Quinoline derivative, pharmaceutical composition and application of pharmaceutical composition in medicine

Abstract

The invention relates to a quinoline derivative as shown in a general formula (I), a pharmaceutical composition and application of the quinoline derivative in medicine. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a quinoline derivative, a pharmaceutical composition and their applications in medicine. Background Art

[0002] Toll-like receptors (TLRs) are a class of pattern recognition receptors that are widely distributed in different tissues, monitor and recognize different pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), and play important roles in both innate immunity and acquired immunity.

[0003] TLRs belong to type I transmembrane proteins. So far, 13 TLR family members have been discovered, 10 of which exist in humans. TLR1, TLR2, TLR4, TLR5, TLR6, TLR10 and TLR11 are located on the cell membrane and can recognize substances such as lipids and lipoproteins of microorganisms; while TLR3, TLR7, TLR8 and TLR9 are located in intracellular vesicular structures (such as lysosomes, endosomes and endoplasmic reticulum, etc.) and recognize nucleic acids of microorganisms.

[0004] TLR7 and TLR8 are the most similar in sequence and function. A large number of studies have shown that the activation of TLR7 / 8 can trigger type I interferon responses and various inflammatory reactions. In the case of autoimmune disorders such as systemic lupus erythematosus (SLE), the abnormal and continuous activation of TLR7 / 8 leads to the deterioration of the disease state. Therefore, developing compounds with selective and potent inhibitory activities to inhibit the over-activated immune response by antagonizing TLR7 / 8 is expected to become a new method for treating autoimmune diseases. Summary of the Invention

[0005] The object of the present invention is to provide a new quinoline derivative or stereoisomer, its pharmaceutical composition and its application in the preparation of drugs for autoimmune diseases.

[0006] One or more embodiments of the present invention provide a compound represented by the general formula (I), or all of its stereoisomers, pharmaceutically acceptable salts or deuterated compounds:

[0007]

[0008] Wherein:

[0009] X is C or N;

[0010] R0 is halogen, D, -OH or C 1-6 alkyl; n is a natural number from 0 to 2.

[0011] One or more embodiments of the present invention provide a compound represented by the general formula (I), or all of its stereoisomers, pharmaceutically acceptable salts or deuterated compounds, and the compounds are selected from one of the following structures:

[0012]

[0013] One or more embodiments of the present invention provide a pharmaceutical composition, and the pharmaceutical composition includes:

[0014] A compound represented by the general formula (I), or its stereoisomers, pharmaceutically acceptable salts or deuterated compounds;

[0015] Optionally one or more other active ingredients; and

[0016] A pharmaceutically acceptable carrier and / or excipient.

[0017] One or more embodiments of the present invention provide the use of the above pharmaceutical composition, the compound of the general formula (I), or its stereoisomers, pharmaceutically acceptable salts or deuterated compounds in the preparation of drugs for autoimmune diseases. Detailed Embodiments

[0018] The following examples illustrate the technical solutions of the present invention in detail, but the protection scope of the present invention includes but is not limited to this.

[0019] Intermediate A

[0020] (3-(8-Cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxylic acid ethyl ester

[0021] ethyl-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxylate

[0022]

[0023] The first step:

[0024] Ethyl 1-benzyl-4-(trifluoromethyl)-2,5-dihydro-1H-pyrrole-3-carboxylate A-1.3

[0025] ethyl-1-benzyl-4-(trifluoromethyl)-2,5-dihydro-1H-pyrrole-3-carboxylate

[0026] Under a N2 atmosphere, dissolve A-1.1 (10 g, 60 mmol) in 30 mL of dichloromethane (DCM). Dropwise add a dichloromethane solution (10 mL) of A-1.2 (14.4 g, 60 mmol) dropwise under an ice bath, and then slowly add a dichloromethane solution (10 mL) of trifluoroacetic acid (684 mg, 6 mmol). Stir at room temperature for 2 h. Add the reaction solution to 30 mL of water, extract with DCM three times, wash the organic phase with 30 mL of saturated brine, dry over anhydrous sodium sulfate, rotary evaporate, and purify by silica gel column chromatography (ethyl acetate: petroleum ether = 1:10) to obtain the target product ethyl 1-benzyl-4-(trifluoromethyl)-2,5-dihydro-1H-pyrrole-3-carboxylate A-1.3 (yellow oily liquid, 15.5 g, yield 86%), which is directly used in the next step of the reaction.

