SGLT2/DPP4 INHIBITOR AND APPLICATION THEREOF
20220242898 · 2022-08-04
Inventors
- Qinghua MAO (Shanghai, CN)
- Tao YU (Shanghai, CN)
- Lu GAN (Shanghai, CN)
- Yi LI (Shanghai, CN)
- Chengde WU (Shanghai, CN)
- Shuhui Chen (Shanghai, CN)
Cpc classification
C07H15/14
CHEMISTRY; METALLURGY
International classification
Abstract
Compounds as SGLT2/DPP4 dual inhibitors, and application in preparation of medicines as the SGLT2/DPP4 dual inhibitors. A compound represented by formula (I), and an isomer or pharmaceutically acceptable salt thereof are specifically involved.
##STR00001##
Claims
1-9. (canceled)
10. A compound of formula (I), an isomer thereof, or a pharmaceutically acceptable salt thereof, ##STR00043## wherein, R.sub.1 is C.sub.1-3 alkyl, which is optionally substituted by 1, 2, or 3 R.sub.a; R.sub.2 and R.sub.3 are each independently selected from H, F, Cl, Br, I, OH, NH.sub.2, C.sub.1-3 alkyl, and C.sub.1-3 alkoxy, wherein the C.sub.1-3 alkyl and the C.sub.1-3 alkoxy are optionally substituted by 1, 2, or 3 R.sub.b; R.sub.5 is selected from H, F, Cl, Br, I, OH, NH.sub.2, and C.sub.1-3 alkyl, wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2, or 3 R.sub.c; R.sub.4 is H, F, or Cl, and when R.sub.4 is Cl, R.sub.2, R.sub.3, and R.sub.5 are not H at the same time; R.sub.a, R.sub.b and R.sub.c are each independently selected from F, Cl, Br, I, OH, and NH.sub.2.
11. The compound, the isomer thereof, or the pharmaceutically acceptable salt thereof as defined in claim 1, wherein, R.sub.1 is CH.sub.3, which is optionally substituted by 1, 2, or 3 R.sub.a.
12. The compound, the isomer thereof, or the pharmaceutically acceptable salt thereof as defined in claim 2, wherein, R.sub.1 is CH.sub.3.
13. The compound, the isomer thereof, or the pharmaceutically acceptable salt thereof as defined in claim 1, wherein, R.sub.2 and R.sub.3 are each independently selected from H, F, Cl, Br, I, OH, NH.sub.2, CH.sub.3, Et, and —OCH.sub.3, wherein the CH.sub.3, Et, and —OCH.sub.3 are optionally substituted by 1, 2, or 3 R.sub.b.
14. The compound, the isomer thereof, or the pharmaceutically acceptable salt thereof as defined in claim 4, wherein, R.sub.2 and R.sub.3 are each independently selected from H, F, Cl, Br, I, OH, NH.sub.2, CH.sub.3, Et, and —OCH.sub.3.
15. The compound, the isomer thereof, or the pharmaceutically acceptable salt thereof as defined in claim 1, wherein, R.sub.5 is selected from H, F, Cl, Br, I, OH, NH.sub.2, CH.sub.3, and Et, wherein the CH.sub.3 and Et are optionally substituted by 1, 2, or 3 R.sub.c.
16. The compound, the isomer thereof, or the pharmaceutically acceptable salt thereof as defined in claim 6, wherein, R.sub.5 is selected from H, F, Cl, Br, I, OH, NH.sub.2, CH.sub.3, and Et.
17. A compound, an isomer thereof, or a pharmaceutically acceptable salt thereof as follows, ##STR00044## ##STR00045##
18. A method of inhibiting SGLT2 and DPP4 in a subject in need thereof, comprising administering the compound, the isomer thereof, or the pharmaceutically acceptable salt thereof according claim 1 into the subject.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0068]
[0069]
[0070]
[0071]
[0072]
[0073]
[0074]
[0075]
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0076] The present disclosure is described in detail by the embodiments below, but it does not mean that there are any adverse restrictions on the present disclosure. The present disclosure has been described in detail herein, wherein specific embodiments thereof are also disclosed, and it will be apparent to those skilled in the art that various variations and improvements can be made to specific embodiments of the present disclosure without departing from the spirit and scope of the present disclosure.
Reference Embodiment 1: Fragment A-1
[0077] ##STR00012##
Synthetic Route
[0078] ##STR00013##
Step 1: Synthesis of Compound A-1-2
[0079] Compound A-1-1 (20 g, 91.32 mmol) was added to a reaction flask containing anhydrous tetrahydrofuran (100 mL). After addition dropwise with a solution of borane in tetrahydrofuran (1 M, 120 mL) into the reaction under nitrogen protection, the mixture was stirred at 15° C. for 16 hours. After the reaction was completed, the reaction was quenched by dropwise with methanol (70 mL) under nitrogen protection. The mixture was concentrated under reduced pressure to obtain a crude product of compound A-1-2, which was directly used in the next reaction step. MS m/z: 229.7 [M+23].sup.+.
Step 2: Synthesis of Compound A-1-3
[0080] Compound A-1-2 (19 g, 92.31 mmol, crude product) was added to a reaction flask containing anhydrous N,N-dimethylformamide (100 mL). After the mixture was cooled to 0° C., sodium hydride (7.00 g, 174.88 mmol) was added to the reaction and the mixture was stirred for 0.5 hours. After the reaction was slowly warmed up to 15° C., allyl bromide (34 g, 281.04 mmol) was added to the reaction and the mixture was continuously stirred for 18 hours. After the reaction was completed, the reaction solution was quenched by dropwise with saturated ammonium chloride aqueous solution (50 mL), the mixture was concentrated under reduced pressure to obtain the residue as a yellow viscous material. Dichloromethane (200 mL) and water (200 mL) were added to the residue and stirred. The organic phase was separated and washed with water (100 mL*2), concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether 100% system) to obtain compound A-1-3.
Step 3: Synthesis of Compound A-1-5
[0081] A solution of n-butyllithium in n-hexane (2.5 M, 33.00 mL) was added dropwise to a solution of compound A-1-3 (15.00 g, 61.20 mmol) in anhydrous tetrahydrofuran (150 mL) at −70° C. under nitrogen protection. After the addition was completed, the reaction was stirred at −70° C. for 0.5 hours to prepare reaction system A. A solution of tert-butylmagnesium chloride in tetrahydrofuran (1.7 M, 69.00 mL) was added dropwise to a solution of compound A-1-4 (16.73 g, 61.20 mmol) in anhydrous tetrahydrofuran (150 mL) at 0° C. under nitrogen protection. After the addition was completed, the reaction was stirred at 0° C. for 0.5 hours and then the mixture was added dropwise to the reaction system A. After the addition was completed, the reaction was stirred at −70° C. for 0.5 hours. The reaction was slowly warmed up to 20° C. and continuously stirred for 2 hours. After the reaction was completed, the mixture was added dropwise with saturated ammonium chloride aqueous solution (100 mL) to quench the reaction, the mixture was concentrated to remove the organic solvent, and the residue was extracted with ethyl acetate (100 mL*3), the organic phases were combined and washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and filtered, then the filtrate was concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate=10:1 to 1:1) to obtain compound A-1-5. .sup.1H NMR (400 MHz, CHCl.sub.3-d) δ ppm 1.37 (s, 3H) 1.58 (s, 3H) 3.07 (d, J=4.27 Hz, 1 H) 4.06-4.11 (m, 2H) 4.59-4.68 (m, 4H) 5.24 (dd, J=10.42, 1.38 Hz, 1 H) 5.30-5.33 (m, 1 H) 5.35 (dd, J=17.32, 1.51 Hz, 1 H) 5.90-6.03 (m, 1H) 6.10 (d, J=3.51 Hz, 1H) 7.15 (t, J=9.03 Hz, 1H) 8.05 (ddd, J=8.22, 5.33, 2.26 Hz, 1H) 8.20 (dd, J=6.90, 2.13 Hz, 1H).
Step 4: Synthesis of Compound A-1-6
[0082] Sodium borohydride (1.20 g, 31.72 mmol) was slowly added in batches into a mixture of Compound A-1-5 (6.20 g, 13.72 mmol), cerium trichloride heptahydrate (6.20 g, 16.64 mmol, 1.58 mL) in anhydrous methanol (100 mL) at 0° C. After addition completed, the reaction was stirred at 20° C. for 16 hours. After the reaction was completed, the reaction was quenched by adding dropwise with saturated ammonium chloride solution (100 mL). The mixture was concentrated to remove the organic solvent, and the mixture became turbid. Citric acid was added to the mixture and adjust the mixture to clarify; then the mixture was extracted with ethyl acetate (100 mL*3), The organic phases were separated and combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and filtered, then the filtrate was concentrated to obtain a crude product of compound A-1-6, which was directly used in the next reaction step. MS m/z: 372.0 [M+18].sup.+.
Step 5: Synthesis of Compound A-1-7
[0083] A mixture of Compound A-1-6 (8.00 g, 17.60 mmol) in acetic acid (42.00 g, 699.42 mmol, 40 mL) and water (40 mL) was stirred at 100° C. for 6 hours. After the reaction was completed, the mixture was concentrated to obtain the crude product as a yellow solid. The crude product was added with toluene (30 mL), and concentrated again. This operation was repeat twice to obtain a crude product of compound A-1-7, which was directly used in the next reaction step. MS m/z: 331.9 [M+18].sup.+.
