COMPOUND HAVING KHK INHIBITORY EFFECT
20230138901 · 2023-05-04
Inventors
- Zhixiang PAN (Shanghai, CN)
- Haiying He (Shanghai, CN)
- Zhigan Jiang (Shanghai, CN)
- Jianhua XIA (Shanghai, CN)
- Lei Zhang (Shanghai, CN)
- Chen ZHANG (Shanghai, CN)
- Jian Li (Shanghai, CN)
- Shuhui Chen (Shanghai, CN)
Cpc classification
C07D403/04
CHEMISTRY; METALLURGY
A61P1/16
HUMAN NECESSITIES
International classification
C07D403/04
CHEMISTRY; METALLURGY
Abstract
A compound having a KHK inhibitory effect, or pharmaceutically acceptable salts thereof, and use thereof in the preparation of a medicament for treating a disease associated with KHK kinase abnormal expression. Provided is a compound as represented by formula (III) or a pharmaceutically acceptable salt thereof.
##STR00001##
Claims
1. A compound represented by formula (III), or a pharmaceutically acceptable salt thereof, ##STR00248## wherein, T.sub.1 is selected from N, and T.sub.2 is selected from CR.sub.a, or T.sub.2 is selected from N, and T.sub.1 is selected from CR.sub.a; R.sub.1 and R.sub.a together with the carbon atoms to which they are directly attached form a ring ##STR00249## wherein the ##STR00250## is optionally substituted with 1 or 2 R; each E.sub.1 and E2 is independently selected from the group consisting of N, NH, O, CH, CH.sub.2, and S; E3 and E4 are each independently selected from the group consisting of CH and N; T3 and T4 are each independently selected from the group consisting of N and CH; each R is independently selected from the group consisting of H, halo, CN, NH.sub.2, OH, C.sub.1-3 alkyl, and C.sub.1-3 alkoxy, wherein the C.sub.1-3 alkyl is optionally substituted with 1, 2, or 3 F; each R.sub.b is independently selected from the group consisting of halo, cyano, and C.sub.1-3 alkyl, wherein the C.sub.1-3 alkyl is optionally substituted with 1, 2, or 3 F; n is selected from 0, 1 and 2; L.sub.1 is selected from the group consisting of a single bond and NH; L.sub.2 is selected from the group consisting of a single bond, —(CH.sub.2).sub.m—, ##STR00251## —CH═CH—,and —O(CH.sub.2).sub.q—, wherein the —(CH.sub.2).sub.m—, ##STR00252## —CH═CH—, and —O(CH.sub.2).sub.q—may be optionally substituted with 1, 2, or 3 R; m is selected from 0, 1 and 2; q is selected from 1 and 2; ring A is selected from the group consisting of 4-8 membered heterocycloalkyl, C3-6 cycloalkyl, phenyl, and 5-6 membered heteroaryl; when R.sub.1 and R.sub.a together with the carbon atoms to which they are directly attached form ring ##STR00253## the ring A is not ##STR00254## and the “4-8 membered heterocycloalkyl and 5-6 membered heteroaryl” comprise 1, 2 or 3 heteroatoms independently selected from the group consisting of O, S and N.
2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the structural unit ##STR00255## is selected from the group consisting of ##STR00256## ##STR00257## are optionally substituted with 1 or 2 R.
3. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the structural unit ##STR00258## is selected from the group consisting of ##STR00259## ##STR00260##
4. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein ring A is selected from the group consisting of ##STR00261## thiazolyl, thienyl, imidazolyl, and pyrazolyl.
5. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein each R is independently selected from the group consisting of H, F, Cl, CN, CH.sub.3, and CF.sub.3.
6. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein each Rb is independently selected from the group consisting of F, Cl, cyano, CH.sub.3, and CF.sub.3.
7. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein L.sub.2 is selected from the group consisting of a single bond, —CH.sub.2—, —CH.sub.2CH.sub.2—, —CH═CH—, and —OCH.sub.2—.
8. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, selected from the group consisting of: ##STR00262## ##STR00263## wherein R.sub.1, R.sub.b, T.sub.1, T.sub.z, n and L.sub.2 are as defined in claim 1.
9. A compound of any of the following formulae, or a pharmaceutically acceptable salt thereof: ##STR00264## ##STR00265## ##STR00266## ##STR00267## ##STR00268## ##STR00269## ##STR00270## ##STR00271## ##STR00272##
10. The compound according to claim 9, or a pharmaceutically acceptable salt thereof, selected from the group consisting of: ##STR00273## ##STR00274## ##STR00275##
11. A method of inhibiting KHK in a subject in need thereof which comprises the administration of a medicament comprising a KHK inhibitor compound according to claim 1, or a pharmaceutically acceptable salt thereof.
12. The method according to claim 11, wherein the subject comprises non-alcoholic fatty liver disease or non-alcoholic steatohepatitis.
13. A medicament composition which comprises a compound according to claim 1 and a pharmaceutically acceptable carrier.
Description
EMBODIMENTS
[0208] The present invention will be described in more detail by way of examples which are not meant to impose any disadvantageous limitation to the invention. While the invention has been described herein in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made to the specific embodiments of the invention, without departing from the spirit and scope of the invention.
Intermediate A-1
[0209] ##STR00111##
[0210] Synthetic Scheme:
##STR00112##
Step 1: Synthesis of Compound A-1_2
[0211] Compound A-1_1 (600.0 mg, 5.30 mmol) was added to a solution of potassium tert-butoxide (655.19 mg, 5.84 mmol) in tetrahydrofuran (15.0 mL) at -40° C., then A-1_1a (730.47 mg, 5.57 mmol) was added dropwise slowly, and the reaction was stirred at 0° C. for 1.5 h. After completion of the reaction, 3 mL acetic acid was slowly added at 0° C., and stirred for 20 min. 80 mL water was added, and the mixed liquid was extracted twice with EtOAc (50 mL). The organic phase was dried and then rotary-evaporated to dryness to remove the solvent, obtaining a crude product. The crude product was purified by automated column chromatography (100-200 mesh, eluant: PE: EtOAc=100:1-100:50) to afford Compound A-1_2. .sub.1H NMR (400 MHz, CDCl.sub.3) δ=10.07 (br s, 1H), 8.31 (s, 1H), 4.46 (q, J=7.2 Hz, 2H), 4.33 (q, J=7.2 Hz, 2H), 1.46 (t, J=7.2 Hz, 3H), 1.36 (t, J=7.2 Hz, 3H).
Step 2: Synthesis of Compound A-1_3
[0212] Compound A-1_2 (826.0 mg, 3.38 mmol) was added to EtOH (0.8 mL) and stirred at 40° C. for 20 min at which time the solid was completely dissolved. H.sub.2O (2.0 mL) was added, and stirred for 10 min. Then, 30% aqueous ammonia (5.92 g, 50.63 mmol) was added, heated to 80° C. and stirred for 2 h, and filtered. The filter cake was washed with ethanol (1×5 mL) and vacuum-dried to afford Compound A-1_3 which was used directly in the next step. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=11.00-10.33 (m, 1H), 8.58 (s, 1H), 7.99-7.66 (m, 2H), 4.23 (q, J=7.2 Hz, 2H), 1.26 (t, J=7.2 Hz, 3H).
Step 3: Synthesis of Compound A-1_4
[0213] Compound A-1_3 (400.0 mg, 1.86 mmol) was dissolved in N-methylpyrrolidinone (5.0 mL), potassium tert-butoxide (625.63 mg, 5.58 mmol) was added, and the mixture was stirred at 110° C. for 1 h. The reaction system was a light pink suspension. After completion of the reaction, 4 mL acetic acid and 20 mL water were added, stirred at room temperature for 1 h, and filtered. The filter cake was dried to afford Compound A-1_4. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=12.64-11.52 (m, 1H), 11.27 (br s, 1H), 8.70 (s, 1H).
Step 4: Synthesis of Compound A-1
[0214] Compound A-1_4 (229.0 mg, 1.35 mmol) was added to phosphorus oxychloride (4.3 mL), DIPEA (2 g, 15.47 mmol) was added, and the reaction was stirred at 110° C. for 30 h. After completion of the reaction, the reaction liquid was cooled to room temperature, and rotary-evaporated to remove the solvent, obtaining a crude product. The crude product was purified by automated column chromatography (100-200 mesh, eluant: PE: EtOAc=100: 1-100:50) to afford Compound A-1.
Intermediate A-8
[0215] Synthetic Scheme:
##STR00113##
Step 1: Synthesis of Compound A-8_2
[0216] To a solution of Compound A-8_1 (2.05 g, 10.00 mmol) in MeOH (50 mL) was added sodium methoxide (3.24 g, 59.98 mmol), and the mixture was stirred at 0° C. for 12 h. TLC (PE: EtOAc=3:1) showed that the reaction was complete, LCMS showed that the reaction was relatively clean, and the target product MS was detected. The reaction liquid was directly rotary-evaporated to dryness. To the residue 100 mL of dichloromethane was added, slurried at room temperature for half an hour, and filtered. The filter cake was washed with dichloromethane (20 mL×2). The filtrate was rotary-evaporated to dryness to afford A-8_2.
Step 2: Synthesis of Compound A-8_3
[0217] To a solution of Compound A-8_2 (1.0 g, 5.10 mmol) in THF (10 mL) was added n-butyl lithium (2.5 M, 3.06 mL) dropwise at −70° C. under N.sub.2 protection, the mixture was stirred for 1 h, then a solution of N-fluorobenzenesulfonimide (NFSI) (3.21 g, 10.19 mmol) in THF (30 mL) was added dropwise, the mixture was further stirred for 15 min, then warmed to 25° C. and stirred for 30 min. TLC (PE: EtOAc=3:1) showed that the reaction was complete, and the target product MS was detected by LCMS. The reaction liquid was carefully quenched with water (50 mL). After extraction with ethyl acetate (30 mL ×3), the organic layers were combined and rotary-evaporated to dryness to afford a crude product. The crude product was purified by a flash column (ISCO®; 24 g SepaFlash® silica column, eluant: 0-18% EtOAc/PE, flow rate: 30 mL/min) to afford Compound A-8_3.
Step 3: Synthesis of Compound A-8_4
[0218] To a solution of Compound A-8_3 (600 mg, 2.60 mmol) in THF (3 mL) was added concentrated hydrochloric acid (12 M, 10 mL), and the mixture was stirred at 90° C. for 12 h. LCMS showed that the reaction was complete and the target product MS was detected. The reaction liquid was filtered to collect the precipitated solid. The filter cake was washed with a small amount of ethyl acetate (0.5 mL ×2), and the water was drained as much as possible. Then, the solid was suspended in 50 mL ethyl acetate and rotary-evaporated to dryness to afford Compound A-8_4. .sup.1H NMR (400 MHz, DMSO-d6) δ=11.86 (s, 1H), 11.27 (s, 1H), 11.32-11.23 (m, 1H), 7.20 (s, 1H).
Step 4: Synthesis of Compound A-8
[0219] To a suspension of Compound A-8_4 (400 mg, 1.97 mmol) in POCl.sub.3 (16.50 g, 107.61 mmol) was added DIPEA (765.44 mg, 5.92 mmol) dropwise, and the mixture turned into reddish brown clear liquid. The mixture was stirred at 110° C. for 12 h. TLC (PE: EtOAc=3:1) showed that the reaction was complete. The reaction liquid was cooled to room temperature, and then concentrated under reduced pressure to remove excess POCl.sub.3. The residue was dissolved with ethyl acetate (50 mL), and washed with 50 mL water. The aqueous phase was extracted with ethyl acetate (20 mL×2). The organic phases were combined, washed with saturated brine (10 mL), and rotary-evaporated to dryness to afford crude product A-8 which was directly used in the next step.
Intermediate A-9
[0220] Synthetic Scheme:
##STR00114##
Step 1: Synthesis of Compound A-9_2
[0221] To a solution of Compound A-8_2 (1 g, 5.10 mmol) in THF (15 mL) was added NCS (1.02 g, 7.64 mmol), and the mixture was reacted under stirring at 80° C. for 16 h. TLC (PE: EtOAc=3:1) showed incomplete consumption of the starting materials with about half left, and LCMS also showed half of the starting materials left. Additional NCS (0.5 g, 0.75 eq) was added, and the mixture was further reacted under stirring at 80° C. for 6 h. TLC (PE: EtOAc=3:1) showed that the reaction was almost complete. The reaction liquid was directly rotary-evaporated to dryness. The crude product was purified by a flash column (ISCO®; 24 g SepaFlash® silica column, eluant: 0-18% EtOAc/PE, flow rate: 20 mL/min) to afford Compound A-9_2. .sup.1H NMR (400 MHz, CDCl.sub.3) δ=7.11 (s, 1H), 4.11 (s, 3H), 4.06 (s, 3H).
[0222] For the remaining two synthetic procedures, reference is made to the procedures of Steps 3 and 4 for Intermediate A-8, and the obtained crude Intermediate A-9 can be directly used in the next step without further purification.
Intermediate A-10
[0223] Synthetic Scheme:
##STR00115##
Step 1: synthesis of Compound A-10_2
[0224] To a solution of Compound A-8_2 (1 g, 5.10 mmol) in THF (15 mL) was added NBS (1.36 g, 7.64 mmol), and the mixture was reacted under stirring at 80° C. for 12 h. The reaction liquid was rotary-evaporated to dryness. The crude product was purified by a flash column (ISCO®; 40 g SepaFlash® silica column, eluant: 0-18% EtOAc/PE, flow rate: 30 mL/min) to afford Compound A-10_2. .sup.1H NMR (400 MHz, CDCl.sub.3) δ=7.19 (d, J=0.8 Hz, 1H), 4.02 (s, 3H), 3.96 (s, 3H).
