GLUCOSIDE DERIVATIVE THAT ACTS AS SGLT1 INHIBITOR AND APPLICATION THEREOF
20220153772 · 2022-05-19
Assignee
Inventors
- Yi LI (Shanghai, CN)
- Tao YU (Shanghai, CN)
- Qinghua MAO (Shanghai, CN)
- Chengde WU (Shanghai, CN)
- Shuhui Chen (Shanghai, CN)
Cpc classification
C07D405/12
CHEMISTRY; METALLURGY
C07H15/14
CHEMISTRY; METALLURGY
C07H15/26
CHEMISTRY; METALLURGY
International classification
Abstract
A glucoside derivative that acts as an SGLT1 inhibitor and an application thereof in the preparation of a drug for SGLT1 related diseases. Specifically disclosed is a compound represented by formula (II), a tautomer thereof or a pharmaceutically acceptable composition thereof.
##STR00001##
Claims
1. A compound represented by formula (II), or an isomer or a pharmaceutically acceptable salt thereof, ##STR00077## wherein R.sub.1 is selected from H, F, Cl, Br, I, OH, NH.sub.2, CN, C.sub.1-6 alkyl and C.sub.1-6 alkoxy, wherein the C.sub.1-6 alkyl and C.sub.1-6 alkoxy are optionally substituted with 1, 2, or 3 R.sub.a; R.sub.2 is selected from F, Cl, Br, I, OH, NH.sub.2 and C.sub.1-3 alkylamino; R.sub.3, R.sub.4 and R.sub.5 are each independently selected from H, F, Cl, Br, I, OH, NH.sub.2, CN, and C.sub.1-3 alkyl optionally substituted with 1, 2, or 3 R.sub.b; L is selected from single bond, —O—, —S—, —C(R.sub.c).sub.2—, and —N(R.sub.d)—; m is selected from 0, 1, and 2; n is selected from 1, 2, and 3; R.sub.a, R.sub.b and R.sub.c are each independently selected from F, Cl, Br, I, OH, NH.sub.2 and CH.sub.3; R.sub.d is selected from H and CH.sub.3.
2. The compound, or an isomer or a pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.1 is selected from H, F, Cl, Br, I, OH, NH.sub.2, CN, CH.sub.3, CH.sub.2CH.sub.3, and ##STR00078## wherein the CH.sub.3, CH.sub.2CH.sub.3, and ##STR00079## are optionally substituted with 1, 2, or 3 R.sub.a.
3. The compound, or an isomer or a pharmaceutically acceptable salt thereof according to claim 2, wherein R.sub.1 is selected from H, F, Cl, Br, I, OH, NH.sub.2, CN, CH.sub.3, CH.sub.2F, CHF.sub.2, CF.sub.3, CH.sub.2CH.sub.3, and ##STR00080##
4. The compound, or an isomer or a pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.2 is selected from F, Cl, Br, I, OH, NH.sub.2, NH(CH.sub.3), and N(CH.sub.3).sub.2.
5. The compound, or an isomer or a pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.3, R.sub.4 and R.sub.5 are each independently selected from H, F, Cl, Br, I, OH, NH.sub.2, CN, and CH.sub.3 optionally substituted with 1, 2, or 3 R.sub.b.
6. The compound, or an isomer or a pharmaceutically acceptable salt thereof according to claim 5, wherein R.sub.3, R.sub.4 and R.sub.5 are each independently selected from H, F, Cl, Br, I, OH, NH.sub.2, CN, CH.sub.3, CH.sub.2F, CHF.sub.2, and CF.sub.3.
7. The compound, or an isomer or a pharmaceutically acceptable salt thereof according to claim 1, wherein L is selected from single bond, —O—, and —S—.
8. The compound, or an isomer or a pharmaceutically acceptable salt thereof according to claim 1, wherein moiety ##STR00081## is selected from ##STR00082##
9. The compound, or an isomer or a pharmaceutically acceptable salt thereof according to claim 1, wherein moiety ##STR00083## is selected from ##STR00084##
10. The compound, or an isomer or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is selected from the group consisting of: ##STR00085## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and L are as defined in claim 1.
11. The compound, or an isomer or a pharmaceutically acceptable salt thereof according to claim 10, wherein the compound is selected from the group consisting of: ##STR00086## ##STR00087## wherein R.sub.2 is selected from the group consisting of F, Cl, Br, I, OH, NH.sub.2, NH(CH.sub.3), and N(CH.sub.3).sub.2; R.sub.1, R.sub.3, R.sub.4, R.sub.5, and L are as defined in claim 10.
12. The compound, or an isomer or a pharmaceutically acceptable salt thereof according to claim 11, wherein the compound is selected from the group consisting of: ##STR00088## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are as defined in claim 11.
13. The compound represented by the following formula, or an isomer or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of: ##STR00089## ##STR00090##
14. The compound, or an isomer or a pharmaceutically acceptable salt thereof according to claim 13, wherein the compound is selected from the group consisting of: ##STR00091## ##STR00092## ##STR00093##
15. A pharmaceutical composition comprising a therapeutically effective amount of the compound, or an isomer or a pharmaceutically acceptable salt thereof according to claim 1 as an active ingredient, and pharmaceutically acceptable carrier (s).
16. A method of treating a disease associated with SGLT1 in a subject in need thereof, comprising administering to the subject the compound, or an isomer or a pharmaceutically acceptable salt thereof according to claim 1.
17. The method according to claim 16, characterized in that the disease is diabetes and obesity.
18. A method of treating a disease associated with SGLT1 in a subject in need thereof, comprising administering to the subject the composition according to claim 15.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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[0109]
[0110]
[0111] Note: {circumflex over ( )}{circumflex over ( )}{circumflex over ( )}{circumflex over ( )} means p<0.0001 relative to vehicle group with chow diet, * means p<0.05 relative to vehicle group with high-sugar and high-fat diet, ** means p<0.01 relative to vehicle group with high-sugar and high-fat diet, *** means p<0.001 relative to vehicle group with high-sugar and high-fat diet, and **** means p<0.0001 relative to vehicle group with high-sugar and high-fat diet.
DETAILED DESCRIPTION OF THE INVENTION
[0112] The present disclosure is described in detail below by means of examples. However, it is not intended that these examples have any disadvantageous limitations to the present disclosure. The present disclosure has been described in detail herein, and the embodiments are also disclosed herein. It will be apparent to those skilled in the art that various changes and modifications may be made to the embodiments disclosed herein without departing from the spirit and scope disclosed herein.
Reference Example 1: Fragment A-1
[0113] ##STR00038##
[0114] Route of Synthesis:
##STR00039##
[0115] Step 1: Synthesis of the Compound A-1-2.
[0116] To a reaction flask were added successively compound A-1-1 (10 g, 57.80 mmol, 1 eq), bis(pinacolato)diboron (16.15 g, 63.58 mmol, 1.1 eq), Pd(dppf)Cl.sub.2 (4.23 g, 5.78 mmol, 0.1 eq), KOAc (17.02 g, 173.40 mmol, 3 eq), and dioxane (120 mL), and the atmosphere was replaced with nitrogen. The mixture was reacted at 100° C. for 2 h. After the reaction was completed, the reaction solution was diluted with 50 mL of water, and extracted with ethyl acetate (50 mL*3). The combined organic phase was washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, and filtered, and the organic phase was rotary-evaporated to dryness under reduced pressure to give a crude product. The crude product was purified by column chromatography (PE:EA=50:1) to give A-1-2. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 7.72 (d, J=8.4 Hz, 2H), 6.83 (d, J=8.4 Hz, 2H), 5.40 (s, 1H), 1.34 (s, 12H).
[0117] Step 2: Synthesis of the Compound A-1-4.
[0118] To a reaction flask were added triphenylphosphine (7151 mg, 27.3 mmol, 1.2 eq), DIAD (5513 mg, 27.3 mmol, 1.2 eq), and THF (40 mL). A-1-3 (5104.7 mg, 27.3 mmol, 1.2 eq) and A-1-2 (5 g, 22.7 mmol, 1 eq) dissolved in THF (40 mL) were then added, and the mixture was reacted at 25° C. for 16 hours. After the reaction was completed, the reaction solution was diluted with 100 mL of water, and extracted with ethyl acetate (100 mL*3). The combined organic phase was dried over anhydrous sodium sulfate, and filtered, and the organic phase was rotary-evaporated to dryness to give a crude product. The crude product was purified by column chromatography (PE:EA=19:1-9:1) to give A-1-4. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 7.72-7.75 (m, 2H), 6.84-6.90 (m, 2H), 4.94 (s, 1H), 3.43-3.69 (m, 4H), 2.07-2.23 (m, 2H), 1.47 (s, 9H), 1.34 (s, 12H).
[0119] Step 3: Synthesis of the Compound A-1-5.
[0120] To a reaction flask were added A-1-4 (1.28 g, 3.29 mmol, 1 eq), EtOAc (10 mL), and hydrogen chloride/EtOAc (4 M, 9.04 mL, 11 eq), and the mixture was stirred at 25° C. for 3 hours. After the reaction was completed, the reaction solution was rotary-evaporated to dryness to give a crude product A-1-5. The crude product A-1-5 was directly used in the next reaction.
[0121] Step 4: Synthesis of the Compound A-1.
[0122] To a reaction flask were added A-1-5 (1.37 g, 4.74 mmol, 1 eq), A-1-6 (1.58 g, 4.74 mmol, 1 eq), HATU (1.80 g, 4.74 mmol, 1 eq), THF (15 mL), and DIEA (612.29 mg, 4.74 mmol, 825.18 μL, 1 eq), and the mixture was stirred at 25° C. for 2.5 hours. After the reaction was completed, the reaction solution was diluted with 20 mL of water, and extracted with ethyl acetate (20 mL*3). The combined organic phase was dried over anhydrous sodium sulfate, and filtered. The filtrate was rotary-evaporated to dryness to give a crude product. The crude product was purified by column chromatography (PE:EA=3:2) to give A-1. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 7.70-7.81 (m, 2H), 7.01-7.13 (m, 1H), 6.81-6.93 (m, 3H), 5.69-5.93 (m, 1H), 4.93-5.06 (m, 1H), 3.48-3.89 (m, 4H), 2.83-3.02 (m, 2H), 2.45-2.61 (m, 2H), 2.21-2.36 (m, 1H), 2.06-2.21 (m, 1H), 1.29-1.41 (m, 21H).
