PREPARATION METHOD FOR BENZOXAZOLEOXAZINE KETONE COMPOUND AND INTERMEDIATE AND CRYSTAL FORM THEREOF

Abstract

A preparation method for a high-purity benzoxazoleoxazine ketone compound, a crystal form thereof, and an intermediate compound for preparing a compound of formula (I), and a preparation method therefor.

##STR00001##

Claims

1. A process for preparing the compound having a structure of formula (I), ##STR00033## which comprises the following step, ##STR00034## wherein, R is an amino-protecting group, or selected from an alkoxycarbonyl, or Cbz, Boc, Fmoc, Alloc, Teco, methoxycarbonyl or ethoxycarbonyl; X is F, Cl, Br or I; R.sup.2 is selected from optionally substituted 5- or 6-membered cyclic amino or heterocyclic amino, “hetero” represents O, N, C(═O) or C(═O)NH, the substituent is independently selected from a C.sub.1-4 alkyl or a heteroalkyl, or, R.sup.2 is selected from ##STR00035##

2. The process of claim 1, wherein the base is selected from an alkali metal base, an alkaline earth metal base or an organometallic base; or, the alkali metal base is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, sodium bicarbonate and/or potassium bicarbonate; or, the alkaline earth metal base is selected from sodium hydride, potassium hydride and/or calcium hydride; or, the organometallic base is selected from sodium methoxide, lithium tert-butoxide, sodium tert-butoxide, potassium tert-butoxide, sodium ethoxide and/or aluminum isopropoxide.

3. The process of claim 1, wherein a molar ratio of the compound (IV) to the base is 1:1 to 5, or 1:2 to 3; a molar ratio of the compound (III) to the compound (IV) is 1:1 to 2; temperature of the reaction is −10 to 50° C., or 0 to 30° C., and/or, the reaction time is 5 to 200 hours, or 10 to 100 hours, or 16 to 48 hours.

4. The process of claim 1, wherein the reaction is carried out in a solvent, the solvent is selected from an amide solvent, an ether solvent or any mixture thereof; or, an amount of the solvent is 10 to 50 times the weight of the compound (IV), or 15 to 20 times, or, the amide solvent is selected from DMF or DMAC, and/or or, the ether solvent is selected from tetrahydrofuran, methyl tetrahydrofuran, dioxane or methyl t-butyl ether.

5. The process of claim 1, comprising: ##STR00036## ##STR00037## ##STR00038## ##STR00039## ##STR00040## ##STR00041## wherein HA is selected from an organic acid or an inorganic acid, or HA is selected from hydrochloric acid, sulfuric acid, oxalic acid, citric acid, maleic acid or fumaric acid.

6. An intermediate for preparing the compound having the structure of formula (I), ##STR00042## wherein, R is an amino-protecting group; or selected from an alkoxycarbonyl, or selected from Cbz, Boc, Fmoc, Alloc, Teco, methoxycarbonyl or ethoxycarbonyl; R.sup.2 is selected from optionally substituted 5- or 6-membered cyclic amino or heterocyclic amino, “hetero” represents O, N, C(═O) or C(═O)NH, the substituent is independently selected from a C.sub.1-4 alkyl or a heteroalkyl; or, R.sup.2 is selected from ##STR00043## HA is selected from an organic acid or an inorganic acid; or HA is selected from hydrochloric acid, sulfuric acid, oxalic acid, citric acid, maleic acid or fumaric acid.

7. A process for preparing the intermediate (IV) of claim 6, comprising: ##STR00044##

8. The process of claim 7, wherein the base is selected from an alkali metal base, an alkaline earth metal base or an organometallic base; or, a molar ratio of the compound (a) to the base is preferably 1:1 to 3; or, a molar ratio of the compound (a) to R.sup.2—H is preferably 1:1 to 2; or, the alkali metal base is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, sodium bicarbonate and/or potassium bicarbonate; or, the alkaline earth metal base is selected from sodium hydride, potassium hydride and/or calcium hydride; or, the organometallic base is selected from sodium methoxide, lithium tert-butoxide, sodium tert-butoxide, potassium tert-butoxide, sodium ethoxide and/or aluminum isopropoxide; or, the reaction is carried out in a solvent, the solvent is selected from a mixed solvent of non-polar solvent and polar solvent; or, the non-polar solvent is selected from benzene, toluene, xylene chlorobenzene or bromobenzene; or, the polar solvent is selected from DMF, DMSO or NMP; or, a volume ratio of the non-polar solvent to the polar solvent is 1:0.5 to 2; and/or, or, an amount of the solvent is 5 to 20 times the weight of the compound (a).

9. The process of claim 7, comprising: ##STR00045## ##STR00046##

10. A crystal form of the compound 1 having the following formula, the structure of which is shown in FIG. 1 or FIG. 2; ##STR00047##

11. A process for preparing the crystal form of claim 10, comprising adding any one of the forms of compound 1 to a solvent and crystallizing, wherein, or, an amount of the solvent is 3 to 50 times the weight of the compound 1, or, the solvent is selected from an alcohol solvent or a mixed solvent containing an alcohol solvent or water; or, the alcohol solvent is selected from methanol, ethanol, isopropanol and/or n-butanol; or, the mixed solvent is selected from a mixed solvent of DMSO and ethanol or water; or, a volume ratio of DMSO to ethanol or water is 1:0.5 to 5.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0214] FIG. 1 is the XRPD spectra of crystal form A of the compound 1 under Cu—K alpha radiation, where the crystal form A is obtained by crystallizing the compound 1 in ethanol.

[0215] FIG. 2 is the XRPD spectra of crystal form B of the compound 1 under Cu—K alpha radiation, where the crystal form B is obtained by crystallizing the compound 1 in DMSO/EtOH system.

