SGLTS INHIBITOR AND APPLICATION THEREOF
20200331950 ยท 2020-10-22
Assignee
Inventors
- Chengde WU (Shanghai, CN)
- Qinghua MAO (Shanghai, CN)
- Yi LI (Shanghai, CN)
- Tao YU (Shanghai, CN)
- Shuhui Chen (Shanghai, CN)
Cpc classification
C07H15/14
CHEMISTRY; METALLURGY
International classification
Abstract
A compound as an SGLT1/SGLT2 dual inhibitor, and an application thereof in the preparation of a drug as the SGLT1/SGLT2 dual inhibitor. The compound is a compound represented by formula (I), an isomer thereof, or a pharmaceutically acceptable salt thereof.
##STR00001##
Claims
1. A compound represented by formula (I), an isomer thereof, or a pharmaceutically acceptable salt thereof, ##STR00067## wherein, m is 1 or 2; n is 0, 1 or 2; D is O or C(R.sub.1)(R.sub.2); ring A is selected from phenyl and 5-6 membered heteroaryl; R.sub.1 is selected from the group consisting of H, F, Cl, Br, I, OH and NH.sub.2; R.sub.2 is selected from the group consisting of H, F, Cl, Br and I; or R.sub.1 and R.sub.2 are connected to form a 5-6 membered heterocycloalkyl; R.sub.3 is selected from the group consisting of 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 C.sub.1-3 alkoxy are optionally substituted by one, two or three R group(s); R.sub.4 is selected from C.sub.1-3 alkyl, wherein the C.sub.1-3 alkyl is optionally substituted by one, two or three R group(s); R is selected from the group consisting of F, Cl, Br, I, OH and NH.sub.2; and the 5-6 membered heteroaryl and 5-6 membered heterocycloalkyl respectively contain one, two, three or four heteroatom(s) or heteroatom group(s) independently selected from the group consisting of NH, O, S and N.
2. The compound, the isomer thereof, or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.3 is selected from the group consisting of H, F, Cl, Br, I, OH, NH.sub.2, CH.sub.3, Et, and OCH.sub.3.
3. The compound, the isomer thereof, or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.4 is selected from CH.sub.3 and Et.
4. The compound, the isomer thereof, or the pharmaceutically acceptable salt thereof according to claim 1, wherein the ring A is selected from phenyl and thienyl.
5. The compound, the isomer thereof, or the pharmaceutically acceptable salt thereof according to claim 4, wherein the ring A is selected from ##STR00068##
6. The compound, the isomer thereof, or the pharmaceutically acceptable salt thereof according to claim 1, wherein the structural unit ##STR00069## is selected from ##STR00070##
7. The compound, the isomer thereof, or the pharmaceutically acceptable salt thereof according to claim 6, wherein the structural unit ##STR00071## is selected from ##STR00072##
8. The compound, the isomer thereof, or the pharmaceutically acceptable salt thereof according to claim 1, wherein the structural unit ##STR00073## is selected from ##STR00074##
9. The compound, the isomer thereof, or the pharmaceutically acceptable salt thereof according to claim 1, selected from the group consisting of ##STR00075## wherein, R.sub.1 and R.sub.2 are as defined in claim 1; R.sub.3 is as defined in claim 1; R.sub.4 is as defined in claim 1.
10. The compound of the following formula, an isomer thereof, or a pharmaceutically acceptable salt thereof according to claim 1, selected from ##STR00076## ##STR00077##
11. A pharmaceutical composition, comprising a therapeutically effective amount of a compound, an isomer thereof or a pharmaceutically acceptable salt thereof according to claim 1 as an active ingredient, and a pharmaceutically acceptable carrier.
12. A method for treating SGLT1/SGLT2 related diseases, comprising a step of administering the compound or a pharmaceutically acceptable salt thereof according to claim 1 to a subject in need.
13. The method according to claim 12, wherein the disease is diabetes.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0087]
[0088]
DETAILED DESCRIPTION
[0089] The present disclosure is described in detail below by referring to the examples, which are not intended to adversely limit the present disclosure. The present disclosure has been described in detail herein, the embodiments of the present disclosure are disclosed herein, and various modifications and changes may be made to the embodiments of the present disclosure without departing from the spirit and scope of the present disclosure, which is obvious for those skilled in the art.
Reference Example 1: Fragment A-1
[0090] ##STR00020##
Synthesis Route:
[0091] ##STR00021##
[0092] Step 1: Synthesis of Compound A-1-3
[0093] Compound A-1-1 (20 g, 84.78 mmol, 10.87 mL, 1 eq) and tetrahydrofuran (125 mL) were successively added to a pre-dried three-necked flask (500 mL). After replaced with nitrogen, the flask was cooled to 78 C., and N-butyllithium (2.5 M, 37.64 mL, 1.11 eq) was slowly added dropwise thereto, and then stirring was performed for 0.5 hours. Finally, compound A-1-2 (12.5 g, 93.26 mmol, 1.1 eq) was added to the flask, and then the temperature was slowly raised to 0 C., and stirring was performed for 0.5 hours. After the reaction was completed, the resulted reaction solution was slowly quenched with a saturated aqueous solution of ammonium chloride (200 mL) at 0-10 C. Then the reaction solution was extracted with ethyl acetate (200 mL2) to obtain organic phases. The combined organic phases were washed with saturated sodium chloride (100 mL), dried with anhydrous sodium sulfate desiccant, filtered to remove the desiccant, and concentrated in vacuo to remove the solvent, obtaining a crude compound A-1-3, which was directly used in the next reaction without purification.
[0094] Step 2: Synthesis of Compound A-1-4.
[0095] Compound A-1-3 (23.2 g, 79.82 mmol, 1 eq) and toluene (600 mL) were successively added to a pre-dried three-necked flask (1000 mL), and then p-toluenesulfonic acid monohydrate (1.82 g, 9.58 mmol, 0.12 eq) was added thereto. After replaced with nitrogen, the flask was heated to 130 C., and stirring was performed for 10 hours (equipped with Dean-Stark). After the reaction was completed, the reaction solution was cooled down, and concentrated in vacuo to remove the solvent to obtain a residue. The residue was subjected to column chromatography (petroleum ether/ethyl acetate system) to separate compound A-1-4. .sup.1H NMR (400 MHz, CHLOROFORM-d) : 7.49-7.43 (m, 2H), 7.27-7.22 (m, 2H), 5.91 (dt, J=1.3, 2.6 Hz, 1H), 2.80-2.63 (m, 4H), 2.19 (tt, J=6.7, 13.7 Hz, 2H).
[0096] Step 3: Synthesis of Compound A-1.
[0097] Compound A-1-4 (2.9 g, 10.62 mmol, 1 eq), pinacol borate (5.39 g, 21.24 mmol, 2 eq), potassium acetate (3.13 g, 31.85 mmol, 3 eq) and 1,4-dioxane (30 mL) were successively added to a pre-dried one-necked 100 mL flask. After replaced with nitrogen, 1,1-bis(diphenylphosphino)ferrocene palladium chloride (776.94 mg, 1.06 mmol, 0.1 eq) was added into the reaction. After replaced with nitrogen again, the flask was heated to 70 C. and stirring was performed for 10 hours. After the reaction was completed, the reaction was cooled down and concentrated in vacuo to remove the solvent to obtain a residue. The residue was purified by column chromatography (petroleum ether/ethyl acetate system) to obtain compound A-1. .sup.1H NMR (400 MHz, CHLOROFORM-d) : 7.78 (d, J=8.4 Hz, 2H), 7.38 (d, J=8.2 Hz, 2H), 5.97 (br s, 1H), 2.79-2.66 (m, 4H), 2.19 (tt, J=6.6, 13.7 Hz, 2H), 1.36 (s, 12H).
[0098] The fragments A2-8 in the following table are synthesized with reference to the steps 1-3 as described in the Reference Example 1. The structures in the table also include their possible isomers.
TABLE-US-00001 Reference Fragments Example A Structure .sup.1H NMR 2 A-2
Reference Example 9: Fragment B-1
[0099] ##STR00029##
[0100] Synthesis Route:
##STR00030## ##STR00031##
[0101] Step 1: Synthesis of Compound B-1-2
[0102] Compound B-1-1 (30 g, 127.41 mmol, 1 eq) and tetrahydrofuran (6 mL) were added to a 3 L three-necked flask, and the borane tetrahydrofuran complex (1 M, 382.23 mL, 3 eq) was added thereto under blowing nitrogen protection. The resulted mixture was reacted at 25 C. for 16 hours. After the reaction was completed, the resulted reaction solution was quenched by dropwise addition with methanol (150 mL) at 25 C. under blowing nitrogen protection. Then the reaction solution was concentrated in vacuo at 45 C. with a water pump to obtain compound B-1-2. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.68 (d, J=2.4 Hz, 1H), 7.37 (dd, J=2.2, 8.6 Hz, 1H), 7.22 (d, J=8.4 Hz, 1H), 4.77 (d, J=5.3 Hz, 2H).