[0123] Referring to the synthesis method of steps 2 to 4 in Reference example 1, fragment A-2 was synthesized.
Reference Example 2: Fragment A-2
[0124] ##STR00040##
[0125] Route of Synthesis:
##STR00041##
[0126] Compound A-2:
[0127] .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 1.33 (s, 12H), 1.37 (d, J=1.76 Hz, 9H), 1.77-1.95 (m, 4H), 2.50-2.65 (m, 2H), 2.86-3.01 (m, 2H), 3.31-3.44 (m, 1H), 3.56-3.84 (m, 3H), 4.06-4.16 (m, 1H), 4.56-4.70 (m, 1H), 5.66 (br d, J=7.53 Hz, 1H), 6.86-6.93 (m, 3H), 7.04-7.12 (m, 1H), 7.76 (d, J=8.28 Hz, 2H).
Reference Example 3: Fragment A-3
[0128] ##STR00042##
[0129] Route of Synthesis:
##STR00043##
[0130] Step 1: Synthesis of the Compound A-3
[0131] To a reaction flask were added the compound A-2 (0.20 g, 323.37 μmol, 1 eq) and anhydrous N,N-dimethylformamide (3 mL). Sodium hydride (30 mg, 750.00 μmol, 60% purity, 2.32 eq) was added at 0° C. The mixture was stirred at 0° C. for 0.5 hours, and methyl iodide (0.15 g, 1.06 mmol, 65.79 μL, 3.27 eq) was then added. The reaction system was stirred at 20° C. for 2 hours. The reaction solution was concentrated to give a crude product. Water (10 mL) was added to the crude product, and the mixture was extracted three times with ethyl acetate (10 mL each time). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to give the compound A-3. The crude product A-3 was directly used in the next reaction.
Reference Example 4: Fragment B-1
[0132] ##STR00044##
[0133] Route of Synthesis:
##STR00045##
[0134] Step 1: Synthesis of the Compound B-1-2
[0135] Lithium aluminum hydride (11 g, 289.82 mmol, 1.25 eq) was dissolved in tetrahydrofuran (200 mL) at 0° C. The atmosphere was replaced with nitrogen three times, and finally filled with nitrogen for protection. Compound B-1-1 (50 g, 232.51 mmol, 1 eq) was dissolved in tetrahydrofuran (200 mL), and slowly added to the reaction solution at 0° C. Generated bubbles were observed. The reaction was heated to 25° C. for 2 hours. At 0° C., water (11 mL) was slowly added dropwise, then 15% aqueous sodium hydroxide solution (11 mL) was added dropwise, and finally water (33 mL) was added. The mixture was filtered, and the filter residue was washed twice with ethyl acetate. The filtrate was rotary-evaporated to dryness to give the crude compound B-1-2.
[0136] Step 2: Synthesis of the Compound B-1-3
[0137] Compound B-1-2 (47.9 g, 238.24 mmol, 1 eq) was dissolved in dimethylformamide (120 mL), and sodium hydride (14.29 g, 357.36 mmol, 60% purity, 1.5 eq) was added at 0° C. The mixture was stirred at 25° C. for 0.5 hours. 3-Bromopropene (57.64 g, 476.47 mmol, 41.17 mL, 2 eq) was slowly added to the reaction solution, and the mixture was reacted at 25° C. for another 2 hours. After the reaction was completed, the reaction was quenched with water (50 mL) at 0° C., and extracted with ethyl acetate (500 mL*2). The organic phase was washed with water (50 mL*2) followed by saturated brine (50 mL*2), and dried over anhydrous sodium sulfate. The crude product was purified by column chromatography to give the title compound B-1-3. The product was confirmed by LCMS, LC-MS (m/z) 263, 265 [M+Na].sup.+.
[0138] Step 3: Synthesis of the Compound B-1-5
[0139] Compound B-1-3 (18.5 g, 76.72 mmol, 1.2 eq) was dissolved in tetrahydrofuran (100 mL) at −78° C., and n-butyl lithium (2.5 M, 33.25 mL, 1.3 eq) was added under nitrogen. The mixture was reacted at −78° C. for 0.5 hours. At the same time, compound B-1-4 (17.47 g, 63.93 mmol, 1 eq) was dissolved in tetrahydrofuran (100 mL). The mixture was cooled to 0° C. and purged with nitrogen. Tert-butyl magnesium chloride (1.7 M, 41.37 mL, 1.1 eq) was then added, and the mixture was reacted at 0° C. for 0.5 hours. The solution of magnesium alkoxy was slowly added to the solution of alkyl lithium at −78° C. The reaction solution was reacted at −78° C. for 0.5 hours, and then heated to 25° C. and reacted for another 15.5 hours. After the reaction was completed, a solution of ammonium chloride (50 mL) was added to the reaction solution at 0° C. The reaction solution was diluted with ethyl acetate (200 mL), and then washed with water (50 mL*2). The organic phases were combined, washed with saturated brine (50 mL*2) to remove water, dried over anhydrous sodium sulfate, and filtered. The filtrate was rotary-evaporated to dryness. The crude product was purified by column chromatography to give the title compound B-1-5. The product was confirmed by LCMS, LC-MS (m/z) 371 [M+Na].sup.+.
[0140] Step 4: Synthesis of the Compound B-1-6
[0141] Compound B-1-5 (17.80 g, 51.09 mmol, 1 eq) was dissolved in methanol (100 mL), and the solution was cooled to 0° C. Cerium trichloride heptahydrate (22.84 g, 61.31 mmol, 5.83 mL, 1.2 eq), and then sodium borohydride (3.87 g, 102.18 mmol, 2 eq) were added. The mixture was heated to 25° C. and reacted for 16 hours. After the reaction was completed, the reaction solution was quenched with water (30 mL), and rotary-evaporated to dryness. The residue was diluted with ethyl acetate (100 mL), washed with water (50 mL*2), then washed with saturated brine (50 mL*2) to remove water, finally dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness under reduced pressure to give the title compound B-1-6. The product was confirmed by LCMS, LC-MS (m/z) 373 [M+Na].sup.+.
[0142] Step 5: Synthesis of the Compound B-1-7
[0143] Compound B-1-6 (10.22 g, 29.17 mmol, 1 eq) was dissolved in water (100 mL) and glacial acetic acid (100 mL), and the mixture was reacted at 100° C. for 16 hours. After the reaction was completed, the mixture was rotary evaporated to dryness at 60° C. under vacuum, and then striped three times with toluene to give compound B-1-7. The product was confirmed by LCMS, LC-MS (m/z) 333 [M+Na].sup.+.
[0144] Step 6: Synthesis of the Compound B-1-8
[0145] Compound B-1-7 (9.52 g, 30.68 mmol, 1 eq) and acetic anhydride (25.05 g, 245.41 mmol, 22.98 mL, 8 eq) were dissolved in pyridine (40 mL), and the solution was stirred at 25° C. for 16 hours. After the reaction was completed, the reaction solution was diluted with ethyl acetate (200 mL), and washed with 1M diluted hydrochloric acid (100 mL*4). The organic phase was washed with water (50 mL*2) and then saturated brine (50 mL*2), finally dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness under reduced pressure. The residue was purified by column chromatography to give the title compound B-1-8. The product was confirmed by LCMS, LC-MS (m/z) 501 [M+Na].sup.+.
[0146] Step 7: Synthesis of B-1-9
[0147] Compound B-1-8 (8.8 g, 18.39 mmol, 1 eq) was dissolved in 1,4-dioxane (100 mL), and thiourea (4.20 g, 55.17 mmol, 3 eq) was added. The atmosphere was replaced with nitrogen three times, and trimethylsilyl trifluoromethanesulfonate (14.31 g, 64.37 mmol, 3.5 eq) was added at 25° C. The mixture was heated to 60° C. and reacted for 2 hours, and then cooled to 25° C. Iodomethane (13.30 g, 93.70 mmol, 5.09 eq) and diisopropylethylamine (19.02 g, 147.13 mmol, 8 eq) were added successively. The mixture was reacted at 25° C. for 14 hours. After the reaction was completed, the reaction solution was diluted with water (80 mL), and extracted with ethyl acetate (80 mL*3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was rotary-evaporated to dryness under reduced pressure to give a crude product. The crude product was purified by column chromatography to give the title compound B-1-9. The product was confirmed by LCMS, LC-MS (m/z) 489 [M+Na].sup.+.
[0148] Step 8: Synthesis of B-1-10
[0149] To a reaction flask were added B-1-9 (2 g, 4.29 mmol, 1 eq), barbituric acid (1.10 g, 8.57 mmol, 2 eq), and ethanol (20 mL). The atmosphere was replaced with nitrogen three times, and then tetrakis(triphenylphosphine)palladium (495.37 mg, 428.68 μmol, 0.1 eq) was added. The mixture was reacted under nitrogen at 70° C. for 16 hours. After the reaction was completed, the reaction solution was diluted with water (20 mL), and extracted with ethyl acetate (20 mL*3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was rotary-evaporated to dryness under reduced pressure to give a crude product. The crude product was purified by column chromatography to give the title compound B-1-10. The product was confirmed by LCMS, LC-MS (m/z) 449 [M+Na].sup.+.
