METHYL-SUBSTITUTED BENZOBISOXAZOLE COMPOUND AND USE THEREOF
20230234968 · 2023-07-27
Inventors
Cpc classification
C07D519/00
CHEMISTRY; METALLURGY
International classification
C07D519/00
CHEMISTRY; METALLURGY
Abstract
Provided are a new methyl-substituted benzobisoxazole compound and the use thereof in the preparation of drugs for treating related diseases. Specifically disclosed is a compound represented by formula (P) or a pharmaceutically acceptable salt thereof.
##STR00001##
Claims
1. A compound represented by formula (P) or a pharmaceutically acceptable salt thereof, ##STR00105## wherein, ring B is selected from ##STR00106## and the ##STR00107## are optionally substituted by 1, 2 or 3 R.sub.5; is selected from a single bond and a double bond, and when T.sub.2 is selected from N,
is selected from the single bond; T.sub.1 and T.sub.2 are selected from N and CH; X.sub.1 and X.sub.2 are each independently selected from CH, N, O and S, and the CH is optionally substituted by one F, Cl, Br and CH.sub.3; L.sub.1 is selected from a single bond and —C.sub.1-3 alkyl-; R.sub.1 is selected from F, Cl, Br, I, OH, NH.sub.2 and CN; u, v, w, r, s and t are each independently 0 or 1; m is selected from 0, 1, 2, 3, 4 and 5; R.sub.2 is selected from ##STR00108## and the ##STR00109## are optionally substituted by 1, 2 or 3 R.sub.a; Y.sub.1 and Y.sub.2 are each independently selected from CH, CH.sub.2, N, NH and O; o and p are each independently selected from 0, 1, 2 and 3; R.sub.3 is selected from —C(═O)—NH—R.sub.b, —C(═O)—R.sub.b, —C(═O)—NH—S(═O).sub.2—R.sub.b, —S(═O).sub.2—NH—R.sub.b, —S(═O).sub.2—R.sub.b, —P(═O)(R.sub.b).sub.2, C.sub.1-3 alkyl, tetrazolyl, isoxazolyl, ##STR00110## and the C.sub.1-3 alkyl, tetrazolyl, isoxazolyl, ##STR00111## are optionally substituted by 1, 2 or 3 R.sub.b, R.sub.5 is selected from F, Cl, Br, I and C.sub.1-3 alkyl; R.sub.4 is selected from H, F, Cl, Br, I and CH.sub.3; R.sub.a is selected from F, Cl, Br and I; R.sub.b is selected from OH, CN, C.sub.1-3 alkyl, C.sub.1-3 alkoxy, C.sub.1-3 alkylamino and oxazolyl, and the C.sub.1-3 alkyl, C.sub.1-3 alkoxy and oxazolyl are optionally substituted by 1, 2 or 3 R; R is selected from F, Cl and Br.
2. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.2 is selected from ##STR00112## and the ##STR00113## are optionally substituted by 1, 2 or 3 R.sub.a.
3. The compound or the pharmaceutically acceptable salt thereof according to claim 2, wherein R.sub.2 is selected from ##STR00114##
4. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein L.sub.1 is selected from the single bond and —CH.sub.2—.
5. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein m is selected from 0, 1 and 2.
6. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.b is selected from OH, CN, CH.sub.3, CF.sub.3 and OCH.sub.3.
7. The compound or the pharmaceutically acceptable salt thereof according to claim 6, wherein R.sub.3 is selected from —COOH, —C(═O)—NH—CN, —C(═O)—NH—OH, —C(═O)—NH—OCH.sub.3, —C(═O)—CF.sub.3, —S(═O).sub.2—NH—CH.sub.3 and —S(═O).sub.2—OH.
8. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein ring B is selected from ##STR00115##
9. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the structural moiety ##STR00116## is selected from ##STR00117##
10. The compound or the pharmaceutically acceptable salt thereof according to claim 1, which is selected from: ##STR00118## wherein, is selected from a single bond and a double bond, and when T.sub.2 is selected from N,
is selected om the single bond; s and v are each independently 0 or 1; R.sub.1, R.sub.2, R.sub.3, L.sub.1, T.sub.1, T.sub.2, X.sub.1, X.sub.2 and m are as defined above.
11. The compound or the pharmaceutically acceptable salt thereof according to claim 10, which is selected from: ##STR00119## wherein, is selected from a single bond and a double bond, and when T.sub.2 is selected from N,
is selected from the single bond; R.sub.1, T.sub.2, X.sub.1 and X.sub.2 are as defined above.
12. A compound represented by the following formula or a pharmaceutically acceptable salt thereof, selected from ##STR00120## ##STR00121## ##STR00122##
13. The compound or the pharmaceutically acceptable salt thereof according to claim 12, which is selected from: ##STR00123## ##STR00124## ##STR00125## ##STR00126##
14. A method for activating GLP-1 receptor in a subject in need thereof, comprising: administering the compound or the pharmaceutically acceptable salt thereof according to claim 1 to the subject.
15. A method for treating diabetes in a subject in need thereof, comprising: administering the compound or the pharmaceutically acceptable salt thereof according to claim 1 to the subject.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0160]
[0161]
[0162]
[0163]
[0164]
[0165]
[0166]
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0167] The present disclosure is described in detail by the embodiments below, but it does not mean that there are any adverse restrictions on the present disclosure. The present disclosure has been described in detail herein, and its specific embodiments have also been disclosed; for one skilled in the art, it is obvious to make various modifications and improvements to the embodiments of the present disclosure without departing from the spirit and scope of the present disclosure.
Reference Embodiment 2: Segment B-2
[0168] ##STR00061##
[0169] Synthetic Route:
##STR00062##
[0170] Step 1: Synthesis of Compound B-2-2
[0171] Under the protection of nitrogen, at 0° C., sodium hydride (9.85 g, 246.18 mmol, content of 60%, 1.5 eq) was added to a solution of compound B-2-1 (23 g, 164.12 mmol, 1 eq) in DMF (115 mL). 2-(Trimethylsilyl)ethoxymethyl chloride (41.04 g, 246.18 mmol, 43.57 mL, 1.5 eq) was added dropwise thereto. After the dropwise addition was completed, the reaction was heated to 25° C. and reacted for 12 hours. After the reaction was completed, the reaction was quenched with ice water (500 mL), extracted with ethyl acetate (200 mL*3), and the organic phases were combined, washed with saturated brine (200 mL*2), dried with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by column chromatography (PE:EA=1:0 to 1:1) to obtain B-2-2. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm −0.07-−0.04 (m, 9H), 0.86-0.93 (m, 2H), 1.40 (t, J=7.13 Hz, 3H), 3.49-3.56 (m, 2H), 4.39 (q, J=7.13 Hz, 2H), 7.17 (s, 1H), 5.76 (s, 2H), 7.24 (d, J=0.63 Hz, 1H).
[0172] Step 2: Synthesis of Compound B-2-3
[0173] Lithium aluminum hydride (6.04 g, 159.21 mmol, 1.5 eq) was dispersed into anhydrous tetrahydrofuran (1000 mL) in batches, cooled to 0° C. under the protection of nitrogen, stirred for 15 minutes, and then compound B-2-2 (28.7 g, 106.14 mmol, 1 eq) was added thereto in batches at 0° C. After the addition was completed, the temperature was raised to 25° C., and the mixture was reacted for 0.5 hours. After the reaction was completed, the mixture was cooled to 0° C., then added with 6 mL of water, 6 mL of 15% sodium hydroxide aqueous solution and 18 mL of water in turn to quench the reaction. The mixture was heated to 25° C. and stirred for 15 minutes, then anhydrous magnesium sulfate was added thereto and stirred for 15 minutes, then filtered. The filtrate was washed with saturated brine (500 mL), dried with anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound B-2-3. .sup.1H-NMR (400 MHz, CDCl.sub.3) δ ppm −0.02-−0.01 (m, 9H), 0.89-0.95 (m, 2H), 3.52 (dd, J=8.76, 7.75 Hz, 2H), 4.72 (s, 2H), 5.37 (s, 2H), 6.93 (d, J=1.13 Hz, 1H), 6.98 (d, J=1.13 Hz, 1H).
[0174] Step 3: Synthesis of Compound B-2-4
[0175] Under the protection of nitrogen, a mixed solution of compound B-2-3 (18.81 g, 82.37 mmol, 1 eq), tert-butylchlorodiphenylsilane (27.17 g, 98.84 mmol, 25.39 mL, 1.2 eq) and imidazole (14.02 g, 205.92 mmol, 2.5 eq) in DMF (188 mL) was reacted for 16 hours at 25° C. After the reaction was completed, the reaction solution was poured into water (1000 mL) to quench the reaction, and ethyl acetate (200 mL*3) was added for extraction. The organic phases were combined, washed with saturated brine (200 mL*3), dried with anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by column chromatography (PE:EA=1:0 to 1:1) to obtain B-2-4. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 0.03 (s, 9H), 0.85-0.91 (m, 2H), 1.06 (s, 9H), 3.41-3.48 (m, 2H), 4.84 (s, 2H), 5.41 (s, 2H), 6.96-7.02 (m, 2H), 7.37-7.46 (m, 6H), 7.66-7.72 (m, 4H).
[0176] Step 4: Synthesis of Compound B-2-5
[0177] Under the protection of nitrogen, N-bromosuccinimide (25.17 g, 141.40 mmol, 44.18 μL, 3 eq) was added to an anhydrous tetrahydrofuran (440 mL) solution of compound B-2-4 (22 g, 47.13 mmol, 1 eq) in batches, and after the addition was completed, the mixture was heated to 25° C. and reacted for 12 hours. After the reaction was completed, the mixture was added with water (440 mL) to quench, and extracted with ethyl acetate (2200 mL*2). After the organic phases were combined, the organic phase was washed with saturated brine (2200 mL), dried with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by column chromatography (PE:EA=1:1 to 2:1) to obtain B-2-5. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 0.02 (s, 9H), 0.85-0.90 (m, 2H), 1.07 (s, 9H), 3.45-3.51 (m, 2H), 4.80 (s, 2H), 5.44 (s, 2H), 7.37-7.48 (m, 6H), 7.64-7.67 (m, 4H).