[0216] FIG. 3 is a DSC spectrum of crystal form A of the compound 1.

[0217] FIG. 4 is a DSC spectrum of crystal form B of the compound 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0218] For better understanding the present invention, the following embodiments further illustrate the present invention, but the present invention is not limited thereto.

Embodiment 1 the Process for Preparing the Compound 1

[0219] Process 1:

##STR00023##

Step 1: Synthesis of 2-(tert-butoxy)-4-fluoro-1-nitrobenzene

[0220] Under an ice-water bath, potassium tert-butoxide (387 g, 3.46 mol) was slowly added to a solution of 2,4-difluoronitrobenzene (500 g, 3.14 mol) in toluene (2 L) in batches, the temperature was kept no more than 20° C. After the addition, the reaction solution was cooled to 0° C. and stirred for 2 hours at this temperature. TLC plate (petroleum ether) was used to detect whether the reaction was complete. The reaction solution was poured into a cold saturated ammonium chloride solution (3 L), the mixture was extracted with ethyl acetate (1 L×3). The organic phases were combined and dried to give 2-(tert-butoxy)-4-fluoro-1-nitrobenzene (670 g, crude, dark red oil, HPLC 90%), which was not further purified and used directly in the next step. .sup.1HNMR (CDCl.sub.3, 400 MHz) δ 7.82 (dd, J=9.2, 6.0 Hz, 1H), 6.93 (dd, J=10.4, 2.8 Hz, 1H), 6.84(m, 1H), 1.48(s. 9H).

Step 2: Synthesis of 4-(3-(tert-butoxy)-4-nitrophenyl)morpholin-3-one

[0221] Under an ice-water bath, potassium tert-butoxide (315 g, 2.81 mol) was slowly added in batches to a solution of 2-(tert-butoxy)-4-fluoro-1-nitrobenzene (600 g, 2.81 mol) and morpholin-3-one (284 g, 2.81 mol) in a mixed solvent of toluene and dimethyl sulfoxide (1:1, 1 L) while the temperature was kept no more than 20° C. After the addition, the reaction solution was heated to 90° C. and stirred at this temperature for 2 hours. TLC (PE) was used to detect whether the reaction was complete. The reaction solution was poured into a cold saturated ammonium chloride solution (4 L), the mixture was extracted with ethyl acetate (1 L×2). The combined organic phase was washed with water (2 L) and evaporated to give 4-(3-(tert-butoxy)-4-nitrophenyl)morpholin-3-one (828 g, crude, dark red oil, HPLC 80%), which was not further purified and used directly in the next step. .sup.1HNMR (CDCl.sub.3, 400 MHz) δ 7.79 (d, J=9.2 Hz, 1H), 7.42 (d, J=2.0 Hz, 1H). 7.10 (dd. J=8.8, 2.4 Hz, 1H), 4.36 (s, 2H), 4.06 (t, J=2.0 Hz, 2H), 3.82 (t, J=2.0 Hz, 2H), 1.45 (s, 9H).

Step 3: Synthesis of 4-(4-amino-3-(tert-butoxy)phenyl)morpholin-3-one

[0222] Iron powder (106 g, 1.90 mol) was added to a vigorously stirred mixed solution of 4-(3-(tert-butoxy)-4-nitrophenyl)morpholin-3-one (140 g, 0.475 mol) and ammonium chloride (101 g, 1.90 mol) in ethanol and water (3:1, 1.2 L). After the addition, the reaction was stirred for about half an hour and became greatly exothermic, cooled with ice water until the heat release stopped, and then warmed to room temperature overnight. TLC (PE:EA=1:1) was used to detect whether the reaction was complete, filtered, and the residue was washed with methanol. TLC (PE:EA=1:1) was used to detect the wash was complete. The filtrates were combined and evaporated to dry, washed with water (500 mL) and extracted with dichloromethane (300 mL×3). The organic phases were combined and dried to give 4-(4-amino-3-(tert-butoxy)phenyl)morpholin-3-one (125 g, crude, black oil, HPLC 70%), which was not further purified and used directly in the next step. .sup.1HNMR (CDCl.sub.3, 400 MHz) δ 6.84 (d, J=2.0 Hz, 1H), 6.75 (m, 1H), 6.65 (m, 1H), 4.76 (brs, 2H), 4.14 (s, 2H), 3.91 (t, J=2.0 Hz, 2H), 3.62 (t, 2.0 Hz, 2H), 1.33 (s, 9H).

Step 4: Synthesis of benzyl (2-hydroxyl-4-(3-ketomorpholine)phenyl) carbamate

[0223] 4-(4-amino-3-(tert-butoxy)phenyl)morpholin-3-one (400 g, 1.51 mol) was dissolved in 4N hydrochloric acid solution (1 L), stirred and reacted overnight. The reaction solution was cooled with ice water, and the pH value of the reaction solution was adjusted to about 7 with 12N sodium hydroxide solution, and then sodium bicarbonate solid (190 g, 2.23 mol) and tetrahydrofuran (600 mL) were added. After cooling to 0° C., a solution of benzyl chloroformate (258 g, 1.51 mol) in tetrahydrofuran (500 mL) was slowly added dropwise and the temperature was kept below 10° C. After the addition, TLC (PE:EA=1:3) was used to detect the reaction was complete. Dichloromethane (2 L) and water (1 L) were added to the reaction solution, layered and the aqueous phase was extracted with dichloromethane (1 L×2). The organic phases were combined and evaporated to dry, and then the ethanol was added to triturate, filtered, the filter cake was washed twice with ethanol (200 mL), the cake was then dried to give benzyl (2-hydroxyl-4-(3-ketomorpholine)phenyl)carbamate (225 g, HPLC 98%), brown solid. .sup.1HNMR (DMSO-d.sub.6, 400 MHz) δ 9.95 (brs, 1H), 8.52 (s, 1H), 7.56 (d, J=9.2 Hz, 1H), 7.45-7.33 (m, 5H), 6.91 (d, J=2.4 Hz, 1H), 6.79 (dd, J=8.4, 2.0 Hz, 1H), 5.15 (s, 2H), 4.18 (s, 2H), 3.95 (t, J=4.8 Hz, 2H), 3.66 (t, J=4.8 Hz, 2H), 1.33 (s, 9H).