Step 2: Synthesis of Compound A-10_3
[0225] To a solution of Compound A-10_2 (0.2 g, 726.95 μmol) in THF (2.5 mL) was added n-butyl lithium (2.5 M, 450 μL) dropwise at −70° C. under N2 protection. After stirring at −70° C. for 1 h, a solution of N-fluorobenzenesulfonimide (NFSI) (460 mg, 1.46 mmol) in THF (5 mL) was added dropwise, the mixture was further reacted under stirring at −70° C. for 15 min, then warmed to 25° C. and stirred for 30 min. TLC (PE: EtOAc=3:1) showed the reaction was complete. The reaction liquid was carefully quenched with water (50 mL), and extracted with ethyl acetate (30 mL ×3). The organic phases were combined and rotary-evaporated to dryness to afford a crude product. The crude product was purified by a flash column (ISCO®; 12 g SepaFlash® silica column, eluant: 0-18% EtOAc/PE, flow rate: 20 mL/min) to afford Compound A-10_3. .sup.1H NMR (400 MHz, CDCl.sub.3) δ=6.53 (br s, 1H), 4.01 (br s, 3H), 3.95 (br s, 3H).
[0226] For the remaining two synthetic procedures, reference is made to the procedures of Steps 3 and 4 for Intermediate A-8, and the obtained crude Intermediate A-10 can be directly used in the next step without further purification.
Intermediate A-11
[0227] Synthetic Scheme:
##STR00116## ##STR00117##
Step 1: Synthesis of Compound A-11_2
[0228] A-11_1 (50 g, 264.49 mmol), NaOMe (100 g, 1.85 mol) were dissolved in MeOH (500 mL), and the reaction was stirred at 80° C. for 12 h under nitrogen protection. LC-MS showed disappearance of starting material signal and generation of product signal, and TLC (petroleum ether: ethyl acetate =3:1) showed formation of a new spot. The reaction liquid was directly rotary-evaporated to dryness, added with water (500 mL), and extracted with EtOAc (400 mL). The organic phase was rotary-evaporated to dryness to afford A-11_2.
Step 2: synthesis of Compound A-11_3
[0229] To a 3000 mL three-necked flask, A-11_2 (90 g, 499.44 mmol) and CHCl.sub.3 (1000 mL) were added, followed by m-chloroperoxybenzoic acid (287.23 g, 1.41 mmol, 85% purity), and the reaction was stirred at 30° C. for 12 h under nitrogen protection. LCMS showed that the starting material signal did not disappear and a product signal was generated, and TLC (dichloromethane: methanol=10:1) showed formation of a new spot. The reaction liquid was filtered, and the filter cake was washed with dichloromethane (500 mL). The filtrate was slowly added to saturated sodium sulfite solution (500 g sodium sulfite was prepared into 2.5 L solution), stirred for one hour to quench the oxidant. The layers were separated, and the aqueous phase was washed with 1000 mL dichloromethane. The organic phases were combined, rotary-evaporated to dryness, added with 1000 mL methyl tent-butyl ether, and washed with saturated sodium carbonate solution (500 mL ×3). The aqueous phases were combined, and washed with 500 mL methyl tent-butyl ether. The (sodium carbonate solution) aqueous phases were combined, and extracted with chloroform (2 L ×4). The chloroform organic phases were combined, and rotary-evaporated to dryness to afford A-11_3. .sup.1H NMR (400 MHz, CDCl.sub.3) δ=4.24-4.11 (m, 3H), 4.06-3.95 (m, 3H), 3.20 (t, J=7.8 Hz, 2H), 2.86 (t, J=7.7 Hz, 2H), 2.28-2.15 (m, 2H).
Step 3: Synthesis of Compound A-11_4
[0230] To a 1000 mL one-neck flask, A-11_3 (59 g, 300.71 mmol) was added, and acetic anhydride (250 mL) was added, and the reaction was stirred at 80° C. for 5 h under nitrogen protection. LCMS showed disappearance of starting material signal and generation of product signal, and TLC (petroleum ether: ethyl acetate=3:1) showed formation of a new spot. The reaction liquid was slowly added to water (500 mL) and extracted with ethyl acetate (300 mL ×2). The organic phase was directly rotary-evaporated to dryness to afford a crude product. The crude product was purified by a flash silica column (ISCO cake, 330 g SepaFlash silica column, eluant: 0-10% EtOAc/PE, flow rate: 100 mL/min) to afford A-11_4. .sub.1H NMR (400 MHz, CDCl.sub.3) δ=6.12-5.90 (m, 1H), 4.02 (d, J=7.0 Hz, 6H), 2.95-2.82 (m, 1H), 2.78-2.57 (m, 2H), 2.14 (s, 3H), 2.08-1.96 (m, 1H).
Step 4: Synthesis of Compound A-11_5
[0231] To a 1000 mL single-neck flask, A-11_4 (40 g, 167.9 mmol) and THF (400 mL)/H20 (100 mL) were added, followed by LiOH.H.sub.2O (14 g, 335.8 mmol), and the reaction was stirred at 20° C. for 12 h under nitrogen protection. LCMS showed disappearance of starting material signal and generation of product signal. The reaction liquid was directly rotary-evaporated to dryness. The crude product was purified by a flash silica column (ISCO 330 g SepaFlash silica column, eluant: 0-20% EtOAc/PE, flow 35mL/min) to afford A-11_5. .sup.1H NMR (400 MHz, CDCl.sub.3) δ=5.10 (t, J=7.0 Hz, 1H), 4.10-3.96 (m, 6H), 2.88 (ddd, J=2.8, 8.9, 15.4 Hz, 1H), 2.70-2.48 (m, 2H), 2.12-1.94 (m, 1H).
Step 5: Synthesis of Compound A-11_6
[0232] To a 5 L three-necked flask, A-11_5 (150 g, 764.52 mmol) and DCM (1500 mL) were added, followed by Dess-Martin periodinane (660 g, 1.56 mol), and the reaction was stirred at 20° C. for 12 h under nitrogen protection. LCMS showed disappearance of starting material signal and generation of product signal, and TLC (petroleum ether: ethyl acetate=3:1) showed formation of a new spot. The reaction liquid was filtered directly, and the filter cake was washed with ethyl acetate (200 mL). The filtrate was directly rotary-evaporated to dryness. Purification by a flash silica column (ISCO cake, 330 g SepaFlash flash silica column, eluant: 0-10% EtOAc/PE, flow rate: 100 mL/min) affords A-11_6. .sup.1H NMR (400 MHz, CDCl.sub.3) δ=4.02 (d, J=8.3 Hz, 6H), 2.92-2.82 (m, 2H), 2.71-2.62 (m, 2H).
Step 6: Synthesis of Compound A-11_7
[0233] To a 1000 mL one-neck flask, A-11_6 (50 g, 257.48 mmol) and DCM (500 mL) were added, followed by DAST (122 g, 756.88 mmol, 100 mL), and the reaction was stirred at 30° C. for 20 h under nitrogen protection. The reaction liquid was slowly added to ice water (2000 mL) to quench, and the filter cake was washed with dichloromethane (2000 mL). The filtrate was directly rotary-evaporated to dryness. Purification by a flash silica column (ISCO cake, 330 g SepaFlash silica column, eluant: 0-10% EtOAc/PE, flow rate: 100 mL/min) affords A-11_7. .sup.1H NMR (400 MHz, CDCl.sub.3) δ=3.98 (d, J=5.1 Hz, 6H), 2.83-2.70 (m, 2H), 2.62-2.41 (m, 2H).
Step 7: Synthesis of Compound A-11_8
[0234] To a 1000 mL one-neck flask, A-11_7 (50 g, 231.28 mmol) and THF (100 mL) were added, followed by concentrated hydrochloric acid (500 mL), and the reaction was stirred at 80° C. for 12 h under nitrogen protection. The reaction liquid was slowly cooled to room temperature, the turbid liquid was filtered, and the filter cake was washed with ethyl acetate (50 mL) to afford A-11_8. .sup.1H NMR (400 MHz, CDCl.sub.3) δ=11.85 (br s, 1H), 11.36-11.12 (m, 1H), 2.61-2.52 (m, 4H).
Step 8: Synthesis of Compound A-11
[0235] To a 1000 mL single-neck flask, A-11_8 (34 g, 180.72 mmol) was added, and POCl3 (206 mL) was added, and the reaction was stirred at 120° C. for 12 h under nitrogen protection. The reaction liquid was rotary-evaporated to dryness, diluted with dichloromethane (500 mL) and then slowly added to water (1500 mL) to quench, and then extracted with dichloromethane (1000 mL ×3). The organic phases were combined, and rotary-evaporated to dryness to afford A-11. .sup.1H NMR (400 MHz, CDCl.sub.3) δ=3.16-3.01 (m, 2H), 2.85-2.65 (m, 2H).
Intermediate B-5
[0236] Synthetic Scheme:
##STR00118##
Step 1: Synthesis of hydrochloride salt of Compound B-5
[0237] Compound B-5_1 (2 g, 8.72 mmol) and HCl/MeOH (4 M, 2.18 mL) were added to MeOH (20 mL), and the reaction was stirred at 70° C. for 1 h. LCMS showed the reaction was complete. The reaction liquid was rotary-evaporated to dryness to afford crude hydrochloride salt of Compound B-5, which was used directly in the next step without further purification.
Intermediate B-6
[0238] Synthetic Scheme:
##STR00119##
Step 1: Synthesis of Compound B-6_2
[0239] In a 50 mL round-bottomed flask, Compound B-6_1 (1.2 g, 4.36 mmol) was dissolved in HCl/MeOH (20 mL), and the reaction flask was stirred at 70° C. for 1h. TLC (PE: EtOAc=1:1) showed the reaction was complete. The reaction liquid was depressurized to afford Compound B-6_2.
Step 2: Synthesis of Compound B-6
[0240] In a 100 mL round-bottomed flask, Compound B-6_2 (600 mg, 2.07 mmol) was dissolved in MeOH (10 mL), the air in the flask was purged with nitrogen, then Pd/C (220.69 mg, 207.38 μmol) was added, the air in the flask was replaced with a hydrogen balloon for three times, and under the hydrogen balloon (15 psi) atmosphere, the mixture was stirred at 10° C. for 10min. TLC (PE: EtOAc=1:1) showed the reaction was complete and the starting material spot disappeared. The reaction liquid was filtered through Celite, the filter cake was washed with MeOH (50 mL), and the collected filtrate was concentrated under reduced pressure. After concentration, Compound B-6 was obtained. .sup.1H NMR (400 MHz, CD.sub.3OD) δ=3.72 (d, J=1.6 Hz, 3H), 3.51-3.39 (m, 3H), 3.27 (br d, J=19.2 Hz, 1H), 2.21-1.97 (m, 3H), 1.41-1.27 (m, 2H).
Intermediate G-2
[0241] Synthetic Scheme:
##STR00120##
Step 1: Synthesis of Compound G-2
[0242] In a 100 mL round-bottomed flask, Compound G-2_1 (500 mg, 2.87 mmol) and potassium carbonate (1.19 g, 8.61 mmol) were dissolved in THF (5 mL), then ethyl bromoacetate (958 mg, 5.74 mmol, 634.44 μL) was added, and the reaction was stirred at 60° C. for 12 h. TLC (PE: EtOAc=1:1) showed the reaction was complete and the starting material spot disappeared. After completion of the reaction, the solvent was removed by rotary-evaporation to dryness to afford a crude product. The crude product was purified by automated column chromatography (100-200 mesh, eluant: PE: EtOAc=100:1-100:50) to afford G-2. .sup.1H NMR (400 MHz, CDCl.sub.3) δ8.35 (s, 1H), 8.27 (d, J=2.5 Hz, 1H), 7.39 (t, J=2.1 Hz, 1H), 4.67 (s, 2H), 4.30 (q, J=7.3 Hz, 2H), 1.34-1.30 (m, 3H).
Intermediate H-1
[0243] Synthetic Scheme:
##STR00121##
Step 1: Synthesis of Compound H-1_1
[0244] A-3 (5 g, 24.38 mmol) and NaOH (4.88 g, 121.91 mmol) were dissolved in THF (25 mL)/H.sub.2O(25 mL), and the reaction was stirred at 20° C. for 12 h under nitrogen protection. LC-MS showed disappearance of starting material signal and generation of product signal, and TLC (petroleum ether: ethyl acetate=3:1) showed formation of a new spot. The reaction liquid was adjusted to pH 2.3 with dilute hydrochloric acid (2 M), added with water (20 mL), and extracted with EtOAc (60 mL). The organic phase was rotary-evaporated to dryness to afford H-1_1.
Step 2: Synthesis of Compound H-1_2
[0245] To a 250 mL three-necked flask, H-1_1 (4.5 g, 24.11 mmol) and acetonitrile (100 mL) were added, followed by C-1 (9.51 g, 31.35 mmol) and potassium carbonate (10.00 g, 72.34 mmol), and the reaction was stirred at 80° C. for 12 h under nitrogen protection. LCMS showed that the starting material signal did not disappear and a product signal was generated, and TLC (petroleum ether: ethyl acetate=3:1) showed formation of a new spot. The reaction liquid was filtered directly. The filter cake was washed with ethyl acetate (30 mL), and the filtrate was directly rotary-evaporated to dryness to afford H-1_2. 1H NMR (400 MHz, CDCl.sub.3) δ11.01 (br s, 1H), 7.24 (d, J=6.0 Hz, 1H), 6.80 (d, J=6.0 Hz, 1H), 4.81-4.58 (m, 1H), 4.32 (dt, J=5.3, 8.9 Hz, 1H), 4.21-4.02 (m, 1H), 2.70-2.49 (m, 1H), 2.15-2.01 (m, 1H), 1.58 (d, J=6.5 Hz, 3H).