[0178] Step 5: Synthesis of Compound B-2-6
[0179] Under the protection of nitrogen, i-PrMgCl—LiCl (1.3 M, 8.13 mL, 1.1 eq) was added dropwise to an anhydrous tetrahydrofuran (60 mL) solution of compound B-2-5 (6 g, 9.61 mmol, 1 eq) at −40° C. stirred for 1.5 hours after the dropwise addition was completed, and then DMF (61.62 g, 843.09 mmol, 64.86 mL, 87.76 eq) was added dropwise thereto. The reaction temperature was raised to 25° C. and the mixture was continued to stir for 30 minutes. After the reaction was completed, water (120 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (50 mL*2). The organic phase was washed with saturated brine (100 mL) after the organic phases were combined, and dried with anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by column chromatography (PE:EA=1:0 to 10:1) to obtain B-2-6. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm −0.03 (s, 9H), 0.84-0.88 (m, 2H), 1.07 (s, 9H), 3.49-3.54 (m, 2H), 4.86 (s, 2H), 5.85 (s, 2H), 7.38-7.42 (m, 5H), 7.64-7.68 (m, 5H), 9.76 (s, 1H).
[0180] Step 6: Synthesis of Compound B-2-7
[0181] Compound B-2-6 (1.23 g, 2.14 mmol, 1 eq) was dissolved in ethanol (61.5 mL), and then sodium ethoxide (2.19 g, 6.43 mmol, content of 20%, 3 eq) and ethyl thioglycolate (273.10 mg, 2.57 mmol, 233.42 μL, 1.2 eq) were added thereto in turn, and the reaction was stirred at 20° C. for 2 hours, then heated to 80° C. and stirred for 12 hours. After the reaction was completed, the mixture was added with water (50 mL) to quench, and extracted with ethyl acetate (25 mL*2). After the organic phases were combined, the organic phase was washed with saturated brine (50 mL), and dried with anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by column chromatography (PE:EA=1:0 to 10:1) to obtain compound B-2-7. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm −0.02 (s, 9H), 0.91-0.96 (m, 2H), 1.40 (t, J=7.13 Hz, 3H), 2.30-2.64 (m, 1H), 3.55-3.61 (m, 2H), 4.39 (q, J=7.13 Hz, 2H), 4.91 (s, 2H), 5.54 (s, 2H), 7.72 (s, 1H).
[0182] Step 7: Synthesis of Compound B-2
[0183] Under the protection of nitrogen, methanesulfonyl chloride (57.84 mg, 504.89 μmol, 39.08 μL, 1.5 eq) was added to a solution of compound B-2-7 (120 mg, 336.59 μmol, 1 eq) and triethylamine (102.18 mg, 1.01 mmol, 140.55 μL, 3 eq) in dichloromethane (2 mL) in batches, and the temperature was raised to 25° C. and the mixture was reacted for 12 hours. After the reaction was completed, the mixture was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by preparative thin layer chromatography (DCM:MeOH=20:1) to obtain compound B-2. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm −0.02 (s, 9H), 0.92-0.97 (m, 2H), 1.40 (t, J=7.13 Hz, 3H), 3.56-3.61 (m, 2H), 4.39 (q, J=7.13 Hz, 2H), 4.85 (s, 2H), 5.57 (s, 2H), 7.72 (s, 1H)
Reference Embodiment 3: Segments B-3 and B-4
[0184] ##STR00063##
[0185] Synthetic Route:
##STR00064##
[0186] Step 1: Synthesis of Compound 001-3
[0187] 001-1 (4 g, 21.16 mmol, 1 eq), toluene (60 mL), 001-2 (3.84 g, 22.25 mmol, 1.05 eq), p-toluenesulfonic acid monohydrate (200.00 mg, 1.05 mmol, 0.05 eq) were added to a reaction flask, and water segregator was used to build a water separator, and the mixture was refluxed at 140-145° C. for 60 hours. After the reaction was completed, the reaction solution was evaporated to dryness by rotary evaporation to obtain a crude product. The crude product was purified by column chromatography (petroleum ether) to obtain 001-3. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 2.12 (d, J=1.2 Hz, 3H), 6.68-6.79 (m, 2H), 6.96 (dd, J=8.0, 1.6 Hz, 1H), 7.12-7.19 (m, 2H), 7.52-7.59 (m, 1H).
[0188] Step 2: Synthesis of Compound 001-5
[0189] 001-3 (0.68 g, 1.98 mmol, 1 eq), 001-4 (660 mg, 2.13 mmol, 1.08 eq), trakis(triphenylphosphine)palladium (114.35 mg, 98.96 μmol, 0.05 eq), potassium carbonate (547.07 mg, 3.96 mmol, 2 eq), water (1.4 mL) and 1,4-dioxane (7 mL) were added to a reaction flask, and the system was replaced with nitrogen, and the mixture was reacted for 16 hours at 100° C. under the protection of nitrogen. After the reaction was completed, the reaction solution was evaporated to dryness by rotary evaporation obtain a crude product. The crude product was purified by column chromatography (PE:EA=27:1) to obtain 001-5. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 1.50 (s, 9H), 2.08 (s, 3H), 2.46-2.66 (m, 2H), 3.57-3.70 (m, 2H), 4.09-4.13 (m, 2H), 6.37 (br s, 1H), 6.72-6.78 (m, 1H), 6.78-6.84 (m, 2H), 7.10-7.18 (m, 2H), 7.51 (t, J=8.4 Hz, 1H).
[0190] Step 3: Synthesis of Compound B-3 and B-4
[0191] 001-5 was purified and separated by supercritical fluid separation (chromatographic column: DAICEL CHIRALCEL OJ (250 mm*50 mm, 10 μm); mobile phase: phase A was CO.sub.2; phase B was [0.1% ammonia water—isopropanol]; B %: 15%-15%) to obtain B-3 (retention time: 2.474 min) and B-4 (retention time: 2.771 min).
Reference Embodiment 4: Segment B-5
[0192] ##STR00065##
[0193] Synthetic Route:
##STR00066## ##STR00067##
[0194] Step 1: Synthesis of compound B-5-2
[0195] B-5-1 (10.00 g, 111.02 mmol, 8.55 mL, 1 eq), tert-butylchlorodiphenylsilane (36.62 g, 133.22 mmol, 34.22 mL, 1.2 eq), imidazole (8.92 g, 131.00 mmol, 1.18 eq), anhydrous DMF (150.00 mL) were added to a reaction flask, and the reaction system was stirred at 0° C. for 3 hours. The reaction solution was concentrated to obtain a crude product, dissolved with ethyl acetate (200 mL). The mixture was washed with water (200 mL*2) and saturated brine (30 mL) in turn, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was separated and purified by column chromatography (PE:EA=1:0 to 10:1) to obtain compound B-5-2. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 1.10 (s, 9H), 3.69 (s, 3H), 4.26 (s, 2H), 7.37-7.45 (m, 6H), 7.69 (dd, J=7.88, 1.38 Hz, 4H).
[0196] Step 2: Synthesis of Compound B-5-3
[0197] In a dry reaction flask, B-5-2 (220 g, 669.76 mmol, 12.82 mL, 1 eq) was added to a methanol solution of ammonia (7 M, 1.58 L, 16.5 eq), and the mixture was stirred at 50° C. for 10 hours. The reaction solution was concentrated, and then separated and purified by column chromatography (PE:EA=1:0 to 3:1) to obtain compound B-5-3. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 1.02 (s, 9H), 3.94 (s, 2H), 7.11 (br s, 1H), 7.40 (br s, 1H), 7.42-7.52 (m, 6H), 7.64 (dd, J=7.91, 1.63 Hz, 4H).
[0198] Step 3: Synthesis of Compound B-5-5
[0199] B-5-4 (68.5 g, 291.37 mmol, 1 eq), B-5-3 (100.47 g, 320.51 mmol, 1.1 eq), cesium carbonate (142.4 g, 437.06 mmol, 1.5 eq), anhydrous 1,4-dioxane (1 L) were added to a reaction flask, and then tris(dibenzylideneacetone)dipalladium (26.68 g, 29.14 mmol, 0.1 eq), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (16.86 g, 29.14 mmol, 0.1 eq) were added thereto under nitrogen atmosphere, and the reaction system was stirred at 110° C. for 1 hour. Water (1 L) was added for dilution, and the mixture was extracted with ethyl acetate (1 L*3). After the phases were separated, the organic phase was collected, washed with saturated brine solution (1 L*3) in turn, dried with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a product. The product was purified by column chromatography (PE:EA=1:0 to 10:1) to obtain compound B-5-5.
[0200] Step 4: Synthesis of Compound B-5-6
[0201] In a dry reaction flask, B-5-5 (25 g, 53.46 mmol, 1 eq) was added to tetrahydrofuran (250 mL), and then N-bromosuccinimide (9.99 g, 56.13 mmol, 1.05 eq) was added thereto, and stirred at 20° C. for 2 hours. The reaction solution was poured into 1.2 L of sodium bicarbonate aqueous solution, added with ethyl acetate (1.2 L*2) for extraction. The organic phases were combined, washed with saturated brine (1.2 L*2), and after the phases were separated, the organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness by rotary evaporation under reduced pressure. Compound B-5-6 was obtained.
[0202] Step 5: Synthesis of Compound B-5-7
[0203] In a dry reaction flask, B-5-6 (100 g, 182.97 mmol, 1 eq) was added to 1,4-dioxane (11 L), and Lawesson reagent (74.00 g, 182.97 mmol, 1 eq) was added thereto, and the mixture was heated to 110° C. and stirred for 3 hours. The reaction solution was concentrated, and then separated and purified by column chromatography (PE:EA=1:0 to 3:1) to obtain compound B-5-7. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 1.20 (s, 9H), 1.39 (t, J=7.15 Hz, 3H), 4.37 (q, J=7.11 Hz, 2H), 4.63 (s, 2H), 7.39-7.52 (m, 6H), 7.67 (dd, J=8.03, 1.25 Hz, 4H), 7.73 (s, 1H), 11.24 (br s, 1H).