Step 6: Synthesis of Compound A-1-8
[0084] To a mixture of Compound A-1-7 (8.00 g, 17.94 mmol), triethylamine (13.81 g, 136.51 mmol, 19.0 mL) in acetonitrile (100 mL) was added acetic anhydride (16.35 g, 160.16 mmol, 15.0 mL), followed by 4-dimethylaminopyridine (35 mg, 286.49 μmol). The reaction was stirred at 20° C. for 8 hours. After the reaction was completed, the reaction was quenched by adding dropwise with saturated sodium bisulfate aqueous solution (10 mL), the mixture was added with water (100 mL) and ethyl acetate (100 mL). The mixture was stirred and then the organic phase was separated; the aqueous phase was extracted twice with ethyl acetate (100 mL). The organic phases were combined, and washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and filtered, then the filtrate was concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate=10:1 to 2:1) to obtain compound A-1-8. MS m/z: 500.1 [M+18].sup.+.
Step 7: Synthesis of Compound A-1-9
[0085] To a mixture of Compound A-1-8 (4.00 g, 6.50 mmol) in anhydrous dioxane (50 mL) were added with thiourea (1.28 g, 16.82 mmol), followed by trimethylsilyl trifluoromethanesulfonate (4.92 g, 22.14 mmol, 4.00 mL) under nitrogen protection. The reaction was stirred at 80° C. for 2 hours. After detecting the formation of intermediate, the reaction was cooled down to 20° C., and iodomethane (3.60 g, 25.36 mmol, 1.58 mL) and diisopropylethylamine (4.20 g, 32.48 mmol, 5.66 mL) were added, and the reaction was stirred at 20° C. for 16 hours. The reaction was quenched by adding with methanol (10 mL), then the mixture was concentrated under reduced pressure. The residue was added with water (50 mL) and the mixture was extracted with ethyl acetate (50 mL*3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered, then the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate=10:1 to 3:1) to obtain compound A-1-9. MS m/z: 493.0 [M+23].sup.+.
Step 8: Synthesis of Compound A-1-10
[0086] To a mixture of Compound A-1-9 (3.20 g, 5.80 mmol), barbituric acid dihydrate (1.50 g, 11.68 mmol, 2.01 eq) in anhydrous ethanol (40 mL) were added with tetrakis(triphenylphosphine) palladium (0.32 g, 276.92 μmol, 5% mol eq) under nitrogen protection and the reaction was stirred at 40° C. for 14 hours. After the reaction was completed, the reaction mixture was cooled and filtered, and the filtrate was concentrated under reduced pressure. The residue was added with water (50 mL) and the mixture was extracted with ethyl acetate (50 mL*3). The organic phases were combined and washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered, then the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate=10:1 to 1:1) to obtain compound A-1-10. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 7.46 (dd, J=7.03, 2.01 Hz, 1H) 7.23-7.27 (m, 1H) 6.97-7.11 (m, 1H) 5.32-5.43 (m, 1H) 5.17-5.28 (m, 1H) 5.12 (t, J=9.66 Hz, 1H) 4.76 (br d, J=5.27 Hz, 2H) 4.55 (d, J=9.79 Hz, 1H) 4.45 (d, J=9.79 Hz, 1H) 2.20 (s, 3H) 2.10 (s, 3H) 2.02 (s, 3H) 1.83 (s, 3H) 1.80-1.86 (m, 1H).
Step 9: Synthesis of Compound A-1
[0087] Phosphorus tribromide (34.59 mg, 127.78 μmol, 12.14 μL) was added dropwise to a solution of compound A-1-10 (0.11 g, 255.55 μmol) in anhydrous tetrahydrofuran (2 mL) under nitrogen protection at 0° C. The reaction was stirred for 16 hours, the temperature was increased from 0° C. to 20° C. during this period. After the reaction was completed, an aqueous solution of potassium carbonate (2 M, 5.5 mL) was added slowly to the reaction and stirring for 10 min to quench the reaction. The reaction solution was stratified after standing and the aqueous phase was extracted with ethyl acetate (10 mL*2), then the organic phases were combined, washed with water (20 mL) and saturated brine (10 mL) sequentially, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. The crude product was stirred with n-hexane (10 mL) for 1 hour at room temperature. The solid was collected by filtration and washed with n-hexane (2 mL×3), and the filter cake was concentrated under reduced pressure to remove the residual solvent to obtain compound A-1. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.39-7.30 (m, 2H), 7.09-7.03 (m, 1H), 5.36 (t, J=9.6 Hz, 1H), 5.22 (t, J=9.6 Hz, 1H), 5.07 (t, J=9.6 Hz, 1H), 4.58-4.52 (m, 2H), 4.48-4.41 (m, 2H), 2.21 (s, 3H), 2.11 (s, 3H), 2.02 (s, 3H), 1.86 (s, 3H).
[0088] Each fragment A-2, A-3, A-4, A-5 in Table 1 was synthesized by referring to synthesis methods of steps 1 to 9 in Reference embodiment 1.
TABLE-US-00001 TABLE 1 Reference embodiment Fragment Structure NMR 2 A-2
Reference Embodiment 6: Fragment A-6
[0089] ##STR00018##
Synthetic Route
[0090] ##STR00019##
Step 1: Synthesis of Compound A-6-2
[0091] Compound A-6-1 (25 g, 133.67 mmol, 1 eq) was dissolved in tetrahydrofuran (250 mL), and sodium hydride (10.69 g, 267.33 mmol, 2 eq) was added at 0° C., the reaction was warmed up to 25° C. and stirred for 0.5 hours. Allyl bromide (48.51 g, 401.00 mmol, 34.65 mL, 3 eq) was added slowly to the reaction and the reaction was continued stirred at 25° C. for 1.5 hours after addition. After the reaction was completed, the reaction was quenched by adding saturated ammonium chloride aqueous solution (100 mL) at 0° C., and the mixture was extracted with ethyl acetate (250 mL×2). The organic phases were combined and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether) to obtain target compound A-6-2. .sup.1H NMR (400 MHz, CD.sub.3OD) δ ppm 4.04 (dt, J=5.5, 1.4 Hz, 2H), 4.45-4.52 (m, 2H), 5.17-5.34 (m, 2H), 5.95 (ddt, J=17.2, 10.7, 5.5, 5.5 Hz, 1H), 7.22-7.32 (m, 2H), 7.43 (d, J=7.5 Hz, 1H), 7.51 (s, 1H).
Step 2: Synthesis of Compound A-6-4
[0092] A solution of n-Butyllithium (2.5 M, 27.12 mL, 1.1 eq) was added dropwise to a solution of compound A-6-2 (14 g, 61.65 mmol, 1 eq) in anhydrous tetrahydrofuran (140 mL) at −78° C. under nitrogen protection. After the dropwise addition was completed, the reaction was stirred at −78° C. for 0.5 hours to obtain reaction system A. A solution of tert-butylmagnesium chloride (1.7 M, 47.14 mL, 1.3 eq) was added dropwise to a solution of compound A-1-4 (18.53 g, 67.81 mmol, 1.1 eq) in anhydrous tetrahydrofuran (180 mL) under nitrogen protection at 0° C. After the dropwise addition was completed, the reaction was stirred at 0° C. for 0.5 hours to prepare reaction system B. The reaction system B was slowly added to the reaction system A at −78° C. under nitrogen protection. The reaction was carried out at −78° C. for 0.5 hours, then the mixture was warmed up to 25° C. and stirred for 15.5 hours. After the reaction was completed, the reaction was quenched by adding an aqueous solution of ammonium chloride (100 mL) to the reaction at 0° C., and the mixture was diluted by ethyl acetate (200 mL). The organic phase was separated and washed with water (50 mL*2), followed by saturated brine (50 mL*2), dried over anhydrous sodium sulfate and concentrated under reduced pressure, then the residue was purified by column chromatography (petroleum ether: ethyl acetate=4:1) to obtain target compound A-6-4, which was confirmed by LCMS, LC-MS (m/z) 357 [M+Na].sup.+.
Step 3: Synthesis of Compound A-6-5
[0093] Compound A-6-4 (13 g, 38.88 mmol, 1 eq) was dissolved in methanol (130 mL) and the mixture was cooled to 0° C. Cerium trichloride (9.58 g, 38.88 mmol, 2.44 mL, 1 eq) and sodium borohydride (2.94 g, 77.76 mmol, 2 eq) were added to the mixture sequentially, the mixture was warmed up to 25° C. and reacted for 16 hours. After the reaction was completed, the reaction was quenched by saturated ammonium chloride aqueous solution (30 mL). The mixture was concentrated under reduced pressure, and the residue was added with ethyl acetate (100 mL) and washed with water (50 mL*2). The organic phase was separated and washed with saturated brine (50 mL*2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain target compound A-6-5, which was confirmed by LCMS, LC-MS (m/z) 359 [M+Na].sup.+.
Step 4: Synthesis of Compound A-6-6
[0094] Compound A-6-5 (10.8 g, 32.11 mmol, 1 eq) was dissolved in a solvent of water (50 mL) and glacial acetic acid (50 mL) and the reaction was reacted at 100° C. for 16 hours. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was added with toluene (150 mL) and concentrated under reduced pressure again to obtain compound A-6-6, which was confirmed by LCMS, LC-MS (m/z) 319 [M+Na].sup.+.