[0150] Step 9: Synthesis of B-1
[0151] To a reaction flask were added compound B-1-10 (1.5 g, 3.52 mmol, 1 eq), triphenylphosphine (1.38 g, 5.28 mmol, 1.5 eq), and dichloromethane (20 mL). The atmosphere was replaced with nitrogen three times, and the mixture was reacted at 25° C. for 0.5 hours. N-bromosuccinimide (938.98 mg, 5.28 mmol, 1.5 eq) was then added at 0° C., and the mixture was reacted at 25° C. for 1.5 hours. After the reaction was completed, the reaction solution was diluted with water (20 mL), and extracted with ethyl acetate (20 mL*3). The combined organic phase was dried over anhydrous sodium sulfate, and filtered. The filtrate was rotary-evaporated to dryness under reduced pressure to give a crude product. The crude product was purified by column chromatography to give the title compound B-1. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.25 (d, J=6.4 Hz, 2H), 7.18 (d, J=8.4 Hz, 1H), 5.38 (t, J=9.6 Hz, 1H), 5.25 (t, J=9.6 Hz, 1H), 5.13 (t, J=9.6 Hz, 1H), 4.56 (d, J=9.6 Hz, 1H), 4.53 (q, J=10.4 Hz, 2H), 4.43 (d, J=9.6 Hz, 1H), 2.40 (s, 3H), 2.21 (s, 3H), 2.11 (s, 3H), 2.02 (s, 3H), 1.84 (s, 3H).
[0152] Referring to the synthesis method of steps 1 to 9 in Reference example 4, each fragment in Table 1 was synthesized.
TABLE-US-00001 TABLE 1 Reference example Fragment Structure NMR 5 B-2
Reference Example 7: Fragment B-4
[0153] ##STR00048##
[0154] Route of Synthesis:
##STR00049##
[0155] Step 1: Synthesis of the Compound B-4-2
[0156] Compound B-4-1 (25 g, 133.67 mmol, 1 eq) was dissolved in tetrahydrofuran (250 mL), and sodium hydride (10.69 g, 267.33 mmol, 60% purity, 2 eq) was added at 0° C. The mixture was stirred at 25° C. for 0.5 hours. Allyl bromide (48.51 g, 401.00 mmol, 34.65 mL, 3 eq) was slowly added to the reaction solution, and the mixture was reacted for another 2 hours at 25° C. After the reaction was completed, the reaction was quenched with saturated aqueous ammonium chloride solution (20 mL) at 0° C., and extracted with ethyl acetate (250 mL*2). The crude product was purified by column chromatography to give the compound B-4-2. .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).
[0157] Step 2: Synthesis of the Compound B-4-3
[0158] Compound B-4-2 (14 g, 61.65 mmol, 1 eq) was dissolved in tetrahydrofuran (140 mL) at −78° C., and n-butyl lithium (2.5 M, 27.12 mL, 1.1 eq) was added under nitrogen. The mixture was reacted at −78° C. for 0.5 hours. At the same time, compound B-1-4 (18.53 g, 67.81 mmol, 1.1 eq) was dissolved in tetrahydrofuran (180 mL). The mixture was cooled to 0° C. and purged with nitrogen. Tert-butyl magnesium chloride (1.7 M, 47.14 mL, 1.3 eq) was then added, and the mixture was reacted at 0° C. for 0.5 hours. The solution of magnesium alkoxy was slowly added to the solution of alkyl lithium at −78° C. The reaction solution was reacted at −78° C. for 0.5 hours, and then heated to 25° C. and reacted for another 15.5 hours. After the reaction was completed, a solution of ammonium chloride (100 mL) was added to the reaction solution at 0° C. The reaction solution was diluted with ethyl acetate (200 mL), and then washed with water (50 mL*2). The combined organic phase was washed with saturated brine (50 mL*2), dried over anhydrous sodium sulfate, and filtered. The filtrate was rotary-evaporated to dryness. The crude product was purified by column chromatography to give the compound B-4-3. The product was confirmed by LCMS, LC-MS (m/z) 357 [M+Na].sup.+.
[0159] Step 3: Synthesis of the Compound B-4-4
[0160] Compound B-4-3 (13 g, 38.88 mmol, 1 eq) was dissolved in methanol (130 mL), and the solution was cooled to 0° C. Cerium trichloride heptahydrate (9.58 g, 38.88 mmol, 1 eq), and then sodium borohydride (2.94 g, 77.76 mmol, 2 eq) were added. The mixture was heated to 25° C. and reacted for 16 hours. After the reaction was completed, the reaction solution was quenched with saturated ammonium chloride aqueous solution (30 mL), and rotary-evaporated to dryness. The residue was diluted with ethyl acetate (100 mL), washed with water (50 mL*2), and then washed with saturated brine (50 mL*2) to remove water. Finally, the organic phase was dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness under reduced pressure to give the title compound B-4-4. The product was confirmed by LCMS, LC-MS (m/z) 359 [M+Na].sup.+.
[0161] Step 4: Synthesis of the Compound B-4-5
[0162] Compound B-4-4 (10.8 g, 32.11 mmol, 1 eq) was dissolved in water (50 mL) and glacial acetic acid (50 mL), and the solution was reacted at 100° C. for 16 hours. After the reaction was completed, the reaction solution was rotary evaporated to dryness under vacuum at 60° C., and then striped three times with toluene to give compound B-4-5. The product was confirmed by LCMS, LC-MS (m/z) 319 [M+Na].sup.+.
[0163] Step 5: Synthesis of the Compound B-4-6
[0164] Compound B-4-5 (9.2 g, 31.05 mmol, 1 eq) was dissolved in 1,4-dioxane (100 mL), and 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) were added. The mixture was stirred at 80° C. for 16 hours. After the reaction was completed, the 1,4-dioxane was removed by concentration under reduced pressure. The reaction solution was diluted with ethyl acetate (100 mL), and washed with 1M diluted hydrochloric acid (100 mL*4). The organic phase was washed with water (50 mL*2), and then saturated brine (50 mL*2). Finally, the organic phase was dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness under reduced pressure. The crude product was purified by column chromatography to give the compound B-4-6, The product was confirmed by LCMS, LC-MS (m/z) 487 [M+Na].sup.+.
[0165] Step 6: Synthesis of B-4-7
[0166] Compound B-4-6 (6.2 g, 13.35 mmol, 1 eq) was dissolved in 1,4-dioxane (62 mL), and thiourea (3.56 g, 46.72 mmol, 3.5 eq) was added. The atmosphere was replaced with nitrogen three times, and trimethylsilyl trifluoromethanesulfonate (11.87 g, 53.40 mmol, 4 eq) was added at 25° C. The mixture was heated to 60° C. and reacted for 1 hour, and then cooled to 25° C. Iodomethane (9.47 g, 66.74 mmol, 5 eq) and diisopropylethylamine (17.25 g, 133.49 mmol, 10 eq) were added successively. The mixture was reacted at 25° C. for 15 hours. After the reaction was completed, the reaction solution was diluted with water (60 mL), and extracted with ethyl acetate (60 mL*3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was rotary-evaporated to dryness under reduced pressure to give a crude product. The crude product was purified by column chromatography to give the title compound B-4-7. The product was confirmed by LCMS, LC-MS (m/z) 475 [M+Na].sup.+.
[0167] Step 7: Synthesis of B-4-8
[0168] To a reaction flask were added B-4-7 (4.4 g, 9.72 mmol, 1 eq), barbituric acid (2.49 g, 19.45 mmol, 2 eq), and ethanol (44 mL). The atmosphere was replaced with nitrogen three times, and tetrakis(triphenylphosphine)palladium (516.80 mg, 486.17 μmol, 0.05 eq) was added. The mixture was reacted under nitrogen at 65° C. for 16 hours. After the reaction was completed, the reaction solution was adjusted to a pH of 7-8 by addition of aqueous sodium bicarbonate solution, and then filtered on a Buchner funnel. The filtrate was collected, and extracted with ethyl acetate (40 mL*2). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was rotary-evaporated to dryness under reduced pressure to give a crude product. The crude product was purified by column chromatography to give the title compound B-4-8. The product was confirmed by LCMS, LC-MS (m/z) 435 [M+Na].sup.+.
[0169] Step 8: Synthesis of B-4
[0170] Compound B-4-8 (300 mg, 727.36 μmol, 1 eq) was dissolved in tetrahydrofuran (3 mL). Phosphorous tribromide (98.44 mg, 363.68 μmol, 34.18 μL, 0.5 eq) was added under nitrogen at 0° C. The mixture was stirred at 0° C. for 3 hours. After the reaction was completed, the reaction solution was washed twice with saturated aqueous potassium carbonate solution. The organic phase was collected, and the aqueous phase was extracted with ethyl acetate (3 mL*2). The organic phases were combined and concentrated to dryness under reduced pressure with a water pump. The crude product was purified by column chromatography to give the title compound B-4. .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 Example 8: Fragment B-5
[0171] ##STR00050##
[0172] Route of Synthesis:
##STR00051##
[0173] Step 1: Synthesis of the Compound B-5-2
[0174] To a reaction flask were added compound B-5-1 (11.00 g, 64.48 mmol, 1 eq) and N-bromosuccinimide (14.30 g, 80.34 mmol, 1.25 eq). Sulfuric acid (202.40 g, 2.06 mol, 110.00 mL, 32.00 eq) was added at 0° C. The mixture was stirred for 1 hour. The reaction solution was added dropwise to ice water (500 mL), and the aqueous phase was extracted three times with ethyl acetate (200 mL*3). The organic phases were combined, and concentrated to give compound B-5-2, which was directly used in the next reaction.
[0175] Step 2: Synthesis of the Compound B-5-3
[0176] To a reaction flask were added compound B-5-2 (19.00 g, 76.16 mmol, 1 eq) and anhydrous tetrahydrofuran (50.0 mL). A solution of borane in tetrahydrofuran (1 M, 160.00 mL, 2.10 eq) was added dropwise, and the reaction system was stirred at 20° C. for 16 hours. Methanol (100 mL) was added dropwise to the reaction solution at 20° C., while bubbling with nitrogen. After quenching the reaction, the mixture solution was refluxed at 70° C. for 1 hour, and concentrated to dryness with a water pump at 45° C. to give a crude product. Water (200 mL) was added, and the mixture was extracted with ethyl acetate (200 mL*3). The organic phases were combined, washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to give compound B-5-3. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 7.66 (s, 1H), 7.24 (s, 1H), 4.73 (d, J=6.02 Hz, 2H), 2.38 (s, 3H), 1.88 (t, J=6.27 Hz, 1H).