Step 3: Synthesis of Compound H-1
[0246] To a 250 mL three-necked flask, H-1_2 (5 g, 22.60 mmol) was added and dissolved with dichloromethane (50 mL). Trifluoroacetic anhydride (6.86 g, 67.79 mmol, 9.44 mL) was slowly added dropwise at 0° C., and the reaction was stirred at 20° C. for 12 h under nitrogen protection. LCMS showed disappearance of starting material signal and generation of product signal, and TLC (petroleum ether: ethyl acetate=3:1) showed formation of a new spot. The reaction liquid was slowly added to ice water (200 mL), adjusted to pH 7-8 with saturated sodium bicarbonate solution, and extracted with ethyl acetate (200 mL ×2). The organic phase was directly rotary-evaporated to dryness to afford a crude product. The crude product was purified by flash silica column (eluant: 0-20% EtOAc/PE, flow rate: 35 mL/min) to afford Compound H-1. H NMR (400 MHz, CDCl.sub.3) δ 7.06-6.91 (m, 2H), 4.55-4.42 (m, 1H), 4.12-3.93 (m, 2H), 2.55-2.37 (m, 1H), 2.06-1.89 (m, 1H), 1.48 (d, J=6.0 Hz, 3H).
Intermediate B-10
[0247] ##STR00122##
Step 1: Synthesis of Compound B-10_2
[0248] B-101 (3 g, 9.76 mmol) was dissolved in DCM (50 mL), the reaction temperature was lowered to −70° C., DIBALH (1 M, 19.52 mL) was slowly added to the reaction liquid at −70° C., and the reaction was stirred at −70° C. for 1 h. LCMS showed disappearance of starting material signal and generation of product signal. Methanol (20 mL) was slowly added dropwise at −70° C. to quench the reaction. After slowly recovering to room temperature, methanol (100 mL) was added, stirred and filtered. The filter cake was washed with 20 mL methanol, and the organic phase was rotary-evaporated to dryness. The crude product was purified by automated column chromatography (100-200 mesh, eluant: PE: EtOAc=100: 1-100: 40) to afford Compound B-10_2. 1H NMR (400 MHz, CDC13) δ7.35 (d, J=7.0 Hz, 4H), 7.24-7.17 (m, 4H), 7.15-7.07 (m, 2H), 4.46 (s, 1H), 3.48 (dd, J=6.3, 11.3 Hz, 1H), 3.33-3.17 (m, 5H), 1.15-1.03 (m, 1H), 0.68 (dd, J=5.5, 8.5 Hz, 1H), 0.31 (t, J=5.5 Hz, 1H).
Step 2: Synthesis of Compound B-10_3
[0249] B-10.sub._2 (1 g, 3.58 mmol) and TEA (543.30 mg, 5.37 mmol, 747.32 μL) were dissolved in DCM (5 mL), the reaction solution was slowly lowered to 0° C., MsCl (0.62 g, 5.41 mmol, 418.92 μL) was slowly added dropwise at 0° C., and the reaction was stirred at 20° C. for 1 h. TLC (petroleum ether: ethyl acetate=3: 1) showed disappearance of starting material signal and formation of a new spot. The reaction liquid was slowly added to ice water (40 mL) to quench, adjusted to about pH 7 by adding saturated aqueous sodium bicarbonate solution, and extracted with DCM (50 mL ×2). The organic phase was rotary-evaporated to dryness to afford Compound B-10_3.
Step 3: Synthesis of Compound B-10_4
[0250] B-10_3 (1.1 g, 1.54 mmol, 50% purity) was dissolved in DMSO (12 mL), NaCN (247.50 mg, 5.05 mmol) was added slowly, and the reaction was stirred at 80° C. for 12 h. LCMS showed disappearance of starting material signal and generation of product signal. TLC (petroleum ether: ethyl acetate=3: 1) showed disappearance of the starting material signal and formation of a new spot. The reaction liquid was slowly added to ice water (30 mL), and extracted with ethyl acetate (40 mL ×2). The organic phases were combined, washed with saturated brine (40 mL). The organic phase was rotary-evaporated to dryness. The aqueous phase was slowly added to basic sodium hypochlorite solution to quench. The crude product was purified by flash silica column (ISCO®; 20 g SepaFlash® silica column, eluant: 0-20% ethyl acetate/petroleum ether, flow rate: 35 mL/min) to afford Compound B-10_4.
Step 4: Synthesis of Compound B-10_5
[0251] To a 50 mL single-neck flask, B-10_4 (0.6 g, 2.19 mmol) was added, then HCl/MeOH (4 M, 6.00 mL) was added slowly, and the reaction was stirred at 80° C. for 12 h. LCMS showed disappearance of starting material signal and generation of product signal. The reaction liquid was directly rotary-evaporated to dryness to afford Compound B-10_5.
Step 5: Synthesis of Compound B-10
[0252] B-10_5 (0.4 g, 1.24 mmol) was dissolved in MeOH (10 mL), Pd/C (0.05 g, 1.24 mmol, 10% palladium content) was added under nitrogen protection, the reaction was set up in a hydrogenation chamber and stirred at 50° C. for 12 h after replaced with hydrogen three times, and H.sub.2 (2.51 mg, 1.24 mmol) pressure was maintained at 45 psi. LCMS showed disappearance of starting material signal. TLC (petroleum ether: ethyl acetate =3: 1) showed disappearance of the starting material signal and formation of a new spot. The reaction liquid was filtered through Celite, and the filter cake was washed with methanol (30 mL). The filtrate was rotary-evaporated to dryness to afford Compound B-10. 1H NMR (400 MHz, CD.sub.3OD) δ 4.28-4.11 (m, 4H), 3.74 (s, 3H), 2.54-2.42 (m, 1H), 2.29-2.16 (m, 1H), 1.35-1.26 (m, 1H), 0.67-0.56 (m, 1H), 0.69-0.54 (m, 1H).
EXAMPLE 1
[0253] ##STR00123##
[0254] Synthetic Scheme:
##STR00124##
Step 1: Synthesis of Compound WX001_1
[0255] Compound B-1 (Intermediate B-1 was synthesized by the method for synthesizing methyl (1R,5S,6S)-3-azabicyclo[3.1.0]hex-6-ylacetate hydrochloride reported on page 53 of the patent WO2017115205A1) (3.7 g, 23.84 mmol) was dissolved in DCM (200.0 mL), lowered to −65° C., and a solution of Compound A-1 (4.9 g, 23.78 mmol) in DCM (100.0 mL) was slowly added dropwise, followed by slow dropwise addition of DIPEA (7.68 g, 59.45 mmol). The reaction was stirred at −65° C. for 1 h, then warmed to 25° C. and stirred for 2 h. After completion of the reaction, the solvent was removed by rotary evaporation to give a crude product. The crude product was purified by automated column chromatography (100-200 mesh, eluant: PE: EtOAc=100: 1-100: 50) to afford WX001_1 .sup.1H NMR (400 MHz, CDCl.sub.3) δ=8.68 (s, 1H), 4.78 (m, 1H), 4.26 (m, 1H), 4.04 (m, 1H), 3.74 (m, 1H), 3.71 (s, 3H), 2.42-2.27 (m, 2H), 1.69 (m, 1H), 1.65-1.50 (m, 1H), 0.96 (m, 1H).
Step 2: Synthesis of Compound WX001_2
[0256] Compound WX001_1 (3.9 g, 12.01 mmol) was added in batches to a solution of the hydrochloride salt of C-1 (1.5 g, 21.09 mmol) in THF (100.0 mL), followed by dropwise addition of DIPEA (3.10 g, 24.02 mmol), and the reaction was stirred at 65° C. for 12 h. After completion of the reaction, the solvent was removed by rotary evaporation to give a crude product. The crude product was purified by automated column chromatography (100-200 mesh, eluant: PE: EtOAc=100: 1-100: 50) to afford compound WX001_2. .sup.1H NMR (400 MHz, CDCl.sub.3) δ=8.27 (s, 1H), 4.71 (m, 1H), 4.51-4.40 (m, 1H), 4.18 (m, 1H), 4.08-3.85 (m, 3H), 3.80-3.60 (m, 4H), 2.42-2.26 (m, 3H), 1.96 (m, 1H), 1.64 (m, 2H), 1.54 (d, J=6.3 Hz, 3H), 1.02-0.93 (m, 1H).
Step 3: Synthesis of Compound WX001
[0257] Compound WX001_2 (4.0 g, 11.13 mmol) was dissolved in THF (100.0 mL) and H.sub.2O (100.0 mL), lithium hydroxide monohydrate (1.40 g, 33.38 mmol) was added, and the reaction was stirred at 20° C. for 4 h. After completion of the reaction, 200 mL water was added, and 1N hydrochloric acid was added to adjust pH to 4-5. The solvent was removed by rotary evaporation. The product was dissolved by addition of 25 mL DMSO, and filtered to remove the insoluble inorganic salts. The solution of the crude product in DMSO was purified by Prep-HPLC (separation method: Welch Xtimate C18 150 mm*25 mm*5 μm; Mobile phase: [water (0.225%FA)-ACN]; B(ACN)%: 45%-75%, 8 min) to afford Compound WX001.
EXAMPLES 35, 42, 43
[0258] ##STR00125##
[0259] Synthetic Scheme:
##STR00126##
Step 1: Synthesis of Compound WX035_1
[0260] Compound B-3 (40 g, 129.28 mmol) was dissolved in DCM (300.0 mL), cooled to 0° C., and a solution of Compound A-11 (27 g, 119.99 mmol) in DCM (200.0 mL) was slowly added dropwise, followed by slow dropwise addition of DIPEA (46.52 g, 359.97 mmol) slowly added dropwise. The reaction was stirred at 0° C. for 3 h. After completion of the reaction, the solvent was removed by rotary evaporation to dryness to give a crude product. The crude product was purified by automated column chromatography (100-200 mesh, eluant: PE: EtOAc=100: 1-100: 50) to afford WX035_ 1. .sup.1H NMR (400 MHz, CDCl3) δ=4.73-4.23 (m, 4H), 3.72 (d, J=5.5 Hz, 3H), 3.07-2.90 (m, 2H), 2.67-2.48 (m, 2H), 1.93 (br d, J=9.5 Hz, 1H), 1.72-1.51 (m, 1H), 1.75-1.49 (m, 1H).
Step 2: Synthesis of Compound WX035_2
[0261] Compound WX035_1 (35 g, 106.15 mmol) was added in batches to a solution of hydrochloride salt of C-1 (38.65 g, 127.38 mmol) in acetonitrile (350.0 mL), then K.sub.2CO.sub.3 (44 g, 318.44 mmol) was added dropwise, and the reaction was stirred at 80° C. for 12 h. The reaction was complete and then directly filtered, and the filtrate was rotary-evaporated to dryness to afford WX035_2. .sup.1H NMR (400 MHz, CDCl3) δ=4.49-4.35 (m, 2H), 4.33-4.17 (m, 3H), 4.07 (dt, J=5.0, 8.8 Hz, 1H), 3.74 (s, 3H), 2.87-2.77 (m, 2H), 2.53-2.36 (m, 3H), 1.97-1.86 (m, 2H), 1.50 (d, J=6.0 Hz, 3H), 1.39 (t, J=5.5 Hz, 1H), 1.32-1.26 (m, 1H), 1.30-1.24 (m, 1H).
Step 3: Synthesis of Compound WX035
[0262] Compound WX035_2 (36 g, 98.8 mmol) was dissolved in THF (350.0 mL) and H.sub.2O (70.0 mL), lithium hydroxide monohydrate (8.29 g, 197.59 mmol) was added, and the reaction was stirred at 20° C. for 12 h. After completion of the reaction, 200 mL water was added, and 1N hydrochloric acid was added to adjust pH to 5-6. Extract was performed with ethyl acetate (300 mL). The solvent was removed by rotary evaporation. The product was dissolved by addition of 25 mL MeOH, and filtered to remove the insoluble inorganic salts. The solution of the crude product in DMSO was purified by Prep-HPLC (separation method: column type: Phenomenex Luna C8 250*50 mm*10 μm; Mobile phase: [H.sub.2O(0.1% TFA)-MeOH]; B%: 5%-60%, 25 min) to afford Compound WX035.
Step 3: Synthesis of Compounds WX042 & 043
[0263] Compound WX035 (35 g, 99.9 mmol) was separated by SFC (column type: DAICEL CHIRALPAK IG (250 mm*30 mm, 10 μm); Mobile phase: B: [0.1% NH.sub.3H.sub.2O MeOH]; B%: 40%-40%, 8 min) to afford Compounds WX042 (ee%: 99.58%, RT =3.061 min) and WX043 (ee%: 98.76%, RT=3.922).
[0264] Examples in the following table were synthesized with reference to the synthesis method in Example 1 (replacing Compound A-1 with the parent core fragment A and replacing Compound B-1 with fragment B in Step 1, respectively).
TABLE-US-00001 Com- Exam- Parent core Structure of the target pound ple fragment A Fragment B compound No. 2
[0265] The compounds shown in the following table were obtained from racemic compounds by SFC resolution
TABLE-US-00002 Com- Retention Racemic Conditions for SFC Exam- pound time compound resolution ple Compound structure No. (min) WX009 Column: DAICEL CHIRALPAK IG (250 mm*30 mm, 10 μm); Mobile phase: A: CO.sub.2, B: [0.1% NH.sub.3H.sub.2O MeOH]; B %: 45%-45%, 8 min 19
Example 23
[0266] ##STR00218##
[0267] Synthetic Scheme:
##STR00219##
Step 1: Synthesis of Compound WX023_2
[0268] Compound A-12 (10 g, 48.77 mmol) and NaOMe (21.08 g, 390.12 mmol) were dissolved in MeOH (200 mL), and the reaction was stirred at 80° C. for 12 h under nitrogen protection. TLC (petroleum ether: ethyl acetate =3: 1) showed formation of a new spot. The reaction liquid was directly rotary-evaporated to dryness, added with water (60 mL), and extracted with ethyl acetate (80 mL×2). The organic phases were combined and rotary-evaporated to dryness to afford WX023_2.