Step 5: Synthesis of Compound A-6-7
[0095] Compound A-6-6 (9.2 g, 31.05 mmol, 1 eq) was dissolved in 1,4-dioxane (100 mL), and then the reaction was added with acetic anhydride (25.36 g, 248.38 mmol, 23.26 mL, 8 eq), pyridine (24.56 g, 310.48 mmol, 25.06 mL, 10 eq), and 4-dimethylaminopyridine (1.90 g, 15.52 mmol, 0.5 eq). The mixture was stirred at 80° C. for 16 hours. After the reaction was completed, the mixture was concentrated under reduced pressure and the residue was diluted with ethyl acetate (100 mL).The organic phase was washed with 1 M hydrochloric acid (100 mL*4), water (50 mL*2), and saturated brine (50 mL*2) sequentially. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to get the residue. The residue was purified by column chromatography (petroleum ether: ethyl acetate=3:1) to obtain target compound A-6-7, which was confirmed by LCMS, LC-MS (m/z) 487 [M+Na].sup.+.
Step 6: Synthesis of A-6-8
[0096] To a solution of Compound A-6-7 (6.2 g, 13.35 mmol, 1 eq) in 1,4-dioxane (62 mL) was added thiourea (3.56 g, 46.72 mmol, 3.5 eq), followed by trimethylsilyl trifluoromethanesulfonate (11.87 g, 53.40 mmol, 4 eq) at 25° C. under nitrogen protection. The reaction was warmed up to 60° C. and reacted for 1 hour, then cooled to 25° C., and added with iodomethane (9.47 g, 66.74 mmol, 5 eq) and diisopropylethylamine (17.25 g, 133.49 mmol, 10 eq) sequentially. The reaction was continuously stirred at 25° C. for 15 hours after addition. After the reaction was completed, the reaction was quenched by water (60 mL), extracted with ethyl acetate (60 mL*3), and the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether: ethyl acetate=4:1) to obtain the target compound A-6-8, which was confirmed by LCMS, LC-MS (m/z) 475 [M+Na].sup.+.
Step 7: Synthesis of A-6-9
[0097] A mixture of A-6-8 (4.4 g, 9.72 mmol, 1 eq), barbituric acid (2.49 g, 19.45 mmol, 2 eq) in ethanol (44 mL) was added with tetrakis(triphenylphosphine)palladium (516.80 mg, 486.17 μmol, 0.05 eq) under nitrogen protection. The reaction was stirred at 65° C. for 16 hours. After the reaction was completed, the pH of the mixture was adjusted to 7 to 8 by aqueous solution of saturated sodium bicarbonate. The mixture was filtered, and the filtrate was extracted with ethyl acetate (40 mL*2). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether: ethyl acetate=3:2) to obtain target compound A-6-9, which was confirmed by LCMS, LC-MS (m/z) 435 [M+Na].sup.+.
Step 8: Synthesis of A-6
[0098] Phosphorus tribromide (98.44 mg, 363.68 μmol, 34.18 μL, 0.5 eq) was added to a solution of compound A-6-9 (300 mg, 727.36 μmol, 1 eq) in anhydrous tetrahydrofuran (3 mL) under nitrogen protection at 0° C., and the reaction was stirred at 0° C. for 3 hours. After the reaction was completed, the reaction solution was washed with 1 N potassium carbonate aqueous solution, and the organic phase was separated, and the aqueous phase was extracted with dichloromethane (2 mL). The organic phases were combined and concentrated under reduced pressure to get the crude product. The crude product was purified by column chromatography (petroleum ether: ethyl acetate=4:1) to obtain target compound A-6. .sup.1H NMR (400 MHz,CDCl.sub.3) δ ppm 1.83-1.86 (m, 3H), 2.02 (s, 3H), 2.10-2.13 (m, 3H), 2.20-2.22 (m, 3H), 4.46-4.49 (m, 2H), 4.54-4.58 (m, 1H), 5.10 (t, J=9.7 Hz, 1H), 5.20-5.27 (m, 1H), 5.31 (s, 1H), 5.34-5.40 (m, 1H), 7.30-7.38 (m, 4H).
Reference embodiment 7: fragment A-7
[0099] ##STR00020##
Synthetic Route
[0100] ##STR00021##
Step 1: Synthesis of Compound A-7-2
[0101] Sodium borohydride (7.28 g, 192.43 mmol, 3.04 eq) was added in batches to a solution of A-7-1 (14 g, 63.35 mmol, 1 eq) in methanol (60 mL) at 0° C. under nitrogen protection. The reaction was stirred for 2 hours at 0° C. After the reaction was completed, an aqueous solution of saturated ammonium chloride (20 mL) was added dropwise at 0° C. and continuously stirred for 30 min to quench the reaction. The mixture was concentrated under reduced pressure to get the residue. Ethyl acetate (300 mL) and an aqueous solution of saturated sodium chloride (100 mL) were added to the residue and stirred. The organic phase was separated and dried over anhydrous sodium sulfate and concentrated under reduced pressure to get the crude product. The crude product was purified by column chromatography (petroleum ether: ethyl acetate=1:0 to 10:1) to obtain compound A-7-2.
Step 2: synthesis of compound A-7-3.
[0102] Sodium hydroxide (20.86 g, 521.45 mmol, 5 eq) was added to water (60 mL) at 0° C. and the mixture was stirred until sodium hydroxide was dissolved completed. To the solution was added toluene (180 mL), followed by compound A-7-2 (24.4 g, 104.29 mmol, 1 eq) and tetrabutylammonium bromide (TBAB) (3.36 g, 10.43 mmol, 0.1 eq). The reaction was stirred at 25° C. for 0.5 hours , and then Allyl bromide (18.92 g, 156.43 mmol, 13.52 mL, 1.5 eq) was added to the mixture. The reaction was warmed up to 50° C. and stirred for 10.5 hours. The mixure was stratified after standing. The organic phase was separated and the aqueous phase was extracted with ethyl acetate (80 mL*2). The organic phases were combined and washed with saturated brine (80 mL*2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a yellow oil. The yellow oil was added with toluene (50 mL) and the mixture was concentrated under reduced pressure again to obtain compound A-7-3, which was directly used in the next step without purification.
Step 3: Synthesis of Compound A-7-5
[0103] To a solution of compound A-1-4 (10.91 g, 39.91 mmol, 1 eq) in anhydrous tetrahydrofuran (200 mL) was added dropwise with a solution of tert-Butylmagnesium chloride (1.7 M, 37.56 mL, 1.6 eq) under nitrogen protection at 0° C. After the dropwise addition was completed, the reaction was stirred at 0 to 5° C. for 0.5 hours. Compound A-7-3 (10.5 g, 39.91 mmol, 1 eq) was added to the reaction under nitrogen protection. The mixture was cooled to −70° C., and a solution of n-butyllithium (2.5 M, 20.75 mL, 1.3 eq) was added dropwise to the reaction. After the dropwise addition was completed, the reaction was stirred at −70° C. for 0.5 hours, then the mixture was warmed up to 25° C. and reacted for 2 hours. After the reaction was completed, a saturated aqueous solution of ammonium chloride solution (50 mL) was added dropwise at 0 to 10° C. to quench the reaction, the mixture was extracted with ethyl acetate (80 mL*2). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. The crude product was separated by column chromatography (petroleum ether: ethyl acetate=10:1 to 2:1) to obtain compound A-7-5, which was confirmed by LCMS, LC-MS (m/z) 393.2 [M+Na].sup.+.
Step 4: Synthesis of Compound A-7-6
[0104] Sodium borohydride (469.29 mg, 12.41 mmol, 0.8 eq) was dissolved in sodium hydroxide aqueous solution (1 M, 4.65 mL, 0.3 eq) and then added to a mixture of Compound A-7-5 (6.5 g, 15.51 mmol, 1 eq) and cerium trichloride (4.59 g, 18.61 mmol, 1.17 mL, 1.2 eq) in methanol (80 mL). The reaction was stirred at 25° C. for 0.5 hours. After the reaction was completed, the reaction was quenched by saturated ammonium chloride aqueous solution (30 mL), the mixture was concentrated under reduce pressure and the residue was added with ethyl acetate (150 mL) and anhydrous magnesium sulfate (20 g). The mixture was filtered through celite, and the filter cake was washed with ethyl acetate (30 mL*3). The combined filtrates were concentrated under reduced pressure to obtain compound A-7-6, which was confirmed by LCMS, LC-MS (m/z) 390.2 [M+H.sub.2O].sup.+.
Step 5: Synthesis of Compound A-7-7
[0105] A mixture of compound A-7-6 (8 g, 21.48 mmol, 1 eq) in acetic acid (80 mL) and water (80 mL) was warmed up to 100° C. and stirred for 16 hours. After the reaction was completed, the reaction was concentrated under reduced pressure to get the residue. The residue was concentrated under reduce pressure twice with toluene (50 mL) to obtain compound A-7-7, which was confirmed by LCMS, LC-MS (m/z) 350.1 [M+H.sub.2O].sup.+.