[0177] Step 3: Synthesis of the Compound B-5-4
[0178] To a reaction flask were added compound B-5-3 (15.60 g, 66.24 mmol, 1 eq) and anhydrous N,N-dimethylformamide (100 mL). Sodium hydride (6.24 g, 156.01 mmol, 60% purity, 2.36 eq) was added at 0° C. The reaction system was stirred at 0° C. for 0.5 hours, and 3-bromopropene (24.04 g, 198.72 mmol, 3 eq) was added. The reaction system was stirred at room temperature (20° C.) for 15.5 hours. Water (200 mL) was added dropwise to the reaction solution to quench the reaction, and the mixture was extracted with ethyl acetate (200 mL*3). The organic phases were combined, washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to give a crude product. The crude product was purified by column chromatography to give the title compound B-5-4, .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 7.66 (s, 1H), 7.23 (s, 1H), 5.91-6.05 (m, 1H), 5.31-5.42 (m, 1H), 5.19-5.29 (m, 1H), 4.55 (s, 2H), 4.10 (dt, J=5.58, 1.35 Hz, 2H), 2.37 (s, 3H).
[0179] Step 4: Synthesis of the Compound B-5-5
[0180] To a reaction flask were added compound B-5-4 (15.30 g, 55.52 mmol, 1 eq) and anhydrous tetrahydrofuran (200 mL). N-butyl lithium (2.5 M, 27 mL, 1.22 eq) was added under nitrogen at −70° C. The reaction system was stirred at −70° C. for 0.5 hours. Compound B-1-4 (15.30 g, 55.99 mmol, 1.01 eq) and anhydrous tetrahydrofuran (200 mL) were added to a reaction flask. Tert-butyl magnesium chloride (1.7 M, 54 mL, 1.65 eq) was added under nitrogen at 0° C., and the reaction system was stirred at 0° C. for 0.5 hours. The solution of magnesium alkoxy was slowly added to the solution of alkyl lithium, and the reaction system was stirred at −70° C. for 0.5 hours. The reaction system was slowly warmed to room temperature (20° C.) and stirred for 1 hour. Saturated ammonium chloride (200 mL) was added dropwise to the reaction solution to quench the reaction, and the reaction solution was concentrated to remove organic solvent. Citric acid was added to adjust the solution to clear. The solution was extracted with ethyl acetate (200 mL*3). The organic phases were combined, washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to give a crude product. The crude product was purified by column chromatography to give the title compound B-5-5. The product was confirmed by LCMS, LC-MS (m/z) 405 [M+Na].sup.+.
[0181] Step 5: Synthesis of the Compound B-5-6
[0182] Compound B-5-5 (10.60 g, 27.69 mmol, 1 eq) was dissolved in anhydrous methanol (200 mL), and the solution was cooled to 0° C. Cerium trichloride heptahydrate (12.38 g, 33.23 mmol, 1.20 eq), and then sodium borohydride (2.1 g, 55.38 mmol, 2 eq) were added. The mixture was heated to 25° C. and reacted for 16 hours. After the reaction was completed, the reaction solution was quenched with saturated aqueous ammonium chloride solution (30 mL), and rotary-evaporated to dryness. The residue was diluted with ethyl acetate (100 mL), washed with water (50 mL*2), and then washed with saturated brine (50 mL*2) to remove water. Finally, the organic phase was dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness under reduced pressure to give the title compound B-5-6, which was used directly in the next reaction.
[0183] Step 6: Synthesis of the Compound B-5-7
[0184] To a reaction flask were added compound B-5-6 (9.85 g, 19.00 mmol, 1 eq), acetic acid (60 mL), and water (60 mL), and the mixture was stirred at 100° C. for 8 hours. The reaction solution was concentrated to give a crude product, and the crude product was then striped with toluene (100 mL) to dryness. This process was repeated twice to give the title compound B-5-7, which was directly used in the next reaction.
[0185] Step 7: Synthesis of the Compound B-5-8
[0186] To a reaction flask were added compound B-5-7 (10.00 g, 29.00 mmol, 1 eq), triethylamine (16.72 g, 165.24 mmol, 23.0 mL, 5.70 eq), acetic anhydride (21.80 g, 213.54 mmol, 20 mL, 7.36 eq), 4-dimethylaminopyridine (40 mg, 327.42 μmol, 1.13e-2 eq), and acetonitrile (100 mL). The mixture was stirred at 25° C. for 2 hours. The reaction solution was concentrated to give a crude product. Ethyl acetate (200 mL) was added, and the mixture was extracted with 50% saturated aqueous sodium hydrogen sulfate solution (200 mL*2). The aqueous phases were combined, and extracted with ethyl acetate (200 mL*2). The organic phases were combined, washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to give a crude product. The crude product was purified by column chromatography to give the title compound B-5-8. The product was confirmed by LCMS, LC-MS (m/z) 535 [M+Na].sup.+.
[0187] Step 8: Synthesis of the Compound B-5-9
[0188] To a reaction flask were added compound B-5-8 (3.60 g, 7.02 mmol, 1 eq), thiourea (1.08 g, 14.19 mmol, 2.02 eq) and anhydrous dioxane (40 mL). The reaction system was stirred at 80° C. for 2 hours. Subsequently, trimethylsilyl trifluoromethanesulfonate (3.90 g, 17.55 mmol, 3.17 mL, 2.50 eq) was added, and the mixture was stirred at 80° C. for another 1 hour. The reaction solution was cooled to room temperature. Iodomethane (3.06 g, 21.56 mmol, 1.34 mL, 3.07 eq) and diisopropylethylamine (4.54 g, 35.09 mmol, 6.11 mL, 5 eq) were added. The reaction system was stirred at 25° C. for 15 hours. Water (50 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (50 mL*2). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to give a crude product. The crude product was purified by column chromatography to give the title compound B-5-9. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 7.48 (s, 1H), 7.15 (s, 1H), 5.98 (ddt, J=17.32, 10.60, 5.36, 5.36 Hz, 1H), 5.22-5.37 (m, 5H), 4.70 (d, J=10.04 Hz, 1H), 4.56 (d, J=2.76 Hz, 2H), 4.54 (d, J=9.79 Hz, 1H), 4.07 (ddt, J=5.49, 2.48, 1.41, 1.41 Hz, 2H), 2.38 (s, 3H), 2.19 (s, 3H), 2.11 (s, 3H), 2.03 (s, 3H), 1.81 (s, 3H).
[0189] Step 9: Synthesis of the Compound B-5-10
[0190] To a reaction flask were added compound B-5-9 (2.70 g, 5.39 mmol, 1 eq), barbituric acid (1.38 g, 10.78 mmol, 2.0 eq), anhydrous ethanol (20 mL), and anhydrous dichloromethane (10 mL). Tetrakis(triphenylphosphine)palladium (622 mg, 0.539 mmol, 0.1 eq) was added under a nitrogen atmosphere, and the reaction system was stirred at 40° C. for 12 hours. The reaction solution was filtered, and the filtrate was concentrated to give a crude product. Water (500 mL) was added, and the mixture was extracted with ethyl acetate (500 mL*3). The organic phases were combined, washed with saturated brine (500 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to give a crude product. The crude product was purified by column chromatography to give the title compound B-5-10. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 7.51 (s, 1H), 7.16 (s, 1H), 5.35-5.41 (m, 1H), 5.26 (dt, J=15.00, 9.57 Hz, 2H), 4.68-4.77 (m, 3H), 4.55 (d, J=10.04 Hz, 1H), 2.38 (s, 3H), 2.17-2.23 (m, 3H), 2.11 (s, 3H), 2.02 (s, 3H), 1.81 (s, 3H).
[0191] Step 10: Synthesis of the Compound B-5
[0192] To a reaction flask were added compound B-5-10 (0.60 g, 468.88 μmol, 1 eq) and anhydrous tetrahydrofuran (10 mL). Phosphorous tribromide (288.00 mg, 1.06 mmol, 0.10 mL, 2.27 eq) was added dropwise under nitrogen at 0° C. The mixture was slowly warmed to 25° C. and stirred for 12 hours. Water (20 mL) was added dropwise to the reaction solution to quench the reaction, and the mixture was concentrated to remove the organic solvent. The remaining aqueous phase was extracted with ethyl acetate (20 mL*3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to give a crude product. The crude product was purified by column chromatography to give the title compound B-5, .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 1.83 (s, 3H), 2.03 (s, 3H), 2.11 (s, 3H), 2.20 (s, 3H), 2.40 (s, 3H), 4.50-4.59 (m, 3H), 4.66 (d, J=10.04 Hz, 1H), 5.20-5.40 (m, 3H), 7.20 (s, 1H), 7.41 (s, 1H).
Reference Example 9: Fragment B-6
[0193] ##STR00052##
[0194] Route of Synthesis:
##STR00053##
[0195] Step 1: Synthesis of the Compound B-6-2
[0196] To a reaction flask were added successively concentrated sulfuric acid (70 mL) and compound B-6-1 (20 g, 132.31 mmol, 1.89 mL, 1 eq), and the mixture was stirred until dissolved. N-bromosuccinimide (28.26 g, 158.77 mmol, 1.2 eq) was added at 0° C. in portions. After the addition was completed, the mixture was stirred at 30° C. until the N-bromosuccinimide was completely dissolved. The solution was further reacted at 30° C. for 0.5 hours. The reaction solution was slowly added to stirred ice water (1 L) to quench the reaction, and then the mixture was stirred at 0° C. for 0.5 hours. After filtration, the filter cake was washed three times with water (100 mL), collected, and then dried to give compound B-6-2, which was directly used in the next reaction.
[0197] Step 2: Synthesis of the Compound B-6-3
[0198] To a reaction flask were added successively water (250 mL), acetonitrile (125 mL), compound B-6-2 (25 g, 97.80 mmol, 1 eq), and concentrated hydrochloric acid (38.55 g, 391.21 mmol, 37.79 mL, 37% purity, 4 eq). A suspension was obtained after stirring. Sodium nitrite (7.09 g, 102.69 mmol, 1.05 eq) was added to the reaction mixture at 0° C., and the mixture was stirred for 0.5 hours. The reaction mixture was added dropwise to a solution of cuprous chloride (10.17 g, 102.69 mmol, 2.46 mL, 1.05 eq), concentrated hydrochloric acid (38.55 g, 391.21 mmol, 37.79 mL, 37% purity, 4 eq) and water (250 mL) at 0° C. After the addition was completed, the reaction solution was reacted at 70° C. for 3 hours. The reaction solution was concentrated to remove acetonitrile. The residue was cooled to room temperature, and then filtered. The filter cake was washed three times with water (100 mL). The filter cake was collected and dissolved in ethyl acetate (200 mL). The layers were separated. The organic phase was dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give compound B-6-3. The product was confirmed by LCMS, LC-MS (m/z) 248.9, 250.8 [M+H].sup.+.