Step 2: Synthesis of Compound WX023_3
[0269] Compound WX023_2 (1 g, 5.10 mmol) and Br.sub.2 (1.22 g, 7.64 mmol, 394.07 μL) were dissolved in HOA.sub.C (20 mL), and the reaction was stirred at 120° C. for 12 h under nitrogen protection. TLC (petroleum ether: ethyl acetate=3: 1) showed formation of a new spot. The reaction liquid was cooled to room temperature, directly rotary-evaporated to dryness, and added with water (20 mL) with stirring for 30 min. The mixture was filtered, and the filter cake was washed with water (10 mL) and collected to afford Compound WX023_3.
Step 3: Synthesis of Compound WX023_4
[0270] To a 50 mL single-neck flask, Compound WX023_3 (900.00 mg, 3.64 mmol) and DIPEA (1.41 g, 10.93 mmol) were added, followed by POCl.sub.3 (14.85 g, 96.85 mmol), and the reaction was stirred at 120° C. for 12 h under nitrogen protection. TLC (petroleum ether: ethyl acetate=3: 1) showed formation of a new spot. The reaction liquid was slowly added to water (50 mL) and extracted with dichloromethane (50 mL×3). The filtrate was directly rotary-evaporated to dryness. The crude product was purified by flash silica column (ISCO®; 40 g SepaFlash® silica column, eluant: 0-15% EtOAc/PE, flow rate: 35 mL/min) to afford Compound WX023_4.
Step 4: Synthesis of Compound WX023_5
[0271] Compound WX023_4 (0.2 g, 704.32 μmol), Intermediate B-1 (131.17 mg, 845.19 μmol), and DIPEA (273.08 mg, 2.11 mmol) were dissolved in DCM (2 mL), and the reaction was stirred at 40° C. for 12 h under nitrogen protection. TLC (petroleum ether: ethyl acetate =3: 1) showed formation of a new spot. The reaction liquid was filtered directly. The filter cake was washed with ethyl acetate (20 mL) and the filtrate was directly rotary-evaporated to dryness to afford Compound WX023_5.
Step 5: Synthesis of Compound WX023_6
[0272] Compound WX023_5 (0.2 g, 496.66 μmol), Intermediate C-1 (166.00 mg, 547.10 μmol), and K.sub.2CO.sub.3 (204.00 mg, 1.48 mmol) were dissolved in acetonitrile (3 mL), and the reaction was stirred at 90° C. for 12 h under nitrogen protection. LCMS showed disappearance of starting material signal and generation of product signal. The reaction liquid was filtered. The filter cake was washed with ethyl acetate (20 mL) and the filtrate was rotary-evaporated to dryness to afford Compound WX023_6.
Step 6: Synthesis of Compound WX023_7
[0273] Compound WX023_6 (0.2 g, 457.30 μmol) and Zn(CN).sub.2 (161.09 mg, 1.37 mmol, 87.08 μL) were dissolved in DMA (2 mL), tri-tert-butylphosphine palladium (233.70 mg, 457.30 μmol) was added under nitrogen protection, and the reaction was stirred at 130° C. for 12 h under nitrogen protection. LCMS showed disappearance of starting material signal and generation of product signal. The reaction liquid was filtered. The filter cake was washed with ethyl acetate (20 mL), and the filtrate was rotary-evaporated to dryness and purified by a flash silica column (ISCO®; 12 g SepaFlash® silica column, eluant: 0-20% EtOAc/PE, flow rate: 35 mL/min) to afford Compound WX023_7.
Step 7: Synthesis of Compound WX023
[0274] Compound WX023_7 (120 mg, 312.93 μmol) and LiOH.H.sub.2O (39.40 mg, 938.80 μmol) were dissolved in THF (2 mL)/H.sub.2O(2 mL), and the reaction was stirred at 20° C. for 12 h under nitrogen protection. LCMS showed disappearance of starting material signal and generation of product signal. The reaction was adjusted to pH 7-8 with 1M dilute hydrochloric acid and extracted with ethyl acetate (20 mL). The organic phase was rotary-evaporated to dryness. The residue was purified by HPLC (column type: Welch Xtimate C18 100 mm*25 mm*3 μm; Mobile phase: [water (0.225% FA)-ACN]; B(ACN)%: 6%-46%, 8 min) to afford Compound WX023.
Example 5
[0275] ##STR00220##
[0276] Synthetic Scheme:
##STR00221##
Step 1: Synthesis of Compound WX005_2
[0277] A-2 (100 mg, 485.31 μmol) was dissolved in DCM (20 mL) at 25° C. and then cooled to −78° C., hydrochloride salt of C-1 (51.93 mg, 485.31 μmol) and DIPEA (188.17 mg, 1.46 mmol, 253.59 mL) were added slowly (within 15 min), and the reaction returned to 25° C. and was stirred for 2 h. Detection by TLC (PE: EtOAc=10: 1) showed the reaction was complete. The solvent was directly concentrated under reduced pressure to afford a crude product. The crude product was purified by automated column chromatography (100-200 mesh, eluant: PE: EtOAc=100: 1 to 100: 50) to afford Compound WX005_1. LCMS (5-95/1.5 min): 0.930 min, [M+H].sup.+=240.9.
Step 2: Synthesis of Compound WX005_2
[0278] WX005_1 (100 mg, 415.43 μmol) was dissolved in THF (15 mL) at 25° C., B-1 (64.47 mg, 415.43 μmol) and DIPEA (161.08 mg, 1.25 mmol, 217.08 mL) were added, and the reaction was stirred at 70° C. for 24 h. Detection by TLC (PE: EtOAc=10: 1) showed the reaction was complete. The solvent was directly concentrated under reduced pressure to afford a crude product. The crude product was purified by automated column chromatography (100-200 mesh, eluant: PE: EtOAc=100: 1-100: 50) to afford Compound WX005_2. LCMS (10-80/7 min): 2.516 min, [M+H].sup.+=360.2.
Step 3: Synthesis of Compound WX005
[0279] Compound WX005_2 (30 mg, 83.46 μmol) was dissolved in H.sub.2O (5 mL) and THF (5 mL) at 25° C., LiOH.H.sub.2O (10.51 mg, 250.39 μmol) was added, and the reaction was stirred at 25° C. for 0.5 h. Detection by TLC (PE: EtOAc=10:1) showed the reaction was complete. The solvent was directly concentrated under reduced pressure to afford a crude product. The crude product was purified by Prep-HPLC (separation method: Welch Xtimate C18 150 mm*25 mm*5 μm; Mobile phase: [water (0.225% FA)-ACN]; B (ACN)%: 45%-75%, 8 min) to afford Compound WX005.
Example 10
[0280] ##STR00222##
[0281] Synthetic Scheme:
##STR00223##
Step 1: Synthesis of Compound WX010_1
[0282] To a 10 mL thumb bottle, A-3 (5 g, 24.38 mmol) and tent-butyl carbamate (4.28 g, 36.57 mmol) were added and dissolved with THF (50 mL), and purged with nitrogen, Cs.sub.2CO.sub.3 (23.83 g, 73.15 mmol), Pd(dba).sub.2 (701.01 mg, 1.22 mmol) and Xantphos (705.41 mg, 1.22 mmol, 0.05 eq) were added, and purged with nitrogen, and the reaction was stirred at 80° C. for 12 h. The reaction was added to 60 mL water, and extracted with DCM (60 mL×2). The organic phases were combined, washed with saturated brine (60 mL), and rotary-evaporated to dryness to afford a crude product. The crude product was purified by automated column chromatography (100-200 mesh, eluant: PE: EtOAc=100: 1-100: 50) to afford WX.sub.010_1.
[0283] .sup.1H NMR (400 MHz, DMSO-d6) δ=10.74 (s, 1H), 7.82 (d, J=6.3 Hz, 1H), 7.74 (d, J=6.0 Hz, 1H), 1.52 (s, 9H).
Step 2: Synthesis of Compound WX010_2
[0284] To a 100 mL eggplant-shaped flask, WX010_1 (5 g, 17.50 mmol) was added and dissolved with DCM (50 mL), and then TFA (71.76 g, 629.32 mmol) was slowly added, and the reaction was stirred at 20° C. for 12 h. The reaction liquid was directly rotary-evaporated to dryness, added with ethyl acetate (50 mL), and washed with saturated sodium bicarbonate solution (30 mL×2). The organic phase was rotary-evaporated to dryness to afford WX010_2. .sup.1H NMR (400 MHz, DMSO-d6) δ=8.02 (br s, 2H), 7.62-7.52 (m, 2H).
Step 3: Synthesis of Compound WX010_3
[0285] To a 10 mL thumb bottle, WX010_2 (3 g, 16.16 mmol) and C-1 (1.24 g, 24.24 mmol) were added and dissolved with DMF (30 mL), the temperature was lowered to 0° C., DIPEA (6.27 g, 48.48 mmol) was added, and the reaction was stirred at 80° C. for 12 h. The reaction liquid was added to 20 mL water, and extracted with DCM (20 mL×2). The organic phases were combined and washed with saturated brine (20 mL). The organic phase was rotary-evaporated to dryness to afford a crude product. The crude product was purified by automated column chromatography (100-200 mesh, eluant: PE: EtOAc =100: 1-100: 50) to afford WX010_3. .sup.1H NMR (400 MHz, CDCl.sub.3) δ=6.96 (d, J=6.0 Hz, 1H), 6.82 (d, J=6.0 Hz, 1H), 5.36-4.97 (m, 2H), 4.59-4.42 (m, 1H), 4.17-3.91 (m, 2H), 2.42 (dtd, J=4.9, 8.6, 10.8 Hz, 1H), 1.97 (tdd, J=6.7, 8.9, 10.7 Hz, 1H), 1.55 (d, J=6.3 Hz, 3H).
Step 4: Synthesis of Compound WX010_4
[0286] Compound WX010_3 (220 mg, 1.01 mmol), D-1 (245 mg, 1.01 mmol), and cesium carbonate (989.60 mg, 3.04 mmol) were added to THF (5 mL) in a 10 mL thumb bottle under N.sub.2 protection, followed by addition of Pd.sub.2(dba).sub.3 (46.35 mg, 50.62 μmol) and Xantphos (29.29 mg, 50.62 μmol), and the reaction was stirred at 70° C. for 12 h. LCMS showed that the reaction was complete. The reaction liquid was filtered and washed with EtOAc (20 mL). After the filtrate was washed with water (20 mL), the organic phase was directly rotary-evaporated to dryness. The crude product was purified by automated column chromatography (100-200 mesh, eluant: PE: EtOAc =100: 1-100: 40) to afford Compound WX010_4.
Step 5: Synthesis of Compound WX010
[0287] To a 10 mL thumb bottle, Compound WX010_4 (68 mg, 183.56 μmol) was added and dissolved with THF (2 mL) and H.sub.2O (2 mL), then LiOH.H.sub.2O (7.70 mg, 183.56 μmol) was added, and the reaction was stirred at 20° C. for 1 h. LCMS showed that the reaction was complete. The reaction liquid was directly rotary-evaporated to dryness, dissolved by adding 3 mL MeOH, and filtered. The filtrate was purified by Prep-HPLC (separation method: Welch Xtimate C18 150 mm*25 mm*5 μm; Mobile phase: [water (0.225% FA)-ACN]; B (ACN)%: 45%-75%, 8 min) to afford Compound WX010.
[0288] Examples in the following table were synthesized with reference to the synthetic method in Example 10 (replacing Compound D-1 with fragment D in Step 1).
TABLE-US-00003 Structure of the target Compound Example Fragment D compound No. 11
Example 16
[0289] ##STR00234##
[0290] Synthetic Scheme:
##STR00235##
Step 1: Synthesis of Compound WX016_1
[0291] To a 50 mL reaction flask, E-1 (0.8 g, 3.29 mmol), F-1 (1.25 g, 4.94 mmol) were added and dissolved with dioxane (10 mL), potassium acetate (968.90 mg, 9.87 mmol) and Pd(dppf)Cl.sub.2 (134.37 mg, 183.64 μmol) were added, and the reaction was stirred at 90° C. for 12 h. LCMS showed that the reaction was completed. The reaction liquid was added with 20 mL water, and extracted with EtOAc (20 mL×2). The organic phases were combined, washed with saturated brine (20 mL), and then rotary-evaporated to dryness. The crude product was purified by automated column chromatography (100-200 mesh, eluant: PE: EtOAc=100: 1-100: 10) to afford Compound WX016_1. .sup.1H NMR (400 MHz, CDCl.sub.3) δ7.61-7.55 (m, 2H), 7.25-7.21 (m, 2H), 3.60 (s, 3H), 2.89 (t, J=8.0 Hz, 2H), 2.62-2.51 (m, 2H), 1.27 (s, 12H).