[0204] Step 6: Synthesis of Compound B-5-8
[0205] In a dry reaction flask, B-5-7 (5 g, 8.89 mmol, 1 eq) was added to DMF (1 mL), and then silver acetate (2.97 g, 17.77 mmol, 910.03 μL, 2 eq) and B-1-8 (1.55 g, 17.77 mmol, 2 eq) was added thereto, and the mixture was stirred at 30° C. for 10 hours. The reaction solution was filtered, added with water (100 mL) to dilute the filtrate, and then ethyl acetate (100 mL*3) was added for extraction. After the phases were separated, the organic phase was collected, washed with saturated brine solution (100 mL*3) in turn, dried with anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was separated and purified by column chromatography (PE:EA=1:0 to 3:1) to obtain compound B-5-8. LCMS, m/z=617.0 [M+1]+.
[0206] Step 7: Synthesis of Compound B-5-9
[0207] In a dry reaction flask, B-5-8 (3.8 g, 6.17 mmol, 1 eq) was added to DMF (40 mL), and then cuprous iodide (352.66 mg, 1.85 mmol, 0.3 eq), N,N′-dimethylethylenediamine (326.45 mg, 3.70 mmol, 398.60 μL, 0.6 eq), cesium carbonate (2.01 g, 6.17 mmol, 1 eq) were added thereto, and the mixture was stirred at 60° C. for 10 hours. Additional cuprous iodide (352.66 mg, 1.85 mmol, 0.3 eq), N,N′-dimethylethylenediamine (326.46 mg, 3.70 mmol, 398.61 μL, 0.6 eq) were added thereto, and the mixture was continued to stir for 4 hours. Water (100 mL) was added for dilution, and ethyl acetate (100 mL*3) was added for extraction. After the phases were separated, the organic phase was collected, washed with saturated brine solution (100 mL*3) in turn, dried with anhydrous sodium sulfate, and concentrated under reduced pressure. Compound B-5-9 was obtained. LCMS, m/z=535.3 [M+1]+.
[0208] Step 8: Synthesis of Compound B-5-10
[0209] In a dry reaction flask, B-5-9 (2 g, 3.15 mmol, purity of 84.334%, 1 eq) was added to tetrahydrofuran (20 mL), and then triethylamine trihydrofluoride (2.54 g, 15.77 mmol, 2.57 mL, 5 eq) was added thereto, and the mixture was stirred at 20° C. for 10 hours. Water (30 mL) was added for dilution, and ethyl acetate (30 mL*3) was added for extraction. After the phases were separated, the organic phase was collected, washed with saturated sodium bicarbonate aqueous solution (30 mL), saturated brine solution (30 mL*3) in turn, dried with anhydrous sodium sulfate, and concentrated under reduced pressure. tert-Butyl methyl ether (10 mL) and ethyl acetate (1 mL) were added to slurry, filtered, and the filter cake was washed with tert-butyl methyl ether (10 mL), and the filter cake was concentrated under reduced pressure. Compound B-5-10 was obtained. LCMS, m/z=297.1 [M+1]+.
[0210] Step 9: Synthesis of Compound B-5-11
[0211] B-5-10 (0.8 g, 2.70 mmol, 1 eq), dichloromethane (10 mL) and triethylamine (819.51 mg, 8.10 mmol, 1.13 mL, 3 eq) were added to a dry reaction flask, and the system was replaced with nitrogen, cooled to 0° C., and added with methanesulfonyl chloride (463.86 mg, 4.05 mmol, 313.42 μL, 1.5 eq), then the mixture was stirred at 20° C. for 3 hours. The reaction solution was poured into water (20 mL) for quenching, added with dichloromethane (20 mL*3) for extraction. After the phases were separated, the organic phase was collected, washed with saturated brine solution (20 mL*3) in turn, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and separated and purified by column chromatography (PE:EA=1:0 to 3:1). Compound B-5 was obtained. LCMS, m/z=315.1 [M+1].sup.+.
Reference Embodiment 5: Segment B-6
[0212] ##STR00068##
[0213] Synthetic Route:
##STR00069## ##STR00070##
[0214] Step 1: Synthesis of Compound B-6-2
[0215] B-6-1 (2.00 g, 12.49 mmol, 1 eq) and anhydrous tetrahydrofuran (100 mL) were added to a reaction flask, and then 2,2,6,6-tetramethylpiperidinylmagnesium chloride lithium chloride complex (1 M, 24.00 mL, 1.92 eq) was added thereto at −40° C., and the reaction system was stirred at −40° C. for 0.5 hours. Carbon tetrabromide (4.14 g, 12.49 mmol, 1 eq) was added thereto, and the reaction system was stirred at −40° C. for 0.5 hours, and the reaction system was stirred at 20° C. for 11 hours. Hydrochloric acid (0.5 M, 10 mL) was added to the reaction solution to quench the reaction, and the mixture was extracted with ethyl acetate (50 mL*3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was separated and purified by column chromatography (PE:EA=1:0-20:1) to obtain compound B-6-2. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 3.88 (s, 3H), 6.89 (s, 1H).
[0216] Step 2: Synthesis of Compound B-6-3
[0217] B-6-2 (1.70 g, 7.11 mmol, 1 eq), B-5-3 (2.23 g, 7.11 mmol, 1.0 eq), potassium carbonate (1.97 g, 14.22 mmol, 2 eq), anhydrous toluene (50 mL) were added to a reaction flask, and then tris(dibenzylideneacetone)dipalladium (0.65 g, 709.82 μmol, 0.10 eq), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (0.82 g, 1.42 mmol, 1.99e.sup.−1 eq) were added thereto under nitrogen atmosphere, and then the reaction system was stirred at 100° C. for 12 hours. The reaction solution was filtered, and water (50 mL) was added to the filtrate, and the mixture was extract with ethyl acetate (50 mL*3). The organic phases were combined, washed with saturated brine (50 mL), dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was separated and purified by column chromatography (PE:EA=1:0-4:1) to obtain compound B-6-3. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 1.16 (s, 9H), 3.87 (s, 3H), 4.31 (s, 2H), 6.42 (s, 1H), 7.41-7.47 (m, 5H), 7.57-7.68 (m, 5H), 9.28 (br s, 1H).
[0218] Step 3: Synthesis of Compound B-6-4
[0219] B-6-3 (2.60 g, 2.84 mmol, purity of 51.53%, 1 eq), N-bromosuccinimide (1.00 g, 5.62 mmol, 1.98 eq) and anhydrous THF (50 mL) were added to a reaction flask, and the reaction system was stirred at 20° C. for 12 hours. Saturated sodium bicarbonate solution (50 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (50 mL*3). The organic phases were combined, washed with saturated brine (50 mL), dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was separated and purified by column chromatography (PE:EA=1:0-10:1) to obtain compound B-6-4. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 1.19 (s, 9H), 3.89 (s, 3H), 4.31 (s, 2H), 7.42-7.50 (m, 6H), 7.66 (br d, J=6.78 Hz, 4H), 9.65 (br s, 1H).
[0220] Step 4: Synthesis of Compound B-6-5
[0221] B-6-4 (1.80 g, 3.27 mmol, 1 eq), Lawesson reagent (1.35 g, 3.34 mmol, 1.02 eq) and anhydrous dioxane (30 mL) were added to a reaction flask, and the reaction system was stirred at 110° C. for 6 hours. The reaction solution was cooled to room temperature and concentrated to obtain a crude product. The crude product was separated and purified by column chromatography (PE:EA=1:0-10:1) to obtain compound B-6-5. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 1.20 (s, 9H), 3.91 (s, 3H), 4.60 (s, 2H), 7.40-7.51 (m, 6H), 7.66 (dd, J=7.91, 1.38 Hz, 4H), 11.18 (br s, 1H).
[0222] Step 5: Synthesis of Compound B-6-6
[0223] B-6-5 (1.50 g, 2.65 mmol, 1 eq), B-1-8 (0.60 g, 6.89 mmol, 2.60 eq), silver acetate (0.90 g, 5.39 mmol, 276.07 μL, 2.04 eq), anhydrous DMF (20 mL) were added to a reaction flask, and the reaction system was stirred at 20° C. for 16 hours. The reaction solution was filtered, and water (50 mL) was added to the filtrate, and the mixture was extracted with ethyl acetate (50 mL*3). The organic phases were combined, washed with saturated brine (50 mL*3), dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was separated and purified by column chromatography (PE:EA=1:0-5:1) to obtain compound B-6-6. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 1.10 (s, 9H), 2.55-2.66 (m, 1H), 2.67-2.78 (m, 1H), 3.56-3.66 (m, 1H), 3.77-3.88 (m, 4H), 4.31-4.45 (m, 2H), 4.53 (dt, J=9.22, 5.93 Hz, 1H), 4.68-4.78 (m, 1H), 5.05-5.17 (m, 1H), 6.95 (br s, 1H), 7.39-7.52 (m, 6H), 7.63 (br t, J=5.65 Hz, 4H).
[0224] Step 6: Synthesis of Compound B-6-7
[0225] B-6-6 (0.80 g, 1.29 mmol, 1 eq), N,N′-dimethylethylenediamine (0.16 g, 1.82 mmol, 195.36 μL, 1.41 eq), acetonitrile (10 mL) were added to a reaction flask, and cuprous iodide (0.16 g, 840.12 μmol, 0.65 eq) was added thereto under nitrogen atmosphere, and the reaction system was stirred at 80° C. for 10 hours. The reaction solution was filtered, added with water (20 mL), and extracted with ethyl acetate (20 mL*3). The organic phases were combined, washed with saturated brine (20 mL*3), dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was separated and purified by thin layer chromatography silica gel plate (PE:EA=3:1) to obtain compound B-6-7.
[0226] Step 7: Synthesis of Compound B-6-8
[0227] B-6-7 (0.40 g, 742.52 μmol, 1 eq) and anhydrous tetrahydrofuran (5 mL) were added to a reaction flask, then tetrabutylammonium fluoride (1 M, 1.00 mL, 1.35 eq) was added thereto, and the reaction system was stirred at 20° C. for 1 hour. The reaction solution was added with water (20 mL) and extracted with ethyl acetate (20 mL*3). The organic phases were combined, washed with saturated brine (20 mL*3), dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was separated and purified by column chromatography (PE:EA=1:0-0:1) to obtain compound B-6-8. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 2.48-2.58 (m, 1H), 2.78-2.89 (m, 1H), 3.91 (s, 3H), 4.40-4.56 (m, 3H), 4.67-4.74 (m, 1H), 4.80-4.90 (m, 2H), 5.19 (qd, J=6.90, 2.89 Hz, 1H).