[0199] Step 3: Synthesis of the Compound B-6-4
[0200] To a reaction flask were added compound B-6-3 (18 g, 64.93 mmol, 1 eq) and tetrahydrofuran (300 mL). Borane dimethyl sulfide (10 M, 19.48 mL, 3 eq) was added dropwise under a nitrogen atmosphere. After the addition was completed, the mixture was reacted with stirring at 30° C. for 16 hours. After the reaction was completed, methanol (100 mL) was added dropwise to the reaction solution to quench the reaction. The reaction solution was refluxed at 70° C. for 1 hour, and then concentrated under reduced pressure to give compound B-6-4, which was directly used in the next reaction.
[0201] Step 4: Synthesis of the Compound B-6-5
[0202] To a reaction flask were added compound B-6-4 (16.5 g, 70.06 mmol, 1 eq) and DMF (180 mL). The mixture was cooled to 0° C., and sodium hydride (5.60 g, 140.12 mmol, 60% purity, 2 eq) was added. After stirring at 0° C. for 0.5 hours, allyl bromide (25.43 g, 210.19 mmol, 3 eq) was added to the reaction solution. The mixture was reacted with stirring at 30° C. for 10 hours. The reaction solution was quenched by adding water (300 mL), and extracted with ethyl acetate (100 mL*3). The organic phases were combined, washed with saturated brine (100 mL), and then concentrated to give a crude product. The crude product was purified by column chromatography to give compound B-6-5. The product was confirmed by LCMS, LC-MS (m/z) 275, 277 [M+H].sup.+.
[0203] Step 5: Synthesis of the Compound B-6-6
[0204] To a reaction flask were added compound B-6-5 (16 g, 52.26 mmol, 1 eq) and tetrahydrofuran (160 mL). The mixture was cooled to −78° C., and n-butyl lithium (2.5 M, 27.17 mL, 1.3 eq) was added dropwise. The mixture was stirred at −78° C. for 0.5 hours. Compound B-1-4 (14.28 g, 52.26 mmol, 1 eq) and tetrahydrofuran (160 mL) were added to a reaction flask, and the mixture was cooled to 0° C. Tert-butyl magnesium chloride (1.7 M, 49.18 mL, 1.6 eq) was added dropwise, and the mixture was stirred at 0-5° C. for 0.5 hours. The solution of magnesium alkoxy was slowly added dropwise to the solution of alkyl lithium at −78° C. The reaction solution was stirred at −78° C. for 0.5 hours, and then reacted with stirring at 25° C. for another 2 hours. A mixed solution of saturated ammonium chloride and saturated brine (volume ratio of 1:1, 150 mL in total) was added dropwise to the reaction solution at 0° C. to quench the reaction. The mixture was extracted with ethyl acetate (100 mL*3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography to give compound B-6-6. The product was confirmed by LCMS, LC-MS (m/z) 383 [M+H].sup.+.
[0205] Step 6: Synthesis of the Compound B-6-7
[0206] Compound B-6-6 (7.40 g, 17.40 mmol, 1 eq) was dissolved in anhydrous methanol (100 mL), and the solution was cooled to 0° C. Cerium trichloride heptahydrate (7.77 g, 20.88 mmol, 1.20 eq), and then sodium borohydride (1.32 g, 34.8 mmol, 2 eq) were added. The mixture was heated to 25° C. and reacted for 16 hours. After the reaction was completed, the reaction solution was quenched with saturated aqueous ammonium chloride solution (30 mL), and rotary-evaporated to dryness. The residue was diluted with ethyl acetate (100 mL), washed with water (50 mL*2), and then washed with saturated brine (50 mL*2) to remove water. Finally, the organic phase was dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness under reduced pressure to give the title compound B-6-7, which was directly used in the next reaction.
[0207] Step 7: Synthesis of the Compound B-6-8
[0208] To a 250 mL single-necked flask were added compound B-6-7 (6.5 g, 16.89 mmol, 1 eq), acetic acid (40 mL) and water (40 mL), and the mixture was reacted at 100° C. for 16 hours. The reaction solution was concentrated to dryness under reduced pressure, and the residue was azeotropically striped with toluene (50 mL*2) to give the compound B-6-8, which was directly used in the next reaction.
[0209] Step 8: Synthesis of the Compound B-6-9
[0210] To a reaction flask were added compound B-6-8 (6 g, 17.40 mmol, 1 eq), triethylamine (11.62 g, 114.85 mmol, 15.99 mL, 6.6 eq), 4-dimethylaminopyridine (212.60 mg, 1.74 mmol, 0.1 eq) and acetonitrile (40 mL). Acetic anhydride (11.73 g, 114.85 mmol, 10.76 mL, 6.6 eq) was then added. The mixture was reacted at 25° C. for 16 hours. The reaction solution was concentrated to remove acetonitrile, and 0.5 N hydrochloric acid (40 mL) was then added. The mixture was extracted with ethyl acetate (50 mL*3). The organic phase was washed with saturated brine (50 mL), and concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography to give compound B-6-9.
[0211] Step 9: Synthesis of the Compound B-6-10
[0212] To a reaction flask were added compound B-6-9 (2.5 g, 4.39 mmol, 1 eq), thiourea (1.17 g, 15.35 mmol, 3.5 eq) and dioxane (30 mL). The atmosphere was replaced with nitrogen, and trimethylsilyl trifluoromethanesulfonate (3.90 g, 17.55 mmol, 3.17 mL, 4 eq) was then added. The mixture solution was reacted at 80° C. for 0.5 hours, and then cooled to 0-5° C. Iodomethane (1.87 g, 13.16 mmol, 819.23 μL, 3 eq) and diisopropylethylamine (2.83 g, 21.93 mmol, 3.82 mL, 5 eq) were added successively, and the mixture was reacted at 25° C. for another 10 hours. Water (30 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (30 mL*3). The organic phases were combined, washed with saturated brine (30 mL), and concentrated to give a crude product. The crude product was purified by column chromatography to give the compound B-6-10. The product was confirmed by LCMS, LC-MS (m/z) 523 [M+Na].sup.+.
[0213] Step 10: Synthesis of the Compound B-6-11
[0214] To a reaction flask were added compound B-6-10 (1.257 g, 2.515 mmol, 1 eq), barbituric acid (644.30 mg, 5.03 mmol, 2 eq), tetrakis(triphenylphosphine)palladium (290.63 mg, 251.50 μmol, 0.1 eq) and EtOH (40 mL). The atmosphere was replaced with nitrogen, and then the mixture was reacted with stirring at 40° C. for 16 hours. Saturated aqueous sodium carbonate solution (50 mL) and ethyl acetate (50 mL) were added to the reaction solution, and a large amount of solid was precipitated out. The mixture was filtered. The layers were separated, and the aqueous phase 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. The filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography to give compound B-6-11. The product was confirmed by LCMS, LC-MS (m/z) 483 [M+Na].sup.+.
[0215] Step 11: Synthesis of B-6
[0216] To a reaction flask were added compound B-6-11 (200 mg, 418.07 μmol, 1 eq) and tetrahydrofuran (5 mL). Phosphorous tribromide (113.17 mg, 418.07 μmol, 39.29 μL, 1 eq) was added at 0° C. The reaction solution was reacted at −10° C. for 0.5 hours, and then poured into an aqueous potassium carbonate solution (20 mL, 1M) at 0° C. The mixture was extracted with ethyl acetate (20 mL*3). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to give a crude product. The crude product was purified by column chromatography to give the title compound B-6. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 1.86 (s, 3H), 2.01 (s, 3H), 2.10 (s, 3H), 2.21 (s, 3H), 2.40 (s, 3H), 4.40 (d, J=10.04 Hz, 1H), 4.51-4.57 (m, 2H), 4.60-4.65 (m, 1H), 5.06 (t, J=9.66 Hz, 1H), 5.17-5.25 (m, 1H), 5.31-5.38 (m, 1H), 7.22 (s, 1H), 7.24 (s, 1H).
Example 1: WXD001 or WXD002
[0217] ##STR00054##
[0218] Route of Synthesis:
##STR00055## ##STR00056##
[0219] Step 1: Synthesis of WXD001-1
[0220] To a reaction flask were added B-2 (0.35 g, 695.27 μmol, 1 eq), A-1 (626.19 mg, 1.04 mmol, 1.49 eq), Na.sub.2CO.sub.3 (147.38 mg, 1.39 mmol, 2 eq), toluene (5 mL), EtOH (1 mL) and H.sub.2O (1 mL). The atmosphere was replaced with nitrogen, and then Pd(PPh.sub.3).sub.4 (160.68 mg, 139.05 μmol, 0.2 eq) was added. The mixture was stirred at 50° C. for 3 hours. After the reaction was completed, the reaction solution was diluted with 20 mL of water, and extracted with ethyl acetate (20 mL*3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was rotary-evaporated to dryness to give a crude product. The crude product was purified by column chromatography to give WXD001-1.
[0221] Step 2: Synthesis of WXD001-2 or WXD001-3
[0222] To a reaction flask were added WXD001-1 (229 mg, 254.16 μmol, 1 eq), LiOH.H.sub.2O (213 mg, 5.08 mmol, 20 eq), THF (0.5 mL), MeOH (1 mL), and H.sub.2O (1 mL), and the mixture was reacted at 25° C. for 1 hour. After the reaction was completed, 10 mL of water was added to the reaction solution. The mixture was extracted with ethyl acetate (10 mL*3). The organic phase was dried over anhydrous sodium sulfate, and filtered. The filtrate was rotary-evaporated to dryness to give a crude product. The crude product was chirally resolved by SFC (chromatographic column: REGIS (s,s) WHELK-O1 (250 mm×30 mm, 5 μm); mobile phase: [0.1% NH.sub.3H.sub.2O-isopropanol]; B (isopropanol) %: 40%-40%, min) to give WXD001-2 (t=4.167 min) or WXD001-3 (t=2.909 min).