[0224] Process 2:

##STR00024##

Step 5: Synthesis of 4-(3-(hydroxyl)-4-nitrophenyl)morpholin-3-one

[0225] 10 mL Con. HCl (aq) was slowly added to a solution of 4-(3-(tert-butoxy)-4-nitrophenyl)morpholin-3-one (10.0 g, 34.0 mmol) in ethyl acetate (30 mL). After the mixture was stirred at room temperature overnight, water (50 mL) was added and the mixture was extracted with ethyl acetate (30 mL×2). The organic phases were combined and evaporated to dry to give a crude, and then ethanol (100 mL) was added to triturate, filtered and evaporated to dry to give 4-(3-(hydroxyl)-4-nitrophenyl)morpholin-3-one (4.12 g, HPLC 98%, yield 51%). .sup.1HNMR (DMSO-d.sub.6, 400 MHz) δ 11.1 (brs, 1H), 7.96 (d, J=8.0 Hz, 1H), 7.31 (d, J=2.4 Hz, 1H), 7.11 (dd, J=8.8, 2.4 Hz, 1H), 4.25 (s, 2H), 3.98 (t, J=4.8 Hz, 2H), 3.79 (t, J=4.8 Hz, 2H), 1.33 (s, 9H).

Step 6: Synthesis of 4-(4-amino-3-(hydroxyl)phenyl)morpholin-3-one

[0226] In a 250 mL hydrogenation flask, under nitrogen gas protection, dry palladium on carbon (200 mg) was added to a solution of 4-(3-(hydroxyl)-4-nitrophenyl)morpholin-3-one (3.5 g, 14.7 mmol) in methanol (100 mL). The reaction solution was stirred at 30° C. for 16 hours at 30 psi hydrogen pressure. TLC (PE:EA=1:1) was used to detect the reaction was complete, and the mixture was filtered, the filter cake was washed with hot methanol (200 mL). The combined organic phase was evaporated to dry to give 4-(4-amino-3-(hydroxyl)phenyl)morpholin-3-one (2.7 g, HPLC 91%, yield 88%). HNMR (DMSO-d.sub.6, 400 MHz) δ 9.22 (brs, 1H), 6.63 (s, 1H), 6.57 (d, J=8.4 Hz, 1H), 6.51 (d, J=8.4 Hz, 1H), 4.59 (brs, 2H), 4.13 (s, 2H), 3.91 (t, J=4.4 Hz, 2H), 3.57 (t, J=4.4 Hz, 2H).

Step 7: Synthesis of benzyl (2-hydroxyl-4-(3-ketomorpholine)phenyl) carbamate

[0227] Under an ice-water bath, sodium bicarbonate solid (1.63 g, 1.9.4 mmol) was added to a mixed solution of 4-(4-amino-3-(hydroxyl)phenyl)morpholin-3-one (2.7 g, 12.9 mmol) in tetrahydrofuran (10 ml) and water (15 mL). After cooling to 0° C., a solution of benzyl chloroformate (1.63 g, 19.4 mmol) in tetrahydrofuran (5 ml) was slowly added dropwise and the temperature was kept no more than 5° C. After the addition, TLC (PE:EA=1:3) was used to detect the reaction was complete. Water (30 ml) was added to the reaction solution and stirred for five minutes and then filtered, the filter cake was washed with water (10 ml), collected and evaporated to dry, triturated with ethanol to give benzyl (2-hydroxyl-4-(3-ketomorpholine)carbamate (3.66 g, white solid, HPLC 96%, yield 82%). .sup.1HNMR (DMSO-d.sub.6, 400 MHz) δ 9.95 (brs, 1H), 8.52 (s, 1H), 7.56 (d, J=9.2 Hz, 1H), 7.45-7.33 (m, 5H), 6.91 (d, J=2.4 Hz, 1H), 6.79 (dd, J=8.4, 2.0 Hz, 1H), 5.15 (s, 2H), 4.18 (s, 2H), 3.95 (t, J=4.8 Hz, 2H), 3.66 (t, J=4.8 Hz, 2H), 1.33 (s, 9H).

[0228] Process 3:

##STR00025##

Step 8: Synthesis of potassium (2R,3R)-2,3,4-trihydroxyl butyrate

[0229] Under an ice-water bath, potassium hydroxide (1.12 kg, 19.98 mol) was slowly added to a mixed solvent of methanol (10 L) and water (2.4 L) and the temperature was kept no more than 45° C. After the addition, oxygen gas was introduced into the reaction solution, when the reaction solution was cooled to 35° C., an aqueous solution of D-(−)-arabinose (1 kg, 6.66 mol, dissolved in 2.4 L water) was added dropwise over more than 6 hours (oxygen gas was introduced during the whole period) and the reaction temperature was kept at 35° C. After the addition, oxygen gas was further introduced for 2 hours, followed by introducing air for 60 hours. The reaction solution was concentrated under reduced pressure to give 2.4 L oil and the oil was slowly added dropwise into the rapidly stirring methanol (IOL) and a large amount of white solid was precipitated. After the addition, the mixture was filtered and dried in vacuum to give white solid (560 g, yield 48%). .sup.1HNMR (D.sub.2O, 400 MHz) δ 4.09 (d, J=4.0 Hz, 1H), 3.97-3.92 (m, 1H), 3.65 (d, J=5.6 Hz, 1H).