[0027] 1 1H NMR (400 MHz, DMSO-d6) δ 7.34 - 7.24 (m, 5H), 4.19 (q, 2H), 3.79 - 3.78 (m, 6H), 1.20 (t, 3H).

[0028] LC-MS m / z (ESI) = 300.1 [M+1].

[0029] The second step:

[0030] ethyl 3-benzyl-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxylate A-1.4

[0031] ethyl-3-benzyl-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxylate

[0032] Under a N2 atmosphere, dissolve trimethylsulfoxonium iodide (3.5 g, 15.8 mmol) in 10 mL of dimethyl sulfoxide. Add a dimethyl sulfoxide solution (5 mL) of sodium hydride (633.6 mg, 15.8 mmol) in batches under an ice bath, and stir at room temperature for 30 minutes. Then add a dimethyl sulfoxide solution (5 mL) of A-1.3 (4.3 g, 14.4 mmol) dropwise, and react at 60 °C for 5 h. Quench the reaction with saturated ammonium chloride, extract with 30 mL of DCM, wash with 30 mL of saturated brine, dry the organic phase over anhydrous sodium sulfate, rotary evaporate, and purify by silica gel column chromatography (ethyl acetate: petroleum ether = 1:10) to obtain the target product ethyl 3-benzyl-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxylate A-1.4 (colorless oily liquid, 3.5 g, yield 78%), which is directly used in the next step of the reaction.

[0033] 11H NMR (400 MHz, DMSO-d6) δ 7.34 - 7.24 (m, 5H), 4.11 (q, 2H), 3.66 (s, 2H), 3.08 - 3.01 (m, 2H), 2.86 - 2.82 (m, 1H), 2.65 - 2.62 (m, 1H), 1.82 - 1.79 (m, 2H), 1.15 (t, 3H).

[0034] LC-MS m / z (ESI) = 314.1 [M+1].

[0035] Step 3:

[0036] ethyl-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxylate A-1.5

[0037] ethyl-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxylate

[0038] Dissolve A-1.4 (1 g, 3.2 mmol) in 50 mL of ethanol, then add Pd / C (681 mg, 0.64 mmol). The reaction system is replaced with 1 atm H2 twice, and the temperature is raised to 60 °C for reaction for 3 h. Filter through diatomaceous earth, rotary evaporate the solvent to obtain the target product ethyl-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxylate A-1.5 (colorless oily liquid, 1 g, yield 88%), which is directly used for the next step.

[0039] 1 1H NMR (400 MHz, DMSO-d6) δ 4.11 (q, 2H), 3.20 - 2.75 (m, 5H), 1.75 (d, 1H), 1.48 - 1.47 (m, 1H), 1.16 (t, 3H).

[0040] LC-MS m / z (ESI) = 224.1 [M+1].

[0041] Step 4:

[0042] (3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxylate A

[0043] ethyl-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxylate

[0044] Under a nitrogen atmosphere, 5-bromoquinoline-8-carbonitrile A-1.6 (654 mg, 2.9 mmol) was dissolved in 30 mL of 1,4-dioxane. Subsequently, A-1.5 (804 mg, 3.5 mmol) was added. The gas was replaced with nitrogen three times. Cesium carbonate (4.3 g, 13.05 mmol) and RuPhosPdG3 (486 mg, 0.58 mmol) were added successively. The gas was replaced with nitrogen three times. The temperature was raised to 90 °C and the reaction was carried out for 2 h. The solvent was evaporated under reduced pressure, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The crude product A was directly used in the next step.

[0045] LC-MS m / z(ESI)=376.1[M+1],398.1[M+23].