[0103] Step 2: Synthesis of Compound B-1-3.
[0104] Compound B-1-2 (27 g, 121.91 mmol, 1 eq) and dimethylformamide (150 mL) were added to a three-necked flask. After protected with nitrogen, sodium hydride (9.75 g, 243.82 mmol, 60% purity, 2 eq) was added thereto at 0 C. Half an hour later, allyl bromide (44.24 g, 365.73 mmol, 32.06 mL, 3 eq) was added thereto, and the resulted mixture was reacted at 25 C. for 15.5 hours. After the reaction was completed, the resulted reaction solution was quenched with a saturated aqueous solution of ammonium chloride (500 mL), and extracted with dichloromethane (100 mL3). The organic phases were washed with a saturated saline solution (500 mL), dried with anhydrous sodium sulfate, and filtered to collect a filtrate. The filtrate was concentrated in vacuo at 45 C. with a water pump, obtaining a crude product. The crude product was purified by passing a rapid column (SiO.sub.2, 100-200 mesh, PE:EA=1:0 to 10:1) to obtain compound B-1-3. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.67 (d, J=2.4 Hz, 1H), 7.35 (dd, J=2.4, 8.4 Hz, 1H), 7.21 (d, J=8.4 Hz, 1H), 6.08-5.91 (m, 1H), 5.39 (q, J=1.6 Hz, 1H), 5.34 (q, J=1.5 Hz, 1H), 5.29-5.24 (q, 1H), 4.57 (s, 2H), 4.13 (td, J=1.3, 5.6 Hz, 2H).
[0105] Step 3: Synthesis of Compound B-1-5.
[0106] Compound B-1-4 (9.9 g, 36.23 mmol, 1 eq) and THF (70.5 mL) were added to a three-necked flask. After replaced with nitrogen, the flask was cooled to 0 C., and tert-butyl Grignard reagent (2M, 29.70 mL, 1.64 eq) was added thereto, and the resulted mixture was reacted at 0 C. for 1 hour to obtain a first reaction solution. Compound B-1-3 (12.32 g, 47.09 mmol, 1.3 eq) and tetrahydrofuran (141 mL) were added to a three-necked flask. After replaced with nitrogen, the flask was cooled to 78 C., and N-butyl lithium (2.5M, 21.74 mL, 1.5 eq) was added thereto, and the resulted mixture was reacted at 78 C. for 0.5 hours to obtain a second reaction solution. The first reaction solution was then added dropwise to the second reaction solution using a syringe to perform reaction at 78 C. for 1 hour and then at 25 C. for 13.5 hours. After the reaction was completed, the resulted reaction solution was quenched with a saturated aqueous solution of ammonium chloride (400 mL), then extracted with ethyl acetate (100 mL3). The organic phases were washed with a saturated saline solution (1000 mL), dried with anhydrous sodium sulfate, and filtered to collect a filtrate. The filtrate was concentrated in vacuo at 45 C. by reducing pressure with a water pump to obtain a crude product. The crude product was purified by passing a rapid column (petroleum ether/ethyl acetate system) to obtain compound B-1-5. .sup.1H NMR (400 MHz, CHLOROFORM-d) =8.21 (s, 1H), 7.94 (dd, J=2.0, 8.4 Hz, 1H), 7.48 (d, J=8.2 Hz, 1H), 6.10 (d, J=3.5 Hz, 1H), 6.05-5.94 (m, 1H), 5.38 (dd, J=1.5, 17.2 Hz, 1H), 5.33 (d, J=2.6 Hz, 1H), 5.28-5.23 (m, 1H), 4.65 (s, 2H), 4.63 (br d, J=3.3 Hz, 1H), 4.61 (d, J=3.5 Hz, 1H), 4.15 (d, J=5.5 Hz, 2H), 2.97 (d, J=4.2 Hz, 1H), 1.59 (s, 3H), 1.38 (s, 3H).
[0107] Step 4: Synthesis of Compound B-1-6.
[0108] Compound B-1-5 (8 g, 21.69 mmol, 1 eq), cerium chloride heptahydrate (9.70 g, 26.03 mmol, 2.47 mL, 1.2 eq) and methanol (180 mL) were added to a reaction flask. After replaced with nitrogen, sodium borohydride (1.64 g, 43.38 mmol, 2 eq) was added thereto at 0 C., and the resulted mixture was reacted at 25 C. for 16 hours. After the reaction was completed, the reaction solution was quenched with a saturated aqueous solution of ammonium chloride (250 mL), and a saturated saline solution (250 mL) was added thereto. Then, the reaction solution was extracted with ethyl acetate (100 mL3) to obtain organic phases (if the liquid is difficult to separate during extraction, the liquid can be separated by filtration with diatomite). The organic phases were dried by anhydrous sodium sulfate and filtered to collect a filtrate. The filtrate was concentrated to dry at 45 C. by reducing pressure with a water pump to obtain compound B-1-6. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.61-7.56 (m, 1H), 7.42-7.31 (m, 2H), 6.05-5.92 (m, 2H), 5.41-5.32 (m, 1H), 5.28-5.18 (m, 2H), 4.64-4.59 (m, 2H), 4.49 (d, J=3.5 Hz, 1H), 4.16-4.03 (m, 5H), 3.36 (br s, 1H), 1.46 (s, 3H), 1.30 (s, 3H).
[0109] Step 5: Synthesis of Compound B-1-7.
[0110] Compound B-1-6 (7.2 g, 19.42 mmol, 1 eq), water (45 mL) and acetic acid (44.31 g, 737.82 mmol, 42.20 mL, 38 eq) were added into a reaction flask to perform reaction at 100 C. for 7 hours. After the reaction was completed, the reaction solution was concentrated to dry at 45 C. by reducing pressure with a water pump, and then subjected to azeotropic drying with toluene (100 mL2), obtaining compound B-1-7. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.42 (br d, J=8.6 Hz, 1H), 7.18 (br s, 1H), 7.09 (br d, J=6.8 Hz, 1H), 5.80 (tt, J=6.0, 16.8 Hz, 1H), 5.54-5.08 (m, 4H), 4.58 (br d, J=5.3 Hz, 1H), 4.43 (br s, 2H), 4.08 (br s, 1H), 4.14-3.80 (m, 3H), 3.62-3.28 (m, 3H), 2.20 (br s, 1H).
[0111] Step 6: Synthesis of Compound B-1-8.
[0112] Compound B-1-7 (6 g, 18.14 mmol, 1 eq), triethylamine (12.11 g, 119.72 mmol, 16.66 mL, 6.6 eq) and acetonitrile (110 mL) were added into a single-necked flask, and then acetic anhydride (12.22 g, 119.72 mmol, 11.21 mL, 6.6 eq) and dimethylaminopyridine (22.16 mg, 181.40 umol, 0.01 eq) were successively added thereto to perform reaction at 25 C. for 16 hours. After the reaction was completed, the reaction solution was quenched with a saturated aqueous solution of sodium bisulfate (100 mL), extracted with ethyl acetate (50 mL3). The organic phases were washed with a saturated saline solution (200 mL), dried with anhydrous sodium sulfate and filtered to collect a filtrate. The filtrate was concentrated to dry at 45 C. by reducing pressure with a water pump to obtain a crude product. The crude product was purified by passing a rapid column (petroleum ether/ethyl acetate system) to obtain compound B-1-8. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.49 (d, J=1.9 Hz, 1H), 7.33 (d, J=8.3 Hz, 1H), 7.25-7.21 (dd, 1H), 5.99 (tdd, J=5.6, 10.4, 17.2 Hz, 1H), 5.87 (d, J=8.3 Hz, 1H), 5.41-5.36 (m, 1H), 5.36-5.31 (m, 1H), 5.30-5.23 (m, 2H), 5.17-5.10 (t, 1H), 4.61-4.52 (m, 3H), 4.12-4.08 (m, 2H), 2.13-2.10 (s, 3H), 2.07 (s, 3H), 2.04-1.99 (s, 3H), 1.85 (s, 3H).
[0113] Step 7: Synthesis of Compound B-1-9.