Step 2: Synthesis of Compound WX016_2
[0292] To a 50 mL reaction flask, Compound WX016_1 (0.2 g, 689.27 μmol) and A-3 (0.19 g, 921.76 μmol) were added and dissolved in toluene (1 mL)/EtOH (0.5 mL)/H2O(0.5 mL), K.sub.2CO.sub.3 (254.79 mg, 1.84 mmol, 2 eq) and Pd(PPh.sub.3).sub.4 (53.26 mg, 46.09 μmol) were added, and the reaction was stirred at 90° C. for 9 h. LCMS detected the product MS. The reaction was added to 20 mL water, and extracted with EtOAc (20 mL×2). The organic phases were combined, washed with saturated brine (20 mL). The organic phase was rotary-evaporated to dryness. The crude product was purified by automated column chromatography (100-200 mesh, eluant: PE: EtOAc=100: 1-100: 40) to afford Compound WX016_2.
Step 3: Synthesis of Compound WX016_3
[0293] To a 10 mL thumb bottle, Compound WX016_2 (115 mg, 348.11 μmol) and hydrochloride salt of C-1 (74.47 mg, 696.23 μmol) were added and dissolved with THF (1 mL), then DIPEA (121.27 mL, 696.23 μmol) was added, and the reaction was stirred at 70° C. for 9 h. LCMS showed that the reaction was complete. The reaction liquid was directly rotary-evaporated to dryness to afford crude WX016-3. The crude product was used directly in the next reaction.
Step 4: Synthesis of Compound WX016
[0294] To a 10 mL thumb bottle, Compound WX016_3 (115 mg, 316.31 μmol) was added and dissolved with THF (2 mL) and H.sub.2O (2 mL), then LiOH.H.sub.2O(13.27 mg, 316.31 μmol) was added, and the reaction was stirred at 20° C. for 12 h. LCMS showed the reaction was complete. The reaction liquid was directly rotary-evaporated to dryness, dissolved by addition of MeOH (3 mL) and filtered. The filtrate was purified by Prep-HPLC (separation method: Welch Xtimate C18 150 mm*25 mm*5 μm; Mobile phase: [water (0.225% FA)-ACN]; B(ACN)%: 45%-75%, 8 min) to afford Compound WX016.
[0295] Examples in the following table were synthesized with reference to the synthetic method in Example 16 (replacing Compound E-1 with fragment E in Step 1).
TABLE-US-00004 Structure of the target Compound Example Fragment E compound No. 17
Example 33
[0296] ##STR00240##
[0297] Synthetic Scheme:
##STR00241##
Step 1: synthesis of Compound WX033_1
[0298] To a 50 mL reaction flask, G-1 (200 mg, 819.39 μmol) and F-1 (312.11 mg, 1.23 mmol) were added and dissolved with dioxane (10 mL), potassium acetate (160.83 mg, 1.64 mmol) and Pd(dppf)Cl2 (59.96 mg, 81.94 μmol) were added, and the reaction was stirred at 90° C. for 12 h. LCMS showed that the reaction was completed. The reaction liquid was added with 20 mL water, and extracted with EtOAc (20 mL×2). The organic phases were combined, washed with saturated brine (20 mL), and rotary-evaporated to dryness. The crude Compound WX033_1 was used directly in the next step without purification.
Step 2: Synthesis of Compound WX033_2
[0299] To a 50 mL reaction flask, Compound WX033_1 (197.62 mg, 416.03 μmol) and H-1 (140 mg, 396.22 μmol) were added and dissolved with dioxane (8 mL), K.sub.3PO.sub.4 (2 M, 396.22 μL) and Pd(dppf)Cl.sub.2 (28.99 mg, 39.62 μmol) were added, and the reaction was stirred at 90° C. for 4 h. LCMS detected the product MS. The reaction was added to 20 mL water, and extracted with EtOAc (20 mL×2). The organic phases were combined, washed with saturated brine (20 mL), and rotary-evaporated to dryness. The crude product was purified by automated column chromatography (100-200 mesh, eluant: PE: EtOAc =100: 1-100: 50) to afford Compound WX033_2. .sup.1H NMR (400 MHz, CDCl.sub.3) δ8.70 (d, J=5.2 Hz, 1H), 7.71-7.67 (m, 1H), 7.63-7.61 (m, 1H), 7.29 (s, 1H), 7.07 (d, J=6.0 Hz, 1H), 4.63-4.58 (m, 1H), 4.25-4.15 (m, 2H), 3.70 (s, 3H), 3.25 (t, J=7.6 Hz, 2H), 2.89 (t, J=7.6Hz, 2H), 2.54-2.49 (m, 1H), 2.04-2.00 (m, 1H), 1.62 (d, J=6.4 Hz, 3H).
Step 3: Synthesis of Compound WX033
[0300] To a 10 mL thumb bottle, Compound WX033_2 (150 mg, 272.76 μmol) was added and dissolved with THF (2 mL) and H.sub.2O (2 mL), then sodium hydroxide (2 M, 681.91μ) was added, and the reaction was stirred at 20° C. for 12 h. LCMS showed that the reaction was complete. The reaction liquid was directly rotary-evaporated to dryness, dissolved by addition of MeOH (3 mL) and filtered. The filtrate was purified by Prep-HPLC (separation method: Welch Xtimate C18 150 mm*25 mm*5 μm; Mobile phase: [water (0.225% FA)-ACN]; B(ACN)%: 45%-75%, 8 min) to afford Compound WX033. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.69-8.60 (m, 1H), 7.88-7.79 (m, 1H), 7.77-7.69 (m, 1H), 7.48-7.39 (m, 1H), 7.32-7.23 (m, 1H), 4.68-4.55 (m, 1H), 4.24-4.04 (m, 2H), 3.22 (t, J=7.2 Hz, 2H), 2.84 (t, J=7.2 Hz, 2H), 2.63-2.48 (m, 1H), 2.14-2.03 (m, 1H), 1.61 (d, J=6.4 Hz, 3H).
[0301] Examples in the following table were synthesized with reference to the synthetic method in Example 33 (replacing Compound G-1 with fragment G in Step 1).
TABLE-US-00005 Compound Example Fragment G Target compound structure No. 39
[0302] The .sup.1H NMR and MS data for each Example are shown in Table 1:
TABLE-US-00006 TABLE 1 .sup.1H NMR and MS Data MS m/z: Compound Calculated (M)/ Example No. .sup.1H NMR Found (M + H) 1 WX001 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.45(s, 1H), 4.76- 345.1/346.3 4.53(m, 2H), 4.49-4.40(m, 1H), 4.14(m, 1H), 4.02- 3.90(m, 2H), 3.60(m, 1H), 2.46-2.35(m, 1H), 2.21(m, 2H), 2.02-1.90(m, 1H), 1.57(m, 2H), 1.52(d, J = 6.0 Hz, 3H), 0.89(m, 1H) 2 WX002 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.47(s, 1H), 4.61(br s, 345.1/346.3 1H), 4.40-4.52(m, 1H), 4.21(br s, 1H), 3.87-4.06(m, 3H), 3.63(br s, 1 H), 2.36-2.53(m, 1H), 2.22(br s, 2H), 1.93-2.04(m, 1H), 1.58(br s, 2H), 1.54(d, J = 6.27 Hz, 3H), 0.91(dt, J = 6.78, 3.64 Hz, 1H) 3 WX003 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.65(s, 1H), 7.80(br d, 355.1/356.4 J = 8.3 Hz, 2H), 7.32(br d, J = 8.3 Hz, 2H), 4.60-4.48(m, 1H), 4.12-3.96(m, 2H), 3.60(s, 2H), )2.57-2.42(m, 1H), 2.07-1.98(m, 1H), 1.57(br d, J = 6.3 Hz, 3H) 4 WX004 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 12.10(br s, 1H), 7.39(d, 344.1/344.9 J = 6.0 Hz, 1H), 6.98(d, J = 6.0 Hz, 1H), 4.37-4.27(m, 1H), 4.03-3.93(m, 2H), 3.90-3.80(m, 2H), 3.80- 3.65(m, 2H), 2.36-2.29(m, 1H), 2.26(br d, J = 7.2 Hz, 2H), 1.90(quin, J = 8.4 Hz, 1H), 1.62(br s, 2H), 1.45(d, J = 6.0 Hz, 3H), 0.75(td, J = 3.6, 6.8 Hz, 1H) 5 WX005 .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.65(s, 1 H), 4.67(br s, 345.1/346.3 1 H), 4.02-4.49(m, 3 H), 3.71-3.87(m, 2 H), 3.39- 3.45(m, 2 H), 2.23(br s, 2 H), 1.99(br s, 1 H), 1.55(br d, J = 6.13 Hz, 3 H), 1.49(br s, 2 H), 0.72(dt, J = 6.72, 3.46 Hz, 1 H) 6 WX006 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.81(d, J = 8.5 Hz, 2H), 345.1/355.2 7.49(d, J = 6.0 Hz, 1H), 7.27(d, J = 8.5 Hz, 2H), 7.01(d, J = 6.0 Hz, 1H), 4.54-4.42(m, 1H), 4.11-3.92(m, 2H), 3.59(s, 2H), 2.55-2.39(m, 1H), 2.07-1.97(m, 1H), 1.54(d, J = 6.0 Hz, 3H) 7 WX007 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.60(d, J = 8.5 Hz, 2H), 368.1/369.0 7.37(d, J = 8.3 Hz, 2H), 6.97(s, 1H), 4.67-4.55(m, 1H), 4.26-4.10(m, 2H), 3.66(s, 2H), 2.72(s, 3H), 2.66- 2.54(m, 1H), 2.15-2.03(m, 1H), 1.45(d, J = 6.3 Hz, 3H) 8 WX008 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 6.97(s, 1H), 4.79- 358.1/359.0 4.68(m, 1H),4.46(br d, J = 11.8Hz, 1H), 4.37(br d, J = 11.8 Hz, 1H), 4.27-4.12(m, 2H), 3.93-3.80(m, 2H), 2.71- 2.61(m, 1H), 2.51(s, 3H), 2.36-2.26(m, 2H), 2.19- 2.08(m, 1H), 1.61(d, J = 6.3 Hz, 5H), 0.63(tt, J = 3.4, 7.0 Hz, 1H) 9 WX009 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.12(d, J = 5.8 Hz, 1H), 330.1/331.1 6.92(d, J = 6.0 Hz, 1H), 4.55-4.28(m, 5H), 4.03-3.88(m, 2H), 2.47-2.33(m, 1H), 2.01-1.91(m, 1H), 1.86- 1.75(m, 1H), 1.50(dd, J = 2.1, 6.1 Hz, 3H), 1.31(t, J = 5.0 Hz, 1H), 1.16(dd, J = 4.8, 8.3 Hz, 1H) 10 WX010 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.96(dd, J = 1.8, 12.8 Hz, 372.1/373.2 1H), 7.58-7.41(m, 2H), 7.24(t, J = 8.5 Hz, 1H), 7.05(d, J = 6.0 Hz, 1H), 4.61-4.46(m, 1H), 4.19-3.93(m, 2H), 3.64(s, 2H), 2.57-2.39(m, 1H), 2.07-1.94(m, 1H), 1.56(d, J = 6.3 Hz, 3H) 11 WX011 .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.90(s, 1H), 8.78(s, 365.1/366.1 1H), 8.13-7.99(m, 2H), 7.68(d, J = 6.0 Hz, 1H), 7.21(d, J = 6.0 Hz, 1H), 4.51-4.40(m, 1H), 4.03-3.92(m, 2H), 2.45-2.38(m, 1H), 2.02-1.90(m, 1H), 1.52(br d, J = 6.0 Hz, 3H) 12 WX012 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.05-7.96(m, 4H), 340.1/341.2 7.57(d, J = 5.8 Hz, 1H), 7.11(d, J = 6.0 Hz, 1H), 4.64- 4.53(m, 1H), 4.21-4.01(m, 2H), 2.63-2.43(m, 1H), 2.14- 1.97(m, 1H), 1.58(d, J = 6.0 Hz, 3H) 13 WX013 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.68(s, 1H), 8.04(dd, 408.1/109.2 J = 2.0, 8.5 Hz, 1H), 7.88(d, J = 8.5 Hz, 1H), 7.55(d, J = 6.0 Hz, 1H), 7.08(d, J = 6.0 Hz, 1H), 4.64-4.47(m, 1H), 4.21- 3.95(m, 2H), 2.61-2.45(m, 1H), 2.11-1.94(m, 1H), 1.55(d, J = 6.3 Hz, 3H) 14 WX014 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.65(br d, J = 10.0 Hz, 390.1/391.2 2H), 7.47(d, J = 6.0 Hz, 1H), 7.03(d, J = 6.0 Hz, 1H), 4.59- 4.40(m, 1H), 4.16-3.91(m, 2H), 3.64(s, 1H), 3.75- 3.58(m, 1H), 3.38-3.28(m, 1H), 3.26(br s, 1H), 2.56- 2.41 (m, 1H), 2.04-1.93(m, 1H), 1.56(d, J = 6.3 Hz, 3H) 15 WX015 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.77(d, J = 11.3 Hz, 2H), 376.1/377.0 7.54(d, J = 6.0 Hz, 1H), 7.1 l(d, J = 6.0 Hz, 1H), 4.65- 4.53(m, 1H), 4.66-4.53(m, 1H), 4.17-4.12(m, 1H), 4.10- 4.02(m, 1H), 2.60-2.50(m, 1H), 2.10-2.03(m, 1H), 1.61(d, J = 6.3 Hz, 3H) 16 WX016 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.78-7.72(m, 2H), 7.50- 353.1/354.1 7.42(m, 2H), 7.37(d, J = 6.4 Hz, 1H), 7.20(d, J = 6.0 Hz, 