[0046] Intermediate B-2: (1R,5S)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxylic acid

[0047]

[0048] The first step:

[0049] 3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxylic acid B

[0050] 3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxylic acid

[0051] The crude product of A (2.02 g, 5.39 mmol) was dissolved in 10 mL of tetrahydrofuran solution. A 10 mL aqueous solution of lithium hydroxide anhydrous (1.29 g, 53.9 mmol) was added dropwise to the reaction solution, and the mixture was stirred at room temperature overnight. After the reaction was completed, the tetrahydrofuran was evaporated under reduced pressure, extracted with ethyl acetate, and the aqueous phase was retained. The pH of the aqueous phase was adjusted to 3-4 with 2M hydrochloric acid aqueous solution, then extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under vacuum. MPLC separation (acetonitrile: water = 47:53) gave the target product 3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxylic acid compound B (yellow solid, 387 mg, 21%).

[0052] 1 1H NMR (400 MHz, DMSO-d6) δ 13.26 (s, 1H), 9.02 - 9.00 (m, 1H), 8.64 - 8.62 (m, 1H), 8.16 (d, J = 8.0 Hz, 1H), 7.61 (dd, 1H), 7.25 (d, 1H), 4.05 - 3.77 (m, 4H), 2.07 - 1.86 (m, 2H).

[0053] LC-MS m / z (ESI) = 348.1 [M+1], 370.1 [M+23].

[0054] Step 6:

[0055] (1S,5R)--3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxylic acid Intermediate B-1

[0056] (1R,5S)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxylic acid Intermediate B-2

[0057] Compound B-1 and Compound B-2 were resolved by chiral prep-HPLC. Analytical method: Chiral column Ig-3, methanol as the mobile phase, flow rate 1 mL / min, retention time of Intermediate B-1 was 3.619 min, and retention time of Intermediate B-2 was 4.741 min.

[0058] Example 1

[0059] (1S,5R)-3-(8-cyanoquinolin-5-yl)-N-((3R,4S)-4-fluoropyrrolidin-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide Compound 1

[0060] (1S,5R)-3-(8-cyanoquinolin-5-yl)-N-((3R,4S)-4-fluoropyrrolidin-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide

[0061]

[0062] Step 1:

[0063] (3R,4S)-3-((1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamido)-4-fluoropyrrolidine-1-carboxylate 1-2

[0064] tert-butyl (3R,4S)-3-((1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamido)-4-fluoropyrrolidine-1-carboxylate

[0065] The raw material (1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxylic acid A-1 (174 mg, 0.5 mmol) was added to dichloromethane (5 ml), then N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (285 mg, 0.75 mmol) and N,N-diisopropylethylamine (129 mg, 1.0 mmol) were added. The reaction mixture was stirred at room temperature for 0.5 h, then (3R,4S)-3-amino-4-fluoropyrrolidine-1-carboxylate tert-butyl ester 1-1 (102 mg, 0.5 mmol) was added. The reaction mixture was stirred at room temperature for 6 h. After the reaction was completed, dichloromethane was directly removed by concentration under reduced pressure, and then purified by reverse chromatography column to obtain (3R,4S)-3-((1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamido)-4-fluoropyrrolidine-1-carboxylate tert-butyl ester 1-2 (200 mg, yield 74.9%).

[0066] LCMS m / z (ESI) = 534.2 [M+1].

[0067] Step 2:

[0068] (1S,5R)-3-(8-cyanoquinolin-5-yl)-N-((3R,4S)-4-fluoropyrrolidin-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide (1S,5R)-3-(8-cyanoquinolin-5-yl)-N-((3R,4S)-4-fluoropyrrolidin-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide

[0069] The raw material (3R, 4S)-3-((1S, 5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide)-4-fluoropyrrolidine-1-carboxylic acid tert-butyl ester 1-2 (200 mg, 0.37 mmol) was added to a mixed solution of dichloromethane (5 ml) and trifluoroacetic acid (5 ml), and the reaction mixture was reacted at room temperature for 2 hours. After the reaction was completed, it was directly concentrated, and the crude product was purified by reverse column chromatography to obtain (1S, 5R)-3-(8-cyanoquinolin-5-yl)-N-((3R, 4S)-4-fluoropyrrolidine-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide compound 1 (60 mg, yield 36.9%).