[0114] Compound B-1-8 (6.5 g, 13.03 mmol, 1 eq), sodium acetate (4.28 g, 52.11 mmol, 4 eq), water (13 mL) and glacial acetic acid (117 mL) were added to a reaction flask. The reaction was cooled to 5 C. after replaced with nitrogen, and palladium dichloride (5.08 g, 28.66 mmol, 2.2 eq) was added thereto. The resulted mixture was reacted at 25 C. for 16 hours. After the reaction was completed, the reaction solution was concentrated to dry at 45 C. by reducing pressure with a water pump to obtain a crude product. The crude product was purified by passing a rapid column (petroleum ether/ethyl acetate system) to obtain compound B-1-9. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.53 (d, J=1.8 Hz, 1H), 7.33 (d, J=8.2 Hz, 1H), 7.21 (dd, J=2.1, 8.3 Hz, 1H), 5.87 (d, J=8.2 Hz, 1H), 5.41-5.34 (t, 1H), 5.30-5.23 (t, 1H), 5.15 (t, J=9.6 Hz, 1H), 4.77 (br d, J=2.4 Hz, 2H), 4.56 (d, J=9.9 Hz, 1H), 2.15-2.10 (s, 3H), 2.07 (s, 3H), 2.02 (s, 3H), 1.85 (s, 3H).
[0115] Step 8: Synthesis of Compound B-1.
[0116] Compound B-1-9 (1 g, 2.18 mmol, 14.04 L, 1 eq), triphenylphosphine (857.44 mg, 3.27 mmol, 1.5 eq) and dichloromethane (20 mL) were added to a reaction flask, and stirred for half an hour after protected with nitrogen, and then N-bromosuccinimide (581.85 mg, 3.27 mmol, 1.5 eq) was added thereto at 0 C. The resulted mixture was reacted at 25 C. for 15.5 hours. After the reaction was completed, the reaction solution was concentrated to dry at 25 C. to obtain a crude product. The crude product was purified by passing a rapid column (petroleum ether/ethyl acetate system) to obtain compound B-1. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.44-7.36 (m, 2H), 7.29 (s, 1H), 5.87 (d, J=8.2 Hz, 1H), 5.41-5.34 (t, 1H), 5.30-5.23 (m, 1H), 5.15-5.03 (m, 1H), 4.68-4.59 (d, 1H), 4.53 (t, J=9.9 Hz, 2H), 2.22 (s, 1H), 2.13 (s, 2H), 2.08-2.05 (m, 3H), 2.04-2.01 (m, 3H), 1.91-1.86 (m, 3H).
Reference Example 10: Fragment B-2
[0117] ##STR00032##
[0118] Synthesis Route:
##STR00033## ##STR00034##
[0119] Step 1: Synthesis of Compound B-2-2
[0120] Lithium aluminum hydride (11 g, 289.82 mmol, 1.25 eq) was dissolved in tetrahydrofuran (200 mL) at 0 C., and filled with nitrogen protection after replaced with nitrogen three times. Compound B-2-1 (50 g, 232.51 mmol, 1 eq) was dissolved in tetrahydrofuran (200 mL), and the resulted solution was slowly added to the reaction solution at 0 C. Bubbles were observed and the reaction solution was heated to 25 C. to continue reaction for 2 hours. Water (11 mL) was slowly added dropwise thereto at 0 C., then 15% aqueous solution of sodium hydroxide (11 mL) was added thereto, and finally water (33 mL) was added thereto. The resulted mixture was filtered to obtain a residue which was then washed twice with ethyl acetate, and the filtrate was concentrated in vacuo to obtain a crude compound B-2-2.
[0121] Step 2: Synthesis of Compound B-2-3
[0122] Compound B-2-2 (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 thereto at 0 C. Stirring was performed at 25 C. for 0.5 hours, and then 3-bromopropene (57.64 g, 476.47 mmol, 41.17 mL, 2 eq) was slowly added to the reaction solution to continue the reaction at 25 C. for 2 hours. After the reaction was completed, the reaction solution was quenched with water (50 mL) at 0 C., extracted with ethyl acetate (500 mL2), washed with water (50 mL2) and then washed with a saturated saline solution (50 mL2), and dried with anhydrous sodium sulfate to obtain a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate system) to obtain the target compound B-2-3.
[0123] Step 3: Synthesis of Compound B-2-4
[0124] Compound B-2-3 (18.5 g, 76.72 mmol, 1.2 eq) was dissolved in tetrahydrofuran (100 mL) at 78 C., and then n-butyllithium (2.5 M, 33.25 mL, 1.3 eq) was added under nitrogen protection to perform reaction at 78 C. for 0.5 hours, obtaining alkyl lithium solution. Compound B-1-4 (17.47 g, 63.93 mmol, 1 eq) was dissolved in tetrahydrofuran (100 mL), cooled to 0 C. and tert-butyl magnesium chloride (1.7 M, 41.37 mL, 1.1 eq) was added dropwise under nitrogen protection to perform reaction at 0 C. for 0.5 hours, obtaining a magnesium alkoxy solution. The magnesium alkoxy solution was slowly added to the alkyl lithium solution at 78 C. to perform reaction at 78 C. for 0.5 hours, then the temperature was raised to 25 C. to continue reaction for 15.5 hours. After the reaction was completed, an amine chloride solution (50 mL) was added to the resulted reaction solution at 0 C. and ethyl acetate (200 mL) was added to dilute the reaction solution. Then the reaction solution was washed with water (50 mL2) to obtain organic phases. The combined organic phases were washed with saturated saline solution (50 mL2) to remove the residue water. Then the resulted was dried with anhydrous sodium sulfate, filtered and dried by rotation to obtain a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate system) to obtain the target compound B-2-4.
[0125] Step 4: Synthesis of Compound B-2-5
[0126] Compound B-2-4 (17.80 g, 51.09 mmol, 1 eq) was dissolved in methanol (100 mL), cooled to 0 C., and cerium trichloride heptahydrate (22.84 g, 61.31 mmol, 5.83 mL, 1.2 eq) and sodium borohydride (3.87 g, 102.18 mmol, 2 eq) were added thereto successively, raised to 25 C. to perform reaction for 16 hours. After the reaction was completed, the resulted reaction solution was quenched with water (30 mL) and concentrated in vacuo. Then the resulted was diluted with ethyl acetate (100 mL) and washed with water (50 mL2). Water was removed by using a saturated saline solution (50 mL2). Finally, the solution was dried with anhydrous sodium sulfate, filtered and concentrated to dry by reducing pressure, obtaining the target compound B-2-5.
[0127] Step 5: Synthesis of Compound B-2-6
[0128] Compound B-2-5 (10.22 g, 29.17 mmol, 1 eq) was dissolved in water (100 mL) and glacial acetic acid (100 mL) to perform reaction at 100 C. for 16 hours. After the reaction was completed, the solvent was vacuum dried by rotation at 60 C., and then dried with toluene three times to obtain compound B-2-6.
[0129] Step 6: Synthesis of Compound B-2-7
[0130] Compound B-2-6 (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 stirred at 25 C. for 16 hours. After the reaction was completed, the reaction solution was diluted with ethyl acetate (200 mL), washed with 1M dilute hydrochloric acid (100 mL4). The organic phase was collected and washed with water (50 mL2), and then washed with a saturated saline solution (50 mL2), and finally was dried with anhydrous sodium sulfate, and filtered and concentrated to dry by reducing pressure, obtaining a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate system) to obtain the target compound B-2-7.
[0131] Step 7: Synthesis of Compound B-2-8
[0132] Compound B-2-7 (7 g, 14.63 mmol, 1 eq) and potassium acetate (5.74 g, 58.52 mmol, 4 eq) were dissolved in acetic acid (135 mL) and water (15 mL). Palladium dichloride (5.71 g, 32.18 mmol, 2.2 eq) was added in an ice bath under nitrogen protection to perform reaction at 25 C. for 16 hours. After the reaction was completed, the reaction solution was vacuum dried by rotation at 45 C. to obtain a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate system) to obtain the target compound B-2-8.