Step 9: Synthesis of methyl (2S,3R)-2,4-dibromo-3-hydroxylbutyrate

[0230] A solution of hydrogen bromide/acetic acid (33%, 2 L) was added to a reactor containing potassium (2R,3R)-2,3,4-trihydroxylbutyrate (560 g, 3.21 mol). The mixture was stirred at room temperature for 24 hours, and then poured into anhydrous methanol (14 L) and further stirred at room temperature for 18 hours. The reaction solution was heated to 65° C. and refluxed for 4 hours and then concentrated under reduced pressure. Water (1 L) and ethyl acetate (1.5 L) were added to extract, the organic phase was separated, dried over sodium sulfate, concentrated to give a mixture of (2S,3R)-2,4-dibromo-3-hydroxylbutyric acid and methyl (2S,3R)-2,4-dibromo-3-hydroxylbutyrate as an oil (706 g). Sulfuric acid (60 mL) was slowly added to a solution of the mixture (680 g) in methanol (3.5 L), heated to reflux for 6 hours. TLC (PE:EA=10:1) was used to detect the reaction was complete, the reaction solution was concentrated, the residue was dissolved in ethyl acetate (800 ml), washed with water (500 ml), the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give the product as an off-white solid (650 g, yield 74%). .sup.1HNMR (CDCl.sub.3, 400 MHz) δ 4.71 (d, J=3.6 Hz, 1H), 4.20-4.15 (m, 1H), 3.83 (s, 3H), 3.55-3.48 (m, 2H).

Step 10: Synthesis of methyl (2R,3S)-3-bromomethyl ethylene oxide-2-carboxylate

[0231] Potassium carbonate (503 g, 3.65 mol) was added to a solution of methyl (2S,3R)-2,4-dibromo-3-hydroxylbutyrate (200 g, 0.729 mol) in acetone (2.5 L), the reaction was carried out at room temperature for 4 hours, TLC (PE:EA=10:1) showed that the raw material disappeared. The reaction solution was filtered and concentrated under reduced pressure to give the product as a pale yellow oil, which was distilled to give the product as a colorless liquid (126 g, 90%) by using a vacuum distillation apparatus. .sup.1HNMR (CDCl.sub.3, 400 MHz) δ 3.83 (s, 3H), 3.67-3.62 (m, 2H), 3.57-3.52 (m, 1H), 3.46-3.43 (m, 1H).

[0232] Process 4:

##STR00026##

Step 11: Synthesis of (3R,4R)-3,4-dihydroxyl dihydrofuran-2(3H)-one

[0233] The isoascorbic acid (17.6 g, 0.1 mol) was dissolved in 250 mL water and the mixture was cooled to 0 to 6° C. Anhydrous sodium carbonate powder (21.2 g, 0.2 mol) was added to the reaction flask in batches. After the addition, 30% hydrogen peroxide (22 mL) was added while stirring, and the internal temperature was raised from 6° C. to 19° C. The mixture was further stirred for 5 minutes under an ice bath and the internal temperature was raised to 27° C. The reaction solution was heated to 42° C. and stirred for 30 minutes. The zinc powder (1.0 g, 0.015 mol) was added to the reaction solution to quench the excess hydrogen peroxide, starch potassium iodide test paper showed negative result. The pH value of the reaction solution was adjusted to 1.0 with 6N hydrochloric acid. The mixture was concentrated at 50° C. under reduced pressure until little amount of white solid was precipitated. Extracted with ethyl acetate (150 ml×3). The organic phase was concentrated to 200 mL (10 to 15 vol); the temperature was cooled to 15 to 25° C. and the mixture was stirred for 5 to 8 hours, (a large amount of white solid precipitated) filtered and dried to give 8.26 g (3R,4R)-3,4-dihydroxyl dihydrofuran-2(3H)-one, yield 70%. .sup.1HNMR (DMSO-d.sub.6, 400 MHz) δ 5.78 (brs, 1H), 5.37 (brs, 1H), 4.39 (d, J=4.4 Hz, 1H), 4.30-4.22 (m, 2H), 4.05 (d, J=10.0 Hz, 1H).

Step 12: Synthesis of methyl (2S,3R)-2,4-dibromo-3-hydroxylbutyrate

[0234] (3R,4R)-3,4-dihydroxyldihydrofuran-2(3H)-one (26.0 g, 221 mmol) and 182.0 mL 33% hydrobromic acid in acetic acid were added to a 1 L flask. After the mixture was stirred at room temperature for 24 hours, 959.0 mL methanol was added and the mixture was further stirred for 36 hours. The resulting reaction solution was heated to 65 to 75° C. and refluxed for 4 hours. The reaction solution was concentrated under reduced pressure, water (70.0 mL) and ethyl acetate (105.0 mL) were added and extracted. The organic phase was separated, dried over sodium sulfate, concentrated to give 51 g crude oil mixture. Sulfuric acid (4.1 mL) was added slowly into a solution of the mixture (51.0 g) in methanol (239.0 mL), the mixture was heated to reflux for 6 hours (60 to 70° C.). The reaction solution was concentrated, the residue was dissolved in ethyl acetate (60.0 mL), washed with water (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered to give the product as white-off solid (51 g, yield 83%). .sup.1HNMR (D.sub.2O, 400 MHz) δ 3.98 (d, J=4.0 Hz, 1H), 3.86-3.81 (m, 2H), 3.54-3.51 (m, 2H).