[0070] LCMS m / z (ESI) = 434.2 [M+1].

[0071] 1 H NMR(400MHz,DMSO-d6)δ9.01(dd,1H),8.64(dd,1H),8.29(d,1H),8.17(d,1H),7.60(dd,1H),7.23(d,1H),4.93(dt,1H),4.27–4 .08(m,1H),4.06–3.79(m,4H),3.63–3.39(m,1H),3.29–3.11(m,1H),3.07–2.76(m,2H),2.64(t,1H),1.95(d,1H),1.66(d,1H).

[0072] 19 F NMR(377MHz,DMSO-d6)δ-63.67,-189.76.

[0073] Example 2

[0074] (1S,5R)-3-(8-cyanoquinolin-5-yl)-N-((3R,4R)-4-fluoropyrrolidin-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide compound 2

[0075] (1S,5R)-3-(8-cyanoquinolin-5-yl)-N-((3R,4R)-4-fluoropyrrolidin-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide

[0076]

[0077] Step 1:

[0078] tert-butyl(3R,4R)-3-((1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamido)-4-fluoropyrrolidine-1-carboxylate 2-2

[0079] tert-butyl(3R,4R)-3-((1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamido)-4-fluoropyrrolidine-1-carboxylate

[0080] Dissolve (1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxylic acid A-1 (695 mg, 2 mmol) in 10 mL of DMF, then add HATU (1.5 g, 4 mmol) and DIEA (388 mg, 6 mmol), stir at room temperature for 15 minutes, and then add tert-butyl(3R,4R)-3-amino-4-fluoropyrrolidine-1-carboxylate 2-1 (408 mg, 2 mmol). Monitor the reaction by LCMS until completion. Extract with ethyl acetate and water. Evaporate the organic phase to dryness to obtain the crude product tert-butyl(3R,4R)-3-((1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamido)-4-fluoropyrrolidine-1-carboxylate 2-2 (pale yellow oil, 1 g).

[0081] LC-MS m / z(ESI)=534.10[M+H] + .

[0082] Step 2:

[0083] (1S,5R)-3-(8-Cyanoquinolin-5-yl)-N-((3R,4R)-4-fluoropyrrolidin-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide Compound 2

[0084] (1S,5R)-3-(8-cyanoquinolin-5-yl)-N-((3R,4R)-4-fluoropyrrolidin-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide

[0085] Dissolve tert-butyl (3R,4R)-3-((1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamido)-4-fluoropyrrolidine-1-carboxylate 2-2 (1 g, 2 mmol) in 20 mL of dichloromethane, then add 10 mL of trifluoroacetic acid, stir at room temperature for 1 hour, monitor the reaction by LCMS until completion, directly concentrate the reaction solution, and purify by reverse-phase C18 column chromatography (alkali method) to obtain the target compound (1S,5R)-3-(8-cyanoquinolin-5-yl)-N-((3R,4R)-4-fluoropyrrolidin-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide Compound 2 (pale yellow solid, 380 mg, 45%).

[0086] 1 H NMR (400 MHz, DMSO-d6) δ 9.01 (m, 1H), 8.63 (m, 1H), 8.26 (m, 1H), 8.17 (d, 1H), 7.60 (m, 1H), 7.23 (d, 1H), 4.98–4.75 (m, 1H), 4.15 (d, 1H), 4.05–3.92 (m, 3H), 3.81 (d, 1H), 3.15 (m, 1H), 3.00–2.86 (m, 2H), 2.61–2.53 (m, 2H), 1.99 (m, 1H), 1.67 (d, 1H).

[0087] LC-MS m / z (ESI) = 434.10 [M+H] + .