[0223] Step 3: Synthesis of WXD001 or WXD002
[0224] To a reaction flask were added WXD001-2 (60 mg, 77.43 μmol, 1 eq), EtOAc (3 mL), and HCl/EtOAc (4 M, 1 mL, 51.66 eq), and the mixture was reacted at 25° C. for 1 hour. After the reaction was completed, the reaction solution was rotary-evaporated to dryness under reduced pressure to give a crude product. The crude product was subjected to preparative high performance liquid chromatography (chromatographic column: Boston Prime C18 150×30 mm, 5 μm; mobile phase: [water (0.05% ammonium hydroxide v/v)-acetonitrile]; B (acetonitrile) %: 47%-77%, 8 min) to give WXD001. Corresponding SFC (method: chromatographic column: Chiralpak AD-3 100×4.6 mm I.D., 3 μm; mobile phase: CO.sub.2-40% methanol (0.05% DEA); flow rate: 2.8 mL/min; column temperature: 40° C.), retention time t=4.404 min.
[0225] To a reaction flask were added WXD001-3 (60 mg, 77.43 μmol, 1 eq), EtOAc (3 mL), and HCl/EtOAc (4 M, 1 mL, 51.66 eq), and the mixture was reacted at 25° C. for 1 hour. After the reaction was completed, the reaction solution was rotary-evaporated to dryness under reduced pressure to give a crude product. The crude product was subjected to preparative high performance liquid chromatography (chromatographic column: Boston Prime C18 150×30 mm 5 μm; mobile phase: [water (0.05% ammonium hydroxide v/v)-acetonitrile]; B (acetonitrile) %: 47%-77%, 8 min) to give WXD002. Corresponding SFC (method: chromatographic column: Chiralpak AD-3 100×4.6 mm I.D., 3 μm, mobile phase: CO.sub.2-40% methanol (0.05% DEA); flow rate: 2.8 mL/min; column temperature: 40° C.), retention time t=5.921 min.
Example 2: WXD003
[0226] ##STR00057##
[0227] Route of Synthesis:
##STR00058##
[0228] Step 1: Synthesis of WXD003-1
[0229] To a reaction flask were firstly added A-2 (319.44 mg, 516.49 μmol, 1.3 eq), B-2 (200 mg, 397.30 μmol, 1 eq), Na.sub.2CO.sub.3 (65.95 mg, 794.59 μmol, 2 eq), and Pd(PPh.sub.3).sub.4 (91.82 mg, 79.46 μmol, 0.2 eq). The atmosphere was replaced with nitrogen three times, and then toluene (4 mL), EtOH (1 mL), and H.sub.2O (1 mL) were added successively. The mixture was heated to 50° C. and reacted for 16 hours. After the reaction was completed, the reaction solution was rotary-evaporated to dryness. The residue was then diluted with water (30 mL), and extracted with ethyl acetate (100 mL*3). The organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was rotary-evaporated to dryness. The residue was purified by preparative chromatography plate to give WXD003-1.
[0230] Step 2: Synthesis of WXD003-2
[0231] WXD003-1 (320 mg, 349.72 μmol, 1 eq) was dissolved in a mixed solvent of MeOH (2 mL), THF (1 mL), and H.sub.2O (2 mL), and LiOH.H.sub.2O (293.48 mg, 6.99 mmol, 20 eq) was added. The mixture was stirred at 25° C. for 1 hour. After the reaction was completed, ethyl acetate (30 mL) was added to the reaction solution. The layers were separated. The organic phase was collected, dried over anhydrous sodium sulfate, and filtered. The filtrate was rotary-evaporated to dryness to give a crude product of WXD003-2, which was directly used in the next reaction.
[0232] Step 3: Synthesis of WXD003
[0233] WXD003-2 (270 mg, 342.24 μmol, 1 eq) was dissolved in ethyl acetate (5 mL), and HCl/EtOAc (4 M, 5 mL, 58.44 eq) was added. The mixture was stirred at 25° C. for 1 hour. After the reaction was completed, the reaction solution was directly rotary-evaporated to dryness. The residue was purified by preparative high performance liquid chromatography (chromatographic column: Xtimate C18 150×25 mm×5 μm; mobile phase: [water (0.225% formic acid)-acetonitrile]; B (acetonitrile) %: 25%-55%, 7 min) to give the product WXD003.
[0234] Referring to the synthesis method of steps 1 to 3 in Example 2, compound WXD004 in Table 2 below was synthesized using fragment B-1 in place of B-2, compound WXD005 in Table 2 below was synthesized using A-3 in place of A-2, compound WXD006 in Table 2 below was synthesized using fragment B-3 in place of B-2, compound WXD007 in Table 2 below was synthesized using fragment B-4 in place of B-2, compound WXD008 in Table 2 below was synthesized using fragment B-5 in place of B-2, and compound WXD009 in Table 2 below was synthesized using fragment B-6 in place of B-2.
TABLE-US-00002 TABLE 2 Example Fragment A Fragment B Compound Structure 3
[0235] The data of .sup.1H NMR spectrum and mass spectrum of each example were shown in Table 3.
TABLE-US-00003 TABLE 3 Example Compound NMR MS m/z 1 WXD001 .sup.1H NMR (400 MHz, CD.sub.3OD) δ ppm 7.04-7.29 (m, 7 675 H), 6.77-6.85 (m, 2 H), 4.94-5.05 (m, 1 H), 4.39 (d, [M + H].sup.+ J = 9.6 Hz, 1 H), 4.08-4.17 (m, 1 H), 3.97 (d, J = 4.0 Hz, 2 H), 3.35-3.77 (m, 8 H), 2.65-2.83 (m, 2 H), 2.60 (qd, J = 7.2, 2.4 Hz, 2 H), 2.20-2.55 (m, 3 H), 2.06-2.14 (m, 4 H), 1.08 (td, J = 7.6, 2.0 Hz, 3 H). WXD002 .sup.1H NMR (400 MHz, CD.sub.3OD) δ ppm 7.03-7.30 (m, 7 675 H), 6.81 (dd, J = 8.8, 4.0 Hz, 2 H), 4.95-5.05 (m, 1 [M + H].sup.+ H), 4.39 (dd, J = 9.6, 2.0 Hz, 1 H), 4.13 (dd, J = 9.2, 2.0 Hz, 1 H), 3.92-4.02 (m, 2 H), 3.34-3.74 (m, 8 H), 2.68-2.83 (m, 2 H), 2.60 (qd, J = 7.2, 3.2 Hz, 2 H), 2.29-2.52 (m, 2 H), 2.02-2.27 (m, 5 H), 1.09 (td, J = 7.2, 3.6 Hz, 3 H). 2 WXD003 .sup.1H NMR (400 MHz, CD.sub.3OD) δ ppm 1.09 (t, J = 7.53 689 Hz, 3 H), 1.62-1.83 (m, 2 H), 1.85-2.04 (m, 2 H), [M + H].sup.+ 2.14 (s, 3 H), 2.54-2.68 (m, 3 H), 2.71-2.83 (m, 1 H), 2.99 (d, J = 7.28 Hz, 2 H), 3.34-3.49 (m, 4 H), 3.55-3.89 (m, 4 H), 3.92-4.02 (m, 2 H), 4.13 (d, J = 9.03 Hz, 1 H), 4.39 (d, J = 9.54 Hz, 1 H), 4.54- 4.65 (m, 1 H), 6.86 (d, J = 8.53 Hz, 2 H), 7.06 (d, J = 8.53 Hz, 2 H), 7.13-7.38 (m, 5 H), 8.52 (s, 1 H). 3 WXD004 .sup.1H NMR (400 MHz, CD.sub.3OD) δ ppm 1.60-1.82 (m, 2 675 H), 1.84-2.06 (m, 2 H), 2.14 (s, 3 H), 2.21 (s, 3 H), [M + H].sup.+ 2.50-2.65 (m, 1 H), 2.69-2.80 (m, 1 H), 2.95 (br d, J = 7.28 Hz, 2 H), 3.34-3.49 (m, 4 H), 3.50-3.86 (m, 4 H), 3.87-3.97 (m, 2 H), 4.13 (d, J = 9.03 Hz, 1 H), 4.39 (d, J = 9.54 Hz, 1 H), 4.57 (br d, J = 3.51 Hz, 1 H), 6.86 (d, J = 8.53 Hz, 2 H), 7.06 (d, J = 8.53 Hz, 2 H), 7.11-7.26 (m, 4 H), 7.27-7.37 (m, 1 H), 8.54 (s, 1 H). 4 WXD005 .sup.1H NMR (400 MHz, CD.sub.3OD) δ ppm 8.49 (br s, 2 H), 703 7.31-7.43 (m, 1 H), 7.12-7.30 (m, 4 H), 7.05 (d, [M + H].sup.+ J = 8.28 Hz, 2 H), 6.85 (br d, J = 7.03 Hz, 2 H), 4.49- 4.63 (m, 1 H), 4.39 (d, J = 9.54 Hz, 1 H), 4.13 (d, J = 9.29 Hz, 1 H), 3.96 (s, 2 H), 3.56-3.89 (m, 4 H), 3.34-3.54 (m, 5 H), 3.20 (br dd, J = 13.80, 5.27 Hz, 1 H), 2.98-3.12 (m, 1 H), 2.76 (s, 5 H), 2.60 (q, J = 7.61 Hz, 2 H), 2.14 (s, 3 H), 1.82-2.00 (m, 2 H), 1.59-1.80 (m, 2 H), 1.08 (t, J = 7.53 Hz, 3 H). 5 WXD006 .sup.1H NMR (400 MHz, CD.sub.3OD) δ ppm 1.75 (br d, 679 J = 14.05 Hz, 2 H), 1.93 (br d, J = 15.56 Hz, 2 H), [M + H].sup.+ 2.15 (s, 3 H), 2.40-2.64 (m, 2 H), 2.70-2.91 (m, 2 H), 3.34-3.52 (m, 6 H), 3.66-3.84 (m, 2 H), 3.93-3.96 (m, 2 H), 4.15 (d, J = 9.54 Hz, 1 H), 4.41 (d, J = 9.54 Hz, 1 H), 4.61 (br d, J = 11.80 Hz, 1 H), 6.89 (d, J = 8.78 Hz, 2 H), 7.05 (t, J = 9.03 Hz, 1 H), 7.12- 7.20 (m, 3 H), 7.23-7.33 (m, 3 H). 