[0228] Step 8: Synthesis of Compound B-6
[0229] B-6-8 (30 mg, 99.90 μmol, 1 eq) and anhydrous dichloromethane (2 mL) were added to a reaction flask, and methanesulfonyl chloride (30 mg, 261.89 μmol, 20.27 μL, 2.62 eq) and triethylamine (30 mg, 296.47 μmol, 41.27 μL, 2.97 eq) were added thereto at 0° C., then the reaction system was stirred at 20° C. for 1 hour. Saturated sodium bicarbonate solution (0.5 mL) was added to the reaction solution for quenching the reaction, and the mixture was concentrated to obtain compound B-6. LCMS: m/z=318.8 [M+1].sup.+.
Reference Embodiment 6: Segments B-7 and B-8
[0230] ##STR00071##
[0231] Synthetic Route:
##STR00072##
[0232] Step 1: Synthesis of Compound 001-6
[0233] 001-5 (0.76 g, 1.70 mmol, 1 eq), Zn(CN).sub.2 (340 mg, 2.90 mmol, 183.78 μL, 1.70 eq), tetrakis(triphenylphosphine)palladium (197 mg, 170.48 μmol, 0.1 eq), DMF (8 mL) were added to a reaction flask, reacted for 16 hours at 120° C. under the protection of nitrogen. After the reaction was completed, the reaction solution was filtered, and the filtrate was added with 25 mL of water, extracted with ethyl acetate (25 mL*3). The organic phases were combined, washed with 25 mL of saturated brine, dried with anhydrous sodium sulfate, filtered and evaporated to dryness by rotary evaporation to obtain a crude product. The crude product was purified by column chromatography (PE:EA=19:1) to obtain 001-6.
[0234] Step 2: Synthesis of Compounds B-7 and B-8
[0235] 001-6 (520 mg, 1.19 mmol, 1 eq) was separated by supercritical fluid (chromatographic column: DAICEL CHIRALPAK AD (250 mm*30 mm, 10 m); mobile phase: phase A was CO.sub.2, phase B was [0.1% NH.sub.3H.sub.2O EtOH]; B %: 15%-15%) to obtain compound B-7 (the retention time was 0.742 min) and compound B-8 (the retention time was 0.846 min). LCMS: m/z=381.0 [M-55].sup.+.
Embodiment 4
[0236] ##STR00073##
[0237] Synthetic Route:
##STR00074## ##STR00075##
[0238] Step 1: Synthesis of Compounds 003A-1 and 003B-1
[0239] Compound B-3 (130 mg, 346.71 μmol, 1 eq), compound B-2 (150 mg, 433.78 μmol, 1.25 eq), potassium carbonate (143.00 mg, 1.03 mmol, 2.98 eq) and acetonitrile (4 mL) were added to a reaction flask, heated to 50° C. and stirred for 16 hours. After the reaction was completed, the reaction solution was filtered, and the filter cake was washed with dichloromethane (10 mL*2), and then the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified and separated by preparative thin layer chromatography silica gel plate separation (PE:EA=3:1) to obtain compound 003A-1. LCMS: m/z=684.3 [M+1].sup.+.
[0240] Compound B-4 (140 mg, 373.38 μmol, 1 eq), compound B-2 (160 mg, 462.70 μmol, 1.24 eq), potassium carbonate (155 mg, 1.12 mmol, 3.00 eq) and acetonitrile (3 mL) were added to a reaction flask, heated to 50° C. and stirred for 3 hours. After the reaction was completed, the reaction solution was filtered, and the filter cake was washed with dichloromethane (10 mL*2), and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified and separated by preparative thin layer chromatography silica gel plate separation (PE:EA=3:1) to obtain compound 003B-1. LCMS: m/z=684.3 [M+1].sup.+.
[0241] Step 2: Synthesis of Compounds 003A-2 and 003B-2
[0242] Compound 003A-1 (140 mg, 204.59 μmol, 1 eq), tetrabutylammonium fluoride (1 M, 1.02 mL, 5 eq) and tetrahydrofuran (4 mL) were added to a reaction flask, stirred at 50° C. for 24 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified and separated by preparative thin layer chromatography silica gel plate (EA:PE=10/90-60/40) to obtain compound 003A-2. LCMS: m/z=554.1 [M+1].sup.+.
[0243] Compound 003B-1 (180.00 mg, 263.05 μmoL 1 eq), tetrabutylammonium fluoride (1 M, 2.5 mL, 9.50 eq) and tetrahydrofuran (3 mL) were added to a reaction flask, stirred at 60° C. for 24 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure. The residue was added with water (10 mL), and then extracted with ethyl acetate (20 mL*3). The organic phases were combined and washed with saturated brine (10 mL), and concentrated under reduced pressure to obtain a crude product. The crude product was purified and separated by preparative thin layer chromatography silica gel plate (PE:EA=1:1) to obtain compound 003B-2. LCMS: m/z=554.3 [M+1].sup.+.
[0244] Step 3: Synthesis of Compounds 003A-3, 003B-3, 003C-3 and 003D-3
[0245] Compound 003A-2 (120 mg, 216.59 μmol, 1 eq), (S)-oxetan-2-yl methanesulfonate (108.00 mg, 649.84 μmol, 3 eq), cesium carbonate (211.71 mg, 649.78 μmol, 3 eq) and acetonitrile (1.5 mL) were added to a reaction flask, stirred at 80° C. for 16 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure. The residue was added with water (10 mL), and then extracted with ethyl acetate (10 mL*3). The organic phases were combined and concentrated under reduced pressure to obtain a crude product. The crude product was purified and separated by preparative thin layer chromatography silica gel plate (DCM:MeOH=20:1) to obtain a mixture of compounds 003A-3 and 003C-3. LCMS: m/z=624.1 [M+1].sup.+.
[0246] Compound 003B-2 (107.00 mg, 193.13 μmol, 1 eq), (S)-oxetan-2-yl methanesulfonate (96.30 mg, 579.39 μmol, 3 eq), cesium carbonate (188.78 mg, 579.39 μmol, 3 eq) and acetonitrile (2 mL) were added to a reaction flask, stirred at 80° C. for 11 hours. After the reaction was completed, the reaction solution was filtered, and the filter cake was washed with ethyl acetate (20 mL), then the filtrate and the washing solution were combined, and concentrated under reduced pressure to obtain a crude product. The crude product was purified and separated by preparative thin layer chromatography silica gel plate (DCM:MeOH=20:1) to obtain a mixture of compounds 003D-3A and 003B-3. LCMS: m/z=624.3 [M+1].sup.+.
[0247] Step 4: Synthesis of Compounds 003A, 003B, 003C and 003D
[0248] The mixture of compounds 003A-3 and 003C-3 (82 mg, 131.38 μmol, 1 eq), 1,5,7-triazabicyclo[4.4.0]dec-5-ene (37 mg, 265.81 μmol, 2.02 eq), acetonitrile (1 mL) and water (0.2 mL) were added to a reaction flask, stirred at 25° C. for 16 hours. After the reaction was completed, the pH was adjusted to about 6 by adding citric acid, and the mixture was concentrated under reduced pressure to obtain a crude product. The crude product was purified and separated by preparative thin layer chromatography silica gel plate (DCM:MeOH=10:1) to collect a crude product, and the crude product was purified and separated by preparative chiral separation (chromatographic column: DAICEL CHIRALPAK IG (250 mm*30 mm, 10 μm); mobile phase: phase A was CO.sub.2, phase B was [0.1% NH.sub.3H.sub.2O-EtOH]; B %: 50%-50%) to obtain 003A (retention time: 2.282 min) and 003C (retention time: 2.818 min).
[0249] 003A: LCMS: m/z=596.1 [M+1].sup.+; .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 2.05 (s, 3H), 2.38 (br s, 2H), 2.60-2.62 (m, 1H), 2.66 (br s, 1H), 3.05 (br s, 2H), 3.52 (br s, 2H), 4.02-4.19 (m, 2H), 4.35 (br s, 1H), 4.47-4.66 (m, 3H), 5.11 (br s, 1H), 6.42 (br s, 1H), 6.68-6.84 (m, 3H), 7.04-7.17 (m, 2H), 7.43-7.63 (m, 2H).
[0250] 003C: LCMS: m/z=596.1 [M+1].sup.+; .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 2.04 (s, 3H), 2.43 (br s, 1H), 2.52-2.66 (m, 4H), 2.90 (br s, 1H), 3.36 (br s, 2H), 4.01 (br s, 2H), 4.43 (br d, J=16.31 Hz, 2H), 4.55 (br d, J=18.32 Hz, 2H), 5.18 (br s, 1H), 6.39 (br s, 1H), 6.65-6.83 (m, 3H), 7.02-7.18 (m, 2H), 7.48 (t, J=8.28 Hz, 1H), 7.82 (br s, 1H).
[0251] The mixture of compounds 003D-3 and 003B-3 (90 mg, 144.20 μmol, 1 eq), 1,5,7-triazabicyclo[4.4.0]dec-5-ene (40.61 mg, 291.74 μmol, 2.02 eq), acetonitrile (2 mL) and water (0.4 mL) were added to a reaction flask, stirred at 25° C. for 15 hours. After the reaction was completed, the pH was adjusted to about 6 by adding citric acid, and the mixture was concentrated under reduced pressure to obtain a crude product. The crude product was purified and separated by preparative thin layer chromatography silica gel plate (DCM:MeOH=20:1) to collect a crude product, and the crude product was purified and separated by preparative chiral separation (chromatographic column: DAICEL CHIRALPAK IG (250 mm*30 mm, 10 μm); mobile phase: phase A was CO.sub.2; phase B was [0.1% NH.sub.3H.sub.2O-EtOH]; B %: 50%-50%) to obtain 003D (retention time: 2.236 min) and 003B (retention time: t=2.679 min).
[0252] 003D: LCMS: m/z=596.1 [M+1].sup.+; .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 2.05 (s, 3H), 2.40 (br s, 1H), 2.65 (br s, 3H), 2.94 (br s, 2H), 3.41 (br s, 2H), 4.02 (br s, 2H), 4.34 (br s, 1H), 4.54 (br s, 3H), 5.13 (br s, 1H), 6.41 (br s, 1H), 6.70-6.82 (m, 3H), 7.05-7.16 (m, 2H), 7.44-7.54 (m, 1H), 7.63 (br s, 1H).