[0088] Example 3

[0089] (1S,5R)-3-(8-Cyanoquinolin-5-yl)-N-((3S,4S)-4-fluoropyrrolidin-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]

[0090] Hexane-1-carboxamide compound 3

[0091] (1S,5R)-3-(8-cyanoquinolin-5-yl)-N-((3S,4S)-4-fluoropyrrolidin-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide

[0092]

[0093] Step 1:

[0094] tert-butyl(3S,4S)-3-((1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamido)-4-fluoropyrrolidine-1-carboxylate 3-2

[0095] tert-butyl(3S,4S)-3-((1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamido)-4-fluoropyrrolidine-1-carboxylate

[0096] Dissolve (1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxylic acid A-1 (695 mg, 2 mmol) in 10 mL of DMF. Subsequently, add HATU (1.5 g, 4 mmol) and DIPEA (388 mg, 6 mmol) in an ice bath and stir at low temperature for 15 minutes. Then add tert-butyl(3S,4S)-3-amino-4-fluoropyrrolidine-1-carboxylate 3-1 (408 mg, 2 mmol) and stir for 1 hour. Monitor the reaction by LCMS until completion. Extract with ethyl acetate and water. Rotate the organic phase to dryness to obtain the crude product tert-butyl(3S,4S)-3-((1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamido)-4-fluoropyrrolidine-1-carboxylate 3-2 (pale yellow oil, 1 g).

[0097] LC-MS m / z(ESI) = 534.10[M+H] + .

[0098] Step 2:

[0099] (1S,5R)-3-(8-cyanoquinolin-5-yl)-N-((3S,4S)-4-fluoropyrrolidin-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide

[0100] Hexane-1-carboxamide compound 3

[0101] (1S,5R)-3-(8-cyanoquinolin-5-yl)-N-((3S,4S)-4-fluoropyrrolidin-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide

[0102] (1S,5R)-3-(8-cyanoquinolin-5-yl)-N-((3S,4S)-4-fluoropyrrolidin-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide was obtained as follows: Dissolve tert-butyl (3S,4S)-3-((1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamido)-4-fluoropyrrolidine-1-carboxylate 3-2 (1 g, 2 mmol) in 20 mL of dichloromethane, then add 10 mL of trifluoroacetic acid, stir at room temperature for 1 hour, monitor the reaction by LCMS until completion, directly concentrate the reaction solution, and purify by reverse-phase C18 column chromatography (alkali method) to obtain the target compound (1S,5R)-3-(8-cyanoquinolin-5-yl)-N-((3S,4S)-4-fluoropyrrolidin-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide (pale yellow solid, 430 mg, 50%).

[0103] 1 H NMR (400 MHz, DMSO-d6) δ 9.01 (m, 1H), 8.64 (m, 1H), 8.28 (d, 1H), 8.17 (d, 1H), 7.60 (m, 1H), 7.23 (d, 1H), 4.87 (m, 1H), 4.15 (m, 1H), 3.97 (m, 3H), 3.82 (d, 1H), 3.15 (m, 1H), 2.97 (d, 1H), 2.89 (d, 1H), 2.57 (m, 1H), 1.98 (d, 1H), 1.67 (d, 1H).

[0104] LC-MS m / z (ESI) = 434.20 [M+H] + .

[0105] Example 4

[0106] (1S,5R)-3-(8-cyanoquinolin-5-yl)-N-(4,4-difluoropyrrolidin-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide compound 4

[0107] (1S,5R)-3-(8-cyanoquinolin-5-yl)-N-(4,4-difluoropyrrolidin-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide

[0108]

[0109] Step 1:

[0110] tert-butyl 4-((1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamido)-3,3-difluoropyrrolidine-1-carboxylate 4-2

[0111] tert-butyl 4-((1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamido)-3,3-difluoropyrrolidine-1-carboxylate

[0112] Dissolve (1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxylic acid A-1 (695 mg, 2 mmol) in 10 mL of DMF. Subsequently, add HATU (1.5 g, 4 mmol) and DIPEA (388 mg, 6 mmol) under ice bath and stir at low temperature for 15 minutes. Then add tert-butyl 4-amino-3,3-difluoropyrrolidine-1-carboxylate (444 mg, 2 mmol) and stir for 1 hour. Monitor the reaction by LCMS until completion, and purify by reverse-phase C18 column chromatography (acid method) to obtain the target compound tert-butyl 4-((1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamido)-3,3-difluoropyrrolidine-1-carboxylate 4-2 (white solid, 1 g, 90%).