[0235] Process 5:

##STR00027##

Step 13: Synthesis of (3R,3aS)-methyl-1-one-7-(3-ketomorpholine)-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazine-3-carboxylic acid

[0236] At 0° C., methyl (2S,3R)-2,4-dibromo-3-hydroxylbutyrate (46.8 g, 0.24 mol), cesium carbonate (130.3 g, 0.40 mol) were added sequentially at one time to a solution of benzyl (2-hydroxyl-4-(3-ketomorpholine)phenyl)carbamate (68.5 g, 0.20 mol) in DMF (700 mL) while stirring. The reaction solution was stirred at 0° C. for 10 hours. Cesium carbonate (65.2 g, 0.20 mol) was further added at 0° C. after the detection result showed (2-hydroxyl-4-(3-ketomorpholine)phenyl)carbamate completely disappeared. The reaction solution was gradually warmed to room temperature and stirred for 12 hours. The reaction mixture was cooled to 0° C., methanol (700 mL) was added and the pH value was adjusted to 1 with 4 N HCl/methanol solution. Sulfoxide chloride (58 mL, 0.80 mol) was slowly added dropwise over about 1 hour at 0° C. The reaction was further stirred at 0° C. for 1 hour and then gradually warmed to room temperature and reacted for 16 hours. When the detection result showed the reaction was complete, the reaction solution was concentrated under reduced pressure at 40° C. to remove methanol. The resulting reaction mixture was cooled and added to a cold aqueous hydrochloric acid solution whose pH value was 2. Solid was collected after filtration to give the crude product and the crude product was washed twice with cold aqueous hydrochloric acid solution (40 mL, pH=2) and twice with methanol (40 mL), and then dried in vacuum to give the target product as a white solid (38 g, yield 54%). .sup.1HNMR (DMSO-d.sub.6, 400 MHz) δ 7.81 (d, J=8.4 Hz, 1H), 7.08-7.02 (m, 2H), 5.15 (d, J=6.8 Hz, 1H), 4.58 (d, J=9.2, 3.2 Hz, 1H), 4.40 (m, 1H), 4.18 (s, 2H), 4.15 (m, 1H), 3.95 (t, J=4.4 Hz, 1H), 3.80 (s, 2H), 3.69 (t, J=4.4 Hz, 1H).

Step 14: Synthesis of (3R,3aS)-3-(hydroxylmethyl)-7-(3-ketomorpholine)-3a,4-dihydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-1(3H)-one

[0237] At 0° C., sodium borohydride (5.0 g, 0.13 mol) was added to a suspension of (3R,3aS)-methyl-1-one-7-(3-ketomorpholine)-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazine-3-carboxylic acid (38.3 g, 0.11 mol) in methanol/dichoromethane (660 mL, 2:1) in batches while stirring over about 40 minutes. The reaction was stirred at 0° C. for 20 minutes. The reaction solution was warmed to room temperature, and the solvent was removed under reduced pressure at 40° C. to obtain a crude. The crude was triturated in water (40 mL) for 10 minutes, filtered, washed with water (20 mL) once and dried in vacuum to give the desired product 28 g. The aqueous phase was extracted with methanol/dichloromethane (1:10), dried over anhydrous sodium sulfate and concentrated to give 5 g the desired product, total yield 90%. .sup.1HNMR (DMSO-d.sub.6, 400 MHz) δ 7.84 (d, J=8.8 Hz, 1H), 7.05-6.99 (m, 2H), 5.34 (brs, 1H), 4.54 (d, J=7.2 Hz, 1H), 4.45 (m, 1H), 4.18 (s, 2H), 4.02 (t, J=4.4 Hz, 1H), 3.95 (s, 2H), 3.77-3.68 (m, 4H).

Step 15: Synthesis of methyl (3R,3aS)-1-one-7-(3-ketomorpholine)-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)methanesulfonate

[0238] At 0° C., triethylamine (25 mL, 180 mmol) was added in one portion and methanesulfonyl chloride (9 mL, 120 mmol) was added dropwise over about 20 minutes to a suspension of (3R,3aS)-3-(hydroxylmethyl)-7-(3-ketomorpholine)-3a,4-dihydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-1(3H)-one (19.2 g, 60 mmol) in dichloromethane (600 mL) while stirring. The reaction was stirred at 0° C. for 1 hour and gradually warmed to room temperature and reacted for 16 hours. When the detection showed the reaction was substantially complete, saturated sodium bicarbonate solution (200 mL) was added. The mixture was filtered and the filtrate was portionated. The aqueous phase was extracted with dichloromethane (100 mL×2). The organic phases were combined, washed twice with saturated brine (200 mL), dried over anhydrous sodium sulfate, and combined with the solid after concentration to give the target product (21 g, yield 87%). .sup.1HNMR (DMSO-d.sub.6, 400 MHz) δ 7.84 (d, J=8.4 Hz, 1H), 7.07-7.01 (m, 2H), 4.79 (m, 1H), 4.64-4.58 (m, 3H), 4.18 (s, 2H), 4.07 (d, J=5.6 Hz, 2H), 3.95 (t, J=4.8 Hz, 1H), 3.69 (t, J=4.8 Hz, 1H), 3.28 (s, 2H).