[0253] 003B: LCMS: m/z=596.1 [M+1].sup.+; .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 2.05 (br s, 3H), 2.35-2.75 (m, 4H), 2.86 (br s, 2H), 3.32 (br s, 2H), 3.97 (br s, 2H), 4.27-4.74 (m, 4H), 5.20 (br s, 1H), 6.39 (br s, 1H), 6.69-6.86 (m, 3H), 7.04-7.17 (m, 2H), 7.49 (t, J=8.28 Hz, 1H), 7.85 (br s, 1H).
[0254] With reference to the synthetic steps of embodiment 4, the starting material in step 1 was replaced with B-7/B-8, and the following compounds 011A, 011B, 011C and 011D were synthesized.
TABLE-US-00001 Embodi- ment 12 Structure Spectrogram 011A
Embodiment 5
[0255] ##STR00080##
[0256] Synthetic Route:
##STR00081##
[0257] Step 1: Synthesis of Compound 004-1
##STR00082##
[0258] Under the protection of nitrogen, wet palladium carbon (0.2 g, purity of 10%) was added to a solution of B-3 (retention time: 2.474 min) (2 g, 4.49 mmol, 1 eq) in ethyl acetate (40 mL), and reacted at 25° C. for 18 hours under hydrogen atmosphere (15 psi). The reaction solution was filtered, and the filter cake was washed with ethyl acetate (5 mL*3), and then the filtrate was combined and evaporated to dryness by rotary evaporation to obtain a crude product. The crude product was purified and separated by column chromatography (EA/PE=0-5%) to obtain compound 004-1. LCMS: m/z=392.2 [M-55].sup.+.
[0259] Step 2: Synthesis of Compound 004-2
##STR00083##
[0260] 004-1 (0.5 g, 1.12 mmol, 1 eq) was dissolved in ethyl acetate (10 mL), and then added with p-toluenesulfonic acid monohydrate (0.25 g, 1.31 mmol, 1.18 eq), protected by nitrogen, and reacted at 50° C. for 18 hours. Saturated sodium bicarbonate aqueous solution (25 mL) was added to the reaction solution, stirred for 2 minutes. The phases were separated, and the organic phase was washed with water (25 mL) and saturated brine (25 mL) respectively, dried with anhydrous sodium sulfate, filtered, and evaporated to dryness by rotary evaporation to obtain compound 004-2. LCMS: m/z=347.9 [M+1].sup.+.
[0261] Step 3: Synthesis of Compound 004-3
##STR00084##
[0262] 004-2 (80 mg, 230.01 μmol, 1.19 eq) and B-5 (61 mg, 193.78 μmol, 1 eq) were dissolved in acetonitrile (2 mL), and then added with potassium carbonate (80 mg, 578.85 μmol, 2.99 eq), protected by nitrogen atmosphere, and reacted at 50° C. for 16 hours. The reaction solution was filtered, and the filtrate was concentrated to obtain a crude product. The crude product was purified by preparative thin layer chromatography silica gel plate (DCM:MeOH=20:1) to obtain compound 004-3. LCMS, m/z=626.2 [M+1].sup.+.
[0263] Step 4: Synthesis of Compound 004
##STR00085##
[0264] 004-3 (40 mg, 63.88 μmol, 1 eq) was dissolved in a mixture of tetrahydrofuran (0.25 mL), methanol (0.25 mL) and water (0.5 mL), and added with lithium hydroxide (7.65 mg, 319.42 μmol, 5 eq), protected by nitrogen atmosphere, and reacted at 25° C. for 1 hour. The pH was adjusted to about 6 by adding citric acid to the reaction solution, and the mixture was concentrated to obtain a crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Phenomenex Gemini-NX 80*40 mm*3 m; mobile phase: [water (0.05% ammonia water)-acetonitrile]; B (acetonitrile) %: 21%-51%, 8 min) to obtain 004. LCMS, m/z=598.1 [M+1].sup.+; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 7.55 (t, J=8.4 Hz, 1H), 7.45-7.51 (m, 2H), 7.30 (br d, J=7.0 Hz, 1H), 6.73-6.78 (m, 2H), 6.67-6.73 (m, 1H), 5.08 (br d, J=3.5 Hz, 1H), 4.52-4.59 (m, 1H), 4.43-4.50 (m, 2H), 4.32-4.39 (m, 1H), 3.73-3.79 (m, 1H), 3.67 (br d, J=13.6 Hz, 1H), 2.97 (br d, J=10.5 Hz, 1H), 2.87 (br d, J=10.5 Hz, 1H), 2.58-2.71 (m, 2H), 2.43 (br d, J=8.8 Hz, 1H), 2.13 (br d, J=11.5 Hz, 2H), 2.01 (s, 3H), 1.65-1.78 (m, 4H).
Embodiment 6
[0265] ##STR00086##
[0266] Synthetic Route:
##STR00087## ##STR00088##
[0267] Step 1: Synthesis of Compounds 005A-2 and 005B-2
[0268] 001-3 (0.61 g, 1.78 mmol, 1 eq), 005-1 (436.51 mg, 1.96 mmol, 1.1 eq), cesium carbonate (852.18 mg, 2.62 mmol, 1.47 eq), 2,2-bis(diphenylphosphino)-1,1-binaphthyl (83.21 mg, 133.64 μmol, 7.51e.sup.−2 eq) and palladium acetate (20.05 mg, 89.31 μmol, 0.05 eq) were suspended in toluene (20 mL), protected by nitrogen atmosphere, reacted at 100° C. for 16 hours. The solvent was evaporated to dryness by rotary evaporation, then dichloromethane (40 mL) was added to fully dissolve the residue, and the mixture was filtered, and the filtrate was evaporated to dryness by rotary evaporation to obtain a crude product. The crude product was separated by column chromatography (PE:EA=20:1) to obtain a racemate. The racemate was separated and purified by supercritical fluid chromatography (DAICEL CHIRALPAK AD (250 mm*30 mm, 10 μm): mobile phase: phase A was CO.sub.2, phase B was [0.1% ammonia water-ethanol]; B %: 15%-15%) to obtain 005A-2 (retention time: 2.836 min), LCMS: m/z=449.0 [M+1].sup.+; and 005B-2 (retention time: 2.664 min), LCMS: m/z=449.0 [M+1].sup.+.
[0269] Step 2: Synthesis of Compounds 005A-3 and 005B-3
[0270] 005A-2 (210.00 mg, 467.80 μmol, 1 eq) was added to ethyl acetate (5 mL), and added with p-toluenesulfonic acid monohydrate (0.1 g, 525.72 μmol, 1.12 eq), protected by nitrogen atmosphere, reacted at 50° C. for 18 hours. The reaction solution was washed with saturated sodium carbonate aqueous solution (5 mL), water (10 mL) and saturated brine (10 mL) respectively. The organic phase was dried with anhydrous sodium sulfate, filtered, and evaporated to dryness by rotary evaporation to obtain compound 005A-3. LCMS: m/z=348.9 [M+1].sup.+.
[0271] 005B-2 (180.00 mg, 400.97 μmol, 1 eq) was dissolved in ethyl acetate (5 mL), and added with p-toluenesulfonic acid monohydrate (0.1 g, 525.72 μmol, 1.31 eq), protected by nitrogen atmosphere, reacted at 50° C. for 34 hours. The reaction solution was washed with saturated sodium carbonate aqueous solution (5 mL), water (10 mL) and saturated brine (10 mL) respectively. The organic phase was dried with anhydrous sodium sulfate, filtered, and evaporated to dryness by rotary evaporation to obtain compound 005B-3. LCMS: m/z=348.9 [M+1].sup.+.
[0272] Step 3: Synthesis of Compounds 005A-4 and 005B-4
[0273] 005A-3 (150.00 mg, 430.05 μmol, 1.2 eq), B-5 (0.11 g, 349.41 μmol, 0.98 eq) and potassium carbonate (0.15 g, 1.09 mmol, 3.03 eq) were suspended in acetonitrile (4 mL), protected by nitrogen atmosphere, and reacted at 50° C. for 16 hours. The reaction solution was filtered, and the filtrate was evaporated to dryness by rotary evaporation to obtain a crude product. The crude product was separated by column chromatography (DCM:MeOH=20:1) to obtain compound 005A-4. LCMS: m/z=627.1 [M+1].sup.+.
[0274] 005B-3 (100 mg, 286.70 μmol, 1.2 eq), B-5 (75 mg, 238.20 μmol, 1.00 eq) and potassium carbonate (0.1 g, 723.56 μmol, 3.03 eq) were suspended in acetonitrile (3 mL), protected by nitrogen, and reacted at 50° C. for 16 hours. The reaction solution was filtered, and the filtrate was evaporated to dryness by rotary evaporation to obtain a crude product. The crude product was separated by column chromatography (DCM:MeOH=40:1) to obtain compound 005B-4. LCMS: m/z=627.0 [M+1].sup.+.
[0275] Step 4: Synthesis of Compounds 005A and 005B
[0276] 005A-4 (0.18 g, 287.02 μmol, 1 eq) was dissolved in tetrahydrofuran (2 mL), and then lithium hydroxide monohydrate (60.00 mg, 1.43 mmol, 4.98 eq), water (1 mL) and methanol (1 mL) were added thereto, and the mixture was stirred at 25° C. for 1 hour. The pH was adjusted to about 6 by adding citric acid to the reaction solution, and the mixture was concentrated to obtain a crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Phenomenex Gemini-NX 80*40 mm*3 m: mobile phase: [water (0.05% ammonia water)—acetonitrile]; B (acetonitrile) %: 23%-53%, 8 min) to obtain compound 005A. ee %=97.08%; LCMS: m/z=599.1 [M+1].sup.+; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 7.51-7.58 (m, 2H), 7.33 (dd, J=8.5, 2.0 Hz, 1H), 7.22 (s, 1H), 6.72-6.78 (m, 1H), 6.54 (d, J=7.8 Hz, 1H), 6.43 (d, J=8.3 Hz, 1H), 5.00-5.09 (m, 1H), 4.40-4.55 (m, 3H), 4.33-4.40 (m, 1H), 3.78 (d, J=13.3 Hz, 1H), 3.65 (d, J=13.3 Hz, 1H), 3.15 (br s, 2H), 3.05 (br s, 2H), 2.63-2.70 (m, 1H), 2.54 (br s, 4H), 2.34-2.43 (m, 1H), 2.01 (s, 3H).
