinase activity with a filter binding assay.

The test results indicated that a compound of formula I was also a potent TYK2 inhibitor and its IC50was less than 10 nM

A person skilled in the art can understand and make some modifications or changes to the invention under the instruction of the present description. These modifications and changes should be in the scope defined in the claims of the invention.