Step 16: Synthesis of 2-(((3S,3aS)-1-one-7-(3-ketomorpholine)-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)methyl)isoindole-1,3-dione

[0239] Methyl ((3R,3aS)-1-one-7-(3-ketomorpholine)-1,3,3a,4-tetrahydrobenzo[b] oxazolo[3,4-d][1,4]oxazin-3-yl)methanesulfonate (5.4 g, 13.5 mmol) was dissolved in DMF (55 mL). At 25° C., potassium phthalimide (3.75 g, 20.3 mmol) was added in one portion while stirring, the mixture was further stirred for 10 minutes and then heated to 70° C. and reacted for 16 hours. When the detection result showed the reaction was substantially complete, the mixture was cooled to room temperature and added to ice water (1.80 mL). The solid was filtered out and collected to give the crude, the crude was washed twice with ice water (20 mL) and dried to give the target product as a white solid (5.4 g, yield 90%). .sup.1HNMR (DMSO-d.sub.6, 400 MHz) δ 7.95-7.85 (m, 4H), 7.80 (d, J=8.8 Hz, 1H). 7.04 (d, J=2.4 Hz, 1H), 7.00 (dd, J=8.8, 2.4 Hz, 1H), 4.73 (m, 1H), 4.72 (m, 1H), 4.18-4.03 (m, 6H), 3.94 (t, J=4.8 Hz, 1H), 3.68 (t, J=4.8 Hz, 1H).

Step 17: Synthesis of (3S,3aS)-3-(aminomethyl)-7-(3-ketomorpholine))-3a,4-dihydrobenzo[b]oxazolo[3,4-d][1,4]-oxazin-1(3H)-one

[0240] MeOH (100 mL) was added to a 250 mL reaction flask, followed by the addition of the compound 2-(((3S,3aS)-1-one-7-(3-ketomorpholine)-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)methyl)isoindole-1,3-dione (10.11 g, 22.5 mmol). After the mixture was fully stirred, a 40% aqueous solution of methylamine (10.5 mL, 135 mmol) was added in one portion, the mixture was further stirred for 10 minutes, and then the temperature was raised to 65° C., further stirred for 4 hours. When the detection result showed the raw material was fully reacted. The reaction solution was then cooled to room temperature and the pH value of which was adjusted to 1 with HCl-MeOH solution (12 mL, 12M) and stirred for 1 hour to form the salt. The reaction solution was concentrated at 40° C. to remove part of methanol (about 50 mL) thereby obtaining a slurry. Followed by the addition of methanol/dichloromethane (60 mL, 1:5) and the mixture was triturated for 16 hours, filtered, the filter cake was washed twice with MeOH (10 mL) and dried to give the hydrochloride salt of the target product (white solid, 7.0 g, yield 87%, purity 98%). .sup.1HNMR (DMSO-d.sub.6, 400 MHz) δ 7.85 (d, J=8.4 Hz, 1H), 6.98-6.95 (m, 2H), 4.82 (m, 1H), 4.59 (d, J=10.4 Hz, 1H), 4.29 (s, 2H), 4.12-4.01 (m, 4H), 3.71 (t, J=5.6 Hz, 1H), 3.52-3.47 (m, 2H), 3.28 (s, 1H).

Step 18: Synthesis of 5-chloro-N-(((3S,3aS)-1-one-7-(3-ketomorpholine)-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)methyl)thiophene-2-carboxamide

[0241] Water (30 mL) and acetone (54 mL) were added to a 250 mL reaction flask, followed by an addition of sodium carbonate (2.48 g, 23.4 mmol), dissolved completely after fully stirred, the mixture was cooled to 0-5° C., (3S,3aS)-3-(aminomethyl)-7-(3-ketomorpholine))-3a,4-dihydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-1(3R)-one hydrochloride (6.40 g, 18.0 mmol) was added over about 10 minutes. At 0 to 5° C., keep stirring until the mixture was completely dissolved (about 30 minutes). And then a solution of 5-chlorothiophene-2-carbonyl chloride (3.90 g, 21.6 mmol) in toluene (11 mL) was added dropwise over about 20 minutes at 0-5° C. After the addition, the reaction was reacted at 0-5° C. for 0.5 hour, then gradually warmed to 20° C. and reacted for 1 hour. During this period, solid was gradually precipitated. TLC (DCM:MeOH=20:1) detected the reaction was complete. Water (100 mL) was added to the reaction solution and stirred for 20 minutes. The mixture was concentrated at 45° C. and the solvent was removed, filtered, and the solid was collected, washed with water (50 ml) and dried to give the target product (white solid, 8.40 g, yield 99%, HPLC 94%, chiral purity 99%). NMR (400 MHz, DMSO-d.sub.6) δ 9.00 (t, J=5.6 Hz, 1 H), 7.85 (d, J=8.8 Hz, 1 H), 7.71 (d, J=4.0 Hz, 1 H), 7.21 (d, J=4.0 Hz, 1 H), 7.05 (d, J=2.0 Hz, 1 H), 7.05 (dd, J=8.8, 2.0 Hz, 1 H), 4.62-4.51 (m, 2 H), 4.18 (s, 2 H), 4.11-4.01 (m, 2 H), 3.95 (t, J=5.2 Hz, 2 H), 3.73 (t, J=5.2 Hz, 2 H), 3.68 (t, J=5.2 Hz, 2 H).

Step 19: Recrystallization of 5-chloro-N-(((3S,3aS)-1-one-7-(3-ketomorpholine)-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)methyl)thiophene-2-carboxamide (crystal form A)

[0242] 2.8 g Crude solid obtained in step 18 was heated to 70° C. and stirred for 30 minutes in 90 mL ethanol, then the mixture was cooled to room temperature and stirred for 16 hours, filtered and 2.4g solid was collected, HPLC 98.3%, yield 86%.