6 WXD007 .sup.1H NMR (400 MHz, CD.sub.3OD) δ ppm 1.72 (br s, 2 H), 661 1.93 (br d, J = 9.0 Hz, 2 H), 2.14 (s, 3 H), 2.59-2.84 [M + H].sup.+ (m, 2 H), 3.01 (br d, J = 6.5 Hz, 2 H), 3.35-3.50 (m, 4 H), 3.52-3.70 (m, 2 H), 3.72-3.84 (m, 2 H), 3.91 (s, 2 H), 4.14 (d, J = 9.3 Hz, 1 H), 4.40 (d, J = 9.8 Hz, 1 H), 4.58 (br s, 1 H), 6.87 (br d, J = 8.3 Hz, 2 H), 7.13 (br d, J = 8.3 Hz, 3 H), 7.19-7.28 (m, 4 H), 7.29- 7.38 (m, 1 H), 8.52 (br s, 1 H). 7 WXD008 .sup.1H NMR (400 MHz, CD.sub.3OD) δ ppm 1.64-1.81 (m, 709 2 H), 1.93 (br d, J = 8.78 Hz, 2 H), 2.11 (s, 3 H), 2.36 [M + H].sup.+ (s, 3 H), 2.54-2.66 (m, 1 H), 2.71-2.82 (m, 1 H), 2.99 (d, J = 7.28 Hz, 2 H), 3.35 (br d, J = 9.54 Hz, 2 H), 3.38-3.45 (m, 1 H), 3.45-3.51 (m, 2 H), 3.51-3.63 (m, 1 H), 3.63-3.86 (m, 3 H), 4.00 (s, 2 H), 4.41 (d, J = 9.54 Hz, 1 H), 4.45 (d, J = 9.03 Hz, 1 H), 4.58 (br d, J = 2.76 Hz, 1 H), 6.87 (d, J = 8.53 Hz, 2 H), 7.12 (d, J = 8.53 Hz, 2 H), 7.17-7.25 (m, 2 H), 7.26 (s, 1 H), 7.28-7.37 (m, 1 H), 8.51 (br s, 1 H). 8 WXD009 .sup.1H NMR (400 MHz, CD.sub.3OD) δ ppm 1.64-1.81 (m, 709 2H), 1.86-2.02 (m, 2H), 2.15 (s, 3H), 2.40 (s, 3H), [M + H].sup.+ 2.42-2.60 (m, 2H), 2.70-2.88 (m, 2H), 3.34-3.41 (m, 2H), 3.42-3.62 (m, 4H), 3.66-3.88 (m, 2H), 4.06 (d, J = 3.01 Hz, 2H), 4.08-4.15 (m, 1H), 4.40 (d, J = 9.54 Hz, 1H), 4.58 (br dd, J = 6.90, 3.39 Hz, 1H), 6.88 (d, J = 8.53 Hz, 2H), 7.10-7.20 (m, 4H), 7.22 (s, 1H), 7.29 (ddd, J = 10.85, 8.97, 7.03 Hz, 1H).
Assay Example 1. In Vitro Cell Activity Assay
Biological Activity Assay 1: SGLT1 Glucose Transport Assay
[0236] 1. Purpose of the Assay:
[0237] The effect of the compounds on the glucose transport activity of the SGLT1 transporter was detected by measuring the amount of [.sup.14C]-labeled glucose entering cells highly expressing Human-SGLT1.
[0238] 2. Method of the Assay
[0239] 2.1. Cell Preparation
[0240] The cells stably expressing Human-SGLT1 used in the assay were constructed by Shanghai WuXi AppTec. The SGLT1 cells were plated on a Cytostar-T (PerkinElmer) 96-well cell culture plate and cultured at 5% CO.sub.2, 37° C. overnight.
[0241] 2.2. SGLT1 Glucose Transport Assay [0242] 1) Assay buffer: 10 mM HEPES buffer (Sigma), 1.2 mM MgCl.sub.2, 4.7 mM KCl, 2.2 mM CaCl.sub.2) and 120 mM NaCl. [0243] 2) The compound with a starting concentration of 1 mM was serially diluted 5-fold with 100% DMSO to 8 concentrations. [0244] 3) 3 μM [.sup.14C]Methyl α-D-glucopyranosid (labeled methyl α-D-glucopyranoside) was prepared with the assay buffer. [0245] 4) The cells were treated with 49 μL of the assay buffer, 1 μL of the serially diluted compound, and 50 μL of the 3 μM [.sup.14C] isotope-labeled sugar solution at 37° C. for 2 hours. [0246] 5) An isotope detector (Micro beta Reader) was used to read. [0247] 6) The data were processed by GraphPad Prism 5.0 software with the calculation formula: log(inhibitor) vs. response—Variable slope to give the IC.sub.50 value of the test compound.
Biological Activity Assay 2: SGLT2 Glucose Transport Assay
[0248] 1. Purpose of the Assay:
[0249] The effect of the compound on the glucose transport activity of the SGLT2 transporter was detected by measuring the amount of [.sup.14C]-labeled glucose entering cells highly expressing Human-SGLT2.
[0250] 2. Method of the Assay
[0251] 2.1. Cell Preparation
[0252] The cells stably expressing Human-SGLT2 used in the assay were constructed by Shanghai WuXi AppTec. The SGLT2 cells were plated on a 96-well cell culture plate (Greiner) and cultured at 5% CO.sub.2, 37° C. overnight.
[0253] 2.2. SGLT2 Glucose Transport Assay [0254] 1) Assay buffer: 10 mM HEPES, 1.2 mM MgCl.sub.2, 4.7 mM KCl, 2.2 mM CaCl.sub.2 and 120 mM NaCl. [0255] 2) Stop buffer: 10 mM HEPES, 1.2 mM MgCl.sub.2, 4.7 mM KCl, 2.2 mM CaCl.sub.2, 120 mM NaCl and 1 μM LX4211. [0256] 3) The compound with a starting concentration of 10 μM was serially diluted 5-fold with 100% DMSO to 8 concentrations. [0257] 4) 6 μM [.sup.14C]Methyl a-D-glucopyranosid was prepared with the assay buffer. [0258] 5) The cells were treated with 49 μL of the assay buffer, 1 μL of the serially diluted compound, and 50 μL of the 6 μM [.sup.14C] isotope-labeled sugar solution at 37° C. for 2 hours. [0259] 6) The liquid in the well was aspirated and the cells were rinsed three times with the stop buffer. [0260] 7) The cells were lysed with 50 μL of 10% sodium hydroxide solution, the cell lysate was pipetted into a scintillation tube, and 2 mL of scintillation fluid was added. [0261] 8) An isotope detector (Tricarb) was used to read. [0262] 9) The data were processed by GraphPad Prism 5.0 software with the calculation formula: log(inhibitor) vs. response—Variable slope to give the IC.sub.50 value of the test compound.
[0263] The assay results were shown in Table 4:
TABLE-US-00004 TABLE 4 Results of the in vitro cell activity assay Human-SGLT1 Human-SGLT2 Compound IC.sub.50 (nM) IC.sub.50 (nM) LX2761 1.1 1.1 WXD001 0.7 6.5 WXD002 0.5 2.9 WXD003 0.7 0.6 WXD004 0.8 0.8 WXD005 3.5 1.4 WXD006 35 1.4 WXD007 69 5.8 WXD008 21 1.5 WXD009 50 3.3
[0264] Conclusion: The compounds of the present disclosure exhibit excellent in vitro inhibitory activity on Human-SGLT1 and Human-SGLT2.
Assay Example 2. In Vivo DMPK Study
[0265] In Vivo DMPK Study in Rats
[0266] Purpose of the assay: Male SD rats were used as test animals to determine the blood concentration of the compound after a single administration and evaluate the pharmacokinetic behavior.
[0267] Procedure of the assay: 4 healthy adult male SD rats were selected, 2 rats in the intravenous injection group, and 2 rats in the oral group. The test compound was mixed with an appropriate amount of vehicle (10% NMP/10% solutol/80% water) for the intravenous injection group, vortexed, and sonicated to prepare a clear solution of 0.5 mg/mL. The clear solution was filtered by a microporous membrane for use. The vehicle for the oral group was 10% NMP/10% solutol/80% water. The test compound was mixed with the vehicle, and then the mixture was vortexed and sonicated to prepare a clear solution of 1 mg/mL. Rats were administered 1 mg/kg intravenously or 10 mg/kg orally, and then whole blood was collected for a certain period. Plasma was prepared. The drug concentration was analyzed by LC-MS/MS method, and the pharmacokinetic parameters were calculated by Phoenix WinNonlin software (Pharsight, USA).
[0268] Note: NMP: N-methylpyrrolidone; solutol: polyethylene glycol-15 hydroxystearate.
[0269] The assay results were shown in Table 5:
TABLE-US-00005 TABLE 5 Results of PK assay of the compounds Oral C.sub.max DNAUC Vd.sub.SS Cl Compound (nM) F % (nM .Math. h/mpk) (L/kg) (mL/min/kg) T.sub.1/2 (h) LX2761 ND ND ND 1.4 37.6 1.9 WXD003 16.5 0.2 2 1.5 32.5 2.3 Note: C.sub.max is maximum concentration; F % is oral bioavailability; DNAUC = AUC.sub.PO/Dose, AUC.sub.PO is oral exposure, Dose is drug dose; Vd.sub.SS is distribution volume; Cl is clearance rate; T.sub.1/2 is half-life; and ND means not detected.