1H), 4.65-4.56(m, 1H), 4.19-4.12(m, 1H), 4.12- 4.05(m, 1H), 3.05(t, J = 7.6 Hz, 2H), 2.70(t, J = 7.6 Hz, 2H), 2.52(dtd, J = 5.2, 8.8, 10.8 Hz, 1H), 2.11-2.04(m, 1H), 1.61(d, J = 6.4 Hz, 3H) 17 WX017 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.10(s, 1H), 7.95(d, J = 351.1/352.1 7.6 Hz, 1H), 7.84-7.74(m, 2H), 7.67-7.59(m, 1H), 7.38(d, J = 6.4 Hz, 1H), 7.25(d, J = 6.0 Hz, 1H), 6.59(d, J = 16.0 Hz, 1H), 4.66-4.57(m, 1H), 4.22-4.08(m, 2H), 2.60-2.47(m, 1H), 2.12-2.05(m, 1H), 1.62(d, J = 6.4 Hz, 3H) 18 WX018 .sup.1H NMR (400 MHz, CD.sub.3OD δ 7.13(d, J = 6.02 Hz, 1H), 318.1/319.0 6.94(d, J = 6.02 Hz, 1H), 4.31-4.62(m, 3H), 3.85- 4.13(m, 4H), 2.97-3.21(m, 1H), 2.74(d, J = 7.78 Hz, 2H), 2.43(dtd, J = 10.85, 8.56, 8.56, 4.89 Hz, 1H), 1.90- 2.08(m, 1H), 1.52(d, J = 6.27 Hz, 3H) 19 WX019 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.12(d, J = 6.0 Hz, 1H), 330.1/331.1 6.93(d, J = 6.0 Hz, 1H), 4.56-4.31(m, 5H), 4.10-3.86(m, 2H), 2.43(dtd, J = 4.8, 8.5, 10.8 Hz, 1H), 2.06-1.89(m, 2H), 1.52(d, J = 6.3 Hz, 3H), 1.34(d, J = 7.3 Hz, 2H) 20 WX020 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.11(d, J = 6.0 Hz, 1H), 330.1/331.1 6.93(d, J = 6.0 Hz, 1H), 4.56-4.31(m, 5H), 4.10-3.84(m, 2H), 2.52-2.35(m, 1H), 2.08-1.85(m, 2H), 1.52(d, J = 6.0 Hz, 3H), 1.34(d, J = 7.5 Hz, 2H) 21 WX021 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 4.52-4.63(m, 1H), 346.1/347.2 4.38(t, J = 2.76 Hz, 2H), 3.98-4.15(m, 6H), 3.69-3.84(m, 2H), 2.64-2.82(m, 1H), 2.49-2.61(m, 1H), 2.33(d, J = 7.03 Hz, 2H), 1.99-2.14(m, 2H), 1.50-1.56(m, 3H), 1.35-1.47(m, 1H) 22 WX022 .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.67(s, 1H), 7.56(br d, 357.1/358.2 J = 8.8 Hz, 1H), 7.43(s, 2H), 7.35(br d, J = 9.5 Hz, 1H), 4.58-4.46(m, 1H), 4.10-3.97(m, 2H), 3.78(s, 2H), 2.48- 2.39(m, 1H), 2.06-1.94(m, 1H), 1.54(d, J = 6.3 Hz, 3H) 23 WX023 .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.13(s, 1H), 4.58-4.41(m, 369.1/370.1 1H), 4.15(t, J = 11.0 Hz, 2H), 4.10-3.96(m, 2H), 3.80(br t, J = 9.5 Hz, 2H), 2.46-2.33(m, 3H), 2.03-1.93(m, 1H), 1.68(br s, 2H), 1.56(d, J = 6.0 Hz, 3H), 1.03(tt, J = 3.5, 7.0 Hz, 1H) 24 WX024 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.94(br d, J = 8.5 Hz, 1H), 356.1/357.1 7.50-7.41(m, 1H), 7.23-7.15(m, 1H), 4.72-4.62(m, 1H), 4.40-4.30(m, 2H), 4.30-4.20(m, 1H), 4.28- 4.08(m, 1H), 4.01(br t, J = 10.2 Hz, 2H), 2.60-2.49(m, 1H), 2.28(br d, J = 7.0 Hz, 2H), 2.10-2.00(m, 1H), 1.71 (br s, 2H), 1.58(d, J = 6.3 Hz, 3H), 0.81(td, J = 3.4, 6.6 Hz, 1H) 25 WX025 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.39(d, J = 6.1 Hz, 1H), 332.1/333.0 6.92(d, J = 6.1 Hz, 1H), 4.57-4.43(m, 1H), 4.12-4.01(m, 2H), 3.94(td, J = 8.4, 16.4 Hz, 2H), 3.76(br d, J = 7.3 Hz, 1H), 3.43(brt, J = 9.4 Hz, 1H), 2.68(td, J = 7.4, 14.8 Hz, 1H), 2.51(d, J = 7.3 Hz, 2H), 2.47-2.39(m, 1H), 2.26(br d, J = 4.8 Hz, 1H), 2.07-1.93(m, 1H), 1.82-1.70(m, 1H), 1.54(d, J = 6.1 Hz, 3H) 26 WX026 .sup.1H NMR (400 MHz, CDCh) δ 4.43-4.25(m, 1H), 4.03- 346.1/347.1 3.90(m, 2H), 3.90-3.82(m, 2H), 3.62-3.36(m, 2H), 3.36- 3.21(m, 2H), 3.14(br t, J = 7.8 Hz, 2H), 2.31(br d, J = 4.9 Hz, 1H), 2.26(br d, J = 7.3 Hz, 2H), 1.99-1.78(m, 1H), 1.44(br s, 2H), 1.40(d, J = 6.1 Hz, 3H), 0.89(td, J = 3.6, 6.8 Hz, 1H) 27 WX027 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.38(t, J = 6.0 Hz, 1H), 344.1/345.1 6.90(d, J = 6.0 Hz, 1H), 4.46(sxt, J = 6.4 Hz, 1H), 4.06- 3.87(m, 5H), 3.85-3.68(m, 1H), 2.49-2.32(m, 1H), 2.30- 1.88(m, 4H), 1.53(d, J = 6.0 Hz, 3H), 1.37-1.33(m, 1H), 1.27(brs, 1H) 28 WX028 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.21(d, J = 3.3 Hz, 1H), 362.1/363.0 4.76-4.64(m, 1H), 4.29-3.78(m, 6H), 2.69-2.58(m, 1H), 2.37(d, J = 7.0 Hz, 2H), 2.17-2.07(m, 1H), 1.77(br s, 1H), 1.87-1.62(m, 1H), 1.60(d, J = 6.3 Hz, 3H), 0.88(tt, J = 3.5, 7.1 Hz, 1H) 29 WX029 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.53(s, 1H), 4.74- 378.1/379.0 4.63(m, 1H), 4.29-3.74(m, 3H), 4.29-3.74(m, 1H), 4.29-3.74(m, 1H), 4.29-3.74(m, 1H), 2.67-2.57(m, 1H), 2.37(d, J = 7.0 Hz, 2H), 2.17-2.07(m, 1H), 1.77(br s, 2H), 1.59(d, J = 6.3 Hz, 3H), 0.88(tt, J = 3.5, 7.0 Hz, 1H) 30 WX030 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.21(d, J = 3.5 Hz, 1H), 362.1/363.0 4.75.4.64(m, 1H), 4.26-3.74(m, 6H), 2.70-2.58(m, 1H), 2.37(d, J = 7.3 Hz, 2H), 2.19-2.07(m, 1H), 1.77(br s, 2H), 1.60(d, J = 6.3 Hz, 3H), 0.88(tt, J = 3.5, 7.0 Hz, 1H) 32 WX032 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.30(d, J = 6.0 Hz, 1H), 318.1/319.1 6.93(d, >6.0 Hz, 1H), 4.88-4.77(m, 1H), 4.54-4.41(m, 1H), 4.08-3.88(m, 2H), 3.19-3.03(m, 1H), 2.79- 2.62(m, 2H), 2.51-2.36(m, 3H), 2.03-1.90(m, 1H), 1.56(d, >6.3 Hz, 3H) 33 WX033 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.69-8.60(m, 1H), 7.88- 354.1/354.8 7.79(m, 1H), 7.77-7.69(m, 1H), 7.48-7.39(m, 1H), 7.32- 7.23(m, 1H), 4.68-4.55(m, 1H), 4.24-4.04(m, 2H), 3.22(t, J = 7.2 Hz, 2H), 2.84(t, J = 7.2 Hz, 2H), 2.63- 2.48(m, 1H), 2.14-2.03(m, 1H), 1.61(d, J = 6.4 Hz, 3H) 34 WX034 .sup.1H NMR (400 MHz, CD.sub.3OD) δ ppm 7.58(d, J = 8.28 Hz, 342.1/343.2 1H), 7.48(dd, >10.79, 8.03 Hz, 1H), 7.22(td, >8.03, 5.02 Hz, 1H), 4.43-4.80(m, 5H), 4.27(td, >9.16, 5.52 Hz, 1H), 4.08-4.19(m, 1H), 2.49-2.65(m, 1H), 1.91- 2.15(m, 2H), 1.57(dd, >6.27, 2.51 Hz, 3H), 1.27- 1.40(m,2H) 35 WX035 H NMR (400 MHz, CD.sub.3OD) δ ppm 4.22-4.50(m, 5H), 350.1/351.1 3.86-4.12(m, 2H), 2.88(br d, >3.76 Hz, 2H), 2.32- 2.55(m,3H), 1.84-2.04(m, 2H), 1.49(dd,>6.02, 1.51 Hz, 3H), 1.22-1.37(m, 2H) 36 WX036 .sup.1H NMR (400 MHz, CDCh) δ 6.84(s, 1H), 4.70(br d, 364.1/365.0 J = 6.0 Hz, 2H), 4.48-4.18(m, 4H), 2.61(br d, >8.3 Hz, 1H), 2.06(br s, 2H), 1.56(dd, >2.9, 6.1 Hz, 3H), 1.51(br d, >3.8 Hz, 1H), 1.46-1.39(m, 1H), 1.46-1.39(m, 1H) 37 WX037 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 6.60(d, J = 4.0 Hz, 1H), 348.1/348.9 4.44.4.19(m, 5H), 4.01-3.85(m, 2H), 2.49-2.35(m, 1H), 2.03-1.89(m, 2H), 1.50(dd, J = 1.6, 6.0 Hz, 3H), 1.37-1.32(m, 2H) 38 WX038 H NMR (400 MHz, CDCl.sub.3) δ 6.84(d, J = 2.5 Hz, 1H), 4.73- 378.1/379.0 4.41(m, 3H), 4.37-4.09(m, 4H), 3.40-3.30(m, 1H), 2.63-2.50(m, 1H), 2.37-2.22(m, 3H), 2.15-1.97(m, 2H), 1.55(dd, J = 4.3, 6.0 Hz, 3H) 39 WX039 H NMR(400 MHz, CD.sub.3OD) δ 8.70(s, 1H), 8.45(d, J = 2.5 356.1/357.2 Hz, 1H), 7.90(s, 1H), 7.40(d, J = 6.1 Hz, 1H), 7.29(d, J = 6.1 Hz, 1H), 4.81-4.94(m, 2H),4.62(br dd, J = 6.6, 13.8 Hz, 1H), 4.21-4.07(m, 2H), 2.59-2.50(m, 1H), 2.14- 2.05(m, 1H), 1.62(d, J = 6.1 Hz, 3H) 40 WX040 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.63(br d, J = 8.78 Hz, 342.1/343.2 1H), 7.36-7.52(m, 1H), 7.17(br d, J = 5.02 Hz, 1H), 4.47- 4.79(m, 5H), 4.03-4.32(m, 2H), 2.52(br s, 1H), 1.84- 2.10(m, 2H), 1.57(d, J = 6.02 Hz, 3H), 1.18-1.41(m, 2 H) 41 WX041 H NMR (400 MHz, CD.sub.3OD) δ 7.57(br d, J = 8.28 Hz, 1H), 342.1/343.2 7.40(dd, J = 10.92, 7.91 Hz, 1H), 7.03-7.25(m, 1 H), 4.34- 4.78(m, 5 H), 4.01-4.26(m, 2 H), 2.44-2.59(m, 1 H), 1.77-2.09(m, 2 H), 1.54(d, J = 6.27 Hz, 3 H), 1.05- 1.36(m, 2H) 42 WX042 H NMR (400 MHz, CD.sub.3OD) δ 4.20-4.67(m, 5H),3.84- 350.1/351.1 4.06(m, 2 H), 2.88(br s, 2 H), 2.31-2.59(m,3 H), 1.80- 2.02(m, 2 H), 1.48(d, J = 6.27 Hz, 3 H), 1.21-1.33(m, 2 H) 43 WX043 H NMR (400 MHz, CD.sub.3OD) δ 4.23-4.67(m, 5 H), 3.84- 350.1/351.1 4.07(m, 2 H), 2.88(br s, 2 H), 2.31-2.57(m, 3 H), 1.79- 2.01 (m, 2 H), 1.48(d, J = 6.27 Hz, 3 H), 1.13-1.35(m, 2 H) 44 WX044 H NMR (400 MHz, CDCl.sub.3) δ 6.71(s, 1H), 4.48-4.35(m, 364.1/365.0 3H), 4.28(q, J = 8.7 Hz, 2H), 4.08-3.90(m, 2H), 2.42- 2.29(m, 1H), 1.95-1.83(m, 2H), 1.43(d, J = 6.0 Hz, 3H), 1.39(t, J = 5.5 Hz, 1H), 1.28(dd, J = 5.5, 8.8 Hz, 1H) 45 WX045 H NMR (400 MHz, CDCl.sub.3) δ 6.73(s, 1H), 4.37-4.19(m, 364.1/365.0 5H), 4.00-3.83(m, 2H), 2.37-2.23(m, 1H), 1.91- 1.79(m, 2H), 1.42(d, J = 6.0 Hz, 3H), 1.40-1.36(m, 1H), 1.28(dd, J = 5.4, 8.7 Hz, 1H) 46 WX046 .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.96(d, J = 5.6 Hz, 344.1/345.1 1H), 7.10(d, J = 5.6 Hz, 1H),4.37-4.23(m, 1H),4.01(br t, J = 10.4 Hz, 2H), 3.87-3.78(m, 2H), 3.74(br d, J = 10.4 Hz, 2H), 2.32(br d, J = 8.0 Hz, 1H), 2.27(br d, J = 7.2 Hz, 2H), 1.95-1.85(m, 1H), 1.63(br s, 2H), 1.46(d, J = 6.4 Hz, 3H), 0.85-0.76(m, 1H) 47 WX047 .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.23(d, J = 7.8 Hz, 1H), 354.1/355.1 8.12(d, J = 6.0 Hz, 1H), 7.79(t, J = 7.8 Hz, 1H), 7.31(d, J = 7.5 Hz, 1H), 7.05(d, J = 6.0 Hz, 1H), 4.70-4.59(m, 1H), 4.27-4.10(m, 2H), 3.30-3.20(m, 2H), 2.96(t, J = 7.2 Hz, 2H), 2.50(dtd, J = 5.0, 8.6, 10.9 Hz, 1H), 2.11-2.02(m, 1H), 1.64(d, J = 6.3 Hz, 3H) 48 WX048 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.91(s, 1H)8.62(s, 1H), 354.1/355.1 8.27(s, 1 H), 7.40(d, J = 6.13 Hz, 1H), 7.28(d, J = 6.00 Hz, 1H), 4.51-4.78(m, 1H), 4.04-4.23(m, 2H), 3.01- 3.21(m, 2H), 2.76(t, J = 7.19 Hz, 2H), 2.47-2.68(m, 1H), 2.02-2.16(m, 1H), 1.62(d, J = 6.25 Hz, 3H) 49 WX049 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 6.87(s, 1H), 4.39- 378.1/379.1 4.18(m, 4H), 4.12(br s, 1H), 3.88(dt, J = 5.2, 8.8 Hz, 1H), 3.80(q, J = 8.4 Hz, 1H), 2.36-2.23(m, 1H), 1.92- 1.79(m, 1H), 1.46(d, J = 5.2 Hz, 1H), 1.39(dd, J = 1.2, 6.0 Hz, 3H), 1.18(s, 3H), 0.91(d, J = 5.2 Hz, 1H) 50 WX050 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.75-7.61(m, 2H), 7.10(t, 358.1/359.1 J = 7.8 Hz, 1H), 4.62(br d, J = 6.0 Hz, 4H), 4.29-4.00(m, 2H), 2.52(dtd, J = 5.3, 8.7, 10.9 Hz, 1H), 2.12-1.91(m, 2H), 1.57(dd, J = 1.8, 6.3 Hz, 3H), 1.40-1.30(m, 2H), 1.26-1.22(m, 1H) 51 WX051 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.27-7.21(m, 1H), 4.80- 378.1/379.1 4.60(m, 1H), 4.47-4.38(m, 1H), 4.09-3.87(m, 2H), 2.64- 2.36(m, 5H), 2.10-1.88(m, 1H), 1.73-1.59(m, 1H), 1.58-1.45(m, 3H), 1.28-1.01(m, 2H) 54 WX054 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.15(d, J = 6.0 Hz, 1H), 344.1/345.1 6.92(d, J = 6.0 Hz, 1H), 4.54-4.26(m, 5H), 4.09-3.87(m, 2H), 2.49-2.33(m, 2H), 2.28-2.16(m, 1H), 2.05- 1.91(m, 1H), 1.52(d, J = 6.0 Hz, 3H), 1.31(quin, J = 7.3 Hz, 1H), 1.01(dd, J = 6.0, 9.0 Hz, 1H), 0.57(t, J = 5.8 Hz, 1H) 55 WX055 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.16(d, J = 6.0 Hz, 1H), 344.1/345.1 6.93(d, J = 6.0 Hz, 1H), 4.53-4.27(m, 5H), 4.10-3.87(m, 2H), 2.49-2.33(m, 2H), 2.28-2.17(m, 1H), 2.06- 1.91(m, 1H), 1.52(d, J = 6.0 Hz, 3H), 1.37-1.26(m, 1H), 1.01(dd, J = 6.0, 9.0 Hz, 1H), 0.57(t, J = 5.8 Hz, 1H) 56 WX056 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.03(s, 1H), 4.53- 378.1/379.1 4.19(m, 5H), 4.10-3.84(m, 2H), 2.49-2.33(m, 2H), 2.29- 2.17(m, 1H), 1.97(tdd, J = 7.0, 8.8, 10.9 Hz, 1H), 1.51(d, J = 6.5 Hz, 3H), 1.37-1.23(m, 1H), 1.01(dd, J = 5.5, 9.0 Hz, 1H), 0.57(t, J = 5.8 Hz, 1H) 57 WX057 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.04(s, 1H), 4.55- 378.1/379.1 4.18(m, 5H), 4.08-3.87(m, 2H), 2.51-2.35(m, 2H), 2.28- 2.18(m, 1H), 2.04-1.90(m, 1H), 1.51(d, J = 6.0 Hz, 3H), 1.36-1.23(m, 1H), 1.01(dd, J = 5.8, 8.8 Hz, 1H), 0.57(t, J = 5.8 Hz, 1H)