Step 6: Synthesis of Compound A-7-8
[0106] A mixture of compound A-7-7 (8 g, 24.07 mmol, 1 eq), triethylamine (19.49 g, 192.60 mmol, 26.81 mL, 8 eq), and 4-dimethylaminopyridine (294.12 mg, 2.41 mmol, 0.1 eq) in acetonitrile (100 mL) were added with acetic anhydride (14.75 g, 144.45 mmol, 13.53 mL, 6 eq). The mixture was stirred at 25° C. for 16 hours. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was diluted with ethyl acetate (100 mL), washed with saturated KHSO.sub.4 solution (80 mL*5), then dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether: ethyl acetate=8:1 to 2:1) to obtain a yellow oil. The yellow oil was dissolved with ethyl acetate (100 mL), and washed with aqueous solution of hydrochloric acid (1 N, 80 mL*4) and saturated brine (80 mL) sequentially. The organic phase was dried over anhydrous sodium sulfate and concentrated, and the residue was concentrated under reduced pressure with toluene (50 mL) twice to obtain compound A-7-8, which was confirmed by LCMS, LC-MS (m/z) 523.1 [M+Na].sup.+.
Step 7: Synthesis of Compound A-7-9.
[0107] Trimethylsilyl trifluoromethanesulfonate (12.44 g, 55.95 mmol, 10.11 mL, 4 eq.) was added to a mixture of compound A-7-8 (7 g, 13.99 mmol, 1 eq.) and thiourea (3.73 g, 48.96 mmol, 3.5 eq.) in anhydrous dioxane (100 mL) under nitrogen protection. The reaction was stirred at 80° C. for 0.5 hours. The reaction was cooled to 10° C., and added with iodomethane (5.96 g, 41.96 mmol, 2.61 mL, 3 eq.) and diisopropylethylamine (9.04 g, 69.94 mmol, 12.18 mL, 5 eq.) sequentially, then the mixture was warmed up to 25° C. and stirred for 10 hours. The reaction was quenched by water (80 mL) and the mixture was extracted with ethyl acetate (100 mL*3). The organic phases were combined, washed with saturated brine (150 mL), and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether: ethyl acetate=10:1 to 3:1) to obtain compound A-7-9, which was confirmed by .sup.1H NMR and LCMS, .sup.1H NMR (400 MHz, CDCl.sub.3) δ 1.86 (s, 3H), 2.04 (s, 3H), 2.12 (s, 3H), 2.21 (s, 3H), 4.00-4.09 (m, 2H), 4.50-4.61 (m, 3H), 4.76-4.83 (m, 1H), 5.18-5.46 (m, 5H), 5.89-6.03 (m, 1H), 6.80 (t, J=9.79 Hz, 1H), 7.49-7.57 (m, 1H), LC-MS (m/z) 534.4 [M+2Na].sup.+.
Step 8: Synthesis of Compound A-7-10
[0108] A mixed of compound A-7-9 (2.5 g, 5.12 mmol, 1 eq), barbituric acid (1.31 g, 10.24 mmol, 2 eq) and Pd(PPh.sub.3).sub.4 (887.08 mg, 767.66 μmol, 0.15 eq) in ethanol (60 mL) was stirred at 50° C. for 20 hours under nitrogen protection. The reaction solution was cooled and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether: ethyl acetate=6:1 to 2:1) to obtain compound A-7-10, which was confirmed by LCMS, LC-MS (m/z) 471.2 [M+Na].sup.+.
Step 9: Synthesis of Compound A-7
[0109] Phosphorus tribromide (271.64 mg, 1.00 mmol, 94.32 μL, 1.5 eq) was added to a solution of compound A-7-10 (300 mg, 668.99 μmol, 1 eq) in anhydrous tetrahydrofuran (5 mL) at 0° C. The reaction was stirred at 20° C. for 3 hours. After the reaction was completed, the reaction was quenched by 1 N potassium carbonate aqueous solution (20 mL) at 0° C., and the mixture was extracted with ethyl acetate (20 mL*2). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether: ethyl acetate=10:1 to 3:1) to obtain compound A-7. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 1.78 (s, 3H), 1.95 (s, 3H), 2.03 (s, 3H), 2.14 (s, 3H), 4.32-4.44 (m, 2H), 4.49 (d, J=10.01 Hz, 1H), 4.68 (d, J=10.01 Hz, 1H), 5.14 (t, J=9.69 Hz, 2H), 5.26-5.33 (m, 1H), 6.74 (t, J=9.69 Hz, 1H), 7.40 (t, J=7.88 Hz, 1H).
[0110] Fragment A-8 in Table 2 was synthesized by referring to the synthesis methods of steps 1 to 9 in reference embodiment 7.
TABLE-US-00002 TABLE 2 Reference embodiment Fragment Structure NMR 8 A-8
Reference Embodiment 9: Fragment B-1
[0111] ##STR00023##
Synthetic Route
[0112] ##STR00024##
Step 1: Synthesis of Compound B-1-2
[0113] Triethylamine (27.02 g, 267.04 mmol, 37.17 mL, 2 eq) was added to a solution of compound B-1-1 (25 g, 133.52 mmol, 1 eq) in dichloromethane (110 mL). The mixture was cooled to 0° C., and methanesulfonyl chloride (15.30 g, 133.52 mmol, 10.33 mL, 1 eq) was added dropwise to the reaction. After the addition was completed, the reaction was warmed from 0° C. to 15° C. and stirred for 3 hours. After the reaction was completed, the reaction was cooled to 0° C. and added slowly with water (100 mL) at 0° C. to quench the reaction. The mixture was extracted with dichloromethane (100 mL*2), and the combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound B-1-2, which was directly used in the next reaction step.
Step 2: Synthesis of Compound B-1-4
[0114] To a mixture of Compound B-1-2 (6.00 g, 22.61 mmol, 1.00 eq) in N,N-dimethylformamide (10.00 mL),was added with cesium carbonate (14.73 g, 45.22 mmol, 2.00 eq) and compound B-1-3 (3.91 g, 22.61 mmol, 1.00 eq). The mixture was stirred at 80° C. for 3 hours. After the reaction was completed, the reaction was quenched by water (50 mL) and extracted with ethyl acetate (30 mL*3). The organic phases were combined, washed with water (30 mL*3) and saturated brine (30 mL) sequentially, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether: ethyl acetate=1:0 to 2:1) to obtain compound B-1-4. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.41-7.34 (m, 2H), 6.78-6.74 (m, 2H), 4.15-4.12 (m, 1H), 3.64-3.52 (m, 4H), 2.18-2.07 (m, 2H), 1.157 (s, 9H).
Step 3: Synthesis of Compound B-1-5
[0115] Compound B-1-4 (3.00 g, 8.77 mmol, 1.00 eq) was added to a solution of hydrogen chloride in ethyl acetate (10 mL, 4 M), and the reaction was carried out at 20 ° C. for 1 hour. After the reaction was completed, water (30 mL) was added to quench the reaction, and the mixture was washed with ethyl acetate (20 mL). The aqueous phase was adjusted to pH=7 with saturated sodium bicarbonate aqueous solution and extracted with ethyl acetate (30 mL). The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product of compound B-1-5. The crude product was used directly in the next reaction step without further purification. .sup.1H NMR (400 MHz, MeOD-d.sub.4) δ 7.39-7.35 (m, 2H), 6.76-6.74 (m, 2H), 5.30 (m, 1H), 3.31-3.13 (m, 4H), 2.15-2.05 (m, 2H).
Step 4: Synthesis of Compound B-1-7
[0116] Compound B-1-6 (24.67 g, 75.38 mmol, 1.05 eq) was dissolved in a solution of anhydrous dichloromethane (200 mL) and anhydrous N,N-dimethylformamide (200 mL) under nitrogen protection, then compound B-1-5 (20 g, 71.79 mmol, 1 eq, HCl) was added at 20° C. and reacted for 1 hour. The reaction was cooled to −70° C. and then continuously stirred for 0.5 hours. Sodium borohydride acetate (30.43 g, 143.60 mmol, 2 eq) was added slowly to the reaction in 30 min, controlling the temperature below −60° C. during the addition. The reaction was stirred at −70° C. for 17.5 hours. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was diluted with water (600 mL) slowly to precipitate a large amount of white flocculent solid, the mixture was stirred for 1 hour and filtered, the filter cake was washed with water (100 mL*3). The filter cake was added with ethanol (500 mL) and stirred for 12 hours. After filtration, the filter cake was collected and washed with ethanol (100 mL), dried under reduced pressure to obtain compound B-1-7. .sup.1H NMR (CDCl.sub.3-d) δ 7.74 (d, J=8.5 Hz, 2H), 7.21 (br s, 1H), 7.02-6.90 (m, 2H), 6.85-6.82 (m, 1H), 6.89-6.82 (m, 2H), 4.88 (br dd, J=7.2, 3.1 Hz, 1H), 4.47 (br d, J=9.5 Hz, 1 H), 4.27 (br d, J=10.0 Hz, 1H), 4.23-4.18 (m, 1H), 3.81-3.68 (m, 1H), 3.38 (br t, J=10.7 Hz, 1H), 3.02-2.89 (m, 3H), 2.64-2.51 (m, 2H), 2.45 (br d, J=11.0 Hz, 1H), 2.39-2.26 (m, 1H), 2.07-1.95 (m, 1H), 1.51 (q, J=11.8 Hz, 1H), 1.33 (s, 12H), 1.26 (br s, 9H).