[0277] 005B-4 (0.12 g, 191.35 μmol, 1 eq) was dissolved in tetrahydrofuran (2 mL), and then lithium hydroxide monohydrate (40.00 mg, 953.21 μmol, 4.98 eq), water (1 mL) and methanol (1 mL) were added thereto, and the mixture was stirred at 25° C. for 1 hour. The pH was adjusted to about 6 by adding citric acid to the reaction solution, and the mixture was concentrated to obtain a crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Phenomenex Gemini-NX 80*40 mm*3 m: mobile phase: [water (0.05% ammonia water)—acetonitrile]; B (acetonitrile) %: 23%-53%, 8 min) to obtain compound 005B. ee %=100%; LCMS: m/z=599.1 [M+1].sup.+; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 7.51-7.58 (m, 2H), 7.33 (dd, J=8.5, 1.8 Hz, 1H), 7.28 (s, 1H), 6.71-6.78 (m, 1H), 6.54 (d, J=7.5 Hz, 1H), 6.43 (d, J=8.3 Hz, 1H), 5.02-5.09 (m, 1H), 4.40-4.55 (m, 3H), 4.34 (dt, J=9.1, 6.0 Hz, 1H), 3.69-3.77 (m, 2H), 3.13 (br s, 2H), 3.04 (br d, J=11.0 Hz, 2H), 2.62-2.70 (m, 1H), 2.55 (br s, 4H), 2.35-2.43 (m, 1H), 2.00 (s, 3H).
Embodiment 7
[0278] ##STR00089##
[0279] Synthetic Route:
##STR00090##
[0280] Step 1: Synthesis of Compound 006-1
##STR00091##
[0281] 004-2 (60.00 mg, 172.51 μmol, 1.10 eq) and B-6 (50 mg, 156.86 μmol, 1 eq) were dissolved in acetonitrile (2 mL), and added with potassium carbonate (64.29 mg, 465.13 μmol, 2.97 eq), protected by nitrogen atmosphere, reacted at 50° C. for 16 hours. The reaction solution was filtered and the filtrate was concentrated to obtain a crude product. The crude product was purified by column chromatography (DCM:MeOH=10:1) to obtain compound 006-1. LCMS: m/z=630.0 [M+1].sup.+.
[0282] Step 2: Synthesis of Compound 006
##STR00092##
[0283] 006-1 (50 mg, 79.35 μmol, 1 eq) was dissolved in a mixture of tetrahydrofuran (1 mL), methanol (0.5 mL) and water (0.5 mL), and added with lithium hydroxide (9.50 mg, 396.76 mol, 5 eq), protected by nitrogen atmosphere, reacted at 25° C. for 1 hour. The pH was adjusted to about 6 by adding citric acid to the reaction solution, and the mixture was concentrated to obtain a crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Phenomenex Gemini-NX 80*40 mm*3 m: mobile phase: [water (0.05% ammonia water)—acetonitrile]; B (acetonitrile) %: 24%-54%, 8 min) to obtain compound 006. LCMS, m/z=616.0 [M+1].sup.+; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 7.53-7.59 (m, 2H), 7.34 (dd, J=8.4, 1.9 Hz, 1H), 6.79 (d, J=4.3 Hz, 2H), 6.71-6.76 (m, 1H), 5.02-5.10 (m, 1H), 4.45-4.63 (m, 3H), 4.39 (dt, J=9.0, 6.1 Hz, 1H), 3.82 (br d, J=13.6 Hz, 1H), 3.69 (br d, J=13.6 Hz, 1H), 2.98 (br d, J=11.0 Hz, 1H), 2.88 (br d, J=11.3 Hz, 1H), 2.58-2.76 (m, 2H), 2.39-2.46 (m, 1H), 2.09-2.23 (m, 2H), 2.02 (s, 3H), 1.66-1.79 (m, 4H).
Embodiment 8
[0284] ##STR00093##
[0285] Synthetic Route:
##STR00094##
[0286] Step 1: Synthesis of Compound 007-1
##STR00095##
[0287] Step 2: Synthesis of Compound 007
##STR00096##
Embodiment 9
[0288] ##STR00097##
[0289] Synthetic Route:
##STR00098##
[0290] Step 1: Synthesis of Compound 008-2
[0291] 001-3 (140.00 mg, 407.48 μmol, 1 eq), 008-1 (98.61 mg, 464.53 μmol, 1.14 eq), toluene (3 mL) and cesium carbonate (195.16 mg, 599.00 μmol, 1.47 eq) were added to a dry reaction flask in turn, and the system was replaced with nitrogen atmosphere, then palladium acetate (4.57 mg, 20.37 μmol, 0.05 eq) and 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (17.76 mg, 28.52 μmol, 0.07 eq) were added thereto, and then the system was replaced with nitrogen again. The temperature was raised to 100° C., and the mixture was stirred for 10 hours. The mixture was diluted with water (20 mL), extracted with ethyl acetate (20 mL*3). After the phases were separated, the organic phases were collected, washed with saturated brine solution (20 mL*3) in turn, dried with anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by thin layer chromatography silica gel plate (DCM:MeOH=20:1) to obtain compound 008-2. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 7.52 (t, J=8.3 Hz, 1H), 7.17-7.09 (m, 2H), 6.72 (t, J=8.0 Hz, 1H), 6.39 (br d, J=7.9 Hz, 1H), 6.04 (br d, J=7.8 Hz, 1H), 3.97-3.81 (m, 4H), 3.53 (s, 2H), 3.47-3.35 (m, 2H), 2.20-2.08 (m, 2H), 2.04 (s, 3H), 1.48 (s, 9H).
[0292] Step 2: Synthesis of Compound 008-3
[0293] 008-2 (100.00 mg, 210.55 μmol, 1 eq), dichloromethane (1 mL) and trifluoroacetic acid (0.1 mL) were added to a dry reaction flask, stirred at 20° C. for 1 hour. Sodium bicarbonate aqueous solution (20 mL) was added for neutralization, and ethyl acetate (20 mL*3) was added for extraction. After the phases were separated, the organic phases were collected, washed with saturated brine solution (20 mL*3) in turn, dried with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound 008-3. LCMS: m/z=375.0 [M+1].sup.+.
[0294] Step 3: Synthesis of Compound 008-4
[0295] 008-3 (92.38 mg, 293.46 μmol, 1 eq), acetonitrile (3 mL) and potassium carbonate (60.84 mg, 440.19 μmol, 1.5 eq) were added to a dry reaction flask, stirred at 60° C. for 10 hours. Water (10 mL) was added for dilution, and ethyl acetate (10 mL*3) was added for extraction. After the phases were separated, the organic phases were collected, washed with saturated brine solution (10 mL*3) in turn, dried with anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by thin layer chromatography silica gel plate (DCM:MeOH=20:1) to obtain compound 008-4. LCMS: m/z=653.3 [M+1].sup.+.
[0296] Step 4: Synthesis of Compound 008
[0297] 008-4 (10 mg, 15.31 μmol, 1 eq), water (0.5 mL), methanol (0.5 mL), tetrahydrofuran (0.5 mL) and lithium hydroxide monohydrate (1.93 mg, 45.93 μmol, 3 eq) were added to a dry reaction flask, stirred at 20° C. for 1 hour. The reaction solution was directly concentrated under reduced pressure. The residue was separated by high performance liquid chromatography (chromatographic column: Phenomenex C18 75*30 mm*3 m; mobile phase: [water (ammonia water+ammonium bicarbonate) acetonitrile]; B (acetonitrile) %: 30%-60%, 8 min) to obtain compound 008. LCMS: m/z=625.1 [M+1].sup.+; .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.68 (s, 1H), 7.56 (t, J=8.3 Hz, 1H), 7.29-7.23 (m, 1H), 7.19 (dd, J=1.5, 8.5 Hz, 1H), 6.69 (t, J=8.0 Hz, 1H), 6.35 (d, J=7.8 Hz, 1H), 6.07 (d, J=8.1 Hz, 1H), 5.17 (dq, J=2.6, 7.0 Hz, 1H), 4.68-4.60 (m, 2H), 4.55 (d, J=2.6 Hz, 1H), 4.45-4.38 (m, 1H), 4.27-4.14 (m, 2H), 3.95-3.92 (m, 1H), 3.88 (s, 2H), 3.82 (d, J=7.4 Hz, 1H), 3.23-3.15 (m, 2H), 3.02-2.96 (m, 2H), 2.83-2.72 (m, 1H), 2.51-2.43 (m, 1H), 2.23 (t, J=7.1 Hz, 2H), 1.99 (s, 3H).
Embodiment 10
[0298] ##STR00099##
[0299] Synthetic Route:
##STR00100##
[0300] Step 1: Synthesis of Compound 009-2
[0301] 001-3 (300 mg, 873.17 μmol, 1.0 eq), 009-1 (203.90 mg, 960.49 μmol, 1.1 eq), 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (38.06 mg, 61.12 μmol, 0.07 eq), palladium acetate (9.80 mg, 43.66 μmoL, 0.05 eq) and cesium carbonate (426.74 mg, 1.31 mmol, 1.5 eq) were added to toluene (10 mL), and reacted for 12 hours at 100° C. under the protection of nitrogen atmosphere. The reaction solution was added with water (20 mL) and ethyl acetate (20 mL*3). After the phases were separated, the organic phases were collected, washed with saturated brine solution (20 mL*3) in turn, dried with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by thin layer chromatography silica gel plate (DCM:MeOH=20:1) to obtain compound 009-2. LCMS: m/z=475.1 [M+1].sup.+.
[0302] Step 2: Synthesis of Compound 009-3
[0303] 009-2 (230 mg, 484.26 μmol, 1 eq) was dissolved in dichloromethane (5 mL), added with trifluoroacetic acid (924.00 mg, 8.10 mmol, 0.6 mL, 16.73 eq), protected by nitrogen atmosphere, reacted for 0.5 hours at 25° C. The reaction solution was directly evaporated to dryness by rotary evaporation to obtain compound 009-3. LCMS: m/z=375.1 [M+1].sup.+.