BIOLOGICAL TEST DATA
Experimental Example 1: Ketohexokinase Assay (KHK assay)
[0303] A. Main materials
[0304] 1. EnVision multilabel reader, Perkin Elmer;
[0305] 2. OptiPlate 384-well microplate, Perkin Elmer, Cat. No. 6007290;
[0306] 3. Recombinant Human Ketohexokinase (KHK), R&D Cat. No.: 8177-HK-020, Lot No.: DDFK0117092;
[0307] 4. Fructose (D(-)-Fructose), SCR Cat. No.: 36003034; and
[0308] 5. ADP-Glo kinase assay kit, Promega_Cat. No.: V9101.
[0309] B. Methods
[0310] a) Kinase reaction
[0311] 1. preparation of Dilution Buffer containing 50 mM hydroxyethylpiperazine ethanethiosulfonic acid (Hepes), 140 mM KCl, 3.5 mM MgCl.sub.2, and 0.01% bovine serum albumin (BSA), pH 7.4.
[0312] 2. A working solution of ketohexokinase at 2.5 times concentration was prepared with Dilution Buffer, with 50 nM ketohexokinase and 12.5 mM fructose.
[0313] 3. A working solution of adenosine triphosphate (ATP) at 2.5 times concentration was prepared with Dilution Buffer, with a concentration of 250 μM.
[0314] 4. Each compound was diluted starting at a concentration of 500 μM, 3-fold diluted for 9 concentration points. The final concentration of the compound in the reaction system started from 10 μM and the final concentration of dimethyl sulfoxide (DMSO) was 2%.
[0315] 5. A 96-well plate was prepared as a reaction plate, and was incubated at room temperature for 5 min after adding 6 μL ketohexokinase working solution at 2.5 times concentration to each well and further adding 3 μL compound working solution to each well.
[0316] 6. The first well of each row was a positive control for a compound, i.e. the same volume of the buffer was added to replace the compound and ketohexokinase. The last well was a negative control for the compound, i.e. the same volume of Dilution Buffer was added instead of the compound.
[0317] 7. 6 μL ATP working solution was added to each well in a 96-well reaction plate to initiate the kinase reaction. The kinase reaction was incubated in a constant temperature heater at 28° C. for 1 h.
[0318] b) ADP-Glo assay
[0319] 1. A384 plate was prepared as a detection plate, and firstly 5 μL ADP-Glo reagent was added.
[0320] 2. 5 μL kinase reaction mixture from the reaction plate was added to each well and incubated in a constant temperature heater at 28° C. for 30 min.
[0321] 3. 10 μL kinase detection reagent was added to each well and incubated in a constant temperature heater at 28° C. for 30 min.
[0322] 4. The detection plate was placed in an EnVision multilabel reader to read the chemiluminescent signal.
[0323] C. Experimental results:
TABLE-US-00007 TABLE 2 results of in vitro activity assay on KHK Compound No. KHK IC.sub.50 WX001 180 nM WX002 190 nM WX003 110 nM WX004 49 nM WX005 650 nM WX006 25 nM WX007 16 nM WX008 210 nM WX009 100 nM WX010 15 nM WX011 17 nM WX012 9.4 nM WX013 17 nM WX014 14 nM WX015 6.7 nM WX016 32 nM WX017 20 nM WX018 390 nM WX019 110 nM WX020 130 nM WX021 160 nM WX022 200 nM WX023 210 nM WX024 61 nM WX025 240 nM WX026 91 nM WX027 250 nM WX028 50 nM WX029 28 nM WX030 31 nM WX032 150 nM WX033 71 nM WX034 110 nM WX035 36 nM WX036 69 nM WX037 150 nM WX038 480 nM WX039 36 nM WX040 320 nM WX041 52 nM WX042 21 nM WX043 28 nM WX044 81 nM WX045 30 nM WX046 700 nM WX047 630 nM WX048 92 nM WX049 210 nM WX050 260 nM WX051 69 nM WX054 1000 nM WX055 650 nM WX056 360 nM WX057 200 nM
[0324] Conclusion: the compounds of the present invention have strong inhibitory activity against human-sourced KHK enzyme.
Experimental Example 2: Assay for Inhibition of hERG potassium Channel
[0325] A. Main materials
[0326] 1. CHO-hERG cell line (Chinese hamster ovary cells stably expressing hERG channel), constructed in-house by Shanghai Institute of Materia Medica, Chinese Academy of Sciences;
[0327] 2. Positive control compound: Cisapride, Sigma Aldrich, Cat. No.: C4740-10 mg;
[0328] 3. Patch clamp amplifier Axopatch 200B, Taser International Inc.
[0329] B. Methods
[0330] a) Cell Culture
[0331] CHO cells stably expressing hERG were cultured in a cell culture dish with a diameter of 35 mm, placed in an incubator at 37° C. and 5% CO.sub.2, and passaged at a ratio of 1: 5 every 48 hours. Formula of the culture medium: 90% F12 (Invitrogen), 10% fetal bovine serum (Gibco), 100 μg/mL G418 (Invitrogen) and 100 μg/mL Hygromycin B (Invitrogen). On the day of the assay, the cell culture medium was aspirated. After rinsed once with extracellular fluid, 0.25% Trypsin-EDTA (Invitrogen) solution was added, and digestion was performed at room temperature for 3-5 min. The digestive fluid was aspirated. After resuspended in extracellular fluid, the cells were transferred to an experimental dish for electrophysiological recording for future use.
[0332] b) Preparation of Intracellular and Extracellular Fkuids
[0333] The extracellular fluid should be prepared once a month. The intracellular fluid must be packed and frozen at −20° C. The components of the intracellular and extracellular fluids are shown in Table 3.
TABLE-US-00008 TABLE 3 Components of the intracellular and extracellular fluids Extracellular Intracellular Components fluid (mM) fluid (mM) NaCl 145 — KCl 4 120 KOH — 31.25 CaCl.sub.2 2 5.374 MgCl.sub.2 1 1.75 Glucose 10 — Na.sub.2ATP — 4 HEPES 10 10 EGTA — 10 pH 7.4 with NaOH 7.2 with KOH Osmolality 295 mOsm 285 mOsm
[0334] c) Preparation of the Compound
[0335] The compound was dissolved into a 20 mM stock solution with DMSO. On the day of the assay, the compound stock solution was 3-fold serially diluted with DMSO, that is, 10 μL the compound stock solution was added to 20 μL DMSO to obtain 6 intermediate concentrations of the compound serially diluted with DMSO, which were 20, 6.66, 2.22, 0.74, 0.24 and 0.082 mM, respectively. Then 10 μL of the compound at the intermediate concentrations was added to 4990 μL extracellular fluid and diluted 500 times to afford the final concentrations to be tested, of which the highest concentration to be tested was 40 μM, and which were 40, 13.3, 4.44, 1.48, 0.49 and 0.16 μM, respectively. Preparation of the positive control compound Cisapride: 150 μM cisapride stock solution was 3-fold serially diluted with 100% DMSO, that is, 10 μL of 150 μM cisapride stock solution was added to 20 μL DMSO to obtain 5 intermediate concentrations of cisapride serially diluted with DMSO, which were 150, 50, 16.7, 5.56 and 1.85 μM, respectively. Then 10 μL of the cisapride at the intermediate concentrations was added to 4990 μL extracellular fluid and diluted 500 times to afford the final concentrations to be tested, of which the highest concentration to be tested was 300 nM and which were 300, 100, 33.3, 11.1 and 3.70 nM, respectively. The content of DMSO in the final test concentration should not exceed 0.2%, and this concentration of DMSO had no effect on hERG potassium channel.
[0336] d) Electrophysiological Recording Process
[0337] In CHO (Chinese Hamster Ovary) cells stably expressing hERG potassium channel, hERG potassium channel currents were recorded at room temperature using whole-cell patch clamp technique. The glass microelectrode was prepared by drawing a glass electrode blank (BF150-86-10, Sutter) by a puller, and the tip resistance after filling intra-electrode liquid is about 2-5 MΩ. The glass microelectrode was inserted into an amplifier head to attach to the Axopatch 200B (Molecular Devices) patch clamp amplifier. Clamping voltage and data recording were controlled and recorded by a computer using pClamp 10 software, with a sampling frequency of 10 kHz and a filtering frequency of 2 kHz. After the whole-cell recordings were obtained, the cells were clamped at −80 mV and the step voltage inducing the hERG potassium current (/hERG) was given a depolarization voltage of 2 s from −80 mV to +20 mV, then repolarized to −50 mV for 1 s and then back to −80 mV. This voltage stimulus was given every 10 s, and a dosing process was started after the hERG potassium current was determined to be stable (1 min). Compound concentrations were administered consecutively starting at low test concentrations, with each test concentration being administered for at least 1 minute. At least 3 cells (n≥3) were tested per concentration of the compound and at least 2 cells (n≥2) were tested per concentration of positive compound.