Step 5: Synthesis of Compound B-1
[0117] Bis(pinacolato)diboron (4.88 g, 19.20 mmol, 1.25 eq), potassium acetate (4.52 g, 46.08 mmol, 3 eq) and Pd(dppf)Cl.sub.2.DCM (3.76 g, 4.61 mmol, 0.3 eq) were added sequentially to a solution of compound B-1-7 (8.5 g, 15.36 mmol, 1 eq) in anhydrous dioxane (170 mL) under nitrogen protection. The reaction was warmed to 100° C. and stirred for 7 hours. The mixture was concentrated under reduced pressure and diluted with dichloromethane (500 mL). The organic phase was washed with water (200 mL×3), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether: ethyl acetate=5:1 to 0:1) to obtain compound B-1. .sup.1H NMR (CDCl.sub.3) δ 7.74 (d, J=8.8 Hz, 2H), 7.25-7.15 (m, 1 H), 7.00-6.87 (m, 2H), 6.84 (d, J=8.8 Hz, 2H), 4.92-4.82 (m, 1H), 4.48 (br d, J=8 Hz, 1H), 4.27 (br d, J=9.8 Hz, 1H), 4.24-4.18 (m, 1H), 3.78-3.66 (m, 1H), 3.38 (br t, J=10.8 Hz, 1H), 3.07-2.84 (m, 3H), 2.65-2.50 (m, 2H), 2.49-2.39 (m, 1H), 2.37-2.27 (br d, J=7.5 Hz, 1H), 2.04-1.98 (m, 1H), 1.69 (s, 3H), 1.52 (q, J=12 Hz, 1H), 1.33 (s, 12 H), 1.26 (s, 9 H).
Embodiment 1: WX001
[0118] ##STR00025##
Synthetic Route
[0119] ##STR00026##
Step 1: Synthesis of Compound WX001-1
[0120] A mixture of B-1 (126.59 mg, 210.81 μmol), A-1 (0.104 g, 210.81 μmol), sodium carbonate (44.69 mg, 421.62 μmol) and tetrakis(triphenylphosphine)palladium (48.72 mg, 42.16 μmol) in a solution of toluene (2.4 mL), ethanol (0.6 mL) and water (0.6 mL) was stirred at 50° C. for 16 hours under nitrogen protection. After the reaction was completed, the mixture was concentrated under reduced pressure and the residue was diluted with dichloromethane (30 mL) and washed with water (20 mL*2). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain crude product. The crude product was stirred with ethanol (9.50 mL) for 3 hours and then the mixture was filtered. The solid was washed with ethanol (2 mL*3) and dried under reduced pressure to obtain compound WX001-1. MS m/z: 887.5 [M+1].sup.+.
Step 2: Synthesis of Compound WX001-2
[0121] A solution of hydrogen chloride in ethyl acetate (4 M, 2.5 mL) was added to a solution of compound WX001-1 (0.128 g, 144.31 μmol) in ethyl acetate (2.5 mL). The reaction was stirred at 20° C. for 16 hours. After the reaction was completed, a crude product of compound WX001-2 was obtained by concentration under reduced pressure. The crude product was used directly in the next reaction step without further purification.
Step 3: Synthesis of Compound WX001
[0122] Potassium carbonate (108.28 mg, 783.42 μmol) was added to a solution of the crude product of compound WX001-2 (0.129 g, 156.68 μmol) in methanol (5 mL). The reaction was stirred at 25° C. for 2 hours. The mixture was filtered after the reaction was completed, and the filtrate was concentrated under reduced pressure and the residue was diluted with dichloromethane (20 mL). The organic phase was washed with water (15 mL*3). The aqueous phases were combined and extracted with dichloromethane (10 mL*2). All organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. The crude product was purified by preparative high performance liquid chromatography (acetonitrile/water/ammonium carbonate/ammonia system) to obtain the compound WX001. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.25-7.17 (m, 1H), 7.17-7.06 (m, 4 H), 7.06-6.92 (m, 3H), 6.77 (brd, J=7.6 Hz, 2H), 4.79 (brs, 1H), 4.34 (brd, J=8.8 Hz, 1 H), 4.24-4.02 (m, 3H), 4.01-3.77 (m, 2H), 3.66-3.52 (m, 1H), 3.52-3.29 (m, 3H), 3.14-3.01 (m, 1H), 2.91-2.82 (m, 1H), 2.79-2.70 (m, 2H), 2.62-2.55 (m, 2H), 2.44-2.30 (m, 2H), 2.27-2.21 (m, 1H), 2.16 (br s, 3H), 2.07-1.89 (m, 1H), 1.49-1.37 (m, 1H).
Embodiment 2: WX002
[0123] ##STR00027##
Synthetic Route
[0124] ##STR00028##
Step 1: Synthesis of WX002-1
[0125] Compound A-2, compound B-1 (309.52 mg, 515.44 μmol, 1 eq), tetrakis(triphenylphosphine)palladium (59.56 mg, 51.54 μmol, 0.1 eq) and potassium carbonate (142.47 mg, 1.03 mmol, 2 eq) were suspended in a mixture of dioxane (4 mL) and water (1 mL) under nitrogen protection, the reaction was stirred at 50° C. for 5 hours. The crude product from reaction was purified by column chromatography (dichloromethane: methanol=1:0 to 4:1) to obtain WX002-1, LC-MS of the product (m/z) 917.8 [M+H].sup.+.
Step 2: Synthesis of WX002-2
[0126] WX002-1 (190 mg, 207.10 μmol, 1 eq) was dissolved in dichloromethane (2.5 mL), and trifluoroacetic acid (770.00 mg, 6.75 mmol, 0.5 mL, 32.61 eq) was added dropwise to the reaction. The reaction was stirred at 25° C. for 2 hours. After the reaction was completed, the mixture was concentrated under reduced pressure, the residue was added with saturated sodium bicarbonate (10 mL) and dichloromethane (10 mL), then the mixture was stirred and the organic phase was separated and concentrated under reduced pressure to obtain WX002-2, LC-MS of the product (m/z) 817.2 [M+H].sup.+.
Step 3: Synthesis of WX002
[0127] Potassium carbonate (135.28 mg, 978.79 μmol, 5 eq) was added to a solution of WX002-2 (160 mg, 195.76 μmol, 1 eq) in methanol (2 mL) at 25° C., then the mixture was stirred for 2 hours. After the reaction was completed, the reaction was filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product.
[0128] The crude product was purified by preparative chromatography to obtain WX002, which was confirmed by LCMS, LC-MS (m/z) 691.1 [M+H].sup.+, .sup.1H NMR (400 MHz, CD.sub.3OD) δ ppm 1.64 (q, J=11.54 Hz, 1H), 1.95-2.04 (m, 1H), 2.14 (s, 3H), 2.28-2.36 (m, 1H), 2.38 (s, 3H), 2.50 (br d, J=14.31 Hz, 1H), 2.62-2.75 (m, 2H), 2.91-3.09 (m, 3H), 3.21-3.30 (m, 1H), 3.35-3.53 (m, 4H), 3.94-4.07 (m, 2H), 4.21-4.31 (m, 1H), 4.42 (d, J=9.54 Hz, 1H), 4.47 (d, J=9.03 Hz, 2H), 4.59 (br s, 1H), 6.82 (d, J=8.78 Hz, 2H), 7.07-7.38 (m, 6H), 8.47 (s, 1H).
[0129] Referring to the synthesis method of steps 1 to 3 in embodiment 2, fragments A-3, A-4, and A-6 were used instead of A-2 to synthesize embodiments 3-5 in Table 3.
TABLE-US-00003 TABLE 3 Embo- diment Fragment A Fragment B 3
Embodiment 6: WX006
[0130] ##STR00036##
Synthetic Route
[0131] ##STR00037##
Step 1: synthesis of WX006-1.
[0132] A mixture of compound A-7 (170 mg, 332.47 μmol, 1 eq), compound B-1 (199.65 mg, 332.47 μmol, 1 eq), tetrakis(triphenylphosphine)palladium (38.42 mg, 33.25 μmol, 0.1 eq) and sodium carbonate (105.71 mg, 997.40 μmol, 3 eq) in a mixture of dioxane (4 mL) and water (1 mL) was stirred at 50° C. for 7 hours under nitrogen protection. After the reaction was completed, the mixture was concentrated under reduced pressure to obtain a crude product of WX006-1, which was confirmed by LCMS, LC-MS (m/z) 906.5 [M+H].sup.+.
Step 2: Synthesis of WX006-2
[0133] Sodium methanol (23.88 mg, 442.01 μmol, 2 eq) was added to a mixture of the crude product of WX006-1 (200 mg, 221.00 μmol, 1 eq) and methanol (5 mL). The reaction was stirred at 25° C. for 2 hours. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was purified twice by silica gel preparative plates (the ratio of developing solvent was dichloromethane: methanol=15:1 and dichloromethane: methanol=10:1 sequentially) to obtain WX006-2, which was confirmed by LCMS, LC-MS (m/z) 779.3 [M+H].sup.+.