[0304] Step 3: Synthesis of Compound 009-4
[0305] 009-3 (170 mg, 453.53 μmol, 1 eq) and B-5 (142.77 mg, 453.53 μmol, 1 eq) were dissolved in acetonitrile (4 mL), added with potassium carbonate (94.02 mg, 680.30 μmol, 1.5 eq) and reacted at 60° C. for 4 hours. The reaction solution was poured into saturated ammonium chloride (20 mL) aqueous solution, and ethyl acetate (20 mL*3) was added for extraction. The organic phases were combined, washed with saturated brine solution (20 mL*2) in turn, dried with anhydrous sodium sulfate, and evaporated to dryness by rotary evaporation under reduced pressure to obtain compound 009-4. LCMS: m/z=653.3 [M+1].sup.+.
[0306] Step 4: Synthesis of Compound 009
[0307] 009-4 (230 mg, 352.13 μmol, 1 eq), tetrahydrofuran (2 mL), methanol (2 mL) and lithium hydroxide monohydrate (44.33 mg, 1.06 mmol, 3 eq) were added to a dry reaction flask, stirred at 20° C. for 2 hours. The reaction solution was directly concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by high performance liquid chromatography separation (chromatographic column: Phenomenex C18 75*30 mm*3 m; mobile phase: [water (ammonia water+ammonium bicarbonate) acetonitrile]; B (acetonitrile) %: 30%-60%) to obtain 009. LCMS: m/z=625.2 [M+1].sup.+; .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.58-7.48 (m, 1H), 7.27-7.23 (m, 1H), 7.17-7.06 (m, 2H), 6.80-6.63 (m, 1H), 6.35 (d, J=7.5 Hz, 1H), 6.13 (br d, J=8.0 Hz, 1H), 5.10-4.96 (m, 1H), 4.61-4.53 (m, 2H), 4.50-4.40 (m, 2H), 4.38-4.29 (m, 2H), 4.04 (br s, 4H), 3.72-3.63 (m, 2H), 3.54-3.47 (m, 1H), 3.45-3.39 (m, 1H), 2.72-2.55 (m, 1H), 2.36-2.17 (m, 3H), 2.03 (s, 3H).
Embodiment 11
[0308] ##STR00101##
[0309] Synthetic Route:
##STR00102## ##STR00103##
[0310] Step 1: Synthesis of Compound 010-2
[0311] 001-3 (0.20 g, 582.11 μmol, 1.00 eq), 010-1 (135.93 mg, 640.33 μmol, 1.10 eq), 2,2-bis(diphenylphosphino)-1,1-binaphthyl (18.12 mg, 29.11 μmol, 0.05 eq), palladium acetate (6.53 mg, 29.11 μmol, 0.05 eq) and cesium carbonate (284.50 mg, 873.17 mmol, 1.50 eq) were suspended in toluene (4 mL) and reacted for 4 hours at 100° C. under the protection of nitrogen. The reaction solution was added with water (5 mL) and ethyl acetate (5 mL) for extraction, and the organic phase was concentrated under reduced pressure to obtain a crude product. The crude product was separated by column chromatography (PE:EA=3:1) to obtain 010-2. LCMS: m/z=475.1 [M+1].sup.+.
[0312] Step 2: Synthesis of Compound 010-3
[0313] 010-2 (90.00 mg, 189.49 μmol, 1.00 eq) was added to ethyl acetate (3.6 mL), and added with p-toluenesulfonic acid monohydrate (39.65 mg, 208.44 μmol, 1.10 eq), reacted at 60° C. for 6 hours. Water and ethyl acetate (2 mL) were added thereto, and the mixture was stirred for 5 min, then the pH was adjusted to 8 by adding sodium bicarbonate solid, and the mixture was stood for phase separation. The aqueous phase was extracted once with ethyl acetate (2 mL). The organic phases were combined, and dried with anhydrous sodium sulfate, filtered, and concentrated to obtain 010-3. LCMS: m/z=375.0 [M+1].sup.+.
[0314] Step 3: Synthesis of Compound 010-4
[0315] B-5 (60.00 mg, 110.49 μmol, 1.00 eq), 010-3 (38.26 mg, 121.54 μmol, 1.10 eq) and potassium carbonate (45.81 mg, 331.46 μmol, 3.00 eq) were suspended in acetonitrile (1.5 mL), reacted at 50° C. for 5 hours. The mixture was filtered, and the filtrate was concentrated to obtain compound 010-4. LCMS: m/z=653.3 [M+1].sup.+.
[0316] Step 4: Synthesis of Compounds 010A and 010B
[0317] 010-4 (0.10 g, 153.10 μmol, 1.00 eq) was dissolved in a mixture of tetrahydrofuran (0.5 mL), methanol (0.5 mL) and water (0.5 mL), and added with lithium hydroxide (18.33 mg, 765.50 μmol, 5.00 eq), reacted at 20° C. for 24 hours. The pH was adjusted to about 5 by adding citric acid, and the mixture was concentrated to obtain a crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Waters Xbridge Prep OBD C18 150*40 mm*10 m: mobile phase: [water (0.05% ammonia water+10 mM ammonium bicarbonate)—acetonitrile]; B (acetonitrile) %: 10%-65%, 8 min) to obtain a racemate. The racemate was separated and purified by prep-SFC (chromatographic column: DAICEL CHIRALPAK IG (250 mm*30 mm, m); mobile phase: phase A was supercritical CO.sub.2, phase B was [0.1% ammonia water-methanol]; B %: 45%-45%, 10 min) to obtain 010A (retention time: 1.307 min), ee %=99.42%; 010B (retention time: 1.657 min), ee %=93.36%.
[0318] 010A: LCMS: m/z=625.1 [M+1].sup.+; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 7.8 (br s, 1H), 7.5-7.6 (m, 2H), 7.3-7.4 (m, 1H), 6.7-6.8 (m, 1H), 6.5 (d, J=7.8 Hz, 1H), 6.3 (d, J=8.5 Hz, 1H), 5.2 (br d, J=4.3 Hz, 1H), 4.7-4.7 (m, 1H), 4.6-4.7 (m, 1H), 4.5-4.5 (m, 1H), 4.4 (dt, J=8.9, 6.0 Hz, 1H), 3.7-3.8 (m, 2H), 3.4 (br d, J=10.4 Hz, 2H), 2.7-2.9 (m, 2H), 2.3-2.4 (m, 2H), 2.0 (m, 5H), 1.8 (br d, J=8.4 Hz, 2H), 1.1-1.3 (m, 2H)
[0319] 010B: LCMS: m/z=625.1 [M+1].sup.+; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 7.7 (br s, 1H), 7.5-7.6 (m, 2H), 7.3 (dd, J=8.4, 1.9 Hz, 1H), 6.7-6.7 (m, 1H), 6.5 (d, J=7.8 Hz, 1H), 6.3 (d, J=8.4 Hz, 1H), 5.1-5.2 (m, 1H), 4.7-4.8 (m, 1H), 4.5-4.6 (m, 1H), 4.4-4.5 (m, 1H), 4.4 (dt, J=8.9, 6.1 Hz, 1H), 3.7-3.8 (m, 2H), 3.4 (br d, J=10.9 Hz, 2H), 2.7-2.9 (m, 2H), 2.3-2.4 (m, 2H), 2.0 (s, 5H), 1.8 (br d, J=7.5 Hz, 2H), 1.1-1.3 (m, 2H).
Experimental Embodiment 1
[0320] Binding simulation of compounds 3-6 to GLP-1 receptor:
##STR00104##
[0321] The molecular docking process was carried out by using the Glide SP [1] and a default option in Maestro (Schrödinger version 2017-2). A co-crystal structure of GLP-1R (PDBID code: 5NX2) was selected as a docking template. To prepare proteins, a hydrogen atom was added using a protein preparation guide module of Maestro [2], and an OPLS3 force field was used. For the preparation of ligands, LigPrep was used to generate the three-dimensional structure of the molecule, and the energy minimization [3] was performed. Using a ligand in a 5NX2 crystal structure as a center of mass, a cubic docking grid with a side length of 30 Å was generated. The ligand was then removed and the embodiment compound was placed during molecular docking process. The interaction type between protein receptor and ligand was analyzed, and then the reasonable docking conformation was selected and saved according to the calculated binding mode and docking score. The binding mode of compound 3-compound 6 to GLP-1 receptor is shown in
[0325] GLP-1 receptor three-dimensional structure (
[0326] GLP-1 binding site of the agonist (
[0327] Antagonist binds to the extracellular regions of the 5th, 6th and 7th helical protein surfaces (
Experimental Embodiment 2: Cell Activity Test In Vitro
[0328] 1. Material
[0329] 1) Cell Line
[0330] The cells were constructed by Wuxi New Drug Development Co., Ltd. Shanghai. Details are shown in the following table.
TABLE-US-00002 Target Host cell GLP-1 HEK293
[0331] 2) Reagent
TABLE-US-00003 cAMP Detection Kit, Cisbio (Cat # 62AM4PEJ) 1M HEPES, Invitrogen (Cat # 15630-106) 1X HBSS, Invitrogen (Cat # 14025) BSA, Sigma (Cat # B2064) IBMX, Sigma (Cat # I5879) Exenatide, Hao Yuan (HY-13443A)
[0332] 3) Equipment
[0333] OptiPlate-384, White, PerkinElmer (Cat #6007290); 384 well plate for Echo, Labcyte (Cat #P-05525); EnVision, PerkinElmer; Vi-cell counter, Beckman (Cat #Vi-CELL™ XR Cell Viability Analyzer)
[0334] 4) Compound Information
[0335] The compound was formulated with DMSO to a working concentration of 30 μM. In this test, the usage amount of each sample was 5 μL.
[0336] 2. Method
[0337] 1) Experimental Material
[0338] Experimental Buffer
TABLE-US-00004 Volume Final concentration 24.5 mL of Hanks Buffer Saline Solution (HBSS) 1x 125 μL of HEPES 1M 5 mM 333 μL of 7.5% BSA Solution 0.1% 25 μL of IBMX 500 mmol/L 0.5 mmol/L
[0339] The pH was adjusted to 7.4 and the volume was fixed to 25 mL with HBSS 1×.