[0338] e) Data Analysis
[0339] In each complete current record, the percent inhibition for each compound concentration can be calculated based on the percentage of the peak current to the negative control. The dose-response curve was obtained by fitting with the standard Hill equation which is particularly as follows:
I.sub.(C)=I.sub.b+(I.sub.fr−I.sub.b)*c.sup.n/(IC.sub.50.sup.n+c.sup.n)
[0340] with C as the compound concentration tested and n as the slope.
[0341] Curve fitting and inhibition rate calculation were performed by Qpatch analysis software. If the inhibition rate at the lowest concentration exceeds half inhibition or the inhibition rate at the highest concentration does not reach half inhibition, the corresponding ICso of the compound is lower than the lowest concentration or the ICso value is higher than the highest concentration.
[0342] Conclusion: the compounds did not show hERG inhibitory activity.
Experimental Example 3: Inhibitory Activity on Cytochrome P450 Isozymes
[0343] A. Experimental Purpose
[0344] The inhibitory effect of test compounds on the activity of human hepatic microsomal cytochrome P450 isozymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) was determined.
[0345] B. Experimental Procedure
[0346] First, the test compound (10 mM) was diluted in gradient to prepare working solutions (100×final concentration) with working solution concentrations: 5, 1.5, 0.5, 0.15, 0.05, 0.015, 0.005 mM, respectively, and at the same time, working solutions of each positive inhibitor of P450 isozymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) and its specific substrate mixture were prepared. Human liver microsomes frozen in a −80° C. refrigerator were thawed on ice, and diluted with PB (phosphate buffer) after all the human liver microsomes were dissolved, to prepare a working solution at a certain concentration (0.253 mg/mL). 20 μl substrate mixture was added to the reaction plate (20 μL PB was added to the Blank wells) while 158 μL human liver microsome working solution was added to the reaction plate which was placed on ice for later use. At this time, 2 μL of each concentration of the test compound (N=1) and the specific inhibitor (N=2) were added to the corresponding wells, and the corresponding organic solvents were added to the groups without inhibitors (the test compound or the positive inhibitor) as control samples (1:1 DMSO:MeOH for the test compound control sample, and 1:9 DMSO:MeOH for the positive control sample). After pre-incubation in 37° C. water bath for 10 min, 20 μL coenzyme factor (NADPH) solution was added to the reaction plate, and incubated in 37° C. water bath for 10 min. 400 μL cold acetonitrile solution (200 ng/mL Tolbutamide and Labetalol as the internal standard) was added to stop the reaction. The reaction plate was placed on a shaker and shaken for 10 min. The mixture was centrifuged at 4,000 rpm for 20 min, and 200 μl the supernatant was added to 100 82 L water for sample dilution. Finally, the plate was sealed, shaken, mixed well and detected by LC/MS/MS.
[0347] C. Experimental Results
[0348] The experimental results are shown in Table 4.
TABLE-US-00009 TABLE 4 Results of the inhibitory effect of the test compounds on the activity of human liver microsomal cytochrome P450 isoenzymes Compound IC.sub.50 (μM) No. CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4 WX001 >50 >50 >50 >50 >50 WX004 >50 >50 >50 >50 >50 WX006 >50 >50 >50 >50 >50 WX042 >50 >50 >50 >50 >50 WX043 >50 >50 >50 >50 >50 Conclusion: the compounds of the present invention showed no CYP inhibitory activity.
Experimental Example 4: Metabolic Stability of Compounds in Liver Microsomes of Human, CD-1 Mouse, Beagle Dog and Cynomolgus Monkey
[0349] A. Experimental Purpose
[0350] Metabolic stability of test compounds was assessed in liver microsomes of human, CD-1 mouse, beagle dog and cynomolgus monkey.
[0351] B. Experimental Procedure
[0352] First, the test compound (10 mM) was subject to a two-step dilution, with an intermediate concentration of 100 μM by dilution with 100% methanol, and the working solution concentration of 10 μM by dilution with potassium phosphate buffer. 8 96-well incubation plates were prepared, named as T0, T5, T10, T20, T30, T60, Blank and NCF60, respectively. The corresponding reaction time points of the first 6 incubation plates were 0, 5, 10, 20, 30 and 60 min, respectively; no test compound or control compound was added to the Blank plate. For the NCF60 plate, the solution of NADPH regeneration system was replaced with potassium phosphate buffer for incubation for 60 min. 10 μl the test compound working solution and 80 μL the microsome working solution (liver microsome protein concentration was 0.625 mg/mL) were separately added to T0, T5, T10, T20, T30, T60 and NCF60 plates, and only the microsome working solution was added to the Blank plate, and then the above plates were placed in a 37° C. water bath for pre-incubation for about 10 min. After the pre-incubation, except for NCF60 plate and TO plate, 10 μL the working solution of the NADPH regeneration system was added to each sample well to start the reaction, and 10 μL potassium phosphate buffer was added to each well in the NCF60 plate. Therefore, in the samples of the test compound or the control compound, the final reaction concentrations of the compound, testosterone, diclofenac and propafenone weres 1 μM, the concentration of liver microsomes was 0.5 mg/mL, and the final concentrations of DMSO and acetonitrile in the reaction system were 0.01% (v/v) and 0.99% (v/v), respectively. After incubation for an appropriate period of time (e.g. 5, 10, 20, 30 and 60 min), 300 μL stop solution (containing 100 ng/mL tolbutamide and 100 ng/mL labetalol in acetonitrile) was added to each sample well to stop the reaction; 300 μL stop solution was added and then 10 μL NADPH working solution was added to the T0 plate. All sample plates were shaken well and centrifuged in a centrifuge (3220×g) for 20 min, then 100 μL supernatant per well was diluted into 300 μL purified water for liquid chromatography tandem mass spectrometry analysis.
[0353] Conclusion: the compounds of the invention have excellent metabolic stability in liver microsomes.
Experimental Example 5: Pharmacokinetic Evaluation of Compounds
[0354] A. Experimental Purpose
[0355] In vivo pharmacokinetics of compounds in SD rats were tested.
[0356] B. Experimental Procedure
[0357] Pharmacokinetic profiles in rodents following intravenous and oral administration of the compounds were tested by standard protocols. In the experiment, candidate compounds were formulated as clear solutions or homogeneous suspensions, and were administered to rats by single intravenous injection (IV, 1 mpk) and oral administration (PO, 3 mpk). The vehicle for intravenous injection was a certain proportion of PEG400 in water, and the vehicle for oral administration was a certain proportion of methylcellulose and Tween 80 in water. Whole blood was collected, and plasma was prepared. Drug concentration was analyzed by LC-MS/MS, and pharmacokinetic parameters were calculated by Phoenix WinNonlin software.
[0358] C. Experimental Results
[0359] The experimental results are shown in Table 5.
TABLE-US-00010 TABLE 5 Pharmacokinetic test results Integral concentration Bioavail- Compound Clearance rate Half-life AUC for PO ability No. (mL/min/kg) T.sub.1/2 (h) (nM .Math. hr) F (%) WX001 1.04 3.12 163046 158 WX004 1.21 2.96 64484 80 WX006 1.05 2.63 39489 44 WX042 8.13 2.27 20515 176 WX043 5.37 5.83 11966 70 Conclusion: the compounds of the present invention have high bioavailability.
Experimental Example 6: Evaluation of the Liver-Blood Ratio of Compounds in Rats
[0360] A. Experimental Purpose
[0361] Tissue distribution of compounds in SD rats was tested.
[0362] B. Experimental Procedure
[0363] In the experiment, candidate compounds were formulated as clear solutions and administered to rats in a single oral dose (PO, 1 mpk). The vehicle for oral administration was a certain proportion of methylcellulose and Tween 80 in water. Whole blood was collected at a certain time and plasma was prepared, and tissues were collected at the corresponding time and prepared into tissue homogenates. Drug concentration was analyzed by LC-MS/MS, and pharmacokinetic parameters were calculated by Phoenix WinNonlin software.
[0364] C. Experimental Results
[0365] The experimental results are shown in Table 6.
TABLE-US-00011 TABLE 6 Pharmacokinetic test results Tested tissue Time Concentration Concentrations in tested tissues (nM) and ratios points (nM) WX001 WX004 WX006 WX012 WX042 WX043 1.5 h Plasma 4880 4670 2010 810 3314 2025 Liver tissue 2443 4420 2415 2100 19900 38250 Liver-blood ratio 0.491 0.943 1.20 2.62 9.53 20.0 Brain tissue 72.8 105 NA NA 76.2 30.6 Brain-blood ratio 0.015 0.022 NA NA 0.0375 0.016 2.0 h Plasma 9090 7755 3100 273 3045 916 Liver tissue 4273 6365 2865 618 20600 27800 Liver-blood ratio 0.469 0.821 0.92 2.32 6.78 30.0 Brain tissue 162 148 NA NA 62.2 13.1 Brain-blood ratio 0.017 0.019 NA NA 0.0211 0.014 6.0 h Plasma 3318 3665 1780 145 259 246 Liver tissue 1790 3900 2180 634 6710 19800 Liver-blood ratio 0.549 1.22 1.25 4.39 26.2 81.5 Brain tissue 91.5 71 NA NA ND ND Brain-blood ratio 0.020 0.023 NA NA ND ND ND indicates that the compound concentration was too low to reach the limit of detection, and NA indicates that the test was not performed. Conclusion: the compounds of the present invention have high hepatic tissue selectivity in mice.
Experimental Example 7: Evaluation of the Liver-Blood Ratio of Compounds in Mice
[0366] A. Experimental Purpose
[0367] Tissue distribution of compounds in C57BL/6 mice was tested.
[0368] B. Experimental Procedure
[0369] In the experiment, candidate compounds were formulated as clear solutions and administered to mice in a single oral dose (PO, 1 mpk). The vehicle for oral administration was a certain proportion of methylcellulose and Tween 80 in water. Whole blood was collected at a certain time and plasma was prepared, tissues were collected at the corresponding time and prepared into tissue homogenates. Drug concentration was analyzed by LC-MS/MS, and pharmacokinetic parameters were calculated by Phoenix WinNonlin software.
[0370] Conclusion: the compounds of the present invention have high hepatic tissue selectivity in mice.
Experimental Example 8: Acute Fructose Feeding Experiment in Mice-in vivo Pharmacodynamic Evaluation
[0371] A. Experimental Purpose
[0372] The effect of test compounds on the fructose content in mice with high fructose intake was investigated.
[0373] B. Experimental Procedure
[0374] All animals were acclimated for one week in an animal house, fed with normal chow and fasted for 16 hours before being given fructose.
[0375] 1) The experiment began at −0.5 hr. A control group was given a certain volume of vehicle (a certain proportion of methylcellulose and Tween 80 in water), and an experimental group was given a certain dose of a solution of the compound (the administered volume was consistent with the vehicle).
[0376] 2) 0.5 hour later and before administration of a fructose solution, a blood sample was collected at 0 hr, followed by oral administration of 1 g/kg of an aqueous fructose solution.
[0377] 3) After the administration of fructose, blood samples were collected from saphenous vein of mice at 0.25, 0.5, 1, 2, 3 and 8 hr, and fructose concentration was analyzed by LC-MS/MS.
[0378] C. Experimental Results
[0379] The experimental results are shown in Table 7.
TABLE-US-00012 TABLE 7 Experimental results of acute fructose feeding in mice WX004, WX024, WX019, WX020, WX026, WX029, WX030, WX041, WX042, WX043, 10 mpk 30 mpk 30 mpk 30 mpk 30 mpk 30 mpk 30 mpk 30 mpk 30 mpk 30 mpk AUC of integral fructose 440.3 139 139 139 112 112 112 263 263 263 concentration for control group (nM .Math. hr) AUC of integral fructose 2194 1858 849 1180 993 1687 1029 1704 3400 3504 concentration for dosed group (nM .Math. hr) AUC of integral fructose 5.0 13.4 6.1 8.5 8.9 15.1 9.2 6.5 12.9 13.3 concentration of dosed group/AUC of Integral fructose concentration of control group Conclusion: the compounds of the present invention have a strong inhibitory effect on fructose metabolism in mice.
[0380] Experimental Example 9: Acute Fructose Feeding Experiment in Rats-in vivo Pharmacodynamic Evaluation
[0381] A. Experimental Purpose
[0382] The effect of test compounds on the fructose content in rats with high fructose intake was investigated.
[0383] B. Experimental Procedure
[0384] All animals were acclimated for at least three days in an animal house, fed with normal chow, and fasted 16 hours before being given fructose.
[0385] 1. The experiment began at −1 hr. A control group was given a certain volume of vehicle and an experimental group was given a certain dose of a solution of the compound (a certain proportion of methylcellulose and Tween 80 in water).
[0386] 2. 1 hour later and before administration of fructose solution, a blood sample was collected at 0 hr, followed by oral administration of 2 g/kg of an aqueous fructose solution.
[0387] 3. After administration of fructose, blood samples were collected from the neck vein of rats at 0.5, 1, 2, 3 and 8 hr, and fructose concentration was analyzed by LC-MS/MS.
[0388] Conclusion: the compounds of the present invention have a strong inhibitory effect on fructose metabolism in rats.