Step 3: Synthesis of WX006
[0134] Trifluoroacetic acid (770.00 mg, 6.75 mmol, 0.5 mL, 58.44 eq) was added to a solution of WX006-2 (90 mg, 115.56 μmol, 1 eq) in dichloromethane (3 mL). The reaction was stirred at 20° C. for 0.5 hours. After the reaction was completed, the mixture was concentrated under reduced pressure. The residue was dissolved in dichloromethane (20 mL) and washed with 1 N potassium carbonate aqueous solution (15 mL). The organic phase was concentrated under reduced pressure to obtain crude product. The crude product was purified by silica gel preparative plate (dichloromethane: methanol=8:1, Rf =0.2), followed by supercritical chiral preparation (column type: DAICEL CHIRALPAK AS (250 mm*30 mm, 10 μm), mobile phase A was supercritical carbon dioxide, phase B: 0.1% ammonia-ethanol solution; ratio B%: 35%-35%) to obtain WX006, which was confirmed by LCMS, LC-MS (m/z) 679.4 [M+H].sup.+, .sup.1H NMR (400 MHz, CD.sub.3OD) δ ppm 1.45-1.56 (m, 1H), 1.93-2.01 (m, 1H), 2.14 (s, 3H), 2.28-2.38 (m, 1H), 2.39-2.47 (m, 1H), 2.57-2.66 (m, 2H), 2.89-2.97 (m, 2H), 2.97-3.05 (m, 2H), 3.34-3.42 (m, 3H), 3.45-3.52 (m, 2H), 3.93 (s, 2H), 4.18-4.24 (m, 1H), 4.30 (d, J=9.54 Hz, 1H), 4.41 (d, J=9.54 Hz, 1H), 4.46-4.53 (m, 1H), 6.83 (d, J=8.53 Hz, 2H), 6.92 (t, J=10.04 Hz, 1H), 7.06-7.18 (m, 4H), 7.18-7.24 (m, 1H), 7.33 (t, J=8.03 Hz, 1H).
[0135] Referring to the synthesis method of steps 1 to 3 in embodiment 6, fragment A-5 was used instead of A-7 to synthesize embodiment 7 in Table 4.
TABLE-US-00004 TABLE 4 Embo- diment Fragment A Fragment B 7
Embodiment 8: WX008
[0136] ##STR00041##
Synthetic Route
[0137] ##STR00042##
Step 1: Synthesis of WX008-1
[0138] A mixture of A-8 (227 mg, 447.41 μmol, 1 eq), B-1 (322.40 mg, 536.89 μmol, 1.2 eq), potassium carbonate (123.67 mg, 894.82 μmol, 2 eq) and tris(dibenzylideneacetone)dipalladium (40.97 mg, 44.74 μmol, 0.1 eq) in a mixture of water (1.5 mL) and dioxane (5 mL) was stirred at 50° C. for 7 hours under nitrogen protection. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain a crude product of WX008-1, which was confirmed by LCMS, LC-MS (m/z) 901.3 [M+H].sup.+.
Step 2: Synthesis of WX008-2
[0139] Sodium methanol (53.36 mg, 987.80 μmol, 2 eq) was added to a mixture of WX008-1 (445 mg, 493.90 μmol, 1 eq) and methanol (3 mL), and the reaction was stirred at 25° C. for 2 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel preparative plate (dichloromethane: methanol=10:1, Rf=0.12) to obtain WX008-2, which was confirmed by LCMS, LC-MS (m/z) 775.3 [M+H].sup.+.
Step 3: Synthesis of WX008.
[0140] Trifluoroacetic acid (1.26 g, 11.02 mmol, 815.72 μL, 40.08 eq) was added to a solution of WX008-2 (213 mg, 274.88 μmol, 1 eq) in dichloromethane (3 mL), and the reaction was stirred at 25° C. for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by preparative high performance liquid chromatography (chromatographic column: Welch Xtimate C18 150*25 mm*5 μm; mobile phase: [H.sub.2O(0.2% FA)-ACN]; B (ACN) %: 10%-40%, 6 min) to obtain WX008 formate, which was confirmed by LCMS, LC-MS (m/z) 675.3 [M+H].sup.+, .sup.1H NMR (400 MHz, CD.sub.3OD) δ ppm 1.29-1.34 (m, 1H), 1.59 (q, J=11.67 Hz, 1H), 1.97 (br dd, J=9.51, 6.63 Hz, 1H), 2.12 (s, 3H), 2.25-2.35 (m, 1H), 2.36 (s, 3H), 2.43-2.50 (m, 1H), 2.59-2.71 (m, 2H), 2.90-2.96 (m, 1H), 2.97-3.05 (m, 2H), 3.10-3.22 (m, 1H), 3.34-3.43 (m, 2H), 3.45-3.54 (m, 2H), 3.89 (s, 2H), 4.19-4.28 (m, 1H), 4.37-4.46 (m, 3H), 6.79 (d, J=8.75 Hz, 2H), 6.87 (d, J=11.01 Hz, 1H), 7.09-7.21 (m, 4H), 7.23-7.26 (m, 2H), 8.49 (s, 1H).
[0141] Proton nuclear magnetic resonance and mass spectrum data of each embodiment are shown in Table 5.
TABLE-US-00005 TABLE 5 Proton nuclear magnetic resonance and mass spectrum data of each embodiment Embodiment Compound .sup.1H NMR MS m/z 1 WX001 .sup.1H NMR(400 MHz, CDCl.sub.3) δ 7.25-7.17 (m, 1 661.4 H),7.17-7.069 (m, 4 H), 7.06-6.92 (m, 3H), 6.77 (br [M + 1].sup.+ d, J = 7.6 Hz, 2H), 4.79 (br s, 1H), 4.34 (br d, J = 8.8 Hz, 1H), 4.24-4.02 (m, 3H), 4.01-3.77 (m, 2H), 3.66-3.52 (m, 1H), 3.52-3.29 (m, 3H), 3.14-3.01 (m, 1H), 2.91-2.82 (m, 1H), 2.79-2.70 (m, 2H), 2.62-2.55 (m, 2H), 2.44-2.30 (m, 2H), 2.27-2.21 (m, 1H), 2.16 (br s, 3H), 2.07-1.89 (m, 1H), 1.49- 1.37 (m,1H) 2 WX002 .sup.1H NMR (400 MHz, CD.sub.3OD) δ8.47 (s, 1H), 7.07- 691.1 7.38 (m, 6H), 6.82 (d, J = 8.78 Hz, 2H), 4.59 (br s, [M + H].sup.+ 1H), 4.47 (d, J = 9.03 Hz, 2H), 4.42 (d, J = 9.54 Hz, 1H), 4.21-4.31 (m, 1H), 3.94-4.07 (m, 2H), 3.35- 3.53 (m, 4H), 3.21-3.30 (m, 1H), 2.91-3.09 (m, 3H), 2.62-2.75 (m, 2H), 2.50 (br d, J = 14.31 Hz, 1H), 2.38 (s, 3H), 2.28-2.36 (m, 1H), 2.14 (s, 3H), 1.95-2.04 (m, 1H), 1.64 (q, J = 11.54 Hz, 1H) 3 WX003 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.49 (s, 1 H), 7.30- 691.1 7.39 (m, 2 H), 7.24 (br d, J = 3.51 Hz, 1 H), 7.17 (dt, [M + H].sup.+ J = 9.22, 4.55 Hz, 2 H), 6.99 (d, J = 8.53 Hz, 2 H), 6.77 (d, J = 8.78 Hz, 2 H), 4.54 (d, J = 9.54 Hz, 2 H), 4.39-4.47 (m, 2 H), 4.19-4.28 (m, 3 H), 3.55- 3.64 (m, 1 H), 3.51 (t, J = 8.78 Hz, 1 H), 3.35-3.45 (m, 2 H), 3.14-3.24 (m, 1 H), 2.90-3.06 (m, 3 H), 2.58-2.74 (m, 2 H), 2.42-2.53 (m, 1 H), 2.25- 2.37 (m, 4 H), 2.15 (s, 3 H), 1.98 (br d, J = 6.78 Hz, 1 H), 1.60 (q, J = 11.54 Hz, 1 H) 4 WX004 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.45 (s, 1 H), 7.09- 691.1 7.30 (m, 7 H), 6.72-6.87 (m, 2 H), 4.50 (d, J = 9.79 [M + H].sup.+ Hz, 1 H), 4.34-4.44 (m, 1 H), 4.26 (br dd, J = 11.04, 2.51 Hz, 1 H), 4.11 (d, J = 9.54 Hz, 1 H), 3.98-4.06 (m, 2 H), 3.33-3.50 (m, 4 H), 2.91-3.11 (m, 3 H), 2.62-2.79 (m, 2 H), 2.52 (br d, J = 13.30 Hz, 1 H), 2.25-2.43 (m, 4 H), 2.10-2.17 (m, 3 H), 1.92- 2.05 (m, 1 H), 1.67 (q, J = 11.63 Hz, 1 H), 1.09 (dd, J = 6.78, 2.76 Hz, 1 H) 5 WX005 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.10-7.33 (m, 9 H), 643.1 6.82 (d, J = 8.53 Hz, 2 H), 4.63 (br s, 1 H), 4.37- [M + H].sup.+ 4.47 (m, 2 H), 4.24 (br d, J = 10.79 Hz, 1 H), 4.16 (d, J = 9.29 Hz, 1 H), 3.93 (s, 2 H), 3.36-3.52 (m, 4 H), 3.12-3.22 (m, 1 H), 2.93-3.09 (m, 3 H), 2.61- 2.71 (m, 2H), 2.49 (br d, J = 12.05 Hz, 1 H), 2.29- 2.41 (m, 1 H), 2.16 (s, 3 H), 1.93-2.04 (m, 1 H), 1.61 (q, J = 11.71 Hz, 1 H) 6 WX006 .sup.1H NMR (400 MHz, CD.sub.3OD) δ7.33 (t, J = 8.03 Hz, 679.4 1H), 7.18-7.24 (m, 1H), 7.06-7.18 (m, 4H), 6.92 (t, [M + H].sup.+ J = 10.04 Hz, 1H), 6.83 (d, J = 8.53 Hz, 2H), 4.46- 4.53 (m, 1H), 4.41 (d, J = 9.54 Hz, 1H), 4.30 (d, J = 9.54 Hz, 1H), 4.18-4.24 (m, 1H), 3.93 (s, 2H), 3.45- 3.52 (m, 2H), 3.34-3.42 (m, 3H), 2.97-3.05 (m, 2H), 2.89-2.97 (m, 2H), 2.57-2.66 (m, 2H), 2.39- 2.47 (m, 1H), 2.28-2.38 (m, 1H), 2.14 (s, 3H), 1.93- 2.01 (m, 1H), 1.45-1.56 (m, 1H) 7 WX007 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.23 (br d, J = 8.88 691.2 Hz, 2H), 7.07-7.19 (m, 4H), 6.72-6.85 (m, 3H), [M + H].sup.+ 4.57-4.67 (m, 2H), 4.29-4.40 (m, 2H), 4.22 (br d, J = 13.26 Hz, 1H), 3.88 (s, 2H), 3.82 (s, 3H), 3.51- 3.57 (m, 1H), 3.44-3.50 (m, 1H), 3.35-3.42 (m, 2H), 2.88-3.07 (m, 4H), 2.57-2.68 (m, 2H), 2.44 (br d, J = 12.88 Hz, 1H), 2.33 (br dd, J = 13.20, 6.57 Hz, 1H), 1.91-2.03 (m, 1H), 2.13 (s, 3H), 1.52 (q, J = 11.76 Hz, 1H) 8 WX008 .sup.1H NMR (400 MHz, CD.sub.3OD) δ8.49 (s, 1 H), 7.23- 675.3 7.26 (m, 2 H), 7.09-7.21 (m, 4 H), 6.87 (d, [M + H].sup.+ J = 11.01 Hz, 1 H), 6.79 (d, J = 8.75 Hz, 2 H), 4.37- 4.46 (m, 3 H), 4.19-4.28 (m, 1 H), 3.89 (s, 2 H), 3.45-3.54 (m, 2 H), 3.34-3.43 (m, 2 H), 3.10- 3.22 (m, 1 H), 2.97-3.05 (m, 2 H), 2.90-2.96 (m, 1 H), 2.59-2.71 (m, 2 H), 2.43-2.50 (m, 1 H), 2.36 (s, 3 H), 2.25 -2.35 (m, 1 H), 2.12 (s, 3 H), 1.97 (br dd, J = 9.51, 6.63 Hz, 1 H), 1.59 (q, J = 11.67 Hz, 1 H), 1.29-1.34 (m, 1 H)