[0113] LC-MS m / z (ESI) = 551.10 [M+H] + .

[0114] Step 2:

[0115] (1S,5R)-3-(8-cyanoquinolin-5-yl)-N-(4,4-difluoropyrrolidin-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide compound 4

[0116] (1S,5R)-3-(8-cyanoquinolin-5-yl)-N-(4,4-difluoropyrrolidin-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide

[0117] Dissolve tert-butyl 4-((1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamido)-3,3-difluoropyrrolidine-1-carboxylate 4-2 (1 g, 1.8 mmol) in 20 mL of dichloromethane, then add 10 mL of trifluoroacetic acid, stir at room temperature for 1 hour, monitor the reaction by LCMS until completion, directly concentrate the reaction solution, and purify by reverse-phase C18 column chromatography (alkali method) to obtain the target compound (1S,5R)-3-(8-cyanoquinolin-5-yl)-N-(4,4-difluoropyrrolidin-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide compound 4 (pale yellow solid, 430 mg, 50%).

[0118] 1 H NMR (400 MHz, DMSO-d6) δ 9.01 (m, 1H), 8.64 (m, 1H), 8.36 (m, 1H), 8.18 (d, 1H), 7.61 (m, 1H), 7.24 (m, 1H), 4.42 (m, 1H), 4.09 - 3.90 (m, 3H), 3.84 (m, 1H), 3.20 (m, 2H), 3.01 - 2.85 (m, 1H), 2.71 (m, 1H), 1.99 (m, 1H), 1.70 (d, 1H). LC-MS m / z (ESI) = 452.10 [M+H] + .

[0119] Example 5

[0120] (1S,5R)-3-(8-cyanoquinolin-5-yl)-N-((3S,4R)-4-fluoropyrrolidin-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide compound 5

[0121] (1S,5R)-3-(8-cyanoquinolin-5-yl)-N-((3S,4R)-4-fluoropyrrolidin-3-yl)-5-

[0122] (trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide

[0123]

[0124] Step 1:

[0125] (3S,4R)-3-((1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamido)-4-fluoropyrrolidine-1-carboxylate 5-2

[0126] tert-butyl (3S,4R)-3-((1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamido)-4-fluoropyrrolidine-1-carboxylate

[0127] The starting material (1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxylic acid A-1 (174 mg, 0.5 mmol) was added to dichloromethane (5 ml), followed by N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (285 mg, 0.75 mmol) and N,N-diisopropylethylamine (129 mg, 1.0 mmol). The reaction mixture was stirred at room temperature for 0.5 h, then (3R,4S)-3-amino-4-fluoropyrrolidine-1-carboxylate tert-butyl ester (102 mg, 0.5 mmol) was added. The reaction mixture was stirred at room temperature for 6 h. After completion of the reaction, dichloromethane was directly removed by concentration under reduced pressure, and then purified by reverse-phase chromatography column to obtain tert-butyl (3S,4R)-3-((1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamido)-4-fluoropyrrolidine-1-carboxylate 5-2 (200 mg, yield 74.9%).

[0128] LCMS m / z (ESI) = 534.2 [M+1].