[0133] Step 8: Synthesis of Compound B-2
[0134] Compound B-2-8 (2.5 g, 5.70 mmol, 1 eq) was dissolved in dichloromethane (40 mL), then triphenylphosphine (2.24 g, 8.55 mmol, 1.5 eq) was added thereto, and stirring was performed under nitrogen protection for 30 minute. The mixture was cooled to 0 C., then N-bromosuccinimide (1.52 g, 8.55 mmol, 1.5 eq) was added thereto, and stirring was performed at 25 C. for 2.5 hours. After the reaction was completed, the reaction solution was concentrated to dry at 25 C. to obtain a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate system) to obtain the target compound B-2. .sup.1H NMR (400 MHz, CHLOROFORM-d) ppm 1.85 (s, 3H), 2.01 (s, 3H), 2.1 (s, 3H), 2.19 (s, 3H), 2.37 (s, 3H) 4.43-4.50 (m, 2H), 4.80-4.83 (d, J=10.4 Hz, 1H), 5.055-5.104 (m, 1H), 5.214-5.249 (m, 1H), 5.553-5.602 (m, 1H), 6.444-6.453 (m, 1H), 7.145-7.165 (m, 1H), 7.209-7.224 (m, 1H), 7.251-7.270 (m, 1H).
Reference Example 11: Fragment B-3
[0135] ##STR00035##
[0136] Synthesis Route:
##STR00036##
[0137] Step 1: Synthesis of Compound B-3-1
[0138] Compound B-2-7 (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 thereto. After replaced with nitrogen three times, trimethylsilyl trifluoromethanesulfonate (14.31 g, 64.37 mmol, 3.5 eq) was added thereto at 25 C., and the resulted was heated to 60 C. to perform reaction for 2 hours, and then cooled to 25 C. Methyl iodide (13.30 g, 93.70 g mmol, 5.09 eq) and diisopropylethylamine (19.02 g, 147.13 mmol, 8 eq) were added successively thereto to perform reaction at 25 C. for 14 hours. After the reaction was completed, the reaction solution was diluted with water (80MI), extracted with ethyl acetate (80 mL3) to collect organic phases. The combined organic phases were with a saturated saline solution (50 mL), dried with anhydrous sodium sulfate, and filtered to obtain a filtrate. The filtrate was dried by rotation under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate system) to obtain the target compound B-3-1, which was confirmed by LCMS.
[0139] Step 2: Synthesis of Compound B-3-2
[0140] Compound B-3-1 (2 g, 4.29 mmol, 1 eq), barbituric acid (1.10 g, 8.57 mmol, 2 eq) and ethanol (20 mL) were added to a reaction flask. After replaced with nitrogen three times, tetra-triphenylphosphine palladium (495.37 mg, 428.68 mol, 0.1 eq) was added thereto to perform reaction at 70 C. under nitrogen atmosphere for 16 hours. After the reaction was completed, the reaction solution was diluted with water (20 mL) and extracted with ethyl acetate (20 mL3). The combined organic phases were washed with a saturated saline solution (20 mL), dried with anhydrous sodium sulfate, and filtered to obtain a filtrate. The filtrate was dried by rotation under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate system) to obtain the target compound B-3-2, which was confirmed by LCMS.
[0141] Step 3: Synthesis of Compound B-3
[0142] Compound B-3-2 (1.5 g, 3.52 mmol, 1 eq), triphenylphosphine (1.38 g, 5.28 mmol, 1.5 eq) and dichloromethane (20 mL) were added to a reaction flask. After replacing nitrogen three times, stirring was performed at 25 C. for 0.5 hours, then N-bromosuccinimide (938.98 mg, 5.28 mmol, 1.5 eq) was added thereto at 0 C. to perform reaction 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 mL3). The combined organic phases were dried with anhydrous sodium sulfate, filtered to obtain a filtrate. The filtrate was dried by rotation under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate system) to obtain the target compound B-3. .sup.1H NMR (400 MHz, CHLOROFORM-d) =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, 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).
[0143] The fragment B-4 in the following table is synthesized with reference to the steps 1-8 in Reference Example 9. The fragment B-5 in the following table is synthesized with reference to the steps 1-8 in Reference Example 10. The structures in the table also include their possible isomers.
TABLE-US-00002 Reference Fragments Example B Structure .sup.1H NMR 12 B-4
[0144] The fragment B-6 in the following table is synthesized with reference to the steps 1-3 in Reference Example 11. The structures in the table also include their possible isomers.
TABLE-US-00003 Reference Example Fragment Structure NMR 14 B-6
Example 1: WXD001
[0145] ##STR00040##
[0146] Synthesis Route:
##STR00041##
[0147] Step 1: Synthesis of Compound WXD001-1.
[0148] Compound B-1 (1 g, 1.92 mmol, 1 eq) was mixed with compound A-1 (797.78 mg, 2.49 mmol, 1 mL, 1.3 eq), sodium carbonate (2 M, 1.92 mL, 2 eq), toluene (20 mL), ethanol (5 mL) and water (5 mL). After purging nitrogen, tetra-triphenylphosphine palladium (221.48 mg, 191.67 mol, 0.1 eq) was added thereto to perform reaction at 50 C. for 16 hours, and the reaction solution turned black. After the reaction was completed, the reaction solution was concentrated with a water pump under reduced pressure at 45 C. to remove ethanol, and then concentrated with an oil pump to remove toluene and water, obtaining a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate system) to obtain the target compound WXD001-1. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.37 (d, J=8.2 Hz, 1H), 7.30 (d, J=8.2 Hz, 2H), 7.20 (dd, J=2.1, 8.3 Hz, 1H), 7.16-7.07 (m, 3H), 5.89 (dd, J=2.1, 3.4 Hz, 1H), 5.84 (d, J=8.2 Hz, 1H), 5.38-5.29 (m, 1H), 5.25 (dd, J=8.3, 9.6 Hz, 1H), 5.09 (t, J=9.6 Hz, 1H), 4.47 (d, J=9.7 Hz, 1H), 4.12-4.00 (m, 2H), 2.76-2.65 (m, 4H), 2.21-2.14 (m, 2H), 2.13-2.08 (m, 3H), 2.07-2.05 (m, 3H), 2.03-2.00 (m, 3H), 1.74-1.69 (m, 3H).
[0149] Step 2: Synthesis of Compound WXD001-2.
[0150] Compound WXD001-1 (1 g, 1.57 mmol, 1 eq), thiourea (239.73 mg, 3.15 mmol, 2 eq) and dioxane (12 mL) were added to a reaction flask. After purging nitrogen, trimethylsilyl trifluoromethanesulfonate (874.97 mg, 3.94 mmol, 711.35 uL, 2.5 eq) was added thereto, and slowly heated to 80 C. to perform reaction for 2 hours. After cooling to 25 C., diisopropylethylamine (1.02 g, 7.87 mmol, 1.37 mL, 5 eq) and methyl iodide (670.52 mg, 4.72 mmol, 294.09 uL, 3 eq) were added successively thereto to perform reaction 25 C. for 14 hours. After the reaction was completed, the reaction solution was diluted with water (5 mL), extracted with dichloromethane (2 mL3) to obtain organic phases. The organic phases were washed with a saturated saline solution (10 mL), dried with anhydrous sodium sulfate, and filtered to obtain a filtrate. The filtrate was concentrated to dry at 45 C. with a water pump to obtain a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate system) to obtain the target compound WXD001-2. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.37 (d, J=8.2 Hz, 1H), 7.30 (d, J=8.2 Hz, 2H), 7.19 (dd, J=2.0, 8.3 Hz, 1H), 7.14-7.08 (m, 3H), 5.89 (br s, 1H), 5.31 (s, 1H), 5.19 (s, 1H), 5.04 (s, 1H), 4.50 (d, J=9.9 Hz, 1H), 4.38 (d, J=9.9 Hz, 1H), 4.08 (d, J=17.0 Hz, 2H), 2.69 (m, J=6.0, 8.1 Hz, 4H), 2.24-2.16 (m, 2H), 2.15 (s, 3H), 2.09 (s, 3H), 2.00 (s, 3H), 1.71 (s, 3H).
[0151] Step 3: Synthesis of Compound WXD001.
[0152] Compound WXD001-2 (760 mg, 1.22 mmol, 1 eq), methanol (6 mL) and tetrahydrofuran (3 mL) were added to a reaction flask, and then lithium hydroxide monohydrate (1.02 g, 24.39 mmol, 20 eq) and water (6 mL) were added thereto to perform reaction at 25 C. for 16 hours. After the reaction was completed, the reaction solution was diluted with water (10 mL), extracted with ethyl acetate (10 mL3) to obtain organic phases. The organic phases were washed with a saturated saline solution (30 mL), dried with anhydrous sodium sulfate, and filtered to obtain a filtrate. The filtrate was concentrated to dry at 45 C. with a water pump to obtain a crude product. The crude product, was purified by preparative high performance liquid chromatography (acetonitrile/water-aqua ammonia system) to obtain the target compound WXD001. SFC showed that the enantiomeric excess ratio was 100%. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) =7.37 (d, J=8.2 Hz, 1H), 7.34-7.30 (m, 2H), 7.28-7.23 (m, 2H), 7.16 (d, J=8.4 Hz, 2H), 6.00-5.84 (m, 1H), 4.38 (d, J=9.5 Hz, 1H), 4.14 (d, J=9.5 Hz, 1H), 4.11-4.04 (d, 2H), 3.48-3.42 (t, 1H), 3.39-3.32 (m, 2H), 2.72-2.63 (m, 4H), 2.23-2.12 (m, 2H), 2.12 (s, 3H).