Experimental Embodiment 1. In Vitro Cell Activity Test
Experimental steps and methods.
Biological Activity Experiment 1: SGLT1 Glucose Transport Assay
1. Experimental Purpose
[0142] To test the effect of compounds on the glucose transport activity of SGLT1 transporters by measuring the amount of [.sup.14C]-labeled glucose entering highly expressed Human-SGLT1 cells.
2. Experimental Methods
2.1. Cell Preparation
[0143] The cells stably expressing Human-SGLT1 used for the experiments were constructed by WuXi AppTec Shanghai. The SGLT1 cells were plated in Cytostar-T (PerkinElmer) 96-well cell culture plates and cultured overnight at 5% CO.sub.2, 37° C.
2.2. SGLT1 Glucose Transport Assay
[0144] Experimental buffer: 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 1.2 mM magnesium chloride (MgCl.sub.2), 4.7 mM potassium chloride (KCl), 2.2 mM calcium chloride (CaCl.sub.2) and 120 mM sodium chloride (NaCl).
[0145] The compounds were diluted with 100% dimethyl sulfoxide (DMSO) at a starting concentration of 1 mM, 8 points of 5-fold serial gradient dilution was made.
[0146] 3 μM [.sup.14C]-labeled methyl α-D-glucopyranoside (Methyl a-D-glucopyranosid) was prepared with experimental buffer.
[0147] Cells were treated with 49 μL of experimental buffer, 1 μL of gradient-diluted compound and 50 μL of 3 μM [14C]- isotope-labeled glucose solution at 37° C. for 2 hours.
[0148] Readings were taken with an isotope detector (Micro beta Reader).
[0149] The data were calculated by GraphPad Prism 5.0 software using the formula: log(inhibitor) vs. response—Variable slope to obtain the IC.sub.50 values of the tested compounds, and the experimental results are shown in Table 5.
Biological Activity Experiment 2: SGLT2 Glucose Transport Assay
1. Experimental Purpose
[0150] To test the effect of compounds on the glucose transport activity of SGLT2 transporters by measuring the amount of [.sup.14C]-labeled glucose entering highly expressed Human-SGLT2 cells.
2. Experimental Methods
2.1. Cell Preparation
[0151] The cells stably expressing Human-SGLT2 used for the experiments were constructed by WuXi AppTec Shanghai. SGLT2 cells were plated in 96-well cell culture plates (Greiner) and cultured overnight at 5% CO.sub.2, 37° C.
2.2. SGLT2 Glucose Transport Assay
[0152] Experimental buffer: 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 1.2 mM magnesium chloride (MgCl.sub.2), 4.7 mM potassium chloride (KCl), 2.2 mM calcium chloride (CaCl.sub.2) and 120 mM sodium chloride (NaCl).
[0153] Termination buffer: 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 1.2 mM magnesium chloride (MgCl.sub.2), 4.7 mM potassium chloride (KCl), 2.2 mM calcium chloride (CaCl.sub.2), 120 mM sodium chloride (NaCl) and 1 μM LX4211.
[0154] The compounds were diluted with 100% dimethyl sulfoxide (DMSO) at a starting concentration of 10 μM, 8 points of 5-fold serial gradient dilution was made.
[0155] 6 μM [.sup.14C]-labeled methyl α-D-glucopyranoside was prepared with experimental buffer.
[0156] Cells were treated with 49 μL of experimental buffer, 1 μL of gradient-diluted compound, and 50 μL of 6 μM [.sup.14C]-isotope-labeled glucose solution at 37° C. for 2 hours.
[0157] The liquid in the wells was aspirated and the cells were washed 3 times with termination buffer.
[0158] The cells were lysed with 50 μL of 10% sodium hydroxide solution, and the cell lysate was aspirated into a scintillation tube, and 2 mL of scintillation solution was added.
[0159] Readings were taken with an isotope detector (Tricarb).
[0160] The data were calculated by GraphPad Prism 5.0 software using the formula: log (inhibitor) vs. response--Variable slope to obtain the IC.sub.50 values of the tested compounds, and the experimental results are shown in Table 5.
Biological Activity Experiment 3: rhDPP4 Inhibitor Screening Experiment
1. Experimental Purpose
[0161] To evaluate the inhibitory activity of compounds against recombinant human dipeptidyl peptidase 4 (rhDPP4) by measuring the half-inhibitory concentration (IC.sub.50) values of the compounds. In the experiment, rhDPP4 was used to catalyze the generation of fluorescein from substrate, the substrate was a luminescent precursor Gly-Pro-aminofluorescein, and the substrate and luciferase were reacted to generate a light signal, the intensity of the light signal was proportional to the enzyme activity.
2. Experimental Methods
[0162] 1) The gradient diluted compounds (4-fold fold-dilution, 10 assay concentrations) were transferred 250 nL into a 384-well plate (PerkingElmer-6007299) using a non-contact nano-acoustic pipetting system (ECHO), and dimethyl sulfoxide (DMSO) concentration was 0.5% in the final reaction system. Blank control wells (containing DMSO, substrate and 10 mM Tris-HCl) and positive control wells (containing DMSO, substrate and rhDPP4) were set up.
[0163] 2) The pre-dispensed frozen buffer containing luciferase was removed and restored to room temperature, and then the substrate was added to configure a working solution with a substrate concentration of 20 μM. RhDPP4 was prepared as a working solution of 0.2 ng/mL using 10 mM Tris-HCl (pH 8.0) aqueous solution.
[0164] 3) 25 μL of working solution containing 20 μM substrate and 25 μL of working solution containing 0.2 ng/mL rhDPP4 were added to a 384-well plate with the addition of the compound, then the mixture was centrifuged for 30 s at 1000 rpm, the plate was sealed with aluminum foil sealing film and incubated for 1 hour at room temperature.
[0165] 4) The intensity of the light signal was detected by EnVision, a multifunctional microplate reader. The raw data were used to calculate the inhibition of rhDPP4 activity by the compound.
% of inhibition activity=100−(compound well signal value−blank control well signal value)/(positive control well signal value−blank control well signal value)*100. [0166] The percent of inhibition was imported into GraphPad Prism software for data processing to derive the corresponding dose-effect curves and to derive the IC.sub.50 values of the tested compounds. The results of the experiments are shown in Table 6.