Step 20: Recrystallization of 5-chloro-N-(((3S,3aS)-1-one-7-(3-ketomorpholine)-1,3,3a,4-tetrahydrobenzo[b]oxazolo[3,4-d][1,4]oxazin-3-yl)methyl)thiophene-2-carboxamide (crystal form B)

[0243] 500 mg Solid of the crude solid achieved in step 18 was dissolved in 0.65 mL DMSO, followed by adding 1.3 mL EtOH, the mixture was heated to 80° C. and fully dissolved, and then naturally cooled to 0 to 10° C. A large amount of solid was precipitated and 400 mg solid was collected after filtration, HPLC 99.3%, yield 80%.

Embodiment 2 the Process for Preparing Compound 2

[0244] ##STR00028##

[0245] The process for preparing compound 2 referred to the process in embodiment 1, where the morpholin-3-one in step 2 was replaced with piperidin-2-one. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.01 (t, J=5.6 Hz, 1 H), 7.83 (d, J=8.8 Hz, 1 H), 7.72 (d, J=4.0 Hz, 1 H), 7.22 (d, J=4.0 Hz, 1 H), 6.87-6.93 (m, 2 H), 4.50-4.64 (m, 2 H), 3.99-4.13 (m, 2 H), 3.73 (t, J=5.6 Hz, 2 H), 3.52-3.60 (m, 2 H), 2.37 (t, J=6.0 Hz, 2 H), 1.77-1.90 (m, 4 H); LCMS (ESI) m/z: 462.1 (M+1).

Embodiment 3 the Process for Preparing Compound 7

[0246] ##STR00029##

[0247] The process for preparing compound 7 referred to embodiment 1, where the morpholin-3-one in step 2 was replaced with tetrahydropyrimidin-2(1H)-one. .sup.1H NMR(400 MHz, DMSO-d.sub.6) δ 9.00 (t, J=6.0 Hz, 1 H), 7.69-7.75 (m, 2H), 7.20 (d, J=4.0 Hz, 1H), 6.85-6.95 (m, 2H), 6.55 (s, 1H), 4.47-4.62 (m, 2H), 3.94-4.10 (m, 2 H), 3.71 (t, J=5.6 Hz, 2H), 3.56 (t, J=5.6 Hz, 2H), 3.20 (td, J=5.2, 2.0 Hz, 2H), 1.91 (t, J=6.0 Hz, 2H); LCMS (ESI) m/z:463.0 (M+1).

Embodiment 4 the Process for Preparing Compound 8

[0248] ##STR00030##

[0249] The process for preparing compound 8 referred to the process in embodiment 1, where the morpholin-3-one in step 2 was replaced with pyridine-2(M)-one. .sup.1H NMR(400 MHz, DMSO-d.sub.6) δ 9.01 (t, J=5.6 Hz, 1 H), 7.95 (d, J=8.8 Hz, 1 H), 7.72 (d, J=4.0 Hz, 1 H), 7.59 (dd, J=6.8, 1.6 Hz, 1 H), 7.49 (ddd, J=9.2, 6.8, 2.0 Hz, 1 H), 7.21 (d, J=4.0 Hz, 1 H), 7.06 (d, J=2.4 Hz, 1 H), 7.01 (dd, J=8.8, 2.4 Hz, 1 H), 6.45 (d, J=9.2 Hz, 1 H), 6.29 (td, J=6.8, 1.2 Hz, 1 H), 4.56-4.69 (m, 2 H), 4.03-4.16 (m, 2 H), 3.75 (t, J=5.6 Hz, 2 H); LCMS (ESI) m/z: 458.1 (M+1).

Embodiment 5 the Process for Preparing Compound 30

[0250] ##STR00031##

[0251] The process for preparing compound 30 referred to the process in embodiment 1, where the morpholine-3-one in step 2 was replaced with (R)-2-(methoxymethyl)pyrrolidine. .sup.1H NMR(400 MHz, DMSO-d.sub.6) δ 9.00 (t, J=5.4 Hz, 1H), 7.71(d, J=4.0 Hz, 1H), 7.57(d, J=9.2 Hz, 1H), 7.21 (d, J=4.0 Hz, 1H), 6.27 (dd, J=9.2, 2.8 Hz, 1H), 6.16 (d, J=2.8 Hz, 1H), 4.53-4.46 (m, 2H), 3.97-3.95 (m, 2H), 3.78-3.73 (m, 1H), 3.70 (t, J=5.6 Hz, 2H), 3.33-3.31 (m, 2 H), 3.27 (s, 3H), 3.21-3.17 (m, 1 H), 3.01-2.95 (m, 1H), 1.99-1.87 (m, 4H); LCMS (ESI) m/z: 478.1 (M+1).

Embodiment 6 the Process for Preparing Compound 34

[0252] ##STR00032##

[0253] The process for preparing compound 34 referred to the process in embodiment 1, where the morpholine-3-one in step 2 was replaced with 1H-1,2,4-triazole. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.27 (s, 1H), 9.05 (t, J=5.6 Hz, 1H), 8.22 (s, 1H), 8.02 (d, J=8.4 Hz, 1H), 7.75 (d, J=4.0 Hz, 3H), 7.52 (m, 2H), 7.22 (d, J=4.0 Hz, 1H), 4.64 (m, 2H), 4.14 (m, 2H), 3.75 (t, J=5.6 Hz, 2H); LCMS (ESI) ink: 432 (M+1).