[0340] Preparation of Detection Reagent
[0341] Preparation of cAMP detection reagent: 250 μL of cAMP-D2 and 250 μL of anti-cAMP cryptate reagent were added to 4 mL of lysis buffer, and mixed gently.
[0342] 2) Experimental Method
[0343] a) Preparation of Compound Plates:
[0344] The test compound was diluted 3 times by 10 points with a starting concentration of 30 μM, and the dilutions were completed by Bravo.
[0345] The reference compound exenatide was diluted 3 times by 10 points with a starting concentration of 500 nM, and the dilutions were completed by Bravo.
[0346] b) Transfer of Compounds:
[0347] 1) 100 nL of compound was transferred to OptiPlate-384 plate using Echo.
[0348] 2) OptiPlate-384 plate was centrifuged at 1000 rpm for 5 seconds.
[0349] c) Preparation of cell suspension
[0350] 1) A GLP-1 cell cryopreservation tube was quickly thawed in 37° C. warm water.
[0351] 2) The cell suspension was transferred to a Transfer 15 mL of centrifuge tube and gently rinsed with 10 mL of HBSS.
[0352] 3) The centrifuge tube was centrifuged at 1000 rpm at room temperature for 1 minute.
[0353] 4) The supernatant was discarded.
[0354] 5) The cells at the bottom were gently dispersed and gently rinsed with 10 mL of HBSS, then the cells were centrifuged for sedimentation, and finally the cells were re-suspended in experimental buffer.
[0355] 6) Cell density and activity were measured by using Vi-cell.
[0356] 7) The concentration of GLP-1 cells was diluted to 2.0*10.sup.5/mL with the experimental buffer.
[0357] 8) 100 nL of diluted cell suspension was transferred into OptiPlate-384 plate.
[0358] 9) The cells were incubated at room temperature for 30 minutes.
[0359] d) Addition of detection reagents:
[0360] 1) 10 μL of 800 nM gradient diluted cAMP standard was added to the empty well of OptiPlate-384 plate.
[0361] 2) 10 μL of cAMP detection reagent was added.
[0362] 3) OptiPlate-384 plate was covered with TopSeal-A film and incubated for 60 min at room temperature.
[0363] The TopSeal-A was removed, and the EnVision reading was performed.
[0364] The experimental results are shown in Table 1:
TABLE-US-00005 TABLE 1 In vitro cell activity test results Compound Human-GLP 1, EC.sub.50 (nM) 003A 1.16 003C 8.63 004 0.20 005A 0.15 006 0.39 011A 0.33 0011B 1.52
[0365] Conclusion: The compound of the present disclosure shows a better agonistic ability to GLP-1 receptor.
Experimental Embodiment 3: Cytochrome P450 Isoenzyme Inhibitory Research Test
[0366] 1. Test Purpose
[0367] To test the inhibitory effects of the test compounds on the activities of human liver microsomal cytochrome P450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4).
[0368] 2. Experimental Method
[0369] Firstly, the test compound (10.0 mM) was diluted in gradient to prepare a working solution (100× final concentration), and the working solution concentrations were 5.00, 1.50, 0.500, 0.150, 0.0500, 0.0150 and 0.00500 mM respectively. At the same time, the working solution of each positive inhibitor and its specific substrate mixture of P450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) was prepared; the human liver microsomes stored in the refrigerator below −60° C. were thawed on ice. When the human liver microsomes were completely dissolved, then diluted with potassium phosphate buffer (PB) to prepare a working solution with a certain concentration (0.253 mg/mL).
[0370] Firstly, 20.0 μL of substrate mixture was added to the reaction plate (20.0 μL of PB was added to the blank well), then 158 μL of human liver microsome working solution was added to the reaction plate, and the reaction plate was placed on ice for later use. At this time, 2.00 μL of test compound (N=1) and specific inhibitor (N=2) of each concentration were added to the corresponding well, and the group without inhibitor (test compound or positive inhibitor) was added with the corresponding organic solvent as a of control sample (control sample of test compound was dimethyl sulfoxide:methanol=1:1, positive control samples were all dimethyl sulfoxide:methanol=1:9). After pre-incubation in 37° C. water bath for 10 minutes, 20.0 μL of a coenzyme factor (NADPH) solution was added to the reaction plate and incubated in 37° C. water bath for 10 minutes. Then 400 μL of pre-cooled acetonitrile solution (internal standard containing 200 ng/mL tolbutamide and labetalol) was added to stop the reaction; the reaction plate was placed in a shaker and shaken for 10 min to mix evenly. Then the reaction plate was centrifuged at 4° C. and 4000 rpm for 20 min. 200 μL of supernatant was taken and added to 100 μL of water for sample dilution. Finally, the plate was sealed, shaken for 10 minutes to mix evenly for LC/MS/MS detection.
TABLE-US-00006 TABLE 2 Results of the inhibitory effect of the compound of the present disclosure on the activity of human liver microsomal cytochrome P450 isoenzymes IC.sub.50 (μM) Compound CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4-M 004 43.7 31.9 23.6 >50 40.8 005A >50 30.8 >50 >50 >50 006 38.8 21.6 >50 >50 48.7
[0371] Conclusion: The compound of the present disclosure has a weak inhibitory effect on human liver microsomal cytochrome P450 isoenzymes and a low risk of “drug-drug interaction”.
Experimental Embodiment 4: Cytochrome P450 Isoenzyme Time-Dependent Inhibitory Research Test
[0372] 1. Test Purpose
[0373] To test the time-dependent inhibitory effect of the test compounds on the activity of human liver microsomal cytochrome P450 isoenzyme CYP2C19.
[0374] 2. Experimental Method
[0375] The experiment was divided into two groups. In the first group, human liver microsomes (HLM) were used as the incubation system, and a series of concentrations of test samples were added to the incubation system, and then coenzyme factor (NADPH) solution was added, and pre-incubated at 37° C. for 30 minutes. After pre-incubation, the probe substrate solution was added, and after incubation for a certain time, the reaction was terminated; the amount of probe substrate metabolites produced in the incubation solution was measured, and the enzyme activity was calculated. The second group of reactions was to use human liver microsomes (HLM) as the incubation system, and a series of concentrations of test samples were added to the incubation system, and then potassium phosphate buffer was added, and pre-incubated at 37° C. for 30 minutes. After pre-incubation, a mixed solution of NADPH and probe substrate was added. After incubation for a certain time, the reaction was terminated, the amount of probe substrate metabolites produced in the incubation solution was measured, and the enzyme activity was calculated.
[0376] Firstly, the test compound (10.0 mM) was diluted in gradient to prepare a working solution (100× final concentration), and the working solution concentrations were 5.00, 1.65, 0.500, 0.165, 0.0500, 0.0165 and 0.00500 mM respectively. At the same time, the positive inhibitor of P450 isoenzyme CYP2C19, probe substrate and NADPH working solution were prepared; the human liver microsomes stored in the refrigerator below −60° C. were thawed on ice. When the human liver microsomes were completely dissolved, then diluted with potassium phosphate buffer (PB) to prepare a working solution with a certain concentration (0.169 mg/mL).
[0377] Then 147.5 μL of human liver microsome working solution was added to the reaction plate, and the reaction plate was placed on ice for use. At this time, 2.50 μL of test compound (N=1) and the working solution of the positive control inhibitor (N=2) of each concentration were added to the corresponding well, and the corresponding organic solvent was added to the group without inhibitor (test compound or positive inhibitor). After incubating the reaction plate at 37° C. for 10 minutes, 50.0 μL of NADPH solution or potassium phosphate buffer solution was added to the first group or the second group of reaction wells respectively to start the reaction. The reaction plate was placed at 37° C. and pre-incubated for 30 minutes; 50 μL of substrate solution or a mixed solution of NADPH and substrate were added to the first group or the second group of reaction wells respectively to start the reaction. After 20 minutes, 250 μL of pre-cooled acetonitrile solution (internal standard containing 200 ng/mL tolbutamide and labetalol) was added to terminate the reaction. The reaction plate was placed on the shaker, shaken for 10 minutes to mix well; then centrifugated at 4° C. and 4000 rpm for 20 minutes. 200 μL of supernatant was taken and added to 200 μL of water for sample dilution; finally, the plate was sealed, shaken for 10 min to mix well for LC/MS/MS detection.
TABLE-US-00007 TABLE 3 Time-dependent inhibition of the compound of the present disclosure on human liver microsomal cytochrome P450 isoenzyme Time- IC.sub.50 dependent IC.sub.50 (μM) offset inhibition Compound CYP −NADPH +NADPH multiple (Yes or No) 005A CYP2C19 44.3 >50 <0.886 No
[0378] Conclusion: The compound of the present disclosure has no time-dependent inhibitory effect on human liver microsomal cytochrome P450 isoenzyme 2C19.
Experimental Embodiment 5: Cynomolgus Monkey In Vivo DMPK Study
[0379] 1. Test Purpose
[0380] Male cynomolgus monkeys were used as test animals, and the plasma concentration of the compound was determined after a single oral administration and the pharmacokinetic behavior was evaluated.
[0381] 2. Experimental Method
[0382] Two healthy male cynomolgus monkeys (fasting) were selected as the oral group. Oral vehicle was 20% hydroxypropyl-p-cyclodextrin aqueous solution. After the test compound was mixed with the vehicle, vortexed and sonicated to prepare 0.1 mg/mL approximately clear solution. The oral dosage of cynomolgus monkey was 0.5 mg/kg. After oral administration, whole blood was collected for a certain time to prepare plasma. The drug concentration was analyzed by LC-MS/MS method, and the pharmacokinetic parameters were calculated by Phoenix WinNonlin 6.3. See Table 4 for the results.
TABLE-US-00008 TABLE 4 PK test results of the compound of the present disclosure Oral DNAUC Compound C.sub.max (nM) (nM .Math. h/mpk) T.sub.1/2 (h) 004 132 1056 3.94 Note: C.sub.max was the maximum concentration; DNAUC = AUC.sub.PO/Dose, AUC.sub.PO was the oral exposure, Dose was the drug dose; T.sub.1/2 was the half-life.
[0383] Conclusion: The compound of the present disclosure has a higher oral exposure in cynomolgus monkeys in vivo.