[0129] Step 2:

[0130] (1S,5R)-3-(8-cyanoquinolin-5-yl)-N-((3S,4R)-4-fluoropyrrolidin-3-yl)-5-

[0131] (Trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide compound 5

[0132] (1S,5R)-3-(8-Cyanoquinolin-5-yl)-N-((3S,4R)-4-fluoropyrrolidin-3-yl)-5-

[0133] (trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide

[0134] The starting material tert-butyl (3S,4R)-3-((1S,5R)-3-(8-cyanoquinolin-5-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamido)-4-fluoropyrrolidine-1-carboxylate 5-2 (200 mg, 0.37 mmol) was added to a mixed solution of dichloromethane (5 ml) and trifluoroacetic acid (5 ml). The reaction mixture was stirred at room temperature for 2 hours. After completion of the reaction, the mixture was directly concentrated, and the crude product was purified by reverse-phase chromatography to obtain (1S,5R)-3-(8-cyanoquinolin-5-yl)-N-((3S,4R)-4-fluoropyrrolidin-3-yl)-5-(trifluoromethyl)-3-azabicyclo[3.1.0]hexane-1-carboxamide compound 5 (118 mg, yield 72.6%).

[0135] LCMS m / z (ESI) = 434.2 [M+1].

[0136] 1 H NMR (400 MHz, DMSO-d6) δ 9.01 (d, 1H), 8.64 (d, 1H), 8.23 (d, 1H), 8.17 (d, 1H), 7.60 (dd, 1H), 7.23 (d, 1H), 4.93 (dt, 1H), 4.15–4.06 (m, 2H), 3.96 - 3.92 (m, 2H), 3.84 - 3.81 (m, 1H), 3.30–3.11 (m, 2H), 3.07–2.76 (m, 2H), 2.64 (t, 1H), 2.04 (d, 1H), 1.65 (d, 1H).

[0137] 19 F NMR (377 MHz, DMSO-d6) δ -63.76, -189.69.

[0138] Biological test

[0139] HEK-Blue-hTLR7 / 8 / 9 cell inhibition assay

[0140] 1. Seed HEK-Blue-hTLR7 / 8 cells (1×10 4 cells / well) and HEK-Blue-hTLR9 cells (1.5×10 4 cells / well) into a 384-well cell culture plate, with a volume of 30 μL per well. Incubate in a 37 °C, 5% CO2 incubator for 4 h.

[0141] 2. Prepare the compounds with DMSO and dilute them 10-fold (1:3 dilution) with DMEM medium to final concentrations of 10000, 3333.3, 1111.1, 370.4, 123.5, 41.2, 13.7, 4.6, 1.5, 0.5 nM, respectively; prepare R848 with DMSO and dilute it with DMEM medium to final concentrations of 0.8 μM (for HEK-Blue-hTLR7) and 3 μM (for HEK-Blue-hTLR8); prepare ODN2006 with endotoxin-free water and dilute it with DMEM medium to a final concentration of 1 μM.

[0142] 3. Use cells treated with DMSO as the blank control group. At the same time, set up cells treated with Resiquimod (R848) alone as the positive control group for HEK-Blue-hTLR7 / 8, and cells treated with ODN2006 alone as the positive control group for HEK-Blue-hTLR9. Use cells treated with the test compound and R848 or ODN2006 as the test groups. Set up 2 parallel wells for each group and incubate in a 37 °C, 5% CO2 incubator.

[0143] 4. After 4 h of incubation, take out the 384-well plate of HEK-Blue-hTLR7 / 8 from the incubator and add R848 alone to each well or add both R848 and the diluted compound simultaneously. Incubate in a 37 °C, 5% CO2 incubator for 16 h; take out the 384-well plate of HEK-Blue-hTLR9 from the incubator and add ODN2006 alone to each well or add both ODN2006 and the diluted compound simultaneously. Incubate in a 37 °C, 5% CO2 incubator for 16 h.

[0144] 5. After 16 h of incubation, take out the 384-well plate from the incubator, centrifuge at 1000 rpm for 1 minute, and use a multi-functional microplate reader to read the optical density value of each well at 620 nm.

[0145] 6. Calculate the cell inhibition rate = (1 - (OD620 test group - OD620 blank group) / (OD620 positive group - OD620 blank group)) × 100%, and calculate the half maximal inhibitory concentration (IC 50 ) by curve fitting.