[0254] In Vitro Activity Assay

[0255] The inhibitory ability of the compounds to be tested on Xa factor or other enzymes, such as thrombin or trypsin, in human or rats were determined by IC.sub.50 values, wherein the IC.sub.50 value was associated with the inhibition constant Ki. The purified enzyme was used in the chromogenic determination. The initial rate of hydrolysis of the chromogenic substrate was determined by the variation of absorbance at 405 nm in the linear part on time (usually 2 to 10 minutes after the addition of the substrate) at 37° C. by using FlexStation III (USA Molecular Instruments). By plotting a curve regarding the relative velocity of the hydrolysis (compared to the control group which was free of inhibition) on the logarithm of the concentration of the compounds and linear regression calculation, the concentration of the inhibitor leading to a 50% reduction of the rate of the hydrolysis of the substrate was determined. The enzyme inhibition constant (Ki) was calculated according to Cheng-Prusoff equation: Ki=IC.sub.50/(1+[S]/Km), where [S] was the substrate concentration and Km was the meter-Mann's constant determined by the double reciprocal mapping method. The IC.sub.50 values of the compounds were obtained by GraphPad Prism software. The curve is fitted by using the “shaped dose effect (variable slope)”.

[0256] Human/Rat Coagulation Factor Xa Assay

[0257] The inhibitory activity on coagulation factor Xa activity in human or rats was measured by using Tris-HCl buffer (50 mM, pH 8.3, 150 mM NaCl). A buffer of 50 mL human coagulation factor Xa (Enzyme Research Laboratories, Inc; final concentration 8.36 nM) or a buffer of 50 μL rat coagulation factor Xa (Enzyme Research Laboratories, Inc; final concentration 57.5 nM) was added dropwise to the appropriate wells of the Greiner 384 microtiter plate to determine IC.sub.50. The buffer containing 2 μL 2% (V/V) DMSO (control group which was free of inhibition) or various concentrations of the compounds to be tested were diluted in the buffer containing 2% (V/V) DMSO, and 48 μL buffer of the supporting base S-2222 (Chromogenix; chemical formula: Bz-IIe-Glu(γ-OR)-Gly-Arg-pNA.HCl R═H (50%), wherein R═CH.sub.3 (50%)) was added, the final concentration was 0.172 mM. In this experiment, the compounds to be tested and the enzyme were incubated for 10 minutes, and then the substrate S-2222 was added to give a final volume of 100 μL to start the assay.

[0258] The compounds to be tested were regarded as being active when K.sub.i<10 μM. The compounds whose K.sub.i<1 μM were preferred in the present invention, more preferably K.sub.i<0.1 μM, further more preferably K.sub.i<0.01 μM, and further preferably K.sub.i<0.001 μM. Determined by the above method, some compounds of the present invention were of K.sub.1<0.1 μM, thus, the compounds of the present invention can be used as effective factor Xa inhibitors.

[0259] Human Thrombin Assay

[0260] The inhibitory activity on human thrombin was determined by using a buffer (10 mM HEPES buffer, pH 7.4, 2 mM CaCl.sub.2). Appropriate wells in the Greiner 384 microtiter plate were used to determine IC.sub.50. A buffer contained 50 μL human thrombin (Sigma; T8885), the final concentration of which was 0.05 NIH unit/mL. A buffer containing 2 μL 2% (V/V) DMSO (control group which was free of inhibition) or various concentration of the compound to be tested was diluted in the buffer containing 2% (V/V) DMSO. A buffer containing 48 μL substrate S-2238 (Chromogenix; Chemical formula: H-D-Phe-Pip-Arg-pNA.2HCl) was added, the final concentration was 30 μM. In this assay, the compound to be tested and the enzyme were pre-incubated for 10 minutes, the substrate was added to give a final volume of 100 μL to start the assay.

[0261] Human Trypsin Assay

[0262] The inhibitory activity on human trypsin was determined by using buffer (50 mM Tris, pH 8.2, and 20 mM CaCl.sub.2). Appropriate wells in the Greiner 384 microtiter plate were used to determine IC.sub.50. A buffer containing 50 μL human trypsin (Sigma; T6424), the final concentration was 0.39 BAEE unit/mL, a buffer containing 2 μL 2% (V/V) DMSO (control group which was free of inhibition) or various concentration of the compound to be tested was diluted in the buffer containing 2% (V/V) DMSO. A buffer containing substrate S-2222 (Chromogenix), the final concentration was 30 μM. In this assay, the compound to be tested and the enzyme were pre-incubated for 10 minutes and then 48 μL substrate was added to give a final volume of 100 μL to start the assay.

[0263] Prothrombin Assay

[0264] The activity of the compound to be tested against prothrombinase was determined by the production of thrombin. In summary, 12.5 μL human factor Xa was incubated in 10 mM HEPES buffer and pH 7.4, 2 mM CaCl.sub.2, the final concentration was 0.5 nM, and 12.5 μL human blood platelets (1×10.sup.7 mL.sup.−1) was added at 37° C. After 10 mins, 25 μL prothrombin was added to initiate the reaction, the final concentration was 0.5 μM, a buffer containing 2 μL 2% (V/V) DMSO (control group which was free of inhibition) or various concentration of the compound was diluted in the buffer containing 2% (V/V) DMSO. After 20 minutes, 48 μL substrate S-2238 (Chromogenix) was added until the final concentration was 50 μM to determine thrombin activity.

TABLE-US-00001 TABLE 1 The results of the compound of the present invention screened in vitro hfXa K.sub.i rfXa K.sub.i Thrombin Trypsin Prothrombin Compound (nM) (nM) K.sub.i (nM) K.sub.i (nM) K.sub.i (nM) 1 1.01 2.87 >1000 >20000 0.47 2 1.93 4.45 >1000 >20000 4.22 7 4.135 16.49 >1000 >20000 4.41 8 1.92 3.17 >1000 >20000 0.9 30 9.71 21.47 >1000 >20000 16.17 34 105.94 / / / /

[0265] Conclusion: the compounds of the present invention showed a potent anticoagulant activity due to the specific inhibitory activity on coagulation factor Xa thereof.