[0270] Conclusion: The compound of the present disclosure exhibits low oral exposure and bioavailability, and exhibits desirable pharmacokinetic properties as a SGLT1 selective inhibitor.
Assay Example 3. In Vivo Efficacy Study
I. In Vivo Efficacy Study of Oral Glucose Tolerance in Rats (OGTT):
[0271] 1. Assay Animals:
TABLE-US-00006 Animals Species SD rats Gender male Weeks of age / About 8 weeks Supplier Shanghai weight old/250 g SLAC Animal feed Ordinary rat and mouse feed
[0272] 2. Assay Grouping
TABLE-US-00007 TABLE 6 Assay grouping information Number of Compound Frequency of Mode of animals in Group grouping dosage administration administration each group 1 Vehicle — Single Gavage 5 control administration group 2 WXD003 5 mg/kg Single Gavage 5 administration
[0273] 3. Assay Process:
1) Animal Adaptation and Preparation
[0274] After the assay animals arrived at the facility, they needed to adapt to the environment in the animal room for 1 week.
2) Fasting and Administration
[0275] After the animals were fasted for 6 hours, they were administered WXD003 or vehicle according to Table 6, and then immediately administered 50% glucose solution (2 g/kg, 4 ml/kg).
3) Blood Glucose Test
[0276] The time for sugar administration was recorded as 0 point. The blood glucose of the animals was detected at 0 minute before sugar administration, and at 15, 30, 60, 90, 120 minutes after sugar administration, respectively. A curve of glucose tolerance was drawn based on the data of time versus blood glucose, and the area under curve (AUC) was calculated.
4) Data Analysis:
[0277] All values were expressed as average values. Statistical analysis was evaluated using Graphpad Prism 6 one-way analysis of variance and Tukey's multiple comparison test. A p value of less than 0.05 was considered statistically significant.
[0278] 4. Assay Results:
TABLE-US-00008 TABLE 7 Results of in vivo efficacy assay of glucose tolerance in rats Vehicle Compound control group WXD003 OGTT blood 1063.4 624.2**** glucose level AUC.sub.0-2 hours (mmol/L × min) Note: ****means p < 0.0001 relative to the vehicle control group.
[0279] Conclusion: Compared with the vehicle control group, the administration group can significantly reduce the blood glucose AUC level of the animals within 2 hours.
II. In Vivo Efficacy Study in Diabetic Mouse Model Induced by STZ Combined with High-Sugar and High-Fat Diet
[0280] 1. Assay Animals
[0281] 7 week-old C57BL/6J male mice, purchased from Jiangsu GemPharmatech Co., Ltd.
[0282] 2. Assay Process
[0283] 1) After the animals adapted to the environment, all mice were divided into two groups according to body weight, fed according to the scheme in Table 8, and injected with vehicle and streptozotocin (STZ) for modeling;
TABLE-US-00009 TABLE 8 Grouping information of STZ modeling assay Group Mouse strain Quantity Feed STZ modeling treatment Normal group C57BL/6J 5 Chow Diet Intraperitoneally injected (Ordinary rat with citrate buffer (pH = 4.5) and mouse feed) for 5 days Model group C57BL/6J 50 High-sugar Intraperitoneally injected (STZ + high-sugar and high-fat with citrate buffer (pH = 4.5) and high-fat feed) feed (Research and STZ (dose: 40 mg/kg) Diet, HFHS, for 5 days D12451)
[0284] 2) According to the random blood glucose test results of the model group, mice with random blood glucose greater than 11 mmol/L were enrolled in the group to continue the administration assay;
[0285] 3) The mice in the model group that meet the criteria for enrollment were divided into 3 groups, 5 in the normal group, and 10 in each model administration group. The mice were administered according to the administration schedule in Table 9;
TABLE-US-00010 TABLE 9 Grouping information of the assay Mode and Dosage Dosage frequency of Group (Week 1 to Week 4) (Week 4 to Week 7) administration The normal Vehicle + Chow diet Vehicle + Chow diet Orally, once group a day The model Vehicle + HFHS Vehicle + HFHS Orally, administration once a day group 1 The model WXD003 WXD003 Orally, administration (3 mg/kg) + HFHS (6 mg/kg) + HFHS once a day group 2
[0286] 4) After starting the administration, the level of change in animal weight was monitored every day, and the blood glucose and glycosylated hemoglobin of the mice in each group after 6 hours of fasting were detected after 4 weeks and 7 weeks of administration, respectively;
[0287] 5) Data analysis: All values were expressed as average values. Statistical analysis was evaluated using Graphpad Prism 6 one-way analysis of variance and Tukey's multiple comparison test, and a p value of less than 0.05 was considered statistically significant.
[0288] 3. Assay Results
[0289] 1) Assay Results after 4 Weeks of Administration:
[0290] a) As shown in Table 10 and
TABLE-US-00011 TABLE 10 The blood glucose of animals after 4 weeks of administration Chow diet + HFHS + HFHS + WXD003 Compound Vehicle Vehicle (3 mg/kg) Blood glucose 7.2 13.7{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}{circumflex over ( )} 10.3*** after 6 hours of fasting (mmol/L)
[0291] b) As shown in Table 11 and
TABLE-US-00012 TABLE 11 The glycosylated hemoglobin of animals after 4 weeks of administration Chow diet + HFHS + HFHS + WXD003 Compound Vehicle Vehicle (3 mg/kg) Glycosylated 4.2 4.9{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}{circumflex over ( )} 4.5*** hemoglobin after 6 hours of fasting (%)
[0292] c) As shown in
[0293] 2) Assay Results after 7 Weeks of Administration
[0294] d) As shown in Table 12 and
TABLE-US-00013 TABLE 12 The blood glucose of animals after 7 weeks of administration Chow diet + HFHS + HFHS + WXD003 Compound Vehicle Vehicle (6 mg/kg) Blood glucose 7.1 15.3{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}{circumflex over ( )} 9.6**** after 6 hours of fasting (mmol/L)
[0295] e) As shown in Table 13 and
TABLE-US-00014 TABLE 13 The glycosylated hemoglobin of animals after 7 weeks of administration Chow diet + HFHS + HFHS + WXD003 Compound Vehicle Vehicle (6 mg/kg) Glycosylated 4.1 5.2{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}{circumflex over ( )} 4.4**** hemoglobin after 6 hours of fasting (%)
[0296] f) As shown in
[0297] Note: {circumflex over ( )}{circumflex over ( )}{circumflex over ( )}{circumflex over ( )} means p<0.0001 relative to the vehicle group with chow diet, * means p<0.05 relative to the vehicle group with HFHS diet, ** means p<0.01 relative to the vehicle group with HFHS diet, *** means p<0.001 relative to the vehicle group with HFHS diet, and **** means p<0.0001 relative to the vehicle group with HFHS diet.
[0298] Conclusion: Compared with the vehicle control group with HFHS diet, the administration group can significantly reduce the blood glucose and glycosylated hemoglobin of animals after 6 hours of fasting, and can effectively control the weight gain of animals.
III. In Vivo Efficacy Study in Obese Mouse Model Induced by HFHS Diet
[0299] 1. Assay animals
[0300] 5 week-old C57BL/6J male mice, purchased from Jiangsu GemPharmatech Co., Ltd.
[0301] 2. Assay process
[0302] 1) After the animals adapted to the environment, they were fed a HFHS diet (Research Diet, HFHS, D12451) for 20 weeks, and then mice weighing more than 40 grams were selected for weight loss assay.
[0303] 2) Animals whose body weight met the standard were adapted for vehicle administration, and then all mice were divided into 5 groups according to body weight for weight loss assay. The effect of the compound in reducing the body weight of animals was detected. The assay grouping was shown in Table 14:
TABLE-US-00015 TABLE 14 Grouping information of the assay Mode and frequency Group Dosage of administration Vehicle group 1 Chow Diet + Vehicle Orally, once a day Vehicle group 2 HFHS + Vehicle Orally, once a day Administration HFHS + WXD003 Orally, once a day group 3 (0.3 mg/kg) Administration HFHS + WXD003 Orally, once a day group 4 (1 mg/kg) Administration HFHS + WXD003 Orally, once a day group 5 (3 mg/kg)
[0304] 3) After starting the administration, the level of change in animal weight was monitored every day, and after 3 weeks of administration, the blood glucose levels of mice in each group after 6 hours of fasting and 1 hour after meal were detected, respectively;
[0305] 4) Data analysis: All values were expressed as average values. Statistical analysis was evaluated using Graphpad Prism 6 one-way analysis of variance and Tukey's multiple comparison test, and a p value of less than 0.05 was considered statistically significant.
[0306] 3. Assay results
[0307] 1) As shown in
[0308] 2) As shown in Table 15 and
TABLE-US-00016 TABLE 15 The blood glucose of animals after 6 hours of fasting after 3 weeks of administration Chow HFHS + HFHS + HFHS + diet + HFHS + WXD003 WXD003 WXD003 Compound Vehicle Vehicle (0.3 mg/kg) (1 mg/kg) (3 mg/kg) Blood 8.3 10.5 9.7 9.6 9.7 glucose after 6 hours of fasting (mmol/L)
[0309] 3) As shown in Table 16 and
TABLE-US-00017 TABLE 16 The blood glucose of animals 1 hour after meal after 3 weeks of administration Chow HFHS + HFHS + HFHS + diet + HFHS + WXD003 WXD003 WXD003 Compound Vehicle Vehicle (0.3 mg/kg) (1 mg/kg) (3 mg/kg) Blood 9.1 11.5 10.5 10.2 9.6* glucose 1 hour after meal (mmol/L) Note: *means p < 0.05 relative to the vehicle group with HFHS diet, ** means p < 0.01 relative to the vehicle group with HFHS diet, *** means p < 0.001 relative to the vehicle group with HFHS diet, and **** means p < 0.0001 relative to the vehicle group with HFHS diet.
[0310] Conclusion: Compared with the vehicle control group with HFHS diet, the administration group can significantly reduce the body weight of animals in a dose-dependent manner, and can also reduce the blood glucose of animals after 6 hours of fasting and 1 hour after meal.