[0146] Table 1: Activity of the Compounds of the Invention in HEK-Blue-hTLR7 / 8 / 9 Cell Assays

[0147] Compound <![CDATA[TLR7IC 50 (nM)]]> <![CDATA[TLR8IC 50 (nM)]]> <![CDATA[TLR9IC 50 (nM)]]> Compound 1 A A C Compound 2 A B C Compound 3 A B C Compound 4 A B C Compound 5 A B C

[0148] Note: A represents 1 nM ≤ IC 50 < 100 nM; B represents 100 nM ≤ IC 50 < 1000 nM; C represents IC 50 ≥ 1000 nM.

[0149] Conclusion: The compounds of the invention have excellent antagonistic activity against HEK-Blue-hTLR7 / 8 cells, with an obvious antagonistic effect, and at the same time have no obvious antagonistic effect on HEK-Blue-hTLR9 cells.

[0150] Human Hepatocyte Stability Experiment

[0151] Experimental Procedure:

[0152] Human hepatocytes (BioIVT, X008000) were used for the hepatocyte stability experiment. The hepatocytes were counted and the viable cell density was determined using AO / PI staining. The cells were diluted to 0.5 × 10 6 viable cells / mL with the culture medium. 198 μL of the hepatocyte suspension was taken and inoculated into a 96-well plate, and then transferred to an incubator for incubation for 10 minutes. Subsequently, 2 μL of a 100 μM test compound or positive control solution was added to the 96-well plate respectively to start the reaction, and then transferred to an incubator for incubation, with 2 replicates set. At 0.5, 15, 30, 60, 90, and 120 minutes after the start of the reaction, 25 μL of the sample was taken out respectively, and 300 μL of acetonitrile containing internal standards IS (100 nM alprazolam, 200 nM caffeine, 200 nM labetalol, and 100 nM tolbutamide) was added to terminate the reaction, and vortexed for 5 minutes. Centrifuge at 3220 g for 45 minutes and take the supernatant. The supernatant was diluted with an equal volume of ultrapure water and then detected by LC / MS / MS.

[0153] Data Analysis:

[0154] The peak area was determined based on the extracted ion chromatogram. Through the regression analysis of the percentage reduction of the test compound versus the time curve, its in vitro half-life (t 1 / 2 ) was determined, and the calculation formula was t 1 / 2 = -0.693 / k. The in vitro intrinsic clearance (in vitro C lint , μL / min / 1 × 10 6 cells) calculation formula was Cl int (in vitro) = -kV / N (V = incubation volume (i.e., 0.2 mL); N = number of hepatocytes per well (i.e., 0.1 × 106 cells)

[0155] The results show that the compounds of the present application have good metabolic stability in human hepatocytes.

[0156] The specification of the present invention describes the specific implementation manners in detail. Those skilled in the art should recognize that the above implementation manners are exemplary and should not be construed as limitations on the present invention. For those skilled in the art, without departing from the principle of the present invention, by making several improvements and modifications to the present invention, the technical solutions obtained by these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A compound represented by the general formula (I), or any stereoisomer, pharmaceutically acceptable salt or deuterated substance thereof: in: X is C or N; R0 is halogen, D, -OH or C 1-6 Alkyl; n is a natural number of 0-2.

2. The compound according to claim 1, or any stereoisomer, pharmaceutically acceptable salt or deuterated substance thereof, wherein the compound is selected from one of the following structures:

3. A pharmaceutical composition, comprising: The compound according to claim 1 or 2, or a stereoisomer, a pharmaceutically acceptable salt or a deuterated substance thereof; optionally one or more other active ingredients; as well as Pharmaceutically acceptable carriers and / or excipients.

4. Use of the compound according to claim 1 or 2, or its stereoisomer, pharmaceutically acceptable salt or deuterated substance, or the pharmaceutical composition according to claim 3 in the preparation of drugs for autoimmune diseases.

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