TABLE-US-00006 TABLE 6 Results of in vitro cell activity test Human-SGLT1 Human-SGLT2 rhDPP4 Compound IC.sub.50 (nM) IC.sub.50 (nM) IC.sub.50 (nM) WX001 1283 21.75 31.03 WX002 187.5 4.84 104 WX003 980.2 332.4 111.6 WX004 33.34 5.96 166 WX005 28.49 5.05 115.9 WX006 297.80 7.02 26.9 WX007 2070 19.73 32.54 WX008 520.60 7.39 — Note: ″—″ indicates that no relevant assay is performed.
Conclusion: The Compounds of the Present Disclosure Exhibit High Selectivity against Human-SGLT2 and Significant Inhibitory Activity against Human-SGLT2 and rhDPP4 In Vitro
Experimental Embodiment 2. In Vivo DMPK Study
[0167] Experimental purpose: Male C57 mice were used as test animals to determine blood concentrations of compounds and assess pharmacokinetic behavior after a single dose.
[0168] Experimental procedure: Six healthy adult male C57 mice were selected, three of them for the intravenous injection group and three of them for the oral group. The compounds to be tested were mixed with an appropriate amount of intravenous injection group solvent (20% polyethylene glycol-400 (PEG400)/10% polyethylene glycol-15 hydroxystearate (solutol)/70% H.sub.2O), the mixture was vortexed and sonicated to obtain 1 mg/mL clarified solution, then the clarified solution was filtered through a microporous membrane and prepared for use; solvent for the oral group was 20% polyethylene glycol-400 (PEG400)/10% polyethylene glycol-15 hydroxystearate (solutol)/70% H.sub.2O, and the compounds to be tested were mixed with the solvent, then the mixture was vortexed and sonicated to obtain 1 mg/mL clarified solution. After 1 mg/kg intravenous administration or 10 mg/kg oral administration in mice, whole blood was collected for a certain period of time, then the plasma was prepared to obtain, and the drug concentration was analyzed by LC-MS/MS method, then the pharmacokinetic parameters were calculated using Phoenix WinNonlin software (Pharsight, USA).
[0169] The results of the experiments are shown in Table 7.
TABLE-US-00007 TABLE 7 Results of PK tests of compounds Com- C.sub.max Oral DNAUC Vd.sub.ss Cl T.sub.1/2 pound (nM) F % (nM.h/mpk) (L/kg) (mL/min/kg) (h) WX001 308 21.4 194.8 12.2 31.4 7.39 WX006 250 18.7 151.3 12.0 29.1 10.5 Note: C.sub.max is the maximum concentration; F % is the oral bioavailability; oral unit exposure is Oral DNAUC = AUC.sub.PO/Dose, AUC.sub.PO is the oral exposure, Dose is the drug dose; Vd.sub.ss is the volume of distribution; Cl is the clearance rate; T.sub.1/2 is the half-life.
Conclusion: The Compounds of the Present Disclosure Exhibit to Possess Certain Oral Exposure and Bioavailability for Mice
Experimental Embodiment 3. In Vivo Pharmacodynamic Study of Oral Glucose Tolerance (OGTT) in Rats
Summary of Experiments
1. Animals
[0170]
TABLE-US-00008 Animal: Species: SD Rat Sex: Male Age/Weight: About 8 weeks/250 g Supplier: Vital River Animal Feed: General rat/mouse feed
2. Experimental Grouping
[0171]
TABLE-US-00009 Frequency of Mode of Number of Compound adminis- adminis- animals per Group grouping Dose tration tration group 1 Vehicle control 0 Single dose Gavage 5 group 2 Positive 3 Single dose Gavage 5 compound mg/kg (Canagliflozin) 3 Test 10 Single dose Gavage 5 compound mg/kg
Experimental Procedure
1. Animal Acclimatization and Preparation
[0172] The animals will be acclimatized in the animal room for 1 week after arrival at the facility.
2. Fasting and Drug Administration
[0173] The animals are fasted for 16 hours in the metabolic cage and given the drug or solvent (5 mL/kg) according to the table above, followed immediately by 50% glucose solution (2 g/kg, 4 mL/kg).
3. Urinary Glucose and Blood Glucose Test
[0174] After 1 hour of glucose administration, the animals resumed feeding, and blood samples were collected at 0 min, 20 min, 40 min, 60 min, 90 min and 120 min for blood glucose measurement; urine at 0 to 24 hours was used for urinary glucose (mg/200 g) and urine volume test, respectively.
4. Data Analysis
[0175] All values were expressed as mean values. Statistical analysis was performed using Graphpad Prism 6 single factor analysis of variance Tukey's multiple comparison test. The p-value less than 0.05 was considered statistically significant.
[0176] The results of the experiments are shown in Table 8:
TABLE-US-00010 TABLE 8 Results of glucose tolerance test in rats Positive Vehicle compound Compound control group (Canagliflozin) WX001 OGTT blood glucose levels 1023.9 945.2 869.0** AUC.sub.0-2 hr (mol/L × min) Urinary glucose level 0.6 1317**** 0.7 (mg/200 gBW) Urine volume 12.5 24.4*** 10.6 (mL/200 gBW) *refers to p < 0.5, **refers to p < 0.01, ***refers to p < 0.001, ****refers to p < 0.0001 vs. the vehicle control group. Note: 200 g BW is 200 g average body weight.
Conclusion: The Compounds of the Present Disclosure Significantly Reduce the Blood Glucose AUC Levels in Animals in 2 Hours Compared to the Vehicle Control Groups; the 24-Hours Urinary Glucose Excretion Levels in Animals are Lower Than Those of the Positive Compound
Experimental Embodiment 4. In Vivo Pharmacodynamic Study of Oral Glucose Tolerance (OGTT) in dbdb Mice
Summary of Experiments
1. Animals
[0177]
TABLE-US-00011 Animal: Species: Dbdb mice Sex: Male Age/Weight: 11-13 weeks/50 g Supplier: Shanghai Model Biology Center Animal General rat/mouse feed Feed:
2. Experimental Grouping
[0178]
TABLE-US-00012 Frequency of Mode of Number of Compound adminis- adminis- animals per Group grouping Dose tration tration group 1 Vehicle control 0 Single dose Gavage 5 group 2 Test compound 50 mg/kg Single dose Gavage 5
Experimental Procedure
1. Animal Acclimatization and Preparation
[0179] The animals will be acclimatized in the animal room for 1 week after arrival at the facility and grouped according to blood glucose and body weight.
2. Fasting and Drug Administration
[0180] The animals are fasted for 6 hours and blood glucose was measured, the drug or solvent were given according to the above table, 50% glucose solution (2 g/kg, 0.4 g/mL) was given after 30 minutes.
3. Test
[0181] The bloods at the time points of -30 min, 0 min, 15 min, 30 min, 60 min, 90 min, 120 min of glucose administration were collected and used for blood glucose measurement. At 1 hour of glucose administration, blood was taken to detect insulin secretion, and at 2 hours of glucose administration, animals were euthanized and blood was taken to detect DPP4 activity and active GLP-1.
4. Data Analysis
[0182] Statistical analysis was performed using Graphpad Prism 8 unpaired T-test for comparison, and the p-values less than 0.05 were considered statistically significant.
[0183] Data are expressed as mean ±standard error, n=4-5. Experimental results are shown in
Conclusion: The Compounds of the Present Disclosure Compared to the Vehicle Control Significantly Reduce the Blood Glucose AUC Levels in Animals in 2 Hours, Significantly Increase Insulin as well as Active GLP-1 Levels, and Reduce DPP4 Activity
Experimental Embodiment 5. In Vivo Pharmacodynamic Study of Oral Glucose Tolerance in BKS-db Mice on a High-Sugar and High-Fat Diet
Summary of Experiments
1. Animals
[0184]
TABLE-US-00013 Animal: Species: BKS-db Sex: Male Age 7 weeks Supplier: Jiangsu Jicui Yaokang Biological Technology Co., Ltd. Animal Feed: 45% HF + 17% HS (RDI #D12451)
2. Experimental Grouping
[0185]
TABLE-US-00014 Frequency of Mode of Number of Compound adminis- adminis- animals per Group grouping Dose tration tration group 1 Normal 0 QD Gavage 3 control group (control) 2 Vehicle 0 QD Gavage 8 control group (Vehicle) 4 Test 50 QD Gavage 8 compound mg/kg
3. Experimental Procedure
[0186] After adaptive feeding of 7-week-old BKS and BKS-db mice, and the blood was collected after fasting for 6 hours, then fasting blood glucose and HbA1c were detected. Mice were grouped according to HbA1c of 6 hours fasting (primary reference index), fasting blood glucose and body weight (secondary reference index).
[0187] At the 2nd week of administration, fasting blood glucose was detected after fasting for 6 hours, and postprandial blood glucose was detected after 1 hour of resumption of feeding; at the 4th week of administration, fasting blood glucose was detected after fasting for 6 hours, and postprandial blood glucose and HbA1c were detected after 1 hour of resumption of feeding; urine was collected from each group of mice. After animals were euthanized, livers were taken for oil red staining and steatosis scoring.
4. Data Analysis
[0188] Statistical analysis was performed using Graphpad Prism 8, One Way ANOVA or unpaired T test for comparison, the p-values less than 0.05 were considered statistically significant.
[0189] Data are expressed as mean±standard error, n=3-8. Experimental results are shown in
Conclusion
[0190]