Example 2: WXD002
[0153] ##STR00042##
[0154] Synthesis Route:
[0155] Step 1: Synthesis of Compound WXD002-1.
[0156] Compound B-3 (40 mg, 81.74 mol, 1 eq), compound A-3 (41.96 mg, 122.61 mol, 1.5 eq), sodium carbonate (17.33 mg, 163.47 mol, 2 eq), toluene (3 mL), ethanol (0.3 mL) and water (0.3 mL) were added to a reaction flask. After purging nitrogen three times, tetra-triphenylphosphine palladium (9.45 mg, 8.17 mol, 0.1 eq) was added thereto to perform reaction at 50 C. for 5 hours under a nitrogen atmosphere. After the reaction was completed, the reaction solution was diluted with water (5 mL), extracted with ethyl acetate (5 mL3) to obtain organic phases. The combined organic phases were dried with anhydrous sodium sulfate, filtered to obtain a filtrate. The filtrate was dried by rotation under reduced pressure to obtain a crude product. The crude product was purified by preparative TLC (petroleum ether/ethyl acetate system) to obtain the target compound WXD002-1. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.30 (d, J=8.0 Hz, 2H), 7.17-7.12 (m, 2H), 7.03-6.99 (m, 3H), 5.96 (t, J=3.6 Hz, 1H), 5.35 (t, J=9.2 Hz, 1H), 5.23 (t, J=9.6 Hz, 1H), 5.14 (t, J=9.6 Hz, 1H), 4.53 (d, 10.0 Hz, 1H), 4.39 (d, 10.0 Hz, 1H), 4.02 (s, 4H), 3.94 (d, J=6.8 Hz, 2H), 2.66-2.62 (m, 2H), 2.46-2.45 (m, 2H), 2.02 (s, 3H), 2.17 (s, 3H), 2.10 (s, 3H), 2.01 (s, 3H), 1.93 (t, J=6.4 Hz, 2H), 1.74 (s, 3H).
[0157] Step 2: Synthesis of WXD002
[0158] Compound WXD002-1 (42 mg, 67.23 mol, 1 eq), methanol (1 mL), tetrahydrofuran (0.5 mL), water (1 mL), and lithium hydroxide monohydrate (56.42 mg, 1.34 mmol, 20 eq) were added to a reaction flask to perform reaction at 25 C. for 1 hour. After the reaction was completed, the reaction solution was diluted with water (5 mL) and extracted with ethyl acetate (5 mL4) to obtain organic phases. The combined organic phases were dried over anhydrous sodium sulfate, and filtered to obtain a filtrate. The filtrate was dried by rotation under reduced pressure to obtain a crude product. The crude product was purified by preparative high performance liquid chromatography mechanical separation (acetonitrile/water-aqua ammonia system) to obtain the target compound WXD002. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.30 (d, J=8.4, 2H), 7.18-7.13 (m, 3H), 7.09 (d, J=8.0 Hz, 2H), 5.95 (t, J=3.6 Hz, 1H), 4.40 (d, J=9.6 Hz, 1H), 4.14 (d, J=9.2 Hz, 1H), 3.99-3.97 (m, 6H), 3.48-3.35 (m, 3H), 2.62-2.58 (m, 2H), 2.41 (s, 2H), 2.21 (s, 3H), 2.13 (s, 3H), 1.89 (t, J=6.4 Hz, 2H).
[0159] Each fragment of the Examples 3-9 in the following table 1 was synthesized with reference to the steps 1-3 in Example 1. The structures in table 1 also include their possible isomers.
TABLE-US-00004 TABLE 1 Reference Example Fragment A Fragment B Compound Structure 3
[0160] Each fragment in Example 10 in the following table 2 was synthesized with reference to the steps 1-2 in Example 2. The structures in table 2 also include their possible isomers.
TABLE-US-00005 TABLE 2 Reference Example Fragment A Fragment B Compound Structure 10
[0161] The hydrogen spectrum and mass spectrum data of each example are shown in Table 3.
TABLE-US-00006 TABLE 3 Reference Example Compound NMR MS m/z 1 WXD001 .sup.1H NMR (400 MHz, METHANOL-d.sub.4) = 7.37 (d, J = 8.2 Hz, 1H), 519 7.34-7.30 (m, 2H), 7.28-7.23 (m, 2H), 7.16 (d, J = 8.4 Hz, 2H), 6.00-5.84 (M + Na) (m, 1H), 4.38 (d, J = 9.5 Hz, 1H), 4.14 (d, J = 9.5 Hz, 1H), 4.11-4.04 (d, 2H), 3.48-3.42 (t, 1H), 3.39-3.32 (m, 2H), 2.72-2.63 (m, 4H), 2.23- 2.12 (m, 2H), 2.12 (s, 3H). 2 WXD002 .sup.1H NMR (400 MHz, CHLOROFORM-d) = 7.30 (d, J = 8.4, 2H), 521 7.18-7.13 (m, 3H), 7.09 (d, J = 8.0 Hz, 2H), 5.95 (t, J = 3.6 Hz, 1H), (M + Na) 4.40 (d, J = 9.6 Hz, 1H), 4.14 (d, J = 9.2 Hz, 1H), 3.99-3.97 (m, 6H), 3.48-3.35 (m, 3H), 2.62-2.58 (m, 2H), 2.41 (s, 2H), 2.21 (s, 3H), 2.13 (s, 3H), 1.89 (t, J = 6.4 Hz, 2H). 3 WXD003 .sup.1H NMR (400 MHz, METHANOL-d.sub.4) ppm 2.07-2.18 (m, 5 H), 499 2.20 (s, 3 H), 2.61-2.74 (m, 4 H), 3.33-3.49 (m, 3 H), 3.98 (s, 2 H), (M + Na) 4.12 (d, J = 9.03 Hz, 1 H), 4.38 (d, J = 9.54 Hz, 1 H), 5.90 (br s, 1 H), 7.06-7.20 (m, 5 H), 7.30 (d, J = 8.28 Hz, 2 H). 4 WXD004 .sup.1H NMR (400 MHz, METHANOL-d.sub.4) ppm 2.14 (s, 3 H), 2.21 (s, 465 3 H), 2.49 (br dd, J = 4.44, 2.69 Hz, 2 H), 3.35-3.49 (m, 3 H), 3.91 (t, J = (M + Na) 5.50 Hz, 2 H), 3.99 (s, 2 H), 4.13 (d, J = 9.01 Hz, 1 H), 4.28 (q, J = 2.79 Hz, 2 H), 4.39 (d, J = 9.38 Hz, 1 H), 6.13 (br s, 1 H), 7.11 (d, J = 8.13 Hz, 2 H), 7.14-7.20 (m, 3 H), 7.33 (d, J = 8.25 Hz, 2 H). 5 WXD005 .sup.1H NMR (400 MHz, METHANOL-d.sub.4) ppm 1.60-1.70 (m, 2 H), 463 1.74-1.82 (m, 2 H), 2.13 (s, 3 H), 2.15-2.20 (m, 2 H), 2.21 (s, 3 H, 2.32-2.42 (M + Na) (m, 2 H), 3.33-3.51 (m, 3 H), 3.96 (s, 2 H), 4.13 (d, J = 9.03 Hz, 1 H), 4.39 (d, J = 9.54 Hz, 1 H), 6.06 (dt, J = 3.58, 2.10 Hz, 1 H), 7.06 (d, J = 8.28 Hz, 2 H), 7.11-7.20 (m, 3 H), 7.26 (d, J = 8.28 Hz, 2 H). 6 WXD006 .sup.1H NMR (400 MHz, CHLOROFORM-d) ppm 7.09-7.17 (m, 3 H), 505 6.68 (d, J = 3.5 Hz, 1 H), 6.50 (d, J = 3.5 Hz, 1 H), 5.77 (br s, 1 H), (M + Na) 4.33 (br d, J = 9.5 Hz, 1 H), 4.13 (br d, J = 9.0 Hz, 1 H), 4.01 (s, 2 H), 3.61-3.70 (m, 1 H), 3.45-3.56 (m, 2 H), 2.78 (br s, 1 H), 2.41-2.66 (m, 5 H), 2.22 (s, 3 H), 2.13 (s, 3 H), 1.99-2.11 (m, 2 H), 1.95 (br s, 1 H) 7 WXD007 .sup.1H NMR (400 MHz, METHANOL-d.sub.4) ppm 2.15 (s, 3 H), 2.28 471 (s, 3 H), 2.45 (br d, J = 1.51 Hz, 2 H), 3.35-3.50 (m, 3 H), 3.86 (t, J = (M + Na) 5.52 Hz, 2 H), 4.10 (s, 2 H), 4.14 (d, J = 9.29 Hz, 1 H), 4.21 (br d, J = 2.51 Hz, 2 H), 4.39 (d, J = 9.54 Hz, 1 H), 5.97 (br s, 1 H), 6.61 (d, J = 3.51 Hz, 1 H), 6.81 (d, J = 3.76 Hz, 1 H), 7.13-7.17 (m, 1 H), 7.18-7.21 (m, 1 H), 7.23 (s, 1 H). 8 WXD008 .sup.1H NMR (400 MHz, METHANOL-d.sub.4) = 7.28 (d, J = 8.2 Hz, 2H), 515 7.23 (dd, J = 2.1, 8.4 Hz, 1H), 7.19-7.12 (m, 3H), 6.93 (d, J = 8.4 Hz, (M + Na) 1H), 5.90 (br s, 1H), 4.37 (d, J = 9.4 Hz, 1H), 4.08 (d, J = 9.0 Hz, 1H), 3.97-3.89 (m, 2H), 3.81 (s, 3H), 3.50-3.36 (m, 3H), 2.71-2.62 (m, 4H), 2.20-2.13 (m, 2H), 2.12 (s, 3H) 9 WXD009 .sup.1H NMR (400 MHz, METHANOL-d.sub.4) ppm 2.06 (s, 3 H), 2.07-2.09 503 (m, 2 H), 2.54-2.69 (m, 4 H), 3.29-3.45 (m, 3 H), 3.94 (d, J = 2.76 Hz, 2 H), (M + Na) 4.09 (d, J = 9.54 Hz, 1 H), 4.34 (d, J = 9.54 Hz, 1 H), 5.87 (br s, 1 H), 6.96-7.04 (m, 1 H), 7.14 (d, J = 8.28 Hz, 2 H), 7.18-7.25 (m, 2 H), 7.28 (d, J = 8.28 Hz, 2 H). 10 WXD010 .sup.1H NMR (400 MHz, METHANOL-d.sub.4) ppm 1.09 (t, J = 7.53 Hz, 3 H), 513 2.10 (m, 1 H), 2.13 (s, 3 H), 2.14-2.22 (m, 1 H), 2.56-2.74 (m, 6 H), 3.34-3.48 (M + Na) (m, 3 H), 4.02 (s, 2 H), 4.13 (d, J = 9.03 Hz, 1 H), 4.39 (d, J = 9.29 Hz, 1 H), 5.91 (br s, 1 H), 7.10 (d, J = 8.28 Hz, 2 H), 7.14-7.21 (m, 2 H), 7.21- 7.25 (m, 1 H), 7.30 (d, J = 8.28 Hz, 2 H).
Experiment Example 1. In Vitro Cell Viability Test
[0162] Experimental Steps and Methods:
[0163] Biological Activity Experiment 1: SGLT1 Glucose Transport Test.
[0164] 1. Purpose of the Experiment:
[0165] The effect of the compound on the glucose transport activity of SGLT1 transporter was detected by measuring the amount of [C]-labeled glucose entering cells with high expression of Human-SGLT1.
[0166] 2. Experimental Methods.
[0167] 2.1. Cell Preparation.
[0168] The cells stably expressing Human-SGLT1 were constructed by Wuxi AppTec(Shanghai) Co., Ltd. SGLT1 cells were laid in Cytostar-T (PerkinElmer) 96-well cell culture plate and cultured overnight in 5% CO.sub.2 environment at 37 C.
[0169] 2.2. SGLT1 Glucose Transport Test.
[0170] 1) Experimental buffer, including: 10 mM 4-hydroxyethylpiperazineethanesulfonic 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).
[0171] 2) Compounds were serially diluted in 100% dimethyl sulfoxide (DMSO) starting at 1 mM and make a 5-folds, 8 points serials compound dilutions.
[0172] 3) 3 M [.sup.4C]-labeled methyl a-D-glucopyranoside was prepared with the experimental buffer.
[0173] 4) The cells were treated with 49 uL of the experimental buffer, 1 L of the gradient diluted compound and 50 L of the 3 M [.sup.14C]-labeled methyl a-D-lucopyranosid solution at 37 C. for 2 hours.
[0174] 5) The data were read with an isotope detector Micro beta Reader.
[0175] 6) The IC.sub.50 value of the tested compound was obtained by the calculation formula: log(inhibitor) vs. responseVariable slope, using GraphPad Prism 5.0 software.
[0176] Biological Activity Experiment 2: SGLT2 Glucose Transport Test.
[0177] 1. Purpose of the Experiment:
[0178] The effect of the compound on the glucose transport activity of SGLT2 transporter was detected by measuring the amount of [.sup.14C]-labeled glucose entering cells with high expression of Human-SGLT2.
[0179] 2. Experimental Methods.
[0180] 2.1. Cell Preparation.
[0181] The cells stably expressing Human-SGLT2 were constructed by Wuxi AppTec(Shanghai) Co., Ltd. SGLT2 cells were laid in 96-well cell culture plate (Greiner) and cultured overnight in 5% CO.sub.2 environment at 37 C.
[0182] 2.2. SGLT2 Glucose Transport Test.
[0183] 1) Experimental buffer, including: 10 mM 4-hydroxyethylpiperazineethanesulfonic 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).
[0184] 2) Stop buffer, including: 10 mM 4-hydroxyethylpiperazineethanesulfonic 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 M LX4211.
[0185] 3) Compounds were serially diluted in 100% dimethyl sulfoxide (DMSO) starting at 10 uM and make a 5-folds, 8 points serials compound dilutions.
[0186] 4) 6 M [.sup.14C]-labeled methyl a-D-lucopyranosid was prepared with the experimental buffer.
[0187] 5) The cells were treated with 49 uL of the experimental buffer, 1 L of the gradient diluted compound and 50 L of the 6 M [.sup.14C]-labeled methyl a-D-lucopyranosid solution at 37 C. for 2 hours.
[0188] 6) The solution was sucked out from holes, and the cells were rinsed with the stop buffer for 3 times.
[0189] 7) The cells were lysed with 50 ul of 10% sodium hydroxide solution, the cell lysate was sucked into a scintillation tube, into which 2 mL scintillation solution was then added.
[0190] 8) The data were read with an isotope detector Tricarb.
[0191] 9) The IC.sub.50 value of the tested compound was obtained by the calculation formula: log(inhibitor) vs. responseVariable slope, using GraphPad Prism 5.0 software.
[0192] The experimental results are shown in Table 4:
TABLE-US-00007 TABLE 4 Results of cell viability test in vitro Human-SGLT1 Human-SGLT2 Compound IC.sub.50 (nM) IC.sub.50 (nM) Sotagliflozin 69.0 1.15 WXD001 210 3.98 WXD002 55.6 1.01 WXD003 49.5 2.04 WXD004 14.1 1.49 WXD005 21.9 1.78 WXD006 39.2 1.02 WXD007 30.1 1.35 WXD008 17.2 5.40 WXD009 233 4.68 WXD010 14.8 2.49
Conclusion: The Compound of the Present Disclosure Exhibits Superior In Vitro Inhibitory Activity Against Human-SGLT1 and Human-SGLT2
Experimental Example 2. Study on the In Vivo Pharmacokinetics in Animals
[0193] Study on the In Vivo Pharmacokinetics in Rats
[0194] The purpose of the experiment: Male SD rats were used as test animals and were given a single administration to determine the plasma concentration of the compound and evaluate the pharmacokinetic behavior.
[0195] Experimental method: Six healthy adult male SD rats were divided into 2 groups, with 3 rats in an intravenous injection group and 3 rats in an oral administration group. In the intravenous injection group, the test compound was mixed with an appropriate amount of vehicle (10% N-methyl pyrrolidone (NMP)/10% polyethylene glycol-15 hydroxystearate (available from solutol)/80% water), then vortexed and sonicated to prepare 0.2 mg/mL clear solution, which was filtered by microporous membrane. In the oral administration group, 10% N-methyl pyrrolidone (NMP)/10% polyethylene glycol-15 hydroxy stearate (available from solutol)/80% water was used as a vehicle and mixed with the test compound, then vortexed and sonicated to prepare 0.40 mg/mL clear solution. The rats were given intravenous administration at a dose of 1 mg/kg or oral administration at a dose of 2 mg/kg. Whole blood was collected at a certain period of time to prepare the plasma. The drug concentration of the plasma was analyzed by LC-MS/MS method, and the pharmacokinetic parameters were calculated using Phoenix WinNonlin software (Pharsight Company, USA).
[0196] The experimental results are shown in Table 5:
TABLE-US-00008 TABLE 5 Results of pharmacokinetic (PK) test of compounds Oral DNAUC C.sub.max (nM .Math. h/ Vd.sub.ss Cl T.sub.1/2 Compound (nM) F % mpk) (L/kg) (mL/min/kg) (h) Sotagliflozin 364 54.8 969 2.01 22.6 1.27 WXD001 410 59.0 1823 3.86 10.1 4.59 WXD003 391 47.7 1013 2.94 14.5 2.56 WXD010 356 56.4 2320 2.48 8.06 4.50 Notes: C.sub.max represents the maximum concentration; F % represents oral bioavailability; Oral DNAUC = AUC.sub.PO/Dose (unit oral exposure), AUC.sub.PO represents oral exposure, Dose is drug dose; Vd.sub.ss is distribution volume; Cl is clearance rate; and T.sub.1/2 is half-life.
Study on the In Vivo Pharmacokinetics in Beagle Dogs
[0197] The purpose of the experiment: Male beagle (Beagle) dogs were used as test animals and were given a single administration to determine the plasma concentration of the compound and evaluate the pharmacokinetic behavior.
[0198] Experimental method: Six male Beagle dogs were divided into 2 groups, with 3 dogs in an intravenous injection group and 3 dogs in an oral administration group. In the intravenous injection group, the test compound was mixed with an appropriate amount of vehicle (20% polyethylene glycol-400 (PEG400)/10% polyethylene glycol-15 hydroxy stearate (available from solutol)/70% water), then vortexed and sonicated to prepare 1 mg/mL clear solution, which was filtered by microporous membrane. In the oral administration group, 20% polyethylene glycol-400 (PEG400)/10% polyethylene glycol-15 hydroxy stearate (available from solutol)/70% water was used as a vehicle and mixed with the test compound, then vortexed and sonicated to prepare 1 mg/mL clear solution. The dogs were given intravenous administration at a dose of 1 mg/kg or oral administration at a dose of 2 mg/kg. Whole blood was collected at a certain period of time to prepare the plasma. The drug concentration of the plasma was analyzed by LC-MS/MS method, and the pharmacokinetic parameters were calculated using Phoenix WinNonlin software (Pharsight Company, USA).
[0199] The experimental results are shown in Table 6:
TABLE-US-00009 TABLE 6 Results of pharmacokinetic (PK) test of compounds Oral DNAUC C.sub.max (nM .Math. h/ Vd.sub.ss Cl T.sub.1/2 Compound (nM) F % mpk) (L/kg) (mL/min/kg) (h) WXD010 1287 82 4051 1.49 6.9 2.4 Notes: C.sub.max represents the maximum concentration; F % represents oral bioavailability; Oral DNAUC = AUC.sub.PO/Dose (unit oral exposure), AUC.sub.PO represents oral exposure, Dose is drug dose; Vd.sub.ss is distribution volume; Cl is clearance rate; T.sub.1/2 is half-life.
Conclusion: The Compound of the Present Disclosure has Good Oral Exposure and Bioavailability
Experimental Example 3. Study on In Vivo Efficacy of Oral Glucose Tolerance Test (OGTT) in Rats: Study on the In Vivo Efficacy of Oral Glucose Tolerance Test (OGTT) in Rats for the First Time
[0200] Summary of the Experiment:
[0201] 1. Animals:
TABLE-US-00010 Animal: Species SD rats Gender: Male Age/weight: About 8 weeks old/250 g Supplier: Shanghai SLAC Animal feed Ordinary feed for rats and mice
[0202] 2. Experimental Grouping:
TABLE-US-00011 Number of Administration Administration animals Groups Compound Dose Frequency method per group 1 Vehicle control group 0 Single Intragastric 5 administration administration 2 Positive compound 10 mg/kg Single Intragastric 5 (Sotagliflozin) administration administration 3 WXD001 10 mg/kg Single Intragastric 5 administration administration 4 WXD003 10 mg/kg Single Intragastric 5 administration administration
[0203] Experiment Procedure:
[0204] 1. Animal Adaptation and Preparation:
[0205] The experimental animals were allowed to adapt to the environment in the animal room for one week after they arrived.
[0206] 2. Fasting and Drug Administration.
[0207] The animals fasted in the metabolic cage for 18 hours, then were given drugs or vehicle (2 ml/kg) according to the above table, and subsequently were immediately given 50% glucose solution (2 g/kg, 4 ml/kg).
[0208] 3. Urine glucose and blood glucose test.
[0209] 2 hours after the administration of glucose solution, feed intake was restored. Blood samples were taken at 0 min, 15 min, 30 min, 45 min, 60 min and 120 min respectively, and urine samples were collected at 0-24 h to test blood glucose, urine glucose (mg/200 g) and urine volume respectively.
[0210] 4. Data Analysis:
[0211] All values are represented as averages. Statistical analysis was performed using Graphpad Prism 6 one-way analysis of variance and Tukey's multiple comparison test. P value of less than 0.05 is considered statistically significant.
[0212] The Experimental Results are Shown in Table 7:
TABLE-US-00012 TABLE 7 Results of glucose tolerance test in rats. Vehicle Positive control compound Compound group (Sotagliflozin) WXD001 WXD003 OGTT blood 1033 823* 832* 797** glucose level AUC.sub.0-2 hr (mol/L min) Urine glucose 1.8 2424.0**** 2181.4**** 1636.4**** level (mg/200 g, BW) Urine volume 21.36 39.74**** 37.58**** 24.58 (mL/200 gBW) Notes: *p < 0.05, **p < 0.01, **p < 0.001, ****p < 0.0001 vs. vehicle control group.
[0213] Study on the In Vivo Efficacy of Oral Glucose Tolerance Test (OGTT) in Rats for the Second Time:
[0214] Summary of the Experiment:
[0215] 1. Animals:
TABLE-US-00013 Animal: Species SD rats Gender: Male Age/weight: About 8 weeks old/250 g Supplier: Shanghai SLAC Animal feed Ordinary feed for rats and mice
[0216] 2. Experiment Grouping:
TABLE-US-00014 Number of Administration Administration animals Groups Compound Dose Frequency method per group 1 Vehicle control group 0 Single Intragastric 5 administration administration 2 Positive compound 10 mg/kg Single Intragastric 5 (Sotagliflozin) administration administration 4 WXD010 10 mg/kg Single Intragastric 5 administration administration
[0217] Experiment Procedure:
[0218] 1. Animal Adaptation and Preparation:
[0219] The experimental animals were allowed to adapt to the environment in the animal room for one week after they arrived.
[0220] 2. Fasting and Drug Administration.
[0221] The animals fasted in the metabolic cage for 18 hours, then were given drugs or vehicle (2 ml/kg) according to the above table, and subsequently were immediately given 50% glucose solution (2 g/kg, 4 ml/kg).
[0222] 3. Urine glucose and blood glucose test.
[0223] 2 hours after the administration of glucose solution, feed intake was restored. Blood samples were taken at 0 min, 15 min, 30 min, 45 min, 60 min and 120 min respectively, and urine samples were collected at 0-24 h to test blood glucose, urine glucose (mg/200 g) and urine volume respectively.
[0224] 4. Data Analysis:
[0225] All values are represented as averages. Statistical analysis was performed using Graphpad Prism 6 one-way analysis of variance and Tukey's multiple comparison test. P value of less than 0.05 is considered statistically significant.
[0226] The experimental results are shown in Table 8:
TABLE-US-00015 TABLE 8 Results of glucose tolerance test in rats. Vehicle Positive control compound Compound group (Sotagliflozin) WXD010 OGTT blood 1134 790**** 720**** glucose level AUC.sub.0-2 hr (mol/L min) Urine glucose 0.8 2843.2**** 2118.7**** level (mg/200 g, BW) Urine volume 11.5 26.4**** 20.0**** (mL/200 g, BW) Notes: *p < 0.05, **p < 0.01, **p < 0.001, ****p < 0.0001 vs. vehicle control group.
Conclusion: Compared with the Vehicle Control Group, the Compound of the Present Disclosure can Significantly Reduce the Blood Glucose AUC Level within 2 Hours and Increase the 24-Hour Urine Glucose Excretion Level of Animals. Compared with the Positive Compound, the Compound of the Present Disclosure has a Lower Level of Urine Glucose Under the Same Hypoglycemic Effect, which is Helpful to Reduce the Side Effects of Urinary Tract Infection
Experimental Example 4: Study on the In Vivo Pharmacodynamics in Diabetic Db/Db Mice
[0227] Summary of the Experiment
[0228] 1. Animal Information:
TABLE-US-00016 Species db/db mice Grade SPF animal Age 5 weeks old The age at which the experiment 6 weeks old started Body weight range about 25 g Gender Male Supplier Model Animal Research Center of Nanjing University Supplier address Nanjing, Jiangsu, China
[0229] 2 Animal Feeding
[0230] The animals were kept in an animal breeding room with strictly controlled environmental conditions after they arrived. The animal breeding room was maintained at a temperature of 20-24 C. and a humidity of 40-70%. The temperature and humidity in the feeding room were monitored in real time by a hygrothermograph, and the temperature and humidity were recorded twice daily (one in the morning and the other in the afternoon). The lighting of the animal feeding room was controlled by an on-off electronic timing system, with lights for 12 hours and dark for 12 hours daily (turned on at 7:00 a.m. and turned off at 19:00 p.m). The mice were raised in separate cages and were given free access to feed (reproductive feed 17053113 for rats and mice, available from Beijing Keao Xieli Feed Co., Ltd.) and water during the experiment.
[0231] 3. Experiment Grouping:
TABLE-US-00017 Administration Number of Administration method and animals Group Treatment cycle @ dose frequency per group 1 Vehicle Weeks 1-4 @0 mg/kg Intragastric 6 control Weeks 5-8 @0 mg/kg administration, group once daily 2 WXD003 Weeks 1-4 @5 mg/kg Intragastric 6 Weeks 5-8 @10 mg/kg administration, once daily 3 WXD010 Weeks 1-4 @5 mg/kg Intragastric 6 Weeks 5-8@10 mg/kg administration, once daily
Experiment Procedure
[0232] 1. Administration of Drug
[0233] During the experiment, the animals were administrated with the corresponding vehicles or drugs according to the group with an administration time of 16:00 and an administration period of 8 weeks.
[0234] The dose was 5 mg/kg from week 1 to week 4; and the dose was 10 mg/kg from week 5 to week 8.
[0235] 2. Blood Glucose Level
[0236] Random and fasting blood glucose levels were measured once a week.
[0237] The random blood glucose level was measured at 10:00 a.m.
[0238] Fasting blood glucose test: The mice fasted from 10:00 a.m., and blood glucose level was firstly measured at 16:00. Then the mice were administered with the drugs, and 2 hours later blood glucose level was measured again, and then feed intake was restored.
[0239] 3. Oral Glucose Tolerance Test (OGTT).
[0240] At the end of the experiment (i.e. the last 3 days of administration), the animals fasted for 6 hours, then were given a single administration of glucose aqueous solution at a dose of 2 g/kg. The time of the glucose administration was recorded as 0 min. Blood glucose levels of the animals were detected at 0 min before the glucose administration, and 15 min, 30 min, 90 min and 120 min after the glucose administration, respectively. The glucose tolerance curve was drawn according to the data of blood glucose levels vs time, and the area under the curve (AUC) was calculated. The administration was given at 16:00.
[0241] 4. Biochemical Detection
[0242] At weeks 4 and 8 of the experiment, the animals fasted for 6 hours, and blood samples were collected to measure glycosylated hemoglobin.
[0243] 5. Body Weight and Food Consumption
[0244] During the experiment, the body weight of the animals was monitored once daily, and food consumption was monitored twice a week.
[0245] 6. Data Processing and Analysis
[0246] All of the data were entered into an Excel document and expressed in the form of meanS.E.M. The differences between groups were compared using graphpad Prism 6 software and one-way analysis of variance (ANOVA). P value of less than 0.05 is considered a significant difference.
[0247] The results of random blood glucose experiment from week 1 to week 8 are shown in Table 9:
TABLE-US-00018 TABLE 9 Results of random blood glucose experiment Vehicle control Group group WXD003 WXD010 Week 1 27.4 2.26 14.3 2.28 *** 16.7 1.00 ** Week 2 21.8 2.09 15.7 0.88 13.5 2.46 * Week 3 25.6 2.65 13.6 1.40 *** 14.1 0.97 *** Week 4 27.4 3.33 13.3 0.81 **** 15.4 1.34 *** Week 5 26.9 3.67 14.1 1.28 ** 13.2 1.09 ** Week 6 28.8 1.84 10.1 1.26 **** 10.6 1.48 **** Week 7 27.2 2.63 12.5 1.23 **** 9.8 0.82 **** Week 8 27.6 2.96 14.4 1.77 *** 9.2 0.68 **** Notes: * p < 0.05, ** p < 0.01, ** p < 0.001, **** p < 0.0001 vs. vehicle control group.
Conclusion: Compared with the Vehicle Control Group, the Compound of the Present Disclosure can Reduce the Random Blood Glucose Level of Animals; the Compound of the Present Disclosure can Further Reduce the Random Blood Glucose Level of Animals with the Increase of Dose
[0248] The results of fasting blood glucose test from week 1 to week 8 are shown in Table 10:
TABLE-US-00019 TABLE 10 Results of fasting blood glucose test Vehicle control Group group WXD003 WXD010 Week 1 21.8 3.30 14.8 2.34 14.7 2.50 Week 2 26.8 3.26 19.9 1.32 14.4 1.47 *** Week 3 28.7 3.00 15.8 1.48 ** 15.8 1.40 ** Week 4 28.9 3.35 14.3 1.29 *** 14.9 1.24 *** Week 5 25.7 2.82 17.4 1.72 * 15.9 0.79 * Week 6 28.5 3.15 10.9 1.42 **** 11.0 1.67 **** Week 7 30.0 2.58 11.7 1.30 **** 9.6 0.91 **** Week 8 31.4 1.86 13.0 1.36 **** 8.1 0.51 **** Notes: * p < 0.05, ** p < 0.01, ** p < 0.001, **** p < 0.0001 vs. vehicle control group.
Conclusion: Compared with the Vehicle Control Group, the Compound of the Present Disclosure can Significantly Reduce the Fasting Blood Glucose Level of Animals; by Increasing the Dose, the Compound of the Present Disclosure can Further Reduce the Fasting Blood Glucose Level of Animals
[0249] The results of oral glucose tolerance test (OGTT) at week 8 are shown in Table 11:
TABLE-US-00020 TABLE 11 Results of oral glucose tolerance test (OGTT) at week 8 Vehicle control Compound group WXD003 WXD010 OGTT blood 4515.5 160.22 2806.5 155.12**** 2118.4 99.17**** glucose level AUC.sub.0-2 hr (mol/L min) Notes: *p < 0.05, **p < 0.01, **p < 0.001, ****p < 0.0001 vs. vehicle control group.
Conclusion: Compared with the Vehicle Control Group, the Compound of the Present Disclosure can Significantly Reduce the Blood Glucose Level AUC within 2 Hours (AUC.SUB.0-2hr.) of Animals
[0250] The results of glycosylated hemoglobin (HbA1c) test at weeks 4 and 8 are shown in Table 12:
TABLE-US-00021 TABLE 12 Results of glycosylated hemoglobin (HbA1c) at weeks 4 and 8 Week 4 Week 8 Rate of Rate of Decline of Decline of Group HbA1c (%) HbA1c (%) HbA1c (%) HbA1c (%) Vehicle 8.4 0 9.6 0 control group WXD003 5.7*** 32 5.9**** 39 WXD010 5.8*** 31 5.1**** 47 Notes: *p < 0.05, **p < 0.01, **p < 0.001, ****p < 0.0001 vs. vehicle control group.
Conclusion: Compared with the Vehicle Control Group, the Compound of the Present Disclosure can Significantly Reduce the Level of Glycosylated Hemoglobin (HbA1c) of Animals; the Compound of the Present Disclosure can Further Reduce the Level of Glycosylated Hemoglobin (HbA1c) of Animals with the Increase of Dose
[0251] The results of body weight